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Published on 28 April 2015 on http://pubs.rsc.org | doi:10.1039/9781782622499-FP001
Drug Discovery for Schizophrenia
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Drug Discovery for

Schizophrenia
Edited by
Tatiana V. Lipina
Institute of Physiology, Novosibirsk, Russia
Email: lipina@physiol.ru
John C. Roder
Lunenfeld–Tanenbaum Research Institute, Toronto, Canada
Email: roder@lunenfeld.ca
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RSC Drug Discovery Series No. 44
Print ISBN: 978-1-78262-026-6

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ISSN: 2041-3203
A catalogue record for this book is available from the British Library
© The Royal Society of Chemistry 2015

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Preface
We wish to thank our friends, family members, and colleagues who encour-
aged us to create this book, “Drug Discovery for Schizophrenia”. We have
carefully included some of the most important directions in the field of
schizophrenia, and are thankful to all contributors, who generously dedi-
cated their time to create their chapters.
This book was motivated by our desire – and that of all the contributors –
to further understand the mechanisms of schizophrenia and envision future
research on this mental disorder at multiple levels; from epigenetics, genetics,
neurochemistry, neuroimmunology, and animal models to opto-/chemo-genet-
ics or protein–protein interactions. Personally, the main motivation was the wish
to help Dr John Roder’s son, Nathan, who suffers from this mental disorder and
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who was diagnosed in his final year of secondary school.

Hopefully, our scientific attempts will ultimately lead to effective treat-
ments for this complex brain disorder. We believe that consistent analyses of
new findings in the field of schizophrenia will benefit psychiatric neurosci-
ence to unlock this complex brain puzzle.
Tatiana V. Lipina and John C. Roder

Edited by Tatiana V. Lipina and John C. Roder
Published by the Royal Society of Chemistry, www.rsc.org
vii
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Contents
Chapter 1 The Genetics of Schizophrenia 1

James N. Samson and Albert H. C. Wong
1.1 Introduction 1
1.2 What Genetics Can Tell Us about Schizophrenia 2
1.2.1 The Heritability of Schizophrenia 2
1.2.2 The Genetic Architecture of Schizophrenia 3
1.3 The Tools of Genomics 5
1.4 What Genetics Has Told Us about Schizophrenia 7
1.4.1 Common Variation 8
1.4.2 Rare Variation 12
1.4.3 The Future of GWASs 15
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1.5 What Genetics is Telling Us about Schizophrenia 16

1.6 The Limitations of Genetic Studies of Schizophrenia 17
1.7 Conclusion 18
1.8 Definitions 19
References 19
Chapter 2 The Impact of Epigenetics in Schizophrenia Research 28

Peter J. Gebicke-Haerter
2.1 Introduction 28
2.2 Genetic Epidemiology: The Hunt for Genes
Associated with Mental Disorders 29
2.3 Where is the missing heritability? 31
2.4 Epigenetics: A New Memory System in Neurobiology 33
2.4.1 DNA Methylation 34
2.4.2 Histone Methylation 35
2.4.3 Histone Acetylation 37

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2.4.4 Epigenetics of GABA-ergic Neurons and
Neocortical Development 39
2.5 Conclusions 40
References 41
Chapter 3 Developmental Neuroimmune Mechanisms

in Schizophrenia 46
Ulrike Stadlbauer and Urs Meyer
3.1 Introduction 46
3.2 Epidemiological and Translational Studies of
Prenatal Infection and Schizophrenia 47
3.3 The Role of Inflammation in Mediating the
Effects of Maternal Infection in the Offspring 49
3.3.1 The Main Components of the Inflammatory
Response System 49
3.3.2 Neurodevelopmental Effects of Cytokines 52
3.3.3 Epidemiological Evidence for the Role of
Inflammation in Mediating the Effects of
Maternal Infection on the Offspring 53
3.3.4 Experimental Evidence for the Role of
Inflammation in Mediating the Effects
of Maternal Infection on the Offspring 53
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3.4 Fetal Brain Development in the Event of Inflammation 55

3.5 Priming of Long-term Neuroinflammation
by Prenatal Infection and Inflammation 56
3.6 (Latent) Neuroinflammation and Disease Progression 57
3.7 Developmental Neuroinflammation as a
Possible Target for Disease Prevention 59
3.8 Conclusions 60
References 60
Chapter 4 The Self-medication Hypothesis in Schizophrenia:

What Have We Learned from Animal Models? 70
Bernard Le Foll, Enoch Ng, José M. Trigo, and Patricia Di Ciano
4.1 Substance Use and Schizophrenia: Clinical Aspects 70

4.1.1 Epidemiology and Clinical Aspects 70
4.1.2 Human Studies Exploring the Impact of
Nicotine on Cognition in Schizophrenia 72
4.1.3 Human Studies Exploring the Impact of
Cannabis on Cognition in Schizophrenia 73
4.2 Human Studies that Explore the Impact of Smoke
on the Side-effects of Antipsychotic Medications 74
4.3 Animal Models to Study the Self-medication
Hypothesis 75
4.3.1 Modelling Schizophrenia in Animals 75
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Contents xi
4.4 Testing the Predictions of the Self-medication
Hypothesis 78
4.5 Review of Studies Evaluating the Impact of
Nicotine on Animal Models of Schizophrenia 79
4.5.1 Animal Models of Dopamine Hyperactivity 79
4.5.2 Neurodevelopmental Models of Schizophrenia 79

4.5.3 The PPI Model of Schizophrenia 80
4.6 Review of Studies Evaluating the Impact of Cannabi-
noid Agonists on Animal Models of Schizophrenia 81
4.6.1 Cognitive Models 81
4.6.2 Neurodevelopmental Models 82
4.6.3 Impact on Neuroleptic Side-effects 82
4.7 Conclusions 82
References 83
Chapter 5 Modelling Schizophrenia: Strategies for Identifying

Improved Platforms for Drug Discovery 89
John L. Waddington and Colm M. P. O’Tuathaigh
5.1 Introduction 89
5.2 Genetic Architecture of Schizophrenia 90
5.3 Behavioural Models of Schizophrenia 91
5.4 Developing Valid Experimental Models of
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Schizophrenia 93
5.5 Phenotypic Characterisation of Mutant Models
of Schizophrenia: Additional Considerations 94
5.5.1 Sex-Specific Phenotypes and Relevance to
Schizophrenia 94
5.5.2 Incorporating Developmental Clinical
Trajectory into the Phenotyping Strategy 95
5.5.3 Importance of Mechanistic Interrogation
of Phenotypic Effects 95
5.6 NRG1 96
5.6.1 NRG1- and ErbB-Deficient Mutant
Mouse Models 96
5.6.2 Mutant Mouse Models of NRG1 and ErbB
Over-Expression 98
5.6.3 NRG1–ErbB Signalling and Antipsychotic
Drug Discovery 100
5.7 DISC1 101
5.8 Dysbindin 102
5.9 Modelling Gene × Environment Interactions
in Schizophrenia Mutant Models 103
5.10 Modelling Gene × Gene Interactions in
Schizophrenia Mutant Models 104
5.11 Conclusions 105
Acknowledgements 106
References 106
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xii Contents
Chapter 6 Drugs that Target the Glutamate Synapse: Implications
for the Glutamate Hypothesis of Schizophrenia 115
Catharine A. Mielnik and Amy J. Ramsey
6.1 The Glutamate Hypothesis of Schizophrenia 115

6.1.1 Molecular and Cellular Components of the
Glutamate Synapse 116

6.1.2 Evidence for a State of NMDA Receptor
Hypofunction 116
6.2 The Integration of Glutamate, Dopamine and
GABA in Schizophrenia 119
6.2.1 Dopamine and Glutamate 119
6.2.2 GABA and Glutamate 121
6.3 Animal Models of Schizophrenia 122
6.3.1 Preclinical Drug Testing in Animal Models 122
6.3.2 CNTRICS-Based Behavioural Paradigms 123
6.4 Pharmacological Targets to Improve
Glutamatergic Signaling 127
6.4.1 NMDA Receptor 127
6.4.2 Glycine and Serine 127
6.4.3 AMPA Receptor 130
6.4.4 Metabotropic Glutamate Receptors 131
References 133
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Chapter 7 Disrupted-in-Schizophrenia-1 (DISC1) Interactome

and Schizophrenia 141
Tatiana V. Lipina and John C. Roder
7.1 Introduction 141

7.2 Functions of DISC1 Interactome in the Brain 142
7.2.1 Neurodevelopment 142
7.2.2 Neuronal Signalling and Synaptic Plasticity 146
7.2.3 Subcellular Functions 148
7.3 The DISC1 Interactome and Schizophrenia 150
7.3.1 Genetics 150
7.3.2 Neuroanatomical and Neurocognitive
Phenotypes 151
7.4 DISC1 Interactome and Mouse Models of
Schizophrenia 151
7.4.1 DISC1 Mouse Models 153
7.4.2 Mouse Models with Modified DISC1
Interactors 157
7.5 Future Directions 162
References 164
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Contents xiii
Chapter 8 GSK3 Networks in Schizophrenia 173
Jivan Khlghatyan, Gohar Fakhfouri, and
Jean-Martin Beaulieu

8.1.1 GSK3-Regulating Pathways 174
8.2 GSK3 in Schizophrenia 179

8.2.1 Biological Evidence for the AKT-GSK3
Pathway in Schizophrenia 179
8.2.2 AKT-GSK3 and the Pathophysiology
of Schizophrenia 181
8.2.3 Wnt-GSK3 Pathway in Schizophrenia 182
8.2.4 NRG1-GSK3 and BDNF-GSK3 in
Schizophrenia 182
8.2.5 DISC1-GSK3 in Schizophrenia 184
8.3 GSK3 and Antipsychotics 185
8.4 How GSK3 Affects Behavior 186
8.4.1 Circadian Rhythms 186
8.4.2 β-Catenin 187
8.4.3 Microtubules 187
8.4.4 AMPA and NMDA Receptors 188
8.4.5 Dynamin I 188
8.5 Biomarkers 188
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8.5.1 Peripheral Blood Cells and Olfactory

Epithelium 189
8.5.2 MRI 189
8.5.3 Electroretinography 189
8.6 Future Prospects 190
References 191
Chapter 9 Protein Interactions with Dopamine Receptors as

Potential New Drug Targets for Treating Schizophrenia 202
Ping Su, Albert H. C. Wong, and Fang Liu

9.2 Dopamine Receptors 204
9.2.1 D1-Like Dopamine Receptor Interacting
Proteins 204
9.2.2 D2-Like Receptor-Interacting Proteins 217
9.3 Targeting Dopamine Receptor Interactions for
Drug Development of Schizophrenia 226
References 227
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xiv Contents
Chapter 10 Optogenetic and Chemogenetic Tools for Drug
Discovery in Schizophrenia 234
Dennis Kätzel and Dimitri M. Kullmann

10.2 Genetically Targeted Manipulation of Neural Activity 235
10.2.1 Optogenetic Activation 237

10.2.2 Chemogenetic Activation 239
10.2.3 Optogenetic Inhibition 239
10.2.4 Chemogenetic Inhibition 240
10.2.5 Silencing of Synaptic Transmission 241
10.2.6 Ablation of Cells 241
10.2.7 Optochemical Genetics and Optical
Pharmacology 241
10.2.8 Optogenetic Interference with Subcellular
Signalling 242
10.2.9 Chemogenetic Interference with Subcellular
Signalling 243
10.3 Getting Started: How to Bring Optogenetics and
Chemogenetics to the Laboratory 243
10.3.1 General Considerations: Which
Molecular Tools? 243
10.3.2 Implementation in the Laboratory 248
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10.4 Application of Optogenetics and Chemogenetics

in Neurological and Psychiatric Diseases 251
10.4.1 Principal Applications: The Road to
Drug Discovery 251
10.4.2 Optogenetic and Chemogenetic
Investigation of Neurological Diseases 253
10.4.3 Optogenetics and Chemogenetics in
Psychiatric Diseases 254
10.4.4 Optogenetic and Chemogenetic
Pharmacology 261
10.4.5 An Optogenetic View on Brain and
Behaviour? 262
10.5 Conclusion 264
Acknowledgements 264
References 264
Subject Index 273
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CHAPTER 1
Published on 28 April 2015 on http://pubs.rsc.org | doi:10.1039/9781782622499-00001
The Genetics of Schizophrenia

JAMES N. SAMSONa AND ALBERT H. C. WONG*a,b
a
Campbell Family Mental Health Research Institute, Centre for Addiction
and Mental Health, Toronto, Ontario, Canada; bDepartment of Psychiatry,
University of Toronto, Toronto, Ontario, Canada
*E-mail: albert.wong@utoronto.ca
1.1 Introduction
If you know the enemy and know yourself, you need not fear the result of a hun-
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dred battles. If you know yourself but not the enemy, for every victory gained you
will also suffer a defeat. If you know neither the enemy nor yourself, you will suc-
cumb in every battle.
Sun Tzu, The Art of War
The greatest difficulty in finding treatments for schizophrenia is that we

do not know the enemy well enough. Revealing the complex etiology and
pathophysiology of schizophrenia has posed a considerable challenge for
researchers, but improving technology is now enhancing our ability to use the
wellspring of information present in the genome to help find these answers.
It is more than three decades since the first development of genome sequenc-
ing technology,1 and we have come to appreciate the intricate way in which
variations in the genome can influence disease. Genetic research has provided
insights into elucidating the pathophysiology of many diseases,2,3 and also
promises to improve clinical outcomes through personalized treatments and
targeted therapeutics.4–6 Studying the genetics of schizophrenia is important

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2 Chapter 1
to discover genes and pathways that contribute to its development. The hope is
that the symptoms of schizophrenia can be prevented or resolved by targeting
therapeutics at these pathways. Still, treatment is most likely to be adminis-
tered late in the development of the disorder, after diagnosable symptoms have
already presented. By this time, the processes leading to the development of
schizophrenia may have caused permanent changes; for example, alterations
in brain morphology. Genetics can also help us to understand the underlying

pathophysiology of the individual symptoms of schizophrenia, allowing for
the development of targeted therapeutics to improve the lives of patients by
treating symptoms after developmental pathways have become fixed. Whether
to understand developmental processes or symptom pathophysiology, the
study of the genetics of schizophrenia has great potential in helping us to
understand the enemy, and hopefully, eventually, to conquer schizophrenia.
1.2 What Genetics Can Tell Us about Schizophrenia

It is now understood that genes and environment work together to influence
the development of disease. The power of genetics to enable us to under-
stand a disease is dependent upon how much of the variance in liability
is contributed by genes compared to other factors. It is also important to
consider the manner in which genes affect phenotype. The heritability and
genetic architecture of schizophrenia tell us how genetic information can be
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used to understand the disorder.
1.2.1 The Heritability of Schizophrenia

The contribution of genes in determining a given phenotype can be quantified by
estimating heritability. Heritability is a mathematical expression of the amount
of variance in phenotype that is explained by genetic variation. This does not
measure how much phenotypic variation is caused by genes; rather, it reflects
the relative contribution of genetic vs. non-genetic factors in determining phe-
notype. Heritability is estimated by comparing the liability of developing a trait
(schizophrenia, for example) between related and unrelated individuals.7,8 Twin
studies have been invaluable for estimating heritability, as it is easier to differ-
entiate between genetics and shared vs. differential environment in such stud-
ies.9 The concordance in phenotype between monozygotic (MZ) and dizygotic
(DZ) twins gives a measure of the correlation between genotypic variation and
presence of a trait. MZ concordance rates for schizophrenia have been reported
between 41% and 65%, with DZ concordance ranging from 0% to 28%.10,11 Since
DZ twins have approximately half the genetic variance of unrelated individuals,
and MZ twins have identical genomes, heritability can be crudely calculated as
twice the difference in concordance (r) between MZ and DZ twins (see eqn 1.1).8
Heritability(h2 ) = 2(r (MZ ) – r (DZ )) (1.1)
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The Genetics of Schizophrenia 3

Hence, the heritability for schizophrenia estimated from twin studies is
81% (95% confidence interval 73–90%).10–13 A limitation of twin studies is
that subjects are usually recruited from restricted environmental settings,
typically from within the same hospital. Heritability estimates using fam-
ily data from national records are lower, at 64–67%.12,14 This difference may
be due to increased variance in environment and diagnostic interpretations
when using subjects from national records. Either way, schizophrenia is

clearly one of the most heritable neuropsychiatric disorders, demonstrating
that genes have a large role to play.
The high estimated heritability for schizophrenia indicates that the
genome contains information explaining much of the underlying patho-
physiology of the disorder. So far, all the variants taken together from cur-
rent genome-wide association study (GWAS) results have been calculated
to account for 20–40% of the variation in liability for schizophrenia.15–18
Even using lower family-based estimates for comparison, current results
do not account for all of the predicted heritability. The term “missing her-
itability” was coined to describe this discrepancy between the proportion
of phenotypic variation explained by results from genetic studies and the
total estimated heritability.19 Current evidence suggests that we may yet
find a large proportion of this missing heritability within the genome. Part
of the missing heritability may also be due to epigenetic DNA and chro-
matin modifications that alter gene expression without changing DNA
sequence. Increased sample sizes and more complete coverage of variants
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with improved genotyping technologies have already uncovered many

new significant schizophrenia-associated genetic loci. We can be optimis-
tic that continuing efforts in interrogating the human genome will reveal
ever increasing numbers of causal variants. This information promises to
provide key insights into the mechanisms underlying the development of
schizophrenia.
1.2.2 The Genetic Architecture of Schizophrenia

Genetic studies have now discovered enough associated risk variants to give
an empirical view of the genetic architecture of schizophrenia.20–22 Schizo-
phrenia is a complex, highly polygenic disorder with multiple variants
conferring risk. Numerous variants with population frequencies >1% have
been associated with schizophrenia. Alongside these common variants,
rare variants with frequencies <0.1%, resulting from de novo mutations and
large-effect structural variations, are implicated in the disorder. The effect
size of schizophrenia associated variants is inversely proportional to their
population frequency. Effect size encompasses the idea of penetrance. Pene-
trance reflects the amount an individual variant contributes to a phenotype,
with completely penetrant variants guaranteeing the presentation of a trait,
and incomplete and low penetrant variants only incrementally increasing
the probability of possessing that trait.
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4 Chapter 1
The allelic spectrum of schizophrenia risk is depicted in Figure 1.1.
Multiple low effect common variants with odds ratios (ORs) typically <1.3
and moderate to high effect, but still incompletely penetrant rare variants
contribute to risk. Risk variants can combine additively where each locus
adds/subtracts a certain amount of risk, or multiplicatively where a certain
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Figure 1.1 Schizophrenia-associated

loci with risk allele population frequency (AF)
plotted against the effect size, given as the odds ratio (OR) for genotypic
relative risk (GRR). Confidence intervals are not shown. The red trian-
gles depict structural variants and the blue circles depict common vari-
ants. Associated variants tend to cluster into two groups: rare variants
with intermediate effect size and common variants with low effect size.
There are no known mendelian variants for schizophrenia (AF<0.001,
GRR>50), with associated rare variants having intermediate to low
GRR. None of the associated common variants have a GRR>1.5. GWAS:
genome-wide association study. This figure is adapted by permission
from Macmillan Publishers Ltd: Nature Reviews Genetics,20 copyright
(2012).
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number or arrangement of loci must be present to reach a threshold to
increase risk. A purely additive model of gene interaction does not explain
the genetics of schizophrenia.23–25 Therefore, the genetic architecture of
schizophrenia probably involves thousands of risk variants which interact
multiplicatively and are highly susceptible to genetic background, pleiot-
ropy, and, of course, environmental effects. Unaffected individuals probably
carry manageable numbers of risk variants, while the probability of devel-

oping schizophrenia rises sharply for individuals with a high burden of risk
alleles. While this picture suggests that common variants are unlikely to be
essential alone, the multiplicative nature of their effects means that single
risk variants can still exert biologically meaningful effects, depending on
the genetic background.24 Hence, associated common variants still warrant
functional investigation alongside higher risk rare variants. The picture so
far suggests that understanding the genetic causes of schizophrenia will
involve understanding not only the functional relevance of individual genes,
but also how multiple genes interact and converge on molecular pathways
to affect behaviour.
1.3 The Tools of Genomics

New technologies are constantly improving our ability to read the human
genome and detect genetic variation between individuals. Genomes vary
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in many ways, from single nucleotide polymorphisms (SNPs) to more

severe structural variations such as copy number variations (CNVs). SNPs
are the most common type of variation.26 Structural variants have a greater
potential to cause disruptions in genes simply due to their size, and hence
large-effect rare variants tend to be of the structural type; however, other
variants can still have large effects.27 A simple schematic of the types of
variation is given in Figure 1.2. Different techniques and technologies are
better for detecting specific types of variation, so study design and genotyp-
ing methods must be tailored to the type of variation relevant to the specific
research question.
Sequencing remains the gold standard to capture all of the variation in the
genome. Next-generation sequencing (NGS) technology has greatly reduced
the cost of sequencing and is now standard practice.6,28 NGS sequences mil-
lions of DNA fragments in parallel, with bases being identified optically in
real time using “sequencing by synthesis” chemistry, greatly reducing time
and costs.29–31 Third-generation sequencing and nanopore based technolo-
gies are on the horizon; these can sequence single DNA molecules without
the need for amplification or cyclic-sequencing steps.32–36 Sequencing is still
too expensive for large samples, so microarray technology remains the most
used genotyping method in GWASs.37 Modern microarrays can now simul-
taneously interrogate up to 1 million SNPs, and are capable of detecting
CNVs and microsatellites; however, inversions and translocations can only
be detected by comparison of fully sequenced genomes.38–40 Only a fraction
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6
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Figure 1.2 A
schematic of DNA variation. A depicts the different types of DNA variation. Single nucleotide polymorphisms (SNPs) are the
most common type of variation, consisting of the substitution of a single nucleotide for another. Single or small numbers of
nucleotide insertions and deletions also occur. Microsatellites are very common variations in the human genome, consist-
ing of varying numbers of consecutively repeating 2–6 base-pair segments. Microsatellites vary greatly between individuals
due to their high mutation rate. Structural variation denotes types of genetic variation typically >1 kb in size. Copy number
variations (CNVs) are large variations in the copy number of segments of DNA, and can include insertions, deletions and
duplications of specific regions. Translocations and inversions are changes in position and orientation of chromosomal seg-
ments, respectively. These can be related to disease when genes are interrupted at the break sites (either end) of the segments.
Chapter 1
Low copy repeats (LCRs), also called segmental duplications, are segments of DNA which occur in two or more copies with
sequence similarity of >90% in a haploid genome. B is a schematic showing how LCRs can cause non-allelic recombination
events resulting in CNVs. Non-allelic recombination is not the only way CNVs are generated, but it is an important mechanism
in which disease-causing de novo mutations can occur.
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of SNPs can be interrogated on a microarray; however, un-interrogated SNPs
can be mathematically predicted from reference genome data by imputa-
tion, greatly increasing coverage.41–46 High coverage is crucial for detecting
disease variants as it is unlikely that the variants on an array are causal;
rather, they are in linkage disequilibrium with true causal variants. Cheaper
sequencing technologies, improved arrays, and better reference informa-
tion for imputation will continue to increase our ability to find disease-as-
sociated variants.
Study design can greatly affect the ability to find genotype–phenotype
associations. There are two major study designs used in human genet-
ics: case–control and pedigree-based. Simpler case–control studies are
better for finding associations with low effect size, but cannot discrim-
inate between inherited and de novo variations.47–49 More complicated
family-based designs can be used to evaluate linkage (co-segregation of
genotypes and phenotypes from parents to offspring), test for associations,
and identify de novo variants.49,50 Subject choice is important as families
with a history of disease (multiplex pedigrees) may be enriched for rare
causal variants, whereas affected subjects whose families have no history
of disease (simplex pedigrees) may be enriched for de novo variants. It is
also important to consider how data are analyzed. False discovery rate pro-
cedures to correct for multiple testing, test-replication designs, and path-
way analysis for enrichment of functionally-related genes are all clever ways
to increase statistical power without relying on massive sample sizes.49–53
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Carefully considered study designs combined with constantly improving

technologies are already generating results in the search for causal variants
for schizophrenia, and we can expect continued progress in schizophrenia
genetics.
1.4 What Genetics Has Told Us about Schizophrenia

Even in the late 2000s there was a worry that genome-wide screens were
finding no true causal variants for schizophrenia.54,55 While early linkage
studies were beginning to find significant loci, many of the most interest-
ing findings were not replicated, and candidate genes tested from signifi-
cant loci yielded no associations.56–59 Past GWASs yielded few strong results
due to lack of power;55,60,61 however, newer GWASs and mega-analyses with
combined sample sizes in the tens of thousands are now finding numerous
significant associations.17,60,62,63 Furthermore, improving genotyping tech-
nology and analysis techniques are making it possible to determine the role
of rare structural variation and de novo mutations in schizophrenia, and
facilitate the identification of rare associated CNVs.64–67 Many schizophre-
nia risk variants are beginning to show biological relevance and potential as
drug targets. There are now too many associated loci to mention in any ade-
quate detail in this section; however, some of the more interesting results
are highlighted.
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8 Chapter 1
1.4.1 Common Variation

Common SNPs account for a large amount of the variance in liability for
schizophrenia. Using statistical models, it was estimated in 2012 that 23%
of the variation in liability for schizophrenia is accounted for by common
SNPs.16 Just 1 year later, that estimate had increased to at least 32%, and
this number should continue to climb as better-powered studies discover

more significant associations.17 A few of the more robust and interesting
GWAS findings are summarized in Table 1.1. Some of the more interest-
ing themes arising from genetic studies in terms of drug discovery are
detailed below.
1.4.1.1 Receptors
Receptors represent obvious potential therapeutic targets. The DRD1 gene
encoding the D1 dopamine receptor gained strong epidemiologic credibil-
ity from early genetic studies.60 While implicating the dopamine system
was not a new finding, this is an example showing that genetic studies were
corroborating established hypotheses. Common SNPs in the receptor genes
CHRNA7 and GRM3 were also associated with schizophrenia, but, as was typ-
ical with early candidate gene studies, many studies also reported no asso-
ciations.60,68 Nevertheless, concordant evidence supported a role for these
receptors.69,70 CHRNA7 encodes a subunit of the ionotropic α-7 nicotinic ace-
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tylcholine receptor (nAChR). This receptor seems to function mainly to mod-

ulate neurotransmitter release in the striatum.71,72 There is evidence that α-7
nAChR agonists have efficacy in improving cognitive deficits in schizophre-
nia, and may be useful in combination with antipsychotics.73–76 Additionally,
rare variants in CHRNA7 show strong associations with schizophrenia.69,77
GRM3 encodes the mGluR3 subunit of the metabotropic glutamate recep-
tor (mGluR). Allosteric and orthosteric modulators of mGluR2/3 are avail-
able,78,79 and evidence from animals and early clinical trials is beginning to
show that some of these agonists may have efficacy in treating the positive
and negative symptoms of schizophrenia.70,80–83 These examples show how
genetic research with concordant biological evidence allows us to tap new
sources with therapeutic potential.
1.4.1.2 The Major Histocompatibility Complex

Associations within an exceptionally complex genomic region on chromo-
some 6 known as the major histocompatibility complex (MHC) are some of
the most robust and consistent findings for schizophrenia.17,18,63,84–86 This
region contains hundreds of genes in high linkage disequilibrium, thus
making it difficult to identify specific genes underlying associated loci.49,87,88
Nevertheless, examining the general role of the MHC in immune function,
autoimmunity, inflammation, and infection in relation to schizophrenia
suggests intriguing new directions in schizophrenia research.89 Animal and
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Table 1.1 Genome-wide

association study (GWAS) findings for schizophrenia.a

Odds
Candidate gene Index SNP Alleles Freq. ratio p-value Function/relevance Ref.
HLA-DRB9 rs114002140 A/G 0.76 1.17 9.1 × 10−14 MHC class II protein *many other genes in LD 17
C10orf32-AS3MT rs7085104 A/G 0.65 1.11 3.7 × 10−13 Read-through transcript. Associated with 17
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blood pressure, CAD, and aneurysm

MAD1L1 rs6461049 T/C 0.57 1.11 5.9 × 10−13 Mitotic spindle-assembly checkpoint 17
component
MIR137 rs1198588 A/T 0.21 0.89 1.7 × 10−12 MicroRNA. rs1198588 is in LD with DPYD, 17
rs1625579 T/G 0.80 1.12 1.6 × 10−11 which is associated with mental retardation 63
CACNA1C rs1006737 A/G 0.33 1.10 5.2 × 10−12 Voltage gated Ca2+ channel subunit. Associ- 17
ated with ASD, BPD, Timothy syndrome
and Brugada syndrome
CACNB2 rs17691888 A/G 0.11 0.86 1.3 × 10−10 Voltage gated Ca2+ channel subunit. Associ- 17
ated with Brugada syndrome and blood
pressure
TSNARE1 rs4129585 A/C 0.44 1.09 2.2 × 10−10 SNARE binding and SNAP receptor activity 17
Intergenic rs10789369 A/G 0.38 1.10 3.6 × 10−10 Unknown. In LD with lincRNA 17
Intergenic rs7940866 A/T 0.51 0.92 1.8 × 10−9 Unknown. In LD with lincRNA and eQTL for 17
SNX19
QPCT rs2373000 T/C 0.40 1.09 6.8 × 10−9 Human pituitary glutaminyl cyclase 17
SLCO6A1 rs6878284 T/C 0.64 0.92 9.0 × 10−9 Member of the solute carrier organic anion 17
transporter family
ITH3-ITH4 rs2239547 1.12 7.8 × 10−9 Inter-alpha-trypsin inhibitors. Associated 63
with BPD
ZEB2 rs12991836 A/C 0.65 0.92 1.2 × 10−8 Zinc-finger binding transcriptional repressor. 17
Associated with Mowat–Wilson syndrome
and mental retardation
AKT3 rs14403 T/C 0.23 0.91 1.8 × 10−8 Serene/threonine protein kinase. Associated 17
with BPD
(continued)
9
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Table 1.1 (continued)
10
Odds
Candidate gene Index SNP Alleles Freq. ratio p-value Function/relevance Ref.
C12orf65 rs11532322 A/G 0.32 1.09 2.3 × 10−8 Mitochondrial matrix protein. Associated with 17
mental retardation
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SDCCAG8 rs1538774 C/G 0.26 0.92 2.5 × 10−8 Centrosome associated protein 17
VRK2 rs2312147 C/T 0.61 1.09 1.9 × 10−9 Serene/threonine kinase 249
ZNF804A rs1344706 G/T 0.41 1.10 2.5 × 10−11 Zinc-finger containing protein. Associated 250
with BPD
PCGEM1 rs17662626 A/G 0.91 1.20 4.6 × 10−8 lincRNA. Associated with prostate cancer 63
MMP16 rs7004635 G/A 0.18 1.10 2.7 × 10−8 Matrix metalloproteinase. Associated with 63
encephalomyelitis and osteochondrosis
CSMD1 rs10503253 A/C 0.19 1.11 4.1 × 10−8 Complement control protein. Associated with 63
epilepsy
CNNM2 rs7914558 G/A 0.59 1.10 1.8 × 10−9 Cyclin M2. Important in Mg2+ homeostasis 63
NT5C2 rs11191580 T/C 0.91 1.15 1.1 × 10−8 Hydrolase involved in purine metabolism 63
NRGN rs12807809 A/G 0.87 1.12 2.8 × 10−9 Neurogranin, PKC substrate. Associated with 249
Jacobsen syndrome and paraneoplastic cer-
ebellar degeneration
CCDC68 rs12966547 G/A 0.58 1.09 2.6 × 10−10 Coiled-coil containing protein 63
TCF4 rs9960767 A/G 0.58 1.20 4.2 × 10−9 Transcription factor. Associated with Pitt– 249
Hopkins syndrome and Fuchs’ endothelial
dystrophy
a
trong results from most recent GWASs. For simplicity, only one major histocompatibility complex (MHC) association is shown. Functions are from
S
the GeneCards summary database (www.genecards.org). ASD: autism spectrum disorder; BPD: bipolar disorder; CAD: coronary artery disease;
freq.: frequency; LD: linkage disequilibrium; PKC: protein kinase C; SNP: single nucleotide polymorphism.
Chapter 1
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in vitro studies are revealing a role for MHC molecules in neurodevelopment,
neuronal and synaptic plasticity, learning, memory, and behavior.90–94 Fur-
thermore, MHC molecule expression is altered in schizophrenia, and risk
loci within the MHC have been associated with functional effects on brain
morphology and cognition in humans.95–100 MHC associated autoimmune
disorders, infections with certain pathogenic microbes, and prenatal mater-
nal immune activation have been associated with increased schizophrenia

risk;101–111 in addition, neuroinflammation and increased inflammatory
markers are seen in patients with the disorder.112–120 Systematic reviews and
meta-analyses show that anti-inflammatory drugs given in combination with
antipsychotics decrease the severity of schizophrenia symptoms.121–123 A
complete review of the intricate relationship between the immune system
and the central nervous system as it relates to the MHC and schizophrenia
is beyond the scope of this chapter; however, it is clear that associations in
the MHC can inform our understanding. Additionally, it is likely that thera-
peutic strategies involving the immune system will improve the treatment of
schizophrenia.
1.4.1.3 Kinases
Genetic studies have implicated a number of protein kinases in schizophre-
nia. Associated SNPs in AKT1 were found in diverse populations with few neg-
ative reports,124–131 and a strong association was recently found in AKT3.17 The
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encoded protein AKT, also called protein kinase B, is activated downstream

of glutamate signaling in neurons, and mediates phosphoinositide (PI)3
kinase-derived signaling.132,133 Additionally, AKT is upstream of glycogen syn-
thase kinase (GSK)3β, which mediates its effects. SNPs in TAOK2 and MAP2K7
have also been associated with schizophrenia.134,135 Thousand-and-one-amino
acid 2 kinase (TAOK2) regulates cortical neuronal morphology.136 Mitogen acti-
vated protein kinase kinase (MAP2K)7 regulates axon development in the cor-
tex, and knocking out MAP2K7 in mice results in schizophrenia-like behavioral
deficits.135,137 Furthermore, TAOK2 and MAP2K7 are both involved in the c-Jun
N-terminal kinase (JNK) signaling pathway, suggesting a role for this pathway
in schizophrenia.137–139 Lastly, linkage regions 8p21–12 and 2q33.3–34, as well
as SNP and microsatellite variants in ERBB4 and NRG1, have been associated
with schizophrenia.56,57,60,140,141 The encoded protein neuregulin (NRG)1 acti-
vates receptor tyrosine-protein kinase erbB-4 (ERBB4) to initiate downstream
signaling involving JNK, extracellular signal-regulated kinase (ERK) and PI3
kinase pathways. NRG1 is involved in neurodevelopment, and NRG1/ERBB4
signaling is implicated in glutamatergic, γ-aminobutyric acid (GABA)ergic and
dopaminergic neurotransmission.142 NRG1 and ERBB4 function in relation to
schizophrenia has been extensively studied in mouse models.143 Rare variants
in TAOK2 and ERBB4 have also been associated with schizophrenia.67,144–149
Each of these kinases, and their related pathways, is an excellent potential tar-
get for therapeutic intervention. The role of these kinases in schizophrenia
will be elucidated with further research.
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12 Chapter 1
1.4.1.4 Calcium Channels
One of the newest findings from GWASs implicates genes encoding l-type
calcium channel subunits CACNA1C and CACNB2 in schizophrenia. These
channels play a role in learning, memory, and synaptic plasticity, and have
also been associated with autism, bipolar disorder, and the calcium chan-
nelopathies Brugada and Timothy syndromes.150–156 Many approved medi-

cations act on calcium channels; for example, some antipsychotics (e.g.,
pimozide) and adjuvants for non-responders in schizophrenia and bipolar
disorder (e.g., verapamil and nifedipine).157,158 Hence, these associations not
only suggest potential mechanisms for the etiology and pathophysiology of
many neuropsychiatric disorders, but also provide hypotheses for quick clin-
ical translation through the repurposing of approved medications.
1.4.1.5 Non-coding RNAs
One of the strongest recent associations is the MIR137 gene coding the
microRNA miR-137. This is particularly notable as miR-137 functions to reg-
ulate multiple genes by binding target sites present on mRNA.159 It is highly
expressed in the brain, and is an important regulator of neurogenesis and
neuronal maturation.160–163 Genes with predicted miR-137 binding sites were
enriched for lower p-values;17 furthermore, many predicted and confirmed
miR-137-regulated targets reached genome-wide significance, including
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HLA-DQA1, CACNA1C, CACNB2, ZEB2, CSMD1, MAD1L1, DPYD, TCF4, and

many others.17,63,164,165 Hence, miR-137 stands at the top of multiple poten-
tial schizophrenia-associated pathways. In addition to miR137, multiple
regions containing long intergenic non-coding RNAs (lincRNAs) recently
reached genome-wide significance.17 The function of these lincRNAs is not
well understood, but they may have roles in epigenetic regulation and devel-
opment.166 These findings provide a myriad exciting new research directions
to understand schizophrenia and find new therapeutic targets.
1.4.2 Rare Variation

Many rare but potent structural variants have been discovered to have a role in
a small proportion of schizophrenia cases; however, these variants tend to be
non-specific and associate with multiple disorders, such as bipolar disorder,
autism, intellectual disability, epilepsy, and others.49 Finding associated rare
variants presents a unique set of challenges in genetic research. Technological
advances have driven much of the search for rare structural variants.39,40,64 With
decreasing sequencing costs, and new microarrays capable of detecting CNVs,
searching for structural variants in large epidemiological studies is becoming
more feasible. Table 1.2 provides a summary of some of the structural varia-
tions associated with schizophrenia, most likely representing the “low-hang-
ing fruit” of rare variations. These tend to be large, centering on structural
variation hotspots.167 Many of these variants span multiple genes, so further
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Table 1.2 Structural
variation associated with schizophrenia.a
Structural Frequency Frequency in Odds
variant Location (Mb) Genes Type in cases controls ratio p-value Other associations Ref.
−4
1q21.1 chr1: 34 Deletion 0.0018 0.0002 9.5 8 × 10 Developmental delay, intellec- 147
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145.0–148.0 Duplication 0.0013 0.0004 4.5 0.02 tual disability, micro and 147
macrocephaly, dysmorphia,
epilepsy, cataracts, cardiac
defects, possibly ASD185,
thrombocytopenia–absent
radius syndrome
2p16.3 chr2: NRXN1 Deletion 0.0018 0.0002 7.5 1 × 10−6 Developmental delay, intellec- 147
50.1–51.2 exons tual disability, epilepsy, ASD,
Pitt–Hopkins-like syndrome 2
3q29 chr3: 19 Deletion 0.0010 0.0 3.8 4 × 10−4 Developmental delay, intellec- 147
195.7–197.3 tual disability, possibly ASD
7q36.3 chr7: VIPR2 Duplication 0.0024 0.0001 16.4 4 × 10−5 196,
158.8–158.9 147
15q13.3 chr15 : 12 Deletion 0.0019 0.0002 12.1 7 × 10−7 Developmental delay, intellec- 147
30.9–33.5 tual disability, epilepsy, ASD,
ADHD
16p11.2 chr16 : 29 Duplication 0.0031 0.0003 9.5 3 × 10−8 ASD 147
29.5–30.2
17q12 chr17 : 18 Deletion 0.0006 0.0 4.49 3 × 10−4 ASD 192
34.8–36.2
22q11.21 chr22 : 53 Deletion 0.0031 0.0 20.3 7 × 10−13 Developmental delay, intellec- 147
18.7–21.8 tual disability, velocardiofa-
cial–DiGeorge syndrome
a
itations refer to the most comprehensive study rather than the initial report. “Genes” refers to the number from the University of California Santa Cruz
C
(UCSC) Known Genes data set. ADHD: attention-deficit hyperactivity disorder; ASD: autism spectrum disorder. Table is adapted by permission from
Macmillan Publishers Ltd: Nature Reviews Genetics,20 copyright (2012).
13
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14 Chapter 1
research is needed to narrow the focus and understand their functional roles
in disease. Nevertheless, some more specific directions have emerged from
the study of rare variation. More rare variations will be found as new technol-
ogy for detecting structural variation is applied to ever larger samples.
1.4.2.1 Disrupted in Schizophrenia 1 (DISC1)

DISC1 was discovered at the breakpoint of a chromosomal translocation
(t1q42.1; 11q14.3) in a Scottish pedigree which presents with severe psychiat-
ric disorders, including schizophrenia, depression, and bipolar disorder.168,169
While this translocation was not found outside this family, additional asso-
ciations with common and rare variants in DISC1 were found.60,170–173 DISC1
encodes a multifunctional scaffolding protein involved in regulating embry-
onic and adult neurogenesis, and neuronal proliferation, differentiation and
migration.170,174–176 Disrupting DISC1 in animal models causes schizophre-
nia-like behavioral deficits.177–180 Indeed, converging evidence from genetics
and functional studies strongly supports a role for DISC1 in schizophrenia.176
DISC1 interacts with many potential target proteins that are involved in syn-
aptic function, and neurodevelopmental, cytoskeletal, and centrosomal
pathways, some of which are also associated with schizophrenia (e.g., AKT,
DPYSL2, GSK3β, PDE4, and TNIK).173,181–186 While DISC1 itself is not the eas-
iest target for therapeutics, it is an example of hypothesis generation from
genetic research greatly contributing to our understanding of schizophrenia.
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1.4.2.2 Neurexin 1 (NRXN1)
Rare CNVs in NRXN1, including a de novo instance, have been associated with
schizophrenia.67,77,187–190 CNVs in NRXN1 are also associated with autism.191,192
NRXN1 encodes members of the neurexin superfamily of proteins, which are
presynaptic cell adhesion molecules which form heterotypic intercellular
junctions with neurologin across synapses.193 NRXN1 is one of the largest
known human genes, and is regulated through alternative splicing at its six
splice sites yielding numerous isoforms, each with unique binding affinities.
Neurexins are thought to be involved in synapse and neuronal maturation
and have been implicated in neurodevelopmental pathways.194,195 Further-
more, neurexins possess an intracellular PDZ domain which can interact
with many presynaptic proteins.194 The potential role of NRXN1 in schizo-
phrenia remains to be elucidated; hence, NRXN1 represents an interesting
future direction in schizophrenia research.
1.4.2.3 Vasoactive Intestinal Peptide Receptor 2 (VIPR2)

Duplications in the VIPR2 locus at 7q36.2 are strongly associated with schizo-
phrenia,77,196 suggesting changes in gene dosage may affect the disorder. Low
copy repeats at the VIPR2 gene may predispose it to structural variations
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197
(see Figure 1.2). VIPR2 encodes the type II vasoactive intestinal peptide
G-protein coupled receptor (also called VPAC2), which is coupled to adenylate
cyclase activity and expressed in the cerebral cortex and thalamus.198 Changes
in gene dosage causing alterations in VIPR2 signaling could hypothetically
be associated with schizophrenia etiology or symptoms; hence, VIPR2 rep-
resents a good potential target for therapeutics. Future research into VIPR2
gene and protein function will reveal its relationship to schizophrenia.
1.4.2.4 Lessons from Sequencing Studies

Sequencing allows researchers to analyze the genome to a level of detail
that is otherwise unattainable; however, high costs continue to limit the
size of sequencing studies.199 While insufficient statistical power is still pre-
cluding the ability to detect significant loci in these studies, clever analysis
techniques are revealing interesting trends for rare variation. Evaluating the
burden of structural variation in cases compared to controls tests a multi-
genic hypothesis in which many rare but different disruptions contribute to
disease risk. Multiple studies report an increased burden of CNVs in schizo-
phrenia.67,200–202 Additionally, a role for de novo mutations in schizophrenia
has been reported in a number of studies.200,203–207 While the evidence for
increased rates of de novo variation is mixed, de novo variants dispropor-
tionately disrupt genes in schizophrenia populations, suggesting a func-
tional role. Pathway analysis has been used to great effect in schizophrenia
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sequencing studies.67,148,208 Genes within rare variants are significantly

enriched in functionally-related gene sets, mostly composed of synaptic pro-
teins; specifically, the voltage gated calcium ion channel, genes within the
activity-regulated cytoskeleton-associated (ARC) protein signaling complex,
N-methyl-d-aspartate (NMDA) receptor complexes, and glutamatergic post-
synaptic proteins.201,203 A caveat of pathway analysis is that there are many
potential biases that result from incomplete data sets, differences in gene
size, or multiple pathway membership.20,52,53,209 Nevertheless, the results for
schizophrenia appear to be etiologically plausible. The future of sequenc-
ing in genetic studies looks promising; however, caution should be taken
as handling the massive amounts of data from fully sequenced genomes
presents challenges for statistical analysis. The falling cost of sequencing
will allow these technologies to be applied to the large samples needed to
detect specific variants.
1.4.3 The Future of GWASs

Moving forward with studies interrogating the genome for schizophrenia-as-
sociated risk variants, a number of important concepts have emerged. The lack
of results from early studies was primarily due to a lack of statistical power,
and increasing sample size is the easiest remedy for this problem.16,17,21,55
Working out the logistics to handle samples in the tens of thousands and
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16 Chapter 1
beyond will be the challenge for future researchers. Evidence suggests that
heterogeneity across diverse groups may be low for schizophrenia;210,211
hence, planned mega-analyses across world populations may be worth pur-
suing.20 As sample size increases, the number of loci reaching genome-wide
significance will climb. In addition, it is important to understand that many
associated loci may not be truly causal, but simply in linkage disequilibrium
with causal variants. Improved microarrays and reference data for imputa-
tion, as well as cheaper sequencing technologies will improve our ability to
find true causal loci, especially rare variants that are less likely to be included
on commercial microarrays.6 Analysis techniques are also improving. The
increasing body of gene function literature, improved categorization and
annotation of functions, and better algorithms will increase the utility of
pathway analysis as a legitimate tool for supporting hypotheses and corrob-
orating evidence of associations.212 Interestingly, many schizophrenia-asso-
ciated loci are also implicated in other disorders, such as autism spectrum
disorders and bipolar disorder.20,213 Furthermore, schizophrenia associations
are enriched in functionally annotated genes.214 While complicating the pic-
ture for schizophrenia genetics, this information can be used beneficially to
estimate the false discovery rate and improve power using purely statistical
methods.212,215,216 These analysis techniques can be applied to existing data
sets as well as new studies to find associations that were previously hidden
by statistical noise. Increasing sample size, improving technology and statis-
tical techniques, and careful study design will facilitate the search for schizo-
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phrenia risk variants in the future.
1.5 What Genetics is Telling Us about Schizophrenia

Long-held distinctions in psychiatric nosology and diagnostic manuals
have attempted to separate psychiatric illness into defined categories hav-
ing discrete boundaries.217,218 Yet the diagnostic taxonomy of schizophrenia
has always necessitated the use of multidimensional criteria, as many core
symptoms transcend the boundaries of multiple disorders.219–223 The picture
emerging from genetic studies suggests that a paradigm shift may be needed,
moving away from discrete classifications. Certainly, one of the recurrent
themes from Section 1.3 is the non-specific nature of associated loci. Many
of the rare variants in Table 1.2 are associated with developmental disorders,
intellectual disability, and autism. Furthermore, multiple schizophrenia-as-
sociated SNPs show cross-disorder associations with autism spectrum dis-
order, attention-deficit hyperactivity disorder, bipolar disorder, and major
depressive disorder.224 For example, SNPs in CACNA1C, ZNF804A, ITH3-ITH4,
and ANK3 were also associated with bipolar disorder.18,20,213
Phenomena such as epistasis, variable expressivity, pleiotropy, or gene–
environment interactions could explain how different disease states arise
from individual genetic variants. It could be the case that these variants are
mediating the same symptoms in different disorders, and are susceptible to
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effects such as epistasis and environmental interactions which modify the
severity of their presentation. Hence, rather than approaching schizophrenia
as a unified disorder, it may be beneficial from a gene discovery perspective
to focus on individual symptom domains that are likely to be more proximal
to the underlying neural substrates, and therefore genes, than the disorder
as a whole.
Segregating analysis by symptom domains is related to the idea of strat-

ifying genetic analysis based on endophenotypes. Endophenotypes can be
defined as a subset of biomarkers which are heritable and increase the risk
of an illness.225,226 These can be any type of trait; for example, neuroimaging,
electrophysiological, and cognitive variables are all considered endopheno-
types for schizophrenia.227–232 There are a number of advantages to using
endophenotypes: they are simpler and closer to the gene level of action, so
associated variants are more likely to show larger effect sizes; they allow for
easier stratification of populations and quantitative ranking within a diag-
nostic category to increase statistical power by decreasing heterogeneity; and
they are more easily translatable to animal models, so it is easier to perform
functional studies of genes.233
Understanding individual symptom domains or endophenotypes also has
consequences for drug development.234 Many schizophrenia risk variants,
particularly rare variants, affect neurodevelopmental pathways that affect
brain structure.20,231,235 Unless intervention occurs pre-emptively during
development, medications are unlikely to be useful in these pathways if the
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changes in brain structure underlie schizophrenia symptoms. Conversely,

much of the functional disability associated with schizophrenia is due
to cognitive impairment.236–238 Therefore, understanding the genetic and
molecular underpinnings of cognitive symptoms is important for develop-
ing therapeutics that will vastly improve the ability of schizophrenic patients
to function.239,240 While overlap is almost guaranteed between domains such
as brain structure and cognitive impairment, consideration of factors that
affect one but not the other is important for informing efforts into drug
discovery. Endophenotypes are already a useful tool for functional analysis
of schizophrenia risk variants.95,230,231,241,242 The use of endophenotypes or
symptom domains to refine future GWAS analyses will aid the search for new
risk variants and facilitate our understanding of the genetic mechanisms
underlying schizophrenia.
1.6 The
Limitations of Genetic Studies of
Schizophrenia
Genetics is an excellent tool for directing research to improve our under-
standing of schizophrenia. The emerging story from genetics has moved
schizophrenia research in interesting new directions. It has also forced us
to abandon some of our past expectations. A longstanding issue in schizo-
phrenia is the reliance on clinical phenomenology for diagnosis.243 Genetic
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18 Chapter 1
research was expected to be a source of biologically valid markers to improve
diagnosis. Sadly, the picture from genetic research shows that this hope may
go unfulfilled. The genetic architecture of schizophrenia, consisting of thou-
sands of low effect variants and variants demonstrating pleiotropy for other
illnesses, demonstrates that genetic variants are, so far, unlikely to be useful
as diagnostic tools.24
While great progress has been made in accounting for the “missing” heri-
tability in schizophrenia, it should be noted that some of the predicted 81%
may remain missing. Gene–environment interactions tend to be attributed
to heritability in epidemiological studies.244 MZ twins, sharing nearly 100%
of their genes, have a concordance of ∼50%, emphasizing the importance
of environmental input. Furthermore, unusual genomic effects, such as
over-dominance,245 and epigenetic factors have been implicated in contrib-
uting to the heritability of schizophrenia.246–248 Gene–environment interac-
tions can be incorporated into genetic research with careful phenotyping,
and would greatly improve our understanding of schizophrenia risk.243 How-
ever, as it stands, the environment is largely ignored. Finally, it should be
emphasized that genetics, above all, is a tool for hypothesis generation. Bar-
ring unusually strong Mendelian associations, the results of genetic research
give probabilistic associations of varying credibility. Many strongly associ-
ated variants do not fall within genes; rather, they are located in intergenic
regions or within introns, and have unknown functional relevance.17,60,62,63
False positives are always a danger, but rigorous avoidance of potential false
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positives can also eliminate evidence of the most interesting results.20,50,51 It

is important to use discretion when moving forward with results, and as with
all science, multiple lines of convergent evidence are desirable.
1.7 Conclusion
The use of genetic information for drug discovery is still in its early develop-
ment. Progress in schizophrenia research was slow early in the past decade,
but we are now finally approaching sufficient sample sizes to accelerate the
discovery of risk variants. Genetic discoveries have associated a plethora
of genes with schizophrenia, and highlighted the complex, heterogeneous
nature of the disorder. Further research to replicate important findings and
functionally characterize risk genes is important to understand the under-
lying processes involved in schizophrenia. Validation of variants and a func-
tional understanding of how specific mutations lead to the development and
presentation of the disorder are essential for novel drug discovery.
While there is no risk variant yet discovered that has an effect on a signif-
icant number of schizophrenia patients, the major discoveries so far appear
to converge on clinically relevant pathways. These findings have advanced
our understanding of the etiology and pathophysiology of schizophrenia.
Given the large overlap between risk variants for schizophrenia and other
psychiatric disorders, priority should be directed to pathways which underlie
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specific symptom domains that cross current diagnostic categories. Genetic
research will continue to find new associated variants and suggest new direc-
tions for understanding schizophrenia. The challenge for future researchers
will be to weave together the intricate picture from genes to proteins to path-
ways, and finally to the generation of novel therapeutics.
1.8 Definitions
Coverage: the estimated proportion of the genome that can be captured
by SNPs interrogated on an array at a preset correlation threshold.
de novo variant: a causal variant that is the result of a mutation occurring
for the first time within a subject; i.e., it was not inherited.
Epistasis: when the effects of one gene depend on one or more additional
modifier genes (i.e., the genetic background).
Gene–environment interaction: when one gene exerts differential effects
on a phenotype when exposed to different environments.
Odds ratio (OR): the ratio of the probability of an event occurring given
exposure over the probability of an event occurring given no exposure.
Exposure can be to anything; however, in genetic studies it usually refers
to the presence of a certain variant. The OR in the context of genetics is
also called the genotypic relative risk.
Over-dominance: a genetic condition in which the phenotype of the hetero-
zygote lies outside the phenotypic range of the homozygotes at any allele.
10:30:19.
Pleiotropy: when one gene exerts effects on multiple apparently unrelated

phenotypes.
Population prevalence rate: the proportion of a population with a given
trait over a given time.
Statistical power: the probability that a statistical test will reject the null
hypothesis when the alternative hypothesis is true; i.e., the probability of
not committing a type II error. Effectively, it is a measure of the ability of a
study to find true significant results.
Variable expressivity: when individuals with the same genotype express a
phenotype to a different degree from each other.
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206. S. L. Girard, J. Gauthier, A. Noreau, et al., Nat. Genet., 2011, 43, 860.
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209. S. Raychaudhuri, R. M. Plenge, E. J. Rossin, et al., PloS Genet., 2009, 5,
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210. Y. Shi, Z. Li, Q. Xu, et al., Nat. Genet., 2011, 43, 1224.
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212. C. Mitrea, Z. Taghavi, B. Bokanizad, et al., Front. Physiol., 2013, 4, 278.
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214. A. J. Schork, W. K. Thompson, P. Pham, et al., PLoS Genet., 2013, 9,
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215. O. A. Andreassen, S. Djurovic, W. K. Thompson, et al., Am. J. Hum. Genet.,
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216. O. A. Andreassen, W. K. Thompson and A. M. Dale, Schizophr. Bull.,
2014, 40, 13.
217. E. Ivleva, G. Thaker and C. A. Tamminga, Schizophr. Bull., 2008, 34, 734.
218. American Psychiatric Association, Diagnostic and statistical manual of
mental disorders: DSM-5, American Psychiatric Association, Washing-
ton, D.C, 2013.
219. C. Rosen, R. Marvin, J. L. Reilly, et al., Clin. Schizophr. Relat. Psychoses,
2012, 6, 145.
220. J. Endicott, J. Nee, J. Fleiss, J. Cohen, J. B. Williams and R. Simon, Arch.
Gen. Psychiatry, 1982, 39, 884.
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224. Cross-Disorder Group of the Psychiatric Genomics Consortium, Lancet,
2013, 381, 1371.
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226. D. C. Glahn, J. E. Curran, A. M. Winkler, et al., Biol. Psychiatry, 2012, 71, 6.
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Schizophr. Res., 2008, 106, 50.
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237. R. S. E. Keefe, J. A. Sweeney, H. Gu, et al., Am. J. Psychiatry, 2007, 164,
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www.Ebook777.com
CHAPTER 2
The Impact of Epigenetics in

Schizophrenia Research
PETER J. GEBICKE-HAERTER*a,b
a
Central Institute of Mental Health, Institute of Psychopharmacology,
Medical Faculty Mannheim, University of Heidelberg, J5, 68159 Mannheim,
Germany; bProgramme of Molecular and Clinical Pharmacology, ICBM,
Medical Faculty, University of Chile, Av. Independencia 1027,
Santiago 7, Chile
*E-mail: peter.gebicke@zi-mannheim.de, pgebicke-haerter@med.uchile.cl
2.1 Introduction
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More than three decades ago, stimulated by the hype around being able to
sequence the whole genome of any organism and to obtain detailed insights
into its construction plans and errors therein leading to diseases, the hunt
for genes showing mutations, deletions, duplications or copy number vari-
ants specific for a disease was opened. Although it became clear very soon
that the majority of mental disorders are multigenic in origin, the impetus
was unabated and extended to searching for combinations of altered genes
instead of single genes associated with a disorder. Many results have been
published, some of them in highly ranked journals, with huge international
collaborative efforts and comparably large financial support, albeit without
any great breakthrough.

28
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The Impact of Epigenetics in Schizophrenia Research 29
2.2 Genetic
Epidemiology: The Hunt for Genes
Associated with Mental Disorders
This enthusiasm for identifying alterations in the DNA sequence associated
with mental disorders received new support with the advent of next-gen-
eration sequencing methods. The approach tacitly implies that the DNA
sequence entails the complete programme for the development of the brain
and its maintenance, and consequently is the nucleus of any mental illness,
as well. In October 2012, preliminary findings about the 98% of the genome
that does not code for protein were published. These data were assembled
within the so-called ENCODE (Encyclopedia of Functional DNA Elements)
project,1 which involved more than 442 professional scientists from 32 insti-
tutes worldwide. After more than 1600 complex experiments that had been
performed on 147 types of human cells at a cost of more than US $308 mil-
lion, piling up a wealth of new data, but evidently with an open end, this
initiative has been compared to a “runaway train”.2 Nevertheless, the strategy
of sequencing the genome continues with unchecked enthusiasm, especially
with the advent of next-generation sequencing technologies, and is justified
on grounds that the heritability [the proportion of phenotypic variation (VP)
that is due to variation in genetic values (VG)] of mental disorders is high.
However, heritability analysis suffers from the interpretation of high herita-
bility estimates (close to 1.0). These estimates could result from a low sensi-
tivity of the trait to changes in the environment or from a high similarity of
the environment in relevant conditions for the trait. It transpires that <2%
of the 80–90% heritability of major psychiatric diseases, such as schizophre-
nia and manic-depressive disorder, can be attributed to genes identified by
10:30:21.
association or linkage. Some genes have been found in schizophrenia and

autism that link rare mutations with abnormal behavior, but mostly these
are de novo mutations not obtained from a parent. The so-called “missing
heritability”3,4 has caused the value of the first generations of genome-wide
association studies (GWASs) to be questioned and has led to a re-thinking of
standard approaches. One modification of the genetic strategy was the inclu-
sion of gene × environment interactions. Another was the appreciation that
onset and development of these disorders goes beyond the one gene–one
disease approach, encompassing many genes linked to these illnesses (poly-
genic, multifactorial, or multigenic disorders). Over the past three decades,
numerous genetic association and linkage studies have been performed to
understand major psychosis, but they have resulted in poor replication and
identified risk alleles with small effect sizes. Altogether, the results from
these studies have proved to be disappointing until now. There is presently
no single polymorphism that has been reliably associated with any psychi-
atric disorder nor with any aspect of human behavior within the “normal”
range (e.g., differences in “intelligence”). To underline these statements, a
few examples in schizophrenia research are mentioned here. In a large inter-
national collaborative GWAS of 3391 people diagnosed as schizophrenic and
3181 controls, more large chromosomal deletions and duplications were
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30 Chapter 2
5
detected in the schizophrenia samples. There were 13 deletions in chromo-
some 22q11.2 of schizophrenic subjects and none in controls. That region
encompasses about 43 different genes. Nine deletions were identified in
region 15q13.3 of schizophrenic patients and none in controls. Additionally,
10 deletions were found in 1q21.1 (composed of 27 genes) of schizophrenics
and one in control subjects. In another study,6 a three-stage tactic was used.
First, several thousand parent–offspring trios were analyzed to find de novo

copy number variants (CNVs). The 66 CNVs identified in that stage were then
examined again in 1433 schizophrenic subjects and 33 250 controls. Three
of them were enriched in the schizophrenic sample. Those three were then
evaluated in a much larger sample of 3285 schizophrenics and 7951 controls
from six different countries in Europe. Among schizophrenics, 11 (0.23%)
subjects showed deletion of region 1q21.1, 26 (0.55%) subjects had a dele-
tion of 15q11.2, and seven (0.17%) subjects had a deletion of 15q13.3, com-
pared with 0.02%, 0.19%, and 0.02%, respectively among controls. These
findings were reviewed by Sebat et al.7 It transpired that the large chromo-
somal deletions were also identified in a number of other neurological dis-
orders, and the authors concluded that “schizophrenia and other disorders
share overlapping biological pathways”. Additionally, they conceded that
schizophrenia shows marked genetic heterogeneity, that many cases arise
from rare mutations of a gene or region of a chromosome, and “some of
which are unique to a single patient”. These conclusions were supported in
an independent review by St Clair,8 who estimated a probability of 2–4% that
all cases of schizophrenia are traceable to CNVs discovered to date, and that
higher resolution assays may raise this figure to 10–20%.
Furthermore, several recent GWASs using SNPs to search for single gene
10:30:21.
defects in schizophrenia have been published. 3322 schizophrenia cases and

3587 controls were included in the International Schizophrenia Consortium
data set, and analyzed for about 1 million SNPs.9 Another data set (SGENE)
examined 314 868 SNPs in 2663 schizophrenics and 13 498 controls.10 A third
data set (Molecular Genetics of Schizophrenia) investigated 2681 schizo-
phrenics and 2653 controls.11 In these types of studies, performing eval-
uations of high quantities of markers and high numbers of samples, it is
accepted practice to adjust for multiple comparisons and set a criterion for
type I error at p < 5 × 10−8. Not even one marker in any of the three separate
data sets achieved this level. No significant associations were found. The sit-
uation as described here in schizophrenia research is similar to that in other
psychiatric disorders. In conclusion, polygenic inheritance will evade detec-
tion by even the largest practically attainable sample because the individual
genetic effects are so small.12
Comparable to a typical GWAS, hundreds of thousands of polymorphisms
of thousands of persons are scanned in genome-wide complex trait analysis
(GCTA). The method is aimed at assessing whether or not a trait similarity
can be associated with a large number of (unidentified) polymorphisms,
instead of trying to identify individual polymorphisms more common among
those who share a given trait. Investigators using GCTA argue that even larger
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studies involving hundreds of thousands of persons have to be performed to
find the multitude of polymorphisms with subtle effects underlying herita-
bility estimates. However, these types of polymorphisms are like needles in
a haystack, the culprit genes remain hidden, and the search for them is the
last gasp of a failed paradigm.13 Is wasting our time and resources running
after ghosts really worthwhile? Many experienced gene hunters have sifted
through all parts of the genetic forest: they knew where to look, and they had
powerful instruments supporting their search, but eventually they returned
home without the rewarding prey.12 And even if there had been some risk
genes discovered to be associated with a disease, as has been the case in
comparable studies on the periphery, consequences for the clinic in question
turned out to be of limited value. For example, in a 2012 study, researchers
found that incorporating genetic information into clinical diagnoses did not
improve the ability of doctors to predict disease risk of breast cancer, type 2
diabetes, or rheumatoid arthritis.14
In general, epidemiological studies are confounded by complex cause-
and-effect relationships, unclear mechanisms by which non-shared envi-
ronmental factors mediate disease risk, and an inability to reconcile the
“heritable” component embedded within what appears to be an environ-
mental domain.15 If we want to understand human traits that have a genetic
component, we have to abandon an excessive and exclusive focus upon
genetic polymorphisms and choose a more holistic approach that under-
stands health and disease as attributes of plastic, adaptive organisms func-
tioning within particular environments.16 Consequently, development is a
dynamic process regulated by networks of interacting genes that function
in an environmental context. This view invalidates several key assumptions
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of statistical genetic analysis made when estimating heritability (e.g., equal

environments). Along these lines there emerges a need to reform the teach-
ing of genetics and to restrict the funding of further searches for elusive
genes that account for so little variance in normal behaviours.13
2.3 Where is the missing heritability?

During the past decade, evidence has been accumulating that gene expres-
sion is modified by the social environment through epigenetic processes and
independently of the primary DNA sequence.17 Apparently, the environment
substantially affects neocortical development, occurring mainly post-natally,
in an environment that is dominated by the caring guidance of the mother.
Two examples are provided here to illustrate these propositions:

1. Childhood abuse may increase susceptibility to schizophrenia, depres-
sion, and bipolar disorders.18 In studies, abused pups growing up in
the presence of abusive parents transmitted changes in gene meth-
ylation to their offspring. In humans, such inheritance can proceed
from the repetitive infliction of moderate to severe forms of trauma, of
which childhood abuse is only one possibility. A tentative consequence
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32 Chapter 2
on the transcriptional level may be decreased transcription of the hip-
pocampal glucocorticoid receptor (GR), which was observed along
with elevated methylation in the promoter of a neuronal glucocorti-
coid receptor called nuclear receptor subfamily 3, group C, member
1 (NR3C1).19 Increased levels of cortisol also result in extensive necro-
sis and apoptosis-related cell death, observed in the hippocampus of
rodents that have experienced either chronic or acute stress. Due to

the high vulnerability of the hippocampus to the detrimental effects of
cortisol, these events can lead to deficiencies of cognition. Accordingly,
extensive cell destruction in the hippocampus has repeatedly been
observed in post-mortem brains of schizophrenia patients.20
2. In contrast, in another study it was shown that DNA demethylation of
genes in the hippocampus of newborn rat pups was increased when
the pups received sustained grooming stimulation from their mothers.
Specifically, exposure to being groomed induced the activity-dependent
transcription factor, nerve growth factor 1-A (also known as Zif-268), to
bind the methylated GR promoter.21

In general, there is a growing body of literature to support the view that
epigenetic processes play pivotal roles in many, if not all, psychiatric disor-
ders.22–25 Generally speaking, environmental impacts giving rise to mental
illness can be acute, but massive, as in post-traumatic stress disorder; sub-
tle, but repetitive and long-lasting, as in affective disorders; or a combina-
tions of both, i.e., one or a few major environmental impacts followed by
many subtle influences during extended periods of time, as hypothesized in
schizophrenia.
10:30:21.
Schizophrenia is a disease particularly well-suited to investigations of

how environmental conditions early in an organism’s life are translated into
adverse epigenetic regulation that subsequently affects the development of
behavior well into adulthood.26 It has been categorized as a neurodevelop-
mental disorder, in which prenatal/perinatal stress tentatively combined with
abnormally adverse conditions of family life during childhood and adoles-
cence can be strong predictors of the emergence of psychosis in the patient’s
twenties.27 Individual patients may or may not carry a genetic predisposi-
tion for schizophrenia. An epigenetic contribution to the etiology of schizo-
phrenia is well supported by several epidemiological, clinical, and molecular
features associated with the disorder.28 For example, despite sharing the
same DNA sequence, the concordance rate between monozygotic (MZ) twins
for schizophrenia is consistently estimated to be <65%, which potentially
reflects environmentally or stochastically mediated epigenetic variation.26,29
What counts even more is that the environments of MZ twins are typically
more alike than those of dizygotic twins (as indicated by numerous studies).
From this it can be concluded that trait similarities ascribed to the greater
genetic similarity of MZ twins might in fact be due to greater environmental
similarity, which significantly inflates heritability estimates. In support of
this, studies of MZ twins discordant for schizophrenia and bipolar disorder
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have revealed a significant enrichment of epigenetic changes in gene net-
works and pathways directly relevant to psychiatric disorders and neuro-
development. Subsequent assessment in post-mortem brain tissue from
affected individuals and controls revealed marked hypomethylation (25%) in
a subset of psychosis patients in a region of psychosis-associated DNA that
was hypomethylated in affected twins.29
In conclusion, our DNA may define aspects of who we are, but simply
knowing what DNA is present in an individual is insufficient. It has become
very clear during the past decade that our environment and our experiences
alter the way in which that DNA is expressed, and the regulation of activation
or silencing of gene function play pivotal roles in their contribution to patho-
physiology. In other words, there is an immediate urge to know how that
DNA is expressed in a particular organ or cell type, and how its expression
changes over time.
In summary, it can be argued that the absence of consistently replicated
major genetic effects, together with evidence for lasting changes in gene
expression after environmental exposures is consistent with the concept that
the biological underpinnings of the major psychiatric disorders are epigene-
tic in form rather than DNA sequence based.30
Hence, rapidly growing evidence from basic research indicates that epigen-
etic regulation underlies normal cognition, and that cognitive dysfunction
occurs upon epigenetic misregulation. Putative epigenetic misregulation is
consistent with the various clinical and epidemiological features of psychiat-
ric diseases, such as discordance of identical twins, sex and parent-of-origin
effects, coincidence between disease onset and the time of major hormonal
changes in the organism, and major fluctuations in clinical course. Epigen-
10:30:21.
etic modifications such as histone acetylation and deacetylation, as well as

DNA methylation can induce lasting and stable changes in gene expression,
and have therefore been implicated in promoting the adaptive behavioral and
neuronal changes that accompany each of these illnesses. Most importantly,
epigenetic changes can be inherited. It may be for that reason the search for
heritable traits of mental disorders on the level of DNA sequence has resulted
in so little success.
2.4 Epigenetics:
A New Memory System in
Neurobiology
Evidence accumulated over the past few decades leaves little doubt that
dynamic regulation of gene expression is a critical requirement for the for-
mation, storage and recall of memory and subsequent behavior. Therefore,
investigations into molecular mechanisms of learning and memory are both
exciting and necessary approaches to obtain better insight into the develop-
ment of psychiatric disorders. They can be subdivided into studies on short-
term memory (STM) and long-term memory (LTM), where the latter has to
undergo one or more phases of consolidation. Learning experiences can
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34 Chapter 2
induce epigenetic modifications that may be more or less stable and hence
are apt to contribute to processes encompassing STM and LTM formation.
Along these lines, they are probably involved in synaptic long-term potentia-
tion (LTP), the cellular correlate of LTM. In consequence, higher level output,
such as changes in behavior, emotional responses, and eventually mental ill-
ness are, to large extents the results of environmental stimuli modifying the
epigenome. As already alluded to, the effects induced by the environment are
not necessarily adverse, but also potentially contribute to the development of
new positive traits that advance human mental capacities.
2.4.1 DNA Methylation

DNA methylation marks have been found to be relatively stable over time. For
instance, changes in DNA methylation induced by fear conditioning endured
for at least 30 days after training.31 However, active DNA demethylation and
remethylation has been identified even in the adult central nervous system
(CNS), which supports the idea that epigenetic markings set in place by ear-
ly-life experiences can be further modified or reversed by learning events,
and influence human behavior and adaptation to subsequent stimuli.32
Identification of methylation marks and modifications of their pat-
terns in schizophrenic patients have been reported recently on genome-
wide scales.33–36 However, most studies focused on specific genes, such
as RELN,37 serotonin receptor HTR2A,38 HTR1A,39 serotonin transporter
5-HTT,40 catechol-O-methyltransferase (COMT),41,42 BDNF,43,44 SOX10,45
and FOXP2. One of the best-studied changes of DNA methylation contrib-
10:30:21.
uting to schizophrenia is located in the promoter region of the reelin gene

(RELN), leading to its downregulation. Reelin is a glycoprotein that is
expressed in adult γ-aminobutyric acid (GABA)-containing neurons, but is
also important for proper neural positioning during brain development.46
Dysregulation of brain neural connectivity in the developing brain has
been suggested as a possible cause of schizophrenia.47 The downregu-
lation of this gene may well result from hypermethylation of the RELN
promoter,48 and post-mortem studies of patients with schizophrenia have
revealed significant downregulation of RELN expression in several brain
regions.49,50 Increased methylation of the reelin promoter does in fact
correlate with elevated activity of DNA methyltransferase- (DNMT)1 and
decreased levels of reelin mRNA in schizophrenia patients.51 It has to be
mentioned, however, that one study did not confirm hypermethylation of
the reelin promoter in the brains of schizophrenic patients.52 Apparently,
these studies are only the beginning of an understanding of the involve-
ment of DNA methylation/demethylation events in processes of memory
formation. There are certainly many more important memory-related
genes around, and it must be assumed that memory consolidation and
recall are dynamic processes that require investigation on temporary
scales, an aspect that is missing in many research projects.
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Interestingly, members of the growth arrest and DNA damage (GADD)45
enzyme family, known to be involved in DNA repair, are also crucial regu-
lators of DNA demethylation in the CNS, counteracting the activities of
DNMTs.53 Another potential mechanism for active demethylation of DNA in
mature neurons is the deamination of 5-methylcytosine to thymine by activa-
tion-induced deaminase, which triggers normal base-excision repair mech-
anisms.54 This reaction often results in hypomethylated promoter regions

and markedly increased new SNPs—by ∼50% if the neighboring CpG sites
have been methylated.55 Typically, the deamination of 5-methylcytosine
on DNA is detrimental, causing mutations that lead to serious human dis-
eases.56 Interestingly, valproate also induces DNA demethylation in nuclear
extracts of adult mouse brain.57 From this it can be concluded that while
DNA hypermethylation of crucial genes associated with schizophrenia may
also promote the progress of Rett syndrome, autism, and numerous forms
of mental retardation, reducing DNA methylation through systemic admin-
istration of DNA methylation inhibitors could result in the onset of other
disorders, including cancer.58
A number of additional DNA modifications, such as 5-hydroxymethylcy-
tosine, 5-formyl-cytosine, and 5-carboxylcystosine, have recently received
considerable attention. For example, 5-hydroxymethylcytosine is believed to
result from the active demethylation of methylated cytosine and appears to
be abundant in brain tissue, warranting further investigation in the context
of neuropsychiatric phenotypes.59
2.4.2 Histone Methylation

10:30:21.
Post-translational histone modifications (PTM) are another major source

of epigenetic regulation. Apart from a few studies reporting changes in
histone methylation in post-mortem tissue from schizophrenia patients,60
PTMs of histone proteins have been largely neglected in epidemiologically
informative study designs of schizophrenia. Methylation of histone proteins
can serve to both activate and repress gene transcription (Figure 2.1), which
underscores the pivotal role of histone methylation in intellectual disability,
addiction, schizophrenia, autism, depression, and neurodegeneration.
Each lysine residue can bind up to three methyl residues added by histone
methyltransferases (HMTs). Hence, mono-, di-, and tri-methylated states can
be distinguished. Gupta et al. found that certain histone methylation pat-
terns, including the histone H3 methylation marks H3K4me3 and H3K9me2,
serve to activate and repress gene transcription, respectively, in the hippo-
campus during fear–memory consolidation.61 Specifically, H3K4me3 corre-
sponds to an increase in the activity of the H3K4-specific methyltransferase
mixed-lineage leukemia (MLL)1, while H3K9me2 is due to the enzymatic
action of the G9a dimethyltransferase.62–64 For instance, mice lacking Mll
show a contextual fear memory deficit, suggesting that histone methylation
at this site is necessary for memory formation. Indeed, a number of memo-
ry-related genes, including BDNF and zif268, are downstream of H3K4me3.61
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36 Chapter 2
Figure 2.1 Schematic

overview of some selected aspects of regulation of gene
expression by DNA methylation and post-translational modifications
(PTMs) of histone proteins. Both processes are interdependent, as
shown exemplarily by methyl CpG binding protein 2 (MeCP2). Unmet-
hylated H3K4 can interact with DNA methylation or with H3K9/K27
acetylation. The histone deacetylase (HDAC) inhibitor sodium butyrate
(Na+But) increases trimethylation at H3K4 and decreases dimethylation
10:30:21.
at H3K9. In general, acetylation on H3-tails results in transcriptional

activation, in contrast to acetylation on H4-tails. HAT: histone acetyl-
transferase; HMT: histone methyltransferase; MLL; methyltransferase
mixed-lineage leukemia. For further details, see text. It is hypothesized
that the complexity of the histone code and interactions with DNA mod-
ifications specify co-ordinated expression of gene networks in brain
regions (regionally) and over time (dynamically). The environment may
interfere with this balanced gene expression by introducing adverse
alterations on the DNA or histone level that accumulate with time,
and eventually result in mental illness. It is presently premature to use
drugs targeting enzymes in this context to restore normal expression of
gene networks.
Typically, H3K4me3 provides a signal for initiation of transcription,65 while

H3K27me3 is a signal for inhibition of transcriptional elongation.66 Fur-
thermore, unmethylated H3K4 can interact with DNA methylation67 or with
H3K9/K27 acetylation, to mention only two examples.68,69 In this context, it
has been shown that chromatin remodeling mechanisms at GABAergic gene
promoters (for the involvement of GABAergic mechanisms, see later), includ-
ing MLL1-mediated histone methylation, operate throughout an extended
period of normal human prefrontal cortex (PFC) development and play a role
in the neurobiology of schizophrenia.70 Nucleosome remodeling is performed
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by chromatin remodeling complexes (CRCs) that interact with DNA and his-
tones to physically alter chromatin structure, and ultimately regulate gene
expression. Mutations in CRC subunits have been linked to intellectual dis-
ability disorders, autism spectrum disorder, and schizophrenia, and there
appears to be both developmental and adult-specific roles for the neuron-spe-
cific CRC neuronal Brg1/hBrm associated factor (nBAF). nBAF regulates gene
expression required for dendritic arborization during development, and in

the adult, contributes to LTP, a form of synaptic plasticity, and LTM.71 It can
be speculated that there is a memory thresholding mechanism. Below a cer-
tain threshold, environmental impact is not sufficiently important to engage
cellular resources. Thus, these mechanisms prevent storage of irrelevant or
non-valuable information. Similar to increased DNA methylation observed
during a memory task, these memory-filtering histone mechanisms would
need to be balanced with other epigenetic mechanisms turned on for storing
valuable memories. To obtain more detailed insights into the interplay of
multiple histone methylation marks, changes to these PTMs have to be inves-
tigated by comparing different behavioral paradigms at different time points
and in different brain regions; important issues that, unfortunately, can only
be pursued in animal model systems. For instance, it has been shown that
methylation at H3K9 during brain development appears to be highly co-or-
dinated and mediated by SET domain, bifurcated (DB)1 or ERG-associated
protein with SET domain (ESET).72 Moreover, treating mice with the histone
deacetylase (HDAC) inhibitor sodium butyrate increased trimethylation at
H3K4 and decreased di-methylation at H3K9.61 This result suggests an inter-
action between histone methylation and histone acetylation.
10:30:21.
2.4.3 Histone Acetylation

Whereas the acetylation of one histone protein, H3, is generally permissive
for transcription, acetylation of histone H4 is a transcriptional silencer.73 Dif-
ferential regulation of H3 and H4 acetylation suggests that different forms
of behavioral conditioning will activate or inactivate different genes respon-
sible for encoding that memory. Evidently, nothing is known about those
genes. One might be tempted to hypothesize that histone acetyltransferases
(HAT) and HDACs specifically recognize gene-specific patterns of PTMs in
context with DNA modifications to ensure changes of expression only in
the genes required for a task. H3K9/K14 acetylation has been investigated
in a few genes tentatively involved in schizophrenia [glutamic acid decar-
boxylase 1 (GAD1), 5-hydroxytryptamine receptor 2C (HTR2C), translocase
of outer mitochondrial membrane 70 homolog A (TOMM70A) and protein
phosphatase 1E (PPM1E)]. The expression of those genes was significantly
lower in young schizophrenia patients compared to controls. However, the
high acetylation levels in young controls steadily decline with age, and tran-
scriptional rates decrease accordingly. It appears that this does not occur
in schizophrenia brains, at least for those genes, which results in higher
acetylation in the brains of aged schizophrenia patients.74,75 Time-courses
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38 Chapter 2
reminiscent of this dynamic gene expression have been found in the superior
temporal cortex of schizophrenia brains, although no information was gath-
ered about methylation or acetylation status.76 For instance, in contrast to
the declining gene transcription (and concurrent decrease of acetylation) of
GAD, HTR2C, etc. in controls, MLL3 shows clear reverse time-course behavior
with age in controls in our study. Its expression increases with age, whereas
in schizophrenia patients its expression stays unchanged. It could be argued

that MLL3 is already upregulated by the disease and, therefore, is resistant
to age-dependent upregulation, or that it is downregulated relative to the
controls.
Major treatments for schizophrenia over the past 40 years have included
the drugs lithium and valproate, both of which we now know are HDAC
inhibitors. Additionally, trichostatin A and sodium butyrate act as HDAC-in-
hibitors, prolonging acetylation, and enhancing memory acquisition, extinc-
tion, and synaptic plasticity.77 Interestingly, HDAC inhibitors do not increase
global levels of acetylation, but seem to target genes active during the mem-
ory paradigm.78 Moreover, there is speculation that different classes of HDAC
inhibitors may allow targeting of specific HDAC classes, such as those associ-
ated with Alzheimer’s, Huntington’s, and other progressive diseases.79,80 Fur-
thermore, preclinical,81 but not clinical82 studies suggest that drugs such as
the non-specific HDAC inhibitor valproate83 may increase histone acetylation
when given chronically in combination with atypical antipsychotic drugs,
including clozapine or olanzapine. The latter appear to antagonize these
effects by increasing HDAC activities and decreasing histone acetylation. For
instance, clozapine has been shown in a recent study in the post-mortem
human prefrontal cortex of individuals under treatment with second-gener-
10:30:21.
ation antipsychotic drugs to have caused lower levels of histone H3 acetyla-

tion associated with mGlu2 than matched comparison subjects who had not
been treated with antipsychotic drugs, as determined post-mortem. These
effects specifically coincided with higher mRNA and protein expression of
HDAC2, its higher binding to the mGlu2 promoter, and downregulation
of expression of this glutamate receptor in murine PFC.84 All these stud-
ies should be considered withcaution and have to take into account some
important questions: which other genes are regulated in the same manner?
Do those genes carry the same PTMs? Are there combinations of other PTMs
resulting in similar gene regulation, but localized in other genes? It is very
likely that the complexity of the histone code is used to generate specific
PTM patterns on genes differentially expressed in certain cell types, brain
regions, etc., and these patterns are adapted to the requirements of aging
and environmental changes. Conversely, it is very unlikely that HAT or HDAC
inhibitors would be able to target these enzymes specifically bound to genes
needed for a specific brain function and leave these enzymes unaffected at
other genes. Therefore, the application of any of those drugs is supposed to
be accompanied by ample detrimental effects on the transcriptional level.
The resultant damage may be dependent on the brain region and the status
of development. The neocortex appears to be particularly vulnerable during
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development, which touches on a very important issue: it is probably the tim-
ing, duration, and place where epigenetic changes occur, hence their dynam-
ics that cause long-lasting dysfunction. Even if selectivity of HDAC inhibitors
for HDAC2, or any other HDAC, could be achieved, there are a great many
more unexplored substrates for this enzyme family, both in its epigenetic
capacity and probably in a far broader acetylome. Treatments with less spe-
cific HDAC inhibitors would increase the problem.85 These issues have been
raised and discussed in detail.86 In summary, before attempting to correct
epigenetic modifications associated with some mental illness by pharma-
cotherapy, we need to know much more about the molecular mechanisms
of gene regulation by the epigenome, both on the DNA and histone level.
We have to identify genes co-regulated in a specific psychiatric disorder on a
temporary scale and to search for common patterns of PTMs of histones and
DNA modifications in those genes and in those conditions. With the present
level of knowledge, any attempts to use drugs as described above are prema-
ture and will lead nowhere.
2.4.4 Epigenetics
of GABA-ergic Neurons and Neocortical
Development
A good example of epigenetic regulation during the critical phases of neo-
cortical development is the migratory activities of GABA-ergic neurons aris-
ing from the ganglionic eminence, movements that contrast with the radial
migration of principal neurons. GABA-ergic neurons are key players in the
development of localized circuits and in the regulation and synchrony of
10:30:21.
larger network oscillations. The synchronization of those networks is an

important means to co-ordinate firing in various regions of the neocortex.
GABA-ergic neurons in the developing neocortex and striatum are respon-
sible for the normal variance in the development of these structures. To a
large extent, this variance is propagated by epigenetic mechanisms (micro-
RNAs, retrotransposons, and imprinted genes) that co-ordinate this devel-
opment and may also increase the susceptibility of the human forebrain
for a variety of mental disorders (epilepsy, schizophrenia, bipolar disorders,
mental retardation, and autism).87,88 Adverse environmental impact may be
compensated by the individual variability of detailed interconnections of the
neocortical neurons, or by apoptotic elimination of developmental errors
and subsequent neurogenesis, which, evidently, is not always sufficient.
The epigenetic mechanisms establishing such variability may be herita-
ble, but their manifestation is highly dependent upon developmental tim-
ing and neuronal activities, which means the right connections have to be
made at the right time. These temporarily and spatially fine-tuned events
have been referred to as neural Darwinism, a form of natural selection at
the cellular level.89 Consequently, functional deficits of GABA-ergic neurons
are involved in a broad spectrum of neurological and psychiatric disorders.90
The LIM homeobox genes Lhx6 and Lhx7 are a class of genes that regulate
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40 Chapter 2
the differentiation of GABA-ergic and cholinergic interneurons in the stria-
tum.91 The initial excitatory role of GABA-ergic neurons in the development
of localized canonical circuits and their subsequent ability to synchronize
oscillations across widespread areas of the neocortex make them particularly
vulnerable in this context. These same neurons switch from excitatory orga-
nizers of neural networks during development to hyperpolarizing inhibitory
neurons in the adult cortex. Hence, in the adult brain, inhibitory GABA-ergic
interneurons maintain network oscillations and enable widely distributed
regions of the cortex to co-ordinate their firing.92 The linkage of schizophre-
nia to GABA-ergic neurons is particularly strong, especially the chandelier
neurons of the orbital frontal cortex.93 These neurons express parvalbumin,
somatostatin, and Lhx6, the latter playing a significant role in the migra-
tion of these neurons from the ganglionic eminence to the frontal cortex.94
Markers of GABA-ergic transmission between chandelier neurons and their
synaptic targets are also changed in the prefrontal cortex of schizophrenic
brains. Increased expression of DNMTs in GABA neurons of the cortex and
striatum is consistent with the increased methylation that has been reported
in schizophrenia patients.26 Insufficient stimulation of N-methyl-d-aspartate
(NMDA)-selective glutamate receptors [NR1 (GRIN1) and NMDA2A (GRIN2A)
receptor assemblies] on these GABA-ergic interneurons contributes to the
proposed GABA hypofunction at cortical pyramidal neurons. Hence, reduced
signaling at NMDA-selective glutamate receptors present on GABA-ergic
interneurons causes the glutamatergic hypofunction, which then facilitates
reduced GABA release onto the main output neurons (pyramidal neurons).95
In keeping with this, the GABA membrane transporter 1 is decreased in pre-
synaptic terminals, whereas the GABAA receptor (α2 subunit) is increased in
10:30:21.
the initial segment of the post-synaptic axon.93 An interesting and proba-

bly important twist, showing interdependency between methylation status
of histones (open or closed chromatin) and DNA methylation in the GAD1
gene, has been reported, showing markedly reduced DNA methylation in the
closed state (H3K27me3) of chromatin in schizophrenia patients.96 Reduced
expression of GAD1 by epigenetic mechanisms was confirmed later in a com-
mentary.97 A tentative pharmacotherapy suggested to normalize GABA-ergic
promoter hypermethylation and adjust the downregulation of GABA-ergic
gene expression, as observed in the post-mortem brain of schizophrenia
patients, by activating DNA demethylation, using combinations of clozapine
or its derivatives with valproic acid or other more potent and selective HDAC
inhibitors.81 In the light of the comments made earlier, such suggestions
need to be considered with extreme caution.
2.5 Conclusions
In conclusion, different gene regulation mechanisms, and presumably
different genes, are active at different time points in different parts of the
brain, even within the same structure, such as the hippocampus. Therefore,
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identifying and controlling for age-related changes of gene expression will
be an important consideration in longitudinal studies of peripheral epigen-
etic marks associated with psychiatric illness. A host of genes are actively
regulated in response to different learning paradigms, such that each type
of learning produces a unique gene–regulation profile. Simply knowing what
DNA is present in an individual is insufficient to know how that DNA will be
expressed in a particular cell or at a specific time.

Statement of conflict of interest: The author confirms that there is no con-
flict of interest.
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J. J. Meana, S. J. Russo and J. González-Maeso, Nat. Neurosci., 2012, 15,
91245.
85. S. E. Hyman, Nat. Neurosci., 2012, 15(9), 1180.
86. D. R. Grayson, M. Kundakovic and R. P. Sharma, Mol. Pharmacol., 2010,
77, 2126.
87. E. B. Keverne, Epigenomics, 2011, 3, 191.
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88. M. M. McCarthy, C. Moore-Kochiacs, X. Gu, E. S. Boyden, X. Han and N.
Kopell, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 11620.
89. G. M. Edelman, Neuron, 1993, 10, 115.
90. E. B. Keverne, Brain Res. Bull., 1999, 49, 467.
91. P. Liodis, M. Demaxa, M. Grigoriou, C. Akufo-Addo, Y. Yanagawa and V.
Pachnis, J. Neurosci., 2007, 27, 3078.
92. V. Volman, M. M. Behrens and T. J. Sejnowski, J. Neurosci., 2011, 31,

18137.
93. D. A. Lewis, Dev. Neurobiol., 2011, 71, 118.
94. D. W. Volk, T. Matsubara, S. Li, E. J. Sengupta, D. Georgiev, Y. Minabe, A.
Sampson, T. Hashimoto and D. A. Lewis, Am. J. Psychiatry, 2012, 169, 1082.
95. D. R. Grayson, Epigenomics, 2010, 2, 3341.
96. H. S. Huang and S. Akbarian, PLoS One, 2007, 2(8), e809.
97. J. Peedicayil, Am. J. Psychiatry, 2009, 166(4), 493.
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CHAPTER 3
Developmental Neuroimmune
Mechanisms in Schizophrenia
ULRIKE STADLBAUERa AND URS MEYER*a
a
Physiology and Behaviour Laboratory, ETH Zurich, Schorenstrasse 16, 8603
Schwerzenbach, Switzerland
*E-mail: urmeyer@ethz.ch
3.1 Introduction
Schizophrenia is a chronic form of psychotic illness which affects approx-
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imately 1% of the population worldwide.1 It undermines the basic human

processes of perception and judgment, as it is characterized by profound
disturbances in mental functions, emotions and behavior. The onset of full-
blown disease typically emerges in late adolescence or early adulthood and
involves the expression of distinct symptom classes, referred to as positive,
negative and cognitive symptoms.2–4 Positive symptoms are features that are
normally not present in healthy individuals but appear as a result of the dis-
ease, including visual and/or auditory hallucinations, delusions, paranoia,
and major thought disorders. Negative symptoms refer to features that are
normally present, but are reduced or absent as a result of the disease pro-
cess, including social withdrawal, apathy, anhedonia, alogia, and behavioral
perseveration. Cognitive symptoms of schizophrenia typically involve distur-
bances in executive functions, working memory impairment, and inability to
sustain attention.

46
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Developmental Neuroimmune Mechanisms in Schizophrenia 47

Despite remarkable advances in the understanding of the neurobiological
and neurochemical aspects of schizophrenia,5–7 the underlying mechanisms
of this disabling disease remain challenging for clinicians and researchers.
In this context, the neurodevelopmental hypothesis of schizophrenia has
received converging support from various research fields since its initial
formulation in the 1980s.8,9 It suggests that the etiology of schizophrenia
involves altered neurodevelopmental processes, in which primary insults

occur in early brain development, long before the illness becomes clinically
manifest.
There is increasing evidence that altered neuroimmune mechanisms
might play a role in the development of schizophrenia and related illnesses.
Stimulated by various epidemiological findings reporting an elevated risk of
schizophrenia following prenatal infection,10,11 one line of research aims to
explore the potential contribution of immune-mediated disruption of early
brain development in the precipitation of long-term psychotic disease.12–15 In
this context, one of the immunological mechanisms studied is developmen-
tal neuroinflammation, which may predispose the organism to schizophre-
nia-relevant brain and behavioral abnormalities.16,17 This chapter summarizes
existing evidence for this hypothesis and discusses the role of developmental
neuroimmune mechanisms with regards to their impact on brain develop-
ment, disease progression, and possible preventive interventions.
3.2 Epidemiological
and Translational Studies of
Prenatal Infection and Schizophrenia
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A variety of retrospective studies have revealed an association between

maternal infection and increased risk of schizophrenia in the offspring,10,11,18
although negative reports also exist.19,20 The link between prenatal exposure
to infection and enhanced schizophrenia risk does not seem to be patho-
gen-specific. Indeed, numerous viral infectious agents have been implicated
in this association, including influenza,21,22 rubella,23 measles,24 polio,25 and
herpes simplex,6 as well as bacterial pathogens causing sinusitis, tonsillitis
and pneumonia,27 genital and/or reproductive infections,28 and the proto-
zoan parasite Toxoplasma gondii.29,30
Furthermore, there is clear serologic evidence for some of the infectious
agents implicated in the prenatal infectious etiology of schizophrenia from
prospective epidemiological approaches.21,29–31 Despite the fact that prenatal
exposure to infection per se seems to exert relatively modest effects across
large populations,20,32 it is likely to be a relevant factor upon interaction
with other schizophrenia risk factors, including genetic predisposition. This
could possibly explain why a substantially increased effect of prenatal infec-
tion on elevating the risk of schizophrenia is seen in offspring with a positive
family history of psychotic disorders.33
In addition, epidemiological researchers are trying to determine whether
prenatal exposure to infection confers vulnerability to specific features of
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48 Chapter 3
schizophrenia neuropathology. Initial evidence was provided by Brown
and colleagues,10,34 showing that deficits in fine-motor coordination, verbal
memory, executive functions, and working memory are more pronounced in
schizophrenic cases with a positive history of prenatal infection compared
to schizophrenic cases without such a history. Furthermore, a significant
association between increased length of the cavum septum pellucidum and
prenatal infection has been demonstrated in exposed schizophrenia cases

compared to unexposed cases, indicating that in-utero exposure to infection
may contribute to neurodevelopmental morphological abnormalities fre-
quently observed in schizophrenic patients.34
As an association was reported between prenatal influenza infection
and adult schizophrenia, Fatemi and colleagues35–37 have pioneered an
experimental animal model of prenatal exposure to human influenza virus
in mice, which has been adopted by other laboratories.38 In this model,
pregnant mice are infused with a sublethal dose of a neurotropic strain
of human influenza virus, and the long-term brain and behavioral effects
are then evaluated in the resulting offspring relative to control offspring
born to sham-treated mothers. Neuroanatomical and brain morphologi-
cal investigations in this model have shown that maternal influenza infec-
tion leads to a variety of neuropathological signs in the offspring’s brains
postnatally, some of which are critically implicated in the neuropathology
of schizophrenia.34–37 Furthermore, prenatal exposure to influenza virus
in mice also induces a set of behavioral and pharmacological changes in
adulthood, which are implicated in both the positive and negative symp-
toms of schizophrenia,37,38 including deficits in sensorimotor gating,
reduced spatial exploration and social interaction, and enhanced sensitivity
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to pharmacological treatment with N-methyl-d-aspartate (NMDA)-receptor

antagonists and hallucinogens. Importantly, the prenatal infection-induced
deficits in sensorimotor gating can be normalized by acute treatment with
typical or atypical antipsychotic drugs,38 suggesting that at least some of
the long-term behavioral changes induced by prenatal influenza exposure
are sensitive to pharmacological compounds used in the symptomatic
pharmacotherapy of schizophrenia. The findings derived from the prenatal
influenza mouse model have recently been completed with experimental
investigations in rhesus monkeys demonstrating the emergence of reduced
gray and white matter in distinct cortical and parieto-cortical brain regions
of neonates born to influenza-infected mothers.39 This extension of transla-
tional research to rhesus monkeys is especially relevant in the present con-
text, because prenatal corticogenesis is more advanced in primates than in
rodent species, and therefore primate models help to verify the relevance
of the findings of animal models to the human condition. Together, the
experimental data obtained in mouse and primate prenatal viral infection
models can be taken as causal evidence to support human epidemiological
studies suggesting that there may be a causal relationship between in-utero
exposure to infection and the emergence of postnatal brain dysfunctions
pertinent to schizophrenic disease.
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3.3 The
Role of Inflammation in Mediating the
Effects of Maternal Infection in the Offspring
If maternal infection occurs during pregnancy, at least some infectious
pathogens, such as rubella, are capable of penetrating the placental barrier
and infiltrating the fetal environment, thereby initiating direct damage to

the growing organism by interference with cell growth, protein synthesis,
and nutrient supply.40 Regardless of these findings, several lines of research
indicate that the deleterious effects of maternal infection on the offspring
are likely to be attributable to maternal/fetal inflammatory responses rather
than direct viral effects on the developing fetus.14,41,42 This issue is discussed
in detail later (Sections 3.3.3 and 3.3.4), following summaries of the main
components of the inflammatory response system (Section 3.3.1) and of the
known neurodevelopmental effects of cytokines (Section 3.3.2).
3.3.1 The
Main Components of the Inflammatory
Response System
One of the first defense mechanisms of the innate immune system to infec-
tion and other physiological insults, such as stress or tissue damage, is
inflammation. Inflammation is characterized by redness and swelling of the
affected tissue and is promoted by a variety of secreted pro-inflammatory
factors, including prostaglandins, leukotrienes, pro-inflammatory cytokines,
and chemokines. Leukotrienes and chemokines are critical for attracting leu-
kocytes to sites of infection and/or tissue damage, whereas prostaglandins
are mediators of the febrile response and of blood vessel dilation.43 Beside
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their extensive roles in the innate and adaptive immune systems, where
they help regulate the recruitment and activation of lymphocytes as well
as immune cell differentiation and homeostasis,44 in addition, some cyto-
kines exert direct effector mechanisms, including induction of cell apoptosis
and inhibition of protein synthesis.45 Pro-inflammatory cytokines, such as
interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, are necessary for
the inflammatory response as they contribute to febrile reactions, activate
phagocytotic cells such as macrophages or dendritic cells, facilitate vascu-
lar permeability, and promote the release of plasma-derived inflammatory
mediators such as bradykinin and components of the complement system.
In the periphery, pro-inflammatory cytokines are produced and released to
a great extent by activated endothelial cells and cells of the mononuclear
phagocyte system (monocytes, macrophages, and monocyte-derived den-
dritic cells). Activation of the innate immune system strongly stimulates the
synthesis of pro-inflammatory molecules, often occurring upon binding of
microbe-specific components by a special class of receptors known as patho-
gen recognition receptors, or when damaged or infected cells send out alarm
signals, many of which are recognized by the same receptors as those that
recognize pathogens.45 Table 3.1 summarizes the major cellular sources and
main biological activities of pro- and anti-inflammatory cytokines.
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50 Chapter 3
Table 3.1 Major
cellular sources and immunological effects of selected pro- and
anti-inflammatory cytokines and some of their known neurodevelop-
mental effects.a,b
Known
Main cellular Main immunological neurodevelopmental
Cytokine source effects effects
IL-1β Activated mono- Promotion of fever Conversion of mid-

cytes/mac- (endogenous brain progenitor
rophages; pyrogen); stim- cells into dopami-
endothelial cells; ulation of other nergic phenotype;
microglia pro-inflammatory promotion of fetal
cytokines and hemato- midbrain dopamine
poietic growth factors; cell survival; disrup-
induction of acute- tion of neuron den-
phase proteins; stim- drite development
ulation of HPA axis; and outgrowth
activation of T-, B-,
and endothelial cells
sIL-1RA Activated mono- Inhibition of IL-1 activ- Inhibition or promo-
cytes/mac- ity; homeostatic con- tion of neurogen-
rophages; trol of inflammation esis depending
endothelial through anti- on specificity of
cells; fibroblasts; inflammatory actions neuroinflammatory
astroctyes milieu and matura-
tional stage
IL-6 Activated mono- Promotion of fever Decreasing survival
cytes/mac- (endogenous pyro- of fetal serotonin
rophages; T gen); induction of neurons; disruption
cells (TH2 and acute-phase proteins; of neuron dendrite
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TH17 cells); stimulation of development and

hepatocytes; immunoglobulin-G outgrowth; pro-
osteoclasts; fibro- production; activation motion of fetal
blasts; astrocytes of T cells; stimulation midbrain dopamine
of HPA axis and dorsal root gan-
glion cell survival
sIL-6R Activated mono- Augmentation of IL-6 Enhancement of
cytes/mac- responses by acting as neuronal sur-
rophages; an IL-6 agonist vival during
hepatocytes; development
osteoclasts
IL-8 Activated mono- Activation of neutro- Largely unknown
cytes/mac- phils; chemotactic for
rophages; neutrophils, T cells
endothelial cells; and basophils
fibroblasts
IL-10 Activated mono- Inhibition of pro- Promotion of neuro-
cytes/macro- inflammatory cytokine nal survival; trophic
phages; T cells synthesis; inhibition support to develop-
(TH2 cells); B of sepsis; promotion ing neurons
cells of humoral immune
responses involving
antibody secretion
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TNF-α Activated mono- Promotion of fever Neuronal apopto-

cytes/macro- (endogenous pyro- sis; disruption of
phages; T cells gen) and sepsis; neuron dendrite
(TH1 cells); nat- direct cytotoxic development and
ural killer cells; effects by inducing outgrowth
endothelial cells; apoptosis; activation
microglia of monocytes, lym-

phocytes, and endo-
thelial cells
sTNFR Virtually all nucle- Inhibition of TNF activ- Blocking neuronal
ated cells ity; homeostatic con- apoptosis (medi-
trol of inflammation ated by TNF-α)
through anti-inflam-
matory actions
TGF-β Megakaryocytes T Inhibition of pro-in- Ventral midbrain
cells (TH3 cells) flammatory cytokine dopaminergic
synthesis; inhibition development by
of natural killer cell promotion of tyro-
activity and growth of sine hydroxylase
T- and B cells; in the expression; regu-
presence of IL-6 stim- lation of (neuro-
ulation of TH17 cells muscular) synapse
formation
a
PA: hypothalamic–pituitary–adrenal; IL: interleukin; R: receptor; RA: receptor agonist;
H
s: secretory; TGF: transforming growth factor; TH: T-helper; TNF: tumor necrosis factor.
b
Based on data provided in Meyer (2013).124
In normal conditions, inflammation is controlled by homeostatic pro-

cesses that limit or counteract the inflammation once it has been induced by
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a pro-inflammatory stimulus such as infection.46 Such control mechanisms

ensure that inflammatory processes efficiently remove invading pathogens
and contribute to tissue repair and wound healing without inducing collat-
eral damage to non-infected, healthy and unwounded tissue. Dysfunction
of control mechanisms may lead to persistent inflammation, known from
numerous pathological conditions such as rheumatoid arthritis, atheroscle-
rosis, and inflammatory bowel disease.46 Microglia and astrocytes are the
major immunocompetent cells of the central nervous system (CNS), regu-
lating both the induction and the limitation of inflammatory processes.47,48
This is achieved through the synthesis of cytokines, up- or downregulation of
various cell surface receptors such as pathogen recognition receptors, cyto-
kine receptors, and numerous receptors crucial for antigen presentation.
Microglia, acting as the first and main form of active immune defense in
the brain, are considered to be the resident macrophages of the CNS, which
constantly scavenge the CNS for damaged neurons, plaques, and infectious
agents.49 Microglia are often referred to as a “double-edged sword”, as they
appear to play crucial roles in both neuronal protection and their pathol-
ogy.50 They secrete neurotrophic factors pivotal for cellular repair and recruit
immune cells into the brain for clearance of infection or cellular debris on
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52 Chapter 3
the one hand; however, on the other hand, chronic or exaggerated microg-
lial activation is linked to the excessive secretion of pro-inflammatory fac-
tors and has been linked to neurodegenerative processes.50 With regards to
astroctyes, the main roles of these glial cells have long been considered to
be related to neuronal support functions. There is evidence, however, sug-
gesting that astrocytes exert a much wider spectrum of functions, including
regulation of neuronal differentiation, axonal guidance, synapse formation,

and brain plasticity.48 Importantly, astrocytes exert modulatory effects on
microglia cells. They seem to have noticeable inhibitory as well as stimula-
tory influences on microglia functions, depending on the precise immune
milieu in which astroctye–microglia interactions take place.51,52
3.3.2 Neurodevelopmental Effects of Cytokines

Cytokines seem to play an essential role in the regulation and modulation of
normal brain development, as these molecules and their receptors are con-
stitutively expressed during fetal brain development.53,54 Aside from various
roles in the peripheral immune system, cytokines have been recognized to
exert a number of critical neurodevelopmental effects, including neuronal
induction, proliferation, migration, and survival;55,56 some of these effects of
selected inflammatory cytokines are summarized in Table 3.1. As they seem
to be essential for normal brain development, it can be expected that altered
cytokine levels during critical periods of early brain development adversely
affect neurodevelopmental processes and contribute to a higher susceptibility
to complex brain disorders of developmental origins, such as schizophrenia.
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Notably, distinct classes of cytokines can exert differing effects in the

developing CNS. Among the variety of pro- and anti-inflammatory cyto-
kines, for instance, IL-1β is the most capable of inducing the conversion
of rat mesencephalic progenitor cells into a dopaminergic phenotype,57
and IL-6 is highly efficacious in decreasing the survival of fetal brain sero-
tonin neurons.58 In contrast, both IL-1β and IL-6 (and to a lesser extent
TNF-α) appear to regulate the survival of fetal midbrain dopaminergic neu-
rons at low to medium concentrations,58 whereas at higher concentrations
the same cytokines can promote the survival of these cells.59,60 A similar
dependency on cytokine specificity and/or concentration has also been
found for TNF- α,61 as it is able to disrupt dendrite development of cortical
neurons at low concentrations, while the same effects can be achieved by
exposure of fetal cortical neurons to higher concentrations of IL-1β, IL-6,
or TNF-α. The responsiveness and/or sensitivity of developing cells to many
signaling cues, including cytokines, can also vary considerably as neurode-
velopment progresses. For example, during early fetal development TNF-α
is neurotrophic to dopaminergic ventral mesencephalic neurons; at later
stages of fetal brain development it can exert neurotoxic effects on these
cells.62 In the context of maternal infection during pregnancy, this high-
lights that the eventual neurodevelopmental impact of abnormal maternal/
fetal cytokine expression is likely to be determined by the precise stage of
brain development.63–65
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3.3.3 Epidemiological
Evidence for the Role of Inflammation
in Mediating the Effects of Maternal Infection on the
Offspring
Intrauterine infection and subsequent maternal/fetal inflammatory
responses have widely been recognized as major contributors to periven-
tricular leukomalacia (i.e. white matter damage), which is causally linked

to subsequent development of cognitive and neurological disabilities,
especially cerebral palsy.66,67 It is characterized by enhanced pro-inflam-
matory cytokine secretion and microglial activation causing the loss of
oligodendrocyte progenitor cells and immature neurons in periventricu-
lar regions.68,69 As numerous infectious pathogens have been implicated
in the association between prenatal infection and schizophrenia, this has
led to the hypothesis that common immunological factors in general, and
pro-inflammatory cytokines in particular, are the mediators of this asso-
ciation.41 However, in contrast to the broad literature implicating inflam-
matory processes in the development of neonatal white matter damage
and cerebral palsy, direct epidemiological evidence linking enhanced
maternal/fetal expression of inflammatory markers and later develop-
ment of schizophrenia is thus far limited to a small number of investi-
gations. These include reports of a significant association between high
maternal levels of the pro-inflammatory cytokines TNF-α26 and IL-821 and
elevated risk of schizophrenia spectrum disorder in the offspring. In addi-
tion, there is evidence implicating increased prenatal maternal IL-8 levels
in exacerbation of structural brain changes in schizophrenic offspring.70
Even though these findings may be indicative of the abnormal expression
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of specific inflammatory markers in the developmental course of schizo-

phrenia and related disorders, such valuable epidemiological data need to
be interpreted with some caution. Not only are all epidemiological studies
observational in nature and thus cannot prove causality, but furthermore,
the available epidemiological studies reporting a significant association
between enhanced maternal cytokine levels and increased schizophrenia
risk in the offspring have so far not been able to delineate the source of
inflammatory mediators. Hence, they fall short of identifying whether the
presence of enhanced maternal cytokine levels is attributable to prior or
ongoing infectious processes, or to other adverse maternal conditions,
such as preeclampsia, obesity and anemia. As discussed in the next Section
3.3.4, experimental research in animals provides a unique opportunity to
overcome these limitations.
3.3.4 Experimental
Evidence for the Role of Inflammation
in Mediating the Effects of Maternal Infection on the
Offspring
Several experimental approaches have been established to test the hypothe-
sis that the detrimental long-term effects of prenatal infection on offspring
brain and behavioral development may be mediated indirectly by activation
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54 Chapter 3
13,14,71
of the maternal/fetal inflammatory response systems. Two of the best
established models are based on maternal exposure to the bacterial endo-
toxin lipopolysaccharide (LPS) and the synthetic analog of double-stranded
RNA, polyriboinosinic–polyribocytidilic acid (polyI:C). Whereas LPS is recog-
nized by toll-like receptor (TLR)4, polyI:C is recognized primarily by TLR3.72,73
Both TLR3 and 4 belong to a class of pathogen recognition receptors that
recognize invariant structures present on virulent pathogens. After binding

to TLRs, both LPS and polyI:C stimulate the production and release of many
pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α.63,74 Moreover,
polyI:C leads to an induction of the type I interferons (IFN)-α and IFN-β.72,75
LPS exposure leads to a cytokine-associated innate immune response that
is typically seen after infection with Gram-negative bacteria;73 administra-
tion of polyI:C mimics the acute-phase response to viral infection.76 Mater-
nal exposure to LPS or polyI:C during pregnancy is capable of enhancing
pro-inflammatory cytokine levels in the three maternal–fetal compartments,
namely, the placenta, the amniotic fluid, and the fetus, including the fetal
brain.63,71 In-utero exposure to such immunogens further leads to activation
of microglia and expression of pro-inflammatory transcription factors such
as nuclear factor -κB in fetal and neonatal brains,77,78 and this is paralleled
by white matter injury (for example, oligodendrocyte precursor cell loss and
hypomyelination) and neuronal apoptosis during fetal and neonatal brain
development.79,80 Taken together, animal models using inflammatory agents
such as LPS or polyI:C provide multiple lines of evidence that in-utero inflam-
mation induces wide-spread neuroinflammation during critical stages of
fetal and neonatal brain development. Furthermore, numerous behavioral,
cognitive, neurochemical, and brain morphological abnormalities have been
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detected in adult animals following maternal gestational exposure to LPS

or polyI:C.13–15,42,71 Importantly, many of the behavioral, cognitive and phar-
macological dysfunctions in adult animals born to LPS- or polyI:C-exposed
mothers are directly implicated in schizophrenia and other psychosis-re-
lated disorders, including abnormalities in sensorimotor gating, selective
attention, working memory, and sensitivity to psychotomimetic drugs, and
at least some of these functional abnormalities can be normalized by acute
and/or chronic antipsychotic drug treatment.13–15,42,71 In summary, the ability
of prenatal exposure to cytokine-releasing agents such as LPS or polyI:C to
induce fetal and neonatal brain inflammation, together with its long-term
impact on brain and behavioral abnormalities relevant to schizophrenia,
emphasizes the essential role of prenatal cytokine-associated inflammation
in mediating the effects of maternal infection on the offspring. Further-
more, this is supported by findings showing that blocking the actions of the
pro-inflammatory cytokines IL-1β or IL-6 in the pregnant maternal host by
genetic or pharmacological interventions prevents the long-term brain and
behavioral consequences of prenatal polyI:C or LPS treatment,81 and that
over-expression of the anti-inflammatory cytokine IL-10 prevents the emer-
gence of multiple behavioral and pharmacological abnormalities typically
seen after prenatal polyI:C-induced immune challenge.82
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Another valuable model to study the relative contribution of prenatal
inflammation to the development of schizophrenia-related brain disease is
based on maternal intramuscular injection of turpentine oil.83 Following
intramuscular injection, turpentine remains confined to the site of admin-
istration and locally causes tissue damage, recruitment and activation of
immune cells, and secretion of pro-inflammatory cytokines.83 It is therefore
possible to study the effects of circulating inflammatory mediators that are

solely produced by the maternal immune system. Hence, in contrast to the
systemic LPS and polyI:C models, placental secretion of inflammatory mark-
ers is minimal, and this facilitates the delineation of the relative contribution
of maternally produced versus placenta-derived inflammatory factors linking
prenatal inflammation and abnormal brain and behavioral development.84,85
Prenatal turpentine treatment induces long-term behavioral, pharmacologi-
cal, and neurochemical changes implicated in schizophrenic disease, includ-
ing prepulse inhibition deficiency, amphetamine hypersensitivity, deficits
in spatial memory, and dopaminergic imbalances in striatal structures.84,85
This provides further strong support for the hypothesis that induction of
maternal inflammatory responses has a key role in mediating the association
between maternal infection during pregnancy and enhanced risk of schizo-
phrenia-related brain pathology in the offspring.
3.4 Fetal
Brain Development in the Event of
Inflammation
Findings have accumulated with respect to long-term behavioral, cognitive,
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neurochemical, and brain morphological abnormalities induced by prena-

tal infection or immune challenge. However, so far, the effects of prenatal
inflammation in terms of early alterations in fetal brain development have
been less extensively studied. There is evidence from the prenatal polyI:C
model that early prenatal immune challenge disrupts perinatal cortical
laminar formation and comprises the normal development of upper-layer
(but not deeper-layer) cortical neurons86 and impedes the normal course of
neurogenesis during fetal development.87 The latter findings are especially
intriguing in view of the fact that the effects of prenatal immune activation
on reduced (hippocampal) neurogenesis persist postnatally and are even evi-
dent at adult stages of development.63,88 Together, it appears that the per-
sistent impairments in postnatal neurogenesis following prenatal immune
challenge are likely to be of developmental origin starting early in fetal life.
PolyI:C-induced maternal immune challenge exerts short-term effects on
the fetal development of the dopamine system, a neurotransmitter system
highly implicated in schizophrenia and related psychotic disorders.89 Mater-
nal immune stimulation by polyI:C in early/middle gestation increases the
number of dopamine neurons in the fetal midbrain at middle/late and late
stages of prenatal development.90,91 This effect is paralleled by changes in the
fetal expression of several genes known to be involved in the development
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56 Chapter 3
91
of dopamine neurons. Notably, these findings do not provide a direct link
between altered fetal dopaminergic development and the emergence of the
well described dopamine-associated structural and functional abnormali-
ties in the postnatal period. However, these results highlight that postnatal
dopaminergic abnormalities emerging after prenatal immune challenge are
developmentally regulated and start early in utero. In view of this, it seems
that prenatal inflammation-induced abnormalities in the development of

the fetal midbrain dopamine system may represent an important primary
mechanism for the postnatal emergence of functional and structural changes
associated with imbalances in the mesocorticolimbic dopamine system.14 In
addition to its effects on the central dopamine system, the long-term neu-
ropathological deficits induced by prenatal infection and/or inflammation
include pre- and postsynaptic changes in various other neurotransmitter sys-
tems, such as the γ-aminobutyric acid (GABA), glutamate, and serotonin sys-
tems, together with alterations in neuronal and glial cell number, structure,
and positioning.71 Given these multiple effects, it seems feasible that mater-
nal/fetal inflammation and associated physiological insults could directly
induce primary defects in the early fetal development of various neurotrans-
mitter systems and cell populations. However, direct evidence for this possi-
bility is still lacking, so it remains unknown how early neurodevelopmental
abnormalities induced by fetal neuroinflammation are converted into long-
term brain and behavioral pathology in adulthood.71
3.5 Priming
of Long-term Neuroinflammation by
Prenatal Infection and Inflammation
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One important question is whether exposure to prenatal infection or inflam-

mation can permanently alter immune functions across postnatal life.17 This
issue seems particularly relevant in view of the fact that schizophrenia is
associated with various immunological abnormalities,92–94 including periph-
eral low-grade inflammation95,96 and signs of microglia and astrocyte over-
activation.97,98 Experimental evidence indicates that prenatal exposure to
infection or inflammation can indeed lead to long-lasting immune abnormal-
ities, including inflammatory changes in the periphery and CNS. Persistent
alterations in peripheral levels of pro-inflammatory cytokines, together with
increased microglia and/or astrocyte activation, have been demonstrated
in rodent models of prenatal viral influenza exposure,35 chronic gestational
LPS exposure,99,100 sub-chronic prenatal IL-6 treatment in mid-to-late gesta-
tion,101 and acute polyI:C treatment in early/middle gestation.102 Addition-
ally, sub-chronic maternal treatment with IL-2 from mid-to-late pregnancy in
mice has been shown to elevate B- and T-cell counts in response to antigenic
stimulation in the juvenile offspring.103 Acute fetal inflammation may further
induce latent neuroinflammatory abnormalities that can be unmasked by
exposure to certain environmental stimuli throughout postnatal life.16 This
idea of multiple hits with either sensitizing or priming effects is also central
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to several theories of prenatal immune priming, which have been put forward
in the context of peripheral immunity, CNS inflammation and progressive
neurodegeneration, and various forms of learning and memory.104,105 There-
fore, inflammatory exposure in early (prenatal or neonatal) life could cause
the organism to respond differently (and often more vigorously) to subse-
quent immunological or non-immunological challenges such as stress.106,107
Priming of exacerbated neuroinflammatory responses has perhaps been best

established in the context of microglia biology, highlighting that microglia
can be primed by initial infectious or inflammatory stimuli to induce exagger-
ated pro-inflammatory responses to secondary environmental stimuli, such
as peripheral inflammation.105,108 As discussed in more detail in section 3.6,
such priming effects also seem highly relevant in the context of schizophre-
nia, because the disorder’s etiology most likely involves exposure to multiple
environmental and/or genetic insults at various stages of brain development
and maturation.109,110
3.6 (Latent)
Neuroinflammation and Disease
Progression
Many of the behavioral, pharmacological, and cognitive disturbances induced
by prenatal inflammation are progressive in nature: They are often dependent
on maturational processes and are pathologically manifest only once the off-
spring reach adolescence or early adulthood, as demonstrated by longitudinal
rat and mouse studies of prenatal immune challenge.65,91,111 This is consistent
with the progression of symptoms in schizophrenia, which tend to progress
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from premorbid to prodromal signs and finally into overt psychotic disease.
Longitudinal neuroanatomical and in-vivo brain imaging studies in rodent
prenatal immune activation models have further shown that the maturation-
dependent functional brain abnormalities are developmentally paralleled (and
possibly also predicted) by progressive changes in brain morphology and neu-
rochemistry.91,100,112 In summary, it appears that early-life inflammatory events
do not induce static effects on the brain, but instead cause progressive changes
in brain and behavioral development. The underlying cellular and molecular
mechanisms responsible for those progressive changes induced by fetal brain
inflammation remain largely elusive. However, it is important to note that in
several models of prenatal immune challenge,35,88,100,102 signs of activated cen-
tral and peripheral inflammatory responses exist prior to the onset of the full
spectrum of schizophrenia-related behavioral, cognitive, and pharmacological
dysfunctions. For instance, prenatal polyI:C exposure in early/middle gesta-
tion in mice leads to increased activation of microglia in pubescence (i.e. on
postnatal day 30),102 a maturational stage at which prenatally polyI:C-exposed
and control offspring do not differ with respect to various schizophrenia-
relevant behavioral and cognitive functions.65,111,112 Similarly, increased
peripheral TNF-α levels have been shown to precede the onset of sensorimotor
gating deficiency in a rat model of prenatal LPS exposure.100 Together, these
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58 Chapter 3
findings lead to several important implications. First, despite the capacity of
prenatal immune challenge to cause peripheral and central inflammation that
persist into the postnatal lifespan, such inflammatory changes do not neces-
sarily translate into overt behavioral manifestations. Second, and perhaps
even more intriguingly, the presence of activated inflammatory responses,
such as enhanced activation of microglia or systemic pro-inflammatory cyto-
kine elevation, may play an important role in the progression of brain disease
following prenatal exposure to infection and/or inflammation. Possibly, pre-
natal immune challenge primes early pre- and postnatal alterations in periph-
eral and central inflammatory response systems, which in turn may promote
developmental neuroinflammation and may disrupt the normal development
and maturation of neuronal systems from juvenile to adult stages of life.
Such developmental neuroinflammation may adversely affect processes that
are essential for normal brain maturation, including myelination, synaptic
pruning, and neuronal remodeling, all of which occur to a great extent during
peri-pubertal brain maturation.113 Priming of postnatal neuroinflammation
by prenatal immune challenge may therefore contribute to the development of
progressive brain and behavioral pathology following prenatal immune chal-
lenge. Early-life exposure to infection and/or inflammation has also the poten-
tial to induce latent neuroinflammatory abnormalities that can be unmasked
and become biologically relevant by additional exposure to certain environ-
mental stimuli throughout postnatal life.106 Related to this, it is of note that
patients with schizophrenia frequently report phases of stress in the proximity
of or during the transition to full-blown psychosis,114 and exposure to physical
or psychological stressors is well known to activate microglia cells and enhance
the production and release of pro-inflammatory cytokines in the CNS.115 Psy-
10:30:24.
chosocial and/or physical stress in the early phase of schizophrenic disease

may therefore be an important factor with the potential to unmask latent neu-
roinflammatory effects, and to unleash their impact on disease progression.
This concept would be consistent with “multiple-hit” theories of schizophre-
nia, suggesting that the disorder’s etiology most probably involves exposure to
multiple environmental and/or genetic insults at various stages of brain devel-
opment and maturation.109,110 Priming of neuroinflammatory responses by
prenatal infection and/or inflammation may also be relevant for the progressive
reduction in gray matter volume that occurs in the proximity of or during the
onset of full-blown psychosis, which seem to resemble an exaggeration of the
gray matter reduction that occurs as a result of normal adult development.116,117
Findings of increased brain glutamate levels in subjects with ultra-high risk
for schizophrenia and first-episode patients have been taken as circumstantial
evidence to support the possibility of (transient) processes of neurodegenera-
tion in the early stages of schizophrenia,118 because excessive synaptic gluta-
mate levels are highly neurotoxic.119 Interestingly, activated microglia release
substantial levels of glutamate,120 and accumulating evidence suggests that
such microglia-mediated toxicity contributes to neuronal damage in the event
of neuroinflammation.21,50,105 Over-activation of microglia also leads to ele-
vated production of quinolinic acid and 3-hydroxykynurenine (OHKY), both of
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121,122
which have potent neurotoxic properties. Drug-naïve first-episode schizo-
phrenic patients displayed enhanced 3-OHKY levels, and levels of 3-OHKY
predicted clinical improvement following antipsychotic drug treatment in as
much as the lowest concentrations of 3-OHKY were associated with the great-
est improvement in symptoms.123 Taken together, the excess in glutamate and
3-OHKY release during the early (prodromal) stages of schizophrenic disease
would fit with the hypothesis that (prenatal infection/inflammation-induced)

neuroinflammatory processes play a critical role in the progressive development
of overt schizophrenic disease.
3.7 Developmental
Neuroinflammation as a
Possible Target for Disease Prevention
It has been suggested that prophylactic or symptomatic treatments targeting
maternal infection and associated inflammatory processes may be efficient
in reducing the incidence of schizophrenia and related disorders.18 Accord-
ing to estimates,11 such preventive efforts could reduce the number of schizo-
phrenia cases by as much as one-third, depending on which infectious agents
were to be considered and the population studied. Findings from animal
models have already provided initial biological plausibility for this possibil-
ity by showing that at least parts of the deleterious neurodevelopment effects
of prenatal infection/inflammation can be attenuated or even fully prevented
by appropriate interventions targeting activated inflammatory response.81,83
Besides prophylactic or symptomatic treatments targeting the maternal host,
anti-inflammatory interventions may have the potential to attenuate pro-
10:30:24.
gressive brain changes and development of psychosis when applied during

the early phases of the developmental course of schizophrenia.124 There is
evidence from clinical trials using the anti-inflammatory agent celecoxib (a
preferential cyclo-oxygenase-2 inhibitor) given in conjunction with atypical
antipsychotic drugs, demonstrating superior beneficial treatment effects of
such anti-inflammatory add-on therapy (compared with treatment outcomes
using antipsychotic drugs alone), especially when the anti-inflammatory
therapy was initiated in the early phase of schizophrenia, as opposed to later
chronic stages.94,95,125 In another study of the early phase of schizophrenia,
administration of the broad-spectrum antibiotic minocycline in conjunc-
tion with standard antipsychotic drugs has been shown to exert superior
effects in improving negative and cognitive symptoms, compared with treat-
ment outcomes using antipsychotic drugs alone.126 In contrast, such anti-
inflammatory strategies may exert no superior effects in the treatment of
schizophrenia when implemented in patients having a long duration of dis-
ease,127 suggesting that neuroinflammatory processes are especially relevant
for the early phase of the disease. It is also important to note that numerous
antipsychotic drugs are known to exert inhibitory effects on immune func-
tions in general, and on pro-inflammatory cytokine networks in particular.128
In the present context, the recently identified microglia-inhibiting effects
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60 Chapter 3
129,130
of antipsychotic drugs seem to be of special interest. Therefore, anti-
psychotic drugs may add to the therapeutic (or even preventive) effects in the
pharmacotherapy of schizophrenia by dampening on-going inflammatory
processes such as microglia over-activation.
3.8 Conclusions
The fact that “…cytokines generated by the maternal immune system (and/
or the placental or fetal immune system) in response to infection may in
part be responsible for the interaction between maternal infection during
pregnancy, altered neuronal development, and schizophrenia” was proposed
by Gilmore and Jarskog for the first time in 1997.41 Since then, wide-rang-
ing epidemiological studies and remarkable advances in modeling prenatal
immune activation effects in animal models have provided strong support
for this hypothesis by underlining the critical role of inflammatory events,
together with downstream pathophysiological processes, in mediating the
short- and long-term neurodevelopmental effects of prenatal infection.
Furthermore, longitudinal studies in animal models indicate that develop-
mental neuroinflammation induced by prenatal immune challenge may be
pathologically relevant beyond the antenatal period, and may contribute to
disease progression associated with the gradual development of full-blown
schizophrenic disease. Undoubtedly, our understanding of the role of devel-
opmental neuroimmune mechanisms in progressive brain changes relevant
to schizophrenia is still in its infancy. Identification of these mechanisms are
highly warranted as they may represent a valuable target to attenuate or even
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prevent the emergence of full-blown brain and behavioral pathology, espe-

cially in individuals with a history of prenatal complications such as in-utero
exposure to infection and/or inflammation.
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www.Ebook777.com
CHAPTER 4
The Self-medication Hypothesis

in Schizophrenia: What Have
We Learned from Animal
Models?
BERNARD LE FOLL*a,b,c, ENOCH NGd, JOSÉ M. TRIGOa, AND
PATRICIA DI CIANOa
a
Translational Addiction Research Laboratory, Centre for Addiction and
Mental Health, 33 Russell Street, Toronto, Ontario, M5S 2S1, Canada;
b
Ambulatory Care and Structured Treatment Program, Centre for Addiction
and Mental Health, 33 Russell Street, Toronto, Ontario, M5S 2S1, Canada;
c
Departments of Family and Community Medicine, Pharmacology,
10:30:28.
Psychiatry, Institute of Medical Sciences, University of Toronto, Toronto,

Canada; dLunenfeld Tanenbaum Research Institute, Mount Sinai Hospital,
600 University Avenue, Toronto, Ontario, M5G 1X5, Canada
*E-mail: bernard.lefoll@camh.ca
4.1 Substance
Use and Schizophrenia: Clinical
Aspects
4.1.1 Epidemiology and Clinical Aspects
Schizophrenia is characterized by three main classes of symptoms: positive,
negative and cognitive. Positive symptoms are the most obvious symptoms
of schizophrenia. These are the hallucinations, delusions and other florid

70
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The Self-medication Hypothesis in Schizophrenia 71

aspects that represent a prototype of the illness. In addition, people with
schizophrenia experience negative symptoms, such as avolition and anhe-
donia. Although more difficult to observe for a third party, these negative
symptoms are nevertheless disruptive to the life of the patient, making it
difficult to engage in everyday activities. Perhaps the most disabling symp-
toms are the cognitive ones, such as impaired memory and attention. These
prevent patients with schizophrenia from living normal lives, maintaining

employment and interacting with others. Of this triad of symptoms, pos-
itive symptoms are treatable with antipsychotics in those individuals that
respond to pharmacological interventions. By comparison, the negative
and cognitive symptoms do not respond well to medication, and to date,
there are no effective treatments for these aspects of schizophrenia. The
self-medication hypothesis posits that people with schizophrenia smoke
and/or use cannabis to alleviate these treatment-resistant symptoms, par-
ticularly the cognitive ones.
Cannabis, together with nicotine, is one of the most frequently abused
substances among people with schizophrenia.1–3 Lifetime diagnosis of men-
tal disease, and particularly personality disorders and psychotic disorders,
has been associated with higher prevalence of transition from substance use
to substance use disorder.4 Those associations were particularly strong for
nicotine, but also notable for cannabis.4 It has been estimated that approx-
imately 70% of people with schizophrenia smoke tobacco, in comparison
to a rate of smoking of approximately 20–30% in the general population.5,6
National epidemiological surveys have reported increased rates of cannabis
use and cannabis use disorders among individuals with mental illness. It is
estimated that 50% or more of schizophrenics might be dependent on can-
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nabis, and up to 80% of patients report current or recent use.7 In contrast,

based on a large epidemiological survey in the USA, it has been estimated
that among those exposed once to cannabis, 7.0% of males and 5.3% of
females will develop cannabis dependence at some point of their life.8
A number of theories have been proposed to explain the high prevalence
of substance use among people with schizophrenia. The addiction vulner-
ability hypothesis posits that the neurobiological changes associated with
schizophrenia predispose patients to both the illness and a higher vulnera-
bility to addiction.9–11 Alternatively, exposure to antipsychotics could be cre-
ating enhanced vulnerability to substance use and addiction.12,13 However,
perhaps the most widely discussed theory is the self-medication hypothesis
which states that schizophrenics smoke tobacco and/or use cannabis to alle-
viate the symptoms of their disease (especially the cognitive symptoms) and/
or to attenuate antipsychotic side-effects.14 Here, we first present evidence
for the self-medication hypothesis in humans using tobacco and or cannabis
by reviewing the impact of these drugs on cognition and also on the side-
effects of antipsychotics. Then, we describe some common animal models
used to study schizophrenia and discuss the findings of animal studies of
the self-medication hypothesis.
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72 Chapter 4
4.1.2 Human
Studies Exploring the Impact of Nicotine on
Cognition in Schizophrenia
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels
composed of five subunits, labeled α2 to α10 and β2 to β4, located in the cen-
tral nervous system. The combination of subunits determines the func-
tionality of the receptor. In the brain, the most prevalent of these are the
homo-oligomeric α7 and the heteromeric α4β2* nAChRs. The role and func-
tion of those combinations are not yet fully elucidated, but there is clear
evidence that implicates α4β2* nAChRs in nicotine addiction.15,16 Notably,
α4β2* nAChRs located in the ventral tegmental area are able to stimulate
dopaminergic transmission.15,17 nAChRs are also located in the frontal cor-
tex and hippocampus [reviewed by Wing et al. (2012)],11 where they may
exert control over cognitive symptoms,18–21 but midbrain nAChRs could
also participate in some cognitive functions.17 As schizophrenia has been
associated with a hyperdopaminergic state in striatal areas and a hypodopa-
minergic state in cortical areas,22,24 nAChRs are well positioned to modulate
dopamine transmission in those areas. Agonists of α7 nAChRs have been
proposed as cognitive enhancers.25–28 However, a large-scale study failed to
demonstrate effects on cognition,29 so more research is needed to demon-
strate that this strategy will work.
Various studies have indicated that nicotine enhances cognition in human
subjects. The challenge has been to demonstrate whether or not these effects
are due to specific effects of nicotine, or due to the reversal of tobacco with-
drawal occurring in smokers. A recent meta-analysis of 41 double-blind place-
bo-controlled studies concluded that nicotine has clear and significant effects
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on six of nine performance domains that were evaluated in those trials.30 Impor-
tantly, nicotine effects on motor abilities, attention and memory represented
true performance enhancement effects because they were not confounded by
withdrawal.30 The situation is a little bit less clear for schizophrenia as fewer
studies have been performed and not all studies included appropriate controls.
In one study, negative symptoms were decreased after smoking a cigarette con-
taining high levels of nicotine.31 Attenuation of negative symptoms was also
observed after smoking a denicotinized cigarette, suggesting some contribu-
tion of conditioning factors, but the change in symptom scores was not as great
after a denicotinized cigarette as observed following a high-nicotine cigarette. It
should be noted that another study found no difference between denicotinized
and nicotinized cigarettes in measures of negative symptoms.32
Although conditioning factors may contribute to the improvement in
symptoms of schizophrenia, nicotine itself may ameliorate cognition, as
administration of nicotine through a nasal spray increased cognitive perfor-
mance in a number of tasks in subjects with schizophrenia.31 In a subsequent
study, nicotine gum produced a modest improvement in attentional func-
tion in non-smokers while impairing performance in smokers.33 The equiv-
ocal nature of these results may be explained by the short abstinence period
(2 hours) used to study cognitive effects, which is supported by a subsequent
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finding that nicotine nasal spray improved cognitive performance in subjects
who had been abstinent overnight.34
Studies have also investigated the impact of withdrawal on the cogni-
tive-enhancing effects of nicotine. In one study, overnight abstinence from
smoking impaired cognitive measures that were reinstated upon resump-
tion of smoking.35 This result suggests that perhaps nicotine serves to
reverse withdrawal-induced deficits in cognition and does not have any

cognitive-enhancing properties on its own. Some resolution of this issue
was provided by a study by Barr et al. (2008) that reported positive effects of
transdermal nicotine on cognitive measures in healthy controls and also in
subjects with schizophrenia that were non-smokers (lifelong non-smokers
or abstinent from smoking for at least 3 months, a time course that ensures
absence of withdrawal).36 Thus, smoking may enhance cognition through
nicotinic mechanisms distinct from the alleviation of withdrawal both
in people with schizophrenia and in controls. Although in some studies
it appears that nicotine produces effects of higher magnitude in subjects
with schizophrenia, compared to controls,36 it should not be forgotten that
the subjects in those studies were under antipsychotic treatment, which
could be a confounding factor. In a recent study, there were no differences
noted in improvements of attentional task performance induced by nico-
tine in healthy controls versus subjects with schizophrenia.37
Cognitive deficits in schizophrenia are believed to be due, in part, to poor
sensory gating. Incoming sensory information is not properly gated, or filtered,
leading to overstimulation and an inability to process this information.38 Pre-
pulse inhibition (PPI) models this deficit in both humans39 and animals.40 To
do this, a startling stimulus is preceded by a stimulus of sub-threshold inten-
10:30:28.
sity. The startle response to the second, larger, stimulus is smaller (inhibited)
following the weaker pre-pulse than it is in the absence of the pre-pulse. In
people with schizophrenia this prepulse inhibition is deficient,41 presumably
due to an inability to gate, or process, the weaker pre-pulse. Studies have con-
sistently found positive effects of smoking on PPI in people with schizophrenia.
Smoking just prior to a test session increased PPI in people with schizophrenia
who smoked,42,43 compared to a lack of effect of smoking in schizophrenic sub-
jects who did not smoke.44 The comparison to non-smokers is relevant to the
present discussion as it implies that cognitive enhancement may be due to a
reversal of deficits that may be observed during withdrawal from nicotine. This
is an important consideration as it has been shown that PPI is reduced during
withdrawal in schizophrenic subjects but not in controls.45
4.1.3 Human
Studies Exploring the Impact of Cannabis on
Cognition in Schizophrenia
Comprehensive reports, such as the Australian Government National Drug
Strategy,46 have stated that “reasons for cannabis use might include social
isolation, lack of emotion or feeling for others, lack of energy, difficulty
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74 Chapter 4
sleeping, depression, anxiety, agitation, tremor or shaking and boredom”.
The failure of antipsychotic medications to treat these negative symptoms
of schizophrenia has led some researchers to hypothesize that patients use
marijuana as a form of self-medication.47,48 Some authors have further pro-
posed that cannabis use might improve cognition in patients.49–52 Indeed,
superior neurocognitive performance in cannabis-using patients compared
to non-using patients was reported in a recent review.50 This is noteworthy

given that, in general, evidence regarding the acute impairments in memory
following cannabis use in humans is robust [see Schoeler and Bhattacharyya
(2013)53 for a recent review]. Supporting this idea, chronic cannabis use was
also able to reduce P50 suppression (positive event-related potential marker
of sensory gating).54–57 It should be noted that the cannabinoid receptor type
1 (CB1) inverse agonist rimonabant (SR141716A) had no effects on positive or
negative symptoms of schizophrenia.58
Despite some reports that suggest that cannabis may alleviate the symptoms
of schizophrenia, there is much more evidence suggesting that cannabis may
actually exacerbate the positive symptoms of schizophrenia, providing a bitter-
sweet amelioration of negative or cognitive symptoms. Different studies seem
to indicate that cannabis use might be involved in psychoses, schizophrenia
and schizophreniform psychosis cases [see Shrivastava et al. (2014)59 for a recent
review]. The most convincing data come from evidence that the administration
of δ-9-tetrahydrocannabinol (THC) to humans can exacerbate schizophrenia
symptoms.60 Numerous studies have hypothesized that exposure to cannabi-
noids, in particular during adolescence, might prompt dysfunctions, poten-
tially causing vulnerability to psychosis.61–63 As cannabis use exacerbates the
severity of schizophrenia symptoms, it has been proposed that it might actually
10:30:28.
constitute a risk factor for this mental disorder.60,64,65 It is unclear if cannabis is

precipitating the onset of illness or increasing the risk for the illness itself.
4.2 Human
Studies that Explore the Impact of
Smoke on the Side-effects of Antipsychotic
Medications
Patients might not only be self-medicating to alleviate the primary symp-
toms of schizophrenia, but also to alleviate the side-effects of antispychot-
ics.66–68 Antipsychotic medications have significant side-effects, including
extrapyramidal symptoms (various movement disorders, including acute
dystonic reactions, pseudoparkinsonism, tardive dyskinesia or akathisia),
sedation, dizziness, weight changes, sexual dysfunction, anticholinergic
symptoms (blurry vision, dry mouth, drooling and constipation), memory
effects, hyperprolactinaemia and galactorrhoea. It is well known that compo-
nents of smoke (likely present in both tobacco smoke and cannabis smoke)
affect the activity of some cytochrome P450 (CYP450) enzymes and there-
fore can modulate the metabolism of antipsychotics and reduce their plasma
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69
concentrations. Such effects may alleviate side–effects, but may also ulti-
mately dampen antipsychotic effectiveness; indeed, it has been shown to
worsen positive symptoms.70,71
4.3 Animal
Models to Study the Self-medication
Hypothesis
Animal models can play a unique role in testing the self-medication hypoth-
esis against competing hypotheses for a number of reasons.72 First, animal
models allow researchers precise temporal control over the induction of
schizophrenia-like and substance-use-like states, as well as the administra-
tion of antipsychotics, in ways that are impossible in human patient popu-
lations where co-morbidity is common and medication state is uncertain.
Second, the use of animal models allows questions to be asked about the
physiology and molecular mechanisms underlying self-medication in ways
that are difficult or impossible to probe in humans for ethical reasons. Third,
animal models allow for precise control of genetic and/or environmental
manipulations to disentangle their contributions or interactions to self-med-
ication phenomena. Below, we first describe how animals are used to model
schizophrenia, and then describe how such models can be used to test the
self-medication hypothesis.
4.3.1 Modelling Schizophrenia in Animals

10:30:28.
There are three major criteria for evaluating any animal model of psychiatric
disease: face validity, construct validity and predictive validity.73 Face validity
requires that the model recreate important behavioral, anatomical or patho-
physiological markers of the disease. Face validity, in other words, refers to
whether an animal model “looks like” the real disease state that is being mod-
elled. An example of face validity would be the self-administration model of
drug addiction, because in this model animals ‘take’ drug in the same way
as humans. Construct validity refers to whether the underlying construct in
the animal model is actually modelling what it purports to study. It requires
that the model be built using a method relevant to the aetiology or patho-
genesis of the disease. For example, a model could mimic genetic mutations
in genes that are known to confer disease risk in humans, or alter neural
circuits thought to be impaired in disease. If these genetic manipulations of
altered neural circuits are the same as those that are awry in the disease, it is
likely that the model has construct validity. Finally, if a model has predictive
validity, treatment challenges (often pharmacological) produce effects that
are predictive of what would happen in humans. A model of schizophrenia
with predictive validity would demonstrate an improvement in symptom
measures following administration of medications that are known to be
effective in humans.
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76 Chapter 4
4.3.1.1 Models of Positive Symptoms (Dopamine Hyperactivity)

With the widespread use of antipsychotics, the 1950s marked a revolution in
the treatment of schizophrenia. The prototype of the time, chlorpromazine,
was established as a dopamine antagonist and spawned research into the
development of related compounds with a similar mechanism of action. It also
sparked the dopamine theory of schizophrenia, which posits that the disorder
is due to an overactive dopamine system. Lending support to the dopamine
hyperactivity hypothesis of schizophrenia is the observation that the psychosis
induced by high-dose stimulant use is indistinguishable from the psychosis
seen in schizophrenia. Such stimulants, especially amphetamines, produce
their effects notably through elevations in dopamine. Thus, the original animal
models of schizophrenia focused on overstimulation of the dopamine system.
To study positive symptoms in schizophrenia, researchers use a common
number of behavioral tests that model changes in dopamine activity.74 Base-
line hyperlocomotion in the open field and enhanced locomotor responses
to psychostimulant (e.g. amphetamine) injections have been proposed to
model positive symptoms because locomotion in rodents is highly depen-
dent on striatal dopamine levels. Imaging studies have revealed excess
striatal dopamine release in subjects with schizophrenia, especially during
psychotic episodes or in response to amphetamine challenge.23
Administering amphetamine or apomorphine to animals results in hyper-
activity at moderate doses and stereotypies at higher doses. Locomotion is
typically measured using horizontal or vertical beam breaks, while stereotyp-
ies consist of focused sniffing, licking and grooming, among other aspects.
The two are believed to represent a continuum, with stereotypies being
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exhibited at high doses. The attenuation of these changes by antipsychotics

is believed be to an indication of amelioration of symptoms of schizophrenia.
The dopamine transporter is intimately involved in the regulation of extra-
cellular dopamine. Through transporter-mediated re-uptake of dopamine
from the synapse, the dopamine transporter clears dopamine from the syn-
apse and keeps neurotransmission in balance. The development of a dopa-
mine transporter knockout mouse,75 was met with excitement in the scientific
community, as dopaminergic mechanisms in pathological states could now be
explored with greater precision. As predicted, these mice are hyperdopaminer-
gic76 and exhibit behaviours consistent with this, such as perseverative loco-
motor activity, stereotypy and cognitive and behavioral inflexibility [reviewed
in Gainetdinov (2008)].77 It is the existence of the latter two deficits that may
make dopamine transporter knockout mice a better model of schizophrenia
than amphetamine- or apomorphine-induced locomotion and stereotypy.
4.3.1.2 Models of Cognitive Dysfunction

Many tasks are available for modelling cognitive symptoms, which is the
dimension thought to be addressed by self-medication in schizophrenia.
The most common test used is PPI, a measure of sensorimotor gating.
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This is a model that is also studied in humans, and is described above.
Another popular test is latent inhibition, a test of attentional processes
described by some as the ability to ignore stimuli that are no longer rele-
vant.78 Working memory impairments are often modelled using the Mor-
ris water maze. In this task, animals are required to locate an underwater
platform based either on memory or a signal provided by cues in the envi-
ronment, thus providing a test of memory and navigation. Other cognitive

tasks include Y-maze spontaneous alternation, T-maze delayed alterna-
tion or the radial arm maze. All three make use of the fact that rodents
will prefer exploring “new” arms of a maze rather than a previously visited
one. Animals displaying greater re-entry to maze arms visited in the past
few seconds suggest impaired working memory. Behavioral flexibility can
be measured by reversal learning in the water maze or T-maze, or in more
complex manners such as the attentional set-shifting task. Animal mod-
els of schizophrenia may be expected to perseverate on an old rule for
behaviour rather than shifting to a new rule that is now in effect. For
example, in the reversal T-maze, model rodents may perseverate much
more than controls in going down the left arm that was rewarded for the
past 15 trials, even though it is now the right arm that is consistently
baited.
4.3.1.3 Neurodevelopmental Models of Schizophrenia

Increasingly, schizophrenia is considered to be a disorder that is caused
by an event that occurs postnatally, and hence neurodevelopmental mod-
els of schizophrenia attempt to reflect changes that may occur during
10:30:28.
development. The neonatal quinpirole and neonatal ventral hippocam-

pal lesion models provide a means to assess some of these changes that
are caused by a postnatal event and develop through adolescence and
adulthood.
The neonatal quinpirole model has been reviewed elsewhere,79 but, briefly,
this model consists of administering quinpirole to rats neonatally. Quin-
pirole is a D2/3 agonist and this treatment results in increased sensitivity
of the D2 receptor80 and behaviours that are consistent with symptoms of
schizophrenia in humans. For example, administration of quinpirole to rats
when they are young results in increased locomotor activity in adulthood and
dopamine release in the nucleus accumbens.81
Another developmental model is the neonatal ventral hippocampal
lesion model. By lesioning the part of the hippocampus that projects to
the frontal cortex, thought to be involved in schizophrenia, a number of
deficits are seen.82 Importantly, even though these lesions are conducted
when the rats are quite young, deficits do not emerge until later in ado-
lescence and adulthood.82 Specifically, impairments seen include those
that may reflect cognitive symptoms such as working memory,83 nega-
tive symptoms such as social behaviour and positive symptoms such as
hyperlocomotion.84
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78 Chapter 4
4.3.1.4 Negative Symptoms and Side-effects

Negative symptoms such as anhedonia and decreased social motivation can
be modelled using the sucrose preference test and the three-chamber social
approach task, respectively. Finally, apart from modelling the core symp-
toms of schizophrenia, animal models can be used to mimic the behavioural
side-effects of antipsychotic drugs. For example, older antipsychotics can

produce extrapyramidal motor symptoms, which can be modelled in rodents.
One measure is catalepsy, a prolonged state of remaining rigid and immo-
bile rather than correcting an externally imposed posture.85 Catalepsy is a
known side-effect of antipsychotic treatment and also a negative symptom
of schizophrenia.
4.3.1.5 Non-behavioural Models
A wide variety of strategies exist to achieve construct validity. Broadly
categorized, these strategies involve the creation of neurotransmitter/
pharmacological models, genetic models and neurodevelopmental/
environmental insult models. Researchers have used pharmacological
or genetic means to produce, not only hyperdopaminergic states (i.e.
amphetamine sensitization or dopamine transporter knockout), but
also hypoglutamatergia [i.e. phencyclidine (PCP) or ketamine injections
or serine racemase mutant mice] to model schizophrenia [see Poels
et al. (2014)86 for a review of human studies showing glutamate deficit
in schizophrenia]. Based on findings from genetic studies of schizophre-
nia, animals have been genetically modified to mimic the common vari-
10:30:28.
ants (i.e. neurexin, dysbindin and neuroligin) or the rare variants of high
penetrance (i.e. Disc1 and 22a11.2 microdeletions) that increase risk for
schizophrenia. Schizophrenia models can also be created by early-life
insults such as maternal immune activation (i.e. injecting pregnant dams
with polyI:C or lipopolysaccharide).
4.4 Testing
the Predictions of the Self-medication
Hypothesis
The large variety of animal models of schizophrenia available allow for
the detailed testing of the self-medication hypothesis. The main predic-
tion of the self-medication hypothesis can be tested in a variety of animal
models to determine whether administration of drugs such as nicotine
or cannabis can ameliorate the cognitive symptoms of schizophrenia.
For example, according to the self-medication hypothesis, it may be pre-
dicted that nicotine would correct the delayed alternation T-maze deficit
found in a Disc1 genetic model of schizophrenia. Alternative hypotheses
can also be tested. For example, the addiction vulnerability hypothesis
could be tested by seeing if impairing the development of dopaminergic
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circuitry leads to both hyperlocomotion and greater likelihood of self-
administration of nicotine. These specific tests remain to be performed, but
below is a summary of those tests that have been conducted. The fact that
each model may mimic only part of the aetiology or pathogenesis of schizo-
phrenia may allow for a more nuanced understanding of co-morbidity
than offered by the existing competing theories.
4.5 Review
of Studies Evaluating the Impact of
Nicotine on Animal Models of Schizophrenia
The next part of this review focuses on investigations into the role of nico-
tine in animal models of schizophrenia. The evidence from several classes
of models is reviewed to evaluate the self-medication model. First, animal
models of dopamine hyperactivity are reviewed, followed by neurodevelop-
mental models and, finally, PPI, a model of cognitive impairment in schizo-
phrenia. There are over 60 models of schizophrenia, and only the more
established models that have been directly evaluated for the effects of nico-
tine are reviewed.
4.5.1 Animal Models of Dopamine Hyperactivity

In one study, chronic nicotine potentiated both the locomotor stimulating
effects and stereotypy induced by either apomorphine or amphetamine.87
In another study, acute nicotine increased apomorphine-induced licking,88
while it was further reported that chronic, but not acute, nicotine resulted in
10:30:28.
more amphetamine-induced locomotion.89 Nicotine administration reduced

haloperidol-induced catalepsy.87 Antagonists at both the α4β2 nAChRs and the
α7 nACHRs reversed the effects of nicotine on methamphetamine-induced
locomotion,89 implicating both receptor subtypes.
The effects of nicotine on the cognitive function of dopamine transporter
knockout mice were studied using the Morris water maze. Knockout mice
were worse at baseline at both the cued and spatial versions of this task.
Administration of nicotine improved performance such that latencies, dis-
tance travelled and successful trials needed to reach the platform approached
levels seen in the wild-type mice and there was no tolerance to this effect.90
In sum, even though nicotine potentiated stimulant-induced locomotion
and stereotypy, it improved performance in the Morris water maze in knock-
out mice lacking the dopamine transporter.
4.5.2 Neurodevelopmental Models of Schizophrenia

Neonatal quinpirole potentiated locomotor sensitization relative to rats
that were treated neonatally with saline.91 Sensitization has been proposed
by some researchers to be a measure of the addictive potential of drugs.92
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80 Chapter 4
Sensitization measures the ability of repeated drug exposure to increase a
subsequent response to that drug. This is consistent with the finding that
neonatal quinpirole also potentiated measures in a conditioned place-pref-
erence model of addiction.93 In the neonatal ventral hippocampus lesion
model of schizophrenia, sensitization to the locomotor-stimulating effects of
nicotine were enhanced in rats that received neonatal lesions of the ventral
hippocampus, compared with controls.94 There were no differences between

lesioned and control groups on the first day of nicotine administration, sug-
gesting a specific effect on sensitization, rather than the basal response to
nicotine. Importantly, using the intravenous self-administration paradigm,
it was found that rats lesioned in the neonatal ventral hippocampus took
more nicotine infusions, and required fewer trials to criterion than did those
with sham lesions.95 In this same study, lesioned animals were impaired on
the radial arm maze, consistent with this model providing a measure of the
cognitive deficits seen in schizophrenia. However, administration of nicotine
prior to testing on the radial arm maze did not improve performance.
The evidence from different neurodevelopmental models of schizo-
phrenia suggest that there may be an enhancement of the addictive prop-
erties of nicotine in schizophrenia-like states. However, there is little
experimental work conducted so far and it is difficult to conclude anything
with certainty. Extrapolating those findings to humans would suggest that
in schizophrenia, cigarettes are smoked because they are more readily
addictive rather than due to any effects on cognition. However, it should
be noted that only one study used the intravenous self-administration
paradigm, which is considered the most valid model of substance use;
another caveat is that limited work has been undertaken on the cogni-
10:30:28.
tive aspects of these models. Therefore, a more systematic exploration is

warranted to provide some clarity.
4.5.3 The PPI Model of Schizophrenia

In rats, a number of studies have examined the effects of nicotine on PPI.
In these studies, the effects of nicotine on PPI itself have been tested, as
well as the effects of nicotine on manipulations that affect PPI. Here we
review only those that look at nicotine effects on PPI itself, to get a better
measure of the effects of nicotine on cognition per se. In one study, Curzon
et al. (1994) found that an acute nicotine injection increased PPI; this effect
was reversed by mecamylamine,96 a nicotinic antagonist, confirming that
the ability to improve cognition was due to stimulation of nicotine recep-
tors. In a subsequent study, strain differences were seen in the amplitude
of startle, but the percent increase in PPI following chronic nicotine was
consistent across strains,97 further supporting the hypothesis that nicotine
enhances cognition. In contrast, chronic nicotine impaired PPI in Long–
Evans rats98 but enhanced it in Sprague Dawley rats,99 while another study
found that acute nicotine decreased PPI in Sprague Dawley rats.100 Further
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complicating matters, in Lister Hooded rats, chronic nicotine impairs PPI,
while acute injections have no effect.101 The reasons for these discrepancies
are unknown, but together they point to the fact that nicotine may have ame-
liorating effects on cognition, albeit only under certain circumstances.
Underscoring species differences in the PPI response following nicotine,
acute nicotine decreased PPI in Sprague Dawley rats but increased it in
BALB/c mice.100 This effect was found to be due to α4β2 nACHRs receptors,
as an agonist replicated this effect. An α7 nACHR agonist did not have con-
sistent effects on PPI.100
Adding to the complexity, baseline PPI performance significantly impacts
the response to nicotine. Kayir et al. (2011) divided rats into tertiles based
on basal PPI, and the upper and lower thirds were given daily injections of
nicotine.102 Locomotor response to nicotine increased over days, and a chal-
lenge injection given after a few days of withdrawal showed evidence of sen-
sitization. In particular, those rats that exhibited low baseline PPI showed
greater sensitization than those with higher PPI. Given that low PPI is also
found in subjects with schizophrenia, this finding suggests that those with
schizophrenia may be more likely to become addicted to nicotine than those
without schizophrenia. Or, conversely, those with poor cognitive abilities (as
seen in schizophrenia) may develop more addiction to nicotine.
4.6 Review
of Studies Evaluating the Impact of
Cannabinoid Agonists on Animal Models of
Schizophrenia
10:30:28.
The effect of cannabinoids on certain aspects of schizophrenia can be stud-

ied using a variety of animal models, including genetic and neonatal-induced
stress models or pharmacological models that target specific neurotrans-
mitter systems such as dopamine or glutamate. Therefore, there is an abun-
dant literature on how different behavioural abnormalities related to certain
symptoms of schizophrenia (PPI, short-term memory deficits, alterations in
social behaviour, locomotor activity, etc.) are affected following the (acute and
chronic) administration of different types of cannabinoids or a combination of
them [see Arnold et al. (2012)103 for a detailed review]. We focus here on some
of the aspects that are relevant for the self-medication hypothesis.
4.6.1 Cognitive Models

Treatment with the CB1/2 agonist WIN 55,212-2 dose dependently impaired
PPI in non-stressed animals.104 In addition, rats chronically treated with
WIN 55212-2 during puberty displayed impaired sensorimotor gating.105 In
contrast, the acute treatment with WIN 55,212-2 improved PPI in psychoso-
cially stressed mice54,106 and THC induced greater enhancement of PPI in a
mouse model of schizophrenia than in wild-type mice.107 This result should
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82 Chapter 4
be read with caution, as there may be possible confounding factors [see
Boucher et al. (2007)107 for a discussion]. The situation is complex, as CB1
receptor antagonists reverse sensorimotor gating deficits induced by PCP,108
and genetic CB1 disruption in mice counteracted the PCP-induced negative
symptoms.109 A large number of studies have shown deficits in long-term
synaptic plasticity, learning and memory induced by THC exposure, which
are primarily mediated through CB1 expressed in the brain.110–113 Altogether,

preclinical findings clearly indicate that THC has deleterious effects on cog-
nition, but the possibility that THC may produce beneficial effects on some
cognitive aspects in models of schizophrenia warrants further exploration.
4.6.2 Neurodevelopmental Models

Chronic exposure to THC in immature animals seems to have enhanced
effects compared to the chronic treatment of mature rats, causing more
prolonged effects on cognitive performance.114–116 Early exposure to
cannabinoids results in a variety of cognitive deficits in adult animals
resembling those found in schizophrenia models (i.e. disruption of PPI of
startle or working memory dysfunction).117 Indeed, maternal exposure to
even low doses of cannabinoid compounds results in altered emotional
behaviour, and enhanced sensitivity to drugs of abuse in the adult rodent
offspring.118 Pubertal rats treated with WIN 55,212-2 showed persistent
alterations in sensorimotor gating, object recognition memory and social
behaviour, among other symptoms.105,119 In line with these findings, peri-
natal, but not adult, exposure to cannabinoid agonists might alter the
10:30:28.
capacity to cope with environmental stress in adult animals by modifying

corticosterone levels.120–122
4.6.3 Impact on Neuroleptic Side-effects

In non-human primates, the CB1 agonist CP55,940 dose dependently reduced
oral dyskinesias induced by the dopamine D1 receptor agonist SKF81297, while
no effects where observed following the administration of rimonabant.123
Somewhat contradictory to this is the further finding in animal models show-
ing that the CB1 receptor antagonist AVE1625 might be useful in treating
extrapyramidal symptoms associated with neuroleptic treatments.124,125
4.7 Conclusions
There is clear evidence that nicotine and tobacco administration can improve
cognition in human subjects. Some of those effects likely represent some
alleviation of withdrawal-induced impairments in cognition, but there is
also a separate positive effect of nicotine on some measures of cognition in
human control subjects. Administration of nicotine also improves cognition
in subjects with schizophrenia; however, there is no clear evidence that those
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effects are more pronounced than in control subjects. Therefore, there is lim-
ited support for the self-medication by tobacco hypothesis of schizophrenia.
A few animal models of schizophrenia suggest that there is an enhanced vul-
nerability for nicotine addiction, as revealed by the sensitization response or
higher nicotine intake. However, these data should be considered to be pre-
liminary, as very limited experimental work has been conducted, and more
experimental work, especially using intravenous self-administration, would

be required to confirm these impressions.
The situation with regard to cannabis is very different. Cannabis (or canna-
binoid agonist) seems to exacerbate the positive symptoms of schizophrenia
(see Fisher et al.126 for an estimate of this impact on the population). There is
a clear detrimental effect of cannabis and of cannabinoid agonists on cogni-
tion. However, surprisingly, some studies suggest that cannabinoid agonists
may improve some measures of cognition in models of schizophrenia. How-
ever, these findings should be considered to be preliminary and should be
replicated in more models. These findings require further exploration to be
considered valid, but if true, could represent a novel direction for treatment
of cognitive dysfunction in schizophrenia.
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CHAPTER 5
Modelling Schizophrenia:
Strategies for Identifying
Improved Platforms for Drug
Discovery
JOHN L. WADDINGTONa,b AND COLM M. P. O’TUATHAIGH*c
a
Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland,
Dublin, Ireland; bJiangsu Key Laboratory of Translational Research and
Therapy for Neuro-Psychiatric-Disorders and Department of Pharmacology,
College of Pharmaceutical Sciences, Soochow University, Suzhou, China;
c
School of Medicine, University College Cork, Brookfield Health Sciences
Complex, Cork, Ireland
10:30:30.
*E-mail: c.otuathaigh@ucc.ie
5.1 Introduction
Schizophrenia is a psychotic illness that is costly for families and society,
affecting approximately 1% of the worldwide population.1,2 The boundaries
of this diagnostic category are arbitrary and likely reflect the intersection of
several domains of psychopathology found in psychotic illness. Diagnos-
tic symptoms of schizophrenia, which typically emerge during early adult-
hood, include positive, psychotic symptoms (hallucinations, delusions,
and thought disorder). There are also negative symptoms (motivational
deficits, anhedonia, flattening of affect, poverty of speech, and decreased

89
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90 Chapter 5
social functioning) and cognitive deficits (working memory deficits, execu-
tive dysfunction, and attentional impairment) that contribute particularly
to functional impairment. Schizophrenia is a neurodevelopmental disorder
characterised by high heritability. However, while the precise nature of the
genetic defect is still largely unknown, recent genome-wide association stud-
ies (GWASs) and copy number variation (CNV) studies have provided new
insights into the etiology of this and related disorders, as well as indicating
new directions for antipsychotic drug discovery.3,4
The past 10–15 years have seen a progressive disinvestment in neuroscience
drug research, and antipsychotic drug discovery has been particularly impacted
by such industrial reprioritisation.5 This is considered to be largely attribut-
able to high failure rates in clinical trials, incomplete understanding of dis-
ease mechanisms, and the absence of treatment biomarkers.6 Our prevailing
understanding of schizophrenia still lacks unequivocal diagnostic pathobiol-
ogy and strong causative genetic mutations.7 Therefore, while our understand-
ing of the nature of psychotic illness continues to progress in an incremental
fashion, it has yet to lead to major advances in the development of novel anti-
psychotic drugs. This continued stasis vis-à-vis antipsychotic drug discovery
may also reflect continued lack of clarity in the field as to current concepts
of psychosis, exemplified by evidence for psychopathological, genetic, and
pathobiological overlaps between schizophrenia, bipolar disorder, and other
forms of psychotic illness.1,8,9 However, given that negative and, particularly,
cognitive symptoms have proven resistant to both first- and second-generation
antipsychotic drugs, there is a greater imperative for both human and preclin-
ical genetic studies to contribute to the development of new antipsychotics.10
As noted above, some authors have emphasised the degree of overlap between
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the symptoms of schizophrenia and other psychotic disorders, including bipo-

lar disorder, and there is ongoing debate regarding the future classification and
diagnosis of neuropsychiatric disorders.11 Adding to this complexity (as well as
having implications for modelling the disorder in animals), psychotic disor-
ders like schizophrenia are not a homogenous entity, but rather characterised
by phenotypic heterogeneity. No two patients with schizophrenia or bipolar
disorder are exactly alike in their clinical presentations, with often overlapping
psychotic, affective, and cognitive symptom clusters. It has been suggested that
ongoing genetic studies might interact with nosological refinements in an iter-
ative fashion, so that we can more accurately dissect their genetic underpin-
nings.12 One pertinent recent development is the National Institute of Mental
Health (NIMH) Research Domain Criteria initiative, which involves the devel-
opment of a research-based classification system for mental disorders that is
informed by the genetics, physiology, and neural circuitry underpinning biobe-
havioural constructs, which cut across current disorder categories.13
5.2 Genetic Architecture of Schizophrenia

Schizophrenia is characterised by a complex genetic architecture, likely
involving a number of very rare genes of large effect that operate on a back-
ground of much more common risk genes of small effect. Therefore, a patient
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Modelling Schizophrenia: Strategies for Identifying Improved Platforms 91

diagnosed with schizophrenia will probably have one or more higher-
penetrance rare alleles and potentially several common alleles of lower pen-
etrance, which modify their effect.14 Additionally, epigenetic mechanisms, as
well as gene × environment [G × E], and gene × gene [G × G] or epistatic interac-
tions, are also implicated. As outlined in section 5.1, schizophrenia and bipo-
lar disorder may share common genetic susceptibility, and this contention
is supported by evidence from studies implicating common linkage regions,

gene expression patterns, and molecular mechanisms in schizophrenia and
bipolar disorder.15 Recent cross-phenotypic genetic studies have revealed
common susceptibility loci [e.g. calcium channel, voltage-dependent, L type,
alpha 1C subunit (CACNA1C), zinc finger protein (ZNF)804A, polybromo-1,
and neurogranin (NRGN)], suggesting that schizophrenia and bipolar disor-
der may lie on a continuum with distinct and overlapping features.16 The
presence of several diverse signals crossing genome-wide significance may
suggest phenotypically and/or mechanistically distinct contributions from
multiple genes, as well as complex epistatic interactions.
To date, GWAS data have corroborated some existing susceptibility targets
(e.g. ErbB417), while also identifying hitherto unknown targets [e.g. the major
histocompatibility complex (MHC) region, transcription factor (TCF)4, neu-
rogranin, and micro-RNA (miR)13718]. With respect to GWASs in schizophre-
nia, few genetic signals have fulfilled stringent criteria, except for ZNF804A,
TCF4, the MHC region and NRGN.19
Several loci have been consistently shown to harbour CNVs in patients
with schizophrenia, including deletions of 1q21.1,20,21 3q29,21 15q11.2,21,23
15q13.11,20,21,24 and 22q11.2,22 as well as microdeletions at 16p11.2 and
16p13.1. Interestingly, CNV deletions, such as that affecting Neurexin 1 or
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Neuroexophilin 2 are also observed in neurodevelopmental disorders such

as autism, possibly implicating some continuum of psychosis with neuro-
developmental conditions.12 In summary, based on existing linkage, GWAS,
and CNV findings, a combination of common alleles of small effect sizes, rare
alleles of variable effect sizes, and CNVs with epistatic interactions and epi-
genetic mechanisms may contribute to the actual genetic risk component.12
Among the more replicably linked common risk genes of small effect identi-
fied in candidate gene association studies, the genetic and biological data indi-
cate the following genes to be associated with increased risk for schizophrenia:
Neuregulin-1 (NRG1), disrupted-in-schizophrenia-1 (DISC1), and dysbindin
(dystrobrevin binding protein-1, DTNBP1). In the present chapter, we consider
the phenotypic data, primarily behavioural and specific to schizophrenia, aris-
ing from mutant mouse models for these three genes, with the aim of discuss-
ing selective preclinical genetic models relating to antipsychotic activity.
5.3 Behavioural Models of Schizophrenia

It is generally recognised that the psychotic symptoms, including hallu-
cinations and delusions, as well as negative symptoms including poverty
of speech and possibly blunted affect, may be intrinsically inaccessible in
non-human species, thereby limiting the potential to develop valid animal
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92 Chapter 5
25–29
models for this disorder. Behavioural models of positive symptoms
have traditionally employed indirect dopamine (DA)-linked motor-based
measures (e.g. novelty- and psychostimulant-induced hyperactivity), and/
or pre-attentional and attentional phenomena such as prepulse inhibi-
tion (PPI) or latent inhibition (LI), paradigms whichmeasure, respectively,
sensorimotor gating and defective salience attribution processes known
to be disturbed in schizophrenia.30–34 Phenotypic modelling of negative

symptoms has focused primarily on a highly restricted set of behavioural
features that are accessible in both humans and animals, e.g. social with-
drawal and avolition.29
With respect to rodent models of cognitive dysfunction in schizophrenia,
earlier reviews of the field emphasised the challenges that result from a lack
of clarity arising from clinical studies of cognitive deficits in schizophre-
nia.27,35 The past decade has witnessed some progress in the generation of
a consensus set of cognitive domains that are disrupted in schizophrenia.
The NIMH established Measurement and Treatment Research to Improve
Cognition in Schizophrenia, and they identified the following seven, con-
sensually agreed, areas of deficiency: attention/vigilance; working mem-
ory; reasoning and problem solving; processing speed; visual learning and
memory; verbal learning and memory; and social cognition. Subsequent
working groups, including Cognitive Neuroscience Treatment Research to
Improve Cognition in Schizophrenia and Treatment Units for Research on
Neurocognition in Schizophrenia have further operationally defined these
areas of impairment, with the latter particularly emphasising the impor-
tance of developing a matching set of animal tasks which might facilitate
modelling of these core domains.26 To this end, large-scale phenotyping
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consortia, for example the European Union-supported Novel Methods lead-

ing to New Medications in Depression and Schizophrenia (NEWMEDS) con-
sortium, made up of academic partners and eight key industry partners,
have been established to standardise a set of behavioural tasks that can be
used in preclinical testing of new drugs to treat schizophrenia and other
neuropsychiatric disorders. To date, NEWMEDS, which is more focused on
standardisation of the behavioural paradigms employed in industry testing,
has made some progress with respect to the development of shared proto-
cols.36 The utility of shared protocols is not restricted to industry-based
research; the problems generated by the diversity of tests applied to mea-
sure the same or different aspects of the same cognitive construct include
difficulties in interpreting contradictory findings that emerge from differ-
ent laboratories using the same mutant line, and reduced comparability of
phenotypes.28
Some authors have questioned the cross-species comparability of exist-
ing tasks that purport to measure similar cognitive processes in rodents
and humans.26,37 However, since the advent of genetically engineered mouse
models for psychotic disorders, researchers have focused their efforts
on the development of murine analogues for learning and memory par-
adigms originally developed in the rat, taking into account any necessary
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stimulus- or response-related adaptations. Additionally, recent developments
in high-throughput computer-automated behavioural testing for rodents
have involved optimisation of touchscreen technology for a variety of visual
modality-based learning and attentional tasks.38–40
5.4 Developing
Valid Experimental Models of
Schizophrenia
Until recent years, mutant models for genes implicated in neuropsychiatric
disorders largely consisted of mice with knockout of a single risk-associated
gene through a combination of gene targeting and trapping or, less com-
monly, transgenics (i.e. gain-of-function mutants and dominant-negative
mutants). The strength of the constitutive mutant approach lies in the abil-
ity to vary gene dosage (e.g. heterozygous vs. homozygous mutants), as well
as the ability to achieve molecular specificity where existing pharmacolog-
ical agents lack sufficient selectivity. The well-rehearsed limitations associ-
ated with constitutive mutants include potential induction of compensatory
mechanisms, redundancy, and increased risk for embryonic or perinatal
lethality where the target gene is functionally pleiotropic.27,35 Specifically in
relation to schizophrenia, some authors have also questioned the heuristic
value of knockout models, as there is no evidence for null mutations in this
disorder.41 Heterozygous knockout involving an approximately 50% reduc-
tion in expression may in fact be more appropriate, particularly for genes
where there is evidence for hemizygocity or a similar degree of down-reg-
ulation in schizophrenia.42 Conversely, some genes are up-regulated in
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schizophrenia or in association with disease risk alleles, and a more rele-

vant manipulation is transgenic over-expression. However, the magnitude of
over-expression in a mouse can differ markedly from the clinical situation,
complicating interpretation of phenotypic data.
Schizophrenia is a polygenic disorder, and the lack of a single causative
mutation precludes the development of a single gene mutant model that
recapitulates the entirety of the disorder, and calls into question the value of
pursuing such an approach.41 Additionally, functional variants have not been
identified for several genes associated with schizophrenia, precluding any
attempt to generate a model with direct clinical relevance to the role of the
gene in the disorder. Thus, many mutant studies constitute investigation of
the functional roles of genes associated with risk for psychosis, rather than
homologous or isomorphic models of psychotic illness itself.
The availability of multiple-gene knockout mutant models can partially
address these concerns, but these models are costly and difficult to produce.43
Where it is not clear which gene combinations should be manipulated, genes
with a well-characterised and disease-relevant biochemical pathway, or genes
within candidate genomic loci, represent good targets.44 There are many spe-
cies differences in the pattern of gene isoforms, and for some schizophrenia
risk genes there may appear to be human-specific isoforms.41
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94 Chapter 5
Recent articles attest to the heuristic value and potential of conditional
models in which the location and timing of expression of the target gene
can be manipulated using various techniques and strategies.7,45 Conditional
mutants, by providing greater control over the induction and region-spec-
ificity of the mutation, represent a powerful approach for modelling disor-
ders that involve neurodevelopmental perturbation, such as schizophrenia.
However, some authors have noted that the etiological validity of certain
mutant models may be questioned on the basis that some human genes (or
splice variants) implicated in schizophrenia may not have murine homo-
logues.25,28,46 This challenge relates to the possibility that one or more
human disease gene(s) might generate more isoforms than the mouse gene,
making it human-specific; in a similar vein, as described later in relation to
NRG1 (see section 5.5), the majority of genetic mouse models have failed
to take into account isoform-specific characteristics that might be crucial
to undertanding the role of genes in the disorder.28 The future model will
likely involve more “humanised” mice, in which schizophrenia-related risk
polymorphisms are introduced into the mouse gene, increasing construct
validity.34,41
An alternative approach consists of chemical mutagenesis using N-ethyl-
N-nitrosourea (ENU).47 Treatment with the mutagen ENU, usually administered
via a series of systemic injections in adult male mice, induces point mutations
in a genome-wide fashion, enabling the identification of previously unknown
genes implicated in disease-relevant phenotypes, and resulting in the develop-
ment of large-scale mutagenesis projects.48,49
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5.5 Phenotypic
Characterisation of Mutant Models
of Schizophrenia: Additional Considerations
5.5.1 Sex-Specific
Phenotypes and Relevance to
Schizophrenia
There is a wealth of clinical evidence for material sex differences in the onset
and psychopathology of schizophrenia, as well as in outcome, but the major-
ity of mutant data discussed in this chapter have been demonstrated in males
only. The general absence of phenotypic studies that have systematically
compared male and female murine phenotypes has been discussed in detail
elsewhere.50 The practical reasons for this approach include increased exper-
imental and labour costs associated with phenotypic assessments conducted
in both sexes, including larger mutant breeding colonies, and increased
running costs for experiments where the sample size is effectively doubled.
Scientific reasons include lack of clarity regarding whether the clinical phe-
notype-specific sex differences reflect biological processes rather than envi-
ronmental and/or social factors and our incomplete understanding of the
basic pathogenesis of schizophrenia, as well as the fact that many of the most
commonly used behavioural and psychopharmacological assays have been
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developed and optimised using male experimental subjects. Adding to the
complexity, existing research has shown that the sexes may differ also in the
nature of and sensitivity to environmental adversities.51,52 Evidence for sex-
ually dimorphic effects of susceptibility gene mutation on psychosis-linked
behavioural endophenotypes may implicate sex hormonal regulation of risk
gene function in a manner that may contribute to the pathogenesis of psy-
chotic illness.27,29,35
5.5.2 Incorporating
Developmental Clinical Trajectory into
the Phenotyping Strategy
Schizophrenia is characterised by subtle impairments over infancy, child-
hood and adolescence, to include increasing recognition of early, non-clin-
ical, psychotic-like experiences, but with the appearance of diagnostic
psychotic symptoms most typically only during adolescence/early adult-
hood; this process is best captured by a lifetime trajectory model of psychotic
illness.9 Its complex developmental trajectory, comprising early neurodevel-
opmental impairments, followed by a subtle pattern of functional deficits,
with the full-blown disorder emerging only in early adulthood,1,9 greatly
exacerbates the challenge of modelling this disorder at a preclinical level.
Typically, mutant models of schizophrenia involve phenotypic assessment
over a period approximating to young adulthood, for valid reasons such as
a desire to avoid multiple testing or the necessity of large subject numbers
that challenges both funding and labour resources. A comprehensive schizo-
phrenia-focused phenotyping strategy must involve assessments over devel-
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opment and maturation that would resolve the sequential emergence of and
interplay between phenotypic effects. In other, non-genetic animal models of
schizophrenia, such as the rat ventral hippocampal lesion model53 and some
mutant models related to schizophrenia,54 it has been common to study phe-
notypes at two or more ages, especially pre- vs. post-pubertal.
5.5.3 Importance
of Mechanistic Interrogation of Phenotypic
Effects
It is generally agreed that mutant models should seek to address and model
the widely espoused clinical strategy of early intervention to ameliorate
poor functional outcome in psychotic illness. A recent study reported syn-
aptic deficits and sensorimotor gating deficts accompanying prolonged
knockdown of the schizophrenia-associated gene DISC1 in mice; this was
achieved via in-utero electroporation of DISC1 short hairpin (sh)RNA selec-
tively targeted to the cells in a lineage of the pyramidal neurons in layer II/
III of the prefrontal cortex (PFC).55 A novel inhibitor to p21-activated kinases,
FRAX486, when administered during adolescence, prevented progressive
synaptic deterioration and ameliorated PPI deficits in adulthood. For many
of the preclinical genetic models discussed below, pharmacological agents
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96 Chapter 5
have been used to probe neurotransmitters implicated either directly or indi-
rectly in genetic mutant phenotypes. Evaluation of pharmacological rescue
of schizophrenia-related phenotypes in mutant models of the disorder is an
important step towards understanding the neurobiological bases of these
gene–phenotype associations, as well as proving of great heuristic value in
drug discovery.3 Where a study incorporates the administration of a prophy-
lactic intervention which might ameliorate the emergence of disease-rele-

vant phenotypes in psychosis-relevant mutant models, this approach has the
potential to inform at a mechanistic level on the relevant phenotypic feature.
5.6 NRG1
Neuregulin-1 is one of a large family of epidermal growth factor (EGF)-do-
main-containing trophic factors, and has more than 30 isoforms, grouped
into six types (I–VI) that are differentiated on the basis of N-terminal
sequence, expression of the α or β EGF-like domain, and if they contain a
transmembrane (TM) region.56 They signal by binding to the ErbB tyrosine
kinase receptor family, in particular ErbB4.56 NRG1 is expressed in diverse
brain areas, including the PFC, hippocampus, cerebellum, and substantia
nigra in both humans and rodents.57,58 The NRG1 isoforms differ in domain
structure and expression levels in various tissues/cells during neural devel-
opment and in adulthood.59 Functionally pleiotropic, NRG1/ErbB4 signalling
has been associated with various neurodevelopmental and plasticity-related
processes, including synapse formation, neuronal migration, and neu-
rotransmitter receptor development and function.60
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NRG1 has been confirmed on meta-analysis to be a replicable risk gene for

schizophrenia.61–63 Supporting neuropathological evidence indicating dis-
ruption to NRG1/ErbB signalling in schizophrenia derives from studies in
human post-mortem brain tissue and cell lines.60,64 There is continued debate
in the field regarding whether up- or down-regulation of NRG1/ErbB signal-
ling is implicated in brains from patients with schizophrenia.65,66 There have
been reports of over-expression of specific NRG1/ErbB4 splice variants;67–70
another study reported increased expression of the ErbB1 protein;71 other
studies have reported either decreased isoform-specific expression of NRG1
transcripts70,72 or no change therein.73 The widespread expression of NRG1
and ErbB, as well as interactions between these molecules with γ-aminobutyric
acid (GABA)ergic,74 glutamatergic,65,75 and dopaminergic neurons76,77 further
supports a role for disrupted NRG1–ErbB signalling in schizophrenia.
5.6.1 NRG1- and ErbB-Deficient Mutant Mouse Models

Several groups have employed various NRG1 knockout mouse lines to study
the relationship between decreased NRG1 and NRG1–ErbB signalling, and
the role of these genes in schizophrenia. Mice lacking NRG1 or ErbB4 exhibit
deficits in behavioural measures relevant to positive symptomatology.
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Heterozygous deletion of the TM-domain truncation of exon 11 NRG1 (the
homozygous knockout being lethal) is associated with hyperactivity, includ-
ing sexually dimorphic effects among individual topographies of exploratory
behaviour, that is normalised by antipsychotic treatment.78–81 PPI deficits
have also been reported in TM-domain (exon 11) NRG1 mutants, although
the magnitude of this phenotype is highly dependent upon protocol-related
environmental factors.32,82,83 NRG1 genotype-related disruption of vari-

ous aspects of social interaction and social cognition has been reported in
mice with heterozygous deletion of the EGF-like domain (pan-isoform) of
NRG1,84 TM-domain (exon 11) NRG1 heterozygotes,80 both constitutive and
conditional ErbB2, ErbB3 and ErbB4 deletion lines,84,85 as well as mutants
with selective loss of ErbB signalling in oligodendrocytes.86 Importantly,
these differences were observed independent of hyperactivity, changes in
anxiety-like behaviour or deficits in olfactory perception. The same negative
symptom-like profile was not observed in EGF-like domain NRG1 mice87 or
type-III isoform-specific NRG1 mutants.88
A recent study has described an alternative TM-domain mutant line with
a truncation from exon 9.89 In contrast with findings obtained with the exon
11 TM-domain line, this study failed to observe any significant NRG1-related
behavioural impairments, aside from mild impairment in cognitive func-
tion. Specifically, no change in exploratory, social, or sensorimotor gating
was observed, but the study demonstrated a sex-specific (male only) deficit
in contextual and cued fear conditioning, as well as decreased object recog-
nition memory performance. No phenotype effect of NRG1 deficiency was
observed on Morris water maze performance.
Neuromorphological analyses failed to reveal any effect of NRG1 deletion
10:30:30.
on the architecture of hippocampal CA1 pyramidal neurons in the exon 11

TM-domain line.89 However, in line with reported involvement of NRG1 in
GABAergic and glutamatergic function, this study also reported a reduc-
tion of glutamic acid decarboxylase (GAD)67 and parvalbumin expression
in the hippocampus of male NRG1 mutants, and a differential behavioural
response to pentylenetetrazol (a GABAA receptor antagonist). Additionally,
chronic administration of valproate, in part a GABA transaminase inhibitor,
was shown to rescue the mild cognitive deficits observed, as well as reverse
the increase in hippocampal GAD67 expression in male NRG1 mutants.
These data suggest that cognitive deficits resulting from NRG1 haploin-
sufficiency in this line, and their amelioration by putative up-regulation of
the GABAergic system by chronic valproate, were associated with reduction
in GABAergic and glutamatergic transmission. Comparisons of differen-
tial phenotypes across both TM-domain NRG1 mutant lines might suggest
that differences in the general targeting strategy and the truncation of dif-
ferent domains or exon location in the NRG1 gene have different effects on
behavioural performance.89
Partial deletion of TM-domain NRG1 is associated with the disruption of
several NRG1 isoforms. Focusing on isoform-specific NRG1 deletion lines,
partial deletion of type III NRG1 was associated with significant disruption
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98 Chapter 5
of PPI (more so than that observed forTM-domain (exon 9) NRG1 mutants).88
Mutants with targeted disruption of type I/type II NRG1 do not demonstrate
a hyperactive phenotype,90 although they demonstrate a deficit in LI, which
may reflect disruption of salience attribution processes. Mice harbouring a
heterozygous deletion in the EGF-like domain (pan-isoform) demonstrated
a transitory increase in exploratory activity, and more rapid habituation, of
exploration in a novel environment. Despite exhibiting no change in basal

PPI levels, these mutants showed differential sensitivity to psychostim-
ulant-induced disruption of PPI.91 These apparently conflicting findings
across the various NRG1 hypomorphic models likely reflects differences in
the biological activities of NRG1 splicing variants.60
As genetic variation in ErbB4 influences GABA levels in humans, these
observations suggest a plausible link between abnormal ErbB4 signalling
and GABAergic function in schizophrenia.92 Selective loss of ErbB4 from GAB-
Aergic interneurons during development caused cellular, physiological, and
behavioural deficits reminiscent of schizophrenia phenotypes. They exhib-
ited impaired hippocampal–prefrontal synchrony, and a decreased number
of dendritic spines in hippocampal CA1 pyramidal cells.7 These findings
suggest that ErbB4 function is required for normal baseline rhythms within
local cortical networks. They demonstrated increased exploratory behaviour,
reduced anxiety as measured in the buried marbles test and elevated plus
maze test, as well as heightened response to novelty as measured in the
social novelty preference test. They also displayed reduced sociability, nest-
ing behaviour deficits, PPI disruption, as well as working memory deficits in
the spontaneous alternation paradigm.
In a related study, in mice with either constitutive or parvalbumin inter-
10:30:30.
neuron-selective disruption of ErbB4, no changes in the saccharin preference

test of anhedonia were observed, nor were any alterations in social behaviour
observed in the resident–intruder paradigm.93
5.6.2 Mutant
Mouse Models of NRG1 and ErbB
Over-Expression
The mechanism by which NRG1 and ErbB4 variation increases risk for
schizophrenia is unknown, perhaps suggesting that splice variant-specific
NRG1/ErbB4 over-expression and hypersignalling of NRG1 may be involved.94
Specific mRNA transcripts for NRG1 and ErbB4 are increased in the frontal
cortex and hippocampus of patients with schizophrenia.68 Weickert et al.
(2012) demonstrated a correlation between the NRG1 HapICE risk haplotype
and increased NRG1 type III mRNA, which was in turn associated with earlier
age of onset.94 They proposed that the HapICE haplotype increases expres-
sion of the most brain-abundant form of NRG1, which, in turn, elicits an ear-
lier clinical presentation, providing a novel mechanism through which this
genetic association may increase risk for schizophrenia. Interestingly, this
increase was not related to antipsychotic treatment, suggesting a link with
the disorder rather than an effect of medication.66,95
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Kato et al. (2010) described the establishment of two mouse transgenic
over-expressor lines carrying the transgene of mouse type 1 NRG1 cDNA.96
These mutant lines demonstrated increased hyperactivity in a novel envi-
ronment and disruption of social interaction, but no change in sensorimo-
tor gating. Both lines were also examined for presence of a social behaviour
phenotype in an isolation-induced resident–intruder test. Male NRG1 hyper-
morphs demonstrated less time in social investigative behaviours com-

pared to sex-matched wild-type residents, while simultaneously displaying
increased frequency of aggressive behaviour towards the unfamiliar mouse.
Ex-vivo monoamine analysis in relevant brain areas revealed a decrease in
DA and dihydroxyphenylacetic acid (DOPAC) levels, as well as decreased
tyrosine hydroxylase (TH) protein levels in the hippocampus of transgenic
mutants over-expressing NRG1. In the PFC, the authors reported a trend
towards decreased DA content, but no change in DA or its metabolites was
observed in the striatum. They also observed an increase in D2 receptor
protein levels in the PFC.
A separate population of transgenic mice with enhanced NRG1 type 1 iso-
form expression, driven by a Thy-1 promoter in brain projection neurons,
demonstrated startle deficits and PPI disruption, novelty-induced hyperac-
tivity, and an age-dependent disruption of working and short-term memory.
Interpretation of these behavioural data is complicated by the co-presence
of whole-body tremor in these mice. Synaptic transmission and long-term
potentiation were unaffected in these animals, but altered hippocampal
oscillations were reported.54
A conditional NRG1 type-1 over-expressing mouse has recently been
described, where the mutation was restricted to the forebrain region,
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including the PFC and hippocampus, areas implicated in schizophrenia.95

These mice carry the type 1 NRG1β cDNA under the control of the tetracy-
cline-responsive promoter element tetO, crossed with CamKII2. Mutants
displayed behavioural deficits that included deficient spatial working
memory and reference memory, increased hyperactivity, impaired PPI,
reduced sociability and social interaction time, as well as being impaired
in glutamatergic and GABAergic transmission. Clozapine administration
ameliorated deficits in the open field, PPI, and social interaction tests.
Interestingly, both synaptic dysfunction and behavioural deficits disap-
peared when expression of the NRG1 transgene was switched off in adult
mice. Furthermore, turning on the mutation in adulthood alone was suf-
ficient to cause impaired glutamatergic function and behavioural defi-
cits, suggesting that the presence of such deficits requires continuous
NRG1 abnormality in adulthood. No change in ErbB4 levels was observed,
regardless of the status of the transgene; however, the study did report
that impaired glutamatergic transmission in this mutant may depend on
Lim domain kinase (LimK1) activity. In contrast, EF (elongation factor)-
1α-NRG1 mice, where NRG1 was expressed in all cells of the body, despite
being hyperactive, showed normal PPI.96 The differences across these
strains, including the Deakin strain, Kato strain, and ctoNRG1 mice, is
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100 Chapter 5
likely due to variation in expression patterns and levels of the NRG1 trans-
gene (for example, hundred-folds above normal in Thy-1-NRG1 mice).
Another mutant line that is relevant to the discussion is an EGF-over-
expressing transgenic line.97 These mice exhibited reduced PPI that was
normalised by the D2 receptor antagonist antipsychotic risperidone, with
modest deficits in contextual fear conditioning, but no alteration in locomo-
tor activity. These EGF transgenics also showed a decrease in striatal TH and
an increase in striatal catechol-O-methyltransferase (COMT) protein levels,
as well as an increase in DOPA decarboxylase protein levels in the accumbens
and PFC. High-performance liquid chromatography analysis of brain mono-
amine levels in these mutants also demonstrated increased DA and DOPAC
content in the accumbens, with such DA alterations being generally depen-
dent on the region examined. Behaviourally, these EGF mutants also exhib-
ited an increase in sensitised behavioural responsivity to chronic cocaine
administration.97
5.6.3 NRG1–ErbB
Signalling and Antipsychotic Drug
Discovery
Studies in both humans and rodents have shown that antipsychotic treat-
ment is correlated with changes in NRG1/ErbB4 expression in both brain
and serum.98 Dissociation of effects of subchronic vs. chronic treatment
with aripiprazole, risperidone, or haloperidol was observed in relation to
changes in the expression of NRG1 and ErbB4 in rat brains. While both
NRG1 and ErbB4 protein levels were elevated following subchronic treat-
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ment, chronic treatment produced an attenuation of both NRG1 and

ErbB4 expression.98 ErbB1 inhibitors have been shown to reverse schizo-
phrenia-related behavioural and neurochemical abnormalities in animal
models of schizophrenia.99–102 Specifically, the ErbB1 inhibitors ZD1839,
PD153035, and OSI-774 reversed PPI and LI deficits resulting from neona-
tal hippocampal lesions and methamphetamine treatment,100,101 as well as
schizophrenia-related deficits observed following neonatal treatment with
EGF in rats.102
Neonatal administration of EGF was associated with the emergence of
PPI and LI deficits, as well as pallidal changes in DA metabolite levels.102,103
The ErbB1 inhibitor ZD1839 reduced EGF-induced deficits in PPI and LI
and normalised DA content and DOPAC levels in the globus pallidus, in a
manner comparable to that observed following subchronic treatment with
the antipsychotic drugs haloperidol and risperidone. Low-dose adminis-
tration of the ErbB1–4 antagonist PD153035 partially reversed the EGF-in-
duced PPI deficit and associated increase in startle response in EGF-treated
rats; however, PD153035 failed to reverse EGF-induced deficits in social
interaction. Collectively, these data suggest that ErbB inhibitors might be
effective against phenotypes linked with positive symptomatology that has
been associated with subcortical domapinergic hyperfunction.
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5.7 DISC1
DISC1 is a prominent schizophrenia susceptibility gene whose functions
have been widely studied.104 It is an essential synaptic protein, which inter-
acts with a wider molecular network to mediate processes associated with
cellular and synaptic function.104 Disruption of specific interactions induces
cellular phenotypes observed in neuropsychiatric disorders, and specific

DISC1–cofactor interactions may be a target for the treatment of mental dis-
orders.105 One of the proven interactions of DISC1 is with phosphodiesterase
(PDE)4B, which is a risk factor for schizophrenia.106 Several mutant models
of DISC1 gene function have been described in the literature, collectively dis-
playing a diverse set of anatomical, behavioural and pharmacological pheno-
types relevant to several neuropsychiatric disorders, including schizophrenia
and depression.107–113
In one of two DISC1 mutations (mutation L100P) generated via ENU muta-
genesis in exon 2 of DISC1, the mutation was associated with deficits in several
behavioural models related to schizophrenia, including PPI, LI and working
memory, as well as displaying hyperactivity; antipsychotic administration
selectively reversed disruption of PPI and LI in this L100P DISC1 mutant line.107
Both the PDE4 inhibitor rolipram and the glycogen synthase kinase (GSK)-3
inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) syner-
gised to reverse PPI deficits and hyperactivity in the L100P DISC1 mutant.114
The reported efficacy of these inhibitors supports the involvement of GSK-3 in
schizophrenia and suggests a potential therapeutic effect of GSK-3 inhibitors.
In the second ENU-generated DISC1 mutant line (Q31L), the mice showed
increased immobility time in the forced swim test, decreased sucrose con-
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sumption (a putative index of anhedonia) and reduced social approach

behaviours, all reminiscent of a depression-related phenotype. Increased
immobility in the forced swim test in Q31L DISC1 mutants was reversed fol-
lowing treatment with the antidepressant bupropion.107 A recently published
study failed to replicate many of these phenotypic differences, including sen-
sorimotor gating deficits, particularly for L100P, while also demonstrating no
genotypic differences in complementary measures of social functioning.115
This study reported no differences between 31L or 100P and their respective
controls in either sociability or social novelty preference, or in a test of dyadic
social interactions in a novel environment.
Mice lacking exon 2 and 3 via targeted mutation of the DISC1 gene displayed
hyperactivity in a novel environment, increased methamphetamine-induced
hyperactivity, and sex-specific disruption of PPI (females only), but no change
in LI or cognition.116 They also displayed an anxiolytic phenotype in the ele-
vated-plus maze, increased social interactions, and decreased hippocampal
long-term potentiation, supporting a wider role for the DISC1 gene in medi-
ating risk of neuropsychiatric disorders generally.103,117,118
Alternative models include a transgenic line with inducible and reversible
expression of a DISC1 C-terminal fragment under the α-calcium/calmod-
ulin-dependent protein kinase (CAMK)II promoter; these mice demonstrated
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102 Chapter 5
109
reduced sociability and impaired spatial working memory. In a transgenic
line with expression of a dominant-negative truncated form of DISC1 under
the αCaMKII promoter, mutants exhibited hyperactivity but no effects on
PPI, social behaviour, or cognition.108 Double transgenic mice expressing
human DISC1 under the cytomegalovirus promoter with tetracycline under
the αCaMKII promoter showed a hyperactive phenotype, with some evidence
for impaired social behaviour and spatial memory, but no effect on PPI.111 In
a transgenic line with expression of truncated DISC1, mutants demonstrated
disruption of LI.112
These DISC1 mutant data illustrate how diverse mutations in the same gene
can produce different phenotypic outcomes, depending upon the nature of
the mutation, or, in some cases, the involvement of putative environmental
factors. The discrepancies across the various models may be therefore due to
differences in genetic manipulation, extent and nature of DISC1 dysfunction
and/or functional diversity of this gene.
5.8 Dysbindin
Genetic variation in DTNBP1 (dysbindin) has been associated with increased
risk for schizophrenia across diverse samples.119,120 Patients with schizophre-
nia demonstrate lower expression levels of dysbindin-1 mRNA and protein in
the PFC and hippocampus.121,122 Studies have also reported an association
between dysbindin variation and several aspects of cognitive dysfunction in
schizophrenia, including both general and domain-specific cognitive defi-
cits.119,123,124 Modification of dopaminergic and glutamatergic function has
been suggested as a potential mechanism underlying the pathogenic role of
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altered dysbindin expression.28

The sdy mouse arose as a spontaneous mutation in the DBA/2J strain
that is caused by a large in-frame deletion of two exons of the mouse dys-
bindin-1 gene; sdy mice express no dysbindin protein.34,125,126 Studies have
demonstrated contradictory findings in relation to the presence or absence
of novelty-induced hyperactivity in the sdy mutant, as well as measures of
DA turnover; these discrepant findings likely reflect influence of background
strain.127–132 While the DBA/2J strain does not show robust PPI (thereby ren-
dering meaningless any PPI assessment of mutants of this background),
PPI was intact in mice with backcrossing of the sdy mutation onto a C57BL6
line.132 However, another study in which sdy mutants were backcrossed onto
a C57BL6 background demonstrated a moderate PPI deficit;133 this deficit
was accompanied by reduced parvalbumin-positive interneuron activity, in a
manner related to schizophrenia.
Dysbindin mutants also showed a mild deficit in social interaction, as indexed
by a reduction in social contacts and time spent in dyadic interaction.128,129 Dys-
bindin mutants were impaired in performing spatial working memory tasks.28,130
Similarly, dysbindin mutants displayed some deficits in spatial learning and
memory as measured in the Morris water maze.132 Moreover, dysbindin mutants
take longer to acquire a reward-based operant task, with these performance
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134
deficits suggesting impairment in interval timing. Dysbindin mutants show
difficulty encoding or maintaining an item in spatial memory,135 with impaired
spatial reference memory and object recognition memory;128 morphological
changes in excitatory asymmetrical synapses on hippocampal CA1 dendritic
spines, larger but fewer presynaptic glutamatergic vesicles, narrower synaptic
cleft, and broader postsynaptic density are also evident.136
5.9 Modelling
Gene × Environment Interactions in
Schizophrenia Mutant Models
Interplay between genes and the environment (i.e. G × E interaction) exerts
significant influence on risk of neuropsychiatric disorders. Mutant mod-
els provide a means for elucidating the contribution of genes, exposure to
adverse environmental factors (e.g. early prenatal and postnatal life adver-
sity, psychosocial stress, and substance abuse), and their interaction in the
development of neuropsychiatric phenotypes.137–139 Studies using mutant
mice may inform on relationships between candidate risk genes and specific,
experimentally controlled environmental interventions. G × E interactions
may exert the following different effects: biological interaction, where the
genetic effects depends on the presence or absence of an environmental fac-
tor; quantitative interaction, resulting in the same direction of effect in both
genotype groups; and qualitative interaction, producing opposite direction
of effects in different genotype carriers.
On a background of established genetic risk, epidemiological studies
have indicated an association between maternal bacterial and viral infec-
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tions during pregnancy and higher incidence in adult offspring of psycho-

sis in general, and of schizophrenia in particular.140,141 Interactions between
genetic risk and environmental stressors in early stages of life appear to be
important in the development of schizophrenia.138,142,143 Preclinical mod-
els of G × E interactions related to schizophrenia have involved examin-
ing the phenotypic consequences of prenatal/maternal or early postnatal
immune challenge in various DISC1, NRG1, and other risk gene mutant
models.144–147 These studies have reported an array of schizophrenia-as-
sociated behavioural abnormalities, including increased novelty-induced
hyperactivity and PPI deficits, in mice with DISC1 mutation that were sub-
jected to immune challenge.144,145 Other studies have examined interactions
between mouse gene manipulations and other epidemiologically relevant
environmental adversities, administered at critical developmental periods,
on the later emergence of schizophrenia-related endophenotypes in adult-
hood; these include the psychotomimetic effects of adolescent cannabis
in COMT mutants,50,148 and adolescent exposure to social defeat stress in
NRG1 mutants.83
In a recent study, Chohan et al. (2014) found that acute but not repeated
restraint stress increased anxiety-related behaviour, in a task-specific man-
ner, in TM-domain NRG1 heterozygous mutant mice.149 Adolescent NRG1
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104 Chapter 5
mutants but not wild type mice exposed to repeated restraint stress failed
to develop PPI. Repeated restraint stress also decreased corticosterone levels
in adolescent NRG1 mutants relative to wild type mice. Repeated restraint
stress increased apical dendritic spine density and decreased apical den-
dritic lengths and complexity in layer II/III pyramidal neurons of the medial
PFC in adolescent NRG1 mutants but not in the wild type.
Cannabis consumption during adolescence is associated with increased

risk of schizophrenia.150 Studies have shown that risk for psychosis is high-
est among those who used cannabis during adolescence and are COMT
Val/Val (low activity allele) carriers;151 the COMT gene encodes COMT
enzyme, one of the major enzymes to degrade DA in the prefrontal cortical
areas. We have recently shown that in COMT knockout mice, the COMT
genotype exerted specific modulation of responsivity to chronic cannabi-
noid administration during adolescence in terms of exploratory activity,
working memory, and PPI;50,148 these deficits were accompanied in a gen-
otype-dependent manner by disturbance in dopaminergic and GABAergic
function.152
Each of the studies outlined above has explored the interaction between
genetic background and a single environmental manipulation, but an alter-
native approach has involved experimental modelling of multiple envi-
ronmental exposures against a background of genetic risk. In a recently
published study, which examined the unique and combined effects of pre-
natal immune challenge and postnatal cross-fostering (a control procedure
which can also function as a stressor) in mice with partial TM-domain
(exon 9) deletion of NRG1, distinct phenotypic effects across schizophre-
nia-related behavioural measures (social interaction, sensorimotor gating,
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and open-field exploration) were observed for both individual environmen-

tal variables as well as interactions between these factors and genotype.147
This study suggests that concepts of gene × environment interaction in risk
for schizophrenia should be elaborated to include multiple interactions
that involve individual genes interacting with multiple biological and psy-
chosocial environmental factors.
5.10 Modelling
Gene × Gene Interactions in
Schizophrenia Mutant Models
An additional and increasingly important approach is to study gene × gene
interactions or epistasis, when the combination of two or more variants
results in marked biological differences among the groups. Clinical genetic
examination of such epistatic interactions requires enormous statistical
power and places vast demands on sample size. Multiple knockout/knockin
animal studies have demonstrated the existence of additive genetic effects,
where the measured phenotype becomes more severe with an increasing
number of risk alleles, but have also supported the importance of non-ad-
ditive genetic interactions, whereby new phenotypes occur.34,153 In future, it
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is hypothesised that these types of interactions may serve to identify groups
of patients with highly increased or reduced risk for certain drug effects or
environmental factors.
Schizophrenia-relevant epistatic interactions between mutations are likely
to be prevalent, but are still poorly understood mechanistically.154 Based on
the effects of dysbindin on cortical dopaminergic signalling, an epistatic
interaction between genetic variations reducing dysbindin and COMT was

demonstrated using COMT and dysbindin double mutants; in contrast to
effects produced by dysbindin or COMT single gene knockout, the com-
bined reduction of both COMT and dysbindin in the same mouse produced
a dramatic deficit in working memory function.153 Importantly, this same
epistatic effect was found in healthy humans performing an N-back working
memory test in conjunction with functional magnetic resonance imaging.
Individuals homozygous for COMT Met alleles (i.e. with a relative reduction
in COMT) and displaying no reduction in dysbindin activity (as evidenced by
not carrying a specific functional variant for DTNBP1) performed better than
other COMT genotypes. In contrast, individuals with COMT Met/Met geno-
types and who were also homozygous for the low dysbindin expression-as-
sociated haplotype were the poorest-performing compared with other COMT
genotypes.153 These results are consistent with the inverted-U function,
showing nonlinear effects of increasing D2 signalling in PFC-dependent cog-
nitive functions.
Against this background of a consequent lack of clinical studies, preclin-
ical modelling of such G × G interactions is only now emerging due to asso-
ciated demands on resources, both human and financial. However, initial
studies such as COMT × dysbindin153 are emerging and hold great promise
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for illuminating the genetic basis of the disorder and identifying novel anti-
psychotic drug targets.
5.11 Conclusions
Fundamentally, the development of new antipsychotic drugs is dependent
upon the development of new, more valid models of schizophrenia which
tap into the etiological basis of the disease, and which are integrated with
end point assessments that are translationally relevant.3 Mutant mouse
models represent a viable and promising avenue, in terms of both under-
standing signalling processes underlying existing antipsychotic action and
discovering novel drug targets. Scientific and practical constrains mean
that further elaboration of G × E and G × G interactions implicated in
schizophrenia using mutant models will require judicious selection of clin-
ically associated biological and enviornmental risk factors. Additionally,
Licinio and Wong (2004) have emphasised that progress in antipsychotic
drug discovery is contingent upon further fundamental discovery science,
so that proper translation can occur. In this context, the way forward should
include further funding initiatives to support basic discovery research.155
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106 Chapter 5
Acknowledgements
The authors’ studies are supported by Science Foundation Ireland Principal
Investigator grant 07/IN.1/B960.
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130. K. Takao, K. Toyama, K. Nakanishi, S. Hattori, H. Takamura, M. Takeda,
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Arnold, Genes, Brain Behav., 2009, 8, 390–397.
133. G. C. Carlson, K. Talbot, T. B. Halene, M. J. Gandal, H. A. Kazi, L.
Schlosser, Q. H. Phung, R. E. Gur, S. E. Arnold and S. J. Siegel, Proc. Natl.
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www.Ebook777.com
CHAPTER 6
Drugs that Target the

Glutamate Synapse:
Implications for the Glutamate
Hypothesis of Schizophrenia
CATHARINE A. MIELNIKa AND AMY J. RAMSEY*a
a
Department of Pharmacology and Toxicology, Faculty of Medicine, Univer-
sity of Toronto, 1 King’s College Circle, Toronto, Ontario, M5S 1A8, Canada
*E-mail: a.ramsey@utoronto.ca
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6.1 The Glutamate Hypothesis of Schizophrenia

The first indication that glutamate neurotransmission might be altered in
schizophrenia originated from an observation that patients had decreased
levels of glutamate in their cerebrospinal fluid.1 This finding was not repli-
cated;2 however, it was part of a movement to look beyond dopamine neuro-
transmission to understand the disorder. In its original form, the glutamate
hypothesis proposed that an underlying cause of schizophrenia symptoms is
a deficit in glutamate neurotransmission. The hypothesis stemmed from the
behavioural effects of drugs that block a subtype of glutamate receptors, and
was further supported by post-mortem and genetic studies. The proposed
role for glutamate in the pathophysiology of schizophrenia is continually

115
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116 Chapter 6
refined as we gain a greater appreciation of the molecular and cellular com-
ponents of glutamate neurotransmission.
6.1.1 Molecular
and Cellular Components of the
Glutamate Synapse
Glutamate acts as the primary excitatory neurotransmitter in the mamma-

lian brain and activates both ionotropic and metabotropic receptors. The
ionotropic receptors are α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) receptors, kainate receptors, and N-methyl-d-aspartate (NMDA)
receptors.3 AMPA and kainate receptors primarily generate excitatory post-
synaptic currents (EPSCs) that are responsible for triggering action poten-
tials. NMDA receptors also contribute to the long-duration component of the
EPSC, but importantly they play a crucial role in synaptic plasticity, learning,
and memory. The metabotropic receptors (mGluR1-8) are G-protein coupled
receptors that primarily couple to Gq (Group I) or Gi/o (Group II and III). The
mGluRs further modulate glutamatergic neurotransmission by triggering
signaling cascades postsynaptically (Group I) and by regulating glutamate
release presynaptically (Group II and III).4 Figure 6.1 depicts the cellular and
molecular components of the glutamate synapse.
The NMDA receptor is a prime target for pharmacotherapy due to its impli-
cation in schizophrenia and its central role in learning and memory. NMDA
receptors exist as heteromers of two NR1 (GluN1) subunits and two NR2
(GluN2A, B, C, or D) subunits, which combine to form a cation channel.3 For
all of the subunits, there is a large amino-terminal domain, a large ligand bind-
ing domain (LBD), and a relatively smaller transmembrane domain (TM) that
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forms the cation pore.3 The NR1 subunit has the LBD for glycine, and the NR2
subunit has the LBD for glutamate. The complement of NR2 subunits influ-
ences ligand-binding properties, the likelihood of channel opening, and the
duration of channel opening. Since the different NMDA receptors have distinct
biological functions, compounds have the potential to selectively modulate a
subset of receptors. Interestingly, NMDA receptors can also be composed of
NR3 (GluN3A or B) subunits instead of or in addition to NR2 subunits. This
also influences both the channel properties and ligand-binding properties.
NR3 subunits have a glycine-binding domain similar to NR1 subunits. The
presence of an NR3 subunit causes the receptor to be less regulated by magne-
sium blockade, and less permeable to calcium. In addition to the ligand bind-
ing domains, NMDA receptors have modulatory sites for zinc and spermine,
and a modulatory site within the channel that binds the non-competitive
inhibitors phencyclidine (PCP), dizocilpine, and ketamine. These several mod-
ulatory sites make the NMDA receptor a rich pharmacological target.
6.1.2 Evidence for a State of NMDA Receptor Hypofunction

Although there are several receptors that are activated by glutamate, the
mechanistic hypotheses to explain schizophrenia primarily focus on
the NMDA receptor.4,5 The NMDA receptor first reached the forefront of the
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Drugs that Target the Glutamate Synapse 117

Figure 6.1 The

glutamate synapse. The glutamate synapse comprises a presynap-
tic glutamate neuron that releases glutamate, a postsynaptic neuron
with glutamate receptors, and neighbouring astrocytes that regulate
neurotransmitter levels in the synapse. Presynaptically, glutamate
(GLU) is stored in vesicles by the glutamate vesicular transporter
(VGLUT). Once released into the synapse following an action potential,
glutamate binds to a number of postsynaptic and presynaptic targets
within the synapse. Glutamate binds to the α-amino-3-hydroxy-5-meth-
yl-4-isoxazolepropionic acid (AMPA) receptor and triggers the opening
of the channel. This allows entry of Na+ into the postsynaptic neuron,
which depolarizes the membrane and elicits an excitatory postsynap-
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tic potential. Membrane depolarization by AMPA receptor activation is

a necessary step for activation of the second class of glutamate recep-
tors, N-methyl-d-aspartate (NMDA) receptors. The activation of NMDA
receptors requires membrane depolarization and concurrent binding
of co-agonists GLU and glycine (GLY). This triggers the opening of the
nonselective cationic ion channel, causing an influx of both Na+ and
Ca2+ ions into the cell. There are also metabotropic glutamate recep-
tors present in the synapse (both pre- and postsynaptically) that modu-
late glutamate signaling. Finally, to help maintain homeostasis within
the glutamate synapse, neighbouring astrocytes modulate glycine and
glutamate levels through glycine transporters (GlyT1) and glutamate
transporters (excitatory amino-acid transporters; EAAT). Within the
astrocyte, glutamate is reduced to glutamine by the enzyme glutamine
synthetase. Glutamine is released to the extracellular space and is con-
verted to glutamate within neurons by the enzyme glutaminase.
glutamate hypothesis of schizophrenia when studies showed that PCP and

ketamine could induce behaviours that are typically seen in schizophrenia,
including psychosis, social withdrawal and working-memory deficits.6–9 Both
ketamine and PCP block the NMDA receptor by binding non-competitively to
a site within the channel. Although ketamine and PCP bind to other proteins
besides the NMDA receptor, their psychotomimetic effects are induced at low
doses where the NMDA receptor is the principal target.8,10 Similar behavioural
effects are induced by dizocilpine, which binds at the same site as PCP and
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118 Chapter 6
ketamine. Dizocilpine is the most selective of these three drugs and has a
very slow off-rate, which causes a sustained blockade of the channel.11 The
similar behavioural profile of these three non-competitive antagonists, PCP,
ketamine, and dizocilpine, strongly argues that the psychotomimetic effects
are due to NMDA receptor blockade.
Beyond the compelling pharmacology of NMDA receptor anatgonists, the
hypothesis for NMDA receptor hypofunction in schizophrenia is supported

by a number of other lines of investigation. These include postmortem
studies,12–17 genetic studies,18,19 and brain imaging studies.20 In addition,
drugs that enhance NMDA receptor function have shown modest effects
in patients with schizophrenia.21 It is important to note that the genetic
evidence for NMDA receptor hypofunction does not focus on mutations of
the receptor subunits genes per se. In fact, the genes that encode the chan-
nel are among the most conserved in mammals,22 and NMDA receptors are
required for postnatal survival.23,24 Perhaps for this reason, the mutations
that have been discovered in schizophrenia patients are often found in
genes that regulate the activity of the NMDA receptor and its downstream
signaling events.
A more recent line of evidence that demonstrates the potential for NMDA
receptor hypofunction to cause symptoms of schizophrenia comes from the
study of autoimmune encephalitis.25–28 Autoimmune encephalitis occurs in
a small percentage of cancer patients who develop antibodies against the
NMDA receptor.27,28 In its early stages, the clinical manifestations of this
disease include delusions, hallucinations and memory impairments that
are similar to schizophrenia. Based on this discovery, subsequent investiga-
tions have been undertaken to determine whether patients diagnosed with
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schizophrenia or schizoaffective disorder also produce antibodies to the

NMDA receptor.29–31 While there are some conflicting observations, a recent
meta-analysis indicates that patients with schizophrenia or schizoaffective
disorder are three times more likely to be seropositive for NMDA receptor
antibodies than healthy controls when a high-specificity threshold for anti-
body titer is applied.32
An important refinement of the glutamate hypothesis is that NMDA recep-
tor hypofunction may lead to excessive glutamate release and glutamatergic
transmission through other receptors. While this may seem counter-intui-
tive, it may in fact be the case, since a loss of NMDA receptor signaling on
γ-aminobutyric acid (GABA) neurons impairs their ability to quieten gluta-
mate neurons. Therefore, it may be more appropriate to describe the gluta-
mate hypothesis as a dysregulation or imbalance of glutamate transmission,
with impaired NMDA receptor function leading to excessive signaling through
other glutamate receptors.33
In addition, several researchers have emphasized the neurotoxic effects of
NMDA receptor hypofunction using NMDA receptor antagonists. Early in the
formulation of the glutamate hypothesis of schizophrenia, studies by Olney
and Farber demonstrated that high doses of channel-blocking NMDA recep-
tor antagonists caused widespread neuron loss.34 The developing brain was
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particularly sensitive to these NMDA receptor antagonist effects and neuro-
toxicity was observed at lower doses.35
The molecular mechanisms that lead to neuron death are not completely
known, but there is some evidence that NMDA receptor channel blockers
induce oxidative stress that leads to neuron loss.36 Interestingly, parvalbu-
min-positive inhibitory neurons appear to be particularly sensitive to this
antagonist induced oxidative stress.36,37 This may also explain how NMDA
receptor hypofunction would lead to a loss of inhibitory inputs and a general
state of disinhibition.38,39
Through these refinements to the glutamate hypothesis, the glutamate
synapse has emerged as one of the most prominent targets for potential
therapeutic intervention in schizophrenia.40 This is advantageous, as the
glutamate synapse is a target-rich environment that contains a large num-
ber of presynaptic, postsynaptic and regulatory proteins that represent
suitable targets for drug development.41,42 Furthermore, NMDA receptor
hypofunction is consistent with the hypothesis that schizophrenia is a
neurodevelopmental disease, manifesting due to improper circuit for-
mation during brain development.43–45 Due to the fact that NMDA recep-
tors play a critical role in the formation, strengthening, and elimination
of neural connections,46 it is realistic to hypothesize that they play an
important role in the neurodevelopmental hypothesis of schizophrenia
as well.
6.2 The
Integration of Glutamate, Dopamine and
GABA in Schizophrenia
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There exists a large body of evidence that suggests that alterations in a

number of neurotransmitter systems, not just a single one, underlie the
pathophysiology of the symptoms associated with schizophrenia. While
the true etiology of schizophrenia, and its pathology, remains unknown
explicitly, there is a growing body of evidence that the disease mani-
fests as a result of alterations in several neurotransmitter systems. It is
possible that these alterations originate from an insult to one system
(e.g. a disruption in glutamatergic signaling), and as a result, subse-
quently alters other interconnected neurotransmitter systems (e.g. dopa-
mine or GABA).
6.2.1 Dopamine and Glutamate

Alterations in dopamine transmission are considered in all hypotheses about
the etiology and pathophysiology of schizophrenia. This is due to the class
effects of dopamine D2 antagonists in the improvement of psychotic symp-
toms. Indeed, the clinical potency of antipsychotic drugs is directly correlated
with their D2 receptor affinity.47,48 Beyond this, several imaging studies have
shown that amphetamine administration elicits a larger release of dopamine
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120 Chapter 6
49
in schizophrenia patients than it does in healthy controls. While dysregu-
lation of dopamine does not explain all of the features of schizophrenia, it is
a likely cause of positive symptoms and may even explain cognitive impair-
ments.50 Therefore, any discussion of the glutamate hypothesis of schizo-
phrenia must address how NMDA receptor hypofunction affects dopamine
neurotransmission.
Preclinical data suggest that elevated dopamine transmission in subcor-

tical structures can be secondary to a deficit in NMDA receptor function.
Furthermore, dopamine has a modulatory role on glutamate receptor phys-
iology and signaling, further linking these two neurotransmitter systems.51
NMDA receptor antagonists increase the firing rate of dopamine neurons
when they are administered systemically.52,53 Slice recordings from NMDA
receptor deficient transgenic mice also show that reductions in NMDA
receptor levels lead to increases in the tonic firing rate of dopamine neu-
rons.54 Therefore, reductions in NMDA receptor activity alter the firing
rate of dopamine neurons that could lead to increased dopamine neuro-
transmission, particularly in the striatum. However, it is not clear whether
increased tonic firing of dopamine neurons is due to changes in intrinsic
dopamine firing, or due to disinhibition of dopamine neurons (or both).
Figure 6.2 depicts how a loss of NMDA receptors on inhibitory interneu-
rons can lead to an increase in the amount of dopamine in the synapse
through disinhibition.
While systemic NMDA receptor antagonists increase tonic firing of dopa-
mine neurons, they also impair phasic or burst firing patterns. Phasic firing
is impaired when NMDA receptor antagonists are delivered systemically,53 or
applied locally to dopamine cell bodies.55 Phasic firing is also impaired when
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NMDA receptors are genetically ablated from dopamine neurons.56,57 In stud-

ies that measure extracellular levels of dopamine, there are mixed reports on
the effect of NMDA receptor antagonists or NMDA receptor knockdown.
Not only does glutamate transmission affect the activity of dopamine neu-
rons, but dopamine also has modulatory effects on NMDA receptors. A key
region where dopamine and glutamate afferents converge is in the striatum,
where cortical glutamatergic afferents and dopamine projections both form
synapses onto GABAergic medium spiny neurons.58,59 In medium spiny neu-
rons, the stimulation of D1 or D2 dopamine receptors has opposing effects on
NMDA receptor transmission. D2 receptor activation decreases NMDA-medi-
ated glutamate transmission, while D1 receptor activation aids in glutamater-
gic transmission.60,61 It is therefore not surprising that D1 receptor antagonists,
which reduce NMDA receptor function, have no antipsychotic activity and
worsen positive symptoms in schizophrenia patients.62 Similarly, excess activa-
tion of D2 receptors in the striatum disrupts the cortico-striato-thalamic loop
and reduces NMDA-specific glutamatergic signaling. Therefore, antipsychotic
drugs, through D2 receptor antagonism, may improve symptoms, not only
through decreasing dopamine signaling, but also by restoring the processing
of information in the cortico-striato-thalamic circuit by augmenting NMDA
receptor function.51
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Figure 6.2 Disinhibition

of GABA and dopamine neurons by NMDA receptor hypo-
function. In normal physiological conditions (A), there is balanced neu-
rotransmission. Excitatory input (via NMDA receptor) onto inhibitory
neurons causes the release of GABA from the cell, which reduces the excit-
ability of downstream neurons. GABA neurons form synapses with a variety
of neurons, including dopamine (DA) and glutamate (GLU) neurons.
GABA signaling on these neurons causes inhibition of postsynaptic
signaling, and limits the release of downstream neurotransmission.
Thus, these dopamine and glutamate neurons are normally inhib-
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ited from firing and there is a low level of dopamine and glutamate
signaling from the postsynaptic neuron. In a state of NMDA receptor
hypofunction (B), inhibitory neurons lose the excitatory input from
NMDA receptors. The reduced firing rate of inhibitory neurons causes a
decrease in the release of GABA, leading to disinhibition of the postsyn-
aptic neuron. Thus, the reduced activation of inhibitory neurons leads
to increased firing and neurotransmitter release from dopamine and
glutamate neurons.
6.2.2 GABA and Glutamate

Recently, both animal and human studies have identified cortical gluta-
mate and GABA neurotransmission, and the balance between the two, as a
critical component in cognition.63 Deficits in cognition are linked to poor
functional outcomes for schizophrenia patients.64 It is believed that the
neural substrate for cognition involves synchronization of neural circuits
in a number of cortical regions.65 Therefore, the neural substrate for cogni-
tive function is thought to involve synchronization of neural activity at the
gamma frequency, and abnormalities within these gamma oscillations may
contribute to cognitive deficits seen in schizophrenia.66
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122 Chapter 6
The prefrontal cortex is extensively interconnected with cortical and sub-
cortical regions, and exerts a top-down control of neural activity between
brains regions that are integral for cognitive control and the coordination of
incoming sensory and motor information.67 One of the core cognitive defi-
cits in schizophrenia is a dysfunction of working memory, the mechanism
by which information is kept and manipulated while performing complex
tasks.68 Gamma oscillations (30–80 Hz) play an important role in working

memory since gamma synchrony has been seen to increase with an increase
in working memory load.69 Patients with schizophrenia have been shown to
have working memory deficits that are accompanied by altered patterns in
oscillatory activity in the cortex,70 and fail to enhance gamma activity with an
increase in working memory load.71 Therefore, there is strong evidence for a
disruption in gamma oscillations leading to cognitive deficits.
The mechanisms by which GABA inhibition may synchronize postsynaptic
cell activity has been reviewed extensively.72,73 In short, gamma oscillations
within cortical circuits are dependent on GABA-mediated inhibitory neu-
rotransmission, specifically the parvalbumin-containing subclass of GABA
interneurons.74 Parvalbumin-containing interneurons innervate pyramidal
neurons and control their output and synchrony.75,76 Furthermore, activation
of parvalbumin-containing fast-spiking interneurons is known to be critical
for the generation of gamma oscillations, organizing functional groups of
neurons.77,78 These neurons are also highly dependent on NMDA receptor
activation to trigger action potentials and fire.79 When NMDA receptors are
selectively ablated in parvalbumin-containing interneurons, mice exhibit the
same schizophrenia-relevant phenotypes that occur with global reduction
in NMDA receptors.77,80–82 Therefore, in a state of reduced NMDA receptor
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signaling, there is a decrease in the activation of parvalbumin-containing

interneurons, which leads to disinhibition of cortical pyramidal cells and to
cognitive deficits. Figure 6.2 illustrates how reduced NMDA receptor func-
tion on GABAergic interneurons causes disinhibition of pyramidal neurons.
6.3 Animal Models of Schizophrenia

6.3.1 Preclinical Drug Testing in Animal Models
Because schizophrenia is a complex, multi-factoral disorder, several valid
pre-clinical animal models are used in the search for new treatments. The
vast majority are rodent models, and despite their inherent limitations,
these models provide enormous insight into disease mechanisms. They are
also essential tools in the search for new drug therapies.
Based on a recent survey of the literature by Koenig and colleagues, there
are over 160 rodent models of schizophrenia.83 The majority of these models
are genetic, but there are also several models that use pharmacological or
lesion methods to model the disorder. Despite the widely varied methods
that are used to model aspects of schizophrenia, most of the rodent models
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share some (but not all) of the common behavioural phenotypes as described
in Chapter 5. Behaviours predictive for antipsychotic efficacy are increased
locomotor activity, increased stereotypy, and impaired sensorimotor gating.
For example, acute PCP induces increased motor activity and stereotypy and
impairs sensorimotor gating in the pre-pulse inhibition of acoustic startle
response. All currently prescribed antipsychotics are effective in normalizing
the activity, stereotypy, and sensorimotor gating disrupted by PCP.

While these behavioural tests are predictive for the amelioration of pos-
itive symptoms, it remains to be seen whether they will also be predictive
for cognitive and negative symptoms. This is difficult to determine because
there are no clinically available drugs that effectively treat those symp-
toms of schizophrenia. There is now a concerted effort to identify new
treatments that address these symptoms, which includes an agreement on
which behavioural paradigms should be used in preclinical screening. The
CNTRICS initiative (Cognitive Neuroscience Treatment Research to Improve
Cognition in Schizophrenia) has outlined several behavioural assays that are
recommended for their potential to predict clinical efficacy. These assays are
listed in Table 6.1 and briefly discussed below. A more detailed description of
the tests can be found in several review articles.84–89
6.3.2 CNTRICS-Based Behavioural Paradigms

The CNTRICS initiative subdivides cognitive and negative symptoms into
distinct behavioural domains that access distinct and overlapping brain cir-
cuits. Briefly, these domains are: perception, attention, working memory,
long-term memory, executive function, motivation, and social cognition.
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The tests for these behavioural domains are chosen with consideration given
to reproducibility across laboratories and the ability to automate or reduce
experimenter bias. The tests are designed for use in rodents (rats and mice)
and primates (macaque) with some degree of face and construct validity to
human tests of the same domains.
Animal models of impaired glutamate signaling are expected to show
impairments in the cognitive tasks described in the CNTRICS initiative.
Indeed, acute administration of NMDA receptor antagonists impairs the
majority of the behaviours highlighted by the initiative. However, these tasks
have only recently been defined, and the large body of research into genetic
and lesion models has been performed using other tasks that are not based
on the use of operant chambers. For example, cognition in rodents is com-
monly interrogated using the Morris Water Maze for spatial memory and cog-
nitive flexibility, radial arm mazes for working and reference memory, and
novel object recognition for explicit memory. Therefore, there is still a large
information gap where the numerous genetic animal models for schizophre-
nia have yet to be tested in the more sophisticated measures of cognition
such as the five-choice serial reaction time test or Pavlovian conditioning and
reversal learning.
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Table 6.1 Behavioural

tests used to assess pro-cognitive drug effects in preclinical rodent models of schizophrenia.
124
Domain Test Demonstration in animal model
Experience-based Pavlovian Pavlovian autoshaping measures the development of a conditioned response to a stimulus like
decision making autoshaping a tone or light. One conditioned stimulus (CS+) is paired with a food reward, while a second
and motivation stimulus (CS−) is presented without a food reward. The animal is presented these two stimuli
over many trials, and their approach behaviour is recorded in response to the CS + stimulus
and the CS− stimulus. This test can measure the rate of reinforcement learning over several
sessions, and learning can be impaired by drugs that block dopamine transmission or
NMDA receptor activity.145,146
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Probabilistic An important aspect of cognition is the ability to choose the appropriate behaviour based on
learning previous experience, which can be measured in animals in several paradigms of probabilistic
learning. In one variant, animals are presented with two locations to nose-poke. When the
animal nose-pokes at one location, it receives a reward 80% of the time. When it nose-pokes
at the other location, it receives a reward 20% of the time. Over time animals learn to prefer-
entially nose-poke in the location that is more likely to give a reward. Probablistic learning is
impaired in schizophrenia, and can be disrupted in animals by manipulations of dopamine
and NMDA receptor signaling.145
T-maze barrier task Motivation can be assessed with tests that measure the extent to which an animal will exert
an effort to recover a food reward. In the T-maze barrier task, the animal is given a choice
between entering one arm with a small reward and no barrier to entry, or another arm with
a large reward and a physical barrier to entry, such as a short wall. Animal models with
impaired motivation will forgo the effort involved in retrieving the large reward and will
choose arm with the small reward more often than a control animal. This task is most sensi-
tive to disruptions in dopamine transmission.145,147
Fixed- and Animals that have been trained to press a lever for a food reward can be tested for the amount
progressive- of effort that they will expend to retrieve a reward. Fixed ratio (FR) tasks deliver one food
ratio lever reward for a given number of lever presses; for example an FR5 delivers one food pellet
pressing for every five lever presses. A progressive ratio schedule increases the required number of
presses with each successive reward, so that a breakpoint can be determined.88,148
Chapter 6
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Learning and Delayed non-match DNMTP uses lever-based operant chambers to measure spatial working memory. Two levers
Drugs that Target the Glutamate Synapse

memory to position are presented, and the animal must remember which lever gave the reward in the previous
(DNMTP) presentation. Then, the animal must press the opposite lever from the one that gave the pre-
vious reward. During the delay between presentations the animal must store the location of
the reward lever in working memory, and must choose the non-matching location to receive
a food reward in the next presentation. The length of the time-delay can be varied to detect
differences in working memory, and performance on the task is generally impaired with
increasing time-delay.85,88
Trial-unique The test is designed to measure visual or spatial working memory similar to the DNMTP test
delayed but uses a touchscreen operant chamber. The animal is presented with a visual stimulus
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non-matching to in one location, and after delay it is presented with another visual stimulus. The animal is
location rewarded when it touches a stimulus that is presented in a different location than the first. If
it touches a stimulus that is presented in the same location as the first, it does not receive a
reward and is given a “time-out”.84
Executive function Reversal learning Tests that measure reversal learning are used to assess cognitive flexibility, which is the ability
to unlearn one behavioural response and adopt a new behavioural response. In these tests,
animals are first conditioned to perform a behaviour for a food reward. In touchscreen ver-
sions the animal learns that a reward is delivered for touching a specific image in a specific
location. Once the animal reaches a criterion level of conditioned response, the rules of the
reward delivery are changed, such that the animal must touch for a different visual image
or a different location. The rate of reversal learning is measured as the number of trials
that are required to reach the same criterion of conditioned response to the new image or
location.88,149
Social cognition Three-chamber This test is used to measure social behaviour in an automated fashion (a manual version of
social interaction test also exists via video recording and manual scoring). The sociability test arena consists
of a polycarbonate box, with partitions separating the box into three chambers. Cylindrical
chrome wire cages are used to contain a novel (stranger) mouse in one or both of the
chambers. Partitions have openings that allow the experimental animal to move freely
between chambers. Automated recording (via an interface box with an embedded real-time
controller) monitors beam breaks and determines transitions in and out of each chamber.
Time spent in each chamber and transitions between each chamber are recorded. A number
of variations exist in the sociability test; the most common variation allows for a five-minute
habituation period in the apparatus and then exposure for ten-minutes to a novel caged
125
mouse in one chamber and an empty wire cage in the other.87,88,150
(continued)
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126
Domain Test Demonstration in animal model
Perception Pre-pulse inhi- PPI of the acoustic startle response measures the subcortical processing of auditory
bition (PPI) of information, whereby the startle reaction to a loud tone is attenuated when it is preceded
acoustic startle by a non-startling tone. PPI measurements can be acquired with a 30 minute test, and data
response are collected with automated chambers. PPI of the startle response is well-documented in
schizophrenia patients and animal models of the disorder. Sensorimotor gating is disrupted
by drugs that block NMDA receptors, and can be restored by antipsychotic drugs.88,89
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Mismatch negativ- MMN uses non-invasive electrical recording to measure the brain’s response to the deviant
ity (MMN) sound or image that occurs within a series of otherwise identical stimuli. MMN is a type of
event-related potential that is evoked when primary sensory systems detect a change in a
pattern of visual or auditory stimuli, and is sometimes referred to as an “oddball” test.
MMN is well-documented in schizophrenia patients and in rodent and primate models of
the disorder. To measure MMN in mice, for example, a series of identical tones would be
interrupted by a different tone, and the brain’s waveform activity 100–250 ms after the
deviant tone would be compared to the waveform activity in response to the other
identical tones. Deficits in MMN can be induced most reliably by drugs that block
NMDA receptors.88,89
Attention Five choice serial 5-CSRTT uses operant chambers where rodents lever press or nose-poke for a food reward.
reaction time The animal receives a brief visual stimulus in one of five locations, and learns over time that
task (5-CSRTT) a reward is given only if the nose poke occurs in the right location, and only if the animal
waits 30 s before responding. Therefore the 5CSRT test can detect impulsivity as well as
attention deficits. This task requires multiple testing sessions to train the animal, but can be
performed in an automated manner and using touch-screen based systems. Performance on
this test is sensitive to pharmacological manipulation of dopamine and serotonin systems,
and some NMDA receptor antagonists.86,88
Chapter 6
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6.4 Pharmacological
Targets to Improve
Glutamatergic Signaling
6.4.1 NMDA Receptor
Since NMDA receptors are postulated to be less functional in the schizo-
phrenic brain, it would seem logical to administer NMDA to augment this

pathway. However, NMDA induces seizures and excitotoxicity, and thus is not
an appropriate treatment. Despite this potential adverse effect, the availabil-
ity of modulatory sites allows for the development of compounds that can
potentiate NMDA receptors therapeutically. Positive allosteric modulators
(PAMs) potentiate NMDA receptor current by interacting with a site other
than the LBD for glutamate or glycine. PAMs increase either the affinity of
the receptor for its endogenous ligand (glutamate), increase the likelihood
that the channel will open or increase the length of time that the channel
is open once it is activated. Because these bind to sites other than the con-
served orthosteric site, there is an opportunity to identify compounds that
have selectivity for different NMDA receptor subunits.
PAMs for the NMDA receptor have recently been characterized by molecular
pharmacology and electrophysiology. A research group at the University of
Bristol has developed several PAMs with differing selectivity for subpopula-
tions of NMDA receptors. For example, while UBP646 is a PAM for all GluN2
subunit combinations, UBP710 acts as a PAM on NMDA receptors that have
GluN2A and B subunits, but inhibits receptors with GluN2C or D. UBP551 acts
as a PAM for GluN2D, but inhibits GluN2A-C-containing NMDA receptors.
Another compound developed by Traynelis and colleagues is CIQ, which is a
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PAM that is selective for GluN2C and D-containing receptors.90 Since inhibi-
tory interneurons have been shown to express GluN2D receptors, it is possible
that 2D-selective PAMs could be particularly beneficial for schizophrenia. This
is due to evidence that inhibitory interneurons may be more influenced by
reduced NMDA receptor activity, as we discuss later in this chapter.
There have been a number of exciting advances in the structural biology of
the NMDA receptor complex.91,92 Crystal structures of NMDA receptors con-
taining different combinations of NR2 subunits have been resolved,92 which
will undoubtedly aid in the further development of drugs. Currently there are
a number of experimental compounds that now need to be tested in preclin-
ical animal models. Given the multitude of NMDA receptor subunit combi-
nations, it is not clear which drug profile will be most beneficial to improve
schizophrenia symptoms.
6.4.2 Glycine and Serine

In addition to directly manipulating NMDA receptor channel properties,
new treatments for schizophrenia may include the proteins that control
the homeostasis of its ligands glutamate, glycine, and d-serine. As seen
in Figure 6.1, the life cycle of glutamate as a neurotransmitter includes
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128 Chapter 6
its packaging into synaptic vesicles by the vesicular glutamate transporter
(VGluT). Released glutamate is cleared from the synapse by plasma mem-
brane transporters (excitatory amino acid transporter, EAAT1-3). Impor-
tantly, these transporters are expressed on both neurons and astroglia, and
astrocytes play the major role in the clearance of glutamate from the syn-
apse. Within astrocytes, glutamate is metabolized to glutamine so that it
can diffuse across the cell membrane and return to neurons without acti-
vating glutamate receptors. In neurons, the glutamine is converted to glu-
tamate by the action of glutaminase enzyme to complete the life cycle.
Although the NMDA receptor is classified as a glutamate receptor, it is also
a glycine receptor and requires glycine or d-serine binding for activation.
Indeed, receptors composed of NR1/NR3 heterodimers are purely glycinergic
receptors that mediate excitatory transmission. Therefore, the glycine-bind-
ing domain of NMDA receptors represents another site for pharmacological
modulation. It is important to emphasize that both glycine and d-serine serve
as ligands for the NMDA receptor, and in fact d-serine binds with greater
affinity than glycine at the orthosteric site. As is the case for glutamate, the
lifecycles of glycine and d-serine illustrate some of the key pharmacological
targets for new drugs.
The therapeutic effects of glycine have been confirmed in a number of pre-
clinical models that are relevant to schizophrenia. NMDA receptor antagonist-
induced neurochemical and behavioural changes can be attenuated or reversed
by glycine or d-serine.93–98 In a PCP model, high-dose glycine attenuates amphet-
amine-induced dopamine release.93 Since high levels of glycine are difficult to
deliver in vivo, more behavioural studies have been performed with d-serine.
d-Serine reverses NMDA receptor antagonist-induced deficits in latent inhibi-
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tion,94 object recognition,95,96 spatial memory,97 and social behaviour.98

Alternatively, targeting machinery and mechanisms in the brain that
endogenously regulate glycine levels may overcome some of the obstacles
faced in the direct administration of glycine, such as the high doses required
to increase brain glycine levels. Synaptic levels of glycine are regulated by
specific high affinity, sodium/chloride-dependent glycine transporters,
GlyT1 and GlyT2.99 The GlyT1 transporter is expressed predominantly on
glial cells within the central nervous system, and regulates glycine levels
in the forebrain.100 GlyT1 maintains local synaptic glycine at very low levels
(<10 μM), thus playing an important part in regulating the NMDA receptor.101
The NMDA receptor and GlyT1 are highly co-localized, further supporting the
role of GlyT1 in regulation of NMDA receptor activity.102 Therefore, a strat-
egy to improve NMDA receptor function is through GlyT1 inhibition, which
increases glycine levels within the synapse and increases occupancy of the
glycine site at the NMDA receptor. As opposed to previous noncompetitive
agents for GlyT1, newer compounds that target the transporter are compet-
itive antagonists with reversible binding kinetics, increasing tolerability.103
Clinical support for targeting GlyT1 in schizophrenia comes from sarco-
sine (N-methylglycine) studies and a phase II study with a high affinity GlyT1
inhibitor (see Table 6.2).102,104 Sarcosine is an intermediate metabolite of
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Table 6.2 Pharmacological
agents that modulate glutamatergic signaling.
Drugs that Target the Glutamate Synapse

Pharmacological Mechanism of
target action Physiological effects Compounds
NMDA receptor PAM Potentiate NMDA receptor channel by increasing GluN2A – UBP512 (weak), UBP710, UBP646,
open channel probability or length of chan- UBP714152
nel open time. Drug actions may be selective GluN2B – UBP710, UBP646, UBP714152
to channels with specific subunit composi- GluN2C – UBP646, CIQ90,152
tion (GluN1, GluN2A, GluN2B, GluN2C, and GluN2D – UBP646 (strong), UBP551, UBP714,
GluN2D)151 CIQ90,152
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NAM Reduce NMDA receptor channel by decreasing GluN1 – UBP608153

open channel probability or length of channel GluN2A – UBP551, UBP608152,153
open time. Drug actions may be selective to GluN2B – UBP551152
channels with specific subunit composition GluN2C – UBP710, UBP551152
(GluN1, GluN2A, GluN2B, GluN2C, and GluN2D) GluN2D – UBP710, UBP551152
AMPA receptor PAM Potentiate AMPA receptor channel by increasing Cyclothiazide154, aniracetam, CX516110,114
(AMPAkine) open channel probability or length of channel
open time.
GlyT1 Inhibitor Inhibition of glycine transporter leads to an Sarcosine (N-methylglycine) and Bitopertin155
increase in glycine levels within the synapse, ALX-5407, LY2365109, SSR-504734, RG-1678106
increasing occupancy of the glycine site at Org-25935102
the NMDA RECEPTOR and potentiating its
activity.102
mGluR1 PAM Controls post-synaptic release of glutamate Ro01-6128 and VU-71106
and GABA, and interacts with the NMDA
RECEPTOR.106
mGluR2/3 Agonist Inhibits further release of glutamate when there is LY2140023155, LY354740 and LY379268124
PAM an accumulation of glutamate in the perisynap- ADX71148, ADX71149 (mGluR2 specific)124
tic extracellular region, ultimately modulating
glutamatergic neurotransmission.124,155
mGluR5 PAM Activation of mGluR5 is known to enhance NMDA CDPPB, VU0360172, ADX-47273, VU0404251,
receptor function and enhance NMDA cur- CPPZ141
rents, which has excitatory effects that lead to CPPHA, VU0034403 and VU0357121106
129
increased activity in forebrain circuits.106,141
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130 Chapter 6
glycine metabolism, and binds to the GlyT1 transporter with low affinity.105
In disease states of decreased metabolism of sarcosine, extremely high levels
of sarcosine in the periphery have proven not to be toxic. However, due to
the unavailability of sarcosine in a Food and Drug Administration approved
formulation, clinical studies remain limited.102 In contrast, the high-affinity
GlyT1 inhibitor, RG-1678, has entered a number of clinical studies, suggest-
ing an absence of toxicity in phase I clinical trials and overall successful

preclinical development. Furthermore, phase II data shows that the GlyT1
inhibitor has a partial effect on negative symptoms and is generally well toler-
ated at all doses.106 RG-1678 (bitopertin) is currently in phase III clinical trials
as an add-on to antipsychotics for the treatment of negative symptoms.107
A different, small clinical study shows that another GlyT1 inhibitor, Org-
25935, reduces the schizophrenia-like effects and perceptual alterations of
ketamine,108 providing additional evidence of the antipsychotic potential of
GlyT1 inhibitors in humans. Like bitopertin, Org-25935 is well-tolerated, but
a recent clinical trial indicates that it may not be effective as an add-on to treat
negative symptoms.109 Currently, there are more clinical trials using GlyT1
inhibitors than any other class of drugs that target the glutamate synapse.
The strong preclinical evidence indicates that GlyT1 inhibition is an excellent
strategy to improve cognitive symptoms with minimal adverse effects.
6.4.3 AMPA Receptor

The activation of AMPA glutamate receptors is an important trigger for the
subsequent activation of NMDA receptors. Therefore, drugs that positively
modulate AMPA receptor function should in theory also improve the func-
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tion of NMDA receptors. PAMs of the AMPA receptors are called AMPAkines,
and this class of drugs has been shown to have pro-cognitive effects in mice
and rats. However, AMPAkines have not shown appreciable efficacy in human
clinical trials so far.
For example, the AMPAkine CX516 prolongs the length of time that the
AMPA channel remains open.110 It improves working and spatial memory
in wild-type rats,111,112 and in rats treated with PCP.113,114 It was also demon-
strated to have some efficacy to improve attention and memory in a place-
bo-controlled pilot study as an add-on treatment to clozapine.115 However, in
a subsequent placebo-controlled add-on trial, the drug was not effective.116
In this trial, stable schizophrenia patients were assessed before treatment,
and CX516 was given for four weeks in addition to clozapine, olanzapine, or
risperidone treatment. The add-on of CX516 did not significantly improve
the positive and negative syndrome scale rating score.
It is not clear whether the augmentation of AMPA receptor signaling is
in fact beneficial in situations where NMDA receptor signaling is impaired.
Indeed, studies in animals indicate that NMDA receptor hypofunction leads to
excessive AMPA signaling.117 A loss of NMDA receptor function on inhibitory
interneurons can produce a state of disinhibition,79 with a resulting overacti-
vation of AMPA receptors.118 This has led to the suggestion that schizophrenia
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results not from a lack of glutamate signaling, but from a dysregulation of glu-
tamate signaling, with too much AMPA receptor activity and too little NMDA
receptor activity.119 In light of this, augmentation of AMPA receptor function
may not be a fruitful strategy to improve cognition in schizophrenia patients.
6.4.4 Metabotropic Glutamate Receptors

The metabotropic glutamate receptors (mGluRs) are G-protein coupled
receptors (GPCRs). It has been estimated that GPCRs are the therapeutic tar-
gets for over half of all currently prescribed drugs, and are considered highly
“druggable” targets. Although their name is based on their signaling through
the heterotrimeric G-proteins, there is mounting evidence that most, if not
all, GPCRs also signal through other pathways that are mediated by beta-
arrestins. With this discovery has come the appreciation that drugs can
bind to the same site on the receptor and preferentially signal through one
pathway over another. Even more significant is that these different signal-
ing pathways mediate different cellular and behavioural effects. Therefore,
drugs that target GPCRs can have “functional selectivity”, meaning that they
can selectively activate a specific intracellular pathway. An emerging field in
the discovery of drugs for schizophrenia will undoubtedly be to profile the
drugs that target mGluRs for their ability to act on both the canonical G-pro-
tein pathways and noncanonical signaling through beta-arrestins.
mGluRs are Class C GPCRs that have two distinguishable unique structural
features; a large extracellular venus fly-trap amino-terminal agonist binding
site and the ability to form constitutive homo- and heterodimers that result
in diverse activation modes.120,121 Glutamate, along with other known ago-
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nists and competitive antagonists, binds to the orthosteric site of mGluRs,

which is located in the extracellular N-terminal region. Conversely, all of the
PAMs and negative allosteric modulators (NAMs) discovered thus far bind to
the 7TM domain.106 Identification of subgroup-specific ligands for the ortho-
steric site has been met with difficulty, most likely due to the fact that there
existed evolutionary pressure to conserve the glutamate binding site across
all mGluRs, however, the same does not apply to allosteric binding sites,
which were under less pressure for conservation.120,122,123 Allosteric modu-
lators, in general, are able to develop functional selectivity, which allows for
the targeting of select downstream signaling pathways, which can lead to
the optimization of pharmacokinetics and other properties of potential drug
candidates.124
In contrast to NMDA receptors and AMPA receptors, mGluRs modulate cell
excitability and synaptic transmission. mGluRs are expressed in a number
of cell types within the brain, both neurons and glial cells, including astro-
cytes, oligodendrocytes, and microglia.125 In terms of cellular and synaptic
localization, mGluR1 and mGluR5 are expressed postsynaptically, and their
activation increases intra-cellular calcium through Gq coupling. mGluR2
and mGluR3 are expressed presynaptically and their activation decreases
cyclic adenosine monophosphate levels through Gi/o coupling. Activation of
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132 Chapter 6
postsynaptic receptors modulates neuron excitability and triggers signaling
cascades that mediate synaptic plasticity, and presynaptic receptors serve as
autoreceptors to regulate neurotransmitter release.124,125 Drug development
for schizophrenia has primarily focused on the modulation of postsynaptic
mGluR1 and mGluR5 receptors and presynaptic mGluR2/3 receptors.126
6.4.4.1 mGluR1 (Group I)
mGluR1 functions by controlling postsynaptic release of glutamate and
GABA, and also via its interactions with the NMDA receptor.127,128 Evidence
suggests that modulation of mGluR1 through PAMs may have potential for
the treatment of positive and cognitive symptoms in schizophrenia. The
lines of evidence are: 1) activation of mGluR1 has been documented to facil-
itate NMDA receptor and AMPA receptor response,129 2) mGluR1 levels are
altered in schizophrenic patients,130 3) mGluR1 knockout mice show deficits
in sensorimotor gating similar to that observed in schizophrenia patients,131
and 4) mGluR1 modulates dopamine release.132 Despite the aforementioned
rationale that mGluR1 PAMs may provide a promising novel target for schizo-
phrenia treatment, few mGluR1 PAMs have been described (Table 6.2) and
the data at present are limited.
6.4.4.2 mGluR2/3 (Group II)

Of all the drugs targeting the mGluRs, agonists of the mGluR2/3 are the
most developed as a novel treatment strategy for schizophrenia.120,126 In
states of excessive glutamatergic tone, mGluR2/3 autoreceptor activation
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attenuates further release of glutamate to normalize perisynaptic levels.133

Since NMDA receptor hypofunction may cause excessive glutamate neuron
firing, a strategy to normalize glutamate signaling is to attenuate gluta-
mate release through the activation of these autoreceptors. It is likely that
the antipsychotic-like activity seen with mGluR2/3 agonists is mediated in
part by the reduction of glutamate release from neurons projecting from
the thalamus to the prefrontal cortex.126,134 A number of mGluR2/3 agonists
show robust efficacy in multiple rodent models. However, despite early clin-
ical results with LY404039, suggesting efficacy in treating both positive and
negative symptoms,135 a phase II dose-finding study did not show effects
above placebo.136 It has been proposed that orthosteric agonists may lead
to receptor desensitization, reducing or eliminating efficacy over time.10 In
addition, knockout mouse studies indicate that the therapeutic effects of
these drugs are primarily mediated through mGluR2 and not mGluR3.137
Therefore, current drug discovery is focused on identifying mGluR2 PAMs
to avoid receptor desensitization and to ensure specificity. Highly selective
mGluR2 PAMs (see Table 6.2) are currently in development for the treatment
of schizophrenia, as well as other disorders of the central nervous system,
and have promising results. Primary objectives of safety and tolerability in
phase I studies are met and advancement into phase IIa clinical testing is
underway.124
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6.4.4.3 mGluR5 (Group I)
The mGluR5 subtype of the metabotropic glutamate receptors is of particu-
lar interest in the context of schizophrenia due to its functional and physi-
cal association with NMDA receptors.138 mGluR5 is expressed in a number of
brain regions, including the cerebral cortex, hippocampus and hypothalamus,
among others, and is not limited to neurons, being expressed on non-neuro-

nal cells including astrocytes, oligodendrocytes and microglia.139 However,
most abundantly, mGluR5 is expressed on pyramidal cells and interneurons
in the cortex.126 Physically, via anchoring proteins Homer, SRC Homology
3 (SH3), SHANK, synapse-associated protein 90/postsynaptic density-95-
associated protein (SAPAP) and postsynaptic density protein (PSD)-95, there
is a link between the NMDA receptor and mGluR5.140 Furthermore, func-
tionally, the NMDA receptor and mGluR5 are co-localized in a number of
regions within the brain, including the hippocampus, striatum and the neo-
cortex, which are all implicated in schizophrenia, functioning through a pos-
itive-feedback loop to indirectly regulate NMDA receptor function.106,139
Activation of the mGluR5 receptor is known to enhance NMDA receptor
function and enhance NMDA currents.124,141 However, orthosteric agonists of
the mGluR5 are excitotoxic and can lead to the issue of receptor desensitization
and downregulation (as described in the previous section).120,123 Therefore,
drug discovery efforts are primarily focused on positive allosteric modulation
of mGluR5. mGluR5 PAMs have been shown to enhance hippocampal synap-
tic plasticity (long-term potentiation and long-term depression) and poten-
tiate NMDA currents. They show robust efficacy in preclinical models and
pro-cognitive effects in a number of cognitive assays.106,120,124,126,142,143 Three
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distinct allosteric sites have led to the development of mGluR5 PAMs. The
only characterized site of the three is the 2-methyl-6-(phenylethynyl)-pyridine
(MPEP) binding site,106 which has led to the discovery of a number of struc-
turally diverse, highly selective, mGluR5 PAMs (see Table 6.2). Although these
studies advance the potential treatment of schizophrenia, it is important to
note that each allosteric modulator can have drastically different effects, and
that not all mGluR5 PAMs are the same. Recent studies have shown that some
mGluR5 PAMs induce severe seizure activity144 and cytotoxicity, leading to
cell death in certain brain regions.124 Other mGluR5 PAMs have been shown
to be well tolerated, further emphasizing that mGluR5 PAMs most likely dif-
fer in their effects on mGluR5 signaling and understanding these differences
is crucial for further development of therapeutically practical mGluR5 PAMs.
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www.Ebook777.com
CHAPTER 7
Disrupted-in-Schizophrenia-1
(DISC1) Interactome and
Schizophrenia
TATIANA V. LIPINA†*a AND JOHN C. RODERa,b
a
Lunenfeld Tanenbaum Research Institute at Mount Sinai Hospital, Toronto,
Ontario, M5G 1X5, Canada; bDepartments of Medical Biophysics and Molec-
ular & Medical Genetics, University of Toronto, Toronto, Ontario, Canada
*E-mail: lipina@physiol.ru
7.1 Introduction
10:30:36.
Schizophrenia is a severely debilitating form of neurodevelopmental men-

tal illness, typified by positive symptoms of hallucinations and delusions;
negative symptoms of decreased motivation, anhedonia; and selective cog-
nitive deficits.1 It is considered to be a complex genetic disorder, with inter-
play between the effects of multiple genes and environmental factors, which
together shape the brain; the symptoms typically do not emerge until early
adulthood.2 Hence, our understanding of the genes, genetic pathways and
their interactions with environmental stressors coupled with brain imaging
and cellular-behavioural characteristics is essential to understand the etiol-
ogy and pathogenesis of schizophrenia.
The disrupted-in-schizophrenia-1 (DISC1) gene emerged as a convinc-
ing genetic risk factor predisposing individuals to schizophrenia, detected
by genetic and clinical association studies and emerging biology.3–7 In this
†
Present address: Institute of Physiology, Novosibirsk, Russia.

141
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142 Chapter 7
chapter we first describe functional roles of DISC1 and its molecular com-
plexes (DISC1 interactome) in the brain. Second, we review genetic associ-
ation data linking the DISC1 interacome with schizophrenia. Next, we will
describe neuroanatomical and neurocognitive phenotypes associated with
schizophrenia and the DISC1 interactome. Lastly, we will go on to discuss
mouse models for schizophrenia related to DISC1 interactome and achieve-
ments in the discovery of new antipsychotic drugs.
7.2 Functions of DISC1 Interactome in the Brain

Essential progress has been made in outlining the biological functions of
DISC1; much of this knowledge came from understanding how individual
proteins directly interact with DISC1 (DISC1 interactors) and what the roles
are of these molecular complexes with DISC1. The hallmark of DISC1 is its
ability to interact with multiple proteins5,7 at different cellular locations8
and developmental time points in the brain. The most comprehensive list
of DISC1-interacting proteins was identified using a yeast two-hybrid screen9
consisting of 127 proteins and 158 interactions. The “DISC1158 network”
(Figure 7.1), together with the early biochemical studies of DISC1, pio-
neered the concept that DISC1 acts as a scaffold protein in the cell. DISC1
ties together divergent pathways, such as the Akt/mammalian target of
rapamycin (mTOR) pathway, a dopaminergic pathway coupled with glyco-
gen synthase kinase (GSK)-3, D2R, phosphodiesterase (PDE)4B/cAMP, and
GSK-3/β-catenin. These pathways play important roles in the regulation of
neurodevelopment and synapse formation, functioning, and maintenance
10:30:36.
to organize the functional DISC1 interactome; therefore, DISC1 interactors

can be grouped into several subcategories according to their functional roles
in neurodevelopment and within the cell (Figure 7.1).
7.2.1 Neurodevelopment
7.2.1.1 Proliferation
Accumulating studies indicate that DISC1 is critically involved in neuro-
genesis when newborn neurons are generated. DISC1 expression peaks at
E14–15 in the embryonic mouse brain, which accompanies active neurogene-
sis in the cortex.10 Neural progenitor proliferation is controlled by the canon-
ical Wnt signalling pathway, where β-catenin regulates the transcription of
genes critically involved in cell proliferation.11 The N-terminus of DISC1
directly binds to GSK-3β and inhibits its enzymatic activity.10 This in turn tar-
gets β-catenin for phosphorylation and causes its degradation.11 Reduction
of β-catenin inhibits cell proliferation while promoting cell differentiation.10
DIX domain containing 1 (DIXDC1), another DISC1 interactor, is involved
in the co-regulation of Wnt-GSK-3β/β-catenin signalling and cortical neural
progenitor proliferation.12 Lissencephaly-1 (LIS1) and nudE nuclear distri-
bution E homolog 1 (NDE1) [or its paralogue nudE nuclear distribution E
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Disrupted-in-Schizophrenia-1 (DISC1) Interactome and Schizophrenia 143

Figure 7.1 Classification

of the disrupted-in-schizophrenia (DISC)1 network,
which includes 158 protein–protein interactions9 by function. The
diagram illustrates implications of distinct DISC1 interactors into
neurodevelopmental functions (regulation of neural precursor prolifer-
ation, neuronal migration, neuronal integration, and maturation) and
their roles on a subcellular level (regulation of gene expression in the
nucleus, association with neurosignalling and synaptic functioning,
role in the centrosome, mitochondria, and intracellular traffic in asso-
ciation with motor proteins). Adapted from T. V. Lipina and J. C. Roder,
10:30:36.
Neurosci. Biobehav. Rev., 2014, 45, 271–294, copyright (2014), with per-
mission from Elsevier.157 APP: amyloid precursor protein; ATF4/5: acti-
vating transcription factor 4/5; BBS4: Bardet–Biedl syndrome 4; CAMDI:
coiled-coil protein associated with myosin II and DISC1; CHCHD6:
coiled-coil-helix-coiled-coil-helix domain containing 6; CHCM1: coiled-
coil helix cristae morphology 1; DIC: dicarboxylate ion carrier; DIXDC1:
DIX domain containing 1; FEZ1: fasciculation and elongation protein
zeta 1; Grb2: growth factor receptor-bound protein 2; GSK-3: glyco-
gen synthase kinase 3; Kal-7: kalirin 7; KIF5A: kinesin family member
5A; KLC1: kinesin light chain 1; LIS1: lissencephaly-1; N-CoR: nuclear
receptor co-repressor; NDE1: nudE nuclear distribution E homolog 1;
NDEL1: nudE nuclear distribution E homolog 1-like 1; PCM1: pericen-
triolar material 1; PCNT: pericentrin; PDE4: phosphodiesterase 4B or
4D; SRR: serine racemase; TNIK: TNF receptor-associated factor 2 and
Nck interacting kinase.
homolog 1-like 1 (NDEL1)], other DISC1 interactors,13,14 bind to DISC1 at close

regions (727–854 aa and 802–835 aa, respectively). LIS1 deficiency affects cell
proliferation through disruption of the orientation of cleavage, determining
the symmetry of division and causing multiple deleterious effects on microtu-
bule function, which can be rescued by NDEL1.15 It is still unknown how DISC1
modulates proliferation via its interaction with LIS1, NDE1, GSK-3/β-catenin,
and DIXDC1, and whether these pathways are interrelated.
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144 Chapter 7
7.2.1.2 Cell migration
Cell migration is a next step after neurogenesis/proliferation and the pro-
cess includes cytoplasmic displacement at the leading front, which is fol-
lowed by migration of the nucleus and, lastly, retraction of the cell. During
development, pyramid neurons produced in the subventrical zones (SVZ)
migrate along guide fibers provided by glial cells to reach the cortex and
become part of the functional cortical neuronal networks. Granule cells
of the hippocampus produced at the dental notch migrate to the dental
gyrus of the hippocampus. In adults, neuronal migration is limited to neu-
ral precursors moving from the SVZ to the olfactory bulbs via the rostral
migratory stream and from the subgranular zone to the granular zone of
the hippocampus. An understanding of the mechanisms by which cells
migrate may lead to the generation of novel drugs for controlling the onset
of schizophrenia. Cells where DISC1 expression has been silenced express
abnormal detection of guidance cues during migration.16 Cortical neuro-
nal migration was blocked by DISC1 inhibition and reversed by overex-
pression of DISC1 in the developing cortex.17,18 DISC1 is also important
for migration of cortical interneurons in the embryonic brain.19 Recent
evidence shows that DISC1, together with N-methyl-d-aspartate (NMDA)
receptors, modulates neuronal migration.20 The disrupted neuronal radial
migration in the cortex was detected in DISC1 mutant mice,21 support-
ing the role of DISC1 in migration. Moreover, Ishizuka et al. revealed the
essential mechanism that regulates the balance between proliferation and
migration in the developing cortex.22 Once DISC1 is phosphorylated at
S710 (DISC1-pS710), it stops interaction with GSK-3, consequently inhib-
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iting DISC1 activity as a positive regulator of Wnt/β-catenin signalling.

DISC1-pS710 is accumulated at the centrosome and increases affinity to
Bardet–Biedl syndrome 4 (BBS4), which in turn enhances its recruitment
to the centrosome.22 BBS4 accumulation at the centrosome in a DISC1-de-
pendent manner is a key mechanism of neuronal migration in the devel-
oping cortex.23 LIS1 and NDEL1 are also critically involved in neuronal
migration.24 LIS1 is required for normal activation of Rho GTPases and
actin polymerization in response to a calcium influx induced by NMDA
receptors. LIS1 is needed for dynein- and microtubule-dependent pro-
cesses, e.g. the coupling of the nucleus and centrosome, or Golgi integrity.
During brain development, LIS1 is involved in proliferation of neuronal
precursors and migration of newly formed neurons from the ventricular/
subventricular zone towards the cortical plate. Mutated LIS1 causes liss-
encephaly due to impaired neuronal migration.25 DIXDC1, another DISC1
interactor, acts as a “switcher” between neuronal migration and prolifera-
tion. The DISC1/DIXDC1 complex without NDEL1 directs neural progenitor
proliferation via the Wnt/GSK-3/β-catenin pathway, whereas recruitment
of NDEL1 switches DISC1/DIXDC1 function into neuronal migration.12
Amyloid precursor protein (APP), another DISC1 interactor, also regulates
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26
neuronal migration. APP deficiency in mice impairs neuronal migration
that is corrected by overexpression of DISC1.26
7.2.1.3 Neuronal Maturation and Integration

The final step of newborn neurons is the proper neuronal maturation and inte-
gration into functional neuronal circuitry. DISC1 per se remains an important
molecule at this stage. So, inhibition of mouse DISC1 expression in the adult
hippocampus affected the cellular morphology of newborn granular cells
(enlarged cell bodies, increased dendritic length and branching, and accelerated
synapse formation)27 and resulted in impaired axonal targeting to the CA3 sub-
field.28 The neuronal mispositioning was observed in the dental gyrus of 129S6/
SvEv mouse inbred strain, carrying the mutated DISC1 allele,29 which likely
causes problematic integration into neuronal circuits within hippocampus.
The impaired neuronal morphology also was observed in the cortex of DISC1
point mutant mice.21 A recent study demonstrated that silencing DISC1 in the
mouse cortex at an early life stage disrupted postnatal maturation of mesocor-
tical dopaminergic projections to the medial prefrontal cortex, affecting the
function of neuronal circuitry and causing schizophrenia-like behaviour.30
DISC1 forms a complex with NDEL1 and fasciculation and elongation pro-
tein zeta (FEZ)1 to regulate distinct phases of neurodevelopment.27 A study by
Kim et al.31 identified an important role of Girdin (or KIAA1212) in molecular
mechanisms of neuronal maturation. Once DISC1 interacts with Girdin, it
prevents Girdin’s capacity to bind and activate AKT.31 The upregulation of Gir-
din or AKT in its active form resembles the effects of DISC1 deficiency on neu-
10:30:36.
ronal development,31,32 suggesting a common pathway implicated in cellular

maturation. Interestingly, the effects of DISC1 downregulation or Girdin over-
expression were corrected by rapamycin (mTOR inhibitor), but not by GSK-3
inhibition,31,32 which suggests a specific role of the mTOR pathway in neu-
ronal maturation. A further study found synergistic action between DISC1
and γ-aminobutyric acid (GABA) signalling to regulate dendritic outgrowth
through AKT/mTOR pathways.33 Downregulation of NKCC1, an ion trans-
porter, implicated in GABA responses, or silencing of the GABAa receptor γ2
subunit, was able to correct dendritic growth abnormalities induced by DISC1
knockdown.33 PDE4, another DISC1 interactor,34,35 regulates cellular cAMP
levels, which in turn affect expression of cAMP response element-binding
protein (CREB), axon guidance, and dendritic growth.36 Neurons carrying the
mutated 129 DISC1 allele showed deficient dendritic branching and altered
axon pathfinding in vitro, which were rescued by reduced cAMP levels.36
Overall, DISC1 and its interacting proteins regulate key steps of neurode-
velopment, including proliferation of neural precursors (GSK-3, DIXDC1,
LIS1, NDE1, and NDEL1), neuronal migration (LIS1, NDEL1, DIXDC1, BBS4,
and APP), and neural integration and maturation (NDEL1, FEZ1, Girdin, and
PDE4) (Figure 7.1).
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146 Chapter 7
7.2.2 Neuronal Signalling and Synaptic Plasticity

In addition to the important role of DISC1 and its interactome in neurode-
velopment, DISC1 directly regulates intracellular signalling and the ability
of synapses to strengthen or weaken their function in response to neuro-
nal activity. cAMP is a second messenger that translates intracellular signal
transduction, via the cAMP-dependent pathway. DISC1 binds with several

cAMP-specific PDE4B/Ds,34,35 regulating their enzymatic activities via direct
DISC1 × PDE4 interaction. DISC1 interacts with the upstream conserved
region (UCR)2 domain of PDE4B, and the increased level of cAMP causes
dissociation of PDE4B from DISC1, which in turn increases PDE4B activ-
ity34 to metabolize cAMP. PDE4 isoforms differ from each other due to their
unique N-terminal regions, which target interaction with specific protein
complexes to affect cAMP levels in a compartmentalized manner.37 Mice
carrying the 129 DISC1 allele have reduced PDE4 activity and expression in
the hippocampus,36 which in turn increases activation of CREB, required
for axon guidance and dendritic growth.36 The DISC1-Q31L point mutation
also reduced PDE4 activity.38 This influences biochemical signalling at the
level of β-arrestin, CREB and monoamines. It also affects synaptic plasticity
in the nucleus accumbens, a brain region regulating reward, leading to the
expression of depression-related behaviour in this mutant line.39 GSK-3,
another DISC1 interactor, is fundamentally implicated in neurotransmitter
function40,41 and synaptic plasticity.42 Moreover, PDE4 and GSK-3 enzymes
have overlapping binding sites on DISC1,10,35 suggesting that their function
can be regulated via the DISC1 complex. More importantly, both PDE4 and
GSK-3 are implicated in the action of antidepressants and antipsychotic
10:30:36.
drugs,43,44 suggesting their role in eliciting therapeutic effects. A recent in

vitro study shows that GSK-3 has a tonic-activating effect on PDE4.45 We
have demonstrated in vivo that the inhibition of signals from both PDE4
and GSK-3 can be synergistic to induce antipsychotic effects observed in
DISC1-L100P mutant mice.46
Data show that DISC1 is present both pre-47 and post-synaptically,38,48,49
indicating that DISC1 and its interacting proteins can regulate communi-
cations between neurons to achieve synaptic plasticity. DISC1 modulates
morphology and density of dendritic spines through Kalirin (Kal)-7, a DISC1
interactor.50 Dendritic spines are small membranous protrusions from den-
drites that receive input from the synapse; they serve as a storage site for
synaptic strength and help to transmit electrical signals to the cell body.
DISC1 dissociates Kal-7 interaction with Ras-related C3 botulinum toxin
substrate (RAC)1, a well-known molecular regulator of dendritic spines,
and as result, RAC1 activity is reduced in NMDA-dependent manner.50
Furthermore, it was shown that DISC1 modulates glutamatergic synapses
post-synaptically through its interaction with TNF receptor-associated fac-
tor 2 and Nck interacting kinase (TNIK),51 the expression and phosphoryla-
tion of which is glutamate receptor (GluR) activity-dependent. Inhibition of
TNIK activity by peptides that affect its binding to DISC1 decreased synaptic
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expression of postsynaptic density protein-95, α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA) receptor subunit GluR1, and the AMPA
regulator, calcium-channel voltage-dependent γ-subunit (CACNG)2 or star-
gazin.51 A recent study demonstrated that DISC1 directly regulates function-
ing of NMDA receptors.52 Knockdown of DISC1 increased NMDA receptor
currents in cortical cultures, which were reversed by a PDE4 inhibitor or pro-
tein kinase A (PKA) activator. The increased NMDA receptor current induced
by DISC1 deficiency was accompanied by an increase in CREB activity, and
consequent blocking of CREB prevented DISC1’s effects on NMDA synap-
tic plasticity.52 Another study showed physiologically co-localization of
the cAMP-related proteins DISC1, PDE4 and D1R with hyperpolarization-
activated cyclic nucleotide-gated channels at excitatory synapses and at the
spine neck in monkey cortex during the performance of a working memory
task.53
Maher and Loturco’s study47 provided evidence that DISC1 can also act
through a presynaptic site. The overexpression of the truncated DISC1 given
in utero almost doubled miniature excitatory synaptic currents (mEPSCs)
mediated by glutamatergic synapses. To directly test if this effect could be
driven by DISC1 presynaptically, authors applied optogenetic stimulation of
presynaptic neurons and showed that DISC1 overexpression facilitates gluta-
mate release at the synapse, whereas DISC1 inhibition induced the opposite
effect.47 DISC1 also modulates glutamatergic synaptic plasticity though its
interaction with serine racemase,54 the enzyme which converts l-serine into
d-serine, active co-agonist of NMDA receptors. The abolished interaction of
DISC1 with serine racemase caused reduced expression of the enzyme, which
led to behavioural deficits in mutant mice consistent with hypofunction of
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NMDA receptors.
Besides the regulation of glutamatergic synaptic transmission, DISC1
also tightly regulates function of the dopaminergic neural system. DISC1-
L100P mutant mice demonstrated an increased response to the dopa-
mine-releaser amphetamine together with an increase of highly sensitive
D2Rs in the striatum.55 The DISC1 dominant-negative mutant mouse line
also showed alterations in the expression of D2Rs and response to meth-
amphetamine.56 A recent study characterized a DISC1 × ATF4 (activating
transcription factor 4) interaction in detail, determining that this com-
plex is regulated by PKA-induced phosphorylation of DISC1 at serine-58.57
The authors showed that loss of function of DISC1 or ATF4, as well as
stimulation of D1Rs, increased PDE4D9 expression due to the disassocia-
tion of DISC1 from the PDE4D9.57 The release of DISC1-mediated genetic
repression of PDE4D9 acts as feedback inhibition of dopaminergic sig-
nalling. DISC1 is also able to regulate a dopaminergic system during neu-
rodevelopment.30,57 Therefore, inhibition of DISC1 in utero significantly
disrupted development of cortical dopaminergic system in mice,30 and
early-life stressors elicited its effects on the development of dopaminergic
mesocortical neurons via glucocorticoid receptors in DISC1 transgenic
mice.58
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148 Chapter 7
7.2.3 Subcellular Functions

DISC1 is located in several subcellular domains, including cytoskeleton-
associated structures, the nucleus, and the mitochondria (Figure 7.1).
7.2.3.1 Nucleus
DISC1 is partially located at the nucleus and can be detected within pro-
myelocytic leukemia nuclear bodies, sites of active transcription.59 DISC1
nuclear targeting is mediated by three functional cis-elements and a putative
leucine zipper spanning residues 607–628, encoded by DISC1 exon 9.59 DISC1
forms a complex with several transcriptional factors within the nucleus and
thereby can globally modulate gene expression and exert multiple effects on
a behavioural level. Indeed, DISC1 interacts with ATF4/5/CREB257,59 and with
a nuclear receptor co-repressor (N-CoR), modulating cAMP response element
(CRE)-mediated gene transcription.59 Interestingly, ATF4/ATF5 are preferen-
tially translated in response to environmental factors, including nutrient
deprivation, viral infection, or oxidative stress,60,61 suggesting that epigenetic
DNA alterations are driven by environmental stressors through ATF4/ATF5.
Moreover, N-CoR59 mediates the repressive activity of nuclear receptors and
can integrate the activities of various transcription factors with a number of
histone-modifying enzymes.62 There is no overlap in the binding domains of
DISC1 to ATF4/CREB2 (leucine zipper domain in exon 9) and N-CoR (exon
12), suggesting that DISC1 may form a complex with both ATF4/ATF5 and
N-CoR simultaneously to modulate gene transcription and elicit epigenetic
effects.
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7.2.3.2 Centrosome
Centrosomes consist of two centrioles surrounded by pericentriolar mate-
rial (PCM) and serve as the main cytoskeleton-organizing centre in the cell
and as the location for microtubule nucleation and anchoring.63,64 Therefore,
centrosomes regulate such cellular processes as mitosis, differentiation, and
migration. DISC1 is localized in the centrosome9,65–67 with its interacting
proteins such as PCM1, NDE1, NDEL1, LIS1, coiled-coil protein associated
with myosin II and DISC1 (CAMDI), pericentrin (PCNT/kendrin), and BBS
(reviewed by Brandon and Sawa 20115 and Thomson et al. 20137) (Figure 7.1).
Some of these proteins are involved in neurodevelopment as described ear-
lier (NDE1, NDEL1, LIS1, and BBS). Importantly, the early study of Kamiya et
al. (2005)17 demonstrated that DISC1 is a part of the microtubule-associated
dynein motor complex, which is essential for maintaining the complex at the
centrosome. The truncated DISC1, due to chromosomal translocation, dis-
sociates from the DISC1–dynein complex at the centrosome,17 which impairs
neurite outgrowth and neurodevelopment. NDE1, NDEL1, and LIS1 form the
dynein motor complex to regulate organelle positioning and provide the cor-
rect functioning of the microtubule network.68 Localization of DISC1 at the
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centrosome requires its association with kendrin (PCNT), and disruption of
DISC1 × kendrin interaction hampered microtubule formation.69 Moreover,
DISC1 binds BBS to fix PCM1 at the centrosomal location, which is required
for the correct localization of kendrin23 and organization of microtubule
arrays.70
CAMDI is another protein associated with DISC1 and myosin II, which
translocates to the centrosome in a DISC1-dependent manner.71 PDE4 reg-

ulates the association of DISC1 with LIS1, NDE1, and NDEL1 at the centro-
some.4 NDE1 protein is phophorylated by PKA at S306 and T131 sites under
regulation of DISC1 and PDE4.4 The phosphorylated NDE1 (NDE1-pT131)
becomes unavailable to LIS1, but facilitates binding to NDEL1.4 PDE4 by
itself binds to NDEL1 and LIS1 in cAMP-sensitive manner,72,73 showing that
cAMP is an important modulator of the DISC1 × NDE1 × NDEL1 × LIS1 com-
plex at the centrosome.
7.2.3.3 Mitochondria
The mitochondrion is an essential organelle supporting many neuronal
functions, providing the main source of energy for neurons. Mitochondrial
dysfunctions, e.g. bioenergetic defects, mitochondrial DNA mutations, mito-
chondrial morphology, movement, and fusion/fission have been observed
in patients with schizophrenia.74 Expression of DISC1 was detected in the
mitochondrial membrane49 in association with mitofilin,75 which is a mito-
chondrial inner membrane protein. The reduced DISC1 function caused
mitochondrial dysfunction, and reduced nicotinamide adenine dinucle-
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otide (NADH) dehydrogenase activity, amount of adenosine triphosphate

(ATP), and Ca2+ dynamics.75 Interestingly, the aberrant DISC1 × mitofilin
complex reduced enzymatic activity of monoamine oxidase-A, which deam-
inates monoamines. The novel mitochondrial protein, coiled-coil helix cris-
tae morphology 1/coiled-coil-helix-coiled-coil-helix domain containing 6
(CHCM1/CHCHD6), has been revealed to interact with DISC1 and mitofi-
lin.76 Manipulation with genetic expression of either mitofilin or CHCM1/
CHCHD6 suggests co-ordinated regulation between these proteins, which
are implicated in the formation of mitochondrial cristae morphology, cell
growth, ATP synthesis, and oxygen consumption. Overall, these findings pro-
vide evidence that DISC1, with its interacting proteins (mitofilin, CHCM1,
and CHCHD6) can compromise mitochondrial function and contribute to
the psychopathological mechanisms of schizophrenia.
7.2.3.4 Motor Proteins
Motor proteins are molecular motors that move along the surface of a suit-
able substrate. Kinesins and cytoplasmic dyneins regulate intracellular
anterograde and retrograde microtubule-based transport, including traffick-
ing of synthesized proteins or vesicles to synapses or axonal growth cones or
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150 Chapter 7
recycling of synaptic vesicles, and transport of neurotransmitters back to the
neuron’s soma. DISC1 binds to kinesin 1 and its inhibition decreased axonal
transport of such kinesin-associated proteins as NDEL1, LIS1, growth factor
receptor-bound protein (GRB)2 and 14-3-3.77,78 FEZ1, another DISC1 inter-
actor,79 directly binds to kinesin 1 and tubuline in order to facilitate antero-
grade mitochondrial transport.80 Expression of aberrant DISC1 impaired
synaptic vesicle transport mediated by FEZ1 × kinesin 1 complex.81 More-

over, DISC1 facilitates interaction between FEZ1 and synaptotagmin (Syt)1,
thereby recruiting synaptic vesicles into the synapse.81 DISC1 also forms a
complex with the dynein intermediate chain and the dynein adaptor protein,
dynactin,17 linking DISC1 with bidirectional intracellular transport.
In sum, it is evident that DISC1 together with its interacting proteins regu-
late essential steps of neural development and is implicated in multiple func-
tions on a subcellular level (Figure 7.1).
7.3 The DISC1 Interactome and Schizophrenia

7.3.1 Genetics
Originally the DISC1 gene was identified in a unique Scottish pedigree in
which an inherited balanced chromosomal translocation disrupts the gene
and segregates with major depression, schizophrenia and bipolar disor-
der.82,83 Following these findings, independent groups detected linkage of
DISC1 locus to schizophrenia in families from Finland,84 Scotland,85 and
the United Kingdom.86 Numerous studies provided support for association
of various DISC1 single-nucleotide polymorphisms (SNPs) in schizophre-
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nia.87 In contrast, a meta-analysis of 1241 SNPs from a total of 11 626 cases

and 15 237 controls found no evidence that common variants at the DISC1
locus are significantly associated with schizophrenia.88 Moreover, the DISC1
locus has not reached genome-wide significance in large-scale meta-analyses
of linkage studies of schizophrenia.89 To explore DISC1 in more detail, Por-
teous and colleagues sequenced 528 kb of the DISC1 locus in 653 patients
and 889 controls.90 Burden analysis for coding and non-coding variants gave
only nominal associations with diagnosis and cognitive traits. One reason for
these genetic contradictions might be because DISC1 confers a genetic risk
factor at the level of endophenotypes or brain circuitries5 that underlies sev-
eral major mental disorders as defined by the Diagnostic and Statistical Man-
ual of Mental Disorders. Indeed, many association studies reported a genetic
link between DISC1 and specific behavioural (e.g. weak concentration,91 poor
recall in memory, verbal ability or visuospatial ability92), electrophysiolog-
ical (e.g. reduced amplitude and latency of the auditory P300 event-related
potential93), and brain (e.g. hippocampal grey matter volume/function and
connectivity94) endophenotypes.
Initial extensive yeast-2-hybrid screening revealed the DISC1 network of 127
proteins and 158 interactions; a large proportion of the proteins have been
examined and validated.9 Genetic association and linkage studies showed that
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DISC1 and part of its interacome are associated with schizophrenia (reviewed
by Chubb et al.,3 Bradshaw and Porteous,4 and Thomson et al.7). The human
DISC1-S704C variant, but not others (A83V, R264Q, or L607F), which inhibits
neuronal migration in developing cortex, is associated with schizophrenia.95
In addition, the increased nuclear expression of the DISC1 75–85 kDa isoform
was detected in post-mortem brains of patients diagnosed with schizophre-
nia.96 A large proportion of DISC1 interactors, including PDE4B, GSK-3β, PCM1,

TNIK, KIF5A (kinesin family member 5A), Kal-7, dysbindin, FEZ1, 14-3-3,
PCNT, MIPT3 (TNF receptor-associated factor 3 interacting protein 1), serine
racemase (SRR), DIXDC1, ATF4/ATF5, NDE1/NDEL1 and GRB2 is involved in
schizophrenia (Figure 7.2). Because risk for schizophrenia is determined by a
network of gene–gene and gene–environment interactions, one approach to
identify whether variation within the putative DISC1 protein pathway influ-
ences risk for schizophrenia is to look for evidence of statistical interaction
impacting disease risk. Indeed, DISC1, citron ρ-interacting serine/threonine
kinase (CIT) and NDEL1 act in epistasis (i.e. when the effect of one gene
depends on the presence of other gene/s) to influence risk for schizophrenia in
clinical samples assessed by machine learning algorithms. Moreover, 75% of
these significant gene–gene interactions were biologically validated: carriers
of the combinations of schizophrenia risk-associated genotypes performed
less efficiently, similar to schizophrenia patients, during a test of working
memory.97 Synergestic interactions between PDE4B and GSK-3 enzymes in a
DISC1-dependent manner have also been reported in preclinical studies.46,98,99
7.3.2 Neuroanatomical and Neurocognitive Phenotypes

10:30:36.
Several DISC1 variants have been associated with neuroanatomical and neu-
rocognitive phenotypes, including hippocampal grey matter volume and
function, poor concentration in schizophrenics,91 recall and memory,92 ver-
bal ability and memory,92 visuospatial ability,92 psychomotor processing,92
visual working memory,92 and general cognitive ability.100
7.4 DISC1
Interactome and Mouse Models of
Schizophrenia
Animal models of psychiatric disorders should fulfill the following criteria: 1)
face validity (similarity of symptoms); 2) construct validity [similarity of genetic
and environmental causes (etiology) and similarity of physiological, neuro-
chemical, and morphological endophenotypes); and 3) predictive validity (sim-
ilarity of response to relevant drug treatments), as originally suggested.101 The
endophenotype approach was proposed to study complex mental disorders to
outline the relationship between a specific structural or functional endophe-
notype(s) and particular gene(s). Since psychiatric disorders have complex
endophenotypes and heterogeneous etiology, it is likely that animal models
can resemble only some endophenotype(s).
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152 Chapter 7
Figure 7.2 DISC1

interacting proteins and associations with schizophrenia. Dia-
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gram of protein–protein interaction domains mapped onto DISC1

and possible associations of DISC1-interacting proteins with schizo-
phrenia based on the literature and the GeneCards database (www.
genecards.org/). DISC1 predicted protein structure where two con-
served regions of the N-terminal region are highlighted [a conserved
putative nuclear localization signal (NLS) and a short conserved serine/
phenylalanine-rich motif (SF-rich motif]. Several regions of predicted
coiled-coil in the C-terminus are also labelled. Adapted from T. V. Lipina
and J. C. Roder, Neurosci. Biobehav. Rev., 2014, 45, 271–294, copyright
(2014), with permission from Elsevier.157 APP: amyloid precursor pro-
tein; ATF4/5: activating transcription factor 4/5; BBS4: Bardet–Biedl
syndrome 4; CAMDI: coiled-coil protein associated with myosin II and
DISC1; DIC: dicarboxylate ion carrier; DISC1: disrupted-in-schizophre-
nia 1; DIXDC1: DIX domain containing 1; EIF3S3: eukaryotic transla-
tion initiation factor 3 subunit 3; FEZ1: fasciculation and elongation
protein zeta 1; Grb2: growth factor receptor-bound protein 2; GSK-3β:
glycogen synthase kinase 3; IP3R1: inositol 1,4,5-trisphosphate recep-
tor, type 1; Kal-7: kalirin 7; KIF5A: kinesin family member 5A; KLC1/2:
kinesin light chain 1/2; LIS1: lissencephaly-1; MAP1A: microtubule-as-
sociated protein 1A; MIPT3: TNF receptor-associated factor 3 inter-
acting protein 1; N-CoR: nuclear receptor co-repressor; NDE1: nudE
nuclear distribution E homolog 1; NDEL1: nudE nuclear distribution E
homolog 1-like 1; PCM1: pericentriolar material 1; PCNT: pericentrin;
PDE4: phosphodiesterase 4B or 4D; SRR: serine racemase; TNIK: TNF
receptor-associated factor 2 and Nck interacting kinase.
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Advances in genetic techniques allow us to assess whether the single gene
studied is upregulated or downregulated in its expression, removed, and/
or controlled both temporally and regionally to resolve basic functioning of
psychopathological processes. Ample genetically modified mouse lines are
constantly being made and are available from different sources, for example
the Mutant Mouse Regional Resource Centers (MMRRC, www.mmrrc.org/),
the European Mouse Mutant Archive (EMMA, www.infrafrontier.eu), Taconic

Farms (www.taconic.com), RIKEN BioResource Center (www.brc.riken.jp/
rmpd/mouse_phenome_about.html) or The Jackson Laboratory (www.jax.org).
Moreover, hybrid animal models combining discrete genetic and environ-
mental factors (G × E) have been recently appreciated, given that schizophre-
nia is largely multi-factorial, involving complex interactions between genetic
and environmental factors. Several environmental stressors, such as psycho-
social stress, drug abuse, or maternal immune activation have been estab-
lished to provoke schizophrenia-related endophenotypes in rodents.102 The
main aim of the G × E approach is to detect synergistic interactions between
genes and the environment, which trigger a specific pathological endophe-
notype. It is difficult to design human studies to directly probe the interac-
tion between genetic and environmental risk factors, since such interaction
might be masked by genetic background and other uncontrolled environ-
mental factors. Therefore, animal G × E models are useful resources that
allow further support of the studied gene as disease gene that modulates
sensitivity to the environment. Such animal models offer a deeper under-
standing of the etiological mechanisms of mental disorders and generate
effective treatments/preventive medicine.
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7.4.1 DISC1 Mouse Models

The originally detected translocation t(1; 11) on DISC1 was proposed to have
a dominant-negative effect due to the normal expression of the truncated
DISC1 protein. Following this idea, several DISC1 transgenic mice have been
created with specific expression of the truncated DISC1 in the forebrain
regions,40,103,104 in the whole brain,105 and at pre- and post-natal develop-
mental periods.40,106 Recently, DISC1 knockout mice were generated with
transient downregulated expression of DISC1 at the pre-/perinatal stages by
injecting short-hairpin (sh)DISC1 into the prefrontal cortex.30 Perhaps the
most common behavioural endophenotype for these DISC1 mouse lines
with expression of the truncated form of DISC1 restricted to the frontal cor-
tex (Table 7.1) is hyperactivity103,104 and increased sensitivity to the dopamine
releaser.30,106 This suggests that cortical DISC1 directly regulates proper for-
mation and functioning of mesocortical dopaminergic system as was directly
demonstrated.30,106 Notably, transient human (h)DISC1 expression at prena-
tal, postnatal, and at both stages similarly reduced level of cortical dopamine
and density of interneurons106 compared to transient knockdown of DISC1
at embryonic day 14.30 As result of frontal deficiency due to the truncated
DISC1, most of these DISC1 mouse lines demonstrated deficient working
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154 Chapter 7
Table 7.1 Behavioural
phenotypes of DISC1 gene modified mice. a
Behavioural phenotypes
Name of mouse
line Sensorimotor Emotional Cognitive
DISC1 genetic models
CaMK-DN-DISC1 Olfaction is High immobility in PPI deficit; working
tg103 impaired; FST memory in Y-maze

hyperactivity and spatial mem-
ory in MWM are
normal
CaMK-DISC1-cc tg Normal Reduced sociability; Working memory
at PND 740 high immobility is deficient in
in FST DNMTP
DN-DISC1 tg104 Hyperactivity High aggression Spatial memory in
MWM is deficient
Pre- and postnatal Normal Reduced sociability; Normal
Tet-off DN-DISC1 increased aggres-
tg106 sion; increased
immobility in TST
DISC1 KD30 tran- Normal Normal PPI deficit; long-term
sient knockdown OR is impaired;
in the cortical short-term OR is
neurons in utero normal, working
memory in T-maze
is deficient
DISC1-12929,36 Normal Normal PPI, MWM and
object recognition
are normal; work-
ing memory and
fear memory are
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deficient
DISC1-Q31L38,39 Normal High immobility Spatial memory in
in FST; reduced MWM is normal;
sociability; social PPI and LI are
anhedonia deficient; mild
deficit in working
memory in T-maze
DISC1-L100P35,38 Hyperactivity Normal PPI and LI deficits;
working memory in
t-maze is deficient;
spatial memory
in MWM and fear
memory are normal
DISC1 × environmental models
DN-DISC1 tg × poly- Hyperactivity High anxiety, No effect
I:C at E9110 increased
immobility in
FST, reduced
sociability
DN-DISC1 tg × poly- No effect Reduced sociability Working memory,
I:C at PND 2–6111 OR and fear mem-
ory are deficient;
increased sensi-
tivity to psycho-
mimetic; no effect
on PPI
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DISC1-L100P+/− × Reduced rearing Reduced sociability Spatial OR deficit;

polyI:C112 impaired LI and
PPI
DN-DISC1-tg-PrP × Hyperactivity High immobility in PPI deficit
social isolation58 FST
DN-DISC1 tg × Hyperactivity; No effect PPI deficit in
Pb2+ 114 reduced rear- females; no effect

ing in males; on working mem-
increased rear- ory in Y-maze, fear
ing in females memory, and star-
tle response
a
aMK-DISC1-cc tg: transgenic mice expressing C-terminal portion of the human DISC1
C
(hDISC1) under control of the α-calmodulin kinase II promoter; CaMK-DN-DISC1 tg: trans-
genic mice expressing dominant-negative C-terminal truncated hDISC1 under control of the
α-calmodulin kinase II promoter; DISC1-129: 129S6/SvEv inbred mouse strain carries a termi-
nation codon at exon 7 of DISC1 gene, which abolishes production of the full-length DISC1
protein; DISC1 KD: DISC1 knockdown; DISC1-L100P: point mutation in the second exon of
DISC1 leading to the substitution of leucine on proline at 100 amino acid of DISC1 protein;
DISC1-Q31L: point mutation in the second exon of DISC1 leading to the substitution of glycine
on leucine at 31 amino acid of DISC1 protein; DN-DISC1-tg-PrP: transgenic mice with putative
dominant-negative DISC1 under expression control of the prion protein promoter; DN-DISC1
tg: transgenic mice with inducible expression of dominant-negative C-terminal truncated
hDISC1limited to forebrain regions, including cerebral cortex, hippocampus and striatum,
using the Tet-off system under the regulation of the CAMKII promoter; DNMTP: delayed non-
match to position; FST: forced swim test; LI: latent inhibition; OR: object recognition; MWM:
Morris water maze; PND: postnatal day; PPI: pre-pulse inhibition; TST: tail suspension test.
Reprinted from T. V. Lipina and J. C. Roder, Neurosci. Biobehav. Rev., 2014, 45, 271–294,
copyright (2014), with permission from Elsevier.
memory30,40 without influence on spatial or fear learning and memory (Table

7.1). In addition to these frontal deficits, pre-pulse inhibition (PPI) deficiency
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as a measure of sensory-motor gating, an endophenotype of schizophrenia,

was also evident.30,103 The success of animal models having conditional
expression of hDISC1 in the cortex suggests the generation of new condi-
tional DISC1 mouse lines with its altered expression in other neural circuit-
ries to reveal new brain functions of DISC1.
A DISC1 point mutant was originally identified in a reverse screening of the
entire mouse N-ethyl-N-nitrosourea (ENU) genome archive (n = 10 000 mouse
samples).38 The point mutation has been identified: L100P (substitution of
leucine on proline at 100 aa), causing a schizophrenia related behavioural
repertoire (deficit in PPI and latent inhibition; and deficient working mem-
ory and hyperactivity) with increased sensitivity to amphetamine in DISC1-
L100P mutant mice.38,55 A subsequent independent study by Walsh et al.
(2012) confirmed hyperactivity in DISC1-L100P mutants with impairments
in initial exploration and habituation to a novel environment.107 Another
study did not reproduce most of behavioural findings for the DISC1 point
mutant line,108 due to a difference either in the genetic background used in
this study (C57BL/6JJcl strain vs. C57BL/6J used in the original study38 and by
Walsh et al.107) or in experimental conditions.
Notably, in spite of the large proportion of DISC1 interacting proteins that
are encoded by essential genes, once expression of DISC1 was abolished109
or downregulated29,36 in models of germline genomic lesions with lost bind-
ing abilities of most DISC1 interactors, such mutant mice showed modest
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156 Chapter 7
behavioural phenotypes and no severe abnormalities in neurogenesis, neu-
ronal migration, or cortical synaptogenesis; however they did express altered
synaptic plasticity, suggesting that DISC1 is a fine-tuning protein for syn-
aptic plasticity and DISC1 pathways are probably regulated by redundant
mechanisms.
Finally, a variety of DISC1 × environment mouse models have been made
with combinations of maternal immune activation and DISC1,110–112 DISC1 ×

social defeat stress,113 DISC1 × social isolation,58 and DISC1 × chronic expo-
sure to lead.114 To date, the DISC1 × E approach has been applied to mice with
hDISC1 overexpression restricted to the cortex (CaMK-DN-DISC1)110,111,114,115
or in the whole brain58 and to the DISC1-L100P mutant line.112,113
Epidemiological studies have reported that prenatal and early-life
postnatal infections are associated with neurodevelopmental disorders,
including schizophrenia.116 The effects of influenza virus,117 toxoplasma
parasite,118 or bacterial endotoxine lipopolysaccharide119 were used to
induce immune activation in experimental animals. Currently, an artificial
double-stranded RNA, polyriboinosinic-polyribocytidylic acid (polyI:C),
which mimics a strong innate immune response similar to viral infection,
is widely used to model schizophrenia-related conditions in rodents.120
The “time window” is the critical factor for introducing the immune acti-
vation as an environmental stressor and needs to be carefully considered
for proper experimental design. Earlier inflammation at embryonic day 9
(E9) is expected to have a more severe effect on neurodevelopment because
it disrupts cell proliferation/differentiation, cell migration, and synapse
maturation, and can be associated mainly with positive symptoms of
schizophrenia.120 Immune activation given in late pregnancy (E17) caused
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abnormalities related to the negative symptoms of this disorder. There is

also evidence that postnatal immune activation also can increase risk of
psychotic illness.121 The polyI:C approach was applied to CaMK-DN-DISC1
mice110 and DISC1-L100P point mutants at E9 and it was also injected into
CaMK-DN-DISC1 pups in the neonatal period (postnatal days 2–6).111,115
Interestingly, immune activation at E9 and in the neonatal period caused
different effects on the cognition of CaMK-DN-DISC1 mice. It had no effect
on spatial learning and memory, sensorimotor gating and working mem-
ory given at E9.11 In contrast, given during the neonatal period polyI:C
provoked a significant reduction of cortical interneurons in parallel with
deficient working memory and object recognition in CaMK-DN-DISC1
mice111 that was corrected by clozapine.115 Hence, these two studies sup-
port timing as an essential factor in the induction of behavioural abnor-
malities in animal models. Analysis of polyI:C effects on the behaviour
of DISC1-L100P heterozygous (DISC1-L100P+/−) mutants revealed specific
interactions between DISC1-L100P+/− and maternal immune activation,
which exacerbated schizophrenia-related behavioural endophenotypes.112
Moreover, we found a causative role of interleukin (IL)-6 in such prena-
tal polyI:C-induced effects, since co-administration of an antibody against
IL-6 corrected deficient PPI and latent inhibition deficits.112
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Beyond immune activation as a pathogenic environmental factor, social
stress is another powerful trigger of psychiatric disorders in humans. So, risk
of schizophrenia is increased in urban environments122 and among immi-
grants.123 Childhood maltreatment or stressful events also decrease mental
health,124 most likely due to alterations in stress response systems, induced
by epigenetic mechanisms.125 Two recent studies combined social stress-
ors with hDISC1 and DISC1-L100P+/− point mutants to probe interactions

between these genetic and environmental factors. Mice with expression of
hDISC1 widely in the brain (DN-DISC1-Tg-PrP) were socially isolated for 3
weeks from 5 to 8 weeks of age58 and chronic social defeat was applied to
DISC1 mutants at adulthood for 2 weeks.113 The first study demonstrated
interaction between hDISC1 and early-life social isolation with stable hypo-
function of the dopaminergic mesocortical system, accompanied by DNA
methylation of the promoter of tyrosine hydroxylase gene in the ventral
tegmental area, where neurons from the mesocortical, but not from the
mesolimbic, area are projected.58 Moreover, behavioural changes and molec-
ular–cellular alterations were corrected by an antagonist of glucocorticoid
receptor RU38486.58 The second study did not detect any exacerbation of
schizophrenia in DISC1 heterozygous point mutants, probably because the
chronic social stress was applied to adult animals when the stress response
systems are already formed (the wrong “time window”), and also because the
social defeat in itself induces strong effects on control animals (a “ceiling”
effect), making it difficult to detect G × E effects.
Lastly, several epidemiological studies reported an association between
prenatal exposure to Pb2+ and the development of schizophrenia in adult-
hood.126 Pb2+ acts as potential blocker of the NMDA receptor complex,127
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and indeed, could be an environmental trigger for schizophrenia once com-

bined with genes related to the brain glutamate system, including DISC1.54
CaMKII-DN-DISC1 mice were raised and maintained on a Pb2+-enriched diet
that caused gender-dependent effect on behaviour: it triggered hyperactivity
in males and PPI deficits with increased sensitivity to MK-801 in females.114
Altogether, these DISC1 × E animal models open new directions in the psy-
chiatric field to further advance our comprehension of the etiology of mental
disorders.
7.4.2 Mouse Models with Modified DISC1 Interactors

There are approximately 30 different genetic mouse lines with impaired
DISC1-interacting proteins. Here we present mouse models with expression
of schizophrenia-related behavioural phenotypes.
7.4.2.1 Neurodevelopment
First, it must be noted that attempts to completely knock out genes that are
essential to neurodevelopment lead to embryonic lethality (e.g. KIF5–KO,
NCoR-KO, ErbB4-KO, Grb2-KO, PCNT-KO, LIS1-KO, or NDEL1-KO). There are
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158 Chapter 7
no mouse models or behavioural data for eukaryotic translation initiation fac-
tor (EIF)3, Girdin, microtubule-associated protein (MAP)1A, PCM1, Mitofilin,
TNIK, DISC1-binding zinc finger protein (DBZ), MIPT3/TNF receptor-associ-
ated factor 3 interacting protein 1 (TRAF3IP1) and Dixdc1 among validated
DISC1 interacting proteins. Moreover, analysis of the available behavioural
data from genetic mouse models with affected DISC1 interactors involved
in neurodevelopment found severe impairments of motor-sensory functions

(Table 7.2). Hence, only delicate manipulations with such genes could be
applied to reveal their potential impact on the behavioural level, for instance,
the introduction of a single point mutation or temporary alteration of gene
expression in particular brain region(s) and at certain stages of development.
Heterozygous LIS1 mice, although viable and fertile, but have various
developmental impairments, including low body weight, seizures or motor
co-ordination. LIS1 deficiency also caused deficient neuronal migration and
on the behavioural level caused deficient spatial learning and memory in
the Morris water maze128 (Table 7.2), resembling symptoms of human type 1
lissencephaly, supporting the role of LIS1 in developmental delay. Although
there is no strong evidence for the association of LIS1 with schizophrenia
in human studies,129 PCM1, its associated protein, showed strong linkage
to schizophrenia,7 so it would be useful to assess schizophrenia-related
behavioural phenotypes in LIS1-deficient mice.
7.4.2.2 Cargo Kinesin Intracellular Transport

DISC1 downregulation inhibits axonal transport of various cargoes, includ-
ing mitochondria and synaptic vesicles, providing evidence for microtu-
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bule-dependent intracellular transport. Generation of neuronal-specific

kinesin heavy chain, KIF5A-deficient mice revealed motor co-ordination
abnormalities, tremor, and seizures.130 Although genetic study did not reveal
a link between KI5A, as a DISC1 interactor, with schizophrenia,131 a signifi-
cant association was detected for the KIF2 gene.132 It would be of interest to
further characterize the behaviour of KIF5Atm1Gsn mice related to cognitive
and emotional endophenotypes to understand its role in the function of the
central nervous system.
FEZ1 also promotes synaptic vesicle transport in association with kinesin
1, and its polymorphism has recently been associated with schizophrenia.133
Cellular, biochemical and behavioural phenotypes of FEZ1-deficient mice sup-
port its role in psychopathological mechanisms of schizophrenia (Table 7.2).
For the first time it was shown that FEZ1 is mainly located in GABA-con-
taining interneurons,134 an important neuronal substrate of schizophrenia.
A lack of FEZ1 induced hyperactivity in mice, and increased high sensitivity
to the psychomimetics MK-801 and methamphetamine. These alterations
were associated with high levels of dopaminergic transmission in the nucleus
accumbens.134 It would be valuable to further characterize schizophrenia-re-
lated behaviour in FEZ1-knockout mice and the ability of antipsychotics to
correct their impaired behaviour.
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Table 7.2 Schizophrenia-related
behavioural phenotypes of mouse genetic lines
with modified DISC1 interactors.a
Name of mouse
Genetic mouse models with impaired DISC1 interactors involved in neural
development
Transgenic LIS1 Low body weight; n/a Spatial learning/
mice128 impaired motor memory deficit
co-ordination; in MWM
seizures
Genetic mouse models with impaired DISC1 interactors involved in intracellular
transport
Knockout FEZ1 Hyperactivity; high sen- Reduced immo- n/a
mice134 sitivity to amphet- bility in FST
amine and MK-801
Heterozygous Normal Modest increase Working memory
YWHAE mice of anxiety in deficit in eight-
(14-3-3ε) 131 EPM arm maze
Knockout 14-3-3ζ Hyperactivity; high Low anxiety PPI and NOR
mice135 exploration deficits; spatial
learning/mem-
ory deficit in
cross-maze
Genetic mouse models with impaired DISC1 interactors involved in
neurosignalling
Knockout PDE4B Hypoactivity and Increased anxi- PPI deficit; normal
mice137,138 low exploration; ety; reduced FC and MWM
high sensitivity to immobility
amphetamine in FST
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Transgenic GSK-3β Hypophagia; hyperac- Reduced immo- n/a

mice158 tivity; high acoustic bility in FST
startle response
Transgenic GSK-3β Hyperactivity; high Reduced immo- Deficits in spatial
mice with specific startle response bility in FST learning and
overexpression memory in
of GSK-3β in the MWM
hippocampus and
frontal cortex143
Heterozygous GSK-3β Normal High anxiety; Spatial learning/
mice142,159 reduced memory deficit
immobil- after long train-
ity in FST; ing in MWM; FC
reduced is deficient after
aggression reactivation
Mutant mice carrying Normal Reduced PPI and SOR
point mutation at sociability deficits
the start of exon
9 of serine race-
mase gene, a T
to A transversion
resulting in non-
sense mutation -
Tyr269 converted
to a stop codon146
(continued)
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160 Chapter 7

Name of mouse
Mice with targeted Hyperactivity High anxiety Spatial learning/
deletion of exon 1 memory deficit
of serine racemase in MWM

gene147
Mice with loss-of- Reduced habituation; Low sociability Deficient work-
function mutation reduced ambulation ing memory;
in Dtnbp1 (dys- facilitated CFC;
trobrevin binding deficient NOR;
protein 1) gene deficient spatial
on DBA/2J genetic learning/mem-
background148−150 ory in MWM
and Barnes
maze
Mice with loss-of- Hyperactivity; reduced Low anxiety PPI deficit; spatial
function mutation habituation learning/mem-
in Dtnbp1 (dys- ory deficits in
trobrevin binding MWM
protein 1) gene on
C57BL/6J genetic
background160
Genetic mouse models with impaired DISC1 interactors involved in synapse
functioning
KALRN-KO151 Hyperactivity High anxiety; PPI deficit; defi-
Knockout KALRN reduced cient spatial
(kalirin, RhoGEF sociability memory in
kinase)mice MWM; deficient
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working mem-
ory in Y-maze
and MWM; defi-
cient CFC
a
FC: contextual fear conditioning; EPM: elevated plus maze; FC: fear conditioning; FST:
C
forced swim test; MWM: Morris water maze; NOR: novel object recognition; PPI: pre-pulse
inhibition; SOR: spatial object recognition. No behavioural data/no mouse models are
available for NDE1, NDEL1, PCNT, EIF3, Girdin, MAP1A, PCM1, Mitofilin, TNIK, NCOR,
DBZ, MIPT3/TRAF3IP1, PCM1, DIXDC1, CAMDI, CHM1, CHCCHD6, ATF4, Grb2, and LIS1.
Reprinted from T. V. Lipina and J. C. Roder, Neurosci. Biobehav. Rev., 2014, 45, 271–294,
copyright (2014), with permission from Elsevier.
14-3-3ε (YWHAE) is involved in protein trafficking and other cellular

processes. A significant association was identified in a human popula-
tion between YWHAE polymorphism and schizophrenia.131 Heterozygous
YWHAE mice, in which 14-3-3ε levels are reduced by 50%, have a mild
increase in anxiety and deficient working memory131 (Table 7.2). Mice with
full knockout of 14-3-3ζ showed hyperactivity, high exploration with reduced
anxiety, deficient sensorimotor gating, and impaired episodic and spatial
memory, resembling schizophrenia-related behavioural endophenotypes135
(Table 7.2).
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7.4.2.3 Neurosignalling
Most of the drugs currently used in psychiatry elicit action on available neu-
rotransmitters in the synaptic cleft, affecting their synthesis or degradation or
release from vesicles to activate or inhibit receptors; hence, individual molec-
ular players within the same signalling pathways are suitable candidates for
drug development. DISC1 is implicated in divergent signalling pathways as

discussed earlier, including PDE4/cAMP,34,35 the dopamine signalling path-
way,3,55 GSK-3β/β-catenin,10,46 Akt/mTOR,31 and the glutamatergic system.54
All isoforms of PDE4 (A–D) interact with DISC1,35 interacting with specific
sites/complexes, regulating cAMP response in distinct compartments of cells,
and therefore affecting diverse aspects of neuronal functioning. Currently,
efforts are mainly focused on characterizing the behaviour of PDE4B- and
PDE4D-knockout mice (Table 7.2), and a PDE4C-knockout line was recently
created.136 PDE4C-knockout mice showed decreased sensitivity to odours, but
the full behavioural repertoire remains to be characterized in the near future.
PDE4B-knockout mice expressed several psychiatry-related behavioural
phenotypes, including PPI deficit and escalated response to amphetamine,
and increased anxiety but reduced immobility in the forced swim test137,138
(Table 7.2). These findings support the pharmacological effects of a PDE4
inhibitor, rolipram, which elicits antipsychotic effects.38 Surprisingly, specific
schizophrenia-related behavioural phenotypes have not yet been studied in
PDE4B-knockout mice, except the PPI deficit (Table 7.2), despite the reported
genetic associations with this mental disorder.4,7
GSK-3 has been implicated in neurodevelopment,31 neurotransmitter
function,44 neuroinflammation,139 and synaptic plasticity.140 GSK-3β has
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been detected as a novel DISC1 interactor,10,46 and accumulating evidence

indicates deregulation of GSK-3 in the pathology of schizophrenia.141 GSK-
3β is implicated in memory reconsolidation processes,142 supporting its role
in cognitive phenotypes. Indeed, conditional transgenic GSK-3β mice with
overexpression of GSK-3 in hippocampus and cortical neurons showed defi-
cient spatial learning and memory in the Morris water maze.143 Lack of GSK-3
impaired memory formation in GSK-3+/− mice.142 GSK-3 heterozygous mice
learnt normally the location of the escape platform in the water-maze during
the first 3 days of training, but showed spatial learning and memory deficits
after longer training (9 days), suggesting that GSK-3 is required for forma-
tion of long-term memory. Contextual fear memory was normally formed
in GSK-3+/− mice, but became deficient once fear memory was reactivated,
suggesting a role of GSK-3 in memory reconsolidation.142 Lack of the GSK-3α
isoform also impaired fear memory in the mice’s response to fearful context
and tone.144 There is still no report about cognitive phenotypes specifically
related to schizophrenia in GSK-3 mouse lines to directly address GSK-3 in
the pathogenesis of schizophrenia.
A recent study revealed that DISC1 binds and stabilizes SRR, the enzyme
that converts l-serine into its active isoform – d-serine – which activates the
NMDA receptor complex.54 The beneficial effects of d-serine have been shown
in clinical and animal studies, supporting hypofunctionality of the NMDA
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162 Chapter 7
145
receptor in schizophrenia. SRR-mutant mice with loss of enzymatic activ-
ity expressed schizophrenia-related behaviour, including PPI deficits, hyper-
activity, reduced sociability, and deficient spatial memory,146,147 (Table 7.2),
supporting the role of d-serine in schizophrenia. These behavioural impair-
ments in SRRrgsc1872 mutants were further exacerbated by MK-801 and cor-
rected by d-serine and clozapine.146 Next, efforts should be dedicated to the
deeper characterization in these mouse lines of schizophrenia-related cogni-

tive capacities such as attention, executive function, and working memory.
Dysbindin is a dystrobrevin-binding protein, which is a necessary compo-
nent of biogenesis in lysosome-related organelle complex 1. It plays a role in
intracellular and synaptic vesicle trafficking and in neurotransmitter release,
specifically contributing to the regulation of dopamine signalling. A sponta-
neous mutation was found in the dysbindin mouse gene and two mouse lines
were generated by backcrossing mutant mice either to C57BL/6J or DBA/2J
inbred mice148–150 (Table 7.2). Overall, Dtnbp1 mice on both genetic back-
grounds resembled some symptoms of schizophrenia, such as deficient work-
ing memory, altered responses to amphetamine, social avoidance, PPI deficit,
hyperactivity, and impaired spatial learning and memory,148–150 supporting the
implication of dysbindin in psychopathological mechanisms of schizophrenia.
7.4.2.4 Synaptic Plasticity
Synaptic spines are dynamic structures that regulate neuronal activity. Short-
term inhibition of DISC1 with inhibitory RNA (RNAi) increased the forma-
tion of spine and GluR1-expressing synapses50 via the DISC1 interactor,
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kalirin-7. DISC1 regulates access of kalirin-7 to Rac1 and controls the dura-
tion and intensity of Rac1 activation by NMDA receptors.50 Kalirin-knockout
mice express schizophrenia-related behaviour, including hyperactivity, PPI
deficit, impaired working and spatial memories, anxiety, and social avoid-
ance.151 Their deficient working memory in the Y-maze was not corrected
by clozapine. Kalirin-7 is an essential protein in the formation of functional
synapses, since its deficiency impaired spine formation,152 acquisition of a
passive avoidance task, and fear memory.153 Moreover, kalirin-7 deficient
mice showed diminished place preference in response to cocaine, the drug
of abuse, than wild-type littermates153 (Table 7.2), suggesting a potential role
of kalirin-7 in neurobiological mechanisms related to addiction.
Overall, some proportion of genetically modified mice without gross abnor-
malities of sensorimotor function expressed schizophrenia-related behavioural
phenotypes, supporting the DISC1 interactome in schizophrenia (Figure 7.2).
7.5 Future Directions

New ground-breaking technologies in genetics, neuroimaging, optogenetics,
and behaviour will greatly advance our comprehension of the pathogenesis
of schizophrenia and its prevention and treatment. Combination of such new
approaches in animal modelling as the generation of conditional knockout
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mouse lines, with optogenetics, will decipher the implication of specific neu-
ral circuitry into the pathogenesis of certain behavioural endophenotypes.
The use of sophisticated genetic approaches in simpler organisms, such as
flies or worms will allow us to narrow down genetic pathways and neural
circuitries implicated in schizophrenia-related behaviour.59 The generation
of genetic rat lines carrying mutated a DISC1 gene or genes from the DISC1
interactome is desirable, since it will offer the opportunity to assess more

complex cognitive performance. Recent findings on nonhuman primates
exposed to maternal immune activation extended the data accumulated
from rodent models to more human-like behaviours.154
Although the knockout of a gene in mice is a reliable approach to assess the
functions of the gene in vivo, as a model of psychiatric disorders it is missing eti-
ological validity. Introducing a single point mutation or a reduced expression
of the gene of at least 50% in heterozygous animals would closely mimic the
variation in risk polymorphisms in human populations and should be further
investigated. Manipulation of gene expression offers unique opportunities for
behavioural studies. The RNAi method allows for downregulated expression
of the studied gene in the desired brain area(s) or/and developmental stage(s)
to dissect the effects of such manipulations in mouse behaviour. However,
the inhibition of RNA may induce an “off-target” effect, triggering a type I
interferon response,155 and can be partly addressed by designing appropriate
controls. It is a high priority to generate conditional knockout mice of DISC1
or its interactors, which could allow us to dissect brain area-specific functions
of the studied gene. Protein–protein interactions are at the core of the DISC1
interactome and it is a high priority to assess the behavioural effects of syn-
thesized peptides specifically uncoupling two studied proteins. So, it would
10:30:36.
be of interest to probe behavioural effects of synthesized peptides interfering

with DISC1 × GSK-310 or DISC1 × TNIK51 interactions, recently reported.
Environmental factors have an important role in shaping brain func-
tions and are modulated by genetic and epigenetic factors.5 Several animal
studies have demonstrated a DISC1-dependent effect of prenatal immune
activation on the expression of schizophrenia-related behaviour in adult-
hood.110–112 Social isolation given for 3 weeks in an adolescent DISC1 trans-
genic mouse model (dominant-negative DISC1 under expression control of
the prion protein promoter; DISC1-DN-Tg-PrP) impaired mesocortical pro-
jection of dopaminergic neurons and caused epigenetic modifications of
the tyrosine hydroxylase gene.58 Therefore, the genetically modified mouse
models described here present valuable resources as “genetic environmen-
tal sensors”, describing genetic reactivity to environmental pathogens. This
could be incorporated into the “two-hit” hypothesis of psychiatric disorders,
suggesting that critical environments can trigger the occurrence of psychiat-
ric disorders in individuals having a genetic predisposition. Finally, risk for
schizophrenia is determined by a network of gene–gene interactions, and
one approach in the identification of whether variation within the putative
DISC1 protein pathway influences risk for schizophrenia is to look for evi-
dence of statistical interaction impacting disease risk. Indeed, DISC1, CIT,
and NDEL1 act in epistasis to influence risk of schizophrenia in clinical
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164 Chapter 7
samples assessed by machine learning algorithms. Moreover, 75% of these
significant gene–gene interactions have been biologically validated: carriers
of the combinations of schizophrenia risk-associated genotypes performed
less efficiently, similar to schizophrenia patients, during testing for working
memory.97 Hence, generation of mouse genetic models with a combination
of gene × gene interactions is an important step towards our understand-
ing of polygenic brain disorders. Recently, a new method was developed to

generate mice in one step with mutations in multiple genes by the CRISPR
(clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-as-
sociated) proteins system,156 which will greatly accelerate in vivo studies of
functionally redundant genes and of epistatic gene interactions.
In summary, systematic integration of behavioural phenotypes in mice
with affected DISC1 interactors, gene × environment models, genetic com-
binations, and effects of peptides targeting protein–protein interactions will
ultimately contribute to a better resolution of the neurobiological mecha-
nisms of schizophrenia and ultimately lead to the development of more
effective medicine to prevent and/or treat this mental disorder.
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www.Ebook777.com
CHAPTER 8
GSK3 Networks in
Schizophrenia
JIVAN KHLGHATYAN†a,b, GOHAR FAKHFOURI†a,b, AND
JEAN-MARTIN BEAULIEU*a,b
a
Department of Psychiatry and Neuroscience, Faculty of Medicine, Laval
University, 1050, Avenue de la Médecine, Québec City, Québec, Canada;
b
Institut Universitaire en Santé Mentale de Québec, 2601, Chemin de la
Canardière, Québec City, Québec, Canada
*E-mail: martin.beaulieu@crulrg.ulaval.ca
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8.1 Introduction
Schizophrenia is a debilitating psychiatric disorder affecting nearly 1%
of the general population. Manifestations emerge in late adolescence or
early adulthood and are categorized into positive symptoms, including
hallucinations, delusions and disorganized behavior; negative symptoms
comprising avolition, asociability and flattened affect, along with cognitive
impairments.1
Therapeutic agents currently in use for schizophrenia comprise first-
generation (typical) and second-generation (atypical) antipsychotics.2 The
former antagonize dopamine D2 receptors (D2R) while the latter possess
the propensity of acting on serotonin 5HT2 receptors in addition to D2Rs.3–5
†
These authors contributed equally to this work.

173
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174 Chapter 8
The signal transduction of D2 and 5HT2 receptors has glycogen synthase
kinase (GSK)3 as a central component. Intriguingly, all antipsychotics share
the common property of inhibiting brain GSK3 activity.6
GSK3 is a highly conserved serine threonine kinase, which exists as two
isoforms, GSK3α (51 kDa) and β (47 kDa), which are encoded by distinct gene
loci.7 In contrast to several other kinases, GSK3 is constitutively active and
can be negatively regulated through phosphorylation and complex forma-

tion. As the name suggests, it was first discovered as one of the kinases that
phosphorylate and inactivate glycogen synthase. Its association with a mul-
titude of neuronal events and signaling processes has emerged ever since,
and ample evidence now converges on a prominent role for this kinase in
neuropsychiatric disorders such as schizophrenia. The first evidence came
from the observations that many schizophrenia risk genes directly inter-
act with or are the members of cascades signaling through GSK3. The fact
that both antipsychotics and psychosis-inducing agents influence GSK3
activity either directly or indirectly position this regulatory enzyme at the
crossroads of the pathways that regulate behavioral outcomes and cogni-
tive functions.
Here, we first describe the major signal transduction cascades regulat-
ing GSK3 activity. Second, we report the findings from human and animal
studies on alteration or deregulation of the GSK3 signaling partners and net-
works in schizophrenia. We then elaborate on how GSK3 interaction with its
established and putative partners might culminate in behavioral phenotypes
and further speculate how these findings could be exploited to develop novel
diagnostics and therapeutic strategies for schizophrenia that target GSK3
and related molecules.
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8.1.1 GSK3-Regulating Pathways

8.1.1.1 Regulation of GSK3 by AKT
AKT, also designated protein kinase B, is a major serine threonine kinase
which is involved in myriad neuronal events like synaptic plasticity, mono-
amine transporter trafficking, and neuron morphology and fate, to name a
few. AKT has three isoforms (AKT1, 2 and 3), all of which are activated by
phosphatidylinositol 3-kinase (PI3K). Following stimulation of cell surface
receptors, PI3K produces phosphatidylinositol (3,4,5)-trisphosphate (PIP3).
The signaling lipid, in turn, binds the pleckstrin homology (PH) domain of
AKT, thereby recruiting AKT to the cell membrane, where it is activated. In
the case of AKT1 this occurs through sequential phosphorylation at residues
Thr308 and Ser473 by intracellular kinases 3-phosphoinositide-dependent
protein kinase 1 (PDK1) and rictor-mammalian target of rapamycin com-
plex (mTORC)2, respectively.2 Activated AKT phosphorylates other proteins,
including GSK3. The inhibitory phosphorylation of GSK3 occurs at N-termi-
nal serine residues, GSK3α at Ser21 and GSK3β at ser9 by AKT8 (Figure 8.1).
Notably, several other serine threonine kinases can phosphorylate GSK3 at
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GSK3 Networks in Schizophrenia 175

Figure 8.1 GSK3

signaling networks. Green proteins: products of genes associ-
ated with increased risk for schizophrenia; blue arrows activation; red
T-shaped arrows: inhibition.
these sites, including p70 ribosomal S6 kinase,9 p90 ribosomal S6 kinase

(p90rsk-1),10 AGC kinase, p38 mitogen-activated protein kinase (MAPK),11
protein kinase C and phospholipase Cγ1.12
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8.1.1.2 Regulation of GSK3 by Wnt

The Wnt machinery features another well-described GSK3-comprising sig-
naling system.13 The Wnt cascade participates in a host of neuronal pro-
cesses such as neural development, synaptogenesis, synapse specificity
and depolarization-dependent synaptic plasticity.14 The participation of
GSK3 in this pathway was initially uncovered in a series of independent
studies in 1995 whereby a dominant negative mutant of GSK3 was shown to
mimic the ability of Wnt to induce dorsal axis duplication in Xenopus.15–17
This notion was fueled by the observation that β-catenin, the key effector of
the canonical Wnt signaling, is a GSK3 substrate and that its phosphoryla-
tion by GSK3 is crucial to its destabilization and subsequent degradation.18
GSK3-mediated β-catenin phosphorylation destabilization is executed in
a so-called β-catenin destruction complex composed of AXIN1/2, adeno-
matous polyposis coli (APC), WTX, Casein kinase I-alpha (CKIα) and GSK3.
The complex is assembled via interaction of the scaffolding protein AXIN
with all the other components and stabilized by GSK3- and CKIα-mediated
AXIN phosphorylation. In the absence of Wnts, a family of glycopro-
teins, cytoplasmic β-catenin is constitutively captured to the complex via
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176 Chapter 8
interaction with AXIN and APC and undergoes sequential phosphorylation
by CKIα and GSK3. Hyperphosphorylated β-catenin is then subjected to
ubiquitylation and proteosomal degradation.13
During activation of the canonical Wnt pathway, ligands bind to the
seven transmembrane-domain protein Frizzled (Fzl) and its co-receptors
the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6), lead-
ing to the recruitment of Dishevelled (Dvl) to LRP5/6.19 Following activa-

tion, Dvl recruits AXIN, which is associated with GSK3, thereby facilitating
GSK3 translocation to the membrane and its dissociation from β-catenin.
Stabilized β-catenin accumulates in the cytoplasm and translocates to the
nucleus where it forms a complex with co-transcription factors T-cell fac-
tor/lymphoid enhancer factor (TCF/LEF) and initiates the transcription of
Wnt-target genes13 (Figure 8.1). Unlike PI3K signaling, the phosphorylation
status, and therefore, catalytic activity of GSK3 is not affected by Wnt path-
way activation.20
8.1.1.3 Regulation of GSK3 by DISC1

Disrupted-in-schizophrenia 1 (DISC1) is a scaffolding protein that influences
manifold aspects of brain function, including neurodevelopment, neurosig-
naling, and synaptic functioning through modulating myriad signaling path-
ways and interaction with protein partners.21 Recent findings have indicated
that DISC1 binds and regulates GSK3β activity (Figure 8.1).22 Using specific
short hairpin RNAs (shRNAs) that silenced endogenous DISC1 expression in
adult hippocampal progenitors (AHPs) or in E13 mouse brains, Mao et al.
demonstrated that DISC1 positively regulates adult and embryonic neural
10:30:40.
progenitor proliferation through modulation of the canonical Wnt path-

way.22 DISC1 loss of function reduced proliferative neural progenitors in the
ventricular/subventricular zone of embryonic mouse brain and abrogated
Wnt3a-induced AHP proliferation, diminished β-catenin levels and spe-
cifically curtailed TCF/LEF transcriptional activity. The defects in TCF/LEF
activity and proliferation were superseded by either DISC1 overexpression
or expression of a degradation-resistant β-catenin. Intriguingly, a decrease
in β-catenin amount was accompanied by increases in its phosphorylation at
Ser33/37 and Thr41 motifs, suggesting that GSK3β activity might be directly
or indirectly inhibited by DISC1.
More direct evidence for the inhibition of GSK3 by DISC1 came from the
finding that DISC1 knockout augmented GSK3β autophosphorylation at
Tyr216, which is required for its activity,23 while DISC1 overexpression had
the opposite effect. Of note, Ser9 phosphorylation was unaffected by DISC1
silencing, ruling out the involvement of the AKT pathway in DISC1-mediated
regulation of GSK3β. In contrast, DISC1 was found to inhibit GSK3β activity
through a direct physical interaction of its N-terminal region (aa 1–220).
Finally, phosphorylation of other known GSK3β substrates, namely neu-
rogenin 2 (Ngn2)24 and CCAAT/enhancer-binding protein (C/EBP)a,25 was
unaffected by DISC1 knockdown or overexpression. The differentiation
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and maturation of adult neural progenitors by DISC1, however, appears
to be exerted independently of GSK3.26 In adult mouse hippocampus,
DISC1 acts through binding to KIAA1212, an AKT interacting partner and
catalytic activity enhancer,27 thereby inhibiting AKT and one of its down-
stream effectors mTOR. A well-designed study proposed as a working
model that during mid-embryonic stages, when progenitor cell prolifer-
ation is prominent, unphosphorylated DISC1 binds GSK3β and regulates

cell proliferation. However, in later embryonic stages when neuronal
migration predominates, phosphorylated DISC1 dissociates from GSK3β
and switches its role to activating neuronal migration.28 These findings
suggest a developmental stage-dependent role of GSK3 and DISC1 during
adult neurogenesis.26 DISC1 has been shown to be a directly interacting
partner of DIX domain containing (DIXDC)1 and this complex can posi-
tively regulate the canonical Wnt signaling.29
8.1.1.4 Regulation of GSK3 by IP6K1

As an additional route of regulation, it has been reported that inositol
pyrophosphate (IP)7 (5-diphosphoinositol pentakisphosphate), generated
by a family of inositol hexakisphosphate kinases (IP6Ks), is a physiological
inhibitor of AKT signaling (Figure 8.1). In mouse embryonic fibroblasts,
IP7 was found to act at the enzyme’s PH domain to block AKT membrane
translocation as well as Thr308 phosphorylation and activation by PDK1.
In vivo, Ip6k1-knockout (KO) mice manifested a dramatic enhancement of
AKT activity and phospho-GSK3β in peripheral tissues including skeletal
10:30:40.
muscle, white adipose tissue, and liver.30 Beyond AKT-dependent modula-

tion of GSK3 activity by IP7, we demonstrated that IP6K1 physiologically
interacts with GSK3α/β in mouse cortical extracts.31 IP6K1 directly binds
the N-terminal domain of active GSK3 and stimulates its catalytic activ-
ity in vitro. Of note, IP6K1-induced potentiation of GSK3 catalytic activ-
ity is independent of IP6K1’s enzymatic activity and involves suppression
of GSK3 phosphorylation at the N-terminal by AKT. Furthermore, Ip6k1
ablation in mice leads to reduced GSK3 activity in different brain regions.
Behavioral phenotypes of Ip6k1-KO mice mimic those of several mouse
models of GSK3 deficiency as well as of wild-type animals receiving GSK3
inhibitors. Reduced exploratory locomotor activity of Ip6k1-KO mice in
a novel environment is similar to that of Gsk3a-KO mice,32 while their
impaired amphetamine-induced hyperlocomotion mirrors the response
elicited in Gsk3b haploinsufficient mice.33 Gsk3a-KO curtails sociability
while complete ablation of forebrain Gsk3b expression, conversely, results
in increased social interactions,34 suggesting that GSK3 isoforms may dif-
ferentially modulate social behavior. However, the decreased sociability
displayed by Ip6k1-KO mice is reminiscent of wild-type mice treated with
a GSK3 inhibitor, TDZD-8, thus indicating that global inhibition of GSK3
disrupts social interactions.31
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178 Chapter 8
8.1.1.5 Regulation of GSK3 by Dopamine and β-Arrestin-2

Several studies indicate the involvement of AKT-GSK3 pathway in DA signal-
ing, regulation of DA-related behaviors as well as in response to antipsychotics
and psychoactive drugs.6,35 The first evidence for this came from a pioneer-
ing study on DA transporter (DAT)-KO mice. These animals exhibit impaired
DA reuptake leading to an approximately fivefold elevation of extracellular

DA levels in the striatum.36–38 Among other consequences, this state of hyper-
dopaminergia results in a reduction of AKT1 phosphorylation/activity and
activation of GSK3α and GSK3β.33 The reverse effect was observed when DA
was depleted in the striatum following administration of an inhibitor of DA
synthesis.33,39,40 Administration of the DA receptor agonists amphetamine,
methamphetamine or apomorphine to wild-type mice also decreased AKT1
activity and resulted in increased GSK3α and GSK3β activity.33,39,40
Further studies involving the use of DA receptor antagonists and receptor
knockout mice revealed a central contribution of D2R in the regulation of
AKT and GSK3 by dopamine. Furthermore, the D3 DA receptor appears to
act as an enhancer of the action of D2R on this signaling modality.41 Impor-
tantly, data obtained from knockout animals suggest that the postsynaptic
D2RL splice variant of D2R is critically important for this form of regulation
of AKT and GSK3 by dopamine. Indeed, D2RL-KO mice display increased
basal AKT1 phosphorylation, despite the presence of the DA D2RS splice
variant in both pre- and post-synaptic neurons.41 That said, the effect of D2R
receptor agonists on AKT and GSK3 phosphorylation has not been tested in
D2RL-KO mice and the specific contribution of D2RL to this modality of DA
receptor signaling remains to be fully investigated.
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Regulation of AKT1 by D2R is not dependent on cyclic adenosine monophos-

phate (cAMP) and canonical D2R signaling pathway. It is instead mediated
by the scaffolding protein β-Arrestin-2 (βArr2).42 When DA receptor agonists
were administered to mice lacking βArr2 (βArr2-KO), inhibition of AKT was
no longer observed.42 Further characterization of how βArr2 regulates AKT in
response to D2R activation showed that stimulation of the DA receptor facili-
tates the formation of a protein complex comprised of βArr2, AKT1 and PP2A,
which causes the dephosphorylation/inactivation of AKT1 by PP2A (Figure
8.1).42 Consequently, diminished activity of AKT1 results in dephosphorylation
and activation of GSK3. Finally, investigations involving gain or loss of func-
tion in mice revealed that GSK3 contributes to the regulation of this modality
of signaling by facilitating the formation of the βArr2/PP2A/AKT1 complex.43,44
The βArr2-AKT1-GSK3 signaling pathway is also important for regulation of
behaviors by D2R. Indeed, βArr2-KO mice are less active than wild-type litter-
mates and have decreased responses to DA receptor agonists.42 In addition,
DAT-KO mice, which also lack βArr2, display a reduction of hyperactive phe-
notype.42 Furthermore, GSK3 inhibitors can reduce hyperactivity in DAT-KO
mice as well as in normal mice treated with amphetamine.33,45 Finally, mice
that are haploinsufficient for the Gsk3b gene or mice lacking GSK3β expres-
sion specifically in neurons that express D2R also show a decreased locomo-
tor response to amphetamine treatment.33,44
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8.1.1.6 Regulation of GSK3 by Serotonin

Beyond dopamine, serotonergic (5HT) neurotransmission has also been
shown to regulate GSK3 activity (Figure 8.1).46 Evidence for this came from
the finding that d-fenfluramine, an enhancer of serotonin tone, profoundly
alters the phosphorylated GSK3β level in various regions of mouse brain.47
Moreover, diverse classes of serotonergic medications, including tricyclic

antidepressants, selective serotonin reuptake inhibitors and monoamine
oxidase inhibitors inactivate GSK3β in vivo.47 The physiological relevance of
serotonin-mediated regulation of GSK3 was further supported by use of a
knockin mouse model carrying a mutant form of tryptophan hydroxylase 2
(Tph2), the rate-limiting enzyme in brain serotonin synthesis.48,49 In these
mice, a decreased level of serotonin synthesis coexisted with a depressive-
and anxiogenic-like phenotype. Importantly, genetic or pharmacological
inhibition of GSK3β largely corrected the observed behavioral abnormalities,
suggesting the implication of GSK3β in serotonin-mediated behavioral phe-
notypes.50 Nonetheless, the underlying mechanisms of serotonin-induced
GSK3β regulation remain only partially understood. Using receptor-selective
agonists and antagonists, it was found that 5HT1A receptors augment, while
5HT2 subtypes diminish phospho-GSK3β levels.47 However, the precise
mechanisms linking the regulation of GSK3 to the activity of these receptors
remain speculative.
8.1.1.7 GSK3 Regulation, a Final Remark

Both GSK3 isoforms can be regulated by several converging mechanisms
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involving protein:protein interactions and/or the modulation of protein

phosphorylation. Interestingly, the regulation of AKT and GSK3 by D2R pro-
vides a unique mechanism by which an extracellular messenger molecule,
in this case dopamine, can regulate the inactivation of AKT-mediated sig-
naling. Furthermore, in view of the central role that DA and serotonin have
been thought to play in the etiology and treatment of schizophrenia,51,52 this
pathway may also provide important clues into how pharmacological treat-
ments acting on DA receptors may compensate for genetic deficits that are
not obviously associated with the modulation of dopaminergic or serotoner-
gic neurotransmission.
8.2 GSK3 in Schizophrenia

8.2.1 Biological
Evidence for the AKT-GSK3 Pathway in
Schizophrenia
The first evidence of AKT involvement in the disease emerged from genetic
studies of schizophrenic patients. Several single nucleotide polymorphisms
(SNPs) of the AKT1 gene are associated with schizophrenia risk in patients
of Northern European origin.53 Later association of AKT1 gene variants with
schizophrenia risk was confirmed in multiple studies and populations,
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180 Chapter 8
54 55 56 57,58
including Iranian, Japanese, Chinese, European, and British59,60
samples However, a few studies have shown the absence of association of
AKT1 haplotypes with schizophrenia in Japanese,61,62 Taiwanese,63,64 Finn-
ish,65 and Korean66 samples. Discrepancies may arise first of all from the eth-
nic origins of subjects analyzed, as well as from the size of cohorts and the
type of analyses performed. For instance, in contrast to Emamian et al.,53
where family-based samples were used, Ohtsuki et al.61 performed haplotype

construction in population-based samples, which might make confirmation
of genetic association more difficult.
In line with genetic studies, decreased expression of AKT1 was observed
in protein extracts of lymphocyte-derived cell lines (LDCs) obtained from
schizophrenic patients compared to control subjects. Moreover, a similar
decrease in AKT1 expression was detected in the post-mortem cortex and hip-
pocampus of patients with schizophrenia. Interestingly, expression changes
were specific to AKT1, since no differences were found in the expression of
AKT2 and AKT3 isoforms.53 In contrast, some studies failed to document any
changes in expression levels of total and phosphorylated AKT1 (pAKT1) in
the frontal cortex of schizophrenic patients.62 This inconsistency may stem
from the quality of post-mortem samples. For example, an inverse correla-
tion was found between pH and AKT1 phosphorylation in post-mortem brain
samples.62 Phosphorylation of AKT1 in post-mortem brain samples depends
on the terminal medical state of the patient and storage conditions after
death.62 Moreover, different brain regions have been examined over a wide
range of studies, which may have contributed further to inconsistencies.
Efforts in recent years have brought more evidence in favor of AKT1 as a
risk factor for schizophrenia. Decreased AKT1 levels as well as pAKT1/AKT1
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ratio in post-mortem brains of schizophrenic patients were documented in

various independent studies.67,68 In some studies, although expression lev-
els of AKT1 were unaltered, decreased pAKT (S473) levels were present in
the dentate gyrus of schizophrenic patients compared to control individu-
als, indicating an overall decrease in AKT1 activity.69 Furthermore, a signifi-
cant association of five new genetic loci with schizophrenia was reported.70
Decreased pAKT1/AKT1 ratio and AKT1 activity were detected in all risk allele
carriers compared to control patients. This suggests that proteins encoded
by genes that are affected in schizophrenia may converge on a common path-
way having AKT1 as a shared component.71 Ultimately, when the complete
set of genes affected in schizophrenia was analyzed, AKT1 was found among
those genes, which were differentially expressed in schizophrenic patients.
Additionally, when metabolic and signaling pathways were reconstructed
based on the set of affected genes, AKT1 appeared to be involved in 20 out of
35 deregulated pathways.72
Being a known substrate of AKT1, GSK3 has also been the subject of stud-
ies undertaken to explore its potential involvement in the pathophysiology of
the disease. Alterations in the expression and activity of GSK3 in schizophre-
nia have been less clearly demonstrated. No differences were found in the
expression level of GSK3 in LDCs or in frontal cortex lysates of schizophrenic
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patients. However, the relative phosphorylation of GSK3β at Ser9 has been
reported to be significantly lower in both LDCs and frontal cortex of schizo-
phrenic patients, indicating an increased activity of GSK3β. It is worth
mentioning that while phosphorylation of GSK3β at Ser9 was changed, no
difference was observed in its phosphorylation at Tyr216, which is regulated
in an AKT1-independent fashion. These data indicate that changes in GSK3β
activity in schizophrenia may be related to changes in AKT153 However, sev-

eral studies have reported decreased protein levels and activity of GSK3 in
schizophrenic patients.73–76 In some cases, decreased immunoreactivity of
GSK3 was detected along with unchanged levels of its direct substrate β-
catenin, which is suggestive of increased GSK3 activity.77 Finally, several
GSK3b gene variants displayed a significant association with schizophre-
nia.78–80 A SNP located in the promoter region of GSK3b was found to affect
transcription factor binding, leading to an increased expression of GSK3β.81
Nevertheless, while confirming the association of GSK3b polymorphisms
with schizophrenia82,83 some studies reported decreased GSK3 mRNA lev-
els82,83 that were paradoxically accompanied by a reduction in β-catenin that
is suggestive of enhanced GSK3 activity.
8.2.2 AKT-GSK3 and the Pathophysiology of Schizophrenia

Schizophrenia is associated with developmental, structural, and functional
abnormalities of the hippocampus. A decreased pAKT1/AKT1 ratio is well
correlated with the reduced hippocampal volume commonly present in
schizophrenic patients.84 Hippocampal abnormalities are also present in
Akt1 -deficient mice exhibiting alterations in synaptic plasticity along with
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impaired neurogenesis resulting from decreased proliferation and survival

of neural progenitors.85 Grey matter deficits in several brain regions have
been identified using structural magnetic resonance imaging (sMRI), and
these correlate with SNPs of 16 schizophrenia risk genes, including AKT1,
PI3K and DRD2.86 Moreover, patients with a genetic variant of AKT1 related
with schizophrenia have poorer memory performance, particularly in atten-
tion/concentration compared to patients devoid of the variant. The same
individuals also display deficits in sustained attention and vigilance of atten-
tion along with several brain morphological vulnerabilities associated with
the AKT1 variant.87 Similar behavior was observed in Akt1-deficient mice
expressing decreased fear-conditioned learning, modest anxiety phenotype,
and deficiencies in spatial memory along with a significantly attenuated pre-
pulse inhibition (PPI) response.85 Ultimately, epistatic interactions of AKT1
with other risk genes were found to contribute to schizophrenia risk as well
as to cognition and brain volume measures of affected individuals.88,89
GSK3b SNPs correlate with reduced thickness of the dorsolateral pre-
frontal cortex (DLPFC) in patients. Additionally, GSK3b polymorphisms are
associated with attenuated activity of DLPFC and decreased cognitive perfor-
mance and working memory as measured by functional (f)MRI.82 Interest-
ingly, pharmacological and genetic inhibition of GSK3 has been associated
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182 Chapter 8
with cognitive improvements in mice and human in a wide range of neu-
rological disorders such as bipolar disorder, Alzheimer’s disease, and frag-
ile X syndrome. A possible underlying mechanism includes the pivotal role
of GSK3 in the modulation of synaptic plasticity, neurogenesis, and neuro-
protection.90 GSK3b polymorphisms also affect the volume of grey matter in
the temporal lobes of schizophrenic patients. This is the brain parenchymal
region with the most consistently documented morphometric abnormalities

in schizophrenia, and neuropathological processes in these regions develop
early at the onset of the illness.91 All together, data indicate the involvement
of the AKT-GSK3 pathway in behavioral manifestations of schizophrenia.
8.2.3 Wnt-GSK3 Pathway in Schizophrenia

Aberrations have also been reported in the GSK3 incorporating Wnt cas-
cade in schizophrenia. At the level of its effector β-catenin, post-mortem
studies have revealed a reduced protein content in the CA3 and CA4 hippo-
campal subregions of schizophrenic subjects,92 which could potentially be
associated to deregulation of β-catenin degradation resulting from aberrant
GSK3 activity. Wnt1 expression has also been found to increase in the same
regions of schizophrenic brains.93 It is therefore plausible that such upregu-
lation occurs as part of an endogenous compensatory mechanism to blunt
the detrimental effects of increased GSK3-mediated β-catenin instability.
Association studies have identified FZD3, APC, and TCF4 as candidate genes
conferring susceptibility to schizophrenia,94–98 and transgenic mice replicate
certain schizophrenia-like behaviors. Partial knockout of APC leads to work-
ing memory impairment in adulthood and a decrease in locomotor activity,
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but normal PPI,99 while transgenic mice overexpressing TCF4 in the brain
demonstrate a disrupted PPI but normal locomotor activity.100 DIXDC1 is
a positive regulator of Wnt, which interacts with Dvl and AXIN, leading to
an increased TCF-dependent transcription.101,102 DIXDC1 knockout brings
about a complex behavioral phenotype in mice with certain elements of
schizophrenia-like behavior.103 Furthermore, mice lacking Dvl1 have defec-
tive social interaction and PPI, which are typical features of schizophrenia.104
Considering the negative regulation of GSK3 by Dvl1, the implication of aug-
mented GSK3 signaling in disrupted social behavior and sensorimotor gat-
ing when Dvl1 is ablated can be envisaged.
8.2.4 NRG1-GSK3 and BDNF-GSK3 in Schizophrenia

Neuregulin (NRG)1 belongs to a family of growth factors encoded by four
distinct genes (NRG1–4). The NRG1 gene gives rise to at least 31 isoforms in
humans, presumably due to multiple promoters and alternative splicing, with
a common epidermal growth factor-like domain. Upon stimulation by NRG1,
ERBB4 forms a homo- or heterodimer with other ERBB subtypes.105 As growth
factors, both NRG1 and brain-derived neurotrophic factor (BDNF) act on recep-
tors classified as receptor-type tyrosine kinases,106 which transmit intracellular
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signals mainly through the MAPK pathway, consisting of Ras-Raf-MAPK kinase
(Mek)-extracellular signal-regulated kinase (Erk) and the PI3K pathway com-
prising PI3K (with its adaptor subunit p85 and the catalytic subunit p110)-AKT-
GSK3 and other downstream molecules (Figure 8.1).107
NRG1-ERBB4 signaling is crucial in neurodevelopmental events such
as radial glia formation, neuronal migration, axon myelination and guid-
ance,as well as dendritic development.108 Multiple genetic studies and

meta-analyses propose NRG1 along with ERBB4 as prime schizophrenia
susceptibility candidates.109–111 Post-mortem studies have revealed ele-
vated levels of NRG1 and ERBB4 expression in brains from schizophrenic
subjects.112–114 Similar increases were found in neurons derived from
induced pluripotent stem cells of patients with schizophrenia.115 Risk
polymorphisms in ERBB4 are also linked with enhanced expression of the
ERBB4 CYT-1 isoform in lymphoblastoid B-cell lines from schizophrenic
patients.115 ERBB4 CYT-1 possesses a PI3K binding site and is capable of
activating the PI3K pathway.116 The same ERBB4 genotype is associated
with increased p110δ PIK3CD expression in schizophrenia.115 In spite of
increased expressions of ERBB4 CYT-1 and PIK3CD, NRG-induced produc-
tion of PIP3 is diminished in schizophrenia and is linked with ERBB4 risk
polymorphisms. Notably, chronic treatment of rats with haloperidol spe-
cifically reduces PIK3CD gene expression in the brain. In the same vein,
inhibition of p110δ (PIK3CD) with a small molecule (IC87114) antagonizes
amphetamine-induced hyperlocomotion in mice, augments brain AKT
Thr308 phosphorylation, and reverses PPI deficits in a rat neonatal ventral
hippocampal lesion model of schizophrenia.115 A decreased NRG1-induced
PI3K-mediated AKT phosphorylation at Ser473 is also present in both
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first-episode unmedicated and chronic medicated schizophrenia, but not

in other psychotic disorders.117,118 These findings are in line with disrupted
AKT signaling in schizophrenia53 and converge on increased GSK3 activity
as a key event in the cascade of events contributing to schizophrenia.
Of particular interest, NRG1 also affects the development and activity of the
dopaminergic system.105 Several studies demonstrate that systemic or intra-
cerebral administration of NRG1β enhances DA release in brain regions asso-
ciated with dopaminergic neurotransmission.119–121 How such modification
could influence behavior was the subject of an elegant study:122 peripheral
administration of NRG1 to neonatal mice culminated in midbrain ERBB4
activation, increased expression, phosphorylation, and enzyme activity of
tyrosine hydroxylase, leading ultimately to a hyperdopaminergic state in
the medial prefrontal cortex which continued to persist through adulthood.
Furthermore, adult mice displayed schizophrenia-associated behaviors,
namely impaired PPI, latent inhibition (LI), and social behaviors, along with
hypersensitivity to methamphetamine.122 Given the potential significance
of βArr-mediated D2R signaling in schizophrenia etiopathology, it is highly
conceivable that the predisposition of enhanced NRG1-ERBB4 to a schizo-
phrenic state is channeled through deregulation of dopaminergic signaling
and AKT-GSK3 as its integral signaling modality.
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184 Chapter 8
Regarding BDNF, studies propose a finely tuned interplay between BDNF
and GSK3β. Chronic blockade of GSK3β activity has been associated with
increased BDNF. It is inferred by the finding that long-term administration of
lithium, an established inhibitor of GSK3β, enhances BDNF expression both
in vitro123 and in vivo.124 A more direct piece of evidence comes from work
on dopaminergic human neuron-like cells, where protracted inhibition of
GSK3β either genetically or pharmacologically was found to augment BDNF

secretion.125 Phencyclidine (PCP) has long been associated with induction of
a psychotomimetic state reminiscent of schizophrenia, incorporating both
positive and negative symptoms.126 Perinatal exposure of mice to PCP evokes
a schizophrenia-like phenotype in later life, which has been associated with
apoptosis. BNDF-induced protection of corticostriatal cultures against PCP-
evoked apoptosis involved inhibition of GSK3β downstream of the PI3K-AKT
signaling cascade.127
8.2.5 DISC1-GSK3 in Schizophrenia

DISC1, a susceptibility gene for schizophrenia, was originally identi-
fied in a large Scottish pedigree. In all affected members of the family, a
balanced t(1; 11) translocation between exon 8 and 9 of the DISC1 gene
co-segregated with schizophrenia and related psychiatric disorders.128–130
Since then, independent genetic association and linkage studies, as well
as post-mortem studies in different ethnic groups have implicated the
DISC1 locus in schizophrenia.131–140 Although the functional consequence
of DISC1 haplotypes in psychiatric disorders is still far from clear, accu-
mulating information on the roles of DISC1 within the brain has provided
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some valuable mechanistic insights. DISC1 polymorphisms may lead to

diminished expression of its protein to half normal levels, i.e. haploin-
sufficiency.141 This notion is corroborated by the observation that mRNA
levels of DISC1 were decreased in lymphocytes from 57 bipolar pedigrees
and that a higher number of manic symptoms correlated with lower levels
of DISC1 expression.142 Moreover, chronic administration of the atypical
antipsychotics olanzapine and risperidone increases DISC1 mRNA levels
in mouse frontal cortex,143 possibly as part of their therapeutic actions.
Another hypothesis, supported by some experimental observations, is that
a mutation in a DISC1 allele gives rise to a dominant negative protein that
disrupts the function of the wild-type protein. For a detailed discussion of
DISC1 in schizophrenia, the reader is referred to elegant reviews published
in this book (see chapter 7) and elsewhere.21,144 Mice with a missense L100P
mutation display schizophrenia-like behavior, i.e. hyperactivity, drastically
impaired PPI, LI, and working memory. The behavioral phenotypes can be
superseded by typical or atypical antipsychotics. The anatomical abnor-
malities of the brain are reminiscent of those reported in schizophrenic
individuals,145 thus confirming DISC1 implication in schizophrenia and
establishing a reproducible animal model for schizophrenia.
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Regarding the putative role of GSK3, pharmacological inactivation of
GSK3β in DISC1-KO mice rescues impaired TCF/LEF-dependent neural pro-
genitor proliferation and abrogates the novelty-induced hyperlocomotion – a
proxy for positive symptoms of schizophrenia.23
Similar to antipsychotic therapy, genetic or pharmacological suppression of
GSK3 offsets the behavioral phenotypes of DISC1-L100P mutant mice.146–148
Moreover, Gsk3a knockdown reverses dendritic spine defects of the frontal cor-
tical neurons observed in these mice,149 substantiating the relevance of GSK3-in-
corporating pathways as a promising therapeutic target in schizophrenia.
8.3 GSK3 and Antipsychotics

Most antipsychotic drugs prescribed for the management of schizophre-
nia share a common ability to act as D2R antagonists. In addition, second-
generation antipsychotics also antagonize 5HT2A serotonin receptors. The
involvement of the AKT-GSK3 pathway in D2R and 5HT2 receptor signaling as
well as in the pathophysiology of schizophrenia positions it as an attractive tar-
get for the development of novel compounds. Indeed, most existing antipsychot-
ics have been shown to affect AKT-GSK3 signaling either directly or indirectly.
Administration of the first-generation antipsychotic haloperidol increases
AKT1 and GSK3β phosphorylation without affecting their expression levels in
mouse brain.53 In SH-SY5Y cells, clozapine increases Ser9 phosphorylation of
GSK3β along with accumulation of β-catenin and its migration to the nucleus.
Chronic and subchronic, but not acute, treatment with haloperidol, risperidone,
and clozapine also caused increases in β-catenin, GSK3β, and pGSK3β levels in
the striatum, prefrontal cortex, hippocampus, and ventral midbrain of rats.150,151
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Interestingly, the effect of clozapine on GSK3 in SY5Y cells is resistant to

the inhibition of the PI3K pathway. This suggested a possible involvement
of other pathways such as Wnt (see above) in GSK3β regulation in response
to this drug.152 However, the involvement of other PI3K-independent mecha-
nisms, such as the inactivation of AKT by PP2A and βArr2 or by 5HT receptors
have not been explored in vivo.
With regards to D2R- and βArr2-mediated mechanisms, studies using
bioluminescence resonance energy transfer to quantify D2R signaling pro-
cesses have shown that despite their different pharmacological properties,
various antipsychotics have in common the ability to antagonize dopamine-
mediated interaction of D2LR with βArr2. Haloperidol, clozapine, aripipra-
zole, chlorpromazine, quetiapine, olanzapine, risperidone, and ziprasidone
all potently antagonize the βArr2 recruitment to D2LR induced by quin-
pirole.153 This observation led to the synthesis of βArr2-biased compounds
that would selectively target βArr2-mediated D2R signaling. On this prem-
ise, the new aripiprazole derivatives UNC9975, UNC0006, and UNC9994 dis-
played antipsychotic-like activity in rodents.154 These three compounds have
the characteristic of binding to D2R without affecting cAMP signaling while
acting as partial agonist for βArr2 recruitment to D2R in the absence of a
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186 Chapter 8
full agonist. Although this can appear to be a contradiction, it is noteworthy
that aripiprazole behaves as a partial agonist for βArr2 recruitment in some
commercial assays when applied alone on cells,154 while acting as an antag-
onist of βArr2 recruitment when simultaneously applied with quinpirole.153
It is thus possible that the different UNC compounds may display different
pharmacological properties when applied alone in vitro or in the context of
an active DA tone in vivo. Further investigations of these compounds, includ-

ing a characterization of their pharmacological profiles in the presence of
full D2R agonists, may constitute an important first step toward the devel-
opment of a new class of antipsychotics targeting GSK3-mediated signaling.
8.4 How GSK3 Affects Behavior

Despite overwhelming data on the role of GSK3 in signaling pathways and
the pathophysiology of neurological disorders, little is known about the
mechanisms by which GSK3 activity contributes to the regulation of behav-
ior. There are more than 100 known substrates, which can be phosphorylated
and regulated in different fashions by GSK3.155 Here we discuss several GSK3
substrates that might be implicated in neurological functions and how their
dysregulation may contribute to pathological conditions (Figure 8.2).
8.4.1 Circadian Rhythms

Circadian rhythms occur with 24 hour periodicity and are important for organ-
isms to adapt to environmental changes. They are orchestrated by the suprachi-
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asmatic nucleus (SCN) located in the anterior hypothalamus. At the molecular
Figure 8.2 GSK3

targets and their implication in neurological functions.
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level, regulation starts with heterodimerization of the basic helix-loop-helix
(bHLH)-PAS domain proteins BMAL1 and circadian locomotor output cycles
kaput (CLOCK). This complex then activates the clock genes Per1, Per2, Cry1
and Cry2. PER and CRY accumulate in the cytosol before translocating into
the nucleus where the PER–CRY complex inhibits the transcription of its own
components by binding to the BMAL1–CLOCK complex. The orphan nuclear
receptor Rev-erbα also plays a role in the negative feedback loops by repressing
the transcription of Bmal1.156 GSK3 is highly expressed in the SCN. It has been
shown that the nonselective GSK3 inhibitor lithium lengthens the period of
circadian rhythms in vitro and in vivo157,158 and that GSK3 haploinsufficiency
lengthens circadian locomotor activity in mice.159 GSK3 exerts its effect on cir-
cadian rhythms by regulating their components. First, GSK3 directly phosphor-
ylates PER2 and promotes its nuclear translocation. Therefore, an alteration in
the activity of GSK3 would result in phase changes due to temporal changes in
the nuclear transfer of PER2.160 GSK3 phosphorylates CRY2, which results in its
degradation.161 GSK3 also phosphorylates and stabilizes the negative compo-
nent of the circadian clock Rev-erbα. Accordingly, the rapid proteasomal degra-
dation of Rev-erbα and the activation of the gene Bmal1 were observed following
GSK3 inhibition using lithium.162 Finally, GSK3 phosphorylates BMAL1 and con-
trols the amplitude of the circadian oscillation by stabilizing this protein, which
appears to be crucial to maintaining the robustness of the circadian clock.163
8.4.2 β-Catenin
One of the best-described GSK3-incorporating pathways is regulation of
β-catenin downstream of Wnt signaling. Wnt-GSK3 signaling is described
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above, so here we focus on how β-catenin regulation by GSK3 may lead to

behavioral effects. β-catenin acts as a transcription factor and when trans-
located to the nucleus acts with TCF transcription factors to regulate the
transcription of several target genes. Moreover, β-catenin interacts with the
cytoskeletal network and is recruited to dendritic spines following depolar-
ization; hence it may have a role in synaptic plasticity.164 GSK3 phosphory-
lates β-catenin resulting in its degradation by proteasome. Therefore, it is
possible that changes in β-catenin regulation by GSK3 influence gene expres-
sion and synaptic plasticity. In support of this possibility, overexpression of
β-catenin mimics the effect of lithium on locomotor hyperactivity, which has
been shown to be GSK3-dependent.45 However, forebrainspecific β-catenin-
KO mice exhibit little behavioral phenotype, therefore suggesting a minor
role for β-catenin in the regulation of normal behavior by GSK3.165
8.4.3 Microtubules
Cytoskeletal rearrangements are important during neuronal growth, axon
guidance, and synapse formation. Microtubules constitute a major part of
the cytoskeleton and their assembly and stability is regulated by microtubule-
associated proteins (MAPs). Several MAPs are substrates of GSK3, as follows.
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188 Chapter 8
a) Tau protein is phosphorylated by GSK3 and influences the regulation of
microtubule assembly.155 b) Collapsin response mediator proteins (CRMPs)
are phosphorylated in vitro and in vivo by GSK3 and regulate axon growth,
number of axons, and dendritic development.166–168 c) MAP1B, a major com-
ponent of the neuronal cytoskeleton, can be phosphorylated by GSK3, lead-
ing to increased stability and affinity for microtubules. GSK3 regulation of
MAP1B is a crucial link between Wnt signaling and axonal remodeling.169,170

d) MAP2C is another cytoskeletal component abundant in neurons. It can be
phosphorylated by GSK3 and along with tau and MAP1B participates in micro-
tubule stability.171 Although the exact mechanism by which GSK3-mediated
phosphorylation of microtubules gives rise to behavioral changes is not clear,
we speculate that by doing so, GSK3 may regulate neurodevelopment and syn-
aptic plasticity, which can result in the modulation of behavioral responses.
8.4.4 AMPA and NMDA Receptors

One possible scenario leading to acute modulation of behavior regulation
by GSK3 is its action on the ionotropic glutamate receptors α-amino-3-hy-
droxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate
(NMDA), known to be important for synaptic plasticity. It has been shown
that activation of GSK3 inhibits long-term potentiation, while its inhibition
prevents development of long-term depression.172,173 Moreover, GSK3 regu-
lates trafficking and cell surface expression of NMDA receptor subunits.172,174
Since glutamate receptors could potentially be involved in the etiology of
psychiatric disorders,175 their regulation by GSK3 may be implicated in the
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pathophysiology of many diseases, including schizophrenia.
8.4.5 Dynamin I
Large GTPase dynamin I can serve as a GSK3 substrate; the resultant phos-
phorylated form is then involved in activity-dependent bulk endocytosis
(ADBE), but not in clathrin-mediated endocytosis. The activity of GSK3-
dependent phosphorylation of dynamin I is necessary and sufficient for
ADBE to take place. Thus, GSK3 may play an important role in preparing syn-
aptic vesicles for retrieval during elevated neuronal activity.176
8.5 Biomarkers
Schizophrenia has been defined for over a century, but in the absence of
explicit biomarkers, diagnosis has traditionally been based on symptoms.95
In this regard, a major restriction faced by psychiatry research is acquiring
quantitative biological data from the living brain.177 Therefore, there is press-
ing need for the development of a noninvasive biological marker with a high
degree of robustness and discrimination in order to improve diagnosis and
facilitate monitoring of treatment response.
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8.5.1 Peripheral Blood Cells and Olfactory Epithelium

Collection and further processing of peripheral blood cells for biochemical
analyses represents a rapid and noninvasive method of data acquisition from
individuals. mRNA and protein expression profiling in the peripheral blood
can provide useful information about alterations in disease-related signal-
ing networks. Individuals with schizophrenia demonstrate diminished AKT1

and pGSK3β protein levels in their peripheral lymphocytes, which mirror
similar alterations in the brain.53 Moreover, stimulation of B lymphoblasts
from schizophrenic subjects using NRG1a leads to a prominent reduction in
phosphorylated AKT,178 which is specific to schizophrenia and not shared by
other psychiatric disorders.117
Olfactory epithelium (OE) represents a neuronal tissue having the unique
property of safe accessibility in live human subjects while surpassing the
limitations of non-neural tissues and post-mortem brain tissue.179 A certain
degree of similarity in gene expression exists between the OE and central
nervous system.180 Furthermore, the relevance of OE to schizophrenia has
been verified by the correlation of negative symptoms with structural and
functional olfactory deficits.181 However, expression studies are lacking on
GSK3 and its signaling partners in OE obtained from diseased versus healthy
individuals.
8.5.2 MRI
Schizophrenia is accompanied by several structural and functional abnor-
malities which can be traced via noninvasive imaging techniques such as
10:30:40.
MRI. Using sMRI, structural alterations such as those occurring in the hip-
pocampal and subcortical volumes, cerebral white matter, and grey matter
thickness were seen in schizophrenia subjects.182–184 Moreover, functional
aberrations including deficits in working memory, attention, and activity
of different brain regions were detected by fMRI in schizophrenic individu-
als.185 The possibility of using noninvasive imaging techniques for early diag-
nosis will encourage effective treatment. The validity of diagnosis based on
MRI data has been evaluated by its correlation with the presence of risk gene
variants.185 Using sMRI, grey matter deficits in frontal and temporal lobes
as well as in thalamus were correlated with polymorphisms in AKT, PI3K,
D2DR,86 and GSK391 loci. Attenuated prefrontal activity as observed using
fMRI, together with a reduction in prefrontal cortical thickness in schizo-
phrenic subjects, was associated with the GSK3 polymorphisms.82
8.5.3 Electroretinography
Being part of the central nervous system owing to its embryonic origin, the
retina can represent an interesting site of investigation for brain disorders
and its activity may mirror the central neurochemistry. The retinal func-
tions can be assessed noninvasively using the flash electroretinogram (ERG).
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190 Chapter 8
Significantly, ERG anomalies have been observed in schizophrenia subjects;
these patients manifest a decrease in cone a-wave and rod a- and b- waves. In
experimental settings, young offspring at high genetic risk for schizophrenia
and bipolar disorder also display decreased rod maximal b-wave amplitude
(Vmax) as a biological endophenotype.186 Consistent with the putative role of
increased GSK3 activity in schizophrenia, overexpression of neuronal GSK3β
in mice (prpGsk3b mice) replicated the same pattern of decrease in rod

b-wave amplitude and a longer rod b-wave implicit time at Vmax as reported
in ERG of high-risk individuals. Interestingly, the opposite change was
observed in mice having diminished GSK3β expression. Altered cone a-wave
was also detected in Gsk3a-KO mice consistent with that seen in schizophre-
nia patients. These findings associate the alterations in GSK3 expression or
activity with distinct ERG anomalies in high-risk individuals and patients,
thus supporting the potential usefulness of ERG measurements as a valuable
biomarker for psychiatric research.187
8.6 Future Prospects

Schizophrenia is a devastating psychiatric disorder with largely unmet ther-
apeutic needs. Current medications are mostly aimed at modulating the
activity of receptors for the monoamine neurotransmitters DA and sero-
tonin. However, these medications do not cover all the positive, negative,
and cognitive symptoms and inflict numerous adverse effects on patients.
Furthermore, genetic evidence does not support a simple model associat-
ing the activation of these receptors with disease progression. We suggests
10:30:40.
that drugs acting on D2R and 5HT2 receptors may exert their therapeutic
effects in mental disorders by “normalizing” cell signaling mechanisms
that are also affected by genetic or environmental factors in people with
schizophrenia.6
In light of data indicating a dysregulation of AKT-GSK3 as a pivotal signal-
ing pathway in the pathophysiology and/or manifestations of schizophrenia,
this pathway may appear to be an interesting target for future drug devel-
opment. However, given their involvement in a broad array of intracellular
networks, global targeting of AKT and GSK3 would not culminate in bene-
ficial clinical outcomes. Therefore, targeting specific pathways involved in
the behavioral manifestations of the disease, that pass through these sig-
naling integrators, is of crucial relevance. On this premise, one intriguing
strategy would be selective interference with distinct signaling modalities
downstream of neurotransmitter receptors. Given the fact that both classes
of antipsychotic medications, in spite of disparate pharmacological and
adverse effect profiles, share the common property of blocking the βArr2-
mediated AKT-GSK3 pathway, it can be speculated that the development of
biased antagonists for the D2 receptor, which disrupts this signal transduc-
tion pathway while leaving other signaling routes unaffected, could provide
a unique advantage. Additional strategies could be aimed at interfering with
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protein complexes mediating certain pathways involved in disease pathol-
ogy. For instance, lithium has been shown to interfere with βArr2-mediated
AKT-GSK3 signaling.33,188 Understanding the fine mechanism of this may
therefore provide new molecular targets for drug discovery.
Beyond drug development, the intricate involvement of GSK3 at the cross-
road of neuronal plasticity, development, and monoamine neurotransmis-
sion may also inspire a new understanding of the pathology of schizophrenia,

a disease that has been linked to all of these aspects of brain function.
In turn, this may help to develop better diagnostic tools and to segregate
patients into subcategories that would be more emendable to personalized
treatment.
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CHAPTER 9
Protein Interactions with

Dopamine Receptors as
Potential New Drug Targets for
Treating Schizophrenia
PING SUa, ALBERT H. C. WONGa,b, AND FANG LIU*a,b
a
Department of Neuroscience, Centre for Addiction and Mental Health,
Toronto, Ontario, Canada; bDepartment of Psychiatry, University of Toronto,
Toronto, Ontario, M5T 1R8, Canada
*E-mail: f.liu.a@utoronto.ca
10:30:43.
9.1 Introduction
Schizophrenia is an important health issue; almost 1% of the population suf-
fers from this disease,1 which has major effects on social and occupational
functioning.2,3 The main symptoms of schizophrenia include psychotic
symptoms consisting of hallucinations (typically auditory) and delusions,
which frequently involve persecution and/or megalomania, termed as pos-
itive symptoms.4 Deficit or negative symptoms are also common, and these
are defined as flat affect, lack of volition (apathy), poverty of speech, lack of
pleasure (anhedonia), as well as social withdrawal.5 Patients also have signif-
icant cognitive dysfunction that often is the most functionally disabling of
all the symptoms, but for which existing treatments are largely ineffective.

202
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Protein Interactions with Dopamine Receptors as Potential New Drug Targets 203
The cause of schizophrenia is unknown, but it is one of the most herita-
ble complex diseases, with an estimated heritability of 80%. Environmental
influences on susceptibility generally have weak effects, and include prena-
tal viral infection, urban birth, perinatal trauma, and post-natal inflamma-
tory processes. Schizophrenia is defined by the clinical presentation, and is
unlikely to be a single disorder, but instead is a heterogeneous condition
with a variety of causes in each individual patient. This complex and variable
etiology complicates drug development efforts and to date, treatments have
been aimed primarily at reducing the symptoms rather than targeting the
cause.
One strategy for developing new treatments is to focus on neural signal-
ing pathways implicated in the pathophysiology of schizophrenia. Dysfunc-
tion within the dopamine neurotransmitter system has been widely linked
to the pathophysiology of schizophrenia. The classical target of existing
antipsychotic medications for schizophrenia is the D2 dopamine receptor
(D2R). Most effective antipsychotics for schizophrenia principally antagonize
the D2 subtype.6,7 In addition to being the main target of existing antipsy-
chotic medications, D2R mRNA and protein expression levels are elevated
in the brain of patients with schizophrenia, as shown in post-mortem, pos-
itron emission tomography and single-photon emission computed tomography
studies.7,8
The dopamine receptor family is a functionally diverse class of G-protein-
coupled receptors (GPCR), present throughout the nervous system.9,10 The
classical view of GPCR function is that downstream effects are mediated
almost exclusively by G-protein-dependent pathways.10,11 However, the recent
discovery of interactions between the dopamine receptors and various other
10:30:43.
receptors and regulatory proteins points to alternative signaling routes.

Using yeast two-hybrid, co-immunoprecipitation, glutathione S-transferase
(GST) pull-down, and in vitro binding assays, more than 20 dopamine recep-
tor interacting proteins have been determined, many of which are relevant
to schizophrenia.12–14 These proteins selectively regulate specific signaling
pathways and functions of dopamine receptors via protein–protein interac-
tions, without affecting other signaling pathways and dopamine receptor
functions. Thus, targeting protein–protein interactions represents a promis-
ing alternative treatment strategy for schizophrenia, which might avoid the
side-effects of existing antipsychotics that simply block the ligand-binding
site of the dopamine receptor.
Peptides that mimic the binding domain responsible for these receptor–
protein interactions can effectively disrupt these protein complexes and
selectively block a particular signaling pathway. The trans-activator of tran-
scription domain (TAT) of the human immnodeficiency virus (HIV) can be
fused to these small peptides to facilitate entry across the cell membrane.
Treatment with TAT-tagged peptides can alter certain behaviors, with mini-
mal off-target effects, demonstrating the promise of this approach for devel-
oping new and better schizophrenia treatments.15–19 In this review, we discuss
the proteins that interact with dopamine receptors, regulatory mechanisms
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204 Chapter 9
for these interactions, and promising avenues for future research into novel
drugs for schizophrenia.
9.2 Dopamine Receptors

Dopamine receptors are members of the seven transmembrane and trimeric
guanosine-5’-triphosphate (GTP)-binding protein (G-protein)-coupled recep-

tor family and are classified into D1-like (D1 and D5) and D2-like (D2, D3,
and D4) receptors based on pharmacological properties, sequence homol-
ogy, and structures.10 D1-like receptors have a short third cytoplasmic loop
and a long C-terminus (CT). D2-like receptors have a long third cytoplasmic
loop and a rather short C-terminus. D1-like receptors couple to Gαs proteins
and activate adenylyl cyclase, while D2-like receptors inhibit adenylyl cyclase
through other G-proteins.10,11 Furthermore, recent studies show that D2Rs
can regulate the activity of glycogen synthase kinase (GSK) 3 in a β-arrestin
2-dependent and G-protein-independent fashion.20–25
9.2.1 D1-Like Dopamine Receptor Interacting Proteins

9.2.1.1 Dopamine D1R Interacting Proteins
9.2.1.1.1 D1R Homo-Oligomers. It has been reported that dopamine D1
receptors (D1Rs) exists as dimers.26,27 The oligomerization-dependent mod-
ulation of trafficking of D1Rs to the cell surface was investigated by using
variants of this receptor with different conformations. A mutation within
transmembrane domain 3, in which aspartic acid (Asp103) was changed to
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alanine (D103A), produced a constitutively active receptor that trafficked

to the cell surface without ability to bind agonists or antagonists.26 This
observation suggests that D1R homo-oligomers regulate D1R trafficking to
intracellular compartments. Wild-type D1Rs co-translocated with D1-NLS,
a chimeric D1R protein containing a nuclear localization sequence (NLS)
to facilitate nuclear translocation of D1Rs. This indicated the existence
of D1R homo-oligomers. Both the wild-type D1Rs and the D1-NLS were
retained at the cell surface upon exposure to the D1R antagonist/inverse
agonist (+)-butaclamol, whereas both of them translocated to the nucleus
after removal of the drug. However, both the receptors were retained by the
drug at the cell surface membrane without the ability to bind agonist if two
substituted serine residues in transmembrane domain 5 were mutated to
alanines (D1-(S198A/S199A)-NLS), demonstrating that this mutation only
affects the agonist binding of D1Rs, but not homo-oligomerization. Wild-
type D1Rs remained at the cell surface while mutant D1-NLS trafficked to
the nucleus after drug removal, indicating disruption of these oligomeri-
zed receptors.27 Furthermore, another study showed that treatment with
membrane-permeable full and partial agonists could rescue intracellular
retention of D1R oligomers due to induced conformation changes to the
D1R.26
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9.2.1.1.2 Receptor Heterodimers
Adenosine A1 Receptor (A1R). The antagonistic interactions between ade-
nosine A1 receptors (A1Rs) and D1Rs in the brain have been well described
in previous studies.28–34 Locomotor activity and sensorimotor gating (mea-
sured as pre-pulse inhibition of startle), two classical behaviors relevant
to schizophrenia, are regulated by the mesolimbic dopamine tract which

terminates at the nucleus accumbens.35,36 In addition, there is recent evi-
dence of antagonistic interactions between A1Rs and D1Rs involved in the
regulation of pre-pulse inhibition and locomotion. Locomotor activity, but
not pre-pulse inhibition, was reduced with N6-cyclopentyl-adenosine (CPA)
infusion into the nucleus accumbens, indicating that A1Rs are involved
in modulating locomotion. Furthermore, co-administration of CPA or
SCH23390, a specific antagonist of D1Rs, into the nucleus accumbens coun-
teracted the increase of locomotor activity stimulated by a γ-aminobutyric
acid (GABA)A receptor antagonist, picrotoxin, in the ventral tegmental area
(VTA). SCH23390 has a similar effect on pre-pulse inhibition in this brain
region.28 In addition, both D1R-induced motor activation in reserpinized
mice and D1R-dependent oral dyskinesia in rabbits were decreased by A1R
stimulation.29 Taken together, these data suggest an antagonistic functional
interaction between A1Rs and D1Rs in regulating locomotor activity and
pre-pulse inhibition.
Furthermore, A1Rs were found to antagonistically modulate the
D1R-mediated regulation of the GABAergic striatoentopeduncular path-
way.30 In the ventral pallidum and nucleus accumbens, co-activation of
A1Rs attenuated a D1R-induced increase of GABA release by the D1R-
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specific agonist, SKF-82958. At the same time, there were no changes in

GABA release in response to D1R agonists or antagonists, or co-activation
of D1Rs with A1Rs in the basal ganglia.31 These observations suggest that
A1Rs modulate D1R-mediated signaling and behaviors via the antagonistic
interaction.
Dopamine signaling might modulate the non-motor behavioral effects
of adenosine agonists and antagonists through the A1R–D1R interaction.
Examples could include the sedative properties of adenosine analogues and
the psycho-stimulant effects of caffeine.37 The A1R agonist (N6-l-phenyliso-
propyl-adenosine) prevented both phencyclidine (PCP)-induced stereotypy
and ataxia, but this compound has no behavior or electroencephalographic
(EEG) effects in male rabbits when given alone. This implies that A1Rs might
be involved in the behavioral effects of PCP via interactions with the D1R.33
The A1R agonist CPA prevented the EEG arousal effects of SKF38393, a
D1R-specific agonist, demonstrating that A1Rs can modulate D1R-induced
EEG arousal and showing that adenosine–dopamine interactions are involved
in brain functions other than motor activity.34 These behavioral results sug-
gest that the antagonistic interaction between A1Rs and D1Rs may regu-
late schizophrenia-related behavior, and might therefore be involved in the
pathophysiology of schizophrenia.
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206 Chapter 9
The molecular basis for this antagonistic interaction has been studied
in co-transfected cell lines and primary cultured neurons.38,39 Immuno-
fluorescence studies demonstrated that A1Rs and D1Rs co-localize to a
great extent in both co-transfected fibroblast cells and primary cultured
cortical neurons, as well as in medium-sized striatal neurons using in
situ hybridization,32 providing basic evidence for a protein–protein inter-
action between A1Rs and D1Rs. Using co-immunoprecipitation assays,

Gines et al.39 demonstrated a selective A1R–D1R heterodimer in fibroblasts
co-transfected with human A1R and D1R complementary (c)DNA, which
disappears after activation of the D1R. However, this co-immunoprecipita-
tion was enhanced by co-treatment with A1R agonists. Pretreatment with
the A1R agonist causes co-clustering of A1Rs and D1Rs, while this effect
was blocked by combined pretreatment with both D1R and A1R agonists
in both fibroblasts and cortical neurons.39 Thus, the A1R–D1R heterodi-
merization forms the molecular basis for the functional cross-talk between
A1Rs and D1Rs in the brain.
The A1R–D1R interaction plays an important role in modulating D1R func-
tions. D1R-mediated cyclic adenosine monophosphate (cAMP) activation was
reduced by combined pretreatment with D1R and A1R agonists, but not with
either one alone. This could be explained by biochemical data showing that
A1R stimulation produces a GTP-independent40 uncoupling of the rat stri-
atal D1Rs from the G-protein.29 The A1R can also modulate ligand-binding
affinity and second-messenger activation of D1R signaling pathways. In par-
ticular, cAMP-response element recruitment, and the phosphorylation of
DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa)
in mouse striatal neurons is modulated by the A1R–D1R interaction.41,42
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Furthermore, by using quantitative receptor autoradiography, the affinity

of D1Rs for the specific D1 antagonist [125I] SCH23982 was reduced by the
selective A1R agonist CPA in both the nucleus accumbens and the medial
prefrontal cortex of the rat brain.37 D1R desensitization can occur after tonic
stimulation of A1Rs in rats chronically treated with the A1R agonist, N6-[(R)-
1-methyl-2-phenylethyl] adenosine.43 Taken together, these studies show
that A1R regulates D1R functions and D1R-mediated behaviors through a
protein–protein interaction with the D1R.
Recently, bivalent ligands for A1R–D1R heterodimers have been developed.
These ligands have affinities for the A1R 10–100 times greater than that of
monovalent ligands. In fluorescence resonance energy transfer (FRET) exper-
iments, the bivalent ligands significantly increased the heterodimerization
of A1Rs and D1Rs compared with the corresponding monovalent ligands,
which may be useful for designing novel drugs aimed at modifying these
protein–protein interactions44 to treat schizophrenia.
Dopamine D2 Receptor (D2R). Although dopamine D1 and D2 receptors

belong to different subfamilies, there are still several lines of evidence indi-
cating functional cross-talk between these two main dopamine receptors,10,11
suggesting the possibility of a protein–protein interaction between D1 and
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D2 receptors. These receptors are co-expressed and co-localized within neu-
rons of both human and rat brain.45 FRET experiments revealed that the
D1 and D2 receptors exist in close proximity on both the cell surface and
in the endoplasmic reticulum (ER),46 suggesting the existence of D1R–D2R
hetero-oligomers. Using co-immunoprecipitation assays, it has been shown
that D1 and D2 receptors form hetero-oligomers in cells co-expressing both
receptors as well as in rat brains, providing evidence for the D1R–D2R inter-
action. Furthermore, this interaction is mediated via the residues 257–271 in
the third intracellular loop of the D2R long isoform.18
Unlike the A1R–D1R interaction, D1R–D2R hetero-oligomers had a syner-
gistic effect on functions of both receptors. There is a novel phospholipase
C (PLC)-mediated calcium signal upon co-activation of both receptors in the
striatum, which cannot be activated by either receptor alone, and is distinct
from Gsolf or Gi/o activation by the D1R or D2R, respectively.47 The activation
of the D1R–D2R hetero-oligomers resulted in a Ca2+-dependent increase in
total and activated Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) in rat
nucleus accumbens.47 In addition, the calcium signal rapidly desensitized
and both receptors co-internalized after either the D1R or D2R binding
pocket was occupied, leading to increased D2R expression and decreased
D1R expression at the cell surface.48 Importantly, the D1R–D2R interaction
was enhanced in post-mortem brain tissue from patients with major depres-
sion, and peptides disrupting the interaction exhibit antidepressant-like
effects,18 which may be due to the unique dopamine-mediated calcium signal
facilitated by this interaction.
N-Methyl-d-Aspartic Acid (NMDA) Receptors. Dopamine and glutamate

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may be the most extensively studied neurotransmitters in the brain. D2R

hyperfunction and NMDA receptor hypofunction have been two main the-
ories in the pathophysiology of schizophrenia.49 In recent years, this dopa-
mine–glutamate system interaction has become even more interesting.
Abnormalities in both glutamate and dopamine signaling, and the balance
between these two neurotransmitter systems, seem to be involved in many
psychiatric conditions, including schizophrenia. Thus, more knowledge
about the interaction between the glutamatergic and dopaminergic systems
will provide important knowledge to assist the development of new treat-
ments for psychiatric disorders.50
NMDA receptors are members of the ionotropic glutamate receptor family.
As ligand-gated ion channels composed of multiple subunits, NMDA recep-
tors can conduct cations across the cell membrane and thus mediate fast
excitatory synaptic transmission. NMDA receptor hypofunction has been
one of the major theories for the pathophysiology of schizophrenia, and cor-
tical dysfunction of NMDA-CaMKII signaling is associated with dopaminer-
gic hypofunction.51
The functional cross-talk between D1Rs and NMDA receptors has been
extensively studied in previous research. Behavioral studies suggest that
D1R agonists can enhance cognitive function, and reverse NMDA receptor
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208 Chapter 9
antagonist, ketamine, or PCP-induced cognitive deficits with an inverted
U-shaped dose response curve. Both 5-hydroxytryptamine (HT)1A receptor
and metabotropic glutamate receptor (GluR)2/3 signaling facilitate these
D1R agonist effects.52 Conversely, there is less ketamine-induced behavioral
activation in D1R knockout mice than in wild-type mice.53 These results indi-
cate that D1Rs regulate NMDA receptor functions related to pathophysiol-
ogy of schizophrenia, and the development of D1R agonists is a priority to

improve cognitive impairment in schizophrenia.54
Sensorimotor gating as measured by pre-pulse inhibition55 of acoustic star-
tle is disrupted in schizophrenia and in rodents receiving systemic admin-
istration of apomorphine, a dopamine D1R/D2R agonist, or MK-801, an
NMDA receptor antagonist. Pre-pulse inhibition has been a popular behav-
ioral endophenotype for schizophrenia and is commonly used to screen for
antipsychotic effects.56,57 In nucleus accumbens neurons, the NMDA NR1
subunit is co-expressed with the D1R, and the NR1 subunit has been sug-
gested to play a role in D1R trafficking, cAMP accumulation,17 and behavioral
dysfunction resulting from systemic administration of apormorphine. This
suggests that NR1 expression in the nucleus accumbens is essential for apo-
morphine-induced D1R surface trafficking,58 as well as auditory startle and
social behaviors that are impaired in many psychiatric disorders.59
Clozapine, the most effective atypical antipsychotic drug for schizo-
phrenia, can increase the presynaptic release of glutamate and dopamine
in a spike-dependent manner, and facilitates cortical dopaminergic and
NMDA receptor-mediated transmission in rats.60 Dopamine modulates
a lasting potentiation of the NMDA receptor component of glutamate
synaptic responses in the prefrontal cortex by stimulating post-synaptic
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D1Rs.60 Dopamine and D1R activation are required for the facilitation of
NMDA-induced currents in cortical pyramidal cells by clozapine.61 Previous
results show that clozapine prevents and reverses the blockade effect of the
non-competitive NMDA receptor antagonist PCP on NMDA-induced cur-
rents and firing activity in pyramidal cells. Furthermore, both clozapine and
asenapine, a new antipsychotic medication, might improve negative and
cognitive symptoms in schizophrenia.61 Taken together, these behavioral
data suggest that D1Rs and NMDA receptors each bi-directionally modulate
the functions of the other, which may also be mediated by direct a protein–
protein interaction.
The anatomy of the mammalian brain contains a dopaminergic projection
from the VTA to the cortex, and a glutamatergic projection from the cortex
to the striatum. The overlap and convergence of these projections provides
the architectural framework for complex neuronal interactions. At the post-
synaptic level, where both D1Rs and NMDA receptors are expressed, interac-
tions between D1Rs and NMDA receptors appear to be particularly relevant.
Different mechanisms are involved in these interactions: 1) phosphorylation
of NMDA receptor subunits mediated by D1R-dependent second-messen-
gers; 2) co-ordinated regulation of receptor trafficking at synaptic sites; and
3) direct protein–protein interactions between D1Rs and NMDA receptors.62
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Liu et al. have shown that there are physical interactions between the D1R
and the NMDA NR1 subunit, as well as between the D1R and the NMDA NR2A
subunit.16,63–65 Through co-immunoprecipitation, in vitro binding assays,
and GST pull-down assays, two distinct interaction sites have been found to
mediate these interactions. One binding site is in the C-terminus of the NR1
subunit and the C-terminus of the D1R (termed D1-t2). The other interaction
site mediating the D1R–NR2A complex is the C-terminus of the NR2A sub-
unit, and the C-terminus of the D1R (termed D1-t3), which is the C-terminus
region adjacent to the D1-t2 region of D1Rs.
NMDA receptor-mediated currents were reduced by D1R stimulation in
hippocampal neurons and in HEK293 cells co-expressing D1Rs and NR1/
NR2A NMDA receptor subunits. This effect was inhibited by intracellular
administration of a peptide corresponding to the D1-t3 region of the D1R,
indicating that NMDA receptor-mediated currents can be altered by the inter-
action between the D1R and the NR2A subunit. Conversely, NMDA recep-
tor-mediated excitotoxicity was blocked by a peptide mimicking the D1-t2
interaction site, suggesting that the D1R–NR1 complex is also involved in
NMDA receptor-mediated excitotoxicity. Furthermore, this protective effect
may be mediated by an increased association of the NR1 subunit with calm-
odulin (CaM)66 upon D1R activation. CaM has been shown to directly bind
to phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) and interact with
the NR1 subunit, which is important for the activation and accumulation of
PI3K. D1R activation led to decreased D1R–NR1 complex levels, as well as an
increase in NR1–CaM binding.16
Conversely, the D1R–NMDA receptor complex also affects D1R function.
Stimulation of NMDA receptors increases both D1R insertion into the plasma
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membrane and D1R-mediated cAMP accumulation.17 Agonist stimulation of

NMDA receptors recruits D1Rs to the cell surface, specifically to dendritic
spines.64 Allosteric changes of the NMDA receptor occur after agonist bind-
ing, and this allows the NMDA receptor to act as a scaffold, which could also
represent a promising target for antipsychotic drug development.65
D1Rs and NMDA receptors interact bi-directionally and may have great
influence the output of the prefrontal cortex, a region that is important for
higher cognition. Hypofunction of dopamine and glutamate systems in the
prefrontal cortex is found in schizophrenia. Thus, upregulating D1 and/or
NMDA receptor signaling via selective activation of D1Rs or co-activation of
NMDA receptors could improve cognition in schizophrenia.67 Understanding
the interaction between the dopamine and glutamate systems, especially the
interaction of D1Rs and NMDA receptors, may enable a more specific and
effective approach to the treatment of schizophrenia.68
9.2.1.1.3 Post-Synaptic Density-95 (PSD-95). PSD-95 is a well-known scaf-

folding protein that is highly enriched in the post-synaptic density (PSD) and
belongs to the membrane-associated guanylate kinase family.69 PSD-95 con-
tains three homology PDZ domains, a Src homology-3 domain, and a gua-
nylate kinase-like70 domain, which allows for interactions with a diverse set
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210 Chapter 9
of proteins. D1R co-localizes with PSD-95 in primary cultured striatal neu-
rons71 and co-transfected HEK293 cells.72 PSD-95 is also co-precipitated by
D1R antibody in transfected HEK293 cells and brain tissue,72 confirming that
PSD-95 interacts with D1Rs both in vitro and in vivo. It has been shown that
this interaction is mediated by the C-terminus of D1Rs and the N-terminus
of PSD-95.71,72
Using FRET assays, it has been shown that the interaction between D1Rs
and PSD-95 is enhanced by stimulation of D1Rs in co-transfected HEK293
cells and in the frontal cortex.72 There are conflicting reports on whether
this interaction can alter D1R-mediated cAMP accumulation. Zhang et al.
found that co-expression of PSD-95 with D1Rs in mammalian cells inhibited
D1R-mediated cAMP accumulation without altering total D1R expression
level or agonist binding properties.71 However, Sun et al. reported that this
interaction does not change D1R-mediated cAMP accumulation.72 These two
studies used different stimulating drugs (SKF81297 vs. dopamine), which
have different D1R binding affinities. The downregulation of D1R signaling
may be due to reduced D1R expression at the cell surface as a consequence
of an enhanced constitutive, dynamin-dependent endocytosis, since PSD-
95 can enhance membrane D1R recovery via increased receptor recycling.72
Alternatively, PSD-95 may regulate cAMP production by modulating G-protein
coupling to D1Rs and/or uncoupling from the activated receptor resulting in
desensitization71 or resensitization.72 In addition, recent evidence suggests
that PSD-95 interacts with a Gγ subunit and may affect G-protein signaling.73
The D1R directly interacts with NMDA receptors via its C-terminus,16
which may be required for targeting the D1R to the cell surface in vitro.17,63
The association of PSD-95 with NMDA receptors and the functional signifi-
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cance of these PDZ-mediated interactions have also been demonstrated.74,75

Thus, a tertiary protein complex of D1R/NMDA receptor/PSD-95 in the brain
may sort and deliver D1Rs to the synapse. Emerging evidence reveals a posi-
tive feedback loop between the NMDA receptor and the D1R in the heads of
dendritic spines in the glutamatergic synapse.76,77 Specifically, D1R activa-
tion stimulates adenylyl cyclase, increases cAMP production, and activates
protein kinase A (PKA), leading to potentiation of the NMDA receptor via
the PKA/DARPP-32/protein phosphatase (PP)1 cascade.77 Moreover, PSD-
95 is critical for D1R-modulated NR1a/NR2B receptor function, since D1R
activation fails to modulate NR1a/NR2B receptor-mediated Ca2+ influx in the
absence of PSD-95.78,79
NMDA receptor activation can enhance D1R-mediated cAMP accumulation
by recruiting more D1Rs to the plasma membrane.17 This positive feedback
loop can produce excessive activation of both D1Rs and NMDA receptors,
ultimately leading to neurotoxicity and cell death.80,81 The D1R/PSD-95 inter-
action dissociates D1R from the NMDA receptor NR1 subunit, and uncouples
NMDA receptor-mediated enhancement of D1R signaling. The NMDA recep-
tor-dependent inhibition of D1R internalization was abolished by the D1R/
PSD-95 interaction, suggesting that the PSD-95/D1R complex interferes with
NMDA-dependent surface delivery/anchorage of D1R. Thus, the PSD-95/D1R
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complex prevents over-activation of both dopamine and glutamate systems.79
In addition, calcyon, when phosphorylated at Ser169 by protein kinase (PKC)
activator or D1R agonist SKF81297, can enhance D1R internalization via the
D1R/PSD-95/calcyon complex.82 These studies demonstrate a role for a glu-
tamate receptor scaffold in dopamine receptor signaling and trafficking and
suggest a new potential target for the modulation of abnormal dopaminergic
function.
9.2.1.1.4 Proteins that Regulate D1R Trafficking

Neurofilament (NF)-M Protein. The expression level and distribution of
D1R at the cell surface is important for D1R signaling and function. There
are several mechanisms regulating trafficking, membrane insertion, and
targeted anchoring. The most important of these mechanisms is direct
protein–protein interaction with D1Rs. The aa782–846 in the C-terminus of
neurofilament (NF)-M interacts with the D1R family, but not those of the
D2-like family.
This interaction leads to reduced total receptor binding activity of D1R
and decreased D1R-mediated cAMP accumulation in HEK293 cells co-trans-
fected with full-length NF-M and D1R.83 Co-expression of NF-M was shown
to reduce D1R expression on the cell surface and enhance D1R protein levels
in the cytosol. Interestingly, desensitization of D1R is abolished when it is
co-expressed with NF-M.
Furthermore, using immunofluorescence techniques, the D1R has been to
shown to co-localize with NF-M in about 50% of medium sized striatal neu-
rons, as well as a group of pyramidal neurons in layer III and layer V of the
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cortex. Moreover, the fluorescent signal intensity of D1R is reduced in NF-M

knockout mice, and there are fewer neurons expressing D1R in these mice,
while there is no change in D2R expression, suggesting that D1R expression
and function are regulated via interaction with NF-M.83
γ-Subunit of the Coatomer Protein Complex (γ-COP). Another protein reg-

ulating D1R trafficking and functions is γ-COP, which is involved in ER/Golgi
transport, and interacts with cytosolic cdc42 as well as the p24 transmem-
brane cargo receptors.84,85 γ-COP co-immunoprecipitates with FLAG-tagged
D1R in transfected HEK293 cells, and this interaction is abolished when the
residues F333, F337, and F341 are mutated to alanine in D1R. This triple
alanine mutant D1R localized closely with an ER-specific rhodamine B-hexyl
ester stain, and co-localized with ER-specific proteins. Meanwhile, results
of pulse-chase analysis showed that this mutant has endoH-sensitive glyco-
sylation, suggesting that the D1R-γ-COP interaction depends on the trans-
port-competent C-terminus of D1R. Besides, this mutant showed quite poor
affinity for the D1R ligand, and could not mediate cAMP accumulation with
agonist stimulation, which might be due to the reduced expression level of
D1R on the cell surface. Furthermore, γ-COP interacts directly with the D1R
C-terminus, which is specific to the motif containing F341 and L344/345.
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212 Chapter 9
This motif has been shown to play a critical role in D1R cell surface trans-
port.86 Truncations at the carboxyl termini of GPCRs are often involved in
neuronal disorders.87 The γ-COP-D1R interaction suggests that the associa-
tion between D1Rs and cytoskeletal proteins may be important for receptor
trafficking.
Dopamine Receptor-Interacting Protein (DRiP)78. Transport of D1Rs from

the ER to the cell surface is critical for membrane localization and functions
of D1Rs.88 Using site-directed mutagenesis, this process has been shown to
be mediated by a hydrophobic motif, FxxxFxxxF, and this motif is highly con-
served among GPCRs.89 The D1R has poor cell surface expression when the
phenylalanine residues in this motif are mutated to alanine. The mutant D1R
remains in the ER, suggesting that this motif is required for export of D1Rs
from the ER. Moreover, the mutant D1R showed reduced ligand binding
affinity and disabled cAMP accumulation after agonist stimulation, which
might be due to reduced cell surface expression.
Using a yeast two-hybrid system, the D1R was found to interact with a 78
kDa protein called dopamine receptor-interacting protein (DRiP)78. DRiP78
contains conserved transmembrane and zinc-finger domains, and is widely
expressed in the heart, brain, lung, and kidney. GST pull-down assays
showed that the wild-type D1R interacts with DRiP78 through the C-termi-
nus. This interaction is weakened in the phenylalanine mutant D1R, suggest-
ing that hydrophobicity in the proximal C-terminus of the D1R is required
for the interaction. In addition, residues 488-673 of DRiP78 were found to
be required for this interaction, and the zinc-finger domains (CxxCxxxH) are
critical for the interaction with D1Rs. D1Rs shifted from the cell surface to
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intracellular membrane compartments when co-expressed with DRiP78, and

DRiP78 co-localized with the intracellular D1Rs, leading to reduced binding
affinity to D1R ligands.89
In contrast to heterologously expressed DRiP78, endogenous DRiP78
efficiently exports D1R from the ER, as determined by pulse-chase analysis
using competing peptides. However, D1R trafficking from the ER to the cell
surface depends on the level of DRiP78 in the cytosol.89 In summary, DRiP78
regulates D1R export from the ER and transport to the cell surface through
interactions between these two proteins.
Calnexin. The expression levels of GPCRs on the cell surface membrane

are an important part of their functional regulation, and this is controlled by
two major processes. The first is synthesis, maturation in the ER, post-trans-
lational modification in the trans-Golgi network, and trafficking, as well as
insertion into the surface membrane. The other process is internalization,
followed by either lysosomal degradation or recycling back to the surface
membrane. Many adaptor proteins and chaperones regulate these processes
through protein–protein interactions with GPCRs.
Using western blot and co-immunoprecipitation assays, calnexin was
found to interact with both D1Rs and D2Rs by acting as an ER chaperone
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protein, and to regulate the trafficking of D1Rs from the ER to the cyto-
plasmic membrane in transfected HEK293T and CAD cells, a central ner-
vous system-derived catecholaminergic cell line.90 Calnexin is a protein
chaperone molecule in the ER, and it regulates protein folding, as well as
promoting the retention of misfolded and incomplete proteins in the ER,
preventing them from being expressed at the cell surface.91–93 By generating
mutant D1Rs and D2Rs lacking N-linked glycosylation sites, and comparing
the co-immunoprecipitation between these receptors and calnexin, it was
shown that the receptor–calnexin interaction was reduced by about 50% in
mutant receptors. Drugs that inhibit protein glycosylation also reduced the
receptor–calnexin interaction to the same extent, indicating that there are
both glycan-dependent and glycan-independent interactions between D1Rs/
D2Rs and calnexin. Calnexin may interact with receptors via N-linked gly-
cans, as well as direct protein–protein interaction.90
Total expression of D2Rs, but not D1Rs, is reduced after treatment with
tunicamycin, an inhibitor of N-linked glycosylation, and castanospermine, a
glucosidase inhibitor interacting with calnexin. D1R-mediated signaling can
be inhibited by disruption of calnexin–receptor interactions, suggesting that
his interaction enhances cell surface D1R expression. Furthermore, disrupt-
ing the calnexin–receptor interaction prevents D1Rs/D2Rs from trafficking to
the cell surface. Although overexpression of calnexin increased co-immuno-
precipitation with D1Rs/D2Rs, D1R expression and D1R-mediated signaling
were reduced.90 Thus, there may be an optimal level of calnexin required for
optimal regulation of D1R/D2R function.
Caveolin-1. Caveolae are a subtype of lipid rafts, which regulate the inte-
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gration of various signaling molecules by facilitating the formation of cell

surface signaling platforms. Thus, caveolin proteins are important for the
specificity and efficiency of signaling transduction. Caveolin-1 is the most
ubiquitously expressed caveolin protein, which acts as a scaffold for recep-
tors, signaling molecules, and adapter proteins.
As a GPCR, D1R-signaling is also regulated by localization in lipid raft
micro-domains, such as caveolae, facilitating D1R signaling through down-
stream signaling molecules, including G proteins and cAMP. It has been
shown that the D1R directly interacts with caveolin-1 in transfected COS7
cells and in rat brain tissue, and this interaction regulates D1R trafficking.
Caveolin-1 facilitates protein localization in caveolae through its scaf-
folding domain and the putative caveolin binding motif in caveolin-asso-
ciated proteins. D1Rs also contain such a motif in the proximal region of
the seventh transmembrane domain.94 The Gαs-protein is also localized in
the caveolin-enriched fraction, suggesting that D1Rs may be regulated in
caveole.95
The physical association between the D1R and caveolin-1 was con-
firmed using co-immunoprecipitation and bioluminescence resonance
energy transfer (BRET) assays in COS-7 cells and rat brain tissue. In COS-7
cells co-transfected with hemagglutinin-tagged D1Rs and cMyc-tagged
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214 Chapter 9
caveolin-1, the D1R interacts with both the α isoform and the β isoform of
caveolin-1, while D1Rs could only co-precipitate the α isoform of caveolin-1
in rat brain.95
The D1R is found to translocate from the non-caveolin-enriched fraction
to the caveolin-enriched fraction upon stimulation with specific agonist
SKF81297 in HEK293 t cells. This translocation occurs after 5 minutes of
stimulation, and reaches a peak of ∼70% in 20 minutes, without changes

in the total amount of D1R and the localization of endogenously expressed
caveolin-1. It is also reported that D1R can be internalized through a caveolar
pathway, while inhibiting caveolae formation decreases D1R internalization
upon stimulation with SKF81297. D1Rs appear to translocate to caveolae
before undergoing caveolar endocytosis upon binding to an agonist.95 Cave-
olin-1 dependent D1R endocytosis is kinetically slower than clathrin-medi-
ated endocytosis of D1Rs.96,97 However, caveolar endocytosis of D1Rs does
not depend on the PKA, although PKA participates in caveolae-dependent
endocytosis via phosphorylation,98 and PKA dependent phosphorylation of
D1Rs is known to occur upon agonist stimulation.99
The D1R contains a putative caveolin-1 binding motif, and the distal aro-
matic residue Trp321 within the binding motif was found to mediate translo-
cation, surface expression, and ligand-binding affinity. The F313 and W318
residues were also found to mediate the interaction between D1R and cave-
olin-1. Caveolar disruption using cholesterol depletion can cause Gs-protein
activation, which is independent of agonist binding, and this activation did
not alter D1R-mediated cAMP accumulation. However, the F313A and W318A
mutation within the caveolin binding motif of the D1R attenuate desensitiza-
tion, suggesting the D1R-caveolin-1 interaction is required for D1R desensiti-
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zation by regulating autophosphorylation.95
Arrestin. Arrestin is extremely important in the trafficking, activity, and

signaling transduction of GPCRs via interaction with these receptors. When
agonists induce phosphorylation of GPCRs via G-protein-coupled recep-
tor kinases, GPCRs bind to arrestin, leading to disrupted heterotrimeric
G-protein-receptor interactions, and desensitization of receptors. In studies
of β-adrenoceptors, it was shown that GPCR–arrestin interaction also results
in rapid internalization of receptors, which is mediated by clathrin-coated
pits, followed by degradation or recycling back to the cytoplasmic mem-
brane.100,101 For angiotensin II type 1a receptors, arrestin can also facilitate
activation and targeting of extracellular signal-regulated kinase (ERK) by
acting as a scaffold protein.102
As a typical GPCR, the D1R can interact with arrestin3 in neostriatal neu-
rons,103 as well as heterologously expressed cell lines.104–106 In heterologously
expressed cell lines, green fluorescent protein-tagged arrestin3 translocates
to the cell surface membrane upon D1R activation, with greater transloca-
tion for arrestin3 compared to arrestin2.107 Arrestin directly binds to the third
intracellular loop of activated D1Rs, but the carboxyl terminus of the D1R
inhibits this interaction. Upon activation, the carboxyl terminus of the D1R
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is phosphorylated before the third intracellular loop, leading to binding of
arrestin to the third intracellular loop of the D1R.11,105 The amino acids Y239
and Q256 are important for the D1R–arrestin3 interaction. The Y239T muta-
tion reduces binding to D1Rs, and Q256Y showed no effect, while Y239T/
Q256Y showed high selectivity for D1Rs.108
This interaction may play a critical role in regulating D1R functions, and
may be involved in the pathophysiology of some mental disorders. For exam-

ple, in l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia, D1R
protein level and D1R sensitivity increases, suggesting some change in the
D1R–arrestin interaction.109 Arrestin is also involved in morphine-induced
locomotion, with D1R signaling through the arrestin–ERK complex.110 The
arrestin-RAF-mitogen-activated protein kinase (MAPK)/ERK pathway is
also important for long-term changes in synaptic plasticity related to drug
abuse.111
N-Ethylmaleimide-Sensitive Factor (NSF) and Sorting Nexin (SNX)-1. Both

NSF and SNX-1 are involved in the processing of D1Rs after internaliza-
tion.112,113 NSF is an ATPase regulating membrane vesicle trafficking, and it
is found to interact with the β2 adrenergic receptor through binding to the
cytoplasmic tail of the receptor. NSF binds to the SNAP receptor complex
and disassembles them as part of the recycling of receptors.114 The binding
domains within GPCRs that interact with the adaptor proteins are in the
C-terminus, and this region of all five subtypes of dopamine receptors can
interact with NSF and SNX-1.113 Further studies showed that NSF binds to
aa387–401 of the D1R C-terminus, leading to enhanced membrane localiza-
tion and D1R-mediated cAMP accumulation, and treatment with TAT-D1R
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aa387–401 decreases synaptic localization and cell surface expression in

hippocampal neurons.115 In contrast to NSF, SNX-1 regulates the lysosomal
degradation of some subtypes of dopamine receptors. However, SNX-1 only
interacts with the C-terminal tail of D5Rs, while it binds weakly to D1Rs and
D3Rs.113
9.2.1.2 D5 Dopamine Receptor (D5R) Interacting Proteins

9.2.1.2.1 GABAA receptor. GABA is one of the major neurotransmitters in
the central nervous system, mediating the majority of inhibitory synaptic
transmission. Both the direct and indirect pathways of the basal ganglia,
which regulate locomotion, are formed by GABA neurons.116,117 GABA neu-
rons are also involved in the pathophysiology of Parkinson’s disease,118–120
l-DOPA-induced dyskinesia,121–123 and drug addiction.124 Furthermore, dis-
ruption of GABA interneuron migration during prenatal development has
been implicated in schizophrenia.125
GABA receptor functions can be influenced by interactions with dopamine
receptors. For example, the Zn2+-sensitive component of GABAA currents are
enhanced by D5R activation, which is PKA-dependent, and can be reversibly
blocked by a PP1 inhibitor. Furthermore, D2R and D5R mRNAs appear in
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216 Chapter 9
cholinergic interneurons with single-cell reverse transcriptase polymerase
chain reaction analysis, whereas D1R mRNAs do not. These results suggest
that dopamine release in the striatum can modulate GABA signaling, which
might be mediated by D5Rs.126
GABAA receptors mediate the fast, bicuculline-blocked response to GABA
after the opening of bicuculline-sensitive Cl− channels. Thus, GABAA recep-
tors participate in most fast inhibitory synaptic signaling in the brain. GABAA
receptors are heteromeric, consisting of subunits from five different classes:
α, β, γ, δ and ε.127 There are four α-helical hydrophobic transmembrane
domains in each subunit. The subunit composition of GABAA receptors plays
a critical role in their function and pharmacological properties. The α and
β subunits are required for functional GABAA receptors expressed at the cell
surface.128
Although GABAA receptors are not GPCRs, the intracellular domains also
contain consensus phosphorylated sites for protein kinases, and GABAA
currents are regulated through kinase-dependent pathways.129,130 For
example, GABAA receptor-mediated synaptic activity can be modified by
D1-like receptor stimulation, which depends on cAMP kinase pathways and
phosphatases.126,131
The D5R interacts with the GABAA receptor, and both receptors have a sim-
ilar distribution in the dendritic shafts and the cell soma/axon hillock area of
certain cortical and hippocampal neurons.126,132,133 Using co-immunoprecip-
itation, GST pull-down and in-vitro binding assays, a direct protein–protein
interaction between D5Rs and GABAA receptors was seen in hippocampal
neurons as well as co-transfected cells. The interaction is mediated by the
C-terminus (residues 429–477) of D5Rs and the second intracellular loop of
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the GABAA receptor γ2 subunit.134

The interaction is agonist-dependent, and appears only after combined
activation of both receptors, indicating that there might be functional cross-
talk between these receptors. D5R-mediated agonist-stimulated cAMP accu-
mulation was reduced by ∼45% by pretreatment with GABA in co-transfected
HEK293 cells. This reduction of cAMP accumulation can be blocked by the
GABAA receptor antagonist picrotoxin and did not change the ligand binding
affinity and expression level of D5Rs. Conversely, GABAA receptor-mediated
whole-cell currents are reduced by ∼30% by D5R activation in co-transfected
HEK293 cells, and this effect is cAMP-independent but D5R-C-terminus
(CT)-dependent. Thus, there is a bi-directional and reciprocal functional regu-
lation between these two receptors. Activation of both D1Rs and D5Rs signifi-
cantly increased GABAA miniature inhibitory post-synaptic current frequency
in cultured hippocampal neurons. This effect can be prevented by treatment
with GST-D5R-CT fusion peptide, but not GST-D1R-CT fusion peptide, showing
the modulation of synaptic currents in vivo via the D5R–GABAA receptor inter-
action.134 Since schizophrenia is associated with cortical neuron dysfunction,
this finding is highly relevant to the pathophysiology of schizophrenia.130,131
The GABAA–D5R interaction was one of the first such neurotransmitter
interactions to be discovered, and demonstrated that direct receptor–receptor
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interactions could have important functional properties. These receptor–
receptor interactions also provide a potentially new drug target for treating
neuropsychiatric disorders. The interfering peptides used as experimen-
tal tools to investigate the function of receptor–receptor interactions have
shown promising therapeutic effects for symptoms of brain disorders in ani-
mal models.
9.2.2 D2-Like Receptor-Interacting Proteins

Among the dopamine receptor subtypes, the D2R is most extensively stud-
ied, since all clinical antipsychotic drugs act as D2R antagonists in the meso-
limbic dopaminergic system, and the ability of these drugs to block D2Rs
correlate with antipsychotic efficiency. But D2R blockade is also responsi-
ble for side-effects such as extra-pyramidal symptoms and prolactin eleva-
tion. Because D2R interactions with other proteins can mediate specific D2R
signaling pathways, these interactions could represent novel antipsychotic
treatment targets.
9.2.2.1 D2R-Interacting Proteins
Unlike the D1-like family, D2-like receptors (D2R, D3R, and D4R) contain a
quite large third cytoplasmic loop, an extremely short C-terminal tail, and
activate inhibitory G-proteins, leading to inhibition of cAMP accumula-
tion.10 Proteins interacting with the third cytoplasmic loop or the C-terminus
of the D2-like receptors thus regulate receptor signaling, and therefore
modulate the physiological functions of these receptors. Furthermore, for
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D2R and D3R genes, unlike D1R and D5R, mRNA splicing generates iso-
forms with differences in the region of the third cytoplasmic loop due to
the introns between exons. There are two isoforms of D2R: D2LR (the long
isoform) and D2SR (the short isoform), the latter of which lacks 29 amino
acids found within the third cytoplasmic loop of the D2LR. The D3R gene
gives rise to at least seven distinct splicing variants, including D3nf, a iso-
form lacking 98 base pairs in the carboxyl-terminal region of the third intra-
cellular loop.14
9.2.2.1.1 D2 Receptor Dimerization. Receptor dimerization is a common

mechanism for regulating receptor functions in the central nervous sys-
tem. In post-mortem striatal tissue from patients with schizophrenia, there
is significantly enhanced expression of D2R dimers and decreased expres-
sion of D2R monomers. D2R dimerization is also increased in the striatum
of amphetamine-induced sensitized state rats, a common animal model
of psychosis. Furthermore, an interfering peptide that disrupts the D2R–
dopamine transporter (DAT) association blocked amphetamine-induced
upregulation of D2R dimerization, suggesting that amphetamine-induced
D2R dimerization may be associated with the D2R–DAT protein–protein
interaction.135
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218 Chapter 9
9.2.2.1.2 D2 Receptor Heterodimers
NMDA Receptor NR2B Subunit. NMDA receptors are ionotropic GluRs,
which are critically important in synaptic plasticity, learning, and memory.136
These receptors comprise an NR1 subunit, combined with different regula-
tory NR2 subunits (NR2A–D). D1Rs interact with the NMDA receptor, and this
interface between the dopamine and glutamate neurotransmitter systems is

potentially important in the understanding of the pathophysiology of neu-
ropsychiatric disorders and as a novel treatment target. The NR2B subunit
also directly interacts with D2R in the confined post-synaptic density micro-
domain of excitatory synapses.137
Cocaine, a psycho-stimulant that blocks the DAT and elevated dopa-
mine levels in synapses, can increase NR2B phosphorylation, suggesting
a functional connection between dopamine receptors and NR2B subunits.
Cocaine-induced elevation of NR2B phosphorylation can be blocked by D2R
antagonists, and enhanced by D2R agonists, indicating that this effect might
involve the D2R.137 The D2R localizes in the perisynaptic and post-synaptic
membrane of glutamatergic synapses in the corticostriatal projection, mak-
ing the interaction between D2R and NR2B possible in vivo.138,139 Biochemi-
cal studies of the post-synaptic density confirm the presence of D2R, and the
D2R–NR2B interaction has been confirmed by co-immunoprecipitation. This
interaction occurs not only in the striatum, but also in the hippocampus and
cortex.137
The D2R directly interacts with the C-terminus of NR2B through an N-ter-
minal 10-aa motif, T225–A234, of its third cytoplasmic domain, but not
D3Rs137 Cocaine enhances this interaction in the striatum, but not in the
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hippocampus and cortex. Cocaine reduces CaMKII–NR2B binding and CaM-

KII-dependent phosphorylation of NR2B at Ser1303 via the D2R–NR2B inter-
action. Since NR2B does not bind CaM, the NR2B–D2R interaction inhibits
NR2B phosphorylation by disrupting CaMKII–NR2B binding.137 Function-
ally, the NR2B–D2R interaction inhibits NMDA receptor-mediated currents
in medium spiny neurons of the striatum and regulates behavioral respon-
siveness to cocaine. Treatment with a TAT-tagged peptide that encodes the
interacting site of the D2R–NR2B interaction inhibits cocaine-induced loco-
motion, highlighting another dopamine–glutamate system interaction that
may be a useful target for treating addiction.137 Finally, D2Rs regulate spines
by modulating the internalization of NR2B subunits in hippocampal neu-
rons, suggesting that this interaction might also be involved in age-related
changes in synaptic connectivity.140
Dopamine D3 Receptor (D3R). Dopamine receptors can form heterodi-

mers.39,141,142 D2–D3 heterodimers are of interest because both receptors
are within the same subfamily, share a high degree of sequence homology,
and have a similar ligand-binding profile.11 D2 and D3 receptors can both
exist as homodimers. D2 and D3 receptors co-localize on dopaminergic
neurons as autoreceptors94,143,144 and at post-synaptic neurons in the globus
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pallidus, nucleus accumbens, frontal cortex pyramidal cells, and GABAergic
interneurons.145,146
A direct interaction between D2 and D3 receptors has been shown in
cultured cells.147 Functionally, pramipexole and ropinirole reduced forsko-
lin-stimulated cAMP production with greater potency in cells co-transfected
with both receptors, compared to cells transfected with either receptor alone.
Partial agonists of D3Rs act as antagonists when D3Rs is co-transfected with

D2Rs into cells. Since D2–D3 heterodimers form a stronger signaling com-
plex than D3R and D2R homodimers, it is conceivable that this interaction
can influence dopaminergic neurotransmission.147
Cannabinoid (CB)1 Receptors. Dopamine and endogenous cannabi-

noids, two neurotransmitter systems in the basal ganglia, display complex
cross-talk. D2Rs interact with CB1 receptors, and the interaction is regulat-
edby CB1 signaling. This interaction is enhanced upon co-stimulation with
sub-saturating concentrations of agonist. However, the interaction switches
the G-protein-coupled signaling from Gαi/o-protein-coupled receptors to Gαs-
protein-coupled receptors. The receptor complex couples to Gαs proteins, but
not Gαi/o proteins upon co-stimulation of both receptors, leading to increased
cAMP accumulation. In addition, this interaction changes the characteristics
of receptor ligands. For example, the pertussis toxin (PTX)-sensitive cannabi-
noid agonist CP55940, shows PTX-insensitive properties in the activation of
ERK1/2 kinases in cells co-expressing both receptors.148
Adenosine A2 Receptor (A2AR). Adenosine A2 receptors (A2ARs) heterodi-

merize with D2Rs, and these complexes are localized in the dendritic spines of
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striatopallidal GABAergic neurons where they can modulate glutamate signal-

ing. Similar to the A1R–D1R interaction, the discovery of A2AR–D2R heterod-
imers suggests a mechanism underlying the opposing functional interaction
between the two receptors. The A1R–D1R interaction may therefore be a use-
ful new drug target for treatment of neuropsychiatric disorders.
The direct interaction between A2A and D2 receptors was demonstrated
by co-immunoprecipitation in D2R-transfected SH-SY5Y and Ltk cells.112
Both receptors co-aggregate, co-internalize, and co-desensitize upon ago-
nist treatment.112 Recently, the direct interaction of the receptors has been
demonstrated using both FRET and BRET112 techniques in co-transfected
HEK293T cells, which suggests that the third intracellular loop of the D2R
and the C-terminus of the A2AR probably mediates this interaction.112 Stim-
ulation of A2ARs produces a decrease in the affinity of D2R agonists, while
D2R activation inhibits A2AR agonist-induced cAMP accumulation and PKA
activation, phosphorylation of DARPP-32, phosphorylation of (cAMP response
element-binding protein (CREB), and activation of MAPK.149
Somatostatin Receptor (SSTR5). The long isoform of the D2R interacts with
the somatostatin receptor SSTR5. D2R and SSTR5 co-localize in medium
spiny neurons in the striatum and pyramidal neurons in the cerebral
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220 Chapter 9
150
cortex. Using FRET, it has been shown that heterodimerization of SSTR5
and D2Rs is induced by ligand binding. Ligand binding to either receptor
can trigger heterodimer formation, and heterodimers do not exist in the
absence of ligand. There is a synergistic effect of somatostatin on D2Rs at
low agonist concentrations via the heterodimer, which could account for the
enhancement of dopaminergic and somatostatinergic transmission induced
by somatostatin or dopamine agonists.150
Serotonin 5-HT2A Receptor. Co-immunoprecipitation, FRET, and BRET stud-

ies show that the serotonin 5-HT2A receptor (5-HT2AR) and the D2 receptor
can physically interact with each other.151 Both these receptors are also targeted
by antipsychotic medications, and some argue that affinity for the 5-HT2AR
determines atypical antipsychotic effects. While both receptors are G-protein
coupled, they stimulate different G-proteins. The 5-HT2AR couples to Gq/11 and
D2R couples to the Gi/o protein. Using radioactive ligand binding and second
messenger production assays it was found that the 5-HT2AR and D2R can form
a functional complex in brain and HEK293 cells. D2R activation increases hal-
lucinogenic agonist affinity for 5-HT2ARs and decreases the 5-HT2AR-induced
inositol phosphate production. In vivo, 5-HT2AR expression is necessary for the
full effects of D2R antagonists on MK-801-induced locomotor activity.152
9.2.2.1.3 Ion Channels

Inwardly Rectifying Potassium Channels (Kir3). The inwardly rectifying
potassium channel (Kir3) interacts with both D2 and D4 receptors. This
interaction is not affected by receptor activation or by modulation of the
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G-protein α subunit. However, the Gβγ subunit is critical for complex forma-
tion, but not for its maintainence, and this might be a more general feature
of G-protein-coupled signal transduction.153
Transient Receptor Potential Channel (TRPC)1. The D2 receptor interacts

with the transient receptor potential channel (TRPC)1 via the 22-aa-long sec-
ond intracellular domain of the D2R.154 This domain of the D2R also inter-
acts with the calcium-activated protein CAPS1, a presynaptic calcium binding
protein that connects D2Rs to intracellular exocytosis components.155 TRPC1
permits agonist-stimulated Ca2+ influx and store-operated Ca2+ release, pro-
viding a mechanism for D2Rs to modulate intracellular Ca2+ signaling. The
D2R also interacts with TRPC4 and TRPC5, two TRPC family members with
high homology with TRPC1.156
Immunohistochemical analysis of monkey prefrontal cortex showed that
TRPC1 and D2Rs co-localize in postsynaptic compartments, providing an
opportunity for TRPC1 and D2Rs to interact in vivo. By deletion mapping
using yeast two-hybrid screen, the interaction domains within TRPC1 have
been determined: residues 726–745, as well as residues 735–754 for TRPC4
and residues 742–761 for TRPC5. The interactions between D2R and both
TRPC1 and TRPC4 have been confirmed in rat brain.154
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Biotinylation assays showed that the D2R–TRPC1 interaction enhances
TRPC1 cell surface expression. TRPC1 plays a critical role in regulating
intracellular Ca2+ homeostasis, which has been implicated in a variety of
neuropathological conditions, including Parkinson’s disease. TRPC1 overex-
pression in PC12 cells protect the cells from 1-methyl-4-phenylpyridinium-
induced neuronal apoptosis, a classic cell-death model of Parkinson’s
disease.157
9.2.2.1.4 Dopamine Transporter (DAT). DAT is a transmembrane protein

that promotes the reuptake of extracellular dopamine and is a target for
drugs of abuse, including cocaine and amphetamine. Lee et al. reported the
D2R-mediated modulation of DAT through a direct protein–protein interac-
tion. This interaction involves the third cytoplasmic loop of D2Rs (I340–Q373)
and the N-terminus of DAT.15 DAT recruitment to the plasma membrane is
facilitated by this interaction, and DAT must of course be expressed on the
cell surface to function. Injection of peptides containing the interacting site
within D2Rs into mice disrupts the DAT–D2R interaction, and decreases syn-
aptosomal dopamine uptake and increases locomotor activity.15
Furthermore, recent studies show that CaMKIIα can regulate the functions
of DAT via interaction with the C-terminus of DAT and phosphorylation of the
DAT N-terminus.158 This phosphorylation is required for amphetamine-in-
duced behaviors.159 Interestingly, the phosphorylation occurs at aa1–27 in
the N-terminus of DAT, and the interacting domain between D2R and DAT is
aa residues 1–15. Thus, it is possible that CaMKIIα-dependent phosphoryla-
tion of DAT might regulate the interaction between D2Rs and DAT.
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9.2.2.1.5 Gi-Protein. The type of Gi-protein-binding D2R differs, depend-

ing on the cell line examined. In the transfected fibroblast cells, CCL1.3, the
D2LR was found to couple to Gi2 and Gi3, but not Gi1 or Go proteins. However,
in transfected NS20Y neuroblastoma cells, the D2LR coupled to Go protein,
but not to any other subunits. Similarly, in AtT-20 cells, the D2LR coupled to
Go and Gi3 proteins. In transfected Sf9 cells, the D2SR was found to couple Gi1
and Gi2 proteins. But in AtT-20 cells, the D2SR was shown to couple to both
Go and Gi2 proteins. Thus, D2R-mediated inhibition of adenylyl cyclase activ-
ity can proceed through coupling of the receptor to either Gi or Gz proteins,
since both these G-proteins have an inhibitory effect on cyclases.160
9.2.2.1.6 Calmodulin (CaM). Recent studies have found a CaM-binding

motif (residues I210–V223) in the N-terminus of the third cytoplasmic loop
of the human D2R,161 indicating that there might be direct protein–protein
interaction between CaM and D2Rs. This motif contains consensus hydro-
phobic residues (Val, Ile) at I210, I215, and V223.162 CaM binds to aa208–226,
which are Ca2+/CaM-dependent. Furthermore, CaM does not appear to bind
to the rest of the third intracellular loop of D2Rs (R210–P239).163 The binding
affinity of Ca2+/CaM for D2Rs showed a dissociation constant of 80 nM and a
similar value to the median inhibitory concentration for CaM.161
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222 Chapter 9
Activation of D2Rs can elevate intracellular Ca levels via the Gβγ/PLCβ.11
2+
Calcium is important for functions of neurons, especially in maintaining the

action potential and excitability of neurons. The D2R localizes on the presyn-
aptic membrane as an auto-receptor of the nigrostriatal projections, which
controls release of dopamine, and on the post-synaptic membrane of the
medium spiny neurons of the neostriatum.164 NMDA receptor-mediated Ca2+
influx in cortical afferent neurons excite medium spiny neurons in the stria-
tum, and modulate D2R signaling.165 This anatomical connection provides a
location for cross-talk between intracellular Ca2+ and D2Rs.
CaM is a small acidic protein serving as an indicator of intracellular Ca2+
levels, which acts as a switch along with the elevated Ca2+ concentration.166
Ca2+ is a downstream effector of D2Rs through the Gβγ/PLCβ pathway, and
elevated Ca2+ levels elicit similar effects to activation of D2Rs.167–171
CaM may serve to maintain balanced dopamine signaling in striatal nerve
cells.161
9.2.2.1.7 Proteins that Regulating D2R Trafficking

Filamin A. D2R functions are regulated by G-protein coupling, receptor
desensitization and sequestration, and intracellular trafficking among mem-
brane compartments.172–174 D2Rs interact with the actin cross-linking protein
filamin A.175,176 Filamin A, also known as non-muscle filamin/actin-binding pro-
tein 280, is expressed ubiquitously, and acts as a dimeric actin-binding protein
that links glycoproteins in the membrane to the actin cytoskeleton.177
In primary cultured rat striatal neurons, D2Rs and filamin A co-localize
in cell bodies and proximal neurites. These two proteins also co-localize in
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astrocytes. Overexpression of filamin A enhances D2R cell surface expres-

sion, while filamin A knockout results in primarily intracellular localization
of D2Rs.176 Amino acids 211–241 in the N-terminus of the third intracellular
loop of D2Rs are responsible for the interaction with filamin A. The K281–
Q329 fragment in the C-terminal portion of D3Rs may also contribute to
the filamin A–D3R interaction. In filamin A, the repeat unit 19 within the
aa1788–2121 region is required for the D2R/D3R–filamin A interaction.175
Filamin A is required for the cytoplasmic membrane localization of
D2Rs,175 by linking D2R to the actin cytoskeleton.178
Protein 4.1N. A number of proteins regulate D2R trafficking via protein–

protein interactions. Cytoskeletal proteins are key molecules for the intracel-
lular trafficking of proteins. The D2R interacts with protein 4.1N, a homolog
of the erythrocyte membrane cytoskeletal protein 4.1 (4.1R). Protein 4.1N
and related protein family members are highly expressed in neurons and
are associated with the sub-membrane cytoskeleton. The 4.1 family also
includes protein 4.1R, found in erythrocytes and the isoforms 4.1 G and 4.1B.
Similar to filamin A, the 4.1 proteins also form attachments between the cell
membrane and the cytoskeleton, which helps to stabilize the neuronal cell
membrane.179
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The 4.1N protein interacts with the N-terminus of the third cytoplasmic
loop of D2Rs (K211–K241) and D3Rs (I211–Q227) respectively, via its C-termi-
nus,180 a highly conserved domain among all 4.1 family members. Both D2
and D3 receptors also interact with the C-terminus of other members of the
4.1 family. In co-transfected HEK293 cells, D2/D3 receptors and protein 4.1N
are co-expressed at the cell surface membrane. Endogenously expressed D2/
D3 receptors and protein 4.1N also co-localize.180 Mutations in protein 4.1N

decrease the expression levels of D2Rs or D3Rs at the cell surface membrane
in Neuro2A cells, suggesting that this interaction is required for expression
and stability of D2/D3 receptors at the neuronal cell surface. Furthermore,
since both filamin A and protein 4.1N bind to the third intracellular loop
of D2/D3 receptors, these two proteins bind to dopamine receptors simul-
taneously when co-expressed in the HEK293 cells, indicating that the bind-
ing sites of these two proteins within D2/D3 receptors might be the same
residues.180
Prostate apoptosis response (Par)-4. Prostate apoptosis response (Par)-4

is another protein shown to interact with D2Rs.181 Par-4 is a pro-apoptotic
factor containing a leucine zipper motif.182 In the nervous system, Par-4
interacts with various proteins, including PKCζ, and WT1, linking Par-4 to
neuronal cell death in neurodegenerative diseases such as Parkinson’s dis-
ease, Alzheimer’s disease, and amyotrophic lateral sclerosis.183–187
Par-4 was shown to interact with D2Rs at the third intracellular loop of
human D2LRs (I212–Q373),181 but not D3Rs. Par-4 co-localizes with D2Rs
in striatum tissue and primary cultured neurons. The co-localization is
detected primarily in the periphery of the cell soma and neuronal processes,
10:30:43.
where the main pool of functional D2Rs is expressed.138 These two proteins
also co-localize with synaptophysin, and they are found in the synaptosomal
fraction.181 Furthermore, in vitro binding assays identified that this interac-
tion is mediated by the first 30 amino acid residues of the third intracellu-
lar loop (I212–K241) and aa245–342 of Par-4 that harbor the leucine zipper
domain. Interestingly, as CaM binds to D2Rs in the same region (I210–V223),
there is competition between CaM and Par-4 to bind D2Rs, and this competi-
tion results in reduced D2R efficacy and decreased ability of D2Rs to inhibit
cAMP accumulation. Primary neurons from mutant mice lacking the D2R
interaction domain of Par-4 exhibit enhanced dopamine-cAMP-CREB signal-
ing pathway,181 and these mice showed depression-like behaviors.
β-Arrestin. D2Rs mediate their physiological effects via both G-protein

dependent and independent signaling pathways. The D2R regulates GSK3
activities via the β-arrestin/PP2A/Akt pathway.21,24 Antipsychotic medications
are antagonists at the D2R and blockade of nigrostriatal and tuberoinfun-
dibular signaling is responsible for the neurological and prolactin-elevating
side-effects. Thus, selective targeting of the D2R–β-arrestin2 interaction and
related signaling pathways may allow for antipsychotic effects without these
particular side-effects.188,189
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224 Chapter 9
Arrestin is involved in desensitization and internalization of GPCRs. Upon
agonist binding, GPCRs will bind to arrestin, and this interaction will disas-
sociate the binding between the receptors and G-proteins, leading to desen-
sitization. D2R–arrestin interaction results in receptor internalization due to
recruitment of the receptor to clathrin-rich regions. Arrestin2 and arrestin3
endogenously expressed in striatal homogenates bind to the third cytoplas-
mic loop of the D2R, while in vitro purified arrestin2 and arrestin3 bind to
the second and third loops and C-terminus of the D2R. In NS20Y cells trans-
fected with D2Rs, D2Rs showed enhanced co-localization with endogenous
arrestin2 and arrestin3 after agonist stimulation.
In contrast to D1Rs, arrestin2, but not arrestin3, translocates to the cell
surface membrane in neostriatal neurons upon agonist binding. Agonist
stimulation leads to selectively enhanced co-immunoprecipitation of the
D2R and arrestin2.190,191 In transfected HEK293 cells, the sequence IYIV212–
215 in the N-terminal region of the third intracellular loop of D2Rs appears to
play a critical role for the binding of arrestin3. Alanine mutations generated
a signaling-biased receptor, which shows intact ligand binding, G-protein
coupling and agonist-induced desensitization, but deficient receptor-medi-
ated translocation of arrestin3 to the cell surface membrane.192 A C-terminal
residue of the second intracellular loop, Lys149, is important for the pref-
erential binding of arrestin3 to D2Rs, and D2R-IC2-K149C mutation leads
to greatly decreased binding to arrestin3 as compared to wild-type D2R-IC2.
Lys149 is at the junction of IC2 and the fourth membrane-spanning helix,
and has intramolecular interactions that contribute to maintaining an inac-
tive receptor state.190
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N-Ethylmaleimide-Sensitive Factor (NSF). There is a direct protein–protein

interaction between the C-terminal tail of the α-Amino-3-hydroxy-5-meth-
yl-4-isoxazolepropionic acid (AMPA)-receptor GluR2 subunit and NSF, which
facilitates AMPA receptor cell surface membrane expression.193 NSF binds
directly to the third cytoplasmic loop (residues F341–Q373) of D2Rs.194
D2R activation by an agonist promotes D2R binding to NSF via the third
intracellular loop of the receptor.194 NSF is an ATPase that is essential for
various membrane fusion events,195 including intracisternal Golgi protein
transport and exocytosis of synaptic vesicles.196 The D2R–NSF interaction
results in decreased NSF–GluR2 interaction. This leads to decreased AMPA
receptor expression at the cell surface, increased interaction between GluR2
and the p85 and the subsequent activation of PI3K. Furthermore, the D2R-
NSF-GluR2-p85 pathway is also involved in D2R inhibition of ischemia-in-
duced cell death, suggesting that NSF regulates interaction between the two
receptors depending on the activation of the D2R.194
Neuronal Calcium Sensor(NCS)-1. Neuronal calcium sensor (NCS)-1 regu-

lates the phosphorylation, trafficking, and signaling transduction of D2Rs
in neurons. It has been shown that NCS-1 can contribute to the pathology
of schizophrenia and may be involved in the efficacy of antipsychotic drug
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197,198
medication in the brain. NCS-1 and D2Rs co-localize within the area
close to intracellular calcium stores and within sites of synaptic transmis-
sion. There is a direct protein–protein interaction between NCS-1 and D2Rs
through aa1–71 of the NCS-1 and aa437–443 of the D2R.199
NCS-1 mediates desensitization of D2Rs, and attenuates agonist-induced
internalization by reducing D2R phosphorylation. D2R-mediated cAMP inhi-
bition is enhanced by interacting with NCS-1 upon dopamine stimulation.

A mutation in NCS-1, which has been shown to impair the calcium-binding
properties of NCS-1, abolishes the ability of NCS-1 to modulate D2R signal-
ing. This interaction might play a role in dentate gyrus plasticity, exploratory
behavior, and spatial memory acquisition.200
Dynamin 2. Dopamine receptor trafficking is critical to receptor function.

The D2R is internalized in a dynamin-2-dependent manner upon agonist
stimulation. Agonist-induced D2R internalization was abolished when D2Rs
were co-expressed with dynamin-2 mutants defective in GTPase function.
In transfected HEK293 cells and primary striatal neurons, dynamin-2 was
found to co-localize to the sites of D2R internalization. The dynamin-2–D2R
interaction was confirmed in adult rat forebrain, providing a novel mecha-
nism trafficking of D2R.201
G-Protein-Coupled Receptor-Associated Sorting Protein (GASP). D2R deg-

radation is regulated by an interaction with the G-protein-coupled recep-
tor-associated sorting protein (GASP). After interacting with GASP, D2Rs
failed to resensitize after agonist treatment in rat brain. Disruption of the
D2R–GASP interaction facilitates recovery of D2R responses, suggesting that
10:30:43.
modulation of the D2R–GASP interaction is important for the functional

downregulation of D2Rs.202
C-Terminus of GAIP Interacting Protein (GIPC). The C-terminus of GPCRs

is a functional cytoplasmic interface for protein association. D2Rs and
D3Rs, but not D4Rs, can interact with the PDZ domain-containing protein,
GIPC, via its C-terminus. This interaction has been confirmed by using
GST pull-down and affinity chromatography assays with recombinant and
endogenous proteins. GIPC co-localizes with D3Rs in neurons of the islands
of Calleja at plasma membranes and in vesicles. It inhibits D3R signaling,
co-internalizes with D2 and D3 receptors, and sequesters receptors in sort-
ing vesicles to prevent their lysosomal degradation.203,204 These findings
identify a novel mechanism in the maintenance, trafficking, and signaling
of dopamine receptors.
9.2.2.2 CaMKII and D3Rs

The D3R is also a GPCR for which protein kinase-mediated phosphoryla-
tion affects internalization, desensitization, resensitization, and intracellu-
lar trafficking. Liu et al.,163 have found that there is a direct protein–protein
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226 Chapter 9
interaction between CaMKIIα and D3Rs involving CaMKIIα-mediated D3R
phosphorylation.
CaMKIIα is highly expressed in the central nervous system, and it is
enriched at synapses.205 CaMKIIα is an important protein kinase that reg-
ulates synaptic plasticity and the functions of multiple neurotransmitters.
There is an auto-inhibitory domain within CaMKIIα. Elevation of intracel-
lular Ca2+ levels due to Ca2+ influx through NMDA receptors results in phos-
phorylation of T286 (α-subunit) or T287 (β-subunit) in the auto-inhibitory
domain of CaMKIIα. This autophosphorylation converts it to a Ca2+-indepen-
dent, and constitutively active kinase. CaMKIIα activity is essential for synap-
tic function and long-term potentiation in the hippocampus.206
CaMKIIα directly interacts with D3Rs through the R210–P239 fragment
of the N-terminal region of the third intracellular loop of the receptor. The
binding is Ca2+ sensitive, and is sustained by CaMKIIα autophosphorylation.
Furthermore, the binding makes the D3R a substrate of CaMKIIα, and facili-
tates CaMKIIα to phosphorylate D3Rs at Ser229, a residue within the binding
fragment.
The CaMKIIα–D3R interaction plays a critical role in response to cocaine.
The D1R is activated by cocaine, resulting in activation of PLCβ, elevation of
intracellular Ca2+, and activation of CaMKIIα. Activated CaMKIIα binds to
and phosphorylates D3Rs.163 The interaction between CaMKIIα and D3Rs
downregulates D3R-mediated signaling, including D3R-mediated inhibi-
tion of cAMP accumulation, phosphorylation of CREB, phosphorylation of
ERK2, phosphorylation of GluR1 at S845, and expression of c-fos. Finally,
this interaction downregulates D3R function in cocaine-induced locomotor
activity.163
10:30:43.
9.3 Targeting
Dopamine Receptor Interactions for
Drug Development of Schizophrenia
By targeting the interacting domains within a pair of interacting proteins,
which typically are less than 30 aa in length, synthetic peptides can disrupt
protein–protein interactions with high specificity. The trans-activator of
transcription domain of the HIV virus can be fused to these small peptides
to facilitate entry across the cell membrane. This is the first step in testing
whether disrupting a given protein–protein interaction might have potential
as a therapeutic strategy. Because disrupting these interactions can affect
only the targeted pathway, the hope is that treatments based on this approach
could have fewer off-target effects and clinical side-effects.18,19,137,163,194
Repeated dosing with peptides and efficient delivery of peptides can be
problematic, although new technologies such as nasal sprays and alter-
native packaging can also be used. An alternative approach is to attempt
to design more conventional small-molecule drugs that target the same
protein–protein interacting site as the synthetic peptide. Such a strategy
exploits the wealth of existing knowledge on small-molecule drug design,
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while still targeting a novel pathway. The standard screening approaches
can be applied to these novel protein interaction targets, using existing
chemical libraries, in conjunction with modern in silico binding prediction
algorithms.
Because the protein–protein interaction can be detected using high
throughput-capable methods such as FRET and BRET, is it possible to screen
large numbers of candidate molecules for their ability to block the desired
protein–protein interaction in vitro. An example of a successful screen using
this type of approach is the D2R/NCS-1 interaction. In that case fluorescence
anisotropy was used to identify small molecules disrupting the interaction
in cellular systems.13
The final steps for drug development would then be the same for these
small molecules as for other drugs in the past. Testing in animal models
would be necessary at some stage, as well as the normal toxicology, pharma-
cokinetic, and bioavailability studies. These steps are common to all drug
development, but the unique opportunity afforded by novel protein–protein
interactions is in the identification of new potential drug targets that could
have greater physiological specificity.
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CHAPTER 10
Optogenetic and Chemogenetic

Tools for Drug Discovery in
Schizophrenia
DENNIS KÄTZEL*a,b AND DIMITRI M. KULLMANNa
a
Institute of Neurology, University College London, Queen Square, WC1N
3BG, London, UK; bDept of Experimental Psychology, University of Oxford,
9 South Parks Road, OX1 3UD, Oxford, UK
*E-mail: d.kaetzel@ucl.ac.uk
10.1 Introduction
10:30:45.
Schizophrenia, together with autism and a few other conditions, sits at the
interface of an arguably arbitrary distinction between neurological and
psychiatric disorders. An accumulating body of evidence points to subtle
structural abnormalities in the brains of affected individuals, but the symp-
tomatology can only be understood by considering the function of large dis-
tributed neural circuits, and therefore touches on the frontiers of knowledge
of the fundamental workings of the brain. This goes some way to explain our
poor understanding of pathophysiological mechanisms and limited treat-
ment options. Until recently, our ability to study nervous tissue at the circuit
level has been very limited.1 This situation has changed with the advent of
optical and chemical remote control of neuronal firing, using light-sensitive

234
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Optogenetic and Chemogenetic Tools for Drug Discovery in Schizophrenia 235

or chemically gated, heterologously expressed ion channels and pumps
(optogenetics2 and chemogenetics,3,4 respectively).
Within just a few years of this technology being adopted by the pioneering
laboratories, the function of individual cells within intact computing neural
circuits has been demonstrated with unprecedented directness. Optogenetic
actuators targeted to the appropriate cell types can, for example, improve
behavioural deficits in rodents that model conditions such as anxiety,5 anhe-

donia6 or Parkinsonian bradykinesia7,8 upon optical illumination of the tis-
sue. Similar manipulations in either normal or genetically modified animals
are likely to shed light on schizophrenia, both by revealing the origins and
mechanisms of its symptoms and by identifying cellular targets for their
treatment.9 Within this endeavour, the dissection of negative and cognitive
symptoms is an especially important challenge. These include anhedonia,
avolition and apathy, as well as deficits in concentrating, problem-solving
ability and learning, and respond poorly to current treatments. They also
represent the greater share of the disease burden (not least because they are
chronic), and correlate with poor outcome.10,11 Furthermore, they are more
accessible to behavioural phenotyping in animal models of the disease than
positive symptoms.
This chapter will describe the various ways in which optogenetics and che-
mogenetics can be deployed to aid drug discovery for schizophrenia, focusing
on hands-on advice on how to implement those tools in the lab.
10.2 Genetically
Targeted Manipulation of
Neural Activity
10:30:45.
Neural circuits consist of distinct cell types, which differ in their expres-
sion of specific genes.12 Although our understanding of the transcription
factors, adhesion proteins, receptors and ligands that guide the formation
of circuits is incomplete, some of the known genetic variability among dis-
tinct neurons can be harnessed to study their function. Artificial genes can
be expressed under the control of gene-regulatory elements (promoters),
which are only active in a certain type of nerve cell, but not others. When
such genes encode exogenous ion channels, pumps or receptors which can
be activated remotely by light or by an otherwise pharmacologically inert
compound, we have arrived in the world of opto- and chemogenetics –
genetically defined, optical or chemical remote activation or inhibition of
neural activity.
It is this combination of remote activation and genetic targeting that
renders those tools so extraordinarily powerful. It makes them superior to
classical methods of intervention, such as lesion studies, or chemical, phar-
macological or electrical stimulation or inhibition of certain brain areas.
This is because, with a few exceptions, such interventions largely do not
allow discrimination between cell types, or compartments within cells,
making it difficult to determine which particular neurons and pathways are
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236 Chapter 10
responsible for any effect measurable at the whole-organism level. Although
conventional lesion studies or electrical or pharmacological stimulation
experiments can provide regional specificity, this is also the case for optoge-
netics and chemogenetics, if viral vectors are targeted to specific areas of the
brain. Indeed, an even higher degree of spatial specificity can be achieved by
delivering light or chemogenetic ligands to small targets as well as by precise
and well-controlled viral transfection.

The idea of targeting optical circuit manipulations genetically was orig-
inally expressed by Nobel laureate Francis Crick.13 The first experimen-
tal realization of an optogenetic actuator14,15 as well as its genetically
targeted expression within a neural circuit in a behaving organism16 was
achieved by the laboratory of Gero Miesenböck. The first directly light-
gated actuator was derived from an algal opsin discovered independently
by the groups of Peter Hegemann17 and John Spudich18 and characterized
as a light-gated ion channel, termed channelrhodopsin-2 (ChR2) by Georg
Nagel, Ernst Bamberg and others.19 Using ChR2, and later on other opsins,
the groups of Karl Deisseroth and Ed Boyden have pioneered optogenet-
ics in rodents,20,21 and hence paved the way towards its use in preclinical
research. Chemogenetics developed very much in the shadow of optogenet-
ics for many years. Indeed, even the name of this method has taken time
to settle, having also been called “chemical-genetics”, “pharmacosynthet-
ics” or, rather more ambiguously, “pharmacogenetics”. Nevertheless, it is
emerging as a powerful alternative strategy to manipulate the brain as its
advantages over optogenetics become ever more appreciated, especially
by behavioural neuroscientists. Chemogenetic actuators feature a bi-
directionally specific key–lock system of activation, where the synthetic ago-
10:30:45.
nist does not act on any endogenous receptor and the synthetic (modified)
receptor is not activated/inhibited by any endogenous molecule and lacks
basal activity; such a system has been termed “designer receptor exclusively
activated by designer drugs” (DREADD).4 As with optogenetics, pioneering
technology has evolved rapidly, with the inhibitory Drosophila allatosta-
tin receptor, developed by the Callaway laboratory22 in 2002, or the inhibi-
tory ivermectin-gated chloride-channel developed by the Anderson group23
in 2007 (and improved by others24), supplanted by more superior tools –
especially the synthetic muscarinic receptors developed by Bryan Roth et al.3
in 2007 and the PSAM–PSEM (pharmacologically selective actuator module–
pharmacologically selective effector molecule) system from Scott Sternson’s
laboratory25 (2011). An important chimera of opto- and chemogenetics,
termed optochemical genetics26 has developed in parallel, mainly in the lab-
oratories of Ehud Isacoff and Dirk Trauner, starting with the first optogenetic
silencer ever published and termed “SPARK” (synthetic photoisomerizable
azobenzene-regulated potassium channel) (2004)27 and followed by light-
gated excitatory28 and inhibitory29 glutamate receptors (2006 and 2010) as
well as various light-gated potassium channel subtypes in 2011.30 Here we
discuss the plethora of opto- and chemogenetic tools, many of which are
illustrated in Figure 10.1, systematically.
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Figure 10.1 Optogenetic

and chemogenetic actuators. The principal concepts
of the most widely used optogenetic activators (a–c) and inhibitors
(d–f) as well as chemogenetic activators (g, h) and inhibitors (i, j) are
10:30:45.
displayed. For optogenetic tools, the most commonly used colour of

light of excitation (mostly corresponding to the peak in the excitation
spectrum) is indicated by the colour of the font. (a) Microbial opsins,
mostly various derivatives of ChR2, are directly light-gated ion chan-
nels. (b) Heterologously expressed ionotropic receptors can be gated by
uncaging their ligand (green). (c, f) Optochemical-genetic ionotropic
receptors are gated by photoswitchable tethered ligands. Directly light-
gated proton (d) or chloride (e) pumps are the primary tools for opto-
genetic inhibition. Chemogenetic activation or inhibition can either
be achieved indirectly via modified G-protein coupled reeptors (g, i),
or directly via synthetic or heterologously expressed ion channels (h, j).
GR, green receiver; ATP, adenosine-tri-phosphate; GlyR-FA/AG, double-
point mutated glycin-insensitive glycin-receptor.
10.2.1 Optogenetic Activation

The principal optogenetic tool for activating neurons is ChR2 (including its
many variants19,31,32). ChR2 was the first light-gated ion channel to be devel-
oped. Its original version had a very low single channel conductance:19,33 40 fS.
This is 500 times lower than the conductance of another channel used as an
optogenetic activator in the same year, P2X2 (20 pS).15 However, P2X2 requires
photolysis of a caged ligand, making it less convenient. Point mutations to
increase conductance as well as genetic methods to strongly increase functional
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238 Chapter 10
32
expression levels have turned ChR2 into a reliable tool to drive cells with
spike-timing precision up to frequencies that approach the highest firing rates
observed in vivo (200 Hz in some fast-spiking interneurons).34 The most versa-
tile and widely used versions of ChR2 at the time of writing are known as “HR”
(referring to the mutation H134R31) and “ET/TC” (E123T/T159C).34 The latter is
among the strongest and fastest optogenetic exciters, which also include ChIEF,
Fast-Receiver (FR)32 and the recently published “top-performer” Chronos.35

The sensitivity of such opsins to light can be expressed as quantum effi-
ciency and channel conductance, but for practical applications it is often
easier to refer to the light power required to bring ChR2-expressing neu-
rons to firing threshold. Because light is rapidly attenuated and scattered
in the brain, depending on its wavelength, this requirement needs to take
into account the distance from the light source and its colour. Moreover,
neurons differ in their intrinsic excitability and degree of expression of
ChR2. As a rough guide, 5–10 ms pulses of blue (peak activation 473 nm,
although light in the range 405–488 nm can be used) laser-light of 5–10 mW
total optical power are sufficient to drive action potentials in neurons up
to 0.5–1.0 mm away (e.g. from the end of the optical fibre inserted into the
brain36) in vivo. Some derivatives of ChR2 remain in a conducting state after
the light pulse has terminated. Such opsins, commonly known as “stabi-
lized step-function opsins” (SSFOs) are less useful for temporally precise
manipulation of neuronal excitability, and are more akin to chemogenetic
activation, albeit triggered by a light pulse: a blue (473 nm) light pulse of
2–5 s will open SSFO-channels of the state-of-the-art version ChR2 “CS/DA”
(C128S/D156A) for at least 30 min.37,38 SSFOs have two advantages. Firstly,
the light sensitivity is increased by two orders of magnitude, because they
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effectively integrate photons, so that tissue further away from the light
source can be activated. Secondly, animals can be disconnected from the
optical fibre after the initial pulse and, for example, inserted into an oper-
ant box for behavioural tests. Moreover, SSFOs can be switched off by brief
illumination with yellow light.
A third flavour of excitable opsins are red-shifted channelrhodopsins –
two versions of the opsin C1V1 (a chimera of the proton channel chan-
nelrhodopsin-1 (ChR1) and the red-shifted Volvox ChR1)) are commonly
used: C1V1TT E122T/E162T (faster) and E122TT (stronger, more red-
shifted).37 Even more red-shifted and more light-sensitive opsins have
recently been published: ReaChR39 and Chrimson.35 The advantages of
the red-shift are threefold. Firstly, red light is scattered less in tissue36
and hence can penetrate deeper, permitting a larger excitation volume
(some permit excitation even through the closed mouse skull). Secondly,
in combination with ChR2 or SSFOs expressed in a distinct cell type, two
populations of cells can be controlled independently within the same
experiment. Thirdly, due to the much slower deactivation kinetics (at sim-
ilar or larger conductance levels), it allows activation in the two-photon
mode, i.e. permits single-cell resolution,40,41 which for ChR2 is possible
only with complicated optics.
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Further types of optogenetic actuators, which are used less frequently,
include ligand-gated ion channels gated by freely floating uncageable (P2X2
or TRPV115) or tethered photoswitchable (LiGluR28) agonists or antagonists,
which have to be added to the tissue at the beginning of the experiment (see
section 10.2.7).
10.2.2 Chemogenetic Activation

On the chemogenetic side, the Gq-coupled modified human muscarinic recep-
tor 3 hM3Dq is arguably the tool of choice3,4,42 at the time of writing. It has been
rendered insensitive to its natural ligand acetylcholine by changing two amino
acids, but is activated by clozapine-N-oxide (CNO). CNO is a normally pharmaco-
logically inert metabolite of clozapine, which is bioavailable via systemic or oral
application with good central nervous system penetration. In contrast to ChR2,
hM3Dq does not feature a cation conductance, but exerts an excitatory action
indirectly through a phospholipase C and calcium-dependent mechanism.42
An important potential confound when using the hM3D/CNO-system in
schizophrenia research is that the metabolic pathway which converts clozap-
ine into CNO is partially reversible: in both guinea pigs and humans signif-
icant levels of clozapine are detected after the application of single doses of
CNO (in the former, 2 hours after intraperitoneal injection, the brain concen-
tration reaches 40–50% of the levels measured after administering an equal
amount of clozapine43). However, to what extent this reversal occurs in rats
and mice is a matter of debate.44
More recently, a suite of synthetic ligand-gated ion channels has been devel-
oped, which allows the direct (non-metabotropic) excitation of neurons with
10:30:45.
pharmacologically inert compounds.25 Here, a chimera of the nicotinic acetyl-

choline-receptor subunit α7 and the 5-hydroxytryptamine (HT)3 receptor is used
and activated with various synthetic compounds (one of them bioavailable via
systemic injection and termed “PSEM-89S”). Although the ability to harness a
cation conductance directly for excitation is compelling, it is unclear whether this
system is suitable for routine in vivo applications. A potential alternative could
be the use of another ligand-gated cation conductance, the vanilloid receptor 1
(TRPV1).15,45,46 It is normally present in the central nervous system at low levels
and in relatively few neurons, but following expression achieved by Cre-dependent
recombination, systemic application of its agonist capsaicin allows robust acti-
vation of defined populations of neurons. TRPV1 was originally suggested as an
optogenetic actuator, as a caged form of its ligand capsaicin was developed.15 It
was also suggested as a thermogenetic actuator (activatable by local heating) as
well as a chemogenetic actuator (activated by capsaicin45,46).
10.2.3 Optogenetic Inhibition

The suite of genetically targetable inhibitors largely mirrors that of excit-
ers. The first optogenetic inhibitor was the mutated potassium channel
SPARK (see section 10.2.7),27 which is no longer used as it constitutes a
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240 Chapter 10
leak-current. A few years later, the light-driven chloride pump NpHR or
Halorhodopsin was developed,47,48 but needed subsequent refinement to
avoid cytotoxicity and low expression levels at the plasma membrane.49,50
It is currently the most widely used tool for optogenetic silencing, but
is surpassed in performance by the improved version of the light-gated
proton pumps (e)Arch and (e)ArchT,32,51,52 and the recently published
chloride pump Jaws.53 All tools are commercially available as adeno-

associated viruses (AAVs). They are usually activated by continuous illu-
mination of 1–5 mW in the green/yellow/red spectrum [with a spectral
peak around 560–580 nm, e.g. 532 nm, 561 nm, or (if necessary for dual-
colour experiments) 594 nm or 635 nm lasers are used]. A less powerful
but more blue-shifted silencer is the proton pump Mac,52 which has an
excitation peak slightly below 550 nm but can be comfortably activated
by blue light (e.g. 473 nm, mostly used to activate ChR2). It is important
to note that proton pumps (ArchT and Mac) and chloride-pumps (NpHR/
Halo) have different side effects: while the former increase the pH of the
respective cells (as protons are pumped out), the latter shift the chloride
reversal potential to more depolarized levels (as chloride is pumped in),
potentially compromising endogenous γ-aminobutyric acid (GABA)-ergic
inhibition.54
10.2.4 Chemogenetic Inhibition

Likewise, on the chemogenetic side, two distinct strategies have been used.
Firstly, metabotropic signalling via a Gi-coupled modified human muscarinic
receptor 4 (hM4Di), which is activated again by CNO, but not by acetylcholine.
10:30:45.
The downstream cascade of this receptor has at least three potential means
for silencing neuronal activity: activation of G-protein-coupled inwardly rec-
tifying potassium channels (GIRK) via the βγ-subunit,3 a decrease of cyclic
adenosine monophosphate (cAMP) levels55 and inhibition of presynaptic cal-
cium-channels leading to attenuation of synaptic transmission, as observed
for native M4 receptors.56–58 While the hyperpolarization achieved by GIRK
opening may reduce, but not completely abolish, spiking activity, the action
on calcium channels may shut down synaptic transmission, especially when
hM4Di receptors are expressed at high level in the pre-synapse. The latter
effect may be the more important of the two.59
The second strategy makes use of ligand-gated chloride channels. Start-
ing with the two-partite glutamate- and ivermectin-gated chloride channel
of Caenorhabditis elegans,23,60 more practical tools have recently emerged,
both based on mammalian glycine receptors:24 a mutated glycine-receptor
gated by ivermectin (but insensitive to glycine) and a chimera of a mutated
nicotinic acetylcholine receptor subunit α7 and the glycine receptor (gated
again by PSEM-89S).25 In both cases, ligands are pharmacologically inert
and bioavailable from systemic injections; however, to date, little experience
with these tools has been published, and their components are not widely
available.
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10.2.5 Silencing of Synaptic Transmission

As mentioned in the previous section, silencing synaptic output is an alter-
native strategy to silencing neuronal activity. Although global activation of
hM4Di both hyperpolarizes neurons and affects synaptic output, the two
actions can, in principle, be dissociated.57 The key advantage is that the activ-
ity of the presynaptic neuron remains unperturbed, and if synaptic transmis-

sion is reduced in a locally restricted fashion by local infusion of the agonist,
distinct projections from one population of presynaptic neurons can be
modified independently.57
To date, it is unclear how well-suited commonly used light-gated chloride
and proton pumps are for silencing of synaptic transmission: their hyperpo-
larizing effects might be too weak to abolish synaptic transmission effectively.
However, more powerful, third or fourth generation optogenetic silencers
might prove useful in that respect, as illustrated by a pioneering study using
eNpHR3.0.5 Another alternative is the use of light-gated G-protein-coupled
receptors (GPCRs). One of the pioneering publications in the field of opto-
genetics demonstrated that the native rat rhodopsin 4, a Gi/o-coupled GPCR,
leads to activation of potassium and inactivation of voltage-gated calcium
channels in the same way as native M2 and M4 receptors, and that it can
inhibit both neuronal activity and synaptic transmission.61
Other tools to silence synaptic transmission rely on the destruction of com-
ponents of the synaptic release machinery, and hence lack temporal precision
in the order of seconds, minutes or hours. In the context of schizophrenia-
related research, the most notable application of this strategy is the use of
virally expressed tetanus-toxin light chain (which cleaves vesicle-associated
10:30:45.
membrane protein 2), specifically in parvalbumin-positive interneurons of

the dorsal hippocampus, to demonstrate the necessity of these neurons for
spatial working, but not reference, memory.62
10.2.6 Ablation of Cells

Targeted ablation of a genetically defined population of cells has become a
less attractive option, given the refined and temporally controlled approaches
described above. Nevertheless, it might be the most straightforward method
to demonstrate the necessity of a specific cell type for a certain psychologi-
cal or physiological function, without later intervention. Such successful tar-
geted ablation has been achieved using the selective expression of attenuated
diphtheria toxin A63,64 or the diphtheria-toxin65–67 receptor from a genomic
locus. A selectively apoptosis-inducing approach has been described as well.68
10.2.7 Optochemical Genetics and Optical Pharmacology

Some of the very first optogenetic actuators relied on mammalian ion chan-
nels, which were optically controlled either by uncaging of their agonist
or by photoisomerization of a linker, which could move the agonist in and
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242 Chapter 10
out of the binding pocket. The ligand-gated ion-channels P2X2 and TRPV1
are examples for the former,15,16 the modified potassium channel SPARK27
an example of the latter. The photoisomerization strategy, using so-called
photoswitched tethered ligands (PTLs), has the advantages of high tempo-
ral resolution (comparable to that achieved by microbial opsins) and does
not require a continuous supply of the caged agonist – once the tether is
established via bioconjugation upon initial application, the system can be

used just like opsin-based optogenetics.26 Hence, it was developed further
and became known as optochemical genetics: usually, native ion channels
are modified to contain a reactive amino-acid residue (mostly cysteine) and
transfected to the cell types of interest. The PTL is applied as a second com-
ponent, which consists of the agonist of the native channel, a photoswitch
(usually an azobenzene) and a reactive group (e.g. maleimide) to connect to
the reactive residue introduced at the right position of the channel to allow
the agonist to bind to the binding pocket. Using this principle, a fast, light-
gated excitatory ionotropic glutamate-receptor termed LiGluR,28 light-gated
metabotropic glutamate receptors 2, 3 and 6,69 light-gated Kv1.3, Kv3.1,
Kv3.4 and Kv7.2 potassium channels30 and an inhibitory potassium-conduct-
ing glutamate receptor termed HyLighter29 have been developed. The merit
of this rapidly expanding toolbox is not merely to provide alternative tools
for optogenetic excitation or inhibition, but to enable the investigation of
the function of a particular receptor in a particular cell type within operating
neural circuits.
In addition to photoswitched tethered ligands that attach to artificially
introduced amino acid residues, photoswitchable tethered or untethered
ligands attach to unmodified ion channels, including the acetylcholine
10:30:45.
receptor, TRPV1 or potassium channels;26,70,71 although lacking genetic spec-

ificity, such tools may be useful as a kind of optically switchable pharmacol-
ogy, if native receptors are to be switched rapidly within a single experiment.
10.2.8 Optogenetic Interference with Subcellular Signalling

Engineered receptors or other proteins designed to alter specific subcellular
signalling pathways in response to light (optogenetics) or to an inert chem-
ical compound (chemogenetics) provide a plethora of options for a future
search for new drug targets, as they allow a molecular signalling cascade to
be triggered in just one type of cell specifically, which cannot generally be
achieved by normal pharmacology.
Optogenetic tools include the engineered “opto-XRs” as well as natural ver-
sions of rhodopsin (described above). The latter generally signal through the
Gi/o pathway, thereby opening GIRKs, inhibiting cAMP production and inac-
tivating voltage-gated calcium-channels.61 ″Opto-XRs”, in turn, are chimeras
in which intracellular compartments of the respective GPCRs to be imitated
have been exchanged for the homologous regions of vertebrate rhodopsin, so
that the signalling pathways of the former are activated by the light-sensitive
gating machinery of the latter. This principle was invented in the laboratory
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72
of Gobind Khorana, and was applied to neuroscience by the Deisseroth lab-
oratory.73 In this way, the Gs-coupled transduction of the β2-adrenoreceptor,
as well as the Gq-coupled pathway of the α1-adrenoreceptor, have been ren-
dered light-sensitive. Potentially particularly relevant to schizophrenia is the
recent development of light-inducible tropomyosin-related kinases A, B and
C.74 With a different, combined optochemical genetic strategy, metabotropic
glutamate receptors 2, 3 and 6 have been rendered light-sensitive with high

temporal precision69 (see Section 10.2.7). Furthermore, for the modulation of
intracellular calcium signalling, a high-calcium permeable version of ChR2,
termed CatCh, has been developed.75 A different path of development has
led to genetically encodable intracellular signalling molecules, which can be
activated by light directly. This includes synthetic systems to selectively acti-
vate transcription, either based on a plant photoreceptor phytochrome76,77 or
on melanopsin.78 Other examples are the optical control of regulators of the
cytoskeleton (Rac, Rho and phosphatidylinositol-3,4,5-trisphosphate)79–81
and of the Ras/extracellular signal-regulated kinase pathway,82 which exem-
plify generic strategies of optical control of protein–protein interactions.83,84
One class of tools, so far only used to remote-control voltage-gated potas-
sium channels, represents a generic approach to optically control endoge-
nous membrane-bound ion channels or even other receptors in a genetically
targeted fashion: lumitoxins, peptide-toxins tethered to a peptide photore-
ceptor domain which is in turn connected to a transmembrane domain, can
be used to switch the toxin-mediated antagonism of a membrane molecule
on and off by brief flashes of light within a few seconds.85
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10.2.9 Chemogenetic Interference with Subcellular Signalling

Chemogenetics, in turn, offers an equally growing range of tools to modify
specific metabotropic receptors. We have already mentioned the DREADDS
hM3Dq and hM4Di as chemogenetic actuators to manipulate electrical activity.
However, as they are actually metabotropic, not ionotropic receptors, they were
originally developed as tools for genetically targeted manipulation of G-protein
signalling, and as such represent a much larger plethora of older as well as more
recent tools (reviewed by Pei et al.4). They include modified GPCRs coupled to
Gi (κ-opiod, α2A-adrenoceptor, 5-HT4B, human-M4), Gq (H1, human-M3) and Gs
(5-HT4A, 5-HT4B, β2-adrenoceptor, melanocortin-4 receptor, rat-M3) proteins.
10.3 Getting
Started: How to Bring Optogenetics
and Chemogenetics to the Laboratory
10.3.1 General Considerations: Which Molecular Tools?
We will highlight some important factors to consider before choosing the appro-
priate tool below. A prevalent practical constraint, however, is ease of availabil-
ity. Some of the most powerful optogenetic activators (ChR2-HR, ChR2-ET/TC,
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244 Chapter 10
C1V1, Chronos and Chrimson) and inhibitors (eNpHR, (e)Arch, (e)ArchT and
Jaws) as well as the DREADDs hM3Dq, hM4Di and rM3Ds are conveniently avail-
able as ready-to-use conditionally and non-conditionally expressing AAV vectors
from established vector core facilities (such as those of the Universities of North
Carolina, Pennsylvania or Stanford). Likewise, various mouse lines expressing
hM3Dq,42 ChR2, Arch(T) or eNpHR86–88 conditionally or non-conditionally, are
publicly available from the Jackson Laboratory, Bar Harbor, Maine, USA. The
latter also holds for a fast growing rich plethora of Cre (and other) driver lines to
target conditionally expressing constructs to a particular cell type.
10.3.1.1 Temporal Resolution
Temporal resolution is often the key to experiments. We argue below that
the biological effect of a certain stimulation might not occur or even be the
opposite when stimulating at one frequency vs. the other. Optogenetics is the
first tool of choice when resolution on the order of milliseconds is required.
Optogenetic silencers are somewhat slower than exciters, but they are usu-
ally used using continuous (not repetitive) stimulation. It should be noted,
however, that the chemogenetic tool hM3Dq, although providing temporal
resolution only in terms of hours, seems to preferentially produce a burst-
like activation or even induce γ-band-oscillations in excitatory cells.42 Often
such high-frequency bursts are necessary to cause sufficient activation (for
example in the dopaminergic system89), and hence researchers go for opto-
genetics – but in fact, hM3Dq might be suitable as well in terms of sufficient
activation as long as the exact timing of each burst is not critical.
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10.3.1.2 Spatial Resolution and Spatial Mismatch

Spatial resolution, targeting exactly the right volume within a brain region or
brain structure, is one of the most profound problems of circuit manipula-
tion. Generally, there are three levers to achieve spatial resolution, in order of
increasing complexity: (1) the spatial restriction of the genetic specification
deployed, (2) the spread of viral transfection, and (3) the spread of the activa-
tor (e.g. the beam of light).

(1) In some cases, the genetic targeting solves the problem of spatial target-
ing, for example in the pioneering study applying optogenetics in the
behaving rodent, ChR2 was targeted to hypocretin-expressing neurons
in the hypothalamus – in this case, enough virus needs to be injected
into the structure to cover its extent, but the genetic specification alone
determines which neurons get transfected.21 Another example is the
widespread use of the Thy1-ChR2 line 18, in which ChR2 is expressed
in pyramidal cells of layer 5 (as well as of some other brain structures),
so that the output from a neocortical area can be controlled quite easily
and specifically.88 A third example is a mouse line, which expresses the
Gs-coupled DREADD rM3Ds selectively in those medium spiny neurons
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(MSN) of the striatum, that project to the globus pallidus (D2-receptor
positive MSNs).90
(2) On the contrary side, there is the example where researchers tried to
target one specific neocortical layer. Although this could also be solved
genetically, since now there are Cre-driver lines for each neocortical layer
available,91 sometimes this is not an option, because the genetic route of
specification is already used to target a specific cell-type within a layer.

In this situation, the determination of the layer needs to be achieved by
injecting minute volumes of a virus, that does not spread very far (50 nl of
lentivirus, in this case).92 Most applications range somewhere in between.
For example, dopamine neurons of the midbrain can be specified using
a tyrosine-hydroxylase (TH) Cre driver line, which exists for both, mouse
and rat. However, TH also specifies noradrenergic neurons. So, for viral
transfection, injection volumes should not be too large, and expression
of the actuator from a genomic locus may not be an option (depending
on the actuator). Generally, for optogenetic application, AAV transfection
volumes of ∼1 µl are used to target a volume of ∼1 µm diameter, which also
roughly corresponds to the volume that can be effectively manipulated by
a beam of laser light of 5–10 mW, exiting from an optical fibre with a core
diameter of 100–400 µm. Of course, many structures do not allow for such
a large volume of manipulation without affecting neighbouring areas.
Transfection volumes for the medial prefrontal cortex, for example, range
from 200 to 400 µl, depending on how focused the expression should
be on the infra- or the pre-limbic area and to what extent neighbouring
structures such as the cingulum or secondary motor cortex can be par-
tially transfected. In general, the restriction of the area of viral transfec-
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tion – in some cases requiring careful titration of small injection volumes,

and angled injection tracts or multiple injection sites – is the primary
approach to spatial control over circuit manipulations. This is because the
extent and strength of expression can (and must) be examined post-hoc
for every experimental animal, using immunhistochemical visualization
of fluorescent markers or tags expressed together with the actuator.
(3) The restriction of the spread of the light or chemical, which is supposed
to gate the heterologously expressed channel or receptor, is another
powerful means of achieving spatial resolution, but also one of the
greatest sources of failure: the volume of viral transfection is not always
centred spherically around the injection side. Moreover, implanting an
optical fibre or guide cannula at exactly the same spot where the virus
was transfected is not trivial. The resulting spatial mismatch between
both components of the system may render the manipulation inef-
fective in some animals. For optical manipulations, two ways exist to
achieve spatial resolution: on the one hand the spread of light of suffi-
cient power is limited due to spatial extension, scattering and absorp-
tion. For example, beyond 1 mm, 473 nm light has lost >90% of its
power, which is not sufficient to activate normal ChR2 reliably if the
initial power was 5–10 mW. (A convenient light transmission calculator
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246 Chapter 10
for brain tissue, allowing for different optical fibres and wavelengths, is
available on the website of the Deisseroth laboratory93). Of course, these
considerations change when using SSFOs or other high-light-sensitive
actuators (see above). On the other hand, the spread of light can be fur-
ther restricted when using a bevelled cannula guide (e.g. from Plastics
One, Roanoke, VA, USA), in which the optical fibre is inserted: in this
way, light only emits at the shorter side of the bevel.5 Fibre guides can
also be used in chemogenetics: when the activating compound (like
CNO) is not applied systemically but infused locally, a spatial resolu-
tion of within a few hundred micrometres can be achieved.

10.3.1.3 The Choice of the Viral or Non-viral Delivery System

In order to transfect the target cells with actuators, three principal strategies
exist: (1) Viral transfection, (2) trangenic expression and (3) in utero electro-
poration. The first two are the most common, as the latter is more difficult
and has not many advantages. (In essence, in utero electroporation can affect
a larger area of the brain compared to viral transfection, without entailing an
expression that affects the whole brain as in the transgenic case. Also, it is
useful for developmental studies, in which viral transfection can mostly not
be applied, given the difficulty of stereotactic surgery in very young animals
and the time needed for the proper expression of the virus.)
In most cases, viral transfection and transgenic expression are combined:
while the virus carries the actuator embedded in a conditionally express-
ing cassette (e.g. lox-sites), the transgenic mouse line supplies the driver
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(e.g. Cre-recombinase expressed under a cell-type-specific promoter), which

determines the cell type in which the virus can be expressed. The reverse may
be applied: a Cre-expressing virus, that can be transported retrogradely, for
example, may be used to specify the expression of an actuator in upstream
neurons, whereby the actuator itself is supplied from a genomic locus. More
rarely, Cre is expressed from “normal” (non-tracer) viruses. This is because
the whole point of using Cre-mice in combination with viruses is that promot-
er-elements that are sufficiently specific to restrict expression to the desired
subtype of cells are generally too large to be encoded in a virus (or unknown
in the first place).94 In addition, viruses have intrinsic tropism. Generally, len-
tiviruses prefer excitatory pyramidal cells, while AAVs prefer interneurons.95
Hence, even the few promoters that may restrict expression to interneurons
when delivered in AAV-vectors, are probably unspecific when applied in len-
tiviruses. And conversely, the widely used calcium/calmodulin-dependent
protein kinase IIα (CamKIIα)-promoter, which was originally developed for
lentiviruses, is indeed not completely specific for excitatory pyramidal cells
when delivered in AAV-vectors (certainly not in AAV195 or AAV5, in which it is
nevertheless often used).
In general, the viral approach is recommended, when small volumes of
tissue are to be targeted, or retrograde or anterograde transmission of the
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actuator is wanted. In turn, transgenic expression can be useful if larger
volumes need to be manipulated, if expression levels across the cells in the
tissue need to be uniform and to achieve probability of expression close
to 100% (e.g. for mapping of local circuitry87), or if there are other reasons
to avoid viral transfection. Of course, often transgenic mice are simply not
available (a situation that is expected to be temporary, however, given the
efforts of the Allen Institute and other mouse genetics laboratories86,88,96).

Often, expression levels achieved with viruses are substantially higher com-
pared to those achieved with transgenic expression, especially if a Cre-lox
approach is used.87 Hence, axonal or synaptic optogenetic activation or inhi-
bition in vivo is expected to be much less reliable, if at all possible, using
stop-floxed transgenic mice. Conversely, with one exception,87,97 expression
levels in transgenic mice are not low enough to have no axonal activation at
all,86 which would be a precondition, for example, for mapping of local cir-
cuits. Efforts have been undertaken to target the actuator to specific cellular
compartments when using viruses, to avoid this unspecificity of localization:
differential tags, e.g. a neurexin-derived tag57 for presynaptic localization, or
a tag with a myosin-binding domain for dendritic localization98 have been
published.
For both viruses and transgenic mice, a non-conditional expression strat-
egy can be used by driving the actuator directly from a cell-type specific pro-
moter. For viruses, the CamKIIα- and the hypocretin-promoter have already
been mentioned as examples. For transgenics, the first ChR2-expressing
lines published were of that kind – they were driven by the Thy1-promoter
which, depending on the line, can be specific for only a subset of neurons
in the brain. Another successful example is that of mice developed via ran-
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dom insertion of bacterial artificial chromosomes (BACs) containing the

actuator within the genomic context of an endogenous gene promoter: with
this approach, mice that express ChR2 for example in GABAergic and cho-
linergic neurons have been developed.96,99 Often expression levels with these
“direct-promoter” strategies display higher expression levels than condition-
ally expressing lines. For example, in the Thy1-ChR2 mice blue laser light
stimulation of 1–2 mW (e.g. at 10 Hz, 10 ms pulses) in the secondary motor
cortex is sufficient to induce circling behaviour, and axonal fibres can be reli-
ably activated.88
When the viral approach is favoured, several viruses are available, and we
have already mentioned the relative preference of lentivirus and AAVs for
principal cells and interneurons. Of these viral vectors lentivirus has two
advantages over AAVs: its packaging size is at least 2 kB larger than that of
AAVs, which is limited to 5 kB, and it can be produced with relatively simple
cell culture methods in the laboratory,100 as long as an ultracentrifuge is avail-
able, and consequently it is much cheaper. Lentivirus is used in vivo at a titre of
107–109 iu (infectious units) and yields usable expression from ∼4–9 days post-
transfection. Its disadvantages are that it usually does not spread as far as
AAVs, given its larger particle size and that it is more complicated to handle,
as it expires within 6–8 hours of defrosting and cannot be refrozen without
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248 Chapter 10
losing large numbers of infectious particles. It should also be noted that
lentivirus may require higher standards of biological safety compared to
AAVs, and that it comes in versions that integrate their genome into the host
genome or do not do so, while AAVs are generally non-integrating. AAVs are
often the vectors of choice, and are used in vivo at titres of 1011–1013 iu. Sero-
types AAV5 or AAV8 are the best bet for most applications – AAV1/2 often
has lower expression levels and expression coverage, while AAV9, AAV10 and
AAVDJ feature higher and broader expression levels. However, the risk of
overexpression, eventually leading to dysfunction or death of cells, is higher
with the latter versions, which is why AAV5 and 8 are currently the most
widely used serotypes. AAV is stored in the freezer, but can be stored in the
refrigerator for a few days and even weeks after defrosting. Its limitations
are its small packaging size, which often limits the promoter elements or
transgenes that can be transfected, difficulty of synthesis of most serotypes
and its slow expression. Although exceptions exists in individual strongly
overexpressing vectors, which are used at 10–14 days post-transfection (if
not diluted in sterile saline), mostly experiments are not performed before
2–3 weeks post-transfection and full expression can take 1–3 months to be
achieved. Expression in axons or synapses at usable levels takes a minimum
of 6–8 weeks (6 weeks per mm of axon in addition to 3–6 weeks basis is a rule
of thumb in the field).
Two other viruses deserve to be mentioned. Rabies or pseudo-rabies virus
is used for retrograde labelling. Herpes simplex features very rapid expres-
sion (resulting in full expression within 3–4 days), but is eventually cytotoxic,
preventing long-term applications.
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10.3.2 Implementation in the Laboratory

10.3.2.1 Dosing
In both chemogenetics and optogenetics, dosing is an important issue. This
applies firstly to the applied dose of viral transfection: often laboratories start-
ing to implement these tools use too high a volume and/or titre. It is essential
to perform at least two rounds of viral transfection combined with histological
studies to establish these two parameters as well as the proper co-ordinates,
which usually differ from what seems optimal according to the stereotaxic
atlas. Volumes should be minimized to restrict expression to the targeted
structure; often seemingly small volumes such as 100 or 200 nl are appropri-
ate for the specific purpose, and titres need to be established for every new
batch of virus and transfection protocol by applying a sensible dilution series.
Similarly, the amount of light used for illumination during optogenetic
experiments is often too high – it can cause excessive local heating thereby
causing unspecific effects or damage, or can result in seizures (see below).
Usually, not more than 10–15 mW light power applied into the brain is
appropriate for optogenetic activators, and not more than 5–10 mW for opto-
genetic inhibitors. High-light sensitive tools like SSFOs, ReaChR, CatCh or
Chronos will require even lower power.
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Dosing in chemogenetic experiments is equally difficult; as with optogenet-
ics, when targeting remote exciters to excitatory cells the margin between pro-
ducing a visible physiological or behavioural effect and causing a subclinical or
overt seizure is quite small. In the case of the hM4D/hM3D-CNO system, con-
centrations of 0.3 mg kg−1 for activation and 3 mg kg−1 for inhibition are often
used. But while 0.3 mg kg−1 can cause seizures in some cases, when epilep-
togenic tissue is transfected, concentrations more than ten-fold higher have

been used to activate cells in non-epileptogenic tissue. A good overview of CNO
doses applied in the studies published to date has been compiled elsewhere.101
10.3.2.2 Adverse Effects
Opto- and chemogenetics cause various adverse effects, which need to be
considered in advance. Firstly, there are obvious problems relating to ste-
reotactic surgery and the implantation of optical or electrophysiological
hardware, including tissue damage and infection. When guide cannulas are
used to insert the optical fibre into the brain on the occasion of every single
experiment, the risk of infection is quite large. In addition, the lower opening
of the cannula might become overgrown with tissue or clotted with blood,
and impede effective transmission of the light. A further unwanted effect –
during the experiment itself – are seizures, which are rarely reported in pub-
lications, but can occur quite easily in certain stimulation paradigms, mainly
when excitatory cells are activated. For example, stimulation of excitatory
cells in the thalamus, CA3, dentate gyrus, secondary motor cortex, amygdala
or medial prefrontal cortex (mPFC) projections to the amygdala may evoke
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seizures at relatively low stimulation intensities.
10.3.2.3 Viral Transfection
Very detailed protocols for viral transfection have been published and do not
need to be repeated here. However, a few crucial aspects are mentioned. The
essential determinants of a successful viral transfection are:

(1) the right speed of infusion (usually 100 nl min−1);
(2) the number of injection sites and volume of infusion as appropriate to
the brain region to be targeted (usually, no more than 0.5–1.0 µl and
1.5–2.0 µl are infused into mouse and rat brains, respectively);
(3) the waiting time post-infusion before removing the needle (usually at
least as long as the duration of the infusion, but at least 5–10 min);
(4) avoidance of back-flow of the viral suspension, either during or after the
removal of the injection needle; it is crucial to avoid mechanical pertur-
bation/vibration while the needle is in the brain as this will create extra
room alongside the needle; creating a “pouch” by driving the needle
∼100 µm below the target site before the injection might help; and
(5) avoidance of overexpression: it may be necessary to dilute viral suspen-
sions to avoid cytotoxic overinfection or overexpression of the transgene.

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250 Chapter 10
10.3.2.4 Setting Up the Optics

While chemogenetics works just as usual pharmacology after the viral transfec-
tion [e.g. with injections of CNO, dissolved in dimethyl sulfoxide (DMSO) (1–10%
final DMSO concentration) and diluted in saline], optogenetics requires certain
optical elements. Again, useful protocols and descriptions have been published
and a lot of material is available on a dedicated optogenetics-wiki.102 In general,

there are four ways of delivering light into the brain for in vivo experiments:

(1) via direct insertion of optical fibres through a guide cannula (available
from, e.g. Plastics One), described in detail elsewhere;103
(2) through fibre-optic cannulas, which are short optical fibres attached to a
connector, which is inserted into the brain and attached to the skull.
A matching connector attached to the cannula before the experiment
allows connection to an optical fibre which is connected to a light
source. This approach has been pioneered by Ed Boyden’s laboratory as
well as by the company Doric Lenses (Quebec, Canada), but fibre-optic
cannulas can now be obtained from multiple suppliers (e.g. ThorLabs,
Newton, New Jersey, USA);
(3) grids of optical fibres or multi-wave guides, which allow the optical exci-
tation of multiple sites in the brain, including with multiple colours;
these are usually custom-orders from specialised companies such as
Doric Lenses, Neuralynx (Bozeman, Montana, USA), Plexon (Dallas,
Texas, USA), Thorlabs, Kendall Research Systems (Boston, Massachu-
setts, USA) and Blackrock Microsystems (Salt Lake City, Utah, USA); and
(4) directly implanted light-emitting diodes (LEDs), potentially attached
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directly to an optical fibre to deliver the light deeper into the brain. This
option is currently still in development, mostly for fully implantable,
wireless optogenetic stimulation, e.g. by Open Source Instruments or
Kendall Research Systems.

The most widely used applications are (1) and (2). (1) is only used when the
experimental approach requires pharmacological access to the brain or when
the guide is to be used to shield illumination off at one side (or to save money).
Before resorting to the more complicated options (3) and (4), it is usually bet-
ter and cheaper to consider multiple implants of single or dual fibre-optic
cannulas (2). In options (1) and (2), a laser of 50–300 mW (100 mW is opti-
mal) is usually deployed, which can be modulated in an analogue as well as
digital fashion (to allow for electronic control of the output power). The laser
is connected to an optical fibre via a fibre launcher (collimator), and then to
a commutator (e.g. from Doric Lenses), which allows for rotation of the fibre
when the animal turns. (Additionally, swivel mounts may be used to stabilize
the optical fibre in the z-axis when the animal’s head moves up and down.) A
second fibre provides the connection from the commutator to the fibre-optic
cannula (2) or directly into the brain (1). LEDs can be used as an alternative
to lasers – they have the advantage of being cheaper, especially at certain
wavelengths such as in the red spectrum (∼600 nm), and allow easy analogue
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modulation. Their disadvantage is that their light is not collimated, and hence
cannot be channelled efficiently into and through an optical fibre. Therefore,
lasers are mostly the first choice in order to have enough room at the higher
power range. While diode-pumped solid-state (DPSS) lasers (e.g. 532 or 561
nm for single-colour experiments, or 594 or 635 nm lasers for dual-colour
experiments) are the only option for silencers (ArchT and halorhodopsin)
and red-shifted exciters (C1V1), blue-spectrum excitation (e.g. for ChR2 and
SSFOs) can be achieved with diode lasers (e.g. 450 nm). These are cheaper and
more stable in output power compared to the commonly used 473 and 488 nm
DPSS lasers. For most applications, optical fibres of 0.39 NA and a 200 µm core
are used at all stages (before and after the commutator and in the fibre-optic
cannula). This relatively simple set-up gets more complicated when dual-co-
lour experiments are required – in this case the lasers (usually 405 nm and 594
nm) need to be set up on an optical breadboard, which allows the laser beams
to be shuttered individually before focusing and collimation into the fibre.
For in vitro applications, either mercury arc lamps or LEDs are used when
the whole field can be illuminated.104 For optogenetic mapping a laser optic
is used, which deploys galvanometric mirrors to steer the beam in the x and y
axes and acousto-optic modulators or filter wheels to control its power.87 Some
companies, including the pioneer in the field, Rapp Optics (Wedel, Germany),
offer turn-key solutions.
10.4 Application
of Optogenetics and
Chemogenetics in Neurological
and Psychiatric Diseases
10:30:45.
10.4.1 Principal Applications: The Road to Drug Discovery

The optogenetic, chemogenetic and related tools described above allow the
modification of the activity of genetically, and often spatially or anatomically,
defined types neurons. Their application in drug discovery for neurologi-
cal and psychiatric diseases can be broadly grouped into three approaches,
which are complimentary and therefore sometimes combined:

(1) induction of neurological/psychiatric symptoms by modification of the
activity of a specific set of neurons;
(2) rescue of pre-existing neurological/psychiatric symptoms in animal
models of the disease by modification of the activity of a specific set of
neurons; and
(3) the elucidation of connections between neurons or their physiological
properties (especially with respect to the network level), which are relevant
to either the disease process or the efficacy of a drug, and which could not
be studied without remote control of genetically specified types of neurons.

The first approach, which is pursued in the behaving animal, serves two pur-
poses. Firstly, it allows a direct causal relationship to be established between
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252 Chapter 10
the activity of a cell type, specific neural connections or a brain region, on the
one hand, and symptoms of the disease, on the other. Secondly – and this is
an often-mentioned but currently entirely unexplored territory – it can serve
to create useful disease models in the first place, which may then serve drug
screening. As such, it can also be used to identify the anatomical or physio-
logical locus of action of a drug, and thereby guide further drug development.
The second approach, like the first one, may help to establish directly the
causal relationship between the activity of a cell type, specific neural connections
or a brain region and symptoms of the disease. It may therefore permit the iden-
tification of cell populations as therapeutic targets, which may later guide the
discovery of druggable targets using single-cell methods of molecular biology.
The third approach fulfils the purpose of elucidating the physiological or
anatomical basis of the presumed therapeutic actions of a drug, or to iden-
tify “nodes” in the neural network, which might be useful to target, given a
certain understanding of the disease process as a whole.
All three approaches generally allow theoretical circuit models of the
respective disease to be tested. Specifically in schizophrenia, various circuit
models have been developed to explain the disease process as well as the
actions of current drugs – those models can now be directly tested, because all
cells that play a role in these models can be directly activated or inhibited and
their presumed effect on either other cells or behavioural symptoms readily
assessed.105–107 Moreover, animal models of the disease can be studied, namely
with respect to the circuit-basis or -origins of the symptoms they display.
The remaining sections of this chapter describe examples from the recent
optogenetic and chemogenetic literature, to elucidate these three approaches
and their corresponding purposes, to spark the creativity of the reader per-
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haps more than abstract definitions may do.

Before describing the application of this technology to understand such
diseases and thereby guide drug discovery, it is worth noting that optogenet-
ics or chemogenetics also hold the promise to treat some of them directly,
which can be an important goal of this research. This view contends that
some severe conditions are and will remain unresponsive to pharmacological
treatment, and optogenetics or chemogenetics serve as great extensions of
the gene-therapeutic toolbox currently under development to fill the gap left
by pharmacotherapy. This is certainly the case for epilepsy, which is refractory
in close to 30% of patients, and especially in cases of focal epilepsy, which is
particularly amenable to gene-therapeutic interventions. Here, progress has
been made in demonstrating the possibility of decreasing epileptic activity
by means of optogenetic108–110 as well as chemogenetic58 inhibition in vari-
ous models of the disease. Another application is Parkinson’s disease, which
could be shown to benefit from an optogenetic version of deep brain stimu-
lation.7,8 Untreatable pain might be another case.111 However, the most likely
therapeutic application of optogenetics will emerge in the treatment of blind-
ness caused by retinal damage or retinal degeneration. This is because, firstly,
the severity of the disease as well as the lack of pharmacological options is
obvious (and thus may justify this gene therapy); secondly, ophthamologists
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who can safely inject a gene-therapeutic vector into the eye outnumber neuro-
surgeons who can perform gene therapy in the brain; and thirdly, the resulting
optical responsiveness of the eye itself constitutes the therapeutic target, and
is not an intermediate stage, where optical hardware needs to be implanted
into the patient’s brain to make use of that responsiveness, as in other appli-
cations. Consequently, much progress has been made in animal models as
well as human tissue on that front.112–115 Most notable, from a technological

perspective, is an approach that involves click chemistry-based modification
of pre-existing potassium channels rendering them light-sensitive, thereby
entirely circumventing the need for gene-therapy.70 However, it is unclear if
optogenetic or chemogenetic treatment will ever be applied to psychiatric
diseases. Obviously, pharmacoresistance is an issue in schizophrenia and
depression, but the risks and invasiveness of such heterodox therapies might
be offset by the larger complexity of the involved circuitry (and presumed
treatment), and the arguably milder suffering of patients under current drug
treatments. The hope (and vision of the present chapter), expressed by Karl
Deisseroth, a leader in the field of rodent optogenetics, is that those novel
tools may vastly enhance our understanding of such diseases to guide novel
pharmacotherapies, rather than constituting therapeutic options in their own
right. In that sense, the circuit level must be brought to the (pharmacological)
treatment, rather than the treatment conducted at the circuit level.
10.4.2 Optogenetic
and Chemogenetic Investigation of
Neurological Diseases
The first studies in the field were performed on neurological rather than psy-
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chiatric diseases, most notably Parkinson’s and, later, epilepsy. While the
epilepsy studies mainly focused on developing a treatment, it is worth exam-
ining the former studies more closely, as they represent a role model for later
optogenetic research in psychiatric diseases.
In the first study that used optogenetics in a disease model, researchers
deployed hemiparkinsonian rats (which result from unilateral 6-hydroxydo-
pamine injections into the medial forebrain bundle) and targeted optoge-
netic actuators to several types of cells and/or projection tracts, which could
be affected by deep brain stimulation (DBS).7 Thus, the paradigm followed
in this project was the rescue approach (paradigm 2, see above), pursuing
the goal of identifying the locus of action of a specific therapy. In distinct
trials, glutamatergic neurons (targeted with the CamKIIα-promoter in a len-
tiviral vector) of the subthalamic nucleus (STN) were either inhibited with
the chloride pump NpHR, or stimulated at γ- or β-range frequency using the
cation-permeant actuator ChR2. Furthermore, astroglia (targeted with the
glial fibrillary acidic protein promoter) in the same region were activated
using ChR2. Finally, using a transgenic mouse line that expresses ChR2 in a
select set of excitatory neurons, including pyramidal cells of neocortical layer
5 (Thy1-ChR2), researchers were able to activate afferent fibres innervating
the STN, at both, “low” (20 Hz) and high (130 Hz) frequency, as well as some
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254 Chapter 10
putative neurons giving rise to these fibres in the primary motor cortex. They
found that only the high-frequency activation of afferent excitatory fibres at
the STN as well as of the corresponding projection neurons in the primary
motor cortex can strongly ameliorate parkinsonian behaviour as observed in
DBS. While the manipulation of excitatory neurons or glia in the STN had no
effect, low-frequency stimulation of excitatory afferent fibres (but not of their
projection neurons in the primary motor cortex) worsened the symptoms.

In addition to the pioneering character of this report, it illustrates import-
ant principles for the application of optogenetics in the context of brain dis-
eases. Firstly, genetically targeted manipulation of neural circuit elements
is crucial to test circuit models of pathology and therapeutic mechanisms
(in this case, the widely believed hypothesis that DBS would act via the
STN-excitatory neurons was shown to be incorrect); secondly, distinguishing
between the activation of fibres vs. the cell bodies of their projection neurons
or of their target neurons is essential, as, counterintuitively, it can have dis-
tinct effects; thirdly, different stimulation patterns or frequencies can have
different, even opposing, effects on downstream circuitry and behaviour –
and whenever this is the case, optogenetic activation would be clearly supe-
rior over chemogenetic manipulation, which largely lacks temporal control.
Similar conclusions can be drawn from a later study, in which MSNs of the
direct, as well as the indirect, pathway of the basal ganglia were manipulated
independently using Cre-driver lines that could target either dopamine-re-
ceptor 1 (D1R)-positive neurons in the direct pathway, or dopamine-receptor
2 (D2R)-positive neurons in the indirect pathway. In this way, it was shown
that activation of D2R-MSNs could elicit parkinsonian symptoms such as
bradykinesia, while activation of D1R-MSNs resulted in the opposite effect
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and could, in fact, rescue such symptoms in a mouse model of the disease.8
10.4.3 Optogenetics
and Chemogenetics in Psychiatric
Diseases
10.4.3.1 Anxiety and Fear Learning
Pioneering optogenetic investigations in the field of psychiatry were con-
ducted in paradigms of anxiety and fear conditioning. Firstly, three landmark
optogenetics papers dissected circuit elements in the amygdala and auditory
cortex that are essential for the acquisition as well as expression of fear mem-
ory associated with an auditory cue (and would therefore correspond to para-
digm 3 above).60,116,117 One of these studies is instructive, as it presents the first
application of a combination of chemogenetics and optogenetics:60 by means
of optogenetic activation (in combination with genetically targeted tracing),
the connectivity of protein-kinase C-δ-positive neurons in the lateral part of the
central amygdala was determined. Using chemogenetic inhibition by
means of the ivermectin-gated chloride-channel described above,23 their
electrophysiological identity and function during fear-conditioning was
demonstrated in vivo.
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Another optogenetic study, focusing on anxiety, rather than fear-learning,
achieved bidirectional control of anxiety (assessed in the elevated plus maze
and the open field) – activation of excitatory fibres originating from the baso-
lateral amygdala (BLA) and innervating the central amygdala proved to be
anxiolytic, while their optogenetic inhibition increased anxiety. Importantly,
optogenetic stimulation of the somata of the BLA cells that gave rise to these
projections did not suffice to cause this effect, illustrating once more the
need to dissociate the manipulation of fibres and cell bodies. Technically,
this separation was achieved via the insertion of the optical fibre through an
implanted bevelled cannula, which prevented the spread of laser light to lat-
eral regions of the amygdala.5 Subsequent studies indicated that such bidi-
rectional control of anxiety can be achieved by targeting other projections,
both from the BLA as well as from the bed nucleus of the stria terminalis, and
that the latter projections can be decomposed into mediators of different
physiological and psychological aspects of anxiety.118,119
10.4.3.2 Optogenetic Investigation of Depression and Negative

Symptoms of Schizophrenia
Substantial progress has been made in the deconstruction of circuits and pro-
jections that control appetitive or motivational behaviour. Given that in rodent
models behavioural tests of depression and of negative symptoms of schizo-
phrenia largely overlap, some of these studies may be regarded as examples
of optogenetic deconstruction of circuits underlying the latter as well; this is
true specifically for assessments of social interaction, anhedonia and motiva-
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tion.120,121 (A similar argument holds for the investigation of social interaction

that is conducted in the context of autism, rather than schizophrenia research.)
It was indeed the first optogenetic rescue study in a model of psychiat-
ric disease that revealed that activation of the mPFC may reverse depressed
behavioural states.6 Mice were exposed to chronic social defeat stress, and
ChR2, driven by an immediate–early gene promoter, was targeted to the mPFC
using a herpes simplex viral vector. A very sparse optical stimulation mim-
icking brief (40 ms) episodes of 100 Hz burst firing was sufficient to reverse
deficits in social interaction and anhedonia (assessed through sucrose pref-
erence), without impairing locomotor behaviour or social memory. Interest-
ingly, optogenetic stimulation had no effect in resilient mice (i.e. in animals
that did not display depressive behaviour after social defeat stress).
Several subsequent optogenetic studies have generally implicated projec-
tions to the nucleus accumbens (NAc) – e.g. projections originating from the
dopaminergic ventral tegmental area (VTA) (preferentially those activated
by the laterodorsal tegmentum), but also from the ventral hippocampus,
mPFC and the BLA – as appetitive.118,122 Conversely, a circuit involving a loop
from the mPFC to the lateral habenula (LH), which in turn innervates VTA
dopaminergic neurons that project preferentially to the mPFC, has emerged
as correlate of aversive or demotivated behaviour. In fact, activation of the
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256 Chapter 10
projection from mPFC to LH (but not of mPFC itself) decreased motivation
in the forced swim test, a common readout for depressive behaviours and
efficacy of antidepressants. Stimulation of the projection from mPFC (but
not of mPFC itself) to the serotonergic dorsal raphe nucleus (DRN) evoked
the opposite effect.123 While direct stimulation of the DRN also increased
motivation in the forced swim test, in addition, it increased locomotor activ-
ity in general – a side effect that was absent when selectively targeting the
mPFC–DRN projection. This combined application of the induction and res-
cue paradigm of optogenetics has thus revealed a target function at the cir-
cuit level which causes specific antidepressant action.
Two further, somewhat contradictory, papers were published simultane-
ously, which directly assessed the effect of the modulation of dopaminergic
neurons on depression-related behaviour.124,125
One study124 applied both the induction as well as the rescue para-
digms. Firstly, the dopaminergic neurons of the VTA were inhibited (using
eNpHR3.0) during tests of motivational and hedonic behaviour (tail suspen-
sion and sucrose preference), and found that such inhibition of dopamine
signalling acutely and reversibly induces the corresponding depression-
related deficits. Secondly, the opposite manipulation was performed (phasic
activation of dopaminergic neurons using ChR2 directly during testing) in
the chronic mild stress (CMS) model of depression, and found this to be suf-
ficient to rescue the CMS symptoms in motivational and hedonic behaviour.
Infusing dopamine-receptor blockers into the NAc was, in turn, sufficient to
impede this optogenetic rescue, which singles out the phasic activity of the
VTA–NAc dopaminergic projection as an antidepressant circuit pathway.
The second study used the induction approach to model a neural phe-
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notype, which is observed in depressive – but not in resilient – mice after

chronic social defeat stress, in order to identify its causal role in the observed
disease pathology at the circuit level.125 This phenotype is an increased pha-
sic firing of VTA dopaminergic neurons, which was mimicked by optogenetic
stimulation of these neurons at 20 Hz while mice underwent a mild social-de-
feat schedule, which on its own would rarely induce depressive symptoms.
Phasic, but not tonic (0.5 Hz) stimulation of dopaminergic neurons during
10 minutes of non-physical exposure to an aggressor, to whose attacks the
mouse had been exposed immediately before (2 min) was sufficient to reliably
induce depressive symptoms as measured by decreased sucrose preference
and social avoidance. Interestingly, the same optogenetic treatment could
also induce depression in mice that had previously proven resilient in the
suprathreshold social-defeat paradigm. By taking advantage of the ability to
stimulate dopaminergic fibres in two of their target structures separately, the
researchers showed that phasic signalling along the dopaminergic projec-
tions to the NAc, but not to the mPFC, could induce such depressive pheno-
types. Interestingly, inhibition of these projections had the reverse effect: not
only did inhibition of the VTA–NAc projection during social defeat inhibit the
induction of symptoms (which singles out these projections as causal), but
the inhibition of the VTA–mPFC projection promoted them, which suggests
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an active antagonistic crosstalk between those two structures, controlling the
susceptibility to depressive symptoms. Although this study was conducted
in the context of depression, it might, perhaps, retrospectively be viewed as
the most important optogenetic investigation of schizophrenia to date. An
overactive mesostriatal dopaminergic projection, caused by disinhibition of
hippocampal and cortical circuits which drive the VTA neurons, as well as a
hypoactive mesocortical dopaminergic projection, potentially resulting from

an increased cortical drive of midbrain GABAergic neurons that selectively
inhibit such mesocortical projection neurons, are at the core of currently
dominant models of schizophrenia pathology.105,106 Although these models
try to explain positive and cognitive symptoms, this study by the Nestler and
Han laboratory provides a powerful link to explaining negative symptoms as
well. In light of this work, circuit pathologies that overactivate the mesolim-
bic and underactivate the mesocortical dopaminergic projections would ren-
der individuals highly susceptible to depressive symptoms, as they remove
the putative barriers of resilience and promote maladaptive depressive states.
The group followed up on their finding mechanistically, and showed that the
optogenetic stimulation of the VTA–NAc projection leads to an increase in
brain-derived neurotrophic factor (BDNF) release in socially stressed, but not
in unstressed, mice, and that such stress could control such BDNF release
via corticotrophin-releasing factor in the NAc.126 This finding provides addi-
tional, circuit-level insight into how stress can promote symptoms of both
depression and schizophrenia. In a follow-up study, the group revealed that
further and excessive stimulation of the VTA-dopaminergic projection (i.e.
a stimulation of already hyperactive DA neurons) can reverse the depressive
phenotype – a revelation of a counter-intuitive mechanism of treatment by
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enhancement, instead of reversal, of underlying circuit pathologies.127

Apart from the importance of these findings, a couple of methodological
notes are to be taken from these studies. Firstly, the second study represents
the first application of a mix of induction and rescue in a graded (subthresh-
old and suprathreshold) disease model to dissect its circuit mechanism.
Given that many schizophrenia mouse models are also partial with respect
to the range of symptoms they display, the same paradigm might be applied
to them, to uncover vulnerabilities as well as the circuit origin of existing
symptoms. Secondly, this illustrates that circuit manipulation, which would
pinpoint the disease mechanism, might not yield any noticeable effect in a
naive animal. It is the intelligent combination of the application of circuit
manipulation in a disease model that renders the former informative in the
first place. Thirdly, the optogenetic induction in the context of subthreshold
social stress (or other conditions) represents a disease model that is unusu-
ally precisely defined in terms of circuitry. This disease model can now be
subjected to pharmacological studies to identify which existing drugs or
drug candidates may influence the individual circuit elements which were
manipulated, and thereby guide drug discovery in a much more targeted way.
Fourthly, and on a more technical note, once again the essential distinction
between cell-type and pathway-specific, as well as between high-frequency
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258 Chapter 10
and low-frequency stimulation, is illustrated. In this case, however, pathway
specificity is achieved via a different strategy: a retrogradely transported virus
(pseudorabies) expressing Cre recombinase is injected into the target of the
projection (NAc vs. mPFC) while a conditionally ChR2-or NpHR-expressing
virus (AAV) is transfected at the origin of the projection (VTA) at a later stage.
In this way, only neurons that project to a certain target will express the opto-
genetic actuator and those neurons are “comfortably” optically stimulated at

their cell somata. (A note of caution, however; this strategy only works if the
respective projection neurons have only one anatomical projection target,
since otherwise a second pathway, although unspecified by the injection of
the retrograde virus, would be manipulated as well.)
It is central to our understanding and treatment of the negative symptoms
of schizophrenia, perhaps more than to our understanding of depression,
that the discrepancy between these studies124,125 is resolved using further
optogenetic investigation in combination with behavioural pharmacology.
While the first study suggests that phasic activation of the VTA–NAc pathway
rescues depressive symptoms in the chronic mild-stress model, the second
study finds that the same manipulation conducted in the context of a sub-
threshold treatment of social stress induces depressive symptoms, even in
resilient mice. In reverse, while the first study finds that inhibition of the
VTA–NAc pathway can acutely cause depressive symptoms, the second study
finds that the same manipulation rescues depressive symptoms induced by
social defeat. Given the central role of D2-dopamine antagonism in the basal
ganglia in the treatment of schizophrenia, the first paper predicts that neg-
ative symptoms of schizophrenia should be worsened (if not caused) by the
standard antipsychotic treatment, and the second study can provide an expla-
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nation why expected circuit alterations in schizophrenia might also cause

depressive symptoms, although it falls short of explaining why such negative
symptoms are so unresponsive to anti-dopaminergic therapy. While potential
solutions to this discrepancy are not discussed here, these findings are superb
exemplifications of the usefulness – if not necessity – of optogenetic circuit
manipulation in understanding disease pathology and drug action. The use
of novel drug candidates, which manipulate the activity of dopamine neurons
directly, rather than their receptors, within optogenetic experiments promise
further insight into the riddle created by the delineated findings.128–130
10.4.3.3 Optogenetic and Chemogenetic Investigation of Circuit

Models of Schizophrenia
We now turn to studies that have addressed circuit models directly relevant
to schizophrenia.
Malfunction of parvalbumin-positive (PV) interneurons is at the core of
major frameworks explaining schizophrenia.105–107 And in reverse, cortical
interneurons, which were previously hard to study because of their small
number and great diversity, were among the first neurons to which optoge-
netic manipulation (and also previous genetic methods) has been applied.
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This is because, for an (opto-)genetic intervention, interneurons are ideal
candidates: their sparseness ensures that not many cells are activated when
they are targeted with focused light,87,97,131 and their diversity, which largely
translates into molecular diversity of distinct marker proteins and pep-
tides,132,133 is ideal for specific targeting of genetic actuators to distinct cell
types to study their function.134 Currently, and in the near future, the rich-
ness of Cre-driver lines targeting those subtypes in combination with opto-

and chemogenetic tools is harnessed, as in the following example.
One optogenetic study deployed a transgenic model of schizophre-
nia, which replicates a supposed core pathology – N-methyl-d-aspartate
(NMDA)-hypofunction on PV-interneurons, modelled through PV-specific
knock-out of NMDA receptor-subunit 1 (NR1)135,136 – to attempt to rescue its
physiological symptoms: PV-interneurons (which lacked NR1) were trans-
fected virally with ChR2 and driven optically at various frequencies, includ-
ing θ and γ bands, in which PV-NR1-knockout mice displayed a significantly
lower or higher baseline power, respectively. Optogenetic activation at
defined frequencies could increase β and γ power, but not θ, in control and
knockout mice, but the capability of driving these frequencies by PV-inter-
neuron activation was markedly lower in knockout mice.136 Stimulation at
randomized inter-stimulus intervals (covering all frequencies at random) in
turn, was also capable of increasing γ-band power – in agreement with ear-
lier observations137 – but failed to significantly increase γ power in knockout
mice. This observation is in agreement with data from pharmacological ani-
mal models as well as schizophrenia patients, which display higher baseline
γ power, but fail to increase γ oscillations on demand, i.e. in a task-related
fashion, which might translate to attentional or other cognitive deficits.138
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This optogenetic experiment was the first to show directly that PV-interneu-
rons, which replicate NMDA-hypofunction, indeed fail to translate acutely
higher excitatory drive into γ-oscillations (potentially due to Gad1-downregu-
lation and, hence, deficient GABAergic synapses in these neurons105,139). The
authors also show that PV-interneuron activation by individual light pulses
entails lower synchronization among PV-interneurons, indicated by higher
latencies and variance of spike times, and that pyramidal cells are less inhib-
ited in the early phase of the γ-cycle, which could explain the failure of their
on-demand synchronization.136
The first application of chemogenetics in schizophrenia research focuses140
on a similar question: the role of oscillations and their coherence across
connected brain areas. The authors used the chemogenetic silencer hM4Di
(activated by 2 mg kg−1 CNO) to reproduce a reduced activity of the medi-
odorsal thalamus, which has been observed in patients during tests of execu-
tive function. Such inhibition impaired both reversal learning (a measure of
executive function) and working memory, as measured using the rewarded
alternation paradigm in the T-maze. They showed that the latter was accom-
panied by an hM4Di–CNO–induced failure of increase in phase-locking and
coherence of oscillations specifically in the β-frequency range between mPFC
and mediodorsal thalamus during the choice phase (which requires working
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260 Chapter 10
memory). This study is thus the first direct demonstration that cognitive
symptoms of schizophrenia may be due to an impairment of “communica-
tion through coherence”141–143 between brain regions.
A seminal paper made use of newly developed tools which allowed sus-
tained optogenetic activation of one cell population (with SSFOs, see above)
and simultaneous manipulation of a second population of cells via laser
light of a red-shifted wavelength (using C1V1, see above), to investigate the

interplay of excitatory and inhibitory neurons.37 The authors investigated the
effect of shifting the excitation/inhibition (E/I) balance towards more exci-
tation or more inhibition by selectively activating pyramidal neurons or PV-in-
terneurons in the mPFC during a range of tasks that are relevant to autism
and schizophrenia, including locomotor activity, novel object preference
(in a simplified version), non-reciprocal sociability and associative learning
assessed through fear conditioning. They showed that sustained activation of
pyramidal cells (although not inhibition of interneurons) can cause decreased
sociability (i.e. a lack of preference for the chamber that contains a mouse
over an empty chamber) and strongly decreased acquisition of fear memory,
while locomotor activity and novel object preference were unaffected. It also
resulted in a strong increase in basal high-γ-band (60–100 Hz) oscillations,
as observed in both the prefrontal cortex of patients with schizophrenia and
after non-specific pharmacological block or PV-specific knockout of NMDA
receptors in rodents.136,144 The simultaneous activation of PV-interneurons
(which in its own right had no effect on learning or sociability) during pyra-
midal cell excitation could rescue the sociability deficit to a small extent, but
not the learning impairment. [However, given that the mPFC-overexcitation
also decreased anxiety (in the elevated plus-maze test), it is unclear to what
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extent the decreased fear conditioning was due to an actual learning deficit
or rather due to a reduction in fear.] The authors also showed, using patch-
clamp recordings in slices and information theoretical analysis, that overacti-
vation of pyramidal cells decreases synaptic information transmission, while
compensatory excitation of PV cells improves it.145 Although the selectivity of
the impairment of novelty preference for a social vs. a non-social stimulus is
striking, the interpretation of the results in the E/I balance framework might
be distracting, and furthermore, the most meaningful outcomes of the study
for the understanding of schizophrenia might be the negative ones: interest-
ingly, mPFC activation did not induce hyperlocomotion. Given that current
circuit theories that put disinhibition at the core of schizophrenia pathology
disagree with respect to which locus of disinhibition – mPFC106,107 or ven-
tral hippocampus105 – would be crucial in producing a hyperdopaminergic
phenotype, this might be regarded as an important advance. Furthermore,
the only partial rescue of the induced deficits via activation of PV-interneu-
rons, as well as the failure to produce any of the tested impairments via
PV-cell inhibition (which should also increase excitation) suggest that the
E/I-framework is misleading and the manipulation subject to two of the cave-
ats of optogenetic stimulation raised above. Firstly, activation of pyramidal
cells effectively recruits PV-interneurons anyway, and secondly, optogenetic
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stimulation arguably destroys some information by preventing or modifying
endogenous neural activity. Although both problems would be expected to
be more pronounced in pulsed modes of activation, they cannot be excluded
when using step-function opsins, as well. Therefore, instead of fine-tuning
the E/I balance, the authors might have rather crudely disrupted the normal
functioning or information representation with their intervention (which
might explain the limited success of rescue as well as the failure of induc-
tion with PV-cell inhibition), and the “specificity” of the effect for social stim-
uli might be simply due to the fact that the novelty of non-social stimuli is
mediated by the hippocampus146 rather than the mPFC. A third caveat is that
optogenetic inhibition and optogenetic excitation are not equivalent: the
optogenetic inhibitor eNpHR3.0 is a chloride pump, whose sustained acti-
vation can disrupt GABAergic inhibition (and possibly make it excitatory, see
above) after some time, so that PV-interneurons might no longer be effec-
tively inhibited. This, of course, leaves the methodical advancement of the
paper unquestioned, and poses a very instructive example of the difficulty of
conducting and interpreting optogenetic experiments.
A related, methodically instructive study investigated another balance rele-
vant to schizophrenia: that between D1-and D2-receptor mediated dopamine
signalling. Using selective optogenetic inhibition of D1-receptor expressing
neurons in the mPFC, temporal control during a cognitive task was impaired,
while their stimulation improved task performance.147 This manipulation
may thus serve as a very specific animal model of D1-dopamine hypofrontal-
ity seen in schizophrenic patients.
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10.4.4 Optogenetic and Chemogenetic Pharmacology

An important special case of paradigm 3 (see above), is the use of optogenetic
stimulation in lieu of electrical stimulation, e.g. in order to study the phar-
macology of synaptic transmission from a particular cell type.148 The first
application of optogenetics to synaptic plasticity illustrates the merit of this
approach: in the classical paradigm to study long-term potentiation, an elec-
trode is placed in the Schaffer collateral tract to activate synapses of the hip-
pocampal CA3 > CA1 excitatory connection. The problem with this approach
is that this electrical stimulation recruits fibres from both the ipsilateral and
contralateral CA3. Optogenetic stimulation allows this problem to be cir-
cumvented, since only one CA3 subfield can be rendered sensitive to optical
stimulation via unilateral viral transfection. This approach revealed that the
observed plasticity effect – in both hemispheres – is largely due to the fibres
originating in the left CA3, while fibres originating in the right CA3 show
hardly any long-term potentiation.149 Subsequently, the authors repeated this
experiment, while an antagonist of the NMDA-receptor subunit 2B, GluN2B,
(Ro 25-6981) was bath-applied during the optical stimulation. This revealed
that fibres originating from the left (“plastic”) CA3 display a GluN2B-medi-
ated current almost twice as large as those from the right, and that its antag-
onism abolishes their potential of synaptic plasticity. This study illustrates
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262 Chapter 10
the principle of optogenetic pharmacology in great clarity: the selectivity of
optogenetic stimulation enables the isolation of signalling pathways in neu-
ral circuits, which could not be separated by any of the conventional meth-
ods, and therefore opens previously closed doors into the pharmacological
study of signal transduction mechanisms in brain tissue.
A similarly clear and more psychiatry-related example, is the demonstration
of the depressing effect of the muscarinic acetylcholine-receptor 4 (M4) on cor-

tico-striatal excitatory synapses. Here the BAC-transgenic ChAT-ChR2 mouse,
described above, was used to selectively and temporally precisely activate cho-
linergic neurons in the striatum, in conjunction with electrical stimulation
of cortico-striatal fibres (in the corpus callosum) and a novel M4-selective
positive allosteric modulator (PAM) (VU0152100).150 With this paradigm, the
authors could demonstrate the depressing effect of cholinergic activity on cor-
tico-striatal synapses and its selective enhancement by a M4-PAM.
A third example studying the locus of the action of a drug (cocaine) in a tar-
get structure (NAc), used optogenetic stimulation of different afferents (from
the mPFC and from the BLA) to monitor the input-specific change of release
probability over time to reveal a projection- and treatment-specific synaptic
change induced by drug withdrawal.151
Chemogenetic pharmacology might be even closer to direct pharmacolog-
ical research, as explained by one of the pioneers in the field:152 as a plethora
of receptors solely activated by a synthetic ligand (RASSLs) or DREADDs is
being developed,4 we have the ability to activate molecular signalling path-
ways of psychiatrically relevant receptors exclusively in genetically and spa-
tially specified cell populations, but not other cells, which also express the
native receptor and would be equally affected in classical pharmacological
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studies. In this way both the induction (1) and the rescue (2) paradigms can
be applied to identify the circuit-locus of action of a specific drug target, e.g. a
certain receptor. This could provide valuable information to guide the devel-
opment of further drug candidates, which have a bias towards manipulat-
ing certain types of cells or parts of molecular cascades that are particularly
prominent in those cells, but not others. Similar reasoning applies at the
subcellular level: DREADDs can be designed and/or used to activate only a
subset of the signalling cascades originating from native GPCRs on demand
in order to investigate such cascades in isolation or in order to screen for
drugs that have a therapeutically relevant bias to some of the cascades, such
as the antipsychotic aripiprazole.152
10.4.5 An Optogenetic View on Brain and Behaviour?

The pre-optogenetic era has left us with literally thousands of publications
investigating which brain regions are responsible for which behaviours using
electrical or chemical lesions, as well as pharmacological (infusion-based)
or electrical stimulation or inhibition of parts of the brain, and correlating
such manipulations with behavioural readouts. Functional brain imaging
has added to this literature.
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The systems theorist Niklas Luhmann noted that complexity can only be
reduced by complexity. But such pre-optogenetic methods lack the level of
complexity at which the brain seems to operate and, with hindsight, often
appear simply as too coarse to dissect the basis of neural computation; this
is true for multiple dimensions, including temporal precision, spatial reso-
lution, selectivity for the genetic or functional subtype of neuron, selectivity
for specific neural projections, avoidance of the manipulation of fibers of

passage and specificity for the subcellular (axonal vs. dendritic) domain of
neurons.
As a tentative speculation, the first decade of opto- and chemogenetic cir-
cuit dissection might be read as an emerging criticism of the view of the
brain that the earlier technologies have left us to hold. For example, when
the first precise, bimodal optogenetic switching of anxiety was shown to
depend on one specific projection inside the amygdala (see above5), such
improved structure–function correlation appeared as a refinement of the
older knowledge about the importance of the amygdala for anxiety. However,
since then, the same precise bimodal control of anxiety has been achieved by
manipulating entirely different projections (see above119). The same image
emerges in social behaviour, where optogenetic control was first demon-
strated by the modulation of the prefrontal cortex (see above6,37), but was
recently also achieved by manipulating a projection from the amygdala to
the ventral hippocampus.153 A third example is working memory, as assessed
in the T-maze, which could be impaired by chemogenetic inhibition of the
mediodorsal thalamus (see above140), but also by disinhibition of the dorsal
hippocampus/CA1.62
The same argument has also been made in reverse, as the manipulation
10:30:45.
of a specific brain region (the mPFC) had no effect on behavioural moti-

vation in a specific (forced-swim) paradigm, but the isolated manipula-
tion of two projections from these regions had robust, but antagonistic
effects.123
A third source of worry about the earlier data has been exemplified multiple
times in this chapter: optogenetic stimulation at high frequencies can have a
different, even opposite, effect compared to stimulation at low frequencies.
These observations raise doubts about the value of hard-won “insights”
gained by lesions, electrical or chemical manipulations, and leave us with
an emerging concept of the brain in which neither behavioural functions
are very tightly correlated to specific brain regions, or the reverse. Instead,
perhaps most brain regions participate in many behavioural functions
with individual local connection motifs and global projections, as well as
the dynamics of the activity along such neuronal fibres, being the true
correlates of elements of cognitive function. Obviously, this change in
conceptualization will affect our thinking about schizophrenia, as most
circuit models, e.g. the prominent “Lisman/Grace” model105 and its mul-
tiple versions, but also older dopamine-related models, operate largely
within the thinking of anatomical correlates of cognitive function and
dysfunction.
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264 Chapter 10
10.5 Conclusion
Arguably, the two most promising routes towards the discovery of new ther-
apeutics in schizophrenia are genomics and genetically targeted circuit dis-
section.154 In this chapter, we have highlighted how the two primary tools for
circuit dissection, opto- and chemogenetics, have been and can be applied to
understand the circuitry that underlies psychiatric symptoms and its patho-
logical aberrations. We propose that these tools can be applied in one of three
principal paradigms: (1) the induction approach of causing schizophrenia-re-
lated symptoms by targeted circuit manipulation, thereby producing a whole
new class of animal models of the disease; (2) the rescue approach of alleviat-
ing schizophrenia-related deficits in animal models of the disease to directly
establish causality between circuit elements and symptoms; and (3) the ana-
tomical and functional characterization of neurons and neural projections
thought to be relevant to schizophrenia, including opto- and chemogenetic
pharmacology. In recent years, substantial progress has been made along all
three paradigms to dissect the basis of neurological and psychiatric diseases.
However, the direct testing of circuit models of schizophrenia using these tech-
nologies still lies ahead of us, and animal models based on targeted circuit
manipulations still have to emerge. We expect that such selective manipula-
tions will greatly help us to understand how current antipsychotics and emerg-
ing drug candidates act at the circuit level, and that such understanding will
provide instructive knowledge as well as animal models and biomarkers to
guide the development of the next generation of schizophrenia drugs.
Acknowledgements
10:30:45.
Schizophrenia-related research in the groups of the authors is funded by the

Wellcome Trust, the John Fell Fund, the Brain & Behavior Research Foun-
dation and a Roche post-doctoral fellowship. This chapter has benefitted
greatly from the training, advice and research experience D. K. received in
the laboratories of Gero Miesenböck, Karl Deisseroth and Ed Boyden, and in
particular from advice from Boris Zemelman, Markus Wölfel, Dirk Trauner,
Darcy Peterka, Tom Davidson, Maisie Lo, Brian Allen, Anthony Zorzos, Tev
Stachniak, Andrew MacAskill, Michael Kohl, Klaus Wanisch and Rob Wykes.
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10:30:45.
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Subject Index
Locators in bold refer to figures/tables
ablation of cells 241 amphetamines 76, 79

acetylation, histone 33, 36, 37–9 amygdala 255. see also fear
activation, optogenetic/chemoge- conditioning
netic 237, 237–9, 243 anhedonia 71, 235, 255. see also
activity-dependent transcription negative symptoms
factor 1-A 32, 35 animal studies
activity-regulated cytoskeleton- DISC1 interactome 151–62,
associated (ARC) protein 154–5, 159–60
signaling complex 15 dopaminergic system 79
addiction 35, 71, 78. see also glutamate hypothesis of
self-medication hypothesis schizophrenia 122–3, 124–6
adenosine A1 receptor neuroinflammation 54–5, 57
(A1R) 205–6, 219 neuronal maturation and
10:30:47.
AKT1 gene 9, 11 integration 145

AKT-GSK3 pathway 174–5, 175, preclinical drug testing 124–6
179–82, 183, 185, 190–1 prenatal infection 48
allelic spectrum of schizophrenia self-medication hypothe-
risk 4 sis 75–8, 75–82
allosteric modulators 127, 129, 130, see also models of
131, 132 schizophrenia
α-7 nicotinic acetylcholine receptor antibiotic drugs 59
(nAChR) 8 anti-inflammatory drugs 11, 59
Alzheimer’s disease 38, 182 antipsychotic drugs
AMPA (α-amino-3-hydroxy-5- AKT-GSK3 signaling 185
methyl-4-isoxazolepropionic acid) animal studies 76
receptors common genetic variation 12
biological functions of DISC1 DISC1-GSK3 pathway 184
interactome 147 first/second-generation
glutamate hypothesis 116, 173–4, 185
117, 129, 130–1, 188 GSK3 networks 173–4, 185–6
glycogen synthase kinase models of schizophrenia
networks 188 90, 105
AMPAkine/CX516 130 negative symptoms 258
273
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274 Subject Index

antipsychotic drugs (continued) avolition 71. see also negative
neuroinflammation 54, 59–60 symptoms
NRG1-ErbB signaling 100
optogenetics/ bacterial endotoxins 54, 55, 56, 57
chemogenetics 262 basolateral amygdala (BLA), fear
positive symptoms 71 conditioning 255
prenatal influenza BDNF. see brain-derived neuro-

infection 48 trophic factor
substance use to alleviate side behavioral effects
effects 74–5, 82 glycogen synthase kinase net-
see also medication and see works 186, 186–8
specific drugs optogenetics/
anxiety 181, 235, 254–5, 263 chemogenetics 262–3
apathy, optogenetics 235. see also behavioural models of
negative symptoms schizophrenia 91–3
apomorphine 76 behavioural tests, preclinical animal
apoptosis 32, 39, 49, 223 drug testing 124–6
ARC (activity-regulated cytoskele- β-arrestin-2 scaffolding protein
ton-associated) protein signaling glycogen synthase kinase net-
complex 15 works 175, 178
aripiprazole 185–6, 262 protein-protein interactions,
arrestin, protein-protein interac- dopamine receptors 223–4
tions 214–15, 223–4 β-catenin, glycogen synthase kinase
The Art of War (Sun Tzu) 1 networks 182, 187
astrocytes 51, 52, 56 biomarkers, glycogen synthase
attentional deficits 71, 90 kinase networks 188–90
10:30:47.
AKT-GSK3 pathway 181 bipolar disorder 10, 12, 14, 16

CNTRICS initiative 123 AKT-GSK3 pathway 182
latent inhibition test 77 diagnostic taxonomy 90, 91
models of schizophrenia 92 disrupted in Schizophrenia 1
neuroinflammation 46, 54 interactome 150
preclinical animal drug epigenetics 29, 32
testing 126 genetic architecture 91
see also latent inhibition; pre- bitopertin 129, 130
pulse inhibition BLA (basolateral amygdala), fear
attention-deficit hyperactivity disor- conditioning 255
der 13, 16 blood biomarkers 189
atypical antipsychotic drugs brain-derived neurotrophic
173–4, 208 factor (BDNF)
Australian Government, National epigenetics 35
Drug Strategy 73 glycogen synthase kinase
autism 12, 16 networks 175, 182, 184
diagnostic taxonomy 91 optogenetics/
epigenetics 29, 35, 37, 39 chemogenetics 257
autoimmunity, major histocompati- broad-spectrum antibiotic drugs 59
bility complex 8, 11 Brugada syndrome 12
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Subject Index 275

C1V1, optogenetics 238, 244, 251, 260 CHRNA7 gene 8
CACNA1C/CACNB2 calcium channel chromatin remodeling complexes
subunits 12 (CRCs) 37
Caenorhabditis elegans 240 chronic stress. see stress
caffeine 205 Chronos, optogenetics 238, 244,
calmodulin 101, 221–2, 225–6 248
calnexin 212–13 circadian rhythms 186, 186–7

cannabinoid (CB)1 receptors 219 circuit models of
cannabis self-medication 71, 83 schizophrenia 258–61
to alleviate cognitive c-Jun N-terminal kinase signaling
symptoms 73–4 pathway 11
to alleviate medication side classifications of schizophrenia 16,
effects 74–5 90, 91
experimental evidence 81–2 clozapine 38, 185
cannabis use, and risk of schizo- DISC1 interactome 156, 162
phrenia 74, 104 optogenetics/
case-control studies, genetics of chemogenetics 239
schizophrenia 7 protein-protein
catechol-O-methyltransferase interactions 208
(COMT) 100, 103–5 CMS (chronic mild stress) model of
cause-and-effect relationships 31 depression 256
caveolin-1, protein–protein CNO (clozapine-N-oxide) 239
interactions 213–14 CNTRICS initiative (Cognitive Neu-
celecoxib 59 roscience Treatment Research to
cell apoptosis 32, 39, 49, 223 Improve Cognition in
cell migration, DISC1 Schizophrenia) 92, 123
10:30:47.
interactome 143 CNVs. see copy number variations

cell nucleus, DISC1 interactome 148 cocaine 218, 262
cell signaling. see signaling cognitive symptoms 46, 90, 123,
mechanisms 125, 202
centrosomes, DISC1 animal studies 76–7, 124
interactome 148–9 epigenetics 35, 37, 39
channelrhodopsin-2 (ChR2) 236, inadequacy of medication
237–8, 243 for 71
chemical mutagenesis, animal models of schizophrenia 92
models 94 optogenetics/
chemogenetic activation/inhi- chemogenetics 235
bition 239, 240, 243. see also pharmacological targets 130
optogenetics/chemogenetics self-medication 72–4,
chemokines, neuroinflammation 49 81–3
childhood abuse 31–2. see also see also memory deficits
epigenetics complexity, systems theory 263
chlorpromazine 76, 185 COMT (catechol-O-methyltransfer-
ChR2 (channelrhodopsin-2) 236, ase) 100, 103–5
237–8, 243 construct validity, mouse
Chrimson, optogenetics 238, 244 models 151
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276 Subject Index

copy number variations (CNVs) 5, 6, depressive disorder 14, 16
7, 12–15, 13, 28, 30, 90, 91 chronic mild stress model 256
cortical interneurons 258–61 DISC1 interactome 150
cortisol 32 epigenetics 35
coverage, genetics of optogenetics/
schizophrenia 19 chemogenetics 255–8
CRCs (chromatin remodeling designer receptor exclusively acti-

complexes) 37 vated by designer drugs (DRE-
Crick, Francis 236 ADD) 236, 243, 244, 262
cross-cultural studies of development. see neurodevelopmen-
schizophrenia 179–80 tal models
C-terminus of GAIP interacting pro- Diagnostic and Statistical Manual of
tein (GIPC) 225 Mental Disorders 150
CX516/AMPAkine 130 diagnostic taxonomy of schizophre-
cyclic adenosine monophosphate nia 16, 90, 91
(cAMP) dihydroxyphenylacetic acid (DOPAC)
optogenetics/ model 99
chemogenetics 240 dimerization, dopamine D2
protein–protein interac- receptors 217
tions 205, 210 diphtheria-toxin, ablation of
cytochrome P450 (CYP450) cells 241
enzymes 74–5 disease progression, role of
cytokines 60 neuroinflammation 57–9
disease prevention disrupted in schizophrenia 1
strategies 59 (DISC1 gene) 14, 141–2
epidemiological DISC1 network 142, 143
10:30:47.
perspectives 53 DISC1-GSK3 pathway 175,

latent neuroinflammation 58 176–7, 184–5
neurodevelopmental effects 52 future directions 162–4
neuroinflammation 49, 50–1 models of schizophrenia 91,
priming by prenatal 95–6, 101–2
infection 56 mouse models 151–62, 154–5,
159–60
DAT. see dopamine transporter neurodevelopmental func-
DDC (DOPA decarboxylase) 100 tions 142–5, 143
de novo mutations/variants 3, 6, 7, neuronal signaling and synap-
15, 19, 29, 30 tic plasticity 143, 146–7
decision-making impairments 124. nicotine self-medication 78
see also cognitive symptoms role in schizophrenia 150–1,
deep brain stimulation (DBS) 253 152
delayed non-match to posi- subcellular functions 143,
tion (DNMTP), animal drug 148–50
testing 125 DIXDC1 gene, Wnt-GSK3
deletions 6, 28, 29–30, 91. see also pathway 182
copy number variations dizocilpine, glutamate hypothesis of
delusions 70–1. see also positive schizophrenia 117–18
symptoms dizygotic (DZ) twin studies 2–3, 32–3
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Subject Index 277

DNA methylation, epigenetics dopamine D5 receptor
34–5, 36 (D5R) 215–17
DNA methyltransferase dopamine receptor-interacting pro-
(DNMT) 34–5 tein (DRiP) 78, 212
DNMTP (delayed non-match to posi- dopamine transporter (DAT)
tion), animal drug testing 125 knockout mice 178
DOPA decarboxylase (DDC) 100 protein–protein interac-

DOPAC (dihydroxyphenylacetic acid) tions 217, 221
model 99 DRD1 gene 8
dopamine hypothesis/dopaminergic DREADD (designer receptor exclu-
system 76, 92, 99, 100 sively activated by designer
animal studies 76, 79 drugs) 236, 243, 244, 262
anti-dopaminergic ther- DRiP (dopamine receptor-
apy 173–4, 185–6, 203, 258 interacting protein) 78, 212
biological functions of DISC1 drug development 17
interactome 147 animal studies 122–3
first and second-generation common genetic
antipsychotic drugs 173–4 variation 8–12
genetic variation, implications glutamate hypothesis 116,
for drug discovery 8 127–33, 129
and glutamate hypothe- neuroinflammation 59–60
sis 119–21, 121 optogenetics/
glycogen synthase kinase net- chemogenetics 251–3
works 178, 183 protein–protein interactions,
neuroinflammation 50, 51, 52, dopamine receptors 226–7
55–6 rare genetic variation 12–15,
10:30:47.
nicotinic acetylcholine 13
receptors 72 see also medication
self-medication DTNBP1 (dysbindin) gene 91, 102–3,
hypothesis 78–9 162
see also protein–protein duplications, genetic 6, 29–30
interactions, dopamine dynamin I 188, 225
receptors dysbindin (dystrobrevin binding
dopamine D1 receptors (D1Rs) protein-1) 91, 102–3, 162
optogenetics/ DZ (dizygotic) twin studies 2–3,
chemogenetics 261 32–3
protein–protein
interactions 204–15 early intervention 95
dopamine D2 receptors (D2R) effect size, schizophrenia associated
glycogen synthase kinase variants 3
networks 190 electroretinography (ERG) 188–90
optogenetics/ elevated plus-maze test,
chemogenetics 261 optogenetics/chemogenetics 260
protein–protein interactions ENCODE (Encyclopedia of Func-
206–7, 217–18, 222–5 tional DNA Elements) project 29
dopamine D3 receptor (DR3) endophenotypes 17
218–19 endotoxins, bacterial 54, 55, 56, 57
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278 Subject Index

ENU (N-ethyl-N-nitrosourea), models of schizophrenia 97
chemical mutagenesis 94 optogenetics/
environmental factors 31–2, 39, chemogenetics 254–5
163. see also epigenetics; gene- fetal brain development, neu-
environment interactions roinflammation 55–6. see also
epidemiological perspectives infection
DISC1 interactome 156, 157 filamin A, protein–protein

epigenetics 29–31 interactions 222
gene-environment first generation (typical)
interactions 103 antipsychotic drugs 173–4, 185
neuroinflammation 47–8, 53 five choice serial reaction time task
substance use 70–1 (5-CSRTT) 126
epigenetics 28, 34, 40–1 5HT2. see serotonin 5-HT2A
DNA methylation 34–5, 36 receptor
epidemiological 5-hydroxymethylcytosine 35
perspectives 29–31 5-hydroxytryptamine receptor 2C
GABA-ergic neurons 39–40 (HTR2C) gene 37–8
histone acetylation 33, 37–9 5-methylcytosine, DNA
histone methylation 35–7, 36 methylation 35
as memory system 33–4, 37 forced swim test 159–60, 161, 256
missing heritability 31–3, 39 fragile X syndrome 182
epilepsy, optogenetics/ frontal cortex, nicotinic acetylcho-
chemogenetics 253 line receptors 72
epistasis 16–17, 91
definitions 19 G × E models. see gene-environment
DISC1 interactome 163–4 interactions
10:30:47.
genetics of schizophrenia 5 GABA (λ-aminobutyric acid)

models of receptor
schizophrenia 104–5 epigenetics 34, 39–40
EPSCs (postsynaptic currents), and glutamate hypothesis 121,
glutamate synapse 116 121–2
ERBB4 gene 11, 96–100, 182–3 models of schizophrenia 97
ERG (electroretinography) 188–90 neuroinflammation 56
etiology of schizophrenia 31, 203 optogenetics/
executive function deficits 123, 125. chemogenetics 261
see also cognitive symptoms protein–protein interac-
extracellular signal-regulated kinase tions 205, 215–17
(ERK) 214–15 GAD1 gene, epigenetics 37–8, 40
extrapyramidal motor symptoms 78 GADD45. see growth arrest and DNA
damage
face validity, mouse models 151 GAIP (G α interacting protein) 225
false discovery rate, genetics of gamma-subunit of the coatomer
schizophrenia 7, 16 protein complex (λ-COP) 211–12
family-based studies 7 GASP (G-protein-coupled
fear conditioning receptor-associated sorting
epigenetics 34, 35 protein) 225
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Subject Index 279

GCTA (genome-wide complex trait CNTRICS initiative 123
analysis) 30–1 common genetic variation 8
gene-environment interactions 2, 5, DISC1 interactome 146–7, 157
16, 17, 18, 29 and dopamine system 119–21,
definitions 19 121, 207, 211
DISC1 interactome 153, and GABA 121, 121–2
154–5, 156 glycine/serine 127–30, 129

models of glycogen synthase kinase
schizophrenia 103–4 networks 188
gene x gene interactions. see epistasis metabotropic receptors 131–3
genetically targeted manipulation of models of schizophrenia 96–8,
neural activity 235–41, 237. see 99
also optogenetics/chemogenetics molecular and cellular
genetics of schizophrenia 1–5, components 116, 117
16–17, 18–19, 90–1 neuroinflammation 56, 58–9
AKT-GSK3 pathway 179–80 NMDA receptors 116–19, 117,
allelic spectrum of 127–8, 129, 131–3, 188
schizophrenia risk 4 pharmacological targets
common genetic variation 8–12 127–33, 129
definitions 19 postsynaptic proteins 15
DISC1 interactome 150–1, 154 preclinical animal drug test-
epidemiological ing 122–3, 124–6
perspectives 29–31 glutamic acid decarboxylase 1
heritability 2–3, 18 (GAD1) gene 37–8, 40
limitations of genetic glycine 127–30, 129
studies 17–18 glycogen synthase kinase (GSK) 11,
10:30:47.
rare genetic variation 12–15, 13 14, 173–4

schema of DNA variation 6 AKT-GSK3 pathway 174–5,
genome-wide association studies 175, 179–82, 183, 190–1
(GWAS) 3, 5, 7, 9–10, 15–17, antipsychotic drugs 185–6
29–30, 90–1 BDNF-GSK3 pathway 175, 182,
genome-wide complex trait analysis 184
(GCTA) 30–1 behavioral effects 186, 186–8
genomics 5–7, 6 biomarkers 188–90
GIPC (C-Terminus of GAIP circadian rhythms 186, 186–7
interacting protein) 225 DISCI interactome 142–6, 143,
GIRK (G-protein-coupled inwardly 151, 152, 159, 161, 163
rectifying potassium channels), DISC1-GSK3 pathway 175,
optogenetics/chemogenetics 176–7, 184–5
240, 242 future directions 190–1
glucocorticoid receptor (GR), NRG1-GSK3 pathway 175,
epigenetics 31–2 182–3
glutamate hypothesis/glutamate regulating pathways 174–9
receptors 115–16 signaling networks 175
AMPA receptors 116, 117, 129, Wnt-GSK3 pathway 175,
130–1, 188 175–6, 182
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280 Subject Index

glycoprotein, DNA methylation 34 optogenetics/
G-protein-coupled receptors chemogenetics 255
(GPCRs) 203, 204 pathophysiology 181
optogenetics/chemogenetics histone acetylation, epigenetics 33,
241, 242, 243, 262 36, 37–9
protein–protein interac- histone acetyltransferases (HATs )
tions 219, 221, 222 37–8

see also dopamine receptors histone deacetylase (HDAC)
G-protein-coupled inwardly inhibitor 37, 38, 39
rectifying potassium histone methylation,
channels (GIRK), optogenetics/ epigenetics 35–7, 36
chemogenetics 240, 242 histone methyltransferases
G-protein-coupled receptor- (HMTs) 35
associated sorting protein holistic approaches 31
(GASP) 225 homeostatic control 51
gray matter HTR2C (5-hydroxytryptamine
DISC1 interactome 151 receptor 2C) gene 37–8
MRI scanning 189 human immunodeficiency virus
neuroinflammation 48, 58 (HIV) 203
pathophysiology 181, 182 Huntington’s disease 38
GRM3 gene 8 hyperactive phenotypes
growth arrest and DNA circadian rhythms 186
damage (GADD)45 enzyme DISC1 interactome 153,
family 35 154–5, 155, 157–8, 159–60,
GSK. see glycogen synthase kinase 160, 162
GWAS. see genome-wide association models of schizophrenia 92,
10:30:47.
studies 97–9, 101–3

self-medication hypothesis
H3K4, epigenetics 35–6, 36 76, 79
hallucinations 70–1. see also
positive symptoms immune function. see neuroim-
haloperidol 185 mune mechanisms
halorhodopsin 240 incidence/prevalence of
HATs (histone schizophrenia 19, 173, 202
acetyltransferases) 37–8 infection 203
HDAC (histone deacetylase) DISC1 interactome 156
inhibitor 37, 38, 39 epidemiological
Hegemann, Peter 236 perspectives 47–8
heritability of schizophrenia 2–3, major histocompatibility
18, 29, 31–3, 39, 203. see also complex 8, 11
genetics models of schizophrenia 103
hippocampus neuroinflammation 55–7
DISC1 interactome 151 see also neuroimmune
epigenetics 32, 35, 40 mechanisms
lesion studies 77, 80 inflammation. see
nicotinic acetylcholine neuroinflammation
receptors 72 influenza infection 47, 48, 156
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Subject Index 281

inhibitors, optogenetic/ latent neuroinflammation 57–9
chemogenetic 237 LCRs (low copy repeats) 6
inositol hexakisphosphate kinases LDCs (lymphocyte-derived cell
(IP6Ks) 175, 177 lines) 180
insertions 6. see also copy number lead exposure 157
variations learning deficits 92. see also
intellectual disability, epi- cognitive symptoms; memory

genetics 35, 37, 39. see also lesion studies 236
cognitive symptoms leukomalacia ( white matter
interleukins 49, 50, 52, 53, 54, 56 damage), 48, 53, 54
interneurons, circuit models of leukotrienes,
schizophrenia 258–61 neuroinflammation 49
intracellular signaling, DISC1 LI (latent inhibition) paradigms 77,
interactome 143, 146–7, 159–60, 92, 184
161–2 lifetime trajectory model 95. see
intramuscular injection, turpentine also neurodevelopmental models
oil 55 light-sensitive ion channels. see
inversions 5, 6. see also copy optogenetics/chemogenetics
number variations LIM homeobox genes (Lhx),
inwardly rectifying potassium epigenetics 39–40
channels 220 lipopolysaccharide (LPS) 54, 55,
ion channels 56, 57
chemically gated. see lithium 38, 191
optogenetics/chemogenetics long intergenic non-coding RNAs
protein–protein interactions, (lincRNAs) 12
dopamine receptors 220–1 long-term memory (LTM) 33–4, 37,
10:30:47.
IP6Ks (inositol hexakisphosphate 123. see also memory deficits

kinases) 175, 177 long-term potentiation (LTP),
synaptic 34, 37
JNK (c-Jun N-terminal kinase) low copy repeats (LCRs) 6
signaling pathway 11 lumitoxins, optogenetics 243
lymphocyte-derived cell lines
kainate receptors, glutamate (LDCs) 180
synapse 116
ketamine 117, 118 magnetic resonance imaging. see
kinases 9, 11, 243 MRI scanning
kinesin intracellular transport, major histocompatibility complex
DISC1 interactome 158–60, 160 (MHC) 8–11, 91
knockout mice 93, 95–7, 104–5, 184 MAP2K7 gene 11
beta-catenin 187 maturational processes. see
DA transporter 76, 78, 79, 178 neurodevelopmental models
DISC1 interactome 153–7, Measurement and Treatment
154–5, 162–3 Research to Improve Cognition in
MAP2K7 11 Schizophrenia (NIMH) 92
serotonin system 179 medication
latent inhibition (LI), paradigms 77, for cognitive and negative
92, 184 symptoms 71
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282 Subject Index

medication (continued) mitogen activated protein kinase
substance use to alleviate kinase (MAP2K) 11
side-effects 74–5, 82 mixed-lineage leukemia (MLL),
see also antipsychotic drugs; epigenetics 35, 36
drug development; phar- MLL3 gene, epigenetics 38
macology; self-medication models of schizophrenia 89–90, 105
hypothesis behavioural 91–3

memory deficits 71, 90 development of valid
AKT-GSK3 pathway 181 models 93–4
animal studies 77 DISC1 101–2
cannabis self-medication 73 DTNBP1 102–3
CNTRICS initiative 123 dysbindin 105
DISC1-GSK3 pathway 159–60, gene-environment
184 interactions 103–4
epigenetics 33–4, 37 gene × gene interactions
glutamate hypothesis of 104–5
schizophrenia 117 genetic architecture 90–1
models of schizophrenia 92 NRG1 96–100, 103–4
neuroinflammation 46, 48, optogenetics/
54, 55, 57 chemogenetics 252
preclinical animal drug phenotypic
testing 123, 125 characterisation 94–6
see also cognitive symptoms; see also animal studies
working-memory monozygotic (MZ) twin studies
mental retardation. see cognitive 2–3, 32–3
symptoms; intellectual disability Morris water maze 77, 79, 123, 158,
10:30:47.
metabotropic glutamate receptors 159–60

(mGluRs) 129, 131–3 motivational deficits
methylation, DNA/histone 34–7, 36 CNTRICS initiative 123
MHC (major histocompatibility optogenetics/
complex) 8–11, 91 chemogenetics 255
microarrays, genomics 5, 7, 12, 16 preclinical animal drug
microglia 51–4, 56–60 testing 124
microsatellites, genetics of see also negative symptoms
schizophrenia 5, 6 motor proteins, DISC1
microtubules/microtubule interactome 149–50
associated proteins 187–8 mouse models, DISC1 interactome
Miesenböck, Gero 236 151–62, 154–5, 159–60. see also
minocycline (broad-spectrum animal studies
antibiotic) 59 MRI scanning, biomarkers of
MIR137 gene 9, 12 schizophrenia 189
mismatch negativity (MMN), animal multifactorial disorder,
drug testing 126 schizophrenia as 29, 153
missing heritability 3, 18, 29, multigenic disorders 28, 29. see also
31–3, 39 epistasis
mitochondria, DISC1 muscarinic acetylcholine-receptor 4
interactome 149 (M4) 262
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Subject Index 283

muscarinic receptors, synthetic 236 neuroanatomical/neurocog-
mutagenesis, animal models 94 nitive phenotypes, DISC1
mutations 28. see also copy number interactome 151
variations; de novo mutations; neurodegenerative diseases 182
genetics epigenetics 35, 38
MZ (monozygotic) twin studies neuroinflammation 52, 57, 58
2–3, 32–3 optogenetics/

chemogenetics 253–4
nAChRs (nicotinic acetylcholine neurodevelopmental models 77,
receptors) 8, 72 141
NADH (nicotinamide adenine animal models 95
dinucleotide dehydrogenase) 149 DISC1 interactome 142–5,
nanopore based technologies, 143, 157–8, 159
genomics 5 neuroinflammation 47, 52, 57
National Drug Strategy, Australian self-medication
Government 73 hypothesis 79–80, 82
National Institute of Mental Health see also neuroimmune
(NIMH) 90, 92 mechanisms
nBA F (neuronal Brg1/hBrm neuroexophilin (NXPH2) gene 91
associated factor) 37 neurofilament (NF)-M protein 211
NCS-1 (neuronal calcium sensor-1) neurogenesis 142–3, 143, 182
224–5 neuroimmune mechanisms 46–7
negative symptoms 46, 71, 89–90, 202 DISC1 interactome 156
animal studies 78 epidemiological
medication for 71 perspectives 47–8
models of schizophrenia 91 major histocompatibility
10:30:47.
optogenetics/chemogenetics complex 8, 11
235, 255–8 see also infection;
pharmacological targets 130 neuroinflammation
self-medication 73–4 neuroinflammation 47, 49–52,
neocortex, epigenetics 38–40 50–1, 60
neonatal quinpirole model 77 DISC1 interactome 156
neonatal ventral hippocampal disease prevention
lesion model 77, 80 strategies 59–60
N-ethyl-N-nitrosourea (ENU), epidemiological
chemical mutagenesis 94 perspectives 53
N-ethylmaleimide-sensitive factor experimental evidence 53–5
(NSF) 215, 224 fetal brain development 55–6
neural Darwinism 39 inflammatory response
neural progenitor cells 142–3, 143 system 49–52
neuregulin (NRG1) gene 10, 11, 91 latent 57–9
glycogen synthase kinase major histocompatibility com-
networks 175 plex 8, 11
models of schizophrenia 91, neurodevelopmental
94, 96–100, 103–4 effects 47, 52
NRG1-GSK3 pathway 182–3 post-natal inflammatory
neurexin 1 (NRXN1) gene 13, 14, 91 processes 203
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284 Subject Index

neuroinflammation (continued) glutamate hypothesis 116–19,
priming by prenatal 117, 127, 128, 129, 130–3
infection 56–7 glycogen synthase kinase
see also cytokines; infection; networks 188
neuroimmune mechanisms optogenetics/chemogenet-
neurological disease. see ics 259–60, 261
neurodegenerative diseases protein-protein interac-

neuronal activity, genetically tions 207–9, 210, 218
targeted manipulation 235–41, N-methylglycine (sarcosine) 128,
237. see also optogenetics/ 130
chemogenetics non-allelic recombination 6
neuronal calcium sensor (NCS)-1 Novel Methods leading to New
224–5 Medications in Depression and
neuronal maturation/integration/ Schizophrenia (NEWMEDS) 92
migration, DISC1 NRG1 gene. see neuregulin (NRG1)
interactome 143, 144–5 gene
neurosignaling. see signaling NRXN1 (neurexin 1) gene 13, 14, 91
mechanisms NSF (N-ethylmaleimide-sensitive
neurotransmitter receptors factor) 215, 224
animal studies 78 nuclear localization sequence (NLS),
common genetic variation 8 D1R homo-oligomers 204
see also specific receptors nuclear receptor subfamily 3, group
(e.g. glutamate receptors) C, member 1 (NR3C1) 32
NEWMEDS (Novel Methods leading nucleus, cellular, DISC1
to New Medications in Depression interactome 148
and Schizophrenia) 92 nucleus accumbens (NAc) 255
10:30:47.
next-generation sequencing NXPH2 (neuroexophilin) gene 91

(NGS) 5, 29
nicotinamide adenine dinucleotide odds ratios (OR)
(NADH) dehydrogenase 149 allelic spectrum of
nicotine self-medication 71, 82–3 schizophrenia risk 4, 4
to alleviate cognitive definitions 19
symptoms 72–3 OHKY (3-hydroxykynurenine),
to alleviate medication side neuroinflammation 58–9
effects 74–5 olanzapine 38, 184, 185
experimental evidence 79–81 olfactory epithelial (OE)
predictions, testing 78–9 biomarkers 189
nicotinic acetylcholine receptors one gene-one disease approach 29
(nAChRs) 8, 72 opsins, optogenetics 236, 237, 238,
NIMH (National Institute of Mental 240–3, 251, 261
Health) 90, 92 optochemical genetics 236. see also
N-methyl-d-aspartate (NMDA) below
receptors 15 optogenetics/chemogenetics
DISC1 interactome 146–7, 234–5, 264
157, 159–60, 161–2 ablation of cells 241
and dopamine 120, 121 behavioral effects 262–3
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Subject Index 285

chemogenetic activation Pavlovian autoshaping, preclinical
239, 243 animal drug testing 124
chemogenetic inhibition PCP (phencyclidine) 117, 118,
240, 243 123, 128
drug development 251–3 PDE4 (phosphodiesterase) 143,
genetically targeted manipula- 146–7, 149, 151, 152, 159, 161
tion of neural activity pedigree-based studies 7, 150, 184

235–41, 237 penetrance 3, 91
laboratory perception
applications 243–51 CNTRICS initiative 123
neurological disease preclinical animal drug
applications 253–4 testing 126
optochemical genet- perinatal trauma, etiology of
ics and optical schizophrenia 203
pharmacology 241–2 peripheral blood cell
optogenetic activation 237, biomarkers 189
237–9 personalized treatments 1
optogenetic inhibition 239–40 pharmacogenetics. see optogenetics/
pharmacology 261–2 chemogenetics
psychiatric diseases 254–61 pharmacological models 78
silencing synaptic pharmacological targets. see drug
transmission 241 development
subcellular signaling pharmacologically selective actua-
pathways 242–3 tor-effector molecule
opto-XRs, optogenetics 242 (PSAM–PSEM) system 236
OR. see odds ratios pharmacology, optogenetics/
10:30:47.
over-dominance, genetics of schizo- chemogenetics 261–2

phrenia 18, 19 phencyclidine (PCP) 117, 118, 123,
128
P2X2 (purinergic receptor), optoge- phenotypic characterisation
netic activation 237 DISC1 interactome 151,
P50 suppression, sensory gating 74 154–5, 159–60
PAMs. see positive allosteric models of schizophrenia 94–6
modulators phosphodiesterase (PDE4) 143,
Parkinsonian bradykinesia 235 146–7, 149, 151, 152, 159, 161
Parkinson’s disease, optogenetics/ photoswitched tethered ligands
chemogenetics 253–4 (PTLs) 242. see also optogenetics
parvalbumin-positive (PV) plasticity, synaptic. see synaptic
interneurons 258–61 plasticity
pathogen recognition receptors 49 pleiotropy, genetics of schizophre-
pathway analysis 2, 5, 7, 15, 16, nia 5, 16, 18, 19
18–19, 30 polygenic disorders 29, 30, 93
kinases 11 polymorphisms. see single
MIR137 gene 12 nucleotide polymorphisms
NRXN1 gene 14 polyriboinosinic-polyribocytidilic
see also signaling mechanisms acid (polyI:C) 54, 55
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286 Subject Index

population prevalence rate 19. prevalence/incidence of
see also incidence/prevalence of schizophrenia 19, 173, 202
schizophrenia prevention strategies,
positive allosteric modulators neuroinflammation 59–60
(PAMs) 127, 129, 130–2, 262 priming effects,
positive symptoms 46, 70–1, neuroinflammation 56–7
89, 202 probabilistic learning, preclinical

animal studies 76 animal drug testing 124
and cannabis use 74, 83 problem solving deficiency 92. see
models of schizophrenia 91 also cognitive symptoms
post-natal inflammatory processing speed deficiency, models
processes 203 of schizophrenia 92
postsynaptic currents (EPSCs), progression of disease, role of
glutamate synapse 116 neuroinflammation 57–9
post-synaptic density-95 (PSD-95), pro-inflammatory cytokines. see
dopamine receptors 209–11 cytokines
post-translational histone proliferation, neural progenitor
modifications (PTM) 35–7, 36 cells 142–3, 143
post-traumatic stress disorder 32 prostaglandins 49
potassium ion channels, dopamine prostate apoptosis response,
receptors 220 dopamine receptors 223
PPI. see pre-pulse inhibition models protein 4.1N, dopamine
PPM1E (protein phosphatase 1E) receptors 222–3
gene 37 protein kinase B 9, 11
preclinical drug testing, animal protein phosphatase 1E (PPM1E)
studies 122–3, 124–6 gene 37
10:30:47.
predictive validity, mouse protein–protein interactions, dopa-

models 151 mine receptors 202–4
prefrontal cortex (PFC) adenosine A1 receptor 205–6,
epigenetics 36, 38 219
optogenetics/ arrestin 214–15, 223–4
chemogenetics 261 calmodulin 221–2, 225–6
prenatal infection. see infection; calnexin 212–13
neuroimmune mechanisms cannabinoid 1 receptors 219
prenatal/perinatal stress 31–2. see caveolin-1 213–14
also epigenetics cyclic adenosine monophos-
pre-pulse inhibition (PPI) phate 205, 210
models 92, 95, 97–104 dopamine D1
AKT-GSK3 pathway 181 receptors 204–15
DISC1 interactome 159–60, 161 dopamine D2 receptors 206–7,
DISC1-GSK3 pathway 184 217–18, 222–5
dopamine receptors 205, 208 dopamine D3 receptor 218–19,
preclinical animal drug 225–6
testing 126 dopamine D5 receptor 215–17
self-medication hypothesis 73, dopamine transporter 217, 221
76, 80–1 drug development 226–7
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Subject Index 287

GABA receptor 205, 215–17 RG-1678 (bitopertin) 129, 130
gamma-subunit of the coatomer rhodopsin 236, 238, 240, 241,
protein complex 211–12 242, 251
G-protein-coupled rimonabant 74, 82
signaling 203, 204, 221, 222 risk variants, genetics of
ion channels 220–1 schizophrenia 3–5, 4
N-ethylmaleimide-sensitive risperidone 184, 185

factor 215, 224 RNA, common genetic variation 12
neurofilament -M protein 211
NMDA Receptors 207–9, 210, sarcosine (N-methylglycine)
218 128, 130
post-synaptic density-95 209–11 scaffolding proteins
serotonin 5-HT2A glycogen synthase kinase net-
receptor 220 works 175, 178
somatostatin receptor 219–20 post-synaptic density 209–11
PSAM–PSEM (pharmacologically protein-protein interactions,
selective actuator module-effector dopamine receptors 223–4
molecule) system 236 Schizophrenia Consortium data
psychiatric diseases, optogenetics/ set 30
chemogenetics 254–61 SCN (suprachiasmatic nucleus) 186
PTLs (photoswitched tethered Scottish pedigree, DISC1 interac-
ligands) 242 tome 150, 184
PTM (post-translational histone second-generation antipsychotic
modifications) 35–7, 36 drugs 173–4, 208
PV (parvalbumin-positive) segmental duplications 6
interneurons 258–61 self-medication hypothesis 71,
10:30:47.
82–3
quetiapine 185 to alleviate medication side-
quinolinic acid 58–9 effects 74–5, 82
quinpirole model 77, 185–6 animal studies 75–8
cannabis self-medication 71,
ras-related C3 botulinum toxin sub- 73–4
strate (RAC)1 146 epidemiological
reasoning deficiency 92. see also perspectives 70–1
cognitive symptoms epigenetics 35
receptor dimerization, dopamine D2 experimental evidence 79–82
receptors 217 neurodevelopmental
receptors, neurotransmitter. see models 79–80, 82
neurotransmitter receptors nicotine self-medication 71,
receptor-type tyrosine 72–3
kinases 182–3 predictions, testing 78–9
recombination, non-allelic 6 sensitization, nicotine
reelin gene (RELN) 34 addiction 79–80
Rett syndrome 35 sensorimotor gating 48
reversal learning, preclinical animal animal studies 95
drug testing 125 cognitive symptoms 73
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288 Subject Index

sensorimotor gating (continued) social defeat stress
neuroinflammation 54 DISC1 interactome 156, 157
P50 suppression 74 optogenetics/
protein–protein interactions, chemogenetics 255
dopamine receptors 208 sodium butyrate 37. see also histone
see also pre-pulse inhibition deacetylase inhibitors
sequencing by synthesis somatostatin receptor

chemistry 5 (SSTR5) 219–20
serine sorting nexin (SNX)-1 gene 215
DISC1 interactome 143, 147, spatial memory 159–60. see also
151, 152, 159–60, 161–2 memory deficits
glutamate hypothesis of spatial resolution, optogenetics/
schizophrenia 127–30 chemogenetics 244–6
serotonin 5-HT2A receptor Spudich, John 236
and dopamine receptors 220 stabilized step-function opsins
glycogen synthase kinase (SSFOs) 238, 246, 248, 251, 260
networks 175, 179, 190 statistical power, definition 19
neuroinflammation 56 stereotypies, animal studies 76, 79
second-generation STM (short-term memory) 33–4.
antipsychotic drugs see also memory deficits
173–4, 185 stress
sex-specific phenotypes, models of DISC1 interactome 156, 157
schizophrenia 94–5 epigenetics 31–2
short-term memory (STM), model of depression 256
epigenetics 33–4. see also optogenetics/
memory deficits chemogenetics 255
10:30:47.
signaling mechanisms striatum 39, 40

DISC1 interactome 143, structural variations, genetic 5, 6, 7,
146–7, 159–60, 161–2 12–15, 13. see also copy number
glycogen synthase kinase variations
networks 175 subcellular signaling pathways,
optogenetics 242–3 optogenetics 242–3
see also pathway analysis substance use. see self-medication
silencing synaptic transmission 241 hypothesis
single nucleotide polymorphisms sucrose preference test 78
(SNPs) 5–7, 6, 16, 30–1 Sun Tzu (The Art of War) 1
AKT-GSK3 pathway 179–80, suprachiasmatic nucleus (SCN) 186
181 synaptic functioning
common genetic variation 8, 11 DISC1 interactome 160
DISC1 interactome 150 glutamate synapse 116, 117
smoking. see cannabis; nicotine long-term potentiation 34, 37
social cognition deficiency silencing synaptic
CNTRICS initiative 123 transmission 241
glutamate hypothesis 117 see also neurotransmitter
models of schizophrenia 92 receptors
preclinical animal drug synaptic plasticity
testing 125 AKT-GSK3 pathway 182
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Subject Index 289

DISC1 interactome 143, tumor necrosis factor (TNF)-α 49,
146–7, 156, 162 51, 52, 53, 57
glycogen synthase kinase net- turpentine oil 55
works 187, 191 twin studies 2–3, 32–3
optogenetics/ typical (first generation)
chemogenetics 261 antipsychotic drugs 173–4, 185
synthetic muscarinic receptors 236

systems theory, complexity 263 UNC compounds, aripiprazole
derivatives 185–6, 262
TAOK2 (TAO kinase 2) gene 11 urban birth, etiology 203
targeted therapeutics 1
tau protein 188 validity, mouse models 151
TCF4 (transcription factor 4) 91 valproate, epigenetics 38
temporal resolution, optogenetics/ variable expressivity 16, 19
chemogenetics 244 variance 3–5, 4, 18
THC (tetrahydrocannabinol) 74, common genetic
81–2 variation 8–12
third-generation sequencing, rare genetic variation 12–15, 13
genomics 5 schema of DNA variation 6
thousand-and-one-amino acid 2 ventral hippocampal lesion
kinase (TAO K2) 11 model 77, 80
three-chamber social approach ventral tegmental area (VTA )
task 78, 125 optogenetics/
Timothy syndrome 12 chemogenetics 256–8
T-maze barrier task, preclinical protein–protein
animal drug testing 124 interactions 205, 208
10:30:47.
tobacco. see nicotine verbal learning and vigilance,

toll-like receptors, models of schizophrenia 92
neuroinflammation 54 VIPR2 gene (vasoactive intestinal
toxoplasma parasite 156 peptide receptor 2) 13, 14–15
transcription domain (TAT ) 203 viral delivery systems, optogenetics/
transcription factor 4 (TCF4) 91 chemogenetics 246–8, 249
transient receptor potential visual learning, models of
channel (TRPC), protein-protein schizophrenia 92
interactions 220–1
translocase of outer mitochondrial white matter damage 48, 53, 54
membrane 70 homolog A WIN 55212-2 81
(TOMM70A) gene 37 Wnt-GSK3 signaling pathway 175,
translocations 5, 6. see also copy 175–6, 182
number variations working memory. see memory deficits
Treatment Units for Research on
Neurocognition in Schizophrenia zif268 (activity-dependent transcrip-
working group 92 tion factor 1-A) 32, 35
trial-unique delayed non-matching ziprasidone 185
to location, preclinical animal ZNF804A (zinc finger protein)
drug testing 125 gene 91
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Drug Descrubrimiento de Farmacos

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Published on 28 April 2015 on http://pubs.rsc.org | doi:10.1039/9781782622499-FP001

Drug Discovery for Schizophrenia

RSC Drug Discovery Series

Advisor to the Board:

Titles in the Series:

10: Extracellular and Intracellular Signaling

33: Comprehensive Biomarker Discovery and Validation for Clinical

How to obtain future titles on publication:

For further information please contact:

Drug Discovery for

RSC Drug Discovery Series No. 44

Print ISBN: 978-1-78262-026-6

© The Royal Society of Chemistry 2015

All rights reserved

Published by The Royal Society of Chemistry,

Registered Charity Number 207890

For further information see our web site at www.rsc.org

who was diagnosed in his final year of secondary school.

RSC Drug Discovery Series No. 44

Chapter 1 The Genetics of Schizophrenia 1

1.5 What Genetics is Telling Us about Schizophrenia 16

Chapter 2 The Impact of Epigenetics in Schizophrenia Research 28

RSC Drug Discovery Series No. 44

Chapter 3 Developmental Neuroimmune Mechanisms

3.4 Fetal Brain Development in the Event of Inflammation  55

Chapter 4 The Self-medication Hypothesis in Schizophrenia:

4.1 Substance Use and Schizophrenia: Clinical Aspects 70

4.5.2 Neurodevelopmental Models of Schizophrenia 79

Chapter 5 Modelling Schizophrenia: Strategies for Identifying

6.1 The Glutamate Hypothesis of Schizophrenia 115

Glutamate Synapse 116

Chapter 7 Disrupted-in-Schizophrenia-1 (DISC1) Interactome

7.1 Introduction 141

8.1 Introduction 173

8.2 GSK3 in Schizophrenia 179

8.5.1 Peripheral Blood Cells and Olfactory

Chapter 9 Protein Interactions with Dopamine Receptors as

9.1 Introduction 202

10.1 Introduction 234

10.2.1 Optogenetic Activation 237

10.4 Application of Optogenetics and Chemogenetics

Subject Index 273

The Genetics of Schizophrenia

The greatest difficulty in finding treatments for schizophrenia is that we

RSC Drug Discovery Series No. 44

in brain morphology. Genetics can also help us to understand the underlying

1.2 What Genetics Can Tell Us about Schizophrenia

used to understand the disorder.

1.2.1 The Heritability of Schizophrenia

Heritability(h2 ) = 2(r (MZ ) – r (DZ )) (1.1)

The Genetics of Schizophrenia 3

when using subjects from national records. Either way, schizophrenia is

with improved genotyping technologies have already uncovered many

1.2.2 The Genetic Architecture of Schizophrenia

Figure 1.1 Schizophrenia-associated

The Genetics of Schizophrenia 5

carry manageable numbers of risk variants, while the probability of devel-

1.3 The Tools of Genomics

in many ways, from single nucleotide polymorphisms (SNPs) to more

The Genetics of Schizophrenia 7

Carefully considered study designs combined with constantly improving

1.4 What Genetics Has Told Us about Schizophrenia

33: Comprehensive Biomarker Discovery and Validation for Clinical

Chapter 1 The Genetics of Schizophrenia 1

1.5 What Genetics is Telling Us about Schizophrenia 16

Chapter 2 The Impact of Epigenetics in Schizophrenia Research 28

Chapter 3 Developmental Neuroimmune Mechanisms

3.4 Fetal Brain Development in the Event of Inflammation 55

Chapter 4 The Self-medication Hypothesis in Schizophrenia:

4.1 Substance Use and Schizophrenia: Clinical Aspects 70

4.5.2 Neurodevelopmental Models of Schizophrenia 79

Chapter 5 Modelling Schizophrenia: Strategies for Identifying

6.1 The Glutamate Hypothesis of Schizophrenia 115

Glutamate Synapse 116

Chapter 7 Disrupted-in-Schizophrenia-1 (DISC1) Interactome

7.1 Introduction 141

8.1 Introduction 173

8.2 GSK3 in Schizophrenia 179

8.5.1 Peripheral Blood Cells and Olfactory

Chapter 9 Protein Interactions with Dopamine Receptors as

9.1 Introduction 202

10.1 Introduction 234

10.2.1 Optogenetic Activation 237

10.4 Application of Optogenetics and Chemogenetics

Subject Index 273

1.2 What Genetics Can Tell Us about Schizophrenia

1.2.1 The Heritability of Schizophrenia

1.2.2 The Genetic Architecture of Schizophrenia

1.3 The Tools of Genomics

1.4 What Genetics Has Told Us about Schizophrenia

1.4.1 Common Variation

1.4.1.2 The Major Histocompatibility Complex

1.4.2 Rare Variation

1.4.2.1 Disrupted in Schizophrenia 1 (DISC1)

1.4.2.3 Vasoactive Intestinal Peptide Receptor 2 (VIPR2)

1.4.2.4 Lessons from Sequencing Studies

1.4.3 The Future of GWASs

1.5 What Genetics is Telling Us about Schizophrenia

117. A. Monji, T. A. Kato, Y. Mizoguchi, et al., Prog. Neuropsychopharmacol.

2.3 Where is the missing heritability?

2.4.1 DNA Methylation

2.4.2 Histone Methylation

2.4.3 Histone Acetylation