Pharmacological Research, 2009

Methods in Behavioral Pharmacology

Allan V Kalueff, PhD

Jan 26,
26 2009: TUMC Pharmacology

Learning Objectives
U
Understand
d
d basic
b i principles
i i l off neurophenotyping
h
i
research: Why? How? Tests vs. Screens
List the three tiers of behavioral phenotyping of
t
transgenic
i mice
i
Describe multiple assays for motor dysfunction in
rodents
Describe rodent models of depression and anxiety
Describe micro- and macro-behavioral approaches to
neurophenotyping research

Why do we need behavioral tests to

understand human brain disorders?

Whyy use animal models?

Ethical considerations
Time
Fiscal
Fi
l
Experimental control

Behavioral tests are used to study:

Effects of environment (e.g., stressors) on behavior

Adverse effects of genetic mutations
Gene x Environment interactions
Side effects of drugs
Drug interactions
Pharmacological effects of drugs in experimental
models of brain disorders
Drug x Gene x Environment interactions

Why use behavioral models?

Throughput

Cost

Ph i l i l C
Physiological
Complexity
l it

Modified from Kokel and Peterson, 2008

Why the
mouse?

Mice and humans share

over 90% of genes
Easy to breed, can be housed in large #s
Precise gene targeting available
At least 80 strains have abnormal depression or
anxiety-related phenotypes
Similarities to human neural circuits

Quantifying behavioral responses

Scoring customized for specific subjects/test
E
Experimenters
i
t
should
h ld b
be:
1)blind to treatment
2) high in inter/intra rater reliability
3) consistent (time, season, place)

Pletnikov, 2006

Three tiers of behavioral phenotyping

(C
(Crawley,
l
2000)
Tier 1. General Observations

Do animals look healthy?

Do they gain weight normally?
Are theyy g
grooming
g normally?
y
Do they move around the cage properly?
Do they show reflex responses, such as eye
blink, ear twitch, whisker twitch, and righting
reflex?

Three tiers of behavioral phenotyping

(C
(Crawley,
l
2000)
Tier 2. Motor functions and Sensory function

Motor function (e.g. gait analysis or motor activity)

Hearing (e.g.
(e g startle)
Tactile sensation (e.g. von Frey hairs)
Thermal sensation (e.g. paw withdrawal to heat)
Vision. Visual cliff test, with glass floor.
Smell. Novel odor test, i.e. vanilla painted onto a wall.

Tier 3. Specific tests in Basic Science Research on the

pharmacology of Neurological and Psychiatric
Diseases

Model vs. Screen

Model
inducing
i d i a d
depressed
d or anxious
i
state (e.g., Open field test novelty)
Changes
g in p
physiology
y
gy and behavior
Emphasis on construct and etiological
validity
Screen
Often used to test drugs or
Test genetically altered model
Emphasis on face and predictive
validity

Traditional tests of affective states

(emotionality)
Anxiety
y
Open field test
Elevated plus maze

Depression
p
Forced swim test
Tail suspension test

Animal models of depression

Porsolt test (Forced Swim test)
Based on learned helplessness
Quantifies number and duration of
immobility episodes
immobility indicates depression
Tail-suspension test
dry version of the forced
swim test

El
Elevated
t d plus
l and
d zero mazes

Social interaction test

Rat exposure

Novelty
y tests
Open field test
Measures distance moved,
vertical rears, and time
spent in the center vs. the
periphery

Light/dark box
Quantifies
Q
tifi number
b off entries
t i and
d time
ti
spent in the in the lighted area.
Exploratory behaviors vs. anxious
be a o s
behaviors

Open field and exploratory strategies

In addition to assessment of the amount of behavior (i.e., frequency and duration
measures), analyses of quality of behavioral represent an important part of
behavioral phenotyping:
spatial
temporal
spatio-temporal characteristics

What? When? Where?

Center
Periphery
Corners

Anxiety
Increased thigmotaxis (peripheral vs. central activity) in
SERT-/- mice

+/+

Reduced exploration activity in SERT-/- mice

D u r a tio n in C e n te r (s )

F r e q u e n c y in C e n te r

100

40
30
20
10

100

120

50

*#

0
SERT +/+SERT +/- SERT -/-

80
60
40
20

T im e in C e n te r :P e r ip h e r y (% )

+/-

0
SERT +/+SERT +/-SERT -/-

75
50

-/-

25
0

SERT +/+SERT +/- SERT -/-

Kalueff et al., 2007

Hallucinogens: Acid trip (LSD)

20 min

1 h 25 min

I'm having a little trouble controlling this

pencil. It seems to want to keep going

2 h 30 min

2 h 32 min

everything is changing color

Everywhere Patient becomes startled
by something on the floor

2 h 35 min

Patient is generally agitated, and becomes

largely none-verbal. I am... everything is...
changed... they're calling... your face...
interwoven... who is... Patient mumbles

Patient sits on his bed. He

reports that intoxication has worn
off except for the occasional
distorting of faces

4 h 25 min
Running back and forth
across the room

5 h 45 min

8 h 00 min

I can feel my knees again

2 h 45 min

Behavioral effects of LSD in rodents

Open field activity

+/+Saline +/+LSD

Anxiety
(5-HT1a)

+/-Saline
Hyperactivity
5-HT2a/c

30

60

90
120
Time, min

150

+/-LSD

SHIRPA: gross phenotype assessment

SmithKline Beecham Pharmaceuticals

Harwell, MRC Mouse Genome Centre
Imperial College School of Medicine
Royal London Hospital, St. Batholomews
Phenotype

Assessment

Assessing motor phenotypes

SHIRPA battery: a widely
widely-accepted
accepted neurological
battery involving a three-stage protocol
It is very basic, and includes measures of muscle
function, cerebellar function, sensory function,
neuropsychiatric function
function, and autonomic function

http://btc.bol.ucla.edu/shirpa.htm

Behavior recorded in Viewing Jar (I)

Body Position
0 = Inactive
1 = Active
2 = Excessive Activity
Tremor
0 = Ab
Absentt
1 = Present
Palpebral Closure
y open
p
0 = Eyes
1 = Eyes closed
Coat Appearance
0 = Tidy and well groomed coat
1 = Irregularities such as piloerection
Whiskers
0 = Present
1 = Absent (include any further comments

Behavior recorded in Viewing

g Jar ((II))

Lacrimation
0 = Absent
1 = Present
Defecation
0 = Present
1 = Absent
Behaviour recorded in the Arena:
Tansfer Arousal
0 = Extended freeze (over 5 seconds)
1 = Brief freeze followed by movement
2 = Immediate movement
Gross Locomotor Activity
The total number of squares the animal enters with all four feet in 30 s
Gait
0 = Fluid movement and approximately 3-mm pelvic elevation
1 = Lack of fluidity in movement (include comments eg. retropulsion,
more than
th 3 mm pelvic
l i elevation)
l
ti )

Behavior recorded in Viewing

g Jar ((III))

Tail Elevation
gg g
0 = Dragging
1 = Horizontal extension
2 = Elevated/straub tail
Startle Response
0 = None
1 = Preyer reflex (backwards flick of the pinnae)
2 = Reaction in addition to the Preyer reflex (e.g., Startled response)
Touch Escape
0 = No response
1 = Response to touch
2 = Flees prior to touch
Behaviour recorded above the Arena:
Positional passivity
0 = Struggles when held by the tail
1 = Struggles when held by the neck
2 = Struggles when laid supine
3 = No struggle

Behavior recorded in Viewing Jar (IV)

Skin Color
0 = Blanched
1 = Pink
2 = Bright, deep red
flush
T
Trunk
k Curl
C l
0 = Absent
1 = Present
p g
Limb Grasping
0 = Absent
1 = Present
Pinna Reflex
0 = Present
1 = Absent

Corneal Reflex
0 = Present
1 = Absent
Contact Righting Reflex
0 = Present
1 = Ab
Absentt
Evidence of Biting
0 = None
g in response
p
to
1 = Biting
handling
Vocalizations
0 = None
1 = Vocal

Summary:
y SHIRPA batteryy
A battery of tests that can be completed within a few
minutes
Observation for normal and abnormal spontaneous
b h i
behaviors,
and
d measurements
t off activity
ti it llevels,
l
arousal, respiration, gait, muscle tone, reflexes,
aggression, etc.
If a subject group shows unusual behavior or
function, further testing must be done in that domain

Motor problems
Disorders may have both peripheral
and central origins:

Cerebellum
Brain stem
Striatum
Basal ganglia
Motor cortex
Spinal
p
cord
Peripheral nervous system
Musculoskeletal deficits

Gait assessment (I)

Detects walking abnormalities
Easy to perform: place non-toxic paint on mouses feet

Sensitive to atypical patterns due to genetic alterations

(see example below genetic mouse model of
Huntington disease)
A) Wild type
B) Mutant mice

Detloff, 2003

Gait assessment ((II))

Ink is applied to the paw

The animal walks on paper
The footprints are analyzed
for:

Transgenic mice overexpressing

p
g neurotrophin-3
p

step length (SL)

print length (PL)
toe spread (TS)
intermediate toe spread (ITS)
Taylor et al
al, 2001

Problems: May be sensitive to procedure-evoked

anxiety/stress

Swimming

Assess ability to swim

Abnormal patterns (vertical vs.
vs horizontal)
Circling
Diving
g
Sinking

Normal horizontal swimming

www.umt.edu/urelations/rview/s
ummer06/mice.htm

Abnormal vertical swimming

Kalueff et al., 2006

Motor skills tests

Drug-induced
D
i d
d turning
i (R
(Rotation
i T
Test))
Forelimb asymmetry (Cylinder Test)
g
Beam walking
Grip strength
Grid walking
Placing test
Rotorod
Landing Foot Spread Test
Skill d reaching
Skilled
hi (f
(forelimb
li b motor
t control)
t l)

Neurodetective International, 2008

Homecage activity chambers (I)

Normal behaviors to assess:

www med associates com

www.med-associates.com

Di
Digging
i
Grooming
Thigmotaxis
g
((staying
y g close to
the walls)
Rearing
Exploration
p

Homecage activity (II): general hypoactivity

Dramatic reduction of 24-h
motor activity in SERT-/mice

Holmes et al., 2002

Homecage activity (III)

Abnormal behaviors to assess:
Hyperactive running
Stereotypes (jumping, circling, somersault)
Seizures
S i
Freezing/ inactivity episodes
Overactive itching
Over-grooming and self-damage
Overall impulsivity

Behavioral perseverations

Commonly seen behavioral perseverations:

www.aps.uoguelph.ca/~gmason/StereotypicAnimalBehaviour

www.nc3rs.org.uk

Barbering
Repetitive Jumping
Bar-Mouthing
Cage-Top
Cage Top Twirling
Excessive Licking
Excessive Grooming

Specific animal tests for motor ability

Locomotory activity (gross assessment)

Balance (e
(e.g.
g Rotorod)
Reflex testing
Strength testing
Fine motor analysis (FMA)

Straight observation

Open field test (OFT)

High/low
g
activity
y level
Body posture
Movement coordination
www med-associates
www.med
associates.com
com
Rearing, exploring
Additional movements (e.g. head twitches)
Thi
Thigmotaxis
i ((avoidance
id
off open centrall areas))

Open-field
Open
field test

1.
2.
3.
4
4.
5.

Open square or circular arena

Typical parameters:
zones entered
time spent in periphery vs. center
grooming time
Rears
defecation
Often use videotracking software (e.g.
Ethovision, HVS Image) providing distance
traveled, speed, etc.
Measures both locomotor activityy and anxiety
y

Spatial working and long-term memory

Serotonin is involved in the regulation of
memory and other cognitive functions
SSRIs have effects on memory in
habituation tasks

Movem ent duration(% of total)

SERT+/+
10,0

SERT +/-

8,0

SERT -/-

6,0
4,0
2,0
0,0
1

We used
W
d OF and
d EPM habituation
h bit ti tto
assess spatial memory in SERT-/- mice

10 11 12 13 14 15 16 17 18 19 20

Travelled distance (% of total)

10,0

8,0

Normalized mouse activityy (%

( of total))

6,0

4,0

2,0

Conclusions
Increased anxiety (previous slide)
But: Normal spatial memory

0,0

10 11 12 13 14 15 16 17 18 19 20

Vertical rears (% of total)

10,0
8,0
6,0
4,0
2,0
0,0
1

10

11

12

13

14

15

16

17

18

19

20

Rating
g scales
Scoring technique using a number to represent the
d
degree
off b
behavioral
h i l severity
it
0
Normal behavior

1
Intermediate motor
disturbances

2
Consistent abnormal
motor coordination

Potential concerns:

Statistics are non-parametric

More quantitative than qualitative
Pletnikov, 2006

Locomotion
Many potential confounds:

Habituation problems (too much/little)

Testing time (consider circadian rhythms)
Variabilityy ((e.g.
g interstrain))
Problems with housing (e.g. multi-species odors,
sex pheromones)
Effect of sound
Floor/ceiling effects
Pletnikov, 2006

Beam walking test

Use food reward or dark
escape"
p area as incentive
beam width = difficulty
Endpoints recorded:
Time to cross beam
Falls
F ll
Hind-leg slips

Chang et al., 2005

Problems:
Often requires pre-training
May involve motivational
f t
factors

Rotorod (Rotarod)
A rotating
g bar, revolves at
constant or increasing
speeds
Latency to fall is primary
endpoint
Typically, mouse
performance as number
of trials

Van Meer and Raber, 2005

Rotorod
Motor
M
t abnormalities:
b
liti
Coordination
Weakness
Muscle tonicity
Involuntary movements
Other domains:
Sensory function
Cognitive
C
iti ability
bilit
Anxiety
Non-motor seizures
Chronic/systemic problems
Problems: Cognitive phenotypes (e.g.
performance
habituation)) mayy affect motor p
Pletnikov, 2006

Measuring strength
Hanger test:
Time latency to fall from
an upside-down screen

phenome.jax.org/.../Lake3_Protocol

Rope climbing test

Ability to climb rope

Latency to reach a 20 cm
mark

Kalueff et al., 2007

Normal grip strength

Chimney test
Consists of a hollow tube large
g enough
g for a mouse
or rat to fit inside comfortably
The animal is placed in the tube, and then the tube is
positioned vertically, with the animals
animal s snout oriented
downwards
The animal will attempt to keep itself from falling and
will slowly walk backwards up to the top of the
chimney
This measures the animals motor ability and
coordination
di ti

Hind-leg clasping reflex

Normal reaction
in a normal mouse

This mouse will appear normal

in the cage but with you pick it
up, it exhibits "clasping" rather
th the
than
th normall plantar
l t
reaction

Clasping indicates
neurological/motor
i
impairments
i
t iin animals
i l

Ansorge et
al 2006
al.

Herzing, 2008

Davis, 2000

Davis, 2000

Foot-clasping
p g

Example of a foot-clasping phenotype

Tanaka et al., 2004

Assessing other motor reflexes

Ri hti reflex
Righting
fl
Mice right themselves onto feet after put on their
backs, or dropped from some height (e.g. 20 cm) on
a cushioned surface
Generally normal unless movement/vestibular
disorders are present
Tail suspension test (abnormal spinning if vestibular
problems)

Digging behaviors
A very common behavior in rodents
Sensitive to stress, and anxiolytic/ anxiogenic
pharmaceutical compounds
Marble-burying test often used to measure this
behavior

Marble burying/digging
To kick sand in someone's face is an archetypal
agonistic interaction between humans
Rodents have been filmed kicking earth toward an
approaching snake in their burrows
burrows. They also bury
noxious objects such as shock probes
M
Mostt behavioral
b h i l scientists
i ti t would
ld assume th
thatt marbles
bl
are non-aversive to mice
Mice are probably not deliberately burying the marbles;
they simply fall through the displaced bedding (MB test
measures digging behavior)
Deacon, 2006

Digging Test Protocol

Digging is defined as coordinated movements of forefore
or hind limbs that displace the substrate

Fill th
the cage 5 cm d
deep with
ith wood
d chips
hi
Several test cages can be run simultaneously
Place a mouse in each cage and start the test
timer. Test duration is 3 min.
The latency to start digging, the number of digging
bouts and the total duration of digging are
recorded
Deacon, 2006

Marble Burying Protocol

1
1.
2.
3.
4
4.
5.

Fill the cage approximately 5 cm deep with wood

chip bedding
Place a regular pattern of glass marbles on the
surface evenly spaced
surface,
spaced, each about 4 cm apart
Place one animal in each cage and leave for 30
min
Count the number of marbles buried (to 2/3 their
depth) with bedding
Alternatively, count the number of marbles buried
fully partially (2/3 their depths)
fully,
depths), and non-buried
non buried

Deacon, 2006
D
Kalueff et al., 2006

Unusual escape attempts

When animal demonstrates abnormally active escape
attempts
E.g. immediately after being placed on a surface the
animal will jump/run away, rather than freezing
Could indicate hyperactivity, very high overall anxiety,
hyperexcitability or other phenotypes
hyperexcitability,
If animal shows these abnormal behaviors, it needs
further examination before being tested in other
paradigms
di

Reflexes and postural reactions

Common tests:

Trunk curl
Rear-limb withdrawal
Low/flat bodyy
Tremor
Hind-leg abduction
F li b positioning
Forelimb
iti i

Ethograms: behavioral microstructure

(patterning): SERT-/- mice

5 min, observation cylinder test: ha, horizontal activity (number of ha episodes); va,
vertical activity (protected rears); f, freezing episodes; g, grooming bouts; d,
defecation; st, Straub tail. Line width reflects frequency of behaviors (circles) or their
transitions (arrows).
(arrows)
Kalueff et al., 2007

Macro-behavioral and Micro-behavioral

Levels of Analysis

Stress
Drugs
Genetic Mutations

Quality

Quantity

Both

Micro-behavioral Video Tracking

Modern tracking systems can analyze an individual animal in a full spectrum,
recording the movement of specified body parts
It is also possible to assess regional distribution of physiological markers, such as
regional body temperature

www.cleversysinc.com

Advantages of Micro-behavioral Analyses

Certain abnormal behaviors can be
detected, such as differences in
swimming pattern in the Forced
Swim Test

Video-tracking algorithmic
computation reduces effects of
manual scoring on reproducibility of
data

Micro-behavioral analysis
complements macro-behavioral
endpoints, resulting in higher
throughput models
Juszczak et al., 2008

Animal Models of Psychiatric Disorders

MacroAnxiety
OCSD
Depression

Micro-

Use
Frequently
Sometimes

Schizophrenia

Rarely
Epilepsy
Serotonin Syndrome
Tourettes Syndrome
Rett S
Syndrome
ndrome

None

Expanding neurophenotyping batteries

OCD screens
Perseverations and stereotypies
Autism/sociability screens
Aggression screens
Chronic stress
Social defeat paradigm
Anhedonic depression
Maternal phenotypes
Cross-fostering
Nest-building phenotypes
Oto-vestibular phenotypes
Bipolar depression
Models of mania

Cognitive screens
Within- and between-trial habituation tasks
Barnes maze,, 3D-maze
Spontaneous alternation tasks
Mismatch negativity
Schizophrenia-related
p
tests
Early life stress
Behavioral effects of enrichment
Neurotoxicity syndromes
Spontaneous (serotonin syndrome)
Drug-potentiated
Drug abuse phenotypes
Ethanol-withdrawal anxiety
Ethanol-related behaviors
g preference
Drug
Screens for hallucinogenic drugs

Ethics: The three Rs

Rs
1) Reduce
R d
- Using
U i ffewer animals
i l iin experiments
i
t
2)) Replace
p
- replace
p
the animal model in vivo with
in vitro or mathematical models, or with a species
lower on the phylogenic scale.
3) Refine - eliminating or relieving pain suffered by the
animals in experiments