Professional Documents
Culture Documents
of Functional
Activity in the Brain
Immunohistochemistry
Teresa L. Krukoff
1. Introduction
Neuroscientists are often faced with the choice of obtaining
quantitative data at the expense of morphological information or
vice versa. The identification of a pathway in the brain offers a
potential anatomical basis for a given physiological function, for
example, but does not directly address the physrological significance of the pathway. Conversely, measurements made from tissue homogenates provide clues to the response of cells to a
stimulus but cannot tell us anything about the specific cells m
which changes occur. This chapter describes an approach that
bridges the gap between morphological data and physiological
significance. The presence of Fos, the protein product of the
immediate-early
gene c-jbs, in a neuron has become a popular
means to identify neurons that participate in a given function
without losing the ability to know precisely where these neurons
are. A brief description of the techniques that were supplanted by
Fos immunohistochemistry
will be followed by a discussion of
c-f& and why its expression as a marker of functional activity has
gained such popularity in the neurosciences. Techniques for Fos
immunohistochemistry,
their compatibility with other techniques,
and important considerations regarding analyses of data obtained
with these approaches will be presented. The chapter will be concluded with a discussion of the advantages and disadvantages of
using c-fos expression as a marker of functional activity in the brain.
Ed
From
Neuromethods,
vol 33
A Boulton,
G B Baker,
and
Cell
A
213
Neurobfology
N
Bateson
Techmques
Q Humana
Press
Inc
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2. Background
2.2.
Markers
of Metabolic
Activity
One of the first popular techniques that was used to metabolically identify regions of the brain that were involved in
specific functions was the use of 2-deoxyglucose (2DG, Kennedy
et al., 1975; Sokoloff, 1977,1981; Krukoff and Scott, 1983,1984).
As 2DG is the structural analog of glucose, the primary source
of energy in the brain, 2DG is transported into cells via the glucose transport system and is phosphorylatecl
to 2DG-6-phosphate. Unlike glucose-6-phosphate,
however, 2DG-6-phosphate
cannot be metabolized further and becomes trapped within the
cell. Therefore, after administration
of a bolus of radioactively
labeled 2DG, regional variations in labeled 2DG-6-phosphate
can be used to autoradiographically
quantify local cerebral glucose utilization. The technical difficulties and expense of the
2DG technique, however,
prompted
investigators
to seek
alternative means to address the question of cellular involvement in specific functions.
The histochemical localization of metabolic enzymes has been
used as a means to identify populations of neurons that respond
to physiological stimuli. For example, the activity of cytochrome
oxidase has been used as a means to study the involvement
of
brain regions in visual processing, and body fluid and cardiovascular regulation (Wong-Riley and Riley, 1983; Krukoff and
Calaresu, 1984a,b). We have also used densitometry to obtain
relative measurements of the activity of hexokinase in studies
in which we were interested in identifying specific regions of
the brain that respond to changes in regulation of body fluids
(Krukoff et al., 1986; Krukoff and Vincent, 1989a), cardiovascular activity (Krukoff,
1988; Krukoff and Vincent, 1989b,
Krukoff and Weigel, 1989), diabetes mellitus (Krukoff and Patel,
1990), and heart failure (Pate1 et al.. 1993). Although inexpensive and technically straightforward,
both the cytochrome oxidase and hexokinase techniques were limited to chronic (days)
experiments because longer periods of stimulus application
time were necessary for significant changes m enzyme levels
to be detected. It was not until the development of the new field
of immediate-early
gene molecular biology that we could
finally identify the participation of individual neurons in acute
experiments
c-fos Expression
215
Considerations
Anesthetics have important effects on Fos expression in neurons, with some anesthetics such as sodium pentobarbital and
ketamine suppressing Fos expression, and others, such as urethane, a-chloralose, or methoxyfluorane stimulating Fos expression (Marota et al., 1992; Krukoff, 1993; Takayama et al., 1994). To
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avoid the complicating effects of anesthesia, therefore, it is recommended that experiments be done in conscious animals On
the other hand, stresses caused by handling or restraint (Ceccatelli
et al., 1989; Melia et al., 1994; Cullinan et al., 1995; Krukoff and
Khalili, 19971, or even exposure to a novel environment (Handa
et al., 1993) are themselves potent stimuli for expression of Fos.
These effects can be greatly reduced by repeatedly exposing
experimental animals to the environmental and handling conditions of the experiment on a daily basis for about a week leading
up to the day of experimentation. In this way, the animals become
habituated to the conditions of the experiment. The most important consideration of these factors is that carefully controlled
experiments will allow the investigator to eliminate the effects of
these extraneous factors on the results.
3. I .2. Types
and Duration
of Tissues
3.2.1.
Conslderatlons
Important
When the experiment has been concluded, experimental animals should be deeply anesthetized with a suitable anesthetic. As
anesthetics themselves have a variety of effects on Fos expression
in the brain (see Subheading 3.1.1.), it is important to keep the
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c-fos Expression
Preparation
of Tjssue
3.3. hm7unohisfochemica/
3.3.1. Important
Sfaiffing
Considerations
Krukoff
218
Preparation
for Light
Microscopy
c-fos Expression
219
Preparation
for Fluorescence
Microscopy
In Combination
IMPORTANT
with
Other
Techniques
CONSIDERATIONS
IMMUNOHISTOCHEMISTRY
FOR OTHER
PROTEINS
Visualization of neurotransmitter proteins or enzymes in activated neurons (express Fos) can be accomplished with double
immunohistochemistry
using primary antibodies (one for the neurotransmitter and one for the Fos) that are raised in different species. For example, we use antibodies to Fos that have been raised
220
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c-fos Expression
221
Krukoff
c-fos
Expression
223
Fig. 3. Bright-field
photomicrographs
through the PVN (delineated
by a dashed line in (A) and the ipsilateral NTS (B) and VLM (C) at the
level of area postrema from an experimental
animal. The asterisk in (A)
indicates the center of the PHA-L injection. In B and C, PHA-L fibers
and terminals that are apposed on TH neurons with Fos-IR nuclei (*I are
indicated by arrows. Single-labeled
Fos-IR nuclei can also be observed
in (B) and (C). fx, fornix; OT, optic tract; III, third ventricle. Bars = 500
pm (A) and 10 pm (B,C) (from Petrov et al., 1995 with permission).
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Kru koff
1995) (Fig. 3.). Tracers for fluorescence and light microscopy are
available and include fluorescent dextrans (Molecular Probes,
Eugene, OR) and Phaseolus vulgaris leucoagglutinin (Vector Labs,
Burlingame, CA) respectively.
3.3.4.4.
IN SITU HYBRIDIZATION
c-fos Express/on
225
4. Analyses
4.1. Choosing
Cells
Avolding
Artifacts
Expressing
Results
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Krukoff
5. Advantages
5.1.
and Disadvantages
Advantages
c-fos
227
Expression
and Disadvantages
Acknowledgments
The author gratefully acknowledges the support of the Medical Research Council of Canada, the Heart and Stroke Foundation
of Alberta, and the Alberta Heritage Foundation for Medical
Research. Review of the manuscript by J. Jhamandas, K. Harris,
and D. MacTavish is appreciated.
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Krukoff
References
Asanuma, M and Ogawa, N (1994) Pitfalls m assessment of c-fos mRNA expression m the brain effects of animal handling
Rmews Neurosct 5,171-178
Ceccatelh, S , Vlllar, M J , Goldstem, M , ancl Hokfelt, T (1989) Expression of cfos immunoreactlvity
m transmitter-characterized
neurons after stress PNAS
86,9569-9573
Coggeshall,
R E and Lekan, H A (1996) Methods for determmmg
numbers of
cells and synapses a case for more umform standards of review
J Camp
Neural
364,6-15
c-fos
Expression
229
J Neurochem 29,13-26
230
Krukoff