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c-fos Expression as a Marker

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
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Neurobfology
N

Bateson

Techmques
Q Humana

Press

Inc

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Kru koff

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

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215

2.3. What is c-fos?


Immediate-early
genes, originally described in the field of
growth regulation, are rapidly induced by extracellular stimuli
and encode proteins that are required for subsequent events to
occur in the cell (Curran and Morgan, 1995). The first of these
genes to be described, c-fos, encodes a protein (Fos) that participates with products of the related Jun family as a component of
the protein complex that binds to the activator-protein-l
(AP-1)
binding site of DNA (Curran and Teich, 1982; Lee et al., 1988; Sheng
and Greenberg, 1990). Genes that contain the AP-1 complex are
activated by the Fos/Jun complex, thereby allowing the expression of the so-called late-onset genes that encode differentiated
neuronal products such as neurotransmitters. Transcription of c-fos
occurs within minutes of application of a stimulus, amounts of
mRNA peak at 30-45 min, and the half-life of the Fos protein is
about 2 h (Muller et al., 1984).
The expression of c-fos in neurons has provided a useful and
popular marker of activated neurons. Its basal expression is low
in most neurons (Herdegen et al., 1995; Krukoff and Khalili, 1997),
but it can be rapidly induced by a broad range of stimuli. The
most common approach is to visualize the presence of Fos within
nuclei of neurons using immunohistochemistry
although mRNA
levels for the gene can be measured with in situ hybridization,
Northern blots, and so on. In addition to the ease and relative low
cost required to demonstrate and count labeled neurons, Fos
immunohistochemistry
allows the investigator to pose questions
related to individual neurons that participate in specific physiological functions. Whereas the procedures in this chapter will be
described for Fos expression, the reader is reminded that other
immediate-early genes can be studied in similar ways.
3. Methods
3.1. Stimulus Application
3.1. I. Important

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

As stated in Subheading 2.3 , c-fos is expressed within minutes


of stimulus application and protein can be detected immunohistochemically within 15-30 min. Most published reports using
Fos immunohistochemistry
describe stimulus application from 30
min to several hours with 1 h being the most common time period
used. In our own experiments we typically apply a stimulus for
60-90 min (Krukoff et al., 1995; Petrov et al., 1995b).
Chronic application of stimulus (i.e., days or more) may not
result in the presence of Fos at the end of the experiment because
expression of c-fos may only be required when the stimulus is first
applied. Once the AP-1 site on a gene has been activated, its continued activation may be unnecessary or suppressed, and c-fos
expression may cease. Therefore, the effects of chronic diseases
on neuronal activation in the brain, for example, may not prove
successful. The best guideline is that pilot experiments should be
carried out to determine whether a chronic stimulus leads to C--OS
expression.
3.2. Preparation

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

length of anesthesia before fixation to a minimum. We have found


that anesthesia not exceeding 10 min in duration is satisfactory
for avoiding anesthesia-induced artifacts.
Postfixation (after perfusion) is a common step used in standard histology and immunohistochemistry.
For Fos immunohistochemistry, we recommend no longer than 1 h of postfixation in
half-strength fixative, as prolonged fixation decreases immunoreactivrty for Fos.
Sections can be processed either as free-floating or thaw-mounted
tissues. Free-floating sections must be the thicker of the two types
(25-60 pm vs 5-20 urn) so that the tissue can be manipulated.
Notwithstanding
the greater thickness, however, immunohistochemical results are generally crisper in free-floating sections
because the solutions used in the processing have access to both
faces of the sections.
3.2.2.

Preparation

of Tjssue

1. Perfuse approx 100 mL of saline through the left ventricle of


the heart to displace the blood.
2. Perfuse with 500 mL of ice-cold 4% paraformaldehyde in 0.1 M
phosphate-buffered
saline (PBS), pH 7.2. To prepare fixative,
wet 20 g paraformaldehyde
in loo-150 mL of distilled water.
Heat to 60C with continuous stirring. Clear the solution with
5-10 N NaOH added dropwise. Filter and top up to 500 mL
with PBS.
3. Remove the brain and place in half-strength fixative: 10%
sucrose (in water). After 1 h transfer the brain to 10% sucrose.
At approx 12-h intervals place the brain into 20% and 30%
sucrose. The sucrose displaces some of the water in the tissue
and reduces the freezing artifact that can occur during sectioning.
4. Cut sections in a cryostat (-20C). For free-floating processing,
cut sections at 25-60 pm in thickness and collect in 0.1 M PBS
(pH 7.2). For thaw-mount processing, cut sections at 5-20 pm.

3.3. hm7unohisfochemica/
3.3.1. Important

Sfaiffing

Considerations

As discussed in Subheading 2.3., c-fos is one in a family of genes


called immediate-early genes. Because several of the protein products of these genes are similar in structure, commercially-available
antibodies will sometimes recognize Fos and Fos-related antigens.

Krukoff

218

It is important to the interpretation of results to be aware of the


antibodys specificity for Fos alone or for Fos-related antigens;
this information is available from the supplier.
The appropriate dilutions will be unique to the antibody and
should be empirically determined with each new stock. Too high
a dilution will lead to high cytoplasmic background and too low
a dilution will not sufficiently demonstrate the protein that is
present.
All antibody solutions used in processing should contain 0.3%
of the detergent, Triton X-100, which permeabilizes the membranes
and enhances penetration of the antibodies.
3.3.2.

Preparation

for Light

Microscopy

Many techniques are available to visualize the presence of an


antibody-antigen
complex in tissue. For the demonstration of Fos
alone, we routinely use the avidin-biotin
immunoperoxidase
method, the reagents for which are provided in the ABC VectaStain
Kit (Vector Labs, Burlingame, CA). Alternatively, each ingredient
necessary for the processing may be purchased separately. The
primary advantage of the ABC method is that the signal is amplified with the avidin-biotin interaction.
1. Incubate sections overnight at 4C or at room temperature with
gentle agitation m anti-Fos antibody. Dilute the antibody in
PBS (pH 7.2) containing 0.3% Triton Xl00 (Triton X- loo/PBS).
2. After two washes in PBS, place sections into biotinylated secondary antibody (1:200 in Triton X-loo/PBS) for 1 h at room
temperature with gentle agitation. The secondary antibody will
be specific for the species in which the primary antibody was
generated
3. Wash in PBS. Incubate sections for 1 h in ABC reagent (1:lOO
in Triton X-lOO/PBS) at room temperature with gentle agitation. The ABC reagent is a complex that contains avidin (with
a very high affinity for biotin) complexed to horseradish peroxidase (HRP) and should be prepared at least 30 min before
use as specified by the manufacturer.
4. Wash twice in PBS. Place tissues in a room temperature solution containing 25 mg diaminobenzidine and 17 uL hydrogen
peroxide in 50 mL of 0.1 M PBS for 5-10 min. The diaminobenzidine will be reduced by the HRP in the ABC complex
and deposited in the tissue as a brown reaction product.

c-fos Expression

219

5. After a rinse in PBS, mount the free-floating


tissues onto
microscope slides using a fine paintbrush. Air-dry the sections
and mount cover slips using a mounting medium of choice.
3.3.3.

Preparation

for Fluorescence

Microscopy

One advantage of demonstrating Fos with fluorescence markers


is that the result is a one-to-one relationship of the fluorescence to
the antigen. The relative amount of antigen can then be measured
by quantitating the strength of the fluorescent signal with confocal
microscopy. If this is the aim, use a secondary antibody to which
the fluorescent marker of choice has been conjugated. A second
advantage is that, in combination with other neuroanatomical techniques, fluorescence microscopy may be the method of choice for
visualizing all or some of the markers. Switching among fluorescent filters is then more straightforward
than switching between
light and fluorescent microscopy during analysis.
If one wishes to amplify the signal with fluorescence in the same
way as described for light microscopy (Subheading 3.3.2.), a
biotinylated
secondary antibody should be followed
by a
streptavidin-fluorescent
marker complex which, like avidin, has
a high affinity for biotin.
3.3.4.
3.3.4.1.

In Combination
IMPORTANT

with

Other

Techniques

CONSIDERATIONS

One of the greatest strengths of using expression of C-$X as a


marker for activated neurons is that immunohistochemistry
for
Fos can be combined with other neuroanatomical techniques, The
presence of Fos adds a powerful functional dimension to anatomical data and, at the same time, preserves the morphological characteristics of analysis that are lost with other approaches. In the
following discussion, I describe three techniques that can be combined with Fos immunohistochemistry,
but it is also important to
remember that triple and even quadruple labeling can be accomplished by combining the techniques further.
3.3.4.2.

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

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Krukoff

in sheep and antibodies raised to neurotransmittern


proteins of
choice that have been raised in rabbit (Petrov et al., 1994) (Fig. 1).
Processing is then carried out for two-color visualization with light
or fluorescence microscopy by following the protocols described
above with the following modifications:
1. Because primary antibodies have been raised in different species, tissues can be incubated overnight in a cocktail containing both antibodies (in PBS) at the appropriate dilutions.
2. On the second day, processing for each antigen should proceed individually. For example, complete the Fos immunohistochemistry and then process for the neurotransmitter protein,
beginning at the step in which the tissues are incubated in the
secondary antibody.
3. For light microscopy, a common approach is to render the
nuclear Fos reaction product black with nickel intensification
(Wouterlood et al., 1987) and the neurotransmitter
protein
brown with regular diaminobenzidine
processing (see Subheading 3.3.2.). Nickel intensification is carried out as follows:
a. Following incubation in the avidin-HRP complex (see Subheading 3.3.2.1, wash tissues in 0.05 M Tris buffer (pH 8.0) at
room temperature.
b. Place tissues in a solution containing 300 mg nickel-ammonium
sulfate, 15 mg diaminobenzidine,
17 uL hydrogen peroxide
in 50 mL 0.05 M Tris buffer (pH 8.0) for 5-10 min. Rinse m
Tris buffer and mount sections onto microscope slides.
4. For fluorescence microscopy, two fluorophores of dissimilar
wavelengths (e.g., fluorescein and rhodamine) are used to tag
the separate antigens.
3.3.4.3. NEUROANATOMICAL TRACERS
To identify the target(s) of a neuron which responds to a given
stimulus (expresses Fos) retrograde tracing techniques can be used
in c:onjunction with Fos immunohistochemistry.
Fluorescent tracers are especially easy to use because it is usually unnecessary to
process the tissue further to make the marker visible. We have
obtained successful results with ZO-nm fluorescent-labeled
latex
microspheres (Krukoff et al., 1995; Petrov et al., 1995) that are available from Lumafluor (Naples, FL). The visualization of Fos can be
accomplished with either light or fluorescence microscopy using
one of the techniques described above. If fluorescence is chosen,
the viewer need only change filters on the fluorescent microscope

c-fos Expression

221

Fig. 1. Flourescent photomicrographs


from the ipsilateral NTS at the
level of area postrema in an experimental
(A,B) and a control (C,D) animal. Double-stained
sections for Fos (A,C) and TH (B,D). Arrowheads
indicate double-stained
cells. Note the increased number of Fos-IR and
double-labeled
profiles in A as compared to C. The borders of the NTS
are delineated by a dashed line. The midline is to the left. TS, solitary
tract. Bar = 40 pm (from Petrov et al., 1995, with permission).

in order to view the tracer (e.g., rhodamine) and to determine if the


same neuron expresses Fos (e.g., fluorescein) (Fig. 2).
In a typical experiment, the retrograde tracer is stereotaxically
injected into the putative central target within the brain of an anesthetized animal. The animal is allowed to recover from anesthesia and to survive for a sufficient period of time (i.e., days) to allow
retrograde transport of the tracer to the cell bodies of origin. The
physiological experiment is carried out to stimulate Fos expression at the end of the survival period and processing is begun as
described previously.
The same techniques can be applied to the combination of
anterograde tracers with Fos immunohistochemistry
(Petrov et al.,

Krukoff

Fig. 2. Flourescent photomicrographs


from the ipsilateral PVN of an
experimental
animal depicting the distribution
of Fos-IR nuclei and cells
containing
the retrograde tracer after injection into the NTS (A-D) or
the VLM (E-H). (A,B) Low-power photomicrographs
of the same field
where Fos IR (A) and the retrograde tracer (B) are viewed with FITC
and RITC filter combinations,
respectively. The locations of the medial

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).

Fig. 2. (continued) (mp), periventricular


(pv), dorsal (dp), and lateral (1~)
parvocellular
parts of the PVN are indicated (nomenclature
according
to Swanson et al., 1981; Sawchenko and Swanson, 1982). In higher-power
photomicrographs,
where Fos IR and the tracer are viewed with RITC
filter combinations
simultaneously,
the arrowheads or asterisks (in a
different animal) indicate double-labeled
cells in the mp 0, pv (D), dp
(E,F), and lp (G,H) PVN. In D, F, and H the asterisks indicate the Fos-IR
nuclei that are surrounded by rhodamine-labeled
latex microspheres.
Single-labeled
Fos-positive nuclei (C, in the pv) and cells containing only
the tracer (E, G, to left of the double-labeled
cells) are also readily distinguishable.
Bars = 40 pm (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

The demonstration of mRNA for a given gene within a neuron


using znsitu hybridization can be combined with Fos immunohistochemistry to obtain results that indicate that an activated neuron (Fos) expresses another gene of interest. Furthermore,
especially if a radioactive probe is used for tn situ hybridization,
the up- or downregulation
of that gene can be measured according to the strength of the signal. The preferred sequence of processing is to complete the immunohistochemistry
for Fos prior to
processing the tissue for in situ hybridization. It is outside the scope
of this chapter to discuss the details of the in situ hybridization
method. Instead, the current discussion will be limited to those
points that must be considered that will allow the investigator to
complete the Fos immunohistochemistry
and make the tissues
ready for in situ hybridization in the same section:
1 As mRNA is susceptible to degradation by RNase, all procedures must be carried out in RNase-free solutions and plasticor glassware. The reader is referred to one of a large number
of available molecular biology manuals to learn more about
avoiding RNase contamination.
2. Free-floating sections are cut at a thickness of 25-30 pm and
processed for Fos immunohistochemistry
as described above.
Sections are then mounted onto microscope slides which are
charged or which have been coated with a substrate (e.g.,
chrome-alum, 3-aminopropyltriethoxysilane
[APTEXI coating)
which ensures that the sections will adhere to the slide during
subsequent processing for in situ hybridization
Chrome-Alum Coatmg:
a. Heat 2 g gelatin in 500 mL water (60C). Add 0.2 g chromium potassium sulfate. Filter.
b. Dip precleaned glass microscope slides into the hot solution.
c. Drain m the vertical position and dry thoroughly in a hot oven.
d. Store at room temperature in a slide box and use as needed.
APTEX Coating:
a. Dip precleaned glass microscope slides for 2 mm in each of
the following solutions at room temperature. acetone, 2%

c-fos Express/on

225

aptex (Sigma, in acetone), two solutions of acetone, two


washes in distilled water.
b. Drain and dry overnight at 37C.
c. Store at room temperature and use as needed.
3. Adequate drying of sections onto slides at least overnight is
required to ensure that adhesion of the section to the slide will
be maintained.
4. Store slides at -70C in slide boxes containing dessicant. Sections in which Fos immunohistochemistry
has been completed
may be stored for up to several months before processing for
in sttu hybridization.

4. Analyses
4.1. Choosing

Sections and Counting

Cells

A major strength of c-fos immunohistochemistry is that activated


(immunoposltive) neurons can be counted in tissue sections. Image
analysis or manual methods can be used for counting of labeled
nuclei.
4.7.1.

Avolding

Artifacts

A potential error that can enter into analyses of labeled proflles


is that one profile can be counted twice when, for example, a
labeled nucleus is cut in half with the two halves appearing in
adjacent sections. To avoid this problem, we derive counts of
labeled profiles from every second or every third section: if sections are 50 pm in thickness, one nucleus cannot span more than
two sections and counts will reflect the actual number of labeled
nuclei.
For multiple labeled cells where the nucleus is labeled for Fos
and the cytoplasm is labeled for a neurotransmitter
or enzyme,
one should count cells from every second or third section as above.
Furthermore, it is important to include in the counts only those
cells whose nuclei (either immunopositive
or immunonegative)
are visible. For further discussion about counting labeled profiles
in tissue sections, see Coggeshall and Lekan (1996).
4.7.2.

Expressing

Results

It is often desirable to express results for a given group (nucleus)


of cells in the brain. Three approaches are most commonly used:

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Krukoff

1. We routinely count labeled neurons (in every second section)


throughout the extent of the cell group, average the values,
and express the results as number per section. The disadvantage of this approach is that any regional differences that
might be present within a group of cells (e.g., rostra1 versus
caudal regions) will not be apparent. To overcome this disadvantage yet continuing to express the results as number per
section, we have subclassified sections into regions (e.g., rostral vs intermediate, and caudal) using arbitrary but reproducible borders among the regions (Krukoff et al., 1994;
Krukoff et al., 1995).
2. Investigators have also used the total count of labeled neurons in a given region of the brain. I consider this approach
less satisfactory because every section must be saved and analyzed, and total numbers of analyzed sections must be identical among animals in order to make comparisons. Furthermore,
a correction factor must be included in the calculations of
labeled profiles to eliminate the artifact associated with counting the same profile twice (Coggeshall and Lekan, 1996).
3. A third means of expressing results is to choose one representative section through a reproducible plane, to count the number of labeled profiles, to express the value as number per
section, and to use this value in order to make comparisons
among animals (Herdegen et al., 1995; Krukoff and Khalili,
1997). As is in item 1, however, a value obtained in this way
will not illustrate differences in counts that may occur in a
rostrocaudal direction through a group of neurons,

5. Advantages
5.1.

and Disadvantages

Advantages

The use of Fos immunohistochemistry


to identify neurons that
belong to functional neural systems has gained wide acceptance
in the neurosciences and is routinely used whenever the investigator requires the morphological data that comes with using tissue sections. The primary reasons that the technique has been so
successful are:
1. Techniques are straightforward
and relatively easy to use. A
corollary of these advantages is that standard equipment and
supplies are required, making the expense reasonable.

c-fos

227

Expression

2. Results can be quantitated either manually or with image


analysis methods. The ability to apply statistical analyses to
the data further strengthens the impact of the results.
3. Standard methods for Fos immunohistochemistry
can be combined with other neuroanatomical techniques. It is now possible, therefore, to assign direct functional significance to the
anatomical data obtained with other means.
4. The presence of Fos illustrates multisynaptic pathways of the
brain, providing a more complete understanding of the brain
areas involved in a given function.
5.2. Points of Caution

and Disadvantages

1. Careful controls are required to eliminate extraneous (and not


always obvious) sources of background activity (e.g., anesthesia, stress, circadian rhythms). Even seemingly mild forms of
handling of conscious animals lead to Fos expression in the
brain (Asanuma and Ogawa, 1994).
2. Although expressed by many systems m response to stimulation, immediate-early genes are not universal markers of neuronal activity (Curran and Morgan, 1995). Therefore, lack of
Fos should not be taken as proof that a neuron has not been
activated. Even with this caveat, however, valuable data can
be obtained about neuronal systems if careful controls are
included in the studies.
3. Neurons that are inhibited do not generally express Fos, limiting study to excitatory pathways.
4. The technique may not be applicable to long-term experiments
if expression of c-fos is not required after the initial stimulus
has been applied.
5. It is not possible to tell whether an identified pathway is unior multisynaptic using the expression of c-fos, and results must
be interpreted with this limitation in mind.
6. The presence of Fos provides no information about which subsequent pathway(s) are also activated.

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

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