6 C-fos Expression as a Marker of Functional Activity in the Brain Immunohistochemistry

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 signifi- cance of the pathway. Conversely, measurements made from tis- sue 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 con- cluded with a discussion of the advantages and disadvantages of using c-fos expression as a marker of functional activity in the brain.

From Neuromethods, vol 33 Cell Neurobfology Techmques Ed A A Boulton, G B Baker, and A N Bateson Q Humana Press Inc

213

214 Kru koff

2. Background

2.2. Markers of Metabolic Activity

One of the first popular techniques that was used to meta- bolically 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 glu- cose transport system and is phosphorylatecl to 2DG-6-phos- phate. 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 glu- cose utilization. The technical difficulties and expense of the 2DG technique, however, prompted investigators to seek alternative means to address the question of cellular involve- ment 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 cardio- vascular 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), cardiovas- cular 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 inexpen- sive and technically straightforward, both the cytochrome oxi- dase 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

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 partici- pates 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 expres- sion 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 physi- ological 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 neu- rons, with some anesthetics such as sodium pentobarbital and ketamine suppressing Fos expression, and others, such as ure- thane, a-chloralose, or methoxyfluorane stimulating Fos expres- sion (Marota et al., 1992; Krukoff, 1993; Takayama et al., 1994). To

216 Krukoff

avoid the complicating effects of anesthesia, therefore, it is rec- ommended 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 condi- tions 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 impor- tant 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 immuno- histochemically 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 con- tinued 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. Important Conslderatlons

When the experiment has been concluded, experimental ani- mals 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

c-fos Expression 277

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 stan- dard histology and immunohistochemistry. For Fos immunohis- tochemistry, we recommend no longer than 1 h of postfixation in half-strength fixative, as prolonged fixation decreases immuno- reactivrty 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, immunohis- tochemical 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 60°C 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 sec- tioning.

4. Cut sections in a cryostat (-20°C). 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/ Sfaiffing

3.3.1. Important 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 prod- ucts of these genes are similar in structure, commercially-available antibodies will sometimes recognize Fos and Fos-related antigens.

218 Krukoff

It is important to the interpretation of results to be aware of the antibody’s 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 ampli- fied with the avidin-biotin interaction.

1. Incubate sections overnight at 4°C 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 sec- ondary 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 agita- tion. The ABC reagent is a complex that contains avidin (with a very high affinity for biotin) complexed to horseradish per- oxidase (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 solu- tion containing 25 mg diaminobenzidine and 17 uL hydrogen peroxide in 50 mL of 0.1 M PBS for 5-10 min. The diamino- benzidine 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 tech- niques, fluorescence microscopy may be the method of choice for visualizing all or some of the markers. Switching among fluores- cent 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. In Combination with Other Techniques

3.3.4.1. IMPORTANT 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 anatomi- cal data and, at the same time, preserves the morphological char- acteristics of analysis that are lost with other approaches. In the following discussion, I describe three techniques that can be com- bined with Fos immunohistochemistry, but it is also important to remember that triple and even quadruple labeling can be accom- plished by combining the techniques further. 3.3.4.2. IMMUNOHISTOCHEMISTRY FOR OTHER PROTEINS

Visualization of neurotransmitter proteins or enzymes in acti- vated neurons (express Fos) can be accomplished with double immunohistochemistry using primary antibodies (one for the neu- rotransmitter and one for the Fos) that are raised in different spe- cies. For example, we use antibodies to Fos that have been raised

220 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 spe- cies, tissues can be incubated overnight in a cocktail contain- ing both antibodies (in PBS) at the appropriate dilutions.

2. On the second day, processing for each antigen should pro- ceed individually. For example, complete the Fos immunohis- tochemistry 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 Sub- heading 3.3.2.). Nickel intensification is carried out as follows:

a. Following incubation in the avidin-HRP complex (see Sub- heading 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 trac- ers 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 avail- able 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) ani- mal. 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 anes- thetized animal. The animal is allowed to recover from anesthe- sia 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 expres- sion 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 dis- tinguishable. Bars = 40 pm (from Petrov et al., 1995 with permission).

224 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 zn situ hybridization can be combined with Fos immunohis- tochemistry to obtain results that indicate that an activated neu- ron (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 accord- ing to the strength of the signal. The preferred sequence of pro- cessing 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 proce- dures must be carried out in RNase-free solutions and plastic- or 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 (60°C). Add 0.2 g chro- mium 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 37°C. 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 -70°C in slide boxes containing dessicant. Sec- tions 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 sec- tions 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:

226 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 disad- vantage 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 disad- vantage yet continuing to express the results as “number per section,” we have subclassified sections into regions (e.g., ros- tral vs intermediate, and caudal) using arbitrary but repro- ducible borders among the regions (Krukoff et al., 1994; Krukoff et al., 1995).

2. Investigators have also used the “total count” of labeled neu- rons in a given region of the brain. I consider this approach less satisfactory because every section must be saved and ana- lyzed, and total numbers of analyzed sections must be identi- cal 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 count- ing the same profile twice (Coggeshall and Lekan, 1996).

3. A third means of expressing results is to choose one represen- tative section through a reproducible plane, to count the num- ber 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 and Disadvantages

5.1. 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 investi- gator requires the morphological data that comes with using tis- sue 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 Expression 227

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 com- bined with other neuroanatomical techniques. It is now pos- sible, 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., anesthe- sia, 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 stimula- tion, immediate-early genes are not universal markers of neu- ronal 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, lim- iting 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 uni- or 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 sub- sequent pathway(s) are also activated.

Acknowledgments The author gratefully acknowledges the support of the Medi-

cal 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.

228 Krukoff

References

Asanuma, M and Ogawa, N (1994) Pitfalls m assessment of c-fos mRNA expres- sion 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 c- fos 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

Cullman, W E , Herman, J P, Battagha, D F. ., Akll, H , and Watson, S J (1995) Pattern and time course of immediate early gene expression m rat brain fol- lowmg acute stress Neurosczence 64,477-505

Curran, T and Morgan, J I (1995) Fos. an immediate-early transcription factor m neurons J Neuroblol 26,403-412

Curran, T and Teach, N M (1982) Candidate product of the FBJ murme osteosarcoma virus oncogene characterization of a 55,000-Dalton phospho- protein J Vlrol 42, 114-122

Handa, R J , Nunley, K. M , and Bollnow, M R (1993) Induction of c-fos mRNA m the brain and anterior pituitary gland by a novel environment Neuroreport 4,1079-1082

Herdegen, T , Kovary, K , Bubl, A, Bravo, R, Zimmermann, M , and Gass, P (1995) Basal expression of the inducible transcription factors c-Jun, JunB, JunD, c-Fos, FosB, and Krox-24 m the adult rat bram J Comp Neural 342, 39-54

Kennedy, C , Des Rosters, M H , Jehle, J W , Relvich, M , Sharpe, F., and Sokoloff L (1975) Mapping of functional neural pathways by autoradiographic sur- vey of local metabolic rate with “C-deoxy-glucose Science 187,850-853

Krukoff, T L (1988) Decreased hexokinase activity m the paraventricular nucleus of the hypothalamus of adult SHR and renal hypertensive rats Am I Phys!ol 254, R508-R512

Krukoff, T L (1994) Expression of c-fos m studies of central autonomic and sensory systems Molec Neurobzol 7,247-263

Krukoff, T L and Scott, T M (1983) The postnatal development of the nucleus commlssurahs and nucleus medialis of the nucleus tractus sohtarlus Dev Brain Res 9,359-367

Krukoff, T L and Scott, T M (1984) A comparison of the development of two subnuclei of the nucleus tractus solltarms in spontaneously hypertensive and normotensive rats Dev Bram Res 12, 39-44

Krukoff, T L. and Calaresu, F R (1984a) Cytochrome oxldase activity m the hypothalamus of SHR and normotensive rats before and after fasting Brazn Res 322,75-82

Krukoff, T L and Calaresu, F R (1984b) Exogenous vasopressm reverses hyperactivity m the hypothalamus of Brattleboro rats Am J Physiol 247, R932-R935

Krukoff, T L and Khallh, P (1997) Stress-induced activation of nitric oxide-producing neurons m the rat bram J Comp Neural 377,509-519

Krukoff, T L. and Patel, K P (1990) Alterations m brain hexokmase activity associated with streptozotocm-induced diabetes melhtus m the rat Brarn Res 522,157-160

c-fos Expression 229

Krukoff, T L and Vincent, D H (1989a) Regional alterations m hexokmase activity within rat bram durmg dehydration and rehydration Am. J Physlol. 256, R1050-R1055.

Krukoff, T L and Vincent, D H (198913) Effects of colchicme on hexokmase activity m the paraventricular and supraoptic nuclei of spontaneously hypertensive and normotensive rats Brain Res Bull 23,47-51

Krukoff, T L and Weigel, M A (1989) Metabolic alterations in discrete regions of the rat brain during development of spontaneous hypertension Brazn Res 491, l-6

Krukoff, T. L , Turton, W E , and Calaresu, F R (1986) Increased hexokmase activity m the forebrain of water deprived and diabetes insipidus rats Am. J Physiol 251, R268-R273

Krukoff, T. L , Harris, K H , Lmetsky, E., and Jhamandas, J H. (1994) Expres- sion of c-fos protein m rat brain elicited by electrical and chemical stimula- tion of the hypothalamic paraventricular nucleus Neuroendocrtnol 59, 590-602

Krukoff, T L., MacTavish, D , Harris, K. H , and Jhamandas, J H (1995) Changes m blood volume and pressure induce c-fos expression m bramstem neurons that prolect to the paraventricular nucleus of the hypothalamus Mol Braw Res 34,99-108

Lee, W M F , Lm, C , and Curran, T (1988) Activation of the transforming potential of the human Fos proto-oncogene requires message stabilization and results in increased amounts of partially modified Fos protein Mol Cell Bzol 8,5521-5527

Marota, J J A, Crosby, G , and Uhl, G R (1992) Selective effects of pentobar- bital and halothane on c-fos and lun-B gene expression m rat brain Anesfhe- stology 77,365-371

Melia, K. R, Ryabmin, A. E, Schroeder, R, Bloom, F E , and Wilson M C (1994) Induction and habituation of immediate early gene expression in rat brain by acute and repeated restraint stress J Neuroscz. 14,5929-5938

Muller, R., Bravo, R., Burckhardt, J , and Curran, T (1984) Induction of c-fos gene and protein by growth factors precedes activation of c-myc Nature 312, 716-720.

Patel, K. I’., Zhang, P L., and Krukoff, T. L (1993) Alterations in brain hexokmase activity associated with heart failure m rat. Am. J Physrol 264, R923-R928

Petrov, T., Jhamandas, J H., and Krukoff, T L. (1994) Electrical stimulation of the central nucleus of the amygdala induces fos-like immunoreactivity m the hypothalamus of the rat a quantitative study. Molec Bratn Res 22,333-340

Petrov, T , Krukoff, T L., and Jhamandas, J. H. (1995a) Convergent Influence of the central nucleus of the amygdala and the paraventricular hypothalamic nucleus upon brainstem autonomic neurons as revealed by c-fos expression and anatomical tracing. J Neurosci Res 42, 835-845

Petrov, T., Harris, K H , MacTavish, D., Krukoff, T. L , and Jhamandas, J H (1995b) Hypotension induces Fos immunoreactivity m NADPH-diaphorase positive neurons m the paraventricular and supraoptic hypothalamic nuclei of the rat Neuropharmacol 34,509-514

Shen, M and Greenberg, M E (1990) The regulation of function of c-fos and other immediate early genes in the nervous system Neuron 41,477-485

Sokoloff, L. (1977) Relation between physiological function and energy metabo- lism m the central nervous system J Neurochem 29,13-26

230 Krukoff

Sokoloff, L. (1981) The relationship between function and energy metabolism Its use m the locahzation of functronal actrvlty m the nervous system Neuroscl 19,159-210

Takayama, K , Suzuki, T , and Mmra, M (1994) The comparison of effects of various anesthetics on expression of Fos protein m the rat brain Neuroscr Lett 176,59-62

Wong-Riley, R and Riley, D. A (1983) The effect of impulse blockage on cyto- chrome oxidase activity in the cat visual system. Brarn Res 261, 185-193

Wouterlood, F G , Bol, J. G J M , and Stembusch, H W M (1987) Double-label rmmunocytochemrstry combmatlon of anterograde neuroanatomlcal trac- ing with Phase&s vulgam leucoagglutmm and enzyme immunohrstochem- lstry of target neurons J Hrstochem Cytochem 35,817-823