basal expression of the inducible transcription factors c-jun, junb, jund, c-fos, fosb, and krox-24...

THE JOURNAL OF COMPARATIVE NEUROLOGY 354:39-56 (1995)

Basal Expression of the Inducible Transcription Factors c-Jun, JunB,

JunD, C-FOS, FosB, and &ox-24 in the Adult Rat Brain

THOMAS HERDEGEN, KARLA KOVARY, ANJA BUHL, RODRIGO BRAVO, MANFRED ZIMMERMA", AND PETER GASS

11. Institute of Physiology, Im Neuenheimer Feld 326 (T.H., A.B., M.Z.) and Institute of Neuropathology, Im Neuenheimer Feld 220 (P.G.), University of Heidelberg,

69120 Heidelberg, Germany; Department of Molecular Biology, Bristol-Myers Squibb Pharmacological Research Institute, Princeton, New Jersey, 08543-4000, USA (K.K., R.B.)

ABSTRACT Jun, Fos, and Krox proteins are inducible transcription factors contributing to the control

of gene expression. The elucidation of their individual expression patterns in the nervous system provides new insights into the ability of neurons to react with changes of gene expression to external stimulation under physiological or pathological conditions. The expression of c-Jun, JunB, JunD, c-Fos, FosB, and k-ox-24 was investigated in the brain of untreated male Sprague-Dawley and female BDIX rats by immunocytochemistry using specific antibodies.

JunD immunoreactivity (IR) labeled the highest number of neurons, being present in almost all neurons of the brain. JunD was expressed at high levels in those areas that also exhibit c-Jun, JunB, c-Fos, and FosB-IR, such as locus coeruleus, periolivary nuclei (ncl.), pontine and central gray, lateral lemniscal ncl., inferior and superior colliculi, leaflet of geniculate ncl., midline nuclei of thalamus, dorsomedial and paraventricular ncl. of hypothalamus, ncl. supraopticus, dorsolateral part of caudate putamen and lateral septal ncl. In contrast to the high number of JunD-positive neurons, c-Jun, JunB, c-Fos, and FosB proteins were detected in rather low numbers of neurons in these brain areas; the rank of the number of immunopositive neurons was c-Fos > JunB > c-Jun > FosB. Particularly high levels of expression were observed for c-Jun in medullary motoneurons, medial geniculate ncl., arcuate ncl., and dentate gyrus, and for JunB in the CA-1 area of the hippocampus and islands of Calleja. The zinc finger protein Krox-24 was expressed in many neurons of these brain areas, with only discrete Jun- and Fos-IR; additionally, many intensely labeled nuclei were present in spinal ncl. of the trigeminal ventromedial ncl. of the hypothalamus and the CA-1 area of the hippocampus. In the cerebellum, nuclear labeling was detected only for c-Jun, JunD, and %ox-24 in granule cells. JunD-IR was also found in glial cells of gray matter and fiber tracts, whereas glial c-Jun-IR was observed only in fiber tracts. Apart from a weak JunD-IR, some areas did not express Jun, Fos, and Krox proteins such as cuneate and gracile ncl., venterobasal complex of thalamus, globus pallidum, and Purkinje cells of the cerebellum.

Our data indicate that inducible transcription factors of the fos, jun, and krox gene families show patterns of individual expression in untreated animals, thereby reflecting different mechanisms and/or thresholds for induction under physiological conditions. o 1995 Wiley-Liss, Inc.

Indexing terms: activator protein-1, immediate-early genes, proto-oncogenes, plasticity, zinc finger

Accepted July 28,1994. Address reprint requests to Thomas Herdegen, 11. Institute of Physiology,

Im Neuenheimer Feld 326,69120 Heidelberg, Germany.

o 1995 WILEY-LISS, INC.

40 T. HERDEGEN ET AL.

Jun, Fos and Krox proteins are cellular transcription factors encoded by immediate-early genes (IEGs) that are rapidly induced in vitro by extracellular stimulation (Almen- dral et al., 1988; Chavrier et al., 1988; Curran et al., 1984; Curran and Morgan, 1985; Kovary and Bravo, 1991a,b, 1992; Hirai et al., 1989; Lau and Nathans, 1985; Milbrandt, 1987; Nakabeppu et al., 1988; Ryder et al., 1989; Ryseck et al., 1988; Zerial et al., 1989). The expression and function of these inducible transcription factors (ITFs) have been reviewed extensively (Angel and Karin, 1991; Bravo, 1990; He and Rosenfeld, 1991; Herschman, 1991; Morgan and Curran, 1991; Sheng and Greenberg, 1990).

Numerous reports have demonstrated that individual areas of the central nervous system express jun, fos, and krox genes in the absence of any intentional stimulation (Cole et al., 1989, 1990; Gass et al., 1993a,b; Herdegen et al., 1990, 1991b, 1993c; Hirai et al., 1989; Honkaniemi et al., 1992; Mack et al., 1990; Mellstrom et al., 1991, Mil- brandt, 1987; Rusak et d., 1992; Schlingensiepen et d . , 1991, 1994; Wisden et al., 1990; Worley et al., 1991; Zerial et al., 1989). This “basal” expression suggests a functional role for ITFs in the brain of untreated rats. However, apart from the c-Fos protein (Bullitt, 1989, 19901, few data have been published on the basal expression of ITFs at the single-cell level in the rat brain. We did not find any comprehensive and/or comparative studies on the expression of c-Jun, JunB, JunD, FosB, and &ox-24 proteins in the brain. Moreover, studies about the expression of c-jun, junB, andjunD mRNAs yielded contradictory results (Cole et al., 1990; Hirai et al., 1989; Mellstrom et al., 1991; Schlingensiepen et al., 1994; Wisden et al., 1990). There- fore, the present study was designed to investigate in parallel the basal expression of c-Jun, JunB, JunD, c-Fos, FosB, and Krox-24 (also termed Egr-1, NGFI-A, Zif1268, Tis 8) proteins in consecutive sections of the rat brain by using specific antisera for each individual protein. Such a comparative approach excludes the variations of animal strains, immunocytochemical procedures, and the collec- tion of data among different laboratories. In addition, we also studied how the expression of ITFs depends on the rat strain and sex.

In recent years a lot of information has been collected on the selective induction of ITFs following physiological and pathophysiological conditions such as epileptic seizures, kindling, and long-term potentiation (Cole et al., 1989; Demmer et al., 1993; Dragunow and Robertson, 1987; Dragunow et al., 1992; Gass et al., 1992, 1993a; Morgan et al., 1987; Sagar et al., 1988; Wisden et al., 1990), hypoxic- ischemic insults (Gass et al., 1992; Gubits et al., 1993; Kiessling et al., 1993; Wessel et al., 1991), noxious peripheral stimulation (Herdegen et al., 1991a,b; Hunt et al., 1987; Lanteri-Minet et al., 1993; Menetrey et al., 1989; Walther et al., 1993; Wisden et al., 1990), stimulation of the autonomic nervous system (Lafarga et al., 1992; Sharp et al., 19911, and axotomy (Dragunow, 1992; Haas et al., 1993; Herdegen et al., 1993a-c; Jenkins et al., 1993; Koistinaho et al., 1993; Leah et al., 1991; Rutherfurd et al., 1992a); some

Abbreviations

CNS central nervous system IEG immediate-early gene ITF inducible transcription factor IR immunoreactivities ncl. nucleus

of these findings have been reviewed (Coderre et al., 1993, Dragunow et al., 1989; Herdegen and Zimmermann, 1994; Kiessling and Gass, 1993,1994; Morgan and Curran, 1991; Zimmermann and Herdegen, 1994). In vitro experiments have revealed that the proteins investigated in the present study differentially influence growth, differentiation, or survival of neurons (Pfarr et al., 1994; Schlingensiepen et al., 1993,1994). Therefore, the investigation of congruent and exclusive expression patterns provides insight into possible nuclear cooperation and may reflect the extent of plasticity on the genetic level in the untreated rat brain.

MATERIALS AND METHODS Housing conditions

Male Sprague-Dawley rats (250-300 g, n = 6) and female BDIX rats (200-250 g, n = 3) were kept under constant housing conditions for 5-7 days (12-hour dayinight cycle at 18”C, cages were changed every second day). Animals were removed from the animal house 2 hours after beginning the day cycle. On these days, admittance to the housing room was prohibited before the experiment. Cages were changed 2 days before. Within 15 minutes after removal, rats were deeply anesthetized with sodium pentobarbital (100 mgikg b.w., i.p.) and transcardially perfused with 100 ml of phosphate buffered saline, followed by 200 ml of 4% parafor- maldehyde in phosphate buffer.

The male Sprague-Dawley rats were investigated in three groups of two rats each in 3-month intervals to obtain representative patterns of ITF expression within the strain. The female BDIX rats were investigated in 2-month intervals.

Immunocytochemistry Brains were postfixed for 24 hours in the same fixative,

followed by immersion in 30% sucrose for 48 hours. Coro- nal sections 40-km thick were cut on a cryostat, and every sixth section was processed for a given antiserum. Sections were incubated with the first antibody for 48 hours, followed by conventional avidin-biotin-complex labeling and visualization with diaminobenzidine intensified by nickel ammonium sulfate and cobalt chloride as described elsewhere (Herdegen et al., 1991a).

All antibodies were generated in rabbits immunized with bacterially expressed fusion proteins as described in detail elsewhere (Kovary and Bravo, 1991b). Specificity of the antibodies was demonstrated by immunoprecipitation in vitro and by preadsorption experiments in vivo (Herdegen et al., 1991a, 199313; Kovary and Bravo, 1991a). Antibodies were diluted as follows: anti-c-Jun (code number 60713) 1:1,500; anti-JunB (72515) 1:5,000; anti-JunD (78314) 1:8,000; anti-c-Fos (68915) 1:25,000; anti-FosB (61411) 1:2,500; and anti-Krox-24 (8021 1) 1:6,000.

Statistics and anatomical mapping From each rat the two sections with the highest number

of labeled nuclei of an individual neuroanatomical area were quantitated; the means of labeled nuclei are listed in Table 1. For identification and designation of the neuroanatomical areas, we used standard maps of the rat brain (Paxinos and Watson, 1989; Swanson, 1992). Swanson’s (1992) anatomical nomenclature was used.

BASAL EXPRESSION OF JUN, FOS, AND KROX PROTEINS 41

TABLE 1. Basal Expression of c-Jun, JunB, JunD, c-Fos, FosB, and &ox-24 Proteins in the CNS of Adult Male Sprague-Dawley Rats'

c-Jun JunB JunD2 c-Fos FosB &-ox-24

Forebrain areas Caudate putamen Glohus pallidum Bed ncl. stria terminalis Ncl. accumhens Lateral septal ncl Medial septal ncl.

Amygdala Central ncl. Basolateral ncl

Hippocampus CA1 area CA!2 area CA3 area Dentate gyrus

Granule cell layer Polymorph layer

Thalamus Paraventricular ncl. Anterior complex Ventral posterior ncl Ventral anterior ncl. Lateral dorsal ncl. Medial dorsal ncl. Central medial ncl. Central lateral ncl. Lateral hahenula n. Medial hahenula n. Zona incerta Reticular ncl. Ncl. reuniens Parafascicular ncl

Hypothalamus Rostra1 division

Paraventricular ncl. Medial preoptic area Median preoptic ncl. Lateral preoptic ncl. Anterior area Lateral area Suprachiasmatic ncl. Supraoptic ncl.

Caudal division Ventromedial complex Dorsomedial ncl. Posterior ncl. Arcuate ncl.

Mammillary region Tuheromammillary ncl Premammillary ncl. Medial mammillary ncl Supramammillary n

Cerebellum Granular layer Purkinje cell layer

Midbrain, hindbrain, and medulla

Ventral tegmental area Suhstantia nigra compact

Suhstantia nigra reticular

Medial pretectal area Anterior pretectal ncl. Olivary pretectal ncl.

Part

p a d

Geniculate nuclei Medial complex Lateral complex Intergeniculate leaflet

Superior colliculus Superficial gray layer Intermediate gray layers

Inferior colliculus Central ncl. Dorsal-xternal ncl

++++ h

++ +++ +++

+

++ ++

++++ ++

++++ ++++

h

+++ b h h h h

+++ +++

+ b + h

h +

+++ ++

+++ h + + + +

+ +++ +++ ++

+ h b

++

+++ -

+ h

h +++

h b

h +

++

+ &

+++ +++

+ - +

++ t++ -

++ ++

+ - +

++ -

+ - - - - - + + + - - - - -

+ + + + - + + +

+ + + +

+ - - +

- -

-

-

- ++ - +

- +

++

++ ++

+++ +++

++++ - +

++ +++ ++

++++ +++

++++ + -

++ +

+ - - - - - + + + - + - - -

+ + + - + - + -

++ ++ ++ ++

+ - - +

+++ -

-

-

- ++ - +

- ++++

++

+++ ++++

++ ++++

TABLE 1. (continued)

c-dun JunB JunD2 c-Fos FosB box-24

Raphe nuclei Dorsal ncl. Magnus ncl. Pallidus ncl.

Olivary nuclei Superior complex Periolivary ncl.

Reticular nuclei Gigantocellular ncl. Paragigantocellular ncl. Lateral ncl. Mesencephalic reticular

ncl. Central gray Pontine reticular ncl. Pontine gray Edinge-Westphal ncl. Red ncl. Parahrachial ncl. Lemniscal ncl. Ncl. incertus Locus coeruleus Tegmental ncl. Dorsal cochlear ncl. Ventral cochlear ncl. Medial vestibular ncl. Lateral vestibular ncl Ncl. of solitary tract Spinal ncl of the tri-

gemmal Gracile ncl. Cuneate ncl. External cuneate ncl. Area postrema

Motor nuclei of cranial nerves

Oculomotor ncl. Motor ncl. of trigeminal Abducens ncl. Facial ncl. Dorsal motor ncl. vagus n Ncl. ambirrnus Hypoglossal ncl. ++

+ h -

b ++

h h h

+++ ++ ++ + +

++ +++

h ++ h h h h h

h

+ ++ b h h h

h h h h h h b

'Means of bilaterally labeled neuronal nuclei in defined neuroanatomical areas for each 40-pm-thick coronal section. The means are ranked in five categories: -, absence of immunoreactivities; +, 1-21); + +, 20-50; + + +, 50-100 + + + +, > 100 labeled nuclei/ section. %For JunD we discriminated hetween the moderate, hasic JunD labeling present in numerous neurons of almost every area that is marked by "h" and intensely stained nuclei above the hasic labeling that were given as numbers according to the above categories.

RESULTS Each antibody evoked a prominent nuclear immunoreac-

tivity (IR) in selective neuroanatomical areas. Table 1 summarizes the different patterns of regional expression of all antibodies investigated in 40-pm-thick coronal sections of untreated adult Sprague-Dawley rats.

Expression of individual proteins c-Jun. The specific anti-c-Jun antiserum evoked a dis-

crete pattern of immunoreactivity in defined areas of the rat brain. In the medulla oblongata, c-Jun was expressed in somatosensory and viscerosensory motoneurons (Fig. 1A) and in the nuclei (ncl.) of the solitary tract. Scattered neurons with c-Jun-IR were present in the trigeminal, pontine, and medullary gray, as well as in the inferior and superior colliculi. This restricted pattern was obviously due to selective expression rather than to low affinity of the antibody because, as shown Figure 1G for comparison, an intense c-Jun-IR was present in many neurons in the adjacent caudal levels of the entorhinal, temporal, and occipital cortex. Areas with intensely labeled neuronal


nuclei were the medial geniculate ncl. (Fig. lB), geniculate leaflet ncl. (Fig. 10, and arcuate ncl. (Fig. 1D). Some scattered c-Jun-positive nuclei were present in the midline nuclei of the thalamus and hypothalamus as well as in the amygdala. The most prominent expression of c-Jun was seen in the dentate gyrus and in the caudal, but not rostral, CA-1 field of the hippocampus (Figs. 1E-F), confirming previous findings on the mRNA level (Cole et al., 1990). In the caudate putamen, a rather low number of c-Jun immunoreactive neurons was present in the dorsolateral part. In the cerebellum, the granule cell layer showed a distinct c-Jun labeling (Fig. 7A).

In contrast to c-Jun, JunB was completely absent in the medulla oblongata. Scattered neurons could be reproduced in the dorsal tegmental ncl., lemniscal ncl., central gray, inferior and superior colliculi (Fig. 2A), arcuate ncl., and in the midline areas of hypothalamus and thalamus. JunB was expressed at high levels in the CA-1 and to a somewhat lesser extent in the CA-3 area of the hippocampus (Fig. 2B-C), whereas its expression in the dentate gyms was almost absent. Intense nuclear labeling of many neuronal nuclei was also prominent in selective forebrain areas, such as caudate putamen (Fig. 2D), ncl. accumbens, lateral septal ncl. (Fig. 2E), and islands of Calleja (Fig. F). In addition to the nuclear distribution, we observed a granular cytoplasmic JunB-IR in medullary motoneurons, particularly in motoneurons of the facial nerve.

In comparison with other transcription factors investigated in the present study, JunB presented a distribution pattern that was generally similar to that of FosB, apart from the JunB expression in the hippocampus.

Of all the proteins investigated, JunD-IR was visible in the highest number of neuronal nuclei throughout the neuraxis. In almost every area of the rat brain, at least a moderate intensity of JunD-IR was present in most of the glial and neuronal populations. In addition to this weak, ubiquitous labeling, discrete patterns of nuclei with intense JunD-IR could be detected reproducing in somatosensory and viscerosensory motoneurons, periolivary ncl. (Fig. 3A), locus coeruleus (Fig. 3B), pontine areas, dorsal raphe ncl. (Fig. 3 0 , lemniscal ncl., and central gray and lateral complex of geniculate ncl. In the hypothalamus, this intense JunD-IR was reproduced in the dorsomedial (Fig. 3D), paraventricular (Fig. 9E-F), and preoptic and supraoptic nuclei (Fig. 31). In the thalamus, strong neuronal labeling was found in the paraventricular, central-medial, and central-lateral nuclei, whereas only a weak basal JunD-IR could be detected in the ventral posterior and ventral anterior complex (Fig. 3G-H). In the rostral brain, intense JunD-IR was present in the lateral septal ncl., ncl. accumbens, and in the dorsolateral part of caudate putamen, whereas the globus pallidum showed only a weak expression, corresponding to that of ventrobasal complex of thalamus, as shown in Figure 3H. JunD was also present in many glial cells throughout the brain (Fig. 3E,H,K). In the cerebellum, moderate JunD-IR was detectable in neurons of the granule cell layer, but not in Purkinje cells (Fig. 7C).

In general, the areas with intense JunD-IR were fairly congruent with those areas that expressed c-Jun, JunB, c-Fos and, FosB proteins. Within a given neuroanatomical area, we found clusters of neurons with different intensities of JunD-IR, suggesting the existence of (functionally divergent) neuronal subpopulations.

Throughout the brain, numerous neuroanatomical areas showed a fairly low number of scattered neurons

JunB.

JunD.

c-Fos.

with a moderate nuclear c-Fos expression (Table 1). How- ever, we also found areas with enhanced labeling (number and intensity), such as the periolivary ncl. (Fig. 4A), dorsal raphe ncl. (Fig. 4B), pontine and central gray, inferior colliculus (Fig. 4C), intermediate layers of superior colliculus (Fig. 4D), and leaflet of geniculate ncl. (Fig. 4E). Some immunoreactive neurons were also seen in the midline areas of the hypothalamus (arcuate ncl., dorsal-medial area, lateral habenulae and paraventricular ncl.) and thalamus (paraventricular and centromedial ncl.), the dentate gyrus, amygdala, and ncl. accumbens. In the rostral brain, expression was restricted to the lateral septal ncl. (Fig. 4H). In contrast to Jun-B-IR and FosB-IR, c-Fos-IR was absent in the caudate putamen.

This protein was absent in the medulla and hindbrain, whereas in the same coronal section a distinct and intense FosB-IR was visible in the outer layers of the caudal cortex. FosB-IR neurons was observed reproducing in the central gray (Fig. 5A), inferior colliculus, supramamil- lary ncl. (Fig. 5B), the upper blade of dentate gyms (Fig. 5C), the caudal, but not rostral, CA-1 field of hippocampus (Fig. 5D), central ncl. of amygdala and the paraventricular ncl. of hypothalamus. The strongest level of FosB expression was visible in the caudate putamen (Fig. 5E) and the lateral septal ncl. (Fig. 5H). In general, FosB-IR was present in those areas of the rostral brain that also showed basal levels of JunB and c-Fos. The number of FosB-labeled neurons was lower compared with those labeled by JunB and c-Fos. The intensity of FosB-IR was high in the stained nuclei, suggesting that the restricted pattern is due to a restricted expression rather than to a low affinity of the antiserum used.

This protein showed an intense labeling of many neurons in various neuronal populations throughout the central nervous system (CNS). However, in contrast to JunD, &-ox-24 was not ubiquitously expressed and was clearly restricted to defined brain areas. In the brainstem, %ox-24 protein was found in the superficial and deep layers of the trigeminal nucleus, in the ncl. of the solitary tract, and in the area of the ncl. raphe pallidus (Fig. 6A). In the rostral medulla, Krox-24 was expressed in the locus coeruleus, tegmental ncl., ncl. incertus, and in many neurons of the pontine complex and lemniscal ncl. In the central gray, numerous Krox-24-labeled nuclei were present in the ventral part at caudal levels but were localized predominantly in the dorsal part at rostral levels. Addi- tional areas with strong and widespread Krox-24-IR were the mesencephalic reticular ncl., the inferior and superior colliculus (Figs. 6B-C), lateral geniculate ncl., ventromedial ncl. of the hypothalamus (Fig. 6D), caudate putamen (predominantly dorsolateral part; Fig. 6E), ncl. accumbens, and the lateral septal ncl. Within the hippocampus, the CA-1 region presented a dense and strong &-ox-24 expression, wherea only few nuclei exhibited Krox-24-IR in the remaining hippocampal fields. In the rostral brain, &-ox-24 was expressed in many neurons of the gray matter (Fig. 6F).

The pattern of &-ox-24 expression was also selective within defined areas: thus, at the level of the geniculate ncl., many Krox-24-positive nuclei were visible in the dorsal part of the section and scarce in the ventral part. Similarly, the ventrobasal complex of thalamus did not show any immuno- staining. In the forebrain, the presence of Krox-24 in the caudate putamen contrasted with its absence in the globus pallidum. In the cerebellum, &-ox-24 expression was restricted to few granule cells (Fig. 7F). The anti-Krox-24

FosB.

Krox-24.


Fig. 1. c-Jun-IR. A: Motonucleus of the facial nerve. B: Medial almost absent in the rostral CA-1; dorsal at right and medial at top. G. geniculate ncl.; dorsal at right and medial at top. C: Intergeniculate Superficial layers of the caudal entorhinal cortex. H: Glial cells in the leaflet. D: Arcuate ncl. of the hypothalamus; the third ventricle is cerebellar peduncle. Unless stated otherwise, dorsal is up, medial is left. visible at left. E,F: Upper blade of the dentate gyrus (dg) and Magnifications: 400x (A), 200x (B), 300x (C-F), 150x (G), 600x (H). hippocampal CA-1 area at Bregma (E) -6.00 and (F) -3.70; c-Jun is


Fig. 2. JunB-IR. A Inferiorcolliculus. B: CA-3 areaofthe hippocampus. C: CA-1 area of the hippocampus. D. Dorsomedial part of the caudate putamen. E: Ventral part ofthe lateral septa1 nucleus: dorsal at

right, the ventricle at top. F Islands of Calleja. Unless stated otherwise, dorsal is up, medial is left. Magnifications: 200x (A,F), 400x (B,C), 1 5 0 ~ (D), 3 0 0 ~ (El.

antiserum also evoked a nuclear labeling of brainstem motoneurons; however, we were unable to reproduce this finding in all the animals investigated, so it was not included in Table 1.

Expression in neurons and glial cells Apart from some meningeal and ependymal cells, expres-

sion of JunB, c-Fos, FosB, and %ox-24 was found exclusively in neurons. In addition to neuronal nuclei, JunD-IR was present in nuclei of glial cells in white and gray matter as well as in fiber tracts (Fig. 3G-H,K), whereas glial c-Jun-IR was detected predominantly in fibers tracts (Fig. 1H).

Examples for different and congruent patterns of immunoreactivities: Hippocampus,

thalamus, striatum, cerebellum, and cortex Different and congruent distributions of Jun, Fos, and

%ox-24 proteins could be demonstrated in neuroanatomical areas with well-defined neuronal subpopulations. Within the hippocampus, each protein showed its individual preferential expression: c-Jun in the upper and lower blades of the dentate gyrus; JunB in CA-1; c-Fos and FosB in the upper blade of the dentate gyrus; and %ox-24 in CA-1; JunD was homogenously distributed over the hippocampus. In the dentate gyrus, c-Jun, JunD, and %ox-24 labeled hilar and


granule neurons, whereas JunB, c-Fos, and FosB were expressed only in the granule neurons.

We observed that expression patterns depend on the rostrocaudal extension of a neuroanatomical area: in the CA-1 subfield of the hippocampus c-Jun, c-Fos and FosB were expressed in caudal, but not rostral, levels. With progressing rostral levels, the patterns of Jun, Fos, and %ox-24 expression shifted in the central gray from ventral to dorsal, and in the caudate putamen from dorsolateral to dorsomedial.

Congruent distribution was also observed within the thalamus: the common labeling of the midline nuclei (paraventricular, medial-central, and medial-lateral nuclei) contrasted with the absence of immunoreactivities in the ventrobasal complex for all proteins investigated.

The striatum showed an exceptional pattern of selective expression because it represents the only area where c-Fos was absent inspite of the presence of c-Jun, JunB, JunD, FosB, and Krox-24. However, the distribution of the stainings were congruent in that all proteins were expressed in the dorsal part and, apart from the weak JunD-IR, absent in the globus pallidum.

In the cerebellum, immunoreactivities were visible only for c-Jun, JunD, and %ox-24 in neurons of the granule cell layer (Fig. 7A,C,F), whereas Purkinje cells were immu- nonegative for all proteins investigated (Fig. 7A-F).

Finally, a striking congruence was also found in the entorhinal and piriform cortices and the superficial layers of the neocortical layers, as described in detail elsewhere (Herdegen et al., 1993~). Figure 8A-F shows an example of the completeness of this study.

Expression in male and female rats of different strains

To determine the validity of the IEG expression patterns in male Sprague-Dwaley rats, we performed an additional series of stainings in female BDIX rats, which represent another rat strain used widely in neuroscience. We could not detect substantial differences in immunoreactivities between these experimental groups. Figure 9 demonstrates almost identical patterns of c-Jun, JunB, and JunD expression between male Sprague-Dawley and female BDIX rats, even in areas that exhibit a high level of protein biosynthe- sis, plasticity, and humoral activities such as hippocampus and paraventricular ncl. of hypothalamus.

DISCUSSION The expression of the IEG encoded proteins c-dun, JunB,

JunD, c-Fos, FosB, and c o x - 2 4 was studied in the CNS of nonstimulated adult male and female rats. Through the use of specific antisera and the investigation of immunoreactivities in consecutive sections, the present study virtually excludes the variability in expression patterns that arise from differences in animal strains and experimental procedures of individual laboratories. Our results demonstrate a different extent of basal expression and a selective neuroanatomical location for each member of thejun, fos, and Krox gene families. The validity of the patterns obtained could be demonstrated by comparing different male and female rat strains that showed strikingly congruent patterns. The immunoreactivities were exclusively nuclear, in accordance with the function of the proteins as transcription factors (extensively reviewed by Angel and Karin, 1991; Morgan and Curran, 1991; Ransone and Verma, 1990). Only the

JunB-IR in medullary motoneurons suggested the possibility of a cytoplasmic presence without nuclear translocation. Recently, a distinct cytoplasmic immunoreactivity of the zinc finger protein Krox-20, another ITF, was also detected in CNS neurons (Herdegen et al., 1993b; Mack et al., 1992).

The “basal” expression of ITFs in untreated rats refers to the absence of intentional stimulation. The standard housing conditions can still provoke a variety of sensory and social stimuli that contribute to the basal expression of ITFs, but this situation reflects the physiological context of daily experience.

According to their neuroanatomical distribution, the proteins of the present study can be divided into three general categories: (1) transcription factors, such as c-Jun, JunB, c-Fos, and FosB, with the predominant pattern of a restricted and somewhat variable expression in single, scattered neurons in combination with a constant expression in few areas; (2) transcription factors, such as JunD, with an ubiquitous, constant expression in many or all neurons of numerous CNS neuronal subpopulations, suggesting a robust constitutive expression; (3) transcription factors, such as &ox-24, that are present in a large number of neurons in several selective neuroanatomical areas, probably induced by ongoing transynaptic input.

Expression of cJun, JunB, C-FOS, and FosB c-Jun, JunB, c-Fos, and FosB are visible in single,

scattered neurons throughout the CNS, with an emphasis on the ncl. of solitary tract, trigeminal ncl., pontine and central gray, colliculi, midline thalamic and hypothalamic ncl., and lateral septal ncl., as well as in the outer layers of the cortex (Herdegen et al., 1993~). These expressions most likely are due to a momentary excitation of single neurons in the course of transferring physiological “everyday” information. Central neurons of untreated rats receive permanent input from, e.g., extero- and proprioceptive receptors, the autonomic nervous system, and intracerebral networks. Neuroanatomical areas with basal ITF expression are involved in (1) specific somato- and viscerosensory information processing, such as the ncl. of solitary tract, spinal trigeminal ncl., colliculi, ncl. geniculate, and the suprachiasmatic nucleus, and (2) collateral and integrative information processing, such as the midline thalamic nuclei and the central gray.

The reason for the preferential expression of ITFs in specific neuronal subpopulations is still not clear, The mere input of proprio- and exteroceptive information is not a sufficient stimulus in adult mammals. The de novo synthesis of ITFs depends on the pathophysiological context of experience, as was shown in spinal and supraspinal neurons following somatosensory stimulation (Herms et al., 1994; Lanteri-Minet et al., 1993; Molander et al., 1992; Mower, 1994; Umemoto et al., 1994; Wisden et al., 1990). For example, the induction of c-Fos in the visual cortex following visual stimuli is reduced substantially with increasing age (Mower, 1994). However, epileptic seizures or cortical spreading depression evoke a strong expression of c-Fos in the majority of neurons in the visual cortex (Herdegen et al., 1993~1, demonstrating the capability of these neurons to express ITFs. Thus, depending on the stimulus and/or neuronal subpopulations, certain (permanently activated?) pathways within the stimulus-transcription coupling can become habituated to stimuli, whereas activation of other pathways might still respond with ITF expression, as

Figure 3


Fig. 4. c-Fos-IR. A Periolivary ncl. B: Dorsal raphe ncl. C: Inferior colliculus. D: Superior colliculus; the labeled nuclei are locatedpredomi- nantly in the intermediate layer. E: Intergeniculate leaflet. F: Lateral

septal ncl.; dorsal at right, ventricle at top. Unless otherwise stated, dorsal is up, medial is left. Magnifications: 3 0 0 ~ (A,F), 1 5 0 ~ ( E D ) , 500X (E).

shown in non-neuronal cells in vitro (Buscher et al., 1988; Devary et al., 1991).

Major parts of the rat brain do not express ITFs despite their integration in sensory-information transfer, such as

Fig. 3. JunD-IR. A Periolivary ncl. B: Locus coeruleus and mesencephalic ncl. of trigeminal. C: Dorsal raphe ncl. and oculomotoric nuclei (arrows). D Dorsomedial ncl. of the hypothalamus. E: Dentate gyrus. F CA-1 area of the hippocampus. G: Medial and ventral-posterior areas of thalamus; arrows indicate the paraventricular, central-medial and central-lateral nuclei, and the rectangle indicates the ventral-lateral area and is shown (H) at higher magnification; labeling is prominent in glial cells and weak in neurons. I: Supraoptic ncl.. K Labeled glial cells of fimbria. Dorsal is up, medial is left. Magnifications: 5 0 0 ~ (A,C,F, H,I,K), 300x (B,D,E).

the ventrobasal complex of the thalamus and the Purkinje cells of the cerebellum. Interestingly, these areas, including as the hippocampus, do not respond with ITF expression following somatosensory noxious stimulation, despite their electricophysiological excitation (Bullitt, 1989, 1990; Per- tovaara et al., 1993; Zimmermann and Herdegen, 1994). However, basal expression was most prominent in areas that demonstrated the strongest expression after sensory stimulation, stimulation of cardiovascular reflexes, and changes of osmotic pressure, e.g., medulla oblongata, central gray, and midline nuclei of the thalamus and hypothalamus (Bullitt, 1989, 1990; Friauf, 1992; Honkaniemi et al., 1992; McKitrick et al., 1992; Pertovaara et al., 1993; Rutherfurd et al., 1992b; Sharp et al., 1991; Zimmermann and Herdegen, 1994).


Fig. 5. FosB-IR. A: Scattered neurons in the ventral area of the central gray; the third ventricle is on the left. B: Supramammillar ncl. C: Upper blade of dentate gyrus; the labeled nuclei are indicated by arrows. D: CA-1 part of the caudal hippocampus at Bregma -6.00;

dorsal at left, lateral at top. E: Dorsal caudate putamen. F: Lateral septal ncl.; dorsal at right, ventricle at top. Unless stated otherwise, dorsal is up, medial is left. Magnifications: 3 0 0 ~ (A-C,F), 5 0 0 ~ (D), 1 5 0 ~ (El.

Apart from the hippocampus and the caudate putamen, the number of c-Fos-labeled neurons exceeded that of c-Jun-, JunB-, and FosB-labeled neurons, suggesting that the mechanism of stimulus-transcription coupling may be similar for these proteins in numerous regions of the brain but with a different threshold for induction. This argument is supported by in vitro findings that demonstrate a co- induction, to varying degrees, of ITF expression in fibroblasts and pheochromocytoma cells following humoral and trophic stimulation (Almendral et al., 1988; Bartel et al., 1989; Hirai et al., 1989; Kovary and Bravo, 1991a,b; Lau and Nathans, 1985; Zerial et al., 1989).

Mechanisms that determine the basal expression of IEGs

In addition to the input of physiological information, various mechanisms contribute to the presence or absence of ITFs under basal conditions. Pharmacological experiments revealed a control of ITFs by the presynaptic release of neurotransmitters. Thus, application of MK-801, a non- competitive antagonist for NMDA receptors, increases the expression of Jun and Fos, but not Krox-24, proteins in the untreated rat brain. This finding indicates an NMDA- receptor-mediated suppression of ITFs (Dragunow and


Fig. 6. Krox-24-IR. A Spinal ncl. of the trigeminal. B: Inferior colliculus; the dorsalexternal areas reveal a higher density of labeled nuclei compared with the central area. C: Superior colliculus; labeled nuclei are visible in both the superficial and intermediate gray layers.

D: Ventromedial ncl. of hypothalamus. E: Dorsolateral part of caudate putamen. F: Area of olfactory tubercle and ncl. accumbens at Bregma +1.70. Unless stated otherwise, dorsal is up, medial is left. Magn~f~ca- tions: 3 0 0 ~ (A), 1 0 0 ~ (B,F), 2 0 0 ~ (C,D), 1 5 0 ~ (E).

Faull, 1990; Gass et al., 1993b). In contrast, activation of a- and P-adrenoreceptors induces jun, fos, and krox genes (Bing et al., 1991; Carter, 1992; Pertovaara et al., 1993).

The investigated ITFs are encoded by genes that belong to the IEG group. By definition, IEGs can be transcribed with the presence of protein synthesis inhibitors, and their transcription therefore depends on the activation of (consti- tutively) present transcription factors such as the serum- response factor (SRF), calcium/cAMP-response element- binding protein (CREB), or activating transcription factor 2 (ATF-8; Ginty et al., 1993; Hagmeyer et al., 1993; Lamph et al., 1990; Rivera et al., 1993). Because of their rather ubiquitous expression, the mere presence of CREB, SRF,

and ATF-2 in the rat brain (Gonzalez et al., 1989; Herdegen et al., 1993c; unpublished observations) is not indicative for neurons with preferential ITF expression. Investigations into the selective distribution of phosphorylated, i.e., activated forms of CREB and SRF proteins in neurons of untreated rats could provide further information. More- over, certain transcription factors can suppress the transcription of ITF genes and therefore can determine the distribution of basal ITF expression. For example, isoforms of calcium-response element-modulator protein (CREM) and SRF-containing complexes prevent the expression of c-fos (Foulkes et al., 1991; Mellstrom et al., 1993; Shaw et al., 1989).


Fig. 7. Selective expression of Jun, Fos, and c o x proteins in the cerebellum. (A) c-Jun, (C) JunD, and (F) %ox-24 are present in the granule cells, but not in the Purkinje cells; (B) JunB, (D) c-Fos, and (E) FosB are not detectable in cerebellar neurons. Magnification: 300 x.

Congruent and complementary patterns of c-Jun and JunB expression

In many CNS regions the distribution of c-dun- and JunB-labeled neurons resembles that of Fos proteins. In specific neuronal subpopulations, however, c-Jun or JunB show a distinct basal expression in almost all neurons of this area, e.g., c-Jun in spinal preganglionic sympathetic and parasympathetic neurons (Herdegen et al., 1991b), spinal and medullary motoneurons, medial geniculate ncl., and dentate granule cells of hippocampus, and JunB in CA-1 area of the hippocampus. The constant expression of c-Jun in the dentate gyrus has been related to the particular plasticity of these neurons, which have the capacity for synaptogenesis and sprouting and are the basis for memory

and learning (Anoukihn and Rose, 1991; Heurteaux et al., 1993; Rose, 1991; Schlingensiepen et al., 1994). Interest- ingly, the Jun proteins that are present in the hippocampus of untreated rat are selectively induced following long-term potentiation in awake rats, whereas c-Fos and FosB, which are absent under basal conditions, are not expressed (Dem- mer et al., 1993).

JunB could be a functional antagonist of c-Jun (Deng and Karin, 1993; Schutte et al., 1989), and this might underlie their divergent effects on neuronal survival, growth, and differentiation (Schlingensiepen et al., 1993, 1994). Accord- ing to this antagonism, the expression patterns of c-jun and junB mRNAs are inverted in the rat brain (Schlingensiepen et al., 1994), but our observations indicate that this is only


Fig. 8. Congruent expression patterns in the rostra1 piriform area at Bregma +2.80 for (A) c-Jun, (B) JunB, (C) JunD, (D) c-Fos, (El FosB, and (F) Krox-24. Ventral is up, medial is right. Magnification: 300x.

partially true. In the dentate gyrus, the area with the strongest c-Jun expression, weak but distinct nuclear JunB-IR is detectable in neurons. Similarly, the majority of neuronal populations with a high expression of JunB also exhibit at least some c-Jun labeling. Of course, we cannot exclude the possibility of complementary expression in single neurons in areas with both c-Jun-IR and JunB-IR. Moreover, an inverse expression of c-jun and junB as an endogenous genetic property might be true in the hippocampus and striatum, but transynaptic stimulation paradigms could control this gene selection. Coexpression of c-jun and junB mRNA and proteins have been reported following numerous stimulation paradigms. However, nerve transection induces a lasting c-Jun expression without increase of JunB-IR in axotomized neurons (Herdegen et al., 1993a).

Finally, the function of ITFs depends on the various forms of their phosphorylation state, which is governed by intracy- toplasmic second-messenger systems, as described in detail for the c-Jun protein (Boyle et al., 1991; Papavassiliou et al., 1992; Pulverer et al., 1991).

Constitutive expression of JunD JunD is present in the majority of neuronal and glial

nuclei in almost all CNS regions. By analogy, high levels of junD mRNA were also found in quiescent fibroblasts (Hirai et al., 1989; Kovary and Bravo, 1991a,b; Pfarr et al., 1994; Ryder et al., 1989). The fairly ubiquitous presence of JunD protein is most likely due to constitutive expression, i.e., a permanent activation of the junD promotor. There are


Fig. 9. Congruent expression patterns of Jun proteins in male Sprague-Dawley (A,C,E) and female BDIX (B,D,F) rats. c-Jun (A,B) and JunB (C,D) in the hippocampus: c-Jun is expressed mainly in the dentate gyrus, and JunB is expressed mainly in CA-1 and, to a lesser

extent, in CA-3. E,F: JunD in the paraventricular ncl. of the hypothalamus around the third ventricle. Magnifications: lOOx (A-D), 300x (E-F).

reports of a specific octamer motif localized at -97 to -90 in thejunD promotor that forms the major determinant of the constitutivejunD expression (De Groot et al., 1991). In view of its highly constitutive expression junD is considered the “house-keeping gene” for the transcriptional control supporting basic cell metabolism (Bravo, 1990). Some brain regions exhibit an intense JunD-IR. These areas coincide with brain regions that express basal c-Jun, JunB, c-Fos, and FosB and respond with a pronounced induction of ITFs following noxious or stressful stimuli, as discussed earlier. JunD-IR was weak in neurons of the ventrobasal complex and globus pallidum and absent from cerebellar Purkinje cells. Thus, the weak or strong basal expression of JunD

might be indicative of those neurons that react to intentional stimulation with the expression of ITFs.

Because the induction of JunD expression shows a de- layed onset and a striking persistence following intentional stimuli in vitro and in vivo (Demmer et al., 1993; Gass et al., 1992, 1993a; Herdegen et al., 1991a, 1993c; Kovary and Bravo, 1991a), basal JunD expression could also reflect physiological events of the last 24 hours.

High levels of Krox-24 due to synaptic activity The =ox-24 protein shows a prominent basal expression

that is most pronounced in the termination areas of (non-nociceptive) the primary derents , colliculi, lateral


geniculate ncl., striatum, and in structures associated with the limbic system such as the piriform and entorhinal cortices, fornix, amygdala, and hippocampal CA-1 neurons. In contrast to the postulated constitutive expression of JunD, basal &-ox-24 expression seems to be a consequence of permanent, ongoing “physiological” synaptic activity (Worley et al., 19911, most likely due to N-methyl-D- aspartate (NMDA) receptor activation and to physiological “everyday” information (Herdegen et al., 1990; Mack and Mack, 1992). Pharmacological blockade of NMDA receptors by MK-801 abolishes the basal Krox-24-IR within a few hours (Gass et al., 199313; Worley et al., 1991). Basal k-ox-% expression markedly exceeds that of c-Fos, indicat- ing a different transcriptional control of these genes in nonstimulated conditions. A differential control is also effective in following axotomy with the induction of %ox-24 but not of c-Fos (Herdegen et al., 1993a). In contrast, a broad variety of external stimuli of neuronal and non- neuronal cells modulate regional and temporal krox-24 and c-fos expression in vivo and in vitro with similar kinetics (Cole et al., 1989; Dragunow et al., 1992; Gass et al., 1992, 1993a; Herdegen et al., 1990, 1991a, 1993c; Lanteri-Minet et al., 1993; Nguyen et al., 1992; Pertovaara et al., 1993; Sukhatme et al., 1988). Both genes share some 5’ regulatory elements that may become activated in parallel by certain types of stimulation.

Comparison of proteins and mRNA expression Only few studies have systematically investigated the

expression ofjun, fos, and krox-24 genes on an mRNA level. Our data demonstrate a rather congruent expression of c-Jun and JunD proteins with the corresponding mRNAs (Cole et al., 1990; Mellstrom et al., 1991; Schlingensiepen et al., 1994; Wisden et al., 1990). In contrast, there is an obvious mismatch between the distribution of junB mRNA and it corresponding protein. A high, widespread transcription of junB was demonstrated in the CNS of the rat (Mellstrom et al., 1991) and in brain homogenates of mice (Hirai et al., 19891, exceeding that of junD. However, our findings were confirmed by a study on c-jun and junB transcription in the rat brain (Schlingensiepen et al., 1994). The &-ox-24 expression described in this study is also congruent with the distribution of its mRNA as described elsewhere (Schlingensiepen et al., 1991). Finally, our data about the low level of FosB expression comfirms previous observations (Dragunow, 1990).

Molecular genetic implications of basal IEG expression

Fos and Jun proteins contain a conserved leucine zipper motif by which these molecules form hetero- or ho- modimers (Cohen et al., 1989; Kovary and Bravo, 1992; Nakabeppu et al., 1988; Ryseck and Bravo, 1991; compre- hensively reviewed by Angel and Karin, 1991). The dimerization is mandatory for their DNA to bind to the AP-1 consensus sequence, a common transcription regulatory element in the promotor region of numerous genes (Gentz et al., 1989; Kouzarides and Ziff, 1988; Ransone et al., 1990). The range of transcriptional control of AP-1 proteins is determined by their potential dimerization with members of different transcription-factor families, such as CRE- BPliATF proteins (Benbrook and Jones, 1990; Hai and Curran, 1991; Macgregor et al., 1990), MyoD (Bengal et al., 1992), and liver regeneration factor-1 (Hsu et al., 1992). This combinatorial multiplicity is of functional relevance

because the different complexes display individual molecular properties that affect DNA binding and transcriptional activity (Hai and Curran, 1991; Konig et al., 1992; Naka- beppu et al., 1988; Ryseck and Bravo, 1991; Shemshedini et al., 1991). Of major importance are the individual transacti- vating effects of AP-1 complexes on their target genes. Promotor regions containing a single AP-1 element are efficiently activated by c-Jun, and this activation is inhib- ited by JunB (Chiu et al., 1989; Deng and Karin, 1993; Schutte et al., 1989). Cis activation might also determine the basal expression of ITFs: the ability of c-Jun to regulate positively c-jun transcription stands in marked contrast to the negative effect of c-Fos on c-fos transcription (Angel et al., 1988; Sassone-Corsi et al., 1988).

The basal presence of c-Jun, JunD, and %ox-24 contrib- utes to the pool of available transcription factors. The low levels of c-Fos and FosB proteins suggest that in untreated animals AP-1 complexes are constituted mainly of Jun proteins. Because AP-1 complexes composed of these dimers have different molecular activities than Fos= Jun heterodimers (Cohen et al., 1989; Nakabeppu et al., 1988; Suzuki et al., 1992; Zerial et al., 1989) that are preferen- tially found after intentional stimulation, Jun dimers might be involved in the regulation of different cellular processes as compared with AP-1 heterodimers.

The dominant basal expression of c-Jun, JunB, JunD, and &-ox-24 in the hippocampus could be a feature of the high plasticity attributed to this brain region. In contrast, neuronal populations without basal and/or reactive expression of ITFs, such as ventrobasal complex of the thalamus, might represent areas with a restricted potency for gene induction. Thus, already in the untreated rat, the cellular level of inducible transcription factors is indicative of the “plasticity” and “stability,” respectively, in terms of reactive changes in gene expression.

ACKNOWLEDGMENTS We thank Mrs. M. Stefan and S. Grimm for excellent

technical assistance. This project was supported by Deut- sche Forschungsgemeinschaft, grants Zi 110122-2.

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basal expression of the inducible transcription factors c-jun, junb, jund, c-fos, fosb, and krox-24...

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