Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study

E L S E V I E R Neuroscience Research 26 (1996) 235--269

NEUROSCIENCE R(SERRCH

Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain" an immunohistochemical and in situ

hybridization study

Masafumi Morimoto, Noriyuki Morita, Hitoshi Ozawa, Keiko Yokoyama, Mitsuhiro Kawata*

Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602, Japan

Received 28 June 1996; accepted 15 August 1996

Abstract

The localization of glucocorticoid receptor (GR) immunoreactivity and mRNA in the adult rat brain was examined by light microscopic and electron microscopic immunohistochemistries, and in situ hybridization. For the purpose of detailed investigation of the distribution and comparison of GR immunoreactivities and mRNAs, specific polyclonal antibodies against a part of the transcription modulation (TR) domain of rat GR were used in the immunohistochemistry, whereas fluorescein-labeled RNA probes, complementary to the TR domain in the GR cDNA were used in the in situ hybridization. In the rat brain, GR immunoreactivity was predominantly localized in the cell nucleus, and the expression of GR mRNA was detected in the cytoplasm. GR-immunoreactive and GR mRNA-containing cells were widely distributed from the olfactory bulb of the forebrain to the gracile-cuneate nuclei of the medulla oblongata. The highest densities of GR-immunoreactive and mRNA-containing cells were observed in the subfields of cerebral cortex, olfactory cortex, hippocampal formation, amygdala, septal region, dorsal thalamus, hypothalamus, trapezoid body, cerebellar cortex, locus coeruleus and dorsal nucleus raphe. The distributional pattern of GR immunoreactivity in many regions was well-correlated with that of GR mRNA, but in the CA3 and CA4 pyramidal layers of the hippocampus, different localization was noted. The present study provides the groundwork for elucidating the role of GRs in brain function.

Keywords: KWD Immunohistochemistry; In situ hybridization; Mapping; mRNA; Polyclonal antibody; Rat brain

I. Introduction

Glucocorticoids from the adrenal cortex are controlled by the brain via the pituitary gland (Keller- Wood and Dallman, 1984; Makara, 1985), and respond to stress and participate in the secretion of corticotropin releasing factor (CRF) and adrenocorti- cotropic hormone (ACTH) through the negative feedback mechanism (Keller-Wood and Dallman, 1984). In addition to this classical role in neuroendocrine function, glucocorticoids have also been shown

* Corresponding author. Tel.: +81 75 2515300; fax: +81 75 2515306: e-mail: [email protected]

tO have various effects on the central nervous system (McEwen et al., 1992; Kawata, 1995); glucocorticoids regulate transmitter levels (Azmitia and McEwen, 1974; Gottesfeld et al., 1978; De Kloet and Reul, 1987), receptor densities (Biegon et al., 1985), signal transduc- tion (Harrelson and McEwen, 1987; Harrelson et al., 1987), neuronal cell birth and death (Sapolsky, 1990; Gould and McEwen, 1993; Sloviter et al., 1993a,b) and neuronal configuration (Kawata et al., 1993, 1994; Nishi et al., 1994). In the peripheral nervous system, glucocorticoids also modulate transmitter choices in developing superior cervical ganglia (McLennan et al., 1980) and in embryonic gut neuroblasts (Jonakait et al., 1980), and modulate the phenotypic differentiation of sympathoadrenal progenitors including chromaffin cells

0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved Pll SO [ 68-0102(96)01105-4

236 M. Morimoto et al. / Neuroscience Research 26 (1996) 235-269

(Michelsohn and Anderson, 1992; Morimoto et al., 1994).

The effects of glucocorticoids are mainly mediated through the adrenal steroid receptors, which can bind to nuclear DNA and influence the transcription of specific genes (Yamamoto, 1985; Beato, 1989). Previous studies have indicated the existence of two adrenal steroid receptors in the brain (De Kloet et al., 1975). One is called the type I receptor, and has a high affinity for corticosterone, the principle glucocorticoid in rodent (Beaumont and Fanestil, 1983; Krozowski and Funder, 1983). The type I receptor has been identified as a mineralocorticoid receptor (MR) by Arriza et al. (1988). MRs generally mediate the action of corticosterone in the brain, because the brain has low content of glucocorticoid-inactivated enzyme, but aldosterone is also the ligand for the receptor (Funder et al., 1988; Rundle et al., 1989). The other receptor is known as the type II receptor with an affinity for corticosterone 6-10 times lower than that of MRs (Reul and De Kloet, 1985; Funder and Sheppard, 1987; Ratka et al., 1989); the type II receptor has been named glucocorticoid receptor (GR).

The blood level of corticosterone shows a circadian rhythm under normal conditions, and stress can markedly increase the blood concentration (Munck et al., 1984). It has been shown that in the brain, MRs mediate the effects of glucocorticoid within its circadian levels, and GRs mediate them beyond the basal levels, because of the difference in the affinity between the two receptors. Therefore, GRs are the principal receptors which mediate the effects of glucocorticoids in response to stress or drastic changes in corticosterone levels (De Kloet and Reul, 1987; Evans and Arriza, 1989).

In previous studies, the distribution of GRs in the rat brain was demonstrated by using receptor autoradiography (Reul and De Kloet, 1985, 1986). The detailed distribution of GR immunoreactivity in the rat brain has been investigated by two groups which used different antibodies (Ahima and Harlan, 1990; Cintra et al., 1994). These immunohistochemical studies showed higher resolutional results than the traditional receptor binding studies did, but there were some discrepancies in the distribution of GR-immunoreactive cells among the studies. In order to solve these discrepancies in the distribution of GR immunoreactivity in the rat brain, we carried out a sensitive immunohistochemical study at both the light and electron microscopic levels by using a newly produced anti-rat GR antiserum which had high specificity, and an in situ hybridization with a fluorescein-labeled probe (Durrant et al., 1993). There have been some in situ hybridization studies of the distribution of GR mRNA-positive neurons in the rat brain (Aronsson et al., 1988; Van Eekelen et al., 1988; Sousa et al., 1989; Whitfield et al., 1990), but these have not provided a detailed distributional pattern of GR mRNA-

containing structures at the cellular level. The cellular resolution obtained with non-radioactive methods is better than that with radioactive methods (Emson, 1993), and sensitivities have been at least equivalent to that of isotopic in situ hybridization (Emson, 1993; Wisden and Morris, 1994). In our present hybridization histochemistry investigation, newly developed RNA probes were used to estimate the detailed distribution of GR mRNA, providing a clear comparison of immunoreactivity and hybridization signal at the more precise cellular basis.

2. Materials and methods

2.1, Production of the GR antibody

2.1.1. Preparation of the antigen The 6RGR vector (provided by Dr K.R. Yamamoto,

Department of Biochemistry and Biophysics, University of California, San Francisco) containing the rat liver GR cDNA was digested at the SalI and the first HindIII sites in the cDNA. The cDNA fragment coding a part of the transcription modulation (TR) domain of the GR was blunted by T4 DNA polymerase (Takara, Japan) and ligated into pGEX-3X (Pharmacia, Sweden), which had been linearized with Smal. Overnight cultures of Es- cherichia coli (E. coli ) strain DH5e transformed with the GR cDNA-inserted plasmid were diluted 1:10 in fresh medium, and then grown for 2 h at 37°C before adding isopropylthio-fl-D-galactoside (IPTG) to 0.1 mM. After a further 6 h growth, cells were pelleted and resuspended in 1/50 culture volume of 0.1 M phosphate buffered saline (PBS) containing 1% Triton X-100. Cells were lysed on ice by mild sonication and were centrifuged at 10 000 x g for 15 min at 4°C. The supernatant was mixed in polypropylene tubes with 1/100 supernatant volume of the 50% slurry of Glutathione Sepharose 4B (Pharmacia) equilibrated with PBS. After absorption overnight at 4°C, the beads were collected by brief centrifugation at 500 x g and washed three times with PBS. They were resuspended in 2 x bed volume of 20 mM Tris-HCl, 100 mM NaC1, 2 mM CaC12 (pH 8.0). After adding Factor Xa (NEB, USA), the tubes were rotated overnight at 4°C. The GR protein was cleaved by Factor Xa from the glutathione-S-transferase (GST) fusion protein bound to the beads. The sequence of the amino-terminal ten amino acids of the protein was determined by an amino acid sequencer (Applied Biosystems, USA).

2.1.2. Immunization Three New Zealand white rabbits (Shimizu, Japan)

were bled before immunization and injected intracuta- neously with 100 p g of the protein emulsified in Fre- und's complete adjuvant (Nakarai, Japan). After 4 weeks, the rabbits had a few more injections of the antigen intramuscularly at 4-week intervals. The rabbits

M. Morirnoto et al. / Neuroscience Research 26 (1996) 235-269 237

were bled on the 7th day after each injection, and the sera were tested for the ability to detect the antigen using immunoblot analysis.

2.13. Immunoblot analysis Male Wistar-ST rats (Shimizu) weighing 250-300 g

were maintained on a 12:12 h light/dark schedule with access to food and water ad lib. The animals (n = 3) were anesthetized with 50 mg/kg sodium pentobarbital and perfused through the heart with 300 ml cold 0.9% NaCI. Whole brains were removed and placed on ice. Hippocampal regions were cut off the brains. Several pieces of liver, from which the GR cDNA was origi- nally cloned, were removed. All tissues were homoge- nized in 4 volume ice-cold buffer, which was 0.1 M PBS containing 1% Triton X-100, 0.5% N-lauroylsarcosine sodium salt (Nakarai), 1 mM Phenylmethylsulfonyl fluoride (Nakarai), 0.1 mM Pepstatin A (Sigma, USA) and 0.1 mM Leupeptin (Sigma). The homogenates were then boiled for 5 min with 1/2 volume of 3 x sample buffer, which contained 0.2 M Tris, 9% sodium dodecyl sulfate (SDS), 30% glycerol, 15% 2-mercaptoethanol and a small amount of BPB. The samples were electrophoresed on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by Laemmli's method (Laemmli, 1970). After electrophoresis, proteins on gel slabs were electrophoretically transferred to polyvinyli- dene difluoride (PVDF) membranes (Immobilon-W ~, Millipore, USA) by using a semi-dry blotting apparatus (Transblot-SD ~*, Bio-Rad, USA). The membranes were soaked in 0.1 M PBS containing 5% skim milk (Difco, USA) overnight at 4°C. They were incubated with the antiserum or the serum before immunization (diluted 1:100 1000) for 90 rain and then with goat anti-rabbit IgG (1:100, Seikagaku, Japan) for 90 rain at 37°C on a shaker. The sheets were incubated with rabbit peroxidase-antiperoxidase (PAP, l:100, DAKO, Denmark) for 60 min at 37°C on a shaker. They were visualized with 3,3'-diaminobenzidine (1.0 mg/ml in 50 mM Tris- HC1, pH 7.6) containing 0.006% hydrogen peroxide for 1 min at room temperature.

2.1.4. Affinity purification The ligand protein, which had been prepared as the

antigen, was coupled with CNBr-activated Sepharose 4B ~ (Pharmacia) according to the manufacturer's instructions. The antiserum was passed through the antigen-coupled Sepharose 4B ® column to bind the antibodies. Subsequently, the antibodies were eluted by 0.1 M glycine-HC1 (pH 2.5).

2.2. Immunohistochemistry

Male Wistar-ST rats weighing 250-300 g were maintained under the same conditions as for the immunoblot analysis. Immunohistochemical procedures

were carried out according to the previous paper (Yuri and Kawata, 1994). The animals (n = 8) were anesthetized with 50 mg/kg sodium pentobarbital and perfused through the heart with cold 0.1 M PBS, followed by 4% paraformaldehyde (PFA) and 0.2% picric acid in 0.1 M phosphate buffer (PB). The brain was removed from the skull, and kept in the same fixative for at least 2 days at 4°C. The brain was then immersed in 0.1 M PB containing 20% sucrose for 2 days at 4°C and sectioned in the frontal plane at 30 pm on a cryostat (Leica Jung CM3000, Germany). The sections were incubated with the antiserum or the serum before immunization (1:1000 10000) for 48 h at 4°C. Subse- quently, the sections were incubated with biotinylated anti-rabbit IgG (1:250, Vector, USA) for 2 h and then placed in avidin-biotin-peroxidase (ABC) complex (1:50, Vector, USA) for 1 h at room temperature. All incubation media containing the primary and secondary antibodies and the ABC reagents were diluted with 0.3% Triton X-100 in 0.I M PBS. The sections were treated with 3,3'-diaminobenzidine (0.2 mg/ml in 50 mM Tris-HC1, pH 7.6) containing 0.006% hydrogen peroxide for 5 rain at room temperature. They were mounted on gelatin-coated glass slides, osmicated, air dried and coverslipped. Absorption tests were performed by using the absorbed serum, which was incubated with the synthetic GR protein (10 /~g/ml in the serum) for 2 h at 37°C.

Bilateral adrenalectomy was carried out in five male Wistar-ST rats which were anesthetized with 25 mg/kg sodium pentobarbital. Two days after adrenalectomy, the rats were perfused and the immunohistochemical analysis was performed as described above.

2.3. In situ hybridization

2.3.1. Preparation of RNA probes A 500-base-pair Bam HI/SalI fragment of rat GR

cDNA corresponding to the 5' portion of the coding region was subcloned from the 6RGR vector and inserted into the pGEM4 vector (Promega Biotec, USA). The plasmid was linearized with BamHI for the template of the antisense probe and with SalI for the template of the sense probe. Both probes were tran- scribed and labeled with the RNA color kit (Amer- sham, UK) according to the manufacturer's instructions (Durrant et al., 1993). The probes were synthesized in 20 /~1 solution containing 1 x transcription buffer (40 mM Tris-HC1, pH 7.5; 6 mM MgCIe; 2 mM spermidine, and 0.01% (w/v) BSA); 10 mM dithio- threitol (DTT); 8 /~l nucleotide (nt) mixture containing fluorescein-ll-UTP, ATP, CTP, GTP and UTP; 20 units human placental ribonuclease inhibitor; 1 lLg linearized DNA template and 25 units RNA polymerase. SP6 RNA polymerase was used for the synthesis of sense probes, and T7 RNA polymerase was used for


that of antisense probes. The mixture was incubated for 2 h at 40°C for SP6 reaction or at 37°C for T7 reaction. The reaction was terminated by the addition of l0 units of RNase-free DNase, followed by incubation for 10 min at 37°C. Thereafter, the probes were hydrolyzed to an average size of 250 nt by treatment with 40 mM NaHCO3 and 60 mM NazCO 3 for the required length of time (in this case, 20 min) at 60°C, and ethanol precipitated. The precipitation was redis- solved in 50 /~1 of RNase-free distilled water, and the amount of RNA probe was estimated using electrophoresis.

2.3.2. In situ hybridization histochemistry Male Wistar-ST rats weighing 250-300 g v~ere

maintained under the same conditions as for the immunoblot analysis. The animals (n = 8) were anesthetized with 50 mg/kg sodium pentobarbital and perfused through the heart with cold 0.1 M PBS, followed by 4% PFA in 0.1 M PB. The brain was removed from the skull, and kept in the same fixative overnight at 4°C. The brain was immersed in 0.1 M PB containing 20% sucrose for 2 days at 4°C, and sectioned in the frontal plane at 14 /zm on the cryostat. The sections were mounted on slides pre- treated with 2% amino propyl-triethoxysilane in ace- tone.

Hybridization histochemistry was carried out according to a slight modification of the manufacturer's protocol and methods published in detail elsewhere (Nomura et al., 1988; Hirota et al., 1992). The sections were washed with 0.1 M PBS and fixed by 4% PFA in 0.1 M PBS for 15 min, followed by a treatment with 20 /~g/ml proteinase K in TE buffer (10 mM Tris- HC1 pH 8.0, 1 mM EDTA) for 5 min at 37°C. The sections were soaked in 4% PFA in 0.1 M PBS for 15 min and washed with 0.1 M PBS. The slides were incubated with 0.25% acetic anhydride in tri- ethanolamine-HC1 pH 8.0 for 10 min and washed with 0.1 M PBS. The slides were left to air dry prior to hybridization. The sections were incubated with 30 /~1 of 500 ng/ml antisense or sense probe in hybridization buffer containing 50% formamide, 10 mM Tris-HC1 pH 7.6, 200 /~g/ml tRNA, 1 × Denhardt's solution, 600 mM NaC1, 0.2% SDS and 1 mM EDTA for 16 h at 55°C. To prevent evaporation, the sections were covered with parafilm ® (American National Can, USA) and the hybridization proceeded in sealed cham- bers saturated with 50% formamide.

After hybridization, the slides were soaked in 5 x SSC at 50°C to dislodge the parafilm, and placed immediately in 2 x SSC containing 50% formamide for 30 min at 50°C. The sections were rinsed in 2 x SSC for 20 rain at 50°C and twice in 0.2 × SSC for 20 min at 50°C.

Subsequently, the slides were soaked in Tris

buffered saline (TBS; 100 mM Tris-HC1 pH 7.5, 400 mM NaC1), and incubated with blocking solution (0.5% (w/v) blocking agent in TBS) for 1 h at room temperature. Then the slides were rinsed in TBS and the sections were incubated with anti-fluorescein alka- line phosphatase conjugate diluted 1:1000 in 0.5% (w/ v) bovine serum albumin in TBS for 1 h at room temperature. The slides were washed three times in TBS for 5.rain and once in detection buffer; 100 mM Tris-HC1 pH 9.5, 100 mM NaC1, 50 mM MgC12, and soaked in the detection buffer containing 360 /zg/ml nitro blue tetrazolium (NBT) and 140 pg/ml 5-bromo- 6-chloro-3-indolyl phosphate p-toluidine salt (BCIP) for 36 h at room temperature. After developing color reaction, the slides were coverslipped with Gelvatol, a mounting media containing polyvinyl alcohol.

2.4. Morphological analysis

Some sections were counterstained with cresyl violet for immunohistochemical study and methyl green or hematoxylin for hybridization histochemistry to iden- tify the cell groups in rat brain based on the atlas of Swanson (1992). Positively-labeled cells were counted under the light microscope (Nikon, Japan) and the image analysis system (Avia EXCEL ®, Nippon Avion- ics, Japan), and expressed as a percentage of total cells in a 22 500-/~m2-square within a cell group. Relative densities were rated according to the same criteria used by Ahima and Harlan (1990): ( - ) if no labeled cells were observed, low ( + ) if less than 30% of the cells were labeled, moderate ( + + ) for 30-70% labeled and high ( + + + ) for more than 70% labeled cells. Intensities for GR-immunoreactivity were rela- tively estimated. The immunoreactivity observed in the CA1 pyramidal layer of the hippocampus and the arcuate nucleus was rated at strong intensity and that in CA4 pyramidal layer and the supraoptic nucleus was rated at weak intensity.

2.5. Electron microscopic immunohistochemical study

Electron microscopic immunohistochemical procedures were carried out according to the method described in previous papers (Tougard and Picart, 1986; Yuri and Kawata, 1992; Shinoda et al., 1994). Male Wistar-ST rats weighing 250-300 g (n = 3) were anesthetized with 50 mg/kg sodium pentobarbital and perfused through the heart with cold 0.1 M PBS, followed by 4% PFA and 0.5% glutaraldehyde in 0.1 M PB. The brain was quickly removed and immersed in the same fixative buffer for 2 h at 4°C. The brain was then immersed in 0.1 M PB containing 30% sucrose for 2 days at 4°C, and the materials were blocked, frozen and sectioned in the frontal plane at 40/zm on the cryostat.

M. Morimoto et al. / Neuroscience Research 26 (1996) 235-269 239

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Fig. 1. A: Induction of the GST fusion protein by IPTG and isolation of the synthetic GR protein. Molecular weight marker (hme 1), total protein of E. coli treated with (lane 3) or without (lane 2) tPTG and the synthetic GR protein after purification (lane 4) were electrophoresed and stained with Coomassie brilliant blue. The fusion protein (arrowhead) appeared after IPTG induction, and the synthetic GR protein was detected as a single band (arrow). Molecular masses are indicated in kDa. B: Immunoblotting for the homogenate of rat brain and liver tissues. Proteins were transferred to a PVDF membrane and immunostained with the antiserum (diluted 1:1000) and the preimmune serum (diluted 1:1000). The antiserum revealed a single band (arrowhead) with a molecular weight identical to rat glucocorticoid receptor in both the brain homogenate (lane 2) and the liver homogenate (lane 3). The preimmune serum detected no specific protein bands in the brain homogenate (lane 1). Molecular masses are indicated in kDa.

The sections were rinsed with 0.01 M sodium meta- periodate in PBS and then rinsed in PBS. The sections were preincubated with PBS containing 1% BSA and 0.03% Triton X-100 (TBSAPBS) for 2 h at room temperature, then incubated free-floating with the antibody against GR diluted 1:7000 in TBSAPBS for 3 days at 4°C. The staining was made by using the biotin-streptoavidin immunoperoxidase technique (Nichirei, Japan) with 3,3'-diaminobenzidine.

After immunostaining, the sections were postfixed with 1% OsO4 for 1.5 h at 4°C. The sections were dehydrated through an ascending ethanol series, and embedded in Epon/Araldite (Nakarai, Japan). Ultra- thin sections were cut with a Reihert Jung Ultracut (Germany) and observed with the electron microscope (JEM CX-200, JEOL, Japan) with or without the staining of uranyl acetate and lead citrate.

2.6. Animals

The animals were maintained according to the Rules and Regulations for Animal Research, Kyoto Prefectural University of Medicine, and the experi- mental procedures were approved by the University Committee for Animal Research.

2. 7. Terminology

To match the distribution of GR-immunoreactive and mRNA-containing cells in the subregions of the rat brain with the name of the nucleus, we adopted the nomenclature from Swanson's atlas (1992).

3. Results

3.1. Characterization and specification of GR antiserum

The GST-GR fusion protein was identified on SDS- PAGE only when E. coli strain DH5c~ transformed with the GR cDNA-inserted plasmid was cultured under induction with IPTG. The identified band showed ca. 50 kDa. The fusion protein was purified by using the affinity beads. The protein which consisted of 172 amino acids, a part of the transcription modulation domain of GR, was cleaved by Factor Xa from the fusion protein. The synthetic GR protein was detected as a single band with the molecular weight of ca. 24 kDa (Fig. 1A). In the analysis with the amino acid sequencer, the sequence of the amino-terminal amino acids of the protein was equal to that of the amino acid coded by the inserted GR cDNA.

An antiserum was collected 7 days after the third injection. Immunoblot analysis from the homogenates of rat brain and liver tissues indicated that the antiserum (diluted 1:1000) revealed a single band with a molecular weight (97 kDa) identical to the rat GR, and the preimmune serum (diluted 1:1000) detected no specific protein bands (Fig. 1B).

By immunohistochemical analysis using this antiserum, GR-immunoreactive cells were demonstrated in many rat brain regions, with the immunoreactivity predominantly localized in the cell nucleus. The crude antiserum had the same specificity and distributional pattern as an affinity-purified antibody which was gen- erated using the synthetic GR protein-coupled Sep- harose 4B ® column. In addition, the antiserum diluted

240 M. Morimoto et al. / Neuroscience Research 26 (1996) 235 269

1:8000 showed high specific immunoreactivity and very low background in the immunohistochemical study (Fig. 2A). The preimmune serum and the absorbed antiserum showed no specific immunoreactivity (Fig. 2B). The serum passed through the affinity column showed no specific immunoreactivity.

Two days after adrenalectomy, the number of GR- immunoreactive cells and the intensity of immunoreactivity decreased and the subcellular location of GR-immunoreactivity was observed within cytoplasm in most brain regions (Fig. 3). However, a few neurons in the parvicellular division of the paraventricular nucleus and the arcuate nucleus still had nuclear immunoreactivity (data not shown).

o O

3.2. Characterization o f R N A probes

The expression of GR mRNA was detected in the cytoplasm of cell bodies by using the antisense fluorescein-labeled probe. The intensity of color reaction in- creased linearly until 48 h development time and after which it showed a plateau (data not shown; Augood et al., 1992). It was therefore difficult to judge obvious differences among the intensities of color reaction in signal-positive cells. The labeled sense probe showed no

Fig. 2. Photomicrographs showing the specificity of the antibody. Immunohistochemistry with the antiserum (diluted 1:8000) showed clear GR immunoreactivity without background (A), and the absorbed antiserum showed no specific staining (B) in the hippocampus. Bar = 500 #m.

Fig. 3. GR immunoreactivity in the pyramidal cells of the cerebral cortex (layer VI). Strong GR immunoreactivity was observed in the nucleus of the intact rat (A), while weak cytoplasmic GR immunoreactivity was observed in the adrenalectomized rat (B). Nomarski modulation contrast, bar = 20/~m.

specific signals against GR m RN A in the rat brain sections, and the antisense probe showed no signals in those sections treated with RNase before hybridization (Fig. 4).

3.3. Distribution o f GR immunoreactivity and m R N A

The relative densities of GR-immunoreactive or GR mRNA-containing cells were estimated in the counterstained sections (Fig. 5), and were summarized in Table 1. In addition, the distribution of G R mRNA-containing cells in the rat brain was summarized in schematic drawings based on the atlas of Swanson (1992) (Fig. 6). G R immunoreactivity was observed predominantly in the cell nuclei of the examined cell groups, except for the CA3 and CA4 pyramidal layers in the hippocampal formation and the supraoptic nucleus in the hypothalamus. G R mRNA-positive signals were recognized only in cytoplasm. This differential distribution at the cellular level provided different images even on the same magnification. The labeled cells were not strictly di- vided into neurons and glial cells, although neurons were clearly distinguished from glial cells by their morphology; but in most of the cell groups where we estimated the ratio of the labeled cells, neurons were the majority ( > 80%).

M. Morimoto et al. / Neuroscience Research 26 (1996) 235 269 241

3.4, GR immunoreactivity at the ultrastructural level

In the electron microscopic study, the GR immunoreactivity was predominantly observed in the cell nucleus, and some of the endoplasmic reticulum (ER) and Golgi apparatus showed a weak immunoreaction. No immunoreactivity was observed in the mitochondria, lyso- some, matrix or cell membrane. The GR immunoreactivity within the nucleus showed no homo- geneous distribution, but did have a speckled pattern

B

Q

(Fig. 7). There was no GR immunoreactivity in the nuclear membrane or the nucleolus. Absorbed antisera did not show any immunoreaction in the nucleus and cytoplasm.

3.5. Forebrain

3.5.1. Isocortex The isocortex generally showed similar distributional

patterns of GR-immunoreactive and mRNA-containing cells among the subregions (Fig. 8A, B). Layer I possessed a small number of GR-immunoreactive and mRNA-containing cells. High densities of GR-immunoreactive and mRNA-containing cells were demonstrated in layers II/III and layer VI. Layer IV and layer V showed a moderate number of both labeled cells. Within posterior parietal association areas (PTLp), ventral temporal association areas (TEv) and visual areas (VIS), layer IV had high densities of GR-immunoreactive and mRNA-containing cells (Fig. 8C, D). Moder- ate densities of both labeled cells were seen in the claustrum (CLA). GR immunoreactivity in the isocortex was almost localized in cell nuclei. The immunoreactivity recognized in layers II/III was strong, but the intensities in the other layers were weak to moderate. In hybridization histochemistry, large cytoplasmic cells, i.e. the pyramidal cells in layer V throughout the isocortex, were clearly recognized among labeled cells, There was a strong correlation in the isocortex between the distribution of GR-immunoreactive and mRNA- containing cells.

C~

Fig. 4. Photomicrographs showing the specificity of in situ hybridization histochemistry by using fluorescein-labeled probes. The antisense fluorescein-labeled probe clearly showed GR mRNA expression signals in the cytoplasm of the pyramidal cells in layers V and VI of the cerebral cortex (A). The labeled sense probe provided no specific signals (B) and the antisense probe showed no signals in the sections treated with RNase before hybridization (C). Bar = 100 /~m.

3.5.2. Olfactory cortex In the main olfactory bulb (MOB), the glomerular,

inner and outer plexiform layers had low densities of GR-immunoreactive and mRNA-containing cells. The mitral layer possessed few immunoreactive cells and a small number of mRNA-containing cells. Moderate densities of both labeled cells were observed in the granule cell layer (Fig. 9). The anterior olfactory nuclei (AON) except the lateral part showed a moderate density of GR-immunoreactive and mRNA-containing cells. The lateral part of the anterior olfactory nuclei (AON1) had a large number of both labeled cells. Layer II of both dorsal and ventral parts of the tenia tecta (TT), the pyramidal layer of the olfactory tubercle (OT), the pyramidal layer of the piriform area (PIR) and the endopiriform nucleus (EP) showed high densities of strong GR-immunoreactive and mRNA-containing cells. Except in the mitral layer, the distributional pattern of GR-immunoreactive cells and that of mRNA-containing cells were the same.

3.5.3. Hippocampal formation Layers I, IV and V of the entorhinal cortex (ENT) in

the hippocampal formation had low densities of GR-

242 M. Morimoto et al. / Neuroscience Research 26 (1996) 235--269

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Fig. 5. Photomicrographs and camera lucida drawings of GR-immunoreactive cells (A, B, C) and GR mRNA-containing cells (D, E, F) which show the criteria of relative densities. A and D show low ( +, labeled cells were less than 30%) density, B and E show moderate ( + +, labeled cells were 30-70%) density and C and F show high (+ + +, labeled cells were more than 70%) density of GR-immunoreactive and mRNA-containing cells. A and D are layer I; B and E are layer V; and C and F are layer I1 in the cerebral cortex. Bar = 25 /~m.

immunoreactive and mRNA-containing cells. In layer III, a moderate density of immunoreactive cells and high density of mRNA-containing cells were recognized. Layers I! and V of the ENT possessed a large number of both labeled cells. Within the subiculum (SUB), the molecular layer and the stratum radiatum had a small number of labeled cells. The pyramidal layer of the SUB showed moderate densities of GR-immunoreactive cells and high densities of GR mRNA- containing cells. In the Ammon's horn, very high densities of GR-immunoreactive ceils were present in the CA1 and CA2 pyramidal layers. In contrast, the CA3 and CA4 pyramidal layers had low densities of weakly GR-immunoreactive cells, and immunoreactivity was observed in both the nucleus and cytoplasm (Fig. 10A, C). A large number of immunoreactive cells were recognized in the granule cell layer of the dentate gyrus (DG). The nuclear immunoreactivity observed in the granule cell layer of the DG and the CA1 and CA2 pyramidal layers had very strong intensity. The distribution pattern of GR mRNA-containing cells in the hippocampus was different from that of immunoreac-

tive cells. High densities of GR mRNA-containing cells were demonstrated in the CA1, CA2, CA3 and CA4 pyramidal layers and the granule cell layer of the DG. The cytoplasms of the pyramidal cells in the CA1, CA2, CA3 and CA4 layers and those of the granule cells in the DG provided strong signals of hybridization (Fig. 10B, D). The stratum lacunosum-moleculare and the stratum oriens of Ammon's horn had a small number of both labeled cells. The molecular and polymorph layers also showed low densities,

3.5.4. A m y g d a l a

The pyramidal layer in the nucleus of lateral olfactory tract (NLOT) and the central nucleus of the amygdala (CEA) possessed a large number of GR-immunoreactive and mRNA-containing cells (Fig. 11). The nuclear immunoreactivity observed in the CEA was strong compared with the other amygdaloid nuclei (Fig. 11A). The medial nucleus of the amygdala (MEA) and the posterior nucleus of the amygdala (PA) had moderate densities of immunoreactive cells and high densities of mRNA-containing cells. Moderate


Table l Relative densities of GR-immunoreactive and GR mRNA-containing cells in the rat brain

Regions Immunohistochemistry In situ hybridization

Present Ahima and Harlan Cintra et al. Present Sousa et al.

ForebraiH Isocortex

Motor areas (MO) Layer I + - + Layer II/III + + + + + / + + + + + / + + + Layer V + + + + + + + Layer Vl + + + + + + + + +

Agranular insular area (AI) Layer I + Layer II / l l I + + + Layer V + + Layer VI + + +

Anterior cingulate area (ACA) Layer I + - + Layer II/Il i + + + + + + + + + Layer V + + + + + LayerVl + + + + + + + +

Auditory areas (AUD) Layer I + Layer II/III + + + Layer IV + + Layer V + + Layer VI + + +

Ectorhinal area (ECT) Layer I + Layer H/III + + + Layer V + + Layer VI + + +

Perirhinal area (PERI) Layer I + Layer II /II l + + + Layer V + + Layer VI + + +

Posterior parietal association areas (PTLp) Layer l + Layer II/ l l l + + + Layer IV + + + Layer V + + Layer VI + + +

Retrosplenial area (RSP) Layer 1 + Layer l l / l II + + + Layer V + + Layer VI + + +

Somatosensory areas (SS) Layer I + - + Layer If/Ill + + + + + / + + + + + / + + + Layer IV + + + + / + + Layer V + + + + + + / + + LayerVI + + + + + + + + +

Ventral temporal association areas (TEv) Layer I + Layer l l / l II + + + Layer IV + + + Layer V + + Layer VI + + +

Visceral area (VISC) Layer I + Layer l l /IlI + + + Layer IV + +

+

+++

++

++4-

+

+++

++

+++

4-

++÷

++

+++

+

+++

++

++

+++

+

+4-+

++

+++

+

+++

+++

+++

+

+++

4-++

+4-

+4-+

+

+++

++

+++

+

+++

++

++

+++

+

+++

+++

++

+++

+

+++

4-+

+++/++

+

+++


Table 1 (continued)



Layer V 4 + Layer V1 + + +

Visual areas (VIS) Layer I + Layer l I / III + + + Layer IV + + + Layer V + + Layer VI + + +

Claustrum (CLA) + + + + +

Olfactory cortex Main olfactory bulb (MOB)

Glomerular layer + - + Outer plexiform layer + Mitral layer - + + - lnner plexiform layer + + Granule cell layer + + + + + +

Anterior olfactory nucleus (AON) Dorsal part + + + + + + External part + + Lateral part + + + + + + + + Medial part + + + + + + Posteroventral part + + + + / + + + + +

Teania tecta (TT) + / + + Dorsal part

Layer 1 + Layer 1I + + + Layer Ill + Layer IV +

Ventral part Layer I + Layer I1 + + + Layer Ill +

Olfactory tubercle (OT) Molecular layer + + Pyramidal layer + + + + + + + Polymorph layer + + Islands of Calleja (isl) + + + + + Major island of Calleja (islm) + +

Piriform area (PIR) Molecular layer + - + Pyramidal layer + + + + + + + + + Polymorph layer + + + / + + +

Endopiriform nucleus (EP) + + + + + + +

Hippocampal formation Retrohippocampal region

Entorhinal area (ENT) Layer I + Layer II + + + Layer Ill + + Layer IV + Layer V + Layer VI + + +

Subiculum (SUB) Molecular layer + Stratum radiatum + Pyramidal layer + + + + + +

Hippocampal region Ammon's horn

CA1 pyramidal layer + + + + + + + + + CA2 pyramidal layer + + + + + + + + + CA3 pyramidal layer + + + + +

+ 4 4 4 4

+

4 4 4 ÷ + 4 4 ÷ + 4 4 4 ÷ + +

4 + + 4 4 + ÷ +

+ + + + + + + + + +

+ ÷ 4 + + + +

÷ + 4 4 + + +

+ + + ÷

+ 4 + 4

÷

4 + 4 + +

÷

÷ 4 + 4 4 4 4 + + + 4

+

4 4 4 4 4 4 4 4

÷

4 4 4 + ÷ ÷ + ÷

4 4 4

4 4 4 4 4

+ ÷ 4 4 4 4 4 4 + ÷ 4 ÷ ÷ 4 4 +


Table 1 (continued)



CA4 pyramidal layer Stratum lacunosum-moleculare Stratum oriens

Dentate gyrus (DG) Molecular layer Granule cell layer Polymorph layer

Amygdala Nucleus of layeral olfactory tract (NLOT)

Molecular layer Pyramidal layer Dorsal cap

Medial nucleus of the amygdala (MEA) Cortical nucleus of the amygdala (COA) Anterior amygdaloid area (AAA) Central nucleus of the amygdala (CEA) Lateral nucleus of the amygdala (LA) Basolateral nucleus of the amygdala (BLA) Basomedial nucleus of the amygdala (BMA) Posterior nucleus of the amygdala (PA)

Septal region Lateral septal nucleus (LS)

Dorsal part (LSd) Intermediate part (LSi) Ventral part (LSv)

Medial septal complex (MSC) Medial septal nucleus (MS) Nucleus of the diagonal band (NDB)

Posterior septal complex (PSC) Septofimbrial nucleus (SF) Triangular nucleus of the septum (TRS)

Bed nuclei of the stria terminalis (BST) Anterior division Oval nucleus Posterior division

Septohippocampal nucleus (SH)

Corpus striatum Striatum

Caudoputamen (CP) Nucleus accumbens (ACB) Fundus of the striatum (FS)

Pallidum Globus pallidus (GP) Substantia innominata (SI)

Thalumus Dorsal Thalmus

Paraventricular nucleus of the thalamus (PVT) Parataenial nucleus (PT) Nucleus reuniens (RE) Anteroventral nucleus (AV) Anteromedial nucleus (AM) Anterodorsal nucleus (AD) lnteranteromedial nucleus (IAM) Interanterodorsal nucleus (IAD) Lateral dorsal nucleus (LD) Mediodorsal nucleus (MD) Submedial nucleus (SMT) Lateral posterior nucleus (LP) Posterior complex (PO) Ventral medial nucleus (VM)

+ + / + + + + + + + + + - / + +

+ - I + +

+ + + + ÷ + + + + + ÷ + + + + +

+ + i + + + + + + + + + + + + + + + + + + + / + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + - - i + + + + + + + + + + + + + + / + + + + + + + + + + + / + + + + + +

+ + + + + + + + + + + + + + + + ÷

+ + - i + + + + / + + - l + +

+ + + + + +

+ + + + / + + + + + + + + +

+ + + + / + + + + I + + + + + + +

+ + + + + + + / + + + + + + + / + + + + + + + + + + + + +

÷ +

+ + + + +

+ +

+ ÷

÷ ÷

÷ +

+ - / + + +

+ + + + +

+ +

+ + + ÷

÷ +

+

+ + ÷ +

+ + + + ÷

÷

4-+

++ ++/+++ + +/++ + + + + ÷ ÷ + + + + / + + + + + + + + + + ÷ + + + + + + + + ÷ ÷ ÷ + + ÷ + + + ÷ + + +

+ + + ÷ + ÷ + ÷ + + + + + + + + + ÷ + + + ÷ + ÷

+ +

÷ + +

+ + + +

+ +

+ + + +

- / +

+ +

÷ + +

+ + +

+ + -/+ + +


Table 1 (continued)



Ventral posterolateral nucleus (VPL) Ventral posteromedial (VPM) Medial geniculate complex (MG) Lateral geniculate complex (LG)

Intralaminar nuclei of the thalamus Rhomboid nucleus (RH) Central medial nucleus (CM) Paracentral nucleus (PCM) Central lateral nucleus (CL) Parafascicular nucleus (PF)

Epithalamus Medial habenula (MH) Lateral habenula (LH)

Ventral thalamus Reticular nucleus (RT) Lateral geniculate complex (LG) Zona incerta (ZI) Subthalmic nucleus (STN)

Hypothalamus Periventricular zone of the hypothalamus

Suprachiasmatic preoptic nucleus (PSCH) Median preoptic nucleus (MEPO) Anteroventral periventricular nucleus (AVPV) Preoptic periventricular nucleus (PVpo) Supraoptic nucleus (SO) Paraventricular nucleus (PVH)

Magnocellular division (PVHm) Paravicellular division (PVhp)

Anterior periventricular nucleus (PVa) Intermediate periventricular nucleus (PVi) Arcuate nucleus (ARH) Posterior periventricular nucleus (PVp)

Medial zone of the hypothalamus Medial preoptic area (MPO) Medial preoptic nucleus (MPN) Anterodorsal preoptic nucleus (ADP) Anteroventral preoptic nucleus (AVP) Suprachiasmatic nucleus (SCH) Subparaventricular zone (SBPV) Anterior hypothalamic area (AHA) Anterior hypothalamic nucleus (AHN) Retrochiasmatic area (RCH) Ventromedial nucleus (VMH) Dorsomedial nucleus (DMH) Ventral premammillary nucleus (PMv) Mammillary body (MBO)

Tuberomammillary nucleus (TM) Supramammillary nucleus (SUM) Dorsal premammillary (PMd) Medial mammillary nucleus (PMd) Lateral mammillary nucleus (LM)

Posterior hypothalmic nucleus (PH) Lateral zone of the hypothalamus

Lateral preoptic area (LPO) Lateral hypothalamic area (LHA)

Brain stem (BS) Sensory

Visual Super colliculus (SC) Superficial gray layer (SCsg) Intermediate gray layer (SCig) Deep gray layer (SCdg)

+ + + +

+ + + + / + + +

+ + + + + + + + + + +

+ + + + + + + + + + + ~ + + +

+ + + +

+ + + / + + + + + + + + + - - / +

+ + + +

+ + + + + + + + +

+ - / + + + + - / + + +

+ - / + - / + + + + + + + + + + + + + + + + + +

+ + / + + - / + + + + + + +

+ + + + + + + + + + + + + + + +

+ + + + + + + + + +

+ + + + + + + +

+ + - - / + + + +

+ + _ _ +

+ + + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + + + + + + + + + + + + + +

+ + + + + + +

+ + + + + + + + + + + +

+ + + + + + + + + + + + + + + +

+ + - / + + + + + + + + +

+ + + + + + +

+ +

+ + + + + / + + + ÷ + + + +

+ + + + + + + + + + +

+ + + + + +

+ +

+ -- +

+-}- ++

+ - / + + + - / + + + +

+ ÷

+ + +

+ + + +

+ ++ --I+ + + +

M. Morimoto et al. I Neuroscience Research 26 (1996) 235-269 247

Table 1 (continued)

Regions Immunohistochemistry

Present Ahima and Harlan Cintra et al.

In situ hybridization

Presenl Sousa et al.

Parabigeminal nucleus (PBG) + + Olivary pretectal nucleus (OP) + Nucleus of the optic tract (NOT) + + Posterior pretectal nucleus (PPT) + Anterior pretectal nucleus (APN) + + Medial pretectal area (MPT) + +

Somatosensory Mesencephalic nucleus of the trigeminal (MEV) + Principal sensory nucleus of the trigeminal (PSV) + + Spinal nucleus of the trigeminal (SPV)

Oral part (SPVO) + + Interpolar part (SPVI) + + Caudal part (SPVC) +

Gracile nucleus (GR) + Cuneate nucleus (CU) +

Auditory Cochlear nuclei (CN)

Dorsal nucleus (DCO) + Ventral nucleus (VCO) + +

Nucleus of the trapezoid body (NTB) + + + Superior olivary complex (SOC) + + Nucleus of the lateral lemniscus (NLL) + Inferior collicullus (IC) +

Vestibular Vestibular nuclei (VNC)

Medial vestibular nucleus (MV) + Lateral vestibular nucleus (LAV) + Superior vestibular nucleus (SUV) + + Spinal vestibular nucleus (SPIV) + Nucleus prepositus (PRP) +

Visceral Nucleus of the solitary tract (NTS) + + Parabrachial nucleus (PB)

Lateral division + + Medial division + +

Motor Eye

Oculomotor nucleus (III) + + Abducens nucleus (VI) + +

Jaw Motor nucleus of the trigeminal (V) +

Face Facial nucleus (VII) + +

Pharynx/Larynx/Esophagus Nucleus ambiguus, dorsal division (AMBd) + +

Tongue Hypoglossal nucleus (XII) +

Viscera + + Edinger-Westphal nucleus (EW) + + Nucleus ambiguus, ventral division (AMBv) +

Extrapyramidal Substantia nigra (SN)

Compact part (SNc) + Reticular part (SNr) +

Ventral tegmental area (VTA) + +

Pre- and post-cerebellar nuclei Tegmental reticular nucleus (TRN) + Inferior olivary complex (IO) + + Lateral reticular nucleus (LRN) + Linear nucleus of the medulla (LIN) + Paramedian reticular nucleus (PMR) + Red nucleus (RN) +

+ + / + + + + + / + + + - t - + 4 . +/++ + 4 -

+/++ 4-4-

4-4-

4 - + +

.1.4. +

+

4-4-

+ + 4 - 4.

+ + / + + +

+/++

+ + / + + +

+ + / + + +

+ + / + + + +

4..1.

+ + / + + +

-t-

4.

+ +

+.1.

.1 .+

,1, +

+/++

-I-

+

4. 4-

.1.4.

+ +

+

.1.+

- / +

+/++

.1.

+ - t - +

4.

-t- .1. +4-

.1.

4. 4. 4. .1. 4.,1, 4-4-

+ ÷

--.1.

4,+ + + + + ÷

-t-

+

4-+ +4-4- +4- +

4.

+ +

+4- + + +

4-4-

+ +

4-+

+ +

+4-

+

+ +

+4-

+

+ + + + ÷

+4- ÷

+4-

+

+4- -t- 4. 4-4- 4.

+

248

Table 1 (continued)

M. Morimoto et al. / Neuroscience Research 26 (1996) 235-269

Regions lmmunohistochemistry

Present Ahima and Harlan Cintra et al.

In situ hybridization

Present Sousa et al.

Cerebellum Deep cerebullar nuclei

Fastigial nucleus (FN) + Interposed nucleus (IP) + Dentate nucleus (DN) +

Cerebellar cortex Molecular cell layer + Purkinje cell layer + + + + Granule cell layer + + + + + +

Reticular core Central gray of the brain + + +

Periaqueductal gray (PAG) + Interstitial nucleus of the Cajal (INC) + Nucleus of Darkschewitsch (ND) + + + + + Dorsal tegmental nucleus (DTN) + Ventral tegmental nucleus (VTN) + Laterodorsal tegmental nucleus (LDT) + Locus coeruleus (LC) + + + Pontine central gray (PCG) + +

Raphe Interfascicular nucleus raphe (IF) + + + Rostral linear nucleus raphe (RL) + + / + + Central linear nucleus raphe (CLI) + + / + + Superior central nucleus raphe (CS) + Dorsal nucleus raph (DR) + + + + + + Nucleus raphe magnus (RM) + + + / + + Nucleus raphe pallidus (RPA) + + + / + + Nucleus raphe obscurus (RO) + + + / + +

Reticular formation Mesencephalic reticular nucleus (MRNfl) + Pedunculopontine nucleus (PPN) + Cuneiform nucleus (CUN) + Pontine reticular nucleus (PRN) + Gigantocellular reticular nucleus (GRN) + + + Paragigantocellular reticular nucleus (PGRN) +

+ + + +

+ / + +

- / +

+ + / + + + + + +

+ +

+ + + + + + + + +

+ ÷

+ + +

+

+ + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + + +

+ + + + + + + +

Relative densities have been rated: - , none; + , low; + + , moderate; + + + , high.

densities of both labeled cells were seen in the dorsal cap of the NLOT, the anterior amygdaloid area (AAA), the cortical nucleus of the amygdala (COA) and the basomedial nucleus of the amygdala (BMA). The lateral nucleus of the amygdala (LA) and the basolateral nucleus of the amygdala (BLA) showed a small number of immunoreactive cells and moderate densities of mRNA-containing cells.

3.5.5. Septal region and corpus striatum In the bed nuclei of the stria terminalis (BST), the

anterior and posterior divisions had moderate densities of GR-immunoreactive and mRNA-containing cells. In the oval nucleus of the BST, a large number of both labeled cells were present. The ventral part of the lateral septal nucleus (LS) showed moderate densities of both positive cells. A small number of GR-immunore-

active and mRNA-containing cells were recognized in the other parts of the LS, the medial septal nucleus (MS), the nucleus of the diagonal band (NDB), the septofimbral nucleus (SF) and the triangular nucleus of the septum (TRS). Moderate densities of both labeled cells were observed in the striatum, i.e. the caudoputamen (CP), the nucleus accumbens (ACB) and the fundus of the striatum (FS) (Fig. 12). Low densities of both labeled cells were present in the pallidum, i.e. the globus paUidus (GP) and the substantia innominata (SI). The nuclear immunoreactivity observed in the pallidum was weak and that in the striatum was moderate.

3.5.6. Thalamus The distributional pattern of GR-immunoreactive

cells in the thalamic nuclei corresponded well with that


A

MOB gr mi opl gl

B

TTd

TTv-

MOs

P

~AONI

AONpv

I s

OT

Fig. 6.

of the GR mRNA-containing cells. The immunoreactivity was localized in the cell nucleus, and its intensities were weak to moderate. The paraventiricular nucleus of the thalamus (PVT), the lateral dorsal nucleus (LD) and the lateral posterior nucleus (LP) possessed high densities of both labeled cells (Fig. 13). The medial geniculate complex (MG) and the lateral geniculate complex (LG) also had a large number of both positive cells. There was no preferential distribution of either labeled cells in

these complexes, but positive cells were evenly distributed in each layer. Moderate densities of immunoreactive and mRNA-containing cells were demonstrated in the nucleus reuniens (RE), anteroventral nucleus (AV), anterodorsal nucleus (AD), mediodorsal nucleus (MD), ventromedial nucleus (VM), central medial nucleus (CM) and parafascicular nucleus (PF). The other areas of the thalamus showed only a few GR-immunoreactive and mRNA-containing cells.


D MOp

l M SSs

j ,v,c= NDB ~ . ~ " ~ .... J~CLA /

~ A { ~ . / PIR I OT

E MOs MOp

fx

SSs MS nt

" % - - a - - ~ ' ~ = ~ ' V I _ _ /h 'IS M E P O ~ ~.~- r3p ~ /~ C LA C \ _ / !

V 3 ~ ~d=~'~f.Ep I'AI "..~'

NDB O T ~ : ~ = . ~ p IR

Fig. 6.

3.5.7. Hypothalamus The intensities of nuclear immunoreactivity observed

in the hypothalamus were moderate to strong. The GR immunoreactivities in the parvicellular division of the paraventricular nucleus (PVHp) and the arcuate nucleus (ARH) were the strongest in the brain, and they were as strong as those observed in the CA1 pyramidal layer of the Ammon's horn. Most subregions in the periventricular zone of the hypothalamus contained a large number of GR-immunoreactive cells. High densities of both labeled cells were found in the median preoptic nucleus (MEPO), anteroventral periventricular

nucleus (AVPV), posterior periventricular nucleus (PVp), ARH and PVHp (Figs. 14 and 15). The suprachiasmatic preoptic nucleus (PSCH), preoptic periventricular nucleus (PVpo), anterior periventricular nucleus (PVa) and intermediate periventricular nucleus (PVi) possessed moderate densities of both positive cells. The supraoptic nucleus (SO) also showed a moderate density of GR-immunoreactive cells, and the weak immunoreactivity was predominantly located in the cytoplasm (Fig. 16A), while a high density of mRNA- containing cells was noted in both the ventral and dorsal part (Fig. 16B). In addition, strongly immunore-


) J.c ,ksc

AHA NLOT~i ~ BMA PIR

G MOs MOp

\

BMA COA~.,.,~ ~ f PIR

Fig. 6.

active, small-sized cells (thought to be glial cells) were distributed in and around the supraoptic nucleus (Fig. 16A). The magnoceIlular division of the paraventricular nucleus (PVHm) had no GR-immunoreactivies, but did have a small number of GR mRNA-containing cells (Fig. 14).

Within the medial zone of the hypothalamus, the medial preoptic nucleus (MPN), ventromedial nucleus (VMH), dorsomedial nucleus (DMH) and ventral pre- mammilary nucleus (PMv) possessed a large number of

immunoreactive and mRNA-containing cells. Moderate densities of both labeled cells were seen in the anterior hypothalamic nucleus (AHN), anterodorsal preoptic nucleus (ADP) and anteroventral preoptic nucleus AVP). The medial preoptic area (MPO) had a low density of GR-immunoreactive cells but a moderate density of GR mRNA-containing cells. There were a few immunoreactive and mRNA-containing cells in the suprachiasmatic nucleus (SCH), subparaventricular zone (SBPV), anterior hypothalamic area (AHA) and


H

L ~ H ~ ~, ~ ~A,UD " ~ " ' ' ~ "~'" ' " " ' ! ' : '

ARH C O A ~ / PIR

MO MOs

~ A U D

PVp COA ~ BMA

BLA

'v. :iii!

Fig. 6. retrochiasmatic area (RCH). The dorsal premammillary nucleus (PMd) of the mammillary body had moderate densities of both labeled cells. The other nuclei of the mammillary body and the lateral zone of the hypothalamus including the lateral preoptic area (LPO) and the lateral hypothalamic area (LHA) showed low densities of GR-immunoreactive and mRNA-containing cells.

3.6. Brain stem

3.6.1. Sensory The superior coUiculus (SC), olivary pretectal nucleus

(OP) and posterior pretectal nucleus (PPT) had a small number of GR-immunoreactive and GR mRNA-containing cells. The parabigeminal nucleus (PBG) and nucleus of the optic tract (NOT) showed moderate

M. Morimoto et al. ! Neuroscience Research 26 (1996) 235-269 253

J PTLp

J.,O' / T Ev

V J ~ " i ~ : ~ l i I 21/11 .,.,/ec7 PeR,

COA

EW}~O'~ M~"~,:~.¢II III I / T E v : :!.

"~ 'q ~ ENTI

Fig. 6.

densities of immunoreactive cells but low densities of mRNA-containing cells. Moderate densities of both labeled cells were observed in the anterior pretectal nucleus (APN) and medial pretectal area (MPT). A few GR-immunoreactive cells were present in the mesencephalic nucleus of the trigeminal (MEV), whereas a moderate density of GR mRNA-containing cells was recognized. The principal sensory nucleus of the trigeminal (PSV) had moderate densities of both positive cells. Low to moderate densities of both labeled cells were

found in the spinal nucleus of the trigeminal (SPV). The gracile nucleus (GR) and cuneate nucleus (CU) showed a small number of labeled cells.

The nucleus of the trapezoid body (NTB) had many GR-immunoreacive and mRNA-containing cells (Fig. 17). Moderate densities of both labeled cells were observed in the superior olivary complex (SOC) and the ventral cochlear nucleus (VCO). A few labeled cells with GR immunoreactivity and mRNA were found in the dorsal cochlear nucleus (DCO), nucleus of the


L VlS

E W ~ ( ~ A G ( J k~ "] ECT

, ~ ENTI

mcp

\

P a ~

Fig. 6.

lateral lemniscus (NLL) and inferior colliculus (IC). Most of the vestibular nuclei had low densities of GR-immunoreactive and mRNA-containing cells. The nucleus of the solitary tract (NTS) and the lateral and medial divisions of the parabrachial nucleus (PB) showed moderate densities of both labeled cells.

3.6.2. Motor The oculomotor nucleus (III) and abducens nucleus

(VI) had moderate densities of GR-immunoreactive and

GR mRNA-containing cells. The facial nucleus (VII) also showed moderate densities of both labeled cells. Several large motor neurons possessed nuclear immunoreactivity with intermediate intensity. Only a small number of GR-immunoreactive and mRNA-containing cells were found in the hypoglossal nucleus (XII). The Edinger-Westphal nucleus (EW) showed a moderate density of GR-immunoreactive ceils and a high density of mRNA-containing cells (Fig. 18). The immunoreactivity was located in both the cytoplasm


N

RPO TRN

~ 'SOC

NTB

O

PG V ~ NTS

PY

Fig. 6.

and nucleus with moderate intensity (Fig. 18A). In the substantia nigra (SN), the compact part (SNc) had low density of immunoreactive cells and moderate density of mRNA-containing cells, whereas the reticular part (SNr) showed low densities of both labeled ceils.

3.6.3. Pre- and post-cerebellar nuclei and cerebellum The inferior olivary complex (IO) possessed moderate

densities of GR-immunoreactive and mRNA-containing cells. A small number of both positive cells were

found in the other areas of the pre- and postcerebellar nuclei. The deep cerebellar nuclei also had low densities of both labeled cells and their immunoreactivity was weak to moderate (Fig. 19). In the cerebellar cortex, the Purkinje cell layer showed a moderate density of weak GR immunoreactivity which was localized in the nucleus. Most of the granule cells showed moderate to strong GR immunoreactivity (Fig. 20A). A high density of GR mRNA-containing cells was demonstrated in the Purkinje cell layer and a moderate density of GR


P

TS

MARN

PY I0

N SPV

py LRN

Fig. 6.

mRNA-containing cells was observed in the granule cell layer. The GR mRNA-containing granule cells showed weak and ringed-shaped hybridization signals because the cytoplasm of the granule cells was very small (Fig. 20B). The molecular layer, in contrast, contained a small number of both labeled cells.

3.6.4. Reticular core The locus coeruleus (LC) had a high density of

GR-immunoreactive cells, and the immunoreactivity was as strong as that observed in the parvicellular

division of the paraventricular nucleus and CA1 pyramidal layer of the hippocampal formation (Fig. 21A). The high density of mRNA-containing cells was also well-correlated in the LC (Fig. 21B). Moderate densities of both labeled cells were seen in the pontine central gray (PCG) and the nucleus of Darkschewitsch (ND). Within the raphe, the dorsal nucleus raphe (DR) had high densities of GR-immunoreactive and mRNA-containing cells (Fig. 22). Moderate densities of GR immunoreactivity and hybridization signal were present in the interfascicular nucleus raphe (IF), nucleus raphe


magnus (RM), nucleus raphe pallidus (RPA) and nucleus raphe obscurus (RO). The reticular formation and the other parts of raphe had only a few GR-immunoreactive cells, while a moderate number of GR mRNA- containing cells were shown in the mesencephalic reticular nucleus (MRN) and cuneiform nucleus (GUN).

3.7. Miscellaneous

The vascular organ of the lamina terminalis, subfornical organ and subcomissural organ showed moderate densities of GR-immunoreactive cells and mRNA-containing cells. Most of the cells in the choroid plexus had GR immunoreactivity in their nuclei, whose intensities

were low to moderate. A high density of GR mRNA- containing cells was also found in the choroid plexus. The area postrema possessed low to moderate densities of both labeled cells, and a few small nuclear GR-immunoreactive cells were observed in the pineal body.

In the white matter, GR immunoreactivity was observed in small nuclear cells. These cells were thought to be glial cells judging from their morphology. The relative density of immunoreactive cells was dependent on the total number of glial cells in the region. The corpus callosum and the anterior commissure had many GR-immunoreactive cells (Fig. 23), and the optic tract had a moderate number of immunoreactive cells. There were a small number of GR-immunoreactive cells in the cerebral peduncle (Fig. 24). Nevertheless, the propotion

Fig. 6. Schematic drawings of the distribution of GR mRNA-containing cells at different rostrocaudal levels of the rat brain based on the atlas of Swanson (1992). AAA, anterior amygdaloid area; ACA, anterior cingulate area; ACB, nucleus accumbens; aco, anterior commissure, olfactory limb; AD, anterodorsal nucleus thalamus; ADP, anterodorsal preoptic nucleus; AHA, anterior hypothalamic nucleus; AHN, anterior hypothalamic nucleus; AI, agranular insular area; AM, anteromedial nucleus thalamus; AMBd, nucleus ambiguus, dorsal division; AMBv, nucleus ambiguus, ventral division; AONd, anterior olfactory nucleus, dorsal part; AONI, anterior olfactory nucleus, lateral part; AONpv, anterior olfactory nucleus, posteroventral part; AP, area postrema; APN, anterior pretectal nucleus; AQ, cerebral aqueduct; ARH, arcuate nucleus hypothalamus; AUD, auditory areas; AV, anteroventral nucleus thalamus; AVP, anteroventral preoptic nucleus; AVPV, anteroventral periventricular nucleus hypothalamus; BLA, basolateral nucleus amygdala; BMA, basomedial nucleus amygdala; BST, bed nuclei stria terminalis; CA1, field CA1, Ammon's horn; CA2, field CA2, Ammon's horn; CA3, field CA3, Ammon's horn; cc, corpus callosum; ccg, corpus callosum, genu; CEA, central nucleus amygdala; chp, choroid plexus; cing, cingulum bundle; CL, central lateral nucleus thalamus; CLA, claustrum; CM, central medial nucleus thalamus; COA, cortical nucleus amygdala; CP, caudoputamen; cpd, cerebral peduncle; CS, superior central nucleus raphe: cst, corticospinal tract; CU, cuneate nucleus; CUN, cuneiform nucleus; DG, dentate gyms; DMH, dorsomedial nucleus hypothalamus; DR, dorsal nucleus raphe; ec, external capsule; ECT, ectorhinal area; ECU, external cuneate nucleus; ENTI, entorhinal area, lateral part; ENTm, entorhinal area, medial part, dorsal zone; ENTmv, entorhinal area, medial part, ventral zone; EP, endopiriform nucleus; EW, Edinger-Westphal nucleus; fa. corpus callosum, anterior forceps; fi, fimbria; FS, fundus of the striatum; fx, columns of the fornix; GR, gracile nucleus; GRN, gigantocellutar reticular nucleus; GU, gustatory area; IA, intercalated nuclei amygdala; IC, inferior colliculus: icp, inferior cerebellar peduncle; int, internal capsule; IO, inferior olivary complex; IPN, interpeduncular nucleus; LA, lateral nucleus amygdala; LC, locus coeruleus; LD, lateral dorsal nucleus thalamus; LG, lateral geniculate complex; LH, lateral habenula; LHA, lateral hypothalarnic area; LM, lateral mammillary nucleus; LP, lateral posterior nucleus thalamus; LPO, lateral preoptic area; LRN, lateral reticular nucleus; LSi, lateral septal nucleus, intermediate part; LSv, lateral septal nucleus, ventral part; MA, magnocellular preoptic nucleus; MARN, magnocellular reticular nucleus; mcp, middle cerebellar peduncle; MD, mediodorsal nucleus thalamus; MDRN, medullary reticular nucleus; MEA, medial nucleus amygdala; MEPO, median preoptic nucleus; MG. medial geniculate complex; MH, medial habenula; MM, medial mammillary nucleus; MOB, main olfactory bulb; MOBgl, main olfactory bulb. glomerular layer; MOBgr, main olfactory bulb, granule cell layer; MOBmi, main olfactory bulb, mitral layer; MOBopl, main olfactory bulb, outer plexiform layer; MOp, primary motor area; MOs, secondary motor area; MPN, medial preoptic nucleus; MRN, mesencephalic reticular nucleus: MS, medial septal nucleus; MV, medial vestibular nucleus; NB, nucleus brachium inferior colliculus; ND, nucleus of Darkschewitsch; NDB nucleus of the diagonal band; NLL, nucleus of the lateral lemniscus; NLOT, nucleus of the lateral olfactory tract; NOT, nucleus of the optic tract; NPC, nucleus of the posterior commissure; NTB, nucleus of the trapezoid body; NTS, nucleus of the solitary tract; och, otpic chiasm; opt, optic tract; OT, olfactory tubercle; PA, posterior nucleus amygdala; PAG, periaqueductal gray; PARN; parvicellular reticular nucleus; PERI, perirhinal area; PG, pontine gray; PGRN, paragigantocellular reticular nucleus; PH, posterior hypothalamic nucleus; PIN, pineal gland; PIR, piriform area: PO, posterior complex thalamus; POR, periolivary region; PPN, pedunculopontine nucleus; PPT, posterior pretectal nucleus; PRN, pontine reticular nucleus; PSV, principal sensory nucleus of the trigeminal; PT, parataenial nucleus; PTLp, parietal region, posterior association areas; PVH, paraventricular nucleus hypothalamus; PVHm, paraventricular nucleus hypothalamus, magnocellular division; PVHp, paraventricular nucleus hypothalamus, parvicellular division; PVp, posterior periventricular nucleus hypothalamus; PVT, paraventricular nucleus thalamus; py, pyramidal tract; RE, nucleus reuniens; RH, rhomboid nucleus; RM, nucleus raphe magnus; RN, red nucleus; RO, nucleus raphe obscurus; RPO, nucleus raphe pontis; RR, mesencephalic reticular nucleus, retrorubral area; RSP, retrosplenial area; RT, reticular nucleus thalamus; SAG~ nucleus sagulum; SC, superior colliculus; SCdg, superior colliculus, deep gray layer; SCH, suprachiasmatic nucleus; SCig, superior colliculus, intermediate gray layer; SF, septofimbrial nucleus; SFO, subfornical organ; SI, substantia innominata; SLC, subcoeruleus nucleus; sin, stria medullaris; SMT, submedial nucleus thalamus; SNc, substantia nigra, compact part; SNr, substantia nigra, reticular part; SO, supraoptic nucleus; SOC, superior olivary complex; SPIV, spinal vestibular nucleus; SPV, spinal nucleus of the trigeminal; SPVI, spinal nucleus of the trigeminal; SPVO, spinal nucleus of the trigeminal, oral part; SSp, primary somatosensory area; SSs, supplemental somatosensory area; STN, subthalamic nucleus; SUB, subiculum; SUM, supramammillary nucleus; TEv, ventral temporal association areas; TRN, tegmental reticular nucleus, pontine gray; TTd, taenia tecta, dorsal part; TTv, taenia tecta, dorsal part; V, motor nucleus of the trigeminal nerve; V3, third ventricle; V4, tburth ventricle; VAL, ventral anterior-lateral complex thalamus; VCO, ventral cochlear nucleus; vhc, ventral hippocampal commissure; VIS, visual areas; VISC, visceral area; VL, lateral ventricle; VM, ventral medial nucleus thalamus; VMH, ventromedial nucleus hypothalamus; Vn, trigeminal nerve; VPL, ventral posterolateral nucleus thalamus; VPM, ventral posteromedial nucleus thalamus; VTA, ventral tegmental area; VTN, ventral tegmental nucleus; XI1, hypoglossal nucleus; ZI, zona incerta.


of GR-immunoreactive cells in those regions was ap- proximately 50%. Only a small number of GR mRNA- containing cells were detectable in the white matter.

4. Discussion

The present study revealed the detailed distribution of GR-imrnunoreactive and mRNA-containing cells in the rat brain by using newly-developed antisera for imrnunohistochemistry and fluorescein-labeled probes for in situ hybridization. We compared the present immunohistochemical data with previous papers (Fuxe et al., 1985; H/irfstrand et al., 1986; Van Eekelen et al., 1987; Kiss et al., 1988; Ahima and Harlan, 1990; Cintra et al., 1994) on the localization of GR immunoreactivity in the brain and explored the differences and simi- larities. Our study was also performed to compare GR mRNA-containing structures identified by in situ hybridization with our immunohistochemical results and with previous papers dealing with GR mRNAs (Sousa et al., 1989). The present results have several points of interest to be discussed.

This study showed that the antibody raised against a part of the transcription modulation domain can be used to demonstrate the GR-irnmunoreactive structures with widespread distribution in the brain. The immunohistochemical specificity of the antisera used in the present study was indicated by the following: (1) Ab- sorption of the antisera with synthetic GR protein blocked all immunostaining. (2) The preirnmune serum and the antisera passed through the affinity column showed no specific immunoreactivity in the brain. (3) Immunoblot analysis indicated the single band corresponding to the GR molecular weight. (4) The antisera had the same specificity and distributional pattern as the affinity-purified antibody. (5) At the electron microscopic and light microscopic levels, GR immunoreactiv-

Fig. 7, Electron micrograph showing the localization of GR immunoreactivity in the nucleus. Pyramidal cell in the CA1 layer of the hippocampal formation. C, cytoplasm; N, nucleus. Bar = 1 /~m.

ity was predominantly localized in the cell nucleus, not in the cytoplasm of the normal conditioned animals. (6) The distribution of GR immunoreactivity was well-correlated with that obtained by in situ hybridization in the whole brain (an exception is CA3 and CA4 pyramidal layers of the hippocarnpal formation, discussed below).

The results of our irnmunohistochemical study are generally in agreement with those from studies by Ahima and Harlan (1990) and Cintra et al. (1994). The highest densities of GR-immunoreactive cells were observed in layers II/III and V! of the isocortex, layer IV of the posterior parietal association areas (PTLp), the ventral temporal association areas (TEv) and visual areas (VIS), the lateral part of the anterior olfactory nucleus (AON), layer II of the teania tecta (TT), the pyramidal layer of the olfactory tubercle (OT) and the piriform area (PIR), the endopiriform nucleus (EP), the layer II and VI of the entorhinal area (ENT), the CA1 and CA2 pyramidal layers of the hippocampal formation, the granule cell layer of the dentate gyrus (DG), the pyramidal layer of the nucleus of lateral olfactory tract (NLOT), the central nucleus of the arnygdala (CEA), the oval nucleus of the bed nuclei of the stria terminalis (BST), the lateral dorsal nucleus (LD), the posterior nucleus (LP), the medial geniculate complex (MG), the lateral geniculate complex (LG), the median preoptic nucleus (MEPO), the anteroventral periventricular nucleus (AVPV), the parvicellular division of the paraventricular nucleus (PVHp), the arcuate nucleus (ARH), the posterior periventricular nucleus (PVp), the medial preoptic nucleus (MPN), the ventromedial nucleus (VMH), the dorsomedial nucleus (DMH), the ventral premammillary nucleus (PMv), the nucleus of the trapezoid body (NTB), the granule cell layer of the cerebellar cortex, the locus coeruleus (LC) and the dorsal nucleus raphe (DR).

There were discrepancies in several regions of the brain between the present results and two previous reports (Ahirna and Harlan, 1990; Cintra et al., 1994). In the isocortex, the two previous studies revealed the GR imrnunoreactivity in the motor, cingulate and so- matosensoty areas. Ahima and Harlan showed no immunoreactivity in layer I, but we confirmed the GR immunoreactivity there, as did Cintra et al. In addition, the distribution of imrnunoreactive cells was examined in more detailed areas by the present study. The distributional pattern was similar in each area, but layer IV of the posterior parietal association areas (PTLp), the ventral temporal association areas (TEv) and the visual areas (VIS) had high densities of GR immunoreactive cells, compared with those of the other areas. The mitral layer of the olfactory bulb was reported to have a moderate density of GR-immunoreactive cells by Ahima and Harlan, but no such immunoreactive cells were recognized in our study or in the report of Cintra


II/111

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Fig. 8. Photomicrographs of immunohistochemistry (A, C) and in situ hybridization histochemistry (B, D) in the auditory area (AUD) (A, B) and the ventral temporal association area (TEv) (C, D) of the isocortex. Layer [ had a small number of GR-immunoreactive and mRNA-containing cells, layers II/II1 showed high densities of both labeled cells, and layer V showed moderate densities. In layer IV, high density of both labeled cells was observed in the TEv (arrow), while a moderate density was observed in the AUD (arrowhead). Bar = 200/~m.

et al. The present study revealed a low density of GR-immunoreact ive cells in the CA3 pyramidal layer of the hippocampal formation. Although Cintra et al. also showed a similar density of immunoreactive cells in the region, Ahima and Harlan showed a high density of immunoreactive cells. In the hypothalamus, there were several discrepancies between our data and the previous reports. It was observed by the present authors and Cintra et al. that high densities of GR-immunoreact ive cells were present in the AVPV, A R H and PVp; in contrast, low densities of immunoreactive cells were

detected in those areas by Ahima and Harlan, The largest difference between the present data and these two previous reports was found in the SO. A moderate density of GR-immunoreact ive cells was found in the SO in our study, but the two previous reports demonstrated little or no immunoreactivity in the area (Ahima and Harlan, 1990; Cintra et al., 1994).

Another group reported that many cells in the SO had G R immunoreactivity by means of the double pH fixation method (Kiss et al., 1988). Receptor autoradio- graphical study and our in situ hybridization study


Fig. 9. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the main olfactory bulb. The mitral layer showed few immunoreactive cells and a small number of mRNA-containing cells. Moderate densities of both labeled cells were observed in the granule cell layer, gl, glomerular layer; mi, mitral layer; gr, granule cell layer. Bar = 100/~m.

Fig. 10. Photomicrographs of GR immunoreactivity (A, C) and GR mRNA expression (B, D) in the hippocampal formation. High densities of GR-immunoreactive cells were present in the CA1 and CA2 pyramidal layers and the granule ceil layer of the dentate gyrus (DG). The CA3 and CA4 pyramidal layers had low densities of weakly GR-immunoreactive cells, and immunoreactivity was also observed in the cytoplasm (C, arrow). High densities of GR mRNA-containing cells were demonstrated in the CA1, CA2, CA3, and CA4 pyramidal layers and the granule cell layer of DG. Bar = 500 pm (A, B), 100 l~m (C, D).

showed the existence o f G R in the SO (Reul and De Kloet , 1986; Van Eekelen et al., 1988). A l t h o u g h our

f ixat ion p rocedure was s imilar to tha t used in the r epor t

o f C in t r a et al. (1994), a m o d e r a t e densi ty o f weak cy top lasmic G R - i m m u n o r e a c t i v e cells was de tec ted in

the SO by using our ant isera. Wi th in the b ra in stem, A h i m a and Har l an (1990) demons t r a t ed tha t m o d e r a t e

to high densit ies o f G R - i m m u n o r e a c t i v e cells were present in the ol ivary pre tec ta l nucleus (OP), the poste-

r ior pre tec ta l nucleus (PPT) and the c o m p a c t pa r t o f

the subs tan t i a n igra (SNc). Those regions had low

densi ty o f G R - i m m u n o r e a c t i v e cells in the present s tudy, and the c o m p a c t pa r t o f the subs tan t i a n igra

(SNc) was also repor ted to have a small number o f

M. Morimoto et al. / Neuroscienee Research 26 (1996) 235 269 26l

Fig. l l. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the central nucleus of the amygdala (CEA). High densities of GR-immunoreactive and mRNA-containing cells were present in the CEA. The nuclear immunoreactivity observed in the CEA was strong compared with the other amygdaloid nuclei. Bar = 200 tLm.

Fig. 12. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the caudoputamen. Moderate densities of both labeled cells were observed, and the nuclear GR immunoreactivity showed moderate intensity• Bar = 100/~m.

, 4

Fig. 13. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the lateral dorsal nucleus of the thalamus (LD). High densities of both labeled cells were observed, and the immunoreactivity was not strong. Bar = 200/~m.

immunoreactive cells by Cintra et al. (1994). The Edinger-Westphal nucleus (EW) had a moderate density of GR-immunoreactive cells in the present study but the study of Ahima and Harlan (1990) showed no immunoreactive cells. Although the nucleus of the trapezoid body was one of the regions which had high density of GR-immunoreactive cells in the present study, the region was reported to show low to moderate density by Cintra et al. (1994). The distributional pat-

tern of GR-immunoreactive cells of the cerebellar cortex in our study was similar to that in the report of Ahima and Harlan (1990).

To date, several polyclonal or monoclonal antibodies have been produced against the rat GR purified from liver cytosol (Eisen, 1980; Okret et al., 1981; Grandics et al., 1982; Westphal et al., 1982; Gametchu and Harrison, 1984; Okret et al., 1984; Robertson et al., 1987). The polyclonal antibodies in general do not


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Fig. 14. Photomicrographs of GR immunoreactivity (A, C) and GR mRNA expression (B, D) at the rostral levels (A, B) and the mid-caudal levels (C, D) in the paraventricular nucleus of the hypothalamus (PVH). The parvicellular division (PVHp) showed high densities of GR-immunoreactive and mRNA-containing cells, while the magnocellular division (PVHm) had no GR-immunoreactivies but a shall number of GR mRNA-containing cells. Bar = 100 gm.

Fig. 15. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the arcuate nucleus (ARH) and ventromedial nucleus of the hypothalamus (VMH). Both nuclei had a large number of GR-immunoreactive and mRNA-containing cells. The nuclear GR immunoreactivity observed in the ARH was very strong, but that in the VMH was moderate. Bar = 200 ~m.

show a high titer, because the rat liver G R protein used for the immunogen was purified under denatur ing conditions; a m m o n i u m sulfate or tr ichloroacetic acid precipitation (Eisen, 1980; Okret et al., 1981). In the present study, we used the GST gene fusion system in order to obtain the immunogen. Various foreign proteins were synthesized as fusion with GST in E. coli,

and were easily purified by glutathione affinity chro- m a t o g r a p h y under non-denatur ing condit ions (Smith

and Johnson, 1988). We were therefore able to obtain high titer antibodies which were used in a 1:8000 dilu- t ion for immunohis tochemistry . The monoc lona l antibodies had high specificity for the rat liver GR, guaranteed by biochemical techniques such as immunoblot t ing; the monoc lona l antibodies can recognize only one epitope (Gametchu and Harr ison, 1984; Okret et al., 1984; Rober t son et al., 1987). We used polyclonal antibodies, which can recognize several epitopes, for


Fig. 16. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the supraoptic nucleus (SO). A moderate density of GR-immunoreactive cells was found and the weak immunoreactivity was predominantly localized in the cytoplasm. Strongly immunoreactive and small-sized cells (--,), thought to be glial cells; arrowhead, pial cells) were observed. A large number of mRNA-containing cells were present in the supraoptic nucleus (SO). Bar = 100/~ m.

Fig. 17. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the nucleus of the trapezoid body (NTB). Many GR-immunoreactive and mRNA-containing cells were noted. Bar = 100 pro.

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Fig. 18. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the Edinger-Westphal nucleus (EW). A moderate density of GR-immunoreactive cells and a high density of mRNA-containing cells were observed in the EW nucleus. The immunoreactivity was located in both the cytoplasm and nucleus with moderate intensity. Bar = 100 lLm.

anatomical pursuits of GR immunoreactivity in various brain regions.

In the present study, antibodies were directed against a portion of the transcription modulation domain within the amino-terminus of rat GR protein. The selected portion showed strong hydrophilicity and thought to have high antigenicity. In addition, the portion had high homology to mouse GR (91.9% by

fasta method) and little homology to any other proteins including other steroid receptors (NBRF PIR Data- base, Release 43.0). Therefore, our polyclonal antibody against the synthesized protein had high sensitivity and specificity for rat GR, both in immunoblot analysis of rat brain and liver tissues and in immunohistochemical analysis of the rat brain.

The differences of the distribution of GR immunore-


Fig. 19. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the interposed nucleus (IP) of the deep cerebellar nuclei. Low densities of GR-immunoreactive and mRNA- containing cells were observed in the nucleus. Bar = 100/~m.

Fig. 20. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the cerebellar cortex. The Purkinje cells (Pu) showed weak nuclear GR immunoreactivity while the GR mRNA expression signals were very strong. A large number of cells in the granule cell layer (Gr) had strong GR immunoreactivity. Nomarski modulation contrast. Bar = 20/~m.

Fig, 21. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the locus coeruleus (LC). High densities of GR-immunoreactive and mRNA-containing cells were observed in the region. The nuclear GR immunoreactivity and cytoplasmic hybridization signals were very strong. Bar = 100/~m.

activity (Ahima and Harlan, 1990; Cintra et al., 1994) might be at tr ibutable to the antibodies used in each study. The monoc lona l an t ibody used by Ah ima and Har lan (1990) recognized an epitope close to the D N A binding domain, whereas that used by Cintra et al. (1994) recognized an epitope in the amino-terminal t ranscript ion modula t ion domain, not D N A and ligand binding domains. Both antibodies detected the rat liver G R at immunoblo t analysis (Gametchu and Harr ison,

1984; Okret et al., 1984) and it had not been reported that any subtypes o f the rat G R existed (Gustafsson et al., 1987). In our immunoblo t analysis, the antiserum detected only a single band with a molecular weight identical to that o f the rat G R for the homogenates o f both liver and brain. Therefore, the distributional dis- crepancy might be related to post- transcript ional loss or modificat ion of the epitopes recognized by the antibodies in various brain regions. Alternatively, the two


Fig. 22. Photomicrographs of GR immunoreactivity (A) and GR mRNA expression (B) in the dorsal nucleus raphe (DR). A large number of both labeled cells were found in the nucleus. Bar = 100 /~m.

Fig. 23. Photomicrographs of GR immunoreactivity of the corpus callosum (cc). A high density of small-sized GR-immunoreactive cells was observed (A). Higher magnification showed the mixed population of immunopositive and immunonegative cells (B). Bar = 500 Itm (A), 50 ~m (B).

Fig. 24. Photomicrographs of GR immunoreactivity of the cerebral peduncle (cpd) (A). A small number of cells had GR immunoreactivity in the area (B). Bar = 500 ~tm (A), 50 ,urn (B).

antibodies might recognize different conformational states of GR (Cintra et al., 1994). Because our polyclonal antibody's immunogen was the synthetic amino- terminal transcription modulation domain of rat GR, the epitope recognized by Cintra's monoclonal antibodies might be included among the epitopes recognized by our antibody, or our antibody might be able to recognize the epitopes close to the epitope recognized by the monoclonal antibody.

By immunohistochemistry in conjunction with this

antiserum, GR-immunoreactive cells were demonstrated in many regions of the rat brain, and the immunoreactivity was predominantly localized in the nucleus. These results were almost similar to those of previous reports (Fuxe et al., 1985, 1987; Van Eekelen et al., 1987; Ahima and Harlan, 1990; Cintra et al., 1994). After adrenalectomy, most of the GR immunoreactivity shifted from the nucleus to the cytoplasm. Because GR protein was generally thought to translocate from the cytoplasm to the nucleus after


ligand binding, these results may indicate that the antibody could recognize not only active ligand-binding GR but also inactive GR.

In the present study, the distribution of GR mRNA expression was examined by means of in situ hybridization using non-radioactive RNA probes. Only a few groups have reported the distribution of GR mRNA- positive cells in the rat brain with the use of radioiso- tope-labeled probes (Aronsson et al., 1988; Van Eekelen et al., 1988; Sousa et al., 1989; Whitfield et al., 1990). The hybridization histochemistry using radioactive probes is advantageous to quantitative analysis, but it is difficult to judge whether each cell shows a positive signal or not. There has been no GR mRNA-mapping study so far using non-radioactive RNA probes in the rat brain. The technique of in situ hybridization using non-radioactive probes took a shorter time and provided higher resolutional basis of the GR mRNA expression at the cellular level in various regions of the rat brain, compared with that using radioactive probes. This technique is valuable for studying the distributional pattern of certain mRNA expression within the heterogeneous population of cells in the brain.

Sousa et al. (1989) have demonstrated high densities of GR mRNA-containing structures in the cerebral cortex, olfactory bulb, CA1 and CA2 pyramidal layers of the hippocampal formation, granule cell layer of the dentate gyrus, anterodorsal nucleus and lateral dorsal nucleus of the thalamus, lateral geniculate complex and Purkinje cell layer and granule cell layer of the cerebellar cortex. The findings of the present study are in good agreement with those of Sousa et al. except in the hippocampal formation, although each riboprobe origi- nated in the different portion of the rat GR cDNA.

The distributional pattern of GR mRNA-containing cells in the rat brain was well-correlated with that of GR-immunoreactive cells. In the CA3 and CA4 pyramidal layers of the hippocampal formation and the SO, high densities of GR mRNA-hybridization signals were observed, while low to moderate densities of weak cytoplasmic or nuclear GR-immunoreactive cells were observed. The differences in the regions between immunoreactivity and mRNA expression might be ex- plained by several possibilities. One possibility is that there might be a significant difference between the amount of GR mRNA and that of GR protein, and GR mRNA could not be perfectly translated into the protein. Alternatively, it is possible that GR immunoreactivity of the antibody might not reflect the genuine amount of GR protein, because the antibody could not detect all conformational types of GR protein with the same sensitivity, or a certain conformation type of GR might be denatured or exude from the cells after the fixative and histochemical procedures (Cintra et al., 1994). Indeed, the cytoplasmic GR immunoreactivity, i.e. the immunoreactivity against the non-ligand bind-

ing form of GR, was generally weaker than the nuclear one, i.e. the immunoreactivity against the ligand binding form of GR; and the special fixation made the GR immunoreactivity of the magnocellular neurons in the SO detectable (Kiss et al., 1988). In addition, the re- gional differences of GR immunoreactivity in the hippocampus might be caused by the existence of MRs (Fuxe et al., 1987). A high density of MR-immunoreactive cells were observed in the hippocampus (Ahima et al., 1991) and MRs had a 6-10 times higher affinity for corticosterone than did GRs (Funder and Sheppard, 1987; Ratka et al., 1989). Therefore, it was possible that MRs might regulate the amount of the ligands which bind to GRs.

In the present hybridization histochemistry, there were several regions in which the percentage of GR mRNA-containing cells were lower than that of GR- immunoreactive cells. The reasons are unclear, but it might be due to the detection method for mRNA-containing cells. It was difficult to detect to mRNA-containing signals in cells whose cytoplasm was very small because the signals were only observed in the cytoplasm. Therefore, the number of GR mRNA-containing cells might be underestimated in the regions which consisted of small cytoplasmic cells. In particular, only a few mRNA-containing glial cells were detectable in the white matter, although low to moderate densities of GR-immunoreactive cells were recognized.

The distribution of MRs has been demonstrated by receptor binding studies (Reul and De Kloet, 1985; McEwen et al., 1986), immunohistochemistry (Ahima et al., 1991) and in situ hybridization histochemistry (Ar- riza et al., 1988; Chao et al., 1989). MRs have been shown to be localized mainly in the limbic brain, hypothalamus and circumventricular organs, but to a lesser extent in the various regions of the brain (Evans and Arriza, 1989; Ahima et al., 1991). Comparing this data with that of the present study, the pyramidal ceils of the CA1 and CA2 layers in the hippocampus and granule cells of the dentate gyrus contain both MR and GR. The colocalization of both receptors in the cell awaits further examination, but these structures could need both MRs and GRs depending on the circulating levels of corticosterone (De Kloet et al., 1994).

GR-immunoreactive and mRNA-containing cells are widely distributed throughout the brain, and the functional role of the GR should: be clarified, but recent data has suggested the ke~/ role of the GR in the hippocampus, hypothalamus, cerebellum and brain stem. In the hippocampus, adrenal steroids have shown to have both degenerative and protective effects (McEwen et al., 1992; Kawata, 1995). The treatment of adult rats with excess corticosterone induces loss of pyramidal cells in the Ammon's horn, and age-related pyramidal loss of pyramidal cells according to the progressive increase in concentration of corticosterone


with age can be reduced by adrenalectomy (Sapolsky et al., 1985, 1986). However, adrenalectomy and the con- sequent loss of corticosterone results in degeneration of the granule cells of the dentate gyrus (Sloviter et al., 1989). Although these areas had high densities of MRs as well as GRs (Ahima et al., 1991), GRs might directly or indirectly mediate those effects.

The hypothalamus through its control of the secretion of releasing and inhibitory factors regulates hormonal synthesis and secretions from the pituitary, and the pituitary hormones regulate hormonal synthesis and secretions from the peripheral target endocrine glands (Dallman et al., 1994). In this hypothalamo-pituitary- adrenal (HPA) axis, corticotropin releasing factor (CRF) is a hypophisiotropic peptide which regulates the secretion of ACTH in the pituitary gland. CRF- containing neurons are localized in the paraventricular nucleus (Kawata et al., 1983; Swanson et al., 1983; Sakanaka et al., 1987), and the majority of these cells also have strong nuclear GR immunoreactivity (Agnati et al., 1985). Furthermore, corticosterone negatively regulates CRF mRNA and peptide expression in these cells (Jingami et al., 1985; Sawchenko, 1987; Beyer et al., 1988; Swanson and Simmons, 1989). Thus, GRs might modulate the corticosterone negative feedback on ACTH secretion through the control of CRF synthesis. The arcuate nucleus has a high density of GR- immunoreactive cells, and the majority of the growth hormone releasing factor (GRF), galanin, somatostatin or neuropeptide Y (NPY)-immunoreactive cells in the region also show strong nuclear GR immunoreactivity (Fuxe et al., 1987). Therefore, glucocorticoids could directly regulate the synthesis and the secretion of these neuropeptides in these neurons via GRs.

In the present study, a high density of GR-immunoreactive cells was observed in the granule cell layer of the cerebellar cortex, and a moderate density of weak nuclear GR-immunoreactive cells was observed in the Purkinje cell layer. In addition, a small number of GR-immunoreactive cells were present in the deep cerebellar nuclei. The detailed functional role of GRs in the cerebellum has not been elucidated, but the implications of the glucocorticoids and GRs for normal rat cerebellar development have been considered (Bohn and Lauder, 1978; Pavlik and Buresova, 1984).

In the reticular core, the locus coeruleus and the dorsal nucleus raphe have a high density of GR-immunoreactive cells (Ahima and Harlan, 1990; Cintra et al., 1994). In the locus coeruleus, the majority of GR- immunoreactive cells are noradrenergic neurons, and in the raphe, the majority of GR-immunoreactive cells are serotoninergic neurons (Hfirfstrand et al., 1986; Fuxe et al., 1987). The colocalization of GR and monoamines in these neurons suggests that glucocorticoids are closely related to the functions of the monoaminergic n e u r o n s .

Acknowledgements

The authors thank Dr Siho Kodama and Dr Tatsuya Kurihara (Suntory Institute for Biomedical Research) for help with the sequencing of the amino acids, Dr Keith R. Yamamoto (Department of Biochemistry and Biophysics, University of California, San Francisco) for his generous gift of the GR cDNA clone, and David T.M. Visser for immunohistochemical assistance.

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Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study