Neuroscience Vol. 39, No. 3, pp. 579-604, 1990 Printed in Great Britain

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CHARTING OF TYPE II GLUCOCORTICOID RECEPTOR-LIKE IMMUNOREACTIVITY IN THE RAT CENTRAL NERVOUS SYSTEM R. S. AHIMA and R. E. HARLAN Department of Anatomy, Tulane University School of Medicine, New Orleans, LA 70112, U.S.A. Abstract-The

rat brain and spinal cord have been mapped for Type II glucocorticoid receptor-like immunoreactivity in neurons and glia, using a monoclonal antibody, BUGRZ, which recognizes an epitope close to the DNA-binding domain of the rat Type II receptor. The study revealed a widespread distribution of Type II-like immunoreactive neurons and glia, and a heterogeneity of densities and intensities of immunoreactive elements. Our results corresponded to a large extent with previous immunocytochemical mapping using Ig2a, a monoclonal antibody against a different epitope in the variable domain, with some notable differences in the hippocampus, hypothalamus and cerebellum. There was also a good correlation between immunocytochemical mapping and binding studies, [jH]steroid autoradiography and mRNA localization of the Type II receptor.

Glucocorticoids have diverse modulatory effects on neurons and glia. Glucocorticoids regulate neuronal transmitter levels,4~7+‘o*4g,50 receptor densities,“‘,” differentiation 28,35,ss vulnersignal transduction, 23~24,39 ability to noxious agents,53 cognition6s38*kand sensory stimuli detection.25 Various glial metabolic processes, including myelination, are regulated by glucocorticoids.27&37 The effects of glucocorticoids are mediated in part through the regulation of the expression of various target genes by the activated glucocorticoid receptor. 22,51,62 Two populations of glucocorticoid receptors have been described, initially through radiolabeled steroid binding and in vivo and in vitro steroid autoradiography,‘4*30~33~45*ti~57~58 and subsequently immunocytochemistry’~“~*’ and detection of mRNA using either in situ hybridization or Northern blotting.‘*3~47,56@’ A Type I glucocorticoid receptor [Type I GR; mineralocorticoid receptor (MR)] is expressed largely by neurons in limbic regions, and to a lesser extent in some thalamic nuclei, brainstem reticular formation, sensory and motor nucIei, cerebellum and spinal cord.‘~‘4*33*45107 The Type I receptor has a high affinity for corticosterone and aldosterone, the principal glucocorticoid and mineralocorticoid, respectively, in rats.2*‘4q33 In limbic regions the receptor is believed to mediate tonic and specific behavioral effects of corticosterone.’ In periventricular regions and the ventrolateral hypothalamus, Type I receptors medi-

BSA, bovine serum albumin; DAB, 3,3’diamino~n~dine; MR, mineralocorticoid receptor; PBS, phosphate-b~ered saline; Type I CR, Type I @ucocorticoid r~ptor/M~, Type II GR, Type II gbzcocorticoid receptor/classical ghzocorticoid receptor.

Abbreviurions:

ate salt intake response and regulation of blood pressure.32su*59The Type I receptor and the renal MR are encoded by the same primary RNA transcripts.’ The receptor protein is expressed in the distal tubules and collecting ducts of the kidney, salivary glands, sweat glands, bladder epithelium and endothelium.‘**2~‘4,30*3’*33,47 Selectivity of the receptor in these latter tissues for aldosterone, which circulates at levels 10~1~ times less than corticosterone, has been ascribed to sequestration of corticosterone by transcortin,‘4,33 and corticosterone inactivation by I l-j? hydroxysteroid dehydrogenase.gv’3 However, transcortin levels are low in the brain and I 1-B hydroxysteroid dehydrogenase has not been demonstrated in the brain.13 A Type II glucocorticoid receptor/classical glucocorticoid receptor (Type II GR), with a IO-fold lower affinity for corticosterone compared to Type I and a high affinity for dexamethasone and specific glucocorticoids, is expressed widely in the CNS by neurons High levels of the receptor are and glia. 3~‘4,2’,33*45,47,56 expressed in the ~p~ampus, neocortex, cerebellum, thalamus, and stress-related nuclei of the hypothalamus and brainstem. The receptor is believed to mediate stress effects of glucocorticoids. Type II receptor-like immunoreactivity has been mapped in the rat CNS using monoclonal antibodies against the rat liver glucocorticoid receptor. IgZa,” an antibody that recognizes an epitope in the variable domain of the receptor, has been used to map immunoreactivity at all levels of the CNS.‘s’7s2’ BUGRl and 2,‘“~‘8~52~58 antibodies that recognize an epitope corresponding to amino acid residues 407-423 in a 16,~mol. wt tryptic fragment which binds to DNA, have been used in limited immunocytochemical studies in the CNS.26~“~58@ The epitope

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R. S. AHIMAand R. E. HARLAN

for BUGR lies in the variable domain, a domain in which the Type I and II receptors share less than 15% identity in amino acid sequence.” In solution binding studies BUGRI does not cross-react with the Type I receptor.44 The present study was aimed at charting Type II receptor-like immunoreactivity at all levels of the rat CNS using BUGRZ, and comparing the patterns with Ig2a. The parameters for comparison were anatomical distribution of immunoreactive neurons and glia, intracellular localization of immunoreactivity, relative densities and intensities of immunoreactive elements. An atlas illustrating relative densities of Type II receptor-like immunoreactivity was also prepared. This study was also aimed at comparing Type II GR distribution obtained from immunocytochemistry (i.e. Ig2a and BUGR) with homogenate binding studies, steroid autoradiography and in situ hybridization of GRmRNA in a semiquantitative tabular form. EXPERIMENTAL PROCEDURES Materials Six male Spragu+Dawley rats (Charles River) weighing 201-225 g, maintained under standard cage conditions and a 12 h-12 h lightdark cycle, and allowed free access to chow and water, were used. BUGR2 mouse anti-rat liver glucocorticoid receptor and P3-Agx-x63 myeloma ceil medium supernatant were kindly suppl&d by RI W. Harrison. L8,58 MINREC2, a polyclonal antibody against Type I GR was supplied Z. S. Krozowski.3’ The Vectastain ABC kit (horse anti-mouse) was purchased from Vector (Burlingame, CA). Immunocytochemistry The rats were given an overdose of sodium pentobarbital i.p., perfused transcardially with 0.025 M phosphatebuffered saline (PBS, pH 7.2) for 5 min followed by an 8 min perfusion with 3% neutral buffered paraformaldehyde. Brains and spinal cords were dissected out, blocked and posttixed in the same fixative for 2 h at room temperature, and cryoprotected in 30% sucrose (w/v) at 4°C until sinking. Tissues were then rapidiy frozen on dry ice and stored wrapped in aluminum foil at -70°C until use. Coronal sections (50 pm) of brain and horizontal sections of spinal cord were cut on a freezing microtome, rinsed in PBS for 1 h, and blocked to reduce non-specific staining in normal horse serum in 0.1% bovine serum albumin (BSA-PBS) according to specifications of the Vectastain ABC kit (Vector). Sections were then permeabilized in 0.2% Triton X-100 (Fisher Diagnostics) in PBS for 20min followed by two 10min rinses in PBS and incubation in I: 125, 1: 250, 1: 500, 1: 1000 or 1: 2000 dilutions of BUGR2 in 0.1% BSA-PBS, equilibrated by orbital shaking at room temperature for IOmin and stored at 4°C for 48 h. Adjacent sections were incubated in normal mouse serum in 0.1% BSA-PBS or P3-Agx-x63 myeloma cell medium su~matant diluted as for BUGRZ, for 48 h at 4°C and served as controls. Sections were next shaken for 10min at room temperature, rinsed twice in PBS for 1Omin each, incubated in biotinylated horse anti-mouse Ik; (Vector) diluted in 0.1% BSA-PBS according to specifications for 45 min, washed twice for 10min each in PBS and incubated for 1 h in avidin-biotinylated horseradish peroxidase (Vector). After washing twice in PBS for lOmin, sections were preincubated in 0.05 M Tris HCI (Sigma) for 5 min, and a

brownish reaction product obtained by incubating in 50 mg% 3,3’diamino~~dine (DAB) tetrahydr~hlo~de (Sigma) and 0.03% hydrogen peroxide (Fisher Scientific) in 0.05 M Tris-HCl, pH 7.6 for 3-6min. The reaction was stopped in water for 5min, and sections transferred into 10 mM sodium acetate (Sigma), and mounted on chromalurn-coated slides. Some sections were stained slightly with 0.25% Cresyl Violet to facilitate identification of neuronal groups. Dehydration of sections was carried out in increasing strengths of ethanol (70, 95 and lOO%), followed by defatting in Histoclear (National Diagnostics) and coverslipping with AccuMount 60 (Baxter). Cross-reactivity between the epitope for BUGRZ and the Type I OR was tested by incubating 20-pm-thick cryostat sections of the parotid gland, a tissue that expresses predorninantly Type I receptor,” in I:500 dilution of BUGRZ. Adjacent sections were incubated in 1: 500 dilution of MINRBC2?’ a polyclonal antibody against the Type I receptor. Detection of immuno~ctivi~ was done with Vectastain ABC kits horse anti-mouse for BUGR2, and goat anti-rabbit for MINREC2. Analysis of sections Sections were examined using a Nikon Optiphot microscope, and GR-like immunoreactive regions photographed with a Nikon FX35A camera and Technical Pan (Kodak) film. Rostrocaudal drawings of coronal sections of the brain and spinal cord were prepared to illustrate Type II GR-like densities in various nuclei. Sections were selected and standardized based on the atlas of Paxinos and Watson.” For the estimation of relative densities of ~munor~ctive neurons, nuclear boundaries were drawn using Cresyl Violet~ounte~~in~ sections. Positively labeled neurons were counted and expressed as a percentage of total neurons within a nuclear boundary. Relative densities were rated 0 if no positively labeled neurons were observed, low (+) if less than 30% of the neurons were positively labeled, moderate (+ +) for 30-70% positively labeled and high (+ + +) for more than 70% positively labeled neurons. In comparing our results with previous immunocytochemical binding studies and Type II mRNA detection, we were faced with the problem of different qualitative and quantitative measures used by those authors. Descriptions of strong/high were scored + t l , moderate + -t-, low + and zero/none 0. Intensities for Type II GR-like immunoreacti~ty were rated strong (as in the granule eel1 layer of the dentate gyrus and locus coeruleus), moderate as in the pyramidal cell layer of CA3, and weak as in the potymorphic layer of the dentate gyrus (CA4) and the globus pallidus. These are illustrated in Figs 1A and 2B, D.

RESULTS Dilution of BUGR2 down to 1: 2000 resulted in a gradual disappearance of specific immunoreactivity in neurons and glia. A dilution of 1: 500 gave optimal specific signal (Figs 1, 2). Control sections incubated with P3-Agx-x63 myeloma cell medium supematant or normal mouse IgG did not show Type II GR-like immunoreactivity (Fig. IB). In the Type I GR crossreactivity studies, parotid gland sections incubated with BUGR2 lacked specific immunoreactivity, while MINREC2 incubation produced specific nuclear and cytoplasmic immunoreactivity in epithelial cells of the striated ducts (data not shown). Type II GR-like immunoreactive elements were observed

Type II glucocorticoid receptors in the brain

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Fig. i . (A) Light micrograph of a coronal section through the dorsal hipp~mpus. Note the high densities of intensely immunoreactive neurons in the pyramidal cell layer of CAL and the granule cell layer of the dentate gyrus (DG). CA3 has a high density of moderately immunoreactive neurons, while CA4 has few weakly immunoreactive neurons. (B) Coronal section of the dorsal hippocampus incubated with P3 Agx-x63 myeloma cell medium su~~ata~t. Note the absence of specific immunoreactivity in the dentate gyrus and CA1 Scale bars in A and B = 200 pm. (C) Horizontal section through the cervical spinal cord showing immunoreactive motor neurons in lamina 9. Note the predominant nuclear immunoreactivity (n) and weak cytoplasmic staining. The nucleolus is devoid of immunoreactivity. Scale bar = 50 ,um. widely dist~buted in both the gray and white matter. Densities as well as intensity of immunoreactivity were variable. In most neuronal groups immunoreacti~ty was predomin~ntiy nuclear (Fig. IC). Where the nucleolus could be resolved, immunoreactivity was

observed to be confined to the rest of the nucleus, the nucleolus showing no immunoreactivity. Some neuronal groups in the facial nucleus, deep cerebellar nuclei and Iaminae 8 and 9 of the spinal cord (Fig. IC) had both nuclear and ~ytoplasmi~ immunoreactivity.

Fig. 2. (A) Light micrograph of a coronal section through the olfactory tubercle (Tu). Note the high density of immunoceactive neurons in layer II, piriform cortex (Pir) and fundus striati (FStr) and relatively low density in the ventral pallidurn (VP). (B) Coronal section through caudate-putamen (CPU) and globus pallidus (GP), showing a high density of immunoreactive neurons in CPU and low density in GP. (C) Coronal section at the level of the medullary pyramid (py) showing high densities of immunoreactive neurons in the dorsal and medial inferior olivary nucleus (IOD, IOM), and relatively fewer neurons in the raphe pallidus (RPa). Note glial immunoreactivity in the pyramid (py). (D) Coronal section through the globus and the bed nucleus of the stria terminalis. Note the relatively low density of immunoreactive neurons in the globus pallidus (GP), high density of strongly immunoreactive neurons in the lateral bed nucleus (BSTL), and low density in the intermediate bed nucleus (BSTI). The internal capsule (ic) does not show any immunoreactive glia. Scale bars in A-D = 200 pm. (E) Coronal section showing strongly immunoreactive neurons in the locus coeruleus (IX), and moderately immunoreactive neurons in the mesencephaiic nucleus of the trigeminal (Me5). (F) Coronal section through the cerebellar cortex showing clusters of immunoreactive Purkinje cells (P) and granule cells (Gr). Note the relative paucity of immunoreactive elements in the molecular layer. Scale bars in E and F = 100 pm. 582

Type II glucccorticoid receptors in the brain Non-specific nuclear and cytoplasmic staining was observed over the pia mater (Figs 1B; 2A, C), ependyma, subfornical region and area postrema. Strongly intense immunoreactivity was observed in the granular layer of the dentate gyrus and pyramidal cell layers of fields CA1 and CA2 of Ammon’s horn (Fig. lA), caudate-putamen (Fig. 2B), locus coeruleus (Fig. 2E), lateral bed nucleus of the stria terminalis (Fig. 2D), central amygdaloid nucleus, motor trigeminal nucleus, granule cells of the olfactory bulb, substantia gelatinosa (lamina 2) of the spinal cord, and selected motor neurons of laminae 8 and 9 of the spinal cord (Fig. 1C). Most other neuronal groups showed moderately intense immunoreactivity. Weak immunoreactivity was observed in the pyramidal cells of CA4 (Fig. lA), globus pallidus (Fig. 2B, D), ventral pallidum (Fig. 2A) and lateral hypothalamus. A large number of small, ovoid to spindleshaped elements with moderate intensity of GR-like immunoreactivity were observed widely distributed in the gray and white matter. These elements showed diffuse immunoreactivity, nuclei were unresolvable, and they were .often found between fiber bundles of myelinated tracts. They were predominantly located in the corpus callosum, pyramids (Fig. 2C), optic tract and chiasm, vestibulocochlear nerve, spinal trigeminal tract and all tracts of the spinal cord. The internal capsule had relatively very few of these immunoreactive elements (Fig. 2D). It was presumed this subgroup of immunoreactive elements associated with myelinated fiber systems represented oligodendroglia. Over wide areas in the gray matter of the brainstem and spinal cord, similar small diffusely immunoreactive elements presumed to be either glia or small neurons were observed. Telencephalon Olfactory bulb. The olfactory nerve layer, glomerular and periglomerular structures lacked immunoreactive elements. The mitral cell layer had a moderate to high density of immunoreactive neurons, predominantly granule cells. The granule cell layer had a high density of immunoreactive cells (Fig. 3A). The anterior olfactory nucleus showed heterogeneity of immunoreactivity. While the ventral and lateral regions had a high density of immunoreactive neurons, the medial, dorsal and posterior regions had moderate densities (Fig. 3B, C). Layer II of the piriform cortex (Fig. 3C-K) showed a high density of moderately to highly immunoreactive neurons, while layer III had moderate to low densities and layer I had no GR-like immunoreactivity. In the hippocampal formation, all granule cells of the dentate gyrus (Figs lA, 31-L) were immunoreactive. Within CAl, the more rostra1 and dorsal pyramidal cell layers had a high density of immunoreactive cells, while more caudal and ventral parts

583

had moderate densities. Moreover, the molecular layer of dorsal CA1 and the adjacent subiculum (Fig. 3L) had a moderate density of immunoreactive neurons. CA2 and CA3 showed high and moderate to high densities of immunoreactive pyramidal cells, respectively, in all regions examined (Figs lA, 31-L). CA4 (PoDG) showed low to moderate densities of immunoreactive neurons (Figs A, 3J-L). In all regions of the neocortex, layers II, III and VI had high densities of immunoreactive neurons, and IV and V relatively lower densities (Fig. 3E). Subcortical and septal regions. High densities of immunoreactive neurons were observed in the caudate-putamen (Figs 2B, 3D-J), core of the nucleus accumbens (Fig. 3D, E), layer II of the olfactory tubercle (Figs 2A, 3DF), corticosmygdaloid transition (Fig. 3F-H), intermediate lateral olfactory nucleus (Fig. 3H), central amygdaloid nucleus (Fig. 31-J), fundus striati and substriatal regions (Figs 2A, 3F-H), medial and lateral parts of the bed nucleus of the stria terminalis (Figs 2D; 3F, G), dorsolateral septum (Fig. 3E, F) and the septohypothalamic nucleus (Fig. 3F). Moderate densities of immunoreactive neurons were observed in layer III of the olfactory tubercle (Figs 2A, 3E), parts of the dorsal nucleus of lateral olfactory tract, bed nucleus of the lateral olfactory tract, medial amygdala, (Fig. 31, J), posterior cortical amygdala (Fig. 35, K), amygdalohippocampal transition (Fig. 3K), basolateral, basomedial and lateral amygdaloid nuclei (Fig. 3J), rostra1 part of the shell of the accumbens nucleus (Fig. 3D, E), substantia innominata (Fig. 3C), endopiriform nucleus and claustrum (Fig. 3D, I), ventral septum and septohippocampal nucleus (Fig. 3D, E), vertical limb of the diagonal band of Broca (Fig. 3E, G), tenia tecta (Fig. 3D) and the Islands of Calleja (Fig. 3E, F). Low densities of immunoreactive neurons were observed in the anterior amygdaloid area (Fig. 3H), intermediate bed nucleus of stria terminalis (Figs 2D, 3F), globus pallidus (Figs 2B, D; 3F-I), caudal part of the shell of the accumbens nucleus (Fig. 3E) horizontal limb of the diagonal band, ventral pallidum (Figs 2A, 3E), basal nucleus of Meynert (Fig. 3H), septofimbrial (Fig. 3F) and triangular septal nuclei (Fig. 3G), intermediate septum, and ventral nucleus of lateral olfactory tract (Fig. 3H). Diencephalon

The habenula nucleus (Fig. 31-J) showed a very low density of immunoreactive neurons. Most of the immunoreactive elements observed appeared to be glia associated with the stria medullaris. Within the thalamus, the ventroposterolateral (Fig. 3J), lateral dorsal (Fig. 31), parts of paraventricular (Fig. 3H), and the lateral and medial geniculate nuclei (Fig. 3K, L) all had high densities of immunoreactive neurons. Moderate densities

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of immunoreactive neurons were observed in the anterodorsal, anteroventral, anteromedial (Fig. 3H), mediodorsal, ventrolateral (Fig. 35) ventroposteromedial (Fig. 35) lateral posterior (Fig. 3K), posterior (Fig. 3J), anterior paraventricular (Fig. 31) paratenial (Fig. 3H), rostra1 reuniens nuclei (Fig. 3H) and the zona incerta (Fig. 31, J). Low densities of immunoreactive neurons were observed in the paracentral (Fig. 31), centromedian, centrolateral (Fig. 3J), rhomboid (Fig. 3H) and rostra1 gelatinosa nuclei (Fig. 31, J). The reticular thalamic nucleus (Fig. 3H, J) had either low densities or no immunoreactive neurons. Within the hypothalamus high densities of immunoreactive neurons were observed in the medial preoptic, striohypothalamic (Fig. 3C) and ventromedial nuclei (Fig. 31, J). Moderate densities of immunoreactive neurons were observed in the medial preoptic area (Fig. 3F), anterior hypothalamic area and nucleus (Fig. 3H, I), paraventricular nucleus (paravocellular) (Fig. 31) lateral anterior (Fig. 3H), tubercinereum (Fig. 31, J), dorsomedial and perifornical nuclei (Fig. 35). Low densities of immunoreactive neurons were observed in the median preoptic (Fig. 3F), suprachiasmatic (Fig. 3G, H), periventricular (Fig. 3F-I-I), lateral mammillary nuclei (Fig. 3K), lateral hypothalamic area (Fig. 3F-H) and the arcuate nucleus (Fig. 31-J). The supraoptic (Fig. 3G, H), medial mammillary (Fig. 3K) and supramammillary nuclei (Fig. 3K) showed virtually no immunoreactivity. Within the pretectum, the olivary and posterior nuclei (Fig. 3K, L), had high densities of immunoreactive neurons while the medial and anterior nuclei (Fig. 3L) had relatively moderate densities. The nucleus of the optic tract (Fig. 3K, L) had moderate to high densities of immunoreactive neurons. Brainstem. In the midbrain, the oculomotor nucleus (Fig. 3K-M), medial accessory oculomotor (Fig. 3L), lateral and dorsal central gray (Fig. 3K-N), Darkschewitsch nucleus (Fig. 3K, L), parts of pars compacta of the substantia nigra (Fig. 3K, L) and dorsal raphe (Fig. 3N) showed high densities of immunoreactive neurons. Moderate densities of immunoreactive neurons were observed in the intermediate gray of the superior colliculus (Fig. 3M, N), pars lateralis of substantia nigra (Fig. 3K), rostra1 and caudal linear raphe, central interpeduncular nucleus (Fig. 3L-M) and ventral tegmental area (Fig. 3K, L). Low densities of immunoreactive neurons were observed in the medial central gray (Fig. 3K-N), superficial gray of superior colliculus (Fig. 3M, N), nucleus of the brachium of inferior colliculus (Fig. 3M), external cortex of inferior colliculus (Fig. 30), pars reticularis of substantia nigra (Fig. 3K, L) and interfascicular nucleus (Fig. 3L). No immunoreactivity was observed in the EdingerWestphal nucleus (Fig. 3L). At pontine levels high densities of GR-immunoreactive neurons were observed in the locus coeruleus

(Figs 2E, 30), medial parabrachial nucleus (Fig. 30), motor trigeminal nucleus (Fig. 30) and dorsal raphe (Fig. 3N, 0). The lateral parabrachial, principal sensory trigeminal, pontine reticular formation (pars oralis) (Fig. 3N) and facial nucleus (Fig. 3P) had moderate densities of immunoreactive neurons. Low densities of GR-immunoreactive neurons were observed in the superior and lateral vestibular (Fig. 3P) and pontine nuclei (Fig. 3N, 0). In the medulla, the inferior olivary subnuclei (Fig. 2C), nucleus ambiguus and pars gelatinosa of the spinal trigeminal nucleus (Fig. 34, R) had moderate to high densities of GR-immunoreactive neurons. Moderate densities of immunoreactive neurons were observed in the solitary nucleus, spinal trigeminal nucleus, pars oralis and interpolaris, medial vestibular nucleus, raphe pallidus, raphe magnus, raphe, obscurus and gigantocellular reticular nuclei (Fig. 3P, Q). Low densities of immunoreactive neurons were observed in the spinal trigeminal nucleus (pars caudalis), dorsal vagal nucleus, nucleus of Roller and prepositus hypoglossal nucleus (Fig. 34, R). No specific immunoreactivity was observed in the hypoglossal, cuneate and gracile nuclei (Fig. 3R). Cerebellum. All regions oft the cerebellar cortex examined showed high densities of immunoreactive neurons in the granule cell layer (Figs 2F, 3P). The Purkinje cell layer had a moderate density of immunoreactive neurons (Figs 2F, 3P). In some folia Purkinje cells were arranged in a columnar fashionalternating patches of immunoreactive and nonimmunoreactive neurons. Very few immunoreactive elements were observed in the molecular layer. Low to moderate densities of moderately immunoreactive neurons were observed in the deep cerebellar nuclei (Fig. 3P, Q). Spinal cord. Lamina 2 had high densities of immunoreactive neurons at all levels (Fig. 3S-U). Lamina 9 had moderate densities of immunoreactive motor neurons at all levels while lamina 8 had low densities (Fig. 3SU). In thoracic segments the intermediolateral cell column had a moderate density of immunoreactive neurons (Fig. 3T). At all levels small groups of immunoreactive elements of low to moderate intensity were observed. They were especially prominent in laminae 4 and 5. They were often small cells, ovoid or spindle-shaped with diffuse immunoreactivity, presumably interneurons or glia. DISCUSSION

In comparing our immunocytochemical map with previous reports based on Ig2a immunoreactivity, binding studies, in vitro and in vivo autoradiography and Type II GR mRNA detection, we were faced with differences in descriptive nomenclature for some nuclear groups, and lack of information on some regions especially in the olfactory tubercle,

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Type II glucocorticoid receptors in the brain brainstem and spinal cord. Where the distribution of Type II GR receptor was described differently from ours, we recorded information provided with appropriate footnotes in the legends accompanying our tables. Regions that were not reported on either, specifically or generally, were represented as blanks in the tables.

Relative densities of BUGR2reactive elements

and IgZa-immuno-

Our BUGR2 map revealed similar distribution patterns to Ig2a with some notable differences in relative densities in the pyramidal cell layer of CA3 (Fig. lA), ventromedial and arcuate hypothalamic

Abbreviationsused in Fig. 3 1-9 3 3PC 7 8vn 10 12 AA

spinal cord laminae/cerebellar lobules oculomotor nucleus oculomotor nucleus-parvocellular part facial nucleus vestibular root of vestibulocochlear nerve dorsal vagal nucleus hypoglossal nucleus anterior amygdaloid area anterior commissure, anterior part ::bC accumbens nucleus, core AcbSh accumbens nucleus, shell aci anterior commissure, intrabulbar part AD anterodorsal thalamic nucleus AHA anterior hypothalamic area, anterior part AHP anterior hypothalamic area, posterior part AHiPM amygdalohippocampal area, posteromedial part AM anteromedial thalamic nucleus Amb nucleus ambiguus AOD anterior olfactory nucleus, dorsal part AOL anterior olfactory nucleus, lateral part AOM ant&or olfactory nucleus, medial part AOP anterior olfactory nucleus, posterior part AOV anterior olfactory nucleus, ventral part AP area postrema APir amygdalopiriform transition APT anterior pretectal nucleus APTD anterior pretectal nucleus, dorsal part APTV anterior pretectal nucleus, ventral part Are arcuate hypothalamic nucleus B basal nucleus of Meynert BAOT bed nucleus of accessory olfactory tract BIC nucleus of brachium of inferior colliculus BLA basolateral amygdaloid nucleus, anterior part BLP basola@al amygdaloid nucleus, posterior part BLV basolateral amygdaloid nucleus, ventral part BM basomedial amygdaloid nucleus, medial part BMA basomedial amygdaloid nucleus, anterior part BSTLD bed nucleus of stria terminalis, lateral division BSTMP bed nucleus of stria terminalis, posteromedial division BSTMPL bed nucleus of stria terminalis, ventral division Cl/Al adrenaline/noradrenaline cells CAl-3 fields of Ammon’s horn corpus callosum :e central amygdaloid nucleus CeL central amygdaloid nucleus, lateral division CG central gray cingulate cortex, area 1 Cgl cingulate cortex, area 2 Cg2 cingulum ZGD central gray, dorsal part CGLD central gray, lateral dorsal part CGLV central gray, lateral ventral part CI caudal interstitial nucleus of medial longitudinal fasciculus CL centrolateral thalamic nucleus Cl claustrum CLi caudal linear nucleus of the raphe CM centromedial nucleus of thalamus cerebral peduncle, basal part cP

CPU

cu

DEn DG Dk DpG DpMe DR &iC EPl EW fi FL fmi Frl Fr2 FStr G Ge5 GI Gi GiA GiV Gl GP Gr Hb HDB I IAD I AM ic ICj icp IF IGr IM IMFLG IMLE InCo InG Int InWh IO IOD IOM IPA IPC IPI IPl IRt LA LaDL

caudate-putamen cuneate nucleus dorsal endopiriform nucleus dentate gyms nucleus of Darkschewitsch deep gray area of superior colliculus deep mesencephalic nucleus dorsal raphe nucleus external capsule external cortex of inferior colliculus external plexiform layer Edinger-Westphal nucleus fimbria of hippocampus forelimb area of cortex forceps minor of corpus callosum frontal cortex, area 1 frontal cortex, area 2 fundus striati gelatinous thalamic nucleus gelatinous layer of caudal spinal trigeminal nucleus granular insular cortex gigantocellular reticular nucleus gigantocellular reticular nucleus, alpha part gigantocellular reticular nucleus, ventral part glomerular layer globus pallidus gracile nucleus habenula nucleus of horizontal limb of the diagonal band intercalated nuclei of the amygdala interanterodorsal thalamic micleus interanteromedial thalamic nucleus internal capsule Islands of Calleja inferior cerebellar peduncle interfascicular nucleus internal granular layer of the bulb intercalated amygdaloid nucleus, main part interstitial nucleus of the medial longitudinal fasciculus, greater part interstitial nucleus of the medial longitudinal fasciculus intercollicular nucleus intermediate gray-superior colliculus interposed cerebellar nucleus intermediate white layer of superior colliculus inferior olivary nucleus inferior olive, dorsal nucleus inferior olive, medial nucleus ititerpeduncular nucleus, apical subnucleus interpeduncular nucleus, caudal subnucleus interpeduncular nucleus, intermediate subnucleus internal plexiform layer intermediate reticular nucleus lateral anterior hypothalamic nucleus lateral amygdaloid nucleus, dorsolateral part conrimed

overleaf

586

R. S. AHIMAand R. E. HARLAN Abbreviations used in Fig. 3Scontinued

Lat LAV LC LDDM LDTg LDVL LH LM lo LOT LP LPO LPB LPGi LPMC LPMR LRt LSD LSI LSO LSV LVe M MA3 MCLH mcp MCPO MD Md Me5 MeA MePD MePV MG MGD

MGV ML md mlf MM MnPO MnR MoS MP MPA MPB MPO MVe MVeV ON OP OPT opt OT P-I PaAP PaMP PaPo Parl PaS PC Pe PeF Pir PL PLCO

lateral cerebellar nucleus lateral amygdaloid nucleus, ventral part locus coeruleus dorsolateral thalamic nucleus, dorsomedial part lateral dorsal tegmental nucleus lateral dorsal thalamic nucleus, ventrolateral part lateral hy~thalamic area lateral m~millary nucleus lateral olfactory tract nucleus of the lateral olfactory tract lateral posterior thalamic nucleus lateral preoptic area lateral parabrachial nucleus lateral paragigantocellular nucleus lateral posterior thalamic nucleus, mediocaudal part lateral posterior thalamic nucleus, mediorostral part lateral reticular nucleus lateral septal nucleus, dorsal part lateral septal nucleus, intermediate part lateral superior olive lateral septal nucleus, ventral part lateral vestibular nucleus mitral ceh layer medial accessory oculomotor nucleus magnocellular nucleus of lateral hypothalamus middle cerebellar peduncle magnocellular preoptic nucleus mediodorsal thalamic nucleus medullary reticular nucleus mesencephalic trigeminal nucleus medial amygdaloid nucleus medial amygdaloid nucleus, posterodorsal part medial amygdaloid nucleus, posteroventral part medial geniculate nucleus medial genicuiate nucleus, dorsal part medial geniculate nucleus, ventral part media1 mamm~l~ary nucleus, lateral part media1 lemniscus medial longitudinal fasciculus medial mammiflary nucleus, medial part median preoptic nucleus median raphe nucleus motor trigeminal nucleus medial mammillary nucleus, posterior part medial preoptic area medial parabrachial nucleus medial preoptic nucleus medial vestibular nucleus medial vestibular nucleus, ventral part olfactory nerve layer optic nerve layer of superior colliculus ohvary pretectal nucleus optic tract nucleus of optic tract perifacial zone paraventricular hypothalamic nucleus, anterior parvocellular part paraventricular hypothalamic nucleus, medial parvocellular part paraventricular hypothalamic nucleus, posterior part parietal cortex, area 1 parasubiculum paracentral thalamic nucleus periventricular hypothalamic nucleus perifornical nucleus piriform cortex para~emniscal nucleus posterolateral cortical amygdaloid nucleus

PLi PMCo PMR Pn PnC PnO PO PoDG PPT PrSVL PrH PrS PT PVA PVP PY Re REth Rh RLi RMg Ro ROb RPU

RSG RE RtTg S S5

Sch SFi SG SGe SHi SHY SI sm SNC SNL SNR so Sol SPO

smg So50

spsc SpVe sP5 st SStr su3 SubB SubCA SuG

SUM SuVe TC TS TT Tu VBD VL VLO VM VMH VP VPL VTA x=P ZI

posterior limitans thalamic nucleus posteromedial cortical amygdaloid nucleus paramedian raphe nucleus pontine nuclei pontine reticular nucleus, caudal part pontine reticular nucleus, oral part posterior thalamic nuclear group polymorph layer of dentate gyrus posterior pretectal nucleus principal sensory trigeminal nucleus, ventroiateral part prepositus hypoglossat nucleus presubiculum paratenial thalamic nucleus paraventricular thalamic nucleus, anterior part paraventricular thalamic nucleus, posterior part pyramidal tract reuniens thalamic nucleus retroethmoid nucleus rhomboid thalamic nucleus rostra1 linear nucleus of the raphe raphe magnus nucleus nucleus of Roller raphe obscurus nucleus raphe pallidus nucleus retrosplenial granular cortex reticular thalamic nucleus reticulotegmental nucleus of the pons subiculum sensory root of trigeminal nerve suprachiasmatic hypothalamic nucleus septofimbrial nucleus suprageniculate thalamic nucleus supragenual nucleus septohippocampal nucleus septohypothalamic nucleus substantia innominata stria medullaris of the thalamus substantia nigra, compact part substantia nigra, laterat part substantia nigra, reticular part supraoptic nucleus solitary nucleus superior paraolivary nucleus subpeduncular tegmental nucleus spinal trigeminal nucleus, oral part spinal trigeminal nucleus, dorsomedial part spinal vestibular nucleus spinal trigeminal tract stria terminalis substriatal nucleus supraoculomotor central gray subbrachial nucleus subcoeruleus nucleus, alpha part superficial gray of superior colliculus supramammilla~ nucleus superior vestibular nucleus tuber cinereum area triangular septal nucleus tenia tecta olfactory tubercle nucleus of vertical limb of diagonal band ventrolateral thalamic nucleus ventrolateral orbital cortex ventromedial thalamic nucleus ventromedial hypothalamic nucleus ventral pallidum ventroposterolateral thalamic nucleus ventral tegmental area decussation of the superior cerebeliar peduncle zona incerta

Type II ghxocorticoid

receptors in the brain

A

B

Fig. 3A,B.

587

6 a

F

k

Type II ~uc~orticoid

receptors in the brain

Fig. 3E,F.

589

590

R. S. AHIMA and R. E. HARLAN

Fig. 3G,H

Type II glucocorticoid

receptors

Fig. 31,J.

in the brain

591

592

R. S. AHIMA and R. E. HARLAN

Fig. 3K,L.

Type II glucucorticoid receptors in the brain

Fig. 3M,N.

593

594

R. S. AHIMA and R. E. HARLAN

Fig. 30,P.

Type II glucocorticoid receptors in the brain

Q

Fig. 3Q,R.

595

R. S. AHMA and R. E. HARLAN

596

S

T

Fig. 3%U. Fig. 3. Charts of GR-immunoreactive neurons and glia at different rostrocaudal levels of the rat brain and spinal cord based on the atlas of Paxinos and Watson.42 (E, P) The distribution of immunoreactive neurons in the neocortex and cerebellar cortex, respectively. This pattern was observed at all levels.

Type II glucocorticoid receptors in the brain nuclei, superficial gray of the superior colliculus, Purkinje cells of the cerebellum (Fig. 2F), nucleus ambiguus and dorsal vagal nucleus. This raised questions concerning the possibility of cross-reactivity between the BUGR2 epitope and similar epitopes in other proteins, especially in the steroid receptor superfamily. BUGRI and 2 recognize the same epitope.% Functional studies on the rat glucocorticoid receptor have localized the BUGR epitope to amino acid residues 407-423, in the variable domain, close to the DNA-binding region.” The epitope for Ig2a has also been localized to amino acids in the variable domain.21~52It is unlikely that BUGR2 recognizes Type I GR for the following reasons. (1) There is less than 15% homology in amino acid sequence between Type II GR and Type I GR and the rest of the superfamily of steroid receptors in the variable domain where the epitope for BUGR2 lies. (2) BUGRl (with the same specificity as BUGRZ) does not cross-react with Type I GR in solutionti (3) BUGR2 does not cross-react with Type I GR in the parotid gland (data not shown). (4) Regions in the rat CNS which express high levels of Type I mRNA, e.g. the polymorphic layer of the dentate gyrus (CA4), intermediate septum, septohippocampal nucleus, reticular and subthalamic nuclei, medial mammillary nucleus, red nucleus, brainstem reticular formation, hypoglossal nucleus and cuneate and gracile nuclei,’ showed little BUGR2 immunoreactivity. However, the possibility of cross-reactivity with unknown epitopes exists. Type

II

g~~cocor~~~o~ receptor ~~lussi~a~ gluco -

eorticoid receptor-lye reports

~~~~oreactiv~ty

and previous

The pyramidal cell layer of CA3 was reported as having only a few cytoplasmically immunoreactive neurons in the first Ig2a map,” and subsequently that its rostra1 part had strong immunoreactivity.” We observed a high density of immunoreactive neurons at all levels of CA3. Our results correspond to results from binding and in vitro and in vivo autoradiographic studies (Table 1). In the cerebellum, our finding of immunoreactive Purkinje cells and neurons in the deep nuclei agreed with Type II CR mRNA dist~bution (Table 4). The high densities of Type II GR-like immunoreactive neurons in the lateral dorsal septum also correspond to findings in the other studies (Table 1). To a large extent distribution patterns of Type II GR-like immunoreactive neurons from BUGR2 and Ig2a maps correspond to Type II GR mRNA distribution (Tables l-5). However, in

597

the mammillary nucleus where little or no immunoreactivity was observed, the Type II GR mRNA level was high (Table 2). In the brainstem, while we observed a heterogeneity in densities of immunoreactive neurons, with high densities in the locus coeruleus, central gray, inferior olivary subnuclei and parts of the spinal trigeminal nucleus (Tables 3-9, Type II GR mRNA levels were reported to be unifo~ly low.” BUGRZ and Ig2a immunoreactivities were equivalent as far as the intracellular location of signal was concerned. Immunoreactivity was predominantly nuclear with a weak cytoplasmic signal. The question of intracellular location of steroid receptors has been discussed by several authors.17*‘9.21,29,43,6L Unlike the estrogen, progesterone and vitamin D receptors which have a strict nuclear location, the glucocorticoid receptors have been identified in both nucleus and cytoplasm in intact rats. Adrenalectomy reduces nuclear signal in favor of cytoplasmic, while treatment with gluc~orticoids restores nuclear signal often above intact levels.‘7~2’*57 This phenomenon may represent transformation and nuclear translocation of the ligand-bound receptor, as in the classical model of GR activation. Both BUGR2 and Ig2a produce a heterogeneity in intensities of immunoreactivity in the CNS. Neurons in the pyramidal cell layers of CA1 and CA2, granule cell layer of the dentate gyrus, central amygdaloid nucleus and locus coeruleus among others, have very intense immunoreactivity compared to other regions. While the functional significance of intensity of immunoreactivity is not known, it is interesting that intensely labeled neuronal groups often have higher Type II GR mRNA levels and show increased binding to glucocorticoids.3J6~‘7~4*~~*56 FunctionaL significance

The widespread distribution of Type II GR as revealed by various methods,‘s’7,45,46,56 and indeed of Type I GR,’ demands a rethinking of the putative roles ascribed to these receptor proteins.‘4,33 Type II GR is not only expressed by neurons and glia in limbic and stress-related regions but also in various neocortical, subcortical, diencephalic, br&&%ir, spinal cord and cerebellar neurons involved in motor and sensory coordination. The roles of the receptor protein in these other regions are yet to be fully understood. We hope our chart of Type II GR-like immunoreactivity will contribute towards increased understanding of the complex and diverse effects of glucocorticoids on CNS neurons and glia.

Tables commence overleaf

R. S. AHIMA and R. E. HARLAN

598

+ + +

+ t

+ + t

+++++

+ g+

+,f,f++

++

t+ +t +t + +

+

++

+

+

+

+

+

+

:s 0

+ +

t + +

++ ++ tt + +

+

+++t; +:+:+:i o++to+t++t+

,TB + +

t

+ + +

t + +

t +

I

:++k + -r t+

t

+++o

t

+

+

fo

L 0

++ +

t t + +

t + +

+ + +

I + 8 +

+t tt ++

+

+ 8 0

+ +

If

:+ !j

to t

+ +

+ +t + +t ++ 20” o”+

s

+

cell layers

+++I/ Oor +v 0

++ or +++

0

+++

+++ +++

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

i-f-b

++

-#--I-+ 0

+t+

-I-+ t+ + +++ I-++

i--C+

++ i-it+ +

Densities have been rated: 0, none; f, low; + +, moderate; + + +, high. *Study based on [)H]RU28362 autoradiography. tStudy based on homogenate binding. iStudy based on in situ hybridization to localize Type II mRNA. §A11nuclei apart from central and basolateral amygdaloid nuclei. //Anterior CA3 of Ammo& horn. gCauda1 CA3 of Ammon’s horn.

CA4 Molecular layer of CA1 Dentate gyrus (granule cell layer) Subicuhun

Hippocampus Fields of Amman’s hog-pyra~dal CA1 CA2 CA3

Cerebral cortex Cingulate cortex Layer I Layer II Layer III Layer IV Layer V Layer VI Front0 parietal cortex (sensory and motor) Layer I Layer II Layer III Layer IV Layer V Layer VI 0

+or+f ++ +++

f++ i-++ +++

or +++ i-S or +++ + +++ +++

++

c + ++

0 +++ +-l-t

+ or +t ++ + “t +++

Cff + ++ ++

Septum Lateral-dorsal Medial Ventral Septohippocampal

Nucleus of diagonal band Horizontal Vertical Septofimbrial Triangular Septohypothalamic

-+++ +++ + ++

Fundus striati Substriatal area Basal nucieus of Meynert Claustrum

+++

+ i+++

++-I-

+++

+or++

++

+I-+

-t-or++

+++ +++

++“I++ ++ + ++

-i-i-

+

-l-i-+

R. S. AHIMA and R. E. HARLAN

600

Table 2. Relative densitives of neurons expressing Type II glucocorticoid receptorjclassical glucocorticoid receptor protein and mRNA in the diencephalon Structure

Refs 16, 17

Thalamus

++ or +++Q

Immunocytochemical Ref. 15

Present

‘H Auto* Ref. 45

Binding? Refs 45, 46

+++p ++** ++tt

+++1

O/l Anterodorsal Anteroventrai Anteromediai Mediodorsal Ventromedial Ventrolateral Ventropasterom~ial Lateral posterior Lateral dorsal Posterior Centromedian Paracentral Paraventricular Paratenial Interanteromedial Rhomboid Reuniens Gelatinosa Lateral geniculate Medial geniculate Habenula Reticular Subthalamic Zona incerta Hypothalamus

t+

Mammillary nucleus Supramammillary nucleus Lateral hypothalamic area

ti

t-k 4-f

or i--l-+-

++

or +++

-I-++

+t+

++t 0 0

+

0 i- or +C$Q.

+ +ttt Median preoptic Medial preoptic area Medial preoptic nucleus Lateral preoptic nucleus Striohypothalamic nucleus Anterior hypothalamic nucleus Anterior hypothalamic area Suprachiasmatic nucleus Supraoptic nucleus Paraventricular nucleus Periventricular nucleus Lateral anterior Tubercinereum Ventromedial nucleus Dorsomedial nucleus Perifornical nucleus Arcuate nucleus

In situ$ Ref. 56

++-a t +ttt

+++

+or++ ++ t-l++ ++ ++ 4++ ++ -!-or++ + ++ or +I-+ +or++ + f ++ -t-or++ +++ -t-+-k 0 or + 0 or + + + or ++

+ ++ if-+ + t++ t+ 3-f f

+t+ +-i--k

i-t+

0 or i-

i-m ++***

0 i-f + ++

f + t IIil

++ 0 or + +I-+

0

Densities have been rated: 0, none; +, low; + +, moderate; + + +, high. *Study based on rH]RU28362 autoradiography. tStudy based on homogenate binding. &Study based on in s&u hybridization to localize Type II mRNA. jjDorsa1 thalamic nuclei. 11 Ventral thalamic nuclei. 9Midline thalamic nuclei. **Parafascicular thalamic nucleus. tjCentrolatera1 thalamic nucleus. ttMediobasa1 hypothalamus. @Preoptic nuclei. 11/~Parvocellular paraventricular hypothalamic nuclei. ‘I/fiMagnocellular arcuate hypothalamic nucleus. ***Parvocellular arcuate hypothalamic nucleus. +ttPeriarcuate hypothalamic nucleus. Yl-lLateral ventral hypothalamus,

+t+ fff

+t +++ +I-+ ++

+++

+t

+++ ++ ++

-i-c+ +tt ++t +t+

++

+or++ -t-t- or +++ ++ ff f 0

or + 0 +

i-4-f

Type II glucocorticoid receptors in the brain

Tabt 3. Relative densities of neurons expressing Type II glucocorticoid receptor/classical glucocorticoid receptor protein and mRNA in the mesencephalon Structure Pretectum Anterior Posterior Olivary Medial Nucleus of optic tract midbrain Oculomotor nucleus Media1 accessory oculomotor Edinger-Westphal nucleus Central gray

Immuno~t~hemical Refs 16, 17 Ref. 15

3H Auto+ Ref. 45

Binding? Refs 45, 46

In siftc$ Ref. 56

+ or t+ +++ +t or +-t-f ++ ti or +-I-+

+§ t-i-i-

i-3-t +++ 0

i-4-t

DON31

Lateral Medial Darkschewitsch nucleus Superior coliicuIu5 Superficial gray Intermediate gray Nucleus of brachium of inferior colliculus External cortex of inferior u3l&3dus Subbrachial nucleus Mes~~phalic trigeminal nucleus Sub&ant& nigra Pars compacta Pars reticulata Pars lateralis Dorsal raphe Rostra1 raphe Central raphe Interfascicular nucleus Red nucleus Interpeduncular nucleus Ventral tegmental area

Present

i--l+

f-t-!0 or -t

+tt tt+

Densities have been rated: 0, none; +, low; t +, moderate; t t +, high. *Study based on [3H]R~~g36~ autoradjogmphy. tStudy based on homo~~at~ binding. tStudy based on in s&u hybridization to localize Type II mRNA. SUniform signal in midbrain.

-I-I-+ + f

f-l+or+-t ++ or ++-I+ ++

t-k-lt or -I-+ +or+f t + f or +-I-I-+

tt tt +t

R. S. AHIMAand R. E. HARLAN

602

Table 4. Relative densities of neurons expressing Type II glu~orti~oid receptor/classical glucocorticoid receptor protein and mRNA in the metencephalon Structure Pons Locus coeruleus Medial ~rabrachial Lateral parabrachial Motor trigeminal nucleus Principal sensory trigeminal nucleus Facial nucleus Abducens nucleus Vestibular nuclei Superior Lateral Dorsal raphe Pontine nuclei Pontine reticular fo~ation Pars oralis Cerebellum Purkinje cells Granule cells Deep nuclei

Refs 16, 17

Imrnun~yt~h~~a~ Ref. 15

Present

‘H Auto* Ref. 45

-!-+ or ffi+++ ff t+ or +++ ++ + or ++ +

t++

Bindingt Refs 45, 46 ++

In siru$ Ref. 56 +§

+ f ++ or tt+ + ++ 0 ++

++ +++ + or++

+

+++ ++ or +++ +I

Densities have been rated: 0, none; +, low; + +, moderate; + + +, high. *Study based on [3H]RU2S362autoradiography. TStudy based on homogenate binding. @tudy based on in situ hyb~di~tjon to localize Type II mRNA. §Uniform signal in pons. IjLow levels in cerebellum.

Table. 5. Relative densities of neurons expressing Type 11ghrcocorticoid receptor/classical glucocorticoid receptor protein and mRNA in the myelen~p~ion and spinal cord Structure

Refs 16, 17

~rnrnun~~~i~~ Ref. 15

Present

“H Auto* Ref. 45

Bindingt Refs 45, 46

+!I Inferior olivary nuclei

+++

Spinal trigeminal nucleus Pars gelatinosa Pars oralis Pars interpolaris Pars caudalis

t-i-t

Nucleus ambiguus Nucleus sohtarius Medial vestibular nucleus Prepositus hypoglossal nucleus Hypoglossal nucleus Dorsal vagal nucleus Raphe pallidus Raphe magnus Raphe obscurus Gigantocellular reticular nucleus Nucleus of Roller Cuneate nucleus Gracile nucleus Spinal cord Lamina 2 Lamina 8 Lamina 9 Intermediolateral

+++

++ or +++

S-l-f

+I-+ ++ ++

+t

+

4-3-t

++t

t or +++ i+

++-I-

+-I+ 0 ++i4 -I-II

+

f

ff

+-or-++

t

fff

Densities have been rated: 0, none; +, low; f f, moderate; + + f , high. *Study based on rH]RU28362 autoradiography. tStudy based on homo~ate binding. Qtudy based on in situ hybridization to localize Type II mRNA. §Dorsal vagal nucleus (rostraI). ilDorsa1vagal nucleus (caudal). lILow levels in hindb~in.

+or++ i-or++ +i-t0 0 +i-+ + ++ ++

i-k -k+ ++

In situ$ Ref. 56

Type II glucocorticoid receptors in the brain Acknowledgements-The authors would like to render their sincere thanks to R. W. Harrison for kindly supplying BUGRZ and P3-Agx-x63 myeloma cell medium super-

603

natant, to Z. S. Krozowski for supplying MINRECZ, and to Debbie Lauff for secretarial help. Research was sup ported by grant NS-24148.

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8. 9.

10. 11. 12.

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