0013.7227/92/1313-1409$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 131, No. 3 Printed in U.S.A.

Sexual Dimorphism in Regulation of Type II Corticosteroid Receptor Immunoreactivity in the Rat Hippocampus* REXFORD S. AHIMA, AARON N. L. LAWSON, RICHARD E. HARLAN

SUZETTE Y. S. OSEI, AND

Departments of Anatomy (R.S.A, R.E.H) and Pharmacology (5’.Y.S.O), and Neuroscience Training Program (R.E.H), Tulane University Medical School, New Orleans, Louisianu 70112; and Department of Anatomy (A.N.L.L.), University of Ghana Medical School, Accra, Ghana ABSTRACT To determine whether there are sex differences in the distribution of type II corticosteroid receptor-immunoreactive (type II-ir) cells in the rat hippocampus, we carried out a quantitative morphometric immunocytochemical study using a mouse monoclonal antibody, BUGRZ. We report that in adrenally intact male and female rats, high densities of cells with nuclear type II-ir were observed in the pyramidal layer of field CA1 and the granular layer of the dentate gyrus. In intact males very few cells, presumably glia, in the stratum oriens showed type II-ir. In contrast, in females, interneurons with diffuse or cytoplasmic type II-ir were observed in the stratum oriens of CA1 and CA3. There were also sex differences in the regulation of type II-ir by corticosterone, the predominant glucocorticoid, and female sex steroids. In male rats the density of cells with nuclear type II-ir in all parts of Ammon’s horn and the dentate gyrus was decreased significantly after adrenalectomy (adx). In contrast, in females such reductions were observed only in the pyramidal layer of CA1 and the granular layer of the dentate gyrus. In both sexes, cells with intense diffuse or mainly

cytoplasmic type II-ir were observed in the pyramidal layer and stratum oriens after adx. The loss of nuclear type II-ir in the hippocampus of adx females was not affected significantly by ovariectomy. In adx males, nuclear Type II-ir was restored in CA1 and the dentate gyrus after treatment with corticosterone or progesterone. Cells in CA3 were, however, unresponsive to treatment with either hormone. In contrast, in adx females, treatment with either corticosterone or progesterone restored nuclear type II-ir to cells in all regions of the hippocampus. In both adx males and females, cytoplasmic type II-ir observed in some cells in the pyramidal layer and stratum oriens, was abolished completely by corticosterone,~ and partially by progesterone treatment. In both adx males and females, estradiol treatment did not affect sign& cantly the pattern of type II-ir. Sex differences in the distribution of type II-ir interneurons in intact rats and the regulation of the intracellular location of type II-ir of adx rats by corticosterone and progesterone, may be important determinants of sex differences in the modulation of hippocampal function by glucocorticoids. (Endocrinology 13 1: 1409-1416,1992)

G

A sexual dimorphism in the binding capacity and affinity of hippocampal corticosteroid receptors has been reported (10). The binding capacity of 3H-corticosterone and 3Hdexamethasone to cytosolic corticosteroid receptors was higher in gonadally intact females than in males. Gonadectomy increased binding capacity in females but not in males. The affinity of hippocampal corticosteroid binding sites for Cort and dexamethasone was lower in gonadally intact females compared to males. The difference in affinity was even more marked when 3H-dexamethasone was used as ligand instead of Cort, suggesting that binding to the lower affinity type II receptor may account for most of the sex differences, Because progesterone (Prog) binds to and enhances the dissociation of glucocorticoids from type II receptors (11, 12), it has been suggested that higher circulating levels of Prog in females may account for reduced affinity of glucocorticoids for type II receptors (10). Type II corticosteroid receptor-like immunoreactivity (type II-ir) has been mapped in the rat hippocampus by several groups (13-17). Using a monoclonal antibody, BUGR2, which recognizes the rat liver type II receptor, we (13) demonstrated that in adrenally intact male rats, most pyramidal cells in field CA1 of Ammon’s horn and granule cells of the dentate gyrus had predominantly nuclear and weak cytoplasmic type II+. Adrenalectomy resulted in the loss of

LUCOCORTICOIDS regulate several processes in the central nervous system (CNS) (1, 2), mainly by activating intracellular corticosteroid receptors, which then act as transcription factors (3, 4). Two types of corticosteroid receptors have been described in the rat CNS (4, 5). A type I receptor, which is identical to the classical mineralocorticoid receptor of the kidney and has a high affinity and low capacity for endogenous glucocorticoids, e.g. corticosterone (Cort), is thought to mediate effects of glucocorticoids on ongoing neural activity. A type II receptor, which is identical to the classical glucocorticoid receptor of the liver, has a low affinity and high capacity for endogenous glucocorticoids, and a high affinity and capacity for synthetic glucocorticoids, e.g dexamethasone. Type II receptors are thought to mediate effects of high circulating levels of glucocorticoids, e.g in stress. The highest levels of both corticosteroid receptors are expressed in the hippocampus (6-9), where they mediate glucocorticoid effects on neuronal proliferation and differentiation, neuronal death, membrane potential, and neuroendocrine regulation of glucocorticoid secretion (1). Received April 1,1992. Address correspondence and requests for reprints to: Richard E. Harlan, Ph.D., Department of Anatomy, Tulane University Medical School, 1430 Tulane Avenue, New Orleans, Louisiana 70112. *This work was supported by NS-24148 (to R.E.H.). 1409

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nuclear type II-ir in most neurons. Cytoplasmic type II-ir was enhanced initially after adrenalectomy (adx) but was unresolvable in most neurons at the light microscopic level 4 weeks after adx. Treatment with Cort, and to a lesser extent, aldosterone, restored nuclear type II-ir. Because the above response of neuronal type II-ir to pertubations in Cort levels had been described by several investigators using different anti-type II receptor antibodies (9, 13-17), we (13) described it as the typical or “type A response.” In a subgroup of pyramidal cells and interneurons in Ammon’s horn, prolonged adx resulted in intense diffuse or cytoplasmic type IIir. These so-called type B cells (13) were not present in adrenally intact, or adx male rats treated with Cort. In a recent study involving the use of antipeptide antibodies, McGimsey et al. (17) showed that in female rats, adx alone did not alter nuclear type II-ir in the hippocampus. Ovariectomy (ovx) and adx, however, resulted in a loss of nuclear type II-ir and an increase in cytoplasmic type II+. In adx male rats, and adx and ovx females, treatment with glucocorticoids and progestins restored nuclear type 11-u. One aim of the present study was to determine whether there are sex differences in the distribution of type II+ cells in hippocampi of intact male and female rats. We also analyzed the regulation of type II+ by corticosterone, the major glucocorticoid in rats, and by female sex steroids using a quantitative morphometric method. We observed sex differences in the distribution of type II-ir intemeurons in hippocampi of intact male and female rats. However, unlike the antipeptide antibodies described recently (16), BUGR2, the monoclonal antibody used in the present study (8, 13, 18-21) revealed a significant loss of nuclear type II+ in hippocampal neurons of both adx male and female rats. Nuclear type II-ir was restored by Cort and Prog treatment in both adx male and female rats, whereas e&radio1 (E2) treatment did not have any significant effect. Materials Animals

and Methods

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vehicle, and the fourth, vehicle only. These rats were killed 2 h after treatment. The fifth group of adx + ovx rats received daily SCinjections of 1 pg/lOO g BW E2 in sesame oil for 1 week and killed. The rats were anaesthesized by injecting them ip with 25 mg/lOO g BW sodium pentobarbital. They were perf&ed transcardially de for 8 min. Brains were di&ectedAout,Apostfiied with the same fixative for 2 h at room temperature, and cryoprotected with 30% sucrose for 24 h. They were then frozen rapidly on dry ice and stored wrapped in aluminum foil at -70 C until sectioning. Coronal sections, 50 pm thick, were cut on a freezing microtome (Reichert, Buffalo, NY), and processed for immunocytochemical detection of Type II-ir with BUGRZ monoclonal antibody, the Vectastain horse antimouse peroxidase kit and nickel-3, 3’ diaminobenzidine tetrahydrochloride as described previously (13). BUGRZ antibody was used at an optimum dilution of 1:lOOO. Control sections were incubated in nonimmune P3 AgX-653 myeloma cell medium (8, 13, 19). Microscopy

and analysis of sections

Sections were examined with a Niion Optiphot microscope equipped with a 10 mm x lo-mm ocular grid, and a Nikon FX35A camera. Photomicrographs were taken with Technical Pan (Kodak) film. Three sections, two showing the dorsal hippocampus and one the dorsal and ventral hippocampus (Fig. 1) were selected from each animal (four animals per treatment group). Cells showing nuclear type II-ir in the pyramidal layer of Ammon’s horn (CAl, CA3), and the granular layer of the dentate gyms (DG) were counted blindly with the aid of 50 pm X 200 pm ocular grids positioned as illustrated in Fig. 1, at a magnification of x200. In each animal, the data were first analyzed for differences in the densities of type II-ir cells per region between hemispheres using the paired Student’s f test, and from one section to another with the unpaired Student’s f test. P < 0.05 was considered significant. There was no significant difference in the density of type II-ir cells per region at the three levels examined; neither did the densities of immunoreactive cells in one hemisphere differ significantly from the other in each animal. Counts of immunoreactive cells were therefore pooled per region in each animal, and densities per region determined by dividing pooled counts by the number of grids sampled. There was no significant difference in the response of adx + ovx female hippocampi to acute or chronic Er treatment (unpaired Student’s f test), therefore data from the latter treatment group were excluded from the analysis. Data from the other treatment groups were analyzed using one-way analysis of variance (ANOVA),-and posthoc comparisons-between-treatment groups carried out with the Student Neuman Keuls test (P < 0.05 was considered significant).

and immunocytochemistry

Adrenally intact male, adrenally intact and randomly cycling female, adx male, adx female, and adx + ovx female Sprague-Dawley rats were purchased from Charles River (Wilmington, MA). Animal care and experimental protocols were approved by the Animal Care and Use Committee of Tulane University. The rats were kept under standard cage conditions with a 12-h light, 12-h dark cycle, and allowed free access to chow. Intact rats were allowed free access to tap water, and adx, and adx + ovx rats, normal saline. Cort, Prop, and Er were purchased from Sigma (St. Louis, MO). Adx male and female rats, respectively, were divided into four groups (n = 4 per group) 2 weeks after adrenalectomy. They were treated acutely with either Cort, Prog, or Ez for 2 h and killed. Two hours was chosen as the duration of acute treatment because we (13) had previously determined that in adx males, optimum intensity of nuclear type II-ir was induced after 2-h treatment with Cort, the cognate endogenous steroid of the type II receptor. One group of adx animals was treated with 1 mg/lOO g BW Cort in 200 ~1 0.005% ethanol-PBS ip. The second group received 1 mg/lOO g BW Prog, the third, 1 pg/lOO g BW EZ in the same vehicle, and the last group vehicle only. Four adrenally intact male and female rats, respectively, were used as controls. Adx + ovx female rats were divided into five ‘groups (n = 4 per group). One group was treated with 1 mg/lOO g BW Cort in 200 ~1 0.005% ethanol-PBS ip. The second group received 1 mg/lOO g BW Prog, and the third, 1 fig/100 g BW Er in the same

Results Type II corticosteroid receptor immunoreactivity was observed only in sections incubated with BUGRZ antibody. Sections incubated with nonimmune P3 AgX-653 myeloma cell medium did not show specific staining (micrograph not provided). The specificity of BUGR2 for type II receptors has been demonstrated by several investigators (8, 13, 18-21). Type II corticosteroid

receptor

immunoreactivity

in hippocampi

of male rats In adrenally intact males, a high density of cells with nuclear immunoreactivity was observed in the pyramidal layer of CA1 (Fig. 2, A, B) and granular layer of the dentate gyms (DG) (Fig. 2A). Fewer cells were observed in CA3 and CA4 (Fig. 2, A, C). In the stratum oriens and molecular layers of Ammon’s horn and the dentate gyms, small cells with nuclear immunoreactivity, presumably glia, were occasionally observed (Fig. 2B). The response of cells in Ammon’s

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of the hippocampus of adx males (Figs. 3D, 4A-D). Cells with intense diffuse or cytoplasmic immunoreactivity (type B cells) were observed in the pyramidal layer and stratum oriens after adx (Fig. 3A). Treatment with Cort abolished immunoreactivity in type B cells in both layers (Fig. 3B). Treatment with Prog abolished immunoreactivity in type B cells in the pyramidal layer (Fig. 3C); a few type B interneurons were, however, observed in the stratum oriens, especially in CA3. E2 treatment did not affect immunoreactivity in type B cells in the pyramidal layer or stratum oriens (Fig. 3D). Treatment with Cort (Fig. 3B) and to a lesser degree, Prog (Fig, 3C), increased the number of immunoreactive glia in the stratum oriens and molecular layers. Type II corticosteroid receptor immunureactivity in hippocampi of female rats

FIG. 1. Rostrocaudal drawings of the hippocampus showing positions of 50 x 100 pm ocular grids used to assess densities of cells with nuclear type II-ir. Bregma = -2.56 mm in A, -3.30 mm in B, and -4.80 mm in C. CAl, CA3, Fields of Ammon’s horn; CA4, polymorphic layer of dentate gyrus; DG, granular layer of dentate gyrus.

horn and the dentate gyrus to adx and steroid treatment is illustrated in Figs. 3 and 4. After 2 weeks of adx the density of type A cells (i.e. cells with nuclear immunoreactivity) was reduced by 75% in CA1 (Figs. 3A, 4A), 63% in CA3 (Fig. 4B), 50% in CA4 (Fig. 4C) and 75% in DG (Fig. 4D). Treatment of adx rats with Cort increased the density of type A cells in the pyramidal layer of CA1 (Figs. 3B, 4A) by 300%, CA4 (Fig. 4C) by 120% and DG (Fig. 4D) by 300%. There was no significant response of type A cells in CA3 (Fig. 4B) to Cort treatment. In contrast, treatment of adx males with Prog increased the density of Type A cells in CA1 by 250%, CA4 by llO%, and DG by 420%. There was no significant response of type A cells in CA3 to Prog treatment. EZ treatment did not affect the density of type A cells in any region

In intact randomly cycling females, high densities of cells with nuclear type II-ir were observed in the pyramidal cell layer of CA1 (Fig. SA, B) and the granular layer of the dentate gyms (Fig. 5A). In CA3 type II-ir cells in the pyramidal layer were less intensely stained compared to males (Figs. 2, A, C; 5, A, C). However, there was no significant difference between the sexes in the densities of immunoreactive cells in CA3 (Fig. 4B). In the stratum oriens, especially in CA3, intemeurons with diffuse or cytoplasmic Type II-ir were observed (Fig. 5, B, C). They were similar to type B intemeurons observed in the stratum oriens of adx males. Two weeks after adx, the density of type A cells in the pyramidal layer of CA1 was decreased by 60% and 48% in DG (Fig. 4, A, D). The density of type A cells in CA3 and CA4 was not affected significantly by adx (Fig. 4, B, C). Similar results were obtained in adx-ovx rats (Fig. 4A-D). Type B cells, i.e. cells with intense diffuse or cytoplasmic type II+, were observed in the pyramidal layer in adx and adx + ovx females (micrographs not provided). Similar cells were observed in the stratum oriens, and were presumed to be a mixture of type B cells and cells observed in that location in intact females. Treatment with Cort or Prog, increased the density of Type A cells in all regions of the hippocampus in adx and adx + ovx females (Fig. 4A-D). In CA1 (Fig. 4A) of adx and adx + ovx females, treatment with Cort increased the density of type A cells by 90%. In contrast, treatment with Prog increased the density of type A cells in CA1 of adx females by 108% and adx + ovx females by 80%. In CA3 (Fig. 4B), treatment with Cort increased the density of type A cells in adx females by 45% and in adx + ovx females by 70%; in contrast, treatment with Prog increased the density of type A cells in adx rats by 48% and in adx + ovx rats by 100%. Treatment with Cort increased the density of type A cells in CA4 (Fig. 4C) of adx females by 94% and 70% in adx + ovx females; in contrast, treatment with Prog increased the density of type A cells by 120% and 160% in adx, and adx + ovx females, respectively. In DG (Fig. 4D), treatment with Cort increased the density of type A cells in adx females by 66%, and adx + ovx females by 140%; this was in contrast to an increase of 140% and 160% in adx and adx + ovx females, respectively, in response to treatment with Prog.

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FIG. 2. Photomicrographs showing type II corticosteroid receptor immunoreactivity (type II-ir) in the dorsal hippocampus of an adrenally intact male rat. A, Note the high density of immunoreactive cells in the pyramidal layer of CA1 and the granular layer of the DG. CAl, CA3, Fields of Ammon’s horn; CA4- polymorphic layer of the DG. B, CAl, Higher magnification showing cells with nuclear type II-ir in the pyramidal layer. Very few immunoreactive cells were observed in the stratum oriens (so) and stratum radiatum (sr). C, CA3- Note pyramidal cells with nuclear type II-ir. Note the near absence of immunoreactive cells in the so and sr. Scale bar, 200 pm in A, 50 pm in B and C.

FIG. 3. Photomicrographs showing regulation of type II-ir in CA1 of male rat hippocampus by Cort, Prog, and Ez. A, Two weeks after adx, note the decrease in the density of pyramidal cells with nuclear immunoreactivity (type A cells) compared to Fig. 2B. Cells with diffuse or predominantly cytoplasmic immunoreactivity (arrows), type B cells, were observed in the stratum oriens (so) and pyramidal layers. sr: stratum radiatum. B, Two weeks a& + 2 h Cort. There was an increase in the density of type A cells and small immunoreactive cells, presumably glia, in the strata oriens and radiaturn. Type B cells were absent. C, Two weeks adx + 2 h Prog. There was an increase in the density of type A cells. Note the absence of type B cells in the pyramidal layer and stratum oriens. D, Two weeks adx + 2 h EO. There was essentially no change compared to adx (A). Arrow marks type B cells in the pyramidal layer. Scale bar, 50 pm.

. In adx and adx + ovx females, the density of cells in the stratum oriens with diffuse type II-ir increased (data not provided). Some of these were presumed to be equivalent to type B cells observed in hippocampi of adx male rats (Fig.

2B), and the others, type II-ir interneurons observed in intact females (Fig. 5, B and C). In addition, as with the male hippocampus after adx, type B cells were observed in the pyramidal layer of adx and adx + ovx rats (micrographs not

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FIG. 4. Bar charts showing mean densities +SEM of cells with nuclear type II-ir (counts/lO,OOO pm’) at x200 magnification in the pyramidal layer of Ammon’s horn @Al, CA3) and the polymorphic (CA4) and DG layers of the dentate gyrus. n = 4 for each treatment group. Int, Adrenally intact; Adx, 2 weeks postadrenalectomy; Adx + Cort, 2 weeks postadrenalectomy and treated with Cort for 2 h; Adx + Prog, 2 weeks postadrenalectomy and treated with Prog for 2 h; Adx + Ez, 2 weeks adrenalectomy and treated with EO for 2 h. By analysis of variance: CAI, F IS.98= 61.8, P < 0.001; CA3, Fia,gg = 11.4, P c 0.001; CA4, Fi3,98 = 8.1, P c 0.001; DG, Fis,gs = 54.5, P C 0.001. By Student Neuman-Keuls test: *. P < 0.05 comnared to adrenallv intact: , t. comnared to adx: &. comnared to adx + Cort. All comparisons were within the same sex group, Le. males, randomly cycling females, and ovx females.

provided). In adx and adx + ovx females treated with Cort or Prog, fewer interneurons with diffuse type II-ir were observed, especially in the stratum oriens of CA3. The number and distribution of these cells was similar to what we observed in intact females (Fig. 5C). No type B cells were observed in the pyramidal layer of rats treated with Cort or Prog (micrographs not provided). Treatment with Cort increased the number of immunoreactive glia in the stratum oriens and molecular layers of Ammon’s horn (micrograph not provided). In summary, whereas Type A cells in all regions of hippocampi of male rats were sensitive to adx, in randomly cycling or ovx females only type A cells in CA1 and DG were sensitive to adx. Type A cells in all regions of hippo-

campi of adx and adx + ovx rats were responsive to Cort and Prog treatment; in contrast, in adx males, type A cells in CA3 were unresponsive to treatment with either hormone. E2treatment did not have any significant effect on the density of type A cells in either sex. The response of type B cells and glia to Cort, Prog, and E2 treatment was similar in males and females. Discussion A similar distribution of cells with predominantly nuclear type II-ir (type A cells) was observed in the pyramidal layer of Ammon’s horn, and the granular layer of the dentate gyrus in both sexes. Although we did not determine the

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Endo. 1992 Voll31. No 3

5. Photomicrographs showing type II-ir in the dorsal hippocampus of an adrenally intact, randomly cycling female rat. A, Note the high density of immunoreactive cells in the pyramidal layer of CA1 and the granular layer of the DG. CAl, CA3, Fields of Ammon’s horn; CA4, polymorphic layer of the DG. B, CAl, Higher magnification showing cells with nuclear type II-ir in the pyramidal layer (p). Interneurons with intense diffuse type II-ir (arrow) were observed in the stratum oriens (so). C, CA3- Note pyramidal cells with nuclear type II-ir. Interneurons with diffuse type II-ir (arrow) were observed in the stratum oriens (so). Scale bar, 200 km in A; 50 pm in B and C. FIG.

phenotype of cells in this study, previous studies (22, 23) have shown that many of the interneurons in these regions are immunoreactive for GABA, CCK, VIP, somatostatin, and the calcium binding proteins, calbindin and parvalbumin. All granule cells of the dentate gyrus, and most pyramidal cells of CA1 are immunoreactive for calbindin. Glucocorticoids regulate the synthesis and release of many of these products (1). The nuclear location of type II-ir in neurons in the pyramidal and granular layers in both sexes is suggestive of direct regulation by glucocorticoids, presumably via a genomic mechanism. The presence of diffuse or predominantly cytoplasmic type II-ir in interneurons in the stratum oriens, especially in CA3, of intact randomly cycling females was intriguing. We speculate that in these neurons, cytoplasmic type II receptors may be involved in the posttranslational processing and transport of neuronal products like GABA, CCK, VIP, and somatostatin. It would be interesting to explore how the sex difference in intemeuronal type II-ir affects the activity of neuronal circuits in the female and male hippocampi. Contrary to the reported inability of adx to abolish nuclear type II-ir in hippocampi of female rats (17), we observed that in both sexes the density of type A cells (i.e. cells with nuclear type II-ir) was reduced in most hippocampal regions after adx. There was however a sex difference in the decrease in the density of Type A cells. Males showed a greater response to adx compared to females. The interpretation of the loss of nuclear type II-ir in response to adx is still a matter for

debate. Whereas some have suggested that it represents translocation of nuclear type II receptors into the cytoplasm in the absence of glucocorticoids (9, 14-16), we (13) have suggested previously that masking of immunogenic sites on the receptor by conformational modification or interaction with other cellular proteins may account, at least in part, for the intracellular location of type II-ir. We did not find any difference in the density of type A neurons between adx and adx + ovx females as reported by McGimsey et al. (17). The difference between the current results and that of McGimsey et al. (17), may be attributed to differences in immunogenic sites for BUGR2 antibody used in this study and the antipeptide antibodies, GR 57 and GR 59 (16). All three antibodies crossreact with both nuclear and cytoplasmic type II corticosteroid receptors. However, whereas cells with both nuclear and cytoplasmic type 11-n can be localized by BUGR2 4 weeks after adx, the antipeptide antibodies cross-react with mainly cytoplasmic type II-ir within a few days after adx (16). We have discussed previously, differences between the localization of type II-ir in the rat CNS by BUGR2 and other monoclonal antibodies (8, 13). In addition to the nuclear-tocytoplasmic translocation of type II-ir in the absence of glucocorticoids, a feature that is shared by most anti-type II receptor antibodies (9, 13, 14-16), BUGR2 localizes a subset of atypical cells with intense diffuse type II-ir in the hippocampus, striatum, habenula, and reticular formation (13). These so-called type B cells were observed in the pyramidal layer and stratum oriens after adx in both sexes, and disap-

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peared after treatment with Cort. It is interesting that type B cells are localized in regions of the hippocampus T*:here neuronal death in response to various noxious stimuli is potentiated by glucocorticoids, i.e. CA1 and CA3 (1). No type B cells were observed in the dentate gyrus, a region which is relatively more resistant to neurotoxic agents (23). The relationship between the occurrence of type B cells and glucocorticoid-potentiated neurotoxicity would be interesting to explore. There was a sex difference in density of type A cells in response to Cort and Prog treatment. In males, Cort and Prog treatment increased the density of type A cells in CAl, CA4, and DG but not in CA3. There was a greater response to treatment with Cort compared to Prog in CAl. The opposite was true in DG. In contrast, type A cells in all regions of the hippocampus of adx and adx + ovx females were responsive to Cort and Prog. Moreover, Cort and Proginduced increases in the density of type A cells were similar in most regions. A possible mechanism underlying cross-regulation of type II-ir in hippocampi of adx rats by noncognate steroids, e.g. Prog in the present study, and aldosterone (13) and androgenie-anabolic steroids (24), is direct binding of these steroids to type II corticosteroid receptors. Androgens and progestins bind to type II corticosteroid receptors in vitro (11, 12, 25, 26). Progestins bind and enhance the dissociation of glucocorticoids to cytosolic type II corticosteroid receptors from liver (11) and AtT-20 cells (12). The process is reversible (12). One can envisage that binding of progestins to type II corticosteroid receptors in the hippocampus may result in a conformational change, and exposure of BUGR immunogenic sites. Increased nuclear type II-ir in response to treatment with progesterone may represent an increase in immunogenicity of nuclear type II corticosteroid receptors and/ or translocation of cytoplasmic type II corticosteroid receptors into the nucleus. The absence of progestin binding sites in the hippocampus (27), a region with high levels of type II corticosteroid receptors (8, 9), is however, inconsistent with regulation of type II-ir by direct binding. We speculate that the discrepancy in the regulation of hippocampal Type II-ir and lack of progestin binding sites may lie in the kinetics of association and dissociation of progestins with type II receptors. Interestingly, EZ, which shows little binding to corticosteroid receptors (l), did not affect type II-ir in type A cells. This finding was in agreement with findings by McGimsey et al. (17). Cross-regulation of Type II+ by Prog in hippocampi of male and female rats may also result from the induction of glucocorticoid-like substances in the CNS or peripheral organs by Prog and/or conversion of Prog into glucocorticoids or glucocorticoid-like substances, resulting in regulation of type II-ir. Such a mechanism would not account for the persistence of some type B cells especially in CA3 in Progtreated rats. Total loss of immunoreactivity in type B cells in adx rats appears to be peculiar to Cort treatment, and not to cross-reacting steroid hormones (13, 24). It is also possible that cross-regulation of type II-ir occurs indirectly via interaction between activated progesterone receptor (PR), type I

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corticosteroid (mineralocorticoid-MR), androgen (AR) receptors, and type II corticosteroid receptors, leading to exposure of immunogenic sites. This hypothesis is attractive because the activated forms of the above receptors bind to the same DNA response element (28), and therefore in cells where they are coexpressed, they are likely to exist in close proximity. This mechanism may be important in ‘the case of aldosterone and androgens, because most hippocampal cells appear to coexpress MR, AR, and type II corticosteroid receptors (7). PR is, however, not expressed in the hippocampus (27); therefore such a mechanism is unlikely to account for cross-regulation of type II+ by Prog. Assuming regulation of type II-ir by Cort and Prog in type A cells is the result of direct binding to type II corticosteroid receptors, our results indicate that most hippocampal pyramidaI cells and granule cells in males and females would bind equally well to Cort and Prog, given the chance. However, since circulating Cort levels are much higher than Prog under normal physiological conditions, and Prog levels are negligible in males, it is likely that most hippocampal type II receptors in both sexes bind to and mediate actions of Cort. This does not, however, exclude the possibility of the mediation of the actions of Prog by female hippocampal type II receptors, especially in phases of the estrous cycle, pregnancy, and lactation characterized by relatively high levels of Prog. We propose that activation of type II receptors by high circulating levels of Prog would result in the expression of specific genes, with consequent alteration in CNS function. Sex differences have been reported in hippocampal morphology (29), dendritic development in response to increased environmental stimulation (30), axonal sprouting (31), excitability by gonadal steroids (32), and complex behaviors, e.g. maze learning (33). Most of these events in the CNS are also regulated by glucocorticoids (1). A sex difference in circulating levels of glucocorticoids has been reported, with females secreting higher basal levels of Cort than males (34). However, female rats have higher levels of transcortin than males, and therefore most of the circulating Cort is bound and not bioavailable (35). Hippocampi of both males and females are likely to be exposed to comparable levels of Cort, and therefore differences in the circulating levels of Cort may not account for sex differences in hippocampal function; rather, sex differences in the binding capacity and affinity of type II corticosteroid receptors, which may be related to differences in circulating levels of Prog (lo), may be more important. Our findings of sex differences in the distribution of type IIir cells, regulation of type II-ir by Cort and cross-regulation of Type II-ir by Prog should provide an anatomical basis for exploring sex differences in the modulation of hippocampal function by glucocorticoids. Acknowledgment Our sincere and P3 AgX-653

thanks to R. Harrison myeloma cell medium.

for

supplying

BUGRP

antibody

References 1.

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C, Rivers N 1983 Sex differences in the dendritic response to differential environments in the granule cells of the dentate gyrus. Sot Neurosci Abstr 9:91 Loy R, Milner T 1980 Sexual dimorphism in extent of axonal sprouting in rat hippocampus. Science 208:1282-1283 Teyler TJ, Vardaris RM, Lewis D, Rawitch AB 1980 Gonadal steroid: effects on excitability of hippocampal pyramidal cells. Science 209:1017-1018 McGivern RF, Clancy AN, Hill MA, Noble EP 1984 Prenatal alcohol exposure alters adult expression of sexually dimorphic behavior in the rat. Science 224:896-898

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in the

Sexual dimorphism in regulation of type II corticosteroid receptor immunoreactivity in the rat hippocampus.

To determine whether there are sex differences in the distribution of type II corticosteroid receptor-immunoreactive (type II-ir) cells in the rat hip...
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