Progress in Histo- and Cytochemistry, Vol. 26 W. Graumann / J. Drukker (Eds.), Histochemistry of Receptors © Fischer Verlag· Stuttgan . Jena . New York· 1992

6.3 Co-localization of brain corticosteroid receptors in the

rat hippocampus

]. A. M. VAN EEKELEN, E. R. DE KLOET Center for Bio-Pharmaceutical Sciences, Leiden University, Leiden (The Netherlands)

Introduction Corticosteroid hormones, which represent the endproduct of the hypothalamic-pituitary-adrenal (HP A) axis, play a critical role in adaptation. These hormones control basal activities throughout the circadian cycle and restore disturbances in homeostasis induced by stress (DE KLOET 1991). Their effects are based on a gene-mediated mechanism of action via binding to intracellular corticosteroid receptors. In the brain we distinguish two distinct types of corticosteroid receptors: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) McEwEN 1986; DE KLOET 1991). Corticosteroid receptor diversity in the brain was initially based on the fundamentally different features of MR and GR with respect to their steroid binding specificity and central topography. In vivo and in vitro autoradiography revealed a predominant neuroanatomicallocalization of MR binding sites in the limbic system as opposed to a more widespread distribution of GR in neurons and glial cells (McEWEN 1968; GERLACH and McEWEN 1972; REUL and DE KLOET 1985, 1986). Both MR and GR bind corticosterone (B), which is the main endogenous corticosteroid in rats (REUL and DE KLOET 1985). However, MR displays a six to tenfold greater affinity than GR does. Consequently, MR is extensively occupied by B at all times, whereas the occupation of GR is contingent on plasma B levels and parallels circadian changes in B. Furthermore, GR occupancy increases immediately after stress-induced elevation of circulating B (REUL et al. 1987). Despite the fact that rat brain MR displays identical affinity for B and aldosterone (ALDO, a pure mineralocorticoid), we can distinguish between MR with ALDo-selective and MR with Bselective properties (KROZOWSKI and FUNDER 1983; WRANGE and Yu 1983). ALDo-selective MR binds predominantly ALDO in brain regions such as the circumventricular organs, which are known to be involved in the control of sodium retention and electrolyte homeostasis (McEWEN et al. 1986). Its preference for ALDO in the face of a 100-1000 fold excess of B may be explained by the activity of specificity conferring factors such as l1~-hydroxysteroid dehydrogenase (EDWARDS et al. 1988; FUNDER et al. 1988) and corticosteroid receptor globulin (DE KLOET et al. 1984; FUNDER and SHEPPARD 1987). The B-selective MR type, which is present in the limbic brain, prefers B to ALDO since the former corticosteroid is circulating in such very high concentrations (REUL and DE KLOET 1985). It is this MR type which has been shown to mediate effects of B on the basal activity of neuroendocrine processes (DALLMAN et al. 1987; DE KLOET 1991).

Corticosteroid receptors in the hippocampus . 251

Thus, the brain contains two essentially different receptor populations, both of which bind B but which differ in affinity by an order of magnitude. This finding has important functional implications (DE KLOET et al. 1987, 1991). We believe that B-selective MR and GR mediated actions of B in the brain are both involved in the maintenance of homeostasis. However, depending on the receptor type different aspects are affected (RATKA et al. 1989). The MR mediated effects of B control the basal activity of circadian-related processes such as the activity of the HPA-axis. In contrast, GR mediated effects cause suppression of stress-induced disturbances of neuroendocrine function and behavior. The recent isolation and cloning of specific human and rat cDNA sequences encoding the MR (ARRIZA et al. 1987, 1988; PATEL et al. 1989) and GR (HOLLENBERG et al. 1985; MIESFIELD et al. 1986) have had a great impact on contemporary corticosteroid receptor research. MR and GR are expressed by different genes. Nevertheless, their primary structure reveals similarity: there is a DNA-binding domain manifesting a high degree of homology among the two receptors (76% for rat and 94% for human corticosteroid receptors); also a hormone-binding domain at the carboxyterminus manifesting less but still considerable homology; and lastly an amino-terminal domain which is highly variable (GREEN and CHAMBON 1986; EVANS 1988). A direct consequence of an almost identical DNA-binding domain within the receptor molecules might well be that MR and GR interact with the same or closely-related hormone-responsive elements on genes regulated by B (EVANS and ARRIZA 1989; BEATO 1989). Results from recent molecular genetic studies using cells transfected with MR and GR gene expression vectors suggest that, if co-expressed, these two receptors are potentially capable of interacting with closely overlapping gene networks (EVANS and ARRIZA 1989). This is of interest considering the fact that radioligand binding studies have shown coincidence in the uptake of receptor type specific 3H-ligands by MR and GR in the hippocampus of the limbic brain (GERLACH and McEWEN 1972; REUL and DE KLOET 1985, 1986; SUTANTO et al. 1988). New developments at the level of the gene (the availability of MR and GR cDNAs) together with the generation of a monoclonal antiserum against the purified rat liver GR-hormone complex (WESTPHAL et al. 1982) allowed us to perform a more detailed cellular analysis of MR and GR gene expression and GR protein in the brain, in particular in the hippocampus, of the intact rat.

Methodology Immunocytochemistry The brains of intact adult male rats (150 g BW) were fixed by transaortic perfusion with different fixatives depending on the brain region studied. For purposes of optimal visualization of GR-immunoreactivity (GRir) in the limbic system, 4% paraformaldehyde and 0.2% picric acid in O.1M phosphate buffer (PB), pH 7.4 at 4°C fixed satisfactorily. The brains were postfixed overnight, sectioned on a vibratome (35 !!m in the coronal plane) and processed for immunocytochemistry. Free-floating brain sections were incubated for 48 h at 4°C with monoclonal antiserum 1 GR .49/4 generated against the rat liver GR (WESTPHAL et al. 1982; diluted 1: 1000 in O.lM PB, pH 7.4). Further incubation with biotinylated antimouse IgG (diluted 1: 500 in 0.05M Tris buffer, pH 7.6) and avidin-biotinperoxidase complex (diluted 1: 250 in 0.05M Tris buffer, pH 7.6) was performed for 1 h each at room temperature. The complex components were obtained in kit form from Vector Laboratories (Burlingame, CA). Inbetween all incubation steps the sections were rinsed twice with Tris buffer. Nonspecific binding of the primary antibody was prevented by incubation with normal horse serum (5% in PB) prior to all other sera

252 .

J. A. M. Van Eekelen, E. R. De Kloet

incubations. To improve the accessibility of the different sera to the cellular compartment, Triton X-l00 (0.1 %) was added to all incubation fluids and washbuffers. 3,3-Aminobenzidine tetrahydrochloride (Polysciences) was applied as the chromogen. Finally, the stained sections were mounted on gelatin coated slides, dehydrated, coverslipped and studied at the light microscopic level. A range of control studies was performed: (1) omission of the primary antibody from the incubation sequence; (2) replacement of the primary antiserum by ascites; and (3) incubation of the sections in the presence of primary antibody preabsorbed with a purified rat liver GR preparation. In all cases the immunoperoxidase reaction was markedly reduced (VAN EEKELEN et al. 1987).

In situ hybridization The rat MR clone was isolated from a rat brain cDNA library (ARRIZA et al. 1988). We used a 513 bp fragment of this clone, representing a specific portion which encodes the last 30 amino acids at the carboxyterminus of MR, and a portion of the 3' -untranslated region. A 500 bp fragment was subcloned from the 2.8 kb rat liver GR clone (MIESFIELD et al. 1986) by M. Bohn (VAN EEKELEN et al. 1988). This fragment encodes the amino-terminus of GR. The MRcDNA inserted in pGEM4 was linearized with HindUI for antisense RNA synthesis and linearized with Eco Rl for RNA synthesis of the sense strand. Similarly, GRcDNA inserted in pGEM3 was linearized with Eco Rl for antisense RNA transcription and with HindUI for synthesis of sense RNA. The (anti)sense probes against tissue MR and GR mRNA were transcribed in vitro with the Riboprobe Gemini system (Promega) and labeled with 35S_UTP (1350 Ci/mM), a suitable isotope for intracellular detection of the hybridization signal. Male adult rats were perfused with 4% paraformaldehyde solution containing 1.4% sodium acetate (pH 6.5 at 4°C), followed rapidly by a 4% paraformaldehyde solution containing 0.4% sodium hydroxide, 4% sodium tetraborate and 0.05% glutaraldehyde (pH 9.5 at 4°C). The brains were postfixed overnight, sectioned (thickness 35 ~m in the coronal plane), mounted on gelatin/poly L-Iysine coated slides, and dried overnight under vacuum. Prior to hybridization, the sections were treated with proteinase K (10 g/ml) for 30 min at 37°C to improve the accessibility of the probe to protein-free tissue mRNA. Furthermore, the tissue had to be neutralized with 0.25% acetic anhydride in O.lM triethanolamine for 10 min. to reduce nonspecific hybridization. The probe (approximately 10 8 dpm/ml) was diluted in a hybridization buffer containing 50% formamide, 300 mM NaCI, 10 mM Tris (pH 8.0),1 mM EDTA, lx Denhardt's solution and 10% dextran sulfate, after being heated for 5 min at 65°C with 500 ~g tRNA, 500 ~g polyARNA and 0.06 M DTT in DEPCtreated ddHzO. 75 ~l of this mix was applied to each slide, which thereafter was covered with siliconized coverslips or parafilm. To prevent evaporation, hybridization was carried out in humidified containers at 55°C overnight. The posthybridization procedure consisted of many washes performed under high stringency conditions (4xSSc - O.lxSSC) to gradually desalt the sections. All washbuffers contained 1 mM DTT to protect the S-S bridges of 35S_UTP. The sections were dehydrated and dried under vacuum conditions before being dipped in Kodak NTB-3 photographic emulsion (1 : 1 diluted in 0.6 M ammonium acetate at 43°C). Following a 10 day exposure time at -80°C, the slides were developed in D19 (Kodak), dehydrated, coverslipped and studied at the light microscopic level. Control conditions ranged from: (1) RNase treatment (20 ~g/ml, 30 min, 37°C) prior to hybridization, (2) hybridization with radiolabeled senseprobe, and (3) hybridization with labeled antisense probe - which was preabsorbed with unlabeled senseprobe (2 h, 55°C). In all conditions no autoradiographic signal above background labelling could be distinguished (VAN EEKELEN et al. 1988).

Results and discussion Recent developments in corticosteroid receptor research allowed characterization of new aspects of the receptor life cycle of MR and GR. It is now feasible to determine mRNA (VAN EEKELEN et al. 1988; ARRIZA et al. 1988; ARONSSON et al. 1988; HERMAN et al. 1989), protein as

Corticosteroid receptors in the hippocampus . 253

antigen (FuxE et al. 1985; VAN EEKELEN et al. 1987) and corticosteroid binding sites measured with ligand binding assays and autoradiography (McEwEN et al. 1968; REUL and DE KLOET 1985, 1986, SUTANTO et al. 1988). Cloning of MR and GR cDNA (MIESFIELD et al. 1986; ARRIZA et al. 1988) provided the tools for in vitro transcription of highly specific radiolabeled single stranded cRNA that is used to hybridize with complementary MR and GR mRNA in situ. Development of the monoclonal antibody 1 GR 49/4 against the rat liver GR-hormone complex (WESTPHAL et al. 1982) allowed detection of the in vivo localization of the form of G R at the cellular and subcellular level. Immunocytochemical analysis of the cellular localization of the GR protein in the adult brain revealed a widespread pattern of GR immunoreactivity (GRir) in the brain (FuxE et al. 1985; VAN EEKELEN et al. 1987). In the hippocampus, the pyramidal neurons of the CAt and CA2 fields and the granular neurons of the dentate gyrus (DG) showed most pronounced immunostaining for GR. A low to undetectable level of GRir was observed in the hippocampal cell fields CA3 and CA4 (Fig. lC). The immunoreactive signal was observed in the cell nuclei of all targets mentioned, whereas cytoplasmic GRir did not exceed background immunostaining (VAN EEKELEN et al. 1987). Specific hybridization of GR antisense RNA with tissue mRNA encoding GR could be detected over the entire nerve cell population of the hippocampal formation of the male adult brain (VAN EEKELEN et al. 1988; ARONSSEN et al. 1988). However, a difference in signal intensity was apparent. The CAt and CA2 pyramidal neurons as well as the granular neurons of the DG revealed a dense accumulation of silver grains, whereas labeling of cell field CA3 and CA4 was considerably less (Fig. ID). Hybridization of MR antisense cRNA with tissue MR mRNA was strong and evenly distributed over all cell fields of the hippocampal formation (Fig. 1B; VAN EEKELEN et al. 1988; ARRIZA et al. 1988; HERMAN et al. 1989). In general, labeling of MR and GR mRNA was characterized by a dense concentration of silver grains over the cytoplasm of the cell. The techniques presently available to study corticosteroid receptors in the brain provide new complementary but independent information on the topography, concentration and properties of MR and GR. Radioligand binding assays, immunocytochemistry and in situ hybridization all serve to detect unique aspects of MR and GR. Radioligand binding studies have revealed the distribution patterns of MR and GR binding sites in the brain. A clear resolution in MR and GR is achieved by the use of tritiated ligands that are highly specific for each receptor type. High resolution autoradiography following a tracer dose of 3H_B showed a cellular topography of MR binding sites evenly distributed over all pyramidal and granular neurons of the hippocampus (Fig.lA; GERLACH and McEWEN 1972; REUL and DE KLOET 1986). In contrast, hippocampal GR binding sites labeled with 3H-RU28362, a potent GR agonist, appeared particularly in the CAt and CA 2 cell fields and in the DG (REUL and DE KLOET 1986; SUTANTO et al. 1988). By in vitro measuring of the total number of receptors in the soluble fraction of the cell, this method allows determination of the binding properties of the two receptor systems, e.g. the maximal binding capacity and ligand affinity (REUL and DE KLOET 1985). However, binding assays, both in vivo and in vitro, require clearance of all endogenous corticosteroids from the receptors, this being accomplished by adrenalectomy at least 24 h prior to sacrifice. Circulating endogenous corticosteroids are a pre-requisite for immunocytochemical detection of GR. It is suggested that the epitope, which is localized in the immunogenic domain of the N-terminus of GR, is only exposed by an GR, since the antiserum 1 GR 49/4 is generated against the purified GRhormone complex (WESTPAL et al. 1982). Thus, in contrast to radioligand binding assays, receptor

Fig. 1. Distribution of MR and GR in the hippocampus. A:MR binding site labeled with 3H-corticosterone (from GERLACH and McEwEN 1972); B: MR mRNA labeled with MR- 35 S-RNA probe; C: Immunoreactive GR antigen; D: GR mRNA labeled with GR_ 35S-RNA probe.

~

"~

tj

?"

r'1

~ ?

~

t"fj

~

?=

!>

':-'

~

'""

N

Corticosteroid receptors in the hippocampus . 255

immunocytochemistry permits cellular and subcellular analysis of the GR protein in the intact animal. Detection of tissue MR and GR mRNA by in situ hybridization is independent of the acutal circulating concentration of corticosteroids. Moreover, the combination of in situ hybridization and subsequent autoradiography permits measurement of optical densities. This latter method provides descriptive as well as quantitative data on MR and GR gene expression in the intact animal (VAN EEKELEN et al. 1991). Note that the topographical patterns of MR and GR gene expression (VAN EEKELEN et al. 1988) coincide with the originally described specific 3H-ligand uptake by MR (Fig. 1A; GERLACH and McEwEN 1972), with GR binding sites (REUL and DE KLOET 1986, SUTANTO et al. 1988), and with the cellular localization of GRir (VAN EEKELEN et al. 1987). The complementory information gathered by such essentially different techniques as those underlines the overlap in distribution patterns of MR and GR within the various cell fields of the hippocampus. Only recently has immunostaining for MR and GR been reported to be co-localized in cultured fetal neurons of the hippocampus (BOHN et al. 1990). Thus, presently available data strongly suggest that pyramidal and granular neurons of the hippocampus co-express MR and GR. Co-expression of MR and GR would allow for interaction of MR and GR mediated effects of B in the brain. This hypothesis is supported by a number of studies at the molecular, cellular and organismic level. The structural homology between MR and GR would permit both receptors to potentially recognize identical hormone responsive elements within promotor regions of genes as being activated by corticosteroid receptor complexes (BEATO 1989; EVANS 1989; EVANS and ARRIZA 1989). Thus, MR and GR may mediate the action of B in the expression of overlapping sets of genes, which would not, however, exclude distinct regulatory function for MR and GR elsewhere. Since the extent of MR and GR occupation varies under physiological conditions (REUL et al. 1987), coordinate functions might be based on partial (and possibly continous) activation of the common gene network by MR under basal conditions. This would then be followed by complete activation via GR at the diurnal surge or in response to stress. This , as postulated by EVANS and ARRIZA (1989) - is likely to be of synergistic character. At the cellular level, MR and GR have been shown to mediate effects of B on the excitability of the hippocampal pyramidal neuron, albeit here in opposite directions OELS and DE KLOET 1989, 1990). The MR mediated action was found to maintain the cellular response to excitatory input, whereas GR mediated action rather suppressed this type of effect when excitability was temporarily augmented by excitatory neurotransmitters. As described in the binary glucocorticoid response theory, the MR mediated action preceded the GR mediated effect in time. Moreover, the MR mediated effect was overridden by the GR mediated event, which was of antagonistic character. It cannot be excluded, though, that MR and GR synergize on other aspects of steroid action, e.g. energy metabolism. Thus, the possible antagonism or synergism of MR and GR mediated action on the part of B needs further attention. Finally, a coordinated mode of action of B via MR and GR has been observed in the regulation of the neuroendocrine response (DALLMAN et al. 1987; RATKA et al. 1989). MR mediated effects of B were shown to control basal activity of the HP A axis and responsiveness to stress, possibly via permissive influences on hippocampal excitability to neurogenic input. In contrast, GR mediated action of B in the hypothalamic and pituitary feedback sites suppresses the stress induced hyperactivity of the HPA axis. In the hippocampus, where GR mediated events are thought to be

a

256 .

J. A. M. Van Eekelen, E. R. De Kloet

involved in stress-induced behavior (DE KLOET et al. 1988), it regulates MR mediated activation of the hippocampus. Thus, corticosteroid control of homeostasis is proposed as depending on a coordinate action of MR and GR mediated effects. For a proper contro!' a balance of both receptor types in the brain may be required. Deviations from this balance may evoke a condition of increased or decreased susceptibility to disturbances in homeostasis (DE KLOET 1991). If the impact of corticosteroid action on brain function and adaptation is regulated at the level of the genome, our data suggest that the criterion of co-localization of MR and GR is met in hippocampal neurons.

Summary New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Cal and CAz and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR. The phenomenon of colocalization of MR and GR in the hippocampus will contribute to a better understanding of the coordinated mode of central action of B via MR and GR.

Acknowledgements We thank Dr. M. C. Bohn (University of Rochester, USA) for sub cloning, including providing the 500 bp GR subclone #2 from the 2.8 Kb GR clone of Drs. K. R. Yamamoto, R. Miesfield and P. J. Godowski (University of California, USA). The MR clone was a gift from Drs. J. L. Arriza and R. M. Evans (The Salk Inst. for BioI. Studies, La Jolla, USA). We are grateful to Dr. H. M. Westphal (Marburg, FRG) for kindly supplying monoclonal antiserum 1 GR 49/4. Our grateful thanks go to Roussel-UCLAF for the gift of its RU-compounds; likewise to ORGANON for the gift of aldosterone, corticosterone and dexamethasone. This research was supported by MEDIGON grant 900-546-058 OAMvE) and 900-546-92 (ERdK) of the Dutch Foundation for Advanced Research.

Corticosteroid receptors in the hippocampus· 257

References ARRIZA, J. L., WEINBERGER, G., CERELLI, T. M., GLASER, B. L., HANDELIN, B. L., HOUSEMAN, D. E., EVANS, R. M.: Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. - Science 237, 268-275 (1987). ARRIZA, J. L., SIMERLY, R. B., SWANSON, L. W.: Neuronal mineralocorticoid as a mediator of glucocorticoid response. - Neuron 1, 887-900 (1988). ARONSSON, M., FUXE, K., DONG, Y., AGNATI, L. F., OKRET, S., GUSTAFSSON, J. A.: Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization. - Proc. natl. Acad. Sci. USA 85, 9331-9335 (1988). BEATO, M.: Gene regulation by steroid hormones. - Cell 56, 335-344 (1989). BOHN, M. C., HOWARD, E., KROZOWSKI, Z.: Mineralocorticoid (MR) and glucocorticoid (GR) receptors are co-expressed in hippocampal neurons. - Progr. 20th Ann. Mtg. Soc. Neurosci. St. Louis, MO 977 Abstr. (1990). DALLMAN, M. F., AKANA, S. F., CASCIO, C. S., DARLINGTON, D. N., JACOBSON, L., LEVIN, N.: Regulation of ACTH secretion: variations on a theme of B. - Progr. Hormone Res. 43, 113-173 (1987). DE KLOET, E. R.: Brain corticosteroid receptor balance and homeostatic control. - Front. Neuroendocrinol. 12, 95-164 (1991). DE KLOET, E. R., DE KOCK, S., SCHILD, V., VELDHUIS, H. D.: Antiglucocorticoid RU38486 attenuates retention of a behaviour and disinhibits the hypothalamic-pituitary-adrenal axis at different sites. Neuroendocrinol. 47, 109-115 (1988). DE KLOET, E. R., RATKA, A., REUL, J. M. H. M., SUTANTO, W., VAN EEKELEN, J. A. M.: Corticosteroid receptor types in the brain: regulation and putative function. - Ann. Rev. New York Acad. Sci. 512, 351-361 (1987). DE KLOET, E. R., VELDHUIS, H. D., WAGENAARS, J. L., BERG INK, E. W.: Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus. - J. Steroid Biochem. 21, 173-178 (1984). EDWARDS, C. R. W., STEWART, P. M., BURT, D., BRETT, L., McINTYRE, M. A., SUTANTO, W., DE KLOET, E. R., MONDER, c.: Localization of l1~-hydroxysteroid dehydrogenase: tissue specific protector of the mineralocorticoid receptor. - Lancet 2, 986-989 (1988). EVANS, R. M.: The steroid and thyroid hormone receptor superfamily. - Science 240, 889-895 (1988). -;-: Molecular characterization of the glucocorticoid receptor. - Rec. Prog. Horm. Res. 45, 1-27 (1989). EVANS, R. M., ARRIZA, J. L.: A molecular framework for the actions of glucocorticoid hormones in the nervous system. - Neuron 2,1105-1112 (1989). FUNDER, J. W., SHEPPARD, K.: Adrenocortical steroids and the brain. - Ann. Rev. Physiol. 49, 397-411 (1987). FUNDER, J. W., PEARCE, P. T., SMITH, R., SMITH, A. 1.: Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. - Science 242, 583-586 (1988). FUXE, K., WIKSTROM, A. C., OKRET, S., AGNATI, L. F., HARFSTRAND, F., Yu, Z. Y., GRANHOLM, L., ZOLl, M., VALE, W., GUSTAFSSON, J. A.: Mapping of the glucocorticoid receptor immunoreactive neurons in the tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptors. - Endocrinol. 117, 1803-1812 (1985). GERLACH, J. L., McEWEN, B. S.: Rat brain binds adrenal steroid hormone: radioautography of hippocampus. - Science 175, 1133-1136 (1972). GREEN, S., CHAMBON, P.: A superfamily of potentially oncogenic hormone receptors. - Nature (Lond.) 324, 615-617 (1986). HERMAN, J. P., PATEL, P. D., AKIL, H., WATSON, S. J.: Location and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat. - Mol. Endocrinol. 3, 1886-1894 (1989). HOLLENBERG, S. M., WEINBERGER, C., ONG, E. S., CERELLI, G., ORO, A., LEBO, R., THOMPSON, E. B., ROSENFELD, M. G., EVANS, R. M.: Primary structure and expression of a functional human glucocorticoid receptor cDNA. - Nature (Lond.) 318,635-641 (1985). JOELS, M., DE KLOET, E. R.: Effects of glucocorticoids and norepinephrine on the excitability in the hippocampus. - Science 245, 1503-1505 (1989).

258 .

J.

A. M. Van Eekelen, E. R. De Kloet

-: Mineralocorticoid receptor mediated effects on membrane properties of rat CAl pyramidal neurons in vitro. - Proc. natl. Acad. Sci. USA 87, 4495-4498 (1990). KROZOWSKI, Z. K., FUNDER, J. W.: Renal mineralocorticoid receptors and hippocampal corticosterone binding species have intrinsic steroid specificiry. - Proc. natl. Acad. Sci. USA 80,6056-6060 (1983). McEwEN, B. S., DE KLOET, E. R., ROSTENE, W.: Adrenal steroid receptors and actions in the central nervous system. - Physiol. Rev. 66, 1121-1188 (1986). McEwEN, B. S., WEISS, J. M., SCHWARTZ, L. S.: Selective retention of corticosterone by limbic structures in the rat brain. - Nature 220,911-912 (1968). MIESFIELD, R., RUSCONI, S., GODOWSKI, P. J., MALER, B. A., OKRET, S., WIKSTROM, A. C., GUSTAFSSON, J. A.: Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. - Cell 46, 389-399 (1986). PATEL, P. D., SHERMAN, T. G., GOLDMAN, D. J., WATSON, S. J.: Molecular cloning of a mineralocorticoid (rype I) receptor complementary bNA from rat hippocampus. - Mol. Endocrinol. 3, 1877-1885 (1989). RATKA, A., SUTANTO, W., BLOEMERS, M., DE KLOET, E. R.: On the role of brain mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in neuroendocrine regulations. - Neuroendocrinol. 50, 117-123 (1989). REUL, J. M. H. M., DE KLOET, E. R.: Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. - Endocrinol. 117, 2505-2512 (1985). -: Anatomical resolution of two rypes of corticosterone receptor sites in rat brain with in vivo autoradiography and computerized image analysis. - J. Steroid Biochem. 24, 269-272 (1986). REUL, J. M. H. M., VAN DEN BOSCH, F. R., DE KLOET, E. R.: Differential response of rype 1 and 2 corticosteroid receptors to changes in plasma steroid level and circadian rhythmiciry. - Neuroendocrinol. 45, 407-412 (1987). SUTANTO, W., VAN EEKELEN, J. A. M., REUL, J. M. H. M., DE KLOET, E. R.: Species specific topography of corticosteroid receptor rypes in rat and hamster brain. - Neuroendocrinol. 47, 398-404 (1988). VAN EEKELEN, J. A. M., BOHN, M. c., DE KLOET, E. R.: Postnatal ontogeny of mineralocorticoid and glucocorticoid receptor gene expression in regions of the rat tel-and diencephalon. - Dev. Brain Res. 61, 33-43 (1991). VAN EEKELEN, J. A. M., KISS, J. Z., WESTPHAL, H. M., DE KLOET, E. R.: Immunocytochemical study on the intracellular localization of the rype 2 glucocorticoid receptor in the rat brain. - Brain Res. 436, 120-128 (1987). VAN EEKELEN, J. A. M., JIANG, W., DE KLOET, E. R., BOHN, M. c.: Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus. - J. Neurosci. Res. 21, 88-94 (1988). WESTPHAL, H. M., MOLDENHAUER, G., BEATO, M.: Monoclonal antibodies to the rat liver glucocorticoid receptor. - EMBO J. 1, 1467-1471 (1982). WRANGE, 0., Yu, Z. Y.: Mineralocorticoid receptor in rat kidney and hippocampus: characterization and quantitation by isoelectric focussing. - Endocrinol. 113, 243-250 (1983).

Co-localization of brain corticosteroid receptors in the rat hippocampus.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in t...
1MB Sizes 0 Downloads 0 Views