0013-7227/91/1291-0226$03.00 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 1 Printed in U.S.A.
Differential Corticosteroid Regulation of Type II Glucocorticoid Receptor-Like Immunoreactivity in the Rat Central Nervous System: Topography and Implications* REXFORD S. AHIMA AND RICHARD E. HARLAN Department of Anatomy and Neuroscience Training Program (R.E.H.), Tulane University School of Medicine, New Orleans, Louisiana 70112
ABSTRACT. Neuronal type II glucocorticoid receptor-like immunoreactivity in the central nervous system shows heterogeneity in intensities and relative densities. This might predict variations in the regional responses of neuronal immunoreactivity to corticosteroids. We investigated changes in the intracellular location of immunoreactivity in the rat central nervous system after adrenalectomy and corticosteroid treatment, and carried out detailed statistical analysis of changes in neuronal nuclear immunoreactivity in the hippocampus and caudateputamen. Three types of responses were observed. The majority of neurons, classified type A, showed a predominant nuclear immunoreactivity in intact rats, lost nuclear and eventually
cytoplasmic immunoreactivity after adrenalectomy, and regained nuclear immunoreactivity within 5 min of corticosterone and 2 h of aldosterone treatment, respectively. A subgroup of neurons in the hippocampus, striatum, septum, and habenula, classified type B, were not immunoreactive in intact rats, showed intense cytoplasmic immunoreactivity after adrenalectomy, and disappeared rapidly after corticosterone treatment and later in response to aldosterone. A subgroup of vermal cerebellar Purkinje neurons, classified type C, developed an intense cytoplasmic immunoreactivity after adrenalectomy, increased in number in response to corticosterone, and did not respond to aldosterone. (Endocrinology 129: 226-236,1991)
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densities and intensities of immunoreactive neurons in limbic, stress-related, motor, and sensory systems. Neuronal type Il-ir is typically nuclear, with a weak cytoplasmic component. Four days to 2 weeks after adrenalectomy, there is reportedly a uniform loss of nuclear immunoreactivity in many regions (4, 7, 8). Treatment with glucocorticoids restores nuclear immunoreactivity (4, 7, 8), while aldosterone treatment reportedly has no effect (7). Considering the marked heterogeneity in the regional distribution of type Il-ir, one would have expected a heterogeneous regional response to glucocorticoids. Moreover, aldosterone has been shown in in vitro experiments to bind to the type II receptor protein (9) and, presumably, could affect type Il-ir in some neurons. In the present study we followed up our previous map of type Il-ir in the rat CNS using BUGR2 monoclonal antibody (6), with a study of the regulation of type Il-ir by corticosteroids. We were interested in regional differences in neuronal response as well as the time courses of such responses to corticosterone and aldosterone, the principal glucocorticoid and mineralocorticoid, respectively, in rats.
LUCOCORTICOIDS exert diverse regulatory effects on neuronal and glial activity in the central nervous system (CNS) (1, 2). Two classes of corticosteroid receptors, types I and II, have been described in the CNS (2). Type I receptor binds preferentially to the predominant glucocorticoid in the species and has an equivalent high affinity for mineralocorticoids. Type II receptor has a high affinity for dexamethasone and other specific glucocorticoids, and a lower affinity for both mineralocorticoids and the species-specific glucocorticoid. Type I receptors presumably mediate tonic and behavioral responses within the diurnal levels of corticosteroids, while type II receptors mediate the stress effects and central negative feedback regulation of glucocorticoids (2, 3). Type II receptor-like immunoreactivity (type Il-ir) has been mapped in the rat CNS using monoclonal antibodies against different epitopes of the variable domain of the rat liver glucocorticoid receptor (4-8). Immunoreactivity is widespread, with a marked heterogeneity in the relative Received January 28,1991. Address requests for reprints to: Richard E. Harlan, Ph.D., Department of Anatomy, Tulane Medical Center School, 1430 Tulane Avenue, New Orleans, Louisiana 70112. * This work was supported by Grant NS-24148 (to R.E.H).
Materials and Methods Adrenalectomized (1, 2, and 4 weeks) and intact SpragueDawley-derived male rats (Charles River, Wilmington, MA), 226
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GLUCOCORTICOID RECEPTOR REGULATION weighing 200-250 g, were used. They were fed standard rat chow. Intact rats were maintained on tap water ad libitum, and adrenalectomized rats on normal saline. All rats were kept under a 12-h light, 12-h dark cycle. BUGR2 mouse antirat liver type II receptor monoclonal antibody and nonimmune P3 AgX-653 myeloma cell medium supernatant were kindly supplied by R. W. Harrison (10, 11). The Vectastain ABC horse antimouse kit was purchased from Vector Laboratories (Burlingame, CA), and corticosterone and d-aldosterone were obtained from Sigma (St. Louis, MO). The adrenalectomized rats were divided into three groups. One group was treated with d-aldosterone (10 Mg/100 g BW) in 200 nl 0.005% (vol/vol) ethanol-PBS, ip. Another group received corticosterone (1 mg/100 g BW in the same vehicle), while a third group received only vehicle. Within each treatment group, four rats were treated for 5 min, 1 h, or 2 h respectively, before death. The adrenalectomized and intact rats were killed with an overdose of sodium pentobarbital ip, perfused transcardially with 0.025 M PBS, pH 7.2, for 5 min, followed by 3% phosphatebuffered paraformaldehyde for 8 min. The abdominal cavities of the adrenalectomized rats were explored to confirm adrenalectomy. Brains and spinal cords were dissected out, blocked, postfixed in the same fixative for 2 h at room temperature, and cryoprotected in 30% sucrose (wt/vol) at 4 C until sinking. They were then rapidly frozen on dry ice and stored wrapped in aluminium foil at —70 C until use. Immunocytochemistry Coronal sections (50 ^m thick) of the brain and spinal cord were cut on a freezing microtome (Reichert, Buffalo, NY), rinsed in PBS for 1 h, blocked with normal horse serum in 0.1% BSA in PBS according to specifications of the Vectastain kit, permeabilized in 0.2% Triton X-100 in PBS for 20 min, incubated in a 1:500 dilution of BUGR2, and processed as described previously (6) with either 3,3'-diaminobenzidine tetrahydrochloride (DAB) or nickel-DAB as chromogen (12). Adjacent control sections were incubated with P3 AgX-653 myeloma cell supernatant or normal mouse immunoglobulin G (IgG). The reaction was stopped in PBS for 5 min, and sections were transferred to 10 mM sodium acetate (Sigma), mounted on chromalum-subbed slides, and dehydrated in increasing strengths of ethanol (70%, 95%, and 100%). Sections were then defatted in Histoclear (National Diagnostics, Somerville, NJ) and coverslipped with Permount (Fisher Scientific, Fairlawn, NJ). Some sections were counterstained with cresyl violet to facilitate delineation of neuronal groups. Sections were examined with a Nikon Optiphot microscope, equipped with a camera lucida drawing tube and a Nikon FX35A camera. Photographs were taken with Technical Pan (Eastman Kodak, Rochester, NY) film. The following parameters were examined: intracellular location of type Il-ir, relative intensities, and relative densities. Relative intensities were determined qualitatively. Three standards were chosen: strong immunoreactivity, as in the pyramidal cell layer of CA1 of intact rats (Fig. 1A); weak immunoreactivity, as in the globus pallidus (Fig. 3A); and moderate immunoreactivity, for immunostaining between these extremes. At least three sections were selected for each region per treatment group per animal.
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Sections from three animals per treatment group, processed under the same conditions, were selected and matched with the help of the atlas of Paxinos and Watson (13) by one experimenter and coded. Relative intensities were then scored blindly by two investigators using the above standards. The effects of adrenalectomy and corticosteroid treatment on the density of cells with nuclear type Il-ir in Ammon's horn and dentate gyrus of the hippocampus and in the caudateputamen were determined by counting neurons with distinct nuclear type Il-ir in grids 100 X 100 /urn at a magnification of X200 with the help of the camera lucida tube. Three nickelDAB-stained sections of the hippocampus, corresponding to Figs. 28, 32, and 35 of the atlas of Paxinos and Watson (13), and three of the caudate-putamen, corresponding to Figs. 17, 21, and 25 of the atlas of Paxinos and Watson (13), were selected, matched, and coded by one investigator from three animals in each treatment group. These were then analyzed blindly by two investigators. Three grids were analyzed for the pyramidal cell layer of CAl, one for CA2, two for CA3, two for CA4, and two for the ventral and dorsal blades, respectively, of the granular layer in each hemisphere. The caudate-putamen in each hemisphere was divided into four quadrants, using selected landmarks. Two grids were analyzed in each quadrant per hemisphere. Counts were corrected for errors using Abercrombie's method (14). The mean counts ± SE per region per treatment were determined from pooled counts. The data were analyzed using one-way analysis of variance, and post-hoc comparisons were carried out using the Student-Neuman-Keuls test.
Results Intact rats Type Il-ir was demonstrated in several neuronal groups. Details of the patterns of distribution have been described previously (6). Immunoreactivity was predominantly nuclear, with a weak cytoplasmic component. Strong immunoreactivity was observed in the pyramidal cell layer of fields CAl and CA2 of Ammon's horn, granule cells of the dentate gyrus, medial and lateral divisions of the bed nucleus of the stria terminalis, central amygdaloid nucleus, layer II of the piriform cortex, locus coeruleus, nucleus ambiguus, substantia gelatinosa, and motor neurons of laminae 8 and 9 of the spinal cord. Sections incubated with normal mouse IgG or nonimmune P3 AgX-653 myeloma cell medium produced only nonspecific signal (e.g. Figs. ID and 5D). The specificity of BUGR2 antibody for the type II receptor in the rat CNS has also been demonstrated by others (11, 15). Type Il-ir and corticosteroids Three types of responses in the intracellular location of type Il-ir and relative densities of type Il-ir neurons were observed after adrenalectomy, adrenalectomy plus corticosterone treatment, and adrenalectomy plus aldosterone treatment.
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FIG. 1. Light micrographs comparing type Il-ir in CAl of the hippocampus. A, Intact (nonadrenalectomized) rat. Note the intense, predominantly nuclear, immunoreactivity in the pyramidal cell layer. B, One week of adrenalectomy. Note the reduction in the number of cells with nuclear type Il-ir and the appearance of mainly cytoplasmic type H-ir in some cells (arrow). C, Four-week postadrenalectomized rat. Note the virtual absence of immunoreactivity in most pyramidal cells (type A response). Arrows mark the limits of the pyramidal cell layer. Note the persistence of cells with intense cytoplasmic immunoreactivity, i.e. type B cells. D, Four-week postadrenalectomized rat. Section incubated with nonimmune P3 AgX-653 myeloma cell supernatant. Note the absence of type Il-ir. Arrows mark the limits of the pyramidal cell layer. E, Four-week postadrenalectomized rat. Five minutes of corticosterone treatment restored some immunoreactivity to type A response neurons, but it was below intact levels. Atypical (type B) neurons have disappeared. F, Five minutes of aldosterone treatment of 4-week postadrenalectomized rats did not completely abolish immunoreactivity in type B neurons. Arrow points to type B neuron in the pyramidal cell layer. G, Two hours of corticosterone treatment of 4-week adrenalectomized rats restored nuclear immunoreactivity (compare to A). H, Two hours of aldosterone treatment restored nuclear immunoreactivity to some type A neurons, but densities were below intact levels (compare to A). No atypical (type B) neurons were seen. Scale bar = 50 nm for A-H.
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Endo • 1991 Voll29«Nol
GLUCOCORTICOID RECEPTOR REGULATION Telencephalon and diencephalon One week after adrenalectomy, type Il-ir neurons in the islands of Calleja; amygdala (except the central nucleus); intermediate and medial divisions of the bed nucleus of the stria terminalis; ventral pallidum; substantia innominata; fundus striati; substriatal area; basal nucleus of Meynert; claustrum; medial, ventral, septofimbrial and triangular septal nuclei; vertical division of the nucleus of the diagonal band; zona incerta; and hypothalamus (except the parvocellular paraventricular nucleus) lost nuclear and cytoplasmic immunoreactivity. Cytoplasmic type Il-ir and some nuclear type Il-ir were still present in the isocortex and all thalamic nuclei by the end of the first week after adrenalectomy. Immunoreactivity was abolished in these regions by the end of the second week after adrenalectomy. Treatment with corticosterone produced weak nuclear immunoreactivity as early as 5 min in most neurons. Two hours of corticosterone treatment increased the intensities of nuclear immunoreactivity and the densities of type Il-ir neurons above intact levels in most regions. Two hours of treatment with aldosterone restored some nuclear immunoreactivity, but below levels seen in intact animals. Because type Il-ir in the vast majority of telencephalic and diencephalic neurons responded to corticosterone treatment in a fashion similar to that described by others (4, 7, 8), these neurons were classified as typical response or type A neurons. Hippocampus Most immunoreactive neurons in Ammon's horn and the dentate gyrus showed a type A response. Nuclear immunoreactivity was abolished in most pyramidal cells of Ammon's horn (compare Fig. 1, A and B) and the granule cells of the dentate gyrus 1 week after adrenalectomy. Some neurons in the pyramidal cell layer of fields CAl and CA2 and the stratum oriens developed predominantly cytoplasmic immunoreactivity 1 week after adrenalectomy (Fig. IB). By 4 weeks postadrenalectomy, similar neurons were observed in CA3. Cytoplasmic immunoreactivity was retained in this subgroup of hippocampal neurons with increasing duration of adrenalectomy, while most hippocampal neurons lost immunoreactivity (Fig. 1C). Adjacent sections of the hippocampus from 1- and 4-week postadrenalectomized rats incubated with either normal mouse IgG or P3 AgX-653 myeloma cell medium did not show type Il-ir (Fig. ID). Nuclear type Il-ir was observed in most hippocampal neurons within 5 min of corticosterone treatment (Fig. IE). The intensity of immunoreactivity was, however, much weaker than that in intact animals (compare Fig. 1, A and E). Two hours of corticosterone treatment increased the intensity of nuclear type Il-ir to intact
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levels (compare Fig. 1, G and A), akin to the type A response observed in the rest of the telencephalon. Two hours of aldosterone treatment restored nuclear type Ilir in most neurons, but the intensities were often weaker compared to those in intact animals (compare Fig. 1, H and A). In the subgroup of hippocampal neurons in which increased cytoplasmic type Il-ir was observed after adrenalectomy, immunoreactivity was abolished after 5 min of corticosterone treatment (compare Fig. 1, B, C, and E) or 2 h of aldosterone treatment (compare Fig. 1, B, C, and H). Five minutes of aldosterone treatment reduced, but did not eliminate, type Il-ir in this subgroup of hippocampal neurons (Fig. IF). This atypical response of type Il-ir to corticosteroids was classified as a type B response. Striatum, septum, and habenula Most immunoreactive neurons in these regions showed a type A response. However, as in the hippocampus, a subgroup of neurons showed an atypical type B response. In the caudate-putamen (Fig. 2, A-F), globus pallidus (Fig. 3, A-C), entopeduncular nucleus, lateral septum, and habenula, a subgroup of neurons showed increased, mainly cytoplasmic, type Il-ir after adrenalectomy (Figs. 2, A-C, and 3, A and B). Immunoreactivity in these neurons was abolished by 5 min of corticosterone treatment (Fig. 2D) or 2 h of aldosterone treatment (Fig. 2F). Adjacent control sections from intact or adrenalectomized rats did not show type Il-ir (data not illustrated). Brainstem, cerebellum, and spinal cord Unlike the telencephalon and diencephalon, nuclear type Il-ir was still present in most parts of the brainstem, cerebellum, and spinal cord 2 weeks after adrenalectomy. By 4 weeks postadrenalectomy, most neurons showed weak cytoplasmic immunoreactivity, even though a few neurons in the locus coeruleus (Fig. 4), inferior olivary nuclei, and spinal motor neurons retained some nuclear type Il-ir. Two hours of corticosterone treatment restored nuclear type Il-ir (compare Fig. 4, A and D), while aldosterone restored some nuclear type Il-ir, but below intact levels. The response of type Il-ir in most neurons in the hindbrain was, thus, a type A response. The gigantocellular reticular nucleus showed a type B response. In the cerebellum a subgroup of Purkinje cells in the vermis lobules 1-3, 9, and 10 also showed increased, predominantly cytoplasmic, type Il-ir after adrenalectomy (compare Fig. 5, A and B). However, unlike type B neurons, immunoreactivity was retained in the cytoplasm of these neurons after corticosterone treatment (Fig. 5, B and C). In addition, nuclear type Il-ir was often resolvable in these Purkinje cells. These neurons, classified type C, did not respond to aldosterone treat-
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Endo«1991 Vol 129 • No 1
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» FIG. 2. Type Il-ir in the caudate-putamen of intact, adrenalectomized, and corticosteroid-treated rats. A, Intact rat. Note the high density of immunoreactive (type A) neurons. Immunoreactivity was primarily nuclear. B, One week of adrenalectomy reduced immunoreactivity in most (type A) neurons; however, note the appearance of atypical (type B) neurons (arrow). C, Four-week postadrenalectomized rat. Note the further decrease in the number of type A neurons and the increase in type B neurons. D, Five minutes of corticosterone treatment of 4week adrenalectomized rats abolished immunoreactivity in type B neurons and restored some nuclear immunoreactivity in type A neurons. E, Four-week postadrenalectomized rat. Two hours of corticosterone treatment increased nuclear immunoreactivity and densities of immunoreactive neurons to or above intact levels (compare with A). F, Four-week postadrenalectomized rat. Two hours of aldosterone treatment restored immunoreactivity in most type A neurons, but the density of labeled neurons was less than that in intact rats (compare with A). Note the absence of type B neurons. Scale bar = 100 ^m (A-F).
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ment. Corticosterone treatment also increased the number of type C Purkinje cells. Incubation of adjacent sections with control myeloma supernatant demonstrated the specificity of immunostaining of type C neurons (Fig. 5D). Changes in the density of neurons with nuclear type IIir in the hippocampus and caudate-putamen in response to adrenalectomy and corticosteroid treatment Figure 6, A-F, illustrates the response of neurons with nuclear type Il-ir in the hippocampus and caudate-putamen to corticosteroids. Hippocampus. One week of adrenalectomy resulted in a
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significant reduction in the numbers of cells with distinct type Il-ir in the pyramidal and granule cell layers (Fig. 6, A-E). Four weeks of adrenalectomy resulted in a further reduction in all of these regions. In 1 week postadrenalectomized rats, 5 min of corticosterone treatment significantly increased counts above adrenalectomy levels in CAl and CA2 (Fig. 6, A and B), but not in CA3 and CA4 or dentate gyrus (Fig. 6, C-E), while 2 h of corticosterone treatment increased counts in all subregions except CA3 (Fig. 6C). Two hours of aldosterone treatment significantly increased densities of type Il-ir cells in CAl (Fig. 6A) and CA2 (Fig. 6B) above adrenalectomy levels. Thus, after 1 week of adrenalectomy, CAl and CA2 were sensitive to the effect of
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GLUCOCORTICOID RECEPTOR REGULATION
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corticosterone, while CA3 and CA4 were more resistant (Fig. 6, C and D). Four weeks after adrenalectomy, 5 min of corticosterone treatment increased the densities of immunoreactive cells in CA1, CA2, and dentate gyrus (Fig. 6, A, B, and E) and resulted in a nonsignificant increase in CA3 and CA4 (Fig. 6, C and D). Two hours of corticosterone treatment significantly increased densities in all regions (Fig. 6, A-E). Two hours of aldosterone treatment significantly increased densities in all regions, except the granule cell layer of the dentate gyrus (Fig. 6, A-E). There was an interesting effect of aldosterone on the pyramidal cells of CA3 and CA4 after 4 weeks of adrenalectomy. While in most subregions, 2 h of corticosterone treatment produced a greater increase in the densities of immunoreactive neurons than did aldosterone (Fig. 6, A, B, and E), the converse was true in CA3 and CA4 (Fig. 6, C and D). In summary, nuclear type Il-ir in the pyramidal cells of CA3 was most resistant to 1 week of adrenalectomy, showed less response to corticosteroid treatment (together with CA4) during this period, and showed a paradoxically increased response to aldosterone (with CA4) 4 weeks after adrenalectomy. Caudate-putamen. One week of adrenalectomy reduced significantly the density of cells with nuclear type Il-ir (Fig. 6F). There was a further reduction 4 weeks after adrenalectomy. Corticosterone treatment produced an increase in density above adrenalectomy levels. The response to corticosterone occurred as early as 5 min and was significantly greater after 2 h. Two hours of aldosterone treatment significantly increased the density above adrenalectomy levels, but significantly less than levels in both intact and corticosterone-treated (2 h) animals. Four weeks after adrenalectomy, corticosterone or aldosterone treatment increased the density of type Il-ir cells. This response was, however, significantly smaller than that to treatment 1 week after adrenalectomy.
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Contrary to previous reports indicating a uniform loss of nuclear and then cytoplasmic immunoreactivity after adrenalectomy (4, 7, 8), we observed three types of responses. In the majority of neurons (type A), there was a progressive loss of type Il-ir with increasing duration FIG. 3. Type Il-ir in the globus pallidus of intact, adrenalectomized, and corticosterone-treated rats. A, Intact rat. Note the few immunoreactive neurons. Immunoreactivity is mainly nuclear and weak. B, Four-week postadrenalectomy. Note the intense mainly cytoplasmic type Il-ir in the neurons (type B response). Immunoreactivity extended into dendrites. C, Four weeks postadrenalectomy. Two hours of corticosterone treatment abolished type Il-ir (compare to B). Scale bar = 50 ^m (A-C).
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FIG. 4. Type Il-ir in the locus coeruleus of intact, adrenalectomized, and corticosterone-treated rats. A, Intact rat. Note the high density of cells showing predominantly nuclear immunoreactivity and a little cytoplasmic immunoreactivity. B, One week of adrenalectomy reduced nuclear immunoreactivity. C, Four-week postadrenalectomized rat. There was a further diminution in the number of cells with nuclear immunoreactivity, but some were still present (arrow). D, Four-week postadrenalectomized rat. Two hours of corticosterone treatment increased nuclear immunoreactivity. Scale bar = 50 ^im (A-D).
Endo • 1991 Vol 129 • No 1
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FIG. 5. Type Il-ir in the vermal cerebellar cortex of intact, adrenalectomized, and corticosterone-treated rats. A, Intact rat. Lobule 2 shows type Il-ir. Note the presence of immunoreactivity in all layers. Purkinje cell immunoreactivity is nuclear (arrow). B, Four weeks postadrenalectomy. Lobule 2 shows the loss of type Il-ir in most cells. Note the persistence of diffuse immunoreactivity in some Purkinje cells. C, Four weeks postadrenalectomy plus 2 h of corticosterone treatment. Lobule 2 shows the restoration of immunoreactivity to all layers. Note two kinds of Purkinje cell immunoreactivity: nuclear (closed arrow) and intensely diffuse (open arrow). D, Four weeks postadrenalectomy plus 2 h of corticosterone treatment. Lobule 2 shows the absence of type Il-ir after incubation with nonimmune medium. Scale bar = 50 urn (AD).
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GLUCOCORTICOID RECEPTOR REGULATION
233
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FIG. 6. Bar charts showing mean numbers ± SEM of cells with distinct nuclear type Il-ir within an area of 104 /*m2 at X200 magnification in the pyramidal cell layer of Ammon's horn (CA1-CA3), the granule cell (DG), and polymorphic (CA4) layers of the dentate gyrus and caudate-putamen (CPu). n = 3 for each treatment group. ADX1, One week of adrenalectomy plus 2 h of vehicle treatment; ADX1C5, 1 week of adrenalectomy plus 5 min of corticosterone treatment; ADX1C2, 1 week of adrenalectomy plus 2 h of corticosterone treatment; ADX1AL2, 1 week of adrenalectomy plus 2 h of aldosterone treatment; ADX4, 4 weeks of adrenalectomy plus 2 h of vehicle treatment; ADX4C5, 4 weeks of adrenalectomy plus 5 min of corticosterone treatment; ADX4C2, 4 weeks of adrenalectomy plus 2 h of corticosterone treatment; ADX4AL2, 4 weeks of adrenalectomy plus 2 h of aldosterone treatment. By analysis of variance: CA1, Fg.,8 = 79.5, P < 0.001; CA2, FH.18 = 138.8, P < 0.001; CA3, F8,18 = 17.4, P < 0.001; CA4, Fg.,8 = 19.8, P < 0.001; DO, Fs.,8 = 76.8, P < 0.001; CPu, F8.,8 = 78.6, P < 0.001. By Student-NeumanKeuls test: *, P < 0.05 compared to intact; t, P < 0.05 compared to ADXl; §, /' < 0.05 compared to ADX4.
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of adrenalectomy. Even though Van Eekelen et al. (7) reported a complete disappearance of nuclear type Il-ir in all regions examined, including the locus coeruleus, by the end of 2 weeks of adrenalectomy, we observed nuclear type Il-ir in a few neurons of the locus coeruleus even after 4 weeks of adrenalectomy. We used a different monoclonal antibody, BUGR2, from that used by Fuxe et al. (4, 5, 8) and Van Eekelen (7). These antibodies have all been well characterized (10,16-18). We recently reported some significant differences between type Il-ir neurons recognized by IgG2a nr 7, the antibody used by Fuxe et al. (4, 5, 8), and BUGR2 in CA3 of the hippocampus, septum, hypothalamus, cerebellum, and spinal cord
in spite of a high degree of correspondence (6). The antibody used by Van Eekelen (7) reportedly showed only weak immunoreactivity in most neurons of the brainstem, apart from the locus coeruleus and nucleus solitarius, even though our previous BUGR2 map (6) and the IgG2a nr 7 map of Fuxe et al. (5, 8) found otherwise. In the present study there was a rostrocaudal gradient in the disappearance of nuclear and cytoplasmic type Ilir after adrenalectomy. Neurons in the telencephalon and diencephalon lost type Il-ir before those in the brainstem and cerebellum. Our discovery of atypically responding type B and C neurons, which developed an increased immunoreactivity
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after adrenalectomy, emphasizes the heterogeneity in neuronal responses to corticosteroids. Unlike type A neurons, which developed increased nuclear immunoreactivity in response to corticosterone treatment, type B neurons were not observed in the presence of corticosterone, while type C neurons of the vermal cerebellar cortex increased in number after corticosterone treatment. Type A neurons responded within a variable time scale to adrenalectomy, and usually within 5 min to corticosterone treatment. Type B neurons also responded to corticosterone treatment within 5 min. We observed an increase in nuclear immunoreactivity in type A neurons in response to 2 h of aldosterone treatment. Van Eekelen (7) reported no response to even higher doses of aldosterone. Aldosterone generally produced a significantly lower increase in nuclear type Il-ir compared to corticosterone or intact levels. However, in the hippocampus, a region with the highest densities of both type I and type II corticosteroid receptors, the pyramidal cells of CA3 and CA4 showed a greater response to aldosterone after long term (4 weeks) adrenalectomy. Functional significance The intracellular distribution of steroid receptor immunoreactivity has attracted a lot of interest. Both type I-ir and type Il-ir have been demonstrated in the cytoplasm and nuclei of cells in nonadrenalectomized rats (4-8, 19). Treatment with glucocorticoids has been shown to increase nuclear type Il-ir in the CNS (4, 7, 8, 11) and various cell lines (20), presumably indicating cytoplasmic to nuclear translocation of the activated receptor (4, 8). Adrenalectomy (4, 7, 8) or depletion of glucocorticoids in growth medium (20), on the other hand, results in a loss of nuclear and, later, cytoplasmic immunoreactivity. In the present study we demonstrated increased nuclear type Il-ir in the majority of neurons within 5 min of corticosterone treatment. This is too rapid for the transcription, translation, and transport of newly synthesized type II receptor into the nucleus. Moreover, glucocorticoids have been shown to downregulate type II receptor synthesis (21, 22). While the increase in nuclear type Il-ir may represent translocation of preexisting cytoplasmic type II receptor, the inability of BUGR2 antibody in the majority of neurons (type A) and other monoclonal antibodies (4, 7, 8) to detect cytoplasmic type Il-ir after prolonged adrenalectomy is suggestive of the masking of immunogenic sites of the receptor in the inactivated state due to a conformational change or interaction with some cytoplasmic factor(s). The presence of a subgroup of neurons (types B and C) that showed increased cytoplasmic immunoreactivity after adrenalectomy and varying responses to corticosteroid treatment further complicates the interpretation of the intracellular location of type Il-ir. Studies on type
Endo • 1991 Vol 129 • No 1
Il-ir involving the use of other antibodies (4, 7, 8,11) did not report similar findings. The heterogeneous response may, therefore, be perculiar to the BUGR epitope of the type II receptor. Perhaps in the type B neurons, the BUGR epitope was exposed by a conformational change in the unactivated receptor or interaction with some cytoplasmic factor(s) expressed selectively by these neurons in the absence of corticosteroids and, hence, the intense, predominantly cytoplasmic immunoreactivity observed after adrenalectomy. Type C Purkinje cells, in addition to showing cytoplasmic type Il-ir in the absence of corticosteroids, showed both nuclear and cytoplasmic type Il-ir in the presence of corticosterone, which means they have the ability to unmask BUGR epitopes in the presence or absence of ligand. Compared to the heterogeneous regulation of type IIir in the CNS, as revealed by BUGR2 antibody, estrogen and progesterone receptor immunoreactivity is nuclear in the presence or absence of ligand (23-29). Blaustein and Turcotte (30), however, reported the presence of cytoplasmic estrogen receptor-like immunoreactivity in the brain of nonovariectomized guinea pigs. Treatment with estradiol resulted in a loss of this immunoreactivity. Androgen receptor-like immunoreactivity is nuclear in intact male rats, but disappears within 3 days after castration (31). Treatment with androgens restores immunoreactivity within 15 min. While differences in the intracellular location of steroid receptor immunoreactivity may be regulated by ligand-dependent changes in immunogenicity, as in the case of type Il-ir (4, 7, 8, 20) and androgen receptor-like immunoreactivity (31), the mechanisms are far from clear. Immunogenicity may be regulated by ligand-dependent changes in the conformation of receptor protein, association with intracellular factors, or stabilization of receptor in particular compartments. Fixation and permeability of tissues to antibody have been shown to influence the location of immunoreactivity (8, 20) and have to be taken into consideration in the interpretation of data. To a large extent the methods of fixation and permeability in the above studies are comparable. The location of steroid receptor immunoreactivity may, therefore, reflect the site of function of these receptors in the cell. Cytoplasmic type Il-ir observed in type A neurons in intact rats, type B neurons in adrenalectomized rats, and type C Purkinje cells after adrenalectomy and corticosterone treatment may signify different roles for the receptor in the cytoplasm, e.g. the regulation of signal transduction pathways, translation, and posttranslational events. The extragenomic role of steroid receptor proteins is attracting considerable interest (32, 33). Nuclear type Il-ir, androgen receptor-like, estrogen receptor-like, and progesterone receptor-like immunoreactivity observed in the presence of ligand are consistent with the genomic role ascribed to ligand-
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GLUCOCORTICOID RECEPTOR REGULATION activated steroid receptors (34-36). The failure of CA3 pyramidal neurons to respond to either corticosterone or aldosterone 1 week after adrenalectomy and the greater response of CA3 and CA4 pyramidal neurons to aldosterone than to corticosterone 4 weeks after adrenalectomy raise some interesting possibilities. CA3 also showed the lowest percent decrease in cells with nuclear type Il-ir in response to 1 week of adrenalectomy. Neurons of CA3 are most vulnerable to noxious stimuli that are dependent on the presence of corticosterone and other glucocorticoids (37); moreover, evidence from steroid autoradiographic studies (1), in situ hybridization of type I mRNA (38), and immunocytochemistry for the type I receptor (Ahima, R. S., Z. Krozowski, and R. Harlan, unpublished) suggest high densities of the type I receptor in CA3. Even though aldosterone has very little binding to type II receptor in vivo compared to type I receptor (9), we have shown type Il-ir regulation by aldosterone in several other regions, including the hippocampus. Whether this has any physiological significance in CA3 and CA4 is unclear. Finally, the heterogeneity in the distribution and corticosteroid regulation of type Il-ir in the CNS may be related to the diverse functions of corticosteroids in the CNS (1, 2). How corticosteroids affect the functions of the few atypical type B and C neurons and how this relates to the function of the majority of neurons (type A) merit further study.
Acknowledgments Special thanks to R. Harrison for supplying BUGR2 antibody and P3 AgX-653 myeloma cell medium, and to Teresa Billiot and Debbie Lauff for secreterial assistance. We thank Louis Lucas for help in the statistical analysis.
References 1. McEwen BS, DeKloet ER, Rostene W 1986 Adrenal steroid receptors and actions in the nervous system. Physiol Rev 66:1121-1187 2. Funder JW, Sheppard K 1987 Adrenocortical steroids and the brain. Annu Rev Physiol 49:397-411 3. DeKloet ER, Reul JMHM 1987 Feedback action and tonic influence of corticosteroids on brain function: a concept arising from heterogeneity of brain receptor systems. Psychoneuroendocrinology 12:83-105 4. Fuxe K, Wikstrom AC, Okret S, Agnati LF, Harfstrand A, Yu ZY, Granholm L, Zoli M, Vale W, Gustafsson J-A 1985 Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor. Endocrinology 117:1803-1812 5. Fuxe K, Harfstrand AP, Agnati LF, Yu Z-Y, Cintra A, Wikstrom AC, Okret S, Cantoni K, Gustafsson J-A 1985 Immunocytochemical studies on the localization of glucocorticoid receptor immunoreactive nerve cells in the lower brainstem and spinal cord of the male rat using a monoclonal antibody against rat liver glucocorticoid receptor. Neurosci Lett 60:1-6 6. Ahima RS, Harlan RE 1990 Charting of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system. Neuroscience 39:579-604 7. Van Eekelen JAM, Kiss JZ, Westphal HM, DeKloet ER 1987
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Immunocytochemical study on the type 2 glucocorticoid receptor in the rat brain. Brain Res 436:120-128 8. Fuxe D, Cintra A, Harfstrand A, Agnati LF, Kalia M, Zoli M, Wikstrom AC, Okret S, Aronsson M, Gustafsson J-A 1987 Central glucocorticoid receptor immunoreactive neurons: new insights into the neuroendocrine regulation of the brain. Ann NY Acad Sci 512:362-393 9. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM 1987 Cloning of human mineralocorticoid receptor cDNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268-275 10. Eisen LP, Reichman ME, Thompson ER, Gametchu B, Harrison RW, Eisen HJ 1985 Monoclonal antibody to the rat glucocorticoid receptor. J Biol Chem 260:11805-11810 11. Uht FM, McKlerry JF, Harrison RW, Bohn MC 1988 Demonstration of glucocorticoid receptor-like immunoreactivity in glucocorticoid-sensitive vasopressin and corticotropin releasing factor neurons in the hypothalamic paraventricular neurons. J Neurosci Res 19:405-411 12. Hancock MB 1984 Visualization of peptide-immunoreactive processes on serotonin-immunoreactive cells using two-color immunoperoxidase staining. J Histochem Cytochem 32:311-316 13. Paxinos G, Watson C 1982 The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney 14. Abercrombie M 1946 Estimation of nuclear populations from microtome sections. Anat Rec 94:239-247 15. Hisano S, Kagotani Y, Tsuruo Y, Daikoku S, Chihara K, Whitnall MH 1988 Localization of glucocorticoid receptor in neuropeptide Y-containing neurons in the arcuate nucleus of the rat hypothalamus. Neurosci Lett 95:13-18 16. Rusconi S, Yamamoto KR 1987 Functional dissection of the hormone and DNA binding activities of the glucocorticoid receptor. EMBO J 6:1309-1315 17. Okret S, Wikstrom A-C, Wrange O, Andersson B, Gustafsson J-A 1984 Monoclonal antibodies against the rat liver glucocorticoid receptor. Proc Natl Acad Sci USA 81:1609-1613 18. Westphal HM, Moldenhauer G, Beato M 1982 Monoclonal antibodies to the rat liver glucocorticoid receptor. EMBO J 1:14671471 19. Krozowski ZS, Rundle SE, Wallace C, Castell MJ, Shen JH, Dowling J, Funder JW, Smith AI1989 Immunolocalization of renal mineralocorticoid receptors with an antiserum against a peptide deduced from the complimentary deoxyribonucleic acid sequence. Endocrinology 125:192-198 20. Wikstrom A-C, Bakke O, Okret S, Bronnegard M, Gustafsson J-A 1987 Intracellular localization of the glucocorticoid receptor: evidence for cytoplasmic and nuclear localization. Endocrinology 120:1232-1242 21. Reul JMHM, Pearce PT, Funder JW, Krozowski ZS 1989 Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration. Mol Endocrinol 3:1674-1680 22. Sheppard KE, Roberts JL, Blum M 1990 Differential regulation of type II corticosteroid receptor messenger ribonucleic acid expression in the rat anterior pituitary and hippocampus. Endocrinology 127:431-439 23. Gorski J, Welshons W, Sakai D 1984 Remodeling the estrogen receptor model. Mol Cell Endocrinol 36:11-15 24. King WJ, Greene GL 1984 Monoclonal antibodies localize estrogen receptor in the nuclei of target cells. Nature 307:745-747 25. Picard D, Kumar V, Chambon P, Yamamoto KR 1990 Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell Regul 1:291-299 26. Blaustein JD, King JC, Toft DO, Turcotte J 1988 Immunocytochemical localization of estrogen-induced progestin receptors in guinea pig brain. Brain Res. 474:1-15 27. Perrot-Applanat M, Logeat F, Groyer-Picard M-T, Milgrom E 1985 Immunocytochemical study of mammalian progesterone receptor using monoclonal antibodies. Endocrinology 116:1473-1484 28. Welshons WV, Lieberman ME, Gorski J 1984 Nuclear localization of unoccupied receptors for glucocorticoids, estrogens, and proges-
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terone in GH3 cells. Endocrinology 117:2140-2147 29. Warembourg M, Logeat F, Milgrom E 1986 Immunocytochemical localization of progesterone receptor in guinea pig central nervous system. Brain Res 384:121-131 30. Blaustein JD, Turcotte JC 1989 Estrogen receptor-immunostaining of neuronal cytoplasmic processes as well as cell nuclei in guinea pig brain. Brain Res 495:75-82 31. Sar M, Lubahn DB, French FS, Wilson EM 1990 Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 127:3180-3186 32. Schumacher M 1990 Rapid membrane effects of steroid hormones: an emerging concept in neuroendocrinology. Trends Neurosci 13:359-361 33. Simons SS, Mercier L, Miller PA, Oshima H, Sistare FD, Thomp-
34. 35. 36. 37. 38.
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son EB, Wasner G, Yen PM 1989 Differential modulation of gene induction by glucocorticoids and antiglucocorticoids in rat hepatoma tissue cells. Cancer Res 49:2244S-2252S Yamamoto KR 1985 Steroid receptor regulated transcription of specific gene networks. Annu Rev Genet 19:209-252 Rousseau GG 1984 Control of gene expression by glucocorticoid hormones. Biochem J 224:1-12 Ringold GM 1985 Steroid hormone regulation of gene expression. Annu Rev Pharmacol Toxicol 25:529-566 Sapolsky RM 1985 A mechanism for glucocorticoid toxicity in the hippocampus: increased neuronal vulnerability to metabolic insults. J Neurosci 5:1228-1232 Arriza J, Simerly R, Swanson L, Evans R 1988 The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response. Neuron 1:887-900
American Thyroid Association 65th Annual Meeting The 65th Annual Meeting of the American Thyroid Association will be held in Boston, Massachusetts, from September 12-15, 1991, at the Hyatt Regency, Cambridge. An International Symposium on Thyroid Autoimmunity will be held September 16 and 17 in Montreal, Quebec, Canada. For meeting information please contact: Leonard Wartofsky, M.D. Secretary, American Thyroid Association Endocrine-Metabolic Service Walter Reed Army Medical Center Washington, DC 20307-5001 Telephone: 301-588-5432 FAX: 301-558-4728.
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Vol. 129, No. 1 Printed in U.S.A.
Differential Corticosteroid Regulation of Type II Glucocorticoid Receptor-Like Immunoreactivity in the Rat Central Nervous System: Topography and Implications* REXFORD S. AHIMA AND RICHARD E. HARLAN Department of Anatomy and Neuroscience Training Program (R.E.H.), Tulane University School of Medicine, New Orleans, Louisiana 70112
ABSTRACT. Neuronal type II glucocorticoid receptor-like immunoreactivity in the central nervous system shows heterogeneity in intensities and relative densities. This might predict variations in the regional responses of neuronal immunoreactivity to corticosteroids. We investigated changes in the intracellular location of immunoreactivity in the rat central nervous system after adrenalectomy and corticosteroid treatment, and carried out detailed statistical analysis of changes in neuronal nuclear immunoreactivity in the hippocampus and caudateputamen. Three types of responses were observed. The majority of neurons, classified type A, showed a predominant nuclear immunoreactivity in intact rats, lost nuclear and eventually
cytoplasmic immunoreactivity after adrenalectomy, and regained nuclear immunoreactivity within 5 min of corticosterone and 2 h of aldosterone treatment, respectively. A subgroup of neurons in the hippocampus, striatum, septum, and habenula, classified type B, were not immunoreactive in intact rats, showed intense cytoplasmic immunoreactivity after adrenalectomy, and disappeared rapidly after corticosterone treatment and later in response to aldosterone. A subgroup of vermal cerebellar Purkinje neurons, classified type C, developed an intense cytoplasmic immunoreactivity after adrenalectomy, increased in number in response to corticosterone, and did not respond to aldosterone. (Endocrinology 129: 226-236,1991)
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densities and intensities of immunoreactive neurons in limbic, stress-related, motor, and sensory systems. Neuronal type Il-ir is typically nuclear, with a weak cytoplasmic component. Four days to 2 weeks after adrenalectomy, there is reportedly a uniform loss of nuclear immunoreactivity in many regions (4, 7, 8). Treatment with glucocorticoids restores nuclear immunoreactivity (4, 7, 8), while aldosterone treatment reportedly has no effect (7). Considering the marked heterogeneity in the regional distribution of type Il-ir, one would have expected a heterogeneous regional response to glucocorticoids. Moreover, aldosterone has been shown in in vitro experiments to bind to the type II receptor protein (9) and, presumably, could affect type Il-ir in some neurons. In the present study we followed up our previous map of type Il-ir in the rat CNS using BUGR2 monoclonal antibody (6), with a study of the regulation of type Il-ir by corticosteroids. We were interested in regional differences in neuronal response as well as the time courses of such responses to corticosterone and aldosterone, the principal glucocorticoid and mineralocorticoid, respectively, in rats.
LUCOCORTICOIDS exert diverse regulatory effects on neuronal and glial activity in the central nervous system (CNS) (1, 2). Two classes of corticosteroid receptors, types I and II, have been described in the CNS (2). Type I receptor binds preferentially to the predominant glucocorticoid in the species and has an equivalent high affinity for mineralocorticoids. Type II receptor has a high affinity for dexamethasone and other specific glucocorticoids, and a lower affinity for both mineralocorticoids and the species-specific glucocorticoid. Type I receptors presumably mediate tonic and behavioral responses within the diurnal levels of corticosteroids, while type II receptors mediate the stress effects and central negative feedback regulation of glucocorticoids (2, 3). Type II receptor-like immunoreactivity (type Il-ir) has been mapped in the rat CNS using monoclonal antibodies against different epitopes of the variable domain of the rat liver glucocorticoid receptor (4-8). Immunoreactivity is widespread, with a marked heterogeneity in the relative Received January 28,1991. Address requests for reprints to: Richard E. Harlan, Ph.D., Department of Anatomy, Tulane Medical Center School, 1430 Tulane Avenue, New Orleans, Louisiana 70112. * This work was supported by Grant NS-24148 (to R.E.H).
Materials and Methods Adrenalectomized (1, 2, and 4 weeks) and intact SpragueDawley-derived male rats (Charles River, Wilmington, MA), 226
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GLUCOCORTICOID RECEPTOR REGULATION weighing 200-250 g, were used. They were fed standard rat chow. Intact rats were maintained on tap water ad libitum, and adrenalectomized rats on normal saline. All rats were kept under a 12-h light, 12-h dark cycle. BUGR2 mouse antirat liver type II receptor monoclonal antibody and nonimmune P3 AgX-653 myeloma cell medium supernatant were kindly supplied by R. W. Harrison (10, 11). The Vectastain ABC horse antimouse kit was purchased from Vector Laboratories (Burlingame, CA), and corticosterone and d-aldosterone were obtained from Sigma (St. Louis, MO). The adrenalectomized rats were divided into three groups. One group was treated with d-aldosterone (10 Mg/100 g BW) in 200 nl 0.005% (vol/vol) ethanol-PBS, ip. Another group received corticosterone (1 mg/100 g BW in the same vehicle), while a third group received only vehicle. Within each treatment group, four rats were treated for 5 min, 1 h, or 2 h respectively, before death. The adrenalectomized and intact rats were killed with an overdose of sodium pentobarbital ip, perfused transcardially with 0.025 M PBS, pH 7.2, for 5 min, followed by 3% phosphatebuffered paraformaldehyde for 8 min. The abdominal cavities of the adrenalectomized rats were explored to confirm adrenalectomy. Brains and spinal cords were dissected out, blocked, postfixed in the same fixative for 2 h at room temperature, and cryoprotected in 30% sucrose (wt/vol) at 4 C until sinking. They were then rapidly frozen on dry ice and stored wrapped in aluminium foil at —70 C until use. Immunocytochemistry Coronal sections (50 ^m thick) of the brain and spinal cord were cut on a freezing microtome (Reichert, Buffalo, NY), rinsed in PBS for 1 h, blocked with normal horse serum in 0.1% BSA in PBS according to specifications of the Vectastain kit, permeabilized in 0.2% Triton X-100 in PBS for 20 min, incubated in a 1:500 dilution of BUGR2, and processed as described previously (6) with either 3,3'-diaminobenzidine tetrahydrochloride (DAB) or nickel-DAB as chromogen (12). Adjacent control sections were incubated with P3 AgX-653 myeloma cell supernatant or normal mouse immunoglobulin G (IgG). The reaction was stopped in PBS for 5 min, and sections were transferred to 10 mM sodium acetate (Sigma), mounted on chromalum-subbed slides, and dehydrated in increasing strengths of ethanol (70%, 95%, and 100%). Sections were then defatted in Histoclear (National Diagnostics, Somerville, NJ) and coverslipped with Permount (Fisher Scientific, Fairlawn, NJ). Some sections were counterstained with cresyl violet to facilitate delineation of neuronal groups. Sections were examined with a Nikon Optiphot microscope, equipped with a camera lucida drawing tube and a Nikon FX35A camera. Photographs were taken with Technical Pan (Eastman Kodak, Rochester, NY) film. The following parameters were examined: intracellular location of type Il-ir, relative intensities, and relative densities. Relative intensities were determined qualitatively. Three standards were chosen: strong immunoreactivity, as in the pyramidal cell layer of CA1 of intact rats (Fig. 1A); weak immunoreactivity, as in the globus pallidus (Fig. 3A); and moderate immunoreactivity, for immunostaining between these extremes. At least three sections were selected for each region per treatment group per animal.
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Sections from three animals per treatment group, processed under the same conditions, were selected and matched with the help of the atlas of Paxinos and Watson (13) by one experimenter and coded. Relative intensities were then scored blindly by two investigators using the above standards. The effects of adrenalectomy and corticosteroid treatment on the density of cells with nuclear type Il-ir in Ammon's horn and dentate gyrus of the hippocampus and in the caudateputamen were determined by counting neurons with distinct nuclear type Il-ir in grids 100 X 100 /urn at a magnification of X200 with the help of the camera lucida tube. Three nickelDAB-stained sections of the hippocampus, corresponding to Figs. 28, 32, and 35 of the atlas of Paxinos and Watson (13), and three of the caudate-putamen, corresponding to Figs. 17, 21, and 25 of the atlas of Paxinos and Watson (13), were selected, matched, and coded by one investigator from three animals in each treatment group. These were then analyzed blindly by two investigators. Three grids were analyzed for the pyramidal cell layer of CAl, one for CA2, two for CA3, two for CA4, and two for the ventral and dorsal blades, respectively, of the granular layer in each hemisphere. The caudate-putamen in each hemisphere was divided into four quadrants, using selected landmarks. Two grids were analyzed in each quadrant per hemisphere. Counts were corrected for errors using Abercrombie's method (14). The mean counts ± SE per region per treatment were determined from pooled counts. The data were analyzed using one-way analysis of variance, and post-hoc comparisons were carried out using the Student-Neuman-Keuls test.
Results Intact rats Type Il-ir was demonstrated in several neuronal groups. Details of the patterns of distribution have been described previously (6). Immunoreactivity was predominantly nuclear, with a weak cytoplasmic component. Strong immunoreactivity was observed in the pyramidal cell layer of fields CAl and CA2 of Ammon's horn, granule cells of the dentate gyrus, medial and lateral divisions of the bed nucleus of the stria terminalis, central amygdaloid nucleus, layer II of the piriform cortex, locus coeruleus, nucleus ambiguus, substantia gelatinosa, and motor neurons of laminae 8 and 9 of the spinal cord. Sections incubated with normal mouse IgG or nonimmune P3 AgX-653 myeloma cell medium produced only nonspecific signal (e.g. Figs. ID and 5D). The specificity of BUGR2 antibody for the type II receptor in the rat CNS has also been demonstrated by others (11, 15). Type Il-ir and corticosteroids Three types of responses in the intracellular location of type Il-ir and relative densities of type Il-ir neurons were observed after adrenalectomy, adrenalectomy plus corticosterone treatment, and adrenalectomy plus aldosterone treatment.
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GLUCOCORTICOID RECEPTOR REGULATION
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FIG. 1. Light micrographs comparing type Il-ir in CAl of the hippocampus. A, Intact (nonadrenalectomized) rat. Note the intense, predominantly nuclear, immunoreactivity in the pyramidal cell layer. B, One week of adrenalectomy. Note the reduction in the number of cells with nuclear type Il-ir and the appearance of mainly cytoplasmic type H-ir in some cells (arrow). C, Four-week postadrenalectomized rat. Note the virtual absence of immunoreactivity in most pyramidal cells (type A response). Arrows mark the limits of the pyramidal cell layer. Note the persistence of cells with intense cytoplasmic immunoreactivity, i.e. type B cells. D, Four-week postadrenalectomized rat. Section incubated with nonimmune P3 AgX-653 myeloma cell supernatant. Note the absence of type Il-ir. Arrows mark the limits of the pyramidal cell layer. E, Four-week postadrenalectomized rat. Five minutes of corticosterone treatment restored some immunoreactivity to type A response neurons, but it was below intact levels. Atypical (type B) neurons have disappeared. F, Five minutes of aldosterone treatment of 4-week postadrenalectomized rats did not completely abolish immunoreactivity in type B neurons. Arrow points to type B neuron in the pyramidal cell layer. G, Two hours of corticosterone treatment of 4-week adrenalectomized rats restored nuclear immunoreactivity (compare to A). H, Two hours of aldosterone treatment restored nuclear immunoreactivity to some type A neurons, but densities were below intact levels (compare to A). No atypical (type B) neurons were seen. Scale bar = 50 nm for A-H.
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Endo • 1991 Voll29«Nol
GLUCOCORTICOID RECEPTOR REGULATION Telencephalon and diencephalon One week after adrenalectomy, type Il-ir neurons in the islands of Calleja; amygdala (except the central nucleus); intermediate and medial divisions of the bed nucleus of the stria terminalis; ventral pallidum; substantia innominata; fundus striati; substriatal area; basal nucleus of Meynert; claustrum; medial, ventral, septofimbrial and triangular septal nuclei; vertical division of the nucleus of the diagonal band; zona incerta; and hypothalamus (except the parvocellular paraventricular nucleus) lost nuclear and cytoplasmic immunoreactivity. Cytoplasmic type Il-ir and some nuclear type Il-ir were still present in the isocortex and all thalamic nuclei by the end of the first week after adrenalectomy. Immunoreactivity was abolished in these regions by the end of the second week after adrenalectomy. Treatment with corticosterone produced weak nuclear immunoreactivity as early as 5 min in most neurons. Two hours of corticosterone treatment increased the intensities of nuclear immunoreactivity and the densities of type Il-ir neurons above intact levels in most regions. Two hours of treatment with aldosterone restored some nuclear immunoreactivity, but below levels seen in intact animals. Because type Il-ir in the vast majority of telencephalic and diencephalic neurons responded to corticosterone treatment in a fashion similar to that described by others (4, 7, 8), these neurons were classified as typical response or type A neurons. Hippocampus Most immunoreactive neurons in Ammon's horn and the dentate gyrus showed a type A response. Nuclear immunoreactivity was abolished in most pyramidal cells of Ammon's horn (compare Fig. 1, A and B) and the granule cells of the dentate gyrus 1 week after adrenalectomy. Some neurons in the pyramidal cell layer of fields CAl and CA2 and the stratum oriens developed predominantly cytoplasmic immunoreactivity 1 week after adrenalectomy (Fig. IB). By 4 weeks postadrenalectomy, similar neurons were observed in CA3. Cytoplasmic immunoreactivity was retained in this subgroup of hippocampal neurons with increasing duration of adrenalectomy, while most hippocampal neurons lost immunoreactivity (Fig. 1C). Adjacent sections of the hippocampus from 1- and 4-week postadrenalectomized rats incubated with either normal mouse IgG or P3 AgX-653 myeloma cell medium did not show type Il-ir (Fig. ID). Nuclear type Il-ir was observed in most hippocampal neurons within 5 min of corticosterone treatment (Fig. IE). The intensity of immunoreactivity was, however, much weaker than that in intact animals (compare Fig. 1, A and E). Two hours of corticosterone treatment increased the intensity of nuclear type Il-ir to intact
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levels (compare Fig. 1, G and A), akin to the type A response observed in the rest of the telencephalon. Two hours of aldosterone treatment restored nuclear type Ilir in most neurons, but the intensities were often weaker compared to those in intact animals (compare Fig. 1, H and A). In the subgroup of hippocampal neurons in which increased cytoplasmic type Il-ir was observed after adrenalectomy, immunoreactivity was abolished after 5 min of corticosterone treatment (compare Fig. 1, B, C, and E) or 2 h of aldosterone treatment (compare Fig. 1, B, C, and H). Five minutes of aldosterone treatment reduced, but did not eliminate, type Il-ir in this subgroup of hippocampal neurons (Fig. IF). This atypical response of type Il-ir to corticosteroids was classified as a type B response. Striatum, septum, and habenula Most immunoreactive neurons in these regions showed a type A response. However, as in the hippocampus, a subgroup of neurons showed an atypical type B response. In the caudate-putamen (Fig. 2, A-F), globus pallidus (Fig. 3, A-C), entopeduncular nucleus, lateral septum, and habenula, a subgroup of neurons showed increased, mainly cytoplasmic, type Il-ir after adrenalectomy (Figs. 2, A-C, and 3, A and B). Immunoreactivity in these neurons was abolished by 5 min of corticosterone treatment (Fig. 2D) or 2 h of aldosterone treatment (Fig. 2F). Adjacent control sections from intact or adrenalectomized rats did not show type Il-ir (data not illustrated). Brainstem, cerebellum, and spinal cord Unlike the telencephalon and diencephalon, nuclear type Il-ir was still present in most parts of the brainstem, cerebellum, and spinal cord 2 weeks after adrenalectomy. By 4 weeks postadrenalectomy, most neurons showed weak cytoplasmic immunoreactivity, even though a few neurons in the locus coeruleus (Fig. 4), inferior olivary nuclei, and spinal motor neurons retained some nuclear type Il-ir. Two hours of corticosterone treatment restored nuclear type Il-ir (compare Fig. 4, A and D), while aldosterone restored some nuclear type Il-ir, but below intact levels. The response of type Il-ir in most neurons in the hindbrain was, thus, a type A response. The gigantocellular reticular nucleus showed a type B response. In the cerebellum a subgroup of Purkinje cells in the vermis lobules 1-3, 9, and 10 also showed increased, predominantly cytoplasmic, type Il-ir after adrenalectomy (compare Fig. 5, A and B). However, unlike type B neurons, immunoreactivity was retained in the cytoplasm of these neurons after corticosterone treatment (Fig. 5, B and C). In addition, nuclear type Il-ir was often resolvable in these Purkinje cells. These neurons, classified type C, did not respond to aldosterone treat-
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Endo«1991 Vol 129 • No 1
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» FIG. 2. Type Il-ir in the caudate-putamen of intact, adrenalectomized, and corticosteroid-treated rats. A, Intact rat. Note the high density of immunoreactive (type A) neurons. Immunoreactivity was primarily nuclear. B, One week of adrenalectomy reduced immunoreactivity in most (type A) neurons; however, note the appearance of atypical (type B) neurons (arrow). C, Four-week postadrenalectomized rat. Note the further decrease in the number of type A neurons and the increase in type B neurons. D, Five minutes of corticosterone treatment of 4week adrenalectomized rats abolished immunoreactivity in type B neurons and restored some nuclear immunoreactivity in type A neurons. E, Four-week postadrenalectomized rat. Two hours of corticosterone treatment increased nuclear immunoreactivity and densities of immunoreactive neurons to or above intact levels (compare with A). F, Four-week postadrenalectomized rat. Two hours of aldosterone treatment restored immunoreactivity in most type A neurons, but the density of labeled neurons was less than that in intact rats (compare with A). Note the absence of type B neurons. Scale bar = 100 ^m (A-F).
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ment. Corticosterone treatment also increased the number of type C Purkinje cells. Incubation of adjacent sections with control myeloma supernatant demonstrated the specificity of immunostaining of type C neurons (Fig. 5D). Changes in the density of neurons with nuclear type IIir in the hippocampus and caudate-putamen in response to adrenalectomy and corticosteroid treatment Figure 6, A-F, illustrates the response of neurons with nuclear type Il-ir in the hippocampus and caudate-putamen to corticosteroids. Hippocampus. One week of adrenalectomy resulted in a
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significant reduction in the numbers of cells with distinct type Il-ir in the pyramidal and granule cell layers (Fig. 6, A-E). Four weeks of adrenalectomy resulted in a further reduction in all of these regions. In 1 week postadrenalectomized rats, 5 min of corticosterone treatment significantly increased counts above adrenalectomy levels in CAl and CA2 (Fig. 6, A and B), but not in CA3 and CA4 or dentate gyrus (Fig. 6, C-E), while 2 h of corticosterone treatment increased counts in all subregions except CA3 (Fig. 6C). Two hours of aldosterone treatment significantly increased densities of type Il-ir cells in CAl (Fig. 6A) and CA2 (Fig. 6B) above adrenalectomy levels. Thus, after 1 week of adrenalectomy, CAl and CA2 were sensitive to the effect of
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GLUCOCORTICOID RECEPTOR REGULATION
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corticosterone, while CA3 and CA4 were more resistant (Fig. 6, C and D). Four weeks after adrenalectomy, 5 min of corticosterone treatment increased the densities of immunoreactive cells in CA1, CA2, and dentate gyrus (Fig. 6, A, B, and E) and resulted in a nonsignificant increase in CA3 and CA4 (Fig. 6, C and D). Two hours of corticosterone treatment significantly increased densities in all regions (Fig. 6, A-E). Two hours of aldosterone treatment significantly increased densities in all regions, except the granule cell layer of the dentate gyrus (Fig. 6, A-E). There was an interesting effect of aldosterone on the pyramidal cells of CA3 and CA4 after 4 weeks of adrenalectomy. While in most subregions, 2 h of corticosterone treatment produced a greater increase in the densities of immunoreactive neurons than did aldosterone (Fig. 6, A, B, and E), the converse was true in CA3 and CA4 (Fig. 6, C and D). In summary, nuclear type Il-ir in the pyramidal cells of CA3 was most resistant to 1 week of adrenalectomy, showed less response to corticosteroid treatment (together with CA4) during this period, and showed a paradoxically increased response to aldosterone (with CA4) 4 weeks after adrenalectomy. Caudate-putamen. One week of adrenalectomy reduced significantly the density of cells with nuclear type Il-ir (Fig. 6F). There was a further reduction 4 weeks after adrenalectomy. Corticosterone treatment produced an increase in density above adrenalectomy levels. The response to corticosterone occurred as early as 5 min and was significantly greater after 2 h. Two hours of aldosterone treatment significantly increased the density above adrenalectomy levels, but significantly less than levels in both intact and corticosterone-treated (2 h) animals. Four weeks after adrenalectomy, corticosterone or aldosterone treatment increased the density of type Il-ir cells. This response was, however, significantly smaller than that to treatment 1 week after adrenalectomy.
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Contrary to previous reports indicating a uniform loss of nuclear and then cytoplasmic immunoreactivity after adrenalectomy (4, 7, 8), we observed three types of responses. In the majority of neurons (type A), there was a progressive loss of type Il-ir with increasing duration FIG. 3. Type Il-ir in the globus pallidus of intact, adrenalectomized, and corticosterone-treated rats. A, Intact rat. Note the few immunoreactive neurons. Immunoreactivity is mainly nuclear and weak. B, Four-week postadrenalectomy. Note the intense mainly cytoplasmic type Il-ir in the neurons (type B response). Immunoreactivity extended into dendrites. C, Four weeks postadrenalectomy. Two hours of corticosterone treatment abolished type Il-ir (compare to B). Scale bar = 50 ^m (A-C).
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FIG. 4. Type Il-ir in the locus coeruleus of intact, adrenalectomized, and corticosterone-treated rats. A, Intact rat. Note the high density of cells showing predominantly nuclear immunoreactivity and a little cytoplasmic immunoreactivity. B, One week of adrenalectomy reduced nuclear immunoreactivity. C, Four-week postadrenalectomized rat. There was a further diminution in the number of cells with nuclear immunoreactivity, but some were still present (arrow). D, Four-week postadrenalectomized rat. Two hours of corticosterone treatment increased nuclear immunoreactivity. Scale bar = 50 ^im (A-D).
Endo • 1991 Vol 129 • No 1
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FIG. 5. Type Il-ir in the vermal cerebellar cortex of intact, adrenalectomized, and corticosterone-treated rats. A, Intact rat. Lobule 2 shows type Il-ir. Note the presence of immunoreactivity in all layers. Purkinje cell immunoreactivity is nuclear (arrow). B, Four weeks postadrenalectomy. Lobule 2 shows the loss of type Il-ir in most cells. Note the persistence of diffuse immunoreactivity in some Purkinje cells. C, Four weeks postadrenalectomy plus 2 h of corticosterone treatment. Lobule 2 shows the restoration of immunoreactivity to all layers. Note two kinds of Purkinje cell immunoreactivity: nuclear (closed arrow) and intensely diffuse (open arrow). D, Four weeks postadrenalectomy plus 2 h of corticosterone treatment. Lobule 2 shows the absence of type Il-ir after incubation with nonimmune medium. Scale bar = 50 urn (AD).
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GLUCOCORTICOID RECEPTOR REGULATION
233
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FIG. 6. Bar charts showing mean numbers ± SEM of cells with distinct nuclear type Il-ir within an area of 104 /*m2 at X200 magnification in the pyramidal cell layer of Ammon's horn (CA1-CA3), the granule cell (DG), and polymorphic (CA4) layers of the dentate gyrus and caudate-putamen (CPu). n = 3 for each treatment group. ADX1, One week of adrenalectomy plus 2 h of vehicle treatment; ADX1C5, 1 week of adrenalectomy plus 5 min of corticosterone treatment; ADX1C2, 1 week of adrenalectomy plus 2 h of corticosterone treatment; ADX1AL2, 1 week of adrenalectomy plus 2 h of aldosterone treatment; ADX4, 4 weeks of adrenalectomy plus 2 h of vehicle treatment; ADX4C5, 4 weeks of adrenalectomy plus 5 min of corticosterone treatment; ADX4C2, 4 weeks of adrenalectomy plus 2 h of corticosterone treatment; ADX4AL2, 4 weeks of adrenalectomy plus 2 h of aldosterone treatment. By analysis of variance: CA1, Fg.,8 = 79.5, P < 0.001; CA2, FH.18 = 138.8, P < 0.001; CA3, F8,18 = 17.4, P < 0.001; CA4, Fg.,8 = 19.8, P < 0.001; DO, Fs.,8 = 76.8, P < 0.001; CPu, F8.,8 = 78.6, P < 0.001. By Student-NeumanKeuls test: *, P < 0.05 compared to intact; t, P < 0.05 compared to ADXl; §, /' < 0.05 compared to ADX4.
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of adrenalectomy. Even though Van Eekelen et al. (7) reported a complete disappearance of nuclear type Il-ir in all regions examined, including the locus coeruleus, by the end of 2 weeks of adrenalectomy, we observed nuclear type Il-ir in a few neurons of the locus coeruleus even after 4 weeks of adrenalectomy. We used a different monoclonal antibody, BUGR2, from that used by Fuxe et al. (4, 5, 8) and Van Eekelen (7). These antibodies have all been well characterized (10,16-18). We recently reported some significant differences between type Il-ir neurons recognized by IgG2a nr 7, the antibody used by Fuxe et al. (4, 5, 8), and BUGR2 in CA3 of the hippocampus, septum, hypothalamus, cerebellum, and spinal cord
in spite of a high degree of correspondence (6). The antibody used by Van Eekelen (7) reportedly showed only weak immunoreactivity in most neurons of the brainstem, apart from the locus coeruleus and nucleus solitarius, even though our previous BUGR2 map (6) and the IgG2a nr 7 map of Fuxe et al. (5, 8) found otherwise. In the present study there was a rostrocaudal gradient in the disappearance of nuclear and cytoplasmic type Ilir after adrenalectomy. Neurons in the telencephalon and diencephalon lost type Il-ir before those in the brainstem and cerebellum. Our discovery of atypically responding type B and C neurons, which developed an increased immunoreactivity
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after adrenalectomy, emphasizes the heterogeneity in neuronal responses to corticosteroids. Unlike type A neurons, which developed increased nuclear immunoreactivity in response to corticosterone treatment, type B neurons were not observed in the presence of corticosterone, while type C neurons of the vermal cerebellar cortex increased in number after corticosterone treatment. Type A neurons responded within a variable time scale to adrenalectomy, and usually within 5 min to corticosterone treatment. Type B neurons also responded to corticosterone treatment within 5 min. We observed an increase in nuclear immunoreactivity in type A neurons in response to 2 h of aldosterone treatment. Van Eekelen (7) reported no response to even higher doses of aldosterone. Aldosterone generally produced a significantly lower increase in nuclear type Il-ir compared to corticosterone or intact levels. However, in the hippocampus, a region with the highest densities of both type I and type II corticosteroid receptors, the pyramidal cells of CA3 and CA4 showed a greater response to aldosterone after long term (4 weeks) adrenalectomy. Functional significance The intracellular distribution of steroid receptor immunoreactivity has attracted a lot of interest. Both type I-ir and type Il-ir have been demonstrated in the cytoplasm and nuclei of cells in nonadrenalectomized rats (4-8, 19). Treatment with glucocorticoids has been shown to increase nuclear type Il-ir in the CNS (4, 7, 8, 11) and various cell lines (20), presumably indicating cytoplasmic to nuclear translocation of the activated receptor (4, 8). Adrenalectomy (4, 7, 8) or depletion of glucocorticoids in growth medium (20), on the other hand, results in a loss of nuclear and, later, cytoplasmic immunoreactivity. In the present study we demonstrated increased nuclear type Il-ir in the majority of neurons within 5 min of corticosterone treatment. This is too rapid for the transcription, translation, and transport of newly synthesized type II receptor into the nucleus. Moreover, glucocorticoids have been shown to downregulate type II receptor synthesis (21, 22). While the increase in nuclear type Il-ir may represent translocation of preexisting cytoplasmic type II receptor, the inability of BUGR2 antibody in the majority of neurons (type A) and other monoclonal antibodies (4, 7, 8) to detect cytoplasmic type Il-ir after prolonged adrenalectomy is suggestive of the masking of immunogenic sites of the receptor in the inactivated state due to a conformational change or interaction with some cytoplasmic factor(s). The presence of a subgroup of neurons (types B and C) that showed increased cytoplasmic immunoreactivity after adrenalectomy and varying responses to corticosteroid treatment further complicates the interpretation of the intracellular location of type Il-ir. Studies on type
Endo • 1991 Vol 129 • No 1
Il-ir involving the use of other antibodies (4, 7, 8,11) did not report similar findings. The heterogeneous response may, therefore, be perculiar to the BUGR epitope of the type II receptor. Perhaps in the type B neurons, the BUGR epitope was exposed by a conformational change in the unactivated receptor or interaction with some cytoplasmic factor(s) expressed selectively by these neurons in the absence of corticosteroids and, hence, the intense, predominantly cytoplasmic immunoreactivity observed after adrenalectomy. Type C Purkinje cells, in addition to showing cytoplasmic type Il-ir in the absence of corticosteroids, showed both nuclear and cytoplasmic type Il-ir in the presence of corticosterone, which means they have the ability to unmask BUGR epitopes in the presence or absence of ligand. Compared to the heterogeneous regulation of type IIir in the CNS, as revealed by BUGR2 antibody, estrogen and progesterone receptor immunoreactivity is nuclear in the presence or absence of ligand (23-29). Blaustein and Turcotte (30), however, reported the presence of cytoplasmic estrogen receptor-like immunoreactivity in the brain of nonovariectomized guinea pigs. Treatment with estradiol resulted in a loss of this immunoreactivity. Androgen receptor-like immunoreactivity is nuclear in intact male rats, but disappears within 3 days after castration (31). Treatment with androgens restores immunoreactivity within 15 min. While differences in the intracellular location of steroid receptor immunoreactivity may be regulated by ligand-dependent changes in immunogenicity, as in the case of type Il-ir (4, 7, 8, 20) and androgen receptor-like immunoreactivity (31), the mechanisms are far from clear. Immunogenicity may be regulated by ligand-dependent changes in the conformation of receptor protein, association with intracellular factors, or stabilization of receptor in particular compartments. Fixation and permeability of tissues to antibody have been shown to influence the location of immunoreactivity (8, 20) and have to be taken into consideration in the interpretation of data. To a large extent the methods of fixation and permeability in the above studies are comparable. The location of steroid receptor immunoreactivity may, therefore, reflect the site of function of these receptors in the cell. Cytoplasmic type Il-ir observed in type A neurons in intact rats, type B neurons in adrenalectomized rats, and type C Purkinje cells after adrenalectomy and corticosterone treatment may signify different roles for the receptor in the cytoplasm, e.g. the regulation of signal transduction pathways, translation, and posttranslational events. The extragenomic role of steroid receptor proteins is attracting considerable interest (32, 33). Nuclear type Il-ir, androgen receptor-like, estrogen receptor-like, and progesterone receptor-like immunoreactivity observed in the presence of ligand are consistent with the genomic role ascribed to ligand-
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GLUCOCORTICOID RECEPTOR REGULATION activated steroid receptors (34-36). The failure of CA3 pyramidal neurons to respond to either corticosterone or aldosterone 1 week after adrenalectomy and the greater response of CA3 and CA4 pyramidal neurons to aldosterone than to corticosterone 4 weeks after adrenalectomy raise some interesting possibilities. CA3 also showed the lowest percent decrease in cells with nuclear type Il-ir in response to 1 week of adrenalectomy. Neurons of CA3 are most vulnerable to noxious stimuli that are dependent on the presence of corticosterone and other glucocorticoids (37); moreover, evidence from steroid autoradiographic studies (1), in situ hybridization of type I mRNA (38), and immunocytochemistry for the type I receptor (Ahima, R. S., Z. Krozowski, and R. Harlan, unpublished) suggest high densities of the type I receptor in CA3. Even though aldosterone has very little binding to type II receptor in vivo compared to type I receptor (9), we have shown type Il-ir regulation by aldosterone in several other regions, including the hippocampus. Whether this has any physiological significance in CA3 and CA4 is unclear. Finally, the heterogeneity in the distribution and corticosteroid regulation of type Il-ir in the CNS may be related to the diverse functions of corticosteroids in the CNS (1, 2). How corticosteroids affect the functions of the few atypical type B and C neurons and how this relates to the function of the majority of neurons (type A) merit further study.
Acknowledgments Special thanks to R. Harrison for supplying BUGR2 antibody and P3 AgX-653 myeloma cell medium, and to Teresa Billiot and Debbie Lauff for secreterial assistance. We thank Louis Lucas for help in the statistical analysis.
References 1. McEwen BS, DeKloet ER, Rostene W 1986 Adrenal steroid receptors and actions in the nervous system. Physiol Rev 66:1121-1187 2. Funder JW, Sheppard K 1987 Adrenocortical steroids and the brain. Annu Rev Physiol 49:397-411 3. DeKloet ER, Reul JMHM 1987 Feedback action and tonic influence of corticosteroids on brain function: a concept arising from heterogeneity of brain receptor systems. Psychoneuroendocrinology 12:83-105 4. Fuxe K, Wikstrom AC, Okret S, Agnati LF, Harfstrand A, Yu ZY, Granholm L, Zoli M, Vale W, Gustafsson J-A 1985 Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor. Endocrinology 117:1803-1812 5. Fuxe K, Harfstrand AP, Agnati LF, Yu Z-Y, Cintra A, Wikstrom AC, Okret S, Cantoni K, Gustafsson J-A 1985 Immunocytochemical studies on the localization of glucocorticoid receptor immunoreactive nerve cells in the lower brainstem and spinal cord of the male rat using a monoclonal antibody against rat liver glucocorticoid receptor. Neurosci Lett 60:1-6 6. Ahima RS, Harlan RE 1990 Charting of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system. Neuroscience 39:579-604 7. Van Eekelen JAM, Kiss JZ, Westphal HM, DeKloet ER 1987
235
Immunocytochemical study on the type 2 glucocorticoid receptor in the rat brain. Brain Res 436:120-128 8. Fuxe D, Cintra A, Harfstrand A, Agnati LF, Kalia M, Zoli M, Wikstrom AC, Okret S, Aronsson M, Gustafsson J-A 1987 Central glucocorticoid receptor immunoreactive neurons: new insights into the neuroendocrine regulation of the brain. Ann NY Acad Sci 512:362-393 9. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM 1987 Cloning of human mineralocorticoid receptor cDNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268-275 10. Eisen LP, Reichman ME, Thompson ER, Gametchu B, Harrison RW, Eisen HJ 1985 Monoclonal antibody to the rat glucocorticoid receptor. J Biol Chem 260:11805-11810 11. Uht FM, McKlerry JF, Harrison RW, Bohn MC 1988 Demonstration of glucocorticoid receptor-like immunoreactivity in glucocorticoid-sensitive vasopressin and corticotropin releasing factor neurons in the hypothalamic paraventricular neurons. J Neurosci Res 19:405-411 12. Hancock MB 1984 Visualization of peptide-immunoreactive processes on serotonin-immunoreactive cells using two-color immunoperoxidase staining. J Histochem Cytochem 32:311-316 13. Paxinos G, Watson C 1982 The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney 14. Abercrombie M 1946 Estimation of nuclear populations from microtome sections. Anat Rec 94:239-247 15. Hisano S, Kagotani Y, Tsuruo Y, Daikoku S, Chihara K, Whitnall MH 1988 Localization of glucocorticoid receptor in neuropeptide Y-containing neurons in the arcuate nucleus of the rat hypothalamus. Neurosci Lett 95:13-18 16. Rusconi S, Yamamoto KR 1987 Functional dissection of the hormone and DNA binding activities of the glucocorticoid receptor. EMBO J 6:1309-1315 17. Okret S, Wikstrom A-C, Wrange O, Andersson B, Gustafsson J-A 1984 Monoclonal antibodies against the rat liver glucocorticoid receptor. Proc Natl Acad Sci USA 81:1609-1613 18. Westphal HM, Moldenhauer G, Beato M 1982 Monoclonal antibodies to the rat liver glucocorticoid receptor. EMBO J 1:14671471 19. Krozowski ZS, Rundle SE, Wallace C, Castell MJ, Shen JH, Dowling J, Funder JW, Smith AI1989 Immunolocalization of renal mineralocorticoid receptors with an antiserum against a peptide deduced from the complimentary deoxyribonucleic acid sequence. Endocrinology 125:192-198 20. Wikstrom A-C, Bakke O, Okret S, Bronnegard M, Gustafsson J-A 1987 Intracellular localization of the glucocorticoid receptor: evidence for cytoplasmic and nuclear localization. Endocrinology 120:1232-1242 21. Reul JMHM, Pearce PT, Funder JW, Krozowski ZS 1989 Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration. Mol Endocrinol 3:1674-1680 22. Sheppard KE, Roberts JL, Blum M 1990 Differential regulation of type II corticosteroid receptor messenger ribonucleic acid expression in the rat anterior pituitary and hippocampus. Endocrinology 127:431-439 23. Gorski J, Welshons W, Sakai D 1984 Remodeling the estrogen receptor model. Mol Cell Endocrinol 36:11-15 24. King WJ, Greene GL 1984 Monoclonal antibodies localize estrogen receptor in the nuclei of target cells. Nature 307:745-747 25. Picard D, Kumar V, Chambon P, Yamamoto KR 1990 Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell Regul 1:291-299 26. Blaustein JD, King JC, Toft DO, Turcotte J 1988 Immunocytochemical localization of estrogen-induced progestin receptors in guinea pig brain. Brain Res. 474:1-15 27. Perrot-Applanat M, Logeat F, Groyer-Picard M-T, Milgrom E 1985 Immunocytochemical study of mammalian progesterone receptor using monoclonal antibodies. Endocrinology 116:1473-1484 28. Welshons WV, Lieberman ME, Gorski J 1984 Nuclear localization of unoccupied receptors for glucocorticoids, estrogens, and proges-
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terone in GH3 cells. Endocrinology 117:2140-2147 29. Warembourg M, Logeat F, Milgrom E 1986 Immunocytochemical localization of progesterone receptor in guinea pig central nervous system. Brain Res 384:121-131 30. Blaustein JD, Turcotte JC 1989 Estrogen receptor-immunostaining of neuronal cytoplasmic processes as well as cell nuclei in guinea pig brain. Brain Res 495:75-82 31. Sar M, Lubahn DB, French FS, Wilson EM 1990 Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 127:3180-3186 32. Schumacher M 1990 Rapid membrane effects of steroid hormones: an emerging concept in neuroendocrinology. Trends Neurosci 13:359-361 33. Simons SS, Mercier L, Miller PA, Oshima H, Sistare FD, Thomp-
34. 35. 36. 37. 38.
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son EB, Wasner G, Yen PM 1989 Differential modulation of gene induction by glucocorticoids and antiglucocorticoids in rat hepatoma tissue cells. Cancer Res 49:2244S-2252S Yamamoto KR 1985 Steroid receptor regulated transcription of specific gene networks. Annu Rev Genet 19:209-252 Rousseau GG 1984 Control of gene expression by glucocorticoid hormones. Biochem J 224:1-12 Ringold GM 1985 Steroid hormone regulation of gene expression. Annu Rev Pharmacol Toxicol 25:529-566 Sapolsky RM 1985 A mechanism for glucocorticoid toxicity in the hippocampus: increased neuronal vulnerability to metabolic insults. J Neurosci 5:1228-1232 Arriza J, Simerly R, Swanson L, Evans R 1988 The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response. Neuron 1:887-900
American Thyroid Association 65th Annual Meeting The 65th Annual Meeting of the American Thyroid Association will be held in Boston, Massachusetts, from September 12-15, 1991, at the Hyatt Regency, Cambridge. An International Symposium on Thyroid Autoimmunity will be held September 16 and 17 in Montreal, Quebec, Canada. For meeting information please contact: Leonard Wartofsky, M.D. Secretary, American Thyroid Association Endocrine-Metabolic Service Walter Reed Army Medical Center Washington, DC 20307-5001 Telephone: 301-588-5432 FAX: 301-558-4728.
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