Up-regulation of androgen receptor immunoreactivity in the rat brain by androgenic-anabolic steroids

226

Brain Research, 622 (1993) 226-236 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19202

Up-regulation of androgen receptor immunoreactivity in the rat brain by androgenic-anabolic steroids C h e r y l S. M e n a r d

and Richard E. Harlan

Department of Anatomy, Tulane University Medical School, New Orleans, LA 70112 (USA)

(Accepted 20 April 1993)

Key words: Testosterone; Steroid receptor; Steroid abuse; Athlete; Castration; Intact; Immunocytochemistry;Optical density

To characterize central nervous system changes that occur with anabolic steroid abuse in humans, immunocytochemicallocalization of androgen receptors in the brains of 10 intact and 10 castrated male rats was conducted after the administration of high levels of androgenic-anabolic steroids (AAS; 14 daily injections of sesame oil or a cocktail of 2 mg/kg testosterone cypionate, 2 mg/kg nandrolone decanoate, and 1 mg/kg boldenone undecylenate). In normal intact oil-treated males, nuclear androgen receptor immunoreactivitywas present in many 'classical' and 'non-classicar androgen target sites in the brain. Administration of AAS increased the intensity of immunoreactivity in most classical androgen target sites and increased both the intensity of immunoreactivityand number of immunoreactive cells in most non-classical androgen target sites. These results may suggest that androgen receptors in the brain are up-regulated by AAS. The simultaneous androgen receptor up-regulation in these regions by AAS may account for the complex anabolic steroid abuse syndrome. Consistently, androgen receptor immunoreactivityin most brain regions was reduced or absent after castration, suggesting that endogenous androgen levels are necessary for normal androgen receptor immunoreactivity. These results identify the distribution of one central nervous system mechanism modified by AAS.

INTRODUCTION Androgenic-anabolic steroids (AAS) are used by as many as one million athletes in the United States for the purpose of increasing body strength, size, and endurance 21. Abuse of these steroids often involves the use of several types of synthetic androgens simultaneously at very high doses 12. While little is known concerning the effects of high doses of A A S on the central nervous system, there is some clinical evidence to suggest that sexual, motor, and psychological disturbances 4'6'7'11'12'22, as well as addiction 12'21, may occur

ventromedial hypothalamus, medial amygdala, hippocampal CA-1 region, and cortex of the male rat brain 15. A n d r o g e n receptor m R N A in the ventral prostate of male rats was found to decrease after castration and increase after androgen replacement 19. Other studies, however, using Northern blot analysis and immunohistochemical techniques, revealed that castration increased and androgen replacement suppressed androgen receptor protein and m R N A levels in various peripheral reproductive tissues of the rat, and in cultured hepatoma and prostate cancer cells from humans 13'14'17'2°. Based on these results, the au-

with abuse of these steroids. Conflicts in the literature exist concerning the regulation of androgen receptors by androgens. Recent investigations have indicated that androgen receptor immunoreactivity in various peripheral reproductive tissues of rat and human males was diminished after castration and restored after androgen replacement 15't9. Similar changes in androgen receptor immunoreactivity also occurred in the medial preoptic area, arcuate,

toregulation of androgen receptors by androgens is not clear. If A A S up-regulate androgen receptors in the brain, however, then central nervous system mechanisms normally regulated by androgens would be modified significantly with the abuse of these steroids. The present study examined the effects of administering very high levels of A A S on androgen receptor immunoreactivity in the brains of castrated and intact male rats. In this study, a combination of three syn-

Correspondence: C.S. Menard, Department of Anatomy, Tulane University Medical School, 1430 Tulane Avenue, New Orleans, LA 70112, USA.

Fax: (1) (504) 584-1687.

227 t h e t i c A A S was a d m i n i s t e r e d daily for 14 days in o r d e r to m i m i c the ' h e a v y u s e ' s e l f - a d m i n i s t r a t i o n r e g i m e u s e d by a t h l e t e s 12. A n d r o g e n r e c e p t o r i m m u n o r e a c t i v ity was e x a m i n e d in b r a i n r e g io n s w h i c h have b e e n extensively c h a r a c t e r i z e d as 'classical' a n d r o g e n t a r g e t sites, as well as o t h e r s w h i c h are ' n o n - c l a s s i c a l ' a n d r o g e n t a r g e t sites. If a n d r o g e n s u p - r e g u l a t e a n d r o g e n r e c e p t o r s as suggested, t h e n a n d r o g e n r e c e p t o r imm u n o r e a c t i v i t y s h o u l d b e i n c r e a s e d by t h e a d m i n i s t r a tion o f A A S an d d e c r e a s e d by castration. MATERIALS AND METHODS

Animal preparation Subjects were 20 Sprague-Dawley male rats (200-250 g) purchased from Charles River. Animals were housed in pairs and maintained on 12 h/12 h light/dark cycle with ad libitum food and water. Two weeks before steroid treatment, 10 male rats were castrated under methoxyflurane anesthesia. During steroid treatment, 5 castrated and 5 intact male rats received 14 daily injections of a cocktail of 2 mg/kg testosterone cypionate, 2 mg/kg nandrolone decanoate, and 1 mg/kg boldenone undecylenate (Sigma Chemical Co.) suspended in sesame oil (Sigma Chemical Co.). As controls, 5 castrated and 5 intact male rats received 14 daily injections of sesame oil only. Approximately 18-24 h after the last injection, all animals were anesthetized with pentobarbital and perfused intracardially with PBS (phosphate-buffered saline solution, pH 7.2) for 3 min, followed by neutral-buffered 3% paraformaldehyde solution (pH 7.2) for 6 min. Brains were removed and post-fixed for 2 h in 3% paraformaldehyde, then cryoprotected with 30% sucrose solution for 48 h at 4°C before freezing with powdered dry ice. Tissue was stored at -70°C until cut. Brains were coronally cut (60/zm) on a freezing microtome and stored in cryoprotectant (30% sucrose, 10% polyvinylpyrrolidone, and 30% ethylene glycol in PBS, pH 7.2) until immunocytochemically processed.

enhanced, thresholded, edited, then measured. Enhancement consisted of a parabola transformation of the image and sharpening of the edges of cells. Thresholding consisted of density slicing each image so that all cells were selected. Editing consisted of removing extraneous particles and separating adjacent cells. Data generated from this analysis consisted of optical density (OD) measurements and cell counts for immunoreactive cells within a 220x230 /zm sample of 'classical' and 'non-classical' regions of the brain. Measurements were standardized across groups by the following: (1) all measurements for a given area were conducted after the same calibration of the image analysis system; (2) all measurements for a given area were conducted after thresholding the images to the same value. Cell count measurements were consistently in agreement with independent manual counts. Furthermore, OD measurements were also consistent with subjective ratings of low, medium, or high staining intensities. After image analysis, data were compiled and statistically analyzed. ANOVA (analysis of variance) and PLSD (Fisher's protected least significant differences) post-hoc comparisons were used to determine group differences in mean OD and cell count measurements for each brain region assessed. Further analysis included partitioning the number of immunoreactive cells for each region according to intensity of staining. Independent ANOVA and PLSD post-hoc comparisons of these partitioned data were used to determine specific differences in the number of cells with low (OD (OD) < 10), moderate (OD 10-20), and intense staining (OD > 20). For comprehensive evaluations of total staining in each brain region analyzed, mean ODs and cell count measurements for each animal in each group were further transformed into integrated ODs (i.e. mean OD x number of labeled cells for each region analyzed). Integrated ODs were also statistically analyzed using ANOVA and PLSD.

RESULTS Androgen

receptor

i m m u n o r e a c t i v i t y was specifi-

cally n u c l e a r t h r o u g h o u t t h e rat b r a i n (Figs. 4 a n d 7 f or examples). F o r m o s t b r a i n regions, t h e d i s t r i b u t i o n a n d intensity o f a n d r o g e n r e c e p t o r i m m u n o r e a c t i v i t y in t h e

Immunocytochemistry Immunocytochemistry was conducted simultaneously on a 1-in-4 series of brain sections of one animal from each group (castrated, steroid-treated; castrated, oil-treated; intact, steroid-treated; intact, oil-treated). During immunocytochemical processing, free-floating sections were rinsed (PBS for 10 min) 3 times, pretreated with 1.5% normal goat serum (Vector Laboratories) in 0.2% Triton-X 100-PBS for 30 min, then incubated with 1 /xg/ml IgG purified polyclonal rabbit antibody (developed by Geoffrey Greene and provided by Gail Prins) in 1% BSA-PBS for 48 h at 4°C. A few sections from each animal which were simultaneously incubated in normal rabbit serum (1 : 880) served as controls. All sections were further processed using the Vectastain rabbit IgG (ABC) peroxidase kit (Vector Laboratories) according to specifications. Immunoreactivity was visualized using 50 mg% 3,3' diaminobenzidine tetrahydrochloride (DAB; Sigma) in Tris (pH 7.6) activated with 0.005% hydrogen peroxide. After immunostaining, sections were mounted on microscope slides, dehydrated in 70%, 95%, then 100% ethanol (1 min each), defatted with Histoclear (Diagnostic Products), and coverslipped. This immunocytochemical procedure was repeated using sections of one animal from each group until all tissue had been processed.

brains

o f rats u n d e r

normal

c o n d i t i o n s (i.e. intact,

o i l - t r e a t e d m a l e rats) was v e r y similar to t h e distribution an d intensity o f a n d r o g e n r e c e p t o r m R N A

ex-

p r e s s i n g cells p r e v i o u s l y r e p o r t e d by S i m e r l y et al. TM However, the steroid regulation of androgen receptor i m m u n o r e a c t i v i t y v a r i e d significantly in t h e O D a n d / o r n u m b e r o f i m m u n o r e a c t i v e ceils f r o m r e g i o n to r e gi on.

'Classical' androgen target sites Six r e g i o n s o f t h e rat f o r e b r a i n an d m i d b r a i n w e r e s t u d i e d as classical t a r g e t sites for a n d r o g e n s b a s e d o n (1) ex t en si v e l i t e r a t u r e i n d i c a t i n g t h e s e s t r u c t u r e s to b e i n v o l v e d in a n d r o g e n - s e n s i t i v e b e h a v i o r s , (2) e x t e n s i v e st u d i es i n d i c a t i n g h i g h levels o f a n d r o g e n r e c e p t o r s , a n d (3) t h e p r e s e n c e o f m o r e t h a n 100 a n d r o g e n r e c e p t o r i m m u n o r e a c t i v e n e u r o n s p e r 220 x 230 ~zm u n i t

Data analysis Sections were matched across groups, then analyzed using an image analysis system (Nikon Joyce Loebl Magiscan) provided by Dr. Ranney Mize, Louisiana State University Medical School, Anatomy Department. The image analysis system and specific techniques used for the analysis of these immunocytochemical data are extensively described in Mize9. Two 40x microscopic images of each sample were captured and averaged. The averaged reconstructed image was

a r e a e x a m i n e d in this study. T h e six r e g i o n s c h o s e n f or study w e r e : t h e dorsal division o f t h e l a t e r a l s e p t a l nucleus (LSD); the posteromedial region of the medial division o f t h e b e d

n u c l e u s o f t h e stria t e r m i n a l i s

( B S T M P M ) ; t h e c e n t r a l an d m e d i a l divisions o f t h e medial preoptic nucleus ( M P O - M P O C ) ; the ventrolat-

228 e r a l division o f t h e v e n t r o m e d i a l h y p o t h a l a m i c n u c l e u s ( V M H V L ) ; t h e d o r s o m e d i a l division o f t h e v e n t r o m e dial h y p o t h a l a m i c n u c l e u s ( V M H D M ) ; a n d t h e post e r o v e n t r a l division o f t h e m e d i a l a m y g d a l o i d n u c l e u s ( M e P V ) (Fig. 1). C o m p a r i s o n s across g r o u p s o f t h e m e a n O D a n d mean number of androgen receptor immunoreactive cells in classical a n d r o g e n r e c e p t o r t a r g e t sites a r e i l l u s t r a t e d in Figs. 2 a n d 3. In i n t a c t m a l e rats, A A S significantly i n c r e a s e d t h e m e a n O D (Fig. 2) in t h r e e o f t h e s e classical a n d r o g e n t a r g e t sites, i.e. t h e L S D , BSTMPM, and MePV, without increasing the mean n u m b e r o f l a b e l e d cells in t h e s e specific r e g i o n s (Fig. 3). H o w e v e r , f u r t h e r analysis d i d i n d i c a t e significant i n c r e a s e s in t h e m e a n n u m b e r o f cells with i n t e n s e

CLASSICAL A N D R O G E N T A R G E T SITES

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Fig. 2. Mean ODs (+S.E.M.) indicating androgen receptor immunostaining intensities in 'classical' androgen target sites in the brains of castrated and intact rats treated with either AAS or sesame oil vehicle. In intact male rats, AAS significantly increased immunostaining in five classical androgen target sites examined (* * P < 0.01) when compared to intact oil-treated controls. In castrated male rats, AAS significantly increased immunostaining in all six classical androgen target sites (* * P < 0.01) when compared to castrated oil-treated controls. In all six classical androgen target sites, immunostaining in castrated male rats treated with AAS was equivalent to immunostaining in intact male rats treated with AAS. Castration significantly decreased immunostaining in three of these regions (* P < 0.01) when compared to intact oil-treated controls. LSD, lateral septal nucleus-dorsal division; BSTMPM, medial division of the bed nucleus of the stria terminalis-posteromedial region; MPO-MPOC, medial preopticnucleus-medial and central divisions; VMHVL, ventromedial hypothalamic nucleus-ventrolateral division; VMHDM, ventromedial hypothalamic nucleus-dorsomedial division; MePV, medial amygdaloid nucleus-posteroventral division.

D

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Fig. 1. Drawings (adapted from Paxinos and Watson 1°) illustrating levels of the rat brain analyzed in this study. Numbered rectangles indicate 220×230 mm areas of the rat brain where OD and cell count measurements were conducted. (Bar = 1 mm). A: bregma 0.20 mm; 1 l , Frl-layer 2 (frontal cortex, area l-layer 2); 21, Frl-layer 4 (frontal cortex, area l-layer 4); 3 m, LSD (lateral septal nucleus-dorsal division); 4 1 , CPu (caudate/putamen-ventrolateral region). B: bregma -0.80 mm; 5n, BSTMPM (medial division of the bed nucleus of the stria terminalis-posteromedial region); 6m, MPOMPOC (medial preoptic nucleus-medial and central divisions). C: bregma -3.14 mm; 7m, CA1 (Ammon's horn-CA1 field); 8m, VMHDM (ventromedial hypothalamic nucleus-dorsomedial division); 91, VMHVL (ventromedial hypothalamic nucleus-ventrolateral division); 1 0 l , MePV, medial amygdaloid nucleus-posteroventral division. D: bregma -5.20 mm; 11 n, CG (central gray); 12 m, SNL (substantia nigra-lateral division); 13 l , SNC (substantia nigra-compact division); 14n, VTA (ventral tegmental area); E: bregma - 10.04 ram; 15 l , LC (locus coeruleus).

staining (i.e. O D > 20) in all six classical r e g i o n s a f t e r s t e r o i d t r e a t m e n t in i n t a c t m a l e s (Fig. 3). T h e r e f o r e , while t h e t o t a l n u m b e r s o f i m m u n o r e a c t i v e cells rem a i n e d r e l a t i v e l y c o n s t a n t in t h e s e p a r t i c u l a r classical t a r g e t sites, t h e n u m b e r s o f i n t e n s e l y s t a i n e d cells w e r e i n c r e a s e d significantly. This general pattern of AAS regulation of androgen r e c e p t o r i m m u n o r e a c t i v i t y was n o t e v i d e n t in two o t h e r classical a n d r o g e n t a r g e t sites, however. I n t h e v e n t r o medial hypothalamus (VMHVL and VMHDM), there w e r e i n c r e a s e s in b o t h t h e m e a n O D a n d t h e m e a n n u m b e r o f i m m u n o r e a c t i v e cells a f t e r s t e r o i d t r e a t m e n t (Figs. 2 a n d 3). T h e r e f o r e , A A S m a y i n c r e a s e a n d r o g e n r e c e p t o r i m m u n o r e a c t i v i t y w i t h i n t h e cell, a n d in c e r t a i n classical a n d r o g e n t a r g e t sites, such as the ventromedial hypothalamus, recruit more androgen r e c e p t o r i m m u n o r e a c t i v e cells in t h a t r e g i o n , as well.

229 These data support the enhanced up-regulation of androgen receptor immunoreactivity in these classical androgen target sites with the administration of high levels of AAS. In one other classical androgen target site, the MPO-MPOC, increases in the mean OD after steroid treatment were quite varied and, therefore, not significant when compared to intact, oil-treated controls (Fig. 2). These data further support the differential regulation of androgen receptor immunoreactivity across these classical androgen target sites.

In all classical androgen target sites, castration significantly decreased the mean number of immunoreactive cells, particularly the intensely stained cells (OD > 20), when compared to normal intact, oil-treated males (Fig. 3). However, the mean OD was significantly decreased only in the ventromedial hypothalamus and amygdala after castration (Fig. 2). These data further support a general up-regulation of androgen receptor immunoreactivity in classical androgen target sites by endogenous androgens. As before, decreases in

CLASSICAL ANDROGEN RECEPTOR TARGET SITES B. Intact, Steroid-Treated Male Rats

A. Intact, Oil-Treated Male Rats

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Fig. 3. M e a n n u m b e r ( + S.E.M.) of immunoreactive cells per unit area in 'classical' androgen target sites of the brains of castrated and intact rats treated with either A A S or sesame oil vehicle. The light, medium, and dark partitions of each bar, respectively, represent the m e a n n u m b e r s of immunoreactive cells with low ( O D < 10), moderate ( O D 10-20), and intense ( O D > 20) immunostaining in each area. In intact male rats, A A S significantly increased the n u m b e r of immunoreactive cells in two of the classical androgen target sites examined (B; ** P < 0.01) when compared to intact oil-treated controls (A). In castrated male rats treated with A A S (D), the n u m b e r of immunoreactive cells was significantly increased ( * * P < 0.01) in all six classical target sites over castrated oil-treated controls (C), to levels equivalent to those observed in intact male rats treated with A A S (B). Castration significantly decreased the n u m b e r of immunoreactive cells in all six regions (C; * P < 0.01) when compared to intact oil-treated controls (A). Abbreviations as in Fig. 2.

230 the mean OD in the MPO-MPOC after castration were not significantly different from normal intact, oiltreated males because of the wide variability within groups in this region. Therefore, endogenous androgens also may differentially up-regulate androgen receptor immunoreactivity in various classical androgen target sites. In castrated rats, treatment with A.AS significantly increased the mean ODs and mean number of labeled cells in all classical androgen target sites to levels comparable to that found in intact rats given AAS. Examples of the effects of castration and AAS treatment on immunostaining in the BSTMPM are shown in Fig. 4.

labeled cells per unit area, (2) presence of low levels of androgen receptor mRNA TM, and (3) hypothetical involvement of these brain regions in the behavioral and psychiatric syndromes often displayed by athletes abusing AAS. The nine regions chosen for study were: layer 2 of area 1 of the frontal cortex (Frl-layer 2); layer 4 of area 1 of the frontal cortex (Frl-layer 4); ventrolateral region of the caudate-putamen (CPu); the CA1 field of Ammon's horn (CA1); the compact division of the substantia nigra (SNC); the lateral division of the substantia nigra (SNL); the midbrain central gray (CG); the ventral tegmental area (VTA); and the locus coeruleus (LC) (Fig. 1). Comparisons across groups of the mean OD and mean number of androgen receptor immunoreactive cells in non-classical androgen receptor target sites are illustrated in Figs. 5 and 6. In four of these non-classical androgen target sites, including CA1, SNC, SNL,

'Non-classical' androgen target sites For comparison, several non-classical androgen target sites were also examined. Nine brain regions were chosen on the basis of: (1) presence of fewer than 100

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Fig. 4. Photomicrographs illustrating specific nuclear androgen receptor immunoreactivity in the bed nucleus of the stria terminalis (posteromedial region of the medial division). The intensity of immunostaining in this 'classical' androgen target site was increased after AAS treatment in intact male rats (B) when compared to immunoreactivity after oil treatment in intact male rats (A); however, AAS treatment in intact males did not increase the number of immunoreactive cells in this classical androgen target site. AAS treatment in castrated male rats (D) increased both the immunostaining intensity and the number of immunoreactive cells when compared to castrated oil-treated controls (C); however, immunoreactivity in intact (B) and castrated (D) male rats was equivalent in all respects after AAS treatment. Castration (C) decreased the number of immunoreactive cells in this region when compared to normal intact male rats (A). Bar = 50 ~m.

231 and CG, AAS significantly increased both the m e a n O D (Fig. 5) and the n u m b e r of immunoreactive cells (Fig. 6) in these regions when compared to oil treatm e n t in intact males. Therefore, AAS may increase androgen receptor immunoreactivity within the cell, as well as recruit more androgen receptor immunoreactive cells, in these regions. These data support the enhanced up-regulation of androgen receptor immunoreactivity in these non-classical target sites which occurs with the administration of high levels of AAS.

NON-CLASSICAL ANDROGEN TARGET SITES

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M e a n Optical Density

Fig. 5. Mean ODs (+S.E.M.) indicating androgen receptor immunostaining intensities in 'non-classical' androgen target sites in the brains of castrated and intact rats treated with either AAS or sesame oil vehicle. In intact male rats, A.AS significantly increased the intensity of immunostaining in four non-classical androgen target sites examined (** P<0.01)when compared to intact oil-treated controls. In castrated male rats, AAS significantly increased immunostaining in seven non-classical androgen target sites examined (* * P < 0.01) when compared to castrated oil-treated controls. In all nine non-classical androgen target sites, immunostaining in castrated male rats treated with AAS was equivalent to immunostaining in intact male rats treated with AAS. Castration significantly decreased immunostaining in three of these regions when compared to intact oil-treated controls (* P<0.01). Frl-layer 2, frontal cortex, area l-layer 2; Frl-layer 4, frontal cortex, area l-layer 4; CPu, caudate/putamen-ventrolateral region; CA1, Ammon's horn-CA1 field; SNC, substantia nigra-compact division; SNL, substantia nigra-lateral division; CG, central gray; VTA, ventral tegmental area; LC, locus coeruleus.

In another non-classical target site, Frl-layer 2, steroid administration in intact male rats increased only the n u m b e r of immunoreactive cells (Fig. 6). Therefore, while immunostaining in Frl-layer 2 was maximal under normal conditions, the n u m b e r of androgen receptor immunoreactive cells in this region was not. In comparison, no significant increases in either immunostaining or the n u m b e r of immunoreactive cells in Frl-layer 4 were found after steroid treatment in intact male rats. This may suggest that immunoreactivity in Frl-layer 4 is maximal under normal conditions in both respects. Castration significantly decreased the n u m b e r of immunoreactive cells in four non-classical androgen target sites, including F r l - l a y e r 2, F r l - l a y e r 4, CA1, and SNC (Fig. 6). These data further support the up-regulation of androgen receptor immunoreactivity in these non-classical androgen target sites by endogenous androgens. Because m e a n O D s and numbers of immunoreactive cells in normal intact, oil-treated males were relatively low, however, decreases in immunostaining after castration were difficult to detect in other non-classical androgen target sites. Therefore, the role of endogenous androgens in the regulation of androgen receptors in these regions remains undetermined. With the exception of two areas, i.e. the CPu and VTA, AAS treatment in castrated male rats increased both the m e a n O D (Fig. 5) and m e a n n u m b e r of immunoreactive cells (Fig. 6) in non-classical androgen target sites to levels comparable to that found in intact rats given AAS. In two other non-classical androgen target sites, CPu and VTA, no significant changes in either O D or n u m b e r of immunoreactive cells per region were detected after either steroid administration or castration (Fig. 5 and 6). These data may suggest that androgenic steroids have little or no effect on androgen receptor immunoreactivity in these regions. Examples of the effects of castration and AAS treatment on androgen receptor immunostaining in the SNL and SNC are shown in Fig. 7. Classical vs. non-classical androgen target sites Integrated O D s were used to evaluate the total immunoreactivity in each brain region analyzed. Comparisons across groups of m e a n integrated O D s for classical and non-classical androgen target sites are illustrated in Figs. 8 and 9. Interestingly, there was very little differences in the pattern of m e a n integrated O D s across groups in classical androgen target sites (Fig. 8) when c o m p a r e d to m e a n O D measures in these same regions (Fig. 2). Two pieces of information were obtained from these data, however. First, in classical

232

NON-CLASSICAL ANDROGEN RECEPTOR TARGET SITES

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B. Intact, Steroid-Treated Male Rats

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Fig. 6. Mean number (+ S.E.M.) of immunoreactive cells per unit area in 'non-classical' androgen target sites in the brains of castrated and intact rats treated with either AAS or sesame oil vehicle. The light, medium, and dark partitions of each bar respectively represent the mean number of immunoreactive cells with low (OD < I0), moderate (OD 10-20), and intense (OD > 20) immunostaining in each area. When compared to oil treatment in normal intact males (A), AAS significantly increased the number of immunoreactive cells in four non-classical androgen target sites examined (B; ** P < 0.01). In castrated male rats treated with AAS (D), the number of immunoreactive cells was significantly increased (* * P < 0.01) in seven non-classical target sites over castrated oil-treated controls (C) to levels equivalent to that observed in intact male rats treated with AAS (B). Castration significantly decreased the number of immunoreactive cells in four regions (C; * P < 0.01), when compared to intact oil-treated controls (A). Abbreviations as in Fig. 5.

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Fig. 7. Photomicrographs illustrating specific nuclear androgen receptor immunoreactivity in the substantia nigra (lateral division). In both intact (B) and castrated (D) male rats, AAS increased both the O D and the n u m b e r of immunoreactive cells in this 'non-classical' androgen target site when respectively compared to immunoreactivity in intact (A) and castrated (C) oil-treated controls. Immunoreactivity in intact (B) and castrated (D) male rats was equivalent in all respects after AAS treatment. Castration (C) only slightly decreased the n u m b e r of immunoreactive cells in this region when compared to normal intact male rats (A). (Bar = 50 ~zm).

androgen target sites the mean integrated ODs for intact, oil-treated males ranged from 825 to 3,402, whereas the range for non-classical androgen target sites varied from 154-487. Therefore, naturally occurring differences in total immunoreactivity between classical and non-classical androgen target sites are profound. Second, the mean integrated ODs in nonclassical androgen target sites of intact males treated with AAS revealed an enhanced regulation of total immunoreactivity by AAS relative to the total immunoreactivity in intact, oil-treated males (Fig. 9). This enhanced regulation was especially noticeable in Frllayer 2, CA1, SNC, SNL, and CG, regions where the mean number of labeled cells was increased significantly after AAS treatment (Fig. 3). Furthermore, the enhanced increase in total immunoreactivity in many of these non-classical androgen target sites was substantial enough to increase immunoreactivity to levels near a n d / o r above the mean integrated density staining

normally observed in classical target sites from intact, oil-treated males (Fig. 8). These results further emphasize the enhanced up-regulation of androgen receptor immunoreactivity in non-classical androgen target sites due to the recruitment of more immunoreactive cells by AAS treatment. DISCUSSION In this study, androgen receptor immunoreactivity was specifically nuclear throughout the brain of all rats, regardless of treatment condition; however, the intensity of androgen receptor immunoreactivity in the brains of normal intact, oil-treated male rats varied considerably from region to region. The distribution and intensity of androgen receptor immunoreactivity in intact, oil-treated male rats was generally consistent with previous reports of the distribution and signal intensity of androgen receptor mRNA in the brains of

234 o v a r i e c t o m i z e d , e s t r o g e n - t r e a t e d f e m a l e a n d intact m a l e rats 18. In a d d i t i o n , t h e g e n e r a l p a t t e r n o f imm u n o r e a c t i v i t y o b s e r v e d h e r e was c o n s i s t e n t with t h e a n d r o g e n r e c e p t o r s t e r o i d b i n d i n g o b s e r v e d using [ 3 H ] d i h y d r o t e s t o s t e r o n e a u t o r a d i o g r a p h y 16. In this study, A A S w e r e f o u n d to i n c r e a s e a n d r o g e n r e c e p t o r i m m u n o r e a c t i v i t y in m a n y 'classical' a n d ' n o n - c l a s s i c a l ' a n d r o g e n t a r g e t sites in t h e rat brain. T h i s r e g u l a t i o n v a r i e d c o n s i d e r a b l y f r o m r e g i o n to region, however. Consistently, c a s t r a t i o n d e c r e a s e d a n d r o g e n r e c e p t o r i m m u n o r e a c t i v i t y in m o s t classical a n d m a n y n o n classical a n d r o g e n t a r g e t sites, f u r t h e r s u p p o r t i n g t h e n o t i o n o f an u p - r e g u l a t i o n o f c e n t r a l a n d r o g e n r e c e p tor i m m u n o r e a c t i v i t y by e n d o g e n o u s a n d r o g e n s . S a r a n d a s s o c i a t e s 15, using t h e A R 5 2 r a b b i t p o l y c l o n a l antibody, also r e p o r t t h a t c a s t r a t i o n d e c r e a s e d a n d s t e r o i d replacement restored androgen receptor immunoreactivity in m a n y o f the r e g i o n s e x a m i n e d in this study, i n c l u d i n g t h e m e d i a l p r e o p t i c nucleus, a r c u a t e nucleus, v e n t r o m e d i a l n u c l e u s o f t h e h y p o t h a l a m u s , m e d i a l nucleus o f t h e a m y g d a l a , t h e CA-1 r e g i o n o f t h e hipp o c a m p u s , a n d t h e cortex.

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Me,In Integrated Optical Density

Fig. 9. Mean integrated ODs (+ S.E.M.) representing immunoreactivity relative to changes in both the intensity of staining and the number of immunoreactive cells in 'non-classical' androgen target sites. In intact male rats, AAS significantly increased integrated ODs in five non-classical androgen target sites examined (** P < 0.01) when compared to intact oil-treated controls. In castrated male rats, AAS significantly increased integrated OD in seven non-classical androgen target sites examined (** P <0.01)when compared to castrated oil-treated controls. In all nine non-classical androgen target sites, integrated ODs in castrated male rats treated with AAS were equivalent to immunostaining in intact male rats treated with AAS. Castration significantly decreased integrated ODs in one of these regions when compared to intact oil-treated controls (* P < 0.01). Abbreviations as in Fig. 5.

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Fig. 8. Mean integrated ODs (+ S.E.M.) representing immunoreactivity relative to changes in both the intensity of staining and the number of immunoreactive cells in 'classical' androgen target sites. In intact male rats, AAS significantly increased integrated ODs in five classical androgen target sites examined (** P<0.01)when compared to intact oil-treated controls. In castrated male rats, AAS significantly increased integrated ODs in all six classical androgen target sites (** P < 0.01) when compared to castrated oil-treated controls. In all six classical androgen target sites, integrated ODs in castrated male rats treated with AAS were equivalent to integrated ODs in intact male rats treated with AAS. Castration significantly decreased integrated ODs in four of these regions (* P < 0.01) when compared to intact oil-treated controls. Abbreviations as in Fig. 2.

I n m o s t classical a n d r o g e n t a r g e t sites, with t h e exception of the ventromedial hypothalamus and the medial preoptic area, up-regulation of androgen recept o r i m m u n o r e a c t i v i t y was i n d i c a t e d by an i n c r e a s e only in t h e O D a f t e r a d m i n i s t r a t i o n o f A A S . T h e r e f o r e , while t h e n u m b e r o f ceils w h i c h c o n t a i n a n d r o g e n r e c e p t o r s in m o s t classical a n d r o g e n t a r g e t sites m a y b e m a x i m a l u n d e r n o r m a l c o n d i t i o n s , i n c r e a s e s in t h e i n t e n s i t y o f s t a i n i n g in this a r e a a r e still p o s s i b l e a f t e r a n d r o g e n i c s t e r o i d t r e a t m e n t . U n l i k e o t h e r classical a n d r o g e n t a r g e t sites, t h e v e n t r o m e d i a l n u c l e u s o f t h e h y p o t h a l a m u s r e s p o n d e d to A A S with i n c r e a s e s in b o t h O D a n d n u m b e r s o f l a b e l e d cells. I n o n e o t h e r classical a n d r o g e n t a r g e t site, t h e m e d i a l p r e o p t i c a r e a ,

235 increases in OD were quite varied within the group that received AAS treatment. Collectively, these results support an up-regulation of androgen receptor immunoreactivity in these classical androgen target sites, which varies considerably from region to region. In addition, these results indicate that brain regions which regulate normal androgenic functioning, such as male sexual behavior and aggression, may be altered significantly with the abuse of AAS. In several non-classical androgen target sites, including the substantia nigra, hippocampus, and central gray, up-regulation of androgen receptor immunoreactivity was indicated by increases in both the OD and the number of immunoreactive cells after ALAS administration. Therefore, the number of cells which contain androgen receptors in these non-classical target sites is not maximal under normal conditions, allowing for increases in the number of immunoreactive cells after androgenic steroid treatment. Indeed, in many nonclassical target sites, including Frl-layer 2, CA1, SNC, SNL, and LC, total immunoreactivity, as indicated by the mean integrated density of staining in these regions, was enhanced substantially enough to increase immunoreactivity to levels near a n d / o r above the mean integrated density staining normally observed in most classical target sites of intact, oil-treated animals. These results indicate that brain regions subserving behavioral manifestations which are not typically considered to be regulated by androgenic steroids, are modified by steroid abuse. In one non-classical androgen target site, layer 2 of the frontal cortex, steroid treatment increased the number of immunoreactive ceils without changing the intensity of immunostaining normally seen in intact males. This further illustrates the regionspecific differential regulation of androgen receptor availability and may explain further the complexity of the anabolic steroid abuse syndrome. While several non-classical androgen target sites, including the frontal cortex (layer 4), caudate/putamen (ventrolateral division), ventral tegmental area and locus coeruleus, did not exhibit significant changes in androgen receptor immunoreactivity after steroid treatment, this does not mean these areas are not involved in the anabolicsteroid abuse syndrome. These areas could be activated indirectly by androgen receptors in other regions. Alternatively, because androgens, such as testosterone, are readily aromatized into estrogen in the brain, it is possible that androgenic steroids could be acting on these areas via estrogen receptors. The widespread androgen receptor immunoreactivity in the rat brain which varied considerably in intensity from region to region and in response to AAS may suggest an anatomical substrate for the complex syn-

drome described with the abuse of these steroids. Although little is known about the phenotypes of neurons expressing androgen receptors, these results demonstrated AAS-induced increases in androgen receptor immunoreactivity in several systems which may include dopaminergic neurons (e.g. the substantia nigra) and opioid neurons (e.g. the ventromedial hypothalamus). Therefore, it is possible to hypothesize that AAS up-regulate androgen receptors, which then mediate changes in central dopaminergic a n d / o r opioid mechanisms responsible for the behavioral manifestations associated with abuse of these steroids. By autoregulation of androgen receptors in dopaminergic a n d / o r opioid peptide neurons of the brain, AAS may induce a feed-forward regulation which would amplify the modification of these mechanisms. The currently proposed up-regulation of androgen receptor immunoreactivity by AAS is consistent with previous studies which report that androgen replacement increased androgen receptor immunoreactivity, whereas castration diminished androgen receptor immunoreactivity in various peripheral reproductive tissues of rat and human males 15'19, and in the medial preoptic area, arcuate, ventromedial hypothalamus, medial amygdala, hippocampal CA-1 region, and cortex of the male rat brain 15. These studies collectively may offer evidence for the up-regulation of androgen receptor immunoreactivity by androgens. Moreover, at least one study did report that androgen receptor mRNA in the ventral prostate of male rats was decreased after castration and was increased after androgen replacement ~9. Further investigations of AAS regulation of androgen receptor mRNA in the central nervous system are ongoing by this laboratory. Whether or not the AAS-induced increase in androgen receptor immunoreactivity observed in this study is directly reflective of an actual increase in nuclear androgen receptor protein cannot be determined by the present immunocytochemical analysis alone. Alternatively, steroid-induced conformational changes in the protein structure may account for increased androgen receptor immunoreactivity in the nucleus. For example, estrogen-induced down-regulation of estrogen receptor immunoreactivity has been reported to vary considerably depending on whether or not the specific antibody used recognized the ligand-binding domain, the hingeregion, or the N-terminus of the estrogen receptor 5. In this example, it has been suggested that, particularly with the antibody recognizing the ligand-binding domain, steroid binding may alter the conformation of the estrogen receptor such that the antibody no longer recognizes it. Although this scenario is unlikely in the present study since the antibody used recognizes the

236 first 21 a m i n o acids o f t h e a n d r o g e n r e c e p t o r , it is still possible that other conformational changes may be t a k i n g p l a c e a f t e r s t e r o i d binding. In a d d i t i o n , estrog e n - i n d u c e d c h a n g e s in e s t r o g e n r e c e p t o r i m m u n o r e activity in t h e b r a i n also have b e e n shown to v a r y d e p e n d i n g on w h e t h e r 4 % p a r a f o r m a l d e h y d e fixation o r 4 % p a r a f o r m a l d e h y d e plus 0.1% g l u t a r a l d e h y d e was u s e d 5. T h e s e f i x a t i v e - i n d u c e d a l t e r a t i o n s w e r e reflective o f m o d i f i c a t i o n s in t h e b a c k g r o u n d staining, however. B e c a u s e b a c k g r o u n d staining was m i n i m a l a n d c o n s i s t e n t o v e r a l l g r o u p s in t h e p r e s e n t study, this m a y not b e an issue h e r e . W h i l e t h e s e factors a r e o f conside r a t i o n , t h e y d o n o t w e a k e n the c u r r e n t i n t e r p r e t a t i o n o f t h e p r e s e n t data. T h a t is, using t h e c o n d i t i o n s p r e s e n t e d in this study, specific n u c l e a r a n d r o g e n r e c e p t o r i m m u n o r e a c t i v i t y in m a n y classical a n d n o n - c l a s s i c a l r e g i o n s o f t h e b r a i n was i n c r e a s e d significantly in m a l e rats t h a t r e c e i v e d A A S t r e a t m e n t a n d d e c r e a s e d significantly in a n i m a l s t h a t w e r e c a s t r a t e d . W h i l e this study f o c u s e d o n t h e effects o f A A S a b u s e on a n d r o g e n r e c e p t o r availability, it d i d n o t a d d r e s s a n d r o g e n r e c e p t o r r e g u l a t i o n via t h e a r o m a t i z a t i o n of A A S into e s t r o g e n s . I n d e e d , c i r c u l a t i n g s e r u m e s t r o g e n levels a r e i n c r e a s e d d r a s t i c a l l y in a t h l e t e s w h o a b u s e A A S 2'3, suggesting t h a t a significant a m o u n t of t h e a n a b o l i c s t e r o i d is a r o m a t i z e d . F u r t h e r m o r e , b e cause t h e r e is a g r e a t d e a l o f o v e r l a p in t h e l o c a l i z a t i o n of the estrogen and androgen receptor mRNA-exp r e s s i n g cells in t h e c e n t r a l n e r v o u s system TM, it is also possible t h a t t h e b e h a v i o r a l m a n i f e s t a t i o n s a s s o c i a t e d with a n a b o l i c s t e r o i d a b u s e m a y also b e m e d i a t e d by t h e i n t e r a c t i o n o f a r o m a t a s e - c o n v e r t e d e s t r o g e n s with e s t r o g e n r e c e p t o r s in d o p a m i n e r g i c a n d / o r o p i o i d regions o f t h e brain. O n e o t h e r possibility is a n a b o l i c s t e r o i d r e g u l a t i o n of c e n t r a l m e c h a n i s m s via t h e glucoc o r t i c o i d r e c e p t o r . I n d e e d , A A S have p r e v i o u s l y b e e n shown to i n t e r a c t with g l u c o c o r t i c o i d r e c e p t o r s in t h e h i p p o c a m p u s 1. F u r t h e r m o r e , t h e r e is an extensive litera t u r e on t h e g l u c o c o r t i c o i d activation of c e n t r a l d o p a m i n e r g i c m e c h a n i s m s in t h e b r a i n 8. T h e c o m p r e hensive investigation of anabolic steroid-induced c h a n g e s in d o p a m i n e r g i c a n d o p i o i d m e c h a n i s m s via a n d r o g e n , e s t r o g e n , o r g l u c o c o r t i c o i d r e c e p t o r s is also o n g o i n g by this l a b o r a t o r y . We wish to thank Dr. Gail Prins for generously providing the androgen receptor antibody used in this study. In addition, we gratefully acknowledge Dr. Ranney Mize and his technical staff for their assistance in the image analysis. Finally, thanks to Dr. Meredith Garcia, Louis Lucas, and Dr. Nathaniel Lawson for their advice with this project. This study was supported by NIDA Grant DA-06194 awarded to R.E.H.

Acknowledgements.

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