Brain Research, 585 (1992) 311-314 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
311
BRES 25224
Regulation of glucocorticoid receptor immunoreactivity in the rat hippocampus by androgenic-anabolic steroids a
R e x f o r d S. A h i m a and
a,b
R i c h a r d E. H a r l a n
a Department of Anatomy and b Neuroscience Training Program, Tulane University Medical School, New Orleans, 1.,,4 70112 (USA) (Accepted 3 March 1992)
Key words: Androgenic-anabolic steroid; Glucocorticoid receptor; Hippocampus; Rat
To determine whether androgenic-anabolic steroids (AAS) regulate glucocorticoid receptors (GR), the effects of a mixture of three commonly abused AAS (testosterone cypionate, nandrolone decanoate and boldenone undecylenate) on GR immunoreactivity (Jr), were examined in hippocampi of adrenalectomized male rats. Treatment with AAS for 1 week increased nuclear GRir in pyramidal cells of CAI, and granule cells of the dentate gvrus. These findings suggest a role for GR in the mediation of some of the CNS effects of androgenic-anabolic steroids.
Anabolic steroids are among the most widely abused drugs in the United States 9. Since these steroids have androgenic properties, they are also referred to as androgenic-anabolic steroids (AAS). Apart from affecting muscle mass, AAS have several effects on CNS function, including affect and cognition, and neuroendocrine regulation ~'4'~. it has been suggested that these effects may be mediated by modulation of dopaminergic systems, and/or interaction between AAS and receptors for androgens, estrogens or glucocorticoids ~. In skeletal muscle, where the actions of AAS have been well documented, androgen receptors are saturated by normal circulating androgens. However, the amount of AAS used by athletes is in vast excess of available androgen receptors (AR), raising the question of the receptor system utilized by AAS. In skeletal muscle the actions of AAS are antagonized by glucocorticoids 9'!°. Moreover, because AAS bind competitively to glucocorticoid receptors (GR) in skeletal muscle !°, it is thought that the actions of AAS are mediated by GR m°. Binding of AAS to cytosolic OR has also been demonstrated in the liver ~5 and in HTC cells ~4. Although binding of AAS to GR has not been demonstrated in the CNS, in humans, high doses of anabolic steroids administered chronically have been shown to reduce plasma adrenocorticotropin (ACTH) levels 4'9. Cessation of treatment restores ACTH levels to normal 4'9.
This is akin to negative feedback regulation of ACTH by excess glucocorticoids, a process mediated at least in part by GR in the hippocampus I1. We therefore hypothesized that, as in skeletal muscle I° and liver Is, GR may mediate some of the effects of anabolic steroids in the CNS. To address this question we examined the regulation of GR immunoreactivity (ir) in the hippocampus by a mixture of three commonly abused anabolic steroids--testosterone cypionate, nandrolone decanoate and boldenone undecylenate '~. We will refer to this mixture as TNB. In adrenally intact male rats, pyramidal cells in Ammon's horn, and granule and polymorphic cells of the dentate gyrus show predominantly nuclear GRir I. We have previously described two types of responses of ORir in the hippocampus following adrenalectomy (adx) with or without corticosteroid treatment !. Most GRir cells in Ammon's horn and dentate gyrus showed a so-called Type A response; i.e. these neurons showed a predominantly nuclear GRir in intact rats and lost GRir following prolonged adx. Nuclear GRir was restored by treatment with corticosteroids. A subpopulation of neurons in the pyramidal cell layer of Ammon's horn and the stratum oriens, classified Type B, were not observed in intact rats, showed intense diffuse or cytoplasmic GRir following adx, and lost immunoreactivity following treatment with corticosterone. In the present study we
Correspondence: R.E. Harlan, Department of Anatomy, Tulane University Medical School, New Orleans, LA 70112, USA. Fax: (I) (504) 584-1687.
312 analyzed the effect of TNB treatment on the density of Type A and B neurons in hippocampi of adx male rats. Four adrenally intact, and twelve 2-week-adx male Sprague-Dawley rats were purchased from Charles River, housed under standard cage conditions with a 12 h light-12 h dark cycle and allowed free access to chow. Intact rats were allowed free access to tap water and adx rats to normal saline. The adx rats were divided into 3 groups. One group was treated with TNB (testosterone cypionate, 2 mg/kg; nandrolone, 2 mg/kg; boldenone, 1 mg/kg in 200 ~1 sesame oil i.m) for 2 h, the second, TNB daily for one week, and the third, vehicle only. The intact rats did not receive any treatment and were used as controls. The animals were anaesthesized with sodium pentobarbitai and sacrificed. Brains were dissected out and prepared for immunocytochemical detection of GR using BUGR2 antiGR monoclonal antibody as described previously z. Three sections of the hippocampus corresponding to Fig. 1, were selected from each animal in a treatment group (n = 4), matched with corresponding sections from other animals, and analyzed blindly for densities of cells with nuclear GRir (number of cells per 10,000 /~m") at ×200 magnification with a Nikon Optiphot microscope equipped with ocular grids. Fig. 1 illus. trates how the ocular grids (50 × 200/~m) were positioned. Counts of GRir cells per region were pooled for both hemispheres in each animal, and mean densities of cells with nuclear GRir were calculated for each treatment group. The data were analyzed using one-way analysis of variance, and posthoc comparisons carried out using the Scheffe test. P < 0.05 was considered significant, Fig, 2 illustrates GRir in the pyramidal cell layer of CAI of the dorsal hippocampus of intact, adx, and adx rats treated with TNB. Following adx, there was a reduction in the density of cells with nuclear GRir in the pyramidal cell layer of fields CAI and CA3 of Ammon's horn, and the granular and polyrnorphic (CA4) layers of the dentate gyrus (compare Fig, 2A and B; Table i), Two hours treatment of adx rats with TNB did not alter densities in the above regions significantly (Fig. 2B and C; Table l), Treatment of adx rats with TNB for 1 week increased densities of GRir cells in the pyramidal cell layer of CAI and granular layer of the dentate gyrus (Fig. 2B and D; Table l), The location of GRir cells in both layers was suggestive of both interneurons and projection neurons. Type B GRir cells were observed in both adx and TNB-treated rats (Fig. 2B-D). The restoration of nuclear GRit in some pyramidal cells of CAI and granule cells of the dentate gyrus by TNB could have resulted from (i) direct interaction
A
B
%
•
Il
I
¢
#
/ f
Fig, I, Diagrams showing positions of ,~0x 2(11)pm ocular grids used
to assess deflsiti~sof cells with ,ucl~tlr GRtr, Br~gma- -2,.~5 mm in A, =3.30 mm in Battd ~4,8(1 mm in C, CAI, CA3: fields of Ammon's horm CA4: p~)lymorphic layer of dentate 8yrus: DG: granular layer of dentatu 8yrus,
between TNB and OR as has been described in liver cytosol and muscle, (ii) metabolism of TNB to cross-reacting species capable of binding to and regulating GRir, (iii) stimulation by TNB of the production of glucocorticoid.like material by the brain or gonads, capable of increasing nuclear GRir, and (iv) interaction between TNB-activated androgen receptors (AR) and GR with a resultant increase in GRit. Although the present study does not provide evidence for direct interaction between TNB and GR in the hippoeampus, cross-regulation of GRit and binding to GR by aldosterone 2''~ and progestins 5a2 has been demonstrated in other studies. It is significant that receptors for aidosterone, glucocorticoids, progestins and androgens have the highest degree of homology in amino acid sequence in their DNA and hormone-binding regions
313
~
.
9,
Fig, 2, Light micrographs showing GRir-pyrarnidal cells in CAI of the dorsal hippocampus, A: adrenally intact rat, Note the large number of cells with nuclear GRir. B: adx rat. The number of cells with nuclel~r GRir was reduced, Cells with cytoplasmic GRir were present (arrow). C: adx + 2 h TNB, The pattern of GRir was similar to B, D: adx + I week TNB, The number of cells with nuclear GRir was increased (compare to B and C), Cells with cytoplasmic GRir were present (arrow). TNB= a mixture of testosterone cypionate, nandrolone decanoate and boldenone undecylenate. Bar = 50/~rn.
TABLE i
Comparison of densities of cells with nuclear GRit in hippocampi o.f adrenally intact, adrenalectomized and TNB.treated rats Values represent mean densities of cells with nuclear GRir (number per I(),{]{)f)/zrn: :t:S.E.M). CAI, CA3: pyramidal cell layers of Amman's horn; CA4: polymorphic layer of dentate gyrus; DG: granular layer of dentate gyrus; Adx+TNB 2 h. adtenalectomized and treated fi~12 h with TNB; Adx+TNB 1 w: adrenalectomized and treated for 1 week with TNB. By Scheffe test, * P < 0.05 compared to intact; ~ P < 0.05 compared to Adx; t p < 0.05 compared to Adx + TNB 2 h.
Region
bztact
Adx
CAI CA3 CA4 DG
56.5+3.9 20.8+ 1.3 10.9 4- 0.5 47.4 :t: 1.6
16.6-1-1.1 10.4+0.7 8.3 + 0.6 I 1.8 4-0.9
* * * *
Adx + TNB 2 h
Adx + TNB 1 w
F¢.t:,o~
P
18.3+ 1.2 * 12.84-0.9 * 10.0 4. 0.5 12.9 + 0.8 *
29.4+ 1.3 ,~t 14.1 +0.8 * 10.4 4- 0.6 21.8 4. 1.3 * '~*
163.7 21.3 3.9 201.9
~ < <
311
BRES 25224
Regulation of glucocorticoid receptor immunoreactivity in the rat hippocampus by androgenic-anabolic steroids a
R e x f o r d S. A h i m a and
a,b
R i c h a r d E. H a r l a n
a Department of Anatomy and b Neuroscience Training Program, Tulane University Medical School, New Orleans, 1.,,4 70112 (USA) (Accepted 3 March 1992)
Key words: Androgenic-anabolic steroid; Glucocorticoid receptor; Hippocampus; Rat
To determine whether androgenic-anabolic steroids (AAS) regulate glucocorticoid receptors (GR), the effects of a mixture of three commonly abused AAS (testosterone cypionate, nandrolone decanoate and boldenone undecylenate) on GR immunoreactivity (Jr), were examined in hippocampi of adrenalectomized male rats. Treatment with AAS for 1 week increased nuclear GRir in pyramidal cells of CAI, and granule cells of the dentate gvrus. These findings suggest a role for GR in the mediation of some of the CNS effects of androgenic-anabolic steroids.
Anabolic steroids are among the most widely abused drugs in the United States 9. Since these steroids have androgenic properties, they are also referred to as androgenic-anabolic steroids (AAS). Apart from affecting muscle mass, AAS have several effects on CNS function, including affect and cognition, and neuroendocrine regulation ~'4'~. it has been suggested that these effects may be mediated by modulation of dopaminergic systems, and/or interaction between AAS and receptors for androgens, estrogens or glucocorticoids ~. In skeletal muscle, where the actions of AAS have been well documented, androgen receptors are saturated by normal circulating androgens. However, the amount of AAS used by athletes is in vast excess of available androgen receptors (AR), raising the question of the receptor system utilized by AAS. In skeletal muscle the actions of AAS are antagonized by glucocorticoids 9'!°. Moreover, because AAS bind competitively to glucocorticoid receptors (GR) in skeletal muscle !°, it is thought that the actions of AAS are mediated by GR m°. Binding of AAS to cytosolic OR has also been demonstrated in the liver ~5 and in HTC cells ~4. Although binding of AAS to GR has not been demonstrated in the CNS, in humans, high doses of anabolic steroids administered chronically have been shown to reduce plasma adrenocorticotropin (ACTH) levels 4'9. Cessation of treatment restores ACTH levels to normal 4'9.
This is akin to negative feedback regulation of ACTH by excess glucocorticoids, a process mediated at least in part by GR in the hippocampus I1. We therefore hypothesized that, as in skeletal muscle I° and liver Is, GR may mediate some of the effects of anabolic steroids in the CNS. To address this question we examined the regulation of GR immunoreactivity (ir) in the hippocampus by a mixture of three commonly abused anabolic steroids--testosterone cypionate, nandrolone decanoate and boldenone undecylenate '~. We will refer to this mixture as TNB. In adrenally intact male rats, pyramidal cells in Ammon's horn, and granule and polymorphic cells of the dentate gyrus show predominantly nuclear GRir I. We have previously described two types of responses of ORir in the hippocampus following adrenalectomy (adx) with or without corticosteroid treatment !. Most GRir cells in Ammon's horn and dentate gyrus showed a so-called Type A response; i.e. these neurons showed a predominantly nuclear GRir in intact rats and lost GRir following prolonged adx. Nuclear GRir was restored by treatment with corticosteroids. A subpopulation of neurons in the pyramidal cell layer of Ammon's horn and the stratum oriens, classified Type B, were not observed in intact rats, showed intense diffuse or cytoplasmic GRir following adx, and lost immunoreactivity following treatment with corticosterone. In the present study we
Correspondence: R.E. Harlan, Department of Anatomy, Tulane University Medical School, New Orleans, LA 70112, USA. Fax: (I) (504) 584-1687.
312 analyzed the effect of TNB treatment on the density of Type A and B neurons in hippocampi of adx male rats. Four adrenally intact, and twelve 2-week-adx male Sprague-Dawley rats were purchased from Charles River, housed under standard cage conditions with a 12 h light-12 h dark cycle and allowed free access to chow. Intact rats were allowed free access to tap water and adx rats to normal saline. The adx rats were divided into 3 groups. One group was treated with TNB (testosterone cypionate, 2 mg/kg; nandrolone, 2 mg/kg; boldenone, 1 mg/kg in 200 ~1 sesame oil i.m) for 2 h, the second, TNB daily for one week, and the third, vehicle only. The intact rats did not receive any treatment and were used as controls. The animals were anaesthesized with sodium pentobarbitai and sacrificed. Brains were dissected out and prepared for immunocytochemical detection of GR using BUGR2 antiGR monoclonal antibody as described previously z. Three sections of the hippocampus corresponding to Fig. 1, were selected from each animal in a treatment group (n = 4), matched with corresponding sections from other animals, and analyzed blindly for densities of cells with nuclear GRir (number of cells per 10,000 /~m") at ×200 magnification with a Nikon Optiphot microscope equipped with ocular grids. Fig. 1 illus. trates how the ocular grids (50 × 200/~m) were positioned. Counts of GRir cells per region were pooled for both hemispheres in each animal, and mean densities of cells with nuclear GRir were calculated for each treatment group. The data were analyzed using one-way analysis of variance, and posthoc comparisons carried out using the Scheffe test. P < 0.05 was considered significant, Fig, 2 illustrates GRir in the pyramidal cell layer of CAI of the dorsal hippocampus of intact, adx, and adx rats treated with TNB. Following adx, there was a reduction in the density of cells with nuclear GRir in the pyramidal cell layer of fields CAI and CA3 of Ammon's horn, and the granular and polyrnorphic (CA4) layers of the dentate gyrus (compare Fig, 2A and B; Table i), Two hours treatment of adx rats with TNB did not alter densities in the above regions significantly (Fig. 2B and C; Table l), Treatment of adx rats with TNB for 1 week increased densities of GRir cells in the pyramidal cell layer of CAI and granular layer of the dentate gyrus (Fig. 2B and D; Table l), The location of GRir cells in both layers was suggestive of both interneurons and projection neurons. Type B GRir cells were observed in both adx and TNB-treated rats (Fig. 2B-D). The restoration of nuclear GRit in some pyramidal cells of CAI and granule cells of the dentate gyrus by TNB could have resulted from (i) direct interaction
A
B
%
•
Il
I
¢
#
/ f
Fig, I, Diagrams showing positions of ,~0x 2(11)pm ocular grids used
to assess deflsiti~sof cells with ,ucl~tlr GRtr, Br~gma- -2,.~5 mm in A, =3.30 mm in Battd ~4,8(1 mm in C, CAI, CA3: fields of Ammon's horm CA4: p~)lymorphic layer of dentate 8yrus: DG: granular layer of dentatu 8yrus,
between TNB and OR as has been described in liver cytosol and muscle, (ii) metabolism of TNB to cross-reacting species capable of binding to and regulating GRir, (iii) stimulation by TNB of the production of glucocorticoid.like material by the brain or gonads, capable of increasing nuclear GRir, and (iv) interaction between TNB-activated androgen receptors (AR) and GR with a resultant increase in GRit. Although the present study does not provide evidence for direct interaction between TNB and GR in the hippoeampus, cross-regulation of GRit and binding to GR by aldosterone 2''~ and progestins 5a2 has been demonstrated in other studies. It is significant that receptors for aidosterone, glucocorticoids, progestins and androgens have the highest degree of homology in amino acid sequence in their DNA and hormone-binding regions
313
~
.
9,
Fig, 2, Light micrographs showing GRir-pyrarnidal cells in CAI of the dorsal hippocampus, A: adrenally intact rat, Note the large number of cells with nuclear GRir. B: adx rat. The number of cells with nuclel~r GRir was reduced, Cells with cytoplasmic GRir were present (arrow). C: adx + 2 h TNB, The pattern of GRir was similar to B, D: adx + I week TNB, The number of cells with nuclear GRir was increased (compare to B and C), Cells with cytoplasmic GRir were present (arrow). TNB= a mixture of testosterone cypionate, nandrolone decanoate and boldenone undecylenate. Bar = 50/~rn.
TABLE i
Comparison of densities of cells with nuclear GRit in hippocampi o.f adrenally intact, adrenalectomized and TNB.treated rats Values represent mean densities of cells with nuclear GRir (number per I(),{]{)f)/zrn: :t:S.E.M). CAI, CA3: pyramidal cell layers of Amman's horn; CA4: polymorphic layer of dentate gyrus; DG: granular layer of dentate gyrus; Adx+TNB 2 h. adtenalectomized and treated fi~12 h with TNB; Adx+TNB 1 w: adrenalectomized and treated for 1 week with TNB. By Scheffe test, * P < 0.05 compared to intact; ~ P < 0.05 compared to Adx; t p < 0.05 compared to Adx + TNB 2 h.
Region
bztact
Adx
CAI CA3 CA4 DG
56.5+3.9 20.8+ 1.3 10.9 4- 0.5 47.4 :t: 1.6
16.6-1-1.1 10.4+0.7 8.3 + 0.6 I 1.8 4-0.9
* * * *
Adx + TNB 2 h
Adx + TNB 1 w
F¢.t:,o~
P
18.3+ 1.2 * 12.84-0.9 * 10.0 4. 0.5 12.9 + 0.8 *
29.4+ 1.3 ,~t 14.1 +0.8 * 10.4 4- 0.6 21.8 4. 1.3 * '~*
163.7 21.3 3.9 201.9
~ < <
