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Benzodiazepine receptors in rat brain are altered by adrenalectomy
176
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Benzodiazepine-receptors in rat brain are altered by adrermlectomy E R R O L B. DE S O U Z A . NICK E G O E D E R S * and M I C H A E L J. K U H A R Neuroscience Branch, Addiction Research Center. National Institute on Drug A bus'e. Baltimore. MD 21224 • U. S,A.
( Accepted May 7lb. 1986) Key words." benzodiazepine receptor
adrenalectomy glucocorticoid adrenocorticotropic hormone (ACTH) - corticotropin-releasing factor (CRF) dexamethasone - - neurotransmitter receptor binding
The effects of adrenalectomy on benzodiazepine receptors in discrete regmns of rat brain were examined using |3H]flunitrazepam as a binding ligand. The concentration of benzodiazepine receptors was significantly increased by 25.50 and 71% in hippocampus, striaturn and hypothalamus, respectively, after adrenalectomy. In contrast, adrenalectomy did not affect the concentration of benzodiazepine receptors in cerebral cortex, olfactory bulb and cerebellum. No significant differences in the apparent binding affinity (K;t) value~ were seen following adrenalectomy in any brain region examined, The adrenalectomy-induced increases in [3H]flunitrazepam binding sites were completely reversed by glucocorticoid replacement with dexamethasone. These results demonstrate that adrenalectomv is capable of selectively modulating benzodiazepine receptors in brain regions presumably involved with gtucocorticoid negative feedback. The data further suggest additiona] mechanisms by which endogenous hypothalamic-pltuitary-adrenocorticalhormones may affect 'anxiety' levels.
Stress is associated with a wide variety of physiological and behavioral responses that are characteristic of Selye's "general a d a p t a t i o n s y n d r o m e '36. The m a j o r endocrine response to stress involves stimulation of the h y p o t h a l a m i c - p i t u i t a r y - a d r e n o c o r t i c a l ( H P A ) axis resulting in c o o r d i n a t e d secretion of corticotropin-releasing factor ( C R F ) , a d r e n o c o r t i c o t r o pic h o r m o n e ( A C T H ) , and glucocorticoid, respectively. A variety of data o b t a i n e d in clinical and laboratory settings have d e m o n s t r a t e d that while the fl-carbolines, a class of active b e n z o d i a z e p i n e receptor 'inverse agonists', elicit behavioral and neuroendocrine changes reminiscent of anxiety or stress t2"23"31, b e n z o d i a z e p m e r e c e p t o r agonists such as d i a z e p a m can alleviate anxiety and inhibit stressinduced increases m p i t u i t a r y - a d r e n o c o r t i c a l hormone secretion t'ts'19. A m o r e direct interaction between benzodiazepines and the H P A axis may extst since administration o f C R F 6. A C T H 13'30'4°. and corticosterone .4'2°'21 can alter b e h a v i o r in different animal models of anxiety and these behavioral effects of
the H P A h o r m o n e s can be reversed bv the benzodiazepines 6.t3.14.2~ Benzodiazepine receptors, part of a complex consisting of recognition sites for y-aminobutyric acid (GABA) and an associated chloride i o n o p h o r e with multiple allosteric binding sites 3:'3s. are heterogenously distributed throughout the central nervous system 42 Glucocorticoids have been shown to modulate m vitro GAI~,a ' - and b e n z o d t a z e p m e - receptors and G A B A - m e d i a t e d chloride flux 33 in brain. In addition, a d r e n a l e c t o m y - i n d u c e d changes in [3H]GABAt6 and [3H]muscimolZ4 binding in selective brain regions have b e e n r e p o r t e d . In the present study, we e x a m i n e d the effect of a d r e n a l e c t o m y and glucocorticoid replacement on benzodiazepine receptor binding in various regions of the rat b r a i n . The results d e m o n s t r a t e that a d r e n a l e c t o m y is c a p a b l e of selectively modulating b e n z o d i a z e p i n e receptors in brain areas associated with glucocorticoid negative feedback. F u r t h e r m o r e . these d a t a suggest additional mechanisms by which e n d o g e n o u s H P A h o r m o n e s
" Present address: Department of Pharmacology and Therapeuucs and Psychiatry, Louisiana State University Medical Center, Shreveport, LA 71130, U.S.A. Correspondence." E.B. De Souza. Neuroscience Branch. NIDA Addiction Research Center. P.O. Box 518/), Baltimore, MD 21224. U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. ~Biomedical Division ~
177 may affect anxiety levels under physiological and pathological conditions. Adult male S p r a g u e - D a w l e y rats (Madison, W1) weighing 200-250 g were adrenalectomized bilaterally through two dorsal incisions under ether anesthesia. Sham bilateral adrenalectomy was performed in a similar manner with muscle separation, but the adrenals were not handled. Rats were housed in a room with regular light-dark cycles and received food and water ad libitum, and adrenalectomized animals were provided with 0.9% saline. Two studies (Studies 1 and 2) were carried out in which rats were adrenalectomized, or sham-operated 14 days prior to sacrifice. In a third study, groups of rats were adrenalectomized or sham-operated 24 days prior to sacrifice and received daily subcutaneous injections of either dexamethasone phosphate (Lypho-Med. Inc., Chicago, IL) (0.25 mg/kg) as free base or saline vehicle (1 ml/kg) for 7 days prior to sacrifice. All rats were sacrificed by decapitation, and following dissection, the brain regions were frozen on dry ice and stored at - 7 0 °C. In vitro benzodiazepine receptor binding was studied under standard conditions 4'5. Briefly, the frozen tissue was weighed and homogenized, washed 5 times to remove all the endogenous G A B A , and in-
cubated at 4 °C for 90 min with [3H]flunitrazepam (81.5 Ci/mmol; NEN) (5 x 10 l0 M final concentration) and unlabeled flunitrazepam (5 x 10 -l° to 10-6 M final concentration) in 1 ml of buffer (50 mM TrisHCI, pH 7.7 at 22 °C) at a final tissue concentration of 1.5 mg/ml original wet weight. The final protein concentration of each membrane preparation was determined by the method of Lowry -'2 using bovine serum albumin as a standard. Assays were terminated by filtration. The radioligand binding data obtained from individual displacement curves of [3H]flunitrazepam by unlabeled flunitrazepam were analyzed by the computer program ' E B D A '2s which provides estimates of equilibrium binding parameters by Scatchard, Hill and E a d i e - H o f s t e e analysis. The data from all studies were analyzed using Student's ttest, one-way analysis of variance and Duncan's multiple range test ]7. Saturation studies were carried out with [3H]flunitrazepam to characterize changes in benzodiazepine receptors in discrete brain regions resulting from the treatment conditions. The maximum concentration of benzodiazepine receptors (B~,x) was increased significantly by 25, 50 and 71% in hippocampus, striatum and hypothalamus, respectively, after adrenalectomy when compared to the concentration of
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Fig. 1. Effects of adrenalectomy on [3H]flunitrazepam binding in (A) hypothalamus and (B) striatum. Data points are the mean of triplicate incubations in a representative experiment which varied by less than 1(1%.The cumulative data are summarized in Table I. Note that adrenalectomy alters the maximum concentration (B,n~×)of [3H]flunitrazepam binding sites without significantly affecting the affinity (Kj) of the ligand for its receptor.
178 effects of dexamethasone were noted on either the affinity or concentration of [3H]flunitrazepam binding sites (data not shown). In another study -~5conicosterone and, t o a much lesser extent, dexamethasone potentiated [3H]flunitrazepam binding to crude svnaptosomes when the binding was carried out at 37 C The differentia) effects of dexamethasone in the two studies may relate to different incubation temperatures and/or the brain regton used in the binding as-
receptors in corresponding brain regions of sham-operated control rats (Fig. 1 and Table I). In contrast, adrenalectomy did not significantly alter the concentration of [3H]flunitrazepam binding sites in other brain areas (cerebra) cortex, olfactory bulb and cerebellum) that have high concentrations of benzodiazepine receptors. No significant differences in the apparent binding affinity (Kj) values were seen following adrenalectomy in any brain region examined (Table I). In order to examine if the increase in the concentration of benzodiazepine receptors in hypothalamus, hippocampus and striatum was a consequence of reduced levels of circulating glucocorticoid in adrenalectomized rats, we replaced the lost glucocorticoid with dexamethasone. The regimen of in vivo dexamethasone treatment used in this study did not significantly alter [3H]flunitrazepam binding (Kd or Bmax) in sham-operated rats. but completely reversed the adrenalectomy-induced increase in benzodiazepine receptors (B~a0 in the striatum, hypothalamus and hippocampus (Table I). To examine any possible direct interaction between glucocomcoids and benzodiazepine receptors, we examined the in vitro effects of increasing concentrations of dexamethasone (10 -~ to 10-5 M) on [3H]flunitrazepam binding at 4 °C in homogenates of rat striatum obtained from intact, non-operated control rats. Using the conditions described above, no significant in vitro
sav.
These results represent the first demonstrauon of adrenalectomy-induced changes m benzodiazepine receptors in discrete areas of rat brain: the adrenalectomv-induced changes were completely reversed by glucocorticoid rep)acemem with dexamethasone, Previous data have demonstrated adrenalectomy-induced alterations in other components of the benzodiazepine-GABA receptor complex 1~''2425. Specifically, adrenalectomy decreased binding to high-affinity G A B A receptors in cerebral cortex, cerebellum. thalamus and hippocampus 24 while increasing binding to low-affinity G A B A receptors m midbrain and striatum~% and increasing high-affinity G A B A transport in the hippocampus, but nol frontal cortex, of adrenalectomized rats 29"3~. G A B A has been shown to increase binding of benzodiazepine receptors through an interaction inw~lving low-affinity G A B A receptors 7'~2'3s. In preliminary experiments.
"FABLE I S u m m a r y o f the effects o f adrenalectomy and glucocorticoid replacement on benzodiazelnne receptors in discrete areus ot rat brain T h e pooled data from Studies 1-3 are presented. Values represent the m e a n z S.E.M. of the affinity (Ka) and m a x t m u m concentration (Bin,x) of benzodiazepine receptors obtained from Scatchard plots of [3H]flunitrazepam binding in individual brain areas from experimental groups of 6 - 8 rats/group. T h e Bmax values are expressed as a percent of the m e a n concentration of [3Hlflunitrazepam binding sites in s h a m - o p e r a t e d control rats in each experiment. The Bmax values t fmol/mg protein: m e a n _-2 S.E.M. ~ in sham-operated rats from a representative experiment were 1594 _+ 9 in cortex. 534 _+ 35 in striatum 997 z 93 in hippocampus. 479 ± 55 in hypothalamus. and 3583 _+ 123 in olfactory bulb. In Studies 1 and 2. rats were sham-operated ( S H A M I or bilaterally adrenalectomized [ A D R E X ) 14 days before sacrifice. In Study 3, rats were operated 24 days before sacrifice and received daily subcutaneous m j e o ions of either saline (SAL) (1 ml/kg) or d e x a m e t h a s o n e ( D E X ) (0.25 mg/kg) for "~days prior to sacrifice Brain region
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Fig. 2. EffEcts of adrenalectomy and glucocorticoid replacement on the concentrations of benzodiazepine receptors in (A) hypothalamus (upper panel) and (B) striatum (lower panel). Rats were sham-operated (SHAM) or bilaterally adrenalectomized (ADREX) 24 days before sacrifice and received subcutaneous daily injections of either saline (SAL) 1 ml/kg, or dexamethasone (DEX) 0.25 mg/kg for 7 days before sacrifice. Data represent the maximum concentration (Bmax)of [3H]flunitrazepam binding sites: mean +_S.E.M. (n = 6 rats/group). * Significant difference at P < 0.01 from all other groups.
we found that G A B A ratios 37 were altered in adrenalectomized rats; the changes were found in striaturn, hypothalamus and hippocampus but not in cerebellum or cerebral cortex (data not shown). Thus glucocorticoids may affect coupling of components of the G A B A - b e n z o d i a z e p i n e receptor complex. The effects of adrenalectomy to increase [3H]flunitrazepam binding were not diffused but rather were confined to brain areas (striatum, hippocampus and hypothalamus) that have high concentrations of steroid receptors and may be involved in glucocorticoid negative feedback ~'~1'26.27. Since glucocorticoids are potent inhibitors of protein synthesis 41, the adre-
nalectomy-induced increases in benzodiazepine receptors may result in part from increased protein synthesis in specific cell populations in hippocampus, hypothalamus and striatum resulting from decreased levels of endogenous corticosterone. Although the anatomical evidence discussed above may suggest that the adrenalectomy-induced increases in [3Hlflunitrazepam binding in our study were related to the direct action of glucocorticoids in brain, it is possible that the effect may be a consequence of elevated levels of A C T H that are induced by adrenalectomy and reversed by glucocorticoids. A C T H is known to influence brain neurochemistry and function 31<15. Systemic administration of ACTHL_ > or ACTHa_ m can induce in rats an increase in low-affinity brain G A B A receptor binding similar to that observed after adrenalectomy 1~'. Pharmacological and physiological studies using a variety of animal models have examined the roles of A C T H and corticosterone in behavioral test procedures used to evaluate the 'anxiolytic" and 'anxiogenic' effects of benzodiazepines and fl-carbolines, respectively. In the social interaction test of anxiety, administration of A C T H and an "anxiogenic' effect which was counteracted by chronic administration of chlordiazepoxidel3; the decrease in social interaction was not a consequence of A C T H stimulated corticosterone release since corticosterone administration not only failed to mimic the 'anxiogenic" effect of A C T H but had significant 'anxiolytic" actions 14. On the basis of the avoidance performance of adrenalectomized and hypophysectomized rats, it has been suggested that A C T H increases excitability while corticosterone counteracts this influence because it acts to restore a normal level of activity 4°. In conflict procedures, neither adrenalectomy nor hypophysectomy alone affected conflict responding, but both these treatments greatly potentiated the anti-conflict effect of diazepam 21. Conversely, exogenous administration of corticosterone antagonized the potentiated anti-conflict actions of diazepam 21. Our data demonstrating an upregulation of benzodiazepine receptors in brain provide a mechanism whereby the anti-conflict effects of benzodiazepine agonists may be enhanced in adrenalectomized rats. Evidence from clinical studies suggests a good correlation between the degree of anxiety in humans and in plasma concentrations of cortisol >35. Increase in circulating cortisol, whether endogenously pro-
180 d u c e d b y stress o r p a t h o l o g i c a l s t a t e s s u c h as C u s h -
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c o m p l e x s u g g e s t t h a t t h e efficacy o f b e n z o d i a z e p i n e
tion. T h i s s t u d y was s u p p o r t e d tw U S P H S
a n x i o l y t i c s m a y b e a f f e c t e d in clinical s t a t e s o f alter-
M H 25951 a n d M H 0 9 1 1 t . a n d by g r a n t s f r o m t h e U p -
ed pituitary-adrenocortical hormone secretion. Fur-
john Company and the McKnight Foundation.
1 Barlow, S.M.. Knight, A.G. and Sullivan, F.M., Plasma corticosterone responses to stress following chronic oral administration of diazepam in the rat, J. Pharm. Pharmacol.. 31 (1979) 23-26. 2 Berger, F.M., Effect of antianxiety drugs on fear and stress, Behav. Sci., 25 (1980) 315-325. 3 Bohus, B., Effect of ACTH-like neuropeptides on animal behavior and man, Pharmacology, 18 (1979) 113-122. 4 Braestrup, C. and Squires, R.F., Specific benzodiazepine receptors in rat brain characterized by high-affinity, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3805-3809. 5 Braestrup, C. and Squires, R.F., Pharmacological characterization of benzodiazepine receptors in the brain, Eur. J. Pharmacol., 48 (1978) 263-270. 6 Britton, K,T., Morgan, J., Rivier, J., Vale, W. and Koob, G.F., Chlordiazepoxide attenuates response suppression induced by corticotropin-releasing factor in the conflic! test, Psychopharmacology, 86 (1985) 170-174. 7 Browner, M., Ferkany, J.W. and Enna, S.J., Biochemical identification of pharmacologically and functionally distinct GABA receptors in rat brain, J. Neurosci., 1 (1981) 514-518. 8 Cleghorn, R.A., Steroid hormones in relation to neuropsychiatric disorders. In H. Hoagland (Ed.), Hormones, Brain Function and Behavior. Academic Press, New York, 1957, pp. 3-25. 9 Dallman, M. and Yates, F.. Anatomical and functional mapping of central neural input and feedback pathways of the adrenocortical system, Mem. Soc. Endocrinol., 17 (1968) 39-72. 10 DeWied, D., Witter, A. and Greven, H.M., Behaviorally active ACTH analogues, Biochem. Pharmacol., 24 (1975) 1453-1468. 11 Feldman, S., Neurophysiological changes in the limbic system related to adrenocortical function. In E. Endroczi (Ed.), Integrative Neurohumoral Mechanisms, Elsevier, Amsterdam, 1983, pp. 173-188. 12 File, S.E., Lister, R.G. and Nutt, D.J., The anxio?,enic action of benzodiazepine antagonists, Neurophurmacology, 21 (1982) 1033-1037. 13 File, S.E, and Vellucci, S,V., Studies on the role of ACTH and of 5-HT in anxiety, using an animal model, J. Pharm. Pharmacol., 30 (1978) 105-110. 14 File, S.E., Vellucci, S.V. and Wendland, S., Corticosterone - - an anxiogenic or an an×iolytic agent, J. Pharm. Pharmaeol., 31 (1979) 300-305.
15 Fuxc, K.. Corrodi. H.. Hokfelt, 1", and Jonsson. ().. ( e n tral monoamine neurons m pituitary-adrenal activity. In D, DeWied and J.A.W.M, Weijen (Eds.). Pituitary. Adrenal and the Brain. Progress in Bruin Research. Vol. 32, Elsevier. Amsterdam, 1970. pp. 42-5 t, 16 Kendall, D.A., McEwen. B.S. and E,nna, S.J.. The influence of ACTH and corticosterone on [~H]GABA receptor binding in rat brain. Brain Research 236 (19821 365-374. t7 Klugh, H.E.. Statistics. Wiley, New York. 1971). 18 Lahti. R.A. and Borshun. G.. Effects of various doses oI minor tranquilizers on plasma corticosterone in stressed rats. Res. Commun. Chem. Path Pharmacol. 11 ¢t975) 595-N)3. 19 Le Fur. G.. Mitrani. G.N., Mizoule, J. and Uzan. A., Relationship between plasma corticosteroids and benzodiazcpines in stress J. Pharmacol. Exp Ther., 211 (I979) 3(t5-308 20 Levitt, E.E.. Persky, It., Brady, J.P, and Fitzgerald. J.A., The effect ol hydrocortisone infusion on hypnotically induced anxiety. Psychosom. Med.. 25 ( I963 ) 158-161. 21 Lippa, A,S.. Greenblatt, E.N. and Pelham. R.W., The use of animal models for delineating the mechanisms of action of anxiolytic agent. In I. Hanin and E Usdin /Eds.), Amreal Models in Psychiatry and Neurology, Pergamon. Ne~ York. 1977. pp. 279-292. 22 Lowry, O.H., Rosebrough, N.J,. Farr, A.L. and Randall. R.J.. Protein measurement with the Folin phenol reagent. J. Biol. Chem.. 193 (1951 p265-275. 23 Maiewski. S.F., Larscheid_ P., Cook .I.M. and Mueller. G.P.. Evidence that a benzodiazepme receptor mechanism regulates the secretion of pituitary/3-endorphin in rats. Endocrinology, 117 (19851 474-480. 24 Majewska. M.D.. Bisserbe. 1.-C and Eskay. R.L., Glucocorticoids are modulators of OABA,~ receptors in brain. Brain Research. 339 [ 1985) 178-182. 25 Majewska, M.D., Skolnick, P. and Paul. S.M.. Pregncnolone and corticosterone: possible modulators of the GABA/benzodiazepine receptor complex. Soc. Neurosc~. Abstr.. 11 (1985) 279. 26 McEwen. B.S., Glucocorticoids and hippocampus: receptors m search of the function. In D. Ganten and D, Pfaff (Eds.), Adrenal Actions on Brain. Springer, Berlin. 1982. pp. 1--22. 27 McEwen. B.S.. Davis. P.G., Parsons, B. and Pfaff, D,W,. The brain as a targel for steriod hormone action. Annu Rev. Neurosci,. 2 (1979) 65-112
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Braitl Rcw~;t< J,,. ~gl ! i98¢)) ]7~> I~l
El~ev'icr
BRE 21731
Benzodiazepine-receptors in rat brain are altered by adrermlectomy E R R O L B. DE S O U Z A . NICK E G O E D E R S * and M I C H A E L J. K U H A R Neuroscience Branch, Addiction Research Center. National Institute on Drug A bus'e. Baltimore. MD 21224 • U. S,A.
( Accepted May 7lb. 1986) Key words." benzodiazepine receptor
adrenalectomy glucocorticoid adrenocorticotropic hormone (ACTH) - corticotropin-releasing factor (CRF) dexamethasone - - neurotransmitter receptor binding
The effects of adrenalectomy on benzodiazepine receptors in discrete regmns of rat brain were examined using |3H]flunitrazepam as a binding ligand. The concentration of benzodiazepine receptors was significantly increased by 25.50 and 71% in hippocampus, striaturn and hypothalamus, respectively, after adrenalectomy. In contrast, adrenalectomy did not affect the concentration of benzodiazepine receptors in cerebral cortex, olfactory bulb and cerebellum. No significant differences in the apparent binding affinity (K;t) value~ were seen following adrenalectomy in any brain region examined, The adrenalectomy-induced increases in [3H]flunitrazepam binding sites were completely reversed by glucocorticoid replacement with dexamethasone. These results demonstrate that adrenalectomv is capable of selectively modulating benzodiazepine receptors in brain regions presumably involved with gtucocorticoid negative feedback. The data further suggest additiona] mechanisms by which endogenous hypothalamic-pltuitary-adrenocorticalhormones may affect 'anxiety' levels.
Stress is associated with a wide variety of physiological and behavioral responses that are characteristic of Selye's "general a d a p t a t i o n s y n d r o m e '36. The m a j o r endocrine response to stress involves stimulation of the h y p o t h a l a m i c - p i t u i t a r y - a d r e n o c o r t i c a l ( H P A ) axis resulting in c o o r d i n a t e d secretion of corticotropin-releasing factor ( C R F ) , a d r e n o c o r t i c o t r o pic h o r m o n e ( A C T H ) , and glucocorticoid, respectively. A variety of data o b t a i n e d in clinical and laboratory settings have d e m o n s t r a t e d that while the fl-carbolines, a class of active b e n z o d i a z e p i n e receptor 'inverse agonists', elicit behavioral and neuroendocrine changes reminiscent of anxiety or stress t2"23"31, b e n z o d i a z e p m e r e c e p t o r agonists such as d i a z e p a m can alleviate anxiety and inhibit stressinduced increases m p i t u i t a r y - a d r e n o c o r t i c a l hormone secretion t'ts'19. A m o r e direct interaction between benzodiazepines and the H P A axis may extst since administration o f C R F 6. A C T H 13'30'4°. and corticosterone .4'2°'21 can alter b e h a v i o r in different animal models of anxiety and these behavioral effects of
the H P A h o r m o n e s can be reversed bv the benzodiazepines 6.t3.14.2~ Benzodiazepine receptors, part of a complex consisting of recognition sites for y-aminobutyric acid (GABA) and an associated chloride i o n o p h o r e with multiple allosteric binding sites 3:'3s. are heterogenously distributed throughout the central nervous system 42 Glucocorticoids have been shown to modulate m vitro GAI~,a ' - and b e n z o d t a z e p m e - receptors and G A B A - m e d i a t e d chloride flux 33 in brain. In addition, a d r e n a l e c t o m y - i n d u c e d changes in [3H]GABAt6 and [3H]muscimolZ4 binding in selective brain regions have b e e n r e p o r t e d . In the present study, we e x a m i n e d the effect of a d r e n a l e c t o m y and glucocorticoid replacement on benzodiazepine receptor binding in various regions of the rat b r a i n . The results d e m o n s t r a t e that a d r e n a l e c t o m y is c a p a b l e of selectively modulating b e n z o d i a z e p i n e receptors in brain areas associated with glucocorticoid negative feedback. F u r t h e r m o r e . these d a t a suggest additional mechanisms by which e n d o g e n o u s H P A h o r m o n e s
" Present address: Department of Pharmacology and Therapeuucs and Psychiatry, Louisiana State University Medical Center, Shreveport, LA 71130, U.S.A. Correspondence." E.B. De Souza. Neuroscience Branch. NIDA Addiction Research Center. P.O. Box 518/), Baltimore, MD 21224. U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. ~Biomedical Division ~
177 may affect anxiety levels under physiological and pathological conditions. Adult male S p r a g u e - D a w l e y rats (Madison, W1) weighing 200-250 g were adrenalectomized bilaterally through two dorsal incisions under ether anesthesia. Sham bilateral adrenalectomy was performed in a similar manner with muscle separation, but the adrenals were not handled. Rats were housed in a room with regular light-dark cycles and received food and water ad libitum, and adrenalectomized animals were provided with 0.9% saline. Two studies (Studies 1 and 2) were carried out in which rats were adrenalectomized, or sham-operated 14 days prior to sacrifice. In a third study, groups of rats were adrenalectomized or sham-operated 24 days prior to sacrifice and received daily subcutaneous injections of either dexamethasone phosphate (Lypho-Med. Inc., Chicago, IL) (0.25 mg/kg) as free base or saline vehicle (1 ml/kg) for 7 days prior to sacrifice. All rats were sacrificed by decapitation, and following dissection, the brain regions were frozen on dry ice and stored at - 7 0 °C. In vitro benzodiazepine receptor binding was studied under standard conditions 4'5. Briefly, the frozen tissue was weighed and homogenized, washed 5 times to remove all the endogenous G A B A , and in-
cubated at 4 °C for 90 min with [3H]flunitrazepam (81.5 Ci/mmol; NEN) (5 x 10 l0 M final concentration) and unlabeled flunitrazepam (5 x 10 -l° to 10-6 M final concentration) in 1 ml of buffer (50 mM TrisHCI, pH 7.7 at 22 °C) at a final tissue concentration of 1.5 mg/ml original wet weight. The final protein concentration of each membrane preparation was determined by the method of Lowry -'2 using bovine serum albumin as a standard. Assays were terminated by filtration. The radioligand binding data obtained from individual displacement curves of [3H]flunitrazepam by unlabeled flunitrazepam were analyzed by the computer program ' E B D A '2s which provides estimates of equilibrium binding parameters by Scatchard, Hill and E a d i e - H o f s t e e analysis. The data from all studies were analyzed using Student's ttest, one-way analysis of variance and Duncan's multiple range test ]7. Saturation studies were carried out with [3H]flunitrazepam to characterize changes in benzodiazepine receptors in discrete brain regions resulting from the treatment conditions. The maximum concentration of benzodiazepine receptors (B~,x) was increased significantly by 25, 50 and 71% in hippocampus, striatum and hypothalamus, respectively, after adrenalectomy when compared to the concentration of
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Fig. 1. Effects of adrenalectomy on [3H]flunitrazepam binding in (A) hypothalamus and (B) striatum. Data points are the mean of triplicate incubations in a representative experiment which varied by less than 1(1%.The cumulative data are summarized in Table I. Note that adrenalectomy alters the maximum concentration (B,n~×)of [3H]flunitrazepam binding sites without significantly affecting the affinity (Kj) of the ligand for its receptor.
178 effects of dexamethasone were noted on either the affinity or concentration of [3H]flunitrazepam binding sites (data not shown). In another study -~5conicosterone and, t o a much lesser extent, dexamethasone potentiated [3H]flunitrazepam binding to crude svnaptosomes when the binding was carried out at 37 C The differentia) effects of dexamethasone in the two studies may relate to different incubation temperatures and/or the brain regton used in the binding as-
receptors in corresponding brain regions of sham-operated control rats (Fig. 1 and Table I). In contrast, adrenalectomy did not significantly alter the concentration of [3H]flunitrazepam binding sites in other brain areas (cerebra) cortex, olfactory bulb and cerebellum) that have high concentrations of benzodiazepine receptors. No significant differences in the apparent binding affinity (Kj) values were seen following adrenalectomy in any brain region examined (Table I). In order to examine if the increase in the concentration of benzodiazepine receptors in hypothalamus, hippocampus and striatum was a consequence of reduced levels of circulating glucocorticoid in adrenalectomized rats, we replaced the lost glucocorticoid with dexamethasone. The regimen of in vivo dexamethasone treatment used in this study did not significantly alter [3H]flunitrazepam binding (Kd or Bmax) in sham-operated rats. but completely reversed the adrenalectomy-induced increase in benzodiazepine receptors (B~a0 in the striatum, hypothalamus and hippocampus (Table I). To examine any possible direct interaction between glucocomcoids and benzodiazepine receptors, we examined the in vitro effects of increasing concentrations of dexamethasone (10 -~ to 10-5 M) on [3H]flunitrazepam binding at 4 °C in homogenates of rat striatum obtained from intact, non-operated control rats. Using the conditions described above, no significant in vitro
sav.
These results represent the first demonstrauon of adrenalectomy-induced changes m benzodiazepine receptors in discrete areas of rat brain: the adrenalectomv-induced changes were completely reversed by glucocorticoid rep)acemem with dexamethasone, Previous data have demonstrated adrenalectomy-induced alterations in other components of the benzodiazepine-GABA receptor complex 1~''2425. Specifically, adrenalectomy decreased binding to high-affinity G A B A receptors in cerebral cortex, cerebellum. thalamus and hippocampus 24 while increasing binding to low-affinity G A B A receptors m midbrain and striatum~% and increasing high-affinity G A B A transport in the hippocampus, but nol frontal cortex, of adrenalectomized rats 29"3~. G A B A has been shown to increase binding of benzodiazepine receptors through an interaction inw~lving low-affinity G A B A receptors 7'~2'3s. In preliminary experiments.
"FABLE I S u m m a r y o f the effects o f adrenalectomy and glucocorticoid replacement on benzodiazelnne receptors in discrete areus ot rat brain T h e pooled data from Studies 1-3 are presented. Values represent the m e a n z S.E.M. of the affinity (Ka) and m a x t m u m concentration (Bin,x) of benzodiazepine receptors obtained from Scatchard plots of [3H]flunitrazepam binding in individual brain areas from experimental groups of 6 - 8 rats/group. T h e Bmax values are expressed as a percent of the m e a n concentration of [3Hlflunitrazepam binding sites in s h a m - o p e r a t e d control rats in each experiment. The Bmax values t fmol/mg protein: m e a n _-2 S.E.M. ~ in sham-operated rats from a representative experiment were 1594 _+ 9 in cortex. 534 _+ 35 in striatum 997 z 93 in hippocampus. 479 ± 55 in hypothalamus. and 3583 _+ 123 in olfactory bulb. In Studies 1 and 2. rats were sham-operated ( S H A M I or bilaterally adrenalectomized [ A D R E X ) 14 days before sacrifice. In Study 3, rats were operated 24 days before sacrifice and received daily subcutaneous m j e o ions of either saline (SAL) (1 ml/kg) or d e x a m e t h a s o n e ( D E X ) (0.25 mg/kg) for "~days prior to sacrifice Brain region
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179 A. H Y P O T H A L A M U S 2000
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Fig. 2. EffEcts of adrenalectomy and glucocorticoid replacement on the concentrations of benzodiazepine receptors in (A) hypothalamus (upper panel) and (B) striatum (lower panel). Rats were sham-operated (SHAM) or bilaterally adrenalectomized (ADREX) 24 days before sacrifice and received subcutaneous daily injections of either saline (SAL) 1 ml/kg, or dexamethasone (DEX) 0.25 mg/kg for 7 days before sacrifice. Data represent the maximum concentration (Bmax)of [3H]flunitrazepam binding sites: mean +_S.E.M. (n = 6 rats/group). * Significant difference at P < 0.01 from all other groups.
we found that G A B A ratios 37 were altered in adrenalectomized rats; the changes were found in striaturn, hypothalamus and hippocampus but not in cerebellum or cerebral cortex (data not shown). Thus glucocorticoids may affect coupling of components of the G A B A - b e n z o d i a z e p i n e receptor complex. The effects of adrenalectomy to increase [3H]flunitrazepam binding were not diffused but rather were confined to brain areas (striatum, hippocampus and hypothalamus) that have high concentrations of steroid receptors and may be involved in glucocorticoid negative feedback ~'~1'26.27. Since glucocorticoids are potent inhibitors of protein synthesis 41, the adre-
nalectomy-induced increases in benzodiazepine receptors may result in part from increased protein synthesis in specific cell populations in hippocampus, hypothalamus and striatum resulting from decreased levels of endogenous corticosterone. Although the anatomical evidence discussed above may suggest that the adrenalectomy-induced increases in [3Hlflunitrazepam binding in our study were related to the direct action of glucocorticoids in brain, it is possible that the effect may be a consequence of elevated levels of A C T H that are induced by adrenalectomy and reversed by glucocorticoids. A C T H is known to influence brain neurochemistry and function 31<15. Systemic administration of ACTHL_ > or ACTHa_ m can induce in rats an increase in low-affinity brain G A B A receptor binding similar to that observed after adrenalectomy 1~'. Pharmacological and physiological studies using a variety of animal models have examined the roles of A C T H and corticosterone in behavioral test procedures used to evaluate the 'anxiolytic" and 'anxiogenic' effects of benzodiazepines and fl-carbolines, respectively. In the social interaction test of anxiety, administration of A C T H and an "anxiogenic' effect which was counteracted by chronic administration of chlordiazepoxidel3; the decrease in social interaction was not a consequence of A C T H stimulated corticosterone release since corticosterone administration not only failed to mimic the 'anxiogenic" effect of A C T H but had significant 'anxiolytic" actions 14. On the basis of the avoidance performance of adrenalectomized and hypophysectomized rats, it has been suggested that A C T H increases excitability while corticosterone counteracts this influence because it acts to restore a normal level of activity 4°. In conflict procedures, neither adrenalectomy nor hypophysectomy alone affected conflict responding, but both these treatments greatly potentiated the anti-conflict effect of diazepam 21. Conversely, exogenous administration of corticosterone antagonized the potentiated anti-conflict actions of diazepam 21. Our data demonstrating an upregulation of benzodiazepine receptors in brain provide a mechanism whereby the anti-conflict effects of benzodiazepine agonists may be enhanced in adrenalectomized rats. Evidence from clinical studies suggests a good correlation between the degree of anxiety in humans and in plasma concentrations of cortisol >35. Increase in circulating cortisol, whether endogenously pro-
180 d u c e d b y stress o r p a t h o l o g i c a l s t a t e s s u c h as C u s h -
ther studies on the precise mechanisnts bv which the
ings d i s e a s e , A d d i s o n ' s d i s e a s e , d e p r e s s i o n , or o t h e r
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psychiatric disorders, or exogenously administered,
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p s y c h o l o g i c a l c h a n g e s in a n x i e t y b e h a v i o r s w h i c h t~c-
ized by a s e n s e o f a p p r e h e n s i o n a n d f e a r s'2~'3'~. T h e
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c o m p l e x s u g g e s t t h a t t h e efficacy o f b e n z o d i a z e p i n e
tion. T h i s s t u d y was s u p p o r t e d tw U S P H S
a n x i o l y t i c s m a y b e a f f e c t e d in clinical s t a t e s o f alter-
M H 25951 a n d M H 0 9 1 1 t . a n d by g r a n t s f r o m t h e U p -
ed pituitary-adrenocortical hormone secretion. Fur-
john Company and the McKnight Foundation.
1 Barlow, S.M.. Knight, A.G. and Sullivan, F.M., Plasma corticosterone responses to stress following chronic oral administration of diazepam in the rat, J. Pharm. Pharmacol.. 31 (1979) 23-26. 2 Berger, F.M., Effect of antianxiety drugs on fear and stress, Behav. Sci., 25 (1980) 315-325. 3 Bohus, B., Effect of ACTH-like neuropeptides on animal behavior and man, Pharmacology, 18 (1979) 113-122. 4 Braestrup, C. and Squires, R.F., Specific benzodiazepine receptors in rat brain characterized by high-affinity, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3805-3809. 5 Braestrup, C. and Squires, R.F., Pharmacological characterization of benzodiazepine receptors in the brain, Eur. J. Pharmacol., 48 (1978) 263-270. 6 Britton, K,T., Morgan, J., Rivier, J., Vale, W. and Koob, G.F., Chlordiazepoxide attenuates response suppression induced by corticotropin-releasing factor in the conflic! test, Psychopharmacology, 86 (1985) 170-174. 7 Browner, M., Ferkany, J.W. and Enna, S.J., Biochemical identification of pharmacologically and functionally distinct GABA receptors in rat brain, J. Neurosci., 1 (1981) 514-518. 8 Cleghorn, R.A., Steroid hormones in relation to neuropsychiatric disorders. In H. Hoagland (Ed.), Hormones, Brain Function and Behavior. Academic Press, New York, 1957, pp. 3-25. 9 Dallman, M. and Yates, F.. Anatomical and functional mapping of central neural input and feedback pathways of the adrenocortical system, Mem. Soc. Endocrinol., 17 (1968) 39-72. 10 DeWied, D., Witter, A. and Greven, H.M., Behaviorally active ACTH analogues, Biochem. Pharmacol., 24 (1975) 1453-1468. 11 Feldman, S., Neurophysiological changes in the limbic system related to adrenocortical function. In E. Endroczi (Ed.), Integrative Neurohumoral Mechanisms, Elsevier, Amsterdam, 1983, pp. 173-188. 12 File, S.E., Lister, R.G. and Nutt, D.J., The anxio?,enic action of benzodiazepine antagonists, Neurophurmacology, 21 (1982) 1033-1037. 13 File, S.E, and Vellucci, S,V., Studies on the role of ACTH and of 5-HT in anxiety, using an animal model, J. Pharm. Pharmacol., 30 (1978) 105-110. 14 File, S.E., Vellucci, S.V. and Wendland, S., Corticosterone - - an anxiogenic or an an×iolytic agent, J. Pharm. Pharmaeol., 31 (1979) 300-305.
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Grants
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