Corticosteroid receptors in rat hippocampal sections: Effect of adrenalectomy and corticosterone replacement

.J. steroid Biochem. Vol. 24, No. 3, pp. 721-724, 1986 Printedin Great Britain.All rights reserved

Copyright c

0022-473 1186 $3.00 + 0.00 1986 PergamonPress Ltd

CORTICOSTEROID RECEPTORS IN RAT HIPPOCAMPAL SECTIONS: EFFECT OF ADRENALECTOMY AND CORTICOSTERONE REPLACEMENT ALAIN SARRIEALJ*, MICHELINE VIAL *, BRUCE MCEWENP,

MONIQUE DUSSAILLANT*,

DANIEL PHILIBERT:, and

WILLIAM

MARTINE

YVONNE BROER*.

MOGUILEWSKY$

ROSTENE*

*INSERM U.55, Centre de Recherches Saint-Antoine, 184, rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France, tThe Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A. and JLaboratoires Roussel-UCLAF, 102, Route de Noisy, 93230 Romainville, France (Received 9 A4ay 1985) Summary-Rat brain sections, located at the hippocampal level, were used to study the effect of bilateral adrenalectomy, with or without corticosterone treatment, on the number and affinity of corticosteroid binding sites. Adrenalectomy induces an increase of corticosterone receptor binding sites whereas adrenalectomy followed by in viva corticosterone treatment produces a 50% decrease of binding site number. Increases and decreases of binding site number were not associated with a significant modification of the affinity for corticosterone. The present data show that in VI’L’O corticosterone modulates its own number of binding sites demonstrated by in vifro binding on brain sections, in a manner which is reminiscent of changes in cytosol receptors demonstrated by conventional biochemical methods, Thus, this in vifro method provides an alternative way to study the plasticity of hippocampal glucocorticoid receptors.

INTRODUCTION

Many studies have shown that the limbic system, especially the hippocampus, was the target of corticosteroid actions in the central nervous system [l]. These actions need the binding of the adrenal steroid to specific intracellular receptors [2] and investigations demonstrated that the number of these binding sites were dependent upon the level of corticosteroid [3,4]. In the central nervous system (CNS) the possible up- and down-regulation of corticosteroid receptors is of fundamental importance since these brain receptors have been shown to be involved in the regulation of the hypothalamopituitary axis [5, 6, 71. The aim of the present study is to provide evidence that a methodology we recently developed to investigate the in vitro binding of adrenal steroids directly on rat brain sections [8, 91 coupled with autoradiographic localization of these binding sites in the rat CNS [lo], can be used to examine the modulation by adrenalectomy and corticosterone treatment of adrenal steroid binding sites.

secretion to circulating levels of adrenal cortical hormones have shown a constant increase of plasma ACTH from 4 to 21 days following bilateral adrenalectomy (ADX) in the rat [13]. Moreover, 4 hour-ADX was sufficient to suppress plasma corticosterone levels [14] without changes in the number of steroid binding sites in the CNS [3]. Thus, rats were bilaterally adrenalectomized for 4 h, 4, 14 and 21 days. After surgery, rats were maintained on a diet of Purina chow and 0.9% saline solution or supplemented with 100 pg/ml corticosterone in the drinking water according to Tornello et a1.[4]. Corticosterone treatment was discontinued 24 h before the sacrifice of the rats. Rats were killed by decapitation and the whole brain was quickly removed and frozen on microtome chucks. Thirty two pm frontal thick sections were prepared as previously described in detail [9]. Sections from A 4000 to A 1800 pm (containing the hippocampal formation) were selected according to the stereotaxic atlas of Konig and Klippel[lS]. Thymus weight was measured after decapitation to test the efficacy of adrenalectomy and corticosterone replacement [4].

EXPERIMENTAL

Binding and protein Preparation

of brain sections

assaJ,s

Binding parameters (& and f?,,,,,) were determined by incubating directly [3H]corticosterone ([jH]CS, 58 Ci/mmol, New England Nuclear) on brain sections as previously described [9] and expressed according to Scatchard analysis [16]. Briefly, sections were incu-

Adult male Wistar rats were used in order to avoid ontogenic-induced changes of glucocorticoid binding sites in the rat brain [l 1, 121. Previous studies on the feedback adjustment of adrenocorticotropin (ACTH) 721

122

Table 1. Etfects

ALAIN SARRIEAUet al. of adrenalectomy

and corticosterone-replacement on thymus weight, corticosteronemia (KJ of PH]CS binding Time

of ADX

4 days 4h Thymus weight (mg/l~ g body wt) Corrlcosterone (ng/ml plasma) & x IO-‘M

-CS

and apparent

297 & I3

390&9

280 i_ 21*

1.6 2 0.5 1.03 40.18

3.1 * 1.3 2.08 +_ 0.43

1.9 2 1.3 1.42_irO.81

-cs 389k

constant

.._

14 days +cs

dissociatxm

21 days +cs

15

224 i 10b

0.9 4 0.3 0.73 i 0.81

0.x $0.1 0.78 i: 0.08

-CS

+CS

286 _c 19

151 *4’

0.6 i_ 0. I -4 0.04

0.7 5 0.2 1.20 i_ 0 22

I .54

Rats were adrenalectomtzed for indicated times and received 0.9% saline solution (- CS) or saline solution and 100 @g/ml corticosterone (-t CS) in drinking water. Corticosterone ievels were determined by r~dioimmuno~s~ay as indicated in the text. 4 were performed accordmg to Scatchard anaiysis [ 161. a: P < 0.01 vs 4 days: b: I’ < 0.0 I vs 14 days; c: P < 0.01 YS 2 I days: n = 4.

bated at room temperature for 20 min with 300~1 buffer (50 mM Tris-HCI, pH 7.5, 2 mM EGTA, 5% glycerol, v/v, 6 mM molybdate, 5 mM ATP, 10 mM dithiotreitol) containing increasing concentrations of f3H]CS alone (total binding) or in the presence of an excess (5. lo-’ M) unlabeled CS (non specific binding). After incubation, sections were washed 3 x 5 min with 50 mM Tris-HCl (pH 7.5) at 4°C and removed with filter disks for scintillation counting [9]. Adjacent sections were used to determine total protein concentrations according to Bradford[l7~ using bovine serum albumin as the standard.

After decapitation, blood samples of rats treated or untreated with CS were collected in heparinized tubes to determine residual plasma corticosterone levels by radioimmunoassay according to the method of Krey et al.[lS] using an antiserum generated against cortisol 21-succinate bovine serum albumin (antisera F 21-53, Endocrine Sciences, Tarzona, Calif.. U.S.A.).

Fig. 1. Modification of corticosterone binding sites in rat hippocampus. Rats were adrenalectomized for indicated

times and supplied with 0.9% saline solution without (clear bars) or with 100#g/ml (dark bars) corticosterone in drinking water as indicated in Experimental. The number of binding sites was determined according to Scatchard[l6]. Each point is the mean + SEM of 3-4 experiments.

RESULTS

As shown in Table l* a significant difference can be noticed between thymus weight of rats supplied with saline solution alone or treated with ~OO~g~rnl CS. Corticosterone induces a decrease of thymus weight indicating the efficacy of the steroid treatment [4]. Under our experimental conditions, levels of plasma corticosterone are somewhat identical for all groups of rats (Table 1). The apparent dissociation constant (KJ, determined according to Scatchard[l(i] is quite similar in all experimental conditions (Table 1). In contrast, the number of binding sites (B,,,) is affected by adrenalectomy and corticosterone treatment as shown in Fig. 1. Bilateral adrenalectomy induces an increase in the number of CS binding sites which raises its maximal Ievel as soon as 4 days after surgery. The presence of corticosterone in drinking water produces a significant decrease of about 50% of CS binding sites. Scatchard plots of a representative experiment between 14 days ADX and ADX rats treated for the same time with CS in ciao, are shown on Fig. 2. Incorporation of [3H]leucine determined according to Cezard ed a/.(19] into proteins of brain sections is

0 006

ow4 I&

42 0002

0

Fig. 2. Scatchard representation of I)H]corticosterone binding. Rats were adrenalectomized for 14 days and maintained with 0.9% saline solution alone (e---0) or supplemented with lOOfig/ml corticosterone (O---AJ) for 13 days in drinking water as indicated in Experimental. Data for Scatchard plots were obtained from specific binding curves determined with increasing concentrations of PH]HCS (IO-’ to lo-* M) in the presence or not of an excess of unlabeled CS (5 10-” M). Three other experiments gave similar results.

Modulation

identical in treated or untreated studied (data not shown).

animals

of corticoid

at all times

DISCUSSION

The present paper describes the influence of adrenalectomy and corticosterone on the number of adrenal steroid binding sites in the Iimbic system on rat brain sections. Since corticosterone is near background level in blood and has no influence on the number of its binding sites in the hippocampus for 4 h following bilateral adrenalectomy [14, 31, this delay has been chosen as a control. The absence of the adrenal glands for longer periods induces an increase of [3H]CS binding sites whereas corticosterone, added in drinking water, induces about 50% decrease of the number of binding sites. In both cases, no significant modification of the affinity is observed. The decrease of binding sites for corticosterone-treated rats is not related to the presence of hormone in blood since corticosterone levels are near background levels indicating that cessation of hormone treatment 24 h before killing the animal is sufficient to eliminate plasma steroid (Table 1). Furthermore, Tornello et a/.[41 have shown that 24 h after the injection of [3H]corticosterone, radioactivity remaining in brain was reduced to background levels suggesting that brain receptors were no more occupied by the hormone; this indicates that corticosterone in drinking water, discontinued 24 h before killing the animals, cannot account for the decrease of corticosterone binding sites. The observation that [3H]leucine incorporation into proteins of rat brain sections is similar in treated or untreated rats (data not shown), suggests that upand down-regulation of corticosterone receptors by the steroid is not due to a general effect of corticosterone on total brain proteins but to a specific effect of the hormone on its own binding sites. Thus it appears that corticosterone is able to modulate the number of its own receptors in the rat central nervous system. Our data are in agreement with previous observations on cytosolic preparations showing up- and down-regulation of corticosteroid receptors following bilateral adrenalectomy and treatment with corticosterone, respectively [3,4]. Since we previously showed that the experimental conditions in which brain sections are incubated revealed binding of ‘HCS to cytosolic receptors [9], our data suggest that brain sections provide an alternative way to study cytosolic corticosteroid brain binding sites. Besides, this method demonstrates the same features of corticosteroid receptor localization, dynamics and physiological regulations as those obtained by more conventional steroid binding assays [3,4, 10,20,21]. Acknowledgements-The

authors wish to thank Mrs D. Lhenry for typing this manuscript and Mr Issoulie for

receptors

723

in hippocampus

photographic assistance. This work was supported from

Roussel-Uclaf

and INSERM

(PRC

by grants n”. 133044).

REFERENCES

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