Brain Research, 587 (1992) 313-318 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.0()

313

BRES 17989

Noradrenergic regulation of type-I and type-ll corticosteroid receptors in amygdala and hypothalamus Stefania Maccari ", Pier Vincenzo Piazza ~, Fran~oise R o u g e - P o n t ~, Luciano Angelucci b, Herv6 Simon ~ and Michel le M o a l a '* Laboratoire tie Psychobiologic tlt,s Comportements Adaptatifs, INSERM U259, Unirersitt: de Bordeaw: IL Bordema" (France) and h istiluto di Farmacologia Medica I!, Unirersit~ di Roma "La Sapienza ", Roma (Italy)

(Accepted 17 March 1992)

Key words: Hypothalamo; Amygdala: Corticosteroid receptor; 6-Hydroxydopamine lesion:, Noradrenaline

The effects of glucocorticoids on various brain functions including the negative feedback control of HPA axis are mediated by two types of receptor (type ! or mineralocorticoid and type !1 or glucocorticoid) in the central nervous system, Futhermore, noradrenergie systems have been showed to stimulate the activity of hypothalamo-pituitaw-adrenal (HPA) axis. The neural and receptor controls of HPA axis activity are generally thought to be independent, Although receptor numbers, especially type-ll receptors, are thought to be regulated by circulating levels of corticostemne, they may also be under direct neural control. Thus, it may be suggested that these two types of control are functionally related and that noradrenergic systems may affect HPA axis activity either directly or indirectly via change in receptor characteristics, A major problem in the interpretation of studies examining neurotransmitter regulation of corticosteroid receptors is that the effects of drugs or brain lesions on receptors levels may he secondaw to their effects on adrenocortical function, In order to demonstrate a neuronal control on corticosteroid receptors, we tested the effect of 6-hydroxydopamine lesion of noradrenergic systems in the pedunculus cerebellaris superior in adrenalectomized animals whose corticosterone levels were maintained within normal limits by corticosterone replacement implants, Both types of receptor were assayed in hypothalamus and 'lmygdala, We show tha~: (!) corticosteroid receptors are influenced by noradrenergic systems; (2) this effect depends o n the brain region and the receptor type. After the noradrenergic lesion type-I receptors were reduced in hypothalamus and amygdala, whereas type-II receptor w~rc only increased in hypothalamus while receptor affinities were unaltered, This finding may encourage fl|rther inw:stigations o n , possible distinct physiological role of the two type of receptors in different brain structures, Futhermore, since norudrenaline is activated during stress, these results may throw more light on the mechanisms controlling glucocorticoid secretion,

INTRODUCTION The activity of the hypothalamic-pituitary-adrenai (HPA) axis, culminating in secretion of glucocorticoids, plays an important role in the adaptive response of the organism to physical and psychological stimuli 13. Indeed, certain psychopathological states, such as depression, have been related to pituitary-adrenal cortical hyperactivity 22.4,~and to an attenuated corticosteroid feedback control 4.43.44. The activity of the HPA axis is modulated by the binding capacity of the central receptors for these hormones-~.10.30. Two types of glucocorticoid receptors have been described. Type-! receptors, mainly distributed in the iimbic structures and with a higher affinity for glucocorticoid~ than the typc-ll receptors

which are widely distributed throughout the brain 47. A low density of these receptors, found in aged subjects sc)..~.~ or experimentally induced by corticosterone treatment .~2 and repeated stress 2,,,~1,gives rise to a more sustained secretion of corticosterone under basal conditions or in response to stress. The aminergic systems, projecting to the hypothalamus i~,, have also been showed to modulate glucocorticoid secretion. Both serotonin .~.c,.~1,40,41,46 and noradrenaline i.~.27.4.~.~,~.~,3 stimulate the activity of HPA axis. Dopamine has also been reported to influence the HPA axis 21, although its role is unclear. The neural and receptor controls of HPA axis activity are generally thought to be independent. Corticosteroid receptors respond essentially to changes in plasma corticosterone levels and adjust their capacity

Correspondence: S. Maccari, INSERM U259, Rue Camille Saint Sa~ns, 33077 Bordeaux Cedex, France. Fax: (33) 56.96,68,93.

314

via an autoregulation mechanism ,~o.,~.s~.~.However, some reports indicate that the binding capacity of corticosteroid receptors may also be under a direct neural control t,:7,3~,ss. These two types of control may thus be functionally related and the neural system may affect HPA axis activity either directly or indirectly via a change in receptor characteristics. Unfortunately much of the evidence for neuronal regulation of these receptors L3q.5s is indirect because the effects of drugs or brain lesions on receptor levels may be secondary to their effects on adrenocortical function. Thus, serotoninergic 2.~.40 and noradrenergic lesions '~ which increase hippocampal corticosteroid receptor numbers also lead to an hypoactivity of HPA axis that it could be responsable of the corticosteroid receptors increase. We have recently shown that corticosteroid receptors may be regulated by noradrenergic activity independently of plasma corticosterone levels. in adrenalectomized rats with plasma corticosterone levels maintained stable by exogenous administration, a noradrenergie lesion was found to induce an increase in the number of type.I receptor along with a decrease in the K0 of type-ii receptors in the hippocampus :'~. In the present study, we investigated the effects of a noradrenergic (NA)lesion on corticosteroid receptors in the hypothalamus and amygdala in which plasma corticosterone levels were maintained stable by replacement therapy, This study has been performed in the same set of animals used in a previous work in

which hippocampal corticosteroid receptors were investigated -"J.The present work was prompted by three main considerations, First, both structures contain both receptor types ", Second, these structures are inner. rated by dorsal and ventral NA systems 3~.s.~.u~,f,2.Third, both the hypothalamus and amygdala are known to modulate cortieosterone secretion 12,1t~.54 and these structures are also anatomically related. Axonal transport studies suggest that direct inputs to CRH cell bodies of the paraventricular nucleus from the hippocampal formation and the amygdala, are funnelled through the ventral component of the septal region, the bed nucleus of the stria terminalis, the ventral amygdalofugal pathway. Futhermore, it has been shown that the hypothalamus givesrise to a prominent projection to the amygdaloid complex through the ventral amygdalofugal pathway and stria terminalis f". The results shown that (1) eorticosteroid receptors are influenced by noradrenergic systems; (2) this effect depends on the brain region and the receptor type. Indeed, type-I receptors were reduced in hypothalamus and amygdala, whereas type-ll receptor were only increased in hypothalamus while receptor affinities were unaltered after a NA lesion.

MATERIALS AND METHODS

Exla'rimental animals Twenty-eight male rats of the Wistar strain (lffa Credo, Lyon) were used in this experiment. The animals were housed in individual cages under controlled lighting (lights on from 06.00 h to 20.00 h) and temperature (23°C). Food and water were available ad libitum. The rats weighed 349 + 25 g at the end of the experiments. Lesion of the noradrenergic systems For the lesion of the noradrenergic systems, the rats were anesthetized with chloral hydrate (500 mg/kg i.p.) and placed in a stereotaxic apparatus with the incisor bar 5 mm above the interaural line. Injections were made in the pedunculus cerebellaris superior (PCS) according to the following coordinates: antero-posterior, -5.8 mm and lateral, + 1.4 mm from bregma; dorso-ventral, - 7 mm from the skull. Rats were injected bilaterally with a solution of 6-hydroxydopamine (6-OHDA; 4 p.g/~l) (n = 14) or with vehicle (NaCI 9 g/I containing ascorbic acid, 5 me/100 ml) (n = 14). The solution was injected slowly (3 min 20 s) and the cannulae were left in place for 2 rain to allow for drug diffusion with minimal withdrawal along the cannula paths. Adrenalectomy and corticosterone replacement therapy At the same time as the PCS lesion, the rats were adrenalectomized via the dorsal approach and implanted subcutaneously with solid corticosterone pellets to provide a continuous administration of this hormone 3~,. The solid 100 mg pellets contained 60 mg of corticosterone 21-hemisuccinate (Agar, Rome) adjusted to 100 mg with cholesterol. Plasma corticosterone levels released by corticosterone pellets were 6:1:0.9 #g/100 ml. in addition, the animals received 50 ~tg/ml of corticosterone 21-hemisuccinate in their drinking water which also contained 0,5% NaCI during the dark period in order to simulate the nocturnal corticosterone peak, Plasma corticoslerone levels induced by drinking water replacement were 12,8:t:3 ttg/l()0 ml, We have defined the animals submitted to replacement therapy as 'fixed corticosterone level' group, Twenty-one days after the PCS lesion and adrenalectomy with corticosterone replacement therapy, the pellets were removed (under ether anesthesia) in order to deplete receptor sites of steroids, Twenty-four hours after removal of pullets, the rats were anesthetized with pentobarbital (30 mg/kg), blood samples were taken to check for the absence of steroids and hypothalamus and amygdala were rapidly dissected and frozen on dp/ice, Tissues were stored at -80°C until receptor assay. Plasma corticosterone was measured by a radiocompetitive binding assay method after extraction into dichloromethane '~",

7~pe.l at~d tylx'.ll corticosteroid receptors The binding characteristics of cytosolic type-I and type-ll corticosteroid receptors of the hypothalamus and amygdala were determined on the pooled tissue of t~) rats, The tissue was homogenized in 2 ml of ice-cold 10 mM Tris-HCI (pH 7,4) containing 2 mM EDTA, 2 mM 2-mercaptoethanol, 10 mM sodium molybdate and 10%. glycerol and centrifuged (105,000 R, 15 min in a Beckman TEl(X) ultracentrifuge) at 0-2°C, The supernatant (cytosolic fraction) was taken for assay, For the type-I receptor assay, aliquots of cytosol (140 ttl) were incubated with tritiated corticosterone (['~H]corticosterone ([~H]B), 101,6 Ci/mmol, New England Nuclear) over a concentration range of 0,625-20 nM (six points for each Scatchard) and with a l(X)-foldexce~ of unlabeled R U 28362. Unlabeled R U 28.162was used to displace['~H]B from type-ll receptors,~4,Type-ll receptor binding was evaluated directlyusing pure glucocorticoid ['~H]RU 28362 (specific activity 78 Ci/mmol) over a concentration range of 0,625-20 nM (six points for each Scatchard), Binding equilibrium was reached after 3 h at 0°C, Non-specific binding (NSB) for the ['~H]B binding was determined in the presence of a 500.fold excess of unlabeled corticosterone and for [3H]RU 28362 was assessed in the presence of a 500-fold excess of unlabeled RU 28362. Bound and unbound ['~H]B or [3H]RU 28362 were separated on Sephadex LH-20 columns equilibrated with Tris-HCl buffer at 2°(:, using 60 ~I of the incubates and eluting with 940 #I of Tris-HCl. A

315 milliliter of the eluate containing the bound form was added to 3 ml of scintillation fluid (LipolumaR, Lumac) and radioactivity was counted. Protein concentration was determined according to Lowry et al. 25 using albumin as standard, The apparent maximum binding capacity (Bmax) of [3H]B or ['~H]RU 28362 and dissociation constants (K d) for both types of receptors were evaluated from Scatchard plots 5,

Neurochemical assessment of dee lesion Samples were obtained after the ultracentrifugation of hypothalamus and amygdala tissues (10 /zl of cytosol preparation). These aliquots were stored at -80°C. The amine concentrations were measured by high pressure liquid chromatography with electrochemical detection 35 Statistical analysis Corticosteroid receptor values are characterized by a log-normal distribution, Raw data were thus transformed to logarithmic scores before statistical analysis, Analysis of variance (ANOVA) for repeated measures was employed for amine levels and corticosteroid receptor numbers, The CrunchR statistical package (Crunch Software Co, Oakland, CA) was used throughout. Results are expressed as percentage of controls.

RESULTS

CORTICOSTEROIDRECEPTORSIN THE HYPOTHALAMUS OF ANIMALSWITH RXED CORTICOSTERONELEVEL r-1 SH~ Kd Bmox I

175

o~

150

~

125

~, Ld

too

~

75

~-

50

8

25

i

TYPE I

TYPE II

NA LESION

TYPE I

TYPE II

Fig. 2. Characterization of hypothalamic corticosteroid receptors three weeks after 6-OHDA lesion of the PCS in animals receiving corticosterone replacement. The maximal binding capacity (Bin,,x) of type-! receptors was lower in the lesioned group. The Bma" of type-ll corticosteroid receptors was higher in the lesioned group, in contrast, the apparent affinities (K d) of both types of receptors for corticosterone were unchanged. The results are represented as a percentage of control mean values. * P < 0.05

Neurochemical effects of 6.hydroxydopamine lesions 6-OHDA lesion of the PCS depleted noradrenaline in the hypothalamus (ANOVA Ft,i, = 67.23; P < 35

HYPOTHALAMUS 30

C '-

25

"~

30

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