Interaction between glutamate and GABA on 3H-noradrenaline release from rat hypothalamus

BrainResearchBulletin,Vol. 37, No. 2, pp. 119-122, 1995 Copyright © 1995ElsevierScienceLtd Printedin the USA.All rightsreserved 0361-9230/95 $9.50 + .00

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Interaction Between Glutamate and GABA on aH-Noradrenaline Release from Rat Hypothalamus CARLOS E. NAVARRO,1 RICARDO J. CABRERA AND ALFREDO O. DONOSO

Laboratorio de Investigaciones Cerebrales, LINCE-CONICET, Facultad de Ciencias Medicas, U.N. Cuyo, Casilla de Correo 425, 5500 Mendoza, Argentina [Received 13 June 1994; Accepted 21 October 1994]

ABSTRACT: Glutamate has been shown to stimulate noradrenaline (NA) release from hypothalamic nerve terminals. In the present study, we evaluated the possible interaction between the excitatory amino acid glutamate and gamma-aminobutyric acid (GABA), an inhibitory transmitter, on noradrenaline (NA) release from mediobasal hypothalamus (MBH) of adult male rats. Hypothalamic slices loaded in vitro with aH-NA were superfused and exposed to glutamate, N-methyI-D-aspartic acid (NMDA), or kainate (KA). We found that *H-NA release evoked by the excitatory amino acids glutamate and NMDA was dramatically decreased by GAB~ The facilitatory effects of NMDA and KA were prevented concentration-dependently by the GABAe receptor antagonist 2-hydroxy secinfen which restored the NMDA effect. In addition, baclofen blocked K+-induced *H-NA release. Activation of GAB-At receptors by muscimol and THIP was ineffective. In conclusion, glutamate and GABA, through GABAe receptons, may interact to modulate NA release from the rat mediobasel hypothslamus.

ulatory effects on luteinizing hormone-releasing hormone (LHRH) release to control pituitary gonadotropin secretion [1,3,6,7,11,15].

METHOD Animals and Assays Male Sprague-Dawley rats weighing 280-300 g were killed by decapitation. The brain was removed rapidly, and a tissue block 1 mm depth corresponding to the mediobasal hypothalamus was prepared. Sagital slices at 0.3 mm intervals were obtained. All details of methods, 3H-NA release assay, and calculations have been described prevously [16]. Briefly, slices from two rats (six to eight slices) were preloaded with 200 nM 3H-NA (40-50 Ci/mmol, New England Nuclear, Boston) in gassed Krebs-Ringer bicarbonate-glucose buffer (KRB) or MgE÷-free KRB (experiments with NMDA) at 37°(7. Then, slices were washed and transferred to perfusion chambers, and after a stabilizer period, the experiments were carried out. Glutamate, NMDA, and KA at concentrations close to their EC5o, determined previously [16], or a high concentration of K ÷ (28 mM KCI), were introduced in the superfusion system for a 7.5-min or 3min period (KA) after collection of five basal samples. The flow rate of perfusion for GLU and NMDA was 0.7 ml/min, and for KA, 2 ml/min. GABA, the GABA agonists muscimol, THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol), and baclofen, and the GABA antagonist 2-hydroxysaclofen, were added at the beginning of the fraction collection and maintained until the end of the experiment. At that time, slices were homogenized in 0.2 N perchloric acid, centrifuged, and 1 ml of clear supernatant was mixed with 8 ml of scintillation fluid. Radioactivity of collected fractions and tissue was determined, and percent of fractional release of 3H-NA was calculated. To quantify the effects of drugs on basal efflux, the results were expressed either as percent of tritium evoked release or percent of net release. The evoked tritium overflow from brain slices incubated with 3H-NA reflects the release of radioactively labeled and unlabeled NA from the noradrenergic nerve terminals [20].

KEY WORDS: Noradrenaline release, Hypothalamus, Glutamate, GABA interaction, N-mathyI-D-aspartate (NMDA), Kainate, Baclofen, GABAe agonists.

INTRODUCTION Excitatory amino acids stimulate noradrenaline (NA) release in several areas of the rat brain [8,21,22]. On the other hand, numerous data indicate that activation of GABAergic receptors results in the decrease of noradrenaline release [4,12,17,21 ]. We have recently shown [ 16] that release of NA from superfused slices of mediobasal hypothalamus is increased by glutamate and its ionotropic agonists N-methyl-Daspartic acid (NMDA) and kainate (KA). To the best of our knowledge, there is no evidence on probable interactions between GABA and glutamate affecting the noradrenergic system at the hypothalamus. Therefore, the aim of the present study was to investigate the effects of GABA and its agonists on the release of 3H-NA evoked by NMDA and KA from superfused mediobasal hypothalamic slices. These findings would help to know whether GABA and glutamate are functionally linked to modify noradrenaline release at the hypothalamic locus, a place where these transmitters may exert reg-

Drugs Glutamate and GABA were purchased from Sigma (St. Louis, MO), and the glutamate and GABA agonists from Research Biochemicals Inc. (Natick, MA).

J To whom requests for reprints should be addressed. 119

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N A V A R R O , C A B R E R A AND D O N O S O

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FIG. 1. Inhibition by 20 nM GABA of ~H-NA release evoked by 40 mM glutamate (GLU) or 200 #M NMDA from superfused medioobasal hypothalamic slices. GABA was applied at the beginning of fraction collection and the excitatory amino acids (EAA) for 7.5 min after collection of five basal fractions . Each column represents the mean _+ SEM of seven chambers. ***p < 0.01 vs. EAA alone.

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FIG. 3. Inhibitory dose-related action of baclofen (BAC) on NMDAevoked release of 3H-NA from mediobasal hypothalamic slices, and its reversal by 2-OH-Saclofen (SAC). Baclofen (200/zM) or baclofen plus 2-OH-saclofen (300/~M) were applied at the beginning of fraction collection, and NMDA for 7.5 min after collection of five basal fractions. Each column represents the mean _+ SEM of seven chambers.

were compared with analysis of variance (ANOVA, one-way) and post hoc D u n c a n ' s new multiple range test. The concentrat i o n - r e s p o n s e curve for baclofen was fitted to the data points and IC50 value was calculated using Graph Pad Inplot 4.03 (Graph Pad Software Inc., San Diego, CA). Differences between means with a p < 0.05 were considered significant.

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As illustrated in Fig. 1, the presence of G A B A (20 mM) in the superfusion fluid significantly reduced the enhanced tritium efflux evoked by glutamate (40 m M ) or N M D A (200 # M ) from mediobasal hypothalamic slices. Responses of noradrenaline to N M D A were unaltered by either muscimol (100 #1) or THIP (I 00 #1), two GABAA agonists (Fig. 2). In contrast, baclofen, a G A B A a agonist, restrained in a dose-dependent manner (IC50 = 80 # M ) the effect of N M D A and hindered the effect of KA (200 # M ) (Figs. 3 and 4). G A B A a receptor-mediated inhibition was determined with 2-hydroxysaclofen, a GABAB selective antagonist. In the experiment illustrated in Fig. 3, 2-hydroxysaclofen (300 # M ) added to baclofen (200/.tM) restored N M D A stimulation of 3H-NA release. Both baclofen and muscimol (10, 20, and 100 # M ) lacked effect by themselves on basal 3H-NA re-

GLUTAMATE-GABA LINK AND NORADRENALINE RELEASE

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tors of the inhibitory effect of GABA on glutamate-evoked 3HNA release but, in contrast, in the participation of GABAA receptors it is quite unlikely. As is shown, 3H-NA release evoked by NMDA was not impaired by muscimol and THIP. The somatodendritic postsynaptic sites seem to be the predominant location of amino acid receptors, albeit both glutamatergic and GABAergic presynaptic receptors have also been described in noradrenergic nerve endings in the brain [4,9,10,13,18,21]. In the mediobasai hypothalamus, the presence of presynaptic receptors in NA nerve terminals is supported by the evidence that only noradrenergic axon terminals projecting from the brain lateral tegmentum (AI and A2 areas) and locus coeruleus (A6) are seen in the arcuate and periventricular nuclei and the median eminence (14). Alternatively, the amino acidergic receptors that participate in the response described here could be located on cell bodies or dendrites of interposed nonadrenergic neurons (interneurons) that impinge on NA nerve terminals. In conclusion, our results raise the possibility that glutamatergic and GABAergic receptors may be part of a local modulatory action on noradrenergic functions operating in the hypothalamus. As the present results mirror the previously demonstrated actions of glutamate and GABAn receptor agonists on LHRH release in vitro [7], such mechanism may be involved in the neuroendocrine effects of NA. This hypothesis is consistent with previous studies demonstrating that NA evokes LHRH release from the hypothalamus and, consequently, LH release from the pituitary gland [1,5,11].

FIG. 4. Effect of baclofen (BAC, 200 #M) on kainate (KA; 200 #M)evoked 3H-NA release from mediobasal hypothalamic slices. Mean _+ SEM (n = 6). ***p < 0.01 vs. KA. lease. Results obtained with the 100/zM concentration are depicted in Fig. 5.

Inhibition by GABA and Baclofen of ~H-NA Release Evoked byK ÷ Both GABA (20 mM) and baclofen (100/zM) added to the perfusion medium containing 28 mM KCI blunted the expected increase in tritium overflow, shown in Fig. 5, from hypothalamic slices loaded with 3H-NA (Fig. 6). Muscimol was inactive (results not shown).

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DISCUSSION The present work demonstrates, in agreement with previous results [16], that NMDA and KA markedly stimulate the release of previously accumulated 3H-NA, indicating that glutamatergic pathways converge on the noradrenergic nerve terminals in the mediobasal hypothalamus. Here, we are examining the ability of GABA to modulate the glutamate effects. As it is shown, GABA clearly reduced the evoked release of 3H-NA in response to glutamate and its ionotropic agonists. GABA receptor agonists are classified into two types with different sites of action: muscimol and THIP are selective GABAA compounds that operate inhibitory chloride channels and baclofen is a selective GABAB compound that activates cellular systems typical of the G-protein coupled receptors [19]. The inhibitory action of baclofen in the present experiments is a receptor-mediated process, because 2-hydroxysaclofen, a selective GABAa receptor antagonist [19], restored the NMDA effect. However, because baclofen disrupted K÷-evoked effects, it seems that receptor-independentrelease was also affected. These results support the involvement of GABAB receptors as media-

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FIG. 5. Representative examples of radioactivity release from mediobasal hypothalamicslices previouslyloaded with 3H-NAand exposed to 28 mM KC1,baclofen (100 #M, BAC), or saclofen(300 #M, SAC). Top: enhancement of 3H-NArelease by K+. Bottom: lack of influence of baclofen or saclofen on basal tritium release. KRB: regular Krebs-Ringer -bicarbonate-glucosebuffer. Each point represents the mean _+ SEM of six fractions.

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FIG. 6. Inhibitory effects of GABA (20 mM) and baclofen (BAC) 1100 #M) on net 3H-NA release induced by 28 mM KCI. Mean ÷ SEM In = 6). **p < 0.01 vs. K +. ACKNOWLEDGEMENTS This study was supported by the Consejo de lnvestigaciones de la Universidad Nacional de Cuyo, Mendoza (CIUNC), Programa Latinoamericano de Capacitacion e Investigacione en Reproduccion Humana (PLACIRH), Consejo Nacional de Investigaciones Cientificas y T6cnicas, Argentina (CONICET) and Consejo de Investigaciones de la Provincia de Mendoza (CONICMEN). We thank the skillful technical assistance of Adriana Carri6n.

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