Mechanism by which GABA, through its GABAA receptor, modulates glutamate release from rat cortical neurons in culture

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Abstract

In cortical neurons, the GABAA agonist, muscimol, increases: (a) basal glutamate release (with a EC50 of 99±7 mM); (b) intracellular calcium and (c) membrane potential, all of these in a dose-dependent manner. These muscimol effects were specific since they were reversed by bicuculline, a GABAA antagonist.

When the action of muscimol was measured at different KCl concentrations, an increase or decrease of the glutamate secretion was observed, depending on the KCl concentration in the medium. At low KCI concentration (5.6 mM of KCl), it depolarized, at 20 mM of KCl it had no effect, but at higher KCl concentrations (30–100 mM of KCl), it produced a hyperpolarization in these cells.

The mechanism by which the GABA-Cl-channel permits Cl fluxes, inward or outward, depending on the membrane potential.

Introduction

Although it is known that GABA is the main inhibitory neurotransmitter in the central nervous system (CNS), its behaviour on neurotransmission and hormone release is unclear since it has been detected that GABA may mediate excitatory responses in some cells. GABA binds to brain membranes at three different sites, called GABAA, GABAB and GABAC receptors. The binding of GABA to its GABAA receptors involves the opening of the chloride channel with an influx of Cl through this channel and, as a consequence, with membrane hyperpolarization and the subsequent inhibition of excitatory neurotransmission. Although this is the general mechanism for GABAA action, depolarizing responses have been described, namely in sensory ganglions, in sympathetic neurons (De Groat, 1970; Adams and Brown, 1986; Bowery and Hill, 1986) and in neonatal hippocampal neurons (Cherubini et al., 1991). GABA may also stimulate GnRH secretion in hypothalamic neurons (Hales et al., 1994). Outside the CNS, GABA is able to depolarize chromaffin cells from bovine adrenal glands (Gonzalez et al., 1992; Parramon et al., 1994) and to release luteinizing hormone in female rat pituitary cells (Virmani, 1990), this effect being mediated by a nonclassical GABAA receptor since release was not blocked by bicuculline. On the other hand, GABA may inhibit the KCl stimulated release of somatostatin from rat spinal cord slices without altering the basal release (Vasko et al., 1990).

This dual (excitatory and inhibitory) action of GABA, functioning through the GABAA receptor, has been detected by others, and several mechanisms have been proposed. In the present paper we present evidence which suggests that this dual GABA action may be mediated by an increase in membrane chloride conductance which could lead to an eflux or influx of chloride through the chloride channels, depending on the membrane potential.

Section snippets

Materials

FURA 2AM, (DiBAC (3)bis- (1,3-dibutylbarbituric acid trimethyne oxonol) (bisoxonol) and (6-methoxy-N(3-sulpho-propil)-quinolinium (SPQ) were obtained from Molecular Probes (Eugene, U.S.A.). Minimum Essential Eagles Medium (EMEM) was supplied by Bio-Whittaker and Foetal Calf (FCS) and Horse Serum (HS) by Sera-Lab, Sussex, U.K. Glutamate dehydrogenase (GDH) and NADP came from Boehringer Mannheim, S.A. (Barcelona, Spain) and 4,4-diisothiocyanato-stilbene-2,2-disulphonic acid (DIDS), furosemide and

Effect of muscimol on basal glutamate secretion

Basal glutamate release was increased when cortical neurons in culture were stimulated with muscimol, the GABAA agonist, at a resting membrane potential. The glutamate secretion evoked by muscimol was dose-dependent with a EC50 of 99±7 mM (Fig. 1A) and the effect was specific since it was reversed to a great extent by 500 mM of bicuculline (BMI), a GABAA antagonist (Fig. 1B). When cells were stimulated with muscimol in a medium without external calcium, a low glutamate secretion was observed.

Discussion

Although GABA is principally an inhibitory neurotransmitter which produces hyperpolarizing responses in nerve cells, as indicated previously, it has also been shown to have depolarizing responses in some neural cells, such as in the sensory ganglion, in sympathetic neurons (Bowery and Hill, 1986), in embrionic rat dorsal horn (Reichling et al., 1994), in embrionic and rat cortical slices (Owens et al., 1996) and in chromaffin cells, which are closely related to sympathetic neurons (Gonzalez et

Acknowledgements

This work was supported by CAICYT grant PB-95/006. and FIS 98/0587. M.T. Herrero is a recipient of a fellowship from the Ministerio de Educación y Ciencia. Our thanks to M. García Mauriño for helping us in culture preparation and to J. Sabatini for improving the English of the manuscript.

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