GT1-7 hypothalamic neurons, which synthesize and secrete gonadotropin releasing hormone (GnRH), have recently been developed and immortalized from transgenic mice and are the only cell line to express GABA(A) receptors. Stimulation of GABA(A) receptors in GT1-7 neurons results in membrane depolarization, not hyperpolarization, a unique function characteristic of these cells. Our preliminary experiments, as well as other recent reports, showed that muscimol, a GABA(A) agonist, stimulated C1- efflux, increased cytosolic [Ca2+), and stimulated GnRH release in GT1-7 cells. These effects were inhibited by bicuculline, a GABA(A) antagonist, and nimodipine, an L-type Ca2+ channel antagonist. Ethanol (50 mM) enhanced muscimol-induced GABA(A) function despite the fact that the GABA(A) receptors in these cells do not appear to contain a gamma subunit. Therefore, we hypothesize that acute ethanol exposure affects GABA(A) receptor function in a novel way that may include enhancement of chloride efflux and calcium ion conductance. To test our hypothesis, we propose the following specific aims:
Specific Aim 1. Characterize the basic function characteristics of the GABA(A) receptor system in GT1-7 neurons. We will characterize GABA(A) mediated increase in [Ca2+]cyt, C1- efflux, membrane potential changes, and GnRH release in GT1-7 cells using muscimol as a GABA(A) agonist and bicuculline as a GABA(A) antagonist. We will determine the involvement of L-type Ca2+ channels in GABA(A) mediated increase in [Ca2+]cyt using nimodipine (an L-type Ca2+ channel antagonist) and BayK 8644 )an L-type Ca2+ channel agonist).
Specific Aim 2. Characterize the acute effects of ethanol on GABA(A) function in GT1-7 cells. We will characterize the effects of acute ethanol treatment on GABA(A) mediated increase in [Ca2+]cyt, C1- efflux, membrane potential,, and GnRH release in GT1-7 cells. We will determine the concentration dependence of acute ethanol treatment on these GABA(A) mediated functions.
| Chen, Eileen C; Javors, Martin A; Norris, Catherine et al. (2004) Dependence of 3',5'-cyclic adenosine monophosphate--stimulated gonadotropin-releasing hormone release on intracellular calcium levels and L-type calcium channels in superfused GT1-7 neurons. J Soc Gynecol Investig 11:393-8 |
| Javors, M A; King, T S; Chang, X et al. (1998) Characterization of chloride efflux from GT1-7 neurons: lack of effect of ethanol on GABAA response. Brain Res 780:183-9 |
