GABA HAS EXCITATORY ACTIONS ON GNRH-SECRETING IMMORTALIZED HYPOTHALAMIC (GT1-7) NEURONS

The effects of gamma-aminobutyric acid (GABA) on clonal gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons were investigated using patch-clamp and fura-2 imaging techniques. Local application of GABA (100 mu M) to GT1-7 cells voltage-clamped in the whole-cell configuration immediately increased membrane conductance and noise consistent with activation of the GABA(A) receptor-Cl- channel complex. Depolarization activated transient Na+ currents which were abolished by tetrodotoxin (TTX; 0.5 mu M), and more sustained Ca2+ currents. Under constant current conditions, GT1-7 cells fired spontaneous action potentials, and depending on the Cl- equilibrium potential, GABA either depolarized cells, causing a rapid activation of action potentials, or hyperpolarized cells. In order to determine the effect of GABA (100 mu M), but not the GABA(B) receptor agonist baclofen (10 mu M), immediately evoked multiple action potentials. Measurement of [Ca2+](i) using fluorescence video microscopy and fura-2 revealed spontaneous, transient, repetitive increases in [Ca2+](i) which had a periodicity ranging from 1 to 60 s. These Ca2+ oscillations were abolished by TTX (1 mu M) and by the removal of extracellular Ca2+. Application of GABA (1 and 10 mu M) induced an immediate increase in [Ca2+](i) in all cells and increased the frequency of Ca2+ oscillations in a dose-dependent manner. The GABA-induced increase in [Ca2+](i) was abolished by bicuculline and by the removal of extracellular Ca2+, and was inhibited by TTX. Baclofen (1 mu M) had no effect on [Ca2+](i). These results suggest that activation of GABA(A) receptors has an excitatory action on GnRH-secreting immortalized hypothalamic neurons caused by a Cl--dependent depolarization. GABA has been reported to increase GnRH secretion; a direct stimulatory action of the neurotransmitter on GABA(A) receptors of GnRH-secreting hypothalamic neurons may be responsible for this effect.