Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1–7 neurons in vitro

Abstract

Immortalized GT1–7 neurons were used to characterize the effect of muscimol, a GABA A receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1–7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml·h −1 with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 μM muscimol or 5 μM muscimol+20 μM of the GABA A receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1–7 neurons was pulsatile with an average interpulse interval of 45.4±0.5 min and an average pulse amplitude of 191.5±22.6 pg·min·ml −1. Our results also demonstrated that the GABA A receptor agonist, muscimol, enhances pulsatile GnRH release from GT1–7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC 50 of 0.47 μM. The effects of 5 μM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABA A receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7±1.8 min, 32.5±2.9 min, 30.6±0.7 min and 25.5±0.4 min in the period following exposure to 0.35, 1, 5 and 10 μM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABA A receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABA A receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals.