MODULATION OF GABA(A) RECEPTOR-MEDIATED IPSCS BY NEUROACTIVE STEROIDSIN A RAT HYPOTHALAMOHYPOPHYSEAL COCULTURE MODEL

1. We have used the whole-cell configuration of the patch-clamp techni que to investigate the effects of neuroactive steroids on GABA(A) rece ptor-mediated synaptic transmission between rat hypothalamic neurones and pituitary intermediate lobe (IL) cells grown in coculture. In orde r to discriminate between possible pre- and postsynaptic sites of acti on, the effects of neurosteroids on GABA(A) receptor-mediated synaptic currents (IPSCs) were compared with those on GABA(A) currents (I-GABA ) triggered by local application of 50 or 500 mu M GABA, which yielded approximately half-maximal and maximal responses, respectively.2. In primary cultures of rat pituitary IL cells, allopregnanolone (5 alpha- pregnan-3 alpha-ol-20-one) reversibly potentiated I-GABA in a dose-dep endent manner with a threshold between 0.1 and 1 nM. at a concentratio n of 10 nM, allopregnanolone increased the response evoked by 50 mu M GABA by +21.4 +/- 5.1% (n = 8), but had no effect on I-GABA induced by 500 mu M GABA. The beta-isomer of allopregnanolone, epipregnanolone ( 5 beta-pregnan-3 beta-ol-20-one, 10 nM), had no effect on I-GABA at an d concentration of GABA tested. 3. At concentrations lower than 10 mu M, pregnenolone sulphate (5-pregnen-3 alpha-ol-20-one sulphate) did no t significantly inhibit I-GABA. However, at 10 mu M, a, systematic red uction of I-GABA evoked by 50 and 500 mu M GABA was observed, with mea n values of -80 and -60%, respectively. This blocking effect was rever sible and accompanied by a marked acceleration of the decay of GABA(A) currents during the application of GABA. 4. In isolated pairs of syna ptically connected hypothalamic neurones and IL cells, allo-pregnanolo ne (10 nM) augmented the mean amplitude of spontaneous IPSCs (sIPSCs) and electrically evoked IPSCs (eeIPSCs) by about 40% and also increase d the mean frequency of sIPSCs. Allopregnanolone (10 nM) also markedly increased the frequency of miniature IPSCs (mIPSCs) recorded in the p resence of TTX (0.5 mu M), but without modifying their mean amplitude. Epipregnanolone had no effect on the amplitude or frequency of sIPSCs . Neither epipregnanolone nor allopregnanolone modified the time to pe ak and decay time constants of GABAergic IPSCs. 5. Pentobarbitone (50 mu M), positive allosteric modulator of GABA(A) receptors, did not aff ect the amplitude of sIPSCs or eeIPSCs, but significantly increased th e decay time constants of both types of IPSCs. Pentobarbitone had no e ffect on the frequency of sIPSCs. 6. Pregnenolone sulphate (10 mu M) c ompletely and reversibly blocked sIPSCs and eeIPSCs. Progressive block of IPSCs was correlated with a gradual decrease of the mean decay tim e constant. 7. Our results suggest that, under physiological condition s, allopregnanolone might be a potent modulator of GABAergic synaptic transmission, acting at both pre- and postsynaptic sites. The involvem ent of pregnenolone sulphate as a modulator of GABAergic IPSCs under p hysiological conditions is, however, more questionable. The mechanisms of action of both types of neurosteroids are discussed.