In ovo chronic neurosteroid treatment affects the function and allosteric interactions of GABAA receptor modulatory sites

We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5β-pregnan-3β-ol-20-one) on the GABA_A receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. ³H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 μM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 μM epipregnanolone decreased allopregnanolone EC₅₀ and E_max values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K_d and B_max values for ³H]flunitrazepam and ³H]GABA binding. ³H]GABA binding modulation disclosed an increase in allopregnanolone EC₅₀ value with a decrease in its E_max value. Diazepam EC₅₀ and E_max values were enhanced, while low affinity sodium pentobarbital EC₅₀ value was reduced by epipregnanolone treatment. The investigation of the GABA_A receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce ³⁶Cl⁻ influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on ³H]flunitrazepam and ³H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA_A receptor sites and function during chick optic lobe development.