PHYSIOLOGICAL STUDIES OF NEUROSTEROID ANALOGS

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Neuroactive steroids and their benz[e]indene (BI) tricyclic analogues are potent and effective modulators of the gamma-aminobutyric acid A receptor-chloride channel complex (GABA A R). At low concentration, these agents augment the actions of GABA, markedly increasing responses to sub-EC 50 GABA concentrations, while having little effect on peak responses to saturating GABA concentrations. At micromolar concentrations, steroid analogues directly activate chloride channels in the absence of GABA. These effects are likely to contribute to the clinical actions of neurosteroids as anesthetics, anticonvulsants and anxiolytics. Other steroids, particularly those that are sulfated at the 3alpha-position, are negative modulators of GABAAR and alter the function of inotropic glutamate receptors (GluR). Although steroids are highly lipophilic, these agents are believed to act at specific sites on receptor proteins. Over the past funding period, a series of steroid and BI analogues were studied to examine structure-activity relationships for enhancement of GABAAR function. Included in these studies was the demonstration that steroid- and BI-mediated potentiation and direct gating of GABAAR occur enantioselectively. These studies provide strong support for the hypothesis that steroids act at specific loci on GABAAR and not via effects on membrane lipids. In the present proposal, initial studies of steroids and Bis will be extended by addressing three specific aims: 1. To gain new information about structural requirements for steroid and BI effects on GABAAR and GluR in cultured rat hippocampal neurons. These studies will conducted in collaboration with D. Covey and will examine further the enantioselctivity of steroid actions. 2. To understand mechanisms involved in neurosteroid-mediated modulation of hippocampal GABAergic and glutamatergic synaptic transmission. These studies will address whether synaptic effects of steroids and Bis are mediated primarily via postsynaptic actions and how steroids produce marked prolongation of inhibitory synaptic currents. 3. To examine factors contributing to the heterogeneity of steroid effects on GABAAR in cultured hippocampal neurons with specific emphasis on differences in GABAAR subunits expressed in excitatory and inhibitory hippocampal neurons. These studies have the potential to provide new information about neurosteroid effects in the CNS and a better understanding of mechanisms involved in steroid-induced anesthesia.