Epilepsy is characterized by recurrent unprovoked seizures. Biological rhythms and external stimuli can modulate seizure frequency partly through circulating steroid hormones. Neurosteroids like allopregnanolone are synthesized in the brain from circulating steroids and have powerful anticonvulsant effects. AIIopregnanolone exerts its actions in the central nervous system by acting on GABAA receptors. Physiological concentrations of allopregnanolone (10 -30 nM) potently enhanced whole cell GABA-evoked currents in hippocampal dentate granule cells acutely isolated from naive rats. In contrast, in dentate granule cells acutely isolated from epileptic rats these physiological concentrations of allopregnanolone failed to enhance whole cell GABAA receptor currents. Whole cell GABA-A receptor currents are generated by activation synaptic and extrasynaptic GABAA receptors. In dentate granule cells of naive rats, a1 and g2 subunits are expressed at synapses while a4 and d subunits are extrasynaptic. The extrasynaptic receptors mediate tonic inhibition while synaptic receptors mediate phasic (synaptic) inhibition. The tonic inhibition is insensitive to allopregnanolone, diazepam, and sensitive to Zn2+ while synaptic inhibition is sensitive to allopregnanolone, diazepam, and insensitive to Zn2+. These preliminary findings support the hypothesis that in the hippocampal dentate granule cells of epileptic rats, there is diminished allopregnanolone modulation of GABAergic inhibition. Decreased allopregnanolone modulation is due to increased expression of alpha4 and delta subunit-containing receptors, which in epileptic rats are present extrasynaptically and in subsynaptic membrane. We will test the predictions of our hypotheses by accomplishing the following specific aims: 1) To characterize allopregnanolone, diazepam, zinc, and furosemide modulation of GABA mediated miniature inhibitory synaptic currents (mlPSCs), and of GABAA receptor mediated background tonic inhibition of hippocampal dentate granule cells of epileptic and control rats. 2) To characterize the amount and site (synaptic versus extrasynaptic) of expression of specific GABAA receptor subunits in dentate granule cells in epileptic and control rats by immunohistochemistry and immunoblot studies combined with confocal laser microscopy. Significance: These experiments are of particular relevance to understanding epilepsy-induced alterations in GABA-A receptors and their modulation by neurosteroids.
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