Astrocytic Control of GABA Inhibition in Epilepsy

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DESCRIPTION (provided by applicant): We have recently shown that naturally occurring neuroactive compounds, endozepines related to the protein Diazepam Binding Inhibitor (DBI) in the brain can mimic the activity of benzodiazepines, which are effective treatments in epilepsy. Benzodiazepines are allosteric modulators of GABAA receptors, which mediate the primary form of synaptic inhibition in the brain. This suggests that, through production of endozepines, brain cells and circuits can-self regulate, to dynamically enhance synaptic inhibition as needed to suppress seizures as they arise from ongoing brain activity. While the existence of DBI-related antiepileptic endozepines has now been demonstrated, little is known regarding the cellular source of endozepines, nor of the means through which they are secreted from cells or processed in the extracellular space to exert their action. The proposed experiments have three aims. 1) Determine whether astrocytes are the source of endozepines, as they express very high levels of DBI, and appear to have a high capacity for secretion, 2) determine the pathways through which cells, most likely astrocytes, sense activity and then respond through secretion and processing of DBI, and 3) Identify the final endozepine molecule (or molecules), which does not appear to be DBI itself, but a protein fragment of DBI. We will use electrophysiological assays to document functional endozepine activity in all three aims. Assays will include determining the kinetics of spontaneous inhibitory post-synaptic currents, an effective assay for detection of exogenous or endogenous allosteric modulation of synaptic GABAA receptors, and responses to high-speed iontophoretic GABA application, a sensitive assay for endozepine activity, network analysis of large-scale thalamic networks in vitro, and EEG analysis of seizure susceptibility in vivo. These studies will be facilitated by the availability of mutant mice that alow for targeted deletion of DBI from astrocytes, and by fluorescent reporter mice that allow for detection of the range and extent of deletion. Overall the results of the proposed experiments will provide mechanistic information regarding endozepine signaling and whether this natural brain activity might ultimately be targeted for therapeutic intervention in epilepsy and other neuropsychiatric disorders of altered GABA signaling.