Upon long term or chronic ethanol exposure, a constellation of adaptive responses can result that negatively impacts patient efforts toward drinking cessation. Altered neural functioning, especially following withdrawal, may culminate in outright epileptiform activity and alcohol withdrawal seizures. These critical events encompass a substantial clinical problem and are presently inopportunely treated using substitution therapy with other CNS depressant agents. This proposal focuses upon mechanisms of alcohol withdrawal seizures and the alterations in synaptic transmission that are thought to encompass a major mechanism for neuronal hyperexcitability that occurs following withdrawal from long term ethanol exposure. Two critical cellular components which have been implicated in the induction of withdrawal hyperexcitability are the N-methyl-D-aspartate subtype (NMDAR) of GLuR receptors and the L-subtype of voltage-gated calcium channels (VGCCs). In this continuation application for funding, we propose to assess the cellular alterations in NMDAR function which underlie the initiation and expression of alcohol withdrawal seizures in an in vitro model system. During the previous funding period, we developed a hippocampal explant system which displayed robust tonic-clonic electrographic seizures immediately after withdrawal from several days of ethanol exposure. Data using prolonged field potential recordings suggest that a selective enhancement of NMDAR function underlies the initiation of ethanol withdrawal seizure events in our explant preparation. In this proposal, we wish to more thoroughly and exhaustively investigate this adaptive response using a variety of neurobiological, molecular and biochemical techniques. We propose to measure alterations in NMDAR function associated with chronic exposure and withdrawal by analysis of synchronous (spontaneous) and asynchronous (evoked strontium) pharmacologically-isolated NMDA miniature synaptic events (Aim 1). Molecular and biochemical techniques will be used to assess alterations in expression of NMDAR mRNA, protein and channel subunit composition (Aim 2). Finally, near real-time calcium imaging in combination with field potential recordings will be used to assess synaptic (NMDAR) and calcium-dependent mechanisms of seizure initiation (Aim 3). Taken together, these studies should provide mechanistic information about the alterations underlying alcohol withdrawal hyperexcitability.
