These continuing studies will probe in detail actions of alcohol on CNS neurons that may be related to its intoxicating properties. The overall goal is to identify target sites of alcohol action responsible for alterations in neurotransmission and electrical excitability known to occur in CNS neurons following acute exposure to alcohol. The cerebellar Purkinje neuron, which exhibits alcohol sensitivity in vivo and in vitro, will be used as the experimental model, utilizing both a culture and slice preparation. Membrane ionic conductances, transmitter receptor mechanisms and intracellular second messenger systems are proposed to be sites of alcohol action. These putative sites are also expressed in other CNS neurons, and thus, results from studies in the Purkinje neurons are likely to generalize to other neuronal types. Ethanol sensitivity will be assessed with electrophysiological techniques, including whole cell current clamp, whole cell voltage clamp and single channel recordings, and with microscopic imaging using Ca2+ and Cl- sensitive fluorescent dyes. All studies will involve doses of alcohol known to produce intoxication in animals and humans, 22 (100 mg %) and 44 mM (200 mg %). A variety of neuronal properties will be studied. Alcohol actions on voltage-sensitive Ca2+ conductances and on Ca2+ activated K+ conductances in the cultured Purkinje neurons will be examined because these sites were implicated in studies completed during the current funding period. Responses mediated by the excitatory amino transmitter glutamate and the inhibitory amino acid transmitter GABA will also be investigated for alcohol sensitivity in the cultured neurons, because studies during the last funding period and results from other laboratories implicate these receptor mediated responses as sites of alcohol action. In addition, the effect of alcohol on synaptic transmission, synaptic plasticity and electrical excitability of the Purkinje neurons will be studied in a cerebellar slice preparation under conditions more reflective of the in vivo environment. These detailed studies will define neuronal sites and mechanisms of alcohol action at the molecular level and lead to a more complete understanding of alcohol actions in the CNS that may underlie its intoxicating properties.
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