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Das J1, Pany S1, Xu S2, Wooden J3, Leasure JL3 and Roman G2

Departments of Pharmacological and Pharmaceutical Sciences1, Biology2, and Psychology3, University of Houston, Houston, TX, 77204

Munc13-1 is a pre-synaptic active-zone protein essential for neurotransmitter release and involved in pre-synaptic plasticity in brain. Alcohol binds to its C1 domain, which is homologous to the C1 domain of PKC epsilon, a kinase implicated in the behavioral responses to ethanol. If Munc13-1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss-of-function mutation in the conserved Munc13-1 ortholog, Dunc-13 in Drosophila melanogaster and Munc13-1 knock-out (+/-) mice. The Dunc-13P84200/+ heterozygotes have 50% wild-type levels of Dunc-13 mRNA and display a very robust increase in ethanol self-administration. This phenotype is reversed by the expression of the rat Munc13-1 protein within the Drosophila nervous system. Reducing Dunc13 activity with either heterozygotes for the loss-of-function Dunc-13P84200 allele or the pan-neural expression of a Dunc13 RNAi transgene resulted in a robust resistance to the sedative effects of ethanol. The Dunc-13P84200/+ haploinsufficient flies also had defects in chronic tolerance. The expression of the wild type Munc13-1:EGFP fusion protein is capable of restoring normal sensitivity to the Dunc-13P84200/+ ethanol sensitivity, but the ethanol binding mutant Munc13-1 E582A:EGFP fusion protein does not rescue ethanol sensitivity. Also both wild type and E582A mutant EGFP fusion display similar membrane translocation kinetics when activated by phorbol esters in Neuro-2a mouse neuroblasoma cells. Hence, the Munc13-1 E582A protein is presynaptically localized and responds to C1 domain activation, but does not bind ethanol and does not support normal levels of ethanol sedation sensitivity in Drosophila. In animal studies, adult C57BL/6J mice (N = 27) did not show any significant behavioral differences in measures of anxiety, motor coordination, and object memory as compared to the Munc13-1 knock-outs (+/-). However, the mutant mice self-administered more ethanol solution than wildtype mice did in a 4-day Drinking in the Dark (DID) paradigm. This effect appears to be moderated by sex, with females drinking significantly more than males. These results suggest that Munc13-1 is an effector of alcohol action in the presynaptic zone. Supported by NIH 1R01AA022414-01A1.