The acute sensitivity and long-term effects of ethanol on a wide variety of postsynaptic receptors has been well documented. And, presynaptic facilitation of GABAergic synapses by acute ethanol has been described in many brain regions. But, acute ethanol does not appear alter presynaptic glutamate release. Several labs, including our own, have shown that chronic ethanol exposure robustly up-regulates presynaptic glutamate release at some synapses. But, the presynaptic mechanism responsible for this chronic up-regulation remains undefined. We recently showed that acute ethanol can robustly modulate presynaptic function at glutamate synapses in the lateral/basolateral amygdala. Like many studies, we did not observe any direct effect of ethanol on glutamate release at these synapses. However, using high frequency stimulation to deplete readily- releasable vesicle pool, we found that acute ethanol robustly inhibited synaptic responses represented by the recycling pool of synaptic vesicles. This data thus potentially defines an entirely novel presynaptic effect ethanol: inhibition of synaptic vesicle recycling. Our data also show that ethanol modulation of vesicle recycling is frequency-dependent. This suggests unique contributions by distinct presynaptic recycling pathways. Finally, we present preliminary data showing that the presynaptic Munc proteins, which regulate both vesicle priming and recycling, modulate ethanol inhibition of these recycling pathways. We thus propose the central hypothesis that Munc13-1 and Munc13-2 mediate ethanol inhibition of distinct vesicle recycling pathways. We will directly test this hypothesis by accomplishing the two specific aims. The first specific aim will test the working hypothesis that ethanol inhibits two independent vesicle recycling pathways, clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Published evidence shows differential, strain-specific ethanol modulation of recycling at both moderate stimulation frequencies, dominated by clathrin-dependent endocytosis, and during high-frequency stimulation, when activity- dependent bulk endocytosis controls recycling. To test this working hypothesis, we will integrate shRNA- mediated knockdown of essential presynaptic proteins within each pathway, selective pharmacological modulation, and whole-cell patch clamp electrophysiology in vitro. The second specific aim will test the working hypothesis that Munc13-1 and Munc13-2 differentially regulate ethanol inhibition of distinct vesicle recycling pathways. Our rationale here is that genetic variations in Munc13-1 and Munc13-2 are associated with the differential, mouse strain-specific ethanol sensitivity of low- and high-frequency responses. We will test our working hypothesis for this aim by again integrating shRNA-mediated knockdown of Munc13-1 or Munc13-2 with in vitro electrophysiology. The proposed work is significant because it will characterize both a novel presynaptic effect of acute ethanol and contributions by unique presynaptic proteins.
The focus of this research project is to understand how specific presynaptic proteins regulate the newly described ethanol modulation of synaptic vesicle recycling. Once the project is completed, we will know both the roles of these proteins and will have identified the specific presynaptic pathways involved with ethanol modulation of recycling. Given the novel nature of the ethanol effect and this understudied family of proteins, this research could provide specific molecular targets for alcohol abuse and alcoholism therapeutics development.
