Short-term plasticity of synaptic transmission is recognized as an important element of information processing in neuronal networks. Acetylcholine release at the C. elegans neuromuscular junction is rapidly depressed upon repetitive stimulation and gradually recovers after a short rest. A simple model is that the readily releasable pool is depleted by extensive release and refilled from either newly endocytosed vesicles or from a large reserve pool. To determine the contributions of different pool refilling mechanisms and identify key molecular players, I use electrophysiological assays to screen candidate genes that function in endocytosis or other vesicle traffic pathways for altered synaptic depression and recovery. From this screen I expect to identify essential proteins that regulate synaptic strength and reveal mechanisms underlying synaptic depression. To target the screen specifically on the presynaptic components of synaptic depression, I built a reference strain to eliminate contributions from the postsynaptic acetylcholine receptors desensitization and the inactivation of channelrhodopsin-2. I used ChIEF, a channelrhodopsin-2 variant with less inactivation, in acr-16(-) background which has little receptor desensitization. Almost all the depression measured from this strain (acr-16;Punc-17::ChIEF::mCherry) is by presynaptic mechanisms. The time course of the depression and recovery measured from this control had two time constants indicating multiple underlying mechanisms. Candidate genes were cherry-picked based on their reported functions in endocytosis and vesicle trafficking steps including docking, priming, clustering and so on. Mutant strains of these candidate genes were crossed into the reference strain. Synaptic depression and recovery at the acetylcholine neuromuscular junction under 10Hz 30 sec stimulations were recorded. Preliminary recordings have been obtained for clathrin (chc-1 ts), dynamin-1 (dyn-1 ts), synaptojanin (unc-26), synapsin (snn-1), RIM (unc-10), liprin-alpha (syd-2), CDK5 (cdk-5), synaptotagmin-1 (snt-1), etc.. One interesting result emerged from the preliminary results is that chc-1 ts did not alter the depression nor the recovery rates at the restrictive temperature. This result calls into doubt endocytosis being the rate-limiting step for the synaptic depression and recovery. Alternatively, clathrin-mediated endocytosis may not be the essential endocytic pathway for synaptic vesicles in C. elegans. Clathrin and other mutants with either augmented or diminished depression or recovery rate will be further characterized using electron microscopy in the absence or presence of neuronal stimulation at different time points.

Affiliation:
- Dept Biol, Univ Utah/HHMI, Salt Lake City, UT.