Mechanism Of Munc13Proteins Function In Regulation Of Synaptic Transmission And Short Term Plasticity At The Calyx Of Held Synapse

The Munc13gene family are multi-domains protein which specifically located at the synaptic active zone. In the CNS, the Munc13protein family consists of three different protein:Munc13-1,Munc13-2(ubMunc13-2&bMunc13-2) and Munc13-3. Munc13s are essential for synaptic vesicle priming the process that making synaptic vesicles become fusion competent. Synaptic transmission were completely abolished in Muncl3s-deficient neurons since the synaptic vesicles were not be able to be released. Muncl3s are also critical for regulating synaptic plasticity. It was hypothesized that different Munc13protein can mediate different short term plasticity. Munc13-2and Munc13-3are functionally dispensable at some synapses, their loss neither alters synaptic transmission nor short term plasticity, but in other synapses, their loss indeed leads to altering synaptic plasticity. We addressed this conundrum at the calyx of Held synapse and studied Munc13s role in the regulation of synaptic transmission and their impact on the reliability of information transfer. First, we found that there are three Munc13protein expressed in the calyces, they are Munc13-1, ubMunc13-2and Munc13-3. Through detailed electrophysiological analysis of Munc13-2, Munc13-3, and Munc13-2-3knock-out and their respective wild-type mice, we found that Munc13-2and Munc13-3are dispensable for basic synaptic transmission at the calyx of Held synapse. But combined loss of Munc13-2and Muncl3-3led to faster synaptic transmission, shorter of synaptic delay, decrease of the RRP, not change of the fast-releasing vesicles pool but30%reduction of the slowly-releasing vesicles pool, an increase of calcium dependent recovery after deletion of RRP. By injecting rAd Munc13-1(1-451) virus into the cochlear nucleus of new born Munc13-2-3DKO mice, we found that calyces overexpressed of Munc13-1(1-451) showed strong reduction of synaptic transmission and also abnormal short term plasticity, the reduction of the synaptic transmission was caused by a reduction of RRP size, these data demonstrated that calyx of Held synapse is Munc13dependent and Munc13-1is the major priming factor. Through quantitative immunohistochemistry by using Munc13-fluorescent protein knock-in mice we and our collaborator found that Munc13-1is the most highly expressed Muncl3isoform at the calyx and the only one highly colocalized with Bassoon at the active zone.The summary conclusion for this study as followed:(1) There are three Munc13proteins expressed at the calyces, they are Munc13-1, ubMunc13-2and Munc13-3.(2) Munc13-1protein is the major priming protein at the calyx of Held synapse, Munc13-2and Munc13-3are redundant for basic synaptic transmission.(3) Munc13s may compete to mediate synaptic vesicles priming. Munc13-1primed vesicles can be released more faster since the vesicles are more close to VGCC at the active zone.(4) Muncl3s differentially regulated synaptic vesicle calcium dependent recovery. Muncl3s may compete to mediate vesicles recovery and Mun13-1mediated vesicles can recover more faster.(5) Munc13-1is the most abundant Munc13protein at the calyces and Muncl3s have differential localization at the active zone. Compared with Munc13-2and Munc13-3, most of Munc13-1protein localize at or close to the active zone.(6) Munc13-1may mediate the conversion of slowly releasing vesicles to fast releasing vesicles.