Function of the exocsyt complex in the calyx of Held presynaptic nerve terminal

Synaptic vesicles have to be constantly transported to the active zone to ensure ongoing synaptic transmission. Although some proteins which mediate docking and priming of synaptic vesicles have been identified (Munc13, RIM, SNAREs) the molecular mechanisms that mobilize synaptic vesicles from the recycling or reserve pool to the active zone are not well understood. Here, we investigated the involvement of the exocyst complex, a multiprotein tethering complex involved in directed transport of vesicle cargo, in the trafficking of synaptic vesicles. By means of stereotaxic injection of recombinant adeno-associated viral particles into the ventral cochlear nucleus of 2-day-old Sprague Dawley rats we performed acute genetic perturbation of exocyst function by overexpression of a dominant-negative truncation construct of Exo70, a subunit of the exocyst complex, in globular bushy cells to analyze potential changes in synaptic transmission at the calyx of Held. Additionally, the morphology of presynaptic terminals was reconstructed from confocal image stacks to investigate the influence of exocyst perturbation on synaptic architecture and maturation. Manual reconstruction of 166 calyces of Held and subsequent volume analysis revealed that overexpression of the dominant-negative truncation construct of Exo70 results in a statistically significant decrease in nerve terminal volume at P13 indicating induced defects in membrane addition by acute genetic perturbation. Surprisingly, calyces, which overexpressed the truncation construct of Exo70 (Exo70Nter), had comparable volumes to wildtype cells at a matured state in P21 rats. These findings suggest that while exocyst function is necessary during early maturation of calyces of Held it seems to be dispensable at later developmental stages. The developmental stage dependent function of Exo70 is further supported by the observation of a localization shift of the full-length GFP-fusion protein Exo70 between the two ages investigated. While adult calyces in P21 rats display plasma membrane localization for GFP-Exo70, this plasma membrane association is not evident for maturing calyces from P13 animals. Despite the hypomorphic nature of P13 calyces overexpressing Exo70Nter, low frequency (0.1 Hz) stimulation did not affect EPSC kinetics, suggesting that the fusion machinery of synaptic vesicles remained unaffected. Decay of EPSC amplitudes upon repetitive stimulation (0.3 – 100 Hz) was indistinguishable from wildtype, suggesting that exocyst is not involved in the molecular mechanisms underlying short-term depression. Similarly, readily releasable pool size and kinetics of recovery from depression, frequency of spontaneous release, quantal size and mEPSC kinetics remained unaffected by Exo70Nter overexpression. Thus, perturbation of exocyst function by overexpression of a dominant-negative truncation construct of Exo70 did not affect synaptic transmission, suggesting that exocyst does not contribute to the synaptic vesicle cycle and neurotransmitter release. In summary, our findings show that the exocyst complex is required for presynaptic membrane addition at early stages of calyx maturation, while a role as a tethering factor of synaptic vesicles in local vesicle recycling remains unlikely. Thus, the exocyst complex may be required for the delivery of cargo from the cell soma to synaptic terminals, but not for trafficking steps in the synaptic vesicle cycle.