The mechanism mediating fast neurotransmitter release at the calyx of Held synapse

Abstract

Fast and synchronous neurotransmitter release relies on the time course of calcium influx into the terminal, the availability of fusion competent vesicles and the proteins comprising the release machinery. Studies in glutamatergic synapses have identified a subpopulation of vesicles in the releasable pool that do not fuse in response to action potential (AP) driven calcium influx and release only slowly in response to sustained stimulation. The nature of these reluctant vesicles, however, remained unclear.This question was investigated at the calyx of Held, a large glutamatergic synapse in the auditory brainstem. By combining electrophysiological recordings of the pre and postsynaptic compartments with calcium imaging and uncaging, it is shown that uniform elevations of the intracellular calcium concentration in the presynaptic terminal were able to elicit rapid release after the vesicles mediating fast and synchronous neurotransmitter release had been depleted. The calcium sensitivity of reluctant vesicles was reduced only 2-fold which is insufficient to explain the 10-fold slower release kinetics that is observed in response to sustained depolarizations of the presynaptic terminal. It is concluded, that reluctant vesicles are indeed release competent, but that their distance to calcium channel clusters at the active zone prevents them to release in response to APs. This indicates that the incorporation of vesicles into the vicinity of calcium channels at the active zone (a positional priming step) is rate-limiting in fast neurotransmitter release rather than acquiring fusion competence (a molecular priming step).The accessibility of the presynaptic terminal of the calyx of Held to patch-pipettes allows one to infuse toxins and peptides to further investigate the positional priming step in exocytosis. Here, it is shown that the N-terminal of synaptobrevin is important for mediating this step, which has been indicated in a previous study based on the effect of tetanus neurotoxin (TeNT) on release at the calyx of Held. In contrast, it was found that BoNT/B, which shares the same cleavage site as TeNT but requires a different binding site on synaptobrevin, changes the intracellular calcium sensitivity of vesicles for fusion, likely by interfering with vesicle exocytosis at a step between calcium sensing and fusion. Positional priming was also affected when a C-terminal peptide from rabphilin3a s C2B domain was infused into the calyx of Held, which blocked synaptic transmission, potentially by interacting with SNAP-25. Its effect on the kinetics of release mimics that of TeNT as it preferentially blocked the fast component of release.These results highlight the importance of calcium channel-vesicle coupling as well as the delicate interplay of multiple proteins of the release machinery in mediating fast neurotransmitter release.