Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction

Abstract

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.active zone | exocytosis | synapse | acetylcholine receptor A hallmark of synaptic transmission is the synchrony between the neuronal action potential and the evoked release of transmitter. However, an asynchronous release mode, first described at the nerve muscle junction (1, 2), also participates in neurotransmission at certain synapses (3-5). Although asynchronous release usually contributes less than 10% of the overall synaptic charge at low stimulus frequencies, it often plays a prominent role at higher frequencies (3, 6), prolonging both inhibitory (7) and excitatory (8, 9) postsynaptic responses through sustained release. Indeed, inhibitory deep cerebellar neurons, which are tuned for high-frequency signaling, rely exclusively on asynchronous synaptic transmission at contacts with inferior olive neurons (10). The mechanisms underlying synchronous and asynchronous release appear to be distinct but share a requirement for calcium (6,11). It is generally thought that local transient calcium signals govern synchronous release, whereas elevated residual calcium levels associated with highfrequency stimulation lead to asynchronous release (12, 13).A vesicular calcium sensor, synaptotagmin, couples synchronous release to presynaptic calcium entry in both mammals (14) and flies (15, 16). In hippocampus, the responsible isoform is syt1 (14, 17), whereas at neuromuscular synapses (18) and calyx of Held (11,19), it is syt2. The mechanisms underlying asynchronous release are not known. Our findings from zebrafish neuromuscular junction provide identification of a synaptotagmin isoform as a signaling component in asynchronous release. Results Paired Recordings Reveal a Small Quantal Content with ReleaseProbability Near "1." Paired whole-cell recordings between the caudal primary motor neuron (CaP) and ventral target skeletal muscle were performed on 72-to 96-h-postfertilization (hpf) zebrafish as previously described (20). The motor neuron was current clamped to −80 mV and stepped positive for 2 ms to elicit an action potential. The fast-type skeletal muscle was voltage clamped to −50 mV to inactivate sodium channels. The amplitude for spontaneous unitary events (...

Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release

“…Synaptotagmin 7 (Syt7), another member of Syt family that has been localized to the plasma membrane, was recently implicated in asynchronous release. Knockdown of Syt7 selectively reduced asynchronous neurotransmitter release at zebrafish neuromuscular synapses and in cultured hippocampal neurons, suggesting that Syt7 may act as a plasma membrane Ca 2+ sensor for asynchronous fusion (46,47). Whether Syt7 and myr-Syt1 share common effector interactions to trigger asynchronous release is unclear; however, unlike the observation with Ca 2+ -binding-defective cytoplasmic Syt1, Syt7 does require Ca 2+ binding to function as an asynchronous sensor (47).…”

Synaptic Vesicle-Recycling Machinery Components as Potential Therapeutic Targets

“…VAMP4 did not show robust trafficking under resting conditions, although it was shown that VAMP4-enriched vesicles can respond to elevated presynaptic Ca 2+ signals and promote release (Raingo et al, 2012;Bal et al, 2013). In addition, syt7 has recently emerged as a key Ca 2+ -sensing synaptic protein that maintains asynchronous neurotransmitter release independently of syt1 (Wen et al, 2010;Bacaj et al, 2013;Jackman et al, 2016).…”

N-terminal domain of complexin independently activates calcium-triggered fusion

“…Synaptic vesicle fusion is orchestrated by the neuronal soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) fusion proteins (3,4), in conjunction with synaptotagmin, complexin, and other synaptic proteins. The Ca 2+ -sensor synaptotagmin is essential for Ca 2+ -triggered release (5)(6)(7)(8). Neuronal SNARE proteins form a ternary complex consisting of synaptobrevin/vesicle-associated membrane protein (VAMP2), syntaxin, and synaptosomal-associated protein .…”