Ablation of All Synaptobrevin vSNAREs Blocks Evoked But Not Spontaneous Neurotransmitter Release at Neuromuscular...

Synaptic transmission occurs when an action potential triggers neurotransmitter release via the fusion of synaptic vesicles with the presynaptic membrane, driven by the formation of SNARE complexes composed of the vesicular (v)-SNARE synaptobrevin and the target (t)-SNAREs Snap-25 and syntaxin-1. Neurotransmitters are also released spontaneously, independent of an action potential, through the fusion of synaptic vesicles with the presynaptic membrane. The major neuronal vSNAREs, synaptobrevin-1 and synaptobrevin-2, are expressed at the developing neuromuscular junction (NMJ) in mice, but their specific roles in NMJ formation and function remain unclear. Here, we examine the NMJs in mutant mouse embryos lacking either synaptobrevin 1 (Syb1 ) or synaptobrevin 2 (Syb2^-/-), and those lacking both (Syb1^-/-). We found that, compared with controls: (1) the number and size of NMJs was markedly increased in Syb2^-/- and Syb1^-/- mice, but not in Syb1 mice; (2) synaptic vesicle density was markedly reduced in Syb1^-/- NMJs; and (3) evoked neurotransmission was markedly reduced in Syb2^-/- NMJs and completely abolished in Syb1^-/- NMJs. Surprisingly, however, spontaneous neurotransmission persists in the absence of both Syb1 and Syb2. Furthermore, spontaneous neurotransmission remains constant in Syb1^-/- NMJs despite changing Ca²⁺ levels. These findings reveal an overlapping role for Syb1 and Syb2 (with Syb2 being dominant) in developing NMJs in mice. Moreover, because spontaneous release becomes Ca²⁺-insensitive in Syb1^-/- NMJs, our findings suggest that synaptobrevin-based SNARE complexes play a critical role in conferring Ca²⁺ sensitivity during spontaneous release.SIGNIFICANCE STATEMENT Neurotransmitters can be released at synapses with (evoked) or without (spontaneous) the influence of action potentials. Whereas evoked neurotransmission requires Ca²⁺ influx, those underlying the spontaneous neurotransmission may occur with or without Ca²⁺ Our findings show that, in the absence neuronal vSNARE synaptobrevin-1 and synaptobrevin-2, evoked neurotransmission is completely abolished; however, spontaneous synaptic transmission not only persists but even increased. Furthermore, spontaneous synaptic transmission that is normally highly Ca²⁺-sensitive became Ca²⁺-independent upon deletion of vSNARE synaptobrevin-1 and synaptobrevin-2. These findings reveal distinct mechanisms for evoked and spontaneous neurotransmitter release. Moreover, these findings suggest that synaptobrevin-based SNARE complexes play critical roles in conferring Ca²⁺ sensitivity during spontaneous neurotransmission at developing neuromuscular synapses in mice.