Abstract
Upon the arrival of repetitive stimulation at the presynaptic terminals of neurons, replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained neurotransmitter release. Kinetics of replenishment and the available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability and speed is unknown. Here, based on comprehensive optical imaging of various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, all of which are implicated in organellar membrane fusion in non-neuronal cells, we show that part of the recycling pool bearing the endosomal Q-SNARE, syntaxin 7 (Stx7), is preferentially mobilized for release during high-frequency repetitive stimulation. Recruitment of the SV pool marked with an Stx7-reporter requires actin polymerization, as well as activation of the Ca2+/calmodulin signaling pathway, reminiscent of rapidly replenishing SVs characterized previously in calyx of Held synapses. Furthermore, disruption of Stx7 function by overexpressing its N-terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 forms rapidly replenishing vesicles essential for synaptic responses to high-frequency repetitive stimulation, and also highlight functional diversities of endosomal SNAREs in generating distinct exocytic vesicles in the presynaptic terminals.
Highlights
Upon the arrival of repetitive stimulation at the presynaptic terminals of neurons, replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained neurotransmitter release
When SypHy was lentivirally transduced into cultured hippocampal neurons[19,20], it was sorted to presynaptic compartments, evidenced by immunostaining with anti-Syb[2] antibody (Fig. 1b)
Presynaptic endosomal SNARE-SEPs were colocalized with Syb[2] (Fig. 1e), the vast majority of endosomal SNARESEPs showed relatively smaller responses compared to SypHy (Fig. 1e, f;
Summary
Upon the arrival of repetitive stimulation at the presynaptic terminals of neurons, replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained neurotransmitter release. Based on comprehensive optical imaging of various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, all of which are implicated in organellar membrane fusion in non-neuronal cells, we show that part of the recycling pool bearing the endosomal Q-SNARE, syntaxin 7 (Stx7), is preferentially mobilized for release during high-frequency repetitive stimulation. Our data indicate that endosomal membrane fusion involving Stx[7] forms rapidly replenishing vesicles essential for synaptic responses to high-frequency repetitive stimulation, and highlight functional diversities of endosomal SNAREs in generating distinct exocytic vesicles in the presynaptic terminals. Several endosomal SNAREs including vti1a, Stx[6], and Stx12/13, engage in SV recycling and may supply RRP11, and VAMP4 is directed to the asynchronous SV pool[12]
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