Regulation Of Neuropeptide Exocytosis And Mechanisms Of Endocytosis In Primary Sensory Neurons

Neuropeptide release plays important role in sensory and nociceptive informationtransmission in DRG neurons. However, this process has only been investigated usingbiochemical methods with limited temporal and spatial resolution. Thus the primarysites, kinetics and regulation of neuropeptide release remains unknown.Using real-time TIRF imaging, we examined neuropeptide release from culturedDRG neurons at the single-vesicle level for the first time. NPY-pHluorin was expressedin DRG neurons as a molecular marker of neuropeptide-containing dense-core vesicles(DCVs). Two types of events were identified. The main type exhibited fullNPY-pHluorin release while the other type was characterized by rapid fusion poreopening-closures with confined NPY-pHluorin release. In addition to somatic releasewhich has been identified before, we also recorded robust neuropeptide release fromDRG neuron axon. DCV in the axon exhibited markedly higher release probability,faster release rate and shorter delay than soma although the calcium concentrations inaxon was not higher. Interestingly, activation of temperature sensing channel TRPV1by capsaicin evoked neuropeptide release similarly in soma and axon. Furthermore,capsaicin evoked release exhibited higher percentage of confined events and morestabilized fusion pore. In summury, we provided evidence that neuropeptide release in DRG neurons was highly regulated by different cellular compartments andstimulations.DRG neurons have robust exocytosis upon stimulation, thus precise and efficientendocytosis is essential for the vesicle recycling and membrane balance. Regulation ofendocytosis has been extensively studied but inhibitors have rarely been found. Here,we show that synaptotagmin-11(Syt11) inhibits clathrin-mediated endocytosis andbulk endocytosis without affecting exocytosis. The frequency of these two types ofendocytic event increased in Syt11-knockdown dorsal root ganglion neurons, whereasthe sizes of endocytosed vesicles and the kinetics of individual bulk endocytosisremained unaffected. Furthermore, Syt11also inhibited endocytosis in hippocampalneurons, implying a general role of Syt11in neurons. Taken together, we propose thatSyt11functions to ensure precision in vesicle retrieval by limiting the sites ofmembrane invagination at the early stage of endocytosis.