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Key points Dynamins are GTPases required for pinching vesicles off the plasma membrane once a critical curvature is reached during endocytosis. Here, we probed dynamin function in central synapses by depleting all three dynamin isoforms in postnatal hippocampal neurons down to negligible levels. We found a decrease in the propensity of evoked neurotransmission as well as a reduction in synaptic vesicle numbers. Recycling of synaptic vesicles during spontaneous or low levels of evoked activity were largely impervious to dynamin depletion, while retrieval of synaptic vesicle components at higher levels of activity was partially arrested. These results suggest the existence of balancing dynamin‐independent mechanisms for synaptic vesicle recycling at central synapses. Classical dynamin‐dependent mechanisms are not essential for retrieval of synaptic vesicle proteins after quantal single synaptic vesicle fusion, but they become more relevant for membrane retrieval during intense, sustained neuronal activity. Loss of dynamin 2 does not impair synaptic transmission. Loss of all three dynamin isoforms mostly affects evoked neurotransmission. Excitatory synapse function is more susceptible to dynamin loss. Spontaneous neurotransmission is only mildly affected by loss of dynamins. Single synaptic vesicle endocytosis is largely dynamin independent. [ABSTRACT FROM AUTHOR]

Spontaneous neurotransmitter release is a fundamental property of synapses in which neurotransmitter filled vesicles release their content independent of presynaptic action potentials (APs). Despite their seemingly random nature, these spontaneous fusion events can be regulated by Ca2+ signaling pathways. Here, we probed the mechanisms that maintain Ca2+ sensitivity of spontaneous release events in synapses formed between hippocampal neurons cultured from rats of both sexes. In this setting, we examined the potential role of vesicle-associated membrane protein 4 (VAMP4), a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in spontaneous neurotransmission. Our results show that VAMP4 is required for Ca2+-dependent spontaneous excitatory neurotransmission, with a limited role in spontaneous inhibitory neurotransmission. Key residues in VAMP4 that regulate its retrieval as well as functional clathrin-mediated vesicle trafficking were essential for the maintenance of VAMP4-mediated spontaneous release. Moreover, high-frequency stimulation (HFS) that typically triggers asynchronous release and retrieval of VAMP4 from the plasma membrane also augmentsCa2+-sensitive spontaneous release for up to 30 min in a VAMP4-dependent manner. This VAMP4-mediated link between asynchronous and spontaneous excitatory neurotransmission might serve as a presynaptic substrate for synaptic plasticity coupling distinct forms of release. [ABSTRACT FROM AUTHOR]

Recent studies suggest that spontaneous and action potential-evoked neurotransmitter release processes are independently regulated. However, the mechanisms that uncouple the two forms of neurotransmission remain unclear. In cultured mouse and rat neurons, we show that the two C2 domain-containing protein copine-6 is localized to presynaptic terminals and binds to synaptobrevin2 as well as other SNARE proteins in a Ca2+ -dependent manner. Ca2+-dependent interaction of copine-6 with synaptobrevin2 selectively suppresses spontaneous neurotransmission in a reaction that requires the tandem tryptophan residues at the C-terminal region of synaptobrevin2. Accordingly, copine-6 loss of function augmented presynaptic Ca2+ elevation-mediated neurotransmitter release. Intracellular Ca2+ chelation, on the other hand, occluded copine-6-mediated suppression of release. We also evaluated the molecular specificity of the copine-6-dependent regulation of spontaneous release and found that overexpression of copine-6 did not suppress spontaneous release in synaptobrevin2-deficient neurons. Together, these results suggest that copine-6 acts as a specific Ca2+-dependent suppressor of spontaneous neurotransmission. [ABSTRACT FROM AUTHOR]

Store-operated calcium entry (SOCE) is activated by depletion of Ca 2+ from the endoplasmic reticulum (ER) and mediated by stromal interaction molecule (STIM) proteins. Here, we show that in rat and mouse hippocampal neurons, acute ER Ca 2+ depletion increases presynaptic Ca 2+ levels and glutamate release through a pathway dependent on STIM2 and the synaptic Ca 2+ sensor synaptotagmin-7 (syt7). In contrast, synaptotagmin-1 (syt1) can suppress SOCE-mediated spontaneous release, and STIM2 is required for the increase in spontaneous release seen during syt1 loss of function. We also demonstrate that chronic ER stress activates the same pathway leading to syt7-dependent potentiation of spontaneous glutamate release. During ER stress, inhibition of SOCE or syt7-driven fusion partially restored basal neurotransmission and decreased expression of pro-apoptotic markers, indicating that these processes participate in the amplification of ER-stress-related damage. Taken together, we propose that presynaptic SOCE links ER stress and augmented spontaneous neurotransmission, which may, in turn, facilitate neurodegeneration., Competing Interests: Declaration of interest The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Spontaneous neurotransmitter release is a fundamental property of synapses in which neurotransmitter filled vesicles release their content independent of presynaptic action potentials (APs). Despite their seemingly random nature, these spontaneous fusion events can be regulated by Ca 2+ signaling pathways. Here, we probed the mechanisms that maintain Ca 2+ sensitivity of spontaneous release events in synapses formed between hippocampal neurons cultured from rats of both sexes. In this setting, we examined the potential role of vesicle-associated membrane protein 4 (VAMP4), a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in spontaneous neurotransmission. Our results show that VAMP4 is required for Ca 2+ -dependent spontaneous excitatory neurotransmission, with a limited role in spontaneous inhibitory neurotransmission. Key residues in VAMP4 that regulate its retrieval as well as functional clathrin-mediated vesicle trafficking were essential for the maintenance of VAMP4-mediated spontaneous release. Moreover, high-frequency stimulation (HFS) that typically triggers asynchronous release and retrieval of VAMP4 from the plasma membrane also augmentsCa 2+ -sensitive spontaneous release for up to 30 min in a VAMP4-dependent manner. This VAMP4-mediated link between asynchronous and spontaneous excitatory neurotransmission might serve as a presynaptic substrate for synaptic plasticity coupling distinct forms of release. SIGNIFICANCE STATEMENT Spontaneous neurotransmitter release that occurs independent of presynaptic action potentials (APs) shows significant sensitivity to intracellular Ca 2+ levels. In this study, we identify the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) molecule vesicle-associated membrane protein 4 (VAMP4) as a key component of the machinery that maintains these Ca 2+ -sensitive fraction of spontaneous release events. Following brief intense activity, VAMP4-dependent synaptic vesicle retrieval supports a pool of vesicles that fuse spontaneously in the long term. We propose that this vesicle trafficking pathway acts to shape spontaneous release and associated signaling based on previous activity history of synapses., (Copyright © 2020 the authors.)

Synaptic vesicle fusion is coupled to swift retrieval of vesicle components from the synaptic plasma membrane. Ca 2+ has been assumed to be a key mediator of this coupling. In a recent study, Orlando et al. unequivocally demonstrate that Ca 2+ is not essential for synaptic vesicle retrieval., (Copyright © 2019 Elsevier Ltd. All rights reserved.)