Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size.
Ontology highlight
ABSTRACT:
SUBMITTER: Toonen RF
PROVIDER: S-EPMC1838751 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Neurexins are a diverse family of cell adhesion molecules that localize to presynaptic specializations of CNS neurons. Heterologous expression of neurexins in non-neuronal cells leads to the recruitment of postsynaptic proteins in contacting dendrites of co-cultured neurons, implicating neurexins in synapse formation. However, isoform-specific knockouts of either all α- or all β-neurexins show defects in synaptic transmission but an unaltered density of glutamatergic synapses, a finding that argues against an essential function of neurexins in synaptogenesis. To address the role of neurexin in synapse formation and function, we disrupted the function of all α- and β-neurexins in cultured hippocampal neurons by shRNA knockdown or by overexpressing a neurexin mutant that is unable to bind to postsynaptic neurexin ligands. We show that neurexin perturbation results in an attenuation of neurotransmitter release that is in large part due to a reduction in the number of readily releasable synaptic vesicles. We also find that neurexin perturbation fails to alter the ability of neurons to form synapses, but rather leads to more frequent synapse elimination. These experiments suggest that neurexins are dispensable for the formation of initial synaptic contacts, but play an essential role in the stabilization and functional maturation of synapses.
| S-EPMC5318902 | biostudies-literature
Project description:Recycling of synaptic vesicles (SVs) is a fundamental step in the process of neurotransmission. Endocytosed SV can travel directly into the recycling pool or recycle through endosomes but little is known about the molecular actors regulating the switch between these SV recycling routes. ADP ribosylation factor 6 (Arf6) is a small GTPase known to participate in constitutive trafficking between plasma membrane and early endosomes. Here, we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs. These features were phenocopied by pharmacological blockage of Arf6 activation. The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting. We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV.
| S-EPMC4764570 | biostudies-literature
Project description:Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca(2+) channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca(2+) channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.
| S-EPMC3747298 | biostudies-literature
Project description:The number of neurotransmitter-filled vesicles released into the synaptic cleft with each action potential dictates the reliability of synaptic transmission. Variability of this fundamental property provides diversity of synaptic function across brain regions, but the source of this variability is unclear. The prevailing view is that release of a single (univesicular release, UVR) or multiple vesicles (multivesicular release, MVR) reflects variability in vesicle release probability, a notion that is well-supported by the calcium-dependence of release mode. However, using mouse brain slices, we now demonstrate that the number of vesicles released is regulated by the size of the readily-releasable pool, upstream of vesicle release probability. Our results point to a model wherein protein kinase A and its vesicle-associated target, synapsin, dynamically control release site occupancy to dictate the number of vesicles released without altering release probability. Together these findings define molecular mechanisms that control MVR and functional diversity of synaptic signaling.
| S-EPMC6716946 | biostudies-literature
Project description:IntroductionDynamic physiological changes in brain extracellular calcium ([Ca2+]o) occur when high levels of neuronal activity lead to substantial Ca2+ entry via ion channels reducing local [Ca2+]o. Perturbations of the extracellular microenvironment that increase [Ca2+]o are commonly used to study how [Ca2+] regulates neuronal activity. At excitatory synapses, the Ca2+-sensing receptor (CaSR) and other G-protein coupled receptors link [Ca2+]o and spontaneous glutamate release. Phospholipase C (PLC) is activated by G-proteins and is hypothesized to mediate this process.MethodsPatch-clamping cultured neocortical neurons, we tested how spontaneous glutamate release was affected by [Ca2+]o and inhibition of PLC activity. We used hypertonic sucrose (HS) to evaluate the readily releasable pool (RRP) and test if it was affected by inhibition of PLC activity.ResultsSpontaneous glutamate release substantially increased with [Ca2+]o, and inhibition of PLC activity, with U73122, abolished this effect. PLC-β1 is an abundant isoform in the neocortex, however, [Ca2+]o-dependent spontaneous release was unchanged in PLC-β1 null mutants (PLC-β1-/-). U73122 completely suppressed this response in PLC-β1-/- neurons, indicating that this residual [Ca2+]o-sensitivity may be mediated by other PLC isoforms. The RRP size was substantially reduced after incubation in U73122, but not U73343. Phorbol esters increased RRP size after PLC inhibition.DiscussionTogether these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca2+]o and other extracellular cues, possibly by modifying the size of the RRP.
| S-EPMC10228687 | biostudies-literature
Project description:Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles.
| S-EPMC4379469 | biostudies-literature
Project description:Endocytosis is essential in maintaining exocytosis at secretory cells. Rapid endocytosis with a time course less than a few seconds is widely observed at nerve terminals and non-neuronal secretory cells. It is generally assumed that rapid endocytosis recycles vesicles within the readily releasable pool (RRP) via a kiss-and-run mechanism that involves rapid opening and closure of a fusion pore at the release site. The present work suggests that both rapid (tau less than approximately 2 s) and slow (tau = approximately 10-20 s) endocytosis do not recycle vesicles to the RRP but to a recycling pool at least a few times larger than the RRP at a nerve terminal, the calyx of Held in rat brainstem. Challenging the long-held view that rapid endocytosis offers a rapid, local vesicle recycling within the RRP, our finding calls for reconsideration of the function and the underlying mechanism of rapid endocytosis. We suggest that rapid endocytosis provides the nerve terminal the ability to recycle vesicles rapidly via the recycling pool and to maintain the normal morphology of the nerve terminal, including the release site, by rapidly clearing the fused vesicle membrane from the release site during intense firing.
| S-EPMC2757152 | biostudies-literature
Project description:To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.
| S-EPMC3497746 | biostudies-literature
Project description:Recruitment of release-competent vesicles during sustained synaptic activity is one of the major factors governing short-term plasticity. During bursts of synaptic activity, vesicles are recruited to a fast-releasing pool from a reluctant vesicle pool through an actin-dependent mechanism. We now show that newly recruited vesicles in the fast-releasing pool do not respond at full speed to a strong Ca(2+) stimulus, but require approximately 4 s to mature to a "superprimed" state. Superpriming was found to be altered by agents that modulate the function of unc13 homolog proteins (Munc13s), but not by calmodulin inhibitors or actin-disrupting agents. These findings indicate that recruitment and superpriming of vesicles are regulated by separate mechanisms, which require integrity of the cytoskeleton and activation of Munc13s, respectively. We propose that refilling of the fast-releasing vesicle pool proceeds in two steps, rapid actin-dependent "positional priming," which brings vesicles closer to Ca(2+) sources, followed by slower superpriming, which enhances the Ca(2+) sensitivity of primed vesicles.
| S-EPMC3773744 | biostudies-literature
Project description:The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in ?-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation.
| S-EPMC3482238 | biostudies-literature