Inhibition of exocytosis or endocytosis blocks activity-dependent redistribution of synapsin.
ABSTRACT The synaptic vesicle cycle encompasses the pre-synaptic events that drive neurotransmission. Influx of calcium leads to the fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter, closely followed by endocytosis. Vacated release sites are repopulated with vesicles which are then primed for release. When activity is intense, reserve vesicles may be mobilized to counteract an eventual decline in transmission. Recently, interplay between endocytosis and repopulation of the readily releasable pool of vesicles has been identified. In this study, we show that exo-endocytosis is necessary to enable detachment of synapsin from reserve pool vesicles during synaptic activity. We report that blockage of exocytosis in cultured mouse hippocampal neurons, either by tetanus toxin or by the deletion of munc13, inhibits the activity-dependent redistribution of synapsin from the pre-synaptic terminal into the axon. Likewise, perturbation of endocytosis with dynasore or by a dynamin dominant-negative mutant fully prevents synapsin redistribution. Such inhibition of synapsin redistribution occurred despite the efficient phosphorylation of synapsin at its protein kinase A/CaMKI site, indicating that disengagement of synapsin from the vesicles requires exocytosis and endocytosis in addition to phosphorylation. Our results therefore reveal hitherto unidentified feedback within the synaptic vesicle cycle involving the synapsin-managed reserve pool.
Full-textDOI: · Available from: Daniel Gitler, Aug 12, 2015
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ABSTRACT: Presynaptic terminals are specialized sites for information transmission where vesicles fuse with the plasma membrane and are locally recycled. Recent work has extended this classical view, with the observation that a subset of functional vesicles is dynamically shared between adjacent terminals by lateral axonal transport. Conceptually, such transport would be expected to disrupt vesicle retention around the active zone, yet terminals are characterized by a high-density vesicle cluster, suggesting that counteracting stabilizing mechanisms must operate against this tendency. The synapsins are a family of proteins that associate with synaptic vesicles and determine vesicle numbers at the terminal, but their specific function remains controversial. Here, using multiple quantitative fluorescence-based approaches and electron microscopy, we show that synapsin is instrumental for resisting vesicle dispersion and serves as a regulatory element for controlling lateral vesicle sharing between synapses. Deleting synapsin disrupts the organization of presynaptic vesicle clusters, making their boundaries hard to define. Concurrently, the fraction of vesicles amenable to transport is increased, and more vesicles are translocated to the axon. Importantly, in neurons from synapsin knock-out mice the resting and recycling pools are equally mobile. Synapsin, when present, specifically restricts the mobility of resting pool vesicles without affecting the division of vesicles between these pools. Specific expression of synapsin IIa, the sole isoform affecting synaptic depression, rescues the knock-out phenotype. Together, our results show that synapsin is pivotal for maintaining synaptic vesicle cluster integrity and that it contributes to the regulated sharing of vesicles between terminals.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 03/2012; 32(12):3969-80. DOI:10.1523/JNEUROSCI.5058-11.2012 · 6.75 Impact Factor
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ABSTRACT: Synapsins are abundant synaptic vesicle (SV)-associated proteins thought to mediate synaptic vesicle mobility and clustering at most synapses. We used synapsin triple knock-out (TKO) mice to examine the morphological and functional consequences of deleting all synapsin isoforms at the calyx of Held, a giant glutamatergic synapse located in the auditory brain stem. Quantitative three-dimensional (3D) immunohistochemistry of entire calyces showed lower amounts of the synaptic vesicle protein vGluT1 while the level of the active zone marker bassoon was unchanged in TKO terminals. Examination of brain lysates by ELISA revealed a strong reduction in abundance of several synaptic vesicle proteins, while proteins of the active zone cytomatrix or postsynaptic density were unaffected. Serial section scanning electron microscopy of large 3D-reconstructed segments confirmed a decrease in the number of SVs to approximately 50% in TKO calyces. Short-term depression tested at stimulus frequencies ranging from 10 to 300 Hz was accelerated only at frequencies above 100 Hz and the time course of recovery from depression was slowed in calyces lacking synapsins. These results reveal that in wild-type synapses, the synapsin-dependent reserve pool contributes to the replenishment of the readily releasable pool (RRP), although accounting only for a small fraction of the SVs that enter the RRP. In conclusion, our results suggest that synapsins may be required for normal synaptic vesicle biogenesis, trafficking and immobilization of synaptic vesicles, yet they are not essential for sustained high-frequency synaptic transmission at the calyx terminal.European Journal of Neuroscience 07/2012; 36(8):3005-20. DOI:10.1111/j.1460-9568.2012.08225.x · 3.67 Impact Factor
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ABSTRACT: Studying epileptogenesis in a genetic model can facilitate the identification of factors that promote the conversion of a normal brain into one chronically prone to seizures. Synapsin triple-knockout (TKO) mice exhibit adult-onset epilepsy, thus allowing the characterization of events as preceding or following seizure onset. Although it has been proposed that a congenital reduction in inhibitory transmission is the underlying cause for epilepsy in these mice, young TKO mice are asymptomatic. We report that the genetic lesion exerts long-term progressive effects that extend well into adulthood. Although inhibitory transmission is initially reduced, it is subsequently strengthened relative to its magnitude in control mice, so that the excitation to inhibition balance in adult TKOs is inverted in favor of inhibition. In parallel, we observed long-term alterations in synaptic depression kinetics of excitatory transmission and in the extent of tonic inhibition, illustrating adaptations in synaptic properties. Moreover, age-dependent acceleration of the action potential did not occur in TKO cortical pyramidal neurons, suggesting wide-ranging secondary changes in brain excitability. In conclusion, although congenital impairments in inhibitory transmission may initiate epileptogenesis in the synapsin TKO mice, we suggest that secondary adaptations are crucial for the establishment of this epileptic network.Cerebral Cortex 12/2012; DOI:10.1093/cercor/bhs384 · 8.67 Impact Factor