Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.

Visualizing sequential compound fusion and kiss-and-run in live excitable cells

Vesicle fusion is assumed to occur at flat membrane of excitable cells. In live neuroendocrine cells, we visualized vesicle fusion at Ω-shape membrane generated by preceding fusion, termed sequential compound fusion, which may be followed by fusion pore closure, termed compound kiss-and-run. These novel fusion modes contribute to vesicle docking, multi-vesicular release, asynchronous release, and endocytosis. We suggest modifying current models of exo-endocytosis to include these new fusion modes.

Asynchronous glutamate exocytosis is enhanced in low release probability synapses and is widely dispersed across the active zone.

The balance between fast synchronous and delayed asynchronous release of neurotransmitters has a major role in defining computational properties of neuronal synapses and regulation of neuronal network activity. However, how it is tuned at the single synapse level remains poorly understood. Here, using the fluorescent glutamate sensor SF-iGluSnFR, we image quantal vesicular release in tens to hundreds of individual synaptic outputs from single pyramidal cells with 4 millisecond temporal and 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among synapses supplied by the same axon, and that and that synchronicity of release is reduced at low release probability synapses. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the release area than synchronous sites. Together, our results reveal a universal relationship between the two major functional properties of synapses – the timing and the probability of neurotransmitter release.

Multiscale modeling of presynaptic dynamics from molecular to mesoscale

Chemical synapses exhibit a diverse array of internal mechanisms that affect the dynamics of transmission efficacy. Many of these processes, such as release of neurotransmitter and vesicle recycling, depend strongly on activity-dependent influx and accumulation of Ca2+. To model how each of these processes may affect the processing of information in neural circuits, and how their dysfunction may lead to disease states, requires a computationally efficient modelling framework, capable of generating accurate phenomenology without incurring a heavy computational cost per synapse. Constructing a phenomenologically realistic model requires the precise characterization of the timing and probability of neurotransmitter release. Difficulties arise in that functional forms of instantaneous release rate can be difficult to extract from noisy data without running many thousands of trials, and in biophysical synapses, facilitation of per-vesicle release probability is confounded by depletion. To overcome this, we obtained traces of free Ca2+ concentration in response to various action potential stimulus trains from a molecular MCell model of a hippocampal mossy fiber axon. Ca2+ sensors were placed at varying distance from a voltage-dependent calcium channel (VDCC) cluster, and Ca2+ was buffered by calbindin. Then, using the calcium traces to drive deterministic state vector models of synaptotagmin 1 and 7 (Syt-1/7), which respectively mediate synchronous and asynchronous release in excitatory hippocampal synapses, we obtained high-resolution profiles of instantaneous release rate, to which we applied functional fits. Synchronous vesicle release occurred predominantly within half a micron of the source of spike-evoked Ca2+ influx, while asynchronous release occurred more consistently at all distances. Both fast and slow mechanisms exhibited multi-exponential release rate curves, whose magnitudes decayed exponentially with distance from the Ca2+ source. Profile parameters facilitate on different time scales according to a single, general facilitation function. These functional descriptions lay the groundwork for efficient mesoscale modelling of vesicular release dynamics.

Synaptotagmin 7 is enriched at the plasma membrane through γ-secretase processing to promote vesicle docking and control synaptic plasticity in mouse hippocampal neurons

Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via γ-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity. SYT7 mediated asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment was observed optically at individual hippocampal synapses Localization, trafficking, and stability of SYT7 is dependent on processing by γ-secretase Short term plasticity defects arise in SYT7KOs due to decreased docking of synaptic vesicles after stimulation SYT7 promotes paired-pulse facilitation and asynchronous release via distinct mechanisms

Asynchronous release sites align with NMDA receptors in mouse hippocampal synapses

Neurotransmitter is released synchronously and asynchronously following an action potential. Our recent study indicates that the release sites of these two phases are segregated within an active zone, with asynchronous release sites enriched near the center in mouse hippocampal synapses. Here we demonstrate that synchronous and asynchronous release sites are aligned with AMPA receptor and NMDA receptor clusters, respectively. Computational simulations indicate that this spatial and temporal arrangement of release can lead to maximal membrane depolarization through AMPA receptors, alleviating the pore-blocking magnesium leading to greater activation of NMDA receptors. Together, these results suggest that release sites are likely organized to activate NMDA receptors efficiently.

Asynchronous Glutamate Release at Autapses Regulates Spike Reliability and Precision in Mouse Neocortical Pyramidal Cells

Autapses are self-synapses of a neuron. Inhibitory autapses in the neocortex release GABA in 2 modes, synchronous release and asynchronous release (AR), providing precise and prolonged self-inhibition, respectively. A subpopulation of neocortical pyramidal cells (PCs) also forms functional autapses, activation of which promotes burst firing by strong unitary autaptic response that reflects synchronous glutamate release. However, it remains unclear whether AR occurs at PC autapses and plays a role in neuronal signaling. We performed whole-cell recordings from layer-5 PCs in slices of mouse prefrontal cortex (PFC). In response to action potential (AP) burst, 63% of PCs showed robust long-lasting autaptic AR, much stronger than synaptic AR between neighboring PCs. The autaptic AR is mediated predominantly by P/Q-type Ca2+ channels, and its strength depends on the intensity of PC activity and the level of residual Ca2+. Further experiments revealed that autaptic AR enhances spiking activities but reduces the temporal precision of post-burst APs. Together, the results show the occurrence of AR at PC autapses, the delayed and persistent glutamate AR causes self-excitation in individual PCs but may desynchronize the autaptic PC population. Thus, glutamatergic autapses should be essential elements in PFC and contribute to cortical information processing.

Expression and distribution of synaptotagmin isoforms in the zebrafish retina

ABSTRACTSynaptotagmins belong to a large family of proteins. While various synaptotagmins have been implicated as Ca2+ sensors for vesicle replenishment and release at conventional synapses, their roles at retinal ribbon synapses remain incompletely understood. Zebrafish is a widely used experimental model for retinal research. We therefore investigated the homology between human, rat, mouse, and zebrafish synaptotagmins 1 to 10 using a bioinformatics approach. We also characterized the expression and distribution of various synaptotagmin (syt) genes in the zebrafish retina using RT-PCR and in situ hybridization, focusing on the family members whose products likely underlie Ca2+-dependent exocytosis in the central nervous system (synaptotagmins 1, 2, 5 and 7). We find that most zebrafish synaptotagmins are well conserved and can be grouped in the same classes as mammalian synaptotagmins, based on crucial amino acid residues needed for coordinating Ca2+ binding and determining phospholipid binding affinity. The only exception is synaptotagmin 1b, which lacks 34 amino acid residues in the C2B domain and is therefore unlikely to bind Ca2+ there. Additionally, the products of zebrafish syt5a and syt5b genes share identity with mammalian class 1 and 5 synaptotagmins. Zebrafish syt1, syt2, syt5 and syt7 paralogues are found in the zebrafish brain, eye, and retina, excepting syt1b, which is only present in the brain. The complementary expression pattern of the remaining paralogues in the retina suggests that syt1a and syt5a may underlie synchronous release and syt7a and syt7b may mediate asynchronous release or other Ca2+ dependent processes in different types of retinal neurons.