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Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca2+. However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites. Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts. These fusion events were coupled to postsynaptic NMDA receptor-driven Ca2+ signals, and at elevated Ca2+ concentrations there was an increase in the number of vesicles that would undergo fusion. Furthermore, spontaneous vesicle fusion propensity in a synapse was Ca2+-dependent but regulated autonomously: independent of evoked fusion probability at the same synapse. Taken together, these results expand classical quantal analysis to incorporate endocytic and exocytic phases of single fusion events and uncover autonomous regulation of spontaneous fusion. Neurons communicate with one another at junctions called synapses. When an electrical signal known as an action potential arrives at a synapse, it causes packages called vesicles to fuse with the membrane that surrounds the neuron. The vesicles contain molecules called neurotransmitters, which are then released into the gap between the neurons. When these molecules bind to receptors on the surface of the second neuron, a copy of the action potential is generated and travels along the second neuron. The empty vesicles are then reabsorbed back into the first cell to be refilled with neurotransmitters so that the whole process can be repeated. In addition to releasing neurotransmitters in response to the arrival of an action potential, neurons sometimes release vesicles spontaneously. Such events are relatively rare and occur seemingly at random, making them difficult to study. However, by labeling a synaptic vesicle protein with a fluorescent protein, Leitz and Kavalali have constructed a system in which they can observe spontaneous vesicle fusions in single synapses in cell cultures, and follow the fate of the vesicles as they are reabsorbed back into the cell. The results reveal a number of key differences between the spontaneous events and those triggered by action potentials. Vesicles released spontaneously are retrieved and recycled much more rapidly than those that are released following the arrival of an action potential. Moreover, increases in calcium levels increase the frequency of both types of events. However, it is also clear that the calcium ions influence the two types of events independently of one another. Recent research on flies has suggested that some regions of synapses only ever release vesicles spontaneously, whereas others only ever release vesicles in response to the arrival of an action potential. The work of Leitz and Kavalali now adds to increasing evidence that the spontaneous release of neurotransmitters may have its own role in neuronal signaling that is distinct from the role played by neurotransmitters that are released in response to action potentials.

A well‐posed physics‐based compact model for a three‐terminal silicon–oxide–nitride–oxide–silicon (SONOS) synaptic circuit element is presented for use by neuromorphic circuit/system engineers. Based on technology computer aided design (TCAD) simulations of a SONOS device, the model contains a nonvolatile memristor with the state variable QM representing the memristor charge under the gate of the three‐terminal element. By incorporating the exponential dependence of the memristance on QM and the applied bias V for the gate, the compact model agrees quantitatively with the results from TCAD simulations as well as experimental measurements for the drain current. The compact model is implemented through VerilogA in the circuit simulation package Cadence Spectre and reproduces the experimental training behavior for the source–drain conductance of a SONOS device after applying writing pulses ranging from −12 V to +11 V, with an accuracy higher than 90%. A well‐posed physics‐based compact model of a three‐terminal silicon–oxide–nitride–oxide–silicon (SONOS) synaptic circuit element is presented for neuromorphic circuit designs. Based on technology‐computer‐aided design (TCAD) simulations, a fundamental compact model requiring a memristor was formulated. The model was verified by simulation in Cadence Spectre with VerilogA, which yielded quantitative agreement to experimentally measured channel currents.

Coupling of synaptic vesicle fusion and retrieval constitutes a core mechanism ensuring maintenance of presynaptic function. Recent studies using fast-freeze electron microscopy and capacitance measurements reported an ultrafast mode of endocytosis operating at physiological temperatures. Here, using rat hippocampal neurons, we optically monitored single synaptic vesicle endocytosis with high time resolution using the vesicular glutamate transporter, synaptophysin and the V0a1 subunit of the vacuolar ATPase as probes. In this setting, we could distinguish three components of retrieval operating at ultrafast (~150–250 ms, ~20% of events), fast (~5–12 s, ~40% of events) and ultraslow speeds (>20 s, ~40% of events). While increasing Ca2+ slowed the fast events, increasing temperature accelerated their time course. In contrast, the kinetics of ultrafast events were only mildly affected by these manipulations. These results suggest that synaptic vesicle proteins can be retrieved with ultrafast kinetics, although a majority of evoked fusion events are coupled to slower retrieval mechanisms. Nerve cells or neurons exchange information at junctions called synapses. To send a message to its neighbor, a neuron must release molecules called neurotransmitters into the synapse. These then bind to receptor proteins on the neighboring cell. But neurons do not release neurotransmitter molecules one at a time. Instead they release them in packages called vesicles. Each vesicle contains about 1,000 molecules, which it releases by fusing with the cell membrane. The entire process takes less than one thousandth of a second. Synaptic vesicles are complex structures made up of many different proteins and lipids. To help ensure that neurons do not run out of vesicles, cells retrieve and recycle these components via a process called endocytosis. A number of studies have attempted to measure how long this retrieval process takes. But the studies – which used a variety of different techniques – yielded results ranging from a few hundredths of a second to more than a minute. Chanaday and Kavalali have now resolved this discrepancy by using fluorescence microscopy to study the retrieval process in rat brain cells. By attaching a fluorescent tag to specific molecules within the vesicle membrane, Chanaday and Kavalali were able to track individual vesicles. The results revealed that neurons retrieve vesicles from synapses via three different pathways. At temperatures like those in the rodent or human body, an ‘ultraslow’ pathway takes more than 20 seconds to retrieve vesicles. By contrast, a ‘fast’ pathway takes about 5 to 12 seconds. The quickest option, an ‘ultrafast’ pathway, retrieves vesicles in about 150 to 250 milliseconds. Increasing the temperature speeds up the fast pathway but has no effect on the ultrafast pathway. Neurons can thus retrieve vesicles from synapses in about 200 milliseconds, or one fifth of a second. Nevertheless, they retrieve about 80% of their vesicles using the two slower pathways. Identifying the mechanisms responsible for vesicle retrieval will help reveal how synapses work, as well as what can go wrong. Changes in components of synaptic vesicles contribute to several neurological and psychiatric diseases. Developing drugs that target synaptic vesicle recycling could be a promising therapeutic avenue.

Astrocytes regulate the formation and function of neuronal synapses via multiple signals; however, what controls regional and temporal expression of these signals during development is unknown. We determined the expression profile of astrocyte synapse-regulating genes in the developing mouse visual cortex, identifying astrocyte signals that show differential temporal and layer-enriched expression. These patterns are not intrinsic to astrocytes, but regulated by visually evoked neuronal activity, as they are absent in mice lacking glutamate release from thalamocortical terminals. Consequently, synapses remain immature. Expression of synapse-regulating genes and synaptic development is also altered when astrocyte signaling is blunted by diminishing calcium release from astrocyte stores. Single-nucleus RNA sequencing identified groups of astrocytic genes regulated by neuronal and astrocyte activity, and a cassette of genes that show layer-specific enrichment. Thus, the development of cortical circuits requires coordinated signaling between astrocytes and neurons, highlighting astrocytes as a target to manipulate in neurodevelopmental disorders.

The cytosolic proteins synucleins and synapsins are thought to play cooperative roles in regulating synaptic vesicle (SV) recycling, but mechanistic insight is lacking. Here, we identify the synapsin E-domain as an essential functional binding-partner of α-synuclein (α-syn). Synapsin E-domain allows α-syn functionality, binds to α-syn, and is necessary and sufficient for enabling effects of α-syn at synapses of cultured mouse hippocampal neurons. Together with previous studies implicating the E-domain in clustering SVs, our experiments advocate a cooperative role for these two proteins in maintaining physiologic SV clusters.

Presynaptic neuronal activity requires the localization of thousands of proteins that are typically synthesized in the soma and transported to nerve terminals. Local translation for some dendritic proteins occurs, but local translation in mammalian presynaptic nerve terminals is difficult to demonstrate. Here, we show an essential ribosomal component, 5.8S rRNA, at a glutamatergic nerve terminal in the mammalian brain. We also show active translation in nerve terminals, in situ, in brain slices demonstrating ongoing presynaptic protein synthesis in the mammalian brain. Shortly after inhibiting translation, the presynaptic terminal exhibits increased spontaneous release, an increased paired pulse ratio, an increased vesicle replenishment rate during stimulation trains, and a reduced initial probability of release. The rise and decay rates of postsynaptic responses were not affected. We conclude that ongoing protein synthesis can limit excessive vesicle release which reduces the vesicle replenishment rate, thus conserving the energy required for maintaining synaptic transmission.

Recent research suggests that in central mammalian synapses, active zones contain several docking sites acting in parallel. Before release, one or several synaptic vesicles (SVs) are thought to bind to each docking site, forming the readily releasable pool (RRP). Determining the RRP size per docking site has important implications for short-term synaptic plasticity. Here, using mouse cerebellar slices, we take advantage of recently developed methods to count the number of released SVs at single glutamatergic synapses in response to trains of action potentials (APs). In each recording, the number of docking sites was determined by fitting with a binomial model the number of released SVs in response to individual APs. After normalization with respect to the number of docking sites, the summed number of released SVs following a train of APs was used to estimate of the RRP size per docking site. To improve this estimate, various steps were taken to maximize the release probability of docked SVs, the occupancy of docking sites, as well as the extent of synaptic depression. Under these conditions, the RRP size reached a maximum value close to two SVs per docking site. The results indicate that each docking site contains two distinct SV-binding sites that can simultaneously accommodate up to one SV each. They further suggest that under special experimental conditions, as both sites are close to full occupancy, a maximal RRP size of two SVs per docking site can be reached. More generally, the results validate a sequential two-step docking model previously proposed at this preparation.

While genetically encoded Ca 2+ indicators are valuable for visualizing neural activity, their speed and sensitivity have had limited performance when compared to chemical dyes and electrophysiology, particularly at synaptic compartments. We addressed these limitations by engineering a suite of next-generation GCaMP8-based indicators, targeted to presynaptic boutons, active zones, and postsynaptic compartments at the Drosophila neuromuscular junction. We first validated these sensors to be superior to previous versions and synthetic dyes. Next, we developed a Python-based analysis program, CaFire , which enables the automated quantification of evoked and spontaneous Ca² + signals. Using CaFire , we show a ratiometric presynaptic GCaMP8m sensor accurately captures physiologically relevant presynaptic Ca 2+ changes with superior sensitivity and similar kinetics compared to chemical dyes. Moreover, we test the ability of an active zone-targeted, ratiometric GCaMP8m sensor to report differences in Ca² + between release sites. Finally, a newly engineered postsynaptic GCaMP8m, positioned near glutamate receptors, detects quantal events with temporal and signal resolution comparable to electrophysiological recordings. These next-generation indicators and analytical methods demonstrate that GCaMP8 sensors, targeted to synaptic compartments, can now achieve the speed and sensitivity necessary to resolve Ca 2+ dynamics at levels previously only attainable with chemical dyes or electrophysiology.

Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional composite nanofilm that facilitates visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈ 3.2 µm (full width at half maximum [FWHM]) and are observed with a mean spatial frequency of one hotspot per ≈ 7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots co-localized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals. To form the vast and complex network necessary for an organism to sense and react to the world, neurons must connect at highly specialized junctions. Individual cells communicate at these ‘synapses’ by releasing chemical signals (or neurotransmitters) such as dopamine, a molecule involved in learning and motivation. Despite the central role that synapses play in the brain, it remains challenging to measure exactly where neurotransmitters are released and how far they travel from their release site. Currently, most tools available to scientists only allow bulk measurements of neurotransmitter release. To tackle this limitation, Bulumulla et al. developed a new way to measure neurotransmitter release from neurons, harnessing a technique which uses fluorescent nanosensors that glow brighter when exposed to dopamine. These sensors form a very thin film upon which neurons can grow; when the cells release dopamine, the sensors ‘light up’ as they encounter the molecule. Dubbed DopaFilm, the technology reveals exactly where the neurotransmitter comes from and how it spreads between cells in real time. In particular, the approach showed that dopamine emerges from 'hot spots' at specific sites in cells; it also helped Bulumulla et al. study how dopamine is released from subcellular compartments that have previously not been well characterized. Improving the sensors so that the film could detect other neurotransmitters besides dopamine would broaden the use of this approach. In the future, combining this technology with other types of imaging should enable studies of individual synapses with intricate detail.

The circadian clock regulates various aspects of brain health including microglial and astrocyte activation. Here, we report that deletion of the master clock protein BMAL1 in mice robustly increases expression of complement genes, including C4b and C3 , in the hippocampus. BMAL1 regulates expression of the transcriptional repressor REV-ERBα, and deletion of REV-ERBα causes increased expression of C4b transcript in neurons and astrocytes as well as C3 protein primarily in astrocytes. REV-ERBα deletion increased microglial phagocytosis of synapses and synapse loss in the CA3 region of the hippocampus. Finally, we observed diurnal variation in the degree of microglial synaptic phagocytosis which was antiphase to REV-ERBα expression. This daily variation in microglial synaptic phagocytosis was abrogated by global REV-ERBα deletion, which caused persistently elevated synaptic phagocytosis. This work uncovers the BMAL1-REV-ERBα axis as a regulator of complement expression and synaptic phagocytosis in the brain, linking circadian proteins to synaptic regulation.