Spontaneous Neurotransmitter Release Research Articles

The stability of the primed pool of synaptic vesicles and the clamping of spontaneous neurotransmitter release rely on the integrity of the C-terminal half of the SNARE domain of syntaxin-1A

The SNARE proteins are central in membrane fusion and, at the synapse, neurotransmitter release. However, their involvement in the dual regulation of the synchronous release while maintaining a pool of readily releasable vesicles remains unclear. Using a chimeric approach, we performed a systematic analysis of the SNARE domain of STX1A by exchanging the whole SNARE domain or its N- or C-terminus subdomains with those of STX2. We expressed these chimeric constructs in STX1-null hippocampal mouse neurons. Exchanging the C-terminal half of STX1’s SNARE domain with that of STX2 resulted in a reduced RRP accompanied by an increased release rate, while inserting the C-terminal half of STX1’s SNARE domain into STX2 leads to an enhanced priming and decreased release rate. Additionally, we found that the mechanisms for clamping spontaneous, but not for Ca2+-evoked release, are particularly susceptible to changes in specific residues on the outer surface of the C-terminus of the SNARE domain of STX1A. Particularly, mutations of D231 and R232 affected the fusogenicity of the vesicles. We propose that the C-terminal half of the SNARE domain of STX1A plays a crucial role in the stabilization of the RRP as well as in the clamping of spontaneous synaptic vesicle fusion through the regulation of the energetic landscape for fusion, while it also plays a covert role in the speed and efficacy of Ca2+-evoked release.

The stability of the primed pool of synaptic vesicles and the clamping of spontaneous neurotransmitter release rely on the integrity of the C-terminal half of the SNARE domain of syntaxin-1A.

The SNARE proteins are central in membrane fusion and, at the synapse, neurotransmitter release. However, their involvement in the dual regulation of the synchronous release while maintaining a pool of readily releasable vesicles remains unclear. Using a chimeric approach, we performed a systematic analysis of the SNARE domain of STX1A by exchanging the whole SNARE domain or its N- or C-terminus subdomains with those of STX2. We expressed these chimeric constructs in STX1-null hippocampal mouse neurons. Exchanging the C-terminal half of STX1's SNARE domain with that of STX2 resulted in a reduced RRP accompanied by an increased release rate, while inserting the C-terminal half of STX1's SNARE domain into STX2 leads to an enhanced priming and decreased release rate. Additionally, we found that the mechanisms for clamping spontaneous, but not for Ca2+-evoked release, are particularly susceptible to changes in specific residues on the outer surface of the C-terminus of the SNARE domain of STX1A. Particularly, mutations of D231 and R232 affected the fusogenicity of the vesicles. We propose that the C-terminal half of the SNARE domain of STX1A plays a crucial role in the stabilization of the RRP as well as in the clamping of spontaneous synaptic vesicle fusion through the regulation of the energetic landscape for fusion, while it also plays a covert role in the speed and efficacy of Ca2+-evoked release.

Microtubule depolymerization contributes to spontaneous neurotransmitter release in vitro

Microtubules are key to multiple neuronal functions involving the transport of organelles, however, their relationship to neurotransmitter release is still unresolved. Here, we show that microtubules present in the presynaptic compartment of cholinergic autaptic synapses are dynamic. To investigate how the balance between microtubule growth and shrinkage affects neurotransmission we induced synchronous microtubule depolymerization by photoactivation of the chemical inhibitor SBTub3. The consequence was an increase in spontaneous neurotransmitter release. An analogous effect was obtained by dialyzing the cytosol with Kif18A, a plus-end-directed kinesin with microtubule depolymerizing activity. Kif18A also inhibited the refilling of the readily releasable pool of synaptic vesicles during high frequency stimulation. The action of Kif18A was associated to one order of magnitude increases in the numbers of exo-endocytic pits and endosomes present in the presynaptic terminal. An enhancement of spontaneous neurotransmitter release was also observed when neurons were dialyzed with stathmin-1, a protein with a widespread presence in the nervous system that induces microtubule depolymerization. Taken together, these results support that microtubules restrict spontaneous neurotransmitter release as well as promote the replenishment of the readily releasable pool of synaptic vesicles.

Nanoelectrochemistry reveals how soluble Aβ42 oligomers alter vesicular storage and release of glutamate

Glutamate (Glu) is the major excitatory transmitter in the nervous system. Impairment of its vesicular release by β-amyloid (Aβ) oligomers is thought to participate in pathological processes leading to Alzheimer's disease. However, it remains unclear whether soluble Aβ42 oligomers affect intravesicular amounts of Glu or their release in the brain, or both. Measurements made in this work on single Glu varicosities with an amperometric nanowire Glu biosensor revealed that soluble Aβ42 oligomers first caused a dramatic increase in vesicular Glu storage and stimulation-induced release, accompanied by a high level of parallel spontaneous exocytosis, ultimately resulting in the depletion of intravesicular Glu content and greatly reduced release. Molecular biology tools and mouse models of Aβ amyloidosis have further established that the transient hyperexcitation observed during the primary pathological stage is mediated by an altered behavior of VGLUT1 responsible for transporting Glu into synaptic vesicles. Thereafter, an overexpression of Vps10p-tail-interactor-1a, a protein that maintains spontaneous release of neurotransmitters by selective interaction with t-SNAREs, resulted in a depletion of intravesicular Glu content, triggering advanced-stage neuronal malfunction. These findings are expected to open perspectives for remediating Aβ42-induced neuronal hyperactivity and neuronal degeneration.

Synaptotagmin 9 Modulates Spontaneous Neurotransmitter Release in Striatal Neurons by Regulating Substance P Secretion.

Synaptotagmin 9 (SYT9) is a tandem C2 domain Ca2+ sensor for exocytosis in neuroendocrine cells; its function in neurons remains unclear. Here, we show that, in mixed-sex cultures, SYT9 does not trigger rapid synaptic vesicle exocytosis in mouse cortical, hippocampal, or striatal neurons, unless it is massively overexpressed. In striatal neurons, loss of SYT9 reduced the frequency of spontaneous neurotransmitter release events (minis). We delved into the underlying mechanism and discovered that SYT9 was localized to dense-core vesicles that contain substance P (SP). Loss of SYT9 impaired SP release, causing the observed decrease in mini frequency. This model is further supported by loss of function mutants. Namely, Ca2+ binding to the C2A domain of SYT9 triggered membrane fusion in vitro, and mutations that disrupted this activity abolished the ability of SYT9 to regulate both SP release and mini frequency. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.SIGNIFICANCE STATEMENT Synaptotagmin 9 (SYT9) has been described as a Ca2+ sensor for dense-core vesicle (DCV) exocytosis in neuroendocrine cells, but its role in neurons remains unclear, despite widespread expression in the brain. This article examines the role of SYT9 in synaptic transmission across cultured cortical, hippocampal, and striatal neuronal preparations. We found that SYT9 regulates spontaneous neurotransmitter release in striatal neurons by serving as a Ca2+ sensor for the release of the neuromodulator substance P from DCVs. This demonstrates a novel role for SYT9 in neurons and uncovers a new field of study into neuromodulation by SYT9, a protein that is widely expressed in the brain.

5-HT3 Receptors in Rat Dorsal Root Ganglion Neurons: Ca2+ Entry and Modulation of Neurotransmitter Release.

Rat dorsal root ganglion (DRG) neurons express 5-hydroxytryptamine receptors (5-HT3Rs). To elucidate their physiological role in the modulation of sensory signaling, we aimed to quantify their functional expression in newborn and adult rat DRG neurons, as well as their ability to modulate the Ca2+-dependent neurotransmitter release, by means of electrophysiological techniques combined with fluorescence-based Ca2+ imaging. The selective 5-HT3R agonist mCPBG (10 μM) elicited whole-cell currents in 92.5% of adult DRG neurons with a significantly higher density current than in responding newborn cells (52.2%), suggesting an increasing serotoninergic modulation on primary afferent cells during development. Briefly, 5-HT3Rs expressed by adult DRG neurons are permeable to Ca2+ ions, with a measured fractional Ca2+ current (i.e., the percentage of total current carried by Ca2+ ions, Pf) of 1.0%, similar to the value measured for the human heteromeric 5-HT3A/B receptor (Pf = 1.1%), but lower than that of the human homomeric 5-HT3A receptor (Pf = 3.5%). mCPBG applied to co-cultures of newborn DRG and spinal neurons significantly increased the miniature excitatory postsynaptic currents (mEPSCs) frequency in a subset of recorded spinal neurons, even in the presence of Cd2+, a voltage-activated Ca2+ channel blocker. Considered together, our findings indicate that the Ca2+ influx through heteromeric 5-HT3Rs is sufficient to increase the spontaneous neurotransmitter release from DRG to spinal neurons.

Syntaxin-1A modulates vesicle fusion in mammalian neurons via juxtamembrane domain dependent palmitoylation of its transmembrane domain.

SNAREs are undoubtedly one of the core elements of synaptic transmission. Contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations in cultured mouse hippocampal neurons: (1) elongation of STX1A's JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; (2) charge reversal mutations in STX1A's JMD; and (3) palmitoylation deficiency mutations in STX1A's TMD. We found that both JMD elongations and charge reversal mutations have position-dependent differential effects on Ca2+-evoked and spontaneous neurotransmitter release. Importantly, we show that STX1A's JMD regulates the palmitoylation of STX1A's TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1A's JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.

Changes in PVN Neurons after Low‐Frequency Acute Optogenetic Stimulation

The paraventricular nucleus of the hypothalamus (PVN) is an important autonomic control center. It receives afferent inputs from many brain regions, including the median preoptic nucleus (MnPO). The connection between the MnPO and the PVN is particularly important in generating chronic intermittent hypoxia phenotypes such as increased sympathetic activity leading to the development of hypertension. PVN‐projecting MnPO neurons discharge frequency is <5Hz. To gain more insight into the acute change in PVN neuron properties after low‐frequency stimulation, whole‐cell patch‐clamp recordings were performed on PVN neurons. Adult 250‐350g male rats were injected bilaterally in the PVN with 100 nL retrogradely transported adeno‐associated virus encoding Channelrhodopsin (AAVrg‐CaMKIIa‐hChR2(H134R)‐mCherry) or with AAVrg‐CAG‐tdTomato as control. Three weeks after the injections, the rats were anesthetized with isoflurane (2‐3%) and sacrificed to prepare horizontal brain slices containing the PVN and MnPO. Postsynaptic currents were recorded from PVN neurons in whole‐cell voltage clamp configuration (Vhold=‐60 mV) under constant bath perfusion of normal aCSF (2‐3 ml/min). Axon terminals in the PVN were stimulated with 5Hz LED‐generated blue light (470nM) pulses of 50‐ms duration, 5 seconds off/on for a total of 1 min, delivered using optical fiber directed to the MnPO. Photo‐evoked currents were measured during 5 min before and after the photo‐stimulation train. Amplitude and frequency of spontaneous currents were compared before and after stimulation as well. At the end of each recording, cells were characterized as type I (magnocellular) or type II (parvocellular) PVN neurons based on the presence of transient outward rectification. Data were analyzed offline using Easy Electrophysiology v2.3.3 software. For both cell types, 5 Hz optogenetic stimulation caused a significant reduction in the optogenetic‐evoked current amplitude during the first 15 minutes after stimulation (before stimulation 94.88±2.85 %, n=9 cells vs. 15 minutes after stimulation 67.52±6.15 %, n=9. P=0.0027) before returning to baseline values. Post‐stimulation spontaneous postsynaptic current amplitude and frequency decreased in type II PVN neurons from retrograde ChR2 animals (pre‐stimulation amplitude 100±0.62 % vs. post‐stimulation amplitude 86.50±0.54 %, P=0.0371; pre‐stimulation frequency 3.82±1.7 Hz, post‐stimulation frequency 2.396±1.2 Hz, P= 0.0385) but not in type I PVN neurons from retrograde ChR2 animals or cells from control animals. Kinetic profiles of spontaneous current in type I and II PVN neurons did not change after 5 Hz optogenetic stimulation. The results suggest that low‐frequency stimulation discriminately alters spontaneous presynaptic neurotransmitter release and possibly postsynaptic responsiveness in PVN neurons. Additional experiments will be needed to specifically stimulate PVN afferents from the MnPO.

Effects of K252a and K252b on CIH induced Changes in mEPSCs from PVN‐projecting MnPO

Chronic intermittent hypoxia (CIH) has been shown to alter the response of neurons in Median Preoptic nucleus (MnPO) to activation of cardiovascular inputs. Our previous results indicate that CIH enhances spontaneous presynaptic neurotransmitter release while also increasing postsynaptic responsiveness in MnPO. In other systems, BNDF/TrkB signaling has been shown to facilitate excitatory neurotransmission, so we tested its role in the increased MnPO excitation triggered by CIH. Data were collected from PVN‐projecting MnPO neurons which play an important role in CIH‐induced hypertension. Adult 250‐350g male rats were injected bilaterally in the PVN with 200 nL retrograde adeno‐associated virus (pAAV‐CAG‐tdTomato). Three weeks after the injections, the rats were exposed to a 7‐day intermittent hypoxia protocol (6 min cycles; 3 minutes of 21% oxygen, 3 min of 10% oxygen maintenance at 10% oxygen for 8 h during the light phase). Normoxic controls were exposed to room air. At the end of the protocol, the rats were anesthetized with isoflurane (2‐3%) and horizontal brain slices of MnPO were prepared. Miniature Excitatory Postsynaptic Currents (mEPSCs) were recorded using whole cell voltage clamp configuration from PVN‐projecting MnPO neurons. The mEPSCs were recorded with the holding potential of ‐60 mV in the presence of a GABAa receptor antagonist (SR 95531 hydrobromide, 25 µM), tetrodotoxin (1µM), and a glycine receptor antagonist (strychnine, 3 µM) in aCSF. Data were analyzed off‐line using Synaptosoft software. Differences in mEPSCs properties, such as peak amplitude, frequency, decay time, area, and cell capacitance were analyzed by ANOVA and Tukey multiple comparison tests. The results showed that CIH significantly increased mEPSCs frequency (Hz: CIH 1.32 ± 0.09 vs Norm 0.83 ± 0.09, p=5E‐04), decay time (ms: CIH 8.01 ± 0.28 vs Norm 6.64 ± 0.36, p=0.004), area (pA.ms: CIH 63.85± 3.65 vs Norm 50.27 ± 3.6, p=0.01), and cell capacitance (pF: CIH 31.55 ± 1.8 vs Norm 25.34 ± 1.95, p=0.02). The contribution of BDNF/TrkB signaling to these CIH effects were tested using K252a and K252b, two structurally related kinase receptor inhibitors. MnPO slices were pre‐incubated with either K252a or K252b (200nM) for 30 min before recording. Neither K252a nor K252b influenced mEPSCs of MnPO neurons from normoxic slices. Both K252a and K252b significantly affected CIH‐induced changes mEPSCs but at different magnitudes. The K252a significantly reduced the effects of CIH on frequency (Hz: K252a 0.46 ± 0.05, p=1E‐08), decay time (ms: K252a 6.07 ± 0.34, p=1E‐04), area (pA.ms: K252a 43.26 ± 3.55, p=5E‐04), and cell capacitance (pF: K252a 19.59 ± 1.61, p=1E‐04;). In addition, K252a also significantly decreased mEPSCs peak amplitude in CIH neurons (pA: CIH 14.87 ± 0.41 vs K252a 13.27 ± 0.39, p=0.03). While K252b pretreatment also affected the same parameters, the changes produced by K252a were significantly greater. These studies suggest a role for BDNF/TrkB signaling in CIH induced changes in MnPO but additional experiments will be required to validate the specificity of our results.

Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor.

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release; however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release in primary hippocampal cultures. In nonneuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of nonphotobleached probes with a time constant of ~10 s. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

A subthreshold synaptic mechanism regulating BDNF expression and resting synaptic strength.

SUMMARYRecent studies have demonstrated that protein translation can be regulated by spontaneous excitatory neurotransmission. However, the impact of spontaneous neurotransmitter release on gene transcription remains unclear. Here, we study the effects of the balance between inhibitory and excitatory spontaneous neurotransmission on brain-derived neurotrophic factor (BDNF) regulation and synaptic plasticity. Blockade of spontaneous inhibitory events leads to an increase in the transcription of Bdnf and Npas4 through altered synaptic calcium signaling, which can be blocked by antagonism of NMDA receptors (NMDARs) or L-type voltage-gated calcium channels (VGCCs). Transcription is bidirectionally altered by manipulating spontaneous inhibitory, but not excitatory, currents. Moreover, blocking spontaneous inhibitory events leads to multiplicative downscaling of excitatory synaptic strength in a manner that is dependent on both transcription and BDNF signaling. These results reveal a role for spontaneous inhibitory neurotransmission in BDNF signaling that sets excitatory synaptic strength at rest.

Corticotropin-releasing factor induces functional and structural synaptic remodelling in acute stress

Biological responses to stress are complex and highly conserved. Corticotropin-releasing factor (CRF) plays a central role in regulating these lifesaving physiological responses to stress. We show that, in mice, CRF rapidly changes Schaffer Collateral (SC) input into hippocampal CA1 pyramidal cells (PC) by modulating both functional and structural aspects of these synapses. Host exposure to acute stress, in vivo CRF injection, and ex vivo CRF application all result in fast de novo formation and remodeling of existing dendritic spines. Functionally, CRF leads to a rapid increase in synaptic strength of SC input into CA1 neurons, e.g., increase in spontaneous neurotransmitter release, paired-pulse facilitation, and repetitive excitability and improves synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD). In line with the changes in synaptic function, CRF increases the number of presynaptic vesicles, induces redistribution of vesicles towards the active zone, increases active zone size, and improves the alignment of the pre- and postsynaptic compartments. Therefore, CRF rapidly enhances synaptic communication in the hippocampus, potentially playing a crucial role in the enhanced memory consolidation in acute stress.

CIH Enhances Glutamatergic Transmission in Median Preoptic Nucleus

Studies have shown that the median preoptic nucleus (MnPO) contributes to hypertension associated with chronic intermittent hypoxia (CIH), a model of the hypoxemia associated with sleep apnea. Virally mediated lesions of MnPO neurons prevent the development of a sustained hypertension associated with CIH. To gain more insight into the properties of excitatory transmission to MnPO neurons develops in CIH, whole cell patch clamp recordings were from MnPO neurons brain slices from rats exposed to 7 days of CIH (6 minute cycles; 3 minutes of 21% oxygen room air pumped in, 90 seconds of nitrogen pumped in to lower the chamber O2 to 10% oxygen, and then 90 seconds of maintenance at 10% oxygen) for 8 h during the light phase or were normoxic controls. Slices were perfused with oxygenates artificial cerebrospinal fluid and the MnPO was visually identified. Miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presences of 25 µm SR95531 (GABAA antagonist) and 1 µm TTX (block Na+ channel-mediated action potentials) using a conventional whole cell patch clamp configuration. The mEPSCs were collected over a 4 min period and amplitude and frequency were compared between cells obtained from slice prepared from normoxic control rats (NORM) or rats exposed to CIH. CIH was associated with a significant increase in mean amplitude (CIH 17.15 ± 0.65 pA, n=19 cells; NORM (15.6 ± 0.6 pA; n=18 cells, P <0.05). Similarly, mean frequency was significantly elevated after CIH (CIH 4.05 ± 0.5 Hz, n=19 vs NORM 2.56 ± 0.37 Hz, n=18; p<0.05) and associated with a significant leftward shift in the cumulative probability of inter event intervals. The results suggest that CIH simultaneously increases enhances spontaneous presynaptic neurotransmitter release while also increasing postsynaptic responsiveness in MnPO. These presynaptic and postsynaptic changes in MnPO that are associated with CIH may represent mechanism contributing to CIH hypertension.

Structural and Functional Synaptic Plasticity Induced by Convergent Synapse Loss in the Drosophila Neuromuscular Circuit.

Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question we ask here is to what extent converging inputs influence each other's structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction (NMJ) where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s MNs; we surveyed multiple muscles for this reason. Here, we identified a cell-specific promoter that allows ablation of 1s MNs postinnervation and measured structural and functional responses of convergent 1b NMJs using microscopy and electrophysiology. For all muscles examined in both sexes, ablation of 1s MNs resulted in NMJ expansion and increased spontaneous neurotransmitter release at corresponding 1b NMJs. This demonstrates that 1b NMJs can compensate for the loss of convergent 1s MNs. However, only a subset of 1b NMJs showed compensatory evoked neurotransmission, suggesting target-specific plasticity. Silencing 1s MNs led to similar plasticity at 1b NMJs, suggesting that evoked neurotransmission from 1s MNs contributes to 1b synaptic plasticity. Finally, we genetically blocked 1s innervation in male larvae and robust 1b synaptic plasticity was eliminated, raising the possibility that 1s NMJ formation is required to set up a reference for subsequent synaptic perturbations.SIGNIFICANCE STATEMENT In complex neural circuits, multiple convergent inputs contribute to the activity of the target cell, but whether synaptic plasticity exists among these inputs has not been thoroughly explored. In this study, we examined synaptic plasticity in the structurally and functionally tractable Drosophila larval neuromuscular system. In this convergent circuit, each muscle is innervated by a unique pair of motor neurons. Removal of one neuron after innervation causes the adjacent neuron to increase neuromuscular junction outgrowth and functional output. However, this is not a general feature as each motor neuron differentially compensates. Further, robust compensation requires initial coinnervation by both neurons. Understanding how neurons respond to perturbations in adjacent neurons will provide insight into nervous system plasticity in both healthy and disease states.

The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss-of-function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with controls in a Ca2+-dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous and evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.

Calmodulin Bidirectionally Regulates Evoked and Spontaneous Neurotransmitter Release at Retinal Ribbon Synapses.

For decades, a role for the Ca2+-binding protein calmodulin (CaM) in Ca2+-dependent presynaptic modulation of synaptic transmission has been recognized. Here, we investigated the influence of CaM on evoked and spontaneous neurotransmission at rod bipolar (RB) cell→AII amacrine cell synapses in the mouse retina. Our work was motivated by the observations that expression of CaM in RB axon terminals is extremely high and that [Ca2+] in RB terminals normally rises sufficiently to saturate endogenous buffers, making tonic CaM activation likely. Taking advantage of a model in which RBs can be stimulated by expressed channelrhodopsin-2 (ChR2) to avoid dialysis of the presynaptic terminal, we found that inhibition of CaM dramatically decreased evoked release by inhibition of presynaptic Ca channels while at the same time potentiating both Ca2+-dependent and Ca2+-independent spontaneous release. Remarkably, inhibition of myosin light chain kinase (MLCK), but not other CaM-dependent targets, mimicked the effects of CaM inhibition on evoked and spontaneous release. Importantly, initial antagonism of CaM occluded the effect of subsequent inhibition of MLCK on spontaneous release. We conclude that CaM, by acting through MLCK, bidirectionally regulates evoked and spontaneous release at retinal ribbon synapses.

Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.

Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane-Proximal Regions of VAMP2 and SNAP25

SummaryThe neuronal protein complexin contains multiple domains that exert clamping and facilitatory functions to tune spontaneous and action potential-triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site-and stage-specific photo-cross-linking reveals that, prior to fusion, the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neuro-transmitters in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

VAMP4 Maintains a Ca2+-Sensitive Pool of Spontaneously Recycling Synaptic Vesicles.

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.SIGNIFICANCE STATEMENT Spontaneous neurotransmitter release that occurs independent of presynaptic action potentials (APs) shows significant sensitivity to intracellular Ca2+ 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 Ca2+-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.