Persistence of quantal synaptic vesicle recycling in virtual absence of dynamins.

Dynamins are GTPases required for pinching vesicles off the plasma membrane once a critical curvature is reached during endocytosis. Here, we probed dynamin function in central synapses by depleting all three dynamin isoforms in postnatal hippocampal neurons down to negligible levels. We found a decrease in the propensity of evoked neurotransmission as well as a reduction in synaptic vesicle numbers. Recycling of synaptic vesicles during spontaneous or low levels of evoked activity were largely impervious to dynamin depletion, while retrieval of synaptic vesicle components at higher levels of activity was partially arrested. These results suggest the existence of balancing dynamin-independent mechanisms for synaptic vesicle recycling at central synapses. Classical dynamin-dependent mechanisms are not essential for retrieval of synaptic vesicle proteins after quantal single synaptic vesicle fusion, but they become more relevant for membrane retrieval during intense, sustained neuronal activity. KEY POINTS: Loss of dynamin 2 does not impair synaptic transmission. Loss of all three dynamin isoforms mostly affects evoked neurotransmission. Excitatory synapse function is more susceptible to dynamin loss. Spontaneous neurotransmission is only mildly affected by loss of dynamins. Single synaptic vesicle endocytosis is largely dynamin independent.

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.

The Synaptic Vesicle Cycle Revisited: New Insights into the Modes and Mechanisms.

Neurotransmission is sustained by endocytosis and refilling of synaptic vesicles (SVs) locally within the presynapse. Until recently, a consensus formed that after exocytosis, SVs are recovered by either fusion pore closure (kiss-and-run) or clathrin-mediated endocytosis directly from the plasma membrane. However, recent data have revealed that SV formation is more complex than previously envisaged. For example, two additional recycling pathways have been discovered, ultrafast endocytosis and activity-dependent bulk endocytosis, in which SVs are regenerated from the internalized membrane and synaptic endosomes. Furthermore, these diverse modes of endocytosis appear to influence both the molecular composition and subsequent physiological role of individual SVs. In addition, previously unknown complexity in SV refilling and reclustering has been revealed. This review presents a modern view of the SV life cycle and discusses how neuronal subtype, physiological temperature, and individual activity patterns can recruit different endocytic modes to generate new SVs and sculpt subsequent presynaptic performance.

Copine-6 Binds to SNAREs and Selectively Suppresses Spontaneous Neurotransmission.

Recent studies suggest that spontaneous and action potential-evoked neurotransmitter release processes are independently regulated. However, the mechanisms that uncouple the two forms of neurotransmission remain unclear. In cultured mouse and rat neurons, we show that the two C2 domain-containing protein copine-6 is localized to presynaptic terminals and binds to synaptobrevin2 as well as other SNARE proteins in a Ca2+-dependent manner. Ca2+-dependent interaction of copine-6 with synaptobrevin2 selectively suppresses spontaneous neurotransmission in a reaction that requires the tandem tryptophan residues at the C-terminal region of synaptobrevin2. Accordingly, copine-6 loss of function augmented presynaptic Ca2+ elevation-mediated neurotransmitter release. Intracellular Ca2+ chelation, on the other hand, occluded copine-6-mediated suppression of release. We also evaluated the molecular specificity of the copine-6-dependent regulation of spontaneous release and found that overexpression of copine-6 did not suppress spontaneous release in synaptobrevin2-deficient neurons. Together, these results suggest that copine-6 acts as a specific Ca2+-dependent suppressor of spontaneous neurotransmission.SIGNIFICANCE STATEMENT Synaptic transmission occurs both in response to presynaptic action potentials and spontaneously, in the absence of stimulation. Currently, much more is understood about the mechanisms underlying action potential-evoked neurotransmission compared with spontaneous release. However, recent studies have shown selective modulation of spontaneous neurotransmission process by several neuromodulators, suggesting specific molecular regulation of spontaneous release. In this study, we identify copine-6 as a specific regulator of spontaneous neurotransmission. By both gain-of-function and loss-of-function experiments, we show that copine-6 functions as a Ca2+-dependent suppressor of spontaneous release. These results further elucidate the mechanisms underlying differential regulation of evoked and spontaneous neurotransmitter release.

Loss of Doc2-Dependent Spontaneous Neurotransmission Augments Glutamatergic Synaptic Strength.

Action potential-evoked vesicle fusion comprises the majority of neurotransmission within chemical synapses, but action potential-independent spontaneous neurotransmission also contributes to the collection of signals sent to the postsynaptic cell. Previous work has implicated spontaneous neurotransmission in homeostatic synaptic scaling, but few studies have selectively manipulated spontaneous neurotransmission without substantial changes in evoked neurotransmission to study this function in detail. Here we used a quadruple knockdown strategy to reduce levels of proteins within the soluble calcium-binding double C2 domain (Doc2)-like protein family to selectively reduce spontaneous neurotransmission in cultured mouse and rat neurons. Activity-evoked responses appear normal while both excitatory and inhibitory spontaneous events exhibit reduced frequency. Excitatory miniature postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), increase in amplitude after quadruple knockdown. This increase in synaptic efficacy correlates with reduced phosphorylation levels of eukaryotic elongation factor 2 and also requires the presence of elongation factor 2 kinase. Together, these data suggest that spontaneous neurotransmission independently contributes to the regulation of synaptic efficacy, and action potential-evoked and spontaneous neurotransmission can be segregated at least partially on a molecular level.SIGNIFICANCE STATEMENT Action potential-evoked and spontaneous neurotransmission have been observed in nervous system circuits as long as methods have existed to measure them. Despite being well studied, controversy still remains about whether these forms of neurotransmission are regulated independently on a molecular level or whether they are simply a continuum of neurotransmission modes. In this study, members of the Doc2 family of presynaptic proteins were eliminated, which caused a reduction in spontaneous neurotransmission, whereas action potential-evoked neurotransmission remained relatively normal. This protein loss also caused an increase in synaptic strength, suggesting that spontaneous neurotransmission is able to communicate independently with the postsynaptic neuron and trigger downstream signaling cascades that regulate the synaptic state.

Role of the endoplasmic reticulum in synaptic transmission.

Neurons possess a complex morphology spanning long distances and a large number of subcellular specializations such as presynaptic terminals and dendritic spines. This structural complexity is essential for maintenance of synaptic junctions and associated electrical as well as biochemical signaling events. Given the structural and functional complexity of neurons, neuronal endoplasmic reticulum is emerging as a key regulator of neuronal function, in particular synaptic signaling. Neuronal endoplasmic reticulum mediates calcium signaling, calcium and lipid homeostasis, vesicular trafficking, and proteostasis events that underlie autonomous functions of numerous subcellular compartments. However, based on its geometric complexity spanning the whole neuron, endoplasmic reticulum also integrates the activity of these autonomous compartments across the neuron and coordinates their interactions with the soma. In this article, we review recent work regarding neuronal endoplasmic reticulum function and its relationship to neurotransmission and plasticity.

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.

Synaptobrevin-2 dependent regulation of single synaptic vesicle endocytosis.

Evidence from multiple systems indicates that vesicle SNARE (soluble NSF attachment receptor) proteins are involved in synaptic vesicle endocytosis, although their exact action at the level of single vesicles is unknown. Here we interrogate the role of the main synaptic vesicle SNARE mediating fusion, synaptobrevin-2 (also called VAMP2), in modulation of single synaptic vesicle retrieval. We report that in the absence of synaptobrevin-2, fast and slow modes of single synaptic vesicle retrieval are impaired, indicating a role of the SNARE machinery in coupling exocytosis to endocytosis of single synaptic vesicles. Ultrafast endocytosis was impervious to changes in the levels of synaptobrevin-2, pointing to a separate molecular mechanism underlying this type of recycling. Taken together with earlier studies suggesting a role of synaptobrevin-2 in endocytosis, these results indicate that the machinery for fast synchronous release couples fusion to retrieval and regulates the kinetics of endocytosis in a Ca2+-dependent manner.

Interneuronal exchange and functional integration of synaptobrevin via extracellular vesicles.

Recent studies have investigated the composition and functional effects of extracellular vesicles (EVs) secreted by a variety of cell types. However, the mechanisms underlying the impact of these vesicles on neurotransmission remain unclear. Here, we isolated EVs secreted by rat and mouse hippocampal neurons and found that they contain synaptic-vesicle-associated proteins, in particular the vesicular SNARE (soluble N-ethylmaleimide-sensitive factor [NSF]-attachment protein receptor) synaptobrevin (also called VAMP). Using a combination of electrophysiology and live-fluorescence imaging, we demonstrate that this extracellular pool of synaptobrevins can rapidly integrate into the synaptic vesicle cycle of host neurons via a CD81-dependent process and selectively augment inhibitory neurotransmission as well as specifically rescue neurotransmission in synapses deficient in synaptobrevin. These findings uncover a novel means of interneuronal communication and functional coupling via exchange of vesicular SNAREs.

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

Recent 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.

Presynaptic store-operated Ca2+ entry drives excitatory spontaneous neurotransmission and augments endoplasmic reticulum stress.

Store-operated calcium entry (SOCE) is activated by depletion of Ca2+ from the endoplasmic reticulum (ER) and mediated by stromal interaction molecule (STIM) proteins. Here, we show that in rat and mouse hippocampal neurons, acute ER Ca2+ depletion increases presynaptic Ca2+ levels and glutamate release through a pathway dependent on STIM2 and the synaptic Ca2+ sensor synaptotagmin-7 (syt7). In contrast, synaptotagmin-1 (syt1) can suppress SOCE-mediated spontaneous release, and STIM2 is required for the increase in spontaneous release seen during syt1 loss of function. We also demonstrate that chronic ER stress activates the same pathway leading to syt7-dependent potentiation of spontaneous glutamate release. During ER stress, inhibition of SOCE or syt7-driven fusion partially restored basal neurotransmission and decreased expression of pro-apoptotic markers, indicating that these processes participate in the amplification of ER-stress-related damage. Taken together, we propose that presynaptic SOCE links ER stress and augmented spontaneous neurotransmission, which may, in turn, facilitate neurodegeneration.

Acetylated tubulin associates with the fifth cytoplasmic domain of Na(+)/K(+)-ATPase: possible anchorage site of microtubules to the plasma membrane.

We showed previously that NKA (Na(+)/K(+)-ATPase) interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane-acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular mass (320-400 kDa) during molecular exclusion chromatography. We also found that microtubules assembled in vitro are able to bind to NKA when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated NKA. Furthermore, we determined that CD5 (cytoplasmic domain 5 of NKA) is capable of interacting with acetylated tubulin. Taken together, our results are consistent with the idea that NKA may act as a microtubule-plasma membrane anchorage site through an interaction between acetylated tubulin and CD5.

Is Ca2+ Essential for Synaptic Vesicle Endocytosis?

Synaptic vesicle fusion is coupled to swift retrieval of vesicle components from the synaptic plasma membrane. Ca2+ has been assumed to be a key mediator of this coupling. In a recent study, Orlando et al. unequivocally demonstrate that Ca2+ is not essential for synaptic vesicle retrieval.

Presynaptic origins of distinct modes of neurotransmitter release.

Presynaptic nerve terminals release neurotransmitter synchronously, asynchronously or spontaneously. During synchronous neurotransmission release is precisely coupled to action potentials, in contrast, asynchronous release events show only loose temporal coupling to presynaptic activity whereas spontaneous neurotransmission occurs independent of presynaptic activity. The mechanisms that give rise to this diversity in neurotransmitter release modes are poorly understood. Recent studies have described several presynaptic molecular pathways controlling synaptic vesicle pool segregation and recycling, which in turn may dictate distinct modes of neurotransmitter release. In this article, we review this recent work regarding neurotransmitter release modes and their relationship to synaptic vesicle pool dynamics as well as the molecular machinery that establishes synaptic vesicle pool identity.

Optical detection of three modes of endocytosis at hippocampal synapses.

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.

Time course and temperature dependence of synaptic vesicle endocytosis.

In presynaptic nerve terminals, synaptic vesicles are recycled locally via an evolutionarily conserved process that ensures maintenance of neurotransmission as well as structural integrity of synapses. Temperature is a key environmental factor that impacts critical steps involved in fusion, endocytosis and transport in different vesicle trafficking pathways. In neurons, temperature changes have been shown to impact synaptic vesicle recycling and synaptic efficacy. But contrary to non-neuronal systems, the temperature dependence of the steps involved in fusion, endocytosis and recycling of synaptic vesicles in presynaptic terminals is not completely understood, and the existing data remain highly debated. In this Review, we discuss the implications of biophysical, biochemical and functional findings on temperature dependence of membrane retrieval in multiple systems. We propose that systematic investigation of the temperature dependence of the presynaptic vesicle trafficking process can provide novel insight into poorly understood mechanisms that govern synaptic vesicle trafficking under diverse physiological conditions.

How do you recognize and reconstitute a synaptic vesicle after fusion?

Synaptic vesicle recycling is essential for sustained and reliable neurotransmission. A key component of synaptic vesicle recycling is the synaptic vesicle biogenesis process that is observed in synapses and that maintains the molecular identity of synaptic vesicles. However, the mechanisms by which synaptic vesicles are retrieved and reconstituted after fusion remain unclear. The complex molecular composition of synaptic vesicles renders their rapid biogenesis a daunting task. Therefore, in this context, kiss-and-run type transient fusion of synaptic vesicles with the plasma membrane without loss of their membrane composition and molecular identity remains a viable hypothesis that can account for the fidelity of the synaptic vesicle cycle. In this article, we discuss the biological implications of this problem as well as its possible molecular solutions.

Synaptotagmin-1- and Synaptotagmin-7-Dependent Fusion Mechanisms Target Synaptic Vesicles to Kinetically Distinct Endocytic Pathways.

Synaptic vesicle recycling is essential for maintaining normal synaptic function. The coupling of exocytosis and endocytosis is assumed to be Ca2+ dependent, but the exact role of Ca2+ and its key effector synaptotagmin-1 (syt1) in regulation of endocytosis is poorly understood. Here, we probed the role of syt1 in single- as well as multi-vesicle endocytic events using high-resolution optical recordings. Our experiments showed that the slowed endocytosis phenotype previously reported after syt1 loss of function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss of function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles toward slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin-7. In contrast, after single synaptic vesicle fusion, syt1 acted as an essential determinant of synaptic vesicle endocytosis time course by delaying the kinetics of vesicle retrieval in response to increasing Ca2+ levels.

GABAergic Agonists Modulate the Glutamate Release from Frontal Cortex Synaptosomes of Rats with Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease that mimics many of the clinical and pathological features of multiple sclerosis. We have previously described a significant diminution in the GABAergic regulation of glutamate release from synaptosomes of EAE rats isolated during the acute stage of the disease. In order to explore the possible metabolic pathways responsible for this alteration, in this work we evaluate the direct effect of different GABAergic agonists on the glutamate release and concomitant synapsin I phosphorylation in synaptosomes from the frontal cortex of control and EAE animals. The results show that GABA as well as the GABA receptor agonists Muscimol (GABAA agonist) and Baclofen (GABAB agonist) caused a decrease in glutamate release in control rats paralleled by a similar reduction in synapsin I phosphorylation. Meanwhile synaptosomes from EAE animals are responsive only to Baclofen with respect to nontreated EAE synaptosomes, since glutamate release from the synaptosomes treated with Muscimol was similar to that observed in EAE rat synaptosomes which was already reduced as consequence of the disease. In the case of the benzodiazepines Diazepam and Clonazepam (GABAA allosteric agonists), both of them induced a reduction in glutamate release in synaptosomes from the CFA rats, effect that was only observed in synaptosomes of EAE rats treated with Clonazepam. In all cases both benzodiazepines showed a higher effect on synapsin I phosphorylation than in glutamate release. These results indicate that the extent of GABAergic modulation of presynaptic terminals depends on the type of agonist employed and this regulation is altered in the frontal cortex during the acute phase of EAE with respect to control animals.

Glutamate release machinery is altered in the frontal cortex of rats with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many of the clinical and pathological features of the human disease multiple sclerosis (MS). Both are inflammatory demyelinating and neurodegenerative pathologies of the central nervous system associated with motor, sensory, and cognitive deficits. In MS, gray matter atrophy is related to the emergence of cognitive deficits and contributes to clinical progression. In particular, prefrontal cortex injury and dysfunction have been correlated to the development of fatigue, one of the most common and disabling symptoms in MS. However, the molecular bases of these changes remain unknown. Taking advantage of EAE similitude, we herein analyze functional and morphological changes in isolated cortical presynaptic terminals (synaptosomes) from an acute rat model. We found impaired glutamate release in the frontal cortex from EAE rats. This defect appeared along with the onset of the disease, reversing when clinical signs were no more evident. Biochemical analysis of EAE synaptosomes revealed alterations in the presynaptic release machinery and in the response to depolarization, which was accompanied by abnormal synapsin I phosphorylation and dispersion. These changes were associated with reduced synaptic vesicle mobility, with no alterations in synaptosomal morphology as evidenced by electron microscopy. The present are the first pieces of evidence unraveling the molecular mechanisms of frontal cortex neuronal dysfunction in EAE and, possibly, MS.