Multiple roles for the active zone protein RIM1alpha in late stages of neurotransmitter release

The active zone protein RIM1alpha interacts with multiple active zone and synaptic vesicle proteins and is implicated in short- and long-term synaptic plasticity, but it is unclear how RIM1alpha's biochemical interactions translate into physiological functions. To address this question, we analyzed synaptic transmission in autaptic neurons cultured from RIM1alpha-/- mice. Deletion of RIM1alpha causes a large reduction in the readily releasable pool of vesicles, alters short-term plasticity, and changes the properties of evoked asynchronous release. Lack of RIM1alpha, however, had no effect on synapse formation, spontaneous release, overall Ca2+ sensitivity of release, or synaptic vesicle recycling. These results suggest that RIM1alpha modulates sequential steps in synaptic vesicle exocytosis through serial protein-protein interactions and that this modulation is the basis for RIM1alpha's role in synaptic plasticity.     

Activity-dependent regulation of vesicular glutamate and GABA transporters: a means to scale quantal size

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.     

Endosomal phosphatidylinositol 3‐phosphate controls synaptic vesicle cycling and neurotransmission

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3‐phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2‐mediated hyperactivation of Cdk5 downstream of receptor‐ and activity‐dependent calcium influx. Our results unravel an unexpected function for PI(3)P‐containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P‐producing VPS34 kinase in neurological disease and neurodegeneration.     

Pentylenetetrazol-Induced Epileptiform Activity Affects Basal Synaptic Transmission and Short-Term Plasticity in Monosynaptic Connections

Epileptic activity is generally induced in experimental models by local application of epileptogenic drugs, including pentylenetetrazol (PTZ), widely used on both vertebrate and invertebrate neurons. Despite the high prevalence of this neurological disorder and the extensive research on it, the cellular and molecular mechanisms underlying epileptogenesis still remain unclear. In this work, we examined PTZ-induced neuronal changes in Helix monosynaptic circuits formed in vitro, as a simpler experimental model to investigate the effects of epileptiform activity on both basal release and post-tetanic potentiation (PTP), a form of short-term plasticity. We observed a significant enhancement of basal synaptic strength, with kinetics resembling those of previously described use-dependent forms of plasticity, determined by changes in estimated quantal parameters, such as the readily releasable pool and the release probability. Moreover, these neurons exhibited a strong reduction in PTP expression and in its decay time constant, suggesting an impairment in the dynamic reorganization of synaptic vesicle pools following prolonged stimulation of synaptic transmission. In order to explain this imbalance, we determined whether epileptic activity is related to the phosphorylation level of synapsin, which is known to modulate synaptic plasticity. Using western blot and immunocytochemical staining we found a PTZ-dependent increase in synapsin phosphorylation at both PKA/CaMKI/IV and MAPK/Erk sites, both of which are important for modulating synaptic plasticity. Taken together, our findings suggest that prolonged epileptiform activity leads to an increase in the synapsin phosphorylation status, thereby contributing to an alteration of synaptic strength in both basal condition and tetanus-induced potentiation.     

Bayesian Computation Emerges in Generic Cortical Microcircuits through Spike-Timing-Dependent Plasticity

The principles by which networks of neurons compute, and how spike-timing dependent plasticity (STDP) of synaptic weights generates and maintains their computational function, are unknown. Preceding work has shown that soft winner-take-all (WTA) circuits, where pyramidal neurons inhibit each other via interneurons, are a common motif of cortical microcircuits. We show through theoretical analysis and computer simulations that Bayesian computation is induced in these network motifs through STDP in combination with activity-dependent changes in the excitability of neurons. The fundamental components of this emergent Bayesian computation are priors that result from adaptation of neuronal excitability and implicit generative models for hidden causes that are created in the synaptic weights through STDP. In fact, a surprising result is that STDP is able to approximate a powerful principle for fitting such implicit generative models to high-dimensional spike inputs: Expectation Maximization. Our results suggest that the experimentally observed spontaneous activity and trial-to-trial variability of cortical neurons are essential features of their information processing capability, since their functional role is to represent probability distributions rather than static neural codes. Furthermore it suggests networks of Bayesian computation modules as a new model for distributed information processing in the cortex.     

Network dynamics and synchronous activity in cultured cortical neurons

Neurons extracted from specific areas of the Central Nervous System (CNS), such as the hippocampus, the cortex and the spinal cord, can be cultured in vitro and coupled with a micro-electrode array (MEA) for months. After a few days, neurons connect each other with functionally active synapses, forming a random network and displaying spontaneous electrophysiological activity. In spite of their simplified level of organization, they represent an useful framework to study general information processing properties and specific basic learning mechanisms in the nervous system. These experimental preparations show patterns of collective rhythmic activity characterized by burst and spike firing. The patterns of electrophysiological activity may change as a consequence of external stimulation (i.e., chemical and/or electrical inputs) and by partly modifying the "randomness" of the network architecture (i.e., confining neuronal sub-populations in clusters with micro-machined barriers). In particular we investigated how the spontaneous rhythmic and synchronous activity can be modulated or drastically changed by focal electrical stimulation, pharmacological manipulation and network segregation. Our results show that burst firing and global synchronization can be enhanced or reduced; and that the degree of synchronous activity in the network can be characterized by simple parameters such as cross-correlation on burst events.     

高锶离子亲和力传感蛋白介导CALYX突触囊泡异步和自发释放

突触囊泡在钙离子(Ca2+)触发下释放神经递质普遍存在着同步和异步两种形式.突触囊泡膜蛋白(synaptotagmin 2,Syt-2)已被证实是Calyx of Held突触囊泡同步释放的Ca2+传感蛋白,而相关的异步释放Ca2+传感蛋白还有待于探索.虽然锶离子(Sr2+)因其物理和化学性质都接近Ca2+,且能触发更多的囊泡异步释放成分而成为研究异步释放机制的常用工具,但有关Sr2+触发异步释放的机制存在着争议.本文在胞外以Sr2+替换Ca2+的条件下,通过对野生型(WT)和Syt-2敲除型(Z2B-/-)小鼠Calyx突触囊泡自发和诱发释放的电生理特性分析,发现Syt-2是介导Sr2+诱发的突触囊泡快速释放的传感蛋白,但不是介导Sr2+相关神经递质异步释放和自发释放的传感蛋白;而未知的触发囊泡异步释放的传感蛋白相比Syt-2对Sr2+具有更高的亲和力,同时也介导突触囊泡的自发释放.这一研究为探索并最终发现触发囊泡异步释放的未知传感蛋白提供了新的线索.     

A neural network model of reliably optimized spike transmission

We studied the detailed structure of a neuronal network model in which the spontaneous spike activity is correctly optimized to match the experimental data and discuss the reliability of the optimized spike transmission. Two stochastic properties of the spontaneous activity were calculated: the spike-count rate and synchrony size. The synchrony size, expected to be an important factor for optimization of spike transmission in the network, represents a percentage of observed coactive neurons within a time bin, whose probability approximately follows a power-law. We systematically investigated how these stochastic properties could matched to those calculated from the experimental data in terms of the log-normally distributed synaptic weights between excitatory and inhibitory neurons and synaptic background activity induced by the input current noise in the network model. To ensure reliably optimized spike transmission, the synchrony size as well as spike-count rate were simultaneously optimized. This required changeably balanced log-normal distributions of synaptic weights between excitatory and inhibitory neurons and appropriately amplified synaptic background activity. Our results suggested that the inhibitory neurons with a hub-like structure driven by intensive feedback from excitatory neurons were a key factor in the simultaneous optimization of the spike-count rate and synchrony size, regardless of different spiking types between excitatory and inhibitory neurons.     

Activity-dependent clustering of functional synaptic inputs on developing hippocampal dendrites

During brain development, before sensory systems become functional, neuronal networks spontaneously generate repetitive bursts of neuronal activity, which are typically synchronized across many neurons. Such activity patterns have been described on the level of networks and cells, but the fine-structure of inputs received by an individual neuron during spontaneous network activity has not been studied. Here, we used calcium imaging to record activity at many synapses of hippocampal pyramidal neurons simultaneously to establish the activity patterns in the majority of synapses of an entire cell. Analysis of the spatiotemporal patterns of synaptic activity revealed a fine-scale connectivity rule: neighboring synapses (<16?μm intersynapse distance) are more likely to be coactive than synapses that are farther away from each other. Blocking spiking activity or NMDA receptor activation revealed that the clustering of synaptic inputs required neuronal activity, demonstrating a role of developmentally expressed spontaneous activity for connecting neurons with subcellular precision.     

Dynamin Isoforms Decode Action Potential Firing for Synaptic Vesicle Recycling

Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.     

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release.     

Modelling Feedback Excitation,Pacemaker Properties and Sensory Switching of Electrically Coupled Brainstem Neurons Controlling Rhythmic Activity

What cellular and network properties allow reliable neuronal rhythm generation or firing that can be started and stopped by brief synaptic inputs? We investigate rhythmic activity in an electrically-coupled population of brainstem neurons driving swimming locomotion in young frog tadpoles, and how activity is switched on and off by brief sensory stimulation. We build a computational model of 30 electrically-coupled conditional pacemaker neurons on one side of the tadpole hindbrain and spinal cord. Based on experimental estimates for neuron properties, population sizes, synapse strengths and connections, we show that: long-lasting, mutual, glutamatergic excitation between the neurons allows the network to sustain rhythmic pacemaker firing at swimming frequencies following brief synaptic excitation; activity persists but rhythm breaks down without electrical coupling; NMDA voltage-dependency doubles the range of synaptic feedback strengths generating sustained rhythm. The network can be switched on and off at short latency by brief synaptic excitation and inhibition. We demonstrate that a population of generic Hodgkin-Huxley type neurons coupled by glutamatergic excitatory feedback can generate sustained asynchronous firing switched on and off synaptically. We conclude that networks of neurons with NMDAR mediated feedback excitation can generate self-sustained activity following brief synaptic excitation. The frequency of activity is limited by the kinetics of the neuron membrane channels and can be stopped by brief inhibitory input. Network activity can be rhythmic at lower frequencies if the neurons are electrically coupled. Our key finding is that excitatory synaptic feedback within a population of neurons can produce switchable, stable, sustained firing without synaptic inhibition.     

Contributions of Bcl-xL to acute and long term changes in bioenergetics during neuronal plasticity

Mitochondria manufacture and release metabolites and manage calcium during neuronal activity and synaptic transmission, but whether long term alterations in mitochondrial function contribute to the neuronal plasticity underlying changes in organism behavior patterns is still poorly understood. Although normal neuronal plasticity may determine learning, in contrast a persistent decline in synaptic strength or neuronal excitability may portend neurite retraction and eventual somatic death. Anti-death proteins such as Bcl-xL not only provide neuroprotection at the neuronal soma during cell death stimuli, but also appear to enhance neurotransmitter release and synaptic growth and development. It is proposed that Bcl-xL performs these functions through its ability to regulate mitochondrial release of bioenergetic metabolites and calcium, and through its ability to rapidly alter mitochondrial positioning and morphology. Bcl-xL also interacts with proteins that directly alter synaptic vesicle recycling. Bcl-xL translocates acutely to sub-cellular membranes during neuronal activity to achieve changes in synaptic efficacy. After stressful stimuli, pro-apoptotic cleaved delta N Bcl-xL (ΔN Bcl-xL) induces mitochondrial ion channel activity leading to synaptic depression and this is regulated by caspase activation. During physiological states of decreased synaptic stimulation, loss of mitochondrial Bcl-xL and low level caspase activation occur prior to the onset of long term decline in synaptic efficacy. The degree to which Bcl-xL changes mitochondrial membrane permeability may control the direction of change in synaptic strength. The small molecule Bcl-xL inhibitor ABT-737 has been useful in defining the role of Bcl-xL in synaptic processes. Bcl-xL is crucial to the normal health of neurons and synapses and its malfunction may contribute to neurodegenerative disease. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

Molecular machines in the synapse: overlapping protein sets control distinct steps in neurosecretion

Activity regulated neurotransmission shapes the computational properties of a neuron and involves the concerted action of many proteins. Classical, intuitive working models often assign specific proteins to specific steps in such complex cellular processes, whereas modern systems theories emphasize more integrated functions of proteins. To test how often synaptic proteins participate in multiple steps in neurotransmission we present a novel probabilistic method to analyze complex functional data from genetic perturbation studies on neuronal secretion. Our method uses a mixture of probabilistic principal component analyzers to cluster genetic perturbations on two distinct steps in synaptic secretion, vesicle priming and fusion, and accounts for the poor standardization between different studies. Clustering data from 121 perturbations revealed that different perturbations of a given protein are often assigned to different steps in the release process. Furthermore, vesicle priming and fusion are inversely correlated for most of those perturbations where a specific protein domain was mutated to create a gain-of-function variant. Finally, two different modes of vesicle release, spontaneous and action potential evoked release, were affected similarly by most perturbations. This data suggests that the presynaptic protein network has evolved as a highly integrated supramolecular machine, which is responsible for both spontaneous and activity induced release, with a group of core proteins using different domains to act on multiple steps in the release process.     

SNARE protein recycling by αSNAP and βSNAP supports synaptic vesicle priming

Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.     

Addition of new neurons and the emergence of a local neural circuit for precise timing

During development, neurons arrive at local brain areas in an extended period of time, but how they form local neural circuits is unknown. Here we computationally model the emergence of a network for precise timing in the premotor nucleus HVC in songbird. We show that new projection neurons, added to HVC post hatch at early stages of song development, are recruited to the end of a growing feedforward network. High spontaneous activity of the new neurons makes them the prime targets for recruitment in a self-organized process via synaptic plasticity. Once recruited, the new neurons fire readily at precise times, and they become mature. Neurons that are not recruited become silent and replaced by new immature neurons. Our model incorporates realistic HVC features such as interneurons, spatial distributions of neurons, and distributed axonal delays. The model predicts that the birth order of the projection neurons correlates with their burst timing during the song.     

Synaptic Activity Regulates the Abundance and Binding of Complexin

Nervous system function relies on precise chemical communication between neurons at specialized junctions known as synapses. Complexin (CPX) is one of a small number of cytoplasmic proteins that are indispensable in controlling neurotransmitter release through SNARE and synaptic vesicle interactions. However, the mechanisms that recruit and stabilize CPX are poorly understood. The mobility of CPX tagged with photoactivatable green fluorescent protein (pGFP) was quantified in vivo using Caenorhabditis elegans. Although pGFP escaped the synapse within seconds, CPX-pGFP displayed both fast and slow decay components, requiring minutes for complete exchange of the synaptic pool. The longer synaptic residence time of CPX arose from both synaptic vesicle and SNARE interactions, and surprisingly, CPX mobility depended on synaptic activity. Moreover, mouse CPX-GFP reversibly dispersed out of hippocampal presynaptic terminals during stimulation, and blockade of vesicle fusion prevented CPX dispersion. Hence, synaptic CPX can rapidly redistribute and this exchange is influenced by neuronal activity, potentially contributing to use-dependent plasticity.     

A hemicord locomotor network of excitatory interneurons: a simulation study

Locomotor burst generation is simulated using a full-scale network model of the unilateral excitatory interneuronal population. Earlier small-scale models predicted that a population of excitatory neurons would be sufficient to produce burst activity, and this has recently been experimentally confirmed. Here we simulate the hemicord activity induced under various experimental conditions, including pharmacological activation by NMDA and AMPA as well as electrical stimulation. The model network comprises a realistic number of cells and synaptic connectivity patterns. Using similar distributions of cellular and synaptic parameters, as have been estimated experimentally, a large variation in dynamic characteristics like firing rates, burst, and cycle durations were seen in single cells. On the network level an overall rhythm was generated because the synaptic interactions cause partial synchronization within the population. This network rhythm not only emerged despite the distributed cellular parameters but relied on this variability, in particular, in reproducing variations of the activity during the cycle and showing recruitment in interneuronal populations. A slow rhythm (0.4–2 Hz) can be induced by tonic activation of NMDA-sensitive channels, which are voltage dependent and generate depolarizing plateaus. The rhythm emerges through a synchronization of bursts of the individual neurons. A fast rhythm (4–12 Hz), induced by AMPA, relies on spike synchronization within the population, and each burst is composed of single spikes produced by different neurons. The dynamic range of the fast rhythm is limited by the ability of the network to synchronize oscillations and depends on the strength of synaptic connections and the duration of the slow after hyperpolarization. The model network also produces prolonged bouts of rhythmic activity in response to brief electrical activations, as seen experimentally. The mutual excitation can sustain long-lasting activity for a realistic set of synaptic parameters. The bout duration depends on the strength of excitatory synaptic connections, the level of persistent depolarization, and the influx of Ca²⁺ ions and activation of Ca²⁺-dependent K⁺ current.     

On the dynamics of the spontaneous activity in neuronal networks

Most neuronal networks, even in the absence of external stimuli, produce spontaneous bursts of spikes separated by periods of reduced activity. The origin and functional role of these neuronal events are still unclear. The present work shows that the spontaneous activity of two very different networks, intact leech ganglia and dissociated cultures of rat hippocampal neurons, share several features. Indeed, in both networks: i) the inter-spike intervals distribution of the spontaneous firing of single neurons is either regular or periodic or bursting, with the fraction of bursting neurons depending on the network activity; ii) bursts of spontaneous spikes have the same broad distributions of size and duration; iii) the degree of correlated activity increases with the bin width, and the power spectrum of the network firing rate has a 1/f behavior at low frequencies, indicating the existence of long-range temporal correlations; iv) the activity of excitatory synaptic pathways mediated by NMDA receptors is necessary for the onset of the long-range correlations and for the presence of large bursts; v) blockage of inhibitory synaptic pathways mediated by GABA(A) receptors causes instead an increase in the correlation among neurons and leads to a burst distribution composed only of very small and very large bursts. These results suggest that the spontaneous electrical activity in neuronal networks with different architectures and functions can have very similar properties and common dynamics.