Asynchronous Transmitter Release Research Articles

Triple Function of Synaptotagmin 7 Ensures Efficiency of High-Frequency Transmission at Central GABAergic Synapses

SUMMARYSynaptotagmin 7 (Syt7) is thought to be a Ca2+ sensor that mediates asynchronous transmitter release and facilitation at synapses. However, Syt7 is strongly expressed in fast-spiking, parvalbumin-expressing GABAergic interneurons, and the output synapses of these neurons produce only minimal asynchronous release and show depression rather than facilitation. To resolve this apparent contradiction, we examined the effects of genetic elimination of Syt7 on synaptic transmission at the GABAergic basket cell (BC)-Purkinje cell (PC) synapse in cerebellum. Our results indicate that at the BC-PC synapse, Syt7 contributes to asynchronous release, pool replenishment, and facilitation. In combination, these three effects ensure efficient transmitter release during high-frequency activity and guarantee frequency independence of inhibition. Our results identify a distinct function of Syt7: ensuring the efficiency of high-frequency inhibitory synaptic transmission.

Ageing brains attend a symphony with asynchronous transmitter release

Ageing brains attend a symphony with asynchronous transmitter release

Different output properties of perisomatic region-targeting interneurons in the basal amygdala.

The perisomatic region of principal neurons in cortical regions is innervated by three types of GABAergic interneuron, including parvalbumin-containing basket cells (PVBCs) and axo-axonic cells (AACs), as well as cholecystokinin and type 1 cannabinoid receptor-expressing basket cells (CCK/CB1BCs). These perisomatic inhibitory cell types can also be found in the basal nucleus of the amygdala, however, their output properties are largely unknown. Here, we performed whole-cell recordings in morphologically identified interneurons in slices prepared from transgenic mice, in which the GABAergic cells could be selectively targeted. Investigating the passive and active membrane properties of interneurons located within the basal amygdala revealed that the three interneuron types have distinct single-cell properties. For instance, the input resistance, spike rate, accommodation in discharge rate, or after-hyperpolarization width at the half maximal amplitude separated the three interneuron types. Furthermore, we performed paired recordings from interneurons and principal neurons to uncover the basic features of unitary inhibitory postsynaptic currents (uIPSCs). Although we found no difference in the magnitude of responses measured in the principal neurons, the uIPSCs originating from the distinct interneuron types differed in rise time, failure rate, latency, and short-term dynamics. Moreover, the asynchronous transmitter release induced by a train of action potentials was typical for the output synapses of CCK/CB1BCs. Our results suggest that, despite the similar uIPSC magnitudes originating from the three perisomatic inhibitory cell types, their distinct release properties together with the marked differences in their spiking characteristics may contribute to accomplish specific functions in amygdala network operation.

Selective Extracellular Stimulation of Pharmacologically Distinct CCK/CB1R Positive Interneuron to Pyramidal Cell Perisomatic Inhibitory Synapses

During prolonged whole cell recording, the intracellular contents are progressively dialyzed with the pipette solution; this often leads to significant changes in synaptic efficacy. To overcome this problem, we developed an approach allowing reliable extracellular stimulation of perisomatic synapses formed by CCK+/CB1+ interneurons onto CA1 pyramidal cells. Functional identification of this input was based on the unique features of CCK+/CB1+ terminals: long-lasting asynchronous transmitter release following high-frequency stimulation and exclusive expression of CB1R. Asynchronous release was used as an indication of proper positioning of the theta glass stimulation pipettes. We found that all extracellularly stimulated inputs with characteristic asynchronous release undergo robust DSI in response to 5-s depolarization and could also be almost entirely blocked by application of the CB1R agonist CP55940, which were similar to the data obtained with paired recordings from connected CB1+ and CA1 pyramidal cells. Thus, we have developed an approach allowing the selective and reliable extracellular stimulation of a subtype of hippocampal perisomatic inhibitory synapses.

Synaptic transmission at the endbulb of Held deteriorates during age-related hearing loss.

Synaptic transmission at the endbulb of Held was assessed by whole-cell patch clamp recordings from auditory neurons in mature (2-4months) and aged (20-26months) mice. Synaptic transmission is degraded in aged mice, which may contribute to the decline in neural processing of the central auditory system during age-related hearing loss. The changes in synaptic transmission in aged mice can be partially rescued by improving calcium buffering, or decreasing action potential-evoked calcium influx. These experiments suggest potential mechanisms, such as regulating intraterminal calcium, that could be manipulated to improve the fidelity of transmission at the aged endbulb of Held. Age-related hearing loss (ARHL) is associated with changes to the auditory periphery that raise sensory thresholds and alter coding, and is accompanied by alterations in excitatory and inhibitory synaptic transmission, and intrinsic excitability in the circuits of the central auditory system. However, it remains unclear how synaptic transmission changes at the first central auditory synapses during ARHL. Using mature (2-4months) and old (20-26months) CBA/CaJ mice, we studied synaptic transmission at the endbulb of Held. Mature and old mice showed no difference in either spontaneous quantal synaptic transmission or low frequency evoked synaptic transmission at the endbulb of Held. However, when challenged with sustained high frequency stimulation, synapses in old mice exhibited increased asynchronous transmitter release and reduced synchronous release. This suggests that the transmission of temporally precise information is degraded at the endbulb during ARHL. Increasing intraterminal calcium buffering with EGTA-AM or decreasing calcium influx with ω-agatoxin IVA decreased the amount of asynchronous release and restored synchronous release in old mice. In addition, recovery from depression following high frequency trains was faster in old mice, but was restored to a normal time course by EGTA-AM treatment. These results suggest that intraterminal calcium in old endbulbs may rise to abnormally high levels during high rates of auditory nerve firing, or that calcium-dependent processes involved in release are altered with age. These observations suggest that ARHL is associated with a decrease in temporal precision of synaptic release at the first central auditory synapse, which may contribute to perceptual deficits in hearing.

A Phenomenological Synapse Model for Asynchronous Neurotransmitter Release.

Neurons communicate with each other via synapses. Action potentials cause release of neurotransmitters at the axon terminal. Typically, this neurotransmitter release is tightly time-locked to the arrival of an action potential and is thus called synchronous release. However, neurotransmitter release is stochastic and the rate of release of small quanta of neurotransmitters can be considerably elevated even long after the ceasing of spiking activity, leading to asynchronous release of neurotransmitters. Such asynchronous release varies for tissue and neuron types and has been shown recently to be pronounced in fast-spiking neurons. Notably, it was found that asynchronous release is enhanced in human epileptic tissue implicating a possibly important role in generating abnormal neural activity. Current neural network models for simulating and studying neural activity virtually only consider synchronous release and ignore asynchronous transmitter release. Here, we develop a phenomenological model for asynchronous neurotransmitter release, which, on one hand, captures the fundamental features of the asynchronous release process, and, on the other hand, is simple enough to be incorporated in large-size network simulations. Our proposed model is based on the well-known equations for short-term dynamical synaptic interactions and includes an additional stochastic term for modeling asynchronous release. We use experimental data obtained from inhibitory fast-spiking synapses of human epileptic tissue to fit the model parameters, and demonstrate that our model reproduces the characteristics of realistic asynchronous transmitter release.

The Same Synaptic Vesicles Originate Synchronous and Asynchronous Transmitter Release

Transmitter release and synaptic vesicle exo- and endocytosis during high-frequency stimulation (20 pulses/s) in the extracellular presence of different bivalent cations (Ca(2+), Sr2+ or Ba2+) were studied in frog cutaneous pectoris nerve-muscle preparations. It was shown in electrophysiological experiments that almost only synchronous transmitter release was registered in a Ca(2+)-containing solution; a high intensity of both synchronous and asynchronous transmitter release was registered in a Sr2+-containing solution, and asynchronous transmitter release almost only was observed in a Ba2+-containing solution. It was shown in experiments with a FM 1-43 fluorescent dye that the synaptic vesicles that undergo exocytosis-endocytosis during synchronous transmitter release (Ca-solutions) are able to participate in asynchronous exocytosis in Ba-solutions. The vesicles that had participated in the asynchronous transmitter release (Ba-solutions) could subsequently participate in a synchronous release (Ca-solutions). It was shown in experiments with isolated staining of recycling and reserve synaptic vesicle pools that both types of evoked transmitter release originate from the same synaptic vesicle pool.

Sensitivity and specificity of auditory steady-state response testing

INTRODUCTION:The ASSR test is an electrophysiological test that evaluates, among other aspects, neural synchrony, based on the frequency or amplitude modulation of tones.OBJECTIVE:The aim of this study was to determine the sensitivity and specificity of auditory steady‐state response testing in detecting lesions and dysfunctions of the central auditory nervous system. METHODS:Seventy volunteers were divided into three groups: those with normal hearing; those with mesial temporal sclerosis; and those with central auditory processing disorder. All subjects underwent auditory steady‐state response testing of both ears at 500 Hz and 2000 Hz (frequency modulation, 46 Hz). The difference between auditory steady‐state response‐estimated thresholds and behavioral thresholds (audiometric evaluation) was calculated.RESULTS:Estimated thresholds were significantly higher in the mesial temporal sclerosis group than in the normal and central auditory processing disorder groups. In addition, the difference between auditory steady‐state response‐estimated and behavioral thresholds was greatest in the mesial temporal sclerosis group when compared to the normal group than in the central auditory processing disorder group compared to the normal group. DISCUSSION:Research focusing on central auditory nervous system (CANS) lesions has shown that individuals with CANS lesions present a greater difference between ASSR‐estimated thresholds and actual behavioral thresholds; ASSR‐estimated thresholds being significantly worse than behavioral thresholds in subjects with CANS insults. This is most likely because the disorder prevents the transmission of the sound stimulus from being in phase with the received stimulus, resulting in asynchronous transmitter release. Another possible cause of the greater difference between the ASSR‐estimated thresholds and the behavioral thresholds is impaired temporal resolution.CONCLUSIONS:The overall sensitivity of auditory steady‐state response testing was lower than its overall specificity. Although the overall specificity was high, it was lower in the central auditory processing disorder group than in the mesial temporal sclerosis group. Overall sensitivity was also lower in the central auditory processing disorder group than in the mesial temporal sclerosis group.

Shunting Inhibition Controls the Gain Modulation Mediated by Asynchronous Neurotransmitter Release in Early Development

The sensitivity of a neuron to its input can be modulated in several ways. Changes in the slope of the neuronal input-output curve depend on factors such as shunting inhibition, background noise, frequency-dependent synaptic excitation, and balanced excitation and inhibition. However, in early development GABAergic interneurons are excitatory and other mechanisms such as asynchronous transmitter release might contribute to regulating neuronal sensitivity. We modeled both phasic and asynchronous synaptic transmission in early development to study the impact of activity-dependent noise and short-term plasticity on the synaptic gain. Asynchronous release decreased or increased the gain depending on the membrane conductance. In the high shunt regime, excitatory input due to asynchronous release was divisive, whereas in the low shunt regime it had a nearly multiplicative effect on the firing rate. In addition, sensitivity to correlated inputs was influenced by shunting and asynchronous release in opposite ways. Thus, asynchronous release can regulate the information flow at synapses and its impact can be flexibly modulated by the membrane conductance.

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.

Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.

Chronic intermittent hypoxia affects integration of sensory input by neurons in the nucleus tractus solitarii

The autonomic nervous and respiratory systems, as well as their coupling, adapt over a wide range of conditions. Chronic intermittent hypoxia (CIH) is a model for recurrent apneas and induces alterations in breathing and increases in sympathetic nerve activity which may ultimately result in hypertension if left untreated. These alterations are believed to be due to increases in the carotid body chemoreflex pathway. Here we present evidence that the nucleus tractus solitarii (nTS), the central brainstem termination site of chemoreceptor afferents, expresses a form of synaptic plasticity that increases overall nTS activity following intermittent hypoxia. Following CIH, an increase in presynaptic spontaneous neurotransmitter release occurs under baseline conditions. Furthermore, during and following afferent stimulation there is an augmentation of spontaneous transmitter release that occurs out of synchrony with sensory stimulation. On the other hand, afferent evoked synchronous transmitter release is attenuated. Overall, this shift from synchronous to asynchronous transmitter release enhances nTS cellular discharge. The role of the neurotransmitter dopamine in CIH-induced plasticity is also discussed. Dopamine attenuates synaptic transmission in nTS cells by blockade of N-type calcium channels, and this mechanism occurs tonically following normoxia and CIH. This dopaminergic pathway, however, is not altered in CIH. Taken together, alterations in nTS synaptic activity may play a role in the changes of chemoreflex function and cardiorespiratory activity in the CIH apnea model.

An encounter of the third kind without the usual suspects: coherent activation of asynchronous transmitter release independent of presynaptic excitation

An encounter of the third kind without the usual suspects: coherent activation of asynchronous transmitter release independent of presynaptic excitation

Synchronous and Asynchronous Transmitter Release at Nicotinic Synapses Are Differentially Regulated by Postsynaptic PSD-95 Proteins

The rate and timing of information transfer at neuronal synapses are critical for determining synaptic efficacy and higher network function. Both synchronous and asynchronous neurotransmitter release shape the pattern of synaptic influences on a neuron. The PSD-95 family of postsynaptic scaffolding proteins, in addition to organizing postsynaptic components at glutamate synapses, acts transcellularly to regulate synchronous glutamate release. Here we show that PSD-95 family members at nicotinic synapses on chick ciliary ganglion neurons in culture execute multiple functions to enhance transmission. Together, endogenous PSD-95 and SAP102 in the postsynaptic cell appear to regulate transcellularly the synchronous release of transmitter from presynaptic terminals onto the neuron while stabilizing postsynaptic nicotinic receptor clusters under the release sites. Endogenous SAP97, in contrast, has no effect on receptor clusters but acts transcellularly from the postsynaptic cell through N-cadherin to enhance asynchronous release. These separate and parallel regulatory pathways allow postsynaptic scaffold proteins to dictate the pattern of cholinergic input a neuron receives; they also require balancing of PSD-95 protein levels to avoid disruptive competition that can occur through common binding domains.

Dynamic Modulation of Phasic and Asynchronous Glutamate Release in Hippocampal Synapses

Although frequency-dependent short-term presynaptic plasticity has been of long-standing interest, most studies have emphasized modulation of the synchronous, phasic component of transmitter release, most evident with a single or a few presynaptic stimuli. Asynchronous transmitter release, vesicle fusion not closely time locked to presynaptic action potentials, can also be prominent under certain conditions, including repetitive stimulation. Asynchrony has often been attributed to residual Ca(2+) buildup in the presynaptic terminal. We verified that a number of manipulations of Ca(2+) handling and influx selectively alter asynchronous release relative to phasic transmitter release during action potential trains in cultured excitatory autaptic hippocampal neurons. To determine whether other manipulations of vesicle release probability also selectively modulate asynchrony, we probed the actions of one thoroughly studied modulator class whose actions on phasic versus asynchronous release have not been investigated. We examined the effects of the phorbol ester PDBu, which has protein kinase C (PKC) dependent and independent actions on presynaptic transmitter release. PDBu increased phasic and asynchronous release in parallel. However, while PKC inhibition had relatively minor inhibitory effects on PDBu potentiation of phasic and total release during action potential trains, PKC inhibition strongly reduced phorbol-potentiated asynchrony, through actions most evident late during stimulus trains. These results lend new insight into PKC-dependent and -independent effects on transmitter release and suggest the possibility of differential control of synchronous versus asynchronous vesicle release.

Asynchronous Transmitter Release from Cholecystokinin-Containing Inhibitory Interneurons Is Widespread and Target-Cell Independent

Neurotransmitter release at most central synapses is synchronized to the timing of presynaptic action potentials. Here, we show that three classes of depolarization-induced suppression of inhibition-expressing, cholecystokinin (CCK)-containing, hippocampal interneurons show highly asynchronous release in response to trains of action potentials. This asynchrony is correlated to the class of presynaptic interneuron but is unrelated to their postsynaptic cell target. Asynchronous and synchronous release from CCK-containing interneurons show a slightly different calcium dependence, such that the proportion of asynchronous release increases with external calcium concentration, possibly suggesting that the modes of release are mediated by different calcium sensors. Asynchronous IPSCs include very large (up to 500 pA/7nS) amplitude events, which persist in low extracellular calcium and strontium, showing that they result from quantal transmitter release at single release sites. Finally, we show that asynchronous release is prominent in response to trains of presynaptic spikes that mimic natural activity of CCK-containing interneurons. That asynchronous release from CCK-containing interneurons is a widespread phenomenon indicates a fundamental role for these cells within the hippocampal network that is distinct from the phasic inhibition provided by parvalbumin-containing interneurons.

A General Model of Synaptic Transmission and Short-Term Plasticity

Some synapses transmit strongly to action potentials (APs), but weaken with repeated activation; others transmit feebly at first, but strengthen with sustained activity. We measured synchronous and asynchronous transmitter release at "phasic" crayfish neuromuscular junctions (NMJs) showing depression and at facilitating "tonic" junctions, and define the kinetics of depression and facilitation. We offer a comprehensive model of presynaptic processes, encompassing mobilization of reserve vesicles, priming of docked vesicles, their association with Ca(2+) channels, and refractoriness of release sites, while accounting for data on presynaptic buffers governing Ca(2+) diffusion. Model simulations reproduce many experimentally defined aspects of transmission and plasticity at these synapses. Their similarity to vertebrate central synapses suggests that the model might be of general relevance to synaptic transmission.

Calcium and synaptic dynamics underlying reverberatory activity in neuronal networks

Persistent activity is postulated to drive neural network plasticity and learning. To investigate its underlying cellular mechanisms, we developed a biophysically tractable model that explains the emergence, sustenance and eventual termination of short-term persistent activity. Using the model, we reproduced the features of reverberating activity that were observed in small (50–100 cells) networks of cultured hippocampal neurons, such as the appearance of polysynaptic current clusters, the typical inter-cluster intervals, the typical duration of reverberation, and the response to changes in extra-cellular ionic composition. The model relies on action potential-triggered residual pre-synaptic calcium, which we suggest plays an important role in sustaining reverberations. We show that reverberatory activity is maintained by enhanced asynchronous transmitter release from pre-synaptic terminals, which in itself depends on the dynamics of residual pre-synaptic calcium. Hence, asynchronous release, rather than being a ‘synaptic noise’, can play an important role in network dynamics. Additionally, we found that a fast timescale synaptic depression is responsible for oscillatory network activation during reverberations, whereas the onset of a slow timescale depression leads to the termination of reverberation. The simplicity of our model enabled a number of predictions that were confirmed by additional analyses of experimental manipulations.

Calcium Signaling Pathways Mediating Synaptic Potentiation Triggered by Amyotrophic Lateral Sclerosis IgG in Motor Nerve Terminals

Sporadic amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects particularly motoneurons. Several pieces of evidence suggested the involvement of autoimmune mechanisms mediated by antibodies in ALS. However, the significance of those antibodies in the disease and the underlying mechanisms are unknown. Here we showed that IgG purified from a group of sporadic ALS patients, but not familial ALS patients, specifically interact with the presynaptic membrane of motoneurons through an antigen-antibody interaction and modulated synaptic transmission. Immunoreactivity against nerve terminals showed strong correlation with synaptic modulation ability. In addition, several controls have ruled out the possibility for this synaptic modulation to be mediated through proteases or nonspecific effects. Effective IgG potentiated both spontaneous and asynchronous transmitter release. Application of pharmacological inhibitors suggested that activation of this increased release required a nonconstitutive Ca2+ influx through N-type (Cav2.2) channels and phospholipase C activity and that activation of IP3 and ryanodine receptors were necessary to both activate and sustain the increased release. Consistent with the notion that ALS is heterogeneous disorder, our results reveal that, in approximately 50% of ALS patients, motor nerve terminals constitutes a target for autoimmune response.

Mefloquine selectively increases asynchronous acetylcholine release from motor nerve terminals

Effectiveness against chloroquine-resistant Plasmodia makes mefloquine a widely used antimalarial drug. However, mefloquine's neurologic effects offset this therapeutic advantage. Cellular actions which might contribute to the neurologic effects of mefloquine are not understood. Structural similarity to tacrine suggested that mefloquine might alter cholinergic synaptic transmission. Therefore, we examined mefloquine's effects at a model cholinergic synapse. Triangularis sterni nerve–muscle preparations were isolated from adult mice and examined with sharp electrode current clamp technique. Within 30 min of exposure to 10 μM mefloquine, miniature endplate potentials (mepps) occurred in summating bursts and their mean frequency increased 10-fold. The threshold concentration for the increase of mean mepp frequency was 0.6 μM mefloquine. Mefloquine continued to increase mean mepp frequency for preparations bathed in extracellular solution lacking Ca 2+. In contrast, mefloquine no longer increased mean mepp frequency for preparations pre-treated with the intracellular Ca 2+ buffer BAPTA-AM. Although mefloquine disrupts a thapsigargin-sensitive neuronal Ca 2+ store, pre-treatment with thapsigargin did not alter the mefloquine-induced alterations of mepps. Since mefloquine, like oligomycin, inhibits mitochondrial F OF 1H +ATP synthase we tested the interaction between these two chemicals. Like mefloquine, oligomycin induced bursts and increased mean frequency of mepps. Furthermore, pre-treatment with oligomycin precluded the mefloquine-induced alterations of asynchronous transmsitter release. These data suggest that mefloquine inhibits ATP production which increases the concentration of Ca 2+ within the cytosol of nerve terminals. This elevation of Ca 2+ concentration selectively increases asynchronous transmitter release since 10 μM mefloquine did not alter stimulus-evoked transmsitter release.