The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles

Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca²⁺ channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca²⁺ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.     

A Sequential Vesicle Pool Model with a Single Release Sensor and a Ca2+-Dependent Priming Catalyst Effectively Explains Ca2+-Dependent Properties of Neurosecretion

Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca²⁺ dependence, but also upstream steps depend on Ca²⁺. After deletion of the Ca²⁺ sensor for fast release – synaptotagmin-1 – slower Ca²⁺-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM) driven by alternative Ca²⁺ sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca²⁺-dependent action: a Ca²⁺-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca²⁺-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca²⁺-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca²⁺-dependent fusion from the NRP. We conclude that the elusive ‘alternative Ca²⁺ sensor’ for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca²⁺-dependent properties of secretion without assuming parallel pools or sensors.     

Exocytotic dynamics in human chromaffin cells: experiments and modeling

Chromaffin cells have been widely used to study neurosecretion since they exhibit similar calcium dependence of several exocytotic steps as synaptic terminals do, but having the enormous advantage of being neither as small or fast as neurons, nor as slow as endocrine cells. In the present study, secretion associated to experimental measurements of the exocytotic dynamics in human chromaffin cells of the adrenal gland was simulated by using a model that combines stochastic and deterministic approaches for short and longer depolarizing pulses, respectively. Experimental data were recorded from human chromaffin cells, obtained from healthy organ donors, using the perforated patch configuration of the patch-clamp technique. We have found that in human chromaffin cells, secretion would be mainly managed by small pools of non-equally fusion competent vesicles, slowly refilled over time. Fast secretion evoked by brief pulses can be predicted only when 75% of one of these pools (the “ready releasable pool” of vesicles, abbreviated as RRP) are co-localized to Ca^2?+? channels, indicating an immediately releasable pool in the range reported for isolated cells of bovine and rat (Álvarez and Marengo, J Neurochem 116:155–163, 2011). The need for spatial correlation and close proximity of vesicles to Ca^2?+? channels suggests that in human chromaffin cells there is a tight control of those releasable vesicles available for fast secretion.     

Temperature-dependent differences between readily releasable and reserve pool vesicles in chromaffin cells

Statistical differences between amperometric traces recorded from chromaffin cells using K(+) and Ba(2+) secretagogues support the assertion that readily releasable pool (RRP) and reserve pool (RP) vesicles can be probed with pool-specific secretagogues. Release from the RRP was evoked by K(+) while release from the RP was evoked by Ba(2+). Similar temperature-dependent changes in spike area and half-width for both pools suggest that the content of RRP and RP vesicles is similar and packaged in the same way. Differences between the vesicle pools were revealed in the temperature dependence of spike frequency. While the burst spike frequency of the RRP, which is comprised of pre-docked and primed vesicles, increased 2.8% per degrees C, the RP spike frequency increased 12% per degrees C. This difference is attributed to a temperature-dependent mobilization of the RP. Furthermore, the RP exhibited more foot events at room temperature than the RRP but this difference was not apparent at 37 degrees C. This trend suggests that RP vesicle membranes have a compromised surface tension compared to RRP vesicles. Collectively, the changes of release characteristics with temperature reveal distinctions between the RRP and the RP.     

Independent Regulation of Basal Neurotransmitter Release Efficacy by Variable Ca2+ Influx and Bouton Size at Small Central Synapses

The efficacy of action potential evoked neurotransmitter release varies widely even among synapses supplied by the same axon, and the number of release-ready vesicles at each synapse is a major determinant of this heterogeneity. Here we identify a second, equally important, mechanism for release heterogeneity at small hippocampal synapses, the inter-synaptic variation of the exocytosis probability of release-ready vesicles. Using concurrent measurements of vesicular pool sizes, vesicular exocytosis rates, and presynaptic Ca²⁺ dynamics, in the same small hippocampal boutons, we show that the average fusion probability of release-ready vesicles varies among synapses supplied by the same axon with the size of the spike-evoked Ca²⁺ concentration transient. We further show that synapses with a high vesicular release probability exhibit a lower Ca²⁺ cooperativity, arguing that this is a direct consequence of increased Ca²⁺ influx at the active zone. We conclude that variability of neurotransmitter release under basal conditions at small central synapses is accounted for not only by the number of release-ready vesicles, but also by their fusion probabilities, which are set independently of bouton size by variable spike-evoked presynaptic Ca²⁺ influx.

Author Summary

Synaptic transmission underlies information transfer among neurons in the brain. The probability that a synapse will release neurotransmitter in response to an action potential varies widely, even among synapses supplied by the same axon. The molecular mechanisms underlying this heterogeneity remain poorly understood. At the level of single synapses, release efficacy is determined largely by two factors: (i) the number of neurotransmitter-containing vesicles ready to be released, and (ii) by the fusion probabilities of these vesicles. By using novel imaging techniques at individual hippocampal presynaptic boutons in culture, we distinguish two independent sources of variability of release probability in small central synapses. First, we find differences in the number of releasable vesicles, and second, we find differences in the exocytosis probability of individual vesicles. To our knowledge, this is the first direct experimental demonstration that the fusion probability of release-ready vesicles is variable among synapses supplied by a single axon, and contributes roughly as much to the overall variability in release probability as does the number of release-ready vesicles.     

Two distinct secretory vesicle�Cpriming steps in adrenal chromaffin cells

Priming of large dense-core vesicles (LDCVs) is a Ca²⁺-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca²⁺ concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.     

Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices.

Many neurosecretory preparations display two components of depolarization-induced exocytosis: a phasic component synchronized with Ca2+ channel opening, followed by a slower sustained component. We evaluated possible mechanisms underlying this biphasic behavior by stimulating mouse chromaffin cells in situ with both depolarizations and flash photolysis of caged Ca2+. From a direct comparison of the secretory responses to both stimuli, we conclude that phasic and sustained release components originate from a readily releasable pool (RRP) of equally fusion-competent vesicles, suggesting that differences in the vesicles' proximity to Ca2+ channels underlie the biphasic secretory behavior. An intermediate pool in dynamic equilibrium with the RRP ensures rapid recruitment of release-ready vesicles after RRP depletion. Our results are discussed in terms of a refined model for secretion in chromaffin cells.     

Synaptic activity slows vesicular replenishment at excitatory synapses of rat hippocampus

Short-term synaptic depression mainly reflects the depletion of the readily releasable pool (RRP) of quanta. Its dynamics, and especially the replenishment rate of the RRP, are still not well characterized in spite of decades of investigation. Main reason is that the vesicular storage and release system is treated as time-independent. If it is time-dependent all parameters thus estimated become problematic. Indeed the reports about how prolonged stimulation affects the dynamics are contradictory. To study this, we used patterned stimulation on the Schaeffer collateral fiber pathway and model-fitting of the excitatory post-synaptic currents (EPSC) recorded from CA1 neurons in rat hippocampal slices. The parameters of a vesicular storage and release model with two pools were estimated by minimizing the squared difference between the ESPC amplitudes and simulated model output. This yields the ‘basic’ parameters (release coupling, replenishment coupling and RRP size) that underlie the ‘derived’ and commonly used parameters (fractional release and replenishment rate). The fractional release increases when [Ca⁺⁺]_o is raised, whereas the replenishment rate is [Ca⁺⁺]_o independent. Fractional release rises because release coupling increases, and the RRP becomes less able to contain quanta. During prolonged stimulation, the fractional release remains generally unaltered, whereas the replenishment rate decreases down to ~10 % of its initial value with a decay time of ~15 s, and this decrease in the replenishment rate significantly contributes to synaptic depression. In conclusion, the fractional release is [Ca⁺⁺]_o-dependent and stimulation-independent, whereas the replenishment rate is [Ca⁺⁺]_o-independent and stimulation-dependent.     

Inactivity produces increases in neurotransmitter release and synapse size.

When hippocampal synapses in culture are pharmacologically silenced for several days, synaptic strength increases. The structural correlate of this change in strength is an increase in the size of the synapses, with all synaptic components--active zone, postsynaptic density, and bouton--becoming larger. Further, the number of docked vesicles and the total number of vesicles per synapse increases, although the number of docked vesicles per area of active zone is unchanged. In parallel with these anatomical changes, the physiologically measured size of the readily releasable pool (RRP) and the release probability are increased. Ultrastructural analysis of individual synapses in which the RRP was previously measured reveals that, within measurement error, the same number of vesicles are docked as are estimated to be in the RRP.     

基于成串刺激计算立即释放囊泡池（RRP）大小的改进方法

突触囊泡的立即释放囊泡池（RRP）概念已被广泛用于突触传递的分析. 基于这些囊泡池中囊泡性质是均匀的假设，通过外推成串刺激累积诱发的突触后兴奋性电流，已经开发了几种确定RRP大小的方法. 然而，使用不同刺激频率确定这些成串刺激得到的RRP大小结果不同. 这种频率依赖性显示了这些估算方法的不完备性，与RRP的定义相矛盾. 因此，我们提出了基于成串刺激计算RRP大小的改进算法. 假设RRP的填充率正比于RRP释放的部分，并且矫正RRP的未使用部分，给出RRP释放过程的完整数学描述，得到具体的解析结果. 与已知的两种常用方法做比较，该方法很好地描述了RRP的释放和填充过程，得到了比较良好的RRP大小和囊泡释放概率大小的评估. 该方法不受刺激频率的条件限制，可以很好地适用于不能给予高频刺激的细胞.     

The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release

Previously it demonstrated that in the absence of Ca²⁺ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca²⁺] _i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217–220.). These studies designate Ca²⁺ entry as opposed to [Ca²⁺] _i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca²⁺ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J. 15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248–253). The kinetics of Ca_v1.2 (Lc-type) and Ca_v2.2 (N-type) Ca²⁺ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Ca_v2.2 coupling to the vesicle. The unique modifications of Ca_v1.2 and Ca_v2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca_v1.2 cytosolic domain Lc_753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca²⁺ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca²⁺-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca²⁺-dependent CAV (RRP) release, initiated by the binding of Ca²⁺ to the channel, upstream to intracellular Ca²⁺ sensor thus establishing the Ca²⁺ channel as the Ca²⁺ sensor of neurotransmitter release.     

Delay between fusion pore opening and peptide release from large dense-core vesicles in neuroendocrine cells.

Peptidergic neurotransmission is slow compared to that mediated by classical neurotransmitters. We have studied exocytotic membrane fusion and cargo release by simultaneous capacitance measurements and confocal imaging of single secretory vesicles in neuroendocrine cells. Depletion of the readily releasable pool (RRP) correlated with exocytosis of 10%-20% of the docked vesicles. Some remaining vesicles became releasable after recovery of RRP. Expansion of the fusion pore, seen as an increase in luminal pH, occurred after approximately 0.3 s, and peptide release was delayed by another 1-10 s. We conclude that (1) RRP refilling involves chemical modification of vesicles already in place, (2) the release of large neuropeptides via the fusion pore is negligible and only proceeds after complete fusion, and (3) sluggish peptidergic transmission reflects the time course of vesicle emptying.     

Rapid reuse of readily releasable pool vesicles at hippocampal synapses

Functional presynaptic vesicles have been subdivided into readily releasable (RRP) and reserve (RP) pools. We studied recycling properties of RRP vesicles through differential retention of FM1-43 and FM2-10 and by varying the time window for FM dye uptake. Both approaches indicated that vesicles residing in the RRP underwent rapid endocytosis (tau approximately 1s), whereas newly recruited RP vesicles were recycled slowly (tau approximately 30 s). With repeated challenges (hypertonic or electrical stimuli), the ability to release neurotransmitter recovered 10-fold more rapidly than restoration of FM2-10 destaining. Finding neurotransmission in the absence of destaining implied that rapidly endocytosed RRP vesicles were capable of reuse, a process distinct from repopulation from the RP. Reuse would greatly expand the functional capabilities of a limited number of vesicles in CNS terminals, particularly during intermittent bursts of activity.     

5-HT offsets homeostasis of synaptic transmission during short-term facilitation.

In this study, we approach the topic of vesicle recruitment and recycling by perturbing neurotransmission at the crayfish neuromuscular junction with altered electrical activity and the presence of the neuromodulator serotonin (5-HT). After induction of short-term facilitation (STF) with stimulus pulse trains (40 Hz, 20 pulses), the amount of synaptic transmission can be maintained at a relatively constant level, producing a plateau in the amplitude of the excitatory postsynaptic potentials (EPSPs) throughout the remaining stimuli within a train of a few hundred milliseconds. With an increase in the frequency of the stimuli within a train (60 Hz, 20 pulses), an altered plateau of larger EPSP amplitudes occurs. This suggests that differential rates of vesicle recruitment can be rapidly reached and maintained. Exposure of nerve terminals to 5-HT further enhances the EPSP amplitudes to yet a higher plateau level. The effect of 5-HT is more pronounced for 40-Hz pulse trains than for 60-Hz trains. This suggests that 5-HT can recruit vesicles into the readily releasable pool (RRP) and that the recruitment is limited at higher stimulation frequencies. The attainment of a larger amplitude in the plateaus of the EPSPs at 60 Hz compared with 40 Hz also suggests that the rapid induction of STF enhances the entry of vesicles into the RRP. By direct quantal counts, mean quantal content increases linearly during STF, and 5-HT offsets the linear release. We propose that 5-HT and electrically induced recruitment of vesicles from a reserve pool to the RRP may share similar recruitment mechanisms.     

Heterogeneous presynaptic release probabilities: functional relevance for short-term plasticity

We discuss a model of presynaptic vesicle dynamics, which allows for heterogeneity in release probability among vesicles. Specifically, we explore the possibility that synaptic activity is carried by two types of vesicles; first, a readily releasable pool and, second, a reluctantly releasable pool. The pools differ regarding their probability of release and time scales on which released vesicles are replaced by new ones. Vesicles of both pools increase their release probability during repetitive stimulation according to the buildup of Ca(2+) concentration in the terminal. These properties are modeled to fit data from the calyx of Held, a giant synapse in the auditory pathway. We demonstrate that this arrangement of two pools of releasable vesicles can account for a variety of experimentally observed patterns of synaptic depression and facilitation at this synapse. We conclude that synaptic transmission cannot be accurately described unless heterogeneity of synaptic release probability is taken into account.     

Synaptotagmin-7 controls the size of the reserve and resting pools of synaptic vesicles in hippocampal neurons

Continuous neurotransmitter release is subjected to synaptic vesicle availability, which in turn depends on vesicle recycling and the traffic of vesicles between pools. We studied the role of Synaptotagmin-7 (Syt-7) in synaptic vesicle accessibility for release in hippocampal neurons in culture. Synaptic boutons from Syt-7 knockout (KO) mice displayed normal basal secretion with no alteration in the RRP size or the probability of release. However, stronger stimuli revealed an increase in the size of the reserve and resting vesicle pools in Syt-7 KO boutons compared with WT. These data suggest that Syt-7 plays a significant role in the vesicle pool homeostasis and, consequently, in the availability of vesicles for synaptic transmission during strong stimulation, probably, by facilitating advancing synaptic vesicles to the readily releasable pool.     

Modulation of Synaptic Vesicle Exocytosis in Muscle-Dependent Long-Term Depression at the Amphibian Neuromuscular Junction

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.     

Modulation of neurosecretion and approaches for its multistep analysis

Background

Neurosecretion is the multistep process occurring in separate spatial and temporal cellular boundaries which complicates its comprehensive analysis. Most of the research are focused on one distinct stage of synaptic vesicle recycling. Here, we describe approaches for complex analysis of synaptic vesicle (SV) endocytosis and separate steps of exocytosis at the level of presynaptic bouton and highly purified SVs.

Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells

Synaptotagmin VII (Syt VII), which has a higher Ca²⁺ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca²⁺-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca²⁺ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.     

The reduced release probability of releasable vesicles during recovery from short-term synaptic depression.

Recovery from synaptic depression is believed to depend mainly on replenishment of the releasable pool of vesicles. We observed that during recovery from depression in a calyx-type synapse, part of the releasable pool was replenished rapidly. Half recovery occurred within 1 s, even in the absence of residual calcium. Vesicles that had recently entered the releasable pool had a 7- to 8-fold lower release probability than those that had been in the pool for more than 30 s. These results suggest that the reduction in the release probability of releasable vesicles contributes greatly to the level of depression. How synapses maintain transmission during repetitive firing is in debate. We propose that during repetitive firing, accumulation of intracellular Ca2+ may facilitate release of the rapidly replenished but reluctant vesicles, making them available for sustaining synaptic transmission.