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.     

Synaptic vesicle pools at the frog neuromuscular junction

We have characterized the morphological and functional properties of the readily releasable pool (RRP) and the reserve pool of synaptic vesicles in frog motor nerve terminals using fluorescence microscopy, electron microscopy, and electrophysiology. At rest, about 20% of vesicles reside in the RRP, which is depleted in about 10 s by high-frequency nerve stimulation (30 Hz); the RRP refills in about 1 min, and surprisingly, refilling occurs almost entirely by recycling, not mobilization from the reserve pool. The reserve pool is depleted during 30 Hz stimulation with a time constant of about 40 s, and it refills slowly (half-time about 8 min) as nascent vesicles bud from randomly distributed cisternae and surface membrane infoldings and enter vesicle clusters spaced at regular intervals along the terminal. Transmitter output during low-frequency stimulation (2-5 Hz) is maintained entirely by RRP recycling; few if any vesicles are mobilized from the reserve pool.     

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.     

A Well-Defined Readily Releasable Pool with Fixed Capacity for Storing Vesicles at Calyx of Held

The readily releasable pool (RRP) of vesicles is a core concept in studies of presynaptic function. However, operating principles lack consensus definition and the utility for quantitative analysis has been questioned. Here we confirm that RRPs at calyces of Held from 14 to 21 day old mice have a fixed capacity for storing vesicles that is not modulated by Ca²⁺. Discrepancies with previous studies are explained by a dynamic flow-through pool, established during heavy use, containing vesicles that are released with low probability despite being immediately releasable. Quantitative analysis ruled out a posteriori explanations for the vesicles with low release probability, such as Ca²⁺-channel inactivation, and established unexpected boundary conditions for remaining alternatives. Vesicles in the flow-through pool could be incompletely primed, in which case the full sequence of priming steps downstream of recruitment to the RRP would have an average unitary rate of at least 9/s during heavy use. Alternatively, vesicles with low and high release probability could be recruited to distinct types of release sites; in this case the timing of recruitment would be similar at the two types, and the downstream transition from recruited to fully primed would be much faster. In either case, further analysis showed that activity accelerates the upstream step where vesicles are initially recruited to the RRP. Overall, our results show that the RRP can be well defined in the mathematical sense, and support the concept that the defining mechanism is a stable group of autonomous release sites.     

海马神经元突触囊泡释放特征及可释放囊泡含量定量分析(英文)

中枢神经系统突触前的神经末梢只有少量的突触囊泡存在,突触囊泡数目的多少和融合模式将影响突触传递的效率。对突触囊泡数目的多少和释放模式的研究依赖于有效的研究方法。在本研究中,与膜亲和力不同的荧光染料用于标记体外培养的海马神经元的功能性突触囊泡。通过场电位和高钾刺激,动态观察荧光强度的变化,结果显示在第一轮刺激中,与膜亲和力低的染料FM2-10脱色的比例(93.0%±5.9%)显著大于与膜亲和力高的染料FM1-43(57.9%±3.5%)。但是,第二和第三轮刺激中FM1-43脱色的比例分别为(24.0±2.3)%,(8.6±1.5)%,显著大于FM2-10的脱色比例[(1.4±3.8)%,(2.3±1.6)%]。这个结果提示快速内吞模式不仅存在于囊泡的第一次释放,同时还存在于囊泡回收后的再次释放。另一方面,高频刺激和高渗蔗糖溶液这两种方法同时用于检测体外混合培养13~14天的抑制性神经元的可释放囊泡池(readily releasable pool,RRP)的大小。结果显示,用高渗蔗糖溶液估计的RRP的大小[(200±23.0)pC]显著大于用高频刺激估计的RRP的大小[(51.1±10.5)pC]。分析其可能的...     

Endogenous calcium buffers regulate fast exocytosis in the synaptic terminal of retinal bipolar cells.

Calcium-triggered exocytosis at the synapse is suppressed by addition of calcium chelators, but the effects of endogenous Ca(2+) buffers have not been tested. We find that 80% of Ca(2+) binding sites in the synaptic terminal of retinal bipolar cells were associated with mobile molecules that suppressed activation of Ca(2+)-sensitive K(+) channels with an efficiency equivalent to approximately 1.2 mM BAPTA. Removing these buffers caused a 30-fold increase in the number of vesicles released by Ca(2+) tail currents lasting approximately 0.5 ms and a 2-fold increase in the rapidly releasable pool of vesicles (RRP). The effects of BAPTA and EGTA indicate that vesicles comprising the RRP were docked at variable distances from Ca(2+) channels. We propose that endogenous Ca(2+) buffers regulate the size of the RRP by suppressing the release of vesicles toward the periphery of the active zone.     

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.     

Monitoring synaptic vesicle recycling in frog motor nerve terminals with FM dyes

Ultrastructural observations made in the study of the frog neuromuscular junction (NMJ) almost three decades ago showed that synaptic vesicle cycling functions through a slow pathway, requiring the use of clathrin-coated vesicles and an endosomal compartment. Simultaneously, a conceptually simpler model emerged, postulating rapid retrieval of vesicle membrane through a mechanism similar to a reversal of vesicle fusion. With the advent of fluorescence imaging which allows the investigator to monitor recycling in living nerve-muscle preparations, new data appeared which reconcile at least in part the two models, indicating that both may be important at this synapse. Two different synaptic vesicle pools can be defined, a readily releasable pool (RRP), consisting of quanta that are immediately available for release, and a reserve pool (RP) that is exocytosed only after prolonged stimulation. Vesicles in the RRP recycle through a fast endocytic pathway, which does not rely on an endosomal compartment, while vesicles in the RP cycle more slowly through formation of infoldings and endosomes and their subsequent severance into vesicles. The two pools mix slowly, and their recycling may be regulated by different mechanisms.     

Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire

Two vesicle pools, readily releasable (RRP) and reserve (RP) pools, are present at Drosophila neuromuscular junctions. Using a temperature-sensitive mutant, shibire(ts), we studied pool sizes and vesicle mobilization rates. In shibire(ts), due to lack of endocytosis at nonpermissive temperatures, synaptic currents continuously declined during tetanic stimulation until they ceased as the result of vesicle depletion. By then, approximately 84,000 quanta were released. Vesicles were mobilized from RP at a rate 1/7-1/10 of RRP. Cytochalasin D inhibited mobilization of vesicles from RP, allowing us to estimate the size of RRP as 14%-19% of all vesicles. Vesicle recycling supports synaptic transmission during prolonged tetanic stimulation and the maximum recycling rate was 1000 vesicles/s.     

Properties of synaptic vesicle pools in mature central nerve terminals

Readily releasable and reserve pools of synaptic vesicles play different roles in neurotransmission, and it is important to understand their recycling and interchange in mature central synapses. Using adult rat cerebrocortical synaptosomes, we have shown that 100 mosm hypertonic sucrose caused complete exocytosis of only the readily releasable pool (RRP) of synaptic vesicles containing glutamate or gamma-aminobutyric acid. Repetitive hypertonic stimulations revealed that this pool recycled (and reloaded the neurotransmitter from the cytosol) fully in <30 s and did so independently of the reserve pool. Multiple rounds of exocytosis could occur in the constant absence of extracellular Ca(2+). However, although each vesicle cycle includes a Ca(2+)-independent exocytotic step, some other stage(s) critically require an elevation of cytosolic [Ca(2+)], and this is supplied by intracellular stores. Repetitive recycling also requires energy, but not the activity of phosphatidylinositol 4-kinase, which maintains the normal level of phosphoinositides. By varying the length of hypertonic stimulations, we found that approximately 70% of the RRP vesicles fused completely with the plasmalemma during exocytosis and could then enter silent pools, probably outside active zones. The rest of the RRP vesicles underwent very fast local recycling (possibly by kiss-and-run) and did not leave active zones. Forcing the fully fused RRP vesicles into the silent pool enabled us to measure the transfer of reserve vesicles to the RRP and to show that this process requires intact phosphatidylinositol 4-kinase and actin microfilaments. Our findings also demonstrate that respective vesicle pools have similar characteristics and requirements in excitatory and inhibitory nerve terminals.     

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.     

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.     

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.     

v-SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.     

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 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.     

Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.     

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.     

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.     

Identification of a novel process limiting the rate of synaptic vesicle cycling at hippocampal synapses

During intense presynaptic activity, the readily releasable pool (RRP) of synaptic vesicles empties more quickly than it can be refilled, and short-term depression results. Ordinarily, the pool refills within 20 s, but long, high-frequency trains of action potentials often induce a form of short-term depression that persists for a much longer time. Here, we report that replenishment of the RRP is governed by two simple processes: the previously identified mechanism termed refilling, and another process that appears after extensive exocytosis and produces a transient decrease in the capacity of the pool, lasting for several minutes. The data presented here place stringent constraints on the types of kinetic models that can be used to describe synaptic vesicular cycling and are inconsistent with the traditional multipool models of vesicular mobilization.