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Edited by: Shin-ya Kawaguchi, Kyoto University, Japan

Reviewed by: Stefan Hallermann, Leipzig University, Germany; Hartmut Schmidt, Leipzig University, Germany

This article was submitted to Cellular Neurophysiology, a section of the journal Frontiers in Cellular Neuroscience

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Following action potential invasion in presynaptic terminals, synaptic vesicles are released in a stochastic manner at release sites (docking sites). Since neurotransmission occurs at frequencies up to 1 kHz, the mechanisms underlying consecutive vesicle releases at a docking site during high frequency bursts is a key factor for understanding the role and strength of the synapse. Particularly new vesicle recruitment at the docking site during neuronal activity is thought to be crucial for short-term plasticity. However current studies have not reached a unified docking site model for central synapses. Here I review newly developed analyses that can provide insight into docking site models. Quantal analysis using counts of vesicular release events provide a wealth of information not only to monitor the number of docking sites, but also to distinguish among docking site models. The stochastic properties of cumulative release number during bursts allow us to estimate the total number of releasable vesicles and to deduce the features of vesicle recruitment at docking sites and the change of release probability during bursts. This analytical method may contribute to a comprehensive understanding of release/replenishment mechanisms at a docking site.

Synaptic vesicles fuse with the presynaptic membrane to release neurotransmitter into the synaptic cleft in a specific structure called active zone (AZ), where each AZ contains one or multiple vesicular docking/release sites (DSs). The DS number corresponds to the maximum number of vesicular release events following an action potential (AP) (review:

A simple approach for estimating synaptic parameters using fluctuation analysis is to utilize the number of released vesicles at single AZs instead of PSC amplitude, as the former method needs less corrections and assumptions than the latter. Recently we have developed a new method to detect individual vesicular release events using EPSCs recorded in simple synapses (

The binomial model of vesicular release classically predicts a parabolic variance-mean relationship for synaptic response fluctuations (

so that:

where

^{2+} uncaging with appropriate flash intensity, and local application of α-Latrotoxin at a single bouton may also be able to provide miniature responses in a simple synapse (

Last and cumulative number of detected vesicular release events in a train at a simple synapse.

One key benefit of detecting vesicular release events in synaptic responses is to obtain the cumulative number of events in a train. Statistical analysis of the cumulative release number provides valuable information to evaluate release models. In this chapter, I will review several models derived on the basis of a classical binomial model and discuss the stochastic properties of the cumulative release number. Some models are described in a previous paper (

Variance-Mean plots of last release number and cumulative release number in various models. A series of Monte Carlo simulations of variance-mean plots of last release number (red, open circles) and cumulative release number (gray, filled squares) (_{1} = 0) but release occurs with a probability _{2–8} = 0.4 from the 2nd stimulus.

In this very simple model, there are four DSs that cannot be replenished with vesicles once they become empty (

In this model, an empty DS is replenished with a rate constant

In contrast to model (ii), in the parallel model release occurs following a Poisson process at DSs or somewhere else in the AZ, in addition to release at DSs without vesicle replenishment (

This model introduces an additional site, called replacement site, for each DS (

This model is similar to the previous model (iv), except that a recruitment step is added to refill the replacement site (

A different model also shows similar statistical properties of the cumulative release number. In this model derived from previous reports (e.g.,

In the two-step or the renewable two-step model, a replacement site is associated to each DS. In the present model, a common replacement pool replaces individual replacement sites. Initially four vesicles in the pool are ready to replenish any empty DS (

The last attractive model is a two-state model where a DS can accommodate one bound vesicle in two different states (loosely docked state and tightly docked state). This model was proposed based on the evidence that the docked/primed synaptic vesicle state is very dynamic (

The plots for the cumulative number among models (v), (vi), and (vii) are similar in shape. However the slope of the later points in model (vii) is larger than in models (v) and (vi), indicating more random release later in a train. In addition, a discrepancy between model (v) and (vi) appears in the plot for the last release number (red circle #2). Likewise, in the case of another proposed model where new release site recruit between stimulations instead of replenishment of vesicles to vacant DS (

As described above, there are similarities and discrepancies among models with certain sets of parameter values. In some sets of values the models are hardly distinguished but in the other sets they are distinguishable, since the shape of the plot for the cumulative number changes with the combination of the parameter values. Therefore, models need to be examined in different experimental conditions possibly changing the parameter values.

Stochastic properties of last and cumulative release number at PF-MLI synapses in cerebellum in 2-week-old rats were best fitted with model (v). Further support for this model was provided by pharmacological experiments (

In summary, when performing variance-mean analysis, the last release number provides information about the number of DSs and the release probability per DS; examination of the variance-mean plot for this number during an AP train provides information on whether release follows a binomial or a Poisson process. Comparing the statistical properties of the cumulative release number among various models, some features concerning replenishment steps are extracted. In certain models, the variance-mean plot displays a jump between the 1st and the 2nd point, implying the existence of an associated site (replacement site) or of an additional vesicle pool replenishing DSs for consecutive release. By contrast, a continuous increase in variance is displayed in other simpler models, like the renewable one-step model and the parallel model (

How reliably can we estimate the total number of releasable/suppliable vesicles using variance-mean plots for cumulative release? Further simulations related to the two-step model are shown in

Occupancy at docking and replacement sites affects stochastic properties of cumulative release number.

Recently methods have been developed to count the number of released vesicles at single synapses using deconvolution analysis (^{2+} channels (

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

The author confirms being the sole contributor of this work and has approved it for publication.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

I thank Alain Marty for valuable comments and suggestions on the manuscript.

^{2+}-triggered fusion by C

_{2}B-domain-mediated synaptic-vesicle membrane attachment.

^{2+}channel to synaptic vesicle distance accounts for the readily releasable pool kinetics at a functionally mature auditory synapse.

^{2+}buffer-dependent reliable but plastic transmission at small CNS synapses revealed by direct bouton recording.

^{2+}-sensor for synaptic vesicle replenishment.

^{2+}sensor for vesicle fusion.

^{2+}channel clusters match those of functionally defined vesicular docking sites in single central synapses.

^{2+}channels and its impact on vesicular release during development.