A maximum of two readily releasable vesicles per docking site at a cerebellar single active zone synapse

Reviewer #1 (Public Review):

Summary: By elevating Ca influx and inducing PTP, the authors have maximized the release probability. In this condition, the release probability is nearly one. Under such a condition, the release site can release another vesicle in a short time. By analyzing mean, variance, and covariance, the authors propose a release model that each release site contains a docking site and a replacement site. They excluded the LS-TS model (Neher and Brose) based on a discrepancy between the model and the data (mean and covariance).

Strengths: The authors have used minimal stimulation and modeling nicely to look into the stochastic nature of release sites with good resolution. This cannot be done at other synapses. Overall conclusions are reasonable and convincing.

Weaknesses: The interpretation is somewhat model-dependent, and it is unclear if the interpretation is unique. For example, it is unclear if the heterogeneous release probability among sites, silent sites, can explain the results. N estimates out of variance-mean analysis for example may be limited by the availability of postsynaptic receptors.

Reviewer #2 (Public Review):

Summary:
Silva et al. describe an experimental study conducted on cerebellar parallel fiber-to-molecular interneuron synapses to investigate the size of the readily releasable pool (RRP) of synaptic vesicles (SVs) per docking site in response to trains of action potentials. The study aims to determine whether there are multiple binding sites for SVs at each docking site, which could lead to a higher RRP size than previously thought.

The researchers used this glutamatergic synapse to conduct their experiments. They employed various techniques and manipulations to enhance release probability, docking site occupancy, and synaptic depression. By counting the number of released SVs in response to action potential trains and normalizing the results based on the number of docking sites, they estimated the RRP size per docking site.

The key findings and observations in the manuscript are as follows:

Docking Site Occupancy and Release Probability Enhancement: The researchers used 4-amidopyridine (4-AP) and post-tetanic potentiation (PTP) protocols to enhance the release probability of docked SVs and the occupancy of docking sites, respectively.

Synchronous and Asynchronous Release: Synchronous release refers to SVs released in response to individual action potentials, while asynchronous release involves SVs released after the initial release response due to calcium elevation. The study observed changes in the balance between synchronous and asynchronous release under different conditions, revealing the degree of filling of the RRP.

Modeling of Release Dynamics: The researchers employed a modeling approach based on the "replacement site/docking site" (RS/DS) model, where SVs bind to a replacement site before moving to a docking site and eventually undergoing release. The model was adjusted to experimental conditions to estimate parameters like docking site occupancy and release probabilities.

Comparison of Different Models: The study compared the RS/DS model with an alternative model known as the "loosely docked/tightly docked" (LS/TS) model. The LS/TS model assumes that a docking site can only accommodate one SV at a time, while the RS/DS model considers the possibility of accommodating multiple SVs.

Maximum RRP Size: Through a combination of experimental results and model simulations, the study revealed that the maximum RRP size per docking site reached close to two SVs under certain conditions, supporting the idea that each docking site can accommodate multiple SVs.

Strengths:
The study is rigorously conducted and takes into consideration the previous work on RRP size and SV docking site estimation. The study addresses a long-standing question in synaptic physiology.

Weaknesses:
It remains unclear how generalizable the findings are to other types of synapses.