Two forms of asynchronous release with distinctive spatiotemporal dynamics in central synapses
- Department of Cell Biology and Physiology, Washington University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Two spatially distinct populations of asynchronous release events at individual synaptic boutons.
(Ai) Stimulation and recording protocol. Each square represents a single frame of 50 ms duration. (Aii) Left: examples of raw fluorescence images of a single vesicle release event before (frame 0) …
Figure 1—figure supplement 1
Ectopic asynchronous events are not an artifact of the variable localization precision or insufficient number of detected events.
(A) Error of event localization (localization precision, dx) for synchronous events, asynchronous events inside the active zone (AZ), and ectopic asynchronous events. (B) Distribution of the number …
Figure 2
Detection of two spatially distinct populations of asynchronous events at individual synaptic boutons using glutamate sensor iGluSnFR.
(A) Examples of raw fluorescence images of single glutamate release events detected using SF-iGluSnFR(A184S) before (frame 0) and after (frames 1,2) a single action potential (AP) stimulation, for …
Figure 3
Differential utilization of release sites by synchronous and asynchronous release events.
(A) Cartoon showing a single active zone (AZ) (orange) with two sample clusters/release sites (blue circles) illustrating the assignment of asynchronous events (green dots) as being inside or …
Figure 4
Distinct temporal features of ectopic asynchronous release events.
(A) Cumulative distributions of the time period between each asynchronous event and the immediately preceding synchronous event for asynchronous events inside the active zone (AZ) (yellow, N = 799) …
Figure 5 with 3 supplements
Exo-endocytosis coupling of synchronous and asynchronous release events.
(A) Mean pHluorin signals of synchronous (black, N = 1774) and asynchronous (green, N = 975) release events; SEM displayed as a shading. (B) Cartoon illustrating how the decay of the pHluorin signal …
Figure 5—figure supplement 1
Exo-/endocytosis coupling of different forms of release.
(A) Mean proportion of two components of endocytosis (ultrafast and fast) among synchronous events, asynchronous events inside the active zone (AZ), and ectopic asynchronous events. (B) Mean …
Figure 5—figure supplement 2
Spatial dependence of endocytosis kinetics of different forms of release.
(A) Spatial analysis of the pHluorin signal using normalized distances to the active zone (AZ) center. Mean pHluorin signals are shown for bins at four different normalized distances (0.25, 0.45, …
Figure 5—figure supplement 3
Differential exo-/endocytosis coupling of synchronous and asynchronous release is not caused by differences in localization precision or lateral diffusion.
(A) Error of event localization (localization precision, dx) for all synchronous events, synchronous events in the first concentric spatial bin (bin 1; 0–100 nm from the active zone [AZ] center), or …
Figure 6 with 1 supplement
Spatiotemporal properties of Sr2+-evoked asynchronous release events.
(A) Cumulative distributions of the normalized distances to the active zone (AZ) center for asynchronous events recorded under control conditions (green, N = 1089) and those recorded in 4 mM Sr2+ …
Figure 6—figure supplement 1
Application of Sr2+ causes desynchronization of release.
(A–C) Proportions of synchronous events (A), asynchronous events inside the active zone (AZ) (B), and ectopic asynchronous events (C) per synapse in control conditions or in the presence of 4 mM Sr2+…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Transfected construct (synthetic) | pFU-vGluT1- pHGFP-W | Viral Vectors Core at Washington University | n/a | |
| Transfected construct (synthetic) | pAAV.hSynapsin.SF-iGluSnFR.A184S | Addgene | 106174-AAV1 | |
| Biological sample (rat, Long-Evans) | Hippocampus of rat pups | Charles River | 006 | |
| Chemical compound, drug | APV | Sigma-Aldrich | 79055-68-8 | |
| Chemical compound, drug | CNQX disodium salt hydrate | Sigma-Aldrich | 115066-14-3 | |
| Chemical compound, drug | Minimum Essential Media (MEM) – no phenol red | Gibco | 51200038 | |
| Chemical compound, drug | Characterized fetal bovine serum | Gibco | 10437028 | |
| Chemical compound, drug | Penicillin-streptomycin (5000 U/ml) | Gibco | 15070063 | |
| Chemical compound, drug | N-2 Supplement (100×) | Gibco | 17502048 | |
| Chemical compound, drug | Neurobasal-A Medium | Gibco | 12349015 | |
| Chemical compound, drug | B-27 supplement (50×), serum free | Gibco | 17504044 | |
| Chemical compound, drug | GlutaMAX Supplement | Gibco | 35050061 | |
| Chemical compound, drug | PDL(poly-D-lysine) | BD Biosciences | 40210 | |
| Chemical compound, drug | Trypsin-EDTA (0.05%), phenol red | Gibco | 25300054 | |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 | |
| Software, algorithm | u-track 2.0 | https://www.utsouthwestern.edu/labs/danuser/software/#utrack_anc; (Aguet et al., 2013; Jaqaman et al., 2008) |
Additional files
-
Supplementary file 1
Table containing the statistical information for all figures.
- https://cdn.elifesciences.org/articles/84041/elife-84041-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/84041/elife-84041-mdarchecklist1-v1.docx
