The readily-releasable pool dynamically regulates multivesicular release
- University of Alabama at Birmingham, United States
- Northern (Arctic) Federal University named after M.V. Lomonosov, Russian Federation
Figures
Figure 1 with 2 supplements
cAMP/PKA activation shifts vesicle release mode from UVR to MVR.
(A and B, left). Fsk (50 µM) was used to stimulate cAMP production by adenylyl cyclase (AC, green) and the cAMP analog 6-Bnz (20 µM) was used to activate protein kinase A (PKA, orange). (A and B, …
Figure 1—figure supplement 1
CF-PC EPSCs CV analysis.
CV analysis, plotted as the ratio of CV2 versus the mean EPSC amplitude ratio, normalized to baseline. For increases or decreases in mean amplitude (vertical dotted line is the mean ratio = 1), a …
Figure 1—figure supplement 2
Reducing release site number does not alter KYN block.
(A) Increasing stimulation frequency between 0.1 and 20 Hz reduced EPSCs due to decrease the number of active release sites, confirmed by variance/mean analysis (Foster and Regehr, 2004). (B and C) …
Figure 2 with 1 supplement
cAMP/PKA activation does not change quantal size but increases the RRP.
(A) Representative sweeps of Sr2+-evoked asynchronous EPSCs (aEPSCs) before (black) and after (orange) application of 6-Bnz (20 µM). Bullets denote detected events. (B) Distribution of aEPSC …
Figure 2—figure supplement 1
cAMP activation does not affect short-term plasticity.
(A) Time course of EPSC amplitudes in response to CF stimulation at 100 Hz for 500 ms before (black) and after (orange) 6-Bnz treatment. (B) Normalized EPSC amplitudes during train stimulation …
Figure 3
cAMP/PKA activation is occluded when MVR is prevalent.
(A and B) Time course of CF-PC EPSC amplitude (top: normalized, open circles) and PPR (bottom: open squares) during bath application of fsk (green) or 6-Bnz (orange). Insets: representative traces …
Figure 4 with 3 supplements
PKA-inhibition shifts vesicle release mode from MVR to UVR.
(A, left) The inactive cAMP analog 8-Br-cAMPs (8-Br, brown) was used to prevent activation of PKA and KT5720 (blue), a small molecule that occupies PKA’s ATP-binding site, was used to inhibit …
Figure 4—figure supplement 1
No change in PPR using a simple depletion model.
(A, left) Paired pulse depression (PPD = (1-PPR) x 100)) plotted as a function of ΔT in control (black) and KT5720-treated (blue) slices. Both data sets were fit with two-phase exponential decays. …
Figure 4—figure supplement 2
PKA inhibition and PPR changes with extracellular Ca2+.
The PPR of CF-PC EPSCs (50 ms ISI) was highly sensitive extracellular Ca2+ reflecting changes in Pr; however, PPR was not affected by KT5720 (p=0.0001 for changes with Ca2+ and p=0.3 for PKA …
Figure 4—figure supplement 3
Inhibition of cAMP/PKA does not affect quantal content.
(A) Representative sweeps of Sr2+-evoked asynchronous EPSCs (aEPSCs) recorded from control (black) and KT5720-treated slices (blue). Bullets denote detected events. aEPSCs were recorded in ACSF …
Figure 5
PKA inhibition reduces the size of the RRP.
(A) Representative EPSCs recorded in response to CF stimulation at 50 Hz for 500 ms in control (black) and KT5720-treated (blue) slices. To relieve receptor saturation, 3 mM KYN was added in a …
Figure 6 with 1 supplement
PKA-mediated regulation of MVR is absent in synapsin TKO mice.
(A) Time course of CF-PC EPSC amplitude (top: normalized, circles) and paired pulse ratio (bottom: PPR with an inter-stimulus interval = 50 ms, squares) following bath application of 6-Bnz (orange) …
Figure 6—figure supplement 1
Synaptic release in TKO mice mimics inhibition of PKA signaling, with a smaller RRP but no change in Pr.
(A) The PPR of CF-EPSCs at multiple ISIs was unchanged in synapsin TKO mice. PPR in hets (black): 0.09 ± 0.02, 0.1 ± 0.01, 0.13 ± 0.01, 0.17 ± 0.02, 0.37 ± 0.01, 0.88 ± 0.02, 0.97 ± 0.01 and in TKOs …
Figure 7
Synapsin-mediated regulation of the RRP controls MVR independent of Pr.
(A) Cartoon and schematic of short-term plasticity model showing how synapsin controls the number of release-competent sites per active zone. Synaptic vesicles (gray) are bound by synapsin (green) …
Figure 8
Fsk increases MVR at PF-MLI synapses independent of Pr.
(A) Unitary responses following five parallel fiber stimuli (S1– S5) delivered at 50 Hz. (B left) Individual parallel fibers were identified by a sharp threshold between failures and successes (S1 …
Author response image 1
PKA inhibition reduces the glutamate transient underlying spillover response onto MLIs in mice aged P21-P28.
(A) Superimposed CF-MLI spillover EPSCs before and after bath application of γ-DGG (300 µM) or NBQX (200 nM) in control slices and after incubation with KT5720 (1 µM) or 8-Br-cAMPs (50 µM) for …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gnetic reagent (M. musculus) | wildtype; WT; control | Jackson Laboratories | RRID:IMSR_JAX:000664 | |
| Genetic reagent (M. musculus) | synapsin triple knockout, TKO | MMRC | RRID:MMRRC_041434-JAX | |
| Genetic reagent (M. musculus) | synapsin triple het; het | this paper | WT x TKO cross | |
| Peptide, recombinant protein | PKA inhibitory fragment (6-22) amide, PKi | Tocris | Cat#: 1904; CAS: 121932-06-7 | |
| Chemical compound, drug | 6-Bnz-cAMP; 6-Bnz | BioLog via Axxora | Cat#: B009; CAS: 30275-80-0 | |
| Chemical compound, drug | 8-Br-cAMPs; 8-Br | Santa Cruz | Cat#: B009; CAS: 30275-80-0 | |
| Chemical compound, drug | forskolin; fsk | HelloBio | Cat#: HB1348; CAS: 66575-29-9 | |
| Chemical compound, drug | KT5720; KT | Tocris | Cat#: 1288; CAS: 108068-98-0 | |
| Chemical compound, drug | kynurenic acid, KYN | Abcam | Cat#: ab120256; CAS: 494-27-3 | |
| Chemical compound, drug | NBQX | Abcam | Cat#: ab120045; CAS: 118876-58-7 | |
| Chemical compound, drug | Picrotoxin | Abcam | Cat#: ab120315; CAS: 124-87-8 | |
| Chemical compound, drug | QX-314 | Abcam | Cat#: ab120118; CAS: 5369-03-9 | |
| Software, algorithm | Axograph X, version 1.5.4 | AxoGraph Scientific | https://axograph.com/ | |
| Software, algorithm | Mathematica 11 | Wolfram | http://www.wolfram.com/mathematica/ | |
| Software, algorithm | pCLAMP 10 | Molecular Devices | https://www.moleculardevices.com/ | |
| Software, algorithm | Prism | Graphpad | https://www.graphpad.com/ |
Table 1
Parameters used in FD2 model.
https://doi.org/10.7554/eLife.47434.018| Symbol | Definition | |
|---|---|---|
| CaXF0 | Concentration of Ca2+-bound site XF | 0 |
| CaXD0 | Concentration of Ca2+-bound site XD | 0 |
| ΔF | Incremental increase in CaXF after a stimulus | 5 |
| ΔD | Incremental increase in CaXD after a stimulus | 0.001 |
| τF | Decay time constant of CaXF after an action potential | 0.1 sec−1 |
| τD | Decay time constant of CaXD after an action potential | 0.05 s |
| KF | Affinity of CaXF for site | 2 |
| KD | Affinity of CaXD for site | 2 |
| k0 | Baseline rate of recovery from recovery state | 0.7 sec−1 |
| kmax | Maximal rate of recovery from refractory state | 20 sec−1 |
| D | Fraction of sites that are release-ready | 1 |
| F | Facilitation probability | (0–1) |
| NT | Total number of sites | (1 - 10) |
| Pocc | Probability of site occupancy | (0–1) |
| RRP | Readily releasable pool | NT * Pocc |
| Psucc | Probability that a competent vesicle will release | F * D |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.47434.019
