The readily-releasable pool dynamically regulates multivesicular release

  1. Jada H Vaden
  2. Gokulakrishna Banumurthy
  3. Eugeny S Gusarevich
  4. Linda Overstreet-Wadiche  Is a corresponding author
  5. Jacques I Wadiche  Is a corresponding author
  1. University of Alabama at Birmingham, United States
  2. Northern (Arctic) Federal University named after M.V. Lomonosov, Russian Federation
9 figures, 2 tables and 1 additional file

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, …

https://doi.org/10.7554/eLife.47434.003
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 …

https://doi.org/10.7554/eLife.47434.004
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) …

https://doi.org/10.7554/eLife.47434.005
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 …

https://doi.org/10.7554/eLife.47434.006
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 …

https://doi.org/10.7554/eLife.47434.007
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 …

https://doi.org/10.7554/eLife.47434.008
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 …

https://doi.org/10.7554/eLife.47434.009
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. …

https://doi.org/10.7554/eLife.47434.010
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 …

https://doi.org/10.7554/eLife.47434.011
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 …

https://doi.org/10.7554/eLife.47434.012
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 …

https://doi.org/10.7554/eLife.47434.013
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) …

https://doi.org/10.7554/eLife.47434.014
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 …

https://doi.org/10.7554/eLife.47434.015
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) …

https://doi.org/10.7554/eLife.47434.016
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 …

https://doi.org/10.7554/eLife.47434.017
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
DesignationSource or referenceIdentifiersAdditional
information
Gnetic reagent (M. musculus)wildtype; WT; controlJackson LaboratoriesRRID:IMSR_JAX:000664
Genetic reagent (M. musculus)synapsin triple knockout, TKOMMRCRRID:MMRRC_041434-JAX
Genetic reagent (M. musculus)synapsin triple het; hetthis paperWT x TKO cross
Peptide,
recombinant protein
PKA inhibitory fragment (6-22) amide, PKiTocrisCat#: 1904;
CAS: 121932-06-7
Chemical compound, drug6-Bnz-cAMP; 6-BnzBioLog via AxxoraCat#: B009;
CAS: 30275-80-0
Chemical compound, drug8-Br-cAMPs; 8-BrSanta CruzCat#: B009;
CAS: 30275-80-0
Chemical
compound, drug
forskolin; fskHelloBioCat#: HB1348;
CAS: 66575-29-9
Chemical compound, drugKT5720; KTTocrisCat#: 1288;
CAS: 108068-98-0
Chemical compound, drugkynurenic acid, KYNAbcamCat#: ab120256; CAS: 494-27-3
Chemical compound, drugNBQXAbcamCat#: ab120045; CAS: 118876-58-7
Chemical compound, drugPicrotoxinAbcamCat#: ab120315; CAS: 124-87-8
Chemical compound, drugQX-314AbcamCat#: ab120118; CAS: 5369-03-9
Software, algorithmAxograph X, version 1.5.4AxoGraph Scientifichttps://axograph.com/
Software, algorithmMathematica 11Wolframhttp://www.wolfram.com/mathematica/
Software, algorithmpCLAMP 10Molecular Deviceshttps://www.moleculardevices.com/
Software, algorithmPrismGraphpadhttps://www.graphpad.com/
Table 1
Parameters used in FD2 model.
https://doi.org/10.7554/eLife.47434.018
SymbolDefinition
CaXF0Concentration of Ca2+-bound site XF0
CaXD0Concentration of Ca2+-bound site XD0
ΔFIncremental increase in CaXF after a stimulus5
ΔDIncremental increase in CaXD after a stimulus0.001
τFDecay time constant of CaXF after an action potential0.1 sec−1
τDDecay time constant of CaXD after an action potential0.05 s
KFAffinity of CaXF for site2
KDAffinity of CaXD for site2
k0Baseline rate of recovery from recovery state0.7 sec−1
kmaxMaximal rate of recovery from refractory state20 sec−1
DFraction of sites that are release-ready1
FFacilitation probability(0–1)
NTTotal number of sites(1 - 10)
PoccProbability of site occupancy(0–1)
RRPReadily releasable poolNT * Pocc
PsuccProbability that a competent vesicle will releaseF * D

Additional files

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