Presynaptic Rac1 controls synaptic strength through the regulation of synaptic vesicle priming
- Department of Anatomy and Cell Biology, University of Iowa, United States
- Department of Human Medicine, Carl-von-Ossietzky University Oldenburg, Germany
- Research Center Neurosensory Science, Carl-von-Ossietzky University Oldenburg, Germany
- Electron Microscopy Core Facility, Max Planck Florida Institute for Neuroscience, United States
- Department of Molecular Neurobiology, Max Planck Institute for Multidisciplinary Sciences, Germany
- Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, Germany
- Department of Otolaryngology, Iowa Neuroscience Institute, University of Iowa, United States
Figures
Figure 1
Loss of presynaptic Rac1 after hearing onset does not affect calyx of Held gross morphology or ultrastructure.
(A) Cre recombinase-expressing HdAds were injected into the cochlear nucleus of Rac1fl/fl mice at P14, yielding Rac1−/− calyces of Held. All experiments were performed at around four weeks of age. …
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Figure 1—source data 1
Excel file containing the data shown in Figure 1 and the results of statistical analysis.
- https://cdn.elifesciences.org/articles/81505/elife-81505-fig1-data1-v2.xlsx
Figure 2
Presynaptic Rac1 regulates synaptic vesicles release probability and synaptic strength.
Synaptic transmission at the calyx of Held – MNTB synapse was studied using different stimulation frequencies at P28 after deletion of Rac1 at P14. (A1, B1) Representative evoked EPSCs for Rac1+/+ …
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Figure 2—source data 1
Excel file containing the data shown in Figure 2 and the results of statistical analysis.
- https://cdn.elifesciences.org/articles/81505/elife-81505-fig2-data1-v2.xlsx
Figure 3
Presynaptic loss of Rac1 increases calcium-independent neurotransmitter release.
(A) Representative recordings of mEPSCs for Rac1+/+ (left, black) and Rac1−/− (right, orange). (B1–B4) Rac1 deletion increased mEPSC frequency but did not affect mEPSC amplitude, rise time, or full …
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Figure 3—source data 1
Excel file containing the data shown in Figure 3 and the results of statistical analysis.
- https://cdn.elifesciences.org/articles/81505/elife-81505-fig3-data1-v2.xlsx
Figure 4
Presynaptic loss of Rac1 decreases SV synchronicity and prolongs EPSC onset at high-frequency stimulation.
(A) Experiments were performed at low (50 Hz, A1) and high (500 Hz, A2) stimulation frequencies. Representative recordings of first (EPSC1) and last (EPSC50) EPSC in the stimulus train. Traces are …
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Figure 4—source data 1
Excel file containing the data shown in Figure 4 and the results of statistical analysis.
- https://cdn.elifesciences.org/articles/81505/elife-81505-fig4-data1-v2.xlsx
Figure 5
Loss of presynaptic Rac1 facilitates synaptic vesicle recovery.
Recovery of single EPSC (EPSCtest) and RRP recovery was measured by two consecutive train stimuli (conditioning stimulus and recovery stimulus) at 500 Hz at varying recovery intervals. (A) Single …
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Figure 5—source data 1
Excel file containing the data shown in Figure 5 and the results of statistical analysis.
- https://cdn.elifesciences.org/articles/81505/elife-81505-fig5-data1-v2.xlsx
Figure 6 with 2 supplements
Numerical simulations of 50 and 500 Hz STP and EPSC recovery after conditioning 500 Hz trains are consistent with Rac1-loss induced changes in SV priming.
Experimental observations were equally well reproduced by either of two kinetic schemes of SV priming and fusion: a single-pool model (A) or a recently proposed (Lin et al., 2022) sequential …
Figure 6—figure supplement 1
Model predictions and parameters of simple single-pool models fitted to Rac1+/+ and Rac1−/− STP and recovery data sets.
(A) Model predictions for synaptic STP during regular stimulus trains consisting of 40 APs and delivered at frequencies from 0.5 to 500 Hz for Rac1+/+ (A1), (black) and Rac1−/− (A2), (orange) …
Figure 6—figure supplement 2
Model predictions and parameters of sequential two-step models fitted to Rac1+/+ and Rac1−/− STP and recovery data sets.
(A) Model predictions for synaptic STP during regular stimulus trains consisting of 40 APs and delivered at frequencies from 0.5 to 500 Hz for Rac1+/+ (A1), (black) and Rac1−/− (A2), (orange) …
Figure 7
Alterations in presynaptic release probability did not impair the reliability of postsynaptic action potential generation but decreased temporal precision during in vivo-like activity.
(A1) AP firing was recorded in response to in vivo-like stimulation patterns derived from responses to sinusoidal amplitude-modulated sounds at different modulation frequencies. Raster plot shows …
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Figure 7—source data 1
Excel file containing the data shown in Figure 7 and the results of statistical analysis.
- https://cdn.elifesciences.org/articles/81505/elife-81505-fig7-data1-v2.xlsx
Figure 8
Proposed model of Rac1’s presynaptic role in regulating synaptic transmission.
In the proposed model, loss of Rac1 results in changes in F-actin at the active zone, thereby reducing the physical barrier between SVs and the plasma membrane which increases synaptic strength …
Author response image 1
Graphical representation of the EQ fit to the average data reported in Figure 2B2.
The fitting area was determined by the steepest slope.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Rac1tm1Djk/J (Rac1flox/flox) | Jackson Laboratory; Glogauer et al., 2003 | RRID:IMSR_JAX:005550 | either sex |
| Antibody | Anti-GFP (rabbit polyclonal) | Abcam | Cat# ab6556 RRID:AB_305564 | EM (0.1 µg/mL) |
| Antibody | 6 nm colloidal Gold-AffiniPure anti-rabbit IgG (donkey polyclonal) | Jackson ImmunoResearch | Cat# 711-195-152 RRID:AB_2340609 | EM (1:100) |
| Sequence-based reagent | primer: 5’-TCC AAT CTG TGC TGC CCA TC-3' | Glogauer et al., 2003 | ||
| Sequence-based reagent | primer: 5’-GAT GCT TCT AGG GGT GAG CC-3' | Glogauer et al., 2003 | ||
| Recombinant DNA reagent | HdAd 28E4 hsyn iCre EGFP (viral vector) | Samuel M. Young, Jr., University of Iowa | ||
| Recombinant DNA reagent | HdAd 28E4 hsyn iCre mEGFP (viral vector) | Samuel M. Young, Jr., University of Iowa | ||
| Recombinant DNA reagent | HdAd 28E4 hsyn mEGFP (viral vector) | Samuel M. Young, Jr., University of Iowa | ||
| Chemical compound, drug | Kynurenic acid | Tocris Bioscience | Cat# 0223 | |
| Chemical compound, drug | Lidocaine N-ethyl bromide (QX-314) | Sigma-Aldrich | Cat# L5783 | |
| Chemical compound, drug | D-AP5 | Tocris Bioscience | Cat# 0106 | |
| Chemical compound, drug | (-)-bicuculline methochloride | Tocris Bioscience | Cat# 0131 | |
| Chemical compound, drug | Strychnine hydrochloride | Tocris Bioscience | Cat# 2785 | |
| Chemical compound, drug | Tetraethylammonium chloride | Sigma-Aldrich | Cat# T-2265 | |
| Chemical compound, drug | Tetrodotoxin | Alomone labs | Cat# T-550 | |
| Chemical compound, drug | Cadmium chloride hemi(pentahydrate) | Sigma-Aldrich | Cat# C3141 | |
| Software, algorithm | Matlab | The Mathworks | RRID:SCR_001622; v9.10 | |
| Software, algorithm | Patchmaster | HEKA; Harvard Bioscience | RRID:SCR_000034; v2x90.2 | |
| Software, algorithm | Igor Pro | Wavemetrics | RRID:SCR_000325; v6.37 | |
| Software, algorithm | Fiji | https://fiji.sc/ | RRID:SCR_002285 | |
| Software, algorithm | Patcher’s Power Tools | Max Planck Institute for Biophysical Chemistry; Gottingen; Germany | RRID:SCR_001950; v2.19 | |
| Software, algorithm | StereoDrive | Neurostar | N/A; v3.1.5 | |
| Software, algorithm | Live Acquisition | Thermo Fisher Scientific | N/A; v2.1.0.10 | |
| Software, algorithm | Neuromatic | Rothman and Silver, 2018 | RRID:SCR_004186 |
