Calcium dependence of neurotransmitter release at a high fidelity synapse
- Carl-Ludwig-Institute for Physiology, Medical Faculty, University of Leipzig, Germany
Figures
Figure 1
Action potential-evoked synaptic release critically depends on basal intracellular Ca2+ concentration.
(A) Left: Illustration of the cellular connectivity of the cMFB to GC synapse during simultaneous pre- and postsynaptic patch-clamp recording. The presynaptic terminal was loaded with an …
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Figure 1—source data 1
Action potential-evoked synaptic release critically depends on basal intracellular Ca2+ concentration.
- https://cdn.elifesciences.org/articles/70408/elife-70408-fig1-data1-v1.xlsx
Figure 2 with 1 supplement
Ca2+ uncaging dose-response curve measured with presynaptic capacitance measurements.
(A) Illustration of the experimental setup showing the light path of the two-photon laser illumination (red line), the UV laser illumination (blue line), the electrophysiology amplifier (‘ephys.’), …
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Figure 2—source data 1
Ca2+ uncaging dose-response curve measured with presynaptic capacitance measurements.
- https://cdn.elifesciences.org/articles/70408/elife-70408-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Measurement of the UV energy profile with caged fluorescein.
(A) 3D plot of the fluorescence profile in response to UV uncaging of caged-fluorescein at different z-positions. (B) Magnification of the middle part in panel (A) over a range of 10 μm.
Figure 3 with 4 supplements
Ca2+ uncaging dose-response curve measured with deconvolution of EPSCs.
(A) Illustration of the cellular connectivity in the cerebellar cortex showing the pre- and postsynaptic compartments during paired whole-cell patch-clamp recordings and Ca2+ uncaging with …
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Figure 3—source data 1
Ca2+ uncaging dose-response curve measured with deconvolution of EPSCs.
- https://cdn.elifesciences.org/articles/70408/elife-70408-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Measuring the KD of the Ca2+ sensitive dyes.
(A) Green (OGB-5N) over red (Atto594) fluorescence ratio for different Ca2+ concentrations, measured using either a Ca2+ calibration buffered kit or by clamping the free Ca2+ using EGTA in the …
Figure 3—figure supplement 2
Comparison of brief versus long UV illumination to rule out fast Ca2+ overshoots.
(A) Post-flash Ca2+ concentration obtained from long flashes of 1 ms duration and 10% UV intensity, normalized to post-flash Ca2+ concentration obtained from brief flashes of 0.1 ms duration and …
Figure 3—figure supplement 3
Correction for the post-flash changes in the fluorescent properties of the intracellular solution.
(A) Green over red fluorescence (G/R) ratios measured in situ normalized to G/R ratios measured in cuvettes. Data represent the different solutions used throughout the study. (a–g) represent …
Figure 3—figure supplement 4
Comparison of the time constants obtained from presynaptic capacitance measurements (τCm) and analysis of postsynaptic current recordings (τdeconv).
The time constants were obtained from the initial fast component of exponential fits of the capacitance trace and the cumulative release trace obtained from the deconvolution analysis of the …
Figure 4
Presynaptic and postsynaptic measurements reveal two kinetic processes of neurotransmitter release.
(A) Example of a capacitance trace showing the two components of release observed within the first 10 ms in response to UV-flash-evoked increase in Ca2+ concentration to 24 μM. The solid magenta …
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Figure 4—source data 1
Presynaptic and postsynaptic measurements reveal two kinetic processes of neurotransmitter release.
- https://cdn.elifesciences.org/articles/70408/elife-70408-fig4-data1-v1.xlsx
Figure 5
Fast sustained release with very weak Ca2+-dependence.
(A) Examples of a capacitance trace showing a sustained component of release. (B) Plot of the number of vesicles released between 10 and 100 ms as estimated from capacitance measurements divided by …
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Figure 5—source data 1
Fast sustained release with very weak Ca2+-dependence.
- https://cdn.elifesciences.org/articles/70408/elife-70408-fig5-data1-v1.xlsx
Figure 6
Release schemes with five Ca2+ steps and fast replenishment via parallel or sequential models can explain Ca2+-dependence of release.
(A) Scheme of the chemical reactions that were implemented in the model to calulate the UV-illumination-evoked increase in the free Ca2+ concentration. The model considered Ca2+ (Ca) and Mg2+ (Mg) …
Figure 7
Ca2+ uncaging with different pre-flash Ca2+ concentrations indicates Ca2+-dependent vesicle priming.
(A) Two consecutive recordings from the same cell pair, with the same post-flash Ca2+ concentration but different pre-flash Ca2+ concentration in the presynaptic terminal. Top: postsynaptic current. …
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Figure 7—source data 1
Ca2+ uncaging with different pre-flash Ca2+ concentrations indicates Ca2+-dependent vesicle priming.
- https://cdn.elifesciences.org/articles/70408/elife-70408-fig7-data1-v1.xlsx
Author response image 1
Miniature EPSC frequency with 30 and 180 nM intracellular Ca2+ concentration.
Boxplots show median and 1st/3rd quartiles with whiskers indicating the whole data range superimposed with the data from individual mossy fiber to granule cell pairs (n = 8 and 8 for the low and …
Tables
Table 1
Parameters for weak, middle, and strong post-flash Ca2+ elevations.
| weak Ca2+ elevation | middle Ca2+ elevation | strong Ca2+ elevation | |
|---|---|---|---|
| UV illumination | |||
| Duration (ms) | 0.1 or 1 | 0.1 | 0.1 or 0.2 |
| Intensity (%) | 10–100 | 20–100 | 100 |
| Concentration in intracellular solution (mM) | |||
| ATTO 594 | 0.010 | 0.020 | 0.020 |
| Fluo 5F | 0.050 | 0 | 0 |
| OGB 5N | 0 | 0.200 | 0.200 |
| CaCl2 | 0.500 | 2.000 | 10.000 |
| DM-N | 0.500 | 2.000 | 10.000 |
| Obtained peak post-flash Ca2+ (µM) | |||
| Min | 1.1 | 2.7 | 15.7 |
| Max | 7.1 | 36.0 | 62.6 |
| Median | 2.4 | 8.8 | 25.1 |
| Simulated uncaging fraction of DMn | |||
| α | 0.08–0.5 | 0.15–0.55 | 0.14–0.25 |
Table 2
Parameters for simulations of Ca2+ release from DMN cage.
| Parameters | Values | References number / Notes | |
|---|---|---|---|
| Resting Ca2+ | [Ca2+]rest | 227*10−9 M | Measured |
| Total magnesium | [Mg2+]T | 0.5*10−3 M | Pipette concentration |
| Fluo-5F | [Fluo] | 0 or 50 *10−6 M (see Table 1) | Pipette concentration |
| KD | 0.83 *10−6 M | Delvendahl et al., 2015 | |
| koff | 249 s-1 | ibid | |
| kon | 3*108 M−1s−1 | Yasuda et al., 2004 | |
| OGB-5N | [OGB] | 0 or 200*10−6 M (see Table 1) | Pipette concentration |
| KD | 31.4*10−6 M | Measured (Figure 3—figure supplement 1A) | |
| koff | 6000 s−1 | ibid. | |
| kon | 2.5*108 M−1s−1 | DiGregorio and Vergara, 1997 | |
| ATP | [ATP] | 5 *10−3 M | Pipette concentration |
| Ca2+ binding | KD | 2*10−4 M | Meinrenken et al., 2002 |
| koff | 100 000 s−1 | ibid. | |
| kon | 5*108 M−1s−1 | ibid. | |
| Mg2+ binding | KD | 100*10−6 M | Bollmann et al., 2000; MaxC |
| koff | 1000 s−1 | ibid. | |
| kon | 1*107 M−1s−1 | ibid. | |
| Endogenous buffer | [EB] | 480 *10−6 M | Delvendahl et al., 2015 |
| KD | 32*10−6 M | ibid | |
| koff | 16 000 s−1 | ibid. | |
| kon | 5*108 M−1s−1 | ibid. | |
| Total DM nitrophen | [DMn]T | 500*10−6 – 10*10−3 M (see Table 1) | Pipette concentration |
| Ca2+ binding | KD | 6.5*10−9 M | Faas et al., 2005 |
| koff | 0.19 s−1 | ibid. | |
| kon | 2.9*107 M−1s−1 | ibid. | |
| Mg2+ binding | KD | 1.5*10−6 M | ibid. |
| koff | 0.2 s−1 | ibid. | |
| Uncaging fraction | α | See Table 1 | |
| Fast uncaging fraction | af | 0.67 | Faas et al., 2005 |
| Photoproduct 1 | [PP1] | ||
| Ca2+ binding | KD | 2.38*10−3 M | Faas et al., 2005 |
| koff | 69 000 s−1 | ibid. | |
| kon | 2.9*107 M−1s−1 | ibid. | |
| Mg2+ binding | KD | 1.5*10−6 M | ibid. |
| koff | 300 s−1 | ibid. | |
| kon | 1.3*105 M−1s−1 | ibid. | |
| Photoproduct 2 | [PP2] | ||
| Ca2+ binding | KD | 124.1*10−6 M | Ibid. |
| koff | 3600 s−1 | ibid. | |
| kon | 2.9*107 M−1s−1 | ibid. | |
| Mg2+ binding | KD | 1.5*10−6 M | ibid. |
| koff | 300 s−1 | ibid. | |
| kon | 1.3*105 M−1s−1 | ibid. |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | NaCl | Sigma-Aldrich | Cat. # S9888 | |
| Chemical compound, drug | NaHCO3 | Sigma-Aldrich | Cat. # S6297 | |
| Chemical compound, drug | Glucose | Sigma-Aldrich | Cat. # G8270 | |
| Chemical compound, drug | AP 5 | Sigma-Aldrich | Cat. # A78403 | |
| Chemical compound, drug | KCl | Sigma-Aldrich | Cat. # P9333 | |
| Chemical compound, drug | CaCl2 | Sigma-Aldrich | Cat. # C5080 | For extracellular solution |
| Chemical compound, drug | CaCl2 | Sigma-Aldrich | Cat. # 21115 | For intracellular solution |
| Chemical compound, drug | EGTA | Sigma-Aldrich | Cat. # E0396 | |
| Chemical compound, drug | NaH2PO4 | Merck | Cat. # 106342 | |
| Chemical compound, drug | Tetrodotoxin | Tocris | Cat. # 1078 | |
| Chemical compound, drug | MgCl2 | Sigma-Aldrich | Cat. # M2670 | |
| Chemical compound, drug | TEA-Cl | Sigma-Aldrich | Cat. # T2265 | |
| Chemical compound, drug | HEPES | Sigma-Aldrich | Cat. # H3375 | |
| Chemical compound, drug | NaGTP | Sigma-Aldrich | Cat. # G8877 | |
| Chemical compound, drug | Na2ATP | Sigma-Aldrich | Cat. # A2383 | |
| Chemical compound, drug | DMnitrophen | Synptic systems | Cat. # 510016 | |
| Chemical compound, drug | CsOH | Sigma-Aldrich | Cat. # C8518 | |
| Chemical compound, drug | Atto594 | ATTO-TEC | Cat. # AD 594 | |
| Chemical compound, drug | OGB1 | Thermo Fisher Scientific | Cat. # 06806 | |
| Chemical compound, drug | OGB-5N | Thermo Fisher Scientific | Cat. # 944034 | |
| Chemical compound, drug | Fluo-5F | Thermo Fisher Scientific | Cat. # F14221 | |
| Chemical compound, drug | KOH solution | Roth | Cat. # K017.1 | |
| Chemical compound, drug | Kynurenic acid | Sigma-Aldrich | Cat. # K3375 | |
| Chemical compound, drug | Cyclothiazide | Sigma-Aldrich | Cat. # C9847 | |
| Chemical compound, drug | Ca2+ Calibration Buffer Kit | Thermo Fisher Scientific | Cat. # C3008MP | |
| Chemical compound, drug | Caged fluorescein | Sigma-Aldrich | Cat. # F7103 | |
| Chemical compound, drug | Glycerol | Sigma-Aldrich | Cat. # G5516 | |
| Chemical compound, drug | Isoflourane | Baxter | Cat. # Hdg9623 | |
| Chemical compound, drug | Aqua B. Braun | Braun | Cat. # 00882479E | For extracellular solution |
| Chemical compound, drug | Sterile Water | Sigma-Aldrich | W Cat. # 3500 | For intracellular solution |
| Strain, strain background (mouse C57BL/6N) | Female, male C57BL/6N | Charles river | https://www.criver.com/ | |
| Other | Vibratome | LEICA VT 1200 | https://www.leica-microsystems.com/ | |
| Other | Femto2D laser-scanning microscope | Femtonics | https://femtonics.eu/ | |
| Other | UV laser source | Rapp OptoElectronic | https://rapp-opto.com/ | 375 nm, 200 mW |
| Other | DMZ Zeitz Puller | Zeitz | https://www.zeitz-puller.com/ | |
| Other | Borocilicate glass | Science Products | https://science-products.com/en/ | GB200F-10 With filament |
| Other | HEKA EPC10/2 amplifier | HEKA Elektronik | https://www.heka.com/ | |
| Other | Ti:Sapphire laser | MaiTai, SpectraPhysics | https://www.spectra-physics.com/ | |
| Other | Ca2+ sensitive electrode (ELIT 8041 PVC membrane) | NICO 2000 | http://www.nico2000.net/index.htm | |
| Other | Single junction silver chloride reference electrode (ELIT 001 n) | NICO 2000 | http://www.nico2000.net/index.htm | |
| Other | PH/Voltmeter | Metler toledo | https://www.mt.com/de/en/home.html | |
| Other | Osmomat 3000 | Gonotec | http://www.gonotec.com/de | |
| Other | TC-324B perfusion heat controller | Warner Instruments | https://www.warneronline.com/ | |
| Software, algorithm | MES | Femtonics | https://femtonics.eu/ | |
| Software, algorithm | Igor Pro | Wavemetrics | https://www.wavemetrics.com/ | |
| Software, algorithm | Patchmaster | HEKA Elektronik | https://www.heka.com/ | |
| Software, algorithm | Adobe illustrator | Adobe | https://www.adobe.com/products/illustrator.html | |
| Software, algorithm | Mathematica | Wolfram | https://www.wolfram.com/mathematica/ | |
| Software, algorithm | Maxchelator | Stanford University | https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/ |
Table 3
Parameters for release scheme models.
| Model1 | Model2 | Model3 | |||
|---|---|---|---|---|---|
| kon | 2.95*109 Ca2+(t) M−1 s−1 | kon,init | 5.10*108 Ca2+(t) M−1 s−1 | kon1 | 0.5 kon2 |
| kon,plug | 0.1 kon,init | kon2 | 5.10*108 Ca2+(t) M−1 s−1 | ||
| koff | 4.42*105 s−1 | koff,init | 2.55*104 s−1 | koff1 | 10 koff2 |
| koff,plug | 0.4 koff,init | koff2 | 2.55*104 s−1 | ||
| b | 0.25 | b | 0.25 | b | 0.25 |
| γ | 1.77*104 s−1 | γ | 1.77*104 s−1 | γ | 1.77*104 s−1 |
| kprim | 0.6+30*(Ca2+(t)/(KD,prim +Ca2+(t))) s−1 | kprim1 | 2.5+60*(Ca2+(t)/(KD,prim1 +Ca2+(t))) s−1 | kprim1 | 30 s−1 |
| kunprim | 0.6+30*(Ca2+Rest/(KD,prim + Ca2+Rest)) s−1 | kunprim1 | 2.5+60*(Ca2+Rest/(KD,prim1 + Ca2+Rest)) s−1 | kunprim1 | 30 s−1 |
| KD,prim | 2 µM | KD,prim1 | 2 µM | ||
| kprim2 | 100+800*(Ca2+(t)/(KD,prim2 +Ca2+(t))) s−1 | kprim2 | 0.5+30*(Ca2+(t)/(KD,prim2 +Ca2+(t))) s−1 | ||
| kunprim2 | 100+800*(Ca2+Rest/(KD,prim2 + Ca2+Rest)) s−1 | kunprim2 | 0.5+30*(Ca2+Rest/(KD,prim2 + Ca2+Rest)) s−1 | ||
| KD,prim2 | 2 µM | KD,prim2 | 2 µM |
