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. 2017 Feb 15;595(4):1263-1271.
doi: 10.1113/JP273467. Epub 2016 Nov 29.

Impact of Vesicular Glutamate Leakage on Synaptic Transmission at the Calyx of Held

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Free PMC article

Impact of Vesicular Glutamate Leakage on Synaptic Transmission at the Calyx of Held

Chihiro Takami et al. J Physiol. .
Free PMC article

Abstract

Key points: It is controversial whether glutamate can leak out of vesicles in the nerve terminal. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate or by blocking vacuolar ATPase activity using bafilomycin A1. In the absence of vesicular glutamate uptake, both spontaneous and nerve-evoked EPSCs underwent a rundown, suggesting that vesicular glutamate can leak out of vesicles. However, the rundown of evoked EPSCs was caused mainly by accumulation of unfilled vesicles after exocytic release of glutamate, suggesting a minor influence of glutamate leakage on synaptic transmission.

Abstract: Glutamate leaks out of synaptic vesicles when the transvesicular proton gradient is dissipated in isolated vesicle preparations. In the nerve terminal, however, it is controversial whether glutamate can leak out of vesicles. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate in whole-cell dialysis or by blocking vacuolar ATPase using bafilomycin A1 (Baf) at the calyx of Held in mouse brainstem slices. Presynaptic glutamate washout or Baf application reduced the mean amplitude and frequency of spontaneous miniature (m)EPSCs and the mean amplitude of EPSCs evoked every 10 min. The percentage reduction of mEPSC amplitude was much less than that of EPSC amplitude or mEPSC frequency, and tended to reach a plateau. The mean amplitude of mEPSCs after glutamate washout or Baf application remained high above the detection limit, deduced from the reduction of mEPSC amplitude by the AMPA receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione. Membrane capacitance measurements from presynaptic terminals indicated no effect of glutamate washout on exocytosis or endocytosis of synaptic vesicles. We conclude that glutamate can leak out of vesicles unless it is continuously taken up from presynaptic cytosol. However, the magnitude of glutamate leakage was small and had only a minor effect on synaptic responses. In contrast, prominent rundowns of EPSC amplitude and mEPSC frequency observed after glutamate washout or Baf application are likely to be caused by accumulation of unfilled vesicles in presynaptic terminals retrieved after spontaneous and evoked glutamate release.

Keywords: calyx of held; glutamate; presynaptic terminal; synaptic transmission.

Figures

Figure 1
Figure 1. Rundowns of EPSCs and miniature (m)EPSCs after whole‐cell washout of cytosolic glutamate in the presynaptic terminal
A, sample traces of EPSCs and mEPSCs, before and 10–30 min after glutamate washout (lower traces, superimposed), and controls with 3 mm glutamate in presynaptic pipettes (upper traces). B, mean amplitudes of EPSCs (triangles) and mEPSCs (circles) at different time periods after glutamate washout (filled symbols) or controls with 3 mm glutamate (open symbols). Each data point was derived from five experiments and normalized to the mean amplitudes at time 0 immediately after rupturing presynaptic membrane (ordinate). Error bars indicate ±SEMs in this and the following figures. Exponential curves were best fitted to data points with an equation of I=I0+(1I0)etτ, where the time constant (τ) and I 0 were 22 min and 0, respectively, for EPSCs, whereas they were 9.4 min and 0.65, respectively, for mEPSCs. At time 0, mean amplitudes of evoked EPSCs were 7.5 ± 0.5 nA (3 mm Glu control, n = 5 pairs) and 7.8 ± 0.7 nA (0 mm Glu, n = 5 pairs) and those of mEPSCs were 35 ± 1.4 pA (3 mm Glu control, n = 5 pairs) and 43 ± 7.2 pA (0 mm Glu, n = 5 pairs). Glutamate concentrations ([Glu]) had significant effects on the amplitude of mEPSCs (repeated‐measures ANOVA: main effect of [Glu], F 1,8 = 5.03, P > 0.05; main effect of time, F 3,24 = 8.3, P < 0.001; [Glu] × time interaction, F 3,24 = 4.2, P < 0.05) and that of EPSCs (repeated‐measures ANOVA: main effect of [Glu], F 1,7 = 31, P < 0.001; main effect of time, F 1.4,10 = 35, P < 0.001; [Glu] × time interaction, F 1.4,10 = 18, P < 0.001). Miniature EPSC amplitudes between 0 and 30 min without glutamate in presynaptic pipettes were significantly different (Bonferroni tests, P < 0.05). The difference in rundown magnitude between controls with 3 mm glutamate and those without glutamate was statistically significant for EPSCs at 10, 20 and 30 min (Bonferroni tests, P < 0.01). C, mean frequency of mEPSCs in different time periods after glutamate washout, normalized to the mean frequency at time 0. The mean frequency of mEPSCs at time 0 was 17 ± 1.3 Hz (3 mm Glu control, n = 5 pairs) and 15 ± 4.2 Hz (0 mm Glu, n = 5 pairs). The glutamate concentration had significant effects on the frequency of mEPSCs (repeated‐measures ANOVA: main effect of [Glu], F 1,8 = 7.4, P < 0.05; main effect of time, F 3,24 = 9.7, P < 0.001; [Glu] × time interaction, F 3,24 = 5.4, P < 0.05). The statistical difference between controls and 0 mm glutamate was significant at 20 min (Bonferroni tests, P < 0.05) and 30 min (Bonferroni tests, P < 0.01). D, representative amplitude histograms of mEPSCs (open bars) in different time periods after glutamate washout. Inset traces show mEPSCs at a slow time scale in this figure and in Figs 2 D and 3E. The total number of events is 100 for each histogram. Background noise distributions (filled bars) were obtained from the baselines of records with no clear mEPSC events. Arrows indicate the mean amplitude of mEPSCs in this figure and in Figs 2 D and 3E. Gaussian curves are fitted to the mEPSC amplitude histograms using the least‐squares method. The coefficient of variation of mEPSC amplitudes was 0.47, 0.52, 0.45 and 0.40, respectively, for 0, 10, 20 and 30 min after glutamate washout.
Figure 2
Figure 2. Rundowns of EPSCs and mEPSCs after blocking glutamate uptake with bafilomycin A1 (Baf)
A, sample traces of EPSCs and mEPSCs before and 0–30 min after 100 s bath application (hatched boxes in B and C) of Baf (5 μm with 0.5% DMSO, lower traces, superimposed) or DMSO alone (controls, upper traces). Presynaptic terminals were kept intact without whole‐cell recording. B, mean amplitudes of EPSCs (triangles) and mEPSCs (circles) in different time periods after application of Baf (filled symbols) or DMSO alone (open symbols). Each data point was derived from five experiments and normalized to the amplitudes before application of Baf or DMSO. The mean amplitude of evoked EPSCs before drug application was 7.3 ± 0.8 nA (DMSO, n = 5 cells) and 7.5 ± 0.6 nA (Baf, n = 8 cells) and that of mEPSCs was 38 ± 5.5 pA (DMSO, n = 5 cells) and 38 ± 3.7 pA (Baf, n = 8 cells). Drug application had a significant effect on the amplitude of mEPSCs (repeated‐measures ANOVA: main effect of drug, F 1,11 = 6.0, P < 0.05; main effect of time, F 2,24 = 2.2, P > 0.05; [Glu] × time interaction, F 2,24 = 2.3, P > 0.05) and that of EPSCs (repeated‐measures ANOVA: main effect of drug, F 1,8 = 8.1, P < 0.05; main effect of time, F 4,32 = 16, P < 0.001; [Glu] × time interaction, F 4,32 = 13, P < 0.001). Differences in the magnitude of amplitude reduction between DMSO controls and Baf application data were statistically significant for mEPSCs at 0 min (Bonferroni tests, P < 0.05) and EPSCs at 30 min (Bonferroni tests, P < 0.01). C, mean frequency of mEPSCs in different time periods after application of Baf (filled triangles) or DMSO alone (open symbols) normalized to the initial values before drug application. The mean frequency of mEPSCs before drug application was 5.6 ± 1.4 Hz (DMSO, n = 5 cells) and 8.7 ± 2.0 Hz (Baf, n = 8 cells). Drug application had a significant effect on the frequency of mEPSCs (repeated‐measures ANOVA: main effect of drug, F 1,11 = 0.8, P > 0.05; main effect of time, F 4,44 = 13, P < 0.001; [Glu] × time interaction, F 4,44 = 8.0, P < 0.001). The mEPSC frequency was significantly reduced at 0, 10, 20 and 30 min after Baf application (Bonferroni tests, P < 0.05). D, representative amplitude histograms of mEPSCs (open bars) in different time periods after Baf application. The total number of events is 100 for each histogram. The coefficient of variation of mEPSC amplitudes was 0.28, 0.25, 0.27, 0.23 and 0.35, respectively, for before and 0, 10, 20 and 30 min after application of Baf.
Figure 3
Figure 3. Reductions in the amplitudes of EPSCs and mEPSCs by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) titration
A, sample traces of EPSCs and mEPSCs without or with CNQX at different concentrations (0.2–1.5 μm, superimposed). B, mean amplitudes of EPSCs (filled circles) and mEPSCs (open circles) plotted against CNQX concentrations. Each data point was derived from five experiments and normalized to control values without CNQX (ordinate). In the presence of 1.5 μm CNQX, the mean amplitude of evoked EPSCs was 2.1± 0.5 nA and that of mEPSCs was 19 ± 0.8 pA. C, mean frequency of mEPSCs in the presence of CNQX at different concentrations, normalized to control values. D, relative mEPSC frequency (ordinate) plotted against relative mEPSC amplitude during glutamate washout (open circles) and in the presence of CNQX at different concentrations (filled circles). E, amplitude histograms of mEPSCs recorded from a postsynaptic neuron in the presence of CNQX at different concentrations. The coefficient of variation of mEPSC amplitudes was 0.44, 0.43, 0.32 and 0.23, respectively, in the presence of 0, 0.2, 1.0 and 1.5 μm CNQX.
Figure 4
Figure 4. Exo‐endocytic membrane capacitance changes in presynaptic terminals after glutamate washout
A, presynaptic membrane capacitance changes (lower traces) induced by Ca2+ currents (upper traces) elicited by a depolarizing command pulse (10 ms, from −80 to 0 mV), with 3 mm glutamate in whole‐cell pipettes or 10 or 20 min after washing out glutamate with glutamate‐free pipette solution (middle and right traces). B, bar graphs summarize Ca2+ charges (QCa, left panel), magnitudes of exocytic capacitance changes (ΔC m, middle panel) and endocytic capacitance half‐decay time (right panel). There was no significant difference (NS) in these parameters between controls (3 mm glutamate) and glutamate washout.

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