Article Figures & Data
Figures
- Figure 1.
Two mechanisms are involved in short-term facilitation at MF–CA3 synapses. A, Representative examples (20 consecutive traces with overlaid average) of MF-evoked EPSC trains (50 Hz, 5 stimuli) recorded in CA3 pyramidal cells in the presence of 1.2 mm (red) and 2.5 mm (black) external calcium. B1, Summary graph of EPSC amplitude shows the facilitation of EPSCs at two different release probabilities. B2, Graph of EPSC rise time as a function of stimulus number shows the gradual decrease in EPSCs rise time in low extracellular calcium (*p < 0.05, 1st EPSC vs 5th EPSC) and a slight increase in conditions of elevated extracellular calcium (**p < 0.01, 1st EPSC vs 5th EPSC). B3, Decay τ of EPSCs was constant during trains of EPSCs in both conditions. B4, Normalized EPSC amplitude as a function of stimulus number demonstrates that short-term facilitation of EPSCs does not occur at the same rate at different release probabilities. C1, Accordingly, the PPR of two adjacent stimuli changed in opposite directions during trains of EPSCs in both conditions. C2, The CV was significantly increased during trains evoked in 1.2 mm extracellular calcium (**p < 0.01, 1st EPSC vs 4th EPSC), whereas a decrease was observed in elevated Ca2+ conditions (***p < 0.001, 1st EPSC vs 5th EPSC). D, CV2 ratios within subsequent events in the train plotted against normalized EPSC amplitudes. This analysis illustrates the changes observed in CV and PPR during trains of facilitating EPSCs, revealing the contribution of two different mechanisms to short-term facilitation.
- Figure 2.
Short-term facilitation involves two components during prolonged activity and requires an increase in intracellular calcium. A, Representative examples of MF-evoked EPSCs (10 stimuli, 20 Hz) recorded in a CA3 pyramidal cell (25 consecutive traces and the average) in 1.2 mm external Ca2+. B, Graphs of EPSC amplitude and CV as a function of stimulus number show the initial increase in CV followed by a gradual decrease during trains of facilitating EPSCs (**p < 0.01, 1st EPSC vs 10th EPSC). C, CV analysis reveals that the two distinct mechanisms are successively involved in short-term facilitation. The arrow points to the fifth stimulus, in which a change in data distribution is observed and corresponds to a shift in the facilitation mechanism. D, EPSCs recorded in 2.5 mm external Ca2+ (black traces) and in the presence of the membrane-permeant slow calcium chelator EGTA-AM (100 μm, red traces). E, Graphs of normalized EPSC amplitude and CV during the trains. Note that the gradual decrease in CV is abolished in the presence of EGTA-AM. F, CV analysis in 2.5 mm external Ca2+ and in the presence of EGTA-AM reveals a shift in the mechanism for short-term facilitation.
- Figure 3.
Mechanisms involved in short-term facilitation are of presynaptic origin. A, Two-photon Z-stack maximal projection of a CA3 pyramidal cell filled with Alexa Fluor-594 obtained after the recordings. Inset shows expanded thorny excrescence in which fast multipoint two-photon glutamate uncaging was performed (white points; total uncaging time, 1.1 ms). Scale bar, 50 μm. B, Examples of uncaging-evoked EPSCs (5 traces and their average) triggered at 20 Hz (B1) and 50 Hz (B2). Red lines indicate the uncaging pulse. C, Summary graphs of normalized uncaging-evoked EPSCs amplitude time course showing no changes in amplitude during trains of uncaging pulses at 20 Hz (C1) and 50 Hz (C2).
- Figure 4.
Increases in the number of release sites and in quantal size. A, Examples of EPSC trains recorded in 1.2 mm (red) and 2.5 mm (black) external Ca2+ used to perform nonstationary variance-mean analysis and covariance analysis. B, Amplitudes of the first (open circles) and fifth (filled circles) EPSCs as a function of repetition number in low (top) and normal (bottom) release probability conditions show the stability of the recordings. C, Nonstationary variance-mean analysis in conditions of low (red) and elevated (black) external calcium performed on the variance-mean plots obtained from trains of EPSCs for all cells. Fitting the data with a linear function yielded the quantal sizes associated with the stimulus number in conditions of low and elevated external Ca2+. Error bars indicate SD. D, Variance and covariance of EPSCs during trains as a function of stimulus number. E, Summary bar graph of quantal size measured with covariance analysis as a function of stimulus number indicates a progressive increase in quantal size in conditions of 1.2 mm external Ca2+ (*p < 0.05, EPSC1,2 vs EPSC4,5) and a constant quantal size in 2.5 mm external Ca2+. F, Representative examples of EPSCs recorded in two conditions of release during trains of 10 stimuli at 50 Hz. G, Variance-mean plots of EPSCs amplitude. The data were fitted with a compound binomial function to measure the number of release sites active in both conditions. Error bars represent SD.
- Figure 5.
Increase in cleft glutamate concentration accompanies EPSC facilitation in condition of low release probability. A, Example traces of MF-evoked EPSCs recorded in controls and in the presence of γ-DGG (0.5 and 2 mm). B, Effects of γ-DGG (0.5 and 2 mm) on EPSC amplitude and kinetics (**p < 0.01, control vs γ-DGG). Amp., Amplitude. C, Example recordings of EPSCs evoked by paired-pulse stimulation (50 Hz) before and after application of γ-DGG (0.5 mm) in ACSF containing 1.2 mm (red) and 2.5 mm (black) Ca2+. Bottom, EPSC averages normalized to the first stimulus before (dark traces) and after (light traces) application of γ-DGG in both conditions. D, Summary graphs of γ-DGG effect on the PPR in 2.5 mm (black) and 1.2 mm (red) external Ca2+ (**p < 0.01, control vs γ-DGG). E, TBOA had no effect on the PPR after application of γ-DGG in 1.2 mm external Ca2+. Ctl, Control.
- Figure 6.
Intracellular calcium stores contribute to short-term facilitation in low extracellular Ca2+. A, Representative examples of EPSCs recorded in 2.5 mm (black) and 1.2 mm (red) external Ca2+ before and after application of CPA (30 μm). B, Summary bar graph showing that addition of CPA had no significant effect on EPSC properties evoked by the first stimulus. Succ. rate, Success rate; Ampli, amplitude. C, Examples of EPSCs trains (5 stimuli at 50 Hz) recorded in 2.5 mm (black) and 1.2 mm (red) external Ca2+ in controls and in the presence of CPA. D, Summary plots showing the absence of effect on EPSC amplitude of CPA during trains evoked in 2.5 mm external Ca2+ (D1) and the decrease in short-term facilitation of EPSCs induced by the application of CPA in low calcium conditions (D2; *p < 0.05, 1st EPSC vs 5th EPSC; **p < 0.01, 1st EPSC vs 5th EPSC). E, Representative recording of EPSCs in control conditions and after the application of EGTA-AM (100 μm) and CPA (30 μm; 10 stimuli, 20 Hz). F, Summary plots showing the EPSC amplitude and the normalized EPSC amplitude as a function of stimulus number. Short-term facilitation was abolished after the application of EGTA-AM and CPA (gray trace; *p < 0.05, 1st EPSC vs 10th EPSC; **p < 0.01, 1st EPSC vs 10th EPSC).
- Figure 7.
Calcium compartmentalization in a single MF terminal revealed by simultaneous multisite recordings of calcium elevations. A, Montage of several Z-stack maximal projections obtained by two-photon microscopy of an Alexa Fluor-594-filled granule cell recorded in the whole-cell configuration. The long axonal projection forms large boutons (insets) contacting their pyramidal cell targets in the CA3 region. Hyperpolarization and depolarization of this granule cell showed a regularly spiking firing pattern typically observed in granule cells. Scale bar, 50 μm. B, Single-plane image of a large MF bouton in CA3 filled with Alexa Fluor-594 by somatic whole-cell recording. Boxed region shows the expanded bouton with the location of points where the calcium elevations were recorded in that neuron. Scale bar, 5 μm. C, Calcium elevations recorded in the MF bouton shown in B in response to 1 and 5 APs evoked at the soma by current injection. Note the absence of failures in calcium elevations. Inset shows a single AP. D, Calcium elevations recorded in the MF bouton shown in B according to their location. Traces shown are the average of four trials with 30 s intervals. Differences in calcium elevation amplitude in response to a single AP are clearly visible between the different points investigated [red asterisk indicates a trace (#18) with large calcium influx, and blue asterisk indicates a trace (#11) with weak calcium increase]. Note that, for closely placed points (#15 and #16), the calcium increase observed is nearly identical. Such double sampling of regions corresponding to the same PSF were excluded from additional analysis, but it demonstrates the precision of the imaging method.
- Figure 8.
Compartmentalized calcium elevations in large MF boutons but not in en passant terminals. A1, Calcium transients (32 traces, with overlaid average) obtained by normalizing the change in calcium-sensitive green fluorescence to the red morphological dye. A2, The SE of the traces shown in A1 was used as a measure of the variation from the mean. Elevations in SE correspond to the timing of the single AP, and the 5 APs burst and show non-uniform calcium signals across points in the bouton. B, Examples of two types of synaptic terminals found on the axons of granule cells. Morphological comparison between large MF boutons and small en passant terminals show the larger single-plane area in MF boutons. Scale bars, 5 μm. C1, Calcium elevations (14 traces, with overlaid average) obtained in an en passant terminal. Calculation of the SE between recorded points shows no increase in the deviation from the mean (C2) unlike in giant MF boutons (A1). D, Quantification of the increase in SE by comparing the peak SE observed during the baseline (Base.) to the peak of the SE measured during one AP (**p < 0.01, baseline vs 1 AP). E, Heat map of calcium elevations observed in the bouton shown in Figure 7 obtained by convoluting color-coded pixels according to calcium elevation amplitude. Color scale shows that warmer colors are associated with a larger ΔG/R ratio. Scale bar, 5 μm. F, Summary diagram depicting how compartmentalized intraterminal calcium contributes to short-term facilitation at MF–CA3 synapses, with the synchronized release of additional vesicles at a single release site followed by the recruitment of additional release sites.
