Contributions of residual calcium to fast synaptic transmission

Abstract

Fast neurotransmitter release is driven by high calcium (10-100 microM) near open channels (Ca(local)), followed by a much smaller (<1 microM), longer-lasting residual calcium (Ca(res)). The most prominent component of release, phasic release, lasts several milliseconds and is thought to be triggered by Ca(local). A transient tail of release then continues over the next 20 msec at 1-10% of peak rates. This transient component of release, which we refer to as TR, is poorly understood, and there is conflicting evidence regarding the role of Ca(local) and Ca(res) in its generation. We used optical methods to monitor Ca(res) and whole-cell voltage-clamp recordings to study TR at synapses between granule cells and stellate cells in rat cerebellar slices. After stimulation the probability of release is elevated greatly, peaking at 500 microseconds and then slowly declining to prestimulus levels after tens of milliseconds. After speeding the decay of Ca(res) levels with EGTA, release is confined to a 3 msec interval, and TR is eliminated. Thus, we find that Ca(res) accounts for a transient tail of release on the millisecond time scale that helps to shape the average synaptic current and accounts for at least 20% of the synaptic charge in the 20 msec interval after stimulation. Ca(res)-dependent TR is likely to contribute significantly to fast synaptic transmission under physiological conditions, particularly during high-frequency bursts that elevate Ca(res).

Fig. 1.

EGTA-AM accelerates the decay of Ca_res and affects the EPSC. A, Peak amplitudes (filled circles) and half-decay times (open circles) of magnesium green fluorescence (ΔF/F) transients were monitored during the addition of 100 μm EGTA-AM into the bath solution (left). Time 0 indicates the onset of EGTA application (black bar). Average calcium transients before (thin trace) and after (bold trace) loading with EGTA are superimposed in theright. Each trace is the average of 20 trials.B, Left, The time course of the peak EPSC during bath exposure to 100 μm EGTA-AM. Eachpoint represents the average value of 10 consecutive peak measurements. B, Right, Superimposed average current traces are shown before (thin trace) and after (bold trace) EGTA application. The synaptic currents are normalized to their respective peak amplitude in the far right to demonstrate the acceleration of the time course of the EPSC waveform after exposure to EGTA. Calcium measurements (A) and synaptic currents (B) were obtained from two different experiments.

Fig. 2.

The quantal events underlying the evoked EPSC. A, Ten consecutive trials showing representative responses to low-intensity stimulation at 0.17 Hz. Traces have been displaced by 200 pA. B, Superimposed quantal events (153) that were used to determine the average quantal event.Inset, Amplitude histogram of the quantal events. Detection of the quantal events is described in Materials and Methods.C, The average EPSC produced by low-intensity parallel fiber stimulation (bold trace; 873 trials, 0.17 Hz). The average response (computed as described in Materials and Methods) is aligned to the peak of the average evoked quantal event (thin trace). The time scale in A and Cis the same. The scale bar corresponds to 10 pA for the average synaptic current and to 29 pA for the average quantal event.D, Normalized traces of the evoked EPSC (bold) and average quantal response (thin) are inverted and plotted on a semilogarithmic scale to compare their time courses of decay. The average quantal event is larger than the average EPSC because of failures and asynchrony.

Fig. 3.

Determination of the time course of release probability. Shown are superimposed traces of the 873 consecutive trials (B) that contribute to the average EPSC (A). Same experiment as in Figure 2. Raster plot of the latency of detected quantal events for each trial (C), corresponding latency histogram (D), and the cumulative latency histogram (E). All traces are plotted on the same time scale and aligned to the time of stimulation (arrowhead). Stimulus artifacts are blanked for clarity.

Fig. 4.

EGTA-AM eliminates late evoked quantal events.A, Comparison of magnesium green ΔF/F signals before (thin trace) and after loading with 100 μm EGTA-AM (bold trace) on a fast time scale. B, Average EPSCs in control conditions (thin trace) and after EGTA-AM (bold trace). EPSCs are normalized to the respective peak currents. C, Superimposed plots of 340 consecutive evoked trials each, recorded in control conditions and after loading with EGTA. D, Raster plot of evoked quanta. Stimulus intensity was increased from 19 to 23 μA after EGTA-AM application wash-in (black bar) to maintain an average of 0.5–1.5 quanta per trial. E, Latency histograms for control conditions and after EGTA-AM application.F, Superimposed cumulative latency histograms for control (thin line) and EGTA (thick line) data. Calcium measurements (A) and synaptic physiology (B–F) were obtained from two different experiments. All traces are on the same time scale. The relative timing of calcium influx (A) and the evoked EPSCs (B–F) was adjusted according to Sabatini and Regehr (1998). B–F are all aligned to the beginning of the stimulus (arrowhead in E). Vertical scale bars: A, 3.4%; B, 20 pA for control and 22.7 pA for EGTA; C, 100 pA;E, 0.04 events per trial.

Fig. 5.

Low external calcium reduces peak calcium levels but does not alter the time course of Ca_res or neurotransmitter release. A, Comparison of magnesium green ΔF/F signals in 2 mmCa_e (thin trace) and 1 mmCa_e (bold trace). Each trace is the average of 20 trials. B, Average EPSCs in 2 mmCa_e (thin trace) and in 1 mmCa_e (bold trace). EPSCs are normalized to their respective peak currents. C, Plots of 360 consecutive evoked trials each, recorded in 2 mmCa_e and in 1 mm Ca_e.D, Raster plot of evoked quanta. Stimulus intensity was increased from 7 to 9 μA after switching to 1 mmCa_e (indicated by the black bar) to maintain an average of 0.5–1.5 quanta per trial. E, Latency histograms for 2 mm Ca_e and 1 mmCa_e. F, Superimposed cumulative latency histograms in 2 mm Ca_e (thin line) and 1 mm Ca_e (thick line) data. Calcium measurements (A) and synaptic physiology (B–F) were obtained from two different experiments. All traces are on the same time scale as described in Figure 4. Vertical scale bars: A, 2.4%;B, 8 pA for control and 14.8 pA for low Ca_e; C, 90 pA; E, 0.03 events per trial.

Fig. 6.

The effects of calcium manipulations on the time course of release. A, Summary cumulative probability histograms in control conditions (n = 15), after loading with 100 μm EGTA-AM (n = 10), and in 1 mm Ca_e(n = 5). The dotted horizontal linecorresponds to 100% of the events in the 20 msec after the onset of release. B, Semilogarithmic plot of the normalized probability histograms, which are calculated by differentiating the corresponding cumulative probability histogram and normalizing to peak rates of release. In the bottom panel the averaged traces for the three different conditions are superimposed. Error bars represent ± SEM. Vertical scales are shown on theright of each row.

Fig. 7.

EGTA accelerates the decay of Ca_resand eliminates late evoked quantal events at 34°C. Shown is the effect of introducing EGTA into presynaptic boutons on Ca_res and synaptic physiology at 34°C. Same format as Figure 4. In all, 180 and 225 trials contributed to the analysis in control conditions and after the loading with EGTA, respectively. Vertical scale bars: A, 2.4%; B, 30 pA for control and EGTA; C, 105 pA; E, 0.12 events per trial.

Fig. 8.

Rates of quantal release in the 1 sec after a single stimulus. Semilogarithmic plot of the normalized probability of release in the 1 sec after stimulation. The continuous plot for this time interval was determined by splicing together the release rates shown in the inset. Inset, The normalized probability of release in the 50 msec after stimulation from the present study (dots; average of 15 experiments) and in the 10–1000 msec after stimulation [line; average of 28 experiments, adapted from Atluri and Regehr (1998)]. The 10–1000 msec data were scaled to align as shown.