Post-tetanic increase in the fast-releasing synaptic vesicle pool at the expense of the slowly releasing pool

Abstract

Post-tetanic potentiation (PTP) at the calyx of Held synapse is caused by increases not only in release probability (P(r)) but also in the readily releasable pool size estimated from a cumulative plot of excitatory post-synaptic current amplitudes (RRP(cum)), which contribute to the augmentation phase and the late phase of PTP, respectively. The vesicle pool dynamics underlying the latter has not been investigated, because PTP is abolished by presynaptic whole-cell patch clamp. We found that supplement of recombinant calmodulin to the presynaptic pipette solution rescued the increase in the RRP(cum) after high-frequency stimulation (100 Hz for 4-s duration, HFS), but not the increase in P(r). Release-competent synaptic vesicles (SVs) are heterogeneous in their releasing kinetics. To investigate post-tetanic changes of fast and slowly releasing SV pool (FRP and SRP) sizes, we estimated quantal release rates before and 40 s after HFS using the deconvolution method. After HFS, the FRP size increased by 19.1% and the SRP size decreased by 25.4%, whereas the sum of FRP and SRP sizes did not increase. Similar changes in the RRP were induced by a single long depolarizing pulse (100 ms). The post-tetanic complementary changes of FRP and SRP sizes were abolished by inhibitors of myosin II or myosin light chain kinase. The post-tetanic increase in the FRP size coupled to a decrease in the SRP size provides the first line of evidence for the idea that a slowly releasing SV can be converted to a fast releasing one.

Figure 1.

Induction of PTP using dual whole-cell patch recordings (WCR) at calyx of Held synapses with presynaptic pipette solution free of CaM (A) or supplemented with CaM (B). (A, a) A time course of EPSC amplitudes evoked by a paired AP-like pulses applied to the presynaptic terminal every 10 s. Open circles and gray crosses are the first and the second EPSC amplitudes to paired pulses (inter-stimulus interval, 20 ms), respectively. Blue and red triangles indicate the time points when HFS (a train of AP-like ramp pulses at 100 Hz for 4 s) and a short train of 20 pulses at 100 Hz were applied. (A, b) EPSCs recorded at black and gray filled circles in A (a) are superimposed. (A, c) First 10 EPSCs evoked by HFS (blue) and a short train of 20 pulses at 100 Hz (red) are superimposed. (A, d) Plots of cumulative (cum.) EPSC amplitude evoked by HFS (blue circles) and a short train of pulses (red circles) shown in A (c). The RRP size was estimated at the y intercept of the back extrapolation line fitted to the last five points. Release probability (P_r) was estimated from the first EPSC amplitude divided by the estimate of the RRP size (RRP_cum). (B, a–d) The same experiment as in A (a–d) at a different synapse except supplement of 3 µM recombinant CaM in the presynaptic pipette solution. The symbols and scheme are the same as in A (a–d). (C–E) Mean values for PTP (C), increments in the RRP size (D), and P_r (E) measured at 20 s after HFS under the following conditions: dual whole-cell patch recordings without (WCR and CaM−) or with supplement of 3 µM CaM in the presynaptic pipette solution (WCR and CaM+) and afferent axon fiber stimulation (AFS and TPP−) in control or in the presence of 2 µM tetraphenylphosphonium (TPP) in the bath solution (AFS and TPP+). (F) Average paired-pulse ratio (PPR; inter-stimulus interval, ISI = 20 ms) before HFS (Ctrl) and at the peak of PTP (PTP). Mean ± SEM; *, P < 0.05; **, P < 0.01 (unpaired t test for C–E, paired t test for F).

Figure 2.

Post-tetanic increase in the FRP size at the expense of the SRP size. (A) Simultaneous recordings of presynaptic Ca²⁺ current (I_Ca,pre; top), EPSC (middle), and cumulative quantal release (bottom) are shown in a chronological order: 40 s before HFS (a train of AP-like pulses at 100 Hz for 4 s) and 40 s and 120 s after HFS. I_Ca,pre was evoked by a long depolarization pulse to 0 mV for 50 ms preceded by a predepolarization to +70 mV for 2 ms to ensure fast activation of presynaptic Ca²⁺ current. The second column shows EPSC (bottom) in response to HFS, a train of AP-like ramp pulses for 4 s at 100 Hz applied to presynaptic terminal (top). B and C show presynaptic Ca²⁺ currents (B, top), EPSCs (B, bottom), and cumulative quantal release (C). Each panel illustrates three superimposed traces recorded before HFS (blue solid line) and 40 s (red solid line) and 120 s (broken line) after HFS in the same synapse as in A. (C, inset) Cumulative quantal release on an expanded time scale. (D–K) Bar graphs illustrate statistical mean of relative values before (ctrl) and after HFS (40 s and 120 s) normalized to the control value measured before HFS. (D) EPSC amplitude; (E) decaying time constant of EPSC; (F) amplitude of I_Ca,pre; (G) size of the FRP; (H) size of the SRP; (I) size of total RRP; (J) release time constant (τ) of the FRP; (K) release τ of the SRP. Data of individual synapses are shown as circles connected with a line superimposed on the bar graph (right ordinate). Error bar, SEM; *, P < 0.05; **, P < 0.01; n.s., not significant (P > 0.05, paired t test). (L) Plot of the relative post-tetanic FRP size as a function of the relative post-tetanic amplitude of presynaptic Ca²⁺ current (I_Ca,pre) normalized to control values measured before HFS (n = 9).

Figure 3.

(A) Ca²⁺ transients (CaTs) evoked by a train of AP-like ramp pulses at 100 Hz for 4 s (HFS). CaTs recorded at 6 synapses (green lines) and an averaged Δ[Ca²⁺]_i trace (open circles with error bars) are superimposed. Presynaptic [Ca²⁺]_i during and after HFS were measured by fluorescence imaging the presynaptic terminal loaded with 100 µM Fura-4F via a patch pipette. (B) The mean peak value of Δ[Ca²⁺]_i evoked by HFS measured using dual WCR (3.04 ± 0.27 µM, n = 6) is compared with that evoked by AFS at 100 Hz for 4 s (2.77 ± 0.63 µM, n = 5). (C) CaTs are the same as in A but on expanded scales of time and Δ[Ca²⁺]_i. (D) Mean values for resting [Ca²⁺]_i before and 40 s after HFS (39.5 ± 16.4 nM and 47.8 ± 15.3 nM, respectively, n = 6, P = 0.25, paired t test). The black arrow in C indicates the time point of 40 s after HFS. Mean ± SEM; n.s., not significant (P > 0.05, paired t test).

Figure 4.

MLCK inhibitor peptide-18 (MLCKip) suppresses the post-tetanic changes in FRP and SRP sizes. (A and B) Presynaptic Ca²⁺ current (A, top), EPSCs (A, bottom) and cumulative quantal release (B). Three traces are superimposed in each panel: recordings at 40 s before (blue solid line) and at 40 s (red solid line) and 120 s (broken line) after HFS. (C–H) Bar graphs illustrate statistical mean of relative values at different time points normalized to the control value measured before HFS. Data from an individual synapse are superimposed on the bar graph (open circles, right ordinate). Error bar, SEM; n.s., not significant (P > 0.05, paired t test).

Figure 5.

Summary for I_Ca,pre amplitude and SV pool sizes before and after HFS (40 s) under different presynaptic conditions. (A) Baseline amplitudes of I_Ca,pre and RRP sizes estimated before HFS are compared between different presynaptic conditions: addition of DMSO (DMSO), 3 µM CaM (CaM), CaM plus 100 µM blebbistatin (Bleb), CaM plus 10 µM MLCK inhibitor peptide-18 (Mip) or CaM plus 3 µM wortmannin (WMN) to the presynaptic patch pipette. Mean values for the amplitude of I_Ca,pre (a), the FRP size (b), the SRP size (c), and the total RRP size (d) are shown as bar graphs. (B) Ratios of the same parameters as shown in A at 40 s after HFS (PTP) to those before HFS (Ctrl). Mean values for ratio of the amplitudes of I_Ca,pre (a), the FRP size (b), the SRP size (c), and the total RRP size (d) are shown. Mean ± SEM, * P < 0.05; ** P < 0.01 (unpaired t test).

Figure 6.

A transient increase in the FRP size can be induced by a single conditioning pulse. (A) Experimental protocol is the same as in Fig. 2 except that a train of AP-like pulses were replaced with a single depolarization pulse to 0 mV for 100 ms duration (conditioning pulse, second column). Presynaptic Ca²⁺ current (I_Ca,pre, top), EPSC (middle), and cumulative release (bottom) are shown in a sequential order. (B and C) Presynaptic Ca²⁺ current (B, top), EPSCs (B, bottom), and cumulative quantal release (C). Each panel shows three superimposed traces recorded at the same synapse as in A at the 40 s before (blue solid line) and 40 s (red solid line) and 120 s (broken line) after the conditioning pulse. (C, inset) A trace for the cumulative release on an expanded time scale. (D–I) Statistical mean of relative values before (ctrl) and after HFS (40 s and 120 s) normalized to the control value measured before HFS. (D) EPSC amplitude; (E) decaying time constant of EPSC; (F) size of the FRP; (G) size of the SRP; (H) the amplitude of I_Ca,pre; (I) size of the total RRP. Data of individual synapses are shown as circles connected with a line (right ordinate). Error bar, SEM; *, P < 0.05; **, P < 0.01; n.s., not significant (P > 0.05, paired t test). (J) Relative FRP sizes as a function of relative amplitudes of I_Ca,pre at 40 s after conditioning (n = 6). Each point of data was normalized to the value measured before applying a conditioning pulse.

Figure 7.

The effects of MLCK inhibitors on the baseline synaptic transmission. (A, a and b) Representative EPSCs evoked by a paired pulse stimulation (ISI, 20 ms) of afferent fibers at 0.1 Hz in the presence of 1 mM kynurenate. EPSCs recorded under control conditions (black) and in the presence of ML-9 (20 µM, A, a, red) or ML-7 (20 µM, A, b, blue) are superimposed. (A, c) Percentage change in baseline amplitude of EPSC on application of ML-9 (n = 4) or ML7 (n = 5). (B, a, and C, a) Train of EPSCs (30 pulses at 100 Hz) under control conditions (black) and after bath application of ML9 (B, a, red) or ML-7 (C, a, blue) are superimposed. Inset, enlarged last six EPSCs. Summary graph for the peak amplitude of first EPSC (B, b, and C, b) and for the mean peak amplitude of last 6 EPSCs (B, c, and C, c) under control conditions (ctrl) and in the presence of ML-9 (B) or ML-7 (C). (D, a) EPSCs were evoked by presynaptic step depolarization to 0 mV for 50 ms duration preceded by +70 mV for 2-ms predepolarizing pulse using presynaptic patch pipette containing CaM only (3 µM, black), CaM plus wortmannin (5 µM, red), or CaM plus ML7 (20 µM, blue). EPSCs obtained from different synapses under each intracellular condition were averaged, and the averaged traces are superimposed with error bars (light colored). (D, b and c) Summary graphs for mean sizes of FRP (D, b) and SRP (D, c) estimated from deconvolution analysis of EPSCs evoked by a 50-ms depolarizing pulse with CaM only (CaM), CaM plus wortmannin (WMN), or CaM plus ML-7 (ML7) included in the presynaptic pipette. (D, d) Presynaptic calcium currents (top) and concomitant EPSCs (bottom) evoked by presynaptic depolarization to 0 mV for 1, 2, and 3 ms in duration. Traces under control conditions (black) and those recorded at 5 min after bath application of 5 µM wortmannin are superimposed (red). Presynaptic patch pipette contained 3 µM CaM.

Figure 8.

Conceptual diagram for the post-tetanic transient increase in the FRP size in a calyx of Held. The diagram shows a schematic model for an active zone in a calyx of Held (red box, inset), which consists of a cluster of calcium channels at the center and four docked SVs (gray spheres) with variable distance from the calcium source (Meinrenken et al. 2002). It is assumed that the proximity of a SV to the Ca²⁺ source (positional priming) is a primary determinant of release kinetics of the SV, and thus two docked SVs which belong to an FRP reside inside the calcium microdomain (red dome), while the rest (two SVs in an SRP) resides outside the microdomain. Results of the present and our previous studies (Lee et al., 2008) imply that presynaptic Ca²⁺ elevation in the presence of CaM during tetanic stimulation activates MLCK and then myosin II, which in turn facilitates translocation of slowly releasing SVs toward the Ca²⁺ source. The alternative possibility that MLCK may facilitate the final step of molecular priming is not illustrated.