The endocytic blocker Dynasore or Pitstop-2 inhibits slow, fast-accelerating and fast endocytosis at the calyx of Held

(A) Average traces of slow endocytic membrane capacitance changes (ΔCm) in response to a 5-ms depolarizing pulse (stepping from-70 mV to +10 mV) in the absence (control, black trace) or presence of Dynasore (100 µM, 10-60 min, brown trace) or Pitstop-2 (25 µM, 10-60 min, green trace), recorded from the calyx of Held presynaptic terminal in slices from P13-15 post-hearing mice at physiological temperature (PT, 37°C) and in 2.0 mM Ca2+ aCSF. The 4th panel from the left shows the superimposed average ΔCm traces under control, Dynasore and Pitstop-2. The rightmost bar graph shows the endocytic decay rate (calculated from the slope 0.45-5.45 s after stimulation) that was slower in the presence of Dynasore (10.3 ± 2.8 fF/s; n = 5; p = 0.004, Student’s t-test) or Pitstop-2 (10.1 ± 3.4 fF/s; n = 5; p = 0.006) than control (28.8 ± 3.7 fF/s; n = 5).

(B) Fast-accelerating endocytosis induced by a train of 20-ms depolarizing pulses (repeated 15 times at 1 Hz) in the absence (control) or presence of Dynasore or Pitstop-2. Averaged traces are shown as in (A). The 4th panel shows the endocytic rates (fF/s) calculated from the slope of Cm decay, 0.45-0.95 ms after each stimulation pulse under control, Dynasore, or Pitstop-2 (superimposed). The rightmost bar graph shows the endocytic rate averaged from stimulations #12-15 (bar in 4th panel) that was slower in the presence of Dynasore (121 ± 19 fF/s, n = 6; p = 0.0045, Student’s t-test) or Pitstop-2 (133 ± 16 fF/s, n = 5; p = 0.007) than control (251 ± 28 fF/s; n = 6), indicating significant inhibition of the fast-accelerating endocytosis by Dynasore or Pitstop-2 (also see Table S1).

(C) Fast-endocytosis (average traces) evoked by a train of 20-ms pulses (repeated 10 times at 10 Hz) in the absence (control) or presence of Dynasore or Pitstop-2. The 4th panel shows cumulative ΔCm traces (superimposed) in an expanded timescale during and immediately after the 10-Hz stimulation train. The rightmost bar graph indicates endocytic decay rates (measured 0.45-1.45 s after the 10th stimulation) in Dynasore (204 ± 25.3 fF; n = 4; p = 0.017; Student’s t-test) or in Pitstop-2 (131 ± 24.6 fF; n = 4; p = 0.004) both of which were significantly slower than control (346 ± 40.1 fF; n = 6).

All data in this figure are presented as mean ± sem and the statistical significance level was set at p < 0.05, denoted with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). The significance of effect was evaluated using one-way ANOVA and Student’s t-test, with Bonferroni-Holm method of p level correction.

Endocytic blockers enhance activity dependent rapid synaptic depression, but do not prolong the recovery from depression at the calyx.

(A1, B1) A train of 30 EPSCs were evoked at the calyx of Held by afferent fiber stimulation at 10 Hz (A1) or 100 Hz (B1) in the absence (control, black traces) or presence of Dynasore (10-60 min, brown traces) or Pitstop-2 (10-60 min, green traces) at PT (37°C) in 1.3 mM Ca2+ aCSF. Panels from left to right: left-top: sample EPSC traces; left-bottom: normalized average EPSC amplitudes at each stimulus number; right-top bar graph: steady-state depression of EPSC amplitudes (mean of EPSCs #26-30, bar in the second panel); right-bottom: normalized 1st-3rd EPSC amplitudes in expanded timescale.

(A2, B2) The recovery of EPSCs from STD in control, or in the presence of Dynasore or Pitstop-2 at the calyx of Held measured using a stimulation protocol (shown on top in A2); a train of 30 stimulations at 10 Hz (A2) or 100 Hz (B2) followed by test pulses after different time intervals (Δt: 0.02, 0.1, 0.3, 1, 3, 8, 12, and 20s). The EPSC amplitude after Δt (IΔt) relative to the first EPSC in the stimulus train (I1st) was normalized by subtracting the steady state EPSCs (Iss) to measure the recovery rates.

(A1) During 10-Hz stimulation, the steady-state depression under control (0.55 ± 0.02; n = 11) was unaltered in the presence of either Dynasore (0.48 ± 0.02; n = 12; p = 0.03, not significantly different, One-way ANOVA and Student’s t-test with Bonferroni-Holm correction) or Pitstop-2 (0.53 ± 0.02; n = 7; p = 0.4, no significant difference).

(A2) After 10-Hz stimulation, the time constant of EPSCs recovery in control (2.3 ± 0.4 s; n = 9) was unchanged in the presence of either Dynasore (1.7 ± 0.2 s; n = 8; p = 0.2, Student’s t-test) or Pitstop-2 (1.9 ± 0.3 s; n = 7; p = 0.4).

(B1) During 100-Hz stimulation, EPSCs underwent a significant depression starting at the 2nd stimulation (10 ms) in the presence of both Dynasore (0.6 ± 0.03; n =10; p < 0.001, t-test) or Pitstop-2 (0.65 ± 0.06; n = 7; p = 0.003) than control (0.91 ± 0.05; n = 11). Bar graph indicates steady-state STD magnitudes; control: (0.42 ± 0.025), Dynasore: (0.25 ± 0.02; p < 0.001), and Pitstop-2: (0.26 ± 0.03; p < 0.001).

(B2) After 100-Hz stimulation, both fast and slow recovery time constants were significantly faster in the presence of Dynasore (τfast: 0.05 ± 0.009 s, n = 8, p = 0.008 and τslow: 1.5 ± 0.2 s, p = 0.003) or Pitstop-2 (τfast: 0.04 ± 0.014 s, n = 6, p = 0.02 and τslow: 1.3 ± 0.3 s, p = 0.008) than control (τfast: 0.18 ± 0.05 s, n = 8 and τslow: 3.2 ± 0.5 s; bar graphs, Table S2).

Scaffold machinery inhibitors have no effect on endocytic membrane retrievals at the calyx of Held

The CDC42 inhibitor ML141 (10 µM, 10-60 min, cyan) or actin depolymerizer Latrunculin B (Lat-B, 10-60 min, 15 µM, red) had no effect on slow endocytosis in response to a 5-ms depolarizing pulse (A) or on fast accelerating endocytosis (B; induced by 1-Hz train of 20 ms x 15 pulses) or fast endocytosis (C; evoked by a 10-Hz train of 20 ms x 10 pulses) at PT (37°C) and in 2.0 mM Ca2+ aCSF at the post hearing-calyceal presynaptic terminals (P13-15) in slices.

(A) Averaged and superimposed ΔCm traces in response to a 5-ms pulse. The rightmost bar graph indicates the endocytic decay rate in control (28.8 ± 3.7 fF; n = 5), unchanged by ML141 (23.0 ± 5.8 fF; n = 6; p = 0.44, Student’s t-test) or Lat-B (25.6 ± 5.2 fF; n = 6; p = 0.65, Table S1).

(B) The average endocytic rate (#12-15) in control (251 ± 28 fF/s; n = 6) unaltered by ML141 (214 ± 19 fF/s; n = 5; p = 0.3, Student’s t-test) or Latrunculin-B (233 ± 48 fF/s; n = 4; p = 0.7; Table S1).

(C) Fast endocytic decay rate in the presence of ML141 (242 ± 21.5 fF; n = 4; p = 0.08, t-test) or Lat-B (350 ± 33.3 fF; n = 5; p = 0.95) was not different from control (346 ± 40.1 fF; n = 6; Table S1).

Statistical significance of all data in this figure were tested using one-way ANOVA and Student’s t-test, with Bonferroni-Holm method of p-level correction.

Scaffold machinery inhibitors strongly enhance rapid synaptic depression activity independently, without prolonging the recovery from depression at the calyx

(A1, B1) EPSCs (30x) evoked at the calyx of Held by afferent fiber stimulation at 10 Hz (A1) or 100 Hz (B1) in the absence (control, black) or presence of ML141 (10-60 min, cyan) or Lat-B (10-60 min, red) at PT and in 1.3 mM Ca2+ aCSF. Panels from left to right: like Figure 2.

(A2, B2) The recovery of EPSCs from STD in control, or in the presence of ML141 or Latrunculin-B at the calyx of Held measured at 10 Hz (A2) or 100 Hz (B2) – like figure 2.

(A1) At 10-Hz stimulation, enhancement of depression became significant starting from the 3rd stimulation (200 ms) in the presence of ML141 (0.61 ± 0.03; n = 9; p = 0.016, One-way ANOVA and Student’s t-test with Bonferroni-Holm p level correction) or Lat-B (0.55 ± 0.04; n = 7; p = 0.006) compared to control (0.74 ± 0.04, n = 11). Bar graph indicates the steady-state depression (STD) strongly enhanced from control (0.55 ± 0.02) by ML141 (0.4 ± 0.02; p < 0.001) or Lat-B: (0.36 ± 0.02; p < 0.001).

(A2) After a train of 30 stimulations at 10 Hz, the time constant of EPSCs recovery under control (2.2 ± 0.4 s; n = 9) was unchanged by ML141 (3.0 ± 0.5 s, n = 8; p = 0.21) or Latrunculin-B (3.4 ± 0.6 s, n = 6; p = 0.16; Table S2).

(B1) At 100-Hz stimulation, EPSCs showed significant enhancement of depression starting at the 2nd stimulation (10 ms) in the presence of ML141 (0.65 ± 0.05; n = 10; p < 0.001) or Lat-B (0.58 ± 0.05; n = 8; p < 0.001) from control (0.91 ± 0.05; n = 11). Bar graph indicates strong steady-state STD produced by ML141 (0.22 ± 0.02; p < 0.001) or Lat-B (0.18 ± 0.013; p < 0.001) compared to control (0.42 ± 0.025).

(B2) The time course of EPSC recovery from STD induced by a train of 30 stimulations at 100 Hz, indicating no significant change in recovery from STD time constants caused by ML141 (τfast: 0.13 ± 0.02 s, n = 8, p = 0.4 and τslow: 2.3 ± 0.3 s, n = 8, p = 0.15) or Lat-B (τfast: 0.072 ± 0.02 s, n = 8, p = 0.05 and τslow: 2.5 ± 0.4 s, p = 0.4) from control (τfast: 0.18 ± 0.05 s, n = 8 and τslow: 3.2 ± 0.5 s; Table S2). One-way ANOVA and Student’s t-test with Bonferroni-Holm p level correction was used to evaluate the statistical significance. The stimulation and recovery protocols were the same as those used in Figure 2.

Endocytic and scaffold machineries co-operate for rapid vesicle replenishment during high-frequency transmission at the calyx of Held

(A) Exponential curve fits to the time-course of synaptic depression during 100-Hz stimulation under control and in the presence of either endocytic blockers or scaffold cascade inhibitors. The control time-course was best fit to a single exponential, whereas the time-course in the presence of endocytic blockers or scaffold cascade blockers was fit best to double exponential function with fast and slow time constants.

(B) Parameters for the curve-fit, including fast and slow time constants (τfast, τslow), weighted mean time constant (τmean) and relative ratio of fast and slow components (Af/As). Similar fast time constant (τfast) in the presence of either endocytic inhibitors or scaffold cascade inhibitors suggests simultaneous operation of both endocytic and scaffold mechanisms for countering the synaptic depression. Similar slow decay time constant irrespective of the presence or absence of blockers suggests that the endocytosis and scaffold mechanisms for vesicle replenishment operates predominantly at the beginning of the high frequency stimulations.

(C) Enhancement of synaptic depression by co-application of Dynasore and Latrunculin-B (10-60 min) was like Latrunculin-B alone and stronger than Dynasore alone. Sample EPSC traces and normalized depression time courses are shown on the upper and lower left panels, respectively. The steady-state STD magnitudes are compared in bar graph; control: (0.42 ± 0.025; n = 11), Dynasore: (0.25 ± 0.02; n = 10, p < 0.001 vs control, p = 0.012 vs Lat-B, p = 0.005 vs Dyn + Lat-B), Latrunculin-B: (0.18 ± 0.013; n = 8, p < 0.001 vs control, p = 0.6 vs Dyn + Lat-B), and Dyn + Lat-B together: (0.17 ± 0.02; n = 11; p < 0.001 vs control). Significance of difference was estimated by one-way ANOVA and Student’s t-test, with Bonferroni-Holm method of p level correction.

Endocytic blockers attenuate synaptic facilitation activity dependently at hippocampal CA1 synapses, but scaffold cascade inhibitors show no effect

(A-D) A train of 30 EPSCs recorded from hippocampal CA1 pyramidal cells evoked by Schaffer collaterals stimulations at 10 Hz (A and C) or 25 Hz (B and D) in the absence (control, black) or presence of endocytic blocker Dynasore (100 μM, 10-60 min, brown) or Pitstop-2 (25 μM, 10-60 min, green) (A, B), or scaffold protein inhibitor ML141 (10 µM, 10-60 min, cyan) or Latrunculin-B (15 µM, 10-60 min, red) (C, D) at PT (37°C) and in 1.3 mM Ca2+ aCSF. Top panels show sample EPSC traces. Lower panels show average EPSC amplitudes normalized and plotted against stimulation numbers. Bar graphs show EPSCs amplitudes averaged from #26-30 events.

(A) At 10 Hz stimulation, EPSCs in control showed facilitation reaching a peak at the 7th stimulation (2.34 ± 0.3; n = 17). Thereafter, it gradually reduced. Dynasore significantly reduced the facilitation immediately before the peak at 6th stimulation (1.53 ± 0.097; n = 10; p = 0.016) compared to control (2.32 ± 0.22), but not Pitstop-2. Towards the end of stimulus train (#26-30), synaptic facilitation in control (1.85 ± 0.17) was significantly attenuated by Dynasore (0.83 ± 0.07; p < 0.001) or Pitstop-2 (1.21 ± 0.08; p = 0.003).

(B) At 25 Hz stimulation, synaptic facilitation peaked at the 12th stimulation in control (3.5 ± 0.4; n = 16), at which the facilitation was significantly attenuated by Dynasore (1.6 ± 0.11; n = 11; p < 0.001, t-test) or Pitstop-2 (2.11 ± 0.22; n = 14; p = 0.004). Also, at #26-30, synaptic facilitation in control (2.65 ± 0.3) was strongly attenuated by Dynasore (0.94 ± 0.1; p < 0.001) or Pitstop-2 (1.62 ± 0.2; p = 0.007).

(C) At 10 Hz stimulation, the peak facilitation at the 7th stimulation in control (2.34 ± 0.3; n = 17) was not significantly changed by ML141 (2.5 ±.22, n = 10; n = 0.8, Student’s t-test) or Lat-B (2.3 ± 0.23; n = 12; p = 0.9). Likewise, the facilitation at #26-30 in control (1.85 ± 0.17) was not altered by ML141 (2.03 ± 0.16; p = 0.5) or Lat-B (1.84 ± 0.1; p = 0.96).

(D) At 25 Hz stimulation, peak facilitation at 12th stimulation in control (3.5 ± 0.4; n = 16) was unchanged by ML141 (3.3 ± 0.34; n = 10; p = 0.72, t-test) or Lat-B (3.1 ± 0.4; n = 11; p = 0.42). Facilitation at #26-30 events in control (2.65 ± 0.3) was also unaltered by ML141 (2.53 ± 0.2; p = 0.8) or Lat-B (2.5 ± 0.25; p = 0.7).

The significance of difference of all data in this figure was estimated by one-way ANOVA and Student’s t-test, with Bonferroni-Holm method of p level correction.

Hypothetical vesicle replenishment scheme by endocytosis and scaffold-machineries during repetitive transmission at fast-signaling calyx and slow-plastic hippocampal CA1 synapses.

During high-frequency transmission, endocytosis driven site-clearance allows activity-dependent replenishment of new vesicles to release sites. This endocytic function counteracts synaptic depression caused by vesicle depletion, thereby maintain synaptic strength, enabling high-fidelity fast neurotransmission at sensory relay synapses, like at the calyx of Held. This endocytosis driven synaptic strengthening function augments synaptic facilitation at slow-plastic synapses like hippocampal CA1 synapses that exhibit long-term plasticity, thereby boosting its induction capability for memory formation. Whereas, the presynaptic scaffold machinery plays a powerful direct vesicle replenishment role, independent of endocytosis and activity, thereby rapidly translocating new vesicles to open release sites including those just opened by endocytic site-clearance. This scaffold function is specifically devoted to fast-signaling synapses, with high release probability but not to plastic synapses, where vesicle depletion is minimal due to low release probability.