Fig. 2.
Activity dependent Ca2+ influx through presynaptic Cav1 augments SV recycling. (A) Representative recordings of PSCs from muscle M6/7 during 1 min of motoneuron stimulation at 1 Hz in control (black trace) and following Cav1 RNAi in motoneurons (blue trace) indicate increased synaptic depression with Cav1 RNAi. (B) For quantification, PSC amplitude was normalized to the first PSC and averaged over 6 control (black circles) and 6 motoneuronal Cav1-kd animals (blue triangles). Lines indicate single exponential fits, and gray shaded areas represent the SEM. Steady-state synaptic depression is reached after ∼25 stimuli and increased by 45% upon Cav1-kd. (C) Similar experiment as in B but with endocytosis blocked by dynasore (80 µM). Normalized PSC amplitudes during 1 Hz motoneuron stimulation for 2 min in control (black trace) and following Cav1 RNAi decline to identical values of steady-state synaptic depression. The time course of PSC amplitude decline is faster within Cav1-kd (see inset Ci). (D) Representation EPSC trains in response to a stimulus train of 1 s duration at 60 Hz (2 mM [Ca2+]). Cumulative EPSC charge was calculated for control (black line) and Cav1-kd by back-extrapolation of a linear fit (dotted lines) to the last 15 stimuli of the cumulative EPSC integrals to time point zero. (F) RRP size was estimated by dividing cumulative EPSC charge by the average mEPSC charge for control (black circles) and Cav1-kd (blue squares). Mean values are indicated by horizontal bars and SD by error bar. (G) Representative false color-coded snapshots of synaptopHluorin imaging from boutons on M6/7 before, during, and at different time intervals after RRP depletion with a 1 s train of 60 Hz stimulation (upper row control, n = 35 boutons from 7 animals, lower row Cav1 kd, n = 30 boutons from 6 animals). (H) Quantification as ΔF/F at increasing time intervals shows significantly reduced SV recycling with Cav1 kd (blue) compared with the control (black). Between the 1,475 and 17,775 ms intervals, fluorescence decline is fitted with single exponentials (lines). Time constant of decay is significantly increased by Cav1 kd from 4.4 to 7.8 s. (I) Representative PSCs during RRP depletion induced by 60 Hz stimulation train for 1 s, followed by single evoked responses at 11 different time points after the train (at 25, 75, 175, 475, 1,475, 4,500, 7,700, 10,275, 12,275, 15,275, and 17,775 ms). Train and first 5 PSCs after RRP depletion (Left) and whole trace (Right). (J) Normalized average recovery PSC amplitudes at different posttrain time points divided by first PSC amplitude of the train for control (black circles, n = 8) and Cav1 RNAi (blue triangles, n = 7). Data are fitted biexponentially (lines). Cav1 kd does not affect the fast recovery component significantly but significantly increases the time constant of the slow recovery component from 14 to 36 s. (K) PSC amplitudes during 60 Hz stimulation for 1 s normalized to the first PSC amplitude of the train show similar amounts of synaptic depression at the end of the stimulation train in control (black) and with Cav1 kd. (L–N) Upper panels (FM load) show representative images of FM1-43 dye taken up into recycled SVs in axon terminals on M6/7 after stimulation with high K+ (20 mM) for 3 min followed by 3 min wash in Ca2+ free saline in a control (Left) and with Cav1-kd (Right). Restimulation for 5 min in high K+ causes nearly complete unloading of labeled SVs (Lower panels) in control (Left) and with Cav1-kd (Right). (N) Quantification reveals significantly reduced dye load with Cav1-kd (blue circles) compared with the control (black circles). By contrast, activity-induced SV release (unload) is not significantly affected. **P < 0.01; ns, not significant.