Different effects of EGTA and BAPTA on vesicle endocytosis at the developing calyces of Held. (a) At P7–9 calyceal terminals, presynaptic Ca²⁺ currents (I_Ca, upper insets) and C_m changes were evoked by a 20-ms depolarizing command pulse (from −80 to +10 mV, onset is indicated by an arrow) in the presence of 0.5 mM EGTA (control, black), 10 mM EGTA (red) or 1 mM BAPTA (blue) in the whole-cell patch pipette. Average C_m traces from six to eight presynaptic terminals and representative I_Ca traces are shown. Gray shading indicates s.e.m. (b) C_m traces in a normalized at the peak. (c) The stimulation protocol described in a was applied to P13–14 calyces. Averaged C_m traces were obtained from 5–9 calyceal terminals. (d) C_m traces in c normalized at the peak. (e) C_m changes induced by a train of 20 depolarizing pulses (20-ms duration) at 20 Hz in the presence of 0.5 mM EGTA (control, black, n = 9), 10 mM EGTA (red, n = 8) or 10 mM BAPTA (blue, n = 5) loaded into P13–14 calyces. EGTA (10 mM) had no significant effect on exocytic ΔC_m (1.07 ± 0.10 pF, P = 0.098). (f) C_m traces in e normalized at the peak.

Ca²⁺ dependence of fast and slow endocytosis elicited by repetitive stimulation at the calyx of Held. (a) Top, C_m changes induced by 20-ms depolarizing pulses (arrows indicate onsets) repeated at 1 Hz for 20 s at P7–9 calyceal terminals preloaded with EGTA at 0.5 mM (black, control) or 10 mM (red). Bottom, averaged C_m records, shown at a longer time scale, from P7–9 calyceal terminals with 0.5 mM (n = 7) or 10 mM EGTA (n = 5). Gray shading indicates s.e.m. Black bars indicate the period of stimulation. (b) Endocytic rate during the stimulus train (fF s⁻¹, ordinate), estimated from linear regression line fit to the C_m decay 0.45–1 s after each pulse versus stimulus number (abscissa) in P7–9 calyces with 0.5 mM (black, n = 16) or 10 mM EGTA (red, n = 6). (c) C_m traces from a normalized at the peak (superimposed). (d–f) Data are presented as in a–c for P13–14 calyces (n = 5–9). Data from terminals loaded with 10 mM BAPTA (blue) are also shown. Error bars represent s.e.m.

Effects of inhibitors for CaM and CaN in fast and slow endocytosis at P7–9 calyces. (a) Top, C_m changes induced by a stimulus train (20 pulses of 20-ms depolarization at 1 Hz) in the terminal preloaded with control peptide (0.2 μM, black, P8), MLCK peptide (0.2 μM, red, P8), CBD peptide (20 μM, dark gray, P8) or MLCK peptide (0.2 μM) and 10 mM EGTA (blue, P9). Bottom, averaged C_m records, shown at a slow time scale, from P7–9 calyceal terminals (n = 5 for each). Data from terminals preloaded with 10 mM EGTA () were superimposed (dashed orange line) on data from terminals preloaded with 10 mM EGTA and MLCK peptide (blue) in the bottom right panel. Gray shading indicates s.e.m. Black bars indicate the period of stimulation. (b) Summary data for the C_m decay rate (ordinate) measured after each pulse during the 1-Hz train versus stimulus number (abscissa) in the presence of control peptide (black, n = 6), MLCK peptide (red, n = 6), CBD peptide (dark gray, n = 5) or MLCK peptide and 10 mM EGTA (blue, n = 6). Dashed line indicates data from 10 mM EGTA-loaded terminals (). (c) Averaged C_m traces shown in a normalized at the peak (superimposed). (d–f) Data are presented as in a–c for terminals loaded with 0.1% DMSO (black) or calyceal terminals loaded with FK-506 (0.1 μM, with 0.1% DMSO, red), cyclosporin A (CysA, 1 μM, with 0.1% DMSO, dark gray) or FK-506 and MLCK peptide (0.2 μM, blue) in P7–9 rats. Sample records (top panels of d) were derived from different calyceal terminals in P8 rats. Averaged C_m records (bottom panels of d) were obtained from 6–11 terminals. Error bars represent s.e.m.

No effect of inhibitors for CaM and CaN in fast and slow endocytosis at P13–14 calyces. (a) Top, sample C_m changes induced by a 1-Hz train of 20 depolarizing pulses with the pipette solution containing control peptide (30 μM, black), MLCK peptide (30 μM, red) or CBD peptide (20 μM, dark gray) at P13–14 calyces. Bottom, averaged C_m records from P13–14 calyces loaded with control peptide (black, n = 6), MLCK peptide (red, n = 8) or CBD peptide (dark gray, n = 5). Gray shading indicates s.e.m. Black bars indicate the period of stimulation. (b) Summary data for the C_m decay rate after each pulse during the 1-Hz train from presynaptic terminals loaded with control peptide (black, n = 6), MLCK peptide (red, n = 8) or CBD peptide (dark gray, n = 6). (c) Averaged C_m traces from a normalized at the peak (superimposed). (d–f) Data are presented as in a–c for calyces loaded with 0.1% DMSO (control, black), FK-506 (10 μM, with 0.1% DMSO, red) or CysA (10 μM, with 0.1% DMSO, dark gray) in P13–14 rats (n = 5–6 terminals). Error bars represent s.e.m.

Developmental decline of CaN expression at the calyx of Held presynaptic terminals. (a) CaN immunoreactivity (left, green), synaptophysin immunoreactivity (middle, red) and their overlap (right). Bottom left (P7 + CaN), background after absorbing the primary antibodies with CaN protein. (b) Densitometric measurements of CaN immunofluorescence intensity in the regions that overlapped with synaptophysin immunofluorescence signals in P7–8 and P14–15 preparations (***P < 0.001, Scheffe test) and the background intensity measured after antibody absorption in P7–P15 preparations (+CaN). Error bars represent s.e.m.

Developmental decline of GTP-independent endocytic component. (a) Top, sample C_m changes induced by a train of 20 depolarizing pulses, with low-[ATP] pipette solution containing 20 mM LiCl (control, with 1 mM GTP, black), 5 mM GTPγS (tetralithium salt, red) or 5 mM GTPγS and 0.2 μM MLCK peptide (blue) at P7–9 calyces. Each depolarizing pulse (from −80 to +10 mV for 20 ms) was preceded by a pre-pulse (stepping to +80 mV for 5 ms) to relieve Ca²⁺ currents from trimeric G protein–dependent inhibition by GTPγS. Bottom, averaged C_m records from the calyces loaded with LiCl (black, n = 6), GTPγS (red, n = 5) or GTPγS and MLCK peptide (blue, n = 5) at P7–9 calyces. Gray shading indicates s.e.m. Black bars indicate the period of stimulation. (b) Summary data for the C_m decay rate after each pulse during the 1-Hz train in the terminals loaded with LiCl (black, n = 7), GTPγS (red, n = 6) or GTPγS and MLCK peptide (blue, n = 6) in P7–9 rats. (c) Data in a normalized at the peaks after the train. (d–f) Data are presented as in a–c from P13–14 calyces (n = 5–8). Error bars represent s.e.m.