(A) Example fluorescence image of calyx of Held presynaptic terminals labeled with anti-Syt2-cypHer. Intravesicular anti-Syt2-cypHer emits fluorescence upon excitation at 645 nm.(B) Schematic view of fluorescence changes of anti-Syt2-cypHer during exo-endocytosis. The orange dots show cypHer coupled to antibodies against the luminal domain of Syt2. The cypHer fluorescence is maximal at intravesicular pH 5.5 and almost quenched at the extracellular pH 7.4. Upon exocytosis, the fluorescence is quenched because of the exposure to the extracellular pH. During endocytosis and re-acidification, the fluorescence is de-quenched again. (C) A train of depolarizing pulses (0 mV for 50 ms following a prepulse to +70 mV for 2 ms, 10 stimuli, interstimulus interval 200 ms, Vm) was applied to elicit a Ca2+ current (ICa), and membrane capacitance (Cm) was measured during the sweep. The prepulse (+70 mV) was applied to activate Ca2+ channels maximally without causing Ca2+ influx. A sine wave (30 mV in amplitude, 1,000 Hz in frequency) was superimposed on a holding potential of -80 mV to measure membrane capacitance (Cm). (D) The top panel shows example fluorescence images showing the cypHer fluorescence image (a) before stimulation, (b) after stimulation, and (c) after recovery, shown in a pseudo-colored scale. Each image was taken at the time point shown in the bottom trace. Scale bar, 10 μm. The bottom panel shows an example of a normalized fluorescence trace of anti-Syt2-cypHer in response to a train of depolarizing pulses. The fluorescence intensity was normalized to the first point in the plot. (E) The top panel shows example traces of normalized Cm (black circles, left axis) and cypHer fluorescence (red circles, right axis) at a calyx terminal stimulated by a train of depolarizing pulses. The Cm trace was normalized to the amplitude of the capacitance jump, and the fluorecence trace was normalized to the initial intensity. The fluorescence trace was inverted to compare the time courses of Cm and fluorescence traces.The bottom panel shows average traces of normalized Cm (black circles, n = 7) and cypHer fluorescence change (red circles, n = 10) at the calyx terminal evoked by a train of depolarizing pulses (7 data were obtained from simultaneous measurements of capacitance and cypHer). Cm traces were normalized to the peak capacitance change (left axis), and fluorecence traces were normalized to the peak fluorescence change (right axis).