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	Figure 1. Dependence of GCs and EPSCs on CCh. (A) Representative samples (n = 8) of GCs measured in Xenopus oocytes before (top) and 3 ms after (bottom) flash photolysis of CNB-carbachol (800 µM). “On” = GCs produced during the depolarizing pulse. The flash inhibited the GCs to 61% of control. “Off” = GCs produced upon return to the holding potential. Inset shows the pulse protocol. (B) Samples of GCs at different steady-state [CCh]o measured in the same oocyte. Inset shows the pulse protocol. [CCh]o (bar) was present throughout the experiment. (C) Normalized DIGCs curves for a depolarizing pulse of 40 (•) or 1 (○) ms with steady-state [CCh]o. In each oocyte, GCs were measured in control and with one [CCh]o (n = 3–13 for each point taken from 14 donors). (D) EPSCs recorded in NMJs of WT mice in various [CCh*]local as indicated. Inset shows pulse protocol. The flash (○) was given 1 ms before the action potential. (E) Single quanta (each trace average of 4 quanta) were recorded in control (solid line) and after a flash (dashed line) producing [CCh*]local = 12 µM. (F) EPSCs evoked in M2KO mice before (solid line) and 1 ms after the flash (dashed line) producing 12 µM [CCh*]local. (G) Data as in D presented as percentage of control EPSC (■, n = 4). “○” represents the 1-ms DIGCs curve from B. For all figures, data points represent mean ± SEM.
	Figure 2. [CCh*]local does not affect Ca2+ currents. (A) Experimental protocol. (A1) The ENTC (dashed square) and the resultant EPSC. (A2) The ENTC on a larger scale. (A3) The ENTC after addition of 100 µM Cd2+. (A4) Net Ca2+ current obtained after subtracting the current seen in A3 from the ENTC. (B–G) Bars in the bottom of each column apply to that entire column. (B and C) Representative example (n = 3) of ENTCs and EPSCs (B) and Ca2+ currents (C) measured with a full action potential (dashed line) and with 50 nM TTX (solid line; i.e., smaller action potential). (D–G) In the presence of 50 nM TTX. (D and E) Representative example (n = 5) of ENTCs and EPSCs (D) and Ca2+ currents (E) measured before (solid line) and 1 ms after (dashed line) the flash ([CCh*]local = 12 µM). [CCh*]local reduced release to 19% of control without affecting Ca2+ currents. (F and G) ENTCs and EPSCs (F) and Ca2+ currents (G) with the same flash in normal [Mg2+]o (1 mM; long dashed line) and with elevated [Mg2+]o (5 mM; short dashed line). Representative example, n = 3.
	Figure 3. Gal inhibited the GCs and reduced ACh release. (A) Samples of GCs induced by depolarizing the oocyte from −120 mV to −40 mV at different concentrations of Gal. Inset shows the pulse protocol. (B) Normalized DIGCs curves for Gal. In each oocyte, GCs were measured in control and with one concentration of Gal (n = 4–10 for each point taken from 6 donors). IC50-GCs = 0.89 ± 0.05 µM. (C) A representative experiment depicting the dose-dependent effect of Gal on the EPSC size. Washing Gal restored the control values of the EPSC. The quantum size (×), monitored throughout the experiment by measuring the size of asynchronous single quanta released more than 50 ms after depolarization, remained constant. (D) Evaluation of the quantal content (m) from m=EPSC−mEPSC− (empty bars), where EPSC− is the average EPSC and mEPSC− is the average size of a single quantum, and from m=InNN0 (full bars), where N is the number of pulses applied and N0 is the number of failures. As seen, the two methods produced similar values of m (n = 6). (E) Ca2+ currents without (solid line) and with (dashed line) 10 µM Gal (representative experiment, n = 3). (F) Superimposed average EPSCs (n = 5) recorded in M2KO mice in control (solid line) and with 5 µM Gal (dashed line). (G) DIrelease curve (■, n = 8) for Gal constructed from experiments such as those in C (IC50-release = 0.85 ± 0.07 µM) compared with the DIGCs curve of Gal (taken from B). (H) Rate of spontaneous release (in WT mice) in control (white), and with 5 µM Gal (gray; n = 5).
	Figure 4. The flash reduced release only if applied before or during the depolarizing pulse. [CCh*]local = 4 µM. [TTX]o = 0.5 µM. Experimental protocols beneath EPSCs and corresponding columns. EPSCs without (solid line) and with (dashed line) flash. (A–C) Same site, each trace is an average of five repetitions. Test pulse, −1.0 μA, 0.2 ms. Flash was applied 1 ms before (A), in the middle of (B), and 0.05 ms after (C) the test pulse. (D) Average of 14 experiments as in A–C. From left to right, 100 ± 2%, 54 ± 2%, 83 ± 5%, and 97.5 ± 4% of control (significance indicated above bars).
	Figure 5. The brief (0.1 ms) and strong (−1.0 µA) prepulse does not admit Ca2+. (A) The rate of release during a train (150 Hz, 100 s) of brief “prepulses” (dashed bar) was similar to that of spontaneous release before the train (■, compare rate before and during the train; n = 5). (B) Representative (n = 4) Ca2+ currents derived from ENTCs without (solid line) and with (dashed line) the brief prepulse preceding the ENTC. Inset shows experimental protocol. (C) Representative traces (without selection) showing evoked release (*) produced by a wider (0.2 ms, −1.0 µA) prepulse. (D) Representative (n = 4) Ca2+ currents derived from ENTCs without (solid line), with the wider prepulse (long dashed line), and with the wider prepulse followed immediately by even stronger (−2.0 µA, 0.1 ms) depolarization (short dashed line). Here and in B the prepulses were given such that they terminated 0.15 ms before the ENTC. Inset shows experimental protocols.
	Figure 6. The effect of the flash is prevented if it is preceded by the very brief (0.1 ms) and strong (−1.0 μA) prepulse. (A–D) Test pulse (−0.6 μA, 0.2 ms) EPSCs under various conditions. Recordings from the same site, each trace is an average of five repetitions. Flash was always applied 1 ms before the test pulse. (A) The test pulse is preceded by 1.2 ms (solid line) or not (dashed line) by the prepulse. (B–D) EPSCs without (solid line) and with (dashed line) a flash. (B) The test pulse alone and preceded by the flash. (C) The flash precedes the prepulse by 0.2 ms. (D) The flash follows the prepulse by 0.2 ms. (E) Average of 14 experiments as in A–D. From left to right, 100 ± 4%, 60 ± 5%, 117 ± 5%, 115 ± 5%, and 62 ± 4% of control.
	Figure 7. Initiation of depolarization-evoked release depends on the timing of the GCs. Experiments performed at 10°C (n = 4–5 for each histogram). Protocols shown on the left of each row correspond to the entire row. Test = test pulse (−0.5 μA, 0.5 ms); Pre = prepulse (−1.0 μA, 0.1 ms); Wide pre = wide prepulse (−0.2 μA, 0.9 ms). (A) In WT mice, application of the prepulse 1 ms before the test pulse increased the amount of release (A1) and caused release to initiate earlier (A2, Kolmogorov-Smirnov [KS] test, P < 0.03). Gal (insets) abolished the effect of the prepulse on release. (B) In M2KO mice the strong and brief prepulse did not affect the amount (B1) or the initiation (B2) of release (P > 0.9). (C) In WT mice, application of a wide prepulse, which causes release by itself (C1, short dashed line), increased the amount of test pulse release (C1) but did not affect initiation of release (C2, P > 0.9). Inset shows representative (n = 3) Ca2+ currents derived from ENTCs without (solid line) and with (dashed line) the wide prepulse preceding the ENTC. (D) In M2KO mice, the wide prepulse increased the amount of release (D1) and caused release to initiate earlier (D2, P = 0.008). (E) Initiation of test pulse release in WT mice without (solid line) and with (dashed line) the brief and strong prepulse and in M2KO mice without a prepulse (short dashed line) shown on the same axes.

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