Recovery from open channel block by acetylcholine during neuromuscular transmission in zebrafish

Abstract

At larval zebrafish neuromuscular junctions (NMJs), miniature end plate currents (mEPCs) recorded in vivo have an unusually fast time course. We used fast-flow application of acetylcholine (ACh) onto outside-out patches to mimic the effect of synaptic release onto small numbers of ACh receptor channels (AChRs). Positively charged ACh acted at hyperpolarized potentials and at millimolar concentrations as a fast ("flickering") open channel blocker of AChRs. Because of filtering, the open channel block resulted in reduced amplitude of single channel currents. Immediately after brief (1 msec) application (without significant desensitization) of millimolar ACh at hyperpolarized potentials, a slower, transient current appeared because of delayed reversal of the block. This rebound current depended on the ACh concentration and resembled in time course the mEPC. A simple kinetic model of the AChR that includes an open channel-blocking step accounted for our single channel results, as well as the experimentally observed slowing of the time course of mEPCs recorded at a hyperpolarized compared with a depolarized potential. Recovery from AChR block is a novel mechanism of synaptic transmission that may contribute in part at all NMJs.

Fig. 1.

Currents evoked in outside-out patches by fast-flow application of ACh. A–C, A series of fast-flow perfusion trials (10–15) separated by 2 sec or more were used to generate an average trace under each condition for the same patch. The top traces are recordings of the junctional current recorded from the open pipette upon rupturing the patches at the end of the experiment and indicate the period of ACh application. Current responses during application of 10 (A), 1 (B), and 0.1 (C) mm ACh to the same patch held at +50 (top traces) or −50 (bottom traces) mV. Thebottom trace in A (at 10 mmACh) demonstrates an early plateau (1), followed by a rebound phase (2). D, Summary of the extent of current blocked (n = 5) at −50 (black bars) and +50 (stippled bars) mV for patches exposed to 0.1 (left pair of bars), 1 (middle pair of bars), and 10 (right pair of bars) mm ACh. E, Summary of the rise times (20–80%) for currents evoked at +50 and −50 mV at either 0.1 (left pair of bars) or 10 (right pair of bars) mm ACh, including the onset of the plateau phase (1) and rebound phase (2) observed at −50 mV. F, Summary of the exponential time constants (τ_off) for deactivation of the currents after termination of the ACh application at +50 and −50 mV at 0.1 (left pair of bars) and 10 (right pair of bars) mmACh.

Fig. 2.

Voltage dependence of the effect of 10 mm ACh. A, Series of current responses evoked in the same patch upon exposure to 10 mm ACh at potentials of +40, +20, −10, −20, −30, −40, −50, −60, and −70 mV (from top to bottom traces; averages of 8 trials). The filled circle denotes the early plateau phase, and the open circle denotes the peak of the rebound phase. B, Plot of the current during the early plateau phase (filled circles) and peak of the rebound phase (open circles) at each potential examined for the traces shown in A. C, Summary of the difference between the peak rebound current and early plateau current (Blocked current), plotted as a percentage of the total current observed at each potential. SDs (n = 5) are indicated by the error bars.D, Same results as in C normalized for the maximal block (at −70 mV) on a logarithmic scale and plotted as a function of voltage. The solid line is the best linear fit of the foot of the curve and indicates an e-fold increase in block for a 32 mV hyperpolarization.

Fig. 3.

Open channel block by ACh. A, Thetop line indicates the 100 msec period of application of 10 mm ACh to a patch containing three AChRs. The top trace shows the current (filtered at 2 kHz) elicited during a single application at +50 mV. The baseline is indicated byc on the left; each of three consecutive open channel current levels are indicated by o. Thebottom trace shows a response by the same patch during ACh application at −50 mV. Note the much smaller current amplitude.B, In contrast, the single channel current was insensitive to voltage when 0.1 mm ACh was applied to the same patch. C, Long pulses (100 msec) of 0.1 mm ACh also evoked a voltage-independent desensitization in this patch. In this example, 45 traces were averaged. At both voltages (+50 and −50 mV), the averaged trace decayed with a time course, which was well fitted by a single exponential curve with time constants (●_d) of 43 msec at +50 mV and 64 msec at −50 mV.

Fig. 4.

Model of AChRs and predicted properties of mEPCs during open channel block. A, Model predictions of the effects of a 1 msec application of 0.1, 1, and 10 mm ACh (as indicated). The solid curves are exponential fits of the current decay after application of 0.1 and 10 mm with the indicated time constants. B, Model prediction of the voltage dependency of open channel block evoked by ACh (10 mm applied for 1 msec) at +50 and −50 mV. The solid curves are exponential fits of the current decay at either potential with the indicated time constants. Model prediction of the evolution of the kinetic states during a 1 msec application of 10 mm ACh at +50 (C) and −50 (D) mV. Each trace represents a different state: monoliganded (AC) and diliganded closed states (A₂C), the open channel (A₂O), and the blocked channel (A₃B). E, Prediction of the time course of the mEPC at −50 and +50 mV elicited by a 0.125 msec duration pulse of 10 mm ACh. The mEPCs are scaled to a similar peak. The solid curves are exponential fits of the mEPC decay at either potential with the indicated time constants. The rise times were 0.08 msec at +50 mV and 0.12 msec at −50 mV.

Fig. 5.

Properties of mEPCs recorded in vivo. A, Overlap of 25 mEPCs recorded from the same muscle fiber at +50 (top traces) and −50 (bottom traces) mV. Cumulative histograms of the 20–80% rise times (B) and monoexponential decay time constants (C) for all events recorded at +50 (n = 99) and −50 (n = 85) mV.D, Summary histogram of the values obtained in all (n = 15) recordings. The mean ± SD for the rise times (left) and decay time constants (right) at −50 (black bars) and +50 (gray bars) mV. The asterisksindicate significance at p < 0.01.

Fig. 6.

Effects of model parameters on predicted properties of the mEPC. A, Decay time constants are plotted as a function of the duration of a square pulse of 10 mm ACh. The values for the durations are incremented by 0.025 msec from 0.050 to 0.525 msec at −50 (filled symbols) and +50 (open symbols) mV. Effects of the ACh concentration on decay time constants (B), peak amplitudes (C), and charge (D) during a 0.125 msec pulse. The concentrations are 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, and 10 mm.