Miniature Endplate Current Rise Times <100 mu s from Improved Dual Recordings Can be Modeled with Passive Acetylcholine Diffusion from a Synaptic Vesicle

We recorded miniature endplate currents (mEPCs) using simultaneous voltage clamp and extracellular methods, allowing correction for time course measurement errors. We obtained a 20–80% rise time (tr) of '80 ms at 22°C, shorter than any previously reported values, and tr variability (SD) with an upper limit of 25–30 ms. Extracellular electrode pressure can increase tr and its variability by 2to 3-fold. Using Monte Carlo simulations, we modeled passive acetylcholine diffusion through a vesicle fusion pore expanding radially at 25 nmzms21 (rapid, from endplate V figure appearance) or 0.275 nmzms21 (slow, from mast cell exocytosis). Simulated mEPCs obtained with rapid expansion reproduced tr and the overall shape of our experimental mEPCs, and were similar to simulated mEPCs obtained with instant acetylcholine release. We conclude that passive transmitter diffusion, coupled with rapid expansion of the fusion pore, is sufficient to explain the time course of experimentally measured synaptic currents with trs of less than 100 ms. Neurotransmitter release is a form of exocytosis, a general process of vesicle fusion and pore formation, allowing release of vesicle contents. The small size of transmitter vesicles has prevented direct measurement of pore formation and expansion at synapses, but these processes have been studied extensively for large endocrine vesicles, such as mast cell granules (1–3). These studies have raised interesting questions about whether the pore opening rate and emptying mechanisms underlying mast cell exocytosis would suffice for fast synaptic currents. Khanin and coworkers (4) used analytic methods to model passive diffusion of acetylcholine (ACh) through a cylindrical fusion pore. They assumed that the pore expanded at the slow rate seen in mast cells (,1 nmzms21, 23°C; ref. 2) and predicted that the appearance of ACh in the synaptic cleft would be too slow for miniature endplate current (mEPC) generation. They considered more rapid pore expansion unlikely and concluded that passive diffusion cannot account for ACh release. However, synaptic V figure (fused open vesicle) appearance suggests that pore opening for ACh vesicles is 1–2 orders of magnitude faster than that for mast cell granules. Using rapid freezing of frog endplates after nerve stimulation, Torri-Tarelli and coworkers (5) found that V figures appeared during a single 500-ms interval after the synaptic delay. From this and the pore’s typical size (radius up to '12 nm), the radial expansion rate must be at least 25 nmzms21 (20°C). Furthermore, the V figures did not appear to close, swell, or collapse during a length of time much longer than the duration of mEPCs. These findings led us to ask whether passive ACh diffusion through a pore expanding at 25 nmzms21 could generate mEPCs comparable with those seen experimentally. Resolution of this issue depends on accurate measurement of the mEPC rising phase. The rise time can be estimated using an extracellular (EC) microelectrode (e.g., ref. 6) or voltage clamp (VC; e.g., refs. 7 and 8), but technical artifacts can plague both methods, and present literature values vary by up to 4-fold (9). We used VC together with EC recording to identify and correct for measurement errors and obtained a 20–80% rise time (tr) of only '80 ms (22°C). To model ACh release and mEPC generation, we wrote a program (to be called MCELL) based on our earlier Monte Carlo algorithms (10, 11). Simulated mEPCs obtained with rapid pore expansion closely reproduced both our experimental mEPCs and simulation results obtained with instant ACh release. We conclude that passive diffusion coupled with rapid pore opening is indeed sufficient for efficient generation of fast synaptic currents, and we discuss possible mechanisms that could underlie rapid opening of synaptic vesicles. MATERIALS AND METHODS mEPC Recordings. Lizard (Anolis carolinensis) ribcages were dissected to expose single layers of muscle fibers. The bathing medium was a reptile Ringer’s solution (8). Individual endplates were visualized directly using Hoffman modulation optics. Temperature was held at 22°C. For VC, we built an amplifier and low capacitance headstages based on earlier designs (8). Microelectrode resistances were only 1–2 MV (3 M KCl) after beveling in a silicon carbide slurry. Good penetrations were identified by stable resting potentials of 280 to 290 mV. Such conditions were essential for low noise, wide bandwidth (23 db at '15 kHz while immersed), and fast clamp speed (20–80% time for change in holding potential, Vh). We built a model muscle cell circuit (400 kV in parallel with 5 nF) and injected known test currents (analog mEPCs generated electronically) into it across a 40-MV resistance (ion channel conductance). The model cell was then clamped to record the analog mEPCs, and accuracy was optimized by adjusting the time constant of the series resistive-capacitive (RC) network used to measure clamp current (12). Analog mEPCs were also used to select a low pass filter cutoff of 9 kHz (23 db; Kron-Heit, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: ACh, acetylcholine; mEPC, miniature endplate current; tr, 20–80% rise time; EC, extracellular; VC, voltage clamp; Vh, DCp holding potential; RC, resistive-capacitive; Ld, random walk mean step length; D, diffusion constant; h, vesicular fusion pore height; r, pore radius; DACh, D for ACh in free solution; Dp, D for ACh inside pore; DCp, the concentration across the pore; Cv, the average vesicular concentration. ¶To whom reprint requests should be addressed.