Quantal content was estimated using the failures method, according to the equatio, where m is the quantal content, N is the number of stimuli in the trial and No is the number of failures. Data were averaged into 5 min bins giving 60 stimuli per bin. Concentrations of calcium and magnesium in the extracellular saline were adjusted to ensure that failure rate was sufficiently high in each trial for the number of evoked quantal currents to fit a Poisson distribution. At least 10 min was allowed for the solution to wash though the recording chamber before beginning each experiment. Trials with Methyl-Beta-cyclodextrin were performed in the 23% Ca2+ crayfish saline. Electrophysiological GSK1120212 signals were monitored on a storage oscilloscope. Signals from the intracellular and loose patch amplifiers were digitized and acquired into data files using a computerized data acquisition system equipped with data analysis and recording software. Nerve terminal signals and EJPs were signal-averaged over 30s intervals, with each signal representing the average of 6 successive responses. The number of stimuli failing to elicit quantal currents was assessed by viewing stored recordings, which were also processed automatically to detect peak values for each signal. Peak values were stored in a.txt file and imported into Microsoft Office Excel 2007 for graphical and statistical analyses. Input resistance was measured by inserting two glass microelectrodes filled with 3 M KCl into the same muscle fiber, injecting hyperpolarizing current through one microelectrode and recording voltage responses with the second. The electrodes were Reversine side effects connected to separate electrometers, at least one of which was equipped with a bridge circuit for passing current. Injected currents were 150 ms in duration and were applied at a rate of 0.1 Hz. In the same trials, L-glutamate was applied to the muscle fibers iontophoretically through an extracellular microelectrode filled with 1 M L-glutamate. Iontophoretic current was applied through a Cyot721 Electrometer using a pulse duration of 50 ms. To estimate cord resistance, current was injected into the muscle fibers 450 ms before each iontophoretic application of Lglutamate. In a separate set of trials, slope resistance was estimated by injecting a series of hyperpolarizing currents of varying amplitude, recording voltage responses and estimating the slope of voltage vs. current plots. Signals were acquired using the computerized data acquisition system, and signals were processed automatically to identify the amplitude of the glutamate response as well as the amplitude of the response to hyperpolarizing current. M?CD and hydroxypropyl-beta-cyclodextrin were stored in powder form at room temperature and were dissolved in crayfish saline to a final concentration of 10 mM in each case. Cholesterol loaded HP?CD was prepared as described previously.