Quantal unit populations at the Drosophila larval neuromuscular junction

Focal extracellular recording at visualized boutons of the Drosophila larval neuromuscular junction was used to determine frequency and time course of the spontaneously occurring quantal events. When simultaneous intracellular recordings from the innervated muscle cell were made, more than one class of quantal event occurred at some of the individual boutons. "True" signals (arising at the bouton within the focal macropatch electrode) were often contaminated by additional signals generated outside the lumen of the focal electrode. Inclusion of these contaminating signals gave spuriously low values for relative amplitude, and spuriously high values for spontaneous quantal emission, for the synapses within the focal electrode. The contaminating signals, which appeared to be conducted along the subsynaptic reticulum surrounding the nerve terminals, generally were characterized by relatively small extracellular signals associated with normal intracellular events in the muscle fiber. From plots of simultaneous extracellular and intracellular recordings, the individual data points were classified according to the angles they subtended with the x axis (extracellular signal axis). Statistical procedures were developed to separate the true signals and contaminants with a high level of confidence. Populations of quantal events were found to be well described by Gaussian mixtures of two or three components, one of which could be characterized as the true signal population. Separation of signals from contaminants provides a basis for improving the estimates of quantal size and spontaneous frequency for the synapses sampled by the focal extracellular electrode.