Supplementary Figure 3: Spike identification and cluster evaluation.
From: Emergent tuning for learned vocalizations in auditory cortex
a, Top, A raw voltage trace (black) shows several spikes from different units recorded on a single channel. Middle, A transformed trace (beige) that amplifies fast voltage accelerations was passed through an amplitude threshold to locate potential spikes. Bottom, Snippets of potential spikes from the raw voltage trace were saved, measured in various ways [e.g., minimum baseline (open circle) and peak (filled) amplitudes at 3 phases of the spike; width at half-height of peaks 2 and 3], and sorted using an unsupervised clustering algorithm (WaveClus). Uniformity of spike shape within clusters and separation between clusters (when more than one unit was identified per channel; >3 per channel was rare) were verified by viewing several plots. They included b, the mean (± SD) spike waveforms (red, n = 16,447 spikes; turquoise, n = 12,329 spikes; orange, n = 8244 spikes; purple, n = 3406 spikes; green, n = 1989 spikes); c, distributions of spike properties for each cluster (i.e. minimum voltage (p2), spike width, amplitude ratios between peaks); and d, spike magnitude over time to ensure the unit was held throughout the experiment. e, Top, Signal-to-noise ratio of the same unit shown in a was quantified by the standard separation index D [difference in sample means (p2 – b) divided by the geometric mean of their standard deviations]. Bottom, Histogram of D values across the entire dataset (n = 4392 units) with an arrow showing the separation of the unit above. For the boxplot, the measure of center is the median, box limits show the 25th and 75th percentiles, and whiskers extend up to 1.5× the interquartile range beyond the quartiles. f, Top, Spike waveform variability over time for the same five units shown in b. In general, well-isolated units had p2 SDs greater than or equal to their respective baseline SDs. Bottom, Mean (± SD) ratio of spike waveform SD relative to baseline SD across the entire dataset. g, Top, Inter-spike intervals for the same unit shown in a shows few refractory period violations. Bottom, Histogram of the percent of intervals less than 1 ms per unit across the entire dataset. Some, but relatively few, very well isolated units exhibited ISIs <1 ms.
