Extended Data Fig. 4: Variability of syllable-specific dLight waveforms across mice and experiments.

a) Average dLight fluorescence aligned to syllable onset after time warping (traces were warped using linear interpolation from syllable onset to syllable offset, see Methods). b) Average probability of a syllable transition occurring near a dLight peak across all experiments. Peaks in the dLight trace were identified by first computing and z-scoring the derivative of the ΔF/F0 trace, and identifying peaks as values that exceeded the 90^th (left), 95^th (middle), or 99^th (right) percentile. We then plotted the probability that a syllable transition occurred given a dopamine peak. Gray shading indicates 95% bootstrap confidence interval of the shuffle. For the 95^th percentile threshold, a syllable transition is likeliest to occur 200 ms prior to the dLight peak. However, the estimated dLight peak lags actual dopamine release by 10s of milliseconds (Extended Data Fig. 1e)³¹. c) Left: schematic showing forms of variability in dLight fluorescence measurements across experiments. dLight fluorescence assessed via photometry often exhibits baseline shifts and shifts in fluorescence scaling that can be normalized across experiments by z-scoring the fluorescence trace. Z-scoring dLight per experiment will have the effect of shifting the distribution leftward (and thus producing negative values). Top right: distribution of all syllable-associated dLight peaks across all mice and all experiments (left), and corresponding cumulative distribution (right). Bottom right: distribution of all syllable-associated dLight peaks across all mice and all experiments after z-scoring fluorescence traces from each experiment (left), and corresponding cumulative distribution (right). d) Left: assessing variability of the average dLight transient amplitudes from mouse to mouse. Shown is the average dLight amplitude aligned to syllable transitions Z-scored relative to a shuffle as in Fig. 1f. The thick black line indicates the average across all mice, and per-mouse averages are shown as thin gray lines. Right: same as left except averages across experiments are shown; the thick black line indicates the overall average, and the thin gray lines are per-experiment averages. e) Pseudo-color plots where each row depicts per-experiment average aligned to syllable onset for all experiments, grouped by mouse. Gray lines indicate boundaries between individual mice. f) Left: pseudo-color plot of all per-syllable dLight waveforms as in Fig. 1h, except shown for each mouse. The color bar on the left indicates which rows correspond to which syllables using the same sorting as Fig. 1h. Within a syllable-specific block, individual rows correspond to per-mouse average dLight waveforms (n = 518 syllable/mouse pairs). Average dLight waveforms are z-scored to a shuffle. Right: the syllable-associated peak dLight value for each row, computed from each waveform between 0–300 ms from syllable onset. g) Average dLight fluorescence aligned to syllable onset for three example syllables shown in Extended Data Fig. 4e; the thick black line indicates the mean across all experiments, with the thin grey lines indicating averages from each mouse. h) The probability of observing a syllable-specific dLight peak value across every syllable instance and across all mice and experiments. Syllable-specific peak values are computed using the maximum value in a 300 ms window after syllable onset. Here, color values indicate the likelihood of observing a specific peak dLight amplitude from trial to trial without averaging. Here, dLight is z-scored within each experiment. Cyan bars show the location of the overall average for each syllable. Rows are sorted in the same order as Fig. 1h and Extended Data Fig. 4f. i) The probability of syllable-specific average dLight peak values across experiments. Syllable-specific peak values are computed using the maximum value in a 300 ms window after syllable onset and averaged over the experiment, and thus do not correspond to the average waveform peaks in Extended Data Fig. 4e. Each row corresponds to a given syllable, and color values indicate the likelihood of observing a given peak dLight amplitude, on average, across experiments. Here, average dLight peaks are z-scored within each experiment. Cyan bars show the location of the overall average for each syllable. Rows are sorted in the same order as Fig. 1h and Extended Data Fig. 4f.