Extended Data Fig. 10: Reinforcement of the target syllable is spatiotemporally precise.

a) Top: schematic describing the hypothesis that optogenetically-evoked DA release influences syllable counts of temporally-adjacent non-targeted syllables. Bottom: weighted average of syllable counts over baseline for non-targeted syllables for the first (left) and second (right) stimulation experiments in learner mice (n = 9). Green and gray shading indicates 95% bootstrap CI for weighted average and time-shuffled data, respectively (n = 1000 shuffles, p > 0.05 for all comparisons, two-sided Mann-Whitney U test). b) Top: schematic describing the hypothesis that opto-DA reinforces similar-velocity syllables to the target. Bottom: average syllable counts over baseline for similar-velocity syllables for stimulation experiment 1 (left) and 2 (right) in learner mice. Green and gray shading indicates 95% bootstrap CI for weighted average and time-shuffled data, respectively (n = 1000 shuffles, p > 0.05 for all comparisons, two-sided Mann-Whitney U test). c) Relative usage change (in syllable counts) of syllables of varying behavioural similarity to the target syllable, with syllables grouped into 10 bins given their relative similarity to the target. Shown are per-mouse-and-bin medians. Top: learner mice. Bottom: no-opsin controls. ** Indicates a significant difference between opto-DA learners (n = 9) and control mice (n = 12) (p = 0.006, U = 103, f = 0.95 two-sided Mann-Whitney U test between median change in counts per learner mouse), all other comparisons p > 0.05. d) Left: schematic of velocity modulation experiment (see Methods). Right: mouse/experiment averages of the targeted syllable’s velocity binned by stimulation number (for velocity, p = 0.013, u = 167, f = 0.77; n = 18 up experiments and n = 12 down experiments, two-sided Mann-Whitney U test). Error bars indicate bootstrap SEM. e) Per-mouse and per-target average target syllable duration, comparing learner mice to controls. Shown is the average duration on stim trials relative to catch trials (stim – catch); no statistically significant differences in duration distributions were identified (p = .98 for both sessions; session one U = 1995 and f = .52; session two U = 1804 and f = .46; two-sided Mann-Whitney U test, n = 144 mouse/target control pairs, n = 107 mouse/target learner pairs). f) Kinematic parameters associated with each target syllable were not altered as a result of opto-DA. Top: a linear classifier (linear discriminant analysis) was trained to use syllable-associated pose dynamics (measured using the mean and variance of the 10 principal components derived from the mouse depth data, see Methods) to predict the identity of the 6 target syllables; p < .001 established via a one-sided shuffle test. Bottom: linear classifiers trained on syllable-associated pose dynamics were unable to distinguish between stimulated and catch trials of single syllables in learner mice. Blue shows classifier performance on shuffled data, and red shows classifier accuracy over repeated cross validation splits; p = 0.069 established via a one-sided shuffle test. g) Stimulation of target syllables did not result in fractionated syllables or lowered detection confidence. Top: distribution showing entropy of cross-likelihoods for syllable detection for each frame, averaged across each experiment. Cross-likelihoods are a quantitative measure of confidence in assigning a given frame of behavioural data to a particular syllable. Distributions show density of average entropy of cross-likelihoods for baseline vs. stimulation experiments; these distributions show no evidence of changes in model confidence on experiments where syllables were targeted with optogenetic stimulation, consistent with opto-DA not substantially changing the kinematics associated with any given syllable in mice that learned. Bottom: distributions show probability density across baseline vs. stimulation experiments of entropy across maximum likelihoods of every syllable. No significant differences were found between stimulation and baseline distributions (all comparisons p > .05, two-sided 2-sample Kolmogorov-Smirnov test). h) Spatial histogram of frame occupancy of the centroid of the animal across stimulation and baseline experiments. Opto-DA mice (DAT-IRES-Cre::Ai32) on the left, no-opsin controls on the right. i) Left: Jensen-Shannon Divergence (JSD) of centroid location probability distributions across mice based on locations during stimulation trials (target performance) on stimulation day and simulated stimulation trials on baseline days (n = 192 mouse/target syllable pairs, p = 0.44, U = 4262, f = 0.49, two-sided Mann-Whitney U test across opto-DA mice and no-opsin controls). Right: JSD of centroid location distributions computed over experiment-wide centroid locations for each mouse (n = 32 mice, p = 0.41, U = 114, f = 0.48, two-sided Mann-Whitney U test). j) Distribution of kinematic parameters averaged per-mouse and per-target for the target syllable on both baseline and stimulation experiments. Left: difference between stimulation and catch trials for the targeted syllable on stimulation day. Right: magnitude of kinematic parameters for all trials across baseline and stimulation experiments. No significant differences were observed between learners and controls (p > .05, two-sided Mann-Whitney U test). k) Same as right half of Extended Data Fig. 10j (for velocity and acceleration), but for all non-target syllables. No significant differences were observed between learners and controls (p > .05, two-sided Mann-Whitney U test). l) Average dLight waveform aligned to the onset of 3-second pulsed stimulation (as elicited by ChrimsonR stimulation). Gray line indicates circular shuffle. Shaded error bars indicate 95% CI. Shaded red region indicates the duration of ChrimsonR stimulation.