Extended Data Fig. 3: Role of Uva and Peri-Uva thalamus in song.
From: Holistic motor control of zebra finch song syllable sequences
a, b) Data from birds expressing eGtACR1 in Uva and implanted with optic fibers over HVC: a) Violin plots reporting Entropy of song segments with (gray) and without (white) stimulation (n = 4); two-way ANOVA testing the effect of optogenetic stimulation, interaction F(3,76) = 26.18 P < 0.001, CTRL vs. STIM, F(1,76) = 1.099 P = 0.2979). b) Violin plots reporting accuracy of song segments with (gray) and without (white) stimulation (n = 4) when using 1 s long stimulation (two-way ANOVA testing the effect of optogenetic stimulation, interaction F(3,76) = 7.91 P < 0.001, CTRL vs. STIM, F(1,76) = 0.06636 P = 0.7974). c) Data from birds expressing ChRmine in UvaHVC neurons and implanted with optic fibers over Uva. Violin plots reporting Entropy of song segments with (200 ms stimulation, gray) and without (white) stimulation (n = 3; two-way ANOVA testing the effect of optogenetic stimulation, interaction F(2,57) = 0.3931 P = 0.6768, CTRL vs. STIM, F(1,57) = 3.500 P = 0.0665). d) Schematic and sample image showing non-selective expression of AAV-ChRmine in Uva and peri-Uva thalamus, followed by thalamic song-contingent stimulation. Sample spectrogram (scale bar 200 ms, horizontal lines mark song elements) displaying motif truncation at syllable boundaries caused by thalamic light stimulation (50 ms 532 nm light pulses, red bars).Sample image illustrating ChRmine expression (red, scale bar 200 ms) in Uva (UvaHVC neurons labeled by tracer injection in HVC (green)) and peri-Uva thalamus, stimulated by light delivered through the implanted fiber optic (white dashed lines top of image). e) (left) plot showing amplitude of all the stimulated (red line) motifs, ordered by time of stimulation in the motif. (right) Plot reporting a subset of stimulated motifs’ latency to optogenetic stimulation (red circle), motif truncation (blue “x”), and restart of a motif (orange), intro notes not followed by a motif (purple), calls (grey) or continuation of the motif after a pause (green) normally not present in the unstimulated motif within 1 s following stimulation. f) Box and scatter plot reporting the probability of motif stop (okra), pause and continuation of the motif (green) or absence of syntactic perturbation (gray) after the light stimulation (thalamus stimulated birds, n = 2, filled squares; empty box plots from HVC stimulation in Fig. 1e reported for comparison; two-way ANOVA testing the difference between stimulation outcome probabilities across all experimental groups, interaction F(14,46) = 57.75 P < 0.001, stimulated subpopulation F(7,23) = 1.088 P = 0.4027, Dunnett’s post-hoc pan-HVC vs. thalamus, motif stop P = 0.9883, pause+continuation P = 0.6761, no perturbation P = 0.9728). g) Cumulative probability curves reporting the latency to song truncation in response to the light stimulation (average ±SEM of each bird’s curve; thalamus-stimulated birds (blue), HVC-stimulated birds (black) dataset from Fig. 1 compared against all experimental groups across the manuscript, 10 ms time bins, two-way ANOVA testing the difference between truncation latency distributions, interaction F(255,867) = 2.351 P < 0.001, stimulated subpopulation F(5,17) = 4.142 P = 0.0121, Tukey’s post-hoc pan-HVC vs. thalamus identifies significant (P < 0.05) differences between 60 and 140 ms time bins. (inset) Violin plots reporting the latency of motif truncation computed across all the birds (thalamus-stimulated birds (blue), HVC-stimulated birds (white) dataset from Fig. 1 compared against all experimental groups across the manuscript; one-way ANOVA testing the difference between truncation latencies, Kruskal Wallis test H(5) = 468.9, post-hoc HVC vs. thalamus P < 0.001). h) Average ±SEM latency of motif truncation in response to thalamic light stimulation across the motif (bins= 10% motif advancement). i) Violin plots reporting the latency of motif truncation upon light stimulation, per bird (thalamus stimulation (blue), HVC stimulation (white, dataset from Fig. 1); nested one-way ANOVA comparing all datasets across the manuscript, F(5.17) = 4.175 P = 0.0117, Dunnett’s post-hoc pan-HVC vs. thalamus P = 0.0028). j) plot representing the distribution of all motif truncation times in relation to the nearest syllable (or complex syllable segment) end. 0 ms indicates truncation occurring at the natural end of the syllable or complex syllable segment, as indicated in17. Thalamic stimulation results in significant truncation prevalence at syllable end, compared to pan-HVC stimulation (AVG ± SEM, pan-HVC n = 6 birds, thalamus n = 2 birds). k) plot representing the truncation latency distribution for thalamic or pan-HVC stimulation (AVG ± SEM, pan-HVC n = 6 birds, thalamus n = 2 birds). l) Average ±SEM probability of post-truncation behavior (within 1 s from truncation: no vocalization resumption (black), motif restart with any introductory note or syllable A (orange), intro notes not followed by a motif (purple), calls (grey), resumption of the motif after a pause normally not present in control motifs (green)) following thalamic light stimulation computed based on the time of stimulation through the progression of the motif (bins= 10% motif advancement). m) Box plots (5-95 percentile, 25,50,75 percentile) reporting the probability (left) and normalized probability (right) of motif restart (thalamus-stimulated birds (orange); empty box plots representing data from birds receiving HVC stimulation reported from Extended Data Fig. 1i (left) and Fig. 1i (right) respectively; one-way ANOVA comparing restart probability across groups, F(5,17) = 6.099 P = 0.0021, Dunnett’s post-hoc pan-HVC vs. thalamus P = 0.0011; one-way ANOVA, testing the difference between groups’ normalized restart probabilities F(5,17) = 9.939 P < 0.0001, Dunnett’s post-hoc HVC vs. thalamus: P < 0.001). The underlying shaded areas represent the probability, for each of the birds, of producing a motif after any one motif (see methods, provides the basis for normalization of motif restart probability; dashed lines show the maximum, median, and minimum). n) Violin plots reporting the latency of motif restart (orange: thalamus stimulation, white: HVC stimulation dataset from Fig. 1 compared against all experimental groups across the manuscript; nested one-way ANOVA comparing latency to restart across groups, F(5,17) = 6.119 P = 0.0020, Dunn’s post-hoc pan-HVC vs. thalamus P < 0.001). o) Same as panel (g) but for latency to motif restart. Two-way ANOVA testing the difference between restart latency distributions, interaction F(594,2178) = 3.212 P < 0.001, stimulated subpopulation F(6,22) = 5.966 P = 0.0036, Tukey’s post-hoc pan-HVC vs. thalamus identifies significant difference (p < 0.05) between 70 and 640 ms time bins. (inset) One-Way ANOVA testing the difference between truncation latencies, Kruskal Wallis test H(6) = 244.7, post-hoc HVC vs. thalamus P < 0.001). Brain outline in d adapted with permission from ref. 60, Wiley.
