Extended Data Fig. 5: Optogenetic stimulation of pallial afferents to HVC does not disrupt the song motif.
From: Holistic motor control of zebra finch song syllable sequences
a) Schematic of in-vivo recording of HVC multiunit neuronal activity in anesthetized birds expressing eGtACR1 in NIf; sample trace (top), raster plot (mid, 10 trials) and normalized peri-stimulus time histogram (bottom) reporting the change in multi-unit HVC firing activity in response to light stimulation of eGtACR1-expressing NIf afferents (1 s from 0.5 s to 1.5 s, red bar; two-way ANOVA comparing the curve between the 0 and 5 s versus corresponding 5 s baseline without stimulation, interaction F(499,10978) = 9.255 P < 0.001, stimulation F(1,22) = 12.07 P = 0.0022, Sidak’s post-hoc identifies difference at 0.92-1.79 s). Inset shows magnified PSTH and scatter plot with the average (per hemisphere) response to the first 200 ms light stimulation (red dashed rectangle) compared to the last 200 ms baseline (black dashed rectangle, Wilcoxon test P = 0.0017; n= hemispheres, birds). b) (top) Schematic of song-contingent light stimulation of NIf axonal terminals in HVC (optic fiber implanted over HVC) and sample spectrogram of unstimulated (top) and stimulated song (bottom, red bars, 200 ms ≈10 mW bilateral 473 nm LED, spectrogram scale, 0-11KHz, scale bar 200 ms, horizontal lines identify the bouts’ (black), introductory notes’ (light gray), motifs’ (dark gray) and linker syllable’s (brown) boundaries). c) Violin plots reporting accuracy of the stimulated song segment (gray), or corresponding control unstimulated segment (white) per each bird (n = 4); two-way ANOVA, interaction F(3,76) = 1.795 P = 0.1553, CTRL vs. STIM, F(1,76) = 0.7208 P = 0.3985). d) Same as (c) but for Entropy (n = 4; two-way ANOVA, interaction F(3,76) = 1.882 P = 0.1397, CTRL vs. STIM, F(1,76) = 0.1807 P = 0.6720). e) same as (c) but for goodness of pitch (n = 4; two-way ANOVA, interaction F(3,76) = 4.553 P = 0.0055, CTRL vs. STIM, F(1,76) = 2.301 P = 0.1334). f) same as (c) but for Accuracy of the entire motif for birds receiving 1 s light stimulation (n = 4; two-way ANOVA, interaction F(3,76) = 2.417 P = 0.0728, CTRL vs. STIM, F(1,76) = 3.072 P = 0.0837). g-l) same as (a-f), but for eGtACR1 expression in mMAN: (g) two-way ANOVA comparing the curve between the 0 and 5 s versus corresponding 5 s baseline without stimulation, interaction F(499,14970) = 8.937 P < 0.001, stimulation F(1,30) = 16.68 P < 0.001, Tukey’s post-hoc identifies difference at 0.85–1.76 s; Inset: Wilcoxon test P = 0.00071; n= hemispheres, birds. (i) n = 4 birds, two-way ANOVA, interaction F(3,76) = 4.483 P = 0.006, CTRL vs. STIM, F(1,76) = 0.6008 P = 0.4407. (j) two-way ANOVA, interaction F(3,76) = 6.168 P < 0.001, CTRL vs. STIM, F(1,76) = 0.1036 P = 0.3119. (k) two-way ANOVA, interaction F(3,76) = 2.434 P = 0.0714, CTRL vs. STIM, F(1,76) = 2.828 P = 0.0967. (l) two-way ANOVA, interaction F(3,76) = 1.795 P = 0.1553, CTRL vs. STIM, F(1,76) = 0.2599 P = 0.6117). m-r) same as (a-f), but for eGtACR1 expression in Av. (m) two-way ANOVA comparing the curve between the 0 and 5 s versus corresponding 5 s baseline without stimulation, interaction F(499,9980) = 9.999 P < 0.001, stimulation F(1,20) = 39.56 P < 0.001, Tukey’s post-hoc identifies difference at 0.77 and 0.84-1.9 s; Inset: Wilcoxon test P < 0.001; n= hemispheres, birds. (o) n = 4 birds, two-way ANOVA, interaction F(3,76) = 3.524 P = 0.0189, CTRL vs. STIM, F(1,76) = 0.1304 P = 0.7190. (p) two-way ANOVA, interaction F(3,76) = 0.6458 P = 0.588, CTRL vs. STIM, F(1,76) = 0.7229 P = 0.3979. (q) two-way ANOVA, interaction F(3,76) = 5.05 P = 0.003, CTRL vs. STIM, F(1,76) = 0.1634 P = 0.2050. (r) two-way ANOVA,interaction F(3,76) = 3.936 P = 0.0115, CTRL vs. STIM, F(1,76) = 1.143 P = 0.2883). Brain outlines in a, g and m adapted with permission from ref. 60, Wiley.
