Ectopic activation of the iRO evokes airflow correlated USV types and switches the relationship of the anti-correlated types.
A, Optogenetic activation of the iRO region in Penk-Cre;Vglut2-Flp;CreONFlpON-ReaChR mice evokes USVs (blue box, 5 Hz stimulation). USVs occur during, or shortly after laser onset. B, Percentage of stimulation bouts containing at least one USV and the percentage of breaths within the stimulation window containing a USV. C, Percentage of optogenetically evoked (blue) or endogenously occurring (gray) syllables that are up fm of down fm. **** p-value< 0.001; two-way ANOVA with Sidak’s post-hoc test, p > 0.05 for all other types. D, Box and whisker plot of the correlation coefficient between breathing airflow and pitch (r) for all opto evoked (n=395) and endogenous (n=1850) USVs and all opto evoked and endogenous vocalizations without down fm (n=143 and 1810) from n=4 opto and n=6 endogenous mice. **** p-value < 0.001; Mann-Whitney test. E, Left, example of the expiratory airflow and pitch for an optogenetically evoked up fm USV. Middle, magnification of airflow and sound. Time of breath airflow during the USV is color coded blue to white. Scatter plot of instantaneous expiratory airflow and pitch for the single USV. Compare to endogenous up fm USV in Fig. 2D. Right, box and whisker plot of correlation coefficients (r) for each optogenetically evoked and endogenous up fm USV (n=15 vs. 589). F, Step up USV as in E. box and whisker plot of correlation coefficients (r) for each optogenetically evoked and endogenous step up USV (n=15 vs. 40). G, r value box and whisker plots for the remaining optogenetically evoked USV types (down fm n=242 vs. 40, step down n=38 vs. 293, flat n=34 vs. 337, short n=27 vs. 168, and chevron n=10 vs. 99 from n=4 opto and 6 endogenous mice. E-G, Two-way ANOVA with Sidak’s post-hoc test for two way comparisons was used; all p-values >0.05. H, Schematic illustrating the two mechanisms to pattern the USV pitch. Left, the reciprocal connection between the iRO and breathing pacemaker patterns the USVs with a positive correlation between pitch and airflow. Right, the retroambiguus (RAm) control of the larynx dictates the anti-correlated USV types. Middle, the combination of these two mechanisms within a single breath create additional USV patterns.