Fig. 3: Optogenetic activation of RAm Nts neurons generates vocalizations and controls sound volume.
From: A brainstem circuit for phonation and volume control in mice
a, RAm Nts neuron optogenetic stimulation by bilateral injection of Cre-dependent AAV encoding bReaChES in NtsCre mice, with laser light delivered via optical fiber. b, Respiratory airflow (bottom) and sonogram (top) during optogenetic stimulation of RAm Nts neurons (yellow bar, 30-Hz laser) in an anesthetized mouse. Inspiration (Insp), downward deflections on airflow trace; expiration (Exp), upward deflections. Note ultrasonic vocalizations of increasing volume and duration during optogenetic stimulation, accompanied by breathing changes. Insets (above), induced vocalizations expanded to millisecond timescale. Note ultrasonic vocalizations with motifs that repeat at the same rate as stimulus pulse. c, Airflow trace of single breaths before (top) and during (bottom) optogenetic stimulation of RAm Nts neurons. Note that tidal volume (VT, shaded area) and expiration time (TE) increase during optogenetic stimulation. d, Effect of pulse frequency on vocalizations generated by optogenetic stimulation of RAm Nts neurons in an anesthetized NtsCre mouse. Note lack of vocalization with 10-Hz stimulation, audible (<20 kHz) vocalizations generated with increasing volume from 15 Hz to 20 Hz and ultrasonic (>20 kHz) vocalizations generated with increasing volume from 25 Hz to 30 Hz. Amp., sound amplitude. e,f, Peak syllable amplitude of optogenetically driven vocalizations (n = 9 mice, colored lines) in audible (e) and ultrasonic (f) frequency ranges. Note monotonic increase in peak syllable amplitude of audible vocalizations with increasing stimulation frequency in all mice and similar monotonic increase in ultrasonic vocalization amplitude in a subset of mice that produced ultrasonic vocalizations. g,h, Syllable pitch for the same optogenetic stimulation trials as e,f. Note that syllable pitch does not change. i, Histograms of peak syllable amplitude of all vocalizations generated during RAm Nts neuron ablation experiments (Fig. 2) (n = 7 mice per condition and n = 8,310 total syllables analyzed). Note decrease in peak syllable amplitude of residual syllables after Casp3-mediated ablation of RAm Nts neurons (P = 0.02, Pre-Casp3 versus Post-Casp3). j, Histograms of syllable pitch for the same syllables as i. Note that syllable pitch does not change after RAm Nts ablation (P = 0.7 Pre-Casp3 versus Post-Casp3, n = 7 mice per condition and n = 8,310 total syllables analyzed). *P < 0.05 and NS, not significant, by paired two-tailed t-test.
