Figure 8. The iRO is sufficient to induce neonatal cry bouts and the cry syllables within a breath.

A, Vglut2-Flp Penk-Cre expression of GFP in the RC::FLTG transgene (Flp + Cre > GFP). Coronal section of medulla showing GFP⁺ neurons (green) and Phox2b expression (gray). Right, magnification of annotated area. B, Top, I_c recording of a GFP⁺ neuron (Vglut2⁺Penk⁺ > GFP) in fast synaptic blockers shows the hallmark iRO rhythm (n = 5/7). Bottom, I_c recording of ReaChR⁺ iRO neuron (Vglut2⁺Penk⁺ > ReaChR) with brief light illumination. C, Quantification of the proportion of all ReaChR⁺ neurons (percent ± SEM) that localize to the iRO (red), PiCo (gray- ChAT positive), and BötC, preBötC, and RAm (black) (n=3). D, Representative change in breathing after 1.5 seconds 10Hz unilateral illumination of the iRO in ReaChR⁺ or wildtype (control) neonatal mice. Average number of augmented breaths in each animal (9 ReaChR⁺, 8 wildtype). E, As in D, representative change from basal breathing to complete cry bout in 5/9 ReaChR⁺ animals. F, Left, time to cry bout onset. Note, no cry bouts were induced in wildtype. Blue bar on y-axis indicates the duration of laser stimulation. Right, average stimulated cry bout length in ReaChR⁺ animals. G, Comparison of the percent of cries that show multiple syllables or expiratory airflow peaks that accompany each syllable without light (sham, OFF) versus with light (ON) in ReaChR⁺ and wildtype controls. Right, mean ± SEM of ON : OFF. H, Basal breath length with 50ms light pulse (N) normalized to the length of the preceding breath (N-1) for breaths with light (ON) and sham. One-sided t-test or Wilcoxon rank sum P < 0.01 (**). I, Model indicating that the iRO is sufficient to induce both the entirety of a cry bout and cry syllables in a breath. See also Figure S8 and Table S1.