Inhibitory Subpopulations in preBötzinger Complex Play Distinct Roles in Modulating Inspiratory Rhythm and Pattern

Inhibitory neurons embedded within mammalian neural circuits shape breathing, walking, and other rhythmic motor behaviors. At the core of the neural circuit controlling breathing is the preBötzinger Complex (preBötC), where GABAergic (GAD1/2⁺) and glycinergic (GlyT2⁺) neurons are functionally and anatomically intercalated among glutamatergic Dbx1-derived (Dbx1⁺) neurons that generate rhythmic inspiratory drive. The roles of these preBötC inhibitory neurons in breathing remain unclear. We first characterized the spatial distribution of molecularly defined preBötC inhibitory subpopulations in male and female neonatal double reporter mice expressing either tdTomato or EGFP in GlyT2⁺, GAD1⁺, or GAD2⁺ neurons. We found that the majority of preBötC inhibitory neurons expressed both GlyT2 and GAD2 while a much smaller subpopulation also expressed GAD1. To determine the functional role of these subpopulations, we used holographic photostimulation, a patterned illumination technique, in rhythmically active medullary slices from neonatal Dbx1^tdTomato;GlyT2^EGFP and Dbx1^tdTomato;GAD1^EGFP double reporter mice of either sex. Stimulation of 4 or 8 preBötC GlyT2⁺ neurons during endogenous rhythm prolonged the interburst interval in a phase-dependent manner and increased the latency to burst initiation when bursts were evoked by stimulation of Dbx1⁺ neurons. In contrast, stimulation of 4 or 8 preBötC GAD1⁺ neurons did not affect interburst interval or latency to burst initiation. Instead, photoactivation of GAD1⁺ neurons during the inspiratory burst prolonged endogenous and evoked burst duration and decreased evoked burst amplitude. We conclude that GlyT2⁺/GAD2⁺ neurons modulate breathing rhythm by delaying burst initiation while a smaller GAD1⁺ subpopulation shapes inspiratory patterning by altering burst duration and amplitude.