Inhibitory subpopulations in preBötzinger Complex play distinct roles in modulating inspiratory rhythm and pattern

Inhibitory neurons embedded within mammalian neural circuits shape breathing, walking, chewing, and other rhythmic motor behaviors. At the core of the neural circuit controlling breathing is the preBötzinger Complex (preBötC), a nucleus in the ventrolateral medulla necessary for generation of inspiratory rhythm. In the preBötC, a recurrently connected network of glutamatergic Dbx1-derived (Dbx1 ⁺ ) neurons generates rhythmic inspiratory drive. Functionally and anatomically intercalated among Dbx1 ⁺ preBötC neurons are GABAergic (GAD1/2 ⁺ ) and glycinergic (GlyT2 ⁺ ) neurons, whose roles in breathing remain unclear. To elucidate the inhibitory microcircuits within preBötC, we first characterized the spatial distribution of molecularly-defined inhibitory preBötC subpopulations in double reporter mice expressing either the red fluorescent protein tdTomato or EGFP in GlyT2 ⁺ , GAD1 ⁺ , or GAD2 ⁺ neurons. We found that, in postnatal mice, the majority of inhibitory preBötC neurons expressed a combination of 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 with high spatiotemporal resolution, in rhythmically active medullary slices from neonatal Dbx1 ^tdTomato ;GlyT2 ^EGFP and Dbx1 ^tdTomato ;GAD1 ^EGFP double reporter mice. 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 the majority of preBötC inhibitory neurons express both GlyT2 and GAD2 and modulate breathing rhythm by delaying burst initiation while a smaller GAD1 ⁺ subpopulation shapes inspiratory patterning by altering burst duration and amplitude.