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 preBotzinger Complex (preBotC), a nucleus in the ventrolateral medulla necessary for generation of inspiratory rhythm. In the preBotC, a recurrently connected network of glutamatergic Dbx1-derived (Dbx1+) neurons generates rhythmic inspiratory drive. Functionally and anatomically intercalated among preBotC Dbx1+ neurons are GABAergic (GAD1/2+) and glycinergic (GlyT2+) neurons, whose roles in breathing remain unclear. To elucidate the inhibitory microcircuits within preBotC, we first characterized the spatial distribution of molecularly-defined preBotC inhibitory subpopulations in double reporter mice expressing either the red fluorescent protein tdTomato or EGFP in GlyT2+, GAD1+, or GAD2+ neurons. We found that, in neonatal mice, the majority of preBotC inhibitory 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 Dbx1tdTomato;GlyT2EGFP and Dbx1tdTomato;GAD1EGFP double reporter mice. Stimulation of 4 or 8 preBotC 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 preBotC 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 preBotC 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.