Mapping projections of excitatory and inhibitory preBötzinger Complex neurons

Abstract

Understanding the neural control of breathing requires a detailed neuroanatomical and functional map of the breathing central pattern generator (CPG). The logical entry point in constructing such a map is the “engine” of respiratory rhythm, the preBötzinger Complex (preBötC), a bilateral microcircuit of ~3000 interconnected neurons in the ventrolateral medulla. The preBötC generates respiratory rhythm in rodents (and presumably all mammals, including humans); rhythmic activity in the preBötC is required to produce inspiratory movements. To delineate the breathing CPG circuit in mice, we used a viral strategy to identify projections from molecularly‐defined populations of preBötC neurons. Using stereotaxic delivery of Cre‐dependent AAV expressing EGFP (AAV‐flex‐EGFP) into the preBötC of somatostatin‐Cre or GlyT2‐Cre mice, we located efferent projections emanating from two broad, functionally distinct classes of neurons: excitatory neurons that express somatostatin (SST) and inhibitory neurons that express the glycine transporter GlyT2. Critically, we optimized the viral delivery method to restrict viral transfections within the preBötC, while sufficiently labeling neurons to visualize long distance projections, enabling reliable identification of preBötC projections. Glutamatergic SST+ preBötC neurons project to brainstem nuclei implicated in the control of breathing including the contralateral preBötC, parahypo‐glossal/nucleus of the solitary tract, ventral respiratory group, and parabrachial/Kölliker‐Fuse nuclei, similar to efferent projections in rats; these neurons also had suprapontine projections to multiple hypothalamic nuclei, periaquaductal grey, as well as sparse projections to amygdala, suggesting that the preBötC relays respiratory information to higher brain regions that may modulate physiological or behavioral processes. Strikingly, glycinergic preBötC neurons send projections in parallel with glutamatergic SST+ neurons to the same brainstem and suprapontine targets.Support or Funding InformationA.P. Giannini Foundation, NIH T32 NS007101, NIH F32 HL126522‐01, NIH RO1 HL070029‐13