Project Summary The first breath of air for an infant is a crucial and complex event that requires coordination of many physiological systems; however, the neural circuitry and molecular signals that help mediate the initiation of respiration in this critical time remains unclear. In adults, regulation of breathing occurs through careful coordination of multiple brainstem nuclei which make up the respiratory central pattern generator. Among these brainstem nuclei is the retrotrapezoid nucleus (RTN), a group of central respiratory chemoreceptors located along the ventral lateral medulla. These glutamatergic neurons increase their firing rate in response to elevated levels of arterial CO2 (PCO2), which in turn can drive respiratory output to re-establish and maintain PCO2 homeostasis via input to other brainstem nuclei within the respiratory network. All RTN neurons express the transcription factor Phox2b, and neuropeptides Neuromedin B (Nmb), and pituitary-adenylate cyclase activating peptide (PACAP). These neurons intrinsically respond to changes in CO2/H+ through two proton sensors: TASK-2, and GPR4. GWAS analysis has revealed PACAP variants have been associated with higher incidence of sudden infant death syndrome (SIDS), and PACAP-KO mice have been observed to have a SIDS- like phenotype. Recent work from our laboratory demonstrated a striking upregulation of PACAP expression in the RTN at the time of birth. Further we have shown that neonatal mice (p2-p12) lacking PACAP have significantly more apneas when exposed to thermal stress, and a blunted respiratory response to hypercapnia compared to their control littermates. These findings unveil a novel role for this peptidergic circuit in protecting respiratory rhythm in neonates. It is currently unknown if PACAP is necessary for the initial establishment or protection of a stable respiratory rhythm in the critical period immediately following birth, and further how this dynamic expression of PACAP is regulated in the first moments of life. I hypothesize that PACAP expression in the RTN is mediated through changes in blood gas homeostasis immediately following birth and is necessary for establishing and protecting respiratory stability in neonates. In Specific Aim 1, I use conditional PACAP knockout mice and whole-body plethysmography to test whether PACAP is necessary for protection of stable respiratory rhythmogenesis in the face of thermal stress, and/or for establishment of normal CO2 stimulated breathing over the course of the first hours of life. In Specific Aim 2, I investigate the mechanism by which PACAP expression is stimulated at the time of birth by manipulating the principal RTN chemosensitive feedback in opposing ways, or by genetically eliminating the putative CO2 sensors of the RTN, while assessing PACAP expression through multiplex in situ hybridization or single cell-qPCR. The proposed studies will provide novel information regarding molecular mechanisms which regulate the gene ...