PROJECT SUMMARY A critical feature of the respiratory system is its ability to adapt to increased metabolic demand by recruitment of expiratory muscles to facilitate alveolar ventilation, a process referred to as active expiration. Active expiration is thought to be mediated by a cluster of neurons located in the lateral parafacial region (pFL). However, despite this physiological significance, the identity of expiratory pFL neurons is not known and unique markers of these cells have not been determined. To address this need, proposed experiments will determine the molecular profile of expiratory pFL neurons and identify unique markers of this population that can be used to selectively label and manipulate this population for detailed functional analysis. We will use RNA-seq to perform an unbiased evaluation of transcript expression of neurons from the pFL region and a nearby respiratory center called the retrotrapezoid nucleus (RTN). This analysis will focus on glutamatergic populations that are expected to include expiratory pFL neurons and RTN neurons. We will use the known molecular profile of RTN neurons as a touchstone for identification of unique pFL markers. To correlate gene expression with function, we will characterize baseline activity and CO2/H+ sensitivity of putative expiratory parafacial neurons back-labelled from an expiratory control center, and we will manually harvest these cells for targeted qPCR to identify common and differentiating genes between RTN and pFL populations. Identifying the transcriptome of pFL neurons will represent a major technical advance in the field of respiratory control as it would make possible the development of targeting strategies to selectively manipulate this population. This work will also provide insight into the genetic organization of neurons and circuits that control breathing, and in doing so help understand how these elements are perturbed in disease.