PROJECT SUMMARY Breathing is necessary for survival, and failure to breath results in death. Simultaneously, the precise and flexible control of breathing is crucial for maintaining homeostasis (e.g., blood oxygenation during exertion), and to serve higher order functions such as vocalization. The neural control for both the inexorable and the flexible capabilities of breathing arises from the activity of neurons in the brainstem. The intrinsically rhythmic pre-Bötzinger complex (preBötC) is thought to be the core or “kernel” driving rhythm generation, but a distributed network of neural centers that extend rostrally and caudally from the preBötC, known as the Ventral Respiratory Column (VRC), has been implicated in control of varied aspects of respiration (e.g., inspiration, expiration, sighing, gasping). Lesion experiments and isolated recordings from in-vitro brain slices have given insights into the roles of the different sub-nuclei that govern respiration. However, it remains unknown how the activity of these neuronal populations is dynamically coordinated to adjust respiratory behaviors during normal breathing and gasping. Here, we record simultaneously from large, spatially distributed populations of genetically identified single neurons across the VRC in-vitro and in-vivo. Based on our preliminary data, we propose a novel “dynamic switching” hypothesis: coordinated neural networks recruit discrete, but anatomically overlapping, populations of neurons to drive unique respiratory behaviors. Moreover, the respiratory role (e.g. inspiratory/expiratory) of individual neurons is not static, as is currently thought, but dynamic and changes with respiratory behavior. This hypothesis is analogous to phenomena observed in locomotor gaits in which discrete spatiotemporal muscle patterns give rise to discrete modes of movement (e.g. walking, trotting, galloping). This proposal tests the dynamic switching hypothesis through three specific aims: Aim 1 quantifies the dynamic and coordinated respiratory roles of single neurons in in-vitro brain slices and in-vivo in anesthetized, breathing mice. We employ state-of-the-art high-density electrophysiology (Neuropixels) with optogenetic techniques to identify the functional role and genetic identity of hundreds of simultaneously recorded neurons and compare the coordinated activity of these populations both between in-vitro and in-vivo preparations. Aim 2 describes how these dynamic networks reconfigure during gasping. Lastly, in Aim 3 we corroborate results in freely behaving animals where respiratory behaviors are highly flexible (e.g. sniffing) and are modulated by top-down centers.