PROJECT SUMMARY Our goal is to explain the neurophysiology of breathing. Breathing refers to periodic movements of the chest and airways that ventilate the lungs. To breathe, the brain must: Generate a rhythm, Produce a spatiotemporal pattern for breathing muscles, Integrate sensory feedback. Here we focus on jobs 1 and 2, discovering the ionic mechanisms underlying the rhythm and motor pattern. The brainstem preBötzinger complex (preBötC) generates the inspiratory breathing rhythm. Its core rhythmogenic interneurons are derived from Dbx1-expressing progenitors (i.e., Dbx1 neurons). Because we know the site (preBötC) and the canonical cell-class (Dbx1) at the point of origin for breathing, we are well equipped to discover which ion channels influence normal inspiration (eupnea), ‘sighs’, and gasps in hypoxia. We manipulate ion channels using genetic technologies. We assess breathing phenotypes in intact adult mice. We explain the biophysics of these phenotypes via patch-clamp recordings from adult Dbx1 preBötC neurons. Three types of ion channels are implicated in preBötC function: (1) Na+ channels that engender persistent Na+ current (INaP), (2) Transient receptor potential (Trp) channels that mediate Ca2+-activated nonspecific cationic current (ICAN), and (3) K+ channels that give rise to transient outward K+ current (i.e., A-current, IA). Specific aim 1 evaluates the role of INaP in Dbx1 preBötC neurons. A rhythmogenic role for INaP has been suspected for 27 years. We use intersectional mouse genetics and short-hairpin RNA (shRNA) to knockout or knock-down Na+ channel genes that give rise to INaP and evaluate its role in eupnea, sighing, and gasping. Specific aim 2 evaluates the role of ICAN in Dbx1 preBötC neurons. ICAN is a major charge carrier for inspiratory bursts. Next-generation RNA Seq technology provides us with a suite of Trp channel targets that we will acutely attenuate using shRNA to test the role of ICAN in eupnea, sighing, and gasping. Specific aim 3 evaluates the role of IA in Dbx1 preBötC neurons. IA-expressing preBötC neurons feature other key rhythmogenic properties so they may be specialized. We use intersectional mouse genetics and shRNA to knockout or knock-down K+ channel genes for IA to evaluate its role in eupnea, sighing, and gasping. Then we selectively kill the 56% of IA-expressing Dbx1 preBötC neurons to test whether they serve a specialized rhythmogenic role. The success of this project would be a watershed in terms of understanding the ion channel-level origins of real behavior. The new knowledge could be applicable to treatment and prophylaxis of respiratory pathologies with a central etiology. The new knowledge may provide general insights regarding the neural origins of motor rhythms.