PROJECT SUMMARY Heart rate is a well-established indicator of overall health and well-being, yet little is known about the neurons that control it. Although the heart has its own pacemaker, heart rate is largely regulated by the autonomic nervous system, subjecting it to the interplay between sympathetic neurons and parasympathetic neurons (i.e., cardiac vagal preganglionic neurons (CVNs)). CVNs control many aspects of cardiac function, ranging from heart rate and atrioventricular conductance (via nodal tissue) to contractility and excitability (via ventricular myocardium). Furthermore, these neurons are highly relevant to heart health: cardiac mortality increases whenever cardiovagal activity is diminished. The nucleus ambiguus (nAmb), a region in the medullary reticular formation of the brainstem, houses the majority of CVNs, along with other neurons that are known to control respiratory functions (bronchoconstriction, bronchosecretion) and innervate upper airway and esophageal muscles. This functional diversity of neurons within the nAmb makes it challenging to isolate CVNs for study. This has greatly limited what we know about their gene expression, synaptic circuitry, and specific roles in cardiac function—thus limiting our ability to target these neurons in the context of cardiovascular diseases. To address these issues, I propose a comprehensive approach to identify CVNs molecularly, anatomically, and functionally. I will characterize CVNs based on transcriptome-wide mRNA expression, which will reveal transcriptional markers that provide genetic access to each nAmb subtype. Leveraging the genetic differences between nAmb subtypes, I will trace each subtype’s axonal projections to thoracic organs, revealing the anatomical organization of the CVNs. Our preliminary studies have pointed to three molecularly distinct neuron subtypes localized in the nAmb, one of which innervates multiple sites in the heart. Lastly, to uncover each subtype’s physiological role, I will activate each subtype using intersectional optogenetics while assessing the effect on heart rate, respiration, and upper airway motor function. Taken together, these studies will further uncover the molecular, anatomical, and functional organization of CVNs, providing a multi-dimensional and comprehensive understanding of the neurons that mediate heart rate.