Project Summary Sudden unexpected death in epilepsy (SUDEP) is the leading cause of premature death in persons with epilepsy who do not have satisfactory seizure control. SUDEP results in more years of potential life lost than any other neurological condition with the exception of stroke. There are no known ways of reliably preventing SUDEP. Convergent lines of evidence suggest that respiratory dysfunction is a critical component of SUDEP pathophysiology. There are several potential mechanistic explanations for the respiratory arrest seen in SUDEP. (1) Serotonin signaling is important for stable breathing and increasing serotonergic tone may be protective against seizure-induced respiratory arrest. Unfortunately, seizures disrupt serotonergic neurotransmission. There is a gap in knowledge as to the mechanism responsible for seizure-induced disruption of serotonergic neurotransmission. (2) Seizures also cause a surge in extracellular adenosine throughout the brain. Increases in adenosine levels suppress breathing and inhibit neural activity. Excessive adenosine signaling has been implicated in the respiratory dysfunction seen in SUDEP; however, the mechanism by which adenosine affects breathing after seizures is unknown. (3) Slow moving (2-5 mm/min) waves of spreading depolarization are sometimes triggered by seizures. Spreading depolarization transiently inactivates the brain tissue. Under certain circumstances spreading depolarization can travel into the brainstem where it halts neural activity in nuclei necessary for cardiorespiratory function and causes death. Brainstem spreading depolarization is a potential cause of SUDEP. Spreading depolarization also causes an increase in extracellular adenosine. It is not known whether the increase in adenosine due to spreading depolarization contributes to seizure-induced death. The goal of this proposal is to integrate the serotonergic, adenosinergic, and spreading depolarization explanations of SUDEP etiology by testing the central hypothesis that adenosine surging as the result of seizure activity and spreading depolarization disrupts serotonergic neurotransmission thereby potentiating respiratory failure. In the first aim, adenosine signaling will be augmented in the serotonergic raphe nuclei during seizures to determine if this alters respiratory responsiveness to CO2 and the likelihood of respiratory failure. In the second aim, brainstem spreading depolarization will be induced in mice in which adenosine signaling has been pharmacologically or genetically altered in the raphe nuclei. The proposed experiments will generate compelling evidence for or against the hypothesized interaction between serotonin and adenosine while providing the training in writing, mentorship, and quantitatively rigorous hypothesis-testing necessary for a career in science.