Hyperventilation, breath-holding, central apnea and respiratory dysrhythmia is typical for patients with Rett Syndrome. In these patients, substance P is deficient in brainstem areas that are associated with the central control of breathing. Therefore we hypothesize that the irregular breathing in RS is due to the brainstem deficiency in Substance P ("Substance P hypothesis"). We also hypothesize that an understanding of how substance P (SP) controls breathing will be essential for developing rational therapies for the breathing disorders in RS. The proposed grant application, aimed at investigating the role of substance P in regulating the central nervous control of breathing, will isolate a critical portion of the respiratory network (the "pre- Botzinger complex") in a transverse brainstem slice from mice. The proposed research addresses 3 fundamental questions: (1) What type of ion channel is modulated by SP? Specific aim 1 examines the hypothesis that SP modulates a low-threshold sodium current in respiratory neurons. The hypothesized ion channel causes a long lasting depolarization in inspiratory non-pacemaker and pacemaker neurons resulting in an excitatory response of the respiratory network. (2) How does SP alter membrane properties of respiratory pacemaker neurons? Specific aim 2 tests the hypothesis that the low-threshold sodium current interacts with the ion channels responsible for the generation of pacemaker activity. We specifically examine whether this sodium channel leads to the activation of a CAN current, which dramatically enhances bursting in cadmium-sensitive pacemaker neurons. This aim will lead to a better understanding of the mechanisms responsible for the SP modulation as well as the ionic mechanisms underlying burst generation in respiratory pacemaker neurons. (3) Are pacemaker neurons dependent on the endogenous activation by SP? Specific aim 3 tests the hypothesis that endogenously released SP is required to maintain regular respiratory activity by modulating pacemaker neurons. Decreased levels of SP will lead to weakening of pacemaker activity and thus to irregular breathing. The expected outcome of this research plan will provide important concepts relevant for RS as it will lead to a better understanding of why low levels of SP cause irregular respiratory activity. [unreadable] [unreadable]