PROJECT SUMMARY Neurons, the basic processing and communicating units of the brain, typically use action potentials to transmit messages from a site near the cell body to neurotransmitter releasing terminals via axons. Action potentials can, in some cases, also travel backwards along the axon towards the cell body and dendrites; I refer to these here as ‘ectopics.’ Ectopics have been recorded in models of epilepsy, as well as in a small group of inhibitory interneurons under normal conditions. Recently, I discovered that nearly all parvalbumin positive (PV+) cells of the brain, which account for about half of neocortical inhibitory cells, can fire ectopics. We do not yet know exactly where in the axon ectopics are generated. In addition to not knowing where in the axon they come from, we also do not know what mechanism generates them. Finally, we do not know what conditions lead to ectopics in vivo. Here, I propose a series of experiments designed to answer these questions. In Aim 1, I will test whether receptors, ion channels, and cotransporters capable of depolarizing the presynaptic terminal are involved in ectopic generation. I will also use an ultra-rapid imaging technique to track action potentials as they travel through the axons of PV+ cells to directly confirm both where ectopics are generated and the number of synapses they trigger. In Aim 2 I will determine whether astrocytes, which envelop and modulate PV+ cell terminals, help elicit ectopics by blocking astrocyte-specific transporters and receptors, as well as by modulating intracellular astrocytic calcium stores and optogenetically depolarizing astrocytes. Finally, in Aim 3, I will undertake a series of experiments to detect ectopics in PV+ cells while mice are under anesthesia, and in various awake, behaving conditions. These experiments will discern both how, and in what contexts, ectopics are generated. Understanding the mechanisms of ectopic action potential initiation will yield new insights into the function of an important population of inhibitory interneurons. It may also reveal mechanisms that relate to brain disorders involving cortical hyperexcitability, providing novel targets for future investigations of circuit-level changes related to these brain disorders and, potentially, new approaches to treatment. This mentored award will support the training I need to establish an independent career as an academic physician-scientist. I will develop technical expertise in voltage-sensitive dye imaging and in vivo recording techniques under the direction of my mentor, Dr. Barry Connors, Ph.D., with input from advisory committee members Drs. Judy Liu, M.D., Ph.D. and Christopher Moore, Ph.D., who are leading experts in their fields. I will supplement this training with courses in the responsible conduct of research, molecular neuroscience techniques, and grant writing, along with attendance at local seminars and national meetings to disseminate my findings and foster collaboration...