Kv4 channels have been shown to play important roles in modulating neural activity: regulating the integration of high-frequency trains of synaptic input, regulating backpropagating action potentials, and contributing to long-term potentiation. Consequently, mutations that affect Kv4 function/availability have been shown to result in spatial learning defects, seizure behavior, as well as temporal lobe epilepsy. We have recently shown that expression and turnover of Kv4 channels are also affected in three new contexts: in modulating cholinergic synaptic homeostasis, in response to over-expression of human Aβ42, and during normal aging. In the proposed studies, we investigate the mechanisms underlying Kv4 expression during cholinergic synaptic homeostasis (also referred to as synaptic scaling). Synaptic homeostasis is a form of plasticity that has been heavily studied in the last decade as a protective mechanism that counterbalances changes in global neural activity; this likely occurs during physiological processes, such as learning/memory and development, as well as during pathological conditions. We used Drosophila central neurons as a model, and showed that Drosophila α7 (Dα7) nAChRs are up-regulated after cholinergic blockade, thereby enhancing synaptic currents and providing a homeostatic response. We found that this homeostatic response triggered a novel regulatory mechanism –the up-regulation of Kv4 channels, which we showed prevents an “overshoot” of the homeostatic response. We further showed that the up-regulation of Kv4 channels is blocked by transcriptional inhibitors, and is dependent on Dα7 nAChRs and Ca2+ influx. Drosophila continues to be an ideal model system for these studies because of its cholinergic CNS, the genetic tools it offers, its less redundant genome (eg. there is only a single Drosophila NFAT and Kv4 gene, each of which represents a multi-gene family in mammals), and the ability to go from mechanisms of gene regulation to physiological relevance in the intact brain, and whole animal behavior. The proposed studies will apply new optogenetic approaches to elicit cholinergic synaptic homeostasis in vivo (Aim-1) –something that has not been explored in any system, and which would currently not be feasible in mammalian systems. We will examine underlying molecular mechanisms, including a novel relationship between α7 nAChRs and Kv4 channels (Aim-2), and inactivity-induced transcription of Kv4 (Aim-3) that is mediated by NFAT (Aim-4). We will also test all molecular mechanisms for their physiological relevance in identified neurons in the intact brain and behaving animal (Aims 4-5). Our studies are likely to reveal novel insights into the underlying mechanisms of cholinergic synaptic homeostasis.