PROJECT SUMMARY/ABSTRACT Neuronal Firing Rate Homeostasis (FRH) describes the ability of neurons to maintain their average firing rates at precise set points in the long-term, even in the face of changing synaptic inputs. The stability provided by FRH enables learning and memory. Achieving FRH requires an FRH control circuit that is akin to a thermostat that compares desired to actual room temperatures or an autopilot that compares desired to actual trajectories. Identifying this control circuit will require direct measurement of FRH by perturbing firing rates and observing homeostatic recovery, which has rarely been done. A promising candidate FRH control circuit is the neuronal Activity-Regulated Gene (ARG) program, in which activity-dependent increases in calcium lead to transcription of genes whose mRNA levels remain elevated for many hours. The ARG program is a promising candidate because individual activity-regulated genes protect against seizures, and they also regulate homeostatic effector mechanisms like synaptic scaling that may mediate FRH. However, the molecules that comprise the FRH control circuit are unknown. We hypothesize that the ARG program is the core control mechanism of FRH. Our extensive preliminary data reinforce the importance of testing this hypothesis at genome-scale. The genome-scale approach is important not only because of the sheer number of ARGs but also because different ARGs appear to “interpret” firing rate error in very different -- but as yet undefined -- ways. Relying on our extensive knowledge of the ARG program, we will evaluate its contribution to FRH. For ARG induction to be a core mechanism of FRH, it must be specified quantitatively by firing rates. We will therefore map the coupling of firing rates to ARG expression levels, by controlling firing rates optogenetically and detecting gene expression with RNA-Seq. We will also directly assess the functional contribution of ARG induction to FRH by perturbing firing rates pharmacologically in cortical neurons and observing homeostatic recovery using multi- electrode arrays, with or without perturbation of ARGs. Our approach is innovative because it is a genome- wide investigation of the activity-sensing control system for homeostasis rather than the far better-studied homeostatic effector mechanisms. Our work is significant because it will advance basic knowledge of the control circuit mediating FRH and produce tools for manipulating FRH that will help connect this control circuit to candidate homeostatic mechanisms.