PROJECT SUMMARY Although the neuromodulators dopamine (DA) and acetylcholine (ACh) are extensively implicated in the pathogenesis and treatment of neuropsychiatric diseases, their effects on the activity of brain circuits are typically studied separately, leaving their ability to dynamically cooperate in supporting brain function and dysfunction incompletely understood. This is a significant limitation as (1) neuropsychiatric disorders typically involve disturbances in multiple modulators, (2) existing therapies rarely target a single modulatory system, and (3) the physiological actions of modulators are fundamentally inter-dependent, since many co-exist in individual brain regions and converge on overlapping cells to stimulate receptors that complement, antagonize, or synergize with one another. An influential hypothesis in the field of basal ganglia research is that ACh gates or modifies the physiological actions of DA in the striatum – a subcortical forebrain region important for learning and motivating goal-directed behaviors. However, we still lack a clear understanding of precisely when, how and over what time course ACh interacts with DA to differentially affect striatal activity and behavior. We recently discovered that ACh levels in the striatum of mice constantly undergo large, subsecond fluctuations that are temporally coordinated with DA and modulated in amplitude depending on behavioral state. The main goal of this proposal is therefore to build of this observation and to test the hypothesis that DA acts in concert with ACh, such that the precise timing and amplitude of subsecond ACh transients relative to DA is key for deciphering the effects of DA on striatal circuits and behavior. We will do so by first monitoring the dynamics of DA and ACh with subsecond precision from the striatum of mice engaged in reward-based behaviors using dual-color photometry of red and green DA and ACh sensors (Aim 1). Next, we will mechanistically dissect the functional impact of ACh on DA release and on striatal activity using photometric imaging combined with genetic and pharmacological manipulations (Aim 2). Lastly, we will establish that ACh transients can gate the effects of DA on motivation and learning using calibrated optogenetic manipulations (Aim 3). Together, the proposed work will provide crucial mechanistic insights into the structure and function of coordinated DA and ACh dynamics in the striatum of mice and pave the way for future investigations into how the disruption or potentiation of such coordination in humans contributes to the development or treatment of neuropsychiatric disorders.