Project Summary The neuromodulators acetylcholine (ACh) and dopamine (DA) play important roles in learning. In the striatum cholinergic interneurons (CINs) are modulated co-incident with the release of DA in response to unpredicted rewards and reward predicting cues and both signals have been implicated in coding prediction error signals. Whereas DA neurons are mostly activated by these salient events, CINs often show a multiphasic response with a prominent pause in activity. The time locked occurrence of both signals suggest that they are coordinated but it is still unclear whether they are regulated independently from each other or whether they mutually regulate each other. Moreover, whether their temporal co-incidence is important for learning still needs to be determined. Support for a mutual co-regulation comes from both stimulation and lesion studies. In the slice, DA released from DA terminals inhibits the activity of CINs via activation of D2 receptors. Strikingly, classical 6-OHDA lesion studies in non-human primates suggest that the pause in Tonically Active Neurons (TANs), the primate counterparts of CINs, is fully dependent on DA; although this hypothesis has been challenged by data implicating thalamic or other projections in the pause generation. Slice physiology and in vivo stimulations studies have further shown that ACh released from CINs locally induces DA release. One limitation of stimulation studies is that they do not measure naturally evoked ACh or DA levels. Thus, the importance of this mutual co-regulation during learning must be determined under natural conditions. An ideal way to achieve this is to simultaneously measure behaviorally-evoked changes in DA and ACh levels in the same animal. Since striatal ACh has been found to be important when behavior needs to be adapted to new task rules the application will focus on understanding the importance of the mutual co-regulation of DA and ACh for cognitive flexibility. To this end, we propose to simultaneously record task-evoked DA and ACh transients in the mouse during two flexible learning behaviors, Go/NoGo and reversal learning. Our preliminary data show that both behaviors are affected by striatal CIN function. We then will isolate the DA-dependent component of the ACh signal by enhancing or abolishing the ability of DA to inhibit CINs. Conversely, we will abolish the ability of CINs to release ACh or inhibit CIN activity with high temporal resolution. Determining how these manipulations affect task-evoked changes in ACh/DA levels and performance will establish the importance of mutual co-regulation of DA and ACh signals for flexible learning. Our studies will provide mechanistic insights into DA and ACh co-regulation in the striatum. This has clinical relevance as both neurotransmitters are dysregulated in the striatum of patients with brain disorders including schizophrenia and Parkinson disorder, where both neuromodulator systems are targeted by current therapeutic t...