Project Summary/Abstract Many of the approved drugs used to treat a variety of diseases in children and adults target G-protein coupled receptors (GPCRs). Furthermore, it is reported that two-thirds of those receptors couple to cyclic adenosine monophosphate (cAMP) regulation. Despite the approved use of GPCR-mediated drugs, little is known about the subsequent cAMP signaling in specific neural circuits and their contributions to synaptic transmission and behavior. In the brain, cellular processes important for learning and memory involve the second messenger cAMP, along with other downstream messengers, such as Protein Kinase A (PKA) to decode neuromodulatory signals. The applicant, Anthony James (post-baccalaureate student) will combine the viral expression of a cell- type specific cAMP or PKA sensor to monitor postsynaptic intracellular signaling and presynaptic optogenetics to precisely manipulate neuronal signaling and/or pharmacology to induce changes in synaptic plasticity, focusing on long-term depression (LTD) with two-photon microscopy. Using this approach, he will evaluate the somatic and dendritic cAMP and/or PKA activity organization in response to light-activated induction of synaptic plasticity. Also, Anthony will determine if expression of the cAMP or PKA sensor alters synaptic properties within expressed cells using electrophysiology. We hypothesize that neuromodulatory signals generate distinct spatial and temporal patterns of cAMP-mediated signaling, which mediates synaptic transmission resulting in appropriate behaviors. Also, we further hypothesize that sensor expression will not alter the synaptic properties of the neuron. The results obtained from this study will further elucidate the molecular mechanisms underlying synaptic transmission involving GPCR-mediated cAMP signaling. Our proposal builds on the experiments outlined in the parent award and will further strengthen the link of causality between synapse-specific activation and subsequent cAMP-PKA signaling in mediating synaptic depression. These experiments are within the scope of the parent award, which examines the spatiotemporal dynamics of cAMP-mediated signaling during synaptic plasticity and learned behaviors. Anthony’s proposal employs optogenetic stimulation to activate a specific subset of corticostriatal terminals to examine the resultant cAMP-mediated signaling. Anthony will receive laboratory training in stereotaxic surgery, acute brain slice preparation, two-photon microscopy, electrophysiology, and data analysis, as well as career development training in writing, scientific communication, and networking. For Anthony’s training, I have recruited a co-mentor to provide complementary mentorship. With our combined mentoring efforts, we will help Anthony in reaching his goal of attending graduate school to pursue a PhD in neuroscience.