The serotonin system is critically involved in mood and anxiety and is the target of first line-treatments for mood and anxiety disorders. It is thought that it is organized into distinct subcircuits which influence different behavioral states but the structure and function of these subcircuits are poorly understood. This proposal will investigate two of the densest serotonergic projections of the dorsal raphe nucleus (DRN), the projection to the VTA (DRN-VTA) and the projection to the amygdala (DRN-amygdala) and their role in reward and aversion. I will test the hypothesis that these projections respond differentially during rewarding and aversive experience and drive different effects on reward and fear learning. I will dissect their function through three specific aims that each elucidate a previously unknown circuit feature using cutting edge tools. In (aim 1), I will use endoscopic in vivo two photon microscopy to image the single-cell activity of DRN-VTA and DRN-amygdala projection neurons to test whether DRN-VTA and DRN-amygdala neurons respond differently to rewarding and aversive stimuli. In (aim 2), I will use retrograde viral tracing in intact brain volumes rendered transparent by the CLARITY method to map the brain wide collateral structure of DRN-VTA and DRN-amygdala projections which could coordinate multiple downstream regions during reward and fear learning. In (aim 3), I will use multi-fiber photometry and closed-loop optogenetics to determine the simultaneous activity in DRN-VTA and DRN-amygdala projections and collateral sites during reward and fear learning and test their causal function during reward and fear learning. In the process, I will become proficient in two photon microscopy, viral circuit tracing, multi-fiber photometry, and closed loop optogenetics, techniques that will be essential in my future career as an independent investigator. The work will be accomplished under the supervision of an expert team of mentors, Karl Deisseroth, Alan Schatzberg, Rob Malenka, Liqun Luo, and Conor Liston, who developed these techniques and have extensive experience in teaching others to use them as well as mentoring young investigators during the transition to independence. These aims will represent the first studies to measure the in vivo activity, brain-wide collateral structure, or closed loop modulation of any DRN projection and establish the dynamics of the DRN projections to two key limbic structures. They will also provide evidence of whether the DRN may have a dual reward/aversion valence circuit structure in which rewarding and aversive signals are transmitted to different downstream structures. Together they will significantly advance our understanding of serotonin circuit dynamics and provide a necessary foundation for understanding aberrations in serotonin circuitry which may contribute to mood and anxiety disorders.