Project Summary/Abstract The peptide neurotensin (NTS) is known to be a potent modulator of dopamine neuron activity, and NTS signaling has been linked to various modalities of behavioral reinforcement and reward, as well as to the behavioral response to drugs of abuse and the motivation for drug taking. Pharmacological studies have found that in the ventral tegmental area (VTA), NTS binding to its receptors depolarizes dopamine neurons through a variety of second messenger cascades, resulting in increased dopamine neuron firing and downstream dopamine release. However, much less is known about the physiological release of NTS from endogenous brain circuits and what role these circuits may play in regulating motivated behavior. Using retrograde mapping we identified 23 brain regions that send NTS input to the VTA; however, few of these inputs are well-studied for their role in NTS signaling, and little is known about what stimuli or behavioral actions may activate NTS neurons. Furthermore, though peptidergic neurons typically co-release a fast neurotransmitter, such as glutamate or GABA, current methods for circuit-specific activation fail to distinguish between downstream effects triggered by peptides versus those evoked by fast transmitters. We hypothesize that different NTS projections to the VTA may play distinct roles in modulating motivated behavior, and that NTS acts synergistically with co-released fast transmitters to govern the dynamics of downstream dopamine neuron activation. Here we propose to determine the neurotransmitter co-expression of select NTS inputs to the VTA and map their synaptic connectivity to dopamine and non-dopamine neurons. We also propose to use viral CRISPR gene mutagenesis techniques in combination with optogenetics to isolate the peptide and fast transmitter components of NTS inputs to the VTA. We will dissect the role of these components in regulating different modalities of behavioral reinforcement and will measure the response of dopamine neurons in vivo to stimulation of specific inputs. Finally, we will use fiber photometry to measure the activity profiles of NTS neurons during learning and performance of a cued reinstatement food reward task. Together, these experiments will add unprecedented circuit-specific precision to our understanding of how these critical peptidergic inputs influence the activity state of dopamine neurons and govern reward and reinforcement learning.