ABSTRACT Cue-driven behaviors are actions motivated by salient environmental stimuli. Maladaptive changes in cue- driven behaviors are fundamental to several neuropsychiatric disorders, including substance use disorder. Ventral tegmental area (VTA) dopamine (DA) neurons have often been assumed to homogeneously encode reward prediction errors. However, even within the nucleus accumbens (NAc), which is the major projection target of VTA DA neurons, DA release has been implicated in several behavioral functions, including reward, aversion, motivation, and incentive salience. This discrepancy might be, at least in part, due to the different methodologies used to record DA cell body activity versus DA release at the axon terminal level. Additionally, recent investigations suggest that VTA DA neurons might differentially contribute to reward and aversion dependent on their projection target and mediolateral position within the VTA. Here, we propose to further detail and define, in a circuit- and cell-type specific manner, the functional heterogeneity of the mesoaccumbal DA system. We will employ a three-pronged approach that leverages pharmacological manipulations, fiber photometry-based recordings of DA cell body activity and DA release, as well as in vivo electrophysiological recordings of DA neurons using Neuropixels and optogenetics. Our investigations will that takes the precise neuroanatomical position of DA neurons within the VTA and their corresponding NAc projection target into consideration. Experiments will be performed in head fixed mice during pharmacological manipulation or during reward seeking behavior as well as in freely behaving mice performing a two-armed bandit task. This will allow us to test whether DA dynamics mediated by different mechanisms in different locations (i.e., at the level of cell bodies versus terminals) underlie distinct behavioral functions. The primary goals are to (1) investigate how different doses of nicotine modulate DA release in distinct NAc subregions. Additionally, we will provide a systematic understanding of how nicotinic acetylcholine receptor function contributes to nicotine-induced DA release in different NAc subregions. (2) We will study how DA cell body activity and DA release in separate mesoaccumbal subcircuits contributes to reward learning and motivated behavior. (3) We will establish an in vivo electrophysiological approach that utilizes Neuropixels and optogenetics to record VTA DA neurons in a circuit- and cell-type specific manner in head fixed mice performing a reward seeking task. Together, we anticipate that these experiments will provide further evidence for our hypothesis that VTA DA neurons make specific contributions to reward learning and motivation in a projection-defined manner. Delineating the precise functions of separate mesoaccumbal DA subcircuits for reward learning and motivated behavior is a significant step in improving our understanding of their diverse roles in health an...