Project Summary The central goal of this proposal is to characterize the role of the insular cortex (InsCtx) in encoding and shaping the physiological drive to consume nicotine in nicotine-dependent mice. The InsCtx integrates sensory, visceral, and limbic information and is implicated in interoception: the sensing of internal bodily states. Interoception plays a crucial role in the development of addiction because the rewarding effects of addictive drugs and the aversive aspects of withdrawal are largely experienced as bodily sensations. Functional imaging in human smokers shows that InsCtx responses to smoking-related cues increase with self- reported craving intensity and with the expectancy of receiving nicotine. Intriguingly, after strokes involving the InsCtx, smokers report increased rates of smoking cessation and fewer nicotine cravings. Nevertheless, it remains unclear how the InsCtx encodes nicotine-replete and withdrawal states during the development of nicotine dependence, and if targeted InsCtx manipulations can curb cued nicotine-seeking behaviors. This gap in understanding is due in part to our inability to track the activity of large numbers of neurons throughout the development of dependence on and withdrawal from nicotine (or other addictive drugs). I have overcome this challenge by adapting our lab’s conceptual and technical framework for chronic two-photon calcium imaging of hundreds of InsCtx neurons during shifts in hunger/thirst to investigate InsCtx encoding of nicotine states and nicotine-predicting cues. Specifically, I propose to track InsCtx neurons as mice (i) develop nicotine dependence via home-cage drinking water exposure, and (ii) learn to respond to visual cues in order to receive oral nicotine rewards. In Aim 1, I will test the hypothesis that as an animal becomes dependent on nicotine, the pattern of InsCtx ongoing activity encoding states of satiety will change across days to reflect this new bodily state. Because nicotine has direct effects centrally on the brain as well as peripherally on bodily physiology, I will use natural and pharmacological manipulations to determine if ongoing InsCtx activity patterns reflect the central and/or peripheral actions of nicotine. In Aim 2, I will examine how InsCtx responses to nicotine- predicting cues correlate with concurrent recordings of mesolimbic dopamine release in the nucleus accumbens, which is known to mediate some of the rewarding aspects of addictive drugs and is profoundly altered in states of addiction. I will then test the hypothesis that acute optogenetic silencing of the InsCtx will disrupt learned dopaminergic and behavioral responses to nicotine-predicting cues. These experiments will advance our understanding of how InsCtx activity patterns change during the emergence of nicotine dependence, and will define the role of the InsCtx in drug-seeking behaviors. This work will improve our understanding of how exposure to nicotine modifies the neural represen...