Relapse to heroin use remains a significant challenge in the treatment of heroin addiction, yet our understanding of those neural systems that enable individuals to inhibit heroin seeking and relapse remains poor. The long-term goal of our laboratory is to identify the neural circuits and the changes in those circuits that underlie the inhibition of heroin-seeking behavior, using rat models of heroin seeking. Previous work with cocaine seeking has suggested that the infralimbic cortex is involved in the extinction and inhibition of cocaine seeking. Yet, the role of this prefrontal region in opioid seeking has been unclear, as studies have produced findings indicating that the infralimbic promotes and inhibits opioid seeking. As increasing evidence suggests that different projections from prefrontal regions can mediate distinct functions, we have hypothesized that the ability of the infralimbic to either promote or inhibit heroin seeking is related to the specific projections from this region to the nucleus accumbens shell (NAshell) vs. the amygdala. In addition, we have recently developed preliminary data suggesting that the anterior portion of the insular cortex (rostral agranular insular cortex, RAIC) inhibits heroin seeking. However, other studies with other drugs of abuse indicate that the RAIC also appears to promote drug-seeking behavior. Thus, it seems likely that, as with the IL, the key to understanding the role of the RAIC in heroin seeking depends on targeting different projections. Indeed, like the infralimbic, the RAIC projects to the amygdala, a region that prior studies have found to be involved in promoting reinstatement to heroin seeking. However, the RAIC also provides an input to the infralimbic itself, thus providing a mechanism for how the RAIC may influence and interact with the infralimbic during the extinction and inhibition of heroin seeking. The proposed studies, therefore, will examine these circuits during heroin seeking and will include approaches that have not, to our knowledge, been used in studies of heroin seeking. In particular, our studies will examine the neurophysiological correlates of heroin seeking in the RAIC and IL via ensemble recordings in both regions simultaneously. The findings from this work will likely reveal neuronal subpopulations in both regions related to the extinction and/or inhibition of heroin seeking. Moreover, our proposed work will use optogenetic and chemogenetic manipulations of the specific projections from the infralimbic to the NAshell and amygdala and from the RAIC to the infralimbic cortex and amygdala. Specifically, these manipulations will allow us to interrogate the role of each pathway in the encoding of the extinction learning and the reinstatement of heroin seeking. As a result, this proposal will provide converging lines of evidence regarding this circuitry and the inhibition of heroin seeking. The findings from the studies will furnish critical new basic knowledge of the n...