Project Summary Opioid use has reached epidemic levels in the United States, and despite decades of research, very few effective treatment options are available. In both humans and rodents, a counter-intuitive increase in craving in response to drug-associated cues is observed following extended periods of abstinence, a phenomenon known as incubation of craving. This craving is thought to contribute to the high rate of relapse observed in patients with substance use disorder. Understanding the molecular mechanisms that drive incubation of craving will be vital for the development of new effective therapeutics. Neurons that express dopamine D1 (D1+) or D2 (D2+) receptors define distinct cellular populations, which are thought to play divergent roles in reward-related behaviors. Pharmacological inhibition of D1+ but not D2+ receptors in the nucleus accumbens shell (NAcSh) blocks incubation of morphine craving, highlighting D1+ NAcSh cells as critical drivers of incubation. D1+ and D2+ cells undergo differential synaptic plasticity in response to drugs of abuse, but how these cell types are differentially influenced by incubation of morphine craving remains an important unaddressed question. Additionally, in both humans and rodents, sex differences are observed in substance use and abuse; females escalate to substance abuse faster and are at greater risk of relapse than men. These sex differences can be further modified by estrous cycle in females, where periods of high estrogen during estrus enhance incubation of cocaine craving. However, the role of estrous cycle in incubation of morphine craving and the associated changes in gene expression remain unknown. Preliminary RNA-sequencing data in the NAcSh of rats following 1 or 30 days of forced abstinence from morphine or saline intravenous self- administration revealed that expression of the transcription factor Nr4a1, as well as several of its putative target genes, is upregulated following 30 days of abstinence in males but not females. However, how estrous cycle might impact Nr4a1 expression during incubation of craving remains unknown. Thus, this project will investigate the cell-type specific role of Nr4a1 during incubation of morphine craving in male and female rats. My central hypothesis is that Nr4a1 in D1+ NAcSh cells leads to incubation of morphine craving, and that this will be enhanced during estrus in females. I will test this hypothesis by leveraging new technology to identify cell-type specific changes in gene expression and by tracking the female estrous cycle. The training plan outlined in this proposal will provide me the resources, skills and environment needed to complete this project and to pursue an independent academic research career. Successful completion of these aims will provide a greater understanding of the cell-type specific mechanisms underlying incubation of craving and will provide support for a potential therapeutic target to prevent relapse.