Novel roles for the circadian transcription factor NPAS2 and striatal dopamine D3 receptor signaling in diurnal fentanyl craving and relapse - PROJECT SUMMARY Our proposal is in direct response to NIDA RFA-DA-24-020, “Mechanistic Studies to Investigate the Interrelationship between Sleep and/or Circadian Rhythms and Substance Use Disorders”. The opioid crisis in the United States has led to skyrocketing economics costs and a surge in fatal overdoses due to availability of synthetic opioids like fentanyl. Individuals struggling with opioid use disorder commonly experience severe and persistent disruptions in their sleep and circadian rhythms, which may contribute to increased cravings and risk of relapse to opioids. There are critical gaps in our understanding of the underlying cellular and molecular mechanisms in the brain that link opioid addiction with sleep and circadian rhythm disturbances. We found that synaptic and behavioral responses to fentanyl are controlled by a circadian-dependent, molecular clock mechanism in the medial shell of the nucleus accumbens (mNAcSh), a major nexus for regulating reward and motivated behaviors. Importantly, we discovered that the circadian transcription factor, neuronal PAS domain 2 (NPAS2), is an integral to this circadian regulation of opioid actions and is markedly enriched in a mNAcSh subpopulation of medium spiny neurons (MSNs) that specifically expresses the dopamine D3 receptor (D3R- MSNs). Indeed, ablation of NPAS2 function in the NAc leads to increased fentanyl craving and relapse behaviors. Notably, the effects of NPAS2 on D3R-MSN function and opioid-related behaviors depend both on sex and time of day, resembling a putative sex-specific and circadian-dependent pathway in the brain that contributes to opioid addiction. Our recent work has also established the importance of D3R signaling in the NAc to control effortful, motivated behaviors to obtain rewards. Thus, our central hypothesis is that NPAS2-dependent transcription in D3R-MSNs of the mNAcSh is a key signaling mechanism involved in opioid craving and relapse in males and females. To test this, our proposal employs novel genetic tools combined with cell type-specific RNA-sequencing, and slice electrophysiology. In Aim 1, we will determine the molecular signaling pathways regulated by NPAS2 in D3R-MSNs involved in fentanyl self-administration behaviors at different times of day. In Aim 2, we will examine the impact of fentanyl on D3R-MSN excitability and activity and NPAS2’s roles in in mediating these effects in a diurnal manner. In Aim 3, we will determine whether loss of either NPAS2 function or D3R expression in D3R- MSNs leads to alterations in fentanyl craving and relapse behaviors across the day. Overall, our proposed studies, if successful, will definitively identify the mechanistic role of NPAS2 in D3R-MSNs in opioid reinforcement and relapse in males and females at different times of day. These advances will drive future development of new circadian-dependent interventions that result in more effective treatment of OUD.
