Progesterone and allopregnanolone of prefrontal cortical activity dynamics and heroin seeking

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ABSTRACT: To develop treatments, it is vital to identify neural mechanisms underlying relapse in opioid use disorder. To dissect neural activity underlying relapse, we developed a novel assay allowing for in vivo two- photon calcium imaging while head-fixed mice engage in heroin self-administration. Using this approach, we find global excitatory activity in the prelimbic-prefrontal cortex (PrL) decreases with acquisition of heroin seeking, an effect that persists through extinction, but then resurges during reinstatement. These dysregulated global PrL dynamics mirror clinical observations that are considered hallmarks of substance use disorder. Importantly, our approach has identified, for the first time, that PrL activity dynamics emerging during acquisition, extinction, and reinstatement are heterogenous, with distinct ensembles exhibiting unique excitatory and inhibitory activity dynamics aligned with behavioral epochs. How these unique activity dynamics guide drug seeking, however, is unknown. This implies greater resolution of PrL activity dynamics is necessary to determine how they functionally regulate behavior. Here, under the expert guidance of Drs. Jim Otis and Peter Kalivas, my K99 training in advanced computational neural analyses and single-cell optogenetics paired with in vivo calcium imaging will resolve the function of discrete PrL ensemble dynamics for relapse (Aim 1). Notably, there are subpopulations of individuals with substance use disorder in whom relapse may emerge through different neurobiological mechanisms. Females form one such subpopulation, as they exhibit enhanced relapse vulnerability and greater prefrontal activity during craving and relapse compared to males. However, this vulnerability covaries with the ovarian hormone cycle, such that peak circulating levels of progesterone (PROG) appear protective. These effects are mediated by its 5alpha-reductase neuroactive steroid metabolite, allopregnanolone (ALLO), which can influence prefrontal circuitry and promote adaptive responding in females. As ALLO acts as a positive allosteric modulator at GABA-A receptors, it likely constrains PrL neuronal activity to suppress drug seeking. As I find ALLO can act directly within PrL to suppress heroin-seeking reinstatement, I hypothesize that PROG and ALLO can disrupt PrL activity dynamics which functionally guide reinstatement. During the R00 phase, my career goal to be an independent investigator will involve building a research program wherein I resolve the influence of PROG and ALLO on PrL activity dynamics during reinstatement using in vivo two-photon calcium imaging. As activation of PrL projections to the nucleus accumbens core (NAcC) is necessary for reinstatement, I will retro-label PrL-NAcC neurons to enable simultaneous monitoring of global PrL and PrL-NAcC ensembles (Aim 2). Using single-cell optogenetic and circuit labelling approaches, I will assess the functional influence of PROG and ALLO on PrL and PrL-NAcC ensembles for reinstatement (Aims 2-3). As I find ALLO but not PROG suppresses reinstatement in males, I will assess steroidal effects on PrL and PrL-NAcC ensemble activity dynamics as a function of biological sex.