Molecular mechanisms of incubation of methamphetamine craving

PROJECT SUMMARY Research into the mechanisms underlying methamphetamine craving, a key driver of relapse, is dwarfed in comparison to other drugs of abuse (e.g., cocaine, opioids). Exacerbated drug craving can be modeled in rats using the incubation of methamphetamine craving paradigm. The nucleus accumbens plays a key role in mediating drug seeking which intensifies commensurate with abstinence duration. Approximately 95% of accumbens neurons are GABAergic medium spiny neurons which can be distinguished by expression of the D1 dopamine receptors (D1DRs) or D2 dopamine receptors (D2DRs), although a relatively small proportion of these cells express both of these receptors. Canonically, D1DR-containing neurons were thought to project exclusively to the ventral tegmental area, whereas D2DR-expressing accumbens efferents extended to the ventral pallidum; however, recent evidence suggests that there is some overlap such that D1DR-containing neurons also project to the ventral pallidum and modulate drug seeking. Generally, D1DR-containing neurons promote drug-related behaviors, whereas D2DR-containing neurons inhibit drug-related behaviors. Importantly, the influence of specific projections of these neuronal subtypes on methamphetamine seeking is unknown. Specific Aim 1 will combine D1DR-Cre and D2DR-Cre transgenic rats, optogenetics, and electrophysiology to dissociate the role of D1DR- vs. D2DR-containing striatopallidal and striatotegmental projections in the incubation of methamphetamine craving. The time-dependent molecular adaptations within accumbens D1DR- containing and D2DR-containing neurons that occur during abstinence from methamphetamine are also unexplored. Very recent advances in single cell sequencing methodologies have enabled simultaneous profiling of the transcriptome and chromatin accessibility in defined cell populations. This is particularly relevant for the current proposal since it will allow us to differentiate in silico effects in accumbens neurons which express either the D1DR or D2DR. Specific Aim 2 will define the landscape of accessible chromatin and gene expression by coupling ATAC sequencing and RNA sequencing from the same single nuclei droplets in rats following short or prolonged abstinence from methamphetamine. Functional validation of a candidate gene will be performed by bidirectionally manipulating gene expression in selectively in D1DR-containing or D2DR- containing neurons and assessing the incubation of methamphetamine craving. Collectively, the proposed studies will elucidate the neurocircuitry and molecular mechanisms underlying the incubation of methamphetamine craving.