Multiplexed dissection of neuronal ensembles in circuits that underlie the facilitation and disinhibition of opioid and alcohol seeking

SUMMARY Alcohol and opioid use disorders are characterized by both the facilitation and disinhibition drug seeking and use. Despite this knowledge, the influence of alcohol and opioid use on brain circuits responsible for facilitating and inhibiting natural reward-seeking behaviors remains unclear. Surprisingly, until recently no study had tracked activity in single neurons from the onset of drug use to relapse, limiting our understanding of how these circuits computationally change to prioritize drug-motivated behaviors. We recently overcame this issue by developing a mouse behavioral assay that allows longitudinal tracking of activity in precisely defined neurons across alcohol and opioid self-administration, extinction, and reinstatement. Using this model, we identified precise neuronal ensembles based on in vivo dynamics within key behavioral facilitation and inhibition circuits, namely from dorsomedial prefrontal cortex and paraventricular thalamus to the nucleus accumbens (dmPFCàNAc; PVTàNAc). Ensembles within each projection showed divergent adaptations predictive of drug seeking, whereas functional manipulations could prevent the facilitation (dmPFCàNAc) or disinhibition (PVTàNAc) of drug seeking. However, the transcriptomic identity and function of each neuronal ensemble remains unknown. Here we will test the overarching hypothesis that dmPFCàNAc and PVTàNAc projections are composed of unique neuronal ensembles organized based on multiplexed gradients of activity, gene expression, spatial location, sensitivity to drug use and seeking, and causal function for the facilitation and disinhibition of drug seeking. We test this hypothesis by employing a combination of innovative tools, now in use within our laboratories, such as single-nucleus RNA sequencing (snRNA-seq), Xenium spatial sequencing (spatial-seq), in vivo two-photon calcium imaging, and single-cell holographic optogenetics. Overall, this project will characterize the transcriptomic profile and function of single neurons and computationally unique neuronal ensembles in key brain circuits that causally govern the facilitation and disinhibition of alcohol and opioid seeking. The groundbreaking strategies employed will be transformative for the field of addiction research, providing comprehensive understanding of gene expression and targetable substrates within relevant cell populations for further study and manipulation for treatment of opioid and alcohol use disorders. Large-scale multimodal calcium imaging and RNA sequencing datasets will be made freely available to the public and scientific community for further study.