Nathan Bolen, Colton J. Treadway, Michael Leonardo, Wes Tackett, Aneesh Gupta, Joshua Sisco, Travis B. Salisbury, James Denvir, Sadia Akter, Vini Magalhaes Borges, Alejandro Q. Nato, Jr., Brandon J. Henderson, Price E. Dickson

In 2024, 47,735 people in the United States died from synthetic opioid overdose, primarily due to fentanyl (CDC, 2026). This striking statistic underscores the urgent need to discover and characterize the genetic underpinnings of fentanyl addiction. To this end, we are using genetically diverse mouse strains (N = 40) from the Collaborative Cross (CC) and their founders to discover genetic, transcriptomic, epigenomic, and neurophysiological mechanisms underlying fentanyl addiction-like behaviors and addiction endophenotypes. Notably, the CC founder strains encompass ~90% of known genetic diversity in laboratory mice originating from Mus musculus. We are testing mice on four behavioral assays: intravenous fentanyl self-administration, sign-tracking/goal-tracking, fentanyl locomotor sensitization, and operant sensation seeking. Machine learning enables nuanced analysis of these mouse behaviors. We are collecting nucleus accumbens punches following sign-tracking/goal-tracking and fentanyl locomotor sensitization; gene expression using RNA-seq will be quantified from this tissue. We are using fast-scan cyclic voltammetry, patch-clamp electrophysiology, spatial transcriptomics, single-cell multiomics, and long-read sequencing to characterize CC founder strains, extreme CC strains, or both. Data collection for this study will occur over five years. Here, we report results after ~20 months of phenotyping. Briefly, interim results reveal significant strain effects, GxE effects, and sex effects on many phenotypes. Over the next several years, we will integrate QTL mapping, eQTL mapping, and genetic correlations spanning multiple biological levels. Collectively, these data will provide a foundation for future deep characterization of identified mechanisms, a lasting community resource, and will ultimately contribute to the development of novel, more effective addiction treatments.

Department of Biomedical Sciences,, Joan C. Edwards School of Medicine,, Marshall University,, 1700 3rd Ave.,, Huntington, WV, 25703, USA.