Leveraging Transcriptomics and Behavior to Investigate Potential Neuronal and Glial Drivers of Substance Use Disorder Endophenotypes

Author

Jack V. Keady, University of KentuckyFollow

Author ORCID Identifier

https://orcid.org/0000-0003-1775-2324

Date Available

7-20-2026

Year of Publication

2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

Faculty

Jill Turner

Abstract

Over 54 million Americans needed substance use treatment in 2023. While there are many facets of substance use disorder, two major issues of interest are the negative reinforcing aspects of substances of abuse, manifesting in part as withdrawal symptomologies, and why a subset of people who use substances of abuse escalate to a use disorder. Nicotine exposure models were used to investigate the mechanisms of withdrawal endophenotypes, as smoking is the leading cause of preventable death in the United States, and a fentanyl self-administration model was used to probe individual differences in escalation phenotypes, as 80% of overdose deaths in 2023 involved some type of opioid. These studies aim to combine behavioral techniques with transcriptomic data to highlight potential druggable targets to alleviate nicotine withdrawal endophenotypes and alter fentanyl escalation behavior.

Individuals undergoing nicotine withdrawal report cognitive deficits and affective dysfunction, which are both major predictors of relapse to smoking behavior. Chapter 2 explores the sex specific effects of nicotine and withdrawal on contextual fear memory, a hippocampally dependent aspect of cognition, and alterations in RNA expression of markers for neuronal activation and genes implicated in smoking cessation outcomes (erbb4 and nrg3) in the dorsal and ventral hippocampus. Female mice withdrawn from nicotine displayed impaired extinction learning, but no effect was observed in males. Nicotine withdrawal caused suppression of markers for neuronal activation and increased erbb4 expression in the ventral hippocampus of both sexes, but these changes were correlated to delays in female extinction learning alone.

Affective dysfunction is a common endophenotype of withdrawal from multiple substances of abuse, and previous works have shown microglia mediate nicotine withdrawal induced anxiety-like phenotypes. Chapter 3 examines the impact of nicotine treatment and withdrawal on the transcriptome of microglia isolated from male murine nucleus accumbens tissue and compares these alterations with published single cell RNA-sequencing mouse models of substance use disorders that include microglia. The microglial transcriptomic alterations induced by nicotine treatment and withdrawal overlapped with those observed in cocaine withdrawal and reinstatement, suggesting potential leukotriene signaling as a mechanism contributing to a pro-inflammatory environment during withdrawal.

Opioid use disorder is characterized by compulsion to seek and obtain opioids resulting in escalating opioid intake. Chapter 4 identifies distinct fentanyl escalation phenotypes of Sprague Dawley rats and investigates transcriptomic differences between these phenotypes in the prefrontal cortex, a brain region involved in executive control over drug taking. Weighted gene co-expression network analysis identified a subset of genes correlated with the observed fentanyl escalation phenotypes. This subset was enriched for genes associated with voltage-gated potassium channels, glutamatergic signaling, and substance use disorder. EZH2 was predicted to regulate the expression of this subset of genes, highlighting a potential target to modulate fentanyl intake.

In summary, female mice experience nicotine withdrawal induced deficits in contextual fear extinction, which may in part be due to elevated ventral hippocampal errb4 expression; nicotine withdrawal caused similar impacts to microglia transcriptome in male mice as cocaine, pointing to leukotriene signaling driving inflammatory response; and EZH2 may be a transcriptional regulator contributing to increased fentanyl intake. While further work is needed to validate the role of these alterations in mediating the observed phenotypes, the combination of behavior and transcriptomic approaches used in the presented studies point to mechanisms for future investigation into nicotine withdrawal and fentanyl escalation endophenotypes.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.270

Funding Information

This study was supported by National Institutes of Health Grants (1-R01-DA053070-01 and 5-R01-DA044311) from 2020 to 2025, the American Foundation for Pharmaceutical Education Pre-Doctoral Fellowship in 2023, and the University of Kentucky Substance Use Disorder Faculty Pilot Grant in 2023.

Recommended Citation

Keady, Jack V., "Leveraging Transcriptomics and Behavior to Investigate Potential Neuronal and Glial Drivers of Substance Use Disorder Endophenotypes" (2025). Theses and Dissertations--Pharmacy. 167.
https://uknowledge.uky.edu/pharmacy_etds/167