Whiskers and Withdrawal: Molecular Insights into Opioid Use Disorder in Male Mouse Models 

Summary Mouse models help unravel the mysteries of opioid addiction through analysis of the molecular changes in the brain triggered by heroin use. Comparison of these findings with human data pinpoints shared genetic patterns, shedding light on potential treatments for opioid use disorder.

News and Views by: Ella Wuichet (elwuichet@davidson.edu) Primary Article: Caleb J. Browne et al., 2023

Disclaimer: This web page was produced as an assignment for an undergraduate course at Davidson College.

Opioid use disorder (OUD) is one of the most pressing public health crises, affecting millions across the world(Lee et al., 2024). In the US alone, more than 70,000 people die from opioid-related overdoses every year, and with opioid prescriptions on the rise, this number continues to increase (National Center for Health Statistics 2024). Although therapeutic methods for overcoming opioid addiction exist, these methods often fail to prevent relapse long term (Smyth et al., 2010). Many of the challenges surrounding overcoming opioid addiction stem from a poor understanding of the molecular mechanisms underlying opioid addiction. Previous studies have supported the idea that drugs can cause changes in the brain that motivate drug use and steer away from natural rewards; however, many of these studies have analyzed psychostimulants, and little is known about these changes in opioid use (Browne et al., 2020). A study published in June 2023 sought to fill this gap in knowledge and investigate the molecular mechanisms of OUD using a mouse model (Caleb J. Browne et al., 2023).

Transcriptional mechanisms – the processes that regulate how our DNA is expressed – are essential in understanding the molecular mechanisms of changes caused in our brain by drug use (Bali & Kenny et al., 2019). Each one of the cells in our bodies contains the exact same DNA but is tightly controlled using transcriptional mechanisms so that select genes can be up-regulated, meaning they are expressed and turned into specific proteins, or down-regulated, meaning they are “turned off” and will not create proteins. This control of our DNA happens in extremes, as seen in the creation of different cell types, but can also occur in more minor ways in response to environmental changes, like drug use (Nielsen et al., 2012). This study sought to examine the way that our DNA expression is changed in response to heroin use and withdrawal. 

To study these transcriptional mechanisms, scientists first created environmental conditions to model distinct stages of OUD in mice models. The stages they studied included ongoing heroin intake with short-term withdrawal, first-time heroin exposure, abstinence after chronic intake, and relapse (Figure 1). In order to analyze the molecular effects of these varying conditions, scientists used a method called RNA-seq. This method allowed them to uncover sequences of RNA copied from DNA, which has undergone modification to either up or down-regulate specific genes. With this tool, scientists collected valuable sequencing data from six brain regions involved in reward processing from mice in all of their model environmental conditions, allowing them to link OUD behavioral profiles with specific gene expression.

Figure 1: Schematic describing experimental conditions. Mice were split into two groups and given 4 hours of saline or heroin injections for 15 days. After injections, two groups were isolated for 24 hours without heroin or saline before they were euthanized for analysis to represent the ongoing heroin intake with short-term withdrawal phenotype of OUD. The rest of the injected groups were isolated for another 30 days before they were split again and injected with either saline or heroin to represent first-time heroin, abstinence after chronic intake, and relapse phenotypes of OUD.

Through comparison of the OUD mice genomic data with control mice data, scientists observed distinct and substantial changes in transcriptional regulation due to heroin exposure. One specific example of changing gene expression occurred in a gene called Xlr3b, which was up-regulated in all six brain regions after heroin exposure. This gene, which is thought to control aspects of neurodevelopment and memory-related cognitive processes, serves as a primary target for heroin to influence drug-seeking behavior. 

Although there were a few other genes up-regulated in all brain regions, scientists determined that most of the transcriptional changes seen after drug exposure were region and context-specific. They found that genes in the medial prefrontal cortex, nucleus accumbens, and dorsal striatum regions had expression heavily influenced by the direct effects of heroin, while genes in the basolateral amygdala and ventral hippocampus regions saw changes in expression related to prolonged withdrawal from heroin and were involved in the contextual memory that drives drug-seeking behavior. Many of the most apparent changes in gene expression were seen in the ventral hippocampus and were related to relapse conditions. Relapse conditions are intrinsically tied to reward circuit changes, supported by a network of proteins called the Extracellular Matrix (ECM), which provides structural support and influences circuit function. Many of the observed changes in expression involved up-regulation of ECM functions, which changes the reward circuit to favor heroin intake and presents itself as a primary driver of OUD. 

With a greater understanding of the molecular changes associated with heroin use, scientists then compared their findings in mice to genomic information collected from humans who died of overdose to determine relevant and overlapping interventional targets. This comparison is extremely valuable as human genomic data can often be messy and difficult to sort through. This is because moderate changes in DNA expression happen in response to a plethora of environmental conditions throughout life, including things as simple as diet or climate, making it hard to pinpoint which changes were in response to drug use, and which were the result of other environmental conditions experienced throughout life. 

Through this comparative analysis, scientists identified 31 genes that showed similar up and down regulation in humans and mice with OUD. These similarities were especially prevalent in the medial prefrontal cortex and ventral tegmental, suggesting that transcriptional signatures in these areas capture significant causal mechanisms of OUD. One of these genes, E2F1, is especially significant as it has been implicated in abstinence from fentanyl and morphine within the nucleus accumbens, making it an ideal therapeutic target for OUD.

This comprehensive study enabled deep characterization of heroin-induced molecular changes that may promote drug-taking and relapse, which serves as an invaluable resource when developing future treatments of OUD. However, despite these groundbreaking discoveries, several outstanding questions remain. For one, further investigation is needed to explore the role of sex-specific differences in molecular reprogramming induced by opioids, as this study was conducted exclusively in male mice despite extensive documentation of sex-specific differences in drug experiences (Townsend et al., 2021). Additionally, further investigation into the transcriptional changes that occur between drug use and periods of abstinence is needed to better understand the nature and permanence of these changes. The ethical implications of using animal models to study addiction and translate findings to human populations also raise important considerations for future research.

© Copyright 2024 Department of Biology, Davidson College, Davidson, NC 28036

References 

1. Lee YK, Gold MS, Blum K, Thanos PK, Hanna C, Fuehrlein BS. Opioid use disorder: current trends and potential treatments. Front Public Health. 2024 Jan 25; 11:1274719. [DOI: https://doi.org/10.3389/fpubh.2023.1274719]
2. F. B. Ahmad, J. A. Cisewski, L. M. Rossen, P. Sutton, Provisional Drug Overdose Death Counts (National Center for Health Statistics, 2023).
3. B. P. Smyth, J. Barry, E. Keenan, K. Ducray, Lapse and relapse following inpatient treatment of opiate dependence. Ir. Med. J. 103, 176–179 (2010). 
4. Browne CJ, Godino A, Salery M, Nestler EJ. Epigenetic Mechanisms of Opioid Addiction. Biol Psychiatry. 2020 Jan 1;87(1): 22-33. [DOI: https://doi.org/10.1016/j.biopsych.2019.06.027]
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6. Bali P, Kenny PJ. Transcriptional mechanisms of drug addiction
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8. Townsend EA, Kim RK, Robinson HL, Marsh SA, Banks ML, Hamilton PJ. Opioid withdrawal produces sex-specific effects on fentanyl-vs.-food choice and mesolimbic transcription. Biol Psychiatry Glob Open Sci. 2021 Aug;1(2):112-122. [DOI: https://doi.org/10.1016/j.bpsgos.2021.04.009]