Opioid use disorder (OUD) is a chronic psychiatric condition marked by increasing and out of control drug intake despite negative consequences, frequent periods of abstinence and relapse, and too often, fatal overdose. Despite the tremendous attention that OUD has received in recent years from the medical and research communities, the rates of overdose and relapse have only continued to increase (Hedegaard et al., 2018). While there are currently available pharmacotherapies to reduce the burden of OUD, these treatments remain ineffective or intolerable for too many; additionally, societal stigma, ineffective medical training, and healthcare disparities result in a substantial number of patients unsuccessfully abstaining from opioid use (Kreek et al., 2019; Pessar et al., 2021; Dydyk et al., 2022). Identification of novel targets for development of therapeutics is a critical need for the field.

While the precise neurobiology underlying substance use disorders remains to be fully understood, there is a robust body of research demonstrating a key role for transcriptomic and epigenetic effects underlying behavioral responses to opioids and other drugs of abuse. Prolonged exposure to drugs of abuse leads to transcriptomic changes in key limbic nuclei that can persist long after the last drug exposure (McClung and Nestler, 2003; Robison and Nestler, 2011; Eipper-Mains et al., 2013). These changes in gene expression are associated with functional changes in critical brain regions and can drive drug taking and seeking behaviors. Recent work has attributed the persistent effects of drug use to changes in activity of transcription factors, as well as epigenetic writers and erasers, which alter the propensity of genes to be transcribed in response to a stimulus (Renthal et al., 2009; Walker et al., 2015; Nestler et al., 2016; Browne et al., 2020). The bulk of this mechanistic work has been performed in animal models of OUD and other substance use disorders; however similar effects have also been demonstrated in human patients with OUD (Egervari et al., 2017).

Most research examining transcriptional and epigenetic regulation in models of OUD and other substance use disorders has focused on the central nervous system (CNS). However, recent research suggests peripheral factors can play a key role in maintaining CNS transcriptional homeostasis. Work over the past 10 years has determined that the population of bacteria that occupy the intestinal tract, collectively called the gut microbiome, is an important regulator of the transcriptional landscape in the brain. Germ-free (GF) mice that are raised in a fully sterile environment with no microbiome have markedly altered transcriptional patterns in multiple brain regions, including the frontal cortex and amygdala (Gacias et al., 2016; Hoban et al., 2016, Hoban et al., 2018; Chu et al., 2019). Specifically, the microglia of GF mice have significantly different transcriptomic and epigenetic profiles, which are at least partially reversible by colonization with a normal microbiome (Erny et al., 2015; Thion et al., 2018). This effect is not just seen in mice that have never had a microbiome. Adult mice treated with broad spectrum antibiotics (Abx) to reduce the bulk and complexity of the microbiome have altered transcriptional regulation in multiple brain regions as well. For example, single nucleus sequencing of Abx treated mice shows regulation of numerous genes in all neuronal and glial cell types in the prefrontal cortex (Chu et al., 2019).

Importantly, work from our group and others has identified robust interactions between the microbiome and behavioral and transcriptional response to opioids. Numerous reports have found that depletion of the microbiome alters development of tolerance to opioids and opioid withdrawal symptoms (Lee et al., 2018; Wang et al., 2018; Zhang et al., 2019; Jalodia et al., 2022). We recently reported that an Abx cocktail significantly reduces locomotor sensitization and conditioned place preference for morphine across a wide dose range (Hofford et al., 2021). Depletion of the microbiome with Abx also enhances self-administration of both oxycodone and fentanyl (Hofford et al., 2022; Simpson et al., 2022). In our recent work, we have found significant transcriptional interactions in the nucleus accumbens (NAc) between microbiome status and morphine treatment. Microbiome depletion without drug exposure only produced modest changes in gene expression in the NAc, but combined microbiome depletion and repeated morphine treatment robustly dysregulated transcriptome-wide gene expression (Hofford et al., 2021). Behavioral and transcriptional effects of microbiome depletion were largely reversible via supplementation with a cocktail of microbiome derived metabolites, the short chain fatty acids (SCFAs), suggesting these bacterial byproducts as potential mechanisms of this gut-brain signaling.

While our previous study provided critical information about how manipulations of the microbiome in adult animals can affect transcriptional regulation in response to opioids, mice treated with Abx still have some populations of antibiotic resistant bacteria in the gut, and growth of non-bacterial microorganisms can be affected by depletion of colonic bacteria. Here, we present an analysis of the NAc transcriptome from GF mice treated with saline or morphine for seven days to provide insight into the effects of lifelong total depletion of a microbiome on transcriptional regulation in this key limbic reward structure. Given our previous findings of robust microbiome-induced transcriptional changes in this region (Hofford et al., 2021), and the extensive literature showing the importance of the NAc in driving addiction-like behaviors (Robison and Nestler, 2011), the NAc seemed an appropriate target for more in depth analysis. These new analyses are integrated with the transcriptomic analyses from our previous study to identify patterns of gene expression induced by long-term GF status versus depletion of the microbiome in early adulthood. We find that there are marked changes in the transcriptome of GF mice compared to conventional mice after saline treatment, but show that GF mice have only modest changes in gene expression following morphine. These findings provide critical additional information about the role of the microbiome in modulating gene expression in the CNS, including across development.