Long access heroin self-administration significantly alters gut microbiome composition and structure

Abstract

Introduction: It is well known that chronic opioid use disorder is associated with alterations in gastrointestinal (GI) function that include constipation, reduced motility, and increased bacterial translocation due to compromised gut barrier function. These signs of disrupted GI function can be associated with alterations in the gut microbiome. However, it is not known if long-access opioid self-administration has effects on the gut microbiome.

Methods: We used 16S rRNA gene sequencing to investigate the gut microbiome in three independent cohorts (N=40 for each) of NIH heterogeneous stock rats before onset of long-access heroin self-administration (i.e., naïve status), at the end of a 15-day period of self-administration, and after post-extinction reinstatement. Measures of microbial α- and β-diversity were evaluated for all phases. High-dimensional class comparisons were carried out with MaAsLin2. PICRUSt2 was used for predicting functional pathways impacted by heroin based on marker gene sequences.

Results: Community α-diversity was not altered by heroin at any of the three phases by comparison to saline-yoked controls. Analyses of β-diversity showed that the heroin and saline-yoked groups clustered significantly apart from each other using the Bray-Curtis (community structure) index. Heroin caused significant alterations at the ASV level at the self-administration and extinction phases. At the phylum level, the relative abundance of Firmicutes was increased at the self-administration phase. Deferribacteres was decreased in heroin whereas Patescibacteria was increased in heroin at the extinction phase. Potential biomarkers for heroin emerged from the MaAsLin2 analysis. Bacterial metabolomic pathways relating to degradation of carboxylic acids, nucleotides, nucleosides, carbohydrates, and glycogen were increased by heroin while pathways relating to biosynthesis of vitamins, propionic acid, fatty acids, and lipids were decreased.

Discussion: These findings support the view that long access heroin self-administration significantly alters the structure of the gut microbiome by comparison to saline-yoked controls. Inferred metabolic pathway alterations suggest the development of a microbial imbalance favoring gut inflammation and energy expenditure. Potential microbial biomarkers and related functional pathways likely invoked by heroin self-administration could be targets for therapeutic intervention.

Keywords: acquisition; extinction; gut microbiome; heroin; microbial diversity; reinstatement.

Figure 1

Experimental timeline and indicators of heroin addiction-like behavior. Experimental timeline indicating the three experimental phases of the study (naïve, self-administration, and extinction), self-administration training sessions (green bars), and stool sample collections over the study duration (A). Number of heroin infusions (one infusion = 20 µg/kg of body weight) for male and female rats over 15 training sessions that are indicated with ribbons (mean ± standard error of the mean) (B). Number of heroin infusions by sex during the progressive ratio test (mean ± standard error of the mean) (C). Active lever presses for male and female rats at three distinct timepoints (end of self-administration, extinction and cue induced reinstatement). Data for active lever presses are an average over training sessions 13-15 for the self-administration timepoint, and single day session measurements for the extinction and cued-induced reinstatement timepoints (median ± interquartile range; **p < 0.005, ****p < 0.00005, paired Wilcoxon) (D), similar to the previous panels.

Figure 2

Effects of heroin self-administration on α-diversity. The α-diversity metrics Chao-1 (A), Shannon (B) and Inverse Simpson (C) were determined for 16S rRNA gene sequencing profiles of rat fecal samples from each of the experimental phases. Data are presented as median ± interquartile range (line through box, respectively) and include data from each subject. No statistical significances were detected.

Figure 3

Effects of heroin self-administration on β-diversity. Principal Coordinates Analysis (PCoA) plots of the bacterial profiles from the 16S rRNA gene sequencing at the naïve (A), self-administration (B), and extinction (C) phases. Percentages along the X and Y axes indicate the amount of variance explained for principal coordinates 1 through 3, respectively. PCoAs were generated using the Bray-Curtis index. Ellipses indicate 80% confidence intervals and individual data points are color-coded by group (blue = saline-yoked controls; red = heroin self-administration). The plot orientations were chosen for clarity of the data in three-dimensional space.

Figure 4

Effects of heroin self-administration on the β-diversity of the gut microbiome in males and females. Principal Coordinates Analysis (PCoA) plots of the bacterial profiles from the 16S rRNA gene sequencing at the naïve (A), self-administration (B), and extinction (C) phases. Plots represent the same data as in Figure 2 , but are color-coded by sex (red circles = females; blue boxes = males). Percentages along the X and Y axes indicate the amount of variance explained for principal coordinates 1 through 3, respectively. PCoAs were generated using the Bray-Curtis index. Ellipses indicate 80% confidence intervals and are color-coded by sex. The plot orientations were chosen for clarity of the data in three-dimensional space.

Figure 5

Bacterial taxa differentially abundant after heroin self-administration. Relative abundance of taxa identified as increased or decreased after the heroin self-administration phase when compared to saline-yoked controls (A). Relative abundance of taxa differentially abundant following the extinction phase (B). Bars indicate mean relative abundance ± SEM. All taxa depicted are statistically significant (q < 0.05) as assessed via MaAsLin2.

Figure 6

Bacterial taxa differentially abundant after heroin self-administration by sex. Relative abundance of taxa identified as increased or decreased in males and females after the heroin self-administration phase (A). Relative abundance of taxa differentially abundant in males and females following the extinction phase (B). Bars indicate mean relative abundance ± SEM. All taxa depicted are statistically significant (q < 0.05) as assessed via MaAsLin2.

Figure 7

Effects of different phases of heroin self-administration on bacterial phyla. Relative abundance of the heroin and saline-yoked groups at the naïve (A), self-administration (B), and extinction (C) phases. Data are presented as mean relative abundance. The symbols indicate: *q < 0.05 for Firmicutes at the self-administration phase; ^† q< 0.005 for Deferribacteres at the extinction phase; and ^‡q < 0.05 for Patescibacteria at the extinction phase.

Figure 8

Effects of different phases of heroin self-administration on bacterial phyla in males and females. Relative abundance of bacterial phyla in males and females at the naïve (A), self-administration (B), and extinction (C) phases. Data are presented as mean relative abundance. The symbols indicate: *q < 0.05 for Spirochaetes at the extinction phase; ^†q < 0.005 for Actinobacteria at the self-administration and extinction phases; ^‡q < 0.005 for Verrucomicrobia at the naïve phase, and ^§q < 0.05 for Tenericutes at the extinction phase.

Figure 9

Differences in the predicted functions of the metagenome from 16S rRNA gene sequencing of fecal samples from rats exposed to heroin. Functional pathways were annotated based on the individual MetaCyc Superpathways, showing the mean proportion (%) of the pathways identified (A) and the magnitude of difference in the pathway proportion between heroin and saline-yoked (B). Horizontal lines through individual data points in (B) indicate standard error of the mean. Negative values indicate pathways less abundant in heroin relative to saline-yoked (blue); Positive values indicate pathways more abundant in heroin (red).