The gut microbiota mediates reward and sensory responses associated with regimen-selective morphine dependence

Abstract

Opioid use for long-term pain management is limited by adverse side effects, such as hyperalgesia and negative affect. Neuroinflammation in the brain and spinal cord is a contributing factor to the development of symptoms associated with chronic opioid use. Recent studies have described a link between neuroinflammation and behavior that is mediated by a gut-brain signaling axis, where alterations in indigenous gut bacteria contribute to several inflammation-related psychopathologies. As opioid receptors are highly expressed within the digestive tract and opioids influence gut motility, we hypothesized that systemic opioid treatment will impact the composition of the gut microbiota. Here, we explored how opioid treatments, and cessation, impacts the mouse gut microbiome and whether opioid-induced changes in the gut microbiota influences inflammation-driven hyperalgesia and impaired reward behavior. Male C57Bl6/J mice were treated with either intermittent or sustained morphine. Using 16S rDNA sequencing, we describe changes in gut microbiota composition following different morphine regimens. Manipulation of the gut microbiome was used to assess the causal relationship between the gut microbiome and opioid-dependent behaviors. Intermittent, but not sustained, morphine treatment was associated with microglial activation, hyperalgesia, and impaired reward response. Depletion of the gut microbiota via antibiotic treatment surprisingly recapitulated neuroinflammation and sequelae, including reduced opioid analgesic potency and impaired cocaine reward following intermittent morphine treatment. Colonization of antibiotic-treated mice with a control microbiota restored microglial activation state and behaviors. Our findings suggest that differing opioid regimens uniquely influence the gut microbiome that is causally related to behaviors associated with opioid dependence.

Fig. 1

Morphine withdrawal alters microglial morphology, hyperalgesia, and impaired reward behavior. a Experimental design. Top: theoretical drug plasma levels in intermittent morphine and continuous morphine groups over four days. Bottom: timeline of experiments, starting with four-day opioid treatment regimen followed by (1) ten days of cocaine-conditioned place preference testing, (2) evoked pain testing, or (3) immunostaining. b Morphine dose–response assayed using thermal tail withdrawal compared in animals pretreated with saline injection or pellet and morphine injection or pellet (error bars = S.E.M., *p = 0.04, ***p < 0.0001, n = 7–8). Right histograms show EC₅₀ values for treatment groups compared with Mann–Whitney tests (error bars = S.E.M., n = 7–8). c Baseline thermal withdrawal thresholds assayed 12 h after the last morphine injection in animals pretreated with saline or morphine injection, saline or morphine pellet, or morphine pellet 24 h after pellet removal. Groups compared with Student’s t-test or one-way ANOVA (error bars = S.E.M., n = 7–8). d Conditioned place preference to cocaine in animals pretreated with intermittent and sustained morphine. Left: representative heat maps illustrating time spent in either saline-paired or cocaine-paired chamber. Right: Group means compared using one-way ANOVA followed by Sidak’s multiple comparison tests. e Impact of sustained and intermittent morphine on microglia morphology. Top: representative immunomicrographs of microglia from saline injection and morphine injection groups in the ventral tegmental area (VTA) and dorsal spinal cord (inset box = 50 µm by 50 µm). Bottom: Graphs represent quantification of 3–4 brain and spinal cord sections per animal. Groups were compared using one-way ANOVA (error bars = S.E.M, n = 4–16)

Fig. 2

Intermittent and sustained morphine uniquely influence beta-diversity of the gut microbiota. a Timeline outlining experiment, starting with four-day opioid treatment regimen followed by fecal pellet collection. b Richness of the gut microbiota is illustrated by rarefaction curves plotting observed taxonomic units (OTUs) versus the number of sequences per treatment group, n = 4–8 per group. c Principal coordinates analysis of the fecal microbiota from intermittent and sustained morphine-treated mice, n = 4–8. d Percent relative abundance of OTUs classified at the class level per treatment group. e Relative abundance (ratio) of the most abundant phyla Bacteroidetes and Firmicutes from samples treated with intermittent or sustained morphine. Groups were compared using a Mann–Whitney test (error bars = S.E.M.). f Relative abundance (number of reads/total reads) of key OTUs in the Lactobacillus, Ruminococcus, Clostridium genera, and the Rikenellaceae family from samples obtained from animals treated with intermittent or sustained morphine. Groups were compared using Kruskal–Wallis followed by Bonferroni test (error bars = S.E.M.)

Fig. 3

Oral antibiotics alters microglial morphology and leads to hyperalgesia and impairments in reward behavior. a Timeline outlining experiments, starting with four days of antibiotic and opioid treatment followed by (1) evoked pain testing then immunostaining or (2) ten days of cocaine condition place preference testing, with antibiotic treatment continuing through both experiment. b Fecal microbiome diversity before and after antibiotic treatment c Impact of antibiotic treatment on morphine analgesia. Left: morphine dose–response curves of water-treated (control), antibiotic-treated, and antibiotic-treated + morphine injection groups compared using two-way ANOVA (error bars = S.E.M., *p = 0.0291. ***p < 0.0001, n = 6). Right: Calculated EC₅₀ for each group, and compared with Kruskal–Wallis test followed by post hoc Dunn’s multiple comparisons test (error bars = S.E.M., n = 4–6). d Baseline tail flick responses compared with Kruskal–Wallis test followed by post hoc Dunn’s multiple comparisons test (error bars = S.E.M., n = 6). e Impact of antibiotic treatment on cocaine-conditioned place preference (CPP). Top: representative heat maps illustrating time spent in either saline-paired or cocaine-paired chamber. Bottom: Cocaine place preference scores for each group compared with a one-way ANOVA followed by a post hoc Sidak’s multiple comparison test. f Impact of antibiotic treatment on microglial morphology in the ventral tegmental area (VTA) and dorsal horn of the spinal cord. Top: representative immunomicrographs from water and antibiotic-treated groups (inset box = 50 µm by 50 µm). Bottom: Quantification from 3–6 sections per animal. Groups were compared with one-way ANOVA tests followed by post hoc Dunn’s multiple comparisons tests (error bars = S.E.M., n = 6)

Fig. 4

Gut microbiome recolonization from naive mice donors, but not morphine-dependent donors, restores normal reward behavior, and microglia morphology in antibiotic-treated mice. a Timeline outlining experiments, starting with four days of antibiotic treatment, followed by four days of microbiome recolonization after oral gavage, fecal pellet collection, six days of cocaine-conditioned place preference, and immunostaining. b Fecal microbiome diversity following microbiome recolonization. c Baseline tail withdrawal after antibiotic treatment (microbiome depleted) and after oral recolonization with fecal pellets isolated from saline and intermittent morphine-treated mice. Groups compared with Student’s t-test (error bars = S.E.M, n = 14). d Cocaine place preference in saline and morphine recolonized groups. Top: representative heat maps illustrating time spent in either saline-paired or cocaine-paired chamber. Bottom: Quantification of cocaine place preferences. Groups compared with one-way ANOVA followed by a post hoc Sidak’s multiple comparison test (error bars = S.E.M, n = 6–8). e Impact of microbiome recolonization on microglial morphology in the ventral tegmental area (VTA) and dorsal horn of the spinal cord. Top: representative immunomicrographs from animals orally gavaged with fecal pellets isolated from saline-treated or morphine-dependent host animals (inset box = 50 µm by 50 µm). Bottom: Quantification of microglial morphology and number from 3–6 slices per animal. Groups were compared with Krukal–Wallis tests (error bars = S.E.M., n = 6–8)