Alterations in microbiome composition and metabolic byproducts drive behavioral and transcriptional responses to morphine

Abstract

Recent evidence has demonstrated that the gut microbiome has marked effects on neuronal function and behavior. Disturbances to microbial populations within the gut have been linked to myriad models of neuropsychiatric disorders. However, the role of the microbiome in substance use disorders remains understudied. Here we show that male mice with their gut microbiome depleted by nonabsorbable antibiotics (Abx) exhibit decreased formation of morphine conditioned place preference across a range of doses (2.5-15 mg/kg), have decreased locomotor sensitization to morphine, and demonstrate marked changes in gene expression within the nucleus accumbens (NAc) in response to high-dose morphine (20 mg/kg × 7 days). Replacement of short-chain fatty acid (SCFA) metabolites, which are reduced by microbiome knockdown, reversed the behavioral and transcriptional effects of microbiome depletion. This identifies SCFA as the crucial mediators of microbiome-brain communication responsible for the effects on morphine reward caused by microbiome knockdown. These studies add important new behavioral, molecular, and mechanistic insight to the role of gut-brain signaling in substance use disorders.

Fig. 1. Oral Abx alters the microbiome.

A Experimental timeline. B, C Abx reduces alpha diversity as measured by observed OTUs and the Shannon index. D Mice from Abx-Sal and Abx-Mor possess unique microbiomes, but H₂O-Sal and H₂O-Mor populations overlap as measured using the unweighted Unifrac distance. E Relative phylum abundance in all groups of mice, each phylum shown in a different color. F Heatmap displaying the fold change (left) and −log(p) value (right) of the top ten most abundant phyla in control mice. Fold change values in red have greater abundance in that group compared to H₂O-Sal and blue values have less abundance (left). Negative log(p) values in pink or red are significant (right). G Abx reduces levels of the caecel SCFA. Data shown as fold change from H₂O treated mice. Data presented as means ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.

Fig. 2. Microbiome knockdown reduces locomotor sensitization and morphine place preference.

A Abx did not alter the locomotor response to repeated morphine but did reduce sensitization (B). C Experimental timeline for 2-pairing CPP. D Abx treatment reduced high-dose morphine CPP. E Experimental timeline for 3-pairing CPP. F Abx mice did not demonstrate morphine CPP with three morphine-chamber pairings at a high dose (15 mg/kg). Data presented as means ± SEM. *p < 0.05.

Fig. 3. Morphine and Abx alter the NAc transcriptome.

A Experimental timeline for RNA-sequencing. B–D Volcano plots depicting differential gene expression between H₂O-Sal and Abx-Sal (B), H₂O-Mor (C), and Abx-Mor (D). Colored circles are significantly differentially regulated transcripts identified using an FDR corrected p < 0.05. E Representation of some top gene-ontology terms regulated in morphine groups. The x-axis is the FDR-corrected −log(p) value with the dotted line indicating an FDR corrected p < 0.05, and circle size represents number of genes per term. F Top differentially predicted transcription factors using Chea software analysis demonstrates factors with different predicted effects between groups. Y-axis is the overall transcription factor enrichment score predicted from genes in the dataset. G Cloud diagrams depicting top regulated transcription factors (hubs) and their predicted downstream targets (nodes).

Fig. 4. Microbiome knockdown alters the NAc transcriptional response to morphine.

A Venn diagram depicting number of differentially regulated genes of H₂O-Mor vs. H₂O-Sal (pink) and Abx-Mor vs. H₂O-Sal (purple). B Heatmap of genes that are differentially regulated by both H₂O-Mor and Abx-Mor. C Gene-ontology pathway analysis of overlapping genes. D Volcano plots depicting differential gene expression between Abx-Mor and H₂O-Mor. Colored circles are significantly differentially regulated transcripts identified using an FDR p < 0.05. E Gene-ontology pathways up and downregulated between Abx-Mor and H₂O-Mor. Pink pathways are upregulated, and blue pathways are downregulated. Dotted vertical line is the significance cut-off of FDR p < 0.05. F Heatmap depicting all significantly regulated histone modifiers between Abx-Mor and H₂O-Mor.

Fig. 5. Replenishment of SCFAs reverses reward deficit and gene expression changes caused by microbiome depletion.

A Experimental timeline for CPP after SCFA replenishment. SCFA supplementation restored morphine place preference in Abx-treated mice when trained at 10 mg/kg morphine with 2 conditioning days (B) or at 15 mg/kg morphine with 3 conditioning days (C). SCFA + Abx normalized gene expression for Egr2 (D) and Egr4 (E) but did not change expression of FosB (F) or Irf8 (G). Data are presented as mean ± SEM. *p < 0.05.