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. 2024 Feb 15;11(2):ENEURO.0388-23.2023.
doi: 10.1523/ENEURO.0388-23.2023. Print 2024 Feb.

Microbiome Depletion Increases Fentanyl Self-Administration and Alters the Striatal Proteome Through Short-Chain Fatty Acids

Rebecca S Hofford  1   2   3 , Katherine R Meckel  3   4 , Elizabeth J Wiser  1 , Weiwei Wang  5 , Jonathon P Sens  1 , Michelle Kim  3   4 , Arthur Godino  3   4 , TuKiet T Lam  5   6   7 , Drew D Kiraly  8   2   3   4   9
Affiliations

Microbiome Depletion Increases Fentanyl Self-Administration and Alters the Striatal Proteome Through Short-Chain Fatty Acids

Rebecca S Hofford et al. eNeuro. .

Abstract

Opioid use disorder (OUD) is a public health crisis currently being exacerbated by increased rates of use and overdose of synthetic opioids, primarily fentanyl. Therefore, the identification of novel biomarkers and treatment strategies to reduce problematic fentanyl use and relapse to fentanyl taking is critical. In recent years, there has been a growing body of work demonstrating that the gut microbiome can serve as a potent modulator of the behavioral and transcriptional responses to both stimulants and opioids. Here, we advance this work to define how manipulations of the microbiome drive fentanyl intake and fentanyl-seeking in a translationally relevant drug self-administration model. Depletion of the microbiome of male rats with broad spectrum antibiotics leads to increased drug administration on increased fixed ratio, progressive ratio, and drug seeking after abstinence. Utilizing 16S sequencing of microbiome contents from these animals, specific populations of bacteria from the gut microbiome correlate closely with levels of drug taking. Additionally, global proteomic analysis of the nucleus accumbens following microbiome manipulation and fentanyl administration to define how microbiome status alters the functional proteomic landscape in this key limbic substructure. These data demonstrate that an altered microbiome leads to marked changes in the synaptic proteome in response to repeated fentanyl treatment. Finally, behavioral effects of microbiome depletion are reversible by upplementation of the microbiome derived short-chain fatty acid metabolites. Taken together, these findings establish clear relevance for gut-brain signaling in models of OUD and lay foundations for further translational work in this space.

Keywords: fentanyl; microbiome; proteomics; self-administration.

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Figures

Figure 1.
Figure 1.
Abx increases motivation to self-administer fentanyl. A, Experimental timeline for Experiment 1. B, H2O-IncFR and Abx-IncFR rats acquired FR1 fentanyl administration at equal rates. C, At increasing FR requirements Abx caused enhanced responding. Inset shows number of infusions earned. D, Abx-IncFR rats had higher breakpoints on a progressive ratio task. E, Return to FR1 normalized responding between groups over two sessions. F, Abx treatment increased fentanyl-seeking after withdrawal in Abx-IncFR rats. G–K, Abx did not affect self-administration of saline. L–P, As a control, another group of rats was maintained on FR1 responding throughout (H2O-FR1, Abx-FR1), and there were no differences in acquisition of self-administration, maintenance of self-administration, progressive ratio, post-PR FR1 responding, or fentanyl-seeking. Data presented as means ± SEM. * p < 0.05, ** p < 0.01. Full statistical results for active and inactive lever presses are included in Tables 1 and 2.
Figure 2.
Figure 2.
Abx treatment does not have effects on body weight. Changes in bodyweight for all groups for the first two weeks of all groups was measured in fentanyl (A) and saline (B) administering rats. There were no main effects or interactions seen due to Abx treatment.
Figure 3.
Figure 3.
Abx shifts the dose-response curve. A, Experimental timeline for Experiment 2. B, Abx did not affect acquisition of self-administration. C, However, there was a significant interaction between dose and Abx treatment; post hoc test indicated a significant difference between groups at the 0.25 microg/kg/infusion dose. Data presented as means ± SEM. ***p < 0.001. Full statistical results for active and inactive lever presses are included in Tables 1 and 2.
Figure 4.
Figure 4.
Abx alters the microbiome. A, Experimental timeline for Experiment 1 and microbiome collection. Abx reduced microbiome diversity as measured by the number of OTUs (B) and Simpson (C) indices. (D) The microbiomes of H2O and Abx rats differed markedly when assessed with either an unweighted (left) or weighted (right) Unifrac dissimilarity matrix. Microbial composition was driven primarily by Abx treatment. E, Donut plots of bacterial phyla abundance in all six groups of rats. F, Heatmap of phyla abundance as fold change from H2O Sal in fentanyl-administering groups of rats. ****p < 0.0001. Data presented as means ± SEM. Full statistical results for diversity indices and phylum abundance are included in Extended Data Tables 4-1 and 4-2.
Figure 5.
Figure 5.
Abundance of several genera correlate with fentanyl intake in rats with an intact microbiome. Correlation plots of H2O fentanyl rats’ fentanyl intake with relative abundance of (A) Ruminococcus, (B) Butyricicoccus, (C) Lachnospiraceae_unclassified, and (D) Anaerotignum. Insets: percent abundance of each genus in H2O and Abx groups, collapsed across administration paradigm (FR1 and IncFR combined). ***p < 0.001, ***p < 0.0001. Data presented as means ± SEM. Full list of correlations between fentanyl intake and genus abundance are in Extended Data Table 5-1.
Figure 6.
Figure 6.
Microbiome knockdown alters the nucleus accumbens proteome. A, Experimental timeline for Experiment 1 and NAc collection. B, Volcano plots depicting protein expression changes in rats compared to H2O Sal. Colored points are significantly regulated proteins (FDR-corrected p < 0.1). C, Select pathways that are predicted to be regulated in the same direction in H2O-IncFR and Abx-IncFR groups. Dotted line at 1.3 indicates significance (FDR-corrected p < 0.05). D, Predicted pathways from proteins upregulated in all fentanyl-administering groups but not present in Abx-Sal. E, Heatmap of fold change protein expression of overlapping upregulated proteins in fentanyl-administering rats. F, Select pathways that are oppositely regulated in H2O and Abx groups. Note X-axis is z-score; all pathways in all groups are significant (FDR-corrected p < 0.05). G, Cloud diagrams of proteins in the “synaptogenesis signaling pathway”. Each diagram presents the same configuration of proteins with differences between groups indicated by the colored protein nodes. (H) Transcription factors predicted to be upstream from downregulated proteins (left) and upregulated proteins (right). Full proteomics comparisons, pathway analyses, and predicted transcription factors are available in Extended Data Tables 6 - 1-7.
Figure 7.
Figure 7.
Supplementation with short chain fatty acids (SCFA) reverses behavioral effects of microbiome depletion. A, Experimental schematic for Experiment 3. B, Control and Abx + SCFA treated rats acquired FR1 administration at the same rate. C, When the fixed ratio was increased H2O and Abx + SCFA rats maintained similar levels of responding. Inset is infusions earned. D, Progressive ratio breakpoint was not different between H2O and Abx + SCFA rats. When comparing the H2O control groups from Experiments 1 & 3 there was no difference in responding on increased fixed ratio (E) or progressive ratio (F) responding. Examination of all groups from Experiments 1 & 3 showed main effect of group with increased responding in the Abx group on the increased fixed ratio (G) and progressive ratio (H) tasks. *p < 0.05; **p < 0.01.
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