Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Oral Abx alters the microbiome.
Fig. 2: Microbiome knockdown reduces locomotor sensitization and morphine place preference.
Fig. 3: Morphine and Abx alter the NAc transcriptome.
Fig. 4: Microbiome knockdown alters the NAc transcriptional response to morphine.
Fig. 5: Replenishment of SCFAs reverses reward deficit and gene expression changes caused by microbiome depletion.

Data availability

All RNA-sequencing files will be uploaded to the publicly available Gene Expression Omnibus server and will be linked to this publication. All data in this paper will be made available upon reasonable request.


  1. 1.

    Substance Abuse and Mental Health Services Administration. The NSDUH report. Rockville, Md.: Office of Applied Studies, Substance Abuse and Mental Health Services Administration, Dept. of Health & Human Services; 2008.

  2. 2.

    Hedegaard H, Miniño AM, Warner M. Drug Overdose Deaths in the United States, 1999-2018. NCHS Data Brief. 2020;356:1–8.

    Google Scholar 

  3. 3.

    Schuckit MA. Treatment of opioid-use disorders. N Engl J Med. 2016;375:357–68.

    PubMed  Google Scholar 

  4. 4.

    Evans CJ, Cahill CM. Neurobiology of opioid dependence in creating addiction vulnerability. F1000Research. 2016;5:F1000 Faculty Rev-1748.

  5. 5.

    Chopra N, Marasa LH. The opioid epidemic: challenges of sustained remission. Int J Psychiatry Med. 2017;52:196–201.

    PubMed  Google Scholar 

  6. 6.

    Rea K, Dinan TG, Cryan JF. Gut microbiota: a perspective for psychiatrists. Neuropsychobiology. 2020;79:50–62.

    PubMed  CAS  Google Scholar 

  7. 7.

    Meckel KR, Kiraly DD. A potential role for the gut microbiome in substance use disorders. Psychopharmacology. 2019;236:1513–30.

    PubMed  PubMed Central  CAS  Google Scholar 

  8. 8.

    Adams JB, Johansen LJ, Powell LD, Quig D, Rubin RA. Gastrointestinal flora and gastrointestinal status in children with autism-comparisons to typical children and correlation with autism severity. BMC Gastroenterol. 2011;11:22.

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Cheung SG, Goldenthal AR, Uhlemann A-C, Mann JJ, Miller JM, Sublette ME. Systematic review of gut microbiota and major depression. Front Psychiatry. 2019;10:34.

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Cryan JF, O’Riordan KJ, Sandhu K, Peterson V, Dinan TG. The gut microbiome in neurological disorders. Lancet Neurol. 2020;19:179–94.

    PubMed  CAS  Google Scholar 

  11. 11.

    Cenit MC, Sanz Y, Codoñer-Franch P. Influence of gut microbiota on neuropsychiatric disorders. World J Gastroenterol. 2017;23:5486–98.

    PubMed  PubMed Central  CAS  Google Scholar 

  12. 12.

    Foster JA, Rinaman L, Cryan JF. Stress & the gut-brain axis: regulation by the microbiome. Neurobiol Stress. 2017;7:124–36.

    PubMed  PubMed Central  Google Scholar 

  13. 13.

    Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan TG, et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA. 2011;108:16050–5.

    PubMed  PubMed Central  CAS  Google Scholar 

  14. 14.

    Erny D, Hrabě de Angelis AL, Jaitin D, Wieghofer P, Staszewski O, David E, et al. Host microbiota constantly control maturation and function of microglia in the CNS. Nat Neurosci. 2015;18:965.

    PubMed  PubMed Central  CAS  Google Scholar 

  15. 15.

    Thion MS, Low D, Silvin A, Chen J, Grisel P, Schulte-Schrepping J, et al. Microbiome influences prenatal and adult microglia in a sex-specific manner. Cell. 2018;172:500–16.e16.

    PubMed  PubMed Central  CAS  Google Scholar 

  16. 16.

    Chu C, Murdock MH, Jing D, Won TH, Chung H, Kressel AM, et al. The microbiota regulate neuronal function and fear extinction learning. Nature. 2019;574:543–8.

    PubMed  PubMed Central  CAS  Google Scholar 

  17. 17.

    Hoban AE, Stilling RM, Ryan FJ, Shanahan F, Dinan TG, Claesson MJ, et al. Regulation of prefrontal cortex myelination by the microbiota. Transl Psychiatry. 2016;6:e774.

    PubMed  PubMed Central  CAS  Google Scholar 

  18. 18.

    Kiraly DD, Walker DM, Calipari ES, Labonte B, Issler O, Pena CJ, et al. Alterations of the host microbiome affect behavioral responses to cocaine. Sci Rep. 2016;6:35455.

    PubMed  PubMed Central  CAS  Google Scholar 

  19. 19.

    Cryan JF, Dinan TG. Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nat Rev Neurosci. 2012;13:701–12.

    PubMed  CAS  Google Scholar 

  20. 20.

    Koh A, De Vadder F, Kovatcheva-Datchary P, Bäckhed F. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites. Cell. 2016;165:1332–45.

    PubMed  CAS  Google Scholar 

  21. 21.

    Frost G, Sleeth ML, Sahuri-Arisoylu M, Lizarbe B, Cerdan S, Brody L, et al. The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism. Nat Commun. 2014;5:3611.

    PubMed  CAS  Google Scholar 

  22. 22.

    Sampson TR, Debelius JW, Thron T, Janssen S, Shastri GG, Ilhan ZE, et al. Gut microbiota regulate motor deficits and neuroinflammation in a model of Parkinson’s disease. Cell. 2016;167:1469–80.e12.

    PubMed  PubMed Central  CAS  Google Scholar 

  23. 23.

    Kratsman N, Getselter D, Elliott E. Sodium butyrate attenuates social behavior deficits and modifies the transcription of inhibitory/excitatory genes in the frontal cortex of an autism model. Neuropharmacology. 2016;102:136–45.

    PubMed  CAS  Google Scholar 

  24. 24.

    Davie JR. Inhibition of histone deacetylase activity by butyrate. J Nutr. 2003;133:2485S–93S.

    PubMed  CAS  Google Scholar 

  25. 25.

    Mews P, Egervari G, Nativio R, Sidoli S, Donahue G, Lombroso SI, et al. Alcohol metabolism contributes to brain histone acetylation. Nature. 2019;574:717–21.

    PubMed  PubMed Central  CAS  Google Scholar 

  26. 26.

    Li X, Egervari G, Wang Y, Berger SL, Lu Z. Regulation of chromatin and gene expression by metabolic enzymes and metabolites. Nat Rev Mol Cell Biol. 2018;19:563–78.

    PubMed  PubMed Central  CAS  Google Scholar 

  27. 27.

    Nankova BB, Agarwal R, MacFabe DF, La, Gamma EF. Enteric bacterial metabolites propionic and butyric acid modulate gene expression, including CREB-dependent catecholaminergic neurotransmission, in PC12 cells-possible relevance to autism spectrum disorders. PLoS ONE. 2014;9:e103740.

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Shah P, Nankova BB, Parab S, La Gamma EF. Short chain fatty acids induce TH gene expression via ERK-dependent phosphorylation of CREB protein. Brain Res. 2006;1107:13–23.

    PubMed  CAS  Google Scholar 

  29. 29.

    Parab S, Nankova BB, La Gamma EF. Differential regulation of the tyrosine hydroxylase and enkephalin neuropeptide transmitter genes in rat PC12 cells by short chain fatty acids: concentration-dependent effects on transcription and RNA stability. Brain Res. 2007;1132:42–50.

    PubMed  CAS  Google Scholar 

  30. 30.

    Mally P, Mishra R, Gandhi S, Decastro MH, Nankova BB, Lagamma EF. Stereospecific regulation of tyrosine hydroxylase and proenkephalin genes by short-chain fatty acids in rat PC12 cells. Pediatr Res. 2004;55:847–54.

    PubMed  CAS  Google Scholar 

  31. 31.

    Goswami C, Iwasaki Y, Yada T. Short-chain fatty acids suppress food intake by activating vagal afferent neurons. J Nutr Biochem. 2018;57:130–5.

    PubMed  CAS  Google Scholar 

  32. 32.

    Lal S, Kirkup AJ, Brunsden AM, Thompson DG, Grundy D. Vagal afferent responses to fatty acids of different chain length in the rat. Am J Physiol Liver Physiol. 2001;281:G907–15.

    CAS  Google Scholar 

  33. 33.

    Fernandes AB, Alves da Silva J, Almeida J, Cui G, Gerfen CR, Costa RM, et al. Postingestive modulation of food seeking depends on vagus-mediated dopamine neuron activity. Neuron. 2020;106:778–88.e6.

    PubMed  PubMed Central  CAS  Google Scholar 

  34. 34.

    Dalile B, Van Oudenhove L, Vervliet B, Verbeke K. The role of short-chain fatty acids in microbiota–gut–brain communication. Nat Rev Gastroenterol Hepatol. 2019;16:461–78.

    PubMed  Google Scholar 

  35. 35.

    Taylor AMW, Castonguay A, Ghogha A, Vayssiere P, Pradhan AAA, Xue L, et al. Neuroimmune regulation of GABAergic neurons within the ventral tegmental area during withdrawal from chronic morphine. Neuropsychopharmacology. 2015;41:949.

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Zhang L, Meng J, Ban Y, Jalodia R, Chupikova I, Fernandez I, et al. Morphine tolerance is attenuated in germfree mice and reversed by probiotics, implicating the role of gut microbiome. Proc Natl Acad Sci USA. 2019;116:13523 LP–32.

    Google Scholar 

  37. 37.

    Kang M, Mischel RA, Bhave S, Komla E, Cho A, Huang C, et al. The effect of gut microbiome on tolerance to morphine mediated antinociception in mice. Sci Rep. 2017;7:42658.

    PubMed  PubMed Central  CAS  Google Scholar 

  38. 38.

    Simpson S, Kimbrough A, Boomhower B, McLellan R, Hughes M, Shankar K, et al. Depletion of the microbiome alters the recruitment of neuronal ensembles of oxycodone intoxication and withdrawal. ENeuro. 2020;7:ENEURO.0312-19.2020.

  39. 39.

    Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–3.

    PubMed  PubMed Central  CAS  Google Scholar 

  40. 40.

    Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37:852–7.

    PubMed  PubMed Central  CAS  Google Scholar 

  41. 41.

    Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–6.

    PubMed  CAS  Google Scholar 

  42. 42.

    Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.

    PubMed  CAS  Google Scholar 

  43. 43.

    Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47.

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Torre D, Lachmann A, Ma’ayan A. BioJupies: automated generation of interactive notebooks for RNA-Seq data analysis in the cloud. Cell Syst. 2018;7:556–61.e3.

    PubMed  PubMed Central  CAS  Google Scholar 

  45. 45.

    Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 2019;47:W191–8.

    PubMed  PubMed Central  CAS  Google Scholar 

  46. 46.

    Lu J, Synowiec S, Lu L, Yu Y, Bretherick T, Takada S, et al. Microbiota influence the development of the brain and behaviors in C57BL/6J mice. PLoS ONE. 2018;13:e0201829.

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    Banerjee S, Sindberg G, Wang F, Meng J, Sharma U, Zhang L, et al. Opioid-induced gut microbial disruption and bile dysregulation leads to gut barrier compromise and sustained systemic inflammation. Mucosal Immunol. 2016;9:1418–28.

    PubMed  PubMed Central  CAS  Google Scholar 

  48. 48.

    Wang F, Meng J, Zhang L, Johnson T, Chen C, Roy S. Morphine induces changes in the gut microbiome and metabolome in a morphine dependence model. Sci Rep. 2018;8:3596.

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Lee K, Vuong HE, Nusbaum DJ, Hsiao EY, Evans CJ, Taylor AMW. The gut microbiota mediates reward and sensory responses associated with regimen-selective morphine dependence. Neuropsychopharmacology. 2018;43:2606–14.

    PubMed  PubMed Central  CAS  Google Scholar 

  50. 50.

    Louis P, Flint HJ. Formation of propionate and butyrate by the human colonic microbiota. Environ Microbiol. 2017;19:29–41.

    PubMed  CAS  Google Scholar 

  51. 51.

    Louis P, Flint HJ. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol Lett. 2009;294:1–8.

    PubMed  CAS  Google Scholar 

  52. 52.

    Douglas GM, Maffei VJ, Zaneveld J, Yurgel SN, Brown JR, Taylor CM, et al. PICRUSt2: an improved and extensible approach for metagenome inference. BioRxiv. 2019.

  53. 53.

    Zito KA, Vickers G, Roberts DC. Disruption of cocaine and heroin self-administration following kainic acid lesions of the nucleus accumbens. Pharm Biochem Behav. 1985;23:1029–36.

    CAS  Google Scholar 

  54. 54.

    Kelsey JE, Carlezon WA, Falls WA. Lesions of the nucleus accumbens in rats reduce opiate reward but do not alter context-specific opiate tolerance. Behav Neurosci. 1989;103:1327–34.

    PubMed  CAS  Google Scholar 

  55. 55.

    Lachmann A, Xu H, Krishnan J, Berger SI, Mazloom AR, Ma’ayan A. ChEA: transcription factor regulation inferred from integrating genome-wide ChIP-X experiments. Bioinformatics. 2010;26:2438–44.

    PubMed  PubMed Central  CAS  Google Scholar 

  56. 56.

    Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinform. 2013;14:128.

    Google Scholar 

  57. 57.

    Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016;44:W90–7.

    PubMed  PubMed Central  CAS  Google Scholar 

  58. 58.

    Hulsen T, de Vlieg J, Alkema W. BioVenn—a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genom. 2008;9:488.

    Google Scholar 

  59. 59.

    Nanda JS, Kumar R, Raghava GPS. dbEM: a database of epigenetic modifiers curated from cancerous and normal genomes. Sci Rep. 2016;6:19340.

    Google Scholar 

  60. 60.

    Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M, et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science. 2013;341:569–73.

    PubMed  CAS  Google Scholar 

  61. 61.

    Waldecker M, Kautenburger T, Daumann H, Busch C, Schrenk D. Inhibition of histone-deacetylase activity by short-chain fatty acids and some polyphenol metabolites formed in the colon. J Nutr Biochem. 2008;19:587–93.

    PubMed  CAS  Google Scholar 

  62. 62.

    Soliman ML, Rosenberger TA. Acetate supplementation increases brain histone acetylation and inhibits histone deacetylase activity and expression. Mol Cell Biochem. 2011;352:173–80.

    PubMed  CAS  Google Scholar 

  63. 63.

    Fellows R, Denizot J, Stellato C, Cuomo A, Jain P, Stoyanova E, et al. Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases. Nat Commun. 2018;9:105.

    PubMed  PubMed Central  Google Scholar 

  64. 64.

    Hoban AE, Stilling RM, Moloney G, Shanahan F, Dinan TG, Clarke G, et al. The microbiome regulates amygdala-dependent fear recall. Mol Psychiatry. 2018;23:1134–44.

    PubMed  CAS  Google Scholar 

  65. 65.

    Bardo MT, Bevins RA. Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology. 2000;153:31–43.

    PubMed  CAS  Google Scholar 

  66. 66.

    Kim S, Kaang B-K. Epigenetic regulation and chromatin remodeling in learning and memory. Exp Mol Med. 2017;49:e281.

    PubMed  PubMed Central  Google Scholar 

  67. 67.

    Sanchis-Segura C, Lopez-Atalaya JP, Barco A. Selective boosting of transcriptional and behavioral responses to drugs of abuse by histone deacetylase inhibition. Neuropsychopharmacology. 2009;34:2642.

    PubMed  CAS  Google Scholar 

  68. 68.

    Sheng J, Lv gang Z, Wang L, Zhou Y, Hui B. Histone H3 phosphoacetylation is critical for heroin-induced place preference. Neuroreport. 2011;22:575–80.

    PubMed  CAS  Google Scholar 

Download references


Morphine sulfate was provided by the NIDA Drug Supply Program. We acknowledge the Microbial Culture & Metabolomics Core of the PennCHOP Microbiome Program for performing targeted metabolomics analyses. Parts of Figs. 1, 2, 3, and 5 were created with with full permission to publsih.

Author information




DDK and RSH designed the experiments. RSH, NLM, TJE, KRM, ATO, and DDK performed experiments. RSH, TJE, KRM, ATO, and DDK analyzed data. RSH and DDK wrote the paper. All authors provided critical edits and feedback of the finalized paper.

Corresponding author

Correspondence to Drew D. Kiraly.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hofford, R.S., Mervosh, N.L., Euston, T.J. et al. Alterations in microbiome composition and metabolic byproducts drive behavioral and transcriptional responses to morphine. Neuropsychopharmacol. 46, 2062–2072 (2021).

Download citation

Further reading


Quick links