Bacterial colonisation of the intestine has a major role in the post-natal development and maturation of the immune and endocrine systems. These processes are key factors underpinning central nervous system (CNS) signalling. Regulation of the microbiome–gut–brain axis is essential for maintaining homeostasis, including that of the CNS. However, there is a paucity of data pertaining to the influence of microbiome on the serotonergic system. Germ-free (GF) animals represent an effective preclinical tool to investigate such phenomena. Here we show that male GF animals have a significant elevation in the hippocampal concentration of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, its main metabolite, compared with conventionally colonised control animals. Moreover, this alteration is sex specific in contrast with the immunological and neuroendocrine effects which are evident in both sexes. Concentrations of tryptophan, the precursor of serotonin, are increased in the plasma of male GF animals, suggesting a humoral route through which the microbiota can influence CNS serotonergic neurotransmission. Interestingly, colonisation of the GF animals post weaning is insufficient to reverse the CNS neurochemical consequences in adulthood of an absent microbiota in early life despite the peripheral availability of tryptophan being restored to baseline values. In addition, reduced anxiety in GF animals is also normalised following restoration of the intestinal microbiota. These results demonstrate that CNS neurotransmission can be profoundly disturbed by the absence of a normal gut microbiota and that this aberrant neurochemical, but not behavioural, profile is resistant to restoration of a normal gut flora in later life.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Journal of Biomedical Science Open Access 28 November 2023
Annals of General Psychiatry Open Access 27 September 2023
The long-term gut bacterial signature of a wild primate is associated with a timing effect of pre- and postnatal maternal glucocorticoid levels
Microbiome Open Access 27 July 2023
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Bischoff SC . ‘Gut health’: a new objective in medicine? BMC Med 2010; 9: 24.
Grenham S, Clarke G, Cryan J, Dinan TG . Brain-gut-microbe communication in health and disease. Front Gastrointest Sci 2011; 2: 94.
Lyte M . The microbial organ in the gut as a driver of homeostasis and disease. Med Hypotheses 2010; 74: 634–638.
Forsythe P, Sudo N, Dinan T, Taylor VH, Bienenstock J . Mood and gut feelings. Brain Behav Immun 2010; 24: 9–16.
Mayer EA . Gut feelings: the emerging biology of gut-brain communication. Nat Rev Neurosci 2011; 12: 453–466.
Cryan JF, O’Mahony SM . The microbiome-gut-brain axis: from bowel to behavior. Neurogastroenterol Motil 2011; 23: 187–192.
O’Hara AM, Shanahan F . The gut flora as a forgotten organ. EMBO Rep 2006; 7: 688–693.
Rhee SH, Pothoulakis C, Mayer EA . Principles and clinical implications of the brain-gut-enteric microbiota axis. Nat Rev Gastroenterol Hepatol 2009; 6: 306–314.
Bercik P, Collins SM, Verdu EF . Microbes and the gut-brain axis. Neurogastroenterol Motil 2012; 24: 405–413.
Gaspar P, Cases O, Maroteaux L . The developmental role of serotonin: news from mouse molecular genetics. Nat Rev Neurosci 2003; 4: 1002–1012.
Leonard BE . HPA and immune axes in stress: involvement of the serotonergic system. Neuroimmunomodulation 2006; 13: 268–276.
Sudo N, Chida Y, Aiba Y, Sonoda J, Oyama N, Yu XN et al. Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice. J Physiol 2004; 558 (Part 1): 263–275.
Heijtz RD, Wang S, Anuar F, Qian Y, Bjorkholm B, Samuelsson A et al. Normal gut microbiota modulates brain development and behavior. Proc Natl Acad Sci USA 2011; 108: 3047–3052.
Neufeld KM, Kang N, Bienenstock J, Foster JA . Reduced anxiety-like behavior and central neurochemical change in germ-free mice. Neurogastroenterol Motil 2010; 23: 255–264, e119.
Bercik P, Denou E, Collins J, Jackson W, Lu J, Jury J et al. The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice. Gastroenterology 2011; 141: 599–609.e593.
Goehler LE, Lyte M, Gaykema RP . Infection-induced viscerosensory signals from the gut enhance anxiety: implications for psychoneuroimmunology. Brain Behav Immun 2007; 21: 721–726.
Goehler LE, Park SM, Opitz N, Lyte M, Gaykema RP . Campylobacter jejuni infection increases anxiety-like behavior in the holeboard: possible anatomical substrates for viscerosensory modulation of exploratory behavior. Brain Behav Immun 2008; 22: 354–366.
Desbonnet L, Garrett L, Clarke G, Kiely B, Cryan JF, Dinan TG . Effects of the probiotic Bifidobacterium infantis in the maternal separation model of depression. Neuroscience 2010; 170: 1179–1188.
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 2011; 108: 16050–16055.
Messaoudi M, Lalonde R, Violle N, Javelot H, Desor D, Nejdi A et al. Assessment of psychotropic-like properties of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in rats and human subjects. Br J Nutr 2011; 105: 755–764.
Desbonnet L, Garrett L, Clarke G, Bienenstock J, Dinan TG . The probiotic Bifidobacteria infantis: an assessment of potential antidepressant properties in the rat. J Psychiatr Res 2008; 43: 164–174.
Jones MD, Lucki I . Sex differences in the regulation of serotonergic transmission and behavior in 5-HT receptor knockout mice. Neuropsychopharmacology 2005; 30: 1039–1047.
Maswood S, Truitt W, Hotema M, Caldarola-Pastuszka M, Uphouse L . Estrous cycle modulation of extracellular serotonin in mediobasal hypothalamus: role of the serotonin transporter and terminal autoreceptors. Brain Res 1999; 831: 146–154.
Cryan JF, Mombereau C . In search of a depressed mouse: utility of models for studying depression-related behavior in genetically modified mice. Mol Psychiatry 2004; 9: 326–357.
Palanza P . Animal models of anxiety and depression: how are females different? Neurosci Biobehav Rev 2001; 25: 219–233.
Hansen CH, Nielsen DS, Kverka M, Zakostelska Z, Klimesova K, Hudcovic T et al. Patterns of early gut colonization shape future immune responses of the host. PLoS One 2012; 7: e34043.
O’Connell Motherway M, Zomer A, Leahy SC, Reunanen J, Bottacini F, Claesson MJ et al. Functional genome analysis of Bifidobacterium breve UCC2003 reveals type IVb tight adherence (Tad) pili as an essential and conserved host-colonization factor. Proc Natl Acad Sci USA 2011; 108: 11217–11222.
Harkin A, Connor TJ, Mulrooney J, Kelly JP, Leonard BE . Prior exposure to methylenedioxyamphetamine (MDA) induces serotonergic loss and changes in spontaneous exploratory and amphetamine-induced behaviors in rats. Life Sci 2001; 68: 1367–1382.
O’Mahony S, Chua AS, Quigley EM, Clarke G, Shanahan F, Keeling PW et al. Evidence of an enhanced central 5HT response in irritable bowel syndrome and in the rat maternal separation model. Neurogastroenterol Motil 2008; 20: 680–688.
Clarke G, Fitzgerald P, Cryan JF, Cassidy EM, Quigley EM, Dinan TG . Tryptophan degradation in irritable bowel syndrome: evidence of indoleamine 2,3-dioxygenase activation in a male cohort. BMC Gastroenterol 2009; 9: 6.
Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 2001; 25: 402–408.
O’Mahony CM, Sweeney FF, Daly E, Dinan TG, Cryan JF . Restraint stress-induced brain activation patterns in two strains of mice differing in their anxiety behaviour. Behav Brain Res 2010; 213: 148–154.
Jacobsen JP, Mork A . The effect of escitalopram, desipramine, electroconvulsive seizures and lithium on brain-derived neurotrophic factor mRNA and protein expression in the rat brain and the correlation to 5-HT and 5-HIAA levels. Brain Res 2004; 1024: 183–192.
Nakamura K, Hasegawa H . Developmental role of tryptophan hydroxylase in the nervous system. Mol Neurobiol 2007; 35: 45–54.
Walther DJ, Bader M . A unique central tryptophan hydroxylase isoform. Biochem Pharmacol 2003; 66: 1673–1680.
Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 2003; 299: 76.
Ruddick JP, Evans AK, Nutt DJ, Lightman SL, Rook GA, Lowry CA . Tryptophan metabolism in the central nervous system: medical implications. Expert Rev Mol Med 2006; 8: 1–27.
Wikoff WR, Anfora AT, Liu J, Schultz PG, Lesley SA, Peters EC et al. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proc Natl Acad Sci USA 2009; 106: 3698–3703.
Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS et al. Metagenomic analysis of the human distal gut microbiome. Science 2006; 312: 1355–1359.
Bethea CL, Lu NZ, Gundlah C, Streicher JM . Diverse actions of ovarian steroids in the serotonin neural system. Front Neuroendocrinol 2002; 23: 41–100.
Imwalle DB, Gustafsson JA, Rissman EF . Lack of functional estrogen receptor beta influences anxiety behavior and serotonin content in female mice. Physiol Behav 2005; 84: 157–163.
Painsipp E, Herzog H, Sperk G, Holzer P . Sex-dependent control of murine emotional-affective behaviour in health and colitis by peptide YY and neuropeptide Y. Br J Pharmacol 2011; 163: 1302–1314.
Bangasser DA, Curtis A, Reyes BA, Bethea TT, Parastatidis I, Ischiropoulos H et al. Sex differences in corticotropin-releasing factor receptor signaling and trafficking: potential role in female vulnerability to stress-related psychopathology. Mol Psychiatry 2010; 15: 877, 896–904.
Schrocksnadel K, Wirleitner B, Winkler C, Fuchs D . Monitoring tryptophan metabolism in chronic immune activation. Clin Chim Acta 2006; 364: 82–90.
Myint AM, Kim YK, Verkerk R, Park SH, Scharpe S, Steinbusch HW et al. Tryptophan breakdown pathway in bipolar mania. J Affect Disord 2007; 102: 65–72.
Myint AM, Kim YK, Verkerk R, Scharpe S, Steinbusch H, Leonard B . Kynurenine pathway in major depression: evidence of impaired neuroprotection. J Affect Disord 2007; 98: 143–151.
Brunson KL, Chen Y, Avishai-Eliner S, Baram TZ . Stress and the developing hippocampus: a double-edged sword? Mol Neurobiol 2003; 27: 121–136.
de Kloet ER, Sibug RM, Helmerhorst FM, Schmidt MV . Stress, genes and the mechanism of programming the brain for later life. Neurosci Biobehav Rev 2005; 29: 271–281.
Schmidt MV . Molecular mechanisms of early life stress—lessons from mouse models. Neurosci Biobehav Rev 2010; 34: 845–852.
Bingham B, McFadden K, Zhang X, Bhatnagar S, Beck S, Valentino R . Early adolescence as a critical window during which social stress distinctly alters behavior and brain norepinephrine activity. Neuropsychopharmacology 2011; 36: 896–909.
Neufeld KA, Kang N, Bienenstock J, Foster JA . Effects of intestinal microbiota on anxiety-like behavior. Commun Integr Biol 2011; 4: 492–494.
Ansorge MS, Morelli E, Gingrich JA . Inhibition of serotonin but not norepinephrine transport during development produces delayed, persistent perturbations of emotional behaviors in mice. J Neurosci 2008; 28: 199–207.
Gross C, Hen R . The developmental origins of anxiety. Nat Rev Neurosci 2004; 5: 545–552.
Gordon JA, Hen R . The serotonergic system and anxiety. Neuromolecular Med 2004; 5: 27–40.
Graeff FG . Serotonin, the periaqueductal gray and panic. Neurosci Biobehav Rev 2004; 28: 239–259.
Nutt DJ . Overview of diagnosis and drug treatments of anxiety disorders. CNS Spectr 2005; 10: 49–56.
Borsini F, Podhorna J, Marazziti D . Do animal models of anxiety predict anxiolytic-like effects of antidepressants? Psychopharmacology (Berl) 2002; 163: 121–141.
Browne CA, Clarke G, Dinan TG, Cryan JF . An effective dietary method for chronic tryptophan depletion in two mouse strains illuminates a role for 5-HT in nesting behaviour. Neuropharmacology 2012; 62: 1903–1915.
Cryan JF, Sweeney FF . The age of anxiety: role of animal models of anxiolytic action in drug discovery. Br J Pharmacol 2011; 164: 1129–1161.
Holmes A, Lit Q, Murphy DL, Gold E, Crawley JN . Abnormal anxiety-related behavior in serotonin transporter null mutant mice: the influence of genetic background. Genes Brain Behav 2003; 2: 365–380.
Kaffman A, Meaney MJ . Neurodevelopmental sequelae of postnatal maternal care in rodents: clinical and research implications of molecular insights. J Child Psychol Psychiatry 2007; 48: 224–244.
Simpson J, Kelly JP . An investigation of whether there are sex differences in certain behavioural and neurochemical parameters in the rat. Behav Brain Res 2012; 229: 289–300.
Gangrade BK, Dominic CJ . Studies of the male-originating pheromones involved in the Whitten effect and Bruce effect in mice. Biol Reprod 1984; 31: 89–96.
Whitten WK . Occurrence of anoestrus in mice caged in groups. J Endocrinol 1959; 18: 102–107.
Adlerberth I, Wold AE . Establishment of the gut microbiota in Western infants. Acta Paediatr 2009; 98: 229–238.
The Alimentary Pharmabiotic Centre is a research centre funded by Science Foundation Ireland (SFI), through the Irish Government's National Development Plan. The authors and their work were supported by SFI (grant nos. 02/CE/B124 and 07/CE/B1368) and by GlaxoSmithKline. GC is in receipt of a research grant from the American Neurogastroenterology and Motility Society (ANMS). GC, JFC and TD are also funded by the Irish Health Research Board (HRB) Health Research Awards (grant no HRA_POR/2011/23). We acknowledge the contribution of Ms Frances O’Brien, Dr Monica Tramullas and Mr Kieran Davey to the study.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Molecular Psychiatry website
About this article
Cite this article
Clarke, G., Grenham, S., Scully, P. et al. The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner. Mol Psychiatry 18, 666–673 (2013). https://doi.org/10.1038/mp.2012.77
This article is cited by
Gegen-Qinlian decoction alleviates anxiety-like behaviors in methamphetamine-withdrawn mice by regulating Akkermansia and metabolism in the colon
Chinese Medicine (2023)
Translational Neurodegeneration (2023)
Annals of General Psychiatry (2023)
Journal of Biomedical Science (2023)
The long-term gut bacterial signature of a wild primate is associated with a timing effect of pre- and postnatal maternal glucocorticoid levels