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Article

Microbiota and host determinants of behavioural phenotype in maternally separated mice

  • Nature Communications 6, Article number: 7735 (2015)
  • doi:10.1038/ncomms8735
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Abstract

Early-life stress is a determinant of vulnerability to a variety of disorders that include dysfunction of the brain and gut. Here we exploit a model of early-life stress, maternal separation (MS) in mice, to investigate the role of the intestinal microbiota in the development of impaired gut function and altered behaviour later in life. Using germ-free and specific pathogen-free mice, we demonstrate that MS alters the hypothalamic–pituitary–adrenal axis and colonic cholinergic neural regulation in a microbiota-independent fashion. However, microbiota is required for the induction of anxiety-like behaviour and behavioural despair. Colonization of adult germ-free MS and control mice with the same microbiota produces distinct microbial profiles, which are associated with altered behaviour in MS, but not in control mice. These results indicate that MS-induced changes in host physiology lead to intestinal dysbiosis, which is a critical determinant of the abnormal behaviour that characterizes this model of early-life stress.

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References

  1. 1.

    & Childhood trauma and psychosis: evidence, pathways, and implications. J. Postgrad. Med. 54, 287–293 (2008).

  2. 2.

    et al. Association between early adverse life events and irritable bowel syndrome. Clin. Gastroenterol. Hepatol. 10, 385–390 (2012).

  3. 3.

    , , , & Cognitive, emotional and neurochemical effects of repeated maternal separation in adolescent rats. Brain Res. 1518, 82–90 (2013).

  4. 4.

    & Towards translational rodent models of depression. Cell Tissue Res. 354, 141–153 (2013).

  5. 5.

    , , & Maternal separation as a model of brain-gut axis dysfunction. Psychopharmacology (Berl) 214, 71–88 (2011).

  6. 6.

    , , & New insights in the etiology and pathophysiology of irritable bowel syndrome: contribution of neonatal stress models. Pediatr. Res. 62, 240–245 (2007).

  7. 7.

    , & Pathophysiological mechanisms of stress-induced intestinal damage. Curr. Mol. Med. 8, 274–281 (2008).

  8. 8.

    et al. Antidepressants attenuate increased susceptibility to colitis in a murine model of depression. Gastroenterology 130, 1743–1753 (2006).

  9. 9.

    , , , & Long-term behavioural and molecular alterations associated with maternal separation in rats. Eur. J. Neurosci. 25, 3091–3098 (2007).

  10. 10.

    et al. Neonatal stress affects vulnerability of cholinergic neurons and cognition in the rat: involvement of the HPA axis. Psychoneuroendocrinology 34, 1495–1505 (2009).

  11. 11.

    et al. Neonatal maternal separation enhances central sensitivity to noxious colorectal distention in rat. Brain Res. 1153, 68–77 (2007).

  12. 12.

    et al. Early-life stress induces visceral hypersensitivity in mice. Neurosci. Lett. 512, 99–102 (2012).

  13. 13.

    et al. A distinct subset of submucosal mast cells undergoes hyperplasia following neonatal maternal separation: a role in visceral hypersensitivity? Gut 58, 1029–1030 (2009).

  14. 14.

    , , & Neonatal maternal deprivation triggers long term alterations in colonic epithelial barrier and mucosal immunity in rats. Gut 53, 501–506 (2004).

  15. 15.

    , & Neonatal maternal separation of rat pups results in abnormal cholinergic regulation of epithelial permeability. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G198–G203 (2007).

  16. 16.

    , , , & Neonatal maternal separation in male rats increases intestinal permeability and affects behavior after chronic social stress. Physiol. Behav. 105, 1058–1066 (2012).

  17. 17.

    et al. Neonatal maternal separation predisposes adult rats to colonic barrier dysfunction in response to mild stress. Am. J. Physiol. Gastrointest. Liver Physiol. 283, G1257–G1263 (2002).

  18. 18.

    et al. Early life stress alters behavior, immunity, and microbiota in rats: implications for irritable bowel syndrome and psychiatric illnesses. Biol. Psychiatry 65, 263–267 (2009).

  19. 19.

    et al. Effects of the probiotic Bifidobacterium infantis in the maternal separation model of depression. Neuroscience 170, 1179–1188 (2010).

  20. 20.

    & Role of probiotics in correcting abnormalities of colonic flora induced by stress. Gut 56, 1495–1497 (2007).

  21. 21.

    , , , & Probiotic treatment of rat pups normalises corticosterone release and ameliorates colonic dysfunction induced by maternal separation. Gut 56, 1522–1528 (2007).

  22. 22.

    , & The interplay between the intestinal microbiota and the brain. Nat. Rev. Microbiol. 10, 735–742 (2012).

  23. 23.

    et al. Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice. J. Physiol. 558, 263–275 (2004).

  24. 24.

    et al. Normal gut microbiota modulates brain development and behavior. Proc. Natl Acad. Sci. USA 108, 3047–3052 (2011).

  25. 25.

    , , & Reduced anxiety-like behavior and central neurochemical change in germ-free mice. Neurogastroenterol. Motil. 23, 255–264 (2011).

  26. 26.

    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).

  27. 27.

    et al. The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice. Gastroenterology 141, 599–609 (2011).

  28. 28.

    et al. Repeated exposure to water avoidance stress in rats: a new model for sustained visceral hyperalgesia. Am. J. Physiol. Gastrointest. Liver Physiol. 289, G42–G53 (2005).

  29. 29.

    , , , & Distinct alterations in colonic morphology and physiology in two rat models of enhanced stress-induced anxiety and depression-like behaviour. Stress 13, 114–122 (2010).

  30. 30.

    et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat. Biotechnol. 31, 814–821 (2013).

  31. 31.

    , & Resistance to early-life stress in mice: effects of genetic background and stress duration. Front. Behav. Neurosci. 5, 13 (2011).

  32. 32.

    & Effects of repeated maternal separation on anxiety- and depression-related phenotypes in different mouse strains. Neurosci. Biobehav. Rev. 31, 3–17 (2007).

  33. 33.

    , & Impaired parasympathetic function increases susceptibility to inflammatory bowel disease in a mouse model of depression. J. Clin. Invest. 118, 2209–2218 (2008).

  34. 34.

    , , & Strain and gender differences in the behavior of mouse lines commonly used in transgenic studies. Physiol. Behav. 72, 271–281 (2001).

  35. 35.

    , & The Mouse Defense Test Battery: pharmacological and behavioral assays for anxiety and panic. Eur. J. Pharmacol. 463, 97–116 (2003).

  36. 36.

    , , , & Psychogenic, neurogenic, and systemic stressor effects on plasma corticosterone and behavior: mouse strain-dependent outcomes. Behav. Neurosci. 115, 443–454 (2001).

  37. 37.

    , , & Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice. Curr. Protoc. Neurosci Chapter 8, Unit 8.10A (2011).

  38. 38.

    , , , & Effects of maternal separation on hypothalamic-pituitary-adrenal responses, cognition and vulnerability to stress in adult female rats. Neuroscience 154, 1218–1226 (2008).

  39. 39.

    & Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stress-induced release in adult rats. Brain Res. Mol. Brain Res. 18, 195–200 (1993).

  40. 40.

    , , , & Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats. Psychoneuroendocrinology 32, 256–266 (2007).

  41. 41.

    , , , & Neonatal maternal separation causes colonic dysfunction in rat pups including impaired host resistance. Pediatr. Res. 59, 83–88 (2006).

  42. 42.

    et al. Pathways involved in gut mucosal barrier dysfunction induced in adult rats by maternal deprivation: corticotrophin-releasing factor and nerve growth factor interplay. J. Physiol. 580, 347–356 (2007).

  43. 43.

    et al. Corticotropin-releasing factor receptor 1 mediates acute and delayed stress-induced visceral hyperalgesia in maternally separated Long-Evans rats. Am. J. Physiol. Gastrointest. Liver Physiol. 289, G704–G712 (2005).

  44. 44.

    et al. Corticotropin-releasing hormone mimics stress-induced colonic epithelial pathophysiology in the rat. Am. J. Physiol. 277, G391–G399 (1999).

  45. 45.

    et al. Altered colonic function and microbiota profile in a mouse model of chronic depression. Neurogastroenterol. Motil. 25, 733–e575 (2013).

  46. 46.

    , , , & Increased adult hippocampal brain-derived neurotrophic factor and normal levels of neurogenesis in maternal separation rats. J. Neurosci. Res. 79, 772–778 (2005).

  47. 47.

    et al. Neonatal repetitive maternal separation causes long-lasting alterations in various neurotrophic factor expression in the cerebral cortex of rats. Life Sci. 90, 578–584 (2012).

  48. 48.

    et al. BDNF expression in the hippocampus of maternally separated rats: does Bifidobacterium breve 6330 alter BDNF levels? Benef. Microbes 2, 199–207 (2011).

  49. 49.

    et al. Maternal separation attenuates the effect of adolescent social isolation on HPA axis responsiveness in adult rats. Eur. Neuropsychopharmacol. 24, 1152–1161 (2014).

  50. 50.

    , , , & Early maternal deprivation reduces the expression of BDNF and NMDA receptor subunits in rat hippocampus. Mol. Psychiatry 7, 609–616 (2002).

  51. 51.

    et al. Commensal microbiota modulate murine behaviors in a strictly contamination-free environment confirmed by culture-based methods. Neurogastroenterol. Motil. 25, 521–528 (2013).

  52. 52.

    & Age-dependent deficits in fear learning in heterozygous BDNF knock-out mice. Learn. Mem. 19, 561–570 (2012).

  53. 53.

    et al. Mechanism of age-related and nitrous oxide-associated anesthetic sensitivity: the role of brain catecholamines. Anesthesiology 69, 716–720 (1988).

  54. 54.

    & The mouse light/dark box test. Eur. J. Pharmacol. 463, 55–65 (2003).

  55. 55.

    , , & Increased colonic motility in a rat model of irritable bowel syndrome is associated with up-regulation of L-type calcium channels in colonic smooth muscle cells. Neurogastroenterol. Motil. 22, e162–e170 (2010).

  56. 56.

    The enteric nervous system and neurogastroenterology. Nat. Rev. Gastroenterol. Hepatol. 9, 286–294 (2012).

  57. 57.

    , & Enteroendocrine and neuronal mechanisms in pathophysiology of acute infectious diarrhea. Dig. Dis. Sci. 57, 19–27 (2012).

  58. 58.

    & Autonomic control of gut motility: a comparative view. Auton. Neurosci. 165, 80–101 (2011).

  59. 59.

    & Effect of the normal microbial flora on gastrointestinal motility. Proc. Soc. Exp. Biol. Med. 126, 301–304 (1967).

  60. 60.

    , , , & Gut microbial products regulate murine gastrointestinal motility via Toll-like receptor 4 signaling. Gastroenterology 143, 1006–1016 (2012).

  61. 61.

    et al. The neuroendocrine stress hormone norepinephrine augments Escherichia coli O157:H7-induced enteritis and adherence in a bovine ligated ileal loop model of infection. Infect. Immun. 72, 5446–5451 (2004).

  62. 62.

    , & Stress at the intestinal surface: catecholamines and mucosa-bacteria interactions. Cell Tissue Res. 343, 23–32 (2011).

  63. 63.

    , , & The role of pH in determining the species composition of the human colonic microbiota. Environ. Microbiol. 11, 2112–2122 (2009).

  64. 64.

    PARs for the course: roles of proteases and PAR receptors in subtly inflamed irritable bowel syndrome. Am. J. Gastroenterol. 108, 1644–1646 (2013).

  65. 65.

    et al. Fecal protease activity is associated with compositional alterations in the intestinal microbiota. PLoS ONE 8, e78017 (2013).

  66. 66.

    et al. Increase of faecal tryptic activity relates to changes in the intestinal microbiome: analysis of Crohn's disease with a multidisciplinary platform. PLoS ONE 8, e66074 (2013).

  67. 67.

    , , , & gamma-Aminobutyric acid production by culturable bacteria from the human intestine. J. Appl. Microbiol. 113, 411–417 (2012).

  68. 68.

    et al. Critical role of gut microbiota in the production of biologically active, free catecholamines in the gut lumen of mice. Am. J. Physiol. Gastrointest. Liver Physiol. 303, G1288–G1295 (2012).

  69. 69.

    et al. The anxiolytic effect of Bifidobacterium longum NCC3001 involves vagal pathways for gut-brain communication. Neurogastroenterol. Motil. 23, 1132–1139 (2011).

  70. 70.

    et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451–1463 (2013).

  71. 71.

    , & Anxiogenic effect of subclinical bacterial infection in mice in the absence of overt immune activation. Physiol Behav. 65, 63–68 (1998).

  72. 72.

    , & Alterations in the central CRF system of two different rat models of comorbid depression and functional gastrointestinal disorders. Int. J. Neuropsychopharmacol. 14, 666–683 (2011).

  73. 73.

    et al. Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in rat. Am. J. Physiol. Gastrointest. Liver Physiol. 282, G307–G316 (2002).

  74. 74.

    , & The mechanism of altered neural function in a rat model of acute colitis. Gastroenterology 112, 156–162 (1997).

  75. 75.

    , , & Impaired acetylcholine release from the myenteric plexus of Trichinella-infected rats. Am. J. Physiol. 257, G898–G903 (1989).

  76. 76.

    et al. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66, 297–303 (2000).

  77. 77.

    , , , & Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end illumina reads. Appl. Environ. Microbiol. 77, 3846–3852 (2011).

  78. 78.

    et al. The loss of topography in the microbial communities of the upper respiratory tract in the elderly. Ann. Am. Thorac. Soc. 11, 513–521 (2014).

  79. 79.

    Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10–12 (2011).

  80. 80.

    , , , & PANDAseq: paired-end assembler for illumina sequences. BMC Bioinformatics 13, 31 (2012).

  81. 81.

    Identification and quantification of abundant species from pyrosequences of 16S rRNA by consensus alignment. Proc. IEEE Int. Conf. Bioinformatics Biomed. 153–157 (2010).

  82. 82.

    et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).

  83. 83.

    & phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8, e61217 (2013).

  84. 84.

    & Identifying biologically relevant differences between metagenomic communities. Bioinformatics 26, 715–721 (2010).

  85. 85.

    , & Statistical methods for detecting differentially abundant features in clinical metagenomic samples. PLoS Comput. Biol. 5, e1000352 (2009).

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Acknowledgements

This manuscript is dedicated to the memory and the work of Patricia (Trish) Blennerhasset. We thank Dr Henry Szechtman for his advice. This work was supported by grants from Crohn’s and Colitis Foundation of Canada and the Canadian Institute of Health Research to P.B. and S.M.C. E.F.V. holds a Canada Research Chair. P.B. is a recipient of HHS Early Career Research Award. G.D.P. received postdoctoral fellowship from the Ontario Ministry of Research and Innovation and from the Canadian Institutes of Health Research (joint CAG and Aptalis). This work was also supported by grants AGL2011-25169, Consolider Fun-C-Food CSD2007-00063 from Ministry of Science and Innovation (Spain), and EU grant no 613979 (MyNewGut) from the 7th Framework Program to Y.S.

Author information

Affiliations

  1. Department of Medicine, Farncombe Family Digestive Health Research Institute, McMaster University, HSC 4W8, 1200 Main Street West, Hamilton, Ontario, Canada L8S 4K1

    • G. De Palma
    • , P. Blennerhassett
    • , J. Lu
    • , Y. Deng
    • , A. J. Park
    • , W. Green
    • , E. Denou
    • , M. A. Silva
    • , M. G. Surette
    • , E. F. Verdu
    • , S. M. Collins
    •  & P. Bercik
  2. Institute of Agrochemistry and Food Technology, National Research Council (IATA-CSIC), Valencia 46980, Spain

    • A. Santacruz
    •  & Y. Sanz

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Contributions

G.D.P. performed all GF and colonization experiments, part of SPF experiments and all the colonization experiments, analysed the data and wrote the manuscript; P.Bl. performed superfusion experiments in all SPF experiments and part of GF experiments, analysed the data and provided technical support; A.J.P., J.L. and E.D. provided technical support; W.G. performed behavioural tests and analysis on SPF animals; M.A.S. analysed the data and contributed to the manuscript; A.S. performed some of the microbiological analysis; Y.S. performed some of the microbiological analysis and reviewed the manuscript; M.G.S. provided Illumina platform and support; E.F.V. participated in the conception of the study and reviewing the manuscript; S.M.C. and P.B. conceived the study, contributed to the interpretation of the data and reviewed the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to P. Bercik.

Supplementary information

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  1. 1.

    Supplementary Information

    Supplementary Figures 1-8 and Supplementary Tables 1-2

Excel files

  1. 1.

    Supplementary Data 1

    PICRUSt estimation of metagenome function based on 16S rRNA sequences using a database of reference genomes.

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