Elsevier

Psychoneuroendocrinology

Volume 42, April 2014, Pages 207-217
Psychoneuroendocrinology

Absence of the gut microbiota enhances anxiety-like behavior and neuroendocrine response to acute stress in rats

https://doi.org/10.1016/j.psyneuen.2014.01.014Get rights and content

Abstract

Background and aims

Establishment of the gut microbiota is one of the most important events in early life and emerging evidence indicates that the gut microbiota influences several aspects of brain functioning, including reactivity to stress. To better understand how the gut microbiota contributes to a vulnerability to the stress-related psychiatric disorders, we investigated the relationship between the gut microbiota, anxiety-like behavior and HPA axis activity in stress-sensitive rodents. We also analyzed the monoamine neurotransmitters in the brain upper structures involved in the regulation of stress and anxiety.

Methods

Germfree (GF) and specific pathogen free (SPF) F344 male rats were first subjected to neurological tests to rule out sensorimotor impairments as confounding factors. Then, we examined the behavior responses of rats to social interaction and open-field tests. Serum corticosterone concentrations, CRF mRNA expression levels in the hypothalamus, glucocorticoid receptor (GR) mRNA expression levels in the hippocampus, and monoamine concentrations in the frontal cortex, hippocampus and striatum were compared in rats that were either exposed to the open-field stress or not.

Results

GF rats spent less time sniffing an unknown partner than SPF rats in the social interaction test, and displayed a lower number of visits to the aversive central area, and an increase in latency time, time spent in the corners and number of defecations in the open-field test. In response to the open-field stress, serum corticosterone concentrations were 2.8-fold higher in GF than in SPF rats. Compared to that of SPF rats, GF rats showed elevated CRF mRNA expression in the hypothalamus and reduced GR mRNA expression in the hippocampus. GF rats also had a lower dopaminergic turnover rate in the frontal cortex, hippocampus and striatum than SPF rats.

Conclusions

In stress-sensitive F344 rats, absence of the gut microbiota exacerbates the neuroendocrine and behavioral responses to acute stress and the results coexist with alterations of the dopaminergic turnover rate in brain upper structures that are known to regulate reactivity to stress and anxiety-like behavior.

Introduction

In mammals, the underlying mechanisms of stress, fear and anxiety are shaped in early life both by genetic and environmental factors, and disturbances of brain development and maturation could contribute to a later vulnerability to psychiatric disorders such as hyper-responsiveness to stress, depression and addiction (Vazquez et al., 2005, Heim et al., 2008, Enoch et al., 2010). Also in early life, mammals establish their gut microbiota, and this process is highly influenced by environmental factors. In normal conditions, these gut symbionts, whose collective genomes encode a vast array of diverse functional genes (Lozupone et al., 2012), affect various aspects of host physiology, including brain development and functions (Grenham et al., 2011, Forsythe and Kunze, 2013). In supporting the role of intestinal bacteria in gut–brain connections, it was shown recently in a cohort of healthy adults that chronic consumption of a fermented milk product with probiotic, known to alter gut microbiota metabolism, modulated the activity of several brain areas involved in sensory perception and emotion (Tillisch et al., 2013). Emerging evidence also indicates the connections between the gut microbiota and neurodevelopmental disorders. Gut microbiota composition and metabolism of autistic children are different from those of the general population (Yap et al., 2010, Finegold et al., 2012), and children with regressive-onset autism reported improvement of gastrointestinal disturbances and behavior features following a treatment with a non-absorbable oral antibiotic aimed at modulating the gut microbiota (Sandler et al., 2000).

Recently, animal experiments showed that germfree (i.e. devoid of gut microbiota, GF) adult male BALB/c mice had an elevated basal level of CRF gene expression in the hypothalamus and over-reacted to an acute restraint stress by a hyper-secretion of adrenocorticotropic hormone (ACTH) and corticosterone (CORT), compared with specific pathogen-free (SPF) counterparts (Sudo et al., 2004). Such a maladaptive response in GF mice to stress was partly corrected by gut microbiota reconstitution with fecal bacteria of the SPF mice. Interestingly, this treatment was only effective when the GF mice were colonized at six but not at 14 weeks of age, suggesting the existence of a critical period for programming the reactivity of the hypothalamic–pituitary–adrenal (HPA) axis (Sudo et al., 2004). In another experiment, the same authors showed that GF BALB/c mice expressed a higher anxiety-like behavior than SPF counterparts in the open-field (OF) and marble-burying tests, and had lower turnover rates of monoamines in several brain regions (Nishino et al., 2013). In contrast, studies conducted with other mice models, namely Swiss adult males and females and NMRI adult males, showed that a GF status was associated with reduced anxiety-like behavior in the elevated plus maze and light–dark choice tests, and increased the striatal and hippocampal turnover of monoamines (Neufeld et al., 2011, Diaz-Heijtz et al., 2011, Clarke et al., 2013). In some of those studies, an HPA axis hyper-activity in basal conditions (Neufeld et al., 2011) or in response to stress (Clarke et al., 2013) was described. The joint presence of HPA axis hyper-activity and anxiolysis is unexpected because stress is commonly associated with anxiety (Shekhar et al., 2005, Laryea et al., 2012). Therefore, the aim of the present study was to clarify the impact of the GF status on HPA axis reactivity to stress and on anxiety by testing the neuroendocrine and behavioral responses of animals to novel challenges. Specifically, we subjected GF and SPF adult male F344 rats to a social interaction experience and to an OF test, and we measured (i) the serum CORT concentration, (ii) the expression level of the CRF gene in the hypothalamus and of the glucocorticoid receptor (GR) gene in the hippocampus, and (iii) monoamine concentrations in the brain upper structures involved in the regulation of stress and anxiety (frontal cortex, hippocampus and striatum). In addition, we carried out a neurological examination to ascertain that behavioral differences between GF and SPF rats could not result from sensorimotor development impairments. We opted for the rat as an animal model to obtain additional data in another species than the mouse, which was exclusively used until now, and because of its more complex behavior (Baker, 2011). The strain F344 was chosen as a hyper-responsive strain to stress (Sarrieau et al., 1998) and we focused the study on the male sex to avoid interferences between estrous cycle and sensitivity to stress (Viau and Meaney, 1991, Mourlon et al., 2011).

Section snippets

Animals

GF and SPF pregnant F344 rats were obtained from Anaxem (Germ-free animal facilities of INRA, UMR1319 Micalis) and Charles River Laboratories (L’Arbresle, France), respectively.

The GF females were housed in sterile isolators (Eurobioconcept, Paris, France) and the GF status was monitored every week by microscopic examination and aerobic and anaerobic cultures of freshly voided fecal samples. The SPF females were also housed in isolators to ensure the same sensorial environment to the SPF pups

Absence of the gut microbiota did not influence the sensorimotor functions.

Presence or absence of the gut microbiota had no effect on any of the sensorimotor functions, regardless the age of the neurological examination (Table S1). The composite neurological score was similar in 30-d old GF and SPF rats (18.0 ± 3.0 vs. 18.0 ± 2.5; p = 0.5160), and in 71-d old GF and SPF rats (18.5 ± 1.5 vs. 19.0 ± 1.0; p = 0.4613). Age had an effect on a few tests, but this effect was independent of the rat microbial status. Visual limb-placing reaction improved with age in GF and SPF rats (from

Discussion

Involvement of the gut microbiota in the gut-brain axis has been recently highlighted by an increasing number of studies using GF animals or antibiotic or probiotic administration (Grenham et al., 2011, Foster and McVey Neufeld, 2013). The present study reveals that, in the stress-sensitive F344 rat strain, absence of the gut microbiota enhanced anxiety-like behavior in face of novel challenges, such as a social interaction experience or exposure to an open and intensely-lit environment (OF

Role of funding source

Not applicable.

Conflict of interest

None declared.

Acknowledgments

We thank Pascal Guillaume, Alain Joffre and Fatima Joly (INRA, UMR1319 Micalis, Anaxem) for skillful technical assistance and we are very grateful to Dr. Chieh J. Chou (Nestlé Institute of Health Sciences, Lausanne, Switzerland) for his critical reading and editing of the manuscript. This work was supported in part by funding from INRA, Nutrition, Chemical Food Safety and Consumer Behavior Division (ANSSD-2008 to SR) and from the French Ministry of Higher Education and Research (Ph.D. grant to

References (54)

  • S.L. Andersen et al.

    Stress, sensitive periods and maturational events in adolescent depression

    Trends Neurosci.

    (2008)
  • L. Arborelius et al.

    The role of corticotropin-releasing factor in depression and anxiety disorders

    J. Endocrinol.

    (1999)
  • E. Arzt et al.

    CRF signaling: molecular specificity for drug targeting in the CNS

    Trends Pharmacol. Sci.

    (2006)
  • M. Baker

    Inside the minds of mice and men

    Nature

    (2011)
  • T.P. Beauchaine et al.

    The effects of allostatic load on neural systems subserving motivation, mood regulation, and social affiliation

    Dev. Psychopathol.

    (2011)
  • P. Bercik et al.

    The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice

    Gastroenterology

    (2011)
  • O. Berton et al.

    Behavioral reactivity to social and nonsocial stimulations: a multivariate analysis of six inbred rat strains

    Behav. Genet.

    (1997)
  • J.A. Bravo et al.

    Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve

    PNAS

    (2011)
  • S. Cabib et al.

    Stress, depression and the mesolimbic dopamine system

    Psychopharmacology

    (1996)
  • C. Capdeville et al.

    Methods for evaluating the neurologic deficit induced by transient cerebral ischemia in the unanesthetized rat

    J. Pharmacol. (Paris)

    (1984)
  • G. Clarke et al.

    The microbiome–gut–brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner

    Mol. Psychiatry

    (2013)
  • Y. Clément et al.

    Pharmacological alterations of anxious behaviour in mice depending on both strain and the behavioural situation

    PLoS One

    (2009)
  • S.M. Collins et al.

    The interplay between the intestinal microbiota and the brain

    Nat. Rev. Microbiol.

    (2012)
  • M. Crumeyrolle-Arias et al.

    Isatin binding proteins in rat brain: in situ imaging, quantitative characterization of specific [3H]isatin binding, and proteomic profiling

    J. Neurosci. Res.

    (2009)
  • J.F. Cryan et al.

    Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour

    Nat. Rev. Neurosci.

    (2012)
  • V. Daugé et al.

    Comparison of the behavioural effects induced by administration in rat nucleus accumbens or nucleus caudatus of selective μ and δ opioid peptides or kelatorphan an inhibitor of enkephalin-degrading-enzymes

    Psychopharmacology (Berl.)

    (1988)
  • L. Desbonnet et al.

    Microbiota is essential for social development in the mouse

    Mol. Psychiatry

    (2013)
  • R. Diaz-Heijtz et al.

    Normal gut microbiota modulates brain development and behavior

    PNAS

    (2011)
  • M.A. Enoch et al.

    The influence of GABRA2, childhood trauma, and their interaction on alcohol, heroin, and cocaine dependence

    Biol. Psychiatry

    (2010)
  • S.E. File et al.

    Can social interaction be used to measure anxiety?

    Br. J. Pharmacol.

    (1978)
  • S.M. Finegold et al.

    Microbiology of regressive autism

    Anaerobe

    (2012)
  • G. Flügge et al.

    Perturbations in brain monoamines systems during stress

    Cell Tissue Res.

    (2004)
  • P. Forsythe et al.

    Voices from within: gut microbes and the CNS

    Cell. Mol. Life Sci.

    (2013)
  • J.A. Foster et al.

    Gut–brain axis: how the microbiome influences anxiety and depression

    Trends Neurosci.

    (2013)
  • M.G. Gareau et al.

    Bacterial infection causes stress-induced memory dysfunction in mice

    Gut

    (2011)
  • G. Gerra et al.

    Homovanillic acid (HVA) plasma levels inversely correlate with attention deficit-hyperactivity and childhood neglect measures in addicted patients

    J. Neural Transm.

    (2007)
  • S. Grenham et al.

    Brain–gut–microbe communication in health and disease

    Front. Physiol.

    (2011)
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