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