Disturbance of the gut microbiota in early-life selectively affects visceral pain in adulthood without impacting cognitive or anxiety-related behaviors in male rats
Introduction
Increasing evidence points to a crucial role for the gut microbiota in health and disease (Matamoros et al., 2013, Petschow et al., 2013, Quigley, 2013, Cenit et al., 2014, Moloney et al., 2014, Nylund et al., 2014). Alterations in the microbiota have been shown in a variety of disorders ranging from obesity, diabetes to inflammatory bowel disorder and irritable bowel syndrome (IBS) (Kostic et al., 2014, Mayer et al., 2014, Moreno-Indias et al., 2014, Walsh et al., 2014). IBS is now recognized as a disorder of the brain–gut microbiome axis, characterized by visceral hypersensitivity, enhanced stress and anxiety (Mayer et al., 2001, Mayer et al., 2014, Longstreth et al., 2006, Mayer and Tillisch, 2011, Fashner and Gitu, 2013). The early postnatal period is the most dynamic stage of intestinal microbiota development. During the first three years of life the abundance, composition and diversity of the gut microbiome goes through rapid and large modification (Matamoros et al., 2013, Nylund et al., 2014). Disruption of these developmental patterns prior to the attainment of a more stable adult-like gastrointestinal (GI) tract microbiota is increasingly being linked to adverse health outcomes (Dominguez-Bello et al., 2011, Versalovic, 2013; Clarke et al., 2014; Moloney et al., 2014). Gut microbes play essential roles in the development of pain pathways, immune system development, and the production of short chain fatty acids (Amaral et al., 2008, Cryan and O’Mahony, 2011, Collins et al., 2012). These interactions between microbiota and the host during the early-life period can significantly influence the central nervous system (Sudo et al., 2004, Bravo et al., 2011, Diamond et al., 2011, Diaz Heijtz et al., 2011, Collins et al., 2012, Clarke et al., 2013, Foster and McVey Neufeld, 2013, Farmer et al., 2014). This has led to the concept of the brain–gut–microbiota axis (Rhee et al., 2009, Collins et al., 2012, Cryan and Dinan, 2012). It can, therefore, be readily appreciated that factors perturbing the microbiota during the early-life could result in long-lasting effects on the gut and even brain function that persist into adulthood (Hsiao et al., 2013, Borre et al., 2014). The parallel development of the central nervous system (CNS) and the microbiota in early-life and the bi-directional communication between these organs puts the bacterial components in a position where they may exert substantial influence over the developing brain (Borre et al., 2014, Stilling et al., 2014).
Indeed, in preclinical studies, germ-free mice have an exaggerated stress response and altered anxiety (Sudo et al., 2004, Clarke et al., 2013). Moreover, anxiety-related behavior can be transmissible from one strain of mouse to another via the microbiota (Bercik et al., 2011). Germ-free mice also show memory deficits (Gareau et al., 2011) and social deficits of relevance to autism (Desbonnet et al., 2014) and decreased pain responses (Amaral et al., 2008). On the other hand probiotic bacteria have been shown to reduce anxiety, improve cognition and visceral pain (Eutamene et al., 2007, Rousseaux et al., 2007, McKernan et al., 2010, Bravo et al., 2011, Johnson et al., 2011, Stilling et al., 2014). Moreover transplantation of microbiota from IBS patients induces visceral pain in germ-free rats (Bercik et al., 2012). Together these results highlight the range of complex behaviors and signaling pathways which may be modulated by the maturing GI microbiome during critical neurodevelopmental windows.
Early-life stress in the form of maternal separation induces marked perturbations in fecal microbiota diversity (Bailey and Coe, 1999, O’Mahony et al., 2009). However, it is unclear if there is a direct link between such microbiota disturbances and the other early-life stress-induced alterations such as increased visceral hypersensitivity, increased anxiety, cognitive changes, enhanced pro-inflammatory cytokine production, changes in mast cell phenotype and elevations in corticosterone (O’Mahony et al., 2009).
Thus in this study we use broad-spectrum non-absorbable antibiotics as tools to disrupt the colonizing microbiota in early life and assess behavioral and physiological parameters in adulthood. Antibiotic administration, while essential to modern medicine, disrupts the developing bacterial ecosystem (Penders et al., 2006). Early-life antibiotic exposure is associated with altered microbiota (Fouhy et al., 2012), allergic disease (Marra et al., 2009) and inflammatory bowel disease (Shaw et al., 2010). Administration of broad-spectrum antibiotics, frequently used in pediatric practices, has been shown to reduce the biodiversity of fecal microbiota and delay the colonization by probiotic strains in infants (Bennet et al., 2002). However, the functional consequence of such modifications on health outcomes and disease susceptibility is unclear, especially at the level of the brain–gut axis.
We hypothesized that disruption of the microbial milieu during this critical period may result in long-lasting disturbances in brain–gut axis function relevant to stress-related psychiatric disorders and IBS.
Section snippets
Animals
Pregnant Sprague–Dawley female rats (Harlan, UK) and their offspring were used. They were kept under controlled conditions (21 ± 1 °C) on a 12-h light/dark cycle (lights on 7:00 am) and fed ad libitum. The dam and litter and later the offspring were maintained in animal cages (15 × 22 × 9 cm). All experiments were conducted in accordance with the European Directive 86/609/EEC, the Recommendation 2007/526/65/EC and approved by the Ethics Committee of University College Cork and the Health Department,
Study 1: Impact of early-life vancomycin
No adverse reaction to the treatment was noted from observing the neonatal pups daily.
Discussion
In this study we demonstrate what is to our knowledge the first time that early-life antibiotic-induced disruption of the colonizing microbiota is associated with visceral hypersensitivity and altered spinal signaling in adulthood. These results indicate that a temporary alteration in the composition of the GI microbiota during a crucial time-window in the neonatal rat has a long-lasting impact on nociceptive pathways and that the developing pain systems are subject to modulation by the
Conclusion
Our data show that neonatal microbiota disruption results in a very specific and sustained alteration in GI physiology, namely, the induction of visceral sensitivity and altered pain pathways in male rats. On the other hand, this intervention had limited effects on homeostasis with no alterations in cognitive, motor, anxiety, or depressive-like behavior being observed with vancomycin treatment. These findings further confirm the importance of colonizing GI bacteria in early life in the
Conflict of interest
There are no conflicts of interest.
Acknowledgments
All authors were involved in the research or preparation of the manuscript. The Alimentary Pharmabiotic Centre is a research center funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI (grant no.s 02/CE/B124 and 07/CE/B1368). The center is also funded by GlaxoSmithKline. J.F.C. is also funded by European Community’s Seventh Framework Programme; Grant Number: FP7/2007-2013, Grant Agreement 201714.
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Equal contributions.
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Current address: School of Biosciences, Cardiff University, Cardiff CF10 3AT, UK.
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Current address: Division of Gastroenterology, Houston Methodist Hospital, 6550 Fannin Street, SM 1001, Houston, TX 77030, USA.