Using Germ-Free Animals to Assess the Influence of the Gut Microbiota on Brain and Behavior

International Journal of Neuropsychopharmacology, (2016) 19(8): 1–17 doi:10.1093/ijnp/pyw020 Advance Access Publication: February 23, 2016 Review review Growing up in a Bubble: Using Germ-Free Animals to Assess the Influence of the Gut Microbiota on Brain and Behavior Pauline Luczynski, MSc; Karen-Anne McVey Neufeld, PhD; Clara Seira Oriach, MSc; Gerard Clarke, PhD; Timothy G. Dinan, MD, PhD, DSc; John F. Cryan, PhD APC Microbiome Institute (Ms Luczynski, Dr McVey Neufeld, Ms Oriach, Dr Clarke, Dr Dinan, and Dr Cryan), Department of Psychiatry and Neurobehavioral Science (Ms Oriach, Dr Clarke, and Dr Dinan), and Department of Anatomy and Neuroscience (Dr Cryan), University College Cork, Cork, Ireland. Correspondence: John F. Cryan, PhD, Room 3.86, Western Gateway Building, University College Cork, Cork, Ireland ([email protected]). Abstract There is a growing recognition of the importance of the commensal intestinal microbiota in the development and later function of the central nervous system. Research using germ-free mice (mice raised without any exposure to microorganisms) has provided some of the most persuasive evidence for a role of these bacteria in gut-brain signalling. Key findings show that the microbiota is necessary for normal stress responsivity, anxiety-like behaviors, sociability, and cognition. Furthermore, the microbiota maintains central nervous system homeostasis by regulating immune function and blood brain barrier integrity. Studies have also found that the gut microbiota influences neurotransmitter, synaptic, and neurotrophic signalling systems and neurogenesis. The principle advantage of the germ-free mouse model is in proof-of-principle studies and that a complete microbiota or defined consortiums of bacteria can be introduced at various developmental time points. However, a germ-free upbringing can induce permanent neurodevelopmental deficits that may deem the model unsuitable for specific scientific queries that do not involve early-life microbial deficiency. As such, alternatives and complementary strategies to the germ- free model are warranted and include antibiotic treatment to create microbiota-deficient animals at distinct time points across the lifespan. Increasing our understanding of the impact of the gut microbiota on brain and behavior has the potential to inform novel management strategies for stress-related gastrointestinal and neuropsychiatric disorders. Keywords: microbiota-gut-brain axis, germ-free mouse, stress response, social development, cognitive dysfunction Introduction Medicine is magical and magical is art tract, respiratory tract, skin, and urogenital tract (Costello et al., Thinking of the Boy in the Bubble 2009). Research on the interactions between microorganisms “Boy in the Bubble” by Paul Simon, 1986 and their host has largely and understandably been focused Humans and other animals share an inextricable and mutualis- on those residing in the intestinal lumen, given that this is the tic relationship with a myriad of resident microorganisms, col- most densely colonized and home to approximately 100 trillion lectively known as the microbiota. These microbial species are bacteria (Frank and Pace, 2008). Over the last decade, numerous present on all body surfaces, including the gastrointestinal (GI) scientific discoveries have highlighted the role of the microbiota Received: December 10, 2015; Revised: February 15, 2016; Accepted: February 18, 2016 © The Author 2016. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 1 provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 | International Journal of Neuropsychopharmacology, 2016 in critical processes, including digestion (Hooper and Gordon, 2014) and also the ability to alter intestinal microbial contents 2001), immune responses (Bäckhed et al., 2005; Hooper and following hypothalamic-pituitary-adrenal (HPA) axis activation Macpherson, 2010), absorption of nutrients (Hooper and Gordon, (O’Mahony et al., 2009; De Palma et al., 2015; Golubeva et al., 2015). 2001), growth (Nicholson et al., 2012), and metabolism (Tremaroli and Backhed, 2012). Until recently, little was known about the role The Microbiota in Health and Disease of the microbiota in the regulation of the central nervous system (CNS); however, there is now a burgeoning body of evidence Compelling clinical evidence exists supporting the view that a indicating that the gut microbiota also influences the brain and stable and diverse gut microbial composition is important for the behavior (Rhee et al., 2009; Collins et al., 2012; Cryan and Dinan, maintenance of optimal health. Indeed, altered microbial profiles 2012; Dinan and Cryan, 2012; Dinan et al., 2013; Foster and McVey have been associated with numerous human illnesses, includ- Neufeld, 2013; Foster et al., 2015; Sampson and Mazmanian, ing cardiovascular disease (Koren et al., 2011; Howitt and Garrett, 2015). These fascinating insights have largely been derived from 2012; Karlsson et al., 2012; Tuohy et al., 2014; Hansen et al., 2015), the study of germ-free (GF) animals that effectively grow up in a irritable bowel syndrome (IBS) (Salonen et al., 2010; Tana et al., sterile bubble and have provided conceptual impetus to the pos- 2010; Jeffery et al., 2012; Wu, 2012), inflammatory bowel disease sibility of exploiting the gut microbiota as a therapeutic target. (Ananthakrishnan, 2015; Marchesi et al., 2015; Sheehan et al., 2015), obesity (Ley et al., 2006; Turnbaugh et al., 2009a; Tremaroli The Microbiota-Gut-Brain Axis and Backhed, 2012), and type 2 diabetes (Qin et al., 2012; Vrieze et al., 2012; Karlsson et al., 2013). However, there is presently lit- The concept of the gut-brain axis has been recognized for some tle data detailing a clear link between the gut microbiota and time and has been used as a framework to assess mechanisms the clinical manifestation of CNS diseases, although mounting by which bidirectional communication between these 2 systems preclinical evidence highlights the necessity for such studies. occurs. More recently, this axis has been extended to include The impact of the microbiota on the CNS can be investigated the contents of the intestinal lumen, and we now refer to the by assessing behavior following various deliberate interven- existence of a ‘microbiota-gut-brain axis’ (Rhee et al., 2009; tions. Infection with pathogenic bacteria heightens anxiety-like Bercik, 2011; Cryan and O’Mahony, 2011; Cryan and Dinan, 2012; behavior and reduces cognitive abilities (Lyte et al., 1998, 2006; Burokas et al., 2015; Mayer et al., 2015), highlighting the increas- Goehler et al., 2008; Gareau et al., 2011). Similarly, antibiotic ingly appreciated importance of bacteria in this communication administration alters the gut microbial composition, which is highway. While the necessity of commensal intestinal micro- accompanied by reduced anxiety-like behavior (Bercik et al., biota for the maintenance of optimal gut-brain function is no 2011a). Probiotics, live organisms that provide health benefits longer disputed, the exact mechanisms by which microbiota- when consumed, improve cognition and attenuate heightened gut-brain communication occurs are still largely unknown. It is stress responsivity in both humans and rodents (Bravo et al., probable that a number of systems are simultaneously involved 2011; Messaoudi et al., 2011; Savignac et al., 2015). As such, we in the transmission of information between microbiota and have recently coined the term “psychobiotic” to describe bacte- brain, and research has focused primarily on immune, neural, ria with beneficial effects on mental health (Dinan et al., 2013). endocrine, and metabolic pathways as likely mediators in this Finally, behavioral traits such as anxiety can be transferred via bidirectional communication (Cryan and Dinan, 2012; Foster and fecal transplantation (Bercik et al., 2011a), although this field of McVey Neufeld, 2013; El Aidy et al., 2015; Janssen and Kersten, research is still in its early stages. 2015; Mayer et al., 2015). Research using GF mice has arguably provided the most con- The human gut is extensively innervated, with approxi- vincing evidence for a role of the microbiota in gut-brain signal- mately 50 000 extrinsic and 100 million intrinsic sensory affer- ling. GF studies have generated exciting data with the goal of ent neurons (Furness, 2006; Blackshaw et al., 2007), all of which directly answering the question: does the gut microbiota influ- function in proximity to the trillions of microorganisms housed ence the nervous system? This review will summarize what the in the intestinal lumen. It is thus unsurprising that neural path- GF mouse model has revealed about the importance of the gut ways play a vital role in microbiota-gut-brain communication. microbiota on the development and function of the CNS with The necessity of an intact vagal nerve for information regard- a focus on neuropsychiatric disorders. Tables 1 and 2 detail ing intestinal microbial status seems dependent upon the spe- what we currently know about how growing up in the absence cific bacterial species in question, and while some studies have of microorganisms alters behavior and brain function, respec- shown the vagus is vital for information

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