Niall P. Hyland

Maternal separation as a model of brain–gut axis dysfunction

RationaleEarly life stress has been implicated in many psychiatric disorders ranging from depression to anxiety. Maternal separation in rodents is a well-studied model of early life stress. However, stress during this critical period also induces alterations in many systems throughout the body. Thus, a variety of other disorders that are associated with adverse early life events are often comorbid with psychiatric illnesses, suggesting a common underlying aetiology. Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder that is thought to involve a dysfunctional interaction between the brain and the gut. Essential aspects of the brain–gut axis include spinal pathways, the hypothalamic pituitary adrenal axis, the immune system, as well as the enteric microbiota. Accumulating evidence suggest that stress, especially in early life, is a predisposing factor to IBS.ObjectiveThe objective of this review was to assess and compile the most relevant data on early life stress and alterations at all levels of the brain gut axis.ResultsIn this review, we describe the components of the brain–gut axis individually and how they are altered by maternal separation. The separated phenotype is characterised by alterations of the intestinal barrier function, altered balance in enteric microflora, exaggerated stress response and visceral hypersensitivity, which are all evident in IBS.ConclusionThus, maternally separated animals are an excellent model of brain–gut axis dysfunction for the study of disorders such as IBS and for the development of novel therapeutic interventions.

Breaking down the barriers: the gut microbiome, intestinal permeability and stress-related psychiatric disorders

The emerging links between our gut microbiome and the central nervous system (CNS) are regarded as a paradigm shift in neuroscience with possible implications for not only understanding the pathophysiology of stress-related psychiatric disorders, but also their treatment. Thus the gut microbiome and its influence on host barrier function is positioned to be a critical node within the brain-gut axis. Mounting preclinical evidence broadly suggests that the gut microbiota can modulate brain development, function and behavior by immune, endocrine and neural pathways of the brain-gut-microbiota axis. Detailed mechanistic insights explaining these specific interactions are currently underdeveloped. However, the concept that a “leaky gut” may facilitate communication between the microbiota and these key signaling pathways has gained traction. Deficits in intestinal permeability may underpin the chronic low-grade inflammation observed in disorders such as depression and the gut microbiome plays a critical role in regulating intestinal permeability. In this review we will discuss the possible role played by the gut microbiota in maintaining intestinal barrier function and the CNS consequences when it becomes disrupted. We will draw on both clinical and preclinical evidence to support this concept as well as the key features of the gut microbiota which are necessary for normal intestinal barrier function.

Disturbance of the gut microbiota in early-life selectively affects visceral pain in adulthood without impacting cognitive or anxiety-related behaviors in male rats

Disruption of bacterial colonization during the early postnatal period is increasingly being linked to adverse health outcomes. Indeed, there is a growing appreciation that the gut microbiota plays a role in neurodevelopment. However, there is a paucity of information on the consequences of early-life manipulations of the gut microbiota on behavior. To this end we administered an antibiotic (vancomycin) from postnatal days 4-13 to male rat pups and assessed behavioral and physiological measures across all aspects of the brain-gut axis. In addition, we sought to confirm and expand the effects of early-life antibiotic treatment using a different antibiotic strategy (a cocktail of pimaricin, bacitracin, neomycin; orally) during the same time period in both female and male rat pups. Vancomycin significantly altered the microbiota, which was restored to control levels by 8 weeks of age. Notably, vancomycin-treated animals displayed visceral hypersensitivity in adulthood without any significant effect on anxiety responses as assessed in the elevated plus maze or open field tests. Moreover, cognitive performance in the Morris water maze was not affected by early-life dysbiosis. Immune and stress-related physiological responses were equally unaffected. The early-life antibiotic-induced visceral hypersensitivity was also observed in male rats given the antibiotic cocktail. Both treatments did not alter visceral pain perception in female rats. Changes in visceral pain perception in males were paralleled by distinct decreases in the transient receptor potential cation channel subfamily V member 1, the α-2A adrenergic receptor and cholecystokinin B receptor. In conclusion, a temporary disruption of the gut microbiota in early-life results in very specific and long-lasting changes in visceral sensitivity in male rats, a hallmark of stress-related functional disorders of the brain-gut axis such as irritable bowel disorder.

Prenatal stress-induced alterations in major physiological systems correlate with gut microbiota composition in adulthood

Early-life adverse experiences, including prenatal stress (PNS), are associated with a higher prevalence of neurodevelopmental, cardiovascular and metabolic disorders in affected offspring. Here, in a rat model of chronic PNS, we investigate the impact of late gestational stress on physiological outcomes in adulthood. Sprague-Dawley pregnant dams were subjected to repeated restraint stress from embryonic day 14 to day 20, and their male offspring were assessed at 4 months of age. PNS induced an exaggeration of the hypothalamic-pituitary-adrenal (HPA) axis response to stress, as well as an elevation of blood pressure and impairment of cognitive function. Altered respiratory control was also observed, as demonstrated by increased variability in basal respiratory frequency and abnormal frequency responses to both hypoxic and hypercapnic challenges. PNS also affected gastrointestinal neurodevelopment and function, as measured by a decrease in the innervation density of distal colon and an increase in the colonic secretory response to catecholaminergic stimulation. Finally, PNS induced long lasting alterations in the intestinal microbiota composition. 16S rRNA gene 454 pyrosequencing revealed a strong trend towards decreased numbers of bacteria in the Lactobacillus genus, accompanied by elevated abundance of the Oscillibacter, Anaerotruncus and Peptococcus genera in PNS animals. Strikingly, relative abundance of distinct bacteria genera significantly correlated with certain respiratory parameters and the responsiveness of the HPA axis to stress. Together, these findings provide novel evidence that PNS induces long-term maladaptive alterations in the gastrointestinal and respiratory systems, accompanied by hyper-responsiveness to stress and alterations in the gut microbiota.

A Gut Feeling about GABA: Focus on GABAB Receptors

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the body and hence GABA-mediated neurotransmission regulates many physiological functions, including those in the gastrointestinal (GI) tract. GABA is located throughout the GI tract and is found in enteric nerves as well as in endocrine-like cells, implicating GABA as both a neurotransmitter and an endocrine mediator influencing GI function. GABA mediates its effects via GABA receptors which are either ionotropic GABAA or metabotropic GABAB. The latter which respond to the agonist baclofen have been least characterized, however accumulating data suggest that they play a key role in GI function in health and disease. Like GABA, GABAB receptors have been detected throughout the gut of several species in the enteric nervous system, muscle, epithelial layers as well as on endocrine-like cells. Such widespread distribution of this metabotropic GABA receptor is consistent with its significant modulatory role over intestinal motility, gastric emptying, gastric acid secretion, transient lower esophageal sphincter relaxation and visceral sensation of painful colonic stimuli. More intriguing findings, the mechanisms underlying which have yet to be determined, suggest GABAB receptors inhibit GI carcinogenesis and tumor growth. Therefore, the diversity of GI functions regulated by GABAB receptors makes it a potentially useful target in the treatment of several GI disorders. In light of the development of novel compounds such as peripherally acting GABAB receptor agonists, positive allosteric modulators of the GABAB receptor and GABA producing enteric bacteria, we review and summarize current knowledge on the function of GABAB receptors within the GI tract.

Toll-Like Receptor mRNA Expression Is Selectively Increased in the Colonic Mucosa of Two Animal Models Relevant to Irritable Bowel Syndrome

Background Irritable bowel syndrome (IBS) is largely viewed as a stress-related disorder caused by aberrant brain-gut–immune communication and altered gastrointestinal (GI) homeostasis. Accumulating evidence demonstrates that stress modulates innate immune responses; however, very little is known on the immunological effects of stress on the GI tract. Toll-like receptors (TLRs) are critical pattern recognition molecules of the innate immune system. Activation of TLRs by bacterial and viral molecules leads to activation of NF-kB and an increase in inflammatory cytokine expression. It was our hypothesis that innate immune receptor expression may be changed in the gastrointestinal tract of animals with stress-induced IBS-like symptoms. Methodology/Principal Findings In this study, our objective was to evaluate the TLR expression profile in the colonic mucosa of two rat strains that display colonic visceral hypersensivity; the stress-sensitive Wistar-Kyoto (WKY) rat and the maternally separated (MS) rat. Quantitative PCR of TLR2-10 mRNA in both the proximal and distal colonic mucosae was carried out in adulthood. Significant increases are seen in the mRNA levels of TLR3, 4 & 5 in both the distal and proximal colonic mucosa of MS rats compared with controls. No significant differences were noted for TLR 2, 7, 9 & 10 while TLR 6 could not be detected in any samples in both rat strains. The WKY strain have increased levels of mRNA expression of TLR3, 4, 5, 7, 8, 9 & 10 in both the distal and proximal colonic mucosa compared to the control Sprague-Dawley strain. No significant differences in expression were found for TLR2 while as before TLR6 could not be detected in all samples in both strains. Conclusions These data suggest that both early life stress (MS) and a genetic predisposition (WKY) to stress affect the expression of key sentinels of the innate immune system which may have direct relevance for the molecular pathophysiology of IBS.

Colonic soluble mediators from the maternal separation model of irritable bowel syndrome activate submucosal neurons via an interleukin-6-dependent mechanism

Irritable bowel syndrome (IBS) is characterized by episodic bouts of abdominal pain, bloating, and altered bowel habit. Accumulating evidence has linked immune activation with IBS, including reports of increases in circulating levels of the proinflammatory cytokine interleukin (IL)-6. However, it is unknown whether IL-6 contributes directly to disease manifestation. As enteric nervous activity mediates motility and secretory function, the aims of this study were to determine the effects of IL-6 on submucosal neurons and related gastrointestinal (GI) function. In these studies, we examined the colons of maternally separated (MS) rats, which exhibit elevated circulating levels of IL-6 in addition to GI dysfunction. To our knowledge, these studies are the first to provide evidence of the sensitivity of submucosal neurons to colonic secretions from MS rats (n = 50, P < 0.05), thus recapitulating clinical biopsy data. Moreover, we demonstrated that the excitatory action is IL-6 dependent. Thereafter, the impact of IL-6 on neuronal and glial activation and absorpto/secretory function was pharmacologically characterized. Other proinflammatory cytokines including IL-8 (n = 30, P > 0.05), IL-1β (n = 56, P > 0.05), and TNF-α (n = 56, P > 0.05) excited fewer neurons. Both muscarinic and nicotinic cholinergic receptors participate in the effect and cause downstream activation of ERK, JAK-STAT, and NF-κB signaling cascades. Functionally, IL-6 increases transepithelial resistance and enhances neurally and cholinergically mediated ion transport. These data provide a role for IL-6 in colonic secretory functions and relate these effects to GI dysfunction in an animal model of IBS, thereby elucidating a potential relationship between circulating levels of IL-6 and aberrant GI function.

The effects of repeated social interaction stress on behavioural and physiological parameters in a stress-sensitive mouse strain

Stress can impair the immune, endocrine and nervous systems. Such perturbations can also affect brain-gut axis communication and lead to functional gastrointestinal disorders such as irritable bowel syndrome (IBS). IBS is a common yet poorly understood disorder which is often co-morbid with anxiety and depression. As there are few mouse models of IBS, this study aimed to investigate if a short and intense social stress which involved bouts of physical interaction could induce behavioural and physiological changes similar to those observed in IBS patients in the innately anxious BALB/c mice. Male BALB/c mice were exposed for 2h to an aggressive male intruder for acute (one-day) or chronic (six-day) stress. Behaviour was analyzed and weight monitored. Two hours post stress, trunk blood and tissues were collected. Plasma was analyzed for inflammatory cytokines and corticosterone and morphological damage to the colon was also assessed. Mice displayed either dominant or submissive status following repeated intruder exposure. Behavioural status correlated with an increase in corticosterone and pro-inflammatory cytokines in both acute and chronic submissive groups. Mice from both status groups had body weight loss coupled with mild damage to the colon. Together these data show that short-term social interaction stress exposure was able to induce behavioural and physiological impairments similar to that observed in patients with dysregulated brain-gut axis function. Moreover, these data demonstrate that social stress-based mouse models may be appropriate for interrogating the mechanisms underlying such disorders.

Functional consequences of neuropeptide Y Y2 receptor knockout and Y2 antagonism in mouse and human colonic tissues

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) differentially activate three Y receptors (Y1, Y2 and Y4) in mouse and human isolated colon. The aim of this study was to characterise Y2 receptor‐mediated responses in colon mucosa and longitudinal smooth muscle preparations from wild type (Y2+/+) and knockout (Y2−/−) mice and to compare the former with human mucosal Y agonist responses. Inhibition of mucosal short‐circuit current and increases in muscle tone were monitored in colonic tissues from Y2+/+ and Y2−/− mice±Y1 ((R)‐N‐[[4‐(aminocarbonylaminomethyl)phenyl)methyl]‐N2‐(diphenylacetyl)‐argininamide‐trifluoroacetate (BIBO3304) or Y2 (S)‐N2‐[[1‐[2‐[4‐[(R,S)‐5,11‐dihydro‐6(6H)‐oxodibenz[b,e]azepin‐11‐yl]‐1‐piperazinyl]‐2‐oxoethyl]cyclopentyl]acetyl]‐N‐[2‐[1,2‐dihydro‐3,5(4H)‐dioxo‐1,2‐diphenyl‐3H‐1,2,4‐triazol‐4‐yl]ethyl]‐argininamide (BIIE0246) antagonists. Predictably, Y2−/− tissues were insensitive to Y2‐preferred agonist PYY(3‐36) (100 nM), but unexpectedly Y4‐preferred PP responses were right‐shifted probably as a consequence of elevated circulating PP levels, particularly in male Y2−/− mice ( Sainsbury et al., 2002 ). BIBO3304 and BIIE0246 elevated mucosal ion transport, indicating blockade of inhibitory mucosal tone in Y2+/+ tissue. While BIBO3304 effects were unchanged, those to BIIE0246 were absent in Y2−/− mucosae. Neither antagonist altered muscle tone; however, BIIE0246 blocked NPY and PYY(3‐36) increases in Y2+/+ basal tone. BIBO3304 abolished residual Y1‐mediated NPY responses in Y2−/− smooth muscle. Tetrodotoxin significantly reduced BIIE0246 and PYY(3‐36) effects in Y2+/+ mouse and human mucosae, but had no effect upon Y‐agonist contractile responses, indicating that Y2 receptors are located on submucosal, but not myenteric neurones. Tonic activation of submucosal Y2 receptors by endogenous NPY, PYY or PYY(3‐36) could indirectly reduce mucosal ion transport in murine and human colon, while direct activation of Y2 receptors on longitudinal muscle results in contraction.

Microbiota-host interactions in irritable bowel syndrome: Epithelial barrier, immune regulation and brain-gut interactions

Irritable bowel syndrome (IBS) is a common, sometimes debilitating, gastrointestinal disorder worldwide. While altered gut motility and sensation, as well as aberrant brain perception of visceral events, are thought to contribute to the genesis of symptoms in IBS, a search for an underlying aetiology has, to date, proven unsuccessful. Recently, attention has been focused on the microbiota as a possible factor in the pathogenesis of IBS. Prompted by a number of clinical observations, such as the recognition of the de novo development of IBS following enteric infections, as well as descriptions of changes in colonic bacterial populations in IBS and supported by clinical responses to interventions, such as antibiotics and probiotics, that modify the microbiota, various approaches have been taken to investigating the microbiota-host response in IBS, as well as in animal models thereof. From such studies a considerable body of evidence has accumulated to indicate the activation or upregulation of both factors involved in bacterial engagement with the host as well host defence mechanisms against bacteria. Alterations in gut barrier function, occurring in response, or in parallel, to changes in the microbiota, have also been widely described and can be seen to play a pivotal role in generating and sustaining host immune responses both within and beyond the gut. In this manner a plausible hypothesis, based on an altered microbiota and/or an aberrant host response, for the pathogenesis, of at least some instances of IBS, can be generated.