Social anxiety disorder-associated gut microbiota increases social fear.

Social anxiety disorder (SAD) is a crippling psychiatric disorder characterized by intense fear or anxiety in social situations and their avoidance. However, the underlying biology of SAD is unclear and better treatments are needed. Recently, the gut microbiota has emerged as a key regulator of both brain and behaviour, especially those related to social function. Moreover, increasing data supports a role for immune function and oxytocin signalling in social responses. To investigate whether the gut microbiota plays a causal role in modulating behaviours relevant to SAD, we transplanted the microbiota from SAD patients, which was identified by 16S rRNA sequencing to be of a differential composition compared to healthy controls, to mice. Although the mice that received the SAD microbiota had normal behaviours across a battery of tests designed to assess depression and general anxiety-like behaviours, they had a specific heightened sensitivity to social fear, a model of SAD. This distinct heightened social fear response was coupled with changes in central and peripheral immune function and oxytocin expression in the bed nucleus of the stria terminalis. This work demonstrates an interkingdom basis for social fear responses and posits the microbiome as a potential therapeutic target for SAD.

From hype to hope: Considerations in conducting robust microbiome science.

Microbiome science has been one of the most exciting and rapidly evolving research fields in the past two decades. Breakthroughs in technologies including DNA sequencing have meant that the trillions of microbes (particularly bacteria) inhabiting human biological niches (particularly the gut) can be profiled and analysed in exquisite detail. This microbiome profiling has profound impacts across many fields of research, especially biomedical science, with implications for how we understand and ultimately treat a wide range of human disorders. However, like many great scientific frontiers in human history, the pioneering nature of microbiome research comes with a multitude of challenges and potential pitfalls. These include the reproducibility and robustness of microbiome science, especially in its applications to human health outcomes. In this article, we address the enormous promise of microbiome science and its many challenges, proposing constructive solutions to enhance the reproducibility and robustness of research in this nascent field. The optimisation of microbiome science spans research design, implementation and analysis, and we discuss specific aspects such as the importance of ecological principals and functionality, challenges with microbiome-modulating therapies and the consideration of confounding, alternative options for microbiome sequencing, and the potential of machine learning and computational science to advance the field. The power of microbiome science promises to revolutionise our understanding of many diseases and provide new approaches to prevention, early diagnosis, and treatment.

Critical windows of early-life microbiota disruption on behaviour, neuroimmune function, and neurodevelopment.

Numerous studies have emphasised the importance of the gut microbiota during early life and its role in modulating neurodevelopment and behaviour. Epidemiological studies have shown that early-life antibiotic exposure can increase an individual's risk of developing immune and metabolic diseases. Moreover, preclinical studies have shown that long-term antibiotic-induced microbial disruption in early life can have enduring effects on physiology, brain function and behaviour. However, these studies have not investigated the impact of targeted antibiotic-induced microbiota depletion during critical developmental windows and how this may be related to neurodevelopmental outcomes. Here, we addressed this gap by administering a broad-spectrum oral antibiotic cocktail (ampicillin, gentamicin, vancomycin, and imipenem) to mice during one of three putative critical windows: the postnatal (PN; P2-9), pre-weaning (PreWean; P12-18), or post-weaning (Wean; P21-27) developmental periods and assessed the effects on physiology and behaviour in later life. Our results demonstrate that targeted microbiota disruption during early life has enduring effects into adolescence on the structure and function of the caecal microbiome, especially for antibiotic exposure during the weaning period. Further, we show that microbial disruption in early life selectively alters circulating immune cells and modifies neurophysiology in adolescence, including altered myelin-related gene expression in the prefrontal cortex and altered microglial morphology in the basolateral amygdala. We also observed sex and time-dependent effects of microbiota depletion on anxiety-related behavioural outcomes in adolescence and adulthood. Antibiotic-induced microbial disruption had limited and subtle effects on social behaviour and did not have any significant effects on depressive-like behaviour, short-term working, or recognition memory. Overall, this study highlights the importance of the gut microbiota during critical windows of development and the subtle but long-term effects that microbiota-targeted perturbations can have on brain physiology and behaviour.

Introduction.

The theme of this monograph reflects the growing research interest in the contribution of the microbiome-gut-brain axis to mental health. This chapter introduces readers to the study of the microbiome in psychiatric research and emphasises how research into the gut microbiome has had a significant impact on our understanding of mental health. A brief summary of the historical background for microbiome research in mental health is followed by examples of evidence linking gut microorganisms to changes in brain function. As novel technological developments have played a major role in providing the evidence for microbiome modulation of brain function, an overview of modern techniques and technologies is then provided. These have broadened our understanding of the range of microorganisms, in addition to bacteria, which contribute to the changes initiated by the microbiome. In addition, common experimental models are reviewed in light of the important role that animal studies, particularly in germ-free rodents, have played in establishing microbiome-gut-brain interactions. This introduction concludes with a summary of the challenges for future microbiome research, providing a forward-thinking perspective echoed in many of the following chapters.

The Microbiome-Gut-Brain Axis in Neurocognitive Development and Decline.

Since the beginning of life on earth, microorganisms have played a significant role in evolution. Throughout the history of Homo sapiens and its precursor humanoid forms, microorganisms have been present at birth and proliferated until death. It is at these extremes of life that the microbiome, especially that within the gastrointestinal tract, is most dynamic and perhaps has its greatest influence on host health. Here, we focus on the role of the gut microbiome as a regulator of brain and behaviour through key points in the human lifespan. We first describe trajectories of the microbiome in early life and ageing, before providing evidence for the existence of sensitive periods in the microbiome-gut-brain axis at these extremes of the lifespan. Finally, we briefly examine potential mechanisms for interactions between the microbiome and the brain during development and ageing.

Microbiota from young mice counteracts selective age-associated behavioral deficits.

The gut microbiota is increasingly recognized as an important regulator of host immunity and brain health. The aging process yields dramatic alterations in the microbiota, which is linked to poorer health and frailty in elderly populations. However, there is limited evidence for a mechanistic role of the gut microbiota in brain health and neuroimmunity during aging processes. Therefore, we conducted fecal microbiota transplantation from either young (3-4 months) or old (19-20 months) donor mice into aged recipient mice (19-20 months). Transplant of a microbiota from young donors reversed aging-associated differences in peripheral and brain immunity, as well as the hippocampal metabolome and transcriptome of aging recipient mice. Finally, the young donor-derived microbiota attenuated selective age-associated impairments in cognitive behavior when transplanted into an aged host. Our results reveal that the microbiome may be a suitable therapeutic target to promote healthy aging.

The gut microbiota in anxiety and depression - A systematic review.

Growing evidence indicates the community of microorganisms throughout the gastrointestinal tract, (i.e., gut microbiota), is associated with anxiety and depressive disorders. We present the first systematic review of the gut microbiota in anxiety disorders, along with an update in depression. Consideration of shared underlying features is essential due to the high rates of comorbidity. Systematic searches, following PRISMA guidelines, identified 26 studies (two case-control comparisons of the gut microbiota in generalised anxiety disorder, 18 in depression, one incorporating both anxiety/depression, and five including symptom-only measures). Alpha and beta diversity findings were inconsistent; however, differences in bacterial taxa indicated disorders may be characterised by a higher abundance of proinflammatory species (e.g., Enterobacteriaceae and Desulfovibrio), and lower short-chain fatty acid producing-bacteria (e.g., Faecalibacterium). Several taxa, and their mechanisms of action, may relate to anxiety and depression pathophysiology via communication of peripheral inflammation to the brain. Although the gut microbiota remains a promising target for prevention and therapy, future research should assess confounders, particularly diet and psychotropic medications, and should examine microorganism function.

Molecular, biochemical and behavioural evidence for a novel oxytocin receptor and serotonin 2C receptor heterocomplex.

The complexity of oxytocin-mediated functions is strongly associated with its modulatory effects on other neurotransmission systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Signalling between oxytocin (OT) and 5-HT has been demonstrated during neurodevelopment and in the regulation of specific emotion-based behaviours. It is suggested that crosstalk between neurotransmitters is driven by interaction between their specific receptors, particularly the oxytocin receptor (OTR) and the 5-hydroxytryptamine 2C receptor (5-HTR2C), but evidence for this and the downstream signalling consequences that follow are lacking. Considering the overlapping central expression profiles and shared involvement of OTR and 5-HTR2C in certain endocrine functions and behaviours, including eating behaviour, social interaction and locomotor activity, we investigated the existence of functionally active OTR/5-HTR2C heterocomplexes. Here, we demonstrate evidence for a potential physical interaction between OTR and 5-HTR2Cin vitro in a cellular expression system using flow cytometry-based FRET (fcFRET). We could recapitulate this finding under endogenous expression levels of both receptors via in silico analysis of single cell transcriptomic data and ex vivo proximity ligation assay (PLA). Next, we show that co-expression of the OTR/5-HTR2C pair resulted in a significant depletion of OTR-mediated Gαq-signalling and significant changes in receptor trafficking. Of note, attenuation of OTR-mediated downstream signalling was restored following pharmacological blockade of the 5-HTR2C. Finally, we demonstrated a functional relevance of this novel heterocomplex, in vivo, as 5-HTR2C antagonism increased OT-mediated hypoactivity in mice. Overall, we provide compelling evidence for the formation of functionally active OTR/5-HTR2C heterocomplexes, adding another level of complexity to OTR and 5-HTR2C signalling functionality. This article is part of the special issue on Neuropeptides.

Is good memory always a good thing? An early offset of infantile amnesia predicts anxiety-like behavior throughout development in rats.

Infants tend to forget experiences much more rapidly than older individuals, a phenomenon referred to as infantile amnesia. This robust, cross-species phenomenon is commonly used to examine memory development. However, in this set of experiments, we examined the novel hypothesis that the expression of infantile amnesia is related to resilience/vulnerability. We conditioned infant rats to associate a white noise with shock. Animals were tested for memory of the association ~1 week later. We found that infants that expressed better memory of the aversive association emitted more vocalizations (indicative of higher levels of distress) when separated from their mother earlier in infancy (Experiment 1). Better expression of memory in infancy also predicted higher levels of conditioned fear (Experiment 2) and anxiety-like behavior (in a light-dark box; Experiment 3) in adulthood. Furthermore, probiotic-treatment administered early in development reduced anxiety-like behavior in animals that exhibited good expression of memory for an aversive association learnt in infancy (Experiment 4). However, the same treatment was ineffective if administered in adulthood. Taken together, these results suggest that individual differences in infants' memory for an aversive association predict anxiety-like behavior throughout development, and that early administration of probiotics can reduce anxiety-like behavior in "at-risk" animals.

Annual Research Review: Critical windows - the microbiota-gut-brain axis in neurocognitive development.

The gut microbiota is a vast, complex, and fascinating ecosystem of microorganisms that resides in the human gastrointestinal tract. As an integral part of the microbiota-gut-brain axis, it is now being recognized that the microbiota is a modulator of brain and behavior, across species. Intriguingly, periods of change in the microbiota coincide with the development of other body systems and particularly the brain. We hypothesize that these times of parallel development are biologically relevant, corresponding to 'sensitive periods' or 'critical windows' in the development of the microbiota-gut-brain axis. Specifically, signals from the microbiota during these periods are hypothesized to be crucial for establishing appropriate communication along the axis throughout the life span. In other words, the microbiota is hypothesized to act like an expected input to calibrate the development of the microbiota-gut-brain axis. The absence or disruption of the microbiota during specific developmental windows would therefore be expected to have a disproportionate effect on specific functions or potentially for regulation of the system as a whole. Evidence for microbial modulation of neurocognitive development and neurodevelopmental risk is discussed in light of this hypothesis, finishing with a focus on the challenges that lay ahead for the future study of the microbiota-gut-brain axis during development.

The Microbiota-Gut-Brain Axis.

The importance of the gut-brain axis in maintaining homeostasis has long been appreciated. However, the past 15 yr have seen the emergence of the microbiota (the trillions of microorganisms within and on our bodies) as one of the key regulators of gut-brain function and has led to the appreciation of the importance of a distinct microbiota-gut-brain axis. This axis is gaining ever more traction in fields investigating the biological and physiological basis of psychiatric, neurodevelopmental, age-related, and neurodegenerative disorders. The microbiota and the brain communicate with each other via various routes including the immune system, tryptophan metabolism, the vagus nerve and the enteric nervous system, involving microbial metabolites such as short-chain fatty acids, branched chain amino acids, and peptidoglycans. Many factors can influence microbiota composition in early life, including infection, mode of birth delivery, use of antibiotic medications, the nature of nutritional provision, environmental stressors, and host genetics. At the other extreme of life, microbial diversity diminishes with aging. Stress, in particular, can significantly impact the microbiota-gut-brain axis at all stages of life. Much recent work has implicated the gut microbiota in many conditions including autism, anxiety, obesity, schizophrenia, Parkinson's disease, and Alzheimer's disease. Animal models have been paramount in linking the regulation of fundamental neural processes, such as neurogenesis and myelination, to microbiome activation of microglia. Moreover, translational human studies are ongoing and will greatly enhance the field. Future studies will focus on understanding the mechanisms underlying the microbiota-gut-brain axis and attempt to elucidate microbial-based intervention and therapeutic strategies for neuropsychiatric disorders.

Early-life stress, microbiota, and brain development: probiotics reverse the effects of maternal separation on neural circuits underpinning fear expression and extinction in infant rats.

Early-life stress has pervasive, typically detrimental, effects on physical and mental health across the lifespan. In rats, maternal-separation stress results in premature expression of an adult-like profile of fear regulation that predisposes stressed rats to persistent fear, one of the hallmarks of clinical anxiety. Probiotic treatment attenuates the effects of maternal separation on fear regulation. However, the neural pathways underlying these behavioral changes are unknown. Here, we examined the neural correlates of stress-induced alterations in fear behavior and their reversal by probiotic treatment. Male Sprague-Dawley rats were exposed to either standard rearing conditions or maternal-separation stress (postnatal days [P] 2-14). Some maternally-separated (MS) animals were also exposed to probiotics (Lactobacillus rhamnosus and L. helveticus) via the maternal drinking water during the period of stress. Using immunohistochemistry, we demonstrated that stressed rat pups prematurely exhibit adult-like engagement of the medial prefrontal cortex during fear regulation, an effect that can be prevented using a probiotic treatment. The present results add to the cross-species evidence that early adversity hastens maturation in emotion-related brain circuits. Importantly, our results also demonstrate that the precocious neural maturation in stressed infants is prevented by a non-invasive probiotic treatment.

What can the gut microbiome teach us about the connections between child physical and mental health? A systematic review.

A deeper understanding of the gut-brain axis is of significance in pediatrics, given the influential role of early childhood experiences and exposures in shaping the microbiome, and health, across the life course. This systematic review synthesized evidence on the connection between the gut microbiome and mental health in children with physical illness. Six electronic databases were systematically searched and data extracted according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. Of 1,476 identified articles, 11 articles reporting on nine unique studies (all randomized controlled trials) were included. Most studies examined the gut microbiome in infants with colic, while the remaining studies investigated outcomes in children aged 1 day to 18 years at risk for atopic dermatitis or irritable bowel syndrome. Baseline and postintervention gut microbiome differences varied across studies. Findings on psychological functioning also varied, with only half of the captured studies showing a positive effect of intervention on psychological well-being. Only two studies analyzed the association between the gut microbiome and psychological outcomes, each with a different pattern of results. As the field moves forward, it will be critical to gain a better understanding of the microbiome characteristics that influence mental health outcomes in pediatric populations.

Feeding melancholic microbes: MyNewGut recommendations on diet and mood.

Depression is a highly prevalent disorder which exerts a major economic impact in all European countries. The brain-gut-microbiota axis has been described as a new paradigm for advancing understanding and treatment of the disorder. There is now over-whelming evidence to support the fact that gut microbes have a major impact on central neurochemistry and behaviour, especially stress related disorders such as depression. Recent studies indicate that patients with depression have a gut dysbiosis. The reason for this dysbiosis is uncertain. Over recent decades, dietary patterns in Europe and elsewhere have undergone major compositional changes, with increased intakes of red meat, high fat foods, and refined sugars. Individuals who consume a Mediterranean diet have lower rates of depression and a recent study suggests that a Mediterranean diet may have antidepressant properties. Assuming this to be the case, which components of the Mediterranean diet mediate the effects? Highly levels of polyphenols or polyunsaturated fatty acids are obvious candidates. We in the MyNewGut consortium recommend that patients with depression or vulnerability to depression should be encouraged to enhance a plant-based diet with a high content of grains/fibres and fish.

Early-life stress leads to sex-dependent changes in pubertal timing in rats that are reversed by a probiotic formulation.

Puberty marks the beginning of a period of dramatic physical, hormonal, and social change. This instability has made adolescence infamous as a time of "storm and stress" and it is well-established that stress during adolescence can be particularly damaging. However, prior stress may also shape the adolescent experience. In the present series of experiments, we observed sex-specific effects of early-life maternal separation stress on the timing of puberty onset in the rat. Specifically, stressed females exhibited earlier pubertal onset compared to standard-reared females, whereas stressed males matured later than their standard-reared counterparts. Further, we demonstrated that a probiotic treatment restores the normative timing of puberty onset in rodents of both sexes. These results are in keeping with previous findings that probiotics reverse stress-induced changes in learned fear behaviors and stress hormone levels, highlighting the remarkable and wide-ranging restorative effects of probiotics in the context of early-life stress.

Making Sense of the Microbiome in Psychiatry.

Microorganisms can be found almost anywhere, including in and on the human body. The collection of microorganisms associated with a certain location is called a microbiota, with its collective genetic material referred to as the microbiome. The largest population of microorganisms on the human body resides in the gastrointestinal tract; thus, it is not surprising that the most investigated human microbiome is the human gut microbiome. On average, the gut hosts microbes from more than 60 genera and contains more cells than the human body. The human gut microbiome has been shown to influence many aspects of host health, including more recently the brain.Several modes of interaction between the gut and the brain have been discovered, including via the synthesis of metabolites and neurotransmitters, activation of the vagus nerve, and activation of the immune system. A growing body of work is implicating the microbiome in a variety of psychological processes and neuropsychiatric disorders. These include mood and anxiety disorders, neurodevelopmental disorders such as autism spectrum disorder and schizophrenia, and even neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Moreover, it is probable that most psychotropic medications have an impact on the microbiome.Here, an overview will be provided for the bidirectional role of the microbiome in brain health, age-associated cognitive decline, and neurological and psychiatric disorders. Furthermore, a primer on the common microbiological and bioinformatics techniques used to interrogate the microbiome will be provided. This review is meant to equip the reader with a primer to this exciting research area that is permeating all areas of biological psychiatry research.

A precision medicine approach to pharmacological adjuncts to extinction: a call to broaden research.

There is a pressing need to improve treatments for anxiety. Although exposure-based therapy is currently the gold-standard treatment, many people either do not respond to this therapy or experience a relapse of symptoms after treatment has ceased. In recent years, there have been many novel pharmacological agents identified in preclinical research that have potential as adjuncts for exposure therapy, yet very few of these are regularly integrated into clinical practice. Unfortunately, the robust effects observed in the laboratory animal often do not translate to a clinical population. In this review, we discuss how age, sex, genetics, stress, medications, diet, alcohol, and the microbiome can vary across a clinical population and yet are rarely considered in drug development. While not an exhaustive list, we have focused on these factors because they have been shown to influence an individual's vulnerability to anxiety and alter the neurotransmitter systems often targeted by pharmacological adjuncts to therapy. We argue that for potential adjuncts to be successfully translated from the lab to the clinic empirical research must be broadened to consider how individual difference factors will influence drug efficacy.

A Brief Guide to Studying Fear in Developing Rodents: Important Considerations and Common Pitfalls.

Development is a time of rapid change that sets the pathway to adult functioning across all aspects of physical and mental health. Developmental studies can therefore offer insight into the unique needs of individuals at different stages of normal development as well as the etiology of various disease states. The aim of this overview is to provide an introduction to the practical implementation of developmental studies in rats and mice, with an emphasis on the study of learned fear. We first discuss how developmental factors may influence experimental outcomes for any study. This is followed by a discussion of methodological issues to consider when conducting studies of developing rodents, highlighting examples from the literature on learned fear. Throughout, we offer some recommendations to guide researchers on best practice in developmental studies. © 2018 by John Wiley & Sons, Inc.

Gutsy Moves: The Amygdala as a Critical Node in Microbiota to Brain Signaling.

The amygdala is a key brain area regulating responses to stress and emotional stimuli, so improving our understanding of how it is regulated could offer novel strategies for treating disturbances in emotion regulation. As we review here, a growing body of evidence indicates that the gut microbiota may contribute to a range of amygdala-dependent brain functions from pain sensitivity to social behavior, emotion regulation, and therefore, psychiatric health. In addition, it appears that the microbiota is necessary for normal development of the amygdala at both the structural and functional levels. While further investigations are needed to elucidate the exact mechanisms of microbiota-to-amygdala communication, ultimately, this work raises the intriguing possibility that the gut microbiota may become a viable treatment target in disorders associated with amygdala dysregulation, including visceral pain, post-traumatic stress disorder, and beyond. Also see the video abstract here: https://youtu.be/O5gvxVJjX18.