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.

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.

Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis.

Activated ghrelin receptor GHS-R1α triggers cell signalling pathways that modulate energy homeostasis and biosynthetic processes. However, the effects of ghrelin on mRNA translation are unknown. Using various reporter assays, here we demonstrate a rapid elevation of protein synthesis in cells within 15-30 min upon stimulation of GHS-R1α by ghrelin. We further show that ghrelin-induced activation of translation is mediated, at least in part, through the de-phosphorylation (de-suppression) of elongation factor 2 (eEF2). The levels of eEF2 phosphorylation at Thr56 decrease due to the reduced activity of eEF2 kinase, which is inhibited via Ser366 phosphorylation by rpS6 kinases. Being stress-susceptible, the ghrelin-mediated decrease in eEF2 phosphorylation can be abolished by glucose deprivation and mitochondrial uncoupling. We believe that the observed burst of translation benefits rapid restocking of neuropeptides, which are released upon GHS-R1α activation, and represents the most time- and energy-efficient way of prompt recharging the orexigenic neuronal circuitry.

The impact of psychosocial defeat stress on the bed nucleus of the stria terminalis transcriptome in adult male mice.

The bed nucleus of the stria terminalis (BNST) is a focal point for the convergence of inputs from canonical stress-sensitive structures to fine-tune the response to stress. However, its role in mediating phenotypes of stress resilience or susceptibility is yet to be fully defined. In this study, we carried out unbiased RNA-sequencing to analyse the BNST transcriptomes of adult male mice, which were classified as resilient or susceptible following a 10-day chronic psychosocial defeat stress paradigm. Pairwise comparisons revealed 20 differentially expressed genes in resilience (6) and susceptible (14) mice compared with controls. An in silico validation of our data against an earlier study revealed significant concordance in gene expression profiles associated with resilience to chronic stress. Enrichment analysis revealed that resilience is linked to functions including retinoic acid hydrolase activity, phospholipase inhibitor and tumour necrosis factor (TNF)-receptor activities, whereas susceptibility is linked to alterations in amino acid transporter activity. We also identified differential usage of 134 exons across 103 genes associated with resilience and susceptibility; enrichment analysis for genes with differential exon usage in resilient mice was linked to functions including adrenergic receptor binding mice and oxysterol binding in susceptible mice. Our findings highlight the important and underappreciated role of the BNST in stress resilience and susceptibility and reveal research avenues for follow-up investigations.

Adolescent dietary manipulations differentially affect gut microbiota composition and amygdala neuroimmune gene expression in male mice in adulthood.

Adolescence is a critical developmental period that is characterised by growth spurts and specific neurobiological, neuroimmune and behavioural changes. In tandem the gut microbiota, which is a key player in the regulation of health and disease, is shaped during this time period. Diet is one of the most important regulators of microbiota composition. Thus, we hypothesised that dietary disturbances of the microbiota during this critical time window result in long-lasting changes in immunity, brain and behaviour. C57BL/6 male mice were exposed to either high fat diet or cafeteria diet during the adolescent period from postnatal day 28 to 49 and were tested for anxiety-related and social behaviour in adulthood. Our results show long-lasting effects of dietary interventions during the adolescent period on microbiota composition and the expression of genes related to neuroinflammation or neurotransmission. Interestingly, changes in myelination-related gene expression in the prefrontal cortex following high fat diet exposure were also observed. However, these effects did not translate into overt behavioural changes in adulthood. Taken together, these data highlight the importance of diet-microbiota interactions during the adolescent period in shaping specific outputs of the microbiota-gut-brain axis in later life.

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.

Resilience to chronic stress is associated with specific neurobiological, neuroendocrine and immune responses.

Research into the molecular basis of stress resilience is a novel strategy to identify potential therapeutic strategies to treat stress-induced psychopathologies such as anxiety and depression. Stress resilience is a phenomenon which is not solely driven by effects within the central nervous system (CNS) but involves multiple systems, central and peripheral, which interact with and influence each other. Accordingly, we used the chronic social defeat stress paradigm and investigated specific CNS, endocrine and immune responses to identify signatures of stress-resilience and stress susceptibility in mice. Our results showed that mice behaviourally susceptible to stress (indexed by a reduction in social interaction behaviour) had higher plasma corticosterone levels and adrenal hypertrophy. An increase in inflammatory circulating monocytes was another hallmark of stress susceptibility. Furthermore, prefrontal cortex mRNA expression of corticotrophin-releasing factor (Crf) was increased in susceptible mice relative to resilient mice. We also report differences in hippocampal synaptic plasticity between resilient and susceptible mice. Ongoing studies will interpret the functional relevance of these signatures which could potentially inform the development of novel psychotherapeutic strategies.

Strain differences in behaviour and immunity in aged mice: Relevance to Autism.

The BTBR mouse model has been shown to be associated with deficits in social interaction and a pronounced engagement in repetitive behaviours. Autism spectrum disorder (ASD) is the most prevalent neurodevelopmental condition globally. Despite its ubiquity, most research into the disorder remains focused on childhood, with studies in adulthood and old age relatively rare. To this end, we explored the differences in behaviour and immune function in an aged BTBR T + Itpr3tf/J mouse model of the disease compared to a similarly aged C57bl/6 control. We show that while many of the alterations in behaviour that are observed in young animals are maintained (repetitive behaviours, antidepressant-sensitive behaviours, social deficits & cognition) there are more nuanced effects in terms of anxiety in older animals of the BTBR strain compared to C57bl/6 controls. Furthermore, BTBR animals also exhibit an activated T-cell system. As such, these results represent confirmation that ASD-associated behavioural deficits are maintained in ageing, and that that there may be need for differential interventional approaches to counter these impairments, potentially through targeting the immune system.

Amygdalar corticotropin-releasing factor mediates stress-induced anxiety.

The extended amygdala, including the Central nucleus of the Amygdala (CeA) and the Bed Nucleus of the Stria Terminalis (BNST), is a complex structure that plays a pivotal role in emotional behavior. The CeA and the BNST are highly interconnected, being the amygdala traditionally more associated with fear and the BNST with anxiety. Yet, studies using excitotoxic lesions also show the involvement of the CeA in the development of stress-induced anxiety. Likewise, others have also highlighted the role of corticotropin-releasing factor (CRF), a neuropeptide highly expressed in CeA, as an anxiogenic factor and, consequently, important for in anxiety disorders. Here, we used an inducible RNAi lentiviral system to assess the effects of reducing CRF expression in CeA in the development of anxiety-like behavior in a model of Chronic Unpredictable Stress. In addition, we evaluated CRF RNAi-mediated alterations in the stress-triggered molecular signature in the BNST. Knockdown of CRF in the CeA decreased stress-induced anxiety levels. No differences were found in a fear-potentiated startle paradigm. Additionally, we observed that stress-induced alterations in the expression of CRF receptors within the BNST are attenuated by CRF knockdown in the CeA. These results emphasize the importance of the role that amygdalar CRF plays in the modulation of anxiety-like behavior and in the molecular signature of stress in the BNST.

Enduring neurobehavioral effects induced by microbiota depletion during the adolescent period.

The gut microbiota is an essential regulator of many aspects of host physiology. Disruption of gut microbial communities affects gut-brain communication which ultimately can manifest as changes in brain function and behaviour. Transient changes in gut microbial composition can be induced by various intrinsic and extrinsic factors, however, it is possible that enduring shifts in the microbiota composition can be achieved by perturbation at a timepoint when the gut microbiota has not fully matured or is generally unstable, such as during early life or ageing. In this study, we investigated the effects of 3-week microbiota depletion with antibiotic treatment during the adolescent period and in adulthood. Following a washout period to restore the gut microbiota, behavioural and molecular hallmarks of gut-brain communication were investigated. Our data revealed that transient microbiota depletion had long-lasting effects on microbiota composition and increased anxiety-like behaviour in mice exposed to antibiotic treatment during adolescence but not in adulthood. Similarly, gene expression in the amygdala was more severely affected in mice treated during adolescence. Taken together these data highlight the vulnerability of the gut microbiota during the critical adolescent period and the long-lasting impact manipulations of the microbiota can have on gene expression and behaviour in adulthood.

Enduring Behavioral Effects Induced by Birth by Caesarean Section in the Mouse.

Birth by Caesarean (C)-section impacts early gut microbiota colonization and is associated with an increased risk of developing immune and metabolic disorders. Moreover, alterations of the microbiome have been shown to affect neurodevelopmental trajectories. However, the long-term effects of C-section on neurobehavioral processes remain unknown. Here, we demonstrated that birth by C-section results in marked but transient changes in microbiome composition in the mouse, in particular, the abundance of Bifidobacterium spp. was depleted in early life. Mice born by C-section had enduring social, cognitive, and anxiety deficits in early life and adulthood. Interestingly, we found that these specific behavioral alterations induced by the mode of birth were also partially corrected by co-housing with vaginally born mice. Finally, we showed that supplementation from birth with a Bifidobacterium breve strain, or with a dietary prebiotic mixture that stimulates the growth of bifidobacteria, reverses selective behavioral alterations in C-section mice. Taken together, our data link the gut microbiota to behavioral alterations in C-section-born mice and suggest the possibility of developing adjunctive microbiota-targeted therapies that may help to avert long-term negative consequences on behavior associated with C-section birth mode.

A Key Role for Neurotensin in Chronic-Stress-Induced Anxiety-Like Behavior in Rats.

Chronic stress is a major cause of anxiety disorders that can be reliably modeled preclinically, providing insight into alternative therapeutic targets for this mental health illness. Neuropeptides have been targeted in the past to no avail possibly due to our lack of understanding of their role in pathological models. In this study we use a rat model of chronic stress-induced anxiety-like behaviors and hypothesized that neuropeptidergic modulation of synaptic transmission would be altered in the bed nucleus of the stria terminalis (BNST), a brain region suspected to contribute to anxiety disorders. We use brain slice neurophysiology and behavioral pharmacology to compare the role of locally released endogenous neuropeptides on synaptic transmission in the oval (ov) BNST of non-stressed (NS) or chronic unpredictably stressed (CUS) rats. We found that in NS rats, post-synaptic depolarization induced the release of vesicular neurotensin (NT) and corticotropin-releasing factor (CRF) that co-acted to increase ovBNST inhibitory synaptic transmission in 59% of recorded neurons. CUS bolstered this potentiation (100% of recorded neurons) through an enhanced contribution of NT over CRF. In contrast, locally released opioid neuropeptides decreased ovBNST excitatory synaptic transmission in all recorded neurons, regardless of stress. Consistent with CUS-induced enhanced modulatory effects of NT, blockade of ovBNST NT receptors completely abolished stress-induced anxiety-like behaviors in the elevated plus maze paradigm. The role of NT has been largely unexplored in stress and our findings highlight its potential contribution to an important behavioral consequence of chronic stress, that is, exaggerated avoidance of open space in rats.