Alan E. Hoban

Transferring the blues: Depression-associated gut microbiota induces neurobehavioural changes in the rat.

The gut microbiota interacts with the host via neuroimmune, neuroendocrine and neural pathways. These pathways are components of the brain-gut-microbiota axis and preclinical evidence suggests that the microbiota can recruit this bidirectional communication system to modulate brain development, function and behaviour. The pathophysiology of depression involves neuroimmune-neuroendocrine dysregulation. However, the extent to which changes in gut microbiota composition and function mediate the dysregulation of these pathways is unknown. Thirty four patients with major depression and 33 matched healthy controls were recruited. Cytokines, CRP, Salivary Cortisol and plasma Lipopolysaccharide binding protein were determined by ELISA. Plasma tryptophan and kynurenine were determined by HPLC. Fecal samples were collected for 16s rRNA sequencing. A Fecal Microbiota transplantation was prepared from a sub group of depressed patients and controls and transferred by oral gavage to a microbiota-deficient rat model. We demonstrate that depression is associated with decreased gut microbiota richness and diversity. Fecal microbiota transplantation from depressed patients to microbiota-depleted rats can induce behavioural and physiological features characteristic of depression in the recipient animals, including anhedonia and anxiety-like behaviours, as well as alterations in tryptophan metabolism. This suggests that the gut microbiota may play a causal role in the development of features of depression and may provide a tractable target in the treatment and prevention of this disorder.

Regulation of prefrontal cortex myelination by the microbiota

The prefrontal cortex (PFC) is a key region implicated in a range of neuropsychiatric disorders such as depression, schizophrenia and autism. In parallel, the role of the gut microbiota in contributing to these disorders is emerging. Germ-free (GF) animals, microbiota-deficient throughout life, have been instrumental in elucidating the role of the microbiota in many aspects of physiology, especially the role of the microbiota in anxiety-related behaviours, impaired social cognition and stress responsivity. Here we aim to further elucidate the mechanisms of the microbial influence by investigating changes in the homeostatic regulation of neuronal transcription of GF mice within the PFC using a genome-wide transcriptome profiling approach. Our results reveal a marked, concerted upregulation of genes linked to myelination and myelin plasticity. This coincided with upregulation of neural activity-induced pathways, potentially driving myelin plasticity. Subsequent investigation at the ultrastructural level demonstrated the presence of hypermyelinated axons within the PFC of GF mice. Notably, these changes in myelin and activity-related gene expression could be reversed by colonization with a conventional microbiota following weaning. In summary, we believe we demonstrate for the first time that the microbiome is necessary for appropriate and dynamic regulation of myelin-related genes with clear implications for cortical myelination at an ultrastructural level. The microbiota is therefore a potential therapeutic target for psychiatric disorders involving dynamic myelination in the PFC.

Microbes & neurodevelopment – Absence of microbiota during early life increases activity-related transcriptional pathways in the amygdala

The mammalian amygdala is a key emotional brain region for eliciting social behaviour, critically involved in anxiety and fear-related behaviours, and hence a focus of research on neurodevelopmental and stress-related disorders such as autism and anxiety. Recently, increasing evidence implicates host-microbe interactions in the aetiology of these conditions. Germ-free (GF) mice, devoid of any microbiota throughout organismal maturation, are a well-established tool to study the effects of absence of the microbiota on host physiology. A growing body of independently replicated findings confirm that GF animals demonstrate altered anxiety-related behaviour and impaired social behaviour. However, the underlying mechanisms of this interaction and the nature of the pathways involved are only insufficiently understood. To further elucidate the molecular underpinnings of microbe-brain interaction, we therefore exploited unbiased genome-wide transcriptional profiling to determine gene expression in the amygdala of GF and GF mice that have been colonised after weaning. Using RNA-sequencing and a comprehensive downstream analysis pipeline we studied the amygdala transcriptome and found significant differences at the levels of differential gene expression, exon usage and RNA-editing. Most surprisingly, we noticed upregulation of several immediate early response genes such as Fos, Fosb, Egr2 or Nr4a1 in association with increased CREB signalling in GF mice. In addition, we found differential expression and recoding of several genes implicated in brain physiology processes such as neurotransmission, neuronal plasticity, metabolism and morphology. In conclusion, our data suggest altered baseline neuronal activity in the amygdala of germ-free animals, which is established during early life and may have implications for understanding development and treatment of neurodevelopmental disorders.

Behavioural and neurochemical consequences of chronic gut microbiota depletion during adulthood in the rat

Gut microbiota colonization is a key event for host physiology that occurs early in life. Disruption of this process leads to altered brain development which ultimately manifests as changes in brain function and behaviour in adulthood. Studies using germ-free (GF) mice highlight the extreme impact on brain health that results from life without commensal microbes. However, the impact of microbiota disturbances occurring in adulthood is less studied. To this end, we depleted the gut microbiota of 10-week-old male SpragueDawley rats via chronic antibiotic treatment. Following this marked, sustained depletion of the gut bacteria, we investigated behavioural and molecular hallmarks of gut-brain communication. Our results reveal that depletion of the gut microbiota during adulthood results in deficits in spatial memory as tested by Morris water maze, decreased visceral sensitivity and a greater display of depressive-like behaviours in the forced swim test. In tandem with these clear behavioural alterations we found changes in altered CNS serotonin concentration along with changes in the mRNA levels of corticotrophin releasing hormone receptor 1 and glucocorticoid receptor. Additionally, we found changes in the expression of brain derived neurotrophic factor (BDNF), a hallmark of altered microbiota-gut-brain axis signalling. In summary, this model of antibiotic-induced depletion of the gut microbiota can be used for future studies interested in the impact of the gut microbiota on host health without the confounding developmental influence of early-life microbial alterations.

The microbiome regulates amygdala-dependent fear recall

The amygdala is a key brain region that is critically involved in the processing and expression of anxiety and fear-related signals. In parallel, a growing number of preclinical and human studies have implicated the microbiome–gut–brain in regulating anxiety and stress-related responses. However, the role of the microbiome in fear-related behaviours is unclear. To this end we investigated the importance of the host microbiome on amygdala-dependent behavioural readouts using the cued fear conditioning paradigm. We also assessed changes in neuronal transcription and post-transcriptional regulation in the amygdala of naive and stimulated germ-free (GF) mice, using a genome-wide transcriptome profiling approach. Our results reveal that GF mice display reduced freezing during the cued memory retention test. Moreover, we demonstrate that under baseline conditions, GF mice display altered transcriptional profile with a marked increase in immediate-early genes (for example, Fos, Egr2, Fosb, Arc) as well as genes implicated in neural activity, synaptic transmission and nervous system development. We also found a predicted interaction between mRNA and specific microRNAs that are differentially regulated in GF mice. Interestingly, colonized GF mice (ex-GF) were behaviourally comparable to conventionally raised (CON) mice. Together, our data demonstrates a unique transcriptional response in GF animals, likely because of already elevated levels of immediate-early gene expression and the potentially underlying neuronal hyperactivity that in turn primes the amygdala for a different transcriptional response. Thus, we demonstrate for what is to our knowledge the first time that the presence of the host microbiome is crucial for the appropriate behavioural response during amygdala-dependent memory retention.

Microbial regulation of microRNA expression in the amygdala and prefrontal cortex

BackgroundThere is growing evidence for a role of the gut microbiome in shaping behaviour relevant to many psychiatric and neurological disorders. Preclinical studies using germ-free (GF) animals have been essential in contributing to our current understanding of the potential importance of the host microbiome for neurodevelopment and behaviour. In particular, it has been repeatedly demonstrated that manipulation of the gut microbiome modulates anxiety-like behaviours. The neural circuits that underlie anxiety- and fear-related behaviours are complex and heavily depend on functional communication between the amygdala and prefrontal cortex (PFC). Previously, we have shown that the transcriptional networks within the amygdala and PFC of GF mice are altered. MicroRNAs (miRNAs) act through translational repression to control gene translation and have also been implicated in anxiety-like behaviours. However, it is unknown whether these features of host post-transcriptional machinery are also recruited by the gut microbiome to exert control over CNS transcriptional networks.ResultsWe conducted Illumina® next-generation sequencing (NGS) in the amygdala and PFC of conventional, GF and germ-free colonized mice (exGF). We found a large proportion of miRNAs to be dysregulated in GF animals in both brain regions (103 in the amygdala and 31 in the PFC). Additionally, colonization of GF mice normalized some of the noted alterations. Next, we used a complementary approach to GF by manipulating the adult rat microbiome with an antibiotic cocktail to deplete the gut microbiota and found that this strategy also impacted the expression of relevant miRNAs.ConclusionThese results suggest that the microbiome is necessary for appropriate regulation of miRNA expression in brain regions implicated in anxiety-like behaviours.

Thinking small: towards microRNA-based therapeutics for anxiety disorders.

Introduction: Anxiety disorders are the most frequently diagnosed psychiatric conditions, negatively affecting quality of life and creating a significant economic burden. These complex disorders are extremely difficult to treat, and there is a great need for novel therapeutics with greater efficacy and minimal adverse side effects. Areas covered: In this review, the authors describe the role that microribonucleic acids (microRNA or miRNA) play in the development of anxiety disorders and their potential to serve as biomarkers of disease as well as targets for pharmacological treatment. Furthermore, the authors discuss the current state of miRNA research, including both preclinical and clinical studies of anxiety disorders. Expert opinion: There is mounting evidence that circulating miRNA may serve as biomarkers of disease and play a role in the development of disease, including psychiatric conditions such as anxiety disorders. Great strides have been made in cancer research, with miRNA-based therapies already in use in clinical studies. However, the use of miRNA for the treatment of neurological disorders, and psychiatric disorders in particular, is still in its nascent stage. The development of safe compounds that are able to cross the blood–brain barrier and target specific cell populations, which are relevant to anxiety-related neurocircuitry, is paramount for the emergence of novel, efficacious miRNA-based therapies in clinical settings.

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

Neurobehavioural effects of Lactobacillus rhamnosus GG alone and in combination with prebiotics polydextrose and galactooligosaccharide in male rats exposed to early-life stress

Early life is a period of significant brain development when the brain is at its most plastic and vulnerable. Stressful episodes during this window of development have long-lasting effects on the central nervous system. Rodent maternal separation (MS) is a reliable model of early-life stress and induces alterations in both physiology and behaviour. Intriguingly, the gut microbiota of MS offspring differ from that of non-separated offspring, suggesting a mechanistic role for the microbiota–gut–brain axis. Hence, we tested whether dietary factors known to affect the gut microbiota alter the neurobehavioural effects of MS. The impact of consuming diet containing prebiotics polydextrose (PDX) and galactooligosaccharide (GOS) alone or in combination with live bacteria Lactobacillus rhamnosus GG (LGG) from weaning onwards in rats subjected to early-life MS was assessed. Adult offspring were assessed for anxiety-like behaviour in the open field test, spatial memory using the Morris water maze, and reactivity to restraint stress. Brains were examined via PCR for changes in mRNA gene expression. Here, we demonstrate that diets containing a combination of PDX/GOS and LGG attenuates the effects of early-life MS on anxiety-like behaviour and hippocampal-dependent learning with changes to hippocampal mRNA expression of genes related to stress circuitry, anxiety and learning.

TLX is an intrinsic regulator of the negative effects of IL-1β on proliferating hippocampal neural progenitor cells

Hippocampal neurogenesis is a lifelong process whereby new neurons are produced and integrate into the host circuitry within the hippocampus. It is regulated by a multitude of extrinsic and intrinsic regulators and is believed to contribute to certain hippocampal‐dependent cognitive tasks. Hippocampal neurogenesis and associated cognition have been demonstrated to be impaired after increases in the levels of proinflammatory cytokine IL‐1β in the hippocampus, such as that which occurs in various neurodegenerative and psychiatric disorders. IL‐1β also suppresses the expression of TLX (orphan nuclear receptor tailless homolog), which is an orphan nuclear receptor that functions to promote neural progenitor cell (NPC) proliferation and suppress neuronal differentiation; therefore, manipulation of TLX represents a potential strategy with which to prevent the antiproliferative effects of IL‐1β. In this study, we assessed the mechanism that underlies IL‐1β–induced changes in TLX expression and determined the protective capacity of TLX to mitigate the effects of IL‐1β on embryonic rat hippocampal neurosphere expansion. We demonstrate that IL‐1β activated the NF‐κB pathway in proliferating NPCs and that this activation was responsible for IL‐1β–induced changes in TLX expression. In addition, we report that enhancing TLX expression prevented the IL‐1β–induced suppression of neurosphere expansion. Thus, we highlight TLX as a potential protective regulator of the antiproliferative effects of IL‐1β on hippocampal neurogenesis.—Ó’Leime, C. S., Kozareva, D. A., Hoban, A. E., Long‐Smith, C. M., Cryan, J. F., Nolan, Y. M. TLX is an intrinsic regulator of the negative effects of IL‐1β on proliferating hippocampal neural progenitor cells. FASEB J. 32, 613–624 (2018). www.fasebj.org