Gerard Moloney

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.

Omega-3 polyunsaturated fatty acids critically regulate behaviour and gut microbiota development in adolescence and adulthood

BACKGROUND Neurodevelopment is strongly influenced by maternal and early-postnatal diet. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are vital structural and functional components of the developing brain. The gut microbiota is also influenced by n-3 PUFA status, however, little is known about the role of maternal and early-life n-3 PUFA intake on offspring gut microbiota development and subsequent interactions with central nervous system functioning and behavioural outcomes. METHODS Pregnant female C57BL/6 mice and their male offspring were fed a control (CON), omega-3 deficient (O3-) or omega-3 supplemented (O3+) diet. Cognitive, depressive and social behaviours were assessed through a battery of behaviour tests in the male offspring at both adolescence (week 4-5) and adulthood (week 11-13). Hypothalamic-pituitary-adrenal axis (HPA) activation was assessed by analysis of stress-induced corticosterone production. Fecal microbiota composition was analysed by 16S sequencing at both adolescent and adulthood. In addition, stimulated spleen cytokine levels were assessed. RESULTS n-3 PUFA interventions induced subtle changes in offspring early-life and adolescent behaviours, which were further evident in adulthood, such that O3- animals displayed impaired communication, social and depression-related behaviours and O3+ animals displayed enhanced cognition. O3- mice displayed an elevated Firmicutes:Bacteroidetes ratio and blunted systemic LPS responsiveness. Contrastingly, O3+ mice displayed greater fecal Bifidobacterium and Lactobacillus abundance and dampened HPA-axis activity. CONCLUSIONS Neurobehavioural development related to cognitive, anxiety and social behaviours, is highly dependent upon in utero and lifelong n-3 PUFA availability. In addition, neurobehavioural changes induced by altering n-3 PUFA status are closely associated with comprehensive alterations in gut microbiota composition, HPA-axis activity and inflammation.

Toll-Like Receptor 4 Regulates Chronic Stress-Induced Visceral Pain in Mice

BACKGROUND Functional gastrointestinal disorders, which have visceral hypersensitivity as a core symptom, are frequently comorbid with stress-related psychiatric disorders. Increasing evidence points to a key role for toll-like receptor 4 (TLR4) in chronic pain states of somatic origin. However, the central contribution of TLR4 in visceral pain sensation remains elusive. METHODS With pharmacological and genetic approaches, we investigated the involvement of TLR4 in the modulation of visceral pain. The TLR4-deficient and wild-type mice were exposed to chronic stress. Visceral pain was evaluated with colorectal distension. Protein expression levels for TLR4, Cd11b, and glial fibrillary acidic protein (glial cells markers) were quantified in the lumbar region of the spinal cord, prefrontal cortex (PFC), and hippocampus. To evaluate the effect of blocking TLR4 on visceral nociception, TAK-242, a selective TLR4 antagonist, was administered peripherally (intravenous) and centrally (intracerebroventricular and intra-PFC) (n = 10-12/experimental group). RESULTS The TLR4 deficiency reduced visceral pain and prevented the development of chronic psychosocial stress-induced visceral hypersensitivity. Increased expression of TLR4 coupled with enhanced glia activation in the PFC and increased levels of proinflammatory cytokines were observed after chronic stress in wild-type mice. Administration of a TLR4 specific antagonist, TAK-242, attenuated visceral pain sensation in animals with functional TLR4 when administrated centrally and peripherally. Moreover, intra-PFC TAK-242 administration also counteracted chronic stress-induced visceral hypersensitivity. CONCLUSIONS Our results reveal a novel role for TLR4 within the PFC in the modulation of visceral nociception and point to TLR4 as a potential therapeutic target for the development of drugs to treat visceral hypersensitivity.

Revisiting Metchnikoff: Age-related alterations in microbiota-gut-brain axis in the mouse

Over the last decade, there has been increased interest in the role of the gut microbiome in health including brain health. This is by no means a new theory; Elie Metchnikoff proposed over a century ago that targeting the gut by consuming lactic acid bacteria such as those in yogurt, could improve or delay the onset of cognitive decline associated with ageing. However, there is limited information characterising the relationship between the behavioural and physiological sequelae of ageing and alterations in the gut microbiome. To this end, we assessed the behavioural, physiological and caecal microbiota profile of aged male mice. Older mice (20-21months old) exhibited deficits in spatial memory and increases in anxiety-like behaviours compared to younger mice (2-3months old). They also exhibited increased gut permeability, which was directly correlated with elevations in peripheral pro-inflammatory cytokines. Furthermore, stress exacerbated the gut permeability of aged mice. Examination of the caecal microbiota revealed significant increases in phylum TM7, family Porphyromonadaceae and genus Odoribacter of aged mice. This represents a shift of aged microbiota towards a profile previously associated with inflammatory disease, particularly gastrointestinal and liver disorders. Furthermore, Porphyromonadaceae, which has also been associated with cognitive decline and affective disorders, was directly correlated with anxiety-like behaviour in aged mice. These changes suggest that changes in the gut microbiota and associated increases in gut permeability and peripheral inflammation may be important mediators of the impairments in behavioural, affective and cognitive functions seen in ageing.

Use of bioluminescence imaging to track neutrophil migration and its inhibition in experimental colitis

Inflammatory bowel disease (IBD) is associated with neutrophil infiltration into the mucosa and crypt abscesses. The chemokine interleukin (IL)‐8 [murine homologues (KC) and macrophage inflammatory protein (MIP)‐2] and its receptor CXCR2 are required for neutrophil recruitment; thus, blocking this engagement is a potential therapeutic strategy. In the present study, we developed a preclinical model of neutrophil migration suitable for investigating the biology of and testing new drugs that target neutrophil trafficking. Peritoneal exudate neutrophils from transgenic β‐actin‐luciferase mice were isolated 12 h after intraperitoneal injection with thioglycollate, and were assessed phenotypically and functionally. Exudate cells were injected intravenously into recipients with dextran sodium sulphate (DSS)‐induced colitis followed by bioluminescence imaging of whole‐body and ex vivo organs at 2, 4 and 16–22 h post‐transfer. Anti‐KC antibody or an isotype control were administered at 20 µg/mouse 1 h before transfer, followed by whole‐body and organ imaging 4 h post‐transfer. The peritoneal exudate consisted of 80% neutrophils, 39% of which were CXCR2+. In vitro migration towards KC was inhibited by anti‐KC. Ex vivo bioluminescent imaging showed that neutrophil trafficking into the colon of DSS recipients was inhibited by anti‐KC 4 h post‐cell transfer. In conclusion, this study describes a new approach for investigating neutrophil trafficking that can be used in preclinical studies to evaluate potential inhibitors of neutrophil recruitment.

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.

Microbiota-related Changes in Bile Acid & Tryptophan Metabolism are Associated With Gastrointestinal Dysfunction in a Mouse Model of Autism

Autism spectrum disorder (ASD) is one of the most prevalent neurodevelopmental conditions worldwide. There is growing awareness that ASD is highly comorbid with gastrointestinal distress and altered intestinal microbiome, and that host-microbiome interactions may contribute to the disease symptoms. However, the paucity of knowledge on gut-brain axis signaling in autism constitutes an obstacle to the development of precision microbiota-based therapeutics in ASD. To this end, we explored the interactions between intestinal microbiota, gut physiology and social behavior in a BTBR T+Itpr3tf/J mouse model of ASD. Here we show that a reduction in the relative abundance of very particular bacterial taxa in the BTBR gut – namely, bile-metabolizing Bifidobacterium and Blautia species, - is associated with deficient bile acid and tryptophan metabolism in the intestine, marked gastrointestinal dysfunction, as well as impaired social interactions in BTBR mice. Together these data support the concept of targeted manipulation of the gut microbiota for reversing gastrointestinal and behavioral symptomatology in ASD, and offer specific plausible targets in this endeavor.

MicroRNAs as biomarkers for major depression: a role for let-7b and let-7c

There is a growing emphasis in the field of psychiatry on the need to identify candidate biomarkers to aid in diagnosis and clinical management of depression, particularly with respect to predicting response to specific therapeutic strategies. MicroRNAs are small nucleotide sequences with the ability to regulate gene expression at the transcriptomic level and emerging evidence from a range of studies has highlighted their biomarker potential. Here we compared healthy controls (n=20) with patients diagnosed with major depression (n=40) and who were treatment-resistant to identify peripheral microRNA biomarkers, which could be used for diagnosis and to predict response to electroconvulsive therapy (ECT) and ketamine (KET) infusions, treatments that have previously shown to be effective in treatment-resistant depression (TRD). At baseline and after treatment, blood samples were taken and symptom severity scores rated using the Hamilton Depression Rating Scale (HDRS). Samples were analyzed for microRNA expression using microarray and validated using quantitative PCR. As expected, both treatments reduced HDRS scores. Compared with controls, the baseline expression of the microRNA let-7b was less by ~40% in TRD patients compared with controls. The baseline expression of let-7c was also lower by ~50% in TRD patients who received ECT. Bioinformatic analysis revealed that let-7b and let-7c regulates the expression of 27 genes in the PI3k-Akt-mTOR signaling pathway, which has previously been reported to be dysfunctional in depression. The expression of miR-16, miR-182, miR-451 and miR-223 were similar to that in controls. Baseline microRNA expression could not predict treatment response and microRNAs were unaffected by treatment. Taken together, we have identified let-7b and let-7c as candidate biomarkers of major depression.

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.