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.

Faecal microRNAs: indicators of imbalance at the host-microbe interface?

The enteric microbiota is characterised by a balance and composition that is unique to the host. It is important to understand the mechanisms through which the host can maintain the composition of the gut microbiota. MicroRNAs (miRNA) are implicated in intercellular communication and have been isolated from bodily fluids including stool. Recent findings suggest that miRNA produced by the host's intestinal epithelial cells (IECs) participate in shaping the microbiota. To investigate whether miRNA expression was influenced by the gut microbiota we measured the expression of miRNAs expressed by intestinal epithelial cells in faeces. Specifically, we measured miRNA expression in faeces from germ-free (GF) and conventional mice and similarly in a rat model of antibiotic-mediated depletion of the gut microbiota control rats. In adult male GF and conventional mice and adult Sprague Dawley (SD) rats were treated with a combination of antibiotics for 8 weeks; total RNA was extracted from faecal pellets taken at week 0, 2, 4, 6 week 8 and the expression of let-7b-3p, miR-141-3p, miR-200a-3p and miR-1224-5p (miRNAs known to be expressed in IECs) were measured relative to U6 at each time point using qRT-PCR. In GF animals the expression of let-7b, miR-141 and miR-200a in faeces was lower compared to conventional mice. Following antibiotic-mediated depletion of gut microbiota, rats showed two divergent profiles of miRNA expression. Following two weeks of antibiotic treatment, the expression of let-7b and miR-1224 dropped significantly and remained low for the remainder of the study. The expression of miR-200a and miR-141 was significantly higher at week 2 than before antibiotic treatment commenced. Subsequently, the expression of miR-200a and miR-141 decreased at week 4 and continued to decrease at week 6. This data demonstrates that miRNAs can be used as an independent, non-invasive marker of microbial fluctuations along with gut pathology in the intestine.

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.

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.