Associations between the use of antibiotics and altered brain function and mental illness are now well evidenced from animal models and clinical trials. Based on these findings, emerging research efforts have largely focused on how high-dose antibiotic- mediated perturbations of the gut microbiota result in altered neurophysiological and behavioural outcomes. However, these studies have not investigated whether antibiotics also act directly on the host nervous system. My central hypothesis is that high-dose antibiotics, as used in experimental models testing the modulatory role of the gut microbiome, can induce pathophysiological outcomes by direct interaction with enteric neuronal circuits. I designed two sets of experiments to characterize the acute effect of high-dose antibiotics on gut motility and gut-brain neuronal signalling. The first experimental study aimed to determine whether acute exposure of the gastrointestinal tract to high-dose antibiotics directly modulates enteric neurons, with consequences for gut motility. To test this, I used enteric nervous system dependent motility reflexes, ex vivo, as an index of putative effects on the intestinal nervous system. The results of these experiments have shown that luminal antibiotics alter oral to anal propulsive peristalsis in a system where such motility is dependent on the enteric nervous system. The second study aimed to test whether these local effects modulate brain function and behaviour by altering responses of vagal afferent pathways. I performed single-unit recordings from the mesenteric nerve bundle in ex vivo preparations to test this research question. The results suggest that antibiotics can increase activity of extrinsic vagal afferent neurons largely through cholinergic synapses with myenteric IPANs. The present work offers significant therapeutic implications, although its main relevance is in the interpretation of the experimental use of high-dose antibiotics on animal models and where effects on behaviour and the nervous system are attributed solely to alterations in the microbiome. / Thesis / Master of Science (MSc) / Little is known about the mechanisms by which high-dose antibiotics produce changes in gut-brain signalling to negatively affect brain functions and behaviour. Although the general consensus is that these changes are caused by antibiotic-mediated perturbations of the gut microbiota, whether high-dose antibiotics also act directly on the nervous system remains a topic of debate. I have hypothesized that high-dose antibiotics, as used in experimental models associating pathophysiological outcomes to gut microbial changes, also produce adverse effects by direct modulation of enteric neuronal circuits. Indeed, our findings suggest that high-dose antibiotics directly signal to enteric neurons, which locally regulate gut motility reflexes and can transmit that information further to vagal neurons, to influence homeostatic regulation of brain functions and behaviour. This work offers novel therapeutic potential for antibiotics and advises careful interpretation of studies that have attributed effects of high-dose antibiotics solely to alterations in the gut microbiome.

Chronic stress has a cumulative physiological impact, causing dysregulation of multiple systems due to allostatic overload. There is growing evidence that one such system, the microbiota, is engaged in persistent bidirectional interplay with the brain—a phenomenon that influences neural function and behaviour. However, the functional role of the microbiota in stress-associated changes and the underlying pathways of communication are unknown. Using a murine model of depression, we demonstrate that chronic stress has top-down effects on the structure of the microbiota community, reducing its richness and diversity, altering its profile, and causing differential abundance of various bacterial genera. These structural changes have functional consequences, including in metabolic pathways responsible for the synthesis of short chain fatty acids, tryptophan, and tyrosine. Using a physiologically active bacteria, Lactobacillus rhamnosus (JB-1), we probed for bottom-up signalling in chronic stress. JB-1 attenuated deficits in anxiety-like and social behaviours, and induced systemic immunoregulatory effects, independent of affecting stress-induced changes in the microbiota. In examining possible mechanisms of gut-brain brain signalling, we observed that in unstressed mice, a single dose of JB-1 causes rapid expression of c-Fos—a marker of neuronal activation—in distributed areas of the brain within 165 minutes, absent behavioural changes. No such effects were observed with heat-killed JB-1, despite that both live and heat-killed preparations facilitated vagal activity. Sub-diaphragmatic vagotomy prevented neuronal activation in most but not all brain regions, suggesting that vagal signalling is critical but indicating the presence of additional independent pathways. Finally, only chronic JB-1 treatment increased ΔFosB expression in the brain, which is indicative of long-term neuronal adaptations, in association with behavioural changes. These studies demonstrate a role for bidirectional gut-brain signalling in chronic stress, and highlight the signalling pathways and brain regions through which gut bacteria exert their influence on host behaviour. / Thesis / Candidate in Philosophy / Stress, which is a leading risk factor for mental illnesses such as depression, drastically affects the microbiota—the community of intestinal bacteria. However, this influence is bidirectional as gut bacteria can also influence the brain. Thus, we sought to understand the role of the microbiota in the negative effects of stress and how these microorganisms interact with the brain. We observed that behavioural changes in mice after chronic stress were associated with inflammation and community-wide changes in the microbiota. Treatment with a bacterial strain, Lactobacillus rhamnosus (JB-1), attenuated changes in behaviour and inflammation, but had no effect on the microbiota composition. We observed that the brain rapidly responded to JB-1 via the vagus nerve, and that chronic treatment caused long-term changes in brain regions. This work will allow us to discover novel pathways that can be targeted with greater specificity in clinical settings, providing an innovative approach to treatment of psychiatric conditions.