Selective serotonin reuptake inhibitors (SSRIs) are used as first-line treatments by up to 10% of pregnant women suffering from major depression. However, the effects of prenatal SSRI use on fetal development are still largely unknown. Serotonin plays crucial roles in the early development of the fetal brain, and the fetus depends on a maternal source of serotonin. In humans, 90% of serotonin synthesis occurs in the gut, promoted by specific microbiota. In the present study, we used a pregnant mouse model to assess for changes in the maternal microbiome, after early prenatal fluoxetine gavage. We examined the fetal brain for changes in the development of the dorsal raphe nucleus, as well as its axonal projections to the prefrontal cortex. Our results suggest that prenatal SSRI use does not result in a significant shift in maternal microbiome, and has no direct effect on the development of the endogenous fetal serotonergic system.

While Parkinson’s disease (PD) is a motor symptom disorder due to neurodegeneration of dopaminergic neurons and accumulation of Lewy bodies in the substantia nigra (SN), gastrointestinal dysfunctions are characteristic of prodromal PD. The most common monogenic mutation for PD lies in the gene leucine rich repeat kinase 2 (LRRK2), which harbors a mutation associated with IBD within the same locus. By studying the PD-causing mutation LRRK2 G2019S in transgenic (Tg) mice, we examined the effect that dextran sulfate sodium (DSS)-induced gut inflammation may have on the neuropathology of PD. This includes dopaminergic neuron loss, microglial and lysosomal activation, and alpha-synuclein (αSyn) aggregations in the SN. We found prodromal DSS-induced colitis exacerbates and accelerates neurodegeneration in LRRK2 G2019S Tg mice by 52 weeks of age. Prodromal colitis also promotes microglial activation and lysosomal formation. Interestingly, 23-week-old males showed increased αSyn aggregations in their microglia. Our data suggests that prodromal gut inflammation exacerbates the severity and onset of neurodegeneration and promotes neuroinflammation of PD driven by the LRRK2 G2019S mutation.

The vagus nerve is proposed to enable communication between the gut microbiome and brain, but activity-based evidence is lacking. Herein, we assess the extent of microbial influences on vagal activity and metabolite signaling mechanisms involved. We find that mice reared without microbiota (germ-free, GF) exhibit decreased vagal afferent tone relative to conventionally colonized mice (specific pathogen-free, SPF), which is reversed by colonization with SPF microbiota. Perfusing non-absorbable antibiotics (ABX) into the duodenum of SPF mice, but not GF mice, acutely decreases vagal activity, which is restored upon re-perfusion with bulk lumenal contents or sterile filtrates from the small intestine and cecum of SPF, but not GF, mice. Of several candidates identified by metabolomic profiling, microbiome- and diet-dependent short-chain fatty acids, bile acids, and 3-indoxyl sulfate stimulate vagal activity with varied response kinetics, which is blocked by co-perfusion of pharmacological antagonists of FFAR2, TGR5, and TRPA1, respectively, into the small intestine. At the single-unit level, duodenal serial perfusion of each metabolite class elicits more singly responsive neurons than dually responsive neurons, suggesting distinct neuronal detection of different microbiome- and macronutrient- dependent metabolites. Finally, microbial metabolite-induced increases in vagal activity correspond with activation of neurons in the nucleus of the solitary tract in the corresponding receptor-dependent manner. Results from this study reveal that the gut microbiome regulates select lumenal metabolites that differentially activate chemosensory vagal afferent neurons, thereby enabling microbial signaling across the gut-brain axis.

The gut microbiota varies in a host of human neurological disorders and in murine models is critical to proper neurodevelopment and in neuropathologies. However, current mechanistic evidence is lacking for how microbes related to homeostatic and/or pathological signaling affect these processes. The incidence of neurological disorders without viable treatment options, including epileptic disorders and cognitive impairment, is increasing which raises the impetus in searching for novel biological pathways and mechanisms by which these disorders may be prevented or treated. The gut microbiota has gained attention as a modifier of neurological risk for disorders in development, adulthood, and aging. The work described in this thesis isolates molecular mechanisms by which the gut microbiota mediates environmental protection or risk in murine models of epileptic episodes, cognitive impairment, and host metabolic dysfunction. Specifically, we report that the ketogenic diet, which provides protection against intractable epileptic seizures, mediates this effect in mice through the gut microbiota, more specifically through modifying gamma-glutamylated amino acids. We characterize cognitive and neurophysiological responses in mice mediated by dietary and stressor-induced changes in the gut microbiota, which is mediated by microbial changes in host immune pathways. Together, our findings provide mechanistic evidence that the host microbiota prevents and contributes to murine neurological dysfunction.

Environmental factors like hypoxia and stress are important risk factors for cognitive impairment, but little is known regarding the molecular mechanisms involved. The gut microbiome is emerging as an important host-factor that mediates the effects of environmental factors on host physiologies, including brain function and behavior. As the prevalence of cognitive impairment is rapidly increasing, an active area of research is in studying how changes in the gut microbiome may impact cognitive ability in animals. Work from our laboratory has examined effects of the high fat, low carbohydrate ketogenic diet (KD) and intermittent hypoxia on the composition of the gut microbiome. Here, we highlight how the KD and hypoxia-associated microbiome impacts synaptic and transcriptomic features in the mouse hippocampus. Depletion of the microbiome in mice pre-exposed to KD and hypoxia alters global gene expression profiles in the CA3 region of the hippocampus, an area previously reported as especially vulnerable to environmental insult. Alterations in hippocampal gene expression were also observed in germ-free animals monocolonized with KD-associated bacterium, Bilophila wadsworthia relative to controls. The transcriptomic alterations correlated with alterations in synaptic structure in the hippocampus, as measured by altered number of excitatory synapses, and the expression of markers for adult hippocampal neurogenesis. Altogether, these data reveal that the gut microbiome is an important mediator of the effects of the KD and hypoxia on hippocampal physiology, which could contribute to the ability of the microbiome to modify cognitive behavior.

The gut microbiota directly interacts with dietary components that their host consumes, generating metabolites that can affect host physiology and behavior. For example, gut bacteria digest complex carbohydrates, or fibers, that cannot be digested by the host and produce short chain fatty acids (SCFAs) via fermentation. SCFAs can stimulate gut motility, promote satiety, improve glucose homeostasis, and decrease inflammation. These diverse effects can be explained by many cell types expressing SCFA receptors, including neurons, gut epithelial cells, immune cells, and endothelial cells. However, the molecular mechanisms by which bacterial metabolism of dietary components affect host physiology remain incompletely understood. The work in this dissertation combines genetic engineering, in vitro assays, and mouse models to study these mechanisms in three distinct contexts: host food preference, methylmercury-induced toxicity, and placental development. In Chapter 2, we review evidence in the literature on whether and how the gut microbiota can influence host food preference. In Chapter 3, we show that gut bacterial metabolism of fructose-containing fibers called fructans influence host food preference. Specifically, colonization of germ-free mice with bacteria that selectively ferment fructans with different linkages increases host preference for the non-fermentable fructan. In Chapter 4, we focus on bacterial metabolism of an ingested environmental toxin—methylmercury in fish. We demonstrate that a gut bacterium engineered to demethylate methylmercury can reduce methylmercury levels in mice eating a high mercury fish diet. In Chapter 5, we show that the maternal gut microbiota is necessary to support placental development in mice, particularly vascularization. SCFAs stimulate angiogenesis in both in vitro assays of an umbilical cell line and microbiota-deficient mice. Together, the results of this work reveal novel mechanisms by which gut bacterial metabolism of diet can affect host health and behavior, which will contribute to development of microbiota-directed therapies for metabolic disorders, methylmercury toxicity, and intrauterine growth restriction.

The high-fat low-carbohydrate ketogenic diet (KD) is an established dietary therapy for individuals with refractory epilepsy, whose seizures are resistant to existing anti-epileptic drugs. However, use of the KD to treat refractory epilepsy is challenging because the diet is difficult for patients to implement, manage, and maintain due to its severe restrictiveness and adverse side effects. Exactly how the clinical KD reduces seizure symptoms when other anti-epileptic drugs are ineffective is poorly understood. The gut microbiome has emerged as a key intermediary between diet and host metabolism, neural activity, and behavior. The gut microbiome modulates seizure susceptibility and the anti-seizure effects of the ketogenic diet (KD) in animal models. This dissertation work seeks to understand if these relationships seen in animal models translate to KD therapies for human drug-resistant epilepsy. Herein we report that KD therapy in children with pediatric epilepsy alters the function of the human gut microbiome. In addition, colonizing mice with KD-associated human gut microbes confers increased resistance to 6-Hz psychomotor seizures, as compared to colonization with gut microbes from matched pre-treatment controls. Parallel analysis of human donor and mouse recipient metagenomic and metabolomic profiles identifies subsets of shared functional features that are seen in response to KD treatment in humans and preserved upon transfer to mice fed a standard diet. These include enriched representation of microbial genes and metabolites related to anaplerosis, fatty acid beta-oxidation, and amino acid metabolism. Mice colonized with KD-associated human gut microbes further exhibit altered hippocampal and frontal cortical transcriptomic profiles relative to colonized pre-treatment controls, including differential expression of genes related to ATP synthesis, glutathione metabolism, oxidative phosphorylation, and translation. Integrative co-occurrence network analysis of the metagenomic, metabolomic, and brain transcriptomic datasets identifies features that are shared between human and mouse networks, and select microbial functional pathways and metabolites that are candidate primary drivers of hippocampal expression signatures related to epilepsy. Together, these findings reveal key microbial functions and biological pathways that are altered by clinical KD therapies for pediatric refractory epilepsy and further linked to microbiome-induced alterations in brain gene expression and seizure protection in mice.The high-fat low-carbohydrate ketogenic diet (KD) is an established dietary therapy for individuals with refractory epilepsy, whose seizures are resistant to existing anti-epileptic drugs. However, use of the KD to treat refractory epilepsy is challenging because the diet is difficult for patients to implement, manage, and maintain due to its severe restrictiveness and adverse side effects. Exactly how the clinical KD reduces seizure symptoms when other anti-epileptic drugs are ineffective is poorly understood. The gut microbiome has emerged as a key intermediary between diet and host metabolism, neural activity, and behavior. The gut microbiome modulates seizure susceptibility and the anti-seizure effects of the ketogenic diet (KD) in animal models. This dissertation work seeks to understand if these relationships seen in animal models translate to KD therapies for human drug-resistant epilepsy. Herein we report that KD therapy in children with pediatric epilepsy alters the function of the human gut microbiome. In addition, colonizing mice with KD-associated human gut microbes confers increased resistance to 6-Hz psychomotor seizures, as compared to colonization with gut microbes from matched pre-treatment controls. Parallel analysis of human donor and mouse recipient metagenomic and metabolomic profiles identifies subsets of shared functional features that are seen in response to KD treatment in humans and preserved upon transfer to mice fed a standard diet. These include enriched representation of microbial genes and metabolites related to anaplerosis, fatty acid beta-oxidation, and amino acid metabolism. Mice colonized with KD-associated human gut microbes further exhibit altered hippocampal and frontal cortical transcriptomic profiles relative to colonized pre-treatment controls, including differential expression of genes related to ATP synthesis, glutathione metabolism, oxidative phosphorylation, and translation. Integrative co-occurrence network analysis of the metagenomic, metabolomic, and brain transcriptomic datasets identifies features that are shared between human and mouse networks, and select microbial functional pathways and metabolites that are candidate primary drivers of hippocampal expression signatures related to epilepsy. Together, these findings reveal key microbial functions and biological pathways that are altered by clinical KD therapies for pediatric refractory epilepsy and further linked to microbiome-induced alterations in brain gene expression and seizure protection in mice.

The microbiota-gut-brain (MGB) axis is a complex communication network connecting the central nervous system (CNS) with the gut microbiota. Lifestyle factors, including diet, can have a profound impact on the composition and function of the gut microbiome and hence modulates the MGB axis. The high-fat diet (HFD) is a major contributor to the obesity pandemic and is associated with increased susceptibility to neurological disorders, including anxiety and depression. The HFD is shown to decrease the abundance of beneficial bacteria and increase systemic levels of proinflammatory cytokines and lipopolysaccharides. Animal and human studies have demonstrated that HFD can induce anxiety-like behavior, as measured by elevated plus maze and open field tests in mice and rats. The underlying mechanism of this effect is believed to involve alterations in the MGB axis and changes in the gut microbiota composition and metabolites. Obesity-induced chronic inflammation induces anxiety-like behavior by activating the immune system and altering neurotransmitter signaling. There is increasing focus on the role of limbic system inflammation in the pathogenesis of several metabolic and neurological disorders, especially concerning HFD. However, the biochemical mechanisms and pathways that link high-fat consumption and CNS homeostasis with the pathology of neurological disorders are not well understood. This thesis investigates the relationship between diet, specifically high-fat diet (HFD), and anxiety-like behavior in mice, focusing on the role of the microbiota-gut-brain (MGB) axis. A 10-week experiment was conducted, wherein male C57BL/6 mice were divided into HFD and control diet (CD) groups. The HFD group exhibited weight gain and increased anxiety-like behavior compared to the CD group, as assessed through various behavioral paradigms, including the open field test (OFT), elevated plus maze (EPM) test, and light/dark box (LDB) test. Notably, anxiety-like behavior was observed as early as three weeks into the HFD treatment. Additionally, the study explored the potential involvement of imidazole propionate (ImP), a microbiota-derived metabolite, in anxiety-like behavior. ImP supplementation induced anxiety-like behavior in mice, despite no significant differences in ImP levels between HFD-fed and CD-fed mice. Histological analysis suggests that ImP decreases cFOS expression in the hypothalamus, a brain region important in the regulation of appetite and insulin signaling. There were no changes in microglial morphology in specific brain regions associated with anxiety regulation in response to ImP. However, further investigations are needed to understand the precise mechanisms underlying the effects of ImP on the MGB axis. Overall, this thesis contributes to the understanding of the complex interplay between diet, the gut microbiota, and the CNS, highlighting potential therapeutic strategies targeting the MGB axis for anxiety and neurological disorders.

The gut microbiome interacts with host physiology to influence neurodevelopment during critical periods of life. Brain-gut interactions during these critical periods can occur through direct communication, such as via gut-derived metabolites or vagal activity, or by indirect or secondary body systems, such as immune or endocrine signaling. Regardless of mechanism, these brain-gut interactions shape neurological trajectories for later-life function. The compilation of work making up this dissertation aims to profile mechanisms of developmental brain-gut interactions in response to environmental perturbations, and explore avenues of microbiota-targeted methods for amelioration of subsequent neurological impairment. Chapters 2 and 3 and Appendix 1 investigate how the microbiome responds to, and moderates effects of, perinatal malnutrition on the developing brain. Here, we report that the maternal microbiome modifies effects of protein undernutrition on fetal neurodevelopment and offspring behavior, and that gut-modulated metabolites ameliorate select phenotypes. Chapter 4 investigates signatures of gestational fluoxetine use on transcriptomic disruptions in the developing fetal brain, and reports a mediating role of the maternal microbiome. Chapter 5 is a translational study investigating correlations between metabolomic changes, functional brain connectivity, and psychiatric symptoms in adults following exposure to early life adversity. We find that four gut-modulated metabolites and connectivity of brain networks including sensorimotor, salience, and central executive, are associated both with early life adversity and with current stress, anxiety, and depression symptoms. Together, this body of work supports the notion of sensitivity of not only the brain, but also the gut, and interactions between the two, to environmental perturbations. Results indicates a causal role of the microbiome during critical windows of prenatal or postnatal development in shaping neurodevelopment and influencing persistent trajectories of behavior, and demonstrate that these interactions occur in response to outside influences including diet, medications, and stress. Finally, this work highlights the gut microbiome as a target of potential intervention for critical period neurodevelopmental disruptions.