Individuals living with depression and anxiety show systemic increases of the bacterial endotoxin lipopolysaccharide (LPS), which induces an inflammatory cascade, resulting in negative effects across the gut-brain axis (GBA). LPS administration in mice has previously been used as a rodent model of depression/anxiety. Flaxseed (FS) contains key bioactives, including an omega-3 fatty-acid, dietary fibre, and a poly-phenolic compound which all may attenuate the effects of LPS through modulation of the GBA. The objectives of this thesis were to examine the effects of LPS on the GBA in C57Bl/6 mice and to determine if dietary supplementation with FS and/or FS oil (FO) provided protection against the LPS challenge. The LPS-induced negative effects across the GBA were partially attenuated by dietary supplementation with FS, but not FO, through changes in microbiota composition/function and systemic-/neuro-inflammation. Therefore, the potential benefits of FS are independent of the oil or are synergistic of all bioactives.

Epilepsy is a complex, chronic neurological disorder with diverse underlying etiologies characterized by the spontaneous occurrence of seizures. Epilepsy affects all ages from neonates to elderly adults, with the most recent CDC estimates stating that ~3 million adults and over 400,000 children are currently suffering from active epilepsy in the U.S. alone. In adults, the leading cause of epilepsy worldwide in central nervous system (CNS) infection, while in neonates the most common cause of seizures is hypoxic/ischemic encephalopathy (HIE). However, in both adults and neonates, current antiepileptic drugs (AEDs) are ineffective in 30-50% of patients, despite the availability of over 20 FDA approved AEDs with diverse molecular targets. This disparity highlights a critical need for novel therapeutics in seizure-susceptibility and epilepsy. The microbes that inhabit gut mucosal surfaces, termed the gut microbiota, have been increasingly implicated in the pathology of neurological diseases including epilepsy. This gut-brain axis is an intriguing therapeutic target in epilepsy as gut microbes can affect the CNS through multiple mechanisms including vagus nerve signaling, immune-gut interactions, and through production of microbial-metabolites including neurotransmitters, short chain fatty acids (SCFAs), lactate, vitamins, and S-equol. Furthermore, the gut microbiota is crucial for neurodevelopment, indicating that the gut-brain axis may be involved in pediatric seizure-susceptibility. This dissertation reviews current evidence on the role of gut metabolites in seizure-susceptibility in epilepsy, highlighting the microbial-derived metabolite S-equol as a potential novel AED. We then evaluate gut microbiome alterations in the Theiler's murine encephalomyelitis virus (TMEV) adult mouse model of CNS infection-induced seizures and find decreases in S-equol-producing bacteria in the gut microbiomes of TMEV-infected mice with seizure phenotypes. We characterize the effect of exogenous S-equol on neuronal function in vitro, demonstrating a reduction in neuronal excitation following S-equol exposure. We additionally characterize entorhinal cortex (ECTX) pyramidal neuronal hyperexcitability, and demonstrate the ability of exogenous S-equol to ameliorate CNS-infection-induced ECTX neuronal hyperexcitability ex vivo. Finally, we demonstrate that perinatal and postnatal exposure to antibiotics alters the gut microbiome and increases seizure-susceptibility following HIE exposure in p9/p10 mice, potentially via sex-specific alterations in neuronal function. Together, this dissertation evaluates the gut-brain axis in pediatric and adult mouse models of seizure-susceptibility and identifies the gut metabolite S-equol as a potential target for the treatment of seizures. / Doctor of Philosophy / Epilepsy, a disease defined by the occurrence of two or more spontaneous seizures, affects over 50 million people worldwide. This makes epilepsy one of the most common chronic neurological disorders across the globe. People with epilepsy suffer increased mortality, lower quality of life, and increased social stigma. There is currently a crisis in the treatment and management of epilepsy, because although over 20 different anti-epileptic drugs (AEDs) are available to patients, these drugs only work in ~70% of individuals with epilepsy, leaving 30% of patients with uncontrolled seizures. Currently available AEDs are designed to target classical central nervous system (CNS) components. However, a growing body of evidence suggests that epilepsy is related to complex systems throughout the body. Therefore, in this manuscript we explore novel therapeutic targets outside of the CNS for the management of seizures. Over 1000 species of bacteria live in the in the human gut, and are termed the gut microbiota. Gut microbes produce a variety of chemicals that circulate through the body and can even reach the brain. Interaction of chemicals produced by the gut microbiota and brain chemistry have been shown to affect disease outcomes in Autism Spectrum Disorder, Parkinson Disease, and other brain disorders. However, very few studies have examined the possibility of a role for the gut microbiota in epilepsy. In this dissertation, we review chemicals produced by the gut microbiota that may alter epilepsy biology. We additionally examine gut microbiota alterations in a rodent model of epilepsy, and identify a novel chemical, S-equol, that is produced by the gut microbiota and impacts epilepsy biology in our rodent model. Lastly, we explore how altering the maternal gut microbiota in rodents can influence seizure-susceptibility in infants.

Our gut microbiota is involved in human development, nutrition, and the pathogenesis of gut disorders, but has more recently been implicated as a possible mechanism in the pathophysiology of several brain disorders, including disorders of mood and affect, such as depression. Researchers have referred to this dynamic, bidirectional signaling pathway between the gut and the brain as the “gut-brain axis.” However, most research on this axis has been limited to rodent studies, and there has been little insight into the mechanism behind it. I propose that the kynurenine pathway, where tryptophan is converted to kynurenine, is a compelling mechanism mediating the gut microbiota’s influence on depression. Kynurenine is a metabolite associated with depression, and this pathway has been shown to be manipulated through probiotic (Lactobacillus reuteri) consumption. I propose to study a probiotic intervention in humans, which would assess tryptophan metabolism along the kynurenine pathway by measuring metabolites downstream of this pathway. Urine, feces and blood samples would be collected from two groups, control and probiotic treatment, on day zero and day thirty. Colonic biopsies would be obtained on day thirty, and various analyses would be run to measure metabolite concentrations from the collected samples. The results from this study will help clarify a mechanistic connection between gut microbes and depression via the kynurenine pathway. Additionally, findings could indicate that a probiotic intervention has the ability to influence depressive behavior via a two-pronged approach originating from the kynurenine pathway.

<p>In this study, structural specificity of flavonoids was investigated toselectively inhibit starch digestive enzymes to stimulate the ileal-brake by triggering glucagon-like peptide-1 (GLP-1) through distal small intestine starch digestion which can regulate food intake and appetite. The double bond between C2 and C3 on flavonoid’s chemical structure plays a critical role to inhibit human pancreatic α-amylase, leading to π-staking interaction. Meanwhile, the hydroxyl group at C3 on the backbone benzopyran ring is intimately related to inhibition of the mucosal α-glucosidases. This selective inhibition is likely the result of fundamental differences in the protein structures of α-amylase and α-glucosidases, as they belong to different glycosyl hydrolase Families 13 and 31 (GH13 and GH31). α-Amylase has the catalytic active siteslocated in wide and shallow grooves on the protein structure, while α-glucosidases possess the narrow and deep catalytic pocket. In an acute study done on mice, luteolin, which had thehigher degree of selectivity toward α-amylase, showed a slow and sustained postprandial glycemic response with a reduced blood glucose peak and extended high glucose profile, compared to 3’,4’-dihydroxylflavonol as the selective α-glucosidases specific inhibitor. Quercetin was inhibitory of both α-amylase and α-glucosidases.Glycemic profiles in mice confirmed in vitro analysis of the inhibitory selectivity of the flavonoids tested. Additionally, the extended glycemic response with luteolin was accompaniedthe higher secretion of GLP-1 at extended postprandial times by delivering more starch portion into the distal small intestine where the ileal-brake and gut-brain axis activation takes place. Overall, selective inhibition of α-amylase by flavonoids potentially could be considered as a key approach to control glucose release from starch with slow and extended, but still complete, digestion for improved glycemic response and minimized adverse side effects that result from severely restricting or even shutting down starch digestion by pharmaceutical grade inhibitors.<br></p>

Puberty is a critical period of development characterized by rapid physiological changes and significant brain reorganizing and remodeling. These rapid changes render the developing brain particularly vulnerable to stress and immune challenge. In mice, exposure to an immune challenge (lipopolysaccharide; LPS) during puberty causes enduring effects on stress reactivity, cognitive functioning, and depression- and anxiety-like behaviors later in life. However, the mechanisms underlying these effects are unknown. The gut microbiome can profoundly influence the immune system. There is also close bidirectional communication between the gut microbiome and the central nervous system (CNS) through neural, endocrine and immune signaling pathways, which can alter brain chemistry and emotional behaviour. Thus, we hypothesized that altering microbial composition during puberty could mitigate acute immune responses and prevent enduring outcomes later in life. The current thesis examined the effect of gut manipulation with probiotics during puberty on LPS-induced immune responses and enduring anxiety- and depression-like behaviours, and stress-reactivity in adulthood, in male and female CD1 mice (Article 1). Next, we examined age and sex differences in gut microbial composition before and after exposure to an immune challenge. We also examined the effects of consuming a single strain probiotic bacterium (Lactobacillus Reuteri) during puberty on the immune response and the long-term changes in memory, anxiety-like behavior, and stress reactivity in adulthood (Article 2). Lastly, we examined how microbial colonization between pubertal and adult mice can alter acute peripheral and central inflammatory responses to LPS (Article 3). The current dissertation has addressed sex-specific vulnerabilities to an immune challenge during pubertal development and the moderating influence of the gut microbiome. These studies have demonstrated that manipulating the gut microbiome during puberty can mitigate acute immune responses and prevent enduring neurobehavioural outcomes later in life.

Time-restricted eating is a fasting diet where the food intake is restricted to a short, typically eight-hour, window each day. It is associated with health benefits such as weight loss, improved sleep, protection against cognitive disorders, and improved cognitive function. The cognitive effects of time-restricted eating have primarily been explained by the production of ketogenesis – an alternative energy source produced when calories are restricted – and anti-inflammatory cytokines. The gut microbiota is the trillions of microorganisms inhabiting the intestinal tract and has also been associated with improved mental health through communication via the gut-brain axis. This review aims to investigate whether changes in the microbiota may mediate the effect of time-restricted eating on cognitive function. Studies investigating the effect of time-restricted eating on the microbiota were systematically reviewed. The results indicate that time-restricted eating may alter the microbiome composition and increase butyrate-producing bacteria. Butyrate is a short-chain fatty acid associated with the expression of genes involved in neural development and the reduction of neuroinflammation. Limited by the few studies reviewed, the results may indicate a possible link between time-restricted eating and cognitive function via the microbiota, although more research is needed.

Major depressive disorder (MDD) is rapidly growing and one of the most common causes of disability and mortality worldwide. People with MDD often display brain changes such as adisrupted balance in neurotransmitters, impaired neurogenesis and neuroplasticity. Traditionally has MDD been treated with medications and talking therapies (psychotherapy). Studies have shown that just around 50 % of people with MDD get improvements from common traditional treatments.Therefore is there a great need for a better understanding of MDD and new treatments. There is now an emerging field of research that indicates that the gut microbiota plays a crucial role in disturbing normal brain functioning in MDD. This connection between the gut and the brain is called the gutbrain axis.The thesis aims to investigate if there is a connection between gut microbiota disruption and MDD and if gut microbiota restoration can be a potential effective future treatment for MDD. Key findings of the thesis were, studies show that people with MDD often display gut microbiota disruption and chronic low grade inflammation. Studies also indicate that this inflammation can cause the specific brain change often displayed in people with MDD. One of the most critical findings in the thesis was that gut brain treatments affect tryptophan metabolism, which affects the risk of MDD. The research area of the gut brain axis is still new and many more studies are needed,particularly in humans.

No description available.

<p>The small intestine holds an intrinsic ability to digest and absorb nutrients from the food we intake without intervention from the central nervous system. This ability is made possible by the population of cells that inhabit the gut, particularly interstitial cells of Cajal of the myenteric plexus and sensory primary intrinsic neurons (AH cells), which ultimately influence muscle function and motility. The AH cells are the first neurons in the hierarchy of sensory neurons in the gut and are therefore a perfect candidate to test the effects of <em>Bifidobacterium longum</em> NCC3001 supernatant since in a physiological setting the metabolites secreted by this bacterium can interact with the AH cells directly or indirectly through absorption by the mucosa.</p> <p>The probiotic <em>Bifidobacterium</em> <em>longum</em> NCC3001 has been shown to normalize anxiety-like behaviour and hippocampal brain derived neurotropic factor (BDNF) levels in mice infected with <em>Trichuris</em> <em>muris </em>in a model of infectious colitis. Utilizing a chronic model of colitis, a study was conducted to decipher whether or not the anxiolytic effects of <em>Bifidobacterium longum</em> NCC3001 involved the vagus. My specific objective in this study was to find evidence for interaction between <em>B.longum</em> NCC3001 and myenteric neurons as a potential route for <em>B.longum</em> NCC3001 to influence CNS function. We assessed a cell’s electro-responsiveness through spike discharge, which is the number of action potentials elicited in response to a supra-threshold depolarizing current injection.</p> <p>The electro-responsiveness of neurons perfused with <em>B. longum</em> NCC3001 supernatant (conditioned medium; n = 4) was significantly reduced compared to the control group (those perfused with Krebs solution; n = 5; <em>P</em> = 0.016). The electro-responsiveness of neurons perfused with the conditioned medium was also significantly lower than that of neurons perfused with unconditioned group (MRS growth medium alone) group (n = 4; <em>P</em> = 0.029). In comparing the excitabilities of the neurons in the control group with that of the control media group, there was no statistical difference (<em>P</em> = 0.29).</p> <p>In subsequent studies, the objective was to identify the AH cells and to determine the effect of <em>B. longum</em> NCC3001 conditioned medium on this population of cells. The electro-responsiveness as measured through spike discharge of AH cells perfused with the conditioned medium (n = 5) was significantly reduced compared to neurons perfused with the unconditioned medium (n = 5; <em>P</em> = 0.02). Sensory neurons perfused with the conditioned medium (n = 9) exhibited a significant reduction in their instantaneous input resistances compared to neurons perfused with the unconditioned medium (n = 8; <em>P </em>= 0.01). There was also a significant reduction in the time-dependent input resistance of neurons perfused with the conditioned medium (n = 9) compared to neurons perfused with the unconditioned medium (n = 8; <em>P </em>= 0.02). In addition, perfusion of the conditioned medium over sensory neurons (n = 9) significantly reduced the magnitude of the hyperpolarization-activated cationic current (<em>I</em>_h) compared to neurons perfused with the unconditioned medium (n = 8; <em>P</em> = 0.0003). Furthermore, there was also a significant reduction in the action potential half width duration of myenteric sensory neurons perfused with conditioned medium (n = 5) compared to that exhibited by neurons perfused with the unconditioned medium (n = 5; <em>P</em> = 0.008).</p> <p>In later experiments, we wanted to gain a more comprehensive understanding of the effect of this bacterium on the gut so we evaluated its effects on the gut musculature. Upon full immersion, the supernatant of <em>Bifidobacterium longum</em> NCC3001 (conditioned medium) caused an initial depolarization of the circular smooth muscle cell. This depolarization continued until the slow wave oscillations in these cells ceased and membrane potential would plateau. Several minutes after this plateau, the slow wave oscillations reappeared and the cell was significantly hyperpolarized relative to the conditions before conditioned medium was added. The resting membrane potential of circular smooth muscle cells in Krebs solution was -54.3 mV and -70.3 mV approximately two minutes after full immersion by the supernatant when the cell was hyperpolarized and a stable recorded was achieved (n = 7; <em>P</em> = 0.02). The average time of onset of depolarization was 18.6 s and the average change in membrane potential (depolarization) from onset of effect to its plateau was 14.0 mV (n = 7). Occasionally, the addition of the conditioned medium only caused an immediate but slight depolarization (n = 3) and in other cases caused only a hyperpolarization of the cell (n = 3) with no significant changes in any slow wave characteristics in either case. Furthermore, any cells that exhibited the waxing and waning of the slow wave lost this pattern upon the addition of the conditioned medium (n = 10).</p> <p>In attempts to understand the role of neurotransmission in this system, we conducted several experiments whereby carbachol (acetylcholine agonist) and L-NNA (nitric oxide synthase inhibitor) were administered to the muscle. Prior to the addition of 1μM carbachol or 2e^-4M L-NNA, we would only observe the pacemaker slow wave associated with the interstitial cells of Cajal of the myenteric plexus during the perfusion of Krebs solution. Upon the addition of carbachol (n = 3) or L-NNA (n = 4), we would observe a second slower frequency pattern appear, referred to as a waxing and waning pattern.</p> / Master of Science (MSc)

The aetiology of major depressive disorder (MDD) is poorly understood. Current evidence suggests immune activation and gut microbiota may play a role. Recent studies demonstrated that behavioural traits can be transferred through microbiota transplantation into germ-free (GF) mice. Here we study whether microbiota from patients with MDD can induce depressive-like behaviour. Methods: GF NIH Swiss mice were colonized with stool microbiota from a patient with MDD with elevated faecal β-defensin 2, or a healthy donor (HC). After three weeks, behaviour was assessed using standard tests. Expression of neuroimmune markers was assessed in the gut and brain using gene expression profiling and immunohistochemistry. Microbiota composition was assessed by 16S rRNA sequencing. Results: Microbiota profiles differed between the two groups of mice (p=0.001). Mice colonised with microbiota from a single characterised MDD patient (MDD1), exhibited lower preference for sucrose (p=0.002) and more emotionality (p=0.003) than mice with HC microbiota, however other MDD mice did not display abnormal behaviour. Abnormal MDD1 behaviour was associated with lower BDNF expression in the dentate gyrus of the hippocampus (p=0.02). Mice colonised with another characterised MDD patient (MDD4 mice) did not have differences in BDNF expression in the same region (p=0.20). MDD1 and MDD4 mice had altered hippocampal and gut gene expression for genes associated with the immune and nervous system. In summary, GF mice colonized with MDD1 microbiota exhibit depression-like behaviors. This appears to be accompanied by changes in intestinal permeability and neuroimmune function. These results suggest that gut microbiota has the capacity to influence the expression of MDD in some patients. / Thesis / Master of Science (MSc)