Anatomy and Neuroscience - Doctoral Theses
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Item Unravelling the impact of early-life nutrition on microbiota-gut-brain axis signalling(University College Cork, 2024) Ratsika, Anna; Cryan, John; Codagnone, Martin; Science Foundation IrelandCurrent lifestyle habits such as aberrant diet are becoming disruptive to our health. Nutrition in early life is a key factor mediating immunity, neurodevelopment, and behavioural outcomes across the lifespan. Optimal nutrition in the first 1000 days of life, starting from preconception to early childhood, are essential for optimal growth, brain and immune system function. Growing lines of evidence suggest that gut microbiota play a role in immune system education, brain function and behaviour. Disruptions in the gut microbiota composition during critical periods of development perinatally might influence the developmental trajectory of the brain and the immune system, with implications for their function later in life. Although the effects of diet-induced microbiota alterations have been studied in the context of brain and immune system development, studies often overlook the interactions between these systems in critical time-windows of developmental opportunity. Given that external influences such as diet could be decisive for brain function there is a growing need for investigation of the effects of early-life nutrition on the microbiota-immune-brain axis during critical windows of development. In this thesis, I focused on the investigation of the influence of microbiota disruptions perinatally via 1. High-fat diet (HFD) during pregnancy and lactation, 2. C-section and 3. Early-life antibiotic administration on offspring brain and immune system development, and behaviour. Using a model of maternal HFD we disrupted the maternal microbiota, which led to increased potent neurotoxic metabolites in maternal circulation. The maternal-diet-induced microbiota disruption and associated metabolic signals had consequences for embryonic brain function and were associated with altered glutamate-related metabolites and genes in the fetal mouse brain and hyperactivity in adolescence specifically in the female offspring. Next, the impact of disruption of vertical microbiota transmission via C-section on the immune system education during weaning was investigated with outputs in the gut, the periphery and at the level of neuroimmune interactions in the juvenile brain. Our data shows that immune system priming in the gut and the periphery is aberrant in response to altered delivery mode, with implications for microglia activation in the male juvenile mouse brain. This highlights the timeframe around weaning as being a critical one for unmasking the enduring effects of early life microbiota disturbances. Lastly, the influence of postnatal early-life dietary intervention with human milk oligosaccharides (HMOs) and prebiotics was investigated on its ability to reverse the effects of antibiotic-induced microbiota depletion on brain transcriptome and behaviour in juvenile male mice. The combination of HMOs with prebiotics led to enhanced social recognition memory and learning and memory pathways in transcriptomic results in social brain areas, possibly via enriched abundance and function of the gut microbiota in male juvenile mice. Taken together, this work provides evidence that the diet and gut microbiota in critical windows of development regulate brain and immune system function. Moreover, we identified that dietary interventions targeting the gut microbiota in early life improve brain function and social behaviour, demonstrating that early-life gut microbiota is important for neurobehavioural outcomes.Item Pre-eclampsia and neuronal development: the potential pathogenic role of inflammation(University College Cork, 2024) Barron, Aaron; O'Keeffe, Gerard W.; McCarthy, Cathal; Tuulari, Jetro; Karlsson, Linnea; Karlsson, Hasse; Irish Research Council for Science, Engineering and TechnologyBackground: Pre-eclampsia (PE) is a common and serious hypertensive disorder of pregnancy that occurs in approximately 3-5% of first-time pregnancies. There is increasing evidence that in utero exposure to PE increases the risk for various neurodevelopmental disorders, particularly autism spectrum disorder and attention-deficit/hyperactivity disorder ADHD, although the mechanism(s) mediating this relationship are yet to be elucidated. Chapter 1 of this thesis describes the evidence implicating a causal role for PE exposure in the aetiology of various neurodevelopmental disorders and neuroanatomical alterations in the small number of imaging studies that have been undertaken in this context. It then postulates that inflammation, a prominent pathological feature of PE, may mediate this association, through direct exposure of the foetal brain to higher levels of inflammatory cytokines; to the various physiological ramifications of maternal immune activation (MIA), which has been strongly implicated in neurodevelopmental and psychiatric disorders; and through perpetuation of the angiogenic factor imbalance and exaggerated oxidative stress seen in PE. Through these various mechanisms, inflammation may adversely affect neuronal development, altering the developmental trajectory of the brain. Methods: Chapter 2 investigated the effects of maternal serum from women with PE or a healthy uncomplicated pregnancy on neurite growth and mitochondrial function in neuronally differentiated human SH-SY5Y neuroblastoma cells. Following this, the pleiotropic cytokine IL-6, was investigated as a potential mechanism. IL-6 concentration was measured in maternal sera and placentae; IL_6 signalling was explored in serum-treated cells; neurite growth was investigated while inhibiting IL-6; and. Lastly, cells were treated with recombinant IL-6 alone. Chapter 3 examined the effects of the anti-angiogenic factor soluble Fms-like tyrosine kinase 1 (sFlt-1), which is highly elevated in PE, on neuronal development. The human neural progenitor cell (NPC) line ReNcell® VM was differentiated into a mixed culture of post-mitotic neurons and glia, and exposed to sFlt-1 during development. The effects of sFlt-1 on neurite length, and βIII tubulin mRNA and protein expression was measured. Lastly, VEGFA inhibition was explored as a potential mechanism by co-treating cells with VEGFA and sFlt-1. Chapter 4 examined the association between maternal C-reactive protein (CRP; as a surrogate measure of MIA) on offspring brain development through analysis of human neuroimaging data from the FinnBrain birth cohort study. We studied the association between maternal prenatal CRP and offspring cortical thickness, surface area, and volume, using vertex-wise statistics; white matter fractional anisotropy (FA) and mean diffusivity (MD), using voxel-wise statistics; and neuronal differentiation and morphology in vitro by exposing developing human neurons to low- or high-CRP maternal serum. Chapter 5 explored the relationship between immune system activity and brain structure in typically developing 5-year-old children. This study employed very similar methods as those in Chapter 4, and was also set within FinnBrain. This chapter examined the association between CRP and cerebrocortical thickness surface area, and volume; white matter FA and MD; and neuronal differentiation and morphology. Results: Chapter 2 - Cells exposed to PE serum for 72h exhibited increased neurite growth and mitochondrial respiration compared to controls. IL-6 was elevated in maternal PE sera, and SH-SY5Y cells exposed to PE serum for 24h had increased phospho-STAT3 levels, which is a key intracellular mediator of IL-6 signalling. Furthermore, incubation with an anti-IL-6 neutralizing antibody prevented the effects of PE serum on neurite growth, while treatment with IL-6 promoted neurite growth in SH-SY5Y cells. Collectively, these data show elevated serum levels of maternal IL-6 in PE, which increases neurite growth and mitochondrial function in SH-SY5Y cells. Chapter 3 - sFlt-1 induced a significant reduction in neurite growth up to a concentration of 100 ng/mL. sFlt-1 (100 ng/mL) also reduced βIII-tubulin mRNA and neuronal differentiation of neurospheres. These effects are thought to be elicited by inhibition of endogenous autocrine and paracrine VEGFA signalling undifferentiated NPCs and post-mitotic neurons/glia expressed VEGFA and its receptor VEGFR-2, while sFlt-1 treatment prevented the neurogenic effects of exogenous VEGFA. Taken together, these data provide the first experimental evidence for a direct effect of sFlt-1 on neurite growth and neuronal differentiation in human neurons through inhibition of VEGFA signalling, independent of its previously characterised role in blood vessel formation. Chapter 4 - Maternal CRP within a normal physiological range (< 10 mg/L) was associated with cortical volume in one cluster of the right lingual gyrus in 5-year-old males, and increased white matter FA, and reduced MD, in 5-year-old females. Hyperphysiological maternal CRP (> 10 mg/L), was associated with increased surface area or volume in three clusters of the cortex in 5-year-old females, and increased FA and decreased MD in female infants, particularly in the left external capsule and body of corpus callosum. In line with these neuroimaging findings, high-CRP maternal serum increased neurite growth of human neurons in vitro, particularly from mothers with female offspring. Overall, these data show that maternal CRP is associated with sex-specific cerebrocortical and white matter alterations in typically developing children. Chapter 5 - CRP was associated with increased cortical surface area or volume in distinct, and sex-specific clusters of the cerebral cortex. Serum from children with elevated CRP increased neurite growth and growth cone surface area in human neurons, the latter effect caused by female sera only. Overall, these findings demonstrate that immune activation in 5-year-old children is associated with brain structural parameters in vivo and neuronal development in vitro, with effects differing by biological sex. Conclusions: These data provide converging evidence for both direct and indirect actions of immune mediators on neuronal development at the single cell level, and sex-specific associations between both MIA and childhood immune activation on human brain development. Collectively, they elucidate a mechanistic role for inflammatory mediators in human neuronal and brain development, particularly in the context of pre-eclampsia.Item Peripheral alterations underlying the negative effects of a cafeteria diet on brain and behaviour: exercise as a mitigating strategy(University College Cork, 2024) Nota, Minke H. C.; Nolan, Yvonne M.; O'Leary, Olivia; Irish Research Council; Science Foundation IrelandA Western lifestyle, characterised by inactivity and overconsumption of saturated fats and sugar, increases risk of depression, anxiety, and cognitive impairment. Obesity, metabolic dysfunction, (neuro)inflammation, and gut microbiota alterations, which can result from a Western lifestyle, are associated with mood disorders and cognitive impairment, thus constituting potential mechanisms by which Western lifestyle impacts the brain. Adult hippocampal neurogenesis (AHN), i.e., the birth of new neurons in the dentate gyrus of the hippocampus, is involved in certain forms of memory, including spatial memory and pattern separation, and in regulating emotion through anxiety behaviours and antidepressant action. A cafeteria (CAF) diet, mimicking human Western-style diets, has been shown to decrease AHN, impair memory, and increase anxiety-like behaviour in rodents, whereas exercise has antidepressant effects and has been shown to improve AHN and cognition. However, interactions between these lifestyle factors remain unclear. Furthermore, effects of Western-style diets and exercise on AHN and associated behaviours have primarily been researched in males, whereas depression, certain anxiety disorders, and dementia disproportionally affect women. The aims of this thesis were to investigate whether voluntary running exercise could alter the effects of a CAF diet on AHN and hippocampus-associated behaviour, and the intake of and preference for a CAF diet in adult male and female rats, and to determine if a CAF diet and exercise could impact metabolic markers, inflammation, and gut-derived metabolites in males. Exercise had anxiolytic effects in males and females, induced modest improvements in spatial learning in males, and decreased spatial memory in females. Additionally, exercise mitigated a CAF diet-induced increase in depression-like behaviour, and a CAF diet blunted an exercise-induced increase in AHN, in males but not females. In exercising males and females with access to CAF diet, intake of energy from CAF foods and saturated fat was decreased, and fibre and protein intake was increased compared to sedentary rats with access to a CAF diet. Moreover, compared to sedentary rats, exercising rats had reduced preference for CAF foods over standard chow, which was maintained for 2 and 5 weeks in females and males, respectively. Increased hypothalamic Drd1 gene expression, which has been shown to promote overeating, in exercising males with access to a CAF diet possibly explained reduced preference for CAF foods in exercising rats not being maintained past 5 weeks. Alterations in metabolic hormones and caecal metabolites offer potential explanations for behavioural and neurogenic effects observed in males. Exercise-induced increased PYY potentially contributed to anxiolytic effects of exercise, and a CAF diet attenuating exercise-induced increased GLP-1 possibly explained the blunting of neurogenic effects of exercise. Attenuation of CAF diet-induced increased leptin and insulin and decreased caecal indole-3-carboxylate and deoxyinosine by exercise potentially contributed to exercise mitigating CAF diet-induced increased depression-like behaviour. Furthermore, exercise attenuated a CAF diet-induced decrease in abundance of caecal anserine, a metabolite previously associated with improved cognitive function. No definitive pro- or anti-inflammatory pattern of a CAF diet and exercise emerged that might have contributed to changes observed in behaviour and AHN. However, ventral hippocampal Il-6r gene expression was decreased in exercising males with access to a CAF diet, possibly explaining the finding that in males, exercise attenuated a CAF diet-induced increase in depression-like behaviour, as reduced general IL-6 activity has been associated with antidepressant effects. Ultimately, these data highlight the importance of exercise combined with a healthy diet for hippocampal health, along with sex differences in lifestyle influences on brain and behaviour. Moreover, these data indicate potential mechanisms, including metabolic hormones and gut microbial metabolites, underlying interactions between a CAF diet and exercise on brain and behaviour, thereby aiding advancement of preventative measures for depression and cognitive impairment.Item Unravelling the role of circadian rhythmicity in microbiota-gut-brain axis signalling(University College Cork, 2023) Tofani Sousa e Silva, Gabriel; Cryan, John; Saks-Kavanaugh Foundation; Science Foundation IrelandModern habits are becoming more and more disruptive to health. Our days are often filled with circadian disruption and stress exposure. We need to understand how our responses to these external stimuli are shaped and how they can be targeted to promote health. A growing body of research demonstrates the role of the gut microbiota in influencing brain function and behavior. The stress and circadian systems, which are essential to maintaining appropriate responses to the environment, are known to be impacted by the gut microbiota. Although gut microbes have been shown to modulate circadian rhythms and stress response, such studies were conducted in an independent manner. Since these systems are interconnected through the hypothalamic-pituitary-adrenal (HPA) axis, there is a need to examine how the gut microbiota may play a role in regulating the integration of stress and circadian signals. In this thesis, I aimed to investigate the influence of circadian rhythmicity in the microbiota-gut-brain axis communication and the consequences of that to stress responsivity. To this end we developed a computational tool, Kronos, that allows us to determine rhythmicity of genes and metabolites in brain regions important to stress and circadian regulation under different microbial status. Moreover, we investigate the interplay of the circadian and stress systems in the absence of the microbiota to dissect its role in the modulation of these systems. Using transcriptomics, and applying Kronos, we demonstrate that microbial depletion by antibiotic administration or germ-free status disrupts the molecular clock in the superchiasmatic nucleus (SCN). Such alterations to the master clock were accompanied with disruptions in the rhythmicity of circulating corticosterone. Furthermore, multi-omics analysis in the hippocampus and amygdala, indicated that microbial status disrupted the diurnal oscillations in genes and metabolites that compose pathways important for the stress response. We then investigate the expression of genes related to circadian rhythms and HPA-axis in the paraventricular nucleus, pituitary, and adrenal glands across the day. This, together with alterations in corticosterone demonstrates a hyper-activation of the HPA-axis at the sleep/wake transition in microbial depleted animals. Such disruption to the rhythmic function of the HPA-axis resulted in a time-of-day specific impairment of the stress response and stress-sensitive behaviors. Lastly, we identify changes in the rhythmic profile of the gut microbiota following microbial depletion. This manifested as peak of bacterial load at the same time the impairments in HPA-axis function were observed. Furthermore, by conducting fecal microbiota transplantation we confirm that the diurnal oscillations in gut microbes after antibiotic treatment regulates glucocorticoids secretion and explore the microbial compositional changes underlying it. This work provides compelling evidence that the gut microbiota regulates stress responsivity via the circadian system. Moreover, we identify the gut microbiota as an important regulator of HPA-axis rhythmic function, demonstrating that the microbiota is essential to adaptively respond to psychological stressors throughout the day.Item The bacteriome and virome in social and stress-related disorders(University College Cork, 2023) Ritz, Nathaniel L.; Cryan, John; Dinan, Timothy G.; Science Foundation IrelandThe gastrointestinal microbiota has been shown to influence mammalian health and well-being in many conditions but has also been implicated in a variety of diseases and disorders. The multi-faceted interaction of the microbiota with the host that affects the brain and behaviour is termed the microbiota-gut-brain axis. While this axis plays an important role in stress and social behaviour, research has largely focused on associations with the bacterial fraction of the community. The gut also harbours viruses, fungi, archaea, and other microbial forms of life that can affect the health of the mammalian host. Gut viruses, which are dominated by bacteriophages, are also ubiquitous in the gut and have the ability to infect bacteria and alter their structure and activity. Moreover, these populations of viruses are intimately associated with the bacterial community, but little is known about their involvement within the microbiota-gut-brain axis. Whether bacteriophages are capable of exerting effects on the brain and behaviour is largely unknown. To this end, we tested whether the virome was affected by stress followed by the capability of the virome to prevent stress-related sequelae. We found that the virome is stress sensitive and that restoring the virome to mice undergoing stress reduced anxiety-like behaviour, immune activation, inflammation, and gene expression in the brain. These data provide evidence that the gut virome can be harnessed to improve stress-coping outcomes. Social diseases and disorders have been implicated in the microbiota-gut-brain axis. Social anxiety disorder is a common but understudied psychiatric condition. Whether specific microbiota communities play a role in social fear responsivity or are capable of driving social fear behaviour is also unknown. Preclinical animal models of social fear allow for testing translational hypotheses that can then be returned to the clinic. We sought to characterize associations of the microbiota with social fear resiliency and susceptibility. Then, we transferred microbiota from individuals with social anxiety disorder to mice to test whether the microbiota played a causative role in the disorder. We found a strong correlation between microbiota and social fear variability along with differential expression of amygdalar genes involved in social behaviour, immunity, and host-microbe interaction. We also found that microbiota from individuals with social anxiety disorder could drive social fear sensitivity in mice. Mice that received microbiota from social anxiety disorder donors also had impaired cytokine release in the gut, reduced immune cell populations in the mesenteric lymph nodes and circulation, altered basal HPA axis, and diminished oxytocin and oxytocin-related expression of genes in the brain. These data indicate that social fear and anxiety are linked with the microbiota and that the gut-brain axis is a target for future research for social anxiety disorder therapeutics. Herein we tested whether gut viruses could alter stress response, whether social fear behaviour is associated with differential microbial communities, and whether human microbiota from individuals with social anxiety disorder can drive social fear behaviour in the mouse. Collectively we have shown that stress-related sequelae can be restored by virome transfer and that the microbiota can play a causal role in social anxiety disorder.