Anatomy and Neuroscience - Doctoral Theses
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Item Novel insights into the role of the gut microbiome on hippocampal synaptic plasticity(University College Cork, 2024) Collins, Michael K.; Cryan, John; O'Riordan, Ken; Science Foundation Ireland; Irish Research CouncilSynaptic plasticity especially within the hippocampus is a central process of the learning machinery of the brain. The microbiota gut-brain axis has emerged as an important regulator of hippocampal function yet there exists a paucity of information on how and when this axis can affect hippocampal synaptic plasticity. New strides need to be taken to develop successful interventions for human diseases which feature dysregulation of synaptic plasticity and to understand when normal synaptic plasticity may be susceptible to altered regulation from the microbiota-gut brain axis. In this thesis we focused primarily on how adult synaptic plasticity in the hippocampus may be affected by microbiome interventions. We also assessed the microbiome’s role in altering the developmental trajectory of the hippocampal transcriptome with a special focus on plasticity-related pathways. Firstly, we used germ free mice to examine how growing up without a gut microbiome has altered synaptic plasticity with hippocampal slice electrophysiology. While basal synaptic efficacy and pre-synaptic short-term plasticity appear normal, we found a deficit of hippocampal long-term potentiation in slices from male germ-free mice whereas slices from female animals produced this plasticity process relatively normally. However, the spike output of these neuronal populations remained normal, possibly indicating an altered excitation-inhibition balance or a homeostatic compensatory mechanism. Following this, we began to interrogate whether the microbiota’s ability to alter synaptic plasticity is developmentally wired or remains susceptible in adulthood. To this end we depleted the microbiome in adult life using either an antibiotic cocktail or a single antibiotic. This two-week microbiota intervention in adulthood produced only subtle effects on short-term plasticity and left LTP unchanged, demonstrating the robustness of hippocampal plasticity to the effects of microbiome depletion in adulthood. We then investigated how a local exposure to short-chain fatty acids (SCFAs), an abundantly produced class of microbial metabolite, may affect hippocampal synaptic plasticity in adulthood. Three SCFAs were tested, acetate, propionate, and butyrate at physiologically relevant concentrations. While no effect of acetate or propionate was found, butyrate-treated slices produced a greater degree of LTP in slices from both male and female mice. We hypothesized this effect was occurring through butyrate’s action at free fatty acid receptor 3 (FFAR3). However, when slices were pre-treated with an inhibitor of this receptor only slices from female mice reverted to producing normal levels of LTP, showing once again a sex-specific response to microbiome signals. Finally, we returned to the GF mouse model, examining four putative critical windows in early life, to establish how the microbiota changes the hippocampal transcriptome and metabolome. Metabolomic analysis revealed post-natal day (P) 21 to be the most differentially regulated in GF mice regardless of sex. GF status also significantly altered the hippocampal transcriptome with effects culminating at the oldest examined timepoint, P21. This effect was mirrored in a targeted enrichment analysis of pathways relating to synaptic plasticity. Unlike the metabolomic analysis however, transcriptomic analysis revealed a strong interaction of microbiome and sex with females showing the greatest differences despite a similarly altered metabolome. Overall, the results of this thesis provide novel insights into how the microbiome may alter hippocampal plasticity in adulthood and early life, with microbiome-induced effects often emerging in a sex-specific manner.Item Unravelling the role of the microbiota-gut-brain axis in irritable bowel syndrome and its psychiatric comorbidities(University College Cork, 2024) Wilmes, Lars; Clarke, Gerard; Cryan, John; Irish Research CouncilIrritable bowel syndrome is a stress-related disorder of gut-brain interaction, characterised by altered bowel movements and visceral hypersensitivity, with a profound impact on quality of life. These symptoms occur in the absence of structural abnormalities or validated biomarkers. While the treatment options under consideration for irritable bowel syndrome have expanded, a significant proportion of patients fail to respond to conventional pharmacological interventions. This challenge is compounded by a subpopulation of patients with psychiatric comorbidities, including anxiety and depression, who tend to exhibit increased symptom severity and psychiatric difficulties that are harder to treat. The microbiota-gut-brain axis has emerged as a promising target for intervention, as supported by both preclinical and clinical studies. However, it remains unclear whether irritable bowel syndrome patients with psychiatric comorbidities possess distinct gut microbiota profiles compared to those without such comorbidities, potentially leading to different outcomes and requiring more precise treatment options. Using a well-validated animal model, we initially aimed to determine whether distinct gut microbiota profiles are causally linked to irritable bowel syndrome phenotypes with or without psychiatric comorbidities. In our preclinical studies, we showed that rats exposed to maternal separation could be clustered into subgroups, each representing different phenotypes: one group exhibited primarily visceral hypersensitivity, another displayed negative valence behaviour, a third had a comorbid phenotype, while a fourth group was resilient. Importantly, these subgroups showed distinct gut microbiota configurations corresponding to their specific phenotypes. Following this discovery, we conducted faecal microbiota transplants from these clusters into naïve rats, providing direct evidence that the microbiota plays a causal role in driving aspects of these phenotypes. To assess the clinical relevance of these findings, we isolated gut microbiota from three human populations: healthy individuals, irritable bowel syndrome patients, and irritable bowel syndrome patients with psychiatric comorbidities. After analysing the gut microbiota profiles, we transplanted these bacteria into naïve animals. Interestingly, while the microbiota from both IBS groups increased visceral hypersensitivity, only minor behavioural changes related to psychiatric disorders were observed in the recipient animals. Finally, we tested a novel next-generation psychobiotic, identified from patients with depression, in an animal model. Specifically, we investigated whether its psychobiotic properties exerted disease-specific effects and whether its efficacy surpassed that of traditional probiotics. However, neither the novel psychobiotic nor the traditionally selected probiotic significantly impacted gastrointestinal physiology or behaviour, highlighting the continuing challenges in translating psychobiotics between bench and bedside. Our findings underscore the critical role of the gut microbiota in driving distinct behavioural phenotypes and the potential in stratification of IBS subgroups based on the presence of psychiatric comorbidities. While the translation of these preclinical findings into effective treatments remains challenging, this suggests that targeted gut microbiota interventions may offer more precise cluster-specific therapeutic benefits. This may ultimately pave the way for more nuanced approaches in clinical IBS management where this is currently a large unmet medical need.Item Unravelling the role of the gut microbiota in shaping neurodevelopment and myelination during critical windows of early-life(University College Cork, 2024) Lynch, Caoimhe M. K.; Cryan, John; Clarke, GerardThere is a growing appreciation that the bidirectional communication between our gut microbes and brain, known as the microbiota-gut-brain axis, plays a crucial role in shaping neural development and function, particularly during critical windows of early life. Myelination, the process in which axons are wrapped in a lipid-rich membrane, is essential for efficient neural communication and cognitive function. Recently, research has begun to unravel the dynamic nature of myelin plasticity and its responsiveness to environmental factors, including the gut microbiota. Most of the research to date has employed proof-of-principle experiments using germ-free (GF) rodents to understand the impact of microbial signals on brain health and behaviour. However, recent investigations have begun to leverage simpler model organisms to interrogate microbiota-gut-brain interactions and improve the translational potential of findings across different model species. Deepening our understanding of how microbes regulate key neurodevelopmental processes during sensitive periods will enhance our knowledge of perturbed microbiota-gut-brain axis signalling in disorders linked to altered myelin plasticity. This thesis investigates the complex relationship between the microbiota-gut-brain axis and myelination, aiming to understand how disruptions or changes in this axis during key developmental periods affect myelin formation and related neurological outcomes. To this end, we adopted three models of perturbed microbiota-gut-brain axis signalling: (1) targeted ABX-induced microbiota depletion, (2) GF rodents and (3) GF zebrafish to characterise the mechanisms underlying microbial regulation and the neuroactive potential of microbial metabolites during early life. We demonstrate that targeted microbiota disruption during critical windows of early life has enduring effects on the structure and function of the gut microbiota. These changes were associated with sex- and time-dependent shifts in circulating immune cells, myelin-related gene expression in the prefrontal cortex, and microglial morphology in the basolateral amygdala. Moreover, females treated with antibiotics before weaning exhibited altered anxiety-like behaviours later in life, suggesting that alterations in key neuromodulators may mediate these effects. To further explore the significance of early-life priming for myelin formation, we adopted a cross-species approach using GF mice and zebrafish. We found that the microbiota is essential for neuronal activity and myelination in a sex- and time-dependent manner. Multi-omics analysis revealed that changes in myelin-related transcriptomic profiles were linked to functional shifts in metabolites related to neurotransmission and cellular metabolism. Disruption of myelin and activity-related pathways occurred alongside the maturation of the prefrontal cortex, resulting in subtle variations in myelin growth during development. Similarly, GF zebrafish exhibited changes in myelin, microglia, and activity-related gene expression. Additionally, our findings show that the microbiota influences microglia maturation across species, including the dynamic regulation of microglia-oligodendrocyte contacts in the spinal cord and microglia infiltration in the brain. Lastly, we demonstrate the neuroactive potential of microbial metabolites on glial and myelin-related gene expression. Moreover, behavioural assessments show that the microbiota did not significantly alter anxiety-like behaviour at 5 days post fertilisation (dpf), though microbially derived metabolites produced subtle effects. Additionally, we show for the first time that the microbiota influences the startle response, where GF larvae exhibit hyperactivity, suggesting impaired habituation to stimuli, which can only be rescued by specific microbial metabolites. Overall, this thesis offers compelling evidence regarding the potential mechanisms by which microbes regulate myelination across species. These findings expand on existing research and enhance the translational potential of microbial regulation on myelination and related behaviours. This work also identifies new possibilities for therapeutic interventions that target the microbiota-gut-brain axis to promote healthy myelination and address disorders linked to myelin disruption.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.