Cannabis use may occur during pregnancy to alleviate symptoms of nausea, anxiety, and stress. However, fetal exposure to cannabinoids, such as THC and CBD, poses potential risks to neurodevelopment and long-term health outcomes. The endocannabinoid system (ECS) is involved in neurodevelopment and gastrointestinal (GI) homeostasis and responds to exogenous cannabinoids. This study aimed to investigate both the baseline development of the ECS in the GI tract and the effects of prenatal cannabis exposure on the development of gut-brain axis components and the ECS. Samples from outbred mice were collected at a set of developmentally important time points, ranging from embryonic day 14 to postnatal day 21. The localization of cannabinoid receptor 1 (CB1) expression was examined in the fetal and postnatal small intestine, and mRNA analysis was performed to evaluate changes in gene expression. Under normal conditions, components of the ECS display developmental regulation in the GI tract. A subset of pregnant mice was exposed to cannabis smoke daily, using a strain of cannabis containing high concentrations of THC, simulating ‘real world’ cannabis use. Exposure to cannabis prenatally revealed significant changes in biometric outcomes, and in the transcription of genes associated with the ECS, ENS development, barrier function, and serotonin signaling pathways. In addition, the onset of ambulation was delayed. These findings provide valuable insights into the developmental regulation of ECS during the prenatal period and highlight the potential risks associated with prenatal cannabis exposure, particularly in terms of gut-brain axis development. Further research is required to better understand the underlying mechanisms. / Thesis / Master of Science (MSc) / There have been growing reports of cannabis consumption during pregnancy, to relieve nausea, anxiety, and stress. However, compounds in cannabis, like THC and CBD, can pose risks to the baby's development and long-term health. This project studied how cannabis use during pregnancy affects gastrointestinal (GI) development, and communication between the GI system and the brain. The endocannabinoid system (ECS) plays a role in regulating the normal functioning of the GI tract and is influenced by cannabis compounds. By exposing pregnant mice to cannabis smoke, we observed significant changes in gene expression in the fetal intestine relating to the GI nervous system, the strength of the gut barrier, and various molecular signalling pathways. In addition, prenatally exposed mice had delayed walking ability. These findings highlight the potential risks of cannabis use during pregnancy on gut-brain axis development. Further research is needed to understand the mechanisms causing these disruptions.

Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter of the central and peripheral nervous systems, predominantly secreted in the gastrointestinal tract, especially in the gut. 5-HT is a crucial enteric signaling molecule and is well known for playing a key role in sensory-motor and secretory functions in the gut. Gastroenteropathy is one of the most clinical problems in diabetic patients with frequent episodes of hyperglycemia. Changes in 5-HT expression may mediate gastrointestinal tract disturbances seen in diabetes, such as nausea and diarrhea. Based on the double immunohistochemical staining, this study determined the variability in the population of 5-HT-positive neurons in the porcine small intestinal enteric neurons in the course of streptozotocininduced diabetes. The results show changes in the number of 5-HT-positive neurons in the examined intestinal sections. The greatest changes were observed in the jejunum, particularly within the myenteric plexus. In the ileum, both de novo 5-HT synthesis in the inner submucosal plexus neurons and an increase in the number of neurons in the outer submucosal plexus were noted. The changes observed in the duodenum were also increasing in nature. The results of the current study confirm the previous observations concerning the involvement of 5-HT in inflammatory processes, and an increase in the number of 5-HT -positive neurons may also be a result of increased concentration of the 5-HT in the gastrointestinal tract wall and affects the motor and secretory processes, which are particularly intense in the small intestines.

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Diabetic gastroenteropathy is a common complication, which develops in patients with long-term diabetes. The pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known for its cytoprotective properties and plays an important role in neuronal development, neuromodulation and neuroprotection. The present study was designed to elucidate, for the first time, the impact of prolonged hyperglycaemia conditions on a population of PACAP-like immunoreactive neurons in selected parts of the porcine gastrointestinal tract. The experiment was conducted on 10 juvenile female pigs assigned to two experimental groups: The DM group (pigs with streptozocin-induced diabetes) and the C group (control pigs). Diabetes conditions were induced by a single intravenous injection of streptozocin. Six weeks after the induction of diabetes, all animals were euthanised and further collected, and fixed fragments of the stomach, duodenum, jejunum, ileum and descending colon were processed using the routine double-labelling immunofluorescence technique. Streptozotocin-induced hyperglycaemia caused a significant increase in the population of PACAP-containing enteric neurons in the porcine stomach, small intestines and descending colon. The recorded changes may result from the direct toxic effect of hyperglycaemia on the ENS neurons, oxidative stress or inflammatory conditions accompanying hyperglycaemia and suggest that PACAP is involved in regulatory processes of the GIT function in the course of diabetes.

<p>Commensal intestinal microbiota number in the realm of 10¹⁴organisms per gram of colonic contents. This considerable bacterial load is acquired during birth and in the early postnatal days and has a defining, extensive impact on host physiology. We now have persuasive evidence that the intestinal microbiota influence the development of the nervous system. The following body of work describes alterations in the nervous system of germ free mice – mice bred and maintained with no exposure to bacteria of any kind. Here we examine diverse measures of neural activity, ranging from stress reactivity and stress-associated behaviours, to changes in neurochemistry of brain regions mutually involved in feeding and stress, to electrophysiological measures of sensory cells in the enteric nervous system. We see that in the absence of colonizing microbiota that neural activity is considerably altered both peripherally and centrally. Specifically, germ free mice exhibit a reduction in basal anxiety-like behaviour accompanied by consistent changes in mRNA gene expression of plasticity-related genes in brain tissue, lifelong reduction in circulating plasma leptin, increases in mRNA gene expression of hypothalamic leptin receptors and neuropeptide Y, and decreased excitability in sensory neurons in the myenteric plexus of the enteric nervous system. Furthermore, while it appears that central systems responsible for stress may have an early critical window for bacterial-induced change, it would seem that the peripheral enteric nervous system retains plasticity into adulthood. This novel work provides insight into the microbial-gut-brain axis and suggests potential avenues for therapies aimed at treating the frequently comorbid gastrointestinal and psychiatric illnesses.</p> / Doctor of Philosophy (Medical Science)

The enteric nervous system (ENS) is an incredibly complex neural network that is extensively integrated within the neuroimmunoendocrine system through countless signalling pathways that have yet to be fully characterized. In the last decade we have discovered that many more neurotransmitters are at work in the ENS than was originally thought. This opens up new avenues of research into physiological phenomena traditionally thought to be associated only with the central nervous system, such as NMDA receptor-induced excitotoxicity, and how these may influence immune interactions. In particular, the kynurenine pathway of the tryptophan catabolism produces many neuro-active and immuno-active constituents whose effects are unknown in the ENS but are of great consequence in many neurodegenerative disorders of the CNS. Our study hypothesized that co-culture of the enteric neurons with mast cells would increase neuronal survival through kynurenic acid production in quinolinic acid (QUIN)-induced excitotoxic conditions. This study developed a novel in vitro co-culture system of enteric neurons and glia grown from murine longitudinal muscle-myenteric plexus tissue and bone marrow-derived mast cells. In addition, a pipeline in image analysis software CellProfiler was designed and optimized in order to reduce human bias and error in subsequent immunocytochemical image analysis. Furthermore, we identified the genetic expression of subunits of the NMDA glutamate receptor in cultured enteric neurons via PCR, which suggests that these cultured neurons may be susceptible to excitotoxicity. PCR analysis of cultured mast cells seemed to indicate that our cultured mast cells do not express KAT-III, the enzyme needed to produce the neuroprotective KYNA. Overall, co-culture with mast cells seemed to decrease neuronal survival. This project developed a novel methodology for the in vivo study of mast cell-nerve interactions, and lays the groundwork for future studies in excitotoxicity in the ENS. / Thesis / Master of Science (MSc) / The enteric nervous system is a vast web of nerves and immune cells that innervates the gut and interacts with the central nervous system through the gut-brain axis. An important mediator in this system is the mast cell, a type of immune cell often involved in protective responses to venoms and allergens. Intriguingly, in normal physiological conditions these cells are in close contact with nerves in the periphery, despite their potential to release damaging constituents. While mast cells are well-known for inciting inflammation and releasing toxic granules, they can also synthesize and release potentially beneficial neuroactive compounds, such as neurotransmitters or growth factors. The aim of this study was to characterize mast cell-nerve interactions in neurotoxic conditions, to see if the proximity of mast cells to nerves might serve a neuroprotective purpose.

10-15% of women take antidepressants during pregnancy. Selective serotonin reuptake inhibitors (SSRIs) are most commonly used for perinatal depression. Perinatal exposure to SSRIs has been shown to disrupt the development of serotonergic signaling pathways in the central nervous system (CNS); however, the effects on the developing enteric nervous system (ENS) remain relatively unexplored. We hypothesized that early life exposure to SSRIs would influence the structural development of the gastrointestinal (GI) tract. We further hypothesized that these structural changes could lead to clinically relevant functional outcomes, such as modifications in susceptibility to inflammation and altered GI motility. Female Wistar rats were given the SSRI, fluoxetine, or vehicle from 2 weeks prior to mating through gestation until weaning. At postnatal day 1 (P1), postnatal day 21 (P21; weaning) and 6 months of age (P6 months) intestines were harvested to assess for structural changes. At P6M, intestines were collected to assess motility in vitro and subsets of the offspring were treated with dextran sulfate sodium (DSS) to assess susceptibility to colitis. At P1, there was a significant decrease in serotonergic neurons in the female colon. At P21, there was a significant increase in serotonergic neurons of both sexes in the colon. At P6M, there was a significant increase in the frequency and velocity of long-distance contractions in the colon when both sexes were combined and an increase in ZO-1 in male colon. In conclusion, SSRI exposure in utero appears to have structural and functional consequences on the developing ENS in the SSRI exposed offspring. The structural consequences are seen in both sexes at P21 and although the structural changes to the ENS resolve by 6 months, motility in the colon continues to be significantly altered. There were no significant differences in chemical colitis, however, we did see difference of quantitative mRNA cytokines, chemokines and extracellular matrix components which may suggest differences in mucosal immune response. The mechanisms by which these changes occur remain to be explored. / Thesis / Master of Science (MSc)

The duodenal mucosa is regularly exposed to acid, digestive enzymes and ingested noxious agents. It is thus critical to maintain a protective barrier to prevent the development of mucosal injury and inflammation, which are often observed in situations when barrier function is impaired. The rate of mucosal bicarbonate secretion, the regulation of epithelial paracellular permeability and motility are each key components of duodenal barrier function. The hormone melatonin is present in high levels in the gastrointestinal tract and it has been hypothesized that melatonin exerts protective properties. This thesis aims to investigate the impact of exogenous melatonin on the regulation of duodenal barrier function and motility in anesthetized rats in vivo. In addition, duodenal tissue was examined histologically and the expression levels of tight junction proteins and melatonin receptors were assessed with qRT-PCR. It was found that melatonin stimulated mucosal bicarbonate secretion and decreased basal paracellular permeability. Exposing the duodenal mucosa to the well-characterized barrier breaker ethanol increased mucosal bicarbonate secretion, paracellular permeability and motility. Omission of luminal Clˉ abolished, while pretreatment with a nicotinic receptor antagonist reduced, the ethanol-induced bicarbonate secretion suggesting that the secretory response to ethanol is meditated via Clˉ/HCO3ˉexchangers and enteric neural pathways. Melatonin reduced the ethanol-induced increases in paracellular permeability and motility either when injected intravenously or when administered in drinking water for two weeks. The actions of melatonin were abolished by the melatonin receptor antagonist luzindole and by nicotinic acetylcholine receptor inhibition. Two weeks oral administration of melatonin up-regulated the expression levels of melatonin receptors, down-regulated the expression of ZO-3 while the expression of ZO-1, ZO-2, claudin 2-4, occludin and myosin light chain kinase were unaffected. Superficial epithelial changes in a few villi were seen in response to ethanol exposure, an effect that was histologically unchanged by melatonin pretreatment. In conclusion, the results suggest that melatonin plays an important role in the neurohumoral regulation of gastrointestinal mucosal barrier function and motility via receptor- and enteric neural-dependent pathways in vivo in rats. Melatonin might be a candidate for treatment of barrier dysfunction in humans.

More than 1.2 million people are estimated to be currently living with the human immunodeficiency virus (HIV) in the United States of America. The gastrointestinal (GI) tract is both a major target and an important component of HIV pathogenesis. The GI processes that are dysregulated during HIV infection are controlled by the enteric nervous system (ENS). Indeed, both clinical and experimental studies have implicated the ENS in HIV and simian immunodeficiency virus (SIV) pathogenesis. In addition to direct viral effects, the HIV virus also indirectly affects the GI tract via cellular and/or viral toxins released by infected cells. Trans-activator of transcription (Tat) is a viral toxin that plays an important role in replication of the HIV virus. While, the HIV virus does not directly infect neurons, Tat has been shown to modulate neuronal function. HIV infection in the gut is accompanied by: translocation of bacteria and bacterial products from the gut lumen to peripheral blood, immune activation and inflammation. Lipopolysaccharide (LPS) is a major bacterial product that is used to determine the rate of bacterial translocation and to drive inflammation. Despite reports of enteric ganglionitis in SIV infected monkeys and autonomic denervation in the jejunum of HIV patients, little is known of the mechanism underlying enteric neuropathogenesis in HIV and the role of the ENS in HIV pathogenesis. In the present study, we assessed the effects of Tat on enteric neuronal excitability and how Tat and LPS interact in the ENS to bring about inflammation and GI motility problems observed in HIV patients. We show that Tat significantly increased enteric neuronal excitability by modulating sodium channels expressed on enteric neurons. Tat sensitized ENS cells to LPS-mediated increase in pro-inflammatory cytokines via a TLR4-mediated pathway involving MyD88. Mice expressing the tat transgene (Tat+) had faster GI transit rates and significantly higher frequencies of diameter changes in the proximal ileum than controls (Tat-). Tat+ mice were also more sensitive to LPS-mediated decreases in colonic transit rate. This study highlights the role of viral and bacterial proteins in HIV pathogenesis in the gastrointestinal tract and also demonstrates a critical role of the ENS in HIV pathogenesis.