Physiology - Theses
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ItemInteractions of the enteric nervous system with the gut microbiome in the neuroligin-3 R451C mouse model of autism( 2020)Autism patients are four times more likely to be hospitalized due to gastrointestinal (GI) dysfunction compared to the general public. However, the exact cause of GI dysfunction in individuals with autism is currently unknown. Genetic predisposition to autism spectrum disorder (ASD) has been highlighted in various studies and mutations in genes that affect nervous system function can drive both behavioural abnormalities and GI dysfunction in autism. Neuroligin-3 (NLGN3) is a postsynaptic membrane protein and the R451C missense mutation in the NLGN3 gene is associated with ASD. Recent studies revealed that the NLGN3 R451C mutation induces GI dysfunction in autism patients as well as in mice but, the cellular localization and the effects of this mutation on NLGN3 production in the enteric nervous system (ENS) have not been reported to date. The intestinal mucosal barrier is the interface separating the external environment from the interior of the body. Mucosal barrier functions are directly regulated by the enteric nervous system. Therefore, ENS dysfunction can induce mucosal barrier impairments. An impaired intestinal barrier has been reported in autism patients, but neurally-mediated barrier dysfunctions have not been assessed in transgenic autism mouse models with an altered nervous system. The intestinal mucus layer is the outermost layer of the mucosa which separates the intestinal microbiota from the intestinal epithelium. The mucus layer also serves as an energy source for mucus-residing microbes in the intestine. Although the composition of mucus-residing microbiota is altered in a subset of autism patients, the underlying physiological interactions between the host and these microbes are unclear. Identifying the precise spatial location of microbial populations in the gut is essential in order to understand host-microbial interactions but this has not been investigated in Nlgn3R451C mice. In Chapter 3, I developed a method combining RNAScope in situ hybridization and immunohistochemistry technique to localize Nlgn3 mRNA in enteric neuronal subpopulations and glia. Further, a 3-dimensional quantitative image analysis method was developed to measure the Nlgn3 mRNA expression in the ENS. The same protocol was used to determine the effects of the Nlgn3 R451C mutation on Nlgn3 mRNA synthesis in the enteric nervous system. Findings from this study showed that, Nlgn3 mRNA is expressed in most submucosal and myenteric neurons in the ENS. Interestingly, this study revealed for the first time that Nlgn3 mRNA is expressed in enteric glia. In addition, analysis from this study demonstrated that the R451C mutation reduces Nlgn3 mRNA expression in most enteric neurons in mutant mice compared to WT. In Chapter 4, I investigated the effects of the Nlgn3 R451C mutation on intestinal mucosal barrier functions including the paracellular pathway and mucosal secretion in the small intestine. The Ussing chamber technique was used to measure the paracellular permeability and mucosal secretion ex vivo. Since the paracellular pathway is regulated by tight junctions, effects of this mutation on tight junction protein gene expression were measured using real-time (RT) PCR array and droplet digital (dd) PCR approaches. The impact of the Nlgn3 R451C mutation on the neurochemistry of the submucosal plexus was examined using immunocytochemistry. Results from these experiments indicated that the R451C mutation increases paracellular permeability and decreases transepithelial resistance (TER) in the distal ileum. However, ileal tight junction protein gene expression is unchanged in mutant mice compared to WT. Pharmacological stimulation of submucosal ganglia decreased the neurally-evoked mucosal secretion in mutant mice compared to WT. In addition, immunohistochemistry data revealed increased numbers of non-cholinergic and decreased cholinergic neuronal populations in the submucosal plexus in the distal ileum but not in the jejunum in Nlgn3R451C mutant mice. Given that, I identified altered barrier functions in the distal ileum of Nlgn3R451C mutant mice, in chapter 5, I also analysed the spatial distribution of the mucus-residing microbial populations in this region. To determine the spatial distribution of total bacteria, phylum Bacteroidetes, phylum Firmicutes, Akkermansia muciniphila (A. muciniphila) and Bacteroides thetaiotamicron (B. thetaiotamicron) were labelled using fluorescent in situ hybridization. Immunofluorescence for the mucin-2 protein was incorporated to co-stain the mucus and determine the thickness of the mucus layer. Both the spatial pattern of microbial populations and mucus layer thickness were analysed using MATLAB-based BacSpace software. Immunofluorescence experiments revealed that the R451C mutation increases mucus density adjacent to the epithelium. Along with increased mucus density, the total bacterial density was higher in the mucosa in mutant mice. Further, a decreased ratio of Bacteroidetes/Firmicutes, a decreased A. muciniphila density and increased density of B. thetaiotamicron were observed in mutant mice. Overall, findings from this thesis revealed that NLGN3 is expressed in the enteric nervous system and that the R451C mutation reduces Nlgn3 mRNA expression levels in enteric neurons. Furthermore, the Nlgn3 R451C mutation impairs intestinal mucosal barrier integrity. Findings from this study also revealed that this mutation alters mucus density as well as the spatial distribution and composition of the microbial community in the distal ileum in mice. Therefore, these findings highlight that an autism-associated gene mutation that affects nervous system function impairs the mucosal barrier and may contribute to the pathophysiology of GI dysfunction in ASD.
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ItemSynaptic mechanisms and function in the mouse enteric nervous system( 2018)Virtually all functions of the enteric nervous system (ENS) rely on synaptic transmission, which occurs at specialised sites referred to as synapses. Molecular mechanisms behind synaptic transmission at central synapses have been extensively characterised, studies accordingly show that pre- and post-synaptic proteins localized to these synapses regulate transmission. However, little is known about the synaptic machinery involved in regulating excitatory transmission at enteric synapses. There is growing evidence to suggest that patients with synaptic protein associated neurodevelopmental and neurodegenerative diseases, such as Parkinson’s Disease, also display abnormalities in gastrointestinal function. This suggests there is a commonality between the central nervous system (CNS) and the ENS. Excitatory transmission within the ENS is primarily mediated by acetylcholine (ACh) acting on nicotinic receptors, there are also many other putative excitatory neurotransmitters in the system whose roles remain elusive. Therefore, the aim of my PhD thesis was to elucidate molecular and pharmacological mechanisms underlying excitatory transmission in the ENS. In Chapter 2, I localized synaptic vesicle proteins synaptophysin, synaptotagmin-1 and vesicular acetylcholine transporter (vAChT) to enteric varicosities. I developed two high-throughput analysis methodologies to quantify co-expression in varicosities and their close contact with enteric neurons. Using these analysis tools, I found that synaptic vesicle proteins synaptophysin and synaptotagmin-1, described to be ubiquitous in pre-synaptic terminals, are not found in all cholinergic varicosities (vAChT+) in the myenteric plexus. I found that in the submucosal plexus, all cholinergic varicosities contained synaptophysin, but some lacked synaptotagmin-1. This highlights the sensitivity of the analysis tool developed and the disparity in synaptic protein localization at cholinergic varicosities between the two plexuses. Additionally, using 3D rendering I examined close contacts between varicosities expressing synaptophysin and vAChT on neuronal nitric oxide synthase (nNOS+) neurons. I found that nNOS+ neurons receive three distinct classes of input. This includes varicosities that either contain vAChT, synaptophysin or both. Overall, my findings demonstrate that there is molecular heterogeneity in cholinergic varicosities within the ENS, which will likely transpire into distinct modes of cholinergic transmission or ACh release at enteric synapses. Moreover, this study highlights the use of advanced image analysis tools to examine connectivity and mechanisms of transmission within the ENS. In Chapter 3, I described the expression of post-synaptic density protein PSD93 in the ENS using immunohistochemical methods. I found that most myenteric neurons, including subpopulations of cholinergic and nitrergic neurons express PSD93. The wide spread expression of PSD93 in the cytoplasm and axons of enteric neurons indicates that it is an unsuitable marker for identifying excitatory post-synaptic densities in the myenteric plexus. Instead, PSD93 is likely to be involved in other cytosolic processes in addition to any role as a post-synaptic density protein at excitatory synapses. In Chapter 4, I demonstrate importance of α-synuclein (α-Syn) in cholinergic function within the ENS. α-Syn is a synaptic vesicle protein pathologically linked to neurodegenerative diseases. I show that α-Syn is expressed in varicosities and some neuronal somata within the mouse colon, a result described previously in other species. Using the quantitative method described in Chapter 2, I found that most cholinergic varicosities (vAChT+) contained α-Syn. I also investigated the implications of α-Syn deletion for ENS function using α-Syn knock out (KO) mice. α-Syn KO mice have increased proportions of cholinergic neurons in the myenteric plexus. Additionally, cross-sections of mouse colon preparations also show that α-Syn KO mice have increased cholinergic innervation to the circular muscle. Calcium (Ca2+) imaging studies reveal that fast synaptic transmission mediated by nicotinic receptors is increased in α-Syn KO mice. However, I found that α-Syn KO mice have a reduced incidence of spontaneous circular muscle contractility, suggesting that there are changes in the circuitry underlying motor patterns. Collectively, these findings suggest that there are alterations in the enteric neural circuitry of α-Syn KO mice and that α-Syn is important for cholinergic transmission. In Chapter 5, I used Ca2+ imaging and high-resolution microscopy to elucidate the mechanisms behind glutamatergic transmission within the ENS. Thus far there is conflicting evidence to suggest the involvement of ionotropic receptors and metabotropic glutamate receptors (mGluRs) in synaptic transmission. I show that many myenteric varicosities that contain vesicular glutamate transporter 2 (vGluT2) are non-cholinergic and express synaptic vesicle proteins synaptophysin using tools I developed in Chapter 2. Using 3D rendering I showed that calbindin (calb+) neurons receive more vGluT2 varicosities than nNOS+ neurons. Exogenous application of glutamate predominantly excites calb+ neurons in the myenteric plexus. Calb+ neurons also receive slow synaptic transmission mediated by endogenous release of glutamate excited by a train of electrical stimuli. Using ionotropic and group I metabotropic glutamate receptor (mGluR) antagonists, I found that group I mGluRs are involved in mediating slow synaptic transmission. This study demonstrates a role for glutamate in mediating excitability of myenteric calb+ neurons. Overall, I have developed powerful methodologies that will provide valuable tools to contribute to understanding mechanisms underlying excitatory transmission within the ENS. The molecular heterogeneity of cholinergic varicosities identified in this thesis, provides a foundation for elucidating ACh release at enteric synapses. I have also shown that post-synaptic density markers that identify excitatory synapses in the autonomic nervous system (ANS) are unsuitable for labelling excitatory synapses in the ENS. This indicates that mechanisms underlying excitatory transmission could differ between the ANS and ENS. I have highlighted the difficulty in establishing a marker for post-synaptic densities within the ENS, which is necessary for a detailed understanding of excitatory transmission. Moreover, I have shown that α-Syn is associated with cholinergic synapses and the deletion of the synaptic vesicle protein has consequential effects on cholinergic transmission and function, thus implicating α-Syn in gastrointestinal pathophysiology. I have also identified a role for group I mGluRs in mediating excitatory slow synaptic transmission, indicating that glutamate is an excitatory neurotransmitter within the ENS. These findings provide a foundation for future analyses of synaptic function in the ENS and point to key questions for further investigation of this understudied nervous system.