Synaptic mechanisms and function in the mouse enteric nervous system
Document TypePhD thesis
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© 2018 Dr. Mathusi Swaminathan
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.
KeywordsEnteric nervous system; Synaptic transmission; Synaptic proteins; Glutamate; alpha-Synuclein
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