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Extrinsic nerves to the gut influence the absorption of water and electrolytes and expulsion of waste contents, largely via regulation of enteric neural circuits; they also contribute to the control of blood 45

This is the peer reviewed version of the following article:Sharrad DF, Gai WP, Brookes SJ. Selective coexpression of synaptic proteins, α-synuclein, cysteine string protein-α, synaptophysin, synaptotagmin-1, and synaptobrevin-2 in vesicular acetylcholine transporterimmunoreactive axons in the guinea pig ileum. J Comp Neurol. 2013 Aug 1;521(11):2523-37.which has been published in final form at doi:10.1002/cne.23296.This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for SelfArchiving. Keywords synaptic transmission; neurodegeneration; Parkinson's disease; enteric nervous system ABSTRACT Parkinson's disease is a neurodegenerative disorder characterized by Lewy bodies and neurites composed mainly of the presynaptic protein α-Synuclein. Frequently, Lewy bodies and neurites are identified in the gut of Parkinson's disease patients and may underlie associated gastrointestinal dysfunctions. We recently reported selective expression of α-Synuclein in the axons of cholinergic neurons in the guinea pig and human distal gut; however, it is not clear whether asynuclein expression varies along the gut, nor how closely expression is associated with other synaptic proteins. We used multiple-labeling immunohistochemistry to quantify which neurons in the guinea pig ileum expressed α-Synuclein, cysteine string protein-a (CSPa), synaptophysin, synaptotagmin-1, or synaptobrevin-2 in their axons. Among the 10 neurochemically defined axonal populations, a significantly greater proportion of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) varicosities (80% 6 1.7%, n . 4, P < 0.001) contained α-Synuclein immunoreactivity, and a significantly greater proportion of α-Synuclein-IR axons also contained VAChT immunoreactivity (78% 6 1.3%, n . 4) compared with any of the other nine populations (P < 0.001). Among synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPa-IR varicosities, 98% 6 0.7%, 96% 6 0.7%, 88% 6 1.6%, and 85% 6 2.9% (n . 4) contained α-Synuclein immunoreactivity, respectively. Among α-Synuclein-IR varicosities, 96% 6 0.9%, 99% 6 0.6%, 83% 6 1.9%, and 87% 6 2.3% (n . 4) contained synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPa immunoreactivity, respectively. We report a close association between the expression of α-Synuclein and the expression of other synaptic proteins in cholinergic axons in the guinea pig ileum. Selective expression of α-Synuclein may relate to the neurotransmitter system utilized and predispose cholinergic enteric neurons to degeneration in Parkinson's disease. Page 1

Background Parkinson's disease is a progressive neurodegenerative disorder that results in the widespread loss of select classes of neurons throughout the nervous system. The pathological hallmarks of Parkinson's disease are Lewy bodies and neurites, of which α‐synuclein fibrils are the major component. α‐Synuclein aggregation has been reported in the gut of Parkinson's disease patients, even up to a decade before motor symptoms, and similar observations have been made in animal models of disease. However, unlike the central nervous system, the nature of α‐synuclein species that form these aggregates and the classes of neurons affected in the gut are unclear. We have previously reported selective expression of α‐synuclein in cholinergic neurons in the gut (J Comp Neurol. 2013; 521:657), suggesting they may be particularly vulnerable to degeneration in Parkinson's disease. Methods In this study, we used immunohistochemistry to detect α‐synuclein oligomers and fibrils via conformation‐specific antibodies after rotenone treatment or prolonged exposure to high [K+] in ex vivo segments of guinea‐pig ileum maintained in organotypic culture. Key Results Rotenone and prolonged raising of [K+] caused accumulation of α‐synuclein fibrils in the axons of cholinergic enteric neurons. This took place in a time‐ and, in the case of rotenone, concentration‐dependent manner. Rotenone also caused selective necrosis, indicated by increased cellular autofluorescence, of cholinergic enteric neurons, labeled by ChAT‐immunoreactivity, also in a concentration‐dependent manner. Conclusions & Inferences To our knowledge, this is the first report of rotenone causing selective loss of a neurochemical class in the enteric nervous system. Cholinergic enteric neurons may be particularly susceptible to Lewy pathology and degeneration in Parkinson's disease.

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Running Title:Extrinsic sensory nerves to rectum and colon Abbreviations:DiI; DiIC12(3) (1,1'-Didodecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) or a 1:1 mixture of DiIC12 (3)