Neurochemical coding compared between varicose axons and cell bodies of myenteric neurons in the guinea-pig ileum

This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/.The discrete functional classes of enteric neurons in the mammalian gastrointestinal tract have been successfully distinguished on the basis of the unique combination of molecules and enzymes in their cell bodies (“chemical coding”). Whether the same chemical coding exists in varicose axons of different functional classes has not been systematically tested. In this study, we quantified the coexistence of markers that define classes of nerve cell bodies in the myenteric plexus of the guinea-pig ileum, in varicose axons of the same neurons. Profound differences between the combinations of immunohistochemical markers in myenteric nerve cell bodies and in their varicosities were identified. These discrepancies were particularly notable for classes of neurons that had previously been classified as cholinergic, based on immunoreactivity for choline acetyltransferase (ChAT) in their cell bodies. To detect cholinergic varicose axons of enteric neurons in this study, we used antiserum against the vesicular acetylcholine transporter (VAChT). ChAT-immunoreactivity has been reported to be consistently co-localized with 5-hydroxytryptamine (5-HT) in interneuronal cell bodies, yet only 29 ± 5% (n = 4) of 5-HT-immunoreactive varicosities contained vesicular acetylcholine transporter (VAChT). Somatostatin coexists with ChAT-immunoreactivity in a class of descending interneuron but only 21 ± 1% (n = 4) of somatostatin-immunoreactive varicosities were VAChT-immunoreactive. Comparable discrepancies were also noted for non-cholinergic markers. The results suggest that chemical coding of cell bodies does not necessarily reflect chemical coding of varicose axon terminals and that the assumption that nerve cell bodies that contain ChAT are functionally cholinergic may be questionable.Australian National Health & Medical Research Counci

Selective co-expression of synaptic proteins, α-synuclein, cysteine string protein-α, synaptophysin, synaptotagmin-1, and synaptobrevin-2 in vesicular acetylcholine transporter-immunoreactive axons in the guinea-pig ileum

Author version made available in accordance with publisher's policy.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 α-synuclein 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-α (CSPα), 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% ± 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% ± 1.3%, n = 4) compared with any of the other nine populations (P < 0.001). Among synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPα-IR varicosities, 98% ± 0.7%, 96% ± 0.7%, 88% ± 1.6%, and 85% ± 2.9% (n = 4) contained α-synuclein immunoreactivity, respectively. Among α-synuclein-IR varicosities, 96% ± 0.9%, 99% ± 0.6%, 83% ± 1.9%, and 87% ± 2.3% (n = 4) contained synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPα 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.Australian National Health & Medical Research Counci

Sensory innervation of the guinea pig colon and rectum compared using retrograde tracing and immunohistochemistry.

Embargoed until 2 April 2017 as per publisher's policy

Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon

"This is the peer reviewed version of the following article: [Chen, B. N., Sharrad, D. F., Hibberd, T. J., Zagorodnyuk, V. P., Costa, M. and Brookes, S. J.H. (2015), Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon. J. Comp. Neurol., 523: 742–756. doi: 10.1002/cne.23704], which has been published in final form at [http://dx.doi.org/10.1002/cne.23704]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. http://olabout.wiley.com/WileyCDA/Section/id-820227.html#terms"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 flow. The distal colon is innervated by extrinsic sympathetic and parasympathetic efferent and spinal afferent neurons, via axons in colonic nerve trunks. In the present study, biotinamide tracing of colonic nerves was combined with immunohistochemical labeling for markers of sympathetic, parasympathetic and spinal afferent neurons to quantify their relative contribution to the extrinsic innervation. Calcitonin gene-related peptide, vesicular acetylcholine transporter and tyrosine hydroxylase, which selectively label spinal afferent, parasympathetic and sympathetic axons, respectively, were detected immunohistochemically in 1 ± 0.5% (n = 7), 15 ± 4.7% (n = 6) and 24 ± 4% (n = 7) of biotinamide-labeled extrinsic axons in myenteric ganglia. Immunoreactivity for vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, vesicular glutamate transporters 1 and 2 accounted for a combined maximum of 14% of biotinamide-labeled axons in myenteric ganglia. Thus, a maximum of 53% of biotinamide-labeled extrinsic axons in myenteric ganglia were labeled by antisera to one of these eight markers. Viscerofugal neurons were also labeled by biotinamide, and shown to have distinct morphologies and spatial distributions that correlated closely with their immunoreactivity for nitric oxide synthase and choline acetyltransferase. As reported for the rectum, nearly half of all extrinsic nerve fibers to the distal colon lack the key immunohistochemical markers commonly used for their identification. Their abundance may therefore have been significantly underestimated in previous immunohistochemical studies

Rotenone and elevated extracellular potassium concentration induce cell-specific fibrillation of α-synuclein in axons of cholinergic enteric neurons in the guinea-pig ileum.

Under embargo until 7 Nov 2017, as per publisher policy.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 (1), 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