Regional cytoarchitecture of the adult and developing mouse enteric nervous system

Hamnett, Ryan; Dershowitz, Lori Bowe; Sampathkumar, Vandana; Wang, Ziyue; Gomez-Frittelli, Julieta; Andrade, Vincent De; Kasthuri, Narayanan; Druckmann, Shaul; Kaltschmidt, Julia A.

doi:10.1016/j.cub.2022.08.030

Cited by 22 publications

(53 citation statements)

References 68 publications

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“…Retrieval of Ileum and distal colon segments was performed as in (Hamnett, Dershowitz et al ., 2022) 13 to the point of removing the mesentery from the intestines. The dissection then proceeded with Method 1, Method 2, or Method 3 , described below.…”

Section: Methodsmentioning

confidence: 99%

“…Immunohistochemistry was performed as described previously 13 . All wash steps were at least 10 minutes each.…”

Section: Methodsmentioning

confidence: 99%

“…Images were acquired as described previously 13 . Z-stacks were acquired with 2.5-3 μm between each focal plane.…”

Section: Methodsmentioning

confidence: 99%

“…Image analysis was performed using ImageJ/FIJI (NIH, Bethesda, MD), as described previously 13 . SST was counted manually after being combined with the thresholded HuC/D image stack and identified by its distinctive cytoplasmic expression pattern.…”

Section: Methodsmentioning

confidence: 99%

“…Reports of the proportion of specific subtypes or marker expression in the ENS have sometimes varied, such as reports of 6-23% of small intestine myenteric neurons expressing enkephalin 3,10 . Variability such as this can result from animal model 3 , background 11 , age 12 , or GI tract region analyzed 13 .…”

Section: Introductionmentioning

confidence: 99%

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Comparison of wholemount dissection methods for neuronal subtype marker expression in the mouse myenteric plexus

Gomez-Frittelli

Hamnett

Kaltschmidt

2023

Preprint

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Background: Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI tract function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression. Methods: We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation as well as a rod-mounted peeling technique. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis. Key Results and Conclusions: We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin, neuronal nitric oxide synthase (nNOS), or somatostatin (SST) in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased marker labeling for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in ileum and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling, demonstrating a critical variability in wholemount muscularis dissection methods.

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Section: Methodsmentioning

confidence: 99%

“…Immunohistochemistry was performed as described previously 13 . All wash steps were at least 10 minutes each.…”

Section: Methodsmentioning

confidence: 99%

“…Images were acquired as described previously 13 . Z-stacks were acquired with 2.5-3 μm between each focal plane.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%