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

Area of Science:

• Neuroscience
• Gastroenterology

Background:

• Accurate identification of enteric nervous system (ENS) neuronal subtypes is crucial for understanding gastrointestinal (GI) function.
• Variability in reported subtype proportions may stem from different wholemount dissection techniques.
• This study investigates how GI dissection methods influence the quantification of ENS neuronal subtype marker expression.

Approach:

• Compared three common ENS wholemount dissection methods: two flat-sheet preparations and one rod-mounted peeling technique.
• Utilized immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis to assess marker expression.
• Evaluated labeling of calretinin, nNOS, SST, substance P, and ChAT in myenteric plexus preparations.

Key Points:

• No significant differences in calretinin, nNOS, or SST labeling were observed between the two flat-sheet preparation methods in the ileum.
• The rod-mounted peeling method significantly decreased labeling for calretinin and nNOS.
• This mechanical method also reduced fluorescent protein reporter signals for substance P and choline acetyltransferase.

Conclusions:

• The rod-mounted peeling technique for ENS wholemount muscularis preparations can introduce variability by decreasing the labeling of specific neuronal markers.
• Discrepancies in ENS subtype quantification may arise from differences in dissection methodology.
• Standardizing wholemount dissection techniques is essential for reliable reporting of ENS neuronal populations.