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

Area of Science:

• Neuroscience
• Gastroenterology
• Anatomy

Background:

• Accurate identification of enteric nervous system (ENS) neuronal subtypes is crucial for understanding gastrointestinal (GI) function.
• Varied reports on subtype marker expression may stem from differing ENS wholemount dissection techniques.
• This study investigates dissection method variability in quantifying ENS marker expression.

Purpose of the Study:

• To compare the impact of different enteric nervous system (ENS) wholemount dissection methods on neuronal subtype marker quantification.
• To determine if specific microdissection and fixation orders affect marker expression analysis.
• To identify potential sources of variability in studying the myenteric plexus.

Main Methods:

• Compared three common ENS wholemount dissection techniques: two flat-sheet preparations and one rod-mounted peeling method.
• Evaluated variations including reversed orientation flat-sheet peeling and step-by-step rod peeling.
• Assessed marker expression using immunohistochemistry, genetic reporters, confocal microscopy, and automated image analysis.

Main Results:

• No significant difference in calretinin or nNOS expression proportion between two flat-sheet methods in the ileum.
• Rod-mounted peeling significantly decreased the proportion of calretinin and nNOS labeled neurons.
• Rod-mounted peeling also reduced tdTomato (Substance P) and ChAT labeling in the distal colon and ileum.

Conclusions:

• The rod-mounted mechanical peeling method reduces labeling for several ENS neuronal markers, including native proteins and transgenic reporters.
• Mechanical manipulation during rod peeling appears to be the cause of decreased labeling.
• Significant variability exists in wholemount muscularis dissection methods, impacting the accurate quantification of ENS neuronal subtypes.