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

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.


Introduction
The enteric nervous system (ENS) is an autonomous network of neurons residing in two mesh-like layers (plexuses) within the wall of the gastrointestinal (GI) tract. The submucosal plexus (SMP) lies just below the mucosa, regulating secretions and local blood flow, while the myenteric plexus (MP) resides between the circular (CM) and longitudinal (LM) muscle layers, controlling gut motility patterns 1 . Distinct regions of the GI tract perform different functions, and we are beginning to understand how this is reflected in motility patterns and the structure of the ENS controlling them 2 , especially by the involvement of many enteric neuronal subtypes [3][4][5] . ENS subtypes have been previously defined by their morphology 6 , electrophysiology 7 , and marker expression 3 .
Recently, multiple single cell RNA sequencing (scRNAseq) studies have also defined enteric neuronal subtypes by RNA expression 4,5,8,9 . Associating these RNA signatures with previously defined ENS? subtypes is of pressing interest to the field.
Comparing results between many of the studies reported on ENS subtypes can be difficult, however. 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 . Tissue processing has also been shown to introduce variability in the quality of enteric marker visualization 14 . Many different GI dissection methods and adaptations thereof have been reported over the years, especially for wholemount preparations of the myenteric plexus (see Table 1). Here, we assessed this potential source of variability in myenteric neuron marker expression in a controlled set of experiments testing (i) the order of microdissection and fixation and (ii) the usage of a rod during the microdissection (see Detailed dissection protocols) across two landmark-defined intestinal regions, ileum and distal colon. We find no significant differences in labeling between two flat sheet methods differing in the order of dissection and fixation steps, but report significantly decreased labeling using the rod peeling method across most markers and regions tested.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2023. ; https://doi.org/10.1101/2023.01.17.524014 doi: bioRxiv preprint

Mice
All procedures conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Stanford University Administrative Panel on Laboratory Animal Care. Mice were group housed up to a maximum of five adults per cage. Food and water were provided ad libitum and mice were maintained on a 12:12 LD cycle.

Dissection protocols
Intestinal segment harvest

Method 1: Fix flat then peel
The intestinal segment was opened along the mesenteric border, flipped over (serosaup) and pinned flat along the edges in a Sylgard-bottomed glass Petri dish, stretched under light tension. Segments were fixed in 4% PFA at 4°C for 90 minutes with gentle rocking, followed by three washes with cold PBS for at least 10 minutes each. To obtain muscularis with myenteric plexus, the muscularis was carefully peeled up and away from the submucosa/mucosa, separating the layers along the first 2-3 mm. The segment was flipped over, mucosa side up, and re-pinned. The mucosa/submucosa was carefully pulled away, while a cotton-tipped swab was used to gently hold down the muscularis.
Segments were stored in PBS with 0.1% NaN3 at 4°C until use.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2023. ; https://doi.org/10.1101/2023.01.17.524014 doi: bioRxiv preprint

Method 2: Peel flat then fix
Each segment was prepared as in Method 1 with the following modifications: (i) the order of microdissection and fixation of the pinned segments was reversed and (ii) fine forceps were used to grasp the mucosa/submucosa and gently pull it away, avoiding the use of the cotton swab applying pressure to the muscularis before fixing. Muscularis segments were fixed, washed, and stored as in Method 1.

Method 3: Peel on rod
A glass rod was inserted through the lumen of the full-length intestinal segment. The diameter of the glass rod (2 mm) was chosen to fit the diameter of the segment and slightly distend it. Forceps were used to gently perforate the muscularis (but not the mucosa/submucosa) at the mesenteric attachment. Ice-cold PBS was applied frequently during dissection to maintain moisture and temperature of the segment. A cotton-tipped swab moistened with cold PBS was used to gently peel away the muscularis, swabbing circumferentially from the perforated mesenteric edge while the segment was manually held stable on the rod. Fully peeled muscularis segments were transferred to a Sylgardbottomed glass Petri dish with cold PBS and pinned flat along the edges under light tension. Muscularis segments were fixed, washed, and stored as in Method 1.

Confocal imaging
Images were acquired as described previously 13 . Z-stacks were acquired with 2.5-3 µm between each focal plane.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2023. ; https://doi.org/10.1101/2023.01.17.524014 doi: bioRxiv preprint

Neuronal and marker quantification
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.

Statistical analyses
Statistical tests and graphical representation of data were performed using Prism 9 software (GraphPad). Statistical comparisons were performed using one-way ANOVA followed by Tukey's correction for multiple comparisons to assess if variations in dissection method were a significant factor (p<0.05) for calretinin, nNOS, and SST subtype marker proportion. Two-way ANOVAs followed by Šídák's multiple comparisons tests were used to determine the effect of dissection method on ChAT-Cre-tdT and Tac1-Cre-tdT marker subtype proportion. Asterisks indicate significant differences.

Results
To probe possible effects of different tissue dissection methods on marker expression, we separately tested significantly different aspects of three dissection methods described in the field (detailed in Methods). We focused on (i) the order, chronologically, of peeling and fixation, and (ii) mechanically different methods of peeling the muscularis, while preserving variables such as dissection solution and temperature, type of fixative and duration of fixation across the three methods.
We first analyzed neuronal marker expression via immunohistochemistry. We chose three subtypes of enteric neurons which can be identified by expression of distinct marker proteins for which antibody staining yields easily quantifiable cell body labeling: excitatory motor neurons by calretinin expression, inhibitory motor neurons by neuronal nitric oxide synthase (nNOS) expression, and a subpopulation of interneurons by somatostatin (SST) expression [18][19][20] . For all three of these markers, no significant difference in proportion of neurons labeled was found between the two flat-sheet microdissection methods (Methods 1 and 2) (Fig 1). In the case of SST, there was also no significant difference in proportion of neurons labeled using the rod peeling method (Method 3) compared to the flat-sheet . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2023. ; https://doi.org/10.1101/2023.01.17.524014 doi: bioRxiv preprint methods (Fig 1l). However, for both calretinin (Fig 1d) and nNOS (Fig 1h), the rod peeling method resulted in significantly fewer neurons labeled than either flat sheet method (calretinin, ~35% (Methods 1 and 2) decreased to ~10% (Method 3); nNOS, ~23% (Methods 1 and 2) decreased to ~13% (Method 3)).
We next asked whether the mechanical technique of wholemount peeling would also impact the reporting of genetically labeled neurons in transgenic mice. Transgenic mice that express a reporter fluorescent protein in a gene-dependent manner can be used to quantify neuronal subtype marker expression for markers where antibody staining does not yield easily quantifiable cell body labeling, such as ChAT and substance P. Like calretinin, ChAT and substance P are both markers for excitatory motor neurons. We compared the two mechanically different peeling methods, flat-sheet (Method 1) and rod mounted (Method 3) in ChAT-Cre-tdT and Tac1-Cre-tdT mice, and expanded our analysis to two regions, ileum and distal colon. In Tac1-Cre-tdT ileum the proportion of neurons labeled by tdT was similar between the two methods tested (Fig 2 f-g, j); however, in ChAT-Cre-tdT ileum, the proportion of neurons labeled was significantly decreased with the rod peeling method as compared to the flat-sheet method, from ~62% (Method 1) to ~26% (Method 3) (Fig 2 a-b, e). In the distal colon, the rod peeling method resulted in decreased tdT labeling in ChAT-Cre-tdT (~38% (Method 1) decreased to ~14% (Method 3)) and Tac1-Cre-tdT (~36% (Method 1) decreased to ~3% (Method 3)) myenteric plexus, with labeling in Tac1-Cre-tdT colon almost completely abolished (Fig 2 c-e, h-j).
Importantly, we did not note any differences in overall neuronal density, their general soma morphology revealed by HuC/D staining, or the subcellular localization of HuC/D protein (Fig 2 k- The exact mechanism of the described differences in marker labeling is unclear. There may be some as yet unknown effect introduced by the distension of the intestinal segment on the glass rod or the handling of the muscularis as it is peeled away from the mucosa, which may cause a rapid change in neuronal marker protein expression without obvious evidence of cell death or mechanical damage. In this study we did not explicitly test for any effect of time elapsed before fixation; however, in our hands the time to fixation between the flat-sheet peel pre-fixation method and the rod peeling method was comparable, suggesting that any decrease in marker labeling with the rod peeling method is not due to the time required to process the tissue prior to fixation. Finally, we note that our comparative study is not exhaustive and that future experiments are required to uncover the cause underlying the observed differences in marker expression. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2023. ; https://doi.org/10.1101/2023.01.17.524014 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2023. ; https://doi.org/10.1101/2023.01.17.524014 doi: bioRxiv preprint Muscle relaxants (nicardipine) 6,23,24 Amount of stretch 23,25 Layers peeled 3,26 Transcardial perfusion with fixative 27 Microdissection vs razor blade 27 Peel muscularis, then fix 28 Layers peeled 2,29 No fixation (for fluorescent reporters) 22     (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2023. ; https://doi.org/10.1101/2023.01.17.524014 doi: bioRxiv preprint peel > fix, n = 9. (h) nNOS: fix > peel, n = 4; peel > fix, n = 4; rod, peel > fix, n = 11. (l) SST: fix > peel, n = 4; peel > fix, n = 4; rod, peel > fix, n = 4.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   ileum rod, peel > fix, n = 9; distal colon fix > peel, n = 3; distal colon rod, peel > fix, n = 9.
All tests two-way ANOVA.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2023. ; https://doi.org/10.1101/2023.01.17.524014 doi: bioRxiv preprint