Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

Summary

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Abstract

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3^R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

Introduction

The enteric nervous system (ENS) is the intrinsic neuronal network of the gastrointestinal tract and modulates various functions such as digestion of intestinal content, absorption of nutrients and the secretion and reabsorption of fluid. Neurons of the ENS are located in the myenteric and submucosal plexuses. The myenteric plexus plays a major role in regulating gastrointestinal motility¹ whereas the submucosal plexus is primarily involved in the control of secretion^2,3. The myenteric plexus is situated between the longitudinal and circular muscle layers of the gastrointestinal wall. The contractile activity of the smooth muscle layers of the in....

Protocol

Animal handling and cervical dislocation of animals prior to all experiments were performed strictly according to protocols approved by the Animal Experimentation Committee for the University of Melbourne (Ethics ID: 1212494.7)

1. Tissue Collection and Dissection

1. Euthanize adult mice by cervical dislocation. If possible avoid anesthesia to prevent influences on gut function via receptors located on neuronal populations of interest.
2. Record the animal's total body weight, pin the body (via the four paws, ventral side exposed to experimenter) firmly to a dissection board using hypodermic needles (Size: 20 G).

Results

Up to 90% of patients with ASD experience an array of gastrointestinal disorders, including diarrhea and constipation^18,24,25. However, the underlying causes of these gastrointestinal issues are unknown. Many mutations identified in patients with ASD are associated with synaptic proteins contributing to alterations and disturbances in synaptic transmission or function. One such mutation, in the gene coding for the cell adhesion molecule neuroligin-3 (NL3 R451C), was identified .......

Discussion

Using this video imaging technique, CMMC frequency was measured as an indication of colonic motility in wild type and NL3^R451C mice, a mouse model of autism spectrum disorder¹⁷. Our results indicate a reduction in the number of CMMCs in mutant NL3^R451C mice compared to wild type mice in the presence of the 5HT_3/4 receptor antagonist Tropisetron suggesting that NL3^R451C mice exhibit an increased sensitivity to Tropisetron. Accordingly, we propose that the neuroligin-3.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
NaCl (MW: 58.44)	Sigma-Aldrich	S7653-250G
KCl (MW: 74.55)	Sigma-Aldrich	P9333-500G
NaH2PO4.2H2O (MW: 156.01)	Chem Supply	471-500G
MgSO4.7H20 (MW: 246.48)	Chem Supply	MA048
CaCl2.2H2O (MW: 147.02)	Chem Supply	CA033
D-Glucose anhydrous (MW: 180.16)	Chem Supply	GA018-500G
NaHCO3 (MW: 84.01)	Chem Supply	GA018-500G
Name	Company	Catalog Number	Comments
Materials
Two chambered organ bath Dimentions: 14 cm x 8 cm x 3 cm	Custom Made		Contact Laboratory Directly
732 MULTI -PURPOSE SEALANT CLEAR	Dow Corning Australia Pty Ltd	1890573
SYLGARD 184 SILICONE ELASTOMER KIT	Dow Corning Australia Pty Ltd	1064291
STOPCOCK 3 WAY FEM-ML L/LOCK S	Terumo Medical Corporation	0912-2006
SYRINGES with Luer Lock Tips 50 ml, 20 ml, 10 ml	Terumo Medical Corporation	N/A
1.57 mm (ID) x 3.16 mm (OD) - Silastic Tubing	Masterflex	508-008
1.02 mm (ID) x 2.16 mm (OD) - Silastic Tubing	Masterflex	508-005
1.50 mm (ID) x 2.50 mm (OD) - Silastic Tubing	Masterflex	508-007
1.60 mm (ID) - Platinum cured silicone tubing	Masterflex	96410 - 14
4.40 mm (ID) - Platinum cured silicone tubing	Masterflex	96410 - 15
3.10 mm (ID) - Platinum cured silicone tubing	Masterflex	96410 -16
Graduated Laboratory Glass Bottles - 500 ml	Thermofisher Scientific	100-400
CHEMICAL RUBBER STOPPER 57 x 65 mm
CHEMICAL RUBBER STOPPER 29 mm x 32 mm
Water heater  (thermo regulator)	Ratek	TH7000
Logitech Webcam	Logitech
Name	Company	Catalog Number	Comments
Software
Virtual Dub - 1.9 11	virtualdub.org
MATLAB R2012a	Graph Pad
Logitech Webcam Software	Logitech