Name: video imaging and spatiotemporal maps to analyze gastrointestinal motility in mice
Uploaded: 2016-02-03T15:00:00
Description: Watch this Scientific Journal Video about Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice at JoVE.com

JCB and ELH-Y were supported by the US Department of Defense CDMRP Autism Research Program (AR11034). NHMRC (1047674) to ELH-Y.The May Stewart Bursary-University of Melbourne trust funded scholarship to MS. We thank Ali Taher, F&#225;tima Ramalhosa and Gracia Seger for technical contributions.

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
<ol>
	<li>Euthanize adult mice by cervical dislocation. If possible avoid anesthesia to prevent influences on gut function via receptors located on neuronal populations of interest.</li>
	<li>Record the animal&#39;s total body weigh...

<ol>
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Using this video imaging technique, CMMC frequency was measured as an indication of colonic motility in wild type and NL3R451C mice, a mouse model of autism spectrum disorder17. Our results indicate a reduction in the number of CMMCs in mutant NL3R451C mice compared to wild type mice in the presence of the 5HT3/4 receptor antagonist Tropisetron suggesting that NL3R451C mice exhibit an increased sensitivity to Tropisetron. Accordingly, we propose that the neuroligin-3...

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 secretion2,3. 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 intestinal wall facilitates the primary functions of the gastrointestinal tract by mixing and propelling intestinal content along the length of the intestine3. Although the extrinsic nerve supply to the gastrointestinal tract from the CNS contributes to gastrointestinal function in vivo, the ENS is capable of regulating gastrointestinal function independently. This unique characteristic enables the functional investigation of enteric neuronal circuits and their contribution to gastrointestinal motility ex vivo.
Colonic migrating motor complexes (CMMCs) are spontaneous, neurogenic events that are the predominant motor pattern observed in isolated mouse colon in the absence of fecal pellets4-9. CMMCs are defined as rhythmic contractions that propagate along a horizontal distance that is at least half the total length of the colon (i.e.,&#160;from the cecum to the rectum)10. The relationship between CMMCs and the contractile patterns that propel fecal pellets is yet to be clearly established, however some pharmacological differences have been reported11. Nevertheless, the ability of the ENS to function independently of the CNS and the existence of neural-mediated motor patterns in the isolated colon provides an ideal assay system to examine disturbances in motility resulting from underlying ENS dysfunction. The spontaneity of gastrointestinal motor patterns allows functional changes in response to pharmacological stimuli to be evaluated.
The use of video imaging and spatiotemporal mapping was first developed to quantitatively examine small intestinal peristalsis in guinea pigs12. Here, an ex vivo technique is described that enables the study of mouse colonic motility patterns using video imaging and analysis of these recordings to construct high-resolution (~100 &#181;m, 33 msec) maps of colonic diameter as a function of position along the colon and of time (spatiotemporal maps). Using in-house edge detection software (Analyse2; available on request), data from full length colonic segments contracting in real time are processed to generate spatiotemporal maps for each experiment. In this step, video (AVI) files are summarized and converted to spatiotemporal maps using Analyse2. Spatiotemporal maps (Figure 2) depict contractility over time and enable the measurement of multiple parameters including propagation speed, magnitude, length and duration. Gut diameter is also recorded throughout the duration of the experiment as a measure of the overall contractility of the tissue segment. This method can be applied to identify differences in the point of initiation of contractile complexes which could indicate altered enteric neural connectivity.
A similar video imaging protocol designed to assess pellet propulsion in guinea pigs has been reported13 however here we outline the application of the video imaging approach for quantification of spontaneous colonic motility (i.e., in the absence of pellets). We also provide detailed information to assist in the dissection and preparation of gastrointestinal tissue for the video imaging approach. This protocol provides researchers with an accessible and easily replicated tool for analyzing enteric neural control of gastrointestinal function in animal models of disease including genetic mouse models.
The video imaging technique enables the analysis of colonic motility in response to various pharmacological agents. Drugs can be administered via the gut lumen or the organ bath external to the colonic preparation. Different regions of the mouse gastrointestinal tract exhibit specific motility patterns such as small intestinal segmentation and CMMCs in the colon.
This technique has been used to identify strain differences in small intestinal function; differential sensitivity to 5-HT3 and 5-HT4 antagonists were observed in the jejunum of Balb/c and C57/Bl6 mice due to the polymorphic nature of the tph2 gene expressed in the two strains6. The effect of 5-HT inhibition on motility remains controversial, as conflicting data has been reported on the importance of endogenous 5-HT on colonic peristalsis and CMMCs14,15. Alterations in motility pre- and postnatally during development7, and the effects of gene mutations on gastrointestinal motility in animal models of disease10 can also be examined by utilizing video imaging. Here we illustrate use of the method for a study of colonic motility in the NL3R451C mouse model of autism, which expresses a missense mutation in the Nlgn3 gene encoding the synaptic adhesion protein Neuroligin-316. This mutation was first identified in patients diagnosed with Autism spectrum disorder (ASD)17, which is strongly associated with GI dysfunction18-22. We investigated whether the NL3 R451C synaptic mutation affects neural outputs in the ENS using the video imaging technique. We present data characterizing CMMCs at baseline and in response to the serotonergic 5HT3/4 receptor antagonist tropisetron in the NL3R451C mouse model of autism.

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			<td>Reagents</td>
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			<td>NaCl (MW: 58.44)</td>
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			<td>S7653-250G</td>
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			<td>KCl (MW: 74.55)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500G</td>
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			<td>NaH2PO4.2H2O (MW: 156.01)</td>
			<td>Chem Supply</td>
			<td>471-500G</td>
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			<td>MgSO4.7H20 (MW: 246.48)</td>
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			<td>MA048</td>
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			<td>CaCl2.2H2O (MW: 147.02)</td>
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			<td>CA033</td>
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			<td>D-Glucose anhydrous (MW: 180.16)</td>
			<td>Chem Supply</td>
			<td>GA018-500G</td>
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			<td>NaHCO3 (MW: 84.01)</td>
			<td>Chem Supply</td>
			<td>GA018-500G</td>
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			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Two chambered organ bath 
			Dimentions: 14 cm x 8 cm x 3 cm</td>
			<td>Custom Made</td>
			<td></td>
			<td>Contact Laboratory Directly&#160;</td>
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		<tr>
			<td>&#160;732 MULTI -PURPOSE SEALANT CLEAR</td>
			<td>Dow Corning Australia Pty Ltd</td>
			<td>1890573</td>
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			<td>SYLGARD 184 SILICONE ELASTOMER KIT&#160;</td>
			<td>Dow Corning Australia Pty Ltd</td>
			<td>1064291</td>
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			<td>STOPCOCK 3 WAY FEM-ML L/LOCK S</td>
			<td>Terumo Medical Corporation</td>
			<td>0912-2006</td>
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			<td>SYRINGES with Luer Lock Tips 50mL, 20 mL, 10 mL</td>
			<td>Terumo Medical Corporation</td>
			<td>N/A</td>
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			<td>1.57 mm (ID) x 3.16 mm (OD) - Silastic Tubing</td>
			<td>Masterflex</td>
			<td>508-008</td>
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			<td>508-005</td>
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			<td>1.50 mm (ID) x 2.50 mm (OD) - Silastic Tubing</td>
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			<td>508-007</td>
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			<td>1.60 mm (ID) - Platinum cured silicone tubing&#160;</td>
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			<td>96410 - 15&#160;</td>
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			<td>Name</td>
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			<td>virtualdub.org</td>
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video imaging and spatiotemporal maps to analyze gastrointestinal motility in mice

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-3R451C 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.

Up to 90% of patients with ASD experience an array of gastrointestinal disorders, including diarrhea and constipation18,24,25. 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 ...

Watch this Scientific Journal Video about Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice at JoVE.com

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

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.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

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