A New Functional Zebrafish Gastrointestinal Motility Assay

Abstract

Gastrointestinal (GI) motility is the coordinated contractions of smooth muscles resulting in mixing and propulsion of material through the GI tract. GI motility is influenced by smooth muscle, enteric neurons, and Interstitial cells of Cajal (ICC). New model systems for GI motility are needed because regulation of motility is poorly understood, and the number of drugs which can assist in GI motility disorders is insufficient. The Kit receptor is required for ICC development and maintenance in mammalian model systems and in humans. Mutations in the Kit receptor are associated with GI motility disorders. The objective of these experiments was to determine motility patterns in wildtype zebrafish and compare them to motility patterns in the zebrafish kita null mutant, sparse (spab5). Contraction frequency was measured from 7 minute digital video recordings of anesthetized larvae immobilized in 1.2% agar. Contraction frequency averaged 0.586 ± 0.222 contractions per minute in wildtype larvae at 7 dpf, and 0.329 ± 0.158 contractions per minute in 7 dpf sparse. The apparent contraction intensity was also scored, with a score from 0, no contractions, to 2, complete occlusion of the lumen. Contraction intensity averaged 1.250 ± 0.444 in wildtype larvae and 1.619 ± 0.498 in sparse larvae at 7 dpf. At 11 dpf contraction intensity decreased to 1.000 ± 0.000 and 1.125 ± 0.354, respectively. A functional motility assay was developed to quantify GI motility that contributes to propulsive movement of intestinal contents. Larvae were fed FITC labeled microspheres, washed, and fluorescence intensity of the GI tract was digitally imaged. Larvae were placed in clean system water, and reimaged after 24 hours. Fluorescence intensities were normalized against initial intensity of ingested microspheres at 7 dpf in the intestinal bulb of the anterior GI tract. At 7 dpf the change in fluorescence intensity in the anterior GI tract decreased by 0.371 ± 0.030 and 0.119 ± 0.021 in the posterior GI tract of wildtype larvae. At 11 dpf fluorescence intensity decreased to 0.455 ± 0.056 in the anterior GI tract, and 0.258 ± 0.046 in the posterior GI tract. Peritoneal injection of the ACK-2 anti-Kit antibody results in distention and an increased volume in the GI tract (Figure 12) (Maeda, Yamagata et al. 1992). These effects result from inactivation of Kit function by ACK-2. The size of the GI tract in sparse larvae appeared to be larger compared to wildtype, suggesting that the null kita mutant resulted in a phenotype similar to the ACK-2 effects on mice. We measured the area of a longitudinal cross section of wild type and sparse mutant larvae at 7 dpf. The average area of the GI tract in wildtype larvae was 4491 ± 754 and 5532 ± 880 in sparse larvae. Fluorescence spectroscopy was used to quantify the fluorescence intensity. Total fluorescence emission averaged 62,630 ± 23,780 counts per second immediately after ingestion in 11 dpf larvae. One day later, total fluorescence intensity averaged 53,277 ± 6,333 counts per second, a 15% decrease. 5-hydroxytryptamine (5-HT), a prokinetic agent, was used to validate the assay. One day after loading, the total fluorescence intensity decreased after application of 5-HT to 86,820 ± 21,850. 11 dpf zebrafish larvae ingest fluorescent labeled microspheres, and spectroscopic analysis showed reduced fluorescence intensity one day after loading. Functional studies showed a reduced contraction frequency and an increase in surface area between sparse mutant and wildtype larvae. The decrease in fluorescence intensity in wildtype larvae suggests aboral movement of microspheres, out of the posterior GI tract.