Intestinal Transit Time and Cortisol-Mediated Stress in Zebrafish

Abstract

Intestinal motility, the spontaneous and rhythmic smooth muscle contraction, is a complex process that is regulated by overlapping and redundant regulatory mechanisms. Primary regulators intrinsic to the gastrointestinal tract include interstitial cells of Cajal, enteric neurons, and smooth muscle cells. Extrinsic primary regulators include the autonomic nervous system, immune system, and the endocrine system. Due to this complexity, a reductionist approach may be inappropriate if the ultimate goal is to understand motility regulation in vivo. Motility can be directly visualized in intact zebrafish, with intact regulatory systems, because larvae are transparent. Intestinal motility can therefore be measured in a complete system. However, the intestinal tract may respond to external influences, such as handling, which may invoke a stress response and influence intestinal transit. We used SR4G transgenic zebrafish, which express green fluorescent protein following activation of glucocorticoid receptors, and showed that handling required for the intestinal motility assay induces stress. Separate experiments showed that exogenous application of hydrocortisone did not influence intestinal transit, suggesting that handling may not interfere with transit measurements in intact zebrafish larvae. These experiments contribute to further development of the zebrafish model for intestinal motility research.

Keywords: intestine; motility; stress.

FIG. 1.

SR4G transgenic zebrafish identified by blue fluorescence in the heart. Fluorescent image of a 7 dpf larva (A) showing heart blue fluorescent protein expression used to identify transgenic fish and an overlay with a transmitted light image (B). Zones used to quantify intestinal transit are overlaid on images with rhodamine B-labeled food (C, D). The rostral position of luminal contents was scored. Examples for each zone are shown. (C, D, E, F).

FIG. 2.

Hydrocortisone stimulates green fluorescent protein (GFP) expression after 4 h in 7 dpf SR4G larva. Transmitted light (column A), fluorescence (column B), and composite images (column C) are shown. Row 1 images were captured immediately after 50 μM hydrocortisone and after 4 h (row 2) and 24 h (row 3). GFP expression increased in brain and other tissues after hydrocortisone stimulation (row 2). GFP expression declined to control levels after 24 h in fresh bath solution (row 3). Fluorescence intensity was quantified in the region of interest (ROI) (white boxes, column 2). Fluorescence within the ROI was brightened proportionally and superimposed on composite images for clarity.

FIG. 3.

The stress response in SR4G zebrafish is activated by the GI transit assay. Larvae were immobilized and confined to a propylene tube at T = 0 h (row 1) and again at T = 4 h (middle row). A separate group of fish was manipulated at 0 h and imaged at T = 4 h using methylcellulose immobilization. Fluorescence quantification in an ROI over the brain was performed (column B). Fluorescence within the ROI was brightened proportionally and superimposed on composite images for clarity (Row C). A clear increase in fluorescence was observed at 4 h in confined and in manipulated larva. Bright fluorescence in the intestine results from ingested food.

FIG. 4.

Increased GFP expression in brain ROI during the transit assay (A) or after manipulating larvae (B). Placing 7 dpf larvae in propylene tubes for imaging (A, n = 17) resulted in increased GFP expression. Manipulating larvae without enclosure in propylene tubes (B, n = 10) also increased fluorescence. Increases in average pixel intensity were statistically significant (*).

FIG. 5.

GI transit is not changed with hydrocortisone (50 μM)-induced stress. Intestinal transit was not altered in hydrocortisone-treated larva.

FIG. 6.

GI transit is not changed with hydrocortisone (50 μM) after mifepristone or TALEN-induced blockade of glucocorticoid receptor signaling. Hydrocortisone stimulated GFP expression in control, but not in mifepristone (1 μM) or Talen-treated larva. Intestinal transit was similar in control and hydrocortisone-treated and Talen-injected larvae (top panels).