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First published online October 21, 2004
Journal of Experimental Biology 207, 4085-4094 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01260

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Ontogeny of the gut motility control system in zebrafish Danio rerio embryos and larvae

Anna Holmberg¹, Thorsten Schwerte², Bernd Pelster² and Susanne Holmgren^1,*

¹ Department of Zoophysiology, Göteborg University, Box 463, SE 405 30 Göteborg, Sweden
² Institute for Zoology and Limnology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria

View larger version (79K): [in a new window]   Fig. 1. Zebrafish at 7 d.p.f. stage. Int, intestine; Sw, swimbladder. The arrow indicates the direction of anterograde contraction waves (Ant). Bar, 0.2 mm.

View larger version (22K): [in a new window]   Fig. 2. The effects of acetylcholine (ACh), atropine (Atr) and neurokinin A (NKA) on gut motility in zebrafish larvae over successive 9 min periods (in vivo experiments). (A) ACh was applied to animals expressing no spontaneous motility (4 d.p.f.). (B–D) Atropine and NKA were applied to animals showing spontaneous motility (B, 4 d.p.f.; C, 5 d.p.f.; D, 6 d.p.f.). Application of saline (NaCl; A–D) did not affect the frequency of anterior anterograde waves (cycles min-1) compared to the control period. (A) ACh (10-5 mol l-1) increased the frequency at 4 d.p.f., indicating the presence of functional muscarinic receptors, but was without effect one stage earlier (not shown). (B–D) Atropine (10-6 mol l-1) reduced the frequency of the spontaneous motility from 4 d.p.f. (B), indicating a release of endogenous acetylcholine in the animal. NKA (10-6 mol l-1) increased the frequency from 5 (C) to 6–8 d.p.f. (D) after block by atropine, but had no visible effect if applied before atropine (not shown).

View larger version (9K): [in a new window]   Fig. 3. The effects of PACAP (pituitary adenylate cyclase-activating polypeptide) on gut motility in zebrafish larvae over successive time periods (in vivo experiments). In the control experiment (A) the injection of saline (NaCl) did not affect the frequency of anterior anterograde waves (cycles min-1). At 5 (C) but not 4 d.p.f. (B) the injection of PACAP (20 nl, 10-6 mol l-1) reduced the frequency of the anterior anterograde waves, indicating the presence of functional PACAP receptors.

View larger version (11K): [in a new window]   Fig. 4. The effects of acetylcholine and pituitary adenylate cyclase-activating polypeptide (PACAP) on smooth muscle preparations from adult zebrafish intestine. (A) Acetylcholine increased mean force and amplitude of contractions in both longitudinal (filled squares) and circular (open squares) preparations of the middle intestine. (B) PACAP-27 decreased the mean force and amplitude in circular strip preparations of middle intestine, after blocking nitric oxide synthase formation by L-NAME (which increased the mean force and amplitude). (C) PACAP-27 increased mean force in longitudinal preparations of the middle intestine. cm, circular muscle layer; lm, longitudinal muscle layer.

View larger version (143K): [in a new window]   Fig. 5. Immunohistochemical demonstration of the innervation of the gut of developing zebrafish, showing NKA-like immunoreactivity at 3 d.p.f. (A) and 7 d.p.f. (B; arrows indicate nerve fibres and arrowheads indicate endocrine cells), and PACAP-like immunoreactivity in nerves at 2 d.p.f. (C; arrows), and 7 d.p.f. (D). Bars, 50 µm.