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First published online June 15, 2006
Journal of Experimental Biology 209, 2472-2479 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02272
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The effects of endogenous and exogenous nitric oxide on gut motility in zebrafish Danio rerio embryos and larvae

Anna Holmberg*, Catharina Olsson and Susanne Holmgren

Department of Zoophysiology, Göteborg University, Box 463, SE 405 30 Göteborg, Sweden


Figure 1
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  		Fig. 1. Schematic picture of a zebrafish larva, at approximately 5 d.p.f. The eye, intestine and swimbladder are marked on the drawing. The intestine is divided into proximal intestine (PI; also referred to as the intestinal bulb), middle intestine (MI) and distal intestine (DI). The length of the larva is approximately 3.5–4 mm. The arrows indicate the starting point and direction of anterograde and retrograde contraction waves, respectively.

 

Figure 2
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  		Fig. 2. The development of a nitrergic control system in the gut of zebrafish embryos and larvae. L-NAME (10–3 mol l–1), which blocks NOS activity, or the nitric oxide donor SNP (10–4 mol l–1) were applied, and changes in anterograde and retrograde contraction frequencies were calculated. The effect of L-NAME was calculated in relation to the first saline application, while effects of SNP were calculated as changes in activity compared to the last 3 min of the preceding L-NAME period (referred to as L-NAME before SNP). (A,B) L-NAME increased the anterograde contraction frequencies in comparison to saline at 4 d.p.f. (A; N=10) and 5–6 d.p.f. (B; N=12), indicating endogenous formation of NO. SNP reduced the gut frequency at 4 d.p.f. (N=7) and 5–6 d.p.f. (N=11), confirming the presence of an inhibitory NO pathway. (C,D) Retrograde contraction waves were not affected by L-NAME (N=9) or SNP (N=4) at 4 d.p.f. (C), but at 5–6 d.p.f. L-NAME (N=12) increased and SNP (N=6) decreased the contraction frequency (D). Results are presented as {Delta} cycles min–1 (mean ± s.e.m.). *P<0.05. d.p.f., days post fertilization; L-NAME, NG-nitro-L-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; SNP, sodium nitroprusside.

 

Figure 3
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  		Fig. 3. The effects of L-NAME and SNP on isolated strip preparations from adult zebrafish. Prevention of NO formation by L-NAME (3x10–4 mol l–1, N=7) increased the mean force exerted by the preparations. Subsequent addition of SNP (10–5 mol l–1, N=5) decreased the L-NAME induced activity to 71.2±27.0% of the control. Results are presented as force developed (mean ± s.e.m.). *P<0.05. L-NAME, NG-nitro-L-arginine methyl ester; NO, nitric oxide; SNP, sodium nitroprusside

 

Figure 4
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  		Fig. 4. NOS-like IR (arrows) in neurones in the gut of zebrafish embryos, larvae and adults. At 3 d.p.f., NOS-IR was observed in the distal (A) and middle parts of the gut. One stage later (4 d.p.f.), NOS-IR was found throughout the gut (B, distal gut; C, proximal gut). (D) NOS-IR nerve cells at 5 d.p.f. (E) In adult zebrafish, NOS-IR was observed throughout the gut, with a denser innervation of the myenteric plexus and circular muscle layer. The photo shows the distal gut. Bars, 50 µm. d.p.f., days post fertilization; IR, immunoreactivity; NOS, nitric oxide synthase.

 

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© The Company of Biologists Ltd 2006