No abstract

Using digital motion analysis, the ontogeny of the cholinergic, tachykinin and pituitary adenylate cyclaseactivating polypeptide (PACAP) control systems was studied in zebrafish Danio rerio larvae, in vivo.

The autonomic nervous system develops following migration and differentiation of precursor cells originating in the neural crest. Using immunohistochemistry on intact zebrafish embryos and larvae we followed the development of the intrinsic enteric and extrinsic vagal innervation of the gut. At 3 days postfertilization (dpf), enteric nerve cell bodies and fibers were seen mainly in the middle and distal intestine, while the innervation of the proximal intestine was scarcer. The number of fibers and cell bodies gradually increased, although a large intraindividual variation was seen in the timing (but not the order) of development. At 11-13 dpf most of the proximal intestine received a similar degree of innervation as the rest of the gut. The main intestinal branches of the vagus were similarly often already well developed at 3 dpf, entering the gut at the transition between the proximal and middle intestine and projecting posteriorly along the length of the gut. Subsequently, fibers branching off the vagus innervated all regions of the gut. The presence of several putative enteric neurotransmitters was suggested by using markers for neurokinin A (NKA), pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide, serotonin (5-hydroxytryptamine, 5-HT), and calcitonin gene-related peptide (CGRP). The present results corroborate the belief that the enteric innervation is well developed before the onset of feeding (normally occurring around 5-6 dpf). Further, the more detailed picture of how development proceeds at stages previously not examined suggests a correlation between increasing innervation and more regular and elaborated motility patterns.

For the first time, spontaneous intestinal activity was demonstrated and quantified before the onset of exogenous feeding in zebrafish Danio rerio embryos and larvae in vivo, using digital motion analysis. At 3 days post fertilization (dpf), erratic and spontaneous contraction waves were observed in the gut. Later (4–7 dpf), more distinct contraction patterns were distinguished, and anterograde and retrograde contraction waves projecting anally and orally along the intestine, respectively, as well as local rectal contraction waves could be identified and quantified. The frequency of both anterograde intestinal and local rectal contractions increased significantly during the first days of development. There was a tendency towards shorter anterograde contraction waves in the first dpf stage investigated, but the velocity of the waves did not differ significantly between the different dpf stages. The presence of developing neurones in the gut of zebrafish was established using immunohistochemistry, staining for a suite of marker proteins (Hu C/D, HNK‐1 and acetylated tubulin). Structural neurones were present in the developing gut from the first dpf stage investigated (2 dpf). In conclusion, during the period (3–7 dpf) when erratic contraction waves turn into a more organized pattern of motility there is also a pronounced development of the innervation, suggesting a correlation in time of the development of gut motility and its neuronal control.

SUMMARY Using motion analysis, the ontogeny of the nitrergic control system in the gut was studied in vivo in zebrafish Danio rerio embryos and larvae. For the first time we show the presence of a nitrergic tonus,modulating both anterograde and retrograde contraction waves in the intestine of developing zebrafish. At 4 d.p.f. (days post fertilisation), the nitric oxide synthase (NOS) inhibitor l-NAME (three boluses of 50–100 nl, 10–3 mol l–1) increased the anterograde contraction wave frequency by 0.50±0.10 cycles min–1. Subsequent application of the NO donor sodium nitroprusside (SNP; three boluses of 50–100 nl, 10–4mol l–1) reduced the frequency of propagating anterograde waves (–0.71±0.20 cycles min–1). This coincided with the first appearance of an excitatory cholinergic tonus, observed in an earlier study. One day later, at 5 d.p.f., in addition to the effect on anterograde contraction waves, application of l-NAME increased(0.39±0.15 cycles min–1) and following SNP application reduced (–1.61±0.36 cycles min–1) the retrograde contraction wave frequency. In contrast, at 3 d.p.f., when no spontaneous motility is observed, application of l-NAME did not induce contraction waves in either part of the gut, indicating the lack of a functional inhibitory tonus at this early stage. Gut neurons expressing NOS-like immunoreactivity were present in the distal and middle intestine as early as 2 d.p.f., and at 1 day later in the proximal intestine. In conclusion, the present study suggests that a nitrergic inhibitory tonus develops shortly before or at the time for onset of exogenous feeding.