The early ontogeny of neuronal nitric oxide synthase systems in the zebrafish

110

SUMMARY Spontaneous regular gut motility in zebrafish begins around 4 days post fertilisation (d.p.f.) and is modulated by release of acetylcholine and nitric oxide. The role of intrinsic or extrinsic innervation for initiating and propagating the spontaneous contractions, however, is not well understood. By creating spatiotemporal maps, we could examine spontaneous motility patterns in zebrafish larvae in vivo at 4 and 7 d.p.f. in more detail. Tetrodotoxin (TTX) was added to elucidate the importance of nervous control. Anterograde and retrograde contraction waves originated in the same region,just posterior to the intestinal bulb. This area correlates well with the distribution of Hu (human neuronal protein C/D)-immunoreactive nerve cell bodies. Whereas numerous immunoreactive nerve cells were present in the mid and distal intestine at both 4 and 7 d.p.f., fewer cells were seen anterior to the origin of contractions. The overall frequency of contractions(1.16±0.15 cycles min–1, N=14 at 4 d.p.f.;1.05±0.09 cycles min–1, N=13 at 7 d.p.f.) and the interval between individual anterograde contraction waves (54.8±7.9 s at 4 d.p.f., N=14; 56.9±4.4 s, N=13 at 7 d.p.f.)did not differ between the two stages but the properties of the contractions were altered. The distance travelled by each wave increased from 591.0±43.8 μm at 4 d.p.f. (N=14) to 719.9±33.2 μm at 7 d.p.f. (N=13). By contrast, the velocity decreased from 4 d.p.f.(49.5±5.5 μm s–1, N=12) to 7 d.p.f.(27.8±3.6 μm s–1, N=13). At 4 d.p.f., TTX did not affect any of the parameters whereas at 7 d.p.f. anterograde frequency(control 1.07±0.12 cycles min–1, N=8; TTX 0.55±0.13 cycles min–1, N=8) and distance travelled (control 685.1±45.9 μm, N=8; TTX 318.7±88.7 μm, N=6) were decreased. In conclusion, enteric or extrinsic innervation does not seem to be necessary to initiate spontaneous contractions of the gut in zebrafish larvae. However, later in development,nerves have an increasingly important role as modulators of intestinal activity.)

Section: Introductionsupporting

confidence: 80%

TTX-sensitive and TTX-insensitive control of spontaneous gut motility in the developing zebrafish (Danio rerio) larvae

Holmberg

Olsson

Hennig

2007

110

“…*P<0.05. L-NAME, N G -nitro-L-arginine methyl ester; NO, nitric oxide; SNP, sodium nitroprusside Effects of NO in zebrafish larval intestine et al, 1998) and birds (Balaskas et al, 1995) as well as in amphibian (Holmberg et al, 2001) and teleost embryos and larvae (Villani, 1999;Poon et al, 2003;Holmqvist et al, 2004). The latter studies include zebrafish; however, the difference in time of appearance of NOS positive neurons between the middle-distal and proximal intestine observed in this study has not been reported previously.…”

Section: Discussionmentioning

confidence: 46%

“…A variety of neurotransmitters have been identified in the developing gut in mammals (Gintzler et al, 1980;Rothman and Gershon, 1982;Larsson et al, 1987;Timmermans et al, 1994;Brandt et al, 1996;van Ginneken et al, 1998), birds (Epstein et al, 1985;Rothman et al, 1986;Balaskas et al, 1995), amphibians (Holmberg et al, 2001;Maake et al, 2001;Badawy and Reinecke, 2003) and teleosts (Reinecke et al, 1997;Villani, 1999;Poon et al, 2003;Holmberg et al, 2004;Holmqvist et al, 2004;Pederzoli et al, 2004). In the zebrafish, enteric neurons are present in the gut before the onset of exogenous feeding (Raible et al, 1992;Bisgrove et al, 1997;Holmberg et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

“…NOS is also present in interstitial cells of Cajal (ICCs) in mammals and are thought to be the pacemaker cells of the gut, but also act as relay cells for both excitatory and inhibitory neurotransmission to gut smooth muscle. In developing zebrafish larvae, neuronal NOS is expressed in nerves innervating peripheral organs, including enteric ganglia (Poon et al, 2003;Holmqvist et al, 2004). However, the function of NO in the developing gut is not known.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The effects of endogenous and exogenous nitric oxide on gut motility in zebrafish Danio rerio embryos and larvae

Holmberg

Olsson

Holmgren

2006

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.

“…Previous studies using in situ hybridization of zebrafish larvae revealed that by 55 h postfertilization all cell populations that express nNOS in the adult brain are present in zebrafish larvae (Holmqvist et al, 2000(Holmqvist et al, , 2004, indicating that NO is present during early development in the brain of zebrafish larvae. In both adults and larvae, information from peripheral chemoreceptors is sent to the CNS via afferent nerves in the nucleus tractus solitarius (NTS), where chemoreceptor information is integrated.…”

Section: Discussionmentioning

confidence: 99%

A role for nitric oxide in the control of breathing in zebrafish (Danio rerio)

Porteus

Pollack

Tzaneva

et al. 2015

Nitric oxide (NO) is a gaseous neurotransmitter, which, in adult mammals, modulates the acute hypoxic ventilatory response; its role in the control of breathing in fish during development is unknown. We addressed the interactive effects of developmental age and NO in the control of piscine breathing by measuring the ventilatory response of zebrafish (Danio rerio) adults and larvae to NO donors and by inhibiting endogenous production of NO. In adults, sodium nitroprusside (SNP), a NO donor, inhibited ventilation; the extent of the ventilatory inhibition was related to the pre-existing ventilatory drive, with the greatest inhibition exhibited during exposure to hypoxia (P O2 =5.6 kPa). Inhibition of endogenous NO production using L-NAME suppressed the hypoventilatory response to hyperoxia, supporting an inhibitory role of NO in adult zebrafish. Neuroepithelial cells (NECs), the putative oxygen chemoreceptors of fish, contain neuronal nitric oxide synthase (nNOS). In zebrafish larvae at 4 days post-fertilization, SNP increased ventilation in a concentrationdependent manner. Inhibition of NOS activity with L-NAME or knockdown of nNOS inhibited the hypoxic (P O2 =3.5 kPa) ventilatory response. Immunohistochemistry revealed the presence of nNOS in the NECs of larvae. Taken together, these data suggest that NO plays an inhibitory role in the control of ventilation in adult zebrafish, but an excitatory role in larvae.