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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
The effects of endogenous and exogenous nitric oxide on gut motility in zebrafish Danio rerio embryos and larvae
Department of Zoophysiology, Göteborg University, Box 463, SE 405 30 Göteborg, Sweden
* Author for correspondence (e-mail: A.Holmberg{at}zool.gu.se)
Accepted 18 April 2006
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Summary |
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Key words: enteric nervous system, L-NAME, sodium nitroprusside, ontogeny, zebrafish Danio rerio
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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;
Bisset et al., 1988;
Sase et al., 2000;
Acosta et al., 2002;
Oyachi et al., 2003;
Anderson et al., 2004) and
teleosts (Rønnestad et al.,
2000; Holmberg et al.,
2003). Zebrafish Danio rerio is becoming a valuable model
animal for developmental studies, not the least when studying development of
gut motility in vivo. The external fertilization and development of
zebrafish eggs and embryos, and the transparency of the gut wall of the
developing zebrafish, allow us to use a non-invasive video technique to study
the gut motility from the earliest stages
(Holmberg et al., 2003;
Holmberg et al., 2004). Thus
we have obtained new physiological data that together with existing molecular
and morphological information can contribute to a better understanding of gut
development.
In a previous study on zebrafish, we observed sporadic non-propagating
contractions from the first day investigated (3 days post fertilization,
d.p.f.), and at 4 d.p.f. spontaneous, regular contraction waves were seen
(Holmberg et al., 2003). These
propagating contraction waves coincide with the first occurrence of a
continuous sheet of circular smooth muscle around and along the gut
(Wallace et al., 2005). This
is before the zebrafish start exogenous feeding, but gut motility might be of
importance in young non-feeding zebrafish to transport slugged off intestinal
material or to prevent bacterial overgrowth. A similar pattern of sweeping
contractions occurs in-between meals in adult vertebrates, the so-called
migrating motor complexes (MMCs)
(Szurszewski, 1969).
Gut motility is primarily controlled by the enteric nervous system. 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). Only a
few of the above studies have correlated neurotransmitter findings with their
effect on gut motility at an early stage. However, in zebrafish, neurons
expressing NKA (neurokinin A) and PACAP (pituitary adenylate
cyclase-activating polypeptide) have been detected in the developing gut from
2 d.p.f., and from 5 d.p.f. the frequency of the anterograde contraction waves
were increased and decreased by the application of NKA and PACAP,
respectively. In addition, a cholinergic tonus was observed from 4 d.p.f.
(Holmberg et al., 2004).
Another important transmitter is nitric oxide (NO), which generally has an
inhibitory effect on smooth muscle cells, including those of the teleost gut
(Karila and Holmgren, 1995;
Olsson and Holmgren, 2000)
(reviewed in Olsson and Holmgren,
2001). Further, NOS (nitric oxide synthase), the enzyme
responsible for NO synthesis, has been identified in gut neurons of several
adult vertebrates including teleosts (Li
and Furness, 1993; Olsson and
Karila, 1995) (reviewed in
Olsson and Holmgren, 2001).
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.
Our aim was to study the development of the nitrergic (i.e. nitric oxide releasing) control system in zebrafish gut, in vivo, by using micro-applications and a video technique.
It was hypothesized that endogenous/exogenous NO is inhibitory on gut motility in zebrafish, as in most other vertebrates. If so, and if the appropriate receptors are present, treatment with the NO donor SNP (sodium nitroprusside) will reduce the frequency of gut contraction waves. Furthermore, if the application of the NOS blocker L-NAME (NG-nitro-L-arginine methyl ester), stimulates the gut motility, this would indicate an endogenous nitrergic tonus in the animal.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Adult zebrafish Danio rerio Hamilton were purchased from a local supplier and were kept in a controlled environment (25°C, 12 h:12 h light:dark).
Motion analysis in zebrafish embryos and larvae
Breeding
Embryos (3 and 4 d.p.f.) and larvae (5 and 6 d.p.f.) from several batches
were used in the study (N=6–12 for each d.p.f. stage). Mature
zebrafish were allowed to breed spontaneously in an aquarium with a breeding
box. On the day of fertilization (0 d.p.f.) eggs were collected and
transferred to small beakers kept at a water temperature of 28°C. During
the experimental period, the embryos and larvae were not fed. Active feeding
at 28°C usually starts around 5–6 d.p.f., and at 6 d.p.f. most of
the yolk is depleted.
Mounting of embryos and larvae
Embryos and larvae were anaesthetized in phosphate-buffered MS222
(3-aminobenzoic acid ethyl ester; Sigma, St Louis, MO, USA, 75–100 mg
l–1, pH 7) and embedded lying on one side in an agarose
solution (type VII, Sigma, 1%, gelling point 26–30°C, dissolved in
phosphate-buffered MS222). The agarose was allowed to set at room temperature
and was covered with the MS222 solution in order to keep the fish
anaesthetized. The gut was observed in vivo, using an inverted
microscope (Nikon, 10x magnification).
Optimas imaging system and image analysis
The Optimas imaging system has been described in detail earlier
(Schwerte and Pelster, 2000).
Live video recordings of the gut were made and a digital film sequence was
created by extracting still images (1 s–1). For the making
and analysis of the film sequences, the Optimas program package (Media
Cybernetics, Gleichen, Germany) was used. From the film, the number of
propagating anterograde and retrograde contractions waves along the gut were
counted (Fig. 1) and the
average frequency was calculated as cycles min–1. The data
were exported to Excel for further analysis.
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Application of drugs
To avoid possible activation of sensory systems by stretching the gut, the
drugs were applied immediately outside the body wall and allowed to diffuse
into the experimental animal. Boluses between 150 and 300 nl of drug solution
were applied next to the abdomen at three different locations along the gut.
Effects of the application per se and normally occurring variations
in gut frequency over time were studied by adding the same volume of saline
(NaCl, 0.9%).
After a 5-min control period, saline was added and the activity recorded for 5 min. Subsequently, the NOS inhibitor L-NAME (10–3 mol l–1) was applied and the effects on contraction frequency were recorded for 20 min. Thereafter, the NO donor SNP (10–4 mol l–1) was applied and the effects were recorded for 9 min. For control purposes, the effects of repeated applications and time were studied in a second set of experiments, where saline was applied instead of L-NAME.
Statistical analysis
The basal contraction frequency during the control period as well as after
saline application was calculated over 5 min. No significant change in
contraction frequency was observed before or after saline application.
Therefore the contraction frequency during the first saline application was
subtracted from all further effects obtained, in order to standardise the
response to drug or second saline application. 9 min after L-NAME
or the second saline application, the maximal contraction wave frequency over
a 6-min period was calculated and was statistically analysed in comparison to
the previous saline application.
The inhibitory effect of SNP was compared to the increased frequency obtained after exposure to L-NAME. Hence, animals that showed no activity before the SNP applications were discarded.
To see the effects of the application per se, the frequency during a 3-min period directly after each saline application was compared with the first 3 min of the control period. For normal variation over time, the first 6 min after the second saline application were compared with the maximal contraction frequency 9 min after saline application (during 6 min).
Mean values ± s.e.m. were calculated for each d.p.f. group. Values were analysed statistically by using Wilcoxon Signed Ranks, matched-pairs test (SPSS 12.0 for Windows). Repetitive uses of experimental groups were taken into account. Differences in mean values were regarded as significant at P<0.05.
In vitro recordings of adult zebrafish intestine activity
Preparations
The middle intestine (MI), posterior to the intestinal bulb, was dissected
out (N=7) and placed in cold zebrafish Ringer's solution (composition
in mmol l–1: NaCl 116, KCl 2.9, CaCl2 1.8, Hepes
5, glucose 11, pH 7.2). Ring preparations (3–4 mm wide) were mounted in
organ baths containing zebrafish Ringer's solution (22°C, bubbled with
0.3% CO2 in air). The force developed by the smooth muscle was
recorded using a force displacement transducer (model FT03, Grass Instruments,
West Warwick, RI, USA) connected to a polygraph (model 7, Grass). An initial
force of 0.5 mN was applied and the preparations were left for 1–2 h to
develop a steady baseline (resting tone). A single dose of L-NAME
(3x10–4 mol l–1) was applied to the
organ bath. When maximal response to L-NAME was obtained, after
approximately 20–25 min, SNP was applied at increasing concentrations in
a cumulative fashion, allowing maximal response to each concentration to be
obtained before addition of a higher concentration
(10–7–10–5 mol
l–1).
Statistical analysis
Changes in force developed by the strip preparations were sampled on a
computer (Labview, acquisition software). A control period of 1 min was
recorded before the addition of drug. The basal frequency was subtracted from
all further calculations. The response to each drug added was calculated as
the mean force during 1 min of peak response to the drug. The effects of
L-NAME were calculated in relation to the control (spontaneous)
activity, which was set to 100%. The inhibitory effect of SNP was calculated
in relation to the increased tonus obtained after exposure to
L-NAME. Differences in mean values were regarded as significant at
P<0.05.
Immunohistochemistry
Embryos and larvae from 2, 3, 5 and 7 d.p.f. (N=4 from each stage)
were anaesthetized in 0.01% MS222 and fixed for 24 h in Zamboni's fixative
(1.5% picric acid, 2% formaldehyde in 0.1 mol l–1 phosphate
buffer, pH 7.2). Adult zebrafish were anaesthetized in 0.1% MS222, decapitated
and the proximal (PI), middle (MI) and distal intestine (DI) were dissected
out and fixed as above. The fixative was removed by repeated washing in 80%
ethanol, followed by dehydration in 95 and 99.5% ethanol, xylene treatment,
and rehydration in an ethanol series (99.5%, 95%, 80%, 50%) to
phosphate-buffered saline (0.1 mol l–1 PBS, 0.9% NaCl) for 30
min for each step. The fixed tissues were stored in a PBS with 30% sucrose
solution at least overnight before preparation for sectioning.
Whole embryos and larvae were positioned in 1.5% agarose–5% sucrose solution, which was left to set in room temperature. The agarose blocks were placed in PBS–sucrose solution at 4°C until they had sunk to the bottom and were then quick-frozen in isopentane chilled by liquid nitrogen. Tissue pieces from adult intestines were embedded in embedding medium (OCT; Sakura, Zoeterwoude, The Netherlands) and frozen as above. Preparations were sectioned on a cryostat (Zeiss Micron International GmbH, Walldorf, Germany), at 16 µm (embryos and larvae) or 4 µm (adults) and picked up on gelatine-coated slides, left to dry in darkness overnight and then stored at –20°C.
The sections were pre-incubated with normal donkey serum (10%, Jackson
ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min in a moist
chamber to reduce non-specific staining. They were then incubated with an
antibody raised against rat neuronal NOS (1:100, 31030, Transduction
Laboratories, Pharmingen, Germany) for 2 days in a moist chamber at room
temperature. The specificity of the antisera has been reported in earlier
studies on fish (e.g. Karila et al.,
1997). Excess antibody was washed away with phosphate buffer
containing 2.0% NaCl three times before incubating with a secondary antibody
conjugated to either indocarbocyanine (Cy3; 1:800, 711-165-157, Jackson
ImmunoResearch Laboratories) or fluorescein isothiocyanate (FITC; 1:100,
711-095-152, Jackson ImmunoResearch Laboratories) for 1 h. The sections were
washed as described above, mounted in Vector H-1000 medium and examined with a
Nikon Eclipse E1000 digital fluorescence microscope equipped with a Nikon
digital camera DXM1200 and Nikon's software, ACT1. Contrast and brightness
were adjusted and mounts were made using Adobe PhotoShop 6.0.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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).
In agreement with earlier studies
(Holmberg et al., 2004),
single or repeated applications of saline per se did not affect the
frequency of contraction waves compared to the control period. Furthermore, no
changes in gut motility over time were seen after the second saline
application in comparison with first saline application (anterograde, 4
d.p.f., 0.15±0.13, N=8, P=0.40; 5–6 d.p.f.,
0.42±0.41, N=8, P=0.64: retrograde 4 d.p.f.,
–0.35±0.33, N=8, P=0.54; 5–6 d.p.f.,
0.76±0.33, N=8, P=0.1).
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L-NAME
Prevention of endogenous NO formation by L-NAME (three boluses
of 50–100 nl, 10–3 mol l–1) increased
the anterograde contraction wave frequency (compared to saline application)
from 4 d.p.f. and onward, indicating an endogenous nitrergic tonus before the
zebrafish commence exogenous feeding (Fig.
2). The increase at 4 d.p.f. was 0.50±0.10 cycles
min–1 (N=10)
(Fig. 2A), and at 5–6
d.p.f. the increase was 0.31±0.08 cycles min–1
(N=12) (Fig. 2B). No
effect of L-NAME was seen at 3 d.p.f. (0.15±0.12 cycles
min–1; N=6, P=0.283).
SNP
The application of the NO donor SNP (three boluses of 50–100 nl,
10–4 mol l–1, after a previous application
of L-NAME) reduced the contraction frequency from 4 d.p.f.onward,
showing that functional reaction pathways for NO are present before onset of
exogenous feeding (Fig. 2). At
4 d.p.f. the reduction was –0.71±0.20 cycles
min–1 (N=7) (Fig.
2A) compared to 3 min prior to application (from an increase of
0.60±0.10 cycles min–1 after L-NAME to
–0.12± 0.21 cycles min–1 in comparison to
saline). At 5–6 d.p.f., SNP decreased contraction frequency with
–0.64±0.12 cycles min–1 compared to 3 min prior
to SNP application (from an increase of 0.42±0.14 cycles
min–1 to –0.21±0.12 cycles
min–1 in comparison to saline, N=11)
(Fig. 2B). At 3 d.p.f., no gut
motility was observed; therefore no conclusion of an inhibitory effect of SNP
at this stage can be drawn from the experiments.
Anterior retrograde contraction waves
The anterior retrograde contraction waves originate from the same general
area as the anterior anterograde contraction waves, i.e. immediately behind
the intestinal bulb (Fig. 1).
The retrograde contraction waves travel in an oral direction along the
proximal intestine (intestinal bulb). The basal contraction frequency was
2.06±0.39 cycles min–1 (N=18) and
1.62±0.28 cycles min–1 (N=20) at 4 and
5–6 d.p.f., respectively. No propagating contractions were seen at 3
d.p.f.
L-NAME
At 5–6 d.p.f., application of L-NAME (three boluses of
50–100 nl, 10–3 mol l–1) increased the
frequency of retrograde waves with 0.39±0.15 cycles
min–1 compared to saline (N=12)
(Fig. 2D), indicating the tonic
release of (inhibitory) endogenous nitric oxide in the larvae. In the younger
stages L-NAME did not induce an increase in contraction frequency
in comparison to saline (3 d.p.f., 0.39±0.57 cycles
min–1, N=6, P=0.18; 4 d.p.f.,
0.33±0.26 cycles min–1, N=9, P=26)
(Fig. 2C). At 5–6 d.p.f.,
4 out of 12 individuals were unaffected by L-NAME.
SNP
The application of the NO donor SNP (three boluses of 50–100 nl,
10–4 mol l–1, after a previous application
of L-NAME) reduced the contraction frequency to 1.61±0.36
cycles min–1 compared to the frequency 3 min prior to the SNP
application at 5–6 d.p.f. (from an increase of 0.75±0.20 to a
decrease of 0.86±0.44 in comparison to saline)
(Fig. 2D). Before the stage of
onset of feeding, SNP did not affect the frequency of retrograde contraction
waves (3 d.p.f., from 0.33±1.26 to 0.00±0.00, N=6: 4
d.p.f., from 0.96±0.42 to –0.54±0.34, N=4,
P=0.07, all values are in comparison with saline)
(Fig. 2C).
Strip preparations of adult intestine
Strip preparations adopted a tonus of 12.8±1.7 mN (N=7).
However, spontaneous rhythmic activity was generally not observed in the
preparations. Prevention of NO formation by L-NAME
(3x10–4 mol l–1, N=7)
increased the mean force exerted by the preparations to 130.8±6.7% of
control, mainly by increasing the basal tonus. In the majority of strip
preparations, the rhythmic contraction pattern that was induced by
L-NAME persisted after washout of L-NAME. Subsequent
addition of SNP, 10–5 mol l–1 (N=5)
decreased the L-NAME induced tension to 71.2±27.0% of
control period (Fig. 3) while
lower concentrations had no effects.
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Immunohistochemistry
At 2 d.p.f., no NOS-like immunoreactive material could be detected along
the gut while at 3 d.p.f., neurons showing NOS-like immunoreactivity were
distinguished in the middle and distal parts of the intestine
(Fig. 4A). From 4 d.p.f.,
NOS-positive neurons were found throughout the gut, however, there was a
higher number of neurons in the more distal parts of the gut compared to the
proximal part (Fig.
4B–D). This difference persisted until 7 d.p.f. when no
major difference could be observed between the different parts of the gut. In
the adult animal, NOS-like immunoreactivity was detected in nerve fibres and
myenteric nerve cell bodies throughout the gut. Nerve fibres were seen in all
layers of the gut, with the highest density in the myenteric plexus and the
circular muscle layer (Fig.
4E).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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;
Olsson and Holmgren, 2000)
(reviewed in Olsson and Holmgren,
2001).
In the present study, NOS was detected in neurons throughout the gut before
onset of exogenous feeding. Similar results have been reported from studies in
prenatal mammals (Timmermans et al.,
1994; Brandt et al.,
1996; van Ginneken 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. This variation may be reflected in the physiological
development of motility (see below).
The early appearance of neurotransmitters in the gut seems to be a common
feature to several neurotransmitters, within and between species. The first
observation of detectable levels of NOS in the present study correlates well
with the occurrence of other putative neurotransmitters, both excitatory (NKA,
acetylcholine) and inhibitory (PACAP), in zebrafish larvae
(Holmberg et al., 2004). In
addition, a variety of inhibitory and excitatory neurotransmitters have been
observed in other vertebrate species including mammals, amphibians and
teleosts before or around the onset of exogenous feeding
(Gintzler et al., 1980;
Rothman and Gershon, 1982;
Saffrey et al., 1982;
Epstein et al., 1985;
Huang et al., 1986;
Saffrey and Burnstock, 1988;
Reinecke et al., 1997;
Salvi et al., 1999;
Holmberg et al., 2001;
Maake et al., 2001;
Badawy and Reinecke, 2003;
Holmberg et al., 2004;
Pederzoli et al., 2004).
One limitation of most of the above studies is that they are strictly
morphological, and functional data are lacking. Even if the neurotransmitters
are present at an early stage, they might not be released, or the appropriate
receptors on smooth muscle cells, ICCs or neurons might not be expressed or
fully functional. In contrast, in our previous study we could correlate the
effects of two putative neurotransmitters, with their appearance in neurons.
The results showed that in vivo application of NKA and PACAP did not
affect anterograde contractions until 4 d.p.f., i.e. at least 2 days later
than they were immunohistochemically detected in neurons
(Holmberg et al., 2004).
Similarly, in the present study, NOS is expressed in neurons at least 1 day
before NO has an effect on propagating gut motility. This time lapse between
occurrence and effect of the neurotransmitters might be explained by the fact
that the smooth muscle cells are not aligned to form a continuous circular
smooth muscle sheet until 4 d.p.f.
(Wallace et al., 2005).
Consequently, contraction waves cannot propagate for longer distances at
earlier stages. This is in agreement with our previous work showing that at 3
d.p.f., non-propagating irregular gut activity is seen in the zebrafish gut
(Holmberg et al., 2003).
For a fully functional gut motility, a fine-tuned cooperation between
excitatory and inhibitory mechanisms is needed. Earlier studies have shown the
presence of an excitatory cholinergic tonus that modulates contraction
frequency from 4 d.p.f. in developing zebrafish larvae
(Holmberg et al., 2004).
Likewise, a functioning cholinergic regulation of gut motility was observed in
foetal guinea pig and rabbit (Gintzler et
al., 1980; Acosta et al.,
2002; Oyachi et al.,
2003). In the zebrafish, our present results suggest that an
inhibitory nitrergic tonus starts to modulate anterior anterograde contraction
waves at the same time as the stimulatory cholinergic tonus develops. These
two mechanisms probably cooperate to keep the gut activity at a desired
contraction frequency.
It is notable that the onset of the effects of NO on retrograde contraction waves (spreading orally over the intestinal bulb) is lagging 1 day behind the effects on anterior contraction waves. A similar lag is seen for the appearance of NOS positive neurons. Such cells were first observed in the middle and distal part at 3 d.p.f. while occurrence in the proximal intestine was delayed 1 day. Further studies are needed to elucidate the significance of the difference in reactivity and expression.
The function of spontaneous contractions in non-feeding fish larvae is so
far unknown. They can be considered as training for future mixing and
transport of foodstuff. However, there is probably also a need for
transportation of slugged-off intestinal material and prevention of bacterial
overgrowth, even before feeding starts. The anus and mouth open up at 3
d.p.f., and bacteria, etc. can thus enter the gut before the animal has
started to actively take in food. Similar housekeeping activity in the gut is
known to occur in e.g. non-feeding adult mammals
(Szurszewski, 1969).
Migrating, rhythmic contractions are part of migrating motor complexes or
MMCs. Anterogradely propagating contraction waves have also been identified in
isolated intestine of fasting adult cod
(Karila and Holmgren, 1995).
The migration velocity was similar to mammalian MMCs and the frequency was
approximately 0.5 cycles min–1. In the present study, the
frequency in zebrafish embryos and larva in vivo was somewhat higher,
with approximately 1 cycle min–1 in the anterograde direction
and 2 cycles min–1 for the retrograde contractions. Whether
this just reflects the different experimental set-up (in vitro vs in
vivo) or depends on species differences is so far difficult to tell.
However, it is likely that the gut control system is immature in the embryos
and larvae and hence the differences in contraction frequency are due to age
and developmental stage. In mammals, ICCs, nerves and smooth muscle have not
reached adult maturity at birth, and although slow waves are present at that
stage gut motility will continue to develop during the period immediately
after birth (Daniel and Wang,
1999; Torihashi et al.,
1997).
ICCs are believed to be involved in the control of gut motility in both the
fed and fasted state in mammals. In mouse, ICCs in the muscular layer (ICC-IM)
express NOS and enhance nitrergic neurotransmission by releasing NO
(Ward et al., 2000;
Burns el al., 1996). In the
present study, NOS appears to be present in neurons only but further studies
are needed to elucidate all sources of the endogenous nitric oxide in
zebrafish.
In conclusion, the present study shows that besides an excitatory
cholinergic tonus (Holmberg et al.,
2004) there is a nitrergic inhibiting tonus present in zebrafish
from just before or at the onset of exogenous feeding. There is probably a
co-functionality between these two pathways to balance sweeping gut
contractions to a desired frequency.
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List of abbreviations |
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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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.
