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First published online March 2, 2007
Journal of Experimental Biology 210, 1084-1091 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.000935
TTX-sensitive and TTX-insensitive control of spontaneous gut motility in the developing zebrafish (Danio rerio) larvae
1 Department of Zoophysiology, Göteborg University, SE 405 30
Göteborg, Sweden
2 Department of Physiology and Cell Biology, University of Nevada, Reno,
USA
* Author for correspondence (e-mail: c.olsson{at}zool.gu.se)
Accepted 23 January 2007
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Key words: intestine, enteric nervous system, spatiotemporal map, teleost
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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).
Similar motility patterns, although not as elaborate, have also been seen in
other vertebrates, for example in the intestine of a fasted teleost (Gadus
morhua) (Karila and Holmgren,
1995).
Various factors including enteric and extrinsic innervation, release of
hormones and the endogenous cyclic activity in interstitial cells of Cajal
(ICCs) all play important roles in the initiation and maintenance of gut
motility. For example, in mammals it is believed that ICCs act as pacemaker
cells and are responsible for setting the frequency of contractions
(Sanders, 1996;
Smith et al., 1987).
Nevertheless, in knockout mice lacking pacemaker ICCs, rhythmic contractions
are still present (Spencer et al.,
2003). In addition, the sodium channel blocker tetrodotoxin (TTX)
abolishes gut motility, suggesting that initiation and propagation of MMCs
mainly depend on neuronal activity
(D'Antona et al., 2001;
Sarna et al., 1981;
Spencer et al., 2003). The
importance of a functional enteric nervous system is further emphasised in
conditions such as Hirschsprung's disease, where the absence of neurons in the
distal part of the gastrointestinal tract causes severe constipation as result
of impaired motility (Gershon,
2002). Whereas the intrinsic enteric nervous system is crucial for
the phase containing regular contractions during the MMCs, extrinsic
innervation probably is needed to develop all three phases
(Chung et al., 1994;
Sarna et al., 1981;
Spencer et al., 2003;
Husebye, 1999).
In parallel with the MMCs found in fasted adult mammals, in human foetuses
where no food is present in the gut, spontaneous gastrointestinal motility has
been observed from the 14th week of gestation
(Sase et al., 2000). The
motility pattern gradually develops and by the 37th week of gestation, clearly
defined MMCs are distinguished in preterm infants
(Bisset et al., 1988). There
are only few studies looking at the mechanisms that trigger the onset of
motility or how it is maintained at early (pre-natal) developmental stages. In
foetal mice, anterograde propulsion of gut content is observed at embryonic
day 17.5 (E17.5) (Anderson et al.,
2004). Since mice lacking enteric neurons showed normal
propulsion, it has been suggested that enteric neurons are not needed for this
activity. On the other hand, ICCs are present in the gut of mice and also in
individuals with an impaired enteric nervous system, from the second half of
gestation. In these animals slow waves appear shortly before birth
(Ward et al., 1997;
Ward et al., 1999).
Like unborn mammals, fish larvae rely on internal food sources (the yolk
sac) immediately after hatching. Zebrafish Danio rerio usually start
to eat at around 5–6 d.p.f. (days post fertilisation) but the first
propagating gut contractions are detected from 4 d.p.f.
(Holmberg et al., 2003).
Acetylcholine and nitric oxide already affect the spontaneous gut activity at
this stage (4 d.p.f.), and it has been postulated that there is a cholinergic
and a nitrergic tone present, both of which modulate muscle activity, either
directly or indirectly (Holmberg et al.,
2004; Holmberg et al.,
2006). In addition, regulatory neuropeptides such as NKA
(neurokinin A) and PACAP (pituitary adenylate cyclase activating polypeptide)
have effects on contraction frequency from 5 d.p.f.
(Holmberg et al., 2004). The
presence of enteric neurons at 2 d.p.f. and the expression of nitric oxide
synthase (NOS), indicative of nitrergic neurons, as well as NKA and PACAP from
2–3 d.p.f. further support the theory that intrinsic enteric innervation
is involved in the control of gut motility before the onset of exogenous
feeding (Bisgrove et al., 1997;
Holmberg et al., 2003;
Holmberg et al., 2004;
Holmberg et al., 2006;
Holmqvist et al., 2004;
Poon et al., 2003;
Wallace et al., 2005).
To investigate the possible mechanisms behind these spontaneous propagating contractions in zebrafish gut and how the control mechanisms may develop around the onset of exogenous feeding, TTX was used to block neurotransmission in unfed anesthetized larvae at 4 and 7 d.p.f. Gut motility patterns were studied using spatiotemporal maps of gut flow in control and treated animals. In addition, the distribution of enteric neurons during development was studied in intact zebrafish embryos and larvae.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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In vivo recordings of gut motility
Larvae from 4 and 7 d.p.f. (N=15 and 14) were studied using in
vivo video recording of spontaneous gut movements. Only animals that had
hatched naturally at 3 d.p.f. were included in the study. The larvae were
anaesthetized in phosphate-buffered MS222 (3-aminobenzoic acid ethyl ester,
75–100 mg l–1, pH 7; Sigma, St Louis, MO, USA) and
embedded in 1% agarose (gelling point 26-30°C; Sea Plaque, Sigma)
dissolved in phosphate-buffered MS222. After the agarose had settled, the
entire gastrointestinal tract of the larvae was monitored in vivo and
gut movements were recorded onto videotape, using an inverted microscope
(Nikon, x4 magnification) equipped with a Panasonic WV-350 camera.
After an initial 5 min control period, 10 µl of tetrodotoxin (TTX, 1 mmol l–1; Tocris, Bristol, UK) or NaCl (0.9%) were applied to the experimental chamber and gut activity was recorded for a further 30 min. The effects of TTX or saline applications were calculated over the last 5–10 min.
Data analysis
The video sequences (5 min) were digitised at 4 frames s–1
using the Optimas program package (Media Cybernetics, Germany). Spatiotemporal
maps (STMaps) (Hennig et al.,
1999) of the movement of luminal content and gut walls were
created by averaging the intensity of the darker coloured luminal content
across the diameter of the gut from a point midway along the swim bladder to
the anus. Contractions could be inferred from STMaps, as a narrowing of the
diameter decreased the overall amount of opaque material at the point of
contraction and perturbed luminal contents, appearing as a coloured band (see
Fig. 1). The average background
intensity was subtracted, revealing only dynamic changes in gut opacity.
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t; s), velocity (v; µm
s–1) and distance (d; µm) travelled by individual
contraction waves were calculated. A line of best fit was manually drawn over
a propagating contraction, from which the slope (v=
d/t) and distance (d) were calculated. The
intervals (t) between two consecutive contraction waves were
determined for each pair of contractions initiated within the experimental
period (5 min). By contrast, contraction frequency was calculated as the total
number of contractions over the whole 5 min period (see
Fig. 1). Hence, a change in
frequency is not necessarily followed by a change in interval time and this
could be reflected in the total time of activity, expressed as the time
between the onset of the first contraction and the end of the last
contraction, in proportion to the total experimental period. Results are
presented as mean ± s.e.m.; only animals showing some activity during
the control period were included. Student's t-test (two-sample
assuming equal variances) was used for statistical evaluation of the results,
with P<0.05 regarded as significant.
Immunohistochemistry
Embryos and larvae from 2, 3, 4 and 7 d.p.f. (N=4 from each stage)
were anaesthetized in 0.01% MS222 and fixed in 4% formaldehyde (pH 7.3) for 2
h at room temperature. The embryos and larvae were permeabilized for 3 h in
distilled water, before being incubated with 10% normal donkey serum (Jackson
ImmunoResearch, West Grove, PA, USA) for 1 h in order to reduce non-specific
staining. The preparations were incubated overnight with primary antisera
consisting of a mixture of anti-acetylated tubulin (AcT, diluted 1:1000;
T-6793, Sigma) and anti-human neuronal protein C/D (Hu, 1:100-1:200; A21271,
Molecular Probes, Eugene, OR, USA), both raised in mice. Primary antibodies
were subsequently detected by a common FITC (fluorescein
isothiocyanate)-conjugated secondary antibody (1:100; Jackson ImmunoResearch)
following a further overnight incubation. All sera and antisera were diluted
with phosphate-buffered saline (PBS, 0.9% NaCl) containing 1% bovine serum
albumin (BSA), 1% dimethylsulfoxide (DMSO), 0.1% Triton X-100 and 0.2% sodium
azide (Ungos et al., 2003) and
the preparations were rinsed in PBS. Preparations were mounted in Vector
H-1000 medium and examined with a Nikon Eclipse E1000 digital fluorescence
microscope equipped with a Nikon Digital Camera DXM1200 and the Nikon
software, ACT1. Contrast and brightness were adjusted and montages were made
using Microsoft PowerPoint.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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;
Holmberg et al., 2004;
Holmberg et al., 2006), the
contractions originated in the anterior part of the middle intestine (see
Fig. 1). By analysing the
STMaps, it was possible to discern periods of regular activity interspersed
with longer or shorter periods of low activity
(Fig. 2). In general, the time
including contractions at any point along the gut was approximately
80–95% of the total time. Contractions propagated in a linear fashion
from the site of initiation as shown by the steady slope (see Figs
1,
2). The activity was not
affected by application of NaCl (Table
2).
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However, when comparing the two developmental stages, it was obvious the motility patterns were gradually changing. At 7 d.p.f., the distance each wave travelled along the gut was increased compared with 4 d.p.f. larvae (Fig. 3; see Table 1 for a summary of the results). By contrast, there was an age-dependent decrease in velocity of almost 50% from 4 to 7 d.p.f. (Fig. 3, Table 1). The frequency and the interval between contraction waves were not different between the two age groups.
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In addition, retrograde contractions were seen in 7 out of 17 animals,
originating in the same area as the anterograde contraction waves
(Fig. 2). At 4 d.p.f., the
retrograde frequency was higher (1.93±0.41 cycles
min–1, N=7) than the anterograde (1.16±0.15
cycles min–1, N=14) (cf.
Holmberg et al., 2003;
Holmberg et al., 2006).
Consequently, it appears that contractions in either direction can begin
independently of each other. At 7 d.p.f., retrograde contractions were more
difficult to trace and are hence not included in the results.
Application of TTX did not affect any of the observed parameters (frequency, interval, velocity or distance) of the anterograde contractions at 4 d.p.f., when compared to the control period (Table 2). By contrast, at 7 d.p.f., TTX reduced the distance the contractions travelled by 55±11% compared with the control period (Figs 2, 3). Likewise, the frequency of contractions was reduced by 49±14% as was the proportion of the time the gut was active (from 94±1% to 44±15%, N=8). However, TTX did not affect the interval time or velocity of the anterograde contraction waves at 7 d.p.f. (see Table 2 for a summary of the results).
TTX decreased the frequency of retrograde contraction waves at 4 d.p.f. but had no effect on either of the other parameters measured (Table 3).
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In addition, TTX did not have a significant effect on heart rate (control: 124.5±7.7, TTX: 125.0±6.3 beats min–1, N=8) indicating that the overall status of the animals was not affected by application of the drug.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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).
Development of spontaneous gut activity
When comparing the control periods of 4 and 7 d.p.f. larvae, it was noted
that similar to previous studies, the distance travelled by the anterograde
contraction waves increased while the overall frequency did not change. The
increased distance could not be related to an increase in gut size since there
was no obvious difference in the length of the middle intestine between 4 and
7 d.p.f. The refined method used in the present study showed that the distance
that each contraction wave travelled was even longer than previously reported
(Holmberg et al., 2003).
However, unlike previous studies, we could also see a decrease in velocity
between 4 and 7 d.p.f. This was mainly because the contraction waves at 4
d.p.f. travelled at a higher speed (approximately twice as fast) than has
previously been observed (Holmberg et al.,
2003). The reduced velocity at 7 d.p.f. could be due to several
factors. The intestinal smooth muscle cells are differentiated around 4 d.p.f.
and a longitudinal and a circular layer can be observed
(Wallace et al., 2005). It is
likely however, that the muscle layers continue to thicken between 4 and 7
d.p.f. This may shorten the distance the electrical current will spread and
result in reduced excitability that will affect the contraction velocity. It
could also be suggested that the muscle cells are slightly hyperpolarised,
possibly due to endogenous release of nitric oxide
(Holmberg et al., 2006). As a
consequence, it will take longer until depolarisation reaches the muscle
action potential threshold and, hence, the velocity will decrease. There is
also a possibility that velocity changes along the gut. If there is a decrease
in velocity along the gut, the longer distance travelled by each wave at 7
d.p.f. compared to at 4 d.p.f. would lead to a lower mean velocity. However,
when looking at the STMaps, there were no obvious deviations in the slopes of
individual contractions, suggesting a fairly consistent velocity. The change
in velocity might also be related to an increase in viscosity of the
intestinal contents (see Larson and
Schulze, 2002).
Usually, an individual wave travels the full distance and dies out before the next wave is initiated. However, given the increase in distance in combination with the decrease in velocity between 4 and 7 d.p.f., the total time every individual wave travels along the gut increases. Although the frequency and interval time were unchanged, suggesting that the clock responsible for the timing of contractions is already well-developed at 4 d.p.f., the variance in interval times at this stage is comparatively larger than at 7 d.p.f. This could indicate that the activity becomes more regular with shorter periods of low activity, as the larvae grow older. Since development is a dynamic process, some individuals will have reached a more developed pattern at 4 d.p.f., while others lag behind.
TTX-sensitive neuronal control
It has previously been suggested that the spontaneous contraction waves
seen in the zebrafish larvae might have similar housekeeping functions as the
interdigestive mammalian migrating motor complexes (MMCs)
(Holmberg et al., 2003;
Holmberg et al., 2004;
Holmberg et al., 2006). TTX
inhibits propagating contractions in mammals as well as in fish
(D'Antona et al., 2001;
Karila and Holmgren, 1995;
Spencer et al., 2003).
Further, it has been suggested that this neuronal input, at least in mammals,
depends mainly on the enteric nervous system
(Sarna et al., 1981). In the 7
d.p.f. zebrafish, TTX reduced the distance travelled by the anterograde
contraction waves as well as the overall frequency but did not affect interval
time or velocity. Hence, by blocking the neurotransmission, each contraction
wave travels a shorter distance, indicating that neurons are needed for
propagation of the wave over the last part of the middle intestine at 7 d.p.f.
The distance is even shorter than during control conditions at 4 d.p.f. At the
same time, neurons are involved in the initiation of the contractions,
indicated by the decrease in frequency after TTX is applied. Although the
overall frequency of anterograde contraction waves was reduced compared to the
control period, the velocity and interval time were not affected by TTX at 7
d.p.f. This was mainly because the total time the gut is active is reduced by
TTX whereas when it is active, the contraction waves occur with the same
interval and velocity as before TTX.
At 4 d.p.f., the only parameter that was affected by TTX was the frequency of retrograde contractions. However, this group comprised relatively few individuals and one has to be a bit cautious when drawing any conclusions from these data. It is possible that the retrograde contraction waves are more irregular than the anterograde, as suggested by the fact that only 7 of 17 animals showed any retrograde contractions during the control period (c.f. 14 of 17 that showed anterograde contractions). This could also be reflected in the fact that three of the four control animals were inactive after addition of NaCl. However, it is possible that the initiation of retrograde contraction waves, in contrast to anterograde, is under TTX-sensitive control already at 4 d.p.f.
TTX-insensitive control
The difference in response to TTX between 4 and 7 d.p.f. suggests the
development of a TTX-sensitive neuronal component during this time. It
indicates that before the normal time for onset of feeding, the initiation and
propagation of anterograde contraction waves are not under neuronal control,
or at least not under TTX-sensitive control. This is similar to the situation
in foetal mice, where the absence of enteric neurons does not affect the
propulsion of gut content compared with control mice
(Anderson et al., 2004).
However, whereas TTX abolishes migrating contractions in adult mammals
(D'Antona et al., 2001;
Spencer et al., 2003), in
zebrafish larvae there seem to be a TTX-insensitive part that remains, at
least at 7 d.p.f. Whether this mechanism is present in older larvae or adult
zebrafish has so far not been investigated. Since velocity was not affected by
TTX at 7 d.p.f., but differed between 4 and 7 d.p.f. in control conditions, it
can be suggested that development of this TTX-insensitive control mechanism
also occurs during this period.
The TTX-insensitive component could be either neurons or putative
interstitial cells of Cajal (ICCs). TTX-insensitive sodium channels are
expressed on a subpopulation of enteric neurons in, e.g. guinea pig and rat
(Rugiero et al., 2003).
Further, although lack of myenteric (pacemaker) ICCs in the small intestine of
mice did not abolish MMCs, some parameters of the migrating contractions were
affected (Spencer et al.,
2003). Whereas the interval between each complex was not affected
in vitro, the contraction frequency within each MMC was reduced in
the absence of myenteric ICCs (Spencer et
al., 2003).
So far very little is known about the possible presence of ICCs in
teleosts. ICC-like cells have been reported in the myenteric plexus of some
teleost species, using Methylene Blue
(Kirtisinghe, 1940), whereas
later studies, using Kit as a marker to detect ICCs in zebrafish using either
immunohistochemistry or in situ hybridization, have failed
(Mellgren and Johnson, 2005;
Parichy et al., 1999;
Wallace et al., 2005) (C.O.
and A.H., unpublished results). Furthermore, mutants lacking one of the two
known zebrafish ckit orthologues showed no obvious defects in
intestinal functions, either in larvae or in adults
(Parichy et al., 1999). This
suggests that if ICCs are present in fish, they probably do not depend on the
Kit receptor for their development, or at least Kit is different from the
mammalian counterpart. However, there is one recent report indicating
Kit-positive cells in the gut of zebrafish larvae
(Rich et al., 2006). In
mammals, Kit-positive ICCs have been detected before birth
(Torihashi et al., 1997;
Wester et al., 1999). Slow
waves also occur during foetal life but are preceded by ICCs
(Ward, 1996;
Ward et al., 1997). The
present data indicate the presence of some sort of pacemaker cells in the gut
around the time the zebrafish larvae start to feed.
Anterograde and retrograde contractions originate in a distinct area of the
intestine, just distal to the developing intestinal bulb (present study)
(Holmberg et al., 2003;
Holmberg et al., 2006). This
region of origin was not affected by TTX. Further, although the motility
pattern is less well coordinated very early in development (3 d.p.f.), most
contraction waves still originate in this area
(Holmberg et al., 2003). This
is in contrast to the mammalian small intestine where propagating contractions
may develop in different regions (e.g.
Furness and Costa, 1987). The
area in the zebrafish gut where the contractions originate correlates with an
area of dense innervation.
However, the neuronal network gradually decreases in the proximal part of
the intestinal bulb both at 4 and 7 d.p.f. This probably explains why gut
motility was not observed in this part of the bulb in the present or earlier
studies (Holmberg et al.,
2003). The immunohistochemical data suggest that enteric neurons
are needed for the propagation of a contraction wave along the intestinal
bulb. Although contractions can travel for a shorter distance, neurons are
needed to maintain the propagating contraction as was indicated by the
decreased distance travelled by the anterograde contractions after TTX at 7
d.p.f. During development, the number of nerve cells as well as the length and
number of their processes increase at least until 13 d.p.f. (C.O., A.H. and
S.H., unpublished results). Retrograde activity was not examined at 7 d.p.f.,
and hence the full importance of the posterior development of enteric neurons
could not be established.
In conclusion, the zebrafish gut motility shows an increasing degree of control when comparing the time before and after the larvae normally start to feed. The control mechanisms include both neuronal and non-neuronal pathways as indicated by the effects of TTX treatment on different parameters of the activity. Although propagating contractions probably serve a housekeeping function in the non-feeding larvae, when the fish start to feed the need for a more controlled motility pattern increases.
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List of abbreviations |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Acknowledgments |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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  R. R. Roberts, J. C. Bornstein, A. J. Bergner, and H. M. Young Disturbances of colonic motility in mouse models of Hirschsprung's disease Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G996 - G1008. [Abstract] [Full Text] [PDF]
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900 µm). PI, proximal intestine (intestinal
bulb); MI, middle intestine; DI, distal intestine. (B) STMaps, over the area
within the box in A, were created based on changes in gut opacity as the gut
content was propelled in a retrograde or anterograde direction. Individual
contractions are identified as darker bands, travelling in either direction
over time (downwards in map, 
