Ontogeny of the gut motility control system in zebrafish Danio rerio embryos and larvae
1 Department of Zoophysiology, Göteborg University, Box 463, SE 405 30
Göteborg, Sweden
2 Institute for Zoology and Limnology, and Center for Molecular Biosciences,
University of Innsbruck, A-6020 Innsbruck, Austria
* Author for correspondence (e-mail: S.Holmgren{at}zool.gu.se)
Accepted 18 August 2004
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Summary |
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Key words: enteric nervous system, development, PACAP, tachykinin, acetylcholine, zebrafish, Danio rerio
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Introduction |
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To our knowledge, information about the development of gut motility in
vertebrates is sparse. In mammals, absorptive functions and gut motility are
developed during fetal life, but the first stools are not passed until after
birth and first feeding (e.g. Neu,
1989; Meetze et al.,
1993
; Sherman et al.,
1996
). In contrast, fish and amphibians theoretically may deposit
stools (from slayed off intestinal material) into the surrounding water at an
earlier stage without risk of infection. Consequently, they may benefit from a
propagating motility already functioning between hatching [at 23 days
post fertilization (d.p.f.)] and the first feeding (at 56 d.p.f.). In
agreement with this, in our previous study we observed spontaneous (i.e. not
induced by drugs, food or mechanical stress) motility in the zebrafish gut
before 5 d.p.f. (Holmberg et al.,
2003
). Sporadic activity was present from the first stage
investigated (3 d.p.f.), and a regular motility pattern was present at one
stage later (4 d.p.f.).
Spontaneous motility of the gut is proposed to have a housekeeper function
and is known to occur in between meals in vertebrates. It is believed that the
enteric nervous system (ENS) is central for the coordination of such
spontaneous activity, as well as for mixing and propulsive contractions after
feeding (Kunze and Furness,
1999; Olsson and Holmgren,
2001
). Other factors, such as interstitial cells of Cajal (ICCs)
and hormones, are also known to affect spontaneous motility. However, the ENS
may be particularly important for the control of spontaneous activity at the
earliest stages, suggested from studies in the mouse, where the ENS and gut
are developed before the ICCs (Wu et al.,
2000
).
The development of the ENS has been studied in many vertebrate species,
including the zebrafish. Enteric neurons as such have been detected in the
zebrafish embryo before the onset of feeding
(Bisgrove et al., 1997;
Holmberg et al., 2003
), but so
far there are no reports on the expression of different transmitters in these
nerves. Earlier studies have shown the presence of regulatory neuropeptides in
neurons in the developing gut of another teleost
(Reinecke et al., 1997
), as
well as in amphibians (Holmberg et al.,
2001
; Maake et al.,
2001
), birds (Epstein et al.,
1981
,
1983
;
Epstein and Poulsen, 1991
) and
mammals (Larsson et al., 1987
;
Pham et al., 1991
;
Van Ginneken et al.,
1998
).
The observed parallel development of enteric neurons and regular
propagating contractions of the gut in zebrafish larvae
(Holmberg et al., 2003)
suggests that the ENS could affect this motility. However, in humans and mice,
rhythmic propagating activity can develop in the absence of functional enteric
neurons (Ward et al., 1999
;
Huizinga et al., 2001
) and
furthermore, even if they are present, the enteric neurons might not be
functional, i.e. the transmitters might not be released or the smooth muscle
cells might not express the proper receptors. Therefore, we find it of
interest to investigate the development and interaction of functioning control
systems in the gut of zebrafish. As a first step, the present study aims to
investigate the ontogeny of the cholinergic, tachykinin and pituitary
adenylate cyclase-activating polypeptide (PACAP) control systems of the gut.
Acetylcholine and substance P/neurokinin A (NKA) are major excitatory
transmitters in the ENS in adult animals, and the vasoactive intestinal
polypeptide (VIP)-related peptide PACAP often participates in inhibitory
control (Costa et al., 2000
;
Matsuda et al., 2000
;
Olsson and Holmgren, 2001
).
Furthermore, SP/NKA and VIP/PACAP are neuropeptides that have been detected in
larval gut neurons before the onset of feeding in some other fish and
amphibian species (turbot Scophthalmus maximus,
Reinecke et al., 1997
; axolotl
Ambystoma mexicanum, Maake et
al., 2001
; Xenopus laevis,
Holmberg et al., 2001
). In the
present study, gut motility in vivo in zebrafish embryos and larvae
is investigated following microinjections of acetylcholine, atropine, NKA and
PACAP, in combination with video recordings of the frequency of anterior
anterograde motility waves in the developing gut with subsequent motion
analysis.
The classical transmitter acetylcholine exhibits an excitatory effect on
gut motility in fish (e.g. Nilsson and
Fänge, 1969; Edwards,
1972
; Thorndyke and Holmgren,
1990
; Burka et al.,
1996
; Aronsson and Holmgren,
2000
). Atropine is a general muscarinic receptor antagonist and
blocks the effect of acetylcholine. It is hypothesised that if atropine blocks
spontaneous motility, then endogenous acetylcholine is released and
functioning in the fish. In addition, an effect of acetylcholine applied to
animals not expressing spontaneous activity would suggest that functional
muscarinic receptors are present, even though there may be no endogenous
release of acetylcholine. Furthermore, if functional NKA and PACAP receptors
are present, then exposure to NKA and PACAP, respectively, will affect the gut
motility.
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Materials and methods |
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The study was performed at the University of Göteborg, Sweden and at the University of Innsbruck, Austria. The Swedish National Board for Laboratory Animals' guidelines were followed.
Motion analysis in zebrafish embryos and larvae
Mounting of embryos and larvae
For the experiments, 70 embryos and larvae from several batches were used.
Each animal was anaesthetized in phosphate-buffered MS222 (3-aminobenzoic acid
ethyl ester, 75100 mg l-1, pH 7; Sigma Chemical Co., St
Louis, MO, USA) and embedded on its side in a liquid agarose solution (1%, Sea
Plaque; gelling point 2630°C, dissolved in phosphate-buffered
MS222). The agarose was allowed to settle and was covered with MS222
(75100 mg l-1, phosphate-buffered) in order to maintain the
anaesthetic condition. The animals are transparent at early stages
(Fig. 1), and the gut and its
movements could be observed in vivo using an inverted microscope
(10x magnification).
Imaging system and image analysis
For a detailed description of the imaging system and the image analysis
used see Schwerte and Pelster
(2000) and Holmberg et al.
(2003
). Still images were
extracted (every second) from video recordings of the gut, using the Optimas
program package (Media Cybernetics, Silver Spring, MD, USA). Using the Optimas
software, a film sequence could be created from the still images. From this
film sequence the frequencies of propagating anterograde contraction waves in
the anterior intestine were determined and the average frequency per minute
(cycles min-1) was calculated. The data were exported to Excel for
further analysis.
Application of the drug outside the body wall
The tip of a syringe was placed immediately outside the animal, and a bolus
of between 50 and 100 nl of drug solution was applied next to the abdomen. For
control purposes, the effects of the application per se were studied
by adding the same volume of saline (NaCl 0.9%). The following drugs
(dissolved in saline) were used: atropine (Sigma, A-0257), acetylcholine
(Sigma), neurokinin A (NKA 7359; Diagnostika, Falkenberg, Sweden) and
pituitary adenylate cyclase-activating peptide (PACAP-27, 4031084.0500;
Bachem, Weil an Rein, Germany).
Animals showing spontaneous activity during the control period were treated with atropine and NKA. The following experimental protocol was used (9 min per treatment): control period; saline; atropine (10-6 mol l-1); saline; NKA (10-6 mol l-1). The effect of acetylcholine was studied in specimens showing no spontaneous activity during the control period, using the protocol (9 min per treatment): control period; saline; acetylcholine (10-5 mol l-1).
Application of drug inside the body cavity
PACAP is a fairly large polypeptide that does not easily penetrate the body
wall, and injections were made intraperitoneally. The needle tip of the
syringe was placed just inside the abdominal wall and the peptide (dissolved
in 20 nl saline) was injected into the body cavity using a
micromanipulator/microinjection apparatus. For control purposes, 20 nl of
saline were injected into the animal. The following experimental protocol was
used: control (3 min); injection of saline or PACAP 10-6 mol
l-1 (6 min).
Statistical analysis
Animals that were unaffected by the drug were not included in the
statistical analysis, and are reported separately. The frequency (cycles
min-1) data were pooled and averaged for each d.p.f. group, and
mean values were calculated and presented as the mean of one d.p.f. group
± S.E.M. For control and saline application/injection the
full experimental period was used to analyse the average frequency. The
effects of PACAP, acetylcholine and NKA appeared soon after
application/injection and therefore the first 6 min were used for analysis.
Atropine had a slower effect and the last 6 min of the experimental period
were used for the analysis. Statistical analyses were performed using
WilcoxonMannWhitney test (SPSS 10.0 for Windows); repetitive use
of experimental groups were taken into account. Differences in mean values
were regarded as significant at P<0.05.
In vitro preparations of adult zebrafish intestine
For comparison, the effects of acetylcholine and PACAP were studied in
vitro on strip preparations of the gut from adult zebrafish.
Smooth muscle preparations
The middle intestine was dissected out 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
(34 mm wide; circular muscle preparation) or the whole middle intestine
cut open longitudinally (longitudinal muscle preparations) were mounted in
organ baths containing zebrafish Ringer's solution (22°C, bubbled with
0.3% CO2 in oxygen). 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
tension (circular muscle preparation, 0.2 mN; longitudinal muscle preparation,
1 mN) was applied and the strip preparations were left for 12 h to
develop a steady baseline (resting) tension and spontaneous rhythmic
contractions. PACAP-27 (longitudinal muscle preparations only) and
acetylcholine were applied to the organ bath at increasing concentrations in a
cumulative fashion, starting at 10-10 or 10-9 mol
l-1, and allowing maximal response to be obtained before addition
of a higher concentration. To study the effects of PACAP, circular
preparations of the middle intestine were pre-treated with L-NAME
(NG-nitro-L-arginine methyl ester; Sigma, 26521), and
PACAP-27 was subsequently added to the organ bath in single doses.
Statistical analysis
Alterations in force developed by the strip preparations were recorded on
the Grass polygraph, and at the same time collected on a computer (Labview
Instruments; Austin, Texas, USA; acquisition software). A control period of 1
min was recorded before the addition of drug. The response of each drug
concentration added was calculated as the mean force during the minute of peak
response to the drug, minus mean force during the control minute.
Concentrationresponse curves were constructed for PACAP (longitudinal
muscle preparation) and acetylcholine and the mean EC50 value
± S.E.M. was calculated. The effects of L-NAME
and PACAP (single exposures) were calculated relative to the control
(spontaneous) activity, which was set to 100%. The inhibitory effect of PACAP
was calculated relative 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 whole. For control purposes adult
animals were collected and anaesthetized with 0.1% MS222 and decapitated.
Tissues from the intestine of adult zebrafish were dissected out and fixed.
All tissues were fixed for 24 h in Zambonis' fixative [15% picric acid, 2%
formaldehyde in phosphate buffer (PB) with 2% NaCl, pH 7.2], and then
repeatedly rinsed in 80% ethanol until all fixative was removed. This was
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 (PBS, 0.9% NaCl) (30 min for each step). The fixed embryo and larvae
were stored in PBSsucrose solution (30% sucrose) at least
overnight.
The animals were placed in a fluid agarosesucrose solution (1.5% agarose, 5% sucrose), which was left (at room temperature) to solidify (approximately at 26°C). The agarose blocks were placed at 4°C in PBSsucrose solution until they had sunk to the bottom and were then frozen in isopentane chilled by liquid nitrogen. Sections (16 µm) were cut on a cryostat (Zeiss Micron International GmbH, Walldorf, Germany), picked up on gelatine-coated slides, left to dry in darkness overnight and then stored at 20°C until used for immunohistochemistry.
From adult intestines, tissue pieces of approximately 10 mmx5 mm were cut out, embedded in OCT (Sakura, Zoeterwoude, The Netherlands) and frozen in isopentane chilled by liquid nitrogen. Sections (4 µm) were cut on a cryostat (Zeiss) and picked up on gelatine-coated slides. The slides were kept overnight to dry in the dark and then stored at 20°C until used.
To avoid unspecific staining, normal serum was applied on sections (10% normal donkey serum, 017-000-1; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min in a moist chamber. The sections were then incubated with the primary antibody for 48 h in a moist chamber at room temperature. Excess antibodies were washed away with PB with 2,0% NaCl (3x) and incubated with secondary antibody for 1 h in the moist chamber (at room temperature). The sections were washed, mounted in Vectashield-mounting medium (H-1000, Vector, Burlingame, CA, USA) and examined using a digital fluorescence microscope (Nikon Eclipse E1000, Nikon Digital Camera DXM1200, Nikon, Tokyo, Japan) and the Easy Image package (Nikon's software). Pictures were further processed using Adobe PhotoShop 6.0.
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Results |
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Effects of acetylcholine and atropine
The first or second application of saline did not affect the frequency of
spontaneous activity compared to the control period
(Table 1, Fig. 2).
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Acetylcholine (10-5 mol l-1) was applied to animals that did not show spontaneous gut motility during the control period. Acetylcholine did not initiate gut motility at 3 d.p.f., while at 4 d.p.f., 38% of the animals showed contractile activity after the application (Table 1, Fig. 2). After an initial peak in frequency, the frequency decreased, probably due to desensitisation. From 5 d.p.f. most observed animals expressed spontaneous gut motility and therefore no experiments were performed.
Atropine (10-6 mol l-1) was applied to animals that expressed spontaneous gut motility, in order to determine if endogenous acetylcholine was released in the animal. Atropine reduced the frequency of anterior anterograde waves, in an increasing number of animals, from the first stage investigated (4 d.p.f.) (Table 1, Fig. 2). The effect of atropine persisted after the second application of NaCl (Table 1, Fig. 2). The motility was never completely quenched by atropine (Fig. 2).
Neurokinin A
After reduction of spontaneous contractile activity by atropine,
application of NKA (10-6 mol l-1) had no significant
effect at 4 d.p.f., but increased the frequency of the contraction waves from
5 d.p.f. (Table 1,
Fig. 2).
PACAP
The injection of saline into the body cavity in control experiments did not
affect the frequency of anterior anterograde waves
(Table 1, Fig. 3).
|
After injection of PACAP-27 (20 nl of 10-6 mol l-1) into 4 d.p.f. animals, however, there was a tendency of a decreased frequency of anterior anterograde waves for a short period of time (36 min after injection) but the effect did not persist throughout the whole experimental period. One stage later, at 5 d.p.f., the frequency of contraction waves decreased during the whole experimental set-up after exposure to PACAP (Table 1, Fig. 3). However, motility was still present after injection of PACAP (Fig. 3).
Strip preparations
Acetylcholine increased the mean force of contractions in both circular
(pD2: 5.3±0.1, N=6; where pD2 is the
negative log of EC50) and longitudinal (pD2: 7.0±0.1,
N=5) smooth muscle strip preparations from the middle intestine
(Fig. 4).
|
Prevention of nitric oxide (NO) formation by L-NAME (3x10-4 mol l-1, N=5) increased the mean force to 155.6±23.6% of control rhythmic contractions of the circular preparations, and subsequent addition of PACAP-27 (10-7 mol l-1, N=5) decreased this activity to 126.5±9.8% of control values (Fig. 4). In contrast, PACAP-27 increased the mean force in the (untreated) longitudinal preparation of the middle intestine (pD2: 7.5±0.2, N=6, Fig. 4).
Immunohistochemsitry
We have no reliable method for detecting the occurrence of cholinergic
neurons by immunohistochemistry. Both NKA-like and PACAP-like immunoreactivity
first occurred occasionally in a few weakly stained nerve fibres in 2 d.p.f.
embryos, and were found in increasing numbers and staining intensity in 3
d.p.f. and 57 d.p.f. specimens (Fig.
5, Table 2).
Endocrine cells containing NKA-like immunoreactive material were observed
occasionally in the distal intestine at stage 5 d.p.f., and increased to a
moderate number at 7 d.p.f.(Table
2). Endocrine cells containing PACAP-like immunoreactive material
were observed occasionally in the distal part of the intestine at 7 d.p.f.
(Table 2). Both NKA and
PACAP-like immunoreactivity was observed in nerve fibre and endocrine cells in
adult zebrafish throughout the gut (Table
2).
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Discussion |
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One major aim of the present study was to determine to what extent the
control of gut motility is functional at the onset of exogenous feeding. In
the zebrafish embryo, the intestine is formed from a solid endodermal rod (the
gut primordium), which develops into a hollow tube around 42 h post
fertilization (h.p.f.; Horne-Badovinic et al., 2001;
Ober et al., 2003). Around 2
d.p.f., the pharynx, oesophagus, liver and pancreas are joined to the
intestine into a functional unit (Wallace
and Pack, 2003
). Enteric nerve cell precursors entered the
anterior gut, spreading backwards while maturing, and neurotransmitters can be
observed in nerves at 2 d.p.f. (Bisgrove et
al., 1997
; Holmberg et al.,
2003
). At 3 d.p.f. the mouth, and one stage later the anus, open
(Wallace and Pack, 2003
) and
at 4 d.p.f. the gut mucosa, pancreas and liver secrete digestive enzymes and
bile (Pack et al., 1996
).
These studies indicate that the digestive system might be functional around
the time for onset of feeding (56 d.p.f. at 28°C), even though the
gut cells normally are not completely depleted of egg yolk at this stage.
However, for an effective digestion and absorption, the food has to be
transported along the gut in a controlled manner.
Regular spontaneous propagating contractions (i.e. not induced by food,
anticipation of food, or drugs) are present in zebrafish larvae before the
onset of feeding (Holmberg et al.,
2003). The function of these movements or whether they are induced
by fluid or waste products in the gut is not known. Spontaneous motility in
gut preparations of adult fish, in vitro, has been suggested to be
analogous to the migrating motor complexes (MMC, phase III-like;
Karila and Holmgren, 1995
;
Olsson et al., 1999
) that
occur in between meals in adult mammals (in vivo). Phase III MMCs are
characterised by rhythmic contractions that travel in an anal direction, and
are considered to be important for transportation of waste products and
prevention of bacterial overgrowth of the gut in the inter-digestive phase. It
can be speculated that motility of the gut is important before the larvae
start to feed for a similar reason and that the observed motility in zebrafish
larvae is analogous to phase III MMC activity.
Motility of the gut is controlled by the ICCs, the ENS, hormones, and
extrinsic sensory and extrinsic autonomic innervation, which may act
independently or in interacting systems (e.g. see
Kunze and Furness, 1999;
Olsson and Holmgren, 2001
).
The relative importance of the different factors is difficult to assess. For
example, ICCs are considered to play a role as pacemakers in the initiation of
contractions, and rhythmic propagating activity may occur on the absence of
enteric neurons but in the presence of functional ICCs
(Ward et al., 1999
;
Huizinga et al., 2001
). On the
other hand, in mice, MMCs have been shown also to develop in the absence of
slow waves (which originate from the ICCs in the myenteric plexus;
Spencer et al., 2003
). We have
not been successful in determining the presence of ICCs in the developing
zebrafish, and it is possible that the development of the ENS preceeds that of
the ICCs, as has been found in mice (Wu et
al., 2000
).
At the onset of feeding, neurons expressing NKA and PACAP are present
throughout the gut in the zebrafish larvae, suggesting a possible role in
controlling the regular gut motility that was observed by Holmberg et al.
(2003). Neurotransmitters are
also expressed in other vertebrates at an early stage. A few studies have
related the expression of neurotransmitters to the first feeding. In the
turbot Scophthalmus maximus
(Reinecke et al., 1997
), the
axolotl Ambystoma mexicanum
(Maake et al., 2001
;
Badawy and Reinecke, 2003
) and
chicken (Epstein et al., 1983
;
Epstein and Poulsen, 1991
),
vasoactive intestinal polypeptide (VIP)-containing nerve fibres have been
observed before or around the time of onset of feeding. In the axolotl, PACAP
coexisted with VIP and was found in gut neurons at an early stage
(Badawy and Reinecke, 2003
). In
Xenopus laevis larvae, several transmitters, including PACAP and NKA,
have been detected by immunohistochemistry in neurons before the onset of
feeding (Holmberg et al.,
2001
). These findings suggest that the neuronal control system, if
not already fully functioning, is ready to play a role in the processing of
food from the first feed. However, the presence of neuronally contained
transmitters does not automatically mean a functional nervous control, which
also must include release and inactivation of the transmitter, and the
presence and activation of receptors.
Acetylcholine and atropine
Acetylcholine is a common transmitter in the gut, but there is, to our
knowledge, little information on the distribution and effect of acetylcholine
during gut development in non-mammalian vertebrates. By contrast, a
functioning cholinergic regulation of gut motility before birth has been
demonstrated in several mammalian species
(Gintzler et al., 1980;
Rothman and Gershon, 1982
;
Oyachi et al.,
2003a
,b
).
The excitatory effect of acetylcholine in adult zebrafish (and the
inhibitory effect of atropine in embryos and larvae) agrees with a large
number of previous reports of a cholinergic gut innervation in vertebrates.
Cholinergic excitatory neurons have been estimated to be the most common nerve
population in the adult mammalian gut (e.g. see
Kunze and Furness, 1999), and
there are also reports on cholinergic mechanisms in the gut of non-mammalian
species, including several teleosts (e.g. for references, see
Jensen and Holmgren,
1994
).
The inhibition of contractions already occurring at 4 d.p.f. by blockade of muscarinic receptors suggests that endogenous acetylcholine is released before the onset of feeding (56 d.p.f.) and affects the spontaneous gut activity. Further experiments are needed to determine whether this includes effects directly on the smooth muscle, via ICC cells, or via interneurons. Our findings also indicate the presence of functional muscarinic receptors from 4 d.p.f., but not one day earlier. It is notable that no effect of acetylcholine was observed at 3 d.p.f., nor was any spontaneous motility, but 1 day later approximately 75% of the animals expressed functional muscarinic receptors as well as spontaneous motility.
The early appearance of a functional cholinergic control agrees with
findings from several mammals. In mice, acetylcholine immunoreactive neurons
have been detected before birth (Rothman
and Gershon, 1982), and in guinea pig gut neurons acetylcholine
was present at 25 days gestation (guinea pigs have about 114 days of
gestation), while an excitatory effect on smooth muscle cells was first
established somewhat later, at day 48 of gestation
(Gintzler et al., 1980
).
Functional muscarinic receptors, mainly of the M3 and, to some extent, the M2
types, are present in fetal sheep (Oyachi
et al., 2003a
). In zebrafish, muscarinic M2-receptor mRNA is
expressed in the early embryo at 12 h.p.f. and in neurons of the heart and
vagus motor ganglion at 30 and 48 h.p.f.
(Hsieh and Liao, 2002
), but
this study does not include the intestine.
PACAP
In a previous study in a teleost, the stargazer Uranoscopus
japonica, using both endogenous and mammalian PACAP, Matsuda et al.
(2000) found that PACAP
relaxed the precontracted rectum, but had no effect on the weak spontaneous
activity of (longitudinal preparations) of the intestine and rectum. In the
Atlantic cod Gadus morhua, spontaneous contractions of both
longitudinal and circular preparations of the intestine were reduced by PACAP
(Olsson and Holmgren, 2000
).
The inhibitory effect of PACAP on the zebrafish larval gut, and on circular
muscle preparations of the adult gut, agrees with these previous studies in
adult teleosts. In contrast, the contraction induced in longitudinal
preparations of adult zebrafish gut is opposite to the relaxation obtained in
longitudinal preparations of both the stargazer
(Matsuda et al., 2000
) and the
Atlantic cod (Olsson and Holmgren,
2000
). Taken together, the results suggest species differences,
and that there are several PACAP receptors present in teleosts in general, and
in the zebrafish gut in particular. These receptors may have a different
expression over time (developing versus adult specimen), in different
tissues (longitudinal versus circular muscle, smooth muscle
versus interneuron, etc.), and in different species. Indeed, at least
two PACAP receptors are expressed in zebrafish at 6 d.p.f., one PACAP type 1
and one PACAP type 2 receptor (Wei et al.,
1998
). Our results suggest that at least one of these receptors is
expressed and already functioning from 5 d.p.f., i.e. around the time for the
onset of exogenous feeding. The results also show that the first expression of
PACAP in gut nerves occurs even earlier (from the first d.p.f. stage
investigated, i.e. 2 d.p.f.). A PACAPGRF (growth hormone releasing
factor) gene is present in zebrafish, and a transient expression in the
nervous system during early development (1 d.p.f.) has been described
(Fradinger and Sherwood, 2000
;
Krueckl et al., 2003
).
Neurokinin A
NKA, along with substance P, occurs in most excitatory cholinergic motor
neurons of the adult vertebrate gut, and acts on tachykinin receptors NK2 and
NK1 on smooth muscle cells and enteric neurons, and on NK1 receptors on ICCs
(Pennefather et al.,
2004).
Available information about the ontogeny of a tachykinin control system in
the gut emanates mainly from immunohistochemical studies, and agrees well with
the results of the present study. In birds and mammals, occasional nerves (and
endocrine cells) showing tachykinin-like immunoreactivity occur early during
fetal development, and mature into a more extensive nerve net before birth
(human, Paulin et al., 1986;
Larsson et al., 1987
; sheep,
Wathuta and Harrison, 1987
;
guinea-pig, Saffrey and Burnstock,
1988
; chicken, Saffrey et al.,
1982
; Epstein et al.,
1983
; duck, Lucini et al.,
1993
). Similarly, in the amphibian Xenopus laevis, both
nerves and endocrine cells are present in the gut before the onset of feeding
(Holmberg et al., 2001
).
However, to our knowledge, this is the first demonstration of functional
tachykinin receptors during this period. Whether the effect of NKA is direct
on the muscle cells or indirect via noncholinergic neurons or ICCs is
not known at this stage. It has been shown previously in adult cod Gadus
morhua and rainbow trout Oncorhynchus mykiss that tachykinins
may act both directly and indirectly in the fish intestine, involving
cholinergic and serotonergic pathways
(Jensen et al., 1987
;
Jensen and Holmgren,
1991
).
In conclusion, we present for the first time results suggesting that
functional receptors for PACAP, acetylcholine and NKA are present in the fish
gut before or around the time for onset of feeding. Furthermore, the effects
of atropine show that endogenous acetylcholine is released and acts on
muscarinic receptors in the larvae. It seems that both excitatory and
inhibitory pathways are well developed when the animal starts to feed. For
fish larvae it is of great importance to be able to digest and absorb food
early in development in order to survive. In halibut and turbot larvae, for
example, the survival chances increase if the larvae start feeding even before
the yolk has been completely absorbed
(Gadomiski and Petersen,
1988). A corresponding early development of a functional digestive
system, including gut motility, as our results imply, would of course further
enhance the chances of survival.
List of abbreviations
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Acknowledgments |
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References |
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