Development of oxygen sensing in the gills of zebrafish
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1
* Author for correspondence at present address: Department of Physiology and Biophysics, Dalhousie University, 5859 University Avenue, Halifax, NS, Canada, B3H 4H7 (e-mail: mjonz{at}dal.ca)
Accepted 1 March 2005
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Summary |
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Key words: O2 chemoreceptor, development, gill, hypoxia, neuroepithelial cells, zebrafish, Danio rerio
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Introduction |
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In mammals, developmental shifts occur in functional O2-sensing
pathways. For example, NEBs of the lung and adrenal chromaffin cells are
O2-sensitive during late fetal and neonatal stages, respectively
(Youngson et al., 1993;
Thompson et al., 1997
).
However, during the postnatal period and following the onset of aerial
respiration, NEBs decrease in number
(Reddick and Hung, 1984
;
Cho et al., 1989
) and
chromaffin cells lose their hypoxic chemosensitivity
(Thompson et al., 1997
).
Furthermore, during postnatal life there is an increase in the number of
sensory nerve fibres innervating the carotid body, the primary
O2-chemosensory organ in adults, and this coincides with a rise in
carotid body sensitivity to hypoxia (for reviews, see
González et al., 1994
;
Donnelly, 2000
). Although much
is known about respiratory development in fish and the effects of hypoxia
during early life (Rombough,
1988
; Burggren and Pinder,
1991
), there is currently little information regarding the
ontogenesis of peripheral O2-sensing mechanisms in aquatic
vertebrates. An investigation of the development of O2
chemoreception in the fish gill is of interest because a functional
respiratory system develops much faster than in mammals.
In teleost fish, four gill arches are innervated by the glossopharyngeal
(first arch only) and vagus nerves, and bear numerous gill filaments, where
O2-sensitive NECs reside (Jonz
and Nurse, 2003; Jonz et al.,
2004
), and respiratory lamellae. Despite the relatively late
formation of the gills, however, fish in embryonic and early larval stages
respond to hypoxia (for a review, see
Rombough, 1988
). In zebrafish,
the pharyngeal arches produce gill filament primordia at 3 days
postfertilization (d.p.f.) but the gills, which lack respiratory lamellae, do
not become functional until 14 d.p.f.
(Kimmel et al., 1995
;
Rombough, 2002
). However, the
hyperventilatory response to hypoxia
(Turesson et al., 2003
), and
changes in cardiac activity in larvae raised under chronic hypoxia
(Jacob et al., 2002
), appear
to develop before this time. This suggests the presence of an
O2-sensing mechanism before complete development of the gills.
Based on previous studies that have described the chemoreceptive properties of O2-sensitive NECs in adult zebrafish and a response to hypoxia in larvae, we sought to describe the development of the hyperventilatory response to hypoxia in zebrafish larvae, and to determine if these events were correlated with the appearance of O2-sensitive NECs and associated neural pathways in gill filament primordia. Using confocal immunofluorescence techniques and ventilation frequency analysis, we describe the development of a quinidine-sensitive, hypoxic response in zebrafish larvae that coincided with O2-sensitive NEC innervation. In addition, a quinidine-insensitive response to hypoxia that preceded the appearance and innervation of NECs of the gill filaments was identified.
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Materials and methods |
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Light microscope observations
Zebrafish larvae were lightly anaesthetized with 0.05 mg
ml1 MS 222 (ethyl 3-aminobenzoate methanesulfonate, Sigma)
in dechlorinated system water and placed in the well of modified culture
dishes (see below) on the stage of an inverted microscope (IM-35, Zeiss, Jena,
Germany). Phase-contrast images of developing gill arches and filaments were
captured using a digital camera (Retiga, QImaging, Burnaby, BC, Canada) and
imaging software (Northern Eclipse, Empix Imaging Inc., Mississauga, ON,
Canada). For images of circulating red blood cells in the gill filaments, a
rapid exposure time of 10 ms was used.
Confocal immunofluorescence
Neuroepithelial cells of the gill filaments are O2-sensitive
(Jonz et al., 2004), and were
identified in developing and adult zebrafish using confocal immunofluorescence
and criteria similar to those used by Jonz and Nurse
(2003
). These NECs were: (1)
located within the gill filaments, (2) epithelial, (3) serotonin (5-HT)
immunoreactive (IR) and (4) innervated. Other NECs were identified using
criteria (24). Zebrafish were killed by overdose with 1 mg
ml1 MS 222. Techniques for immunolabelling and confocal
imaging of adult gill tissue were similar to those previously described
(Jonz and Nurse, 2003
). Larvae
between 3 and 9 d.p.f. were fixed by immersion in phosphate-buffered solution
(PBS) containing 4% paraformaldehyde at 4°C overnight. PBS contained the
following: (in mmol l1) NaCl, 137;
Na2HPO4, 15.2; KCl, 2.7; KH2PO4,
1.5; pH 7.8 (Bradford et al.,
1994
). Fixed larvae were rinsed in PBS and permeabilized for
4872 h at 4°C with a solution (PBS-TX) containing 1% fetal calf
serum (FCS) and 0.5% Triton X-100 in PBS (pH 7.8). NECs of developing gill
filaments were identified in whole-mount preparations using antibodies
directed against serotonin (5-HT;
Dunel-Erb et al., 1982
;
Jonz and Nurse, 2003
). Neurons
and nerve fibres of the gill arches and developing filaments were identified
using antibodies against a zebrafish-derived neuron-specific antigen (zn-12).
zn-12 is a general neuronal marker in zebrafish
(Trevarrow et al., 1990
), and
its labelling of neural structures of the gill has been previously
characterized (Jonz and Nurse,
2003
). Polyclonal rabbit 5-HT antibodies (Sigma) were used at a
dilution of 1:200 and localized with goat anti-rabbit secondary antibodies
conjugated to fluorescein isothiocyanate (FITC, 1:50, Jackson ImmunoResearch
Laboratories Inc., West Grove, PA, USA). Monoclonal mouse anti-zn-12
(Developmental Studies Hybridoma Bank, University of Iowa, USA) was used at a
dilution of 1:100 and localized with goat anti-mouse secondary antibodies
conjugated with Alexa 594 (1:100, Molecular Probes, Eugene, OR, USA). All
antibodies were diluted with PBS-TX. Fixed larvae were incubated in primary
antibodies for 2448 h at 4°C and in secondary antibodies at room
temperature (2224°C) for 1 h in darkness. Gill baskets or
individual gill arches were removed from larvae with fine forceps and prepared
as whole mounts on glass microscope slides in Vectashield (Vector Laboratories
Inc., Burlingame, CA, USA) to reduce photobleaching. Whole-mount gill
preparations were examined in the longitudinal plane using an upright
microscope (Eclipse E800, Nikon, Melville, NY, USA) and a confocal scanning
system (Radiance 2000, BioRad, Hercules, CA, USA) equipped with argon (Ar) and
helium-neon (HeNe) lasers with peak outputs of 488 nm and 543 nm,
respectively. Images were detected using a photomultiplier tube and photodiode
array, and were collected using confocal graphics software (LaserSharp 2000,
BioRad). Each image is presented as a composite projection of serial optical
sections. Image processing and manipulation was performed using Corel Draw 9
(Corel Corp., Ottawa, ON, Canada).
Ventilation frequency measurements
Adult zebrafish were lightly anaesthetized with 0.1 mg
ml1 MS 222 dissolved in dechlorinated system water. Initial
tests were performed to determine if quinidine, a blocker of the
O2-sensitive background K+ current in chemoreceptive
NECs of the zebrafish gill (Jonz et al.,
2004), could produce changes in ventilation frequency, and
therefore be used to mimic the effects of hypoxia in whole-animal experiments.
Adult zebrafish were immersed in 500 ml of system water with or without
(control) 1 mmol l1 quinidine. In addition, adults were
exposed to voltage-dependent K+ channel blockers, i.e. 20 mmol
l1 tetraethylammonium (TEA) plus 5 mmol l1
4-aminopyridine (4-AP), or equimolar NaCl (control). TEA plus 4-AP did not
inhibit the hypoxic sensitivity of isolated zebrafish NECs
(Jonz et al., 2004
). The
frequency (min1) of buccal or opercular movements was
visually determined after the application of each drug for 5 min. Some
experiments were performed in the absence of MS 222 to observe general changes
in behaviour induced by these drugs. In experiments where the effects of
different concentrations of quinidine were tested on ventilation frequency in
adults, a fast-flow (30 ml min1) continuous perfusion
chamber was constructed. A small well was carved out of a plastic Petri dish
(60 mm) coated with Sylgard (Dow Corning Corp., Midland, MI, USA) and filled
with
4 ml of solution. Anaesthetized zebrafish were transferred to the
chamber and held lightly in place with a fine nylon mesh pinned to the bottom
of the dish to restrict excessive movements during experiments. The dish was
placed on the stage of a dissecting microscope (M6C-10, Lomo, Prospect
Heights, IL, USA) and the frequency of buccal or opercular movements was
observed during a 3 min perfusion of 0.1 mmol l1, 0.5 mmol
l1 or 1 mmol l1 quinidine. All solutions
contained 0.1 mg ml1 MS 222 at room temperature
(2224°C) and were adjusted to pH 7.4. Drugs were purchased from
Sigma.
The effects of hypoxia and 1 mmol l1 quinidine on
behaviour and ventilation frequency were further tested on embryos and larvae
of various developmental stages (210 d.p.f.). For these experiments,
developing zebrafish were placed in a small chamber and perfused continuously
at 4 ml min1. Embryos and larvae were lightly anaesthetized
with 0.05 mg ml1 MS 222 dissolved in dechlorinated system
water. Zebrafish were transferred in a Pasteur pipette containing a small
volume of system water (100 µl) to the central well (8 mm in diameter)
of modified culture dishes. Polystyrene culture dishes (Falcon, BD
Biosciences, San Jose, CA, USA) were modified by drilling a small central hole
in the bottom and attaching a glass coverslip to the underside with Sylgard. A
piece of fine nylon mesh was placed over the bottom of the dish on the inside,
which confined the embryos/larvae to the well. The dish was fitted with a
stainless steel collar that held the mesh in place and formed a perfusion
chamber. The dish was fixed to the stage of an inverted microscope (Axiovert S
100, Zeiss). Responses of developing zebrafish to hypoxia and quinidine were
determined by observing the rate of body/pectoral fin movements or
buccal/opercular movements, depending on the development stage. Embryonic and
larval fish rely on cutaneous respiration during early developmental stages
and exhibit increased movement of the pectoral fins and body when exposed to
hypoxia to facilitate gas exchange (for a review, see
Rombough, 1988
). In older
larvae (
3 d.p.f.), movement of the gills and operculum, and buccal
pumping, developed and were instead used to determine ventilation frequency.
Hypoxia (PO2=25 mmHg) was produced by bubbling
the solution in the perfusion reservoir with 100% N2 for at least
30 min prior to the experiment. The PO2 of
solution in the perfusion chamber was verified using a carbon fibre electrode
(10 µm, Dagan Corporation, Minneapolis, MN, USA) and an EPC 9 amplifier
(Heka Electronik, Lambrecht, Germany) as described previously
(Jonz et al., 2004
). Control
solution (PO2=150 mmHg) was contained in
another reservoir and bubbled with compressed air. Tubing used to transfer the
perfusate to the chamber was gas impermeable (Tygon, Saint-Gobain Performance
Plastics Corporation, Akron, OH, USA). The responses of zebrafish to hypoxia,
1 mmol l1 quinidine, or 1 mmol l1
quinidine plus hypoxia were determined after perfusing the chamber with each
solution for 3 min. All solutions contained 0.05 mg ml1 MS
222 at room temperature (2224°C) and were adjusted to pH 7.4.
Preliminary experiments indicated that higher doses of MS 222 (e.g. 0.1 mg
ml1) reduced the response of larvae to hypoxia, but 0.5 mg
ml1 did not. In all experiments reported in the present
study, ventilation or response frequency measurements were determined several
minutes after placing the animals in the observation chamber to ensure that
subsequent responses were not affected by handling or confinement with the
nylon mesh.
Statistical analysis
Ventilation frequencies and behavioural responses from all experiments are
reported as mean ± S.E.M. Student's
t-test was used to compare the means of two groups. For data analysis
necessitating multiple comparisons, analysis of variance (ANOVA) followed by
the Bonferroni post-test was employed.
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Results |
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NECs of the gill in developing zebrafish
The gill basket in developing zebrafish is ventrally-situated and is
composed of four bilateral pharyngeal arches that bear gill filament primordia
(Fig. 2AC). Gill arches
were observed in live specimens at 3 and 5 d.p.f., and red blood cells could
be seen moving through the early vasculature of developing filaments
(Fig. 2B). Isolated gills were
examined using confocal immunofluorescence in larvae between 3 and 9 d.p.f.
Developing gill filaments were observed as early as 3 d.p.f. and did not
contain 5-HT-IR NECs at this stage (Fig.
3A,B); however, other 5-HT-IR NECs of the gill arches were present
at 3 d.p.f. and appeared to be innervated by zn-12-IR nerve fibres
(Fig. 3A,B). This organization
of gill arch NECs continued throughout larval development (Figs
4A,B,
5C,D) and resembled that seen
in the adult stage (Fig. 1B).
Inaddition, zn-12-IR neurons were also observed in the gill arch at 3 d.p.f.
(Fig. 3B), and at later
developmental stages (Figs
4B,D,
5B). It is noteworthy that
although filament primordia did not contain NECs at 3 d.p.f., zn-12-IR nerve
fibres were observed in these regions (Fig.
3BD). These may form free nerve endings in the gill
filaments. At 5 d.p.f., 5-HT-IR NECs were observed in developing gill
filaments (Fig. 4A,C) that
resembled those of adults (Fig.
1A). At this stage, innervation of NECs of the filaments by
zn-12-IR nerve fibres was variable, indicating that formation of contacts
between filament NECs and nerve fibres may begin around this time.
Fig. 4B illustrates an example
of a zn-12-IR nerve fibre emanating from the branchial nerve that did not
reach 5-HT-IR NECs of developing filaments. By contrast,
Fig. 4D depicts the close
association or innervation of a NEC by a zn-12-IR nerve fibre at 5 d.p.f.
After 7 d.p.f., gill filaments were longer and primordia of respiratory
lamellae were first observed (Fig.
5A). By this time, NECs of the gill filaments clearly received
innervation from nerve fibres of the branchial nerve or gill arch neurons
(Fig. 5B), as was also observed
at 9 d.p.f. (Fig. 5C,D). NECs
of the respiratory lamellae, however, as described in adult zebrafish
(Jonz and Nurse, 2003), were
not observed in lamellar primordia during these developmental stages. The
major developmental events described in this section are summarized in
Fig. 9.
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Effects of quinidine on ventilation in adult zebrafish
Preliminary experiments (not shown) indicated that unanaesthetized adult
zebrafish immersed in 1 mmol l1 quinidine or 20 mmol
l1 TEA plus 5 mmol l1 4-AP displayed
unbalanced and erratic swimming behaviour. This suggests that these
K+ channel blockers were taken up across the gills. Interestingly,
only 1 mmol l1 quinidine had the additional effects of
inducing hyperventilation and surface-skimming behaviour in larvae,
reminiscent of hypoxia-like responses. As shown in
Fig. 6, in anaesthetized
adults, 1 mmol l1 quinidine significantly increased
ventilation frequency from 161.1±13.4 min1 to
207.8±5.1 min1 (P<0.005). In contrast,
zebrafish exposed to TEA plus 4-AP or an equimolar substitution of NaCl
(control) showed no significant change in ventilation frequency
(P>0.05; ANOVABonferroni test). The dose-dependent effect
of quinidine on ventilation frequency is illustrated in
Fig. 7 for adult zebrafish
studied in continuous-perfusion experiments. Doses of 0.5 and 1 mmol
l1 quinidine significantly increased ventilation frequency
above control values (P<0.05; ANOVABonferroni test).
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Effects of hypoxia and quinidine on ventilation in developing zebrafish
Experiments were performed in which a behavioural response or ventilation
frequency was quantified in zebrafish embryos and larvae (210 d.p.f.)
after stimulation with hypoxia or quinidine, to determine the time during
development when O2-sensitive pathways become functional. Based on
data indicating that gill NECs were consistently innervated by 7 d.p.f.
(Fig. 5), we predicted that
this developmental event would have a significant physiological impact on the
ventilatory response. In a continuously perfused chamber, zebrafish embryos
responded to hypoxia as early as 2 d.p.f. Embryos at this stage exhibited no
observable behaviour under control conditions (normoxia), but after exposure
to hypoxic solution embryos responded with a significant increase in the
frequency of pectoral fin and body movements (11.2±2.8
min1; N=11; P<0.05; Student's
t-test). Buccal or opercular movements were not observed at this
stage. As shown in Fig. 8A, at
3 d.p.f. the response to hypoxia included a significant increase in the
frequency of buccal and opercular movements (i.e. hyperventilation) from
2.8±1.0 min1 during normoxia to 42.5±12.5
min1 during hypoxia (P<0.05; Student's
t-test). This response was irregular in frequency, but synchronous
with movement of the pectoral fins, suggesting that the latter response may
indeed act to improve cutaneous gas exchange in larvae, as previously reported
(see Rombough, 1988). This
coordinated ventilatory response to hypoxia continued throughout development
and became regular in frequency at 8 d.p.f. An increase in basal ventilatory
frequency and a dramatic rise in the hyperventilatory response to hypoxia
occurred at 7 d.p.f. (Fig. 8A).
In normoxia, ventilation frequency was 45.6±15.4
min1, whereas in hypoxia ventilation significantly increased
to 200.8±23.0 min1 (P<0.05; Student's
t-test). This rise in the hyperventilatory response to hypoxia at 7
d.p.f. was significantly greater than the response at earlier stages
(P<0.001; ANOVABonferroni test), and did not increase
further at 9 d.p.f. These results are consistent with the innervation and
involvement of a greater number of O2-sensitive chemoreceptors.
|
Based on previous work, which established that quinidine mimics the hypoxic
response in isolated gill NECs from adult zebrafish by inhibiting background
K+ channels and inducing depolarization
(Jonz et al., 2004), and the
present data indicating that quinidine induced hyperventilation in adult
zebrafish (Fig. 7), we asked if
development of a quinidine-sensitive hypoxic response could be demonstrated at
the whole-animal level in zebrafish larvae. In a continuously perfused
chamber, the ventilatory response of zebrafish larvae to 1 mmol
l1 quinidine was absent at 3 d.p.f.
(Fig. 8B) in larvae that
responded to hypoxia (not shown), indicating the absence of a quinidine
response when NECs of the gill filaments were absent. However, at 7 d.p.f.,
ventilation frequency significantly increased from 45.6±15.4
min1 in controls to 122.4±35.6 after quinidine
application (P<0.05; Student's t-test), and a similar
response was observed at 10 d.p.f. (Fig.
8B). It is also noteworthy that the ventilatory response of 7
d.p.f. larvae to quinidine plus hypoxia (208.5±69.6
min1), compared to hypoxia
(Fig. 8A) or quinidine
(Fig. 8B) alone, did not differ
significantly (P>0.05; ANOVABonferroni test). Taken
together, these data suggest that the effects of hypoxia and quinidine may act
through overlapping pathways in the gill to modulate ventilation, and this
parallels our previous demonstration of the occlusive effect of quinidine on
the hypoxic sensitivity of isolated zebrafish NECs
(Jonz et al., 2004
). Thus,
these results indicate that the appearance of a maximal response to hypoxia,
and quinidine sensitivity, at 7 d.p.f. approximately coincides with
innervation of filament NECs in developing zebrafish. Before this time, the
response to hypoxia is quinidine-insensitive and appears to be independent of
NECs of the gill filaments. The above results are summarized in
Fig. 9.
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Discussion |
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The first 5-HT-immunoreactive (IR) cells to appear during development were
those of the pharyngeal or gill arches, which were present on or before 3
d.p.f., and appeared to receive innervation from a bundle of zn-12-IR nerve
fibres. These cells displayed morphological features characteristic of NECs,
such as their epithelial location, storage of a neurotransmitter (i.e. 5-HT)
and innervation. However, their location in the gill arches instead of the
gill filaments, and their lack of innervation from neurons intrinsic to the
filaments in adults, preclude their designation as O2-sensitive
NECs (Jonz and Nurse, 2003;
Jonz et al., 2004
) at this
point. In addition, NECs of the gill arches were distinct from 5-HT-IR
Merkel-like cells of the gill rakers that associate with taste receptor cells
(M. G. Jonz and C. A. Nurse, unpublished observations; see also
Zaccone et al., 1994
;
Hansen et al., 2002
). However,
since NECs of the gill arches are innervated and persist in adults, it is
plausible that they may play an important physiological role in the gill,
perhaps similar to O2-sensitive NECs of the filaments.
Alternatively, gill arch NECs may well be progenitors or precursors of
O2-sensitive NECs of the filaments.
While gill filament primordia were observed as early as 3 d.p.f. (see also
Kimmel et al., 1995), NECs
were not observed in these structures until 5 d.p.f. By this time, filament
NECs resembled the adult morphology and were close to the external medium and
arterial blood supply, as indicated by the presence of circulating red blood
cells in the filaments. In addition, at 5 d.p.f. some NECs of the filaments
appeared to receive innervation from zn-12-IR nerve fibres emanating from the
branchial nerve of the gill arch, the major supply of sensory innervation to
the gill filaments from the glossopharyngeal and vagus nerves
(Nilsson, 1984
;
Sundin and Nilsson, 2002
). The
present findings confirm a previous report that the cranial nerves innervating
the gill reach the gill arches by 3 d.p.f.
(Higashijima et al., 2000
).
NEC innervation in the filaments was more common in 7 and 9 d.p.f. larvae and,
although not investigated in this study, probably continued to increase with
the number of NECs throughout development. In adult zebrafish, many zn-12-IR
nerve fibres of the branchial nerve originate from a source extrinsic to the
gills and course distally through the filaments via a nerve bundle
and plexus, where they innervate NECs of both the filaments and respiratory
lamellae (Jonz and Nurse,
2003
). While we did not observe formation of the nerve plexus in
this study, this source of innervation appeared to resemble that of the
extrinsic nerve supply in adults. In addition, zn-12-IR neurons that were
found in the gill arches at all larval stages examined may also contribute to
the innervation of NECs of the filaments. In adults, such neurons intrinsic to
the gills are located within the filaments, where they innervate filament NECs
and extend processes that terminate at a proximal region of the efferent
filament artery (Jonz and Nurse,
2003
). It is possible that the neurons of the gill arch in larvae
may be pioneering neurons that migrate into the filaments and innervate these
structures later in development. Therefore, filament NEC morphology and
innervation patterns observed in larvae at approximately 7 d.p.f. are
reminiscent of those observed in adults, and may represent afferent sensory
pathways involved in O2 sensing in the gill (see
Jonz and Nurse, 2003
).
Development of a hyperventilatory response to hypoxia and quinidine
A behavioural response to hypoxia developed relatively early in zebrafish
and presented itself as an increase in frequency of pectoral fin and
whole-body movements in 2 d.p.f. embryos. Such a response has previously been
observed in embryonic and larval fish and may facilitate gas exchange across
the skin before the gills develop (for a review, see
Rombough, 1988). Zebrafish
larvae can rely completely on cutaneous respiration until at least 7 d.p.f.
(Rombough, 2002
). In addition,
the present study also demonstrates the coordinated activity of gill
ventilation with pectoral fin and body movements in larvae. Therefore, these
data suggest that the latter behaviours in embryos and larvae may indeed
improve cutaneous gas exchange and can be used to identify a response to
hypoxia before the hyperventilatory response develops. At 3 d.p.f., a
ventilatory response to hypoxia developed. Since NECs of the gill filaments
were not observed at these stages, this O2-sensitive response must
have originated from elsewhere.
Although the primary O2-sensitive chemoreceptors involved in the
hypoxia response in fish are located within the gills, several studies have
suggested the existence of other populations of extrabranchial O2
chemoreceptors (Burleson et al.,
1992; Milsom et al.,
2002
; Burleson and Milsom,
2003
). Given that convective O2 transport is not needed
in zebrafish larvae until
14 d.p.f.
(Jacob et al., 2002
),
approximately the time that the gills are needed for respiration
(Rombough, 2002
), the early
response to hypoxia observed in the present work is not likely to be the
result of stimulation of central chemoreceptors. Such a central
O2-sensing mechanism would require a functional system of
O2 transport to detect blood hypoxia. In addition, there is
currently no convincing evidence to indicate a role for central O2
chemoreceptors in respiratory regulation. Interestingly, Milsom et al.
(2002
) reported that only
sectioning of the trigeminal and facial nerves, in addition to
glossopharyngeal and vagus denervation, completely abolished the ventilatory
response to hypoxia in the tambaqui, and suggested that an additional group of
O2-sensitive chemoreceptors may be present in the orobranchial
cavity. Since a sensory component of the facial nerve also innervates the gill
arches in fish (Nilsson, 1984
;
Sundin and Nilsson, 2002
), our
results may further suggest that NECs of the gill arches (which also face the
orobranchial cavity) may contribute to O2 chemoreception and detect
changes in O2 tension in embryos and larvae before
O2-sensing pathways in the gill filaments develop. In mammals, for
example, neuroepithelial bodies (NEBs) of the lung and adrenal chromaffin
cells are O2-sensitive during late fetal and neonatal stages, and
may play a significant role in the transition to postnatal life and adaptation
to hypoxia during development (Youngson et
al., 1993
; Thompson et al.,
1997
; Cutz and Jackson,
1999
). Likewise, development of the hyperventilatory response to
hypoxia before complete formation of the gills in zebrafish may act to ensure
that O2-sensing pathways are functional by the time the larvae
become completely dependent on branchial respiration. Moreover, during
mammalian development there is an increase in the number of sensory nerve
fibres innervating type I cells of the carotid body, the primary
O2-chemosensory organ in adults, and this coincides with an
increase in type I cell and carotid body sensitivity to hypoxia (for reviews,
see González et al.,
1994
; Donnelly,
2000
). Similarly, we observed that after the first appearance of
NECs in the gill filaments in 5 d.p.f. zebrafish larvae, their innervation was
not consistently observed until 7 d.p.f. This increase in innervation of NECs
of the gill filaments corresponded to an increase in basal ventilatory
frequency and a rise in sensitivity of the ventilatory response to hypoxia
that reached a maximum at 7 d.p.f. These changes may have been due to an
increase in input to the central nervous system from activation of more
peripheral chemoreceptive pathways.
We further showed that filament NECs are indeed functional at 7 d.p.f., and
that an increase in innervation could account for the rise in the response to
hypoxia, by using the background K+ channel blocker, quinidine, to
inhibit O2-sensitive ion channels of gill NECs in vivo.
Quinidine has previously been shown to produce the same effects as hypoxia,
such as background K+ channel inhibition and membrane
depolarization, in several O2-sensitive cells
(O'Kelly et al., 1999;
Buckler et al., 2000
;
Campanucci et al., 2003
),
including NECs of the zebrafish gill filaments
(Jonz et al., 2004
). The
present experiments performed on adult zebrafish established that whole-animal
application of quinidine elicited a hyperventilatory response in a
dose-dependent manner. Although the specificity of the effects of quinidine
are difficult to determine when applied in this manner, the present data lead
us to propose that quinidine, like hypoxia, can stimulate NECs of the gill
filaments by inhibiting the O2-sensitive background K+
current when exogenously applied, leading to activation of sensory pathways.
This may have occurred via direct stimulation of NECs exposed to
quinidine in the external water, or secondarily, following uptake across the
gills. NECs reside within a permeable epithelium, where they are exposed to
water and the arterial blood supply, and may be capable of responding to
changes in both environments. Furthermore, although exogenous application of
quinidine and the voltage-dependent K+ channel blockers,
tetraethylammonium (TEA) and 4-aminopyridine (4-AP), clearly had other
behavioural effects on unanaesthetized adult zebrafish, only quinidine
produced the additional response of hyperventilation. In parallel with these
findings, quinidine, but not TEA or 4-AP, mimicked and occluded the hypoxic
response in isolated O2-sensitive NECs
(Jonz et al., 2004
). In
developing zebrafish, application of quinidine did not induce hyperventilation
in 3 d.p.f. larvae, indicating the absence of a non-specific quinidine
response when quinidine-sensitive NECs were not present, but induced
hyperventilation in 7 and 10 d.p.f. larvae once innervated NECs had appeard.
Moreover, because the hyperventilatory response of larvae to quinidine plus
hypoxia did not differ from the response to quinidine or hypoxia alone,
quinidine appeared to function through the same pathway as hypoxia to modulate
ventilation.
Thus, the present results, showing the development of a ventilatory
response sensitive to both hypoxia and quinidine, corroborate our findings of
the later appearance and innervation of O2-sensitive NECs of the
gill filaments, and suggest that this innervation is sensory and is required
to produce the ventilatory response to hypoxia. Interestingly, the behavioural
response of pectoral fin and buccal movements remained coordinated during the
stages examined, but did not become regular in frequency until 8 d.p.f. This
may relate more to central rather than peripheral pathways that appear to
develop later still in zebrafish larvae
(Turesson et al., 2003).
Results from this study may also suggest that since significant development of
the ventilatory response to hypoxia in zebrafish takes place in the absence of
NECs of the respiratory lamellae, which are similar to NECs of the filaments
in both their morphology and innervation
(Jonz and Nurse, 2003
),
lamellar NECs may not play a major role in O2 sensing, or are more
important during later developmental stages once the lamellae have completely
formed.
The present study describes the ontogenesis of peripheral O2 chemoreception in the gills of zebrafish and the hyperventilatory response to hypoxia, and is the first account of the correlation between such developmental events and identified functional O2-chemoreceptors in an aquatic vertebrate. We show that the development of an elevated response to hypoxia, and a quinidine-sensitive ventilatory response, occurred within the first week following fertilization and may be attributable to the appearance and innervation of O2-sensitive neuroepithelial cell (NECs) of the gill filaments. Before the appearance of filament NECs, zebrafish responded to hypoxia via another O2-sensing pathway, suggesting that shifts in O2-sensing sites occur with development in fish, as they do in mammals. Moreover, it appears that these developmental changes in functional O2-sensing pathways are not unique to air-breathing mammals, but may have appeared earlier in vertebrate evolution.
The results from this study form a foundation for future investigations in O2 chemoreception involving the use of mutagenesis and large-scale genetic screens. Since larvae can survive without the need for branchial respiration for many days, mutations affecting the gill are not expected to be lethal during this time. Behavioural assays designed to test the ventilatory responses of mutagenized zebrafish larvae to hypoxia and quinidine may facilitate the identification and characterization of mutations that affect the function of O2 sensing in NECs. Such advances may lead to a greater understanding of O2 chemoreception at the cellular level.
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