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First published online March 17, 2006
Journal of Experimental Biology 209, 1261-1273 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02058
Chemoreceptor plasticity and respiratory acclimation in the zebrafish Danio rerio
Department of Biology, University of Ottawa, 10 Marie Curie, Ottawa, ON K1N 6N5, Canada
* Author for correspondence (e-mail: sfperry{at}science.uottawa.ca)
Accepted 21 December 2005
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Fish were exposed for 28 days to hyperoxia (PwO2>350 mmHg), or hypoxia (PwO2=30 mmHg) or hypercapnia (PwCO2=9 mmHg). Their responses to acute hypoxia or hypercapnia were then compared to the response of control fish kept for 28 days in normoxic and normocapnic water. In control fish, the ventilatory response to acute hypoxia consisted of an increase in breathing frequency while the response to acute hypercapnia was an increase in relative breathing amplitude. The stimulus promoting the hyperventilation during hypercapnia was increased PwCO2 rather than decreased pH. Exposure to prolonged hyperoxia decreased the capacity of fish to increase breathing frequency during hypoxia and prevented the usual increase in breathing amplitude during acute hypercapnia. In fish previously exposed to hyperoxia, episodic breathing continued during acute hypoxia until PwO2 had fallen below 70 mmHg. In fish chronically exposed to hypoxia, resting breathing frequency was significantly reduced (from 191±12 to 165±16 min–1); however, the ventilatory responses to hypoxia and hypercapnia were unaffected. Long-term exposure of fish to hypercapnic water did not markedly modify the breathing response to acute hypoxia and modestly blunted the response to hypercapnia.
To determine whether branchial chemoreceptors were being influenced by long-term acclimation, all four groups of fish were acutely exposed to increasing doses of the O2 chemoreceptor stimulant, sodium cyanide, dissolved in inspired water. Consistent with the blunting of the ventilatory response to hypoxia, the fish pre-exposed to hyperoxia also exhibited a blunted response to NaCN. Pre-exposure to hypoxia was without effect whereas prior exposure to hypercapnia increased the ventilatory responses to cyanide.
To assess the impact of acclimation to varying gas levels on branchial O2 chemoreceptors, the numbers of neuroepithelial cells (NECs) of the gill filament were quantified using confocal immunofluorescence microscopy. Consistent with the blunting of reflex ventilatory responses, fish exposed to chronic hyperoxia exhibited a significant decrease in the density of NECs from 36.8±2.8 to 22.7±2.3 filament–1.
Key words: zebrafish, Danio rerio, hypoxia, hyperoxia, hypercapnia, neuroepithelial cell, serotonin, breathing
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; Sundin and Nilsson,
2002). The chemoreceptors, when stimulated by changes in
O2 and/or CO2/pH, initiate a variety of
cardiorespiratory and hormonal responses, including changes in breathing rate
and/or amplitude, heart rate and systemic resistance and plasma catecholamine
levels (Satchell, 1959;
Randall and Smith, 1967;
Holeton and Randall, 1967)
(see reviews by Fritsche and Nilsson,
1993; Smatresk,
1990; Burleson et al.,
1992; Perry and Gilmour,
2002; Sundin and Nilsson,
2002; Reid and Perry,
2003). The chemoreceptor cells share many histological and
cytochemical similarities with mammalian carotid body glomus cells
(Fritsche and Nilsson, 1993)
and indeed they are likely to be the evolutionary precursors of the mammalian
carotid body, the main site for sensing changes in blood gases in mammals
(Gonzalez et al., 1992).
Like other components of the nervous system, respiratory control systems
exhibit marked plasticity. This plasticity can be morphological and/or
functional and is based on prior experience
(Baker et al., 2001;
Mitchell and Johnson, 2003).
There are several potential sites of respiratory neuroplasticity, including
the sensory chemoreceptors themselves, signal transmission pathways, central
rhythm generation or pattern formation control
(Powell et al., 2000).
Together with genotype, age and gender, the partial pressure of respiratory
gases can influence plasticity (Mitchell
and Johnson, 2003). In the wild, fish can be exposed to
fluctuations in environmental O2 and CO2 levels both
diurnally and spatially (Crocker et al.,
2000). Consequently, fish have developed behavioural,
physiological and morphological mechanisms of acclimating to fluctuating
environments, a reflection of their respiratory plasticity
(Perry and Gilmour, 2002;
Burleson et al., 2002;
Sollid et al., 2003;
Jonz et al., 2004).
Ventilatory acclimation to hypoxia (VAH) is one of several examples of
hypoxia-induced respiratory plasticity in mammals. VAH may manifest itself as
an increase in breathing response to subsequent hypoxia owing to heightened
chemoreceptor and central nervous system (CNS) sensitivity
(Forster et al., 1971;
Sato et al., 1992;
Aaron and Powell, 1993;
Bisgard and Neubauer, 1995;
Soulier et al., 1997;
Dwinell and Powell, 1999;
Bisgard, 2000;
Powell et al., 2000;
Baker et al., 2001). Only a
single study (Burleson et al.,
2002) has assessed VAH in fish; that study demonstrated that
catfish Ictalurus punctatus exposed to chronic moderate hypoxia (75
mmHg; 1 mmHg
1 Torr133.3 Pa) exhibited a heightened ventilatory
sensitivity to acute hypoxia.
Hyperoxia-induced respiratory plasticity has been reported in mammals
(Lahiri et al., 1987;
Liberzon et al., 1989;
Torbati et al., 1989) and is
manifested by a reversible blunting of the ventilatory response to hypoxia. To
our knowledge, no studies have yet addressed the potential for respiratory
plasticity in fish exposed to hyperoxia.
In mammals, pre-exposure to hypercapnia does not alter the response to
acute hypoxia or hypercapnia (Remmers and
Lahiri, 1998; Kondo et al.,
2000). Despite the accruing evidence that environmental
CO2 is a potent and specific ventilatory stimulant in fish
(Heisler et al., 1988;
Graham et al., 1990;
Milsom, 1995;
Perry et al., 1999;
Burleson and Smatresk, 2000;
McKendry et al., 2001;
Perry and Reid, 2002;
McKenzie et al., 2003)
(reviewed by Gilmour, 2001),
we are unaware of any studies that have examined respiratory acclimation to
hypercapnia in fish.
The present study focused on evaluating respiratory plasticity in zebrafish
Danio rerio following exposure to chronic hypoxic, hyperoxic or
hypercapnic conditions. This was accomplished using a non-invasive recording
method that registers the change in voltage that is transferred through the
water during opercular movements (Almitras and Larsen, 2000). To implicate
morphological changes of branchial chemoreceptors
(Jonz and Nurse, 2003;
Jonz and Nurse, 2005;
Jonz et al., 2004) as a
mechanism of plasticity, gills were analyzed by confocal immunofluorescence
microscopy.
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Verification of the method for continuous monitoring of breathing frequency and relative breathing amplitude
In a separate experimental series, zebrafish were placed in a breathing
recording chamber constructed at University of Ottawa. The chamber was a
cylindrical transparent plastic tube (length=3 cm, diameter=1 cm). Two
electrodes (standard copper–tin wires) were submerged in the water
inside the chamber and separated by a distance of approximately 2 cm. Coarse
mesh was inserted into each end of the chamber to prevent the fish from making
contact with the electrodes. Each chamber was provided with continuous water
flow (
10 ml min–1). The experiments were filmed using a
Canon digital movie camera (model NTSC ZR70mc) and the images were transferred
to a personal computer using Digital Video Camcorder software (Ulead Video
Studio SE Basic Version 6.0). A scale bar was included in the visual field to
allow measurement of opercular displacement (in mm, the measure of ventilation
amplitude used in the present study). The analog voltage signals associated
with opercular movements were amplified using an amplifier made at University
of Ottawa, converted to digital data and stored on computer by interfacing
with a data acquisition system (Biopac Systems Inc., Goleta, CA, USA) using
AcqKnowledgeTM data acquisition software (sampling rate set at 50 000 Hz)
and a PentiumTM PC. A high pass filter (0.5 Hz) and two low-pass filters
(each 21 Hz) were built into the amplifier. The amplifier had a gain of 40 and
the Biopac system was set to a gain of 50 for a total gain of 2000.
Breathing frequencies (fR) and amplitude were measured independently by analyzing video recordings and AcqKnowledgeTM files. To assess the suitability of the electronic recording technique to reliably provide accurate measurements of breathing frequency and relative amplitude, data obtained from the two techniques were compared and subjected to correlation analysis. Opercular breathing movements produced oscillating voltage changes, with each breathing cycle producing a distinct minimum and maximum voltage. Thus, fR was determined by counting the number of voltage peaks over a set time interval. On the basis of video analysis, it was determined that opercular displacement during normal breathing was 1–3 mm (the sum of both operculae). Thus, for simplicity, the data acquisition was calibrated assuming that the voltage fluctuations at rest represented a 1 mm total opercular deflection. Thus, in all experiments, relative breathing amplitude was calculated as the difference between minimum and maximum values of voltage changes that were continuously recorded for each breathing cycle. Fish were exposed to hypercapnia (3.5 mmHg, see below) to induce changes in breathing amplitude that could be quantified independently by video analysis and electronic recording. To ensure a wide range of breathing frequencies to analyse by each technique, data were obtained from fish allowed to recover for varying periods of time after transfer to the respirometer.
To further prove that the measured voltage oscillations were exclusively related to breathing movements, four 28 day control fish (see below) were anaesthetized in situ with 0.1 ml ml–1 benzocaine. Breathing was absent in the anaesthetized fish but the heart continued to beat and some involuntary body movements remained. The signal obtained during anaesthesia was then compared to the signal obtained before and after the fish was in anaesthesia. These fish were not used for any other experiments.
Pre-exposure of fish to hypercapnia, hypoxia or hyperoxia
Zebrafish were exposed to hypercapnia (PwCO2=7–9
mmHg), hypoxia (PwO2=30–40 mmHg), or hyperoxia
(PwO2=350–450 mmHg) at 28°C in a 2 l tank for 28
days. Control fish were kept under similar conditions for 28 days but were
provided with normoxic and normocapnic water. For each treatment, at least two
groups of fish were pre-exposed at different times. Hypercapnia was achieved
by pumping mixtures of CO2 in air (1–2%) through a gas
equilibration column provided with aerated water. For hypoxia, the water
equilibration column was supplied with a mixture of 95% nitrogen
(N2) and 5% air. The gas mixtures were supplied by a Cameron Gas
Mixer (model GF-3/MP, Port Aransas, TX, USA). To achieve hyperoxia, pure
O2 (100%) was bubbled directly into fish tanks being supplied with
minimal volumes of normoxic-dechloraminated water (30 ml
h–1). Water PCO2 was measured using a
CO2 electrode (Cameron Instrument Company, model E201) connected to
a Cameron BGM 200 blood gas meter. Measurements of O2 were made
using a fiber optic oxygen electrode (Ocean Optics Foxy AL300, Dunedin, FL,
USA) and associated hardware and software (Ocean Optics SD 2000). After 28
days, the fish were tested for their ventilatory responses to acute hypoxia,
hypercapnia or external cyanide. They were then euthanized by overdose of
anaesthetic (1 mg ml–1 ethyl 3-aminobenzoate methanesulfonate
salt; MS 222), and the gills were removed and prepared for immunocytochemistry
(see below).
Ventilatory responses to acute hypoxia
A group of fish from each pre-exposure group was randomly chosen for this
experiment and were not used in any other experiment. Fish were placed in the
breathing recording chamber for 1–3 h prior to beginning of experiments,
to allow their breathing to become uniform. For each fish, resting breathing
amplitude (VAMP) was assumed to be 1 mm and the system was
calibrated accordingly. PwO2 within the fish chamber was
continuously measured by using a PO2 electrode calibrated
with solutions of sodium sulphite (20 mg ml–1;
PO2=0 mmHg) and air-saturated dechlorinated Ottawa
tapwater (PO2=153 mmHg). After taking the breathing
measurements for normoxic water, fish were exposed to hypoxia in seven equal
steps ranging from 130 to 20 mmHg. Hypoxic conditions were achieved by
bubbling N2, progressively increasing flow rates, through a
water–gas equilibration column that provided flowing water to the fish.
Continuous data recordings were obtained for ventilation frequency and
VAMP after the PwO2 had reached the
target value (10 min later). In each case, at least 5 min of breathing
data were analyzed to obtain mean values. Typically, fish were exposed to each
step of hypoxia for 15–20 min.
Ventilatory responses to acute hypercapnia
In a separate series of experiments, groups of fish from each pre-exposure
group were randomly chosen to assess the ventilatory responses to hypercapnia
and were not used in any other experiment. PwCO2 within
the fish chamber was continuously measured by a PCO2
electrode (Cameron Instrument Company, model E201) connected to a Cameron BGM
200 blood gas meter calibrated using mixtures of 0.25 and 1.0% CO2
in water provided by a gas-mixer (Cameron Instrument Company GF-3/MP). After a
1–3 h stabilisation period in normocapnic water
(PwCO2<0.5 mmHg; pH=7.4–7.5), measurements of
ventilation were initiated. Fish were then exposed to hypercapnia in three
steps: 1 mmHg, 2.5 mmHg and 3.5 mmHg, and then returned to normocapnia for a
final set of breathing measurements. Fish were exposed to each level of
hypercapnia for 15–20 min. Hypercapnia was achieved by bubbling
different percentages of CO2 through a water–gas
equilibration column providing flowing water to the fish. Breathing data were
obtained as described above.
To confirm that hypercapnia (elevated PwCO2) rather than the reduction in water pH was initiating the observed breathing changes, ventilation of eight control fish was monitored in acidified (pH=6.3) normocapnic water. A pH of 6.3 was chosen because it corresponded to the pH reached at the highest degree of hypercapnia (PwCO2=3.5 mmHg). Approximately 15 l of water was titrated with 1 mol l–1 HCl until the pH was lowered to 6.3. The water was then bubbled overnight with air having passed through a 10 mol l–1 solution of KOH (which reduces the amount of CO2 in air) and if necessary, the pH was adjusted to 6.3 the next day.
Ventilatory responses to acute hyperoxia
Ten fish were randomly selected from the 28-day control group and were not
used in any other experiment. Four fish exhibiting episodic breathing and six
fish displaying continuous breathing were subjected to acute hyperoxia.
PO2 within the fish chamber was continuously measured by a
fibre optic O2 electrode (see above). After breathing was assessed
in normoxic water, the water supplying the chambers was bubbled with
O2 through a water–gas equilibration column using a gas mixer
(Cameron Instrument Company GF-3/MP). Breathing was again analyzed once the
water PO2 had reached at least 300 mmHg.
Externally administered cyanide
In a separate series of experiments, groups of fish from each pre-exposure
group were randomly chosen for this experiment. Different concentrations
(0.5–200 µg ml–1) of sodium cyanide (NaCN) dissolved
in water were introduced into the water supplying the flow-through recording
chambers, and then flushed with cyanide-free water within 30 s. The NaCN flows
across the gills and interacts with externally oriented (water-sensing)
chemoreceptors and consequently stimulates the O2 chemoreceptors on
the gill arches. After each injection, respiratory values were recorded for
1–2 min. The next dose was administered at least 10 min later. The doses
of NaCN were determined based on pilot experiments in which
dose–response curves for respiratory responses to NaCN were studied.
Confocal immunofluorescence microscopy
The basic protocols for gill extraction, immunolabeling and confocal
imaging were modified from a previous study
(Jonz and Nurse, 2003).
Zebrafish were killed by overdose with anaesthetic (MS 222). Gill baskets were
rinsed in 1 mol l–1 phosphate-buffered saline (PBS; pH 7.4)
and fixed by immersion in 4% paraformaldehyde (prepared in PBS) at 4°C for
4 h. Fixed gills were rinsed in PBS and permeabilized for 24–48 h at
4°C in PBS containing 1% fetal calf serum and 0.5% Triton X-100 (pH
7.8).
Neuroepithelial cells (NEC) of gill filaments were identified in
whole-mount preparations using antibodies directed against serotonin (5-HT;
Jonz and Nurse, 2003) and
against synaptic vesicle protein (SV2;
Jonz and Nurse, 2003), found
in neuronal and endocrine cells. Neurons and nerve fibres of the gill arches
and developing filaments were identified using antibodies against a
zebrafish-derived neuron-specific antigen (ZN-12;
Trevarrow et al., 1990;
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 Alexa 488 (1:600,
Molecular Probes, Eugene, OR, USA). Monoclonal mouse anti-ZN-12 (Developmental
Studies Hybridoma Bank, The University of Iowa, Department of Biological
Sciences, Iowa City, IA 52242) was used at a dilution of 1:25. Monoclonal
mouse anti SV-2 (Developmental Studies Hybridoma Bank, The University of Iowa,
Department of Biological Sciences, Iowa City, IA 52242) was used at a dilution
of 1:100. Both anti-mouse antibodies were localized with goat anti-mouse
secondary antibodies conjugated with Alexa 546 (1:400, Molecular Probes). All
antibodies were diluted with PBS-TX. Fixed gill filaments were incubated in
primary antibodies for 4 days at 4°C and in secondary antibodies at room
temperature (22–24°C) for 1 h in darkness. Gill filaments were
prepared as whole mounts on glass microscope slides in Crystal MountTM
(Sigma) or Vectashield® (Vector Laboratories, Inc, Burlingame, CA,
USA).
Whole-mount gill preparations were examined with a confocal scanning system (Olympus BX50WI, Melville, NY, USA) equipped with an argon (Ar) laser. Images were collected using confocal graphics software (Fluoview 2.1.39, Melville, NY, USA). Each image obtained using a confocal scanning system is presented as a composite projection of serial optical sections size 0.3 µm. Image processing and manipulation was performed using Paint Shop Pro.
Gill baskets were also viewed using a Zeiss Axiphot light microscope and a digital Hamamatsu C5985 chilled CCD camera (East Syracuse, NY, USA). Images were captured using the Metamorph imaging system (Version 4.01).
The assessment of the number of chemoreceptors per filament and the percentage of the area occupied by chemoreceptors was done using Scion Image Beta 4.02 software (Frederick, MD, USA). Each gill was examined for the number of chemoreceptors on the filaments that were captured in their full length, and then the relative area of the filament was compared to the relative area of the chemoreceptors, as seen on the computer.
Statistical analysis
Ventilation frequencies and relative amplitudes from all experiments are
reported as means ± 1 standard error of the mean (s.e.m.). All data
sets were analyzed using two-way repeated-measures analysis of variance
(ANOVA). If a statistical difference was identified, a post hoc
multiple (`all pair wise') comparison test (Bonferroni's t-test) was
applied. Where appropriate, some data were analysed by one-way ANOVA followed
by Bonferroni's t-test (Table
1) or by unpaired Student's t-test
(Fig. 3 and
Fig. 7B). All statistical tests
were performed using a commercial statistical software package (SigmaStat
version 3.0).
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Pausing frequency
Episodic breathing during the 1–3 h pre-experimental period in
normoxic/normocapnic water was exhibited by 20% of all zebrafish that were
tested, and was composed of breathing and non-breathing (apnea or pause)
periods. Episodic breathing in normal water was never observed in the fish
that were pre-exposed to hypoxia or hypercapnia
(Table 1); hyperoxia
pre-exposure was without significant effect on the pattern of episodic
breathing. The average number of pauses (apneic periods) in all control (seven
out of 12 fish tested for acute hypoxia) and hyperoxia pre-exposed fish was 19
and 12 min–1, respectively (P=0.095). The total
non-breathing period in control fish was 28.7±5.2 s
min–1 compared to 35.5± 8.4 s min–1
in the hyperoxia pre-exposed fish (P=0.056).
The pausing frequency in ten control fish exposed to acute hyperoxia was not increased significantly: average for the four fish exhibiting episodic breathing during normoxia= 12.0±1.1 pauses min–1 versus 13.0±2.2 pauses min–1 during hyperoxia) (Fig. 3A) but the duration of time occupied by apnoeic periods was increased from 19.5±2.22 to 43.1±2.2 s min–1 (Fig. 3B). During acute hyperoxia, 80% of fish exhibited episodic breathing. Representative original data recordings from fish during normoxia and acute hyperoxia are illustrated in Fig. 3C,D, respectively. In contrast to acute hyperoxia, acute hypoxia decreased the number of breathing pauses and the total duration of apnoeic periods in control and hyperoxia pre-exposed fish (Fig. 4). However, while pausing frequency and duration of apnea were decreased significantly in control fish at a PwO2 of 130 mmHg and disappeared at 110 mmHg, the episodic breathing pattern in the hyperoxia pre-exposed fish disappeared only when PwO2 had fallen below 70 mmHg (Fig. 4A,B). Unlike acute hyperoxia or hypoxia, acute hypercapnia (Fig. 4C,D) did not significantly alter episodic breathing.
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Pre-exposure to hyperoxia
The respiratory responses of zebrafish that were pre-exposed to hyperoxia
(
350 mmHg for 28 days) to acute hypoxia, hypercapnia or external cyanide
are depicted in Fig. 7. The
breathing frequency response to hypoxia was significantly blunted. Indeed, a
statistically significant increase in frequency was observed only when
PwO2 had fallen to 40 mmHg compared to 110 mmHg in the
control fish. Furthermore, by re-plotting the data between
PwO2 values of 150 and 40 mmHg as linear regressions and
analyzing the slopes, it was possible to demonstrate a significant reduction
in the rate at which ventilation frequency increased during hypoxia in the
fish pre-exposed to hyperoxia (1.26±0.19 versus 0.68±
0.16 breaths min–1 mmHg–1 in control and
hyperoxic fish, respectively, Fig.
7B). The breathing amplitude responses to hypercapnia were
eliminated in fish pre-exposed to hyperoxia
(Fig. 7C) and the response to
external cyanide was blunted (Fig.
7D).
Immunocytochemistry results (representative pictures shown on Fig. 8) demonstrated that the numbers of 5HT-positive cells on the filament were significantly reduced (one-way ANOVA, P=0.04) in the fish pre-exposed to hyperoxia compared to other groups of fish (Table 1).
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Pre-exposure to hypercapnia
Except for increased breathing frequency at a PwO2 of
20 mmHg, the zebrafish pre-exposed to hypercapnia (9 mmHg for 28 days)
displayed similar responses to acute hypoxia as the control fish
(Fig. 10A). The ventilatory
response to hypercapnia was blunted (Fig.
10B) if one considers that breathing amplitude was not
significantly elevated until the final stage (3.5 mmHg) was reached. The
response to cyanide was significantly increased in the fish pre-exposed to
hypercapnia (Fig. 10C).
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)
was exploited to record non-invasively the fR and relative
breathing amplitude in adult zebrafish. By comparing the computer-acquired
data with video recordings taken of the same fish, it was demonstrated that
the voltage oscillations measured in the water were an accurate and
reproducible index of fR. Although this procedure, when
slightly modified, can be used to measure cardiac frequency
(Altimiras and Larsen, 2000),
there was no obvious contribution of the heart contraction to the measured
voltage changes based on a comparison of the data from breathing and
non-breathing (anaesthetized) fish. The technique also proved to be a reliable
indicator of breathing amplitude, assuming that opercular displacement
reflects ventilatory stroke volume. However, because there is no simple method
to measure the absolute magnitude of opercular displacement in resting fish,
the equipment could not be calibrated to obtain absolute data for breathing
amplitude. Thus, without further refinement, this method is only suited to
monitor relative changes in breathing amplitude. In the present experiments,
resting opercular displacement was assumed to be 1 mm in all fish regardless
of body mass and breathing frequency and thus 1 mm was selected as the
calibration factor for converting the peak-to-peak voltage oscillations to
measures of linear displacement. However, as the data in
Fig. 2B show, there can be a
marked difference in the absolute values of opercular displacement from those
calculated using the online recording system. The other significant limitation
with this technique is that any swimming movements beyond those required for
the fish to remain stationary often obscure the signals associated with
breathing. Thus, reliable data could only be obtained from fish that were
stationary within their chambers. In our experience, similar problems are also
encountered when attempting to analyze breathing motions from video recordings
when zebrafish are swimming or struggling.
Having validated the use of the non-invasive recording technique to monitor fR and relative opercular displacement, subsequent experiments were designed to evaluate the pattern of breathing in resting zebrafish and the impact of long-term acclimation to environments of altered gas composition.
Episodic breathing in zebrafish – the influence of acclimation or acute environmental change
Under resting conditions, 20% of the fish examined in this study exhibited
episodic breathing, characterized by periods of regular breathing interspersed
with periods of apnea or breathing pauses. Episodic (or periodic) breathing
has been described under resting conditions in all vertebrates
(Milsom, 1991), including
water-breathing fish (Smith et al.,
1983; Nonnotte et al.,
1993; Reid et al.,
2003). Most water-breathing fish that have been examined, however,
exhibit continuous breathing during normoxia, although under conditions of
lowered respiratory drive (e.g. hyperoxia), episodic breathing may occur (see
review by Milsom, 1991). In
the present study, pausing frequency was increased by hyperoxia and decreased
by hypoxia. These findings reinforce previous studies (e.g.
Reid et al., 2003) and are
consistent with the view that episodic breathing is shaped, at least in part,
by afferent input from peripheral chemoreceptors (see review by
Smatresk, 1990). The original
findings of the present study were that (i) long-term acclimation to hypoxia
or hypercapnia abolished episodic breathing (51 fish were assessed in normal
water) and (ii) acclimation to hyperoxia postponed the disappearance of
episodic breathing in fish exposed to acute hypoxia. Because all studies were
performed at least 3 h after acclimated fish had been returned to normal
water, the changes in breathing patterns presumably reflect a continuing
effect of the prior acclimation. Thus, if driven by changes in afferent
sensory input from peripheral chemoreceptors, the effects appear to endure for
at least 3 h after the chemoreceptors are once again experiencing normoxic and
normocapnic conditions. Further support for a long-term effect on breathing
patterns was the fact that episodic breathing continued in fish acclimated to
hyperoxia even under conditions of increased respiratory drive (hypoxia). It
would be interesting to determine the length of time required to re-establish
normal breathing patterns.
Acute respiratory responses to hypoxia or hypercapnia
As reported for other fish species, zebrafish displayed an increase in
fR in response to acute hypoxia (see table 1 in
Gilmour, 2001). Interestingly,
hypoxia did not elicit an accompanying increase in ventilation amplitude. This
pattern of response to hypoxia is different from that observed in most fish
that have been studied in which both fR and amplitude
increase (Shelton et al.,
1986). Although the common carp Cyprinus carpio also does
not display an increase in ventilation amplitude during hypoxia
(Soncini and Glass, 2000),
this response does not appear to be shared by all Cyprinids because the tench
Tinca tinca increases both fR and opercular
amplitude during hypoxia (Hughes and
Shelton, 1962). Furthermore, the absence of a ventilation
amplitude response to hypoxia in zebrafish cannot be attributed to the high
resting fR because a different group of fish from the same
population fish exhibited a pronounced increase in opercular displacement
during hypercapnia (see below).
External injections of NaCN (to pharmacologically stimulate O2
chemoreceptors) also caused an increase in fR without
altering breathing amplitude. This confirms that, in this species, at least
part of the hyperventilatory response to hypoxia is being mediated by
branchial (most likely external) O2 chemoreceptors (see reviews by
Burleson and Milsom, 1995;
Fritsche and Nilsson, 1993).
Recently, Jonz et al. (Jonz et al.,
2004) provided direct evidence that the neuroepithelial cells of
the zebrafish gill respond to hypoxia in a similar fashion as the glomus cells
of the mammalian carotid body and thus are likely to be the O2
sensors of the fish gill.
This is the first study to examine the respiratory response of zebrafish to
hypercapnia. As documented for other species (see table 2 in
Gilmour, 2001), zebrafish
responded to hypercapnia by increasing ventilation amplitude. However, unlike
in the majority of species previously examined, zebrafish did not display a
concomitant increase in fR
(Gilmour, 2001). Recent
evidence suggests that the cardiorespiratory responses of fish to elevated
CO2 are initiated largely by external branchial receptors that
respond to changes in ambient PCO2 rather than pH
(Burleson and Smatresk, 2000;
McKendry et al., 2001;
McKendry and Perry, 2001;
Perry and Reid, 2002;
Gilmour et al., 2005). The
results of the present study provided additional evidence that it is the
change of PwCO2 and not pH that is responsible for
increasing breathing amplitude during hypercapnia.
It is believed that the glomus cells of the mammalian carotid body sense
changes in both PO2 and PCO2 and thus
act as combined O2/CO2 chemoreceptors (Gonzales et al.,
1994; Zhang and Nurse, 2004;
Prabhakar and Jacono, 2005).
It is not known whether the O2-sensing neuroepithelial cells of the
fish gill are also able to detect changes in PCO2.
Although the ventilatory responses to hypoxia and hypercapnia in zebrafish
were markedly different (increased fR during hypoxia;
increased amplitude during hypercapnia), this does not exclude the presence of
a single receptor type sensing both O2 and CO2. Indeed,
it is plausible that stimulation of single cell type could be linked to varied
responses, given that the nature of the response is likely to be dictated by
downstream signal transduction pathways.
Acclimation to hyperoxia
This is the first study to assess the impact of long-term acclimation to
hyperoxia on ventilatory reflexes in fish. The results demonstrated that
exposure to hyperoxia for 28 days blunted the subsequent ventilatory response
to hypoxia, external cyanide and hypercapnia. Long-term hyperoxia is known to
cause a similar attenuation of the carotid body chemosensitivity to hypoxia
and cyanide in cats (Lahiri et al.,
1987; Lahiri et al.,
1990) and rats (Arieli et al.,
1988) but is apparently without effect on humans
(Gelfand et al., 1998), even
when the levels of hyperoxia approach the limits of toxicity. In those mammals
exhibiting a hyperoxic blunting of the ventilatory response to hypoxia, the
response to hypercapnia is sustained
(Torbati et al., 1989),
reduced (Lahiri et al., 1990)
or even enhanced (Lahiri et al.,
1987). The continuance of CO2 sensitivity in the face
of a severe blunting or abolishment of the response to hypoxia has led to the
idea that hyperoxia in mammals specifically targets O2-sensing
mechanisms of the carotid body. The carotid body also retains its usual
responsiveness to nicotine or dopamine following hyperoxia
(Lahiri et al., 1987;
Lahiri et al., 1990), further
suggesting that the blunting effects of hyperoxia are not caused by general
cellular damage. In the present study, the reflex hyperventilatory response to
hypercapnia was eliminated (at least statistically;
Fig. 7C) by prior exposure to
hyperoxia. The coincident inhibitory effects of hyperoxia on O2-
and CO2-mediated reflexes suggest that a common element of
chemoreception is being affected. The simplest explanation is that the NECs,
functioning as dual O2 and CO2 sensors, are being
influenced by hyperoxia. In support of this idea, the density of gill filament
NECs was significantly reduced after 28 days of hyperoxia. Thus, we speculate
that the sensitivity of the ventilatory response to hypoxia or hypercapnia is
controlled, at least partially, by the numbers of NECs exposed to the inspired
water. This theory does not exclude the possibility that other levels of
levels of respiratory control are being impacted by hyperoxia.
Acclimation to hypoxia
Previous experiments evaluating respiratory acclimation to hypoxia have
focused on mammals. The results have demonstrated that the hypoxic ventilatory
response (HVR) is either increased after continuous chronic hypoxia
(Weil, 1986;
Bisgard and Forster, 1996;
Dwinell and Powell, 1999) or
unaffected (Powell et al.,
2000). To date, only a single study has investigated the
respiratory consequences of chronic hypoxia in fish
(Burleson et al., 2002). The
results of that study on catfish Ictalurus punctatus showed that 7
days of acclimation to moderate hypoxia caused an increase in
fR and increased sensitivity to hypoxia. In contrast, the
results of the present study using zebrafish revealed a significant reduction
of resting fR without any effect on the ventilatory
responsiveness to hypoxia or cyanide. The lack of an effect of hypoxia
acclimation on the acute responses to hypoxia or cyanide is consistent with
the finding that the density of gill filament NECs was unaltered by chronic
hypoxia. In a previous study, Jonz et al.
(Jonz et al., 2004) also
showed that the numbers of 5-HT positive NECs were unchanged by 60 days of
hypoxic exposure (35 mmHg) although their size was increased by 15%. However,
by combining a marker for synaptic vesicle protein (SV-2), it was demonstrated
that the total number of NECs (5-HT-positive and 5-HT-negative) was increased
by chronic hypoxia (Jonz et al.,
2004). Whether or not the 5-HT-negative NECs are also capable of
sensing O2 is unclear. Several explanations for the reduced
breathing frequency in the fish exposed to chronic hypoxia can be offered.
First, an enhancement of O2 transfer and blood O2
transport (Perry and Wood,
1989; Nikinmaa,
2001) may result in a lowering of the ventilatory convection
requirement. Second, the return of the fish to normoxia after 28 days in
hypoxic water may result in the perception of a state of relative hyperoxia by
the branchial O2 chemoreceptors.
Acclimation to hypercapnia
In mammals, studies suggest that pre-exposure to hypercapnia does not alter
the response to acute hypoxia or hypercapnia
(Bisgard and Forster, 1996;
Remmers and Lahiri, 1998;
Kondo et al., 2000). Like in
mammals, the ventilatory response of zebrafish to hypoxia was unaltered by
chronic hypercapnia although the response to external cyanide was increased
and the response to hypercapnia was modestly blunted. The attenuation of the
breathing response to cyanide without affecting the hypoxic response is
particularly interesting and suggests that hypercapnia might be influencing
the responsiveness of external branchial O2 chemoreceptors (thus
explaining an enhanced response to cyanide) without affecting, or even
possibly reducing, the responsiveness of internal O2 receptors.
Although the numbers of branchial filament 5-HT-positive NECs were unaffected
by chronic hypercapnia, we cannot exclude the possibility that 5-HT-negative
NECs were being targeted. Unfortunately, the single laser confocal microscope
used for this study did not allow us to co-localize 5-HT and SV-2 and thus we
were unable to identify the 5-HT-negative/SV-2-positive cells.
Perspectives
Currently, it is unknown whether the gill NECs, demonstrated to be
responsive to hypoxia (Jonz et al.,
2004) and sharing similar properties with the glomus cells of the
carotid body, are also able to sense changes in ambient
PCO2. Although no direct evidence was provided in this
study, the finding that hyperoxia blunted the breathing responses to
hypercapnia (as well as hypoxia), together with the result that hypercapnia
enhanced the response to cyanide, suggests a certain degree of interaction
between O2- and CO2-sensing and suggest that the NECs
may be acting as both O2 and CO2 chemoreceptors. Clearly
this is an area that warrants further investigation.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Statistically significant difference (P<0.05)
between the two groups.
