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First published online March 28, 2008
Journal of Experimental Biology 211, 1231-1242 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.015248
A comparative analysis of putative oxygen-sensing cells in the fish gill
Department of Zoology, University of British Columbia, 6270 University Blvd, Vancouver, BC, V6T 1Z4, Canada
* Author for correspondence (e-mail: ciuhandu{at}zoology.ubc.ca)
Accepted 12 February 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: fish chemoreceptors, neuroepithelial cells, serotonin, gill raker
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
These acute hypoxic responses are reflexly initiated by oxygen chemoreceptors,
but the sites of these receptors have not been definitively established.
Previous researchers have proposed that oxygen chemoreceptors in fish could be
located in the brain (Smatresk et al.,
1986), the arterial/venous vasculature
(Randall, 1982), the
orobranchial cavity (Milsom et al.,
2002), the pseudobranch
(Laurent and Dunel, 1980) or
the gills (Burleson and Milsom,
1993; Reid and Perry,
2003; Sundin et al.,
2000). It has also been proposed that the receptors in the gills
are situated in locations that sense either external oxygen tension
(PO2) in the incident flow of water
(PwO2), internal
PO2 within the gill vasculature itself
(PaO2 or PvO2),
or both (Randall, 1982).
Of note is that the arterial blood supply to the first gill arch of fish is
homologous to the carotid artery where the carotid body, the primary
peripheral chemoreceptive organ in mammals, is found. Furthermore, putative
O2 chemosensing cells in the first gill arch and carotid body have
similar ultrastructure (Bailly et al.,
1992; Jonz and Nurse,
2003), patterns and levels of afferent nerve discharge
(Milsom and Brill, 1986;
Burleson and Milsom, 1993),
embryonic origin (Milsom,
1998), innervation by the glossopharnygeal (CN IX) nerve
(Sundin and Nilsson, 2002;
Gonzalez et al., 1994) and
chemoreceptive mechanisms (Jonz et al.,
2004).
The putative oxygen chemosensing cells in fish gills are neuroepithelial
cells (NECs). Neuroepithelial cells have been characterized in the respiratory
epithelia of all classes of vertebrates. First described in the airway
epithelia of mammals (Feyrter,
1938) as neuroepithelial bodies (NEBs), this general group of
amine-containing cells is characterized by granulated vesicles, proximity to
efferent and afferent nerve fibres, and fluorescence when treated with
formaldehyde vapour (Pearse,
1969). The first report of NECs in fish gills used
formaldehyde-induced fluorescence to reveal both isolated and clustered
populations of cells in the primary epithelium of the fish gill, characterized
by the presence of dense-cored vesicles (DCV, 80–100 nm in diameter)
containing monoamines (serotonin) in proximity to neurons
(Dunel-Erb et al., 1982). Since
this first report, NECs have been detected in every fish studied to date with
the majority of studies focusing exclusively on the first gill arch.
Using antibodies for serotonin (5-hydroxytryptamine; 5-HT), a transmembrane
synaptic vesicle marker (SV2) and a general neuronal marker raised in
zebrafish (zn12), Jonz and Nurse (Jonz and
Nurse, 2003) showed a strong association between NECs and both
intrinsic and extrinsic nerve fibres in zebrafish gills. NECs of zebrafish are
located along the central axis of the gill filament as well as the edges of
the lamellae, locations that would be ideal for sensing both
PaO2 and PwO2,
respectively. NECs have been previously shown to degranulate during acute
hypoxia (Dunel-Erb et al.,
1982), suggesting that neurotransmitter release occurs, and
subsequent studies found that this was probably mediated by inhibition of
background K+ channels leading to membrane depolarization
(Jonz et al., 2004). Chronic
hypoxia, however, resulted in NEC hypertrophy and extension of cell processes
(Jonz et al., 2004) revealing
time–domain effects of hypoxia on NECs and further supporting their role
as chemoreceptors.
Rainbow trout Oncorhynchus mykiss respond more vigorously to
arterial than aquatic hypoxia and have relatively poor hypoxia tolerance
(Tetens and Lykkeboe, 1981;
Burleson and Milsom, 1990;
Burleson and Milsom, 1993).
Carp, on the other hand, respond only to aquatic hypoxia and are extremely
hypoxia tolerant (Hughes et al.,
1983) (A. E. O'Neil, A. L. Lumsden and W.K.M., manuscript in
preparation). Given the data presented above, we hypothesized that such
differences between physiological responses to hypoxia in these two species
would be associated with different distributions of putative
O2-sensing cells in the gills that reflect each species' hypoxia
tolerance. The objective of the present study was to further test the above
hypotheses in several species with varying hypoxia tolerances, and to
comprehensively analyze all putative O2-sensing regions within the
gills. Thus, as well as examining NEC distributions in trout and carp, species
which have been the subject of previous investigations, we collected data from
two previously unstudied species of closely related Amazonian fish that differ
in their hypoxia tolerance (traira Hoplias malabaricus and
trairão Hoplias lacerdae). Based on our hypothesis, we
predicted that the hypoxia-intolerant fish (trout and trairão) would
have NEC distributions that favour sensing arterial oxygen levels, whereas
hypoxia tolerant species (goldfish Carassius auratus and traira)
would have distributions that favour sensing environmental oxygen levels.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Tissue preparation
The first and second gill arches were excised from each fish and washed in
ice-cold phosphate-buffered saline (PBS) containing (in mmol
l–1): NaCl, 137; Na2HPO4, 15.2; KCl,
2.7; KH2PO4, 1.5; buffered to pH 7.8 with 1 mol
l–1 NaOH (Jonz and Nurse,
2003). Individual gill arches were syringe-perfused with
heparinized PBS (1000 i.u. ml–1) until filaments appeared
clear, and fixed by immersion in 4% paraformaldehyde in PBS at 4°C
overnight. Tissues were then rinsed in PBS and cryoprotected in a 30% sucrose
solution. Segments of the first and second gill arches were then frozen in
Tissue-Tek® (Sakura Finetek, Fisher Scientific, Ottawa, ON, Canada) at
–80°C until the blocks were either sectioned longitudinal to the
gill filament at 10–30 µm or transverse to the gill filament at
10–14 µm using a cryostat (Leica CM3050 S, Leica Microsystems,
Nussloch, Germany) and mounted on Superfrost® plus slides (VWR
International, West Chester, PA, USA) for immunohistochemistry.
Immunohistochemistry
Slides were washed in PBS and blocked in 10% normal goat serum (Jackson
Laboratories, distributed by Cedarlane Laboratories, Hornby, ON, Canada) for 1
h. Primary antibodies (used individually or in combination) were diluted in a
permeabilizing solution (PBS, 0.2% Triton X-100, 0.1% sodium azide) according
to the optimal dilutions, determined in preliminary experiments, detailed in
Table 1, and set on the slides
to incubate overnight at room temperature. Following incubation of the primary
antibodies, the slides were again washed in PBS. The slides were then treated
with fluorescently labelled secondary antibodies diluted in PBS
(Table 1) and left to incubate
at room temperature for 2 h in darkness. Following a final wash with PBS,
coverslips were mounted with Vectashield (Vector Laboratories, Burlington, ON,
Canada) to prevent photobleaching, and the edges of the coverslips were sealed
with nail polish. Slides were stored at 4°C until viewed under an
epifluorescence light microscope (Axioplan 2, Zeiss, Jena, Germany). Control
experiments were performed in which the primary antibodies were excluded to
control for non-specific binding of the secondary antibody, and normal serum
(IgG) from primary antibody host species was used (at an equivalent protein
concentration) to test for non-specific reaction of the antibodies' primary
hosts. The results of these control experiments showed only negligible
staining with no specific immunolabelling of the gill structures (data not
shown). Immunolabelled slides were observed under a light microscope (Axioplan
2, Zeiss, Jena, Germany) and digitally captured using a Q-Imaging (Burnaby,
BC, Canada) CCD camera and analyzed using image analysis software (Northern
Eclipse, Empix Imaging, ON, Canada). A few representative sections cut at
16–30 µm were further examined using a confocal scanning system
(Pascal 2, Axioskop X, Zeiss, Jena, Germany) equipped with argon (Ar) and
helium-neon (He-Ne) lasers with peak outputs of 488 nm and 543 nm,
respectively. Images obtained from the epifluorescence light microscope were
subsequently used for quantification of staining intensity and cell
counts.
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Quantification
Distribution patterns of 5-HT immunoreactive (5-HT-IR) cells were obtained
through analysis of average staining intensity from the tips of the lamellae
to the midline of the filament after subtracting background (threshold)
intensity. This distance was expressed relative to lamellar length, where 0
represents the lamellar tip and 100 represents the filament midline, to
standardize the distance measured in each individual fish for varying gill
sizes and across all species examined. Intensity thresholds were created for
each individual fish, and line intensity measurements were made along the
relative lamellar length of 140 individual lamellae from six individual fish
per species. This method of quantification displays the pattern of
immunoreactivity analyzed pixel by pixel from numerous microscope images.
Threshold and intensity analysis were conducted using SigmaScan Pro 5.0 (SPSS,
Chicago, IL, USA). Additionally, the numbers of 5-HT-IR cells were counted per
mm of filament (McCormick et al.,
2003) from six representative images per individual fish.
Transmission electron microscopy
Gill arches were fixed in 2.5% glutaraldehyde in a 0.1 mol
l–1 sodium cacodylate buffer (pH 7.35), rinsed with 0.1 mol
l–1 sodium cacodylate and postfixed in 2% osmium tetroxide in
0.1 mol l–1 sodium cacodylate. After washing in distilled
water, the preparations were dehydrated and embedded in Spurr's resin.
Sections (60 nm thick) were cut using a Leica Ultracut T (Vienna, Austria)
using a 35° Diatome diamond knife, and mounted on 150 mesh copper grids.
Specimens were viewed with a Hitachi H7600 (Hitachi Ltd, Tokyo, Japan)
transmission electron microscope and pictures were captured using a 1K AMT
(Advanced Microscopy Techniques Corp., Danvers, MA, USA) side mounted digital
camera.
Statistics
Data are presented as mean average intensity with 95% confidence limits,
and as mean ± s.e.m. for 5-HT-IR cell number. Differences between cell
numbers in filaments and lamellae within each species were compared using a
Student's paired t-test. One-way analysis of variance (ANOVA)
followed by a Tukey–Kramer post-hoc test was used to test
differences in filament and lamellar cell number among the four species.
P<0.05 was used to reject the null hypothesis. Statistical
analyses were performed using the software package JMP 5.1.2 (SAS Institute
Inc., Cary, NC, USA).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Counts of 5-HT-IR cells produced results similar to the average intensity profiles. The number of cells on the filaments compared with the lamellae of each species were significantly different in all fish (P<0.001) (Fig. 3). As well, significant differences were found between species (Fig. 3). Rainbow trout had a higher number of immunoreactive cells on the filaments than the three more hypoxia-tolerant species. Additionally, there was a significant difference in the numbers of 5-HT-IR cells in the lamellae between the species of Hoplias.
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Confocal images of gill sections doubly-labelled with anti-5-HT and a general neuronal marker (anti-zn12) traced nerve fibres solely down the filament in trout, traira and trairão (Fig. 5A,C,D), whereas in goldfish nerve fibres extended down the filament (Fig. 5B) and into the secondary lamellae (inset). There did not appear to be any nerve fibres extending out of the primary epithelium into the lamellae of trout, traira and trairão. Thus, although the 5-HT-IR cells along the lamellae of goldfish were innervated, in traira and trairão they did not appear to be innervated.
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Although there were distinct differences in the distribution of 5-HT-IR cells along the lamellae and filament, the distribution of 5-HT-IR cells at the tips of the gill filaments was consistent among the four species examined (Fig. 8). In all four species, clusters of 5-HT-SV2-positive cells were found at the tip of every filament, where the lamellae gradually decreased in size and the filament was directly exposed to the ambient environment (Fig. 8). A general neuronal marker (zn12) traced nerve profiles into the tip of the filament, and higher magnification images using confocal microscopy showed a close association between the 5-HT-IR cells and nerves (Fig. 9). This finding was characteristic of all fully intact filaments in all four species.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
McKenzie et al., 1991;
Sundin et al., 2000;
Florindo et al., 2006).
Additionally, electrophysiological recording of chemoreceptive nerve fibres in
the first gill arch (Milsom and Brill,
1986; Burleson and Milsom,
1993) and patch-clamp recording of isolated neuroepithelial cells
(NECs) (Jonz et al., 2004)
provided direct evidence of the chemoreceptive responses of the gill and NECs
to hypoxia. Since their first identification
(Dunel-Erb et al., 1982), gill
NECs have been documented in every fish species studied to date, and their
putative role in oxygen sensing and chemoreception has been supported by
histological and electrophysiological examination. Characterized by the
presence of serotonin stored in vesicles, NECs degranulate and depolarize in
acute hypoxia (Dunel-Erb et al.,
1982; Jonz et al.,
2004), presumably releasing vesicular serotonin either on target
cells, vasculature, or afferent nerves.
Within the fish gill, there are numerous potential sites for oxygen
sensing. Sites capable of monitoring arterial or venous oxygen levels include
the efferent filament artery (eFA), the central venous sinus (cvs) or the
afferent filament artery (aFA); aquatic hypoxia
(PwO2) may be detected where the lamellar,
filament, or gill raker epithelium is exposed to the ambient water.
Oxygen-sensing cells at these different sites could also be either
chemoreceptive or paracrine. A respiratory chemoreceptive cell releases a
neurotransmitter directly onto an afferent nerve thereby transmitting
information to the central nervous system to produce hypoxic reflexes, as in
the case of the glomus cells of the carotid body
(Krammer, 1978;
Gonzalez et al., 1994;
Nurse, 2005). Alternatively,
non-innervated cells releasing chemical signals play a paracrine role
(Nurse, 2005), releasing
neuroendocrine transmitters directly onto a nearby target cell, such as
vascular smooth muscle (Zaccone et al.,
1992). In our comparative analysis of potential
O2-sensing sites, we observed NECs that could serve both
chemoreceptive and paracrine roles based on their location and innervation.
Furthermore, our results indicated a striking difference in the distribution
of putative O2-sensing cells (chemoreceptive and paracrine), as
identified by colocalization of serotonin (5-HT) and synaptic vesicles (SV2),
among the four species examined.
Chemoreceptive NECs
In all species examined in this study, we observed a cluster of innervated
cells containing serotonin in vesicles at the filament tips (Figs
8,
9). NECs at the filament tips
have also been described in cod (Sundin et
al., 1998a), the Antarctic borch Pagothenia borchgrevinki
(Sundin et al., 1998b) and
zebrafish (Jonz and Nurse,
2003), and thus this appears to be a consistent location for
chemoreceptive neuroepithelial cells across species. NECs at the filament tip
are in an ideal location to sense changes in
PwO2, releasing serotonin directly onto an
afferent nerve, transmitting the signal to the central nervous system (CNS)
and producing a reflex hypoxic response. The presence of such a mechanism is
supported by physiological data, indicating that all fish included in this
study reflexly respond (increase ventilation) to aquatic hypoxia
(Burleson and Milsom, 1993;
Sundin et al., 1999) (A. E.
O'Neil, A. L. Lumsden and W.K.M., manuscript in preparation). More
specifically, denervation of the glossopharyngeal and vagus nerves in rainbow
trout (Reid and Perry, 2003)
and traira (Sundin et al.,
1999) abolished the hyperventilation induced by application of
exogenous (aquatic) NaCN, thereby localizing the external chemoreceptors to
the gills. Subsequent studies supported this finding in other fish species
(Milsom et al., 2002;
Florindo et al., 2006),
indicating that external NaCN stimulates hyperventilation by chemoreceptors
located in the gills monitoring the aquatic environment.
Although innervated NECs were consistently present at the filament tip, we found species differences in the distribution of innervated NECs along the centre of the filament. Innervated cells containing serotonergic vesicles were present in high number in the filament core of rainbow trout, less so in trairão and traira, but not at all in goldfish (Figs 4, 5). The filament is flanked by an efferent filament artery (eFA) and afferent filament artery (aFA), with a central venous sinus (cvs) running down the middle of the filament, making it an ideal location to sense internal oxygen levels (PaO2 or PvO2). Examination of cross sections of the filament in trout, traira, and trairão (Fig. 7) revealed that most NECs along the central filament were associated with the eFA, a location that would sense the oxygenated blood returning to the systemic circulation. NECs in the centre of the filament may play a chemoreceptive role, sensing internal (PaO2) hypoxia and producing a reflex response.
Examination of cross sections of goldfish filaments failed to identify any
NECs in the filament (Fig. 7).
This is in contrast to the finding of a previous study that identified
innervated 5-HT-IR cells in the primary filament of goldfish
(Saltys et al., 2006).
Although the discrepancy between our findings and those of Saltys et al.
(Saltys et al., 2006) may be a
reflection of differences in the preparations used in the two studies, this
seems highly unlikely. Saltys et al.
(Saltys et al., 2006) used
whole-mount preparations and we used transverse slices. Although slices are
thinner and there is the possibility that NECs are confined to a specific
plane that we failed to examine this seems unlikely. Furthermore, our findings
are supported by our inability to produce a response in carp to internal
injections of NaCN (A. E. O'Neil, A. L. Lumsden and W.K.M., manuscript in
preparation). Others (Eclancher and
Dejours, 1975), however, have found a response to internal
injections of NaCN. Thus, at the moment we cannot explain the discrepancy.
In goldfish, the NECs in the secondary lamellae were innervated, which is
consistent with previous findings (Saltys
et al., 2006). Furthermore, aquatic hypoxia caused lamellae to
protrude in the crucian carp Carassius carassius, increasing the
respiratory surface area as an adaptive and reversible morphological change
(Sollid et al., 2003). A
similar gross morphological alteration was seen in both crucian carp and
goldfish as a response to temperature
(Sollid et al., 2005), and it
is likely that this morphological change due to environmental factors may be
characteristic of Carassius. Therefore, it would be especially
advantageous for the O2-sensing cells of carp to be located at the
tips of the lamellae as opposed to the middle of the lamellae
(Fig. 4), where they are
continually exposed to the ambient water regardless of increases in
intra-lamellar cell mass associated with hypoxia- and temperature-induced
changes.
The finding of NECs in the filament and lamellae that are innervated
suggests that they release neurotransmitter (serotonin) across a synapse,
transmitting a signal to the CNS. Thus, these cells may serve a chemoreceptive
role, and physiological data supports the presence of hypoxic reflexes
stimulated by internal hypoxia in these species. Altering internal
O2 levels by hypoxemia, injection of hypoxic blood, or reduced
blood flow to the gills stimulates ventilation in the rainbow trout
(Holeton, 1971;
Smith and Jones, 1982). In
traira, ventilatory amplitude increased with both internal hypoxia
(PaO2) and bolus injections of NaCN
(Sundin et al., 1999). As
well, single fibre nerve recordings of the trout gills displayed
chemoreceptive afferent transmission in response to both internal and external
hypoxia (Burleson and Milsom,
1993). However, carp Cyprinus carpio did not respond to
internal injection of NaCN in some studies (A. E. O'Neil, A. L. Lumsden and
W.K.M., manuscript in preparation), which is consistent with our failure to
find innervated filamental NECs in a location able to sense blood oxygen
levels.
Additionally, we found innervated NECs in the gill rakers of goldfish and trout, an ideal location for sensing external hypoxia. However, double immunolabelling of these cells for a synaptical vesicle protein and serotonin identified a strong colocalization in goldfish (Fig. 10B), but not in trout (Fig. 10A). Moreover, when these cells were observed under a higher magnification, staining patterns in goldfish were consistent with those found in lamellar NECs (compare Fig. 4B with Fig. 10B inset), whereas in trout they were different from NECs found in the filament core (compare Fig. 4A with Fig. 11A, inset). These findings imply that 5-HT-IR cells in the gill rakers of goldfish release 5-HT as a neurotransmitter, but those in trout do not. In goldfish, NECs in the gill rakers can be an additional location for sensing changes in PwO2, consistent with the ability to respond to external hypoxia (A. E. O'Neil, A. L. Lumsden and W.K.M., manuscript in preparation). In trout, these 5-HT-IR cells might be Merkel-like cells associated with taste buds (reviewed by Zacconne et al., 1994). Merkel-like cells are serotonergic and innervated, similar to the cells seen in trout gill rakers. However, Merkel cells in teleosts are located at the base of the taste bud, whereas the 5-HT-IR cells found in trout gills are located exclusively in the top layer of the gill raker epidermis. 5-HT-IR cells in the gills were often neighbouring cells containing dense cored vesicles but not serotonin, thus these cells might be taste bud cells.
Paracrine NECs
The cells with serotonin-containing vesicles in the lamellae of traira and
trairão cannot play a direct role in afferent signalling to the CNS, as
there were no nerve fibres extending out of the primary epithelium and into
the lamellae (Figs 5,
7). Saltys et al.
(Saltys et al., 2006), using
the same neuronal marker (zn12), also did not see nerves extending into the
lamellae of juvenile trout using whole-mount fixed samples examined with
confocal microscopy. Jonz and Nurse, however, did see innverated lamellae in
the zebrafish (Jonz and Nurse,
2003). However, re-examination of our data with confocal
microscopy (Fig. 5) still did
not reveal innervated lamellae in trout, traira and trairão.
Furthermore, detailed reviews of gill morphology and branchial innervation do
not describe any innervation extending deep into the lamellae
(Laurent and Dunel, 1980;
Wilson and Laurent, 2002;
Sundin and Nilsson, 2002).
Thus, our data suggest that in addition to the chemoreceptors monitoring
the PwO2 at the filament tip, and
PaO2 in the efferent filament artery (trout,
traira, and trairão), traira, and trairão also have paracrine
cells potentially monitoring PwO2 in the
lamellae (Fig. 4). Acting in a
paracrine fashion, these cells may release serotonin, a potent
vasoconstrictor, directly onto the contractile pillar cells of the lamellae,
thereby optimizing respiratory surface area. Under resting conditions, fish
decrease the loss of ions across the gill by perfusing only two-thirds or less
of the lamellae (Booth, 1978;
Farrell et al., 1979);
however, hypoxic conditions require microcirculatory alterations to increase
the respiratory surface area to its maximum. Pillar cell contraction increases
lamellar blood spaces and drives blood across the full lamellar sheet, which
has a significant effect on matching the respiratory surface area with the
oxygen demands of the fish (Smith and
Johnson, 1977; Stenslokken et
al., 2006). In this way, these lamellar 5-HT-IR cells found in the
traira and trairão potentially monitor the hypoxic waters they
frequently inhabit, functioning to increase the respiratory surface area in
response to lowered PwO2. Lamellar NECs were
not present in the trout, which indicates that this fish species may not
require additional monitoring of the external environment, as rainbow trout
rarely encounter hypoxic waters. Additionally, in vivo studies of the
microcirculation of the rainbow trout gill did not show direct vasodilation in
the lamellae via pillar cell contraction
(Sundin and Nilsson, 1997),
which is consistent with our failure to find NECs in the trout lamellae (Figs
1,
4). In addition to
chemoreceptive responses, the NECs of the filament may act in a paracrine
manner. In vivo examination of rainbow trout gill vasculature exposed
to hypoxia demonstrated constriction of the eFA resulting in overflow to the
cvs and a continuous flow through the lamellae
(Sundin and Nilsson,
1997).
|
),
2.6 mmHg for goldfish (Burggren,
1982), and 8.7 mmHg for traira
(Perry et al., 2004). There is
no available data on the P50 value for trairão, but
an approximation of 13 mmHg can be made based on the critical oxygen tensions
(PcO2) of traira and trairão
(Rantin et al., 1992) and
P50 values of other Erythrinids
(Perry et al., 2004). In
general, there was a positive correlation between the number of 5-HT-IR cells
in the filament and the P50, and a negative correlation
with the number of 5-HT-IR cells in the lamellae
(Fig. 3).
This trend, however, is not supported by other lines of evidence. Single
fibre nerve recording from tuna gills revealed more fibres responsive to
alterations in internal hypoxia than external hypoxia, supporting the
suggestion that active, hypoxia-intolerant fish are more sensitive to a drop
in PaO2 than
PwO2
(Milsom and Brill, 1986).
However, the rainbow trout gill exhibited an equal number of fibres responsive
to internal or external hypoxia (Burleson
and Milsom, 1993), making this generalization speculative at best.
Furthermore, although there was a large difference in the neuroepithelial cell
distribution in the rainbow trout compared to the goldfish, traira and
trairão, we did not observe the hypothesized difference in cell
distribution related to hypoxia tolerance between the two Hoplias
species. Although the traira is well adapted to life in shallow, stagnant,
hypoxic lakes, the trairão inhabits well-aerated waters of central and
southern Brazil (Rantin et al.,
1993). Consequently, the traira has a higher hypoxia tolerance
(PcO2=20 mmHg), higher Hb-O2
affinity (P50=8.7 mmHg), larger respiratory surface area,
lower metabolic rate and higher anaerobic capacity compared with the
trairão (PcO2=35 mmHg)
(Fernandes et al., 1994;
Rantin et al., 1993;
Perry et al., 2004). Filament
and lamellar cell counts were different in the two Hoplias species,
but the specific cell numbers did not produce the expected correlation to
hypoxia tolerance (Fig. 3). We
would have expected the more hypoxia-tolerant traira to have relatively more
putative NECs in the lamellae and fewer in the filament compared with
trairão, which was not the case. However, the difference in hypoxia
tolerances of these two Hoplias species is slight compared to the
larger difference in P50 between trout and goldfish. Based
on the data from the two Hoplias species, however, for now we cannot
distinguish whether the striking difference we see in NEC distribution between
trout and goldfish is due to differences in hypoxia tolerance or differences
in phylogeny. Attributing the difference in distribution to differences in
physiological responses to internal versus external hypoxia, and to
different actions of the putative O2-sensing cells must remain
speculative.
In conclusion, we describe innervated NECs at the filament tips that were present in all species in a prime location to sense PwO2, in agreement with physiological data, indicating that all fish studied to date respond to aquatic hypoxia (Fig. 12). It also appeared that there were putative chemoreceptors monitoring PaO2 surrounding the eFA, but the presence of these internal chemoreceptors were species specific and correlated with the ability to respond to internal (arterial) hypoxia. Additionally, we describe putative O2 chemoreceptors in the gill rakers of goldfish. As well, we propose a paracrine role for the non-innervated NECs we found in the lamellae, acting directly on the pillar cells to enhance respiratory surface area when exposed to aquatic hypoxia. Finally, we speculate that differences in NEC distribution are not due to phylogeny, but instead appear to be adaptations related to hypoxia tolerance.
LIST OF ABBREVIATIONS
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Bailly, Y., Dunel-Erb, S. and Laurent, P. (1992). The neuroepithelial cells of the fish gill filament: indolamine-immunocytochemistry and innervation. Anat. Rec. 233,143 -161.[CrossRef][Medline]
Booth, J. H. (1978). The distribution of blood
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Burleson, M. L. and Milsom, W. K. (1990). Propranolol inhibits O2-sensitive chemoreceptor activity in trout gills. Am. J. Physiol. 258,R1089 -R1091.[Medline]
Burleson, M. L. and Milsom, W. K. (1993). Sensory receptors in the first gill arch of rainbow trout. Respir. Physiol. 93,97 -110.[CrossRef][Medline]
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