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First published online April 18, 2006
Journal of Experimental Biology 209, 1709-1715 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02199
The role of branchial and orobranchial O2 chemoreceptors in the control of aquatic surface respiration in the neotropical fish tambaqui (Colossoma macropomum): progressive responses to prolonged hypoxia
1 Department of Zoology and Botany, São Paulo State
University–UNESP, São José do Rio Preto, SP,
Brazil
2 Department of Physiological Sciences, Federal University of São
Carlos, 13565-905 São Carlos, SP, Brazil
3 Division of Life Sciences, University of Toronto at Scarborough,
Scarborough, OT, Canada
4 Department of Zoology, University of British Columbia, Vancouver, BC,
Canada
* Author for correspondence (e-mail: ftrantin{at}power.ufscar.br)
Accepted 7 March 2006
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Key words: cardiorespiratory reflex, hypoxia, O2 chemoresponse, respiratory frequency, heart frequency, ASR, inferior lip swelling, Colossoma macropomum
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). To alleviate the effects of hypoxia, this species
performs aquatic surface respiration (ASR) facilitated by the development of
lower lip dermal swelling (Val and
Almeida-Val, 1995). The lower lip is not involved in gas exchange
but serves as a mechanical structure that enhances skimming of the
well-aerated surface water across the gills
(Saint-Paul, 1988). Tambaqui
immediately begin ASR even in moderate hypoxia (50–70 mmHg), and the
frequency of ASR increases as the environment becomes more hypoxic. However,
the complete development of the swollen lip takes 3 h or more
(Rantin and Kalinin,
1996).
Several studies have examined the cardio-respiratory responses of tambaqui
to hypoxia. The O2 receptors eliciting the reflex bradycardia and
increase in breathing frequency during hypoxia were distributed on all gill
arches and sensed changes in both arterial blood and inspired water
(Sundin et al., 2000). By
contrast, the O2 receptors that triggered the elevation in systemic
vascular resistance and breathing amplitude during hypoxia were
extra-branchial. In this study (Sundin et
al., 2000), fish were exposed to hypoxia without access to the
surface where they could perform ASR, and for only a short period of time
(10–30 min), a period too short to induce lip swelling. The
cardiorespiratory reflex responses of tambaqui during long-term (6 h) exposure
to hypoxia (PO2=10 mmHg) were subsequently evaluated
(Rantin et al., 2002) and the
role of the various O2 receptors involved in the cardiorespiratory
reflex responses in eliciting ASR and the development of lower lip swelling
examined. Their data suggest that extrabranchial O2 receptors
participated in the initiation of ASR and the swelling of the inferior lip.
The main objective of the present study was to determine whether orobranchial
O2 chemoreceptors innervated by cranial nerves V and VII, could be
the extrabranchial receptors involved in these responses.
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130 mmHg (17.3 kPa)] at a constant temperature
(25°C). The fish were fed ad libitum with commercial food pellets
but were fasted for 2 days prior to experimentation.
Animal preparation
Fish were anesthetized in a benzocaine solution (100 mg
l–1; pre-dissolved in 3 ml of 70% ethanol). After anesthesia,
fish were transferred to a surgical table and their gills were artificially
ventilated with an aerated, weaker benzocaine solution (50 mg
ml–1). Using a Dremel® rotary tool, a hole was
drilled through the snout between the nostrils, and a flared cannula (PE-100)
was fed from inside the mouth out though the hole and secured with a cuff. The
fish were then fitted with ECG electrodes according to the method described
(Glass et al., 1991). One
electrode (+) was inserted and sutured in a ventral position between the gills
and the heart, and a second (–) in a ventrocaudal position close to the
pelvic fins. After surgery, the fish were placed in the experimental chamber
for at least 24 h to recover in normoxic water
(PwO2130 mmHg, 17.29 kPa) at their acclimation
temperature (25°C).
Ventilation
Ventilation rate (fR, breaths min–1)
was recorded by connecting the buccal catheter to a Narco P-1000B pressure
transducer and a universal coupler (Narco 7189) of a Narco Narcotrace 40
physiograph (Narco Bio-Systems, Houston, TX, USA). Ventilation amplitude
(VAMP) was measured in arbitrary units and expressed as
percent change from initial values (Sundin
et al., 1999; Sundin et al.,
2000).
Heart rate
Electrocardiography was used to record heart rate (fH,
beats min–1) by counting the number of QRS complexes
min–1. The ECG electrodes were connected to a universal
coupler and a third electrode (reference) was immersed in the water of the
experimental setup. This preparation produced ECG recordings equivalent to
those obtained from bipolar lead I of a standard human electrocardiograph.
Denervation of cranial nerves IX (glossopharyngeal) and X (vagus)
Fish were anesthetized and placed on a surgical table where they were
artificially ventilated as described above. The denervation followed the
protocol described (Sundin et al.,
2000). Under a stereoscopic microscope (Opto SM 2001, Opto
Electronics, São Carlos, SP, Brazil), the operculum was reflected
forward, and a small incision (2 cm) was made in the epithelium at the dorsal
end of the 1st and 2nd gill arches where they meet the roof of the opercular
cavity. The incision allowed access to cranial nerve IX and the branchial
branches of cranial nerve X. The branchial nerves of all gill arches were
carefully dissected free of connective tissue and cut with fine iris scissors.
The cardiac and visceral branches of the vagus were preserved in all
cases.
Denervation of cranial nerves V (trigeminal) and VII (facial)
This denervation followed the protocol described
(Milsom et al., 2002). Under
the stereoscopic microscope, the opercular and palatine branches of cranial
nerve VII, as well as all mandibular branches of cranial nerve V innervating
the orobranchial cavity were sectioned. This removed sensory information
arising from the mouth and buccal cavity. Two small branches of cranial nerve
VII innervating the opercular muscles were left intact which were sufficient
to produce opercular movements that could be monitored as an indication of the
frequency and amplitude of ventilation. The opercular branches of VII
innervating the floor of the mouth were accessed where they course over the
inner surface of the operculum, the palatine branches of VII were accessed
through a midline incision in the roof of the mouth. The mandibular branches
of V innervating the roof of the mouth were accessed bilaterally by rotating
the eyes and cutting the nerves, where they coursed over the back of the
orbit, through a small incision in the top of the conjunctiva. In all cases,
cranial nerves IX and X to the gills were intact.
The healing process in tambaqui was rapid, and the incisions were covered with `scar tissue' within 24 h. All denervations were documented using a video camera attached to the microscope and connected to an ATI Pro interface of a Pentium IBM PC, and confirmed post mortem by autopsy.
After surgery, fish were ventilated with aerated water, and as soon as they showed signs of arousal from anesthesia, they were transferred to the experimental system where they recovered for 24 h in normoxic water prior to experimentation.
Experimental system
To simultaneously examine the effects of hypoxia on ASR and respiratory and
heart frequencies, an experimental setup similar to that described
(Rantin and Kalinin, 1996;
Rantin et al., 1998) was used.
The system consisted of two chambers; an upper compartment, where the fish was
kept during the experiment, and a lower part, serving to gas the water with
N2. The water was continuously recirculated from the lower to the
upper compartment. The shape of the upper chamber allowed fish to remain on
the bottom or move up to the surface to perform ASR, whereas lateral movements
were restricted. This compartment was also equipped with two ventilators to
maintain a unidirectional air flow above the water surface. This `air tunnel'
removed the excess of N2 released from the water and kept a
constant atmospheric gas concentration on the water surface, so that the
PO2 of the surface layer was about 10 mmHg higher than in
the rest of the tank. The experimental temperature was kept constant
(25±1°C) by a thermostat (TRM 10.40, Terroni Equipments Ltd.,
São Carlos, SP, Brazil) controlling a heating coil placed inside the
lower chamber.
Movements and behavior were continuously monitored by means of a closed circuit TV (Sharp VL-L 310B video camera and Sansung CN-3355Z monitor) and recorded on videotape (Semp X470 VCR) to verify the occurrence of ASR.
Experimental protocol
The experiments were conducted in two phases: the first with the fish
intact, and the second with the fish denervated (groups IX+X, V and V+VII).
Thus, for each fish, the protocol consisted of an initial surgery and recovery
overnight. On the second day they were exposed to severe hypoxia
(PwO2=10 mmHg) for 360 min, following which they were
returned to normoxic water and allowed to recover. Recovery took approximately
1 h but fish were allowed to rest overnight. On the third day, fish were
denervated and kept undisturbed for a post-surgical recovery period of 24 h.
The denervated fish were then subjected to the same level of hypoxia for
another 360 min. Separate groups of 10 fish each were used for each denervated
group. For each of the V and V+VII groups, two animals underwent all surgical
procedures but the nerves were not transected (shams). The responses of these
animals in the second run were no different from those of the intact animals
in the first run and so the first run for each animal was used as the control
for the second trial post-denervation. The fR and
fH values were recorded during the last 10 min of each 30
min interval.
To verify the effects of hypoxia on the swelling of the inferior lip, the dimensions of the inferior lip (length and width) were measured using graduated calipers before and after exposure to hypoxia. An initial measurement in intact fish was made in the anesthetized animals during the implantation of the buccal cannula and EKG electrodes, and in denervated fish during the denervation procedures. Measurements in both groups were again made by re-anesthetizing the fish briefly at the end of the hypoxia protocol, before returning the fish to aerated water. Before and after the hypoxic exposure, the images of the inferior lips were digitized by means of a video camera coupled to the microscope (Opto SM 2001, Opto Electronics) and connected to an ATI Pro interface of a Pentium IBM PC. The digitized images were used to determine the area of the inferior lips by means of a computer program (Jandel Sigma Plot, Image Measurement Software, Version 3.0, Jandel Corporation).
Statistics
To compare changes in each variable over time at each O2
tension, a one-way repeated-measures analysis of variance (ANOVA) was
performed followed by a Tukey–Kramer multiple comparison test.
Differences were considered significant if P<0.05. A paired
t test was employed to compare the areas of the inferior lips of
intact and denervated fish during normoxia and hypoxia. Values are presented
as means ± s.e.m. The commercial package GraphPad InStat v. 3.0
(GraphPad Software Inc.) was used to carry out statistical analyses.
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Fig. 2 depicts the effect of severe hypoxia on the VAMP of intact and IX and X denervated (Fig. 2A), V denervated (Fig. 2B) and V and VII denervated (Fig. 2C) fish during 360 min. In the intact fish VAMP increased significantly during the first 60 min of exposure to severe hypoxia and remained elevated until the end of the experimental time (360 min). The VAMP was not abolished by denervation of cranial nerves IX+X, V and V+VII.
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ASR and inferior lips swellings
The effects of severe hypoxia on ASR and swelling of the inferior lips are
illustrated in Figs 4 and
5, respectively. Both intact
fish and fish following denervation of cranial nerves IX and X, performed ASR
and developed inferior lip swelling during the exposure to severe hypoxia. ASR
frequency (events h–1) increased significantly in intact and
IX and X denervated fish during the first hour and reached peak values after
180 min of exposure to severe hypoxia (17 and 11, respectively)
(P<0.05). While the peak levels of ASR in terms of events
h–1 were not sustained, the amount of time the fish spent at
the water surface was, ranging from 40% to 60% over the last 3 h in both
groups (Fig. 4B).
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ASR was completely abolished by bilateral section of cranial nerve V (Fig. 4) and cranial nervesV+VII (not shown).
Denervation of cranial nerves V, V+VII or IX+X did not affect the development of inferior lip swelling in response to severe hypoxia. All groups of tambaqui developed the same, significant (P<0.05) degree of inferior lip swelling (Fig. 5).
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). They observed discrete increases in both
fR and fH, but without statistical
significance. In the present study, however, these increases in both
fR and fH were significant. This may
reflect the much longer post-surgical recovery period used in the present
study (24 h). In traíra, Hoplias malabaricus, an increase in
fH and a non-significant decrease in blood pressure were
also recorded after complete bilateral gill denervation
(Sundin et al., 1999).
Considering that this denervation abolishes both afferent (sensory) and
efferent (motor) innervation of the gills, it is not clear if the changes in
fH originated from removal of sensory feedback or removal
of motor tonus to systemic arteries (i.e. a reflex increase in heart rate due
to a fall in blood pressure), i.e. whether these change represent cause or
effect.
Gill ventilatory responses to hypoxia
The ventilatory response to severe hypoxia that was observed in the present
study included a significant and sustained increase in breathing frequency. As
in other studies (Sundin et al.,
2000; Milsom et al.,
2002), in the present study we found that total gill denervation
in tambaqui abolished the increase in fR in response to
short-term exposure to hypoxia. We found that it also abolished the long-term
increase in fR. We did not quantify changes in respiratory
amplitude in this study, however. According to Milsom et al., the
hypoxia-induced increase in ventilation amplitude (VAMP)
could only be eliminated following denervation of both the gills and the
orobranchial cavity (Milsom et al.,
2002). This may explain the VAMP variation
observed in all denervation protocols, since in the present study gills and
orobranchial cavity were not denervated simultaneously, as was done in the
earlier study (Milsom et al.,
2002). Total gill denervation did not abolish ventilatory
responses to hypoxia in tench (Hughes and
Shelton, 1962), sea raven
(Saunders and Sutterlin, 1971)
or traíra (Sundin et al.,
1999). By contrast, the hypoxic ventilatory response was
completely abolished by gill denervation in channel catfish
(Burleson and Smatresk, 1990)
and gar (Smatresk, 1989).
Clearly there are large species differences in the role of the branchial
chemoreceptors in this response.
Milsom et al. also suggested that circulating catecholamines did not
contribute to the hypoxic ventilatory response
(Randall and Taylor, 1991) in
tambaqui since the exogenous application of catecholamines inhibited
ventilation (Milsom et al.,
2002). This suggests that if hypoxia becomes severe enough to
cause a release of catecholamines from chromaffin tissue into the circulation,
the net effect would be to depress ventilation. This is consistent with the
ideas advanced by Perry et al. (Perry et
al., 1992). Since no ventilatory depression was observed in the
present study, this suggests that the tambaqui were not undergoing severe
stress even at the levels of hypoxia we applied (10 mm Hg), possibly
reflecting the extreme hypoxia-tolerance of this species
(Perry et al., 2004).
Heart rate responses to hypoxia
With some exceptions, such as the sea raven
(Saunders and Sutterlin, 1971)
and five-bearded rockling (Fritsche,
1990), most teleosts exhibit a reflex bradycardia in response to
hypoxia. Also, in the majority of species so far studied, the bradycardia is
sustained during the entire hypoxic period. In species such as the dogfish
(Butler et al., 1977) and
traíra (Sundin et al.,
1999), however, the heart rate gradually returns to normoxic
levels despite sustained hypoxia. This was also the case with the tambaqui in
the present study; during long-term exposure to severe hypoxia, the
fH of intact tambaqui returned to normoxic values over 300
min of exposure. The O2 chemoreceptors eliciting the bradycardia
during short-term exposure to hypoxia are situated on all gill arches
(Sundin et al., 2000) and they
sense changes in both the blood and inspired water. Hypoxic bradycardia was
completely abolished by denervation of the branchial branches of cranial
nerves IX and X (Sundin et al.,
2000). Our data also confirm this. Interestingly, however, while
denervation of the opercular and palatine branches of cranial nerve VII, as
well as all mandibular branches of cranial nerve V, innervating the
orobranchial cavity, did not affect the initial bradycardia, it did abolish
the slow return of heart rate to starting values. Since heart rates were not
different between intact and denervated fish at 300 min, and the heart rate
did not statistically return to starting values in the intact fish at 360 min
in these trials either, it is difficult to read too much into this. That said,
heart rates were significantly lower in the denervated fish at 360 mincompared
to intact fish and the reasons for this are not clear, but could possibly be
the result of a more severe hypoxaemia in the denervated fish compared to
controls. Without blood gas data we cannot confirm this.
ASR and development of inferior lip swelling in response to severe hypoxia
Severe hypoxia rapidly induced ASR and slowly induced the development of
inferior lip swelling in intact tambaqui. These results are in agreement with
previous studies of others (Saint-Paul,
1988; Val and Almeida-Val,
1995; Rantin and Kalinin,
1996; Rantin et al.,
1998). Furthermore, fish with cranial nerves IX and X denervated
also performed ASR and developed inferior lip swelling, which is in agreement
with the data of Sundin et al. (Sundin et
al., 2000). However, denervation of the mandibular branches of
cranial nerve V innervating the orobranchial cavity completely abolished ASR.
And, while one previous study (Sundin et
al., 2000) also found that the development of inferior lip
swelling in branchial denervated fish was considerably lower than in intact
fish, in the present study it was observed that the development of inferior
lip swelling was practically the same in the intact and denervated fish. The
inferior lip swelling induced in tambaqui by severe hypoxia was also not
abolished by denervation of cranial nerves V and VII to the orobranchial
cavity in the present study. This suggests that the formation of inferior lip
swelling in tambaqui either (1) can be elicited by any one group of the
O2 chemoreceptors at multiple sites (we did not denervate all of V,
VII, IX and X simultaneously), (2) is controlled by O2 receptors
located outside the gills and orobranchial cavity, or (3) results from a
direct effect of hypoxia/hypoxemia on the lip tissue itself. Given the time
course of the response and the data collected to date, we favour the latter
possibility.
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Acknowledgments |
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References |
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