Extrabranchial chemoreceptors involved in respiratory reflexes in the neotropical fish Colossoma macropomum (the tambaqui)
1 Department of Zoology, University of British Columbia, 6270 University
Boulevard, Vancouver, British Columbia, Canada V6T 1Z4
2 Department of Physiological Sciences, Federal University of São
Carlos, 13565-905 São Carlos SP, Brazil
Present address: Physiology Division, Department of Medicine, University of
California, San Diego, La Jolla, CA 92093-0623, USA
Present address: Department of Zoophysiology, Göteborg University, Box
463S-405 30, Göteborg, Sweden
* e-mail: milsom{at}zoology.ubc.ca
Accepted 3 April 2002
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Summary |
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Our results also revealed the presence of receptors in the gills that account for all of the increase in ventilation amplitude and part of the increase in ventilation frequency during hyperoxic hypercarbia, a group or groups of receptors, which may be external to the orobranchial cavity (but not in the central nervous system), that contribute to the increase in ventilation frequency seen in response to hyperoxic hypercarbia and the possible presence of CO2-sensitive receptors that inhibit ventilation frequency, possibly in the olfactory epithelium.
Key words: fish, tambaqui, Colossoma macropomum, chemoreceptor, hypoxia, hypercapnia, ventilation, gills, orobranchial cavity, cranial nerve, catecholamine
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Introduction |
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In a previous study (Sundin et al.,
2000), we showed that the decrease in heart rate and increase in
breathing frequency exhibited by tambaqui exposed to environmental hypoxia
were reflexly elicited by the stimulation of receptors located exclusively
within the gills. The receptors responsible for elevating systemic vascular
resistance, breathing amplitude and swelling of the inferior lip and that
induced aquatic surface respiration during hypoxia, however, were located, at
least in part, at some site outside the gills. Extrabranchial receptors have
also been implicated in the increase in ventilation amplitude associated with
hypoxia in tench (Tinca tinca), sea raven (Hemitripterus
americanus) and traira (Hoplias malabaricus)
(Hughes and Shelton, 1962
;
Saunders and Sutterlin, 1971
;
Sundin et al., 1999
), but not
channel catfish (Ictalurus punctatus) and longnose gar
(Lepisosteus osseus) (Smatresk,
1989
; Burleson and Smatresk,
1990
). Several potential sites have been suggested for
extrabranchial oxygen receptors including the orobranchial cavity (innervated
by cranial nerves V and VII) (Hughes and
Shelton, 1962
; Butler et al.,
1977
), the central nervous system
(Satchell, 1961
;
Saunders and Sutterlin, 1971
)
and the heart and ventral aorta (innervated by visceral branches of the vagus
nerve) (Smatresk et al.,
1986
). It has also been suggested that hypoxia could produce
cardiorespiratory responses by its action on adrenal chromaffin cells, giving
rise to the systemic release of catecholamines
(Randall and Taylor,
1991
).
The goal of the present study was to examine each of these possible sites for the presence of extrabranchial receptors that reflexly contribute to the increase in respiratory amplitude during hypoxia in the tambaqui.
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Materials and methods |
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Animal preparation
All animals were anaesthetised in an aqueous solution of benzocaine (100 mg
l-1) predissolved in 2 ml of 70% ethanol. A surgical level of
anaesthesia was achieved in approximately 5 min. During surgery, the gills
were ventilated with a second solution of benzocaine (50 mg l-1)
gassed with air. Impedance electrodes were sutured to each operculum to
monitor the breath-by-breath displacement of the operculum and to measure
ventilation rate (fv) and an index of ventilation amplitude
(V AMP). Using a Dremel tool, a hole was drilled through the snout
between the nostrils, and a flared cannula (PE 160) was fed from inside the
mouth out through the hole and was secured with a cuff. This allowed
administration of NaCN solutions into the buccal cavity in order to stimulate
O2 chemoreceptors throughout the orobranchial cavity monitoring the
respiratory water.
Series I (denervation studies, unanaesthetized fish)
In one group of fish, following the general preparation, the operculum was
reflected forward, and a small incision (approximately 1.5 cm) was made in the
epithelium at the dorsal end of the gill arches where they meet the roof of
the opercular cavity. This permitted access to cranial nerves IX
(glossopharyngeal) and X (vagus) innervating the gill arches
(Fig. 1). The branchial nerves
to all gill arches were carefully dissected free of connective tissue and cut
with fine iris scissors (gill-denervated, GD, group, N=9). In all
cases, the cardiac and visceral branches of the vagus were left intact.
Tambaqui do not have a pseudobranch, so this produced complete gill
denervation. In a control group (control, C, N=9), all nerves were
exposed but left intact. In a third group (gills and orobranchial cavity
denervated, GOD, N=8), following gill denervation, the opercular and
palatine branches of cranial nerve VII and all mandibular branches of cranial
nerve V innervating the orobranchial cavity were also sectioned
(Fig. 2). This removed sensory
information arising from the mouth and buccal cavity. We left two small
branches of cranial nerve VII intact, and these were sufficient to produce
opercular movements that could be monitored as an indication of the frequency
and amplitude of ventilation. The opercular branches of cranial nerve VII
innervating the floor of the mouth were accessed where they course over the
inner surface of the operculum, the palatine branches of cranial nerve VII
were accessed through a midline incision in the roof of the mouth and the
mandibular branches of cranial nerve V innervating the roof of the mouth were
accessed bilaterally by rotating the eyes and sectioning the nerves, where
they coursed over the back of the orbit, through a small incision in the top
of the conjunctiva.
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All animals in series I were allowed to recover from surgery, and experiments were performed the following day. All denervations were confirmed post mortem by autopsy, and all data presented here are from fish for which complete denervations could be confirmed.
Series II (denervation studies, decerebrate fish)
In this series, following the general preparation, the fish were placed in
a Perspex chamber filled with flowing water and secured into a stereotaxic
apparatus. A mouthpiece fitted with a ventilation tube was sewn into the
mouth, and the fish was placed on artificial ventilation. The fish was secured
in such a way that the gills and operculum were submerged in the water in the
box while the top of the head was above the water. Using a Dremel tool, the
top of the cranium was removed and the brain exposed. The adipose material
surrounding the brain was removed by suction, the forebrain aspirated
(decerebration) and the rostral space so produced was packed with cotton. The
spinal cord was then severed (spinalization) slightly caudal to the second
spinal nerve to prevent the animal from moving. In the control group (DC,
N=12), once the animal was decerebrate and spinalized, anaesthesia
was discontinued and the animal was ventilated with aerated water. In nine of
these animals (decerebrate, gills denervated, DGD group) following the control
experimental run, cranial nerves IX and X were then denervated within the
cranial cavity where they exit the medulla. This produced a total central
denervation of these nerves, denervating not only the branchial branches to
the gills but also all the visceral and lateral-line branches of the vagus
nerve. In a final group of nine fish (decerebrate, gills and orobranchial
cavity denervated, DGOD group), these procedures were repeated in animals in
which the opercular and palatine branches of cranial nerve VII and the
mandibular branches of cranial nerve V had just been sectioned peripherally as
described in series I.
All animals were left for 2 to stabilize before experiments began. Again, all denervations were confirmed post mortem by autopsy.
Series III (central perfusion studies)
Fish used in this study (N=6) were prepared in an identical
fashion to the control fish in series II.
Series IV (pharmacology studies)
In this series (N=6), following the general preparation, a cannula
(PE50) was inserted into the ventral aorta through the afferent branchial
artery of the third gill arch on one side. These fish were then allowed to
recover from anaesthesia, and experiments were performed the following day. No
nerves were denervated in this group.
Experimental protocols
Series I (denervation studies, unanaesthetized fish)
In this group, animals were placed into individual cylindrical tubes housed
within larger experimental tanks (approximately 801) with free-flowing aerated
water. This prevented the fish from swimming to the surface to perform aquatic
surface respiration. Mesh covered the ends of the cylindrical tubes to allow
rapid equilibration between the tubes and the larger tanks. A large slit on
the top of the tubes permitted leads and cannulae to exit the tank. The tank
was covered to maintain a dark and quiet environment for the fish. After at
least 24h of recovery from surgery, the fish were first subjected to a series
of external (into the snout cannula) injections of water (control) and NaCN (1
ml of 1 or 2 mg ml-1 in water). In each case, the cannula was
flushed with 1.0 ml of water to ensure complete drug delivery. After each
injection, respiratory variables were recorded for 3-5 min.
The animals were next subjected to abrupt, progressive environmental hypoxia produced by shutting off the airflow and gassing the tank holding the fish with nitrogen. The water PO2 was lowered from an air-saturated level of 18.6 kPa (140 mmHg; 25 °C) to 1.3 kPa (10 mmHg) over approximately 10 min. At this point, the nitrogen flow was halted, airflow was restored and the water PO2 was gradually returned to normoxic levels.
Animals were then subjected to progressive environmental hyperoxia produced by shutting off the airflow and gassing the tank holding the fish with 100 % O2. The water PO2 was increased from an air-saturated level of 18.6 kPa (140 mmHg; 25 °C) to 80 kPa (approximately 600 mmHg) over approximately 10 min. At this point, the oxygen flow was halted, airflow was restored and the water PO2 gradually returned to normoxic levels.
Finally, the C and GD groups were subjected to abrupt, progressive
environmental hyperoxic hypercarbia by gassing the tank with a mixture of 5 %
CO2 in 100 % O2. We had determined in a previous study
(Sundin et al., 2000) that
exposure to 5 % CO2 was necessary to produce a reliable respiratory
stimulus in this species. With this level of CO2, the water pH fell
from approximately 7.0 to 5.0 over 10 min. The animals were then returned to
normocapnic conditions, and cardiorespiratory variables were monitored until
the water pH returned to at least 6.5. As this procedure failed to stimulate
ventilation in the GD group, it was not repeated in the GOD group.
Series II (denervation studies, decerebrate fish)
In this series, fish were left in the stereotaxic apparatus following
surgery and artificially ventilated with well-aerated water for at least 2 h.
The animals were then exposed to the same protocol used in series I with one
small difference. In this series of experiments, a separate reservoir of water
was pregassed with each gas mixture so that the transition from one gas to
another was immediate. Animals were left on each experimental gas mixture for
at least 10 min before being returned to aerated water. In this study, all
groups (DC, DGD and DGOD) were exposed to all gas mixtures.
Series III (central perfusion studies)
In this series, all fish were artificially ventilated with well-aerated
water throughout. The cranial cavity was initially superfused with
well-aerated saline (0.9 % NaCl) buffered to pH 7.8 by the addition of a small
amount of CO2. Flow was maintained by gravity at a rate of 5 ml
min-1 from a series of pre-gassed reservoirs. After a 15 min
control period, the superfusate was switched to a hypoxic (1.3 kPa or 10 mmHg
PO2), a hyperoxic (80 kPa or approximately 600 mmHg
PO2), a hypercapnic (5 kPa or 38 mmHg
PCO2), an acidic (pH 7.2) or an alkaline (pH 8.4) solution
for 15 min. The hypoxic, hyperoxic and hypercapnic solutions were all buffered
to pH 7.8, while the acidic and alkaline solutions were gassed with air. Test
solutions were presented at random with a 15 min period of superfusion with
well-aerated saline between each.
Series IV (pharmacology studies)
In this series of experiments, animals were maintained in cylindrical tubes
housed within larger experimental tanks with free-flowing, aerated water as
described for series I. The protocol began with an injection of 0.3 ml of
saline (0.9 % NaCl) into the ventral aorta to determine whether the vehicle
would cause any change in ventilation. Adrenaline was then injected in boluses
of 1, 10 and 100 nmol kg-1, and the catheter was flushed with 0.3
ml of saline following each injection. The animals were next subjected to the
same abrupt, progressive environmental hypoxia protocol described for series I
[water PO2 reduced from an air-saturated level
of 18.6 kPa (140 mmHg; 25°C) to 1.3 kPa (10 mmHg) over approximately 10
min]. The beta-blocker sotalol was then administered (3 mg kg-1),
and the highest dose of adrenaline (100 nmol kg-1) and the hypoxia
run were repeated to examine the effects of beta-blockade on the adrenaline-
and the hypoxia-induced ventilatory response.
Data analysis
In all experiments, the opercular impedance leads were connected to an
impedance converter to measure fV (breaths min-1) and
VAMP (arbitrary units). The partial pressure of oxygen in
the water (PWO2) was monitored continuously
(±0.1 mmHg) with an oxygen electrode (FAC 001 O2 electrode
and FAC 204A oxygen analyser) supplied, via a siphon, with a steady
flow of water from each experimental chamber. The electrodes were calibrated
with solutions of sodium bisulphate in borax
(PO2=0 kPa) and air-equilibrated water
(PO2=18.6 kPa; 25°C). Water pH was
continuously measured with a pH electrode calibrated with standard
solutions.
For each gas mixture (hypoxia, hyperoxia and hyperoxic hypercarbia), respiratory variables were analysed for a 1 min control period immediately prior to the run, for the final minute of the run and after 30 min of recovery. During the NaCN injection experiments, data were analysed for a 30s control period immediately prior to an injection of saline or NaCN and at 10 s intervals for the first minute post-injection. During the second and third minute post-injection, data were analysed for a 30 s period each minute. Maximum responses are reported. For the adrenaline injections, data were analysed for a 30 s control period immediately prior to an injection of saline or adrenaline, at 30 s and at 1, 2, 3 and 4 min post-injection.
fV is reported in absolute values. Since VAMP was measured in arbitrary units, VAMP and total ventilation (VTOT=VAMPfV) are reported as the percentage change from the control or starting value.
Statistical analyses
The data are reported as the mean ±1 standard error of the mean
(S.E.M.). Data were compared using one-way repeated-measure analysis of
variance (ANOVA) to test for the significance of changes in response to each
stimulus. If significant differences (P<0.05) were found, a
Dunnett's multiple-comparison test was used as a post hoc test. To
evaluate the effects of selective denervations on the responses to the
different treatments, a two-way repeated-measures ANOVA was used. For series
IV, paired comparisons were made using the Wilcoxon rank sum test.
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Results |
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Breathing frequency also increased during exposure to hyperoxic hypercarbia in both C and DC groups (series I and II respectively). Interestingly, while this increase was eliminated following complete branchial denervation in fish with intact brains (GD), it was not eliminated in the DGD or the DGOD groups (Fig. 3).
Hypoxia and external NaCN significantly elevated VAMP in control and GD groups in series I, while hyperoxia reduced VAMP in these same groups (Fig. 4). Hypoxia also produced an increase in VAMP in the decerebrate control (DC) and DGD groups (series II), but the decrease in VAMP seen during hyperoxia was not significant in any of the decerebrate groups. Following denervation of the gills and orobranchial cavity (GOD and DGOD groups in series I and II respectively), no significant changes in VAMP were seen in response to any of these experimental treatments.
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Ventilatory amplitude also increased during exposure to hyperoxic hypercarbia in both C and DC groups (Fig. 4), and these increases were eliminated by complete branchial denervation alone in both groups.
In series I, the effects of hypoxia, hyperoxia and NaCN on total ventilation (VTOT) were reduced, but still significant, following total branchial denervation but completely abolished following gill and orobranchial denervation (Fig. 5). In the decerebrate fish in series II, the responses to hypoxia and hyperoxia were smaller than those of the fish in series I and were eliminated by complete branchial denervation alone.
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By contrast, while the increase seen in VTOT during hyperoxic hypercarbia in series I was eliminated following complete branchial denervation, it was not eliminated even following complete denervation of the gills and orobranchial cavity in the decerebrate fish in series II (Fig. 5).
Series III
There were no effects of central perfusion of the decerebrate brain on
fV or VAMP with any of the test solutions
(Fig. 6).
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Series IV
Adrenaline at 10 and 100 nmol kg-1 significantly decreased
ventilation rate and amplitude and, hence, total ventilation in
unanaesthetized, intact fish (Fig.
7). When sotalol was present, the decrease in frequency was
abolished, indicating that betareceptors were involved in the
adrenaline-induced depression of respiratory frequency. The reductions in
ventilation amplitude and total ventilation persisted following sotalol
treatment, however, although they were delayed. The increase in breathing
frequency during hypoxic exposure was also altered following sotalol treatment
(Fig. 8). Breathing frequency
now reached peak values at 5.3 kPa (40 mmHg) and then fell as
PWO2 was reduced further; breathing frequency
returned towards normoxic values more quickly during the recovery period. This
is in contrast to the progressive increase in breathing frequency seen in
control fish down to 1.3 kPa (10 mmHg) and the slow return of breathing
frequency to control levels on recovery. The pattern of change in
VAMP on exposure to hypoxia was unaffected by pretreatment
with sotalol.
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Discussion |
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We have previously shown that fish tolerate total gill denervation well.
Indeed, it was the failure of such denervation to remove all the hypoxic
ventilatory response that led to the present study. Our next step was to also
remove any contribution of sensory input arising from the heart and ventral
aorta (innervated by visceral branches of the vagus nerve). To do this, we
opened the cranium, decerebrated and spinalectomized the fish and made initial
recordings with all cranial nerves intact. Cranial nerves IX and X were then
sectioned centrally. This not only denervated the gills but also removed any
sensory information coming from the cardiac, visceral and lateral-line
branches of the vagus nerve. These had been left intact in our previous
studies. The responses to hypoxia obtained from both these groups of fish
(decerebrate, spinalectomized fish with all cranial nerves intact, DC, and
decerebrate, spinalectomized fish with transections of cranial nerves IX and
X, DGD) were extremely similar to the responses obtained from control fish (C)
and from fish that had undergone peripheral denervation of the gills (GD) in
both our previous (Sundin et al.,
2000) and current experiments. This validated our approach.
To eliminate possible extrabranchial oxygen receptors in the orobranchial cavity (innervated by cranial nerves V and VII), we then transected the mandibular branches of cranial nerve V as well as both the opercular and palatine branches of cranial nerve VII. This removed sensory information arising from the mouth and buccal cavity. We left two small branches of cranial nerve VII intact on each side, which were sufficient to produce opercular movements that could be monitored as an indication of the frequency and amplitude of ventilation. These movements were small but appeared to be sufficient to ventilate the fish adequately under resting, normoxic conditions, despite the fact that under these conditions all other motor output to the respiratory muscles had been transected. The primary evidence for this was that the brain-intact fish (GOD group) recovered from surgery, could be run through a full protocol 24 h later and had mean breathing frequencies that did not differ from those of control fish (63.4±7.0 versus 60.6±4.1 breaths min-1 in control fish).
While large decreases in arterial Po2 have been shown
to accompany bilateral gill denervation in the sea raven Hemitripterus
americanus (Saunders and Sutterlin,
1971), the lack of any elevation in resting breathing frequency in
the tambaqui suggests that blood gas levels must have been relatively normal
in the fish in series I (although this could also have resulted from such
offsetting factors as a reduced breathing frequency due to denervation coupled
with an increased breathing frequency due to lower levels of arterial
Po2). This was certainly not a concern for the fish in
series II since they were artificially ventilated. Resting ventilation aside,
the question remains, could these fish respond to respiratory stimuli in a
normal fashion? There is no doubt that the ability to generate forceful
respiratory movements was compromised in these fish, and the question
remained, could they increase breathing amplitude or could they only increase
breathing frequency? Could we distinguish whether the absence of an amplitude
response to hypoxia or hypercarbia was because the sensory receptors involved
in producing this response were removed or because the muscles capable of
generating this response were denervated? This is an important question since,
after this denervation, the only changes seen in breathing amplitude in
response to hypoxia and hyperoxic hypercarbia were small and insignificant.
The critical observation pertinent to this question comes from observation of
the variation that occurred in individual recordings and the responses of fish
to disturbance. This evidence suggests that all the fish were capable of at
least doubling the amplitude of their opercular movements, implying that this
experimental approach was also valid.
Responses to hypoxia, hyperoxia and NaCN
We have shown previously that total denervation of the gills in tambaqui
eliminated the increases in breathing frequency seen during hypoxia and in
response to internal injections of NaCN. However, it neither eliminated the
increase in breathing frequency in response to external cyanide nor the
increase in breathing amplitude that occurred in response to hypoxia or
external injections of NaCN (Sundin et
al., 2000). The results we obtained in the present study are
consistent with this with the exception that, this time, total gill
denervation did eliminate the frequency response to external cyanide. The
results of the present study go on to show, however, that all remaining
responses could be eliminated by further denervation of the orobranchial
cavity. Not surprisingly, the results also show that the receptors involved in
producing ventilatory adjustments of breathing frequency and amplitude in
response to hyperoxia (not examined in earlier studies) are innervated just as
are those involved in producing the responses to hypoxia. The responses of the
fish in series I and II were very similar and, thus, our data do not support
suggestions that O2-sensitive receptors involved in respiratory
responses may be situated in the heart and dorsal aorta, instead suggesting
that such receptors may be confined to the gills and orobranchial cavity.
The picture that emerges from this and our previous study
(Sundin et al., 2000) is that
the receptors involved in producing the hypoxic ventilatory response are
distributed throughout all the gill arches and the orobranchial cavity in the
tambaqui. While the frequency response arises almost exclusively from the
branchial receptors, a significant component of the amplitude response arises
from extrabranchial sites within the orobranchial cavity. It has previously
been shown in tench (Tinca tinca), sea raven (Hemipterus
americanus) and traira (Hoplias malabaricus) that branchial
denervation alone was insufficient to eliminate ventilatory responses to
hypoxia (Hughes and Shelton,
1962
; Saunders and Sutterlin,
1971
; Sundin et al.,
1999
) while it was sufficient to do so in the channel catfish
(Ictalurus punctatus) and the gar (Lepisosteus osseus)
(Smatresk, 1989
;
Burleson and Smatresk, 1990
).
The biological significance of these species differences remains unclear, and
it will also remain to be seen whether these former species in which the
hypoxic ventilatory response was not eliminated by gill denervation alone
share the same receptor distribution as the tambaqui.
Our data do not support a role for central O2 chemoreceptors in
fish, in agreement with several previous studies on other species
(Kawasaki, 1980:
Hedrick et al., 1991
; McKenzie
et al.,
1991a
,b
).
Attempts at central stimulation were without effect (series III). Our data
also do not support a role for the hypoxic release of catecholamines into the
circulation as a causal factor in the hypoxic ventilatory response
(Randall and Taylor, 1991
)
since the exogenous application of catecholamines inhibited ventilation. 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. It appears that the exogenous adrenaline
administered in the present study acted to reduce breathing frequency
via beta-receptor activation while the depression of ventilation
amplitude occurred, at least in part, by some other mechanism. Paradoxically,
while beta-receptor blockade eliminated the frequency depression caused by
exogenous adrenaline, it attenuated the frequency response to hypoxia, leading
to a respiratory depression at levels of hypoxia below 5.3 kPa (40 mmHg). Such
a depression is common during severe hypoxia in bimodal breathers (McKenzie et
al.,
1991a
,b
;
Randall et al., 1980
) but has
not, to our knowledge, been investigated in water-breathing fish.
At present, this result is difficult to explain. Our data indicate that the
entire frequency response arises from the gills and is eliminated by gill
denervation (Sundin et al.,
2000; present study, series I and II). It has previously been
shown that O2-sensitive chemoreceptors on the first gill arch of
trout (Oncorhynchus mykiss) are not stimulated by catecholamines
(Burleson and Milsom, 1995
) and
that exogenous catecholamines infused during hypoxia inhibit breathing in this
species (Kinkead and Perry,
1991
). Sotalol, however, which is a specific antagonist of
beta-adrenoceptors, reduces the magnitude of the frequency response to hypoxia
(series IV data), suggesting that such receptors are involved in the
excitatory response. One possibility is that catecholamines may act as
neurotransmitters at some central site in the pathway by which severe hypoxia
stimulates ventilation. It has been suggested that catecholamines stimulate
breathing by central mechanisms in mammals
(Eldridge et al., 1985
) and
fish (Randall and Taylor,
1991
).
Whatever the situation in tambaqui, our data show that circulating
catecholamines do not stimulate breathing. In previous studies, catecholamine
infusion has been shown to increase or decrease ventilation frequency in fish
depending on the species, the time of the year and the physiological state of
the animal (for a review, see Randall and
Perry, 1992). It has been argued both that circulating
catecholamines do (Randall and Taylor,
1991
) and that they do not
(Perry et al., 1992
) play a
physiological role in regulating breathing, and our data support the latter
view.
Responses to hyperoxic hypercarbia
Total denervation of the gills in fish with intact brains in our previous
work eliminated the ventilatory response to hypercarbia both in this species
(Sundin et al., 2000) and in
the traira (Reid et al.,
2000
). Identical results were obtained again in this study with
hyperoxic hypercarbia. The increases in breathing frequency, amplitude and
total ventilation during hyperoxic hypercarbia in control fish (C) were
eliminated in the GD fish, clearly showing the presence of specific
CO2/pH receptors within the gills. Indeed, total ventilation now
decreased during hyperoxic hypercarbia compared with ventilation in normoxia,
most likely an effect of the hyperoxia, which was not eliminated until the
orobranchial cavity was also denervated (see section above on the responses to
hypoxia and hyperoxia).
Following decerebration and spinalectomy, however, a significant increase
in frequency was still present when fish with complete central transection of
cranial nerves IX and X were exposed to hypercarbic water. Decerebration or
spinalectomy appeared to have removed an inhibitory influence of hypercarbia,
revealing another source of excitation arising from an extrabranchial site.
Thus, breathing rate went from being slightly reduced in the GD fish to being
significantly elevated in the DGD fish. One possible explanation of these
results is that there are receptors present in the olfactory epithelium which,
when stimulated by hypercarbia, inhibit ventilation. Such inhibition has been
shown to be present in many air-breathing lower vertebrates
(Ballam, 1985;
Coates and Ballam, 1989
;
Coates et al., 1991
). The
removal of this inhibition by decerebration (cranial nerves 0, I and II would
be transected) appears to reveal a secondary source of excitation. This
additional excitation was not removed by further transections (DGOD fish).
While the receptors responsible for this excitation may reside within the
brainstem, addition of CO2 to the mock cerebrospinal fluid, as well
as superfusion of the brain with acidic or alkaline solutions, did not affect
ventilation in our study (series III), just as it has not in other studies
(Kawasaki, 1980
;
Hedrick et al., 1991
; McKenzie
et al.,
1991a
,b
).
Thus, if central receptors exist within the brainstem itself in tambaqui and
if they contribute to the hypercapnic ventilatory response, they are not
located near the surface of the brain, the typical location for many central
chemoreceptors in air-breathing vertebrates
(Nattie, 2000
). The response
that remains is almost exclusively a frequency response, and it is also
possible that it arises from receptors with afferent fibres in the two small
opercular branches of cranial nerve VII that we left intact. Unfortunately, we
could not test this in the present study because further denervation of these
branches would have completely eliminated motor innervation of the respiratory
muscles.
In summary, our data reveal the presence of O2-sensitive receptors in the orobranchial cavity innervated by cranial nerves V and VII that are involved in reflex increases in breathing amplitude. Denervation of these and of the O2-sensitive receptors on the gills is required to eliminate the hypoxic and hyperoxic ventilatory responses as well as all responses to internal and external injections of NaCN. In addition, the data confirm the presence of specific CO2/pH receptors within the gills that mediate increases in ventilation amplitude and also reveal the presence of CO2-sensitive receptors, probably in the olfactory epithelium, that inhibit ventilation frequency and a group(s) of extrabranchial receptors that contribute to increases in ventilation frequency in response to hyperoxic hypercarbia. The site(s) of these latter receptors remains to be determined.
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Acknowledgments |
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References |
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