N-methyl-D-aspartate receptors mediate chemoreflexes in the shorthorn sculpin Myoxocephalus scorpius
Department of Zoology, Göteborg University, Box 463, S-405 30 Gothenburg, Sweden
* Author for correspondence (e-mail: jenny.turesson{at}zool.gu.se)
Accepted 10 January 2003
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Summary |
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Fish were equipped with opercular, branchial and snout cannulae for measurements of cardiorespiratory parameters and drug injections. Oxygen chemoreceptor reflexes were evoked by rapid hypoxia, NaCN added into the blood (internal, 0.3 ml, 50 µgml1) and the mouth (external, 0.5 ml, 1 mg ml1), before and after systemic administration of the NMDA receptor antagonist MK801 (3 mg kg1).
Hypoxia produced an MK801-sensitive increase in blood pressure and ventilation frequency, whereas the marked bradycardia and the increased ventilation amplitude were NMDA receptor-independent. The fish appeared more responsive to externally applied cyanide, but the injections and MK801 treatment did not distinguish whether external or internal oxygen receptors were differently involved in the hypoxic responses.
In addition, using single-labelling immunohistochemistry on sections from the medulla and ganglion nodosum, the presence of glutamate and NMDA receptors in the vagal oxygen chemoreceptor pathway was established.
In conclusion, these results suggest that NMDA receptors are putative central control mechanisms that process oxygen chemoreceptor information in fish.
Key words: Fish, hypoxia, NaCN, cardiovascular, respiration, nucleus tractus solitarius, glutamate, N-methyl-D-aspartate (NMDA) receptor
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Introduction |
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In fish, the afferent signals from the branchial oxygen receptors terminate
in the dorsomedial part of the medulla oblongata, in a bilateral, elongated
structure along both sides of the fourth ventricle named the nucleus tractus
solitarius (NTS). In the sculpin the vagal portion of the NTS, the vagal
sensory area (Xs), has recently been defined and it stretches in a
rostrocaudal direction from 2.0 mm rostral to obex to 0.5 mm caudal of obex
(Sundin et al., 2003).
In the NTS the first integrative step of sensory input takes place (for a
review, see van Geirsbergen et al., 1992), and the excitatory amino acid
glutamate is probably the neurotransmitter being released by most of these
sensory terminals (Saha et al.,
1995; Sykes et al.,
1997
; Mizusawa et al.,
1994
; Perrone,
1981
). It has also been established that an ionotropic
N-methyl-D-aspartate (NMDA) glutamate receptor, found within NTS on
both interneurons and afferent terminals
(Aicher et al., 1999
), is
involved in the central mediation of oxygen chemoreceptor inputs and the
subsequent development of chemoreflexes in mammals
(Aylwin et al., 1997
;
Haibara et al., 1995
;
Lin et al., 1996
;
Mizusawa et al., 1994
;
Ohtake et al., 1998
).
While there exists some knowledge regarding hypoxia-induced
cardiorespiratory reflexes and their control via the autonomic
nervous system in fish, much less is known about the sensory system,
particularly the central mechanisms involved in the processing of oxygen
chemoreceptor information. As a first step towards establishing glutamate as
an important transmitter in cardiorespiratory control in fish, Sundin et al.
(2003) showed that glutamate,
microinjected into the rostral end of the Xs in the shorthorn sculpin, could
evoke cardiorespiratory responses that mimicked oxygen chemoreceptor-activated
chemoreflexes. To continue that study, the first aim of this paper was to
determine whether glutamate, via the NMDA receptor, is involved in
the development of oxygen chemoreflexes in fish. The second aim was to
establish the location of glutamate and NMDA receptors in the vagal sensory
system, comprising vagal fibres and cell bodies in the ganglion nodosum, and
the Xs. In addition, we sought to distinguish whether the activation of both
external and/or internal oxygen chemoreceptors utilize NMDA receptors to
produce cardiorespiratory reflexes.
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Materials and methods |
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Immunohistochemical experiments
The presence of glutamate and NMDA receptors in vagal nerve fibres and
sensory cell bodies in ganglion nodosum and the Xs was established using
single-labelling immunohistochemistry (antisera listed in
Table 1).
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Ten animals were killed with an overdose of MS222 (3-aminobenzoic acid ethyl ester, methanesulfonate salt, 300400 mg l1 seawater; Sigma), and the medulla together with the ganglion nodosum were removed and fixed in 4% formaldehyde overnight. After fixation the tissues were rinsed in phosphate buffer (PBS, 0.9% NaCl) for 30 min and then cryoprotected with a PBS-sucrose solution (0.9% NaCl, 30% sucrose) overnight. The tissues were embedded in mounting medium (Tissue-teck, Sakusa, Zoeterwoude, Netherlands) and frozen in isopentane cooled in liquid nitrogen. Cross sections of the medulla and planar sections comprising the ganglion nodosum (12 µm thick) were cut on a cryostat (Cryo-Star HM 560 M. Microm, Walldorf, Germany) and mounted on gelatine-coated slides. Immunostaining was performed by incubating the slides with primary antiserum against glutamate and the NMDAR1 subunit of the NMDA receptor (Table 1) for 24 h at room temperature. Excess primary antiserum was removed by rinsing in PBS (2.0% NaCl) for 3x10 min. The slides were then incubated with the secondary antiserum DaR-CY3 or DaR-FITC (Table 1) for 60 min. After an additional rinse in PBS (2.0% NaCl) for 3x10 min, coverslips were placed over carbonate-buffered glycerol on the slides. Finally, the slides were examined using a fluorescence microscope (Olympus BX 60, Olympus Optical Co. Ltd., Tokyo, Japan). Photographs were taken using a Nikon digital camera DMX 1200. In order to reduce unspecific staining the glass slides were pre-incubated with normal donkey serum (NDS) before the primary antibody incubation was begun.
To visualize the general histology of the labelled sections, some were stained for Nissl substance.
Specificity controls of antisera
To control the specificity of the glutamate primary antiserum, an
absorption test was conducted according to the protocols given in Ottersen et
al. (1986). Primary antiserum
against glutamate was pre-incubated with glutaraldehyde-conjugated glutamate
(102 mol l1) for 20 h at 4°C. This
mixture was added to glass slides with sections from both the medulla and
ganglion nodosum. The specificity of the secondary antiserum was assessed by
adding a `blank' solution (without the primary antiserum) to the slides. Both
the absorption and blank tests were performed in parallel with positive
controls.
Since the NMDAR1 antigen could not be obtained from Chemicon Inc., only the
blank test was performed. However, according to the producer Chemicon Inc. and
Flynn et al. (1999),
this antibody is very specific and does not crossreact with any other
glutamate receptor subunits in fish.
Cardiorespiratory experiments
Surgical procedure
The fish (N=7) were anaesthetised in MS222 (100 mg
l1 seawater) until spontaneous breathing stopped, and then
transferred to an operating table where cooled (10°C) aerated seawater
containing MS222 (50 mg l1 seawater) was passed over the
gills throughout surgery. A polyethylene cannula (PE50), tipped with a thinner
cannula (PE10), was inserted into the afferent branchial artery of the third
gill arch according to the method described by Axelsson and Fritsche
(1994). The cannula, filled
with heparinized (100 i.u. ml1) 0.9% NaCl, was used for
ventral aortic pressure measurements (Pva), heart rate
(fH) and intra-arterial administrations of drugs. A second
cannula (PE90) was inserted through the snout for drug administration into the
respiratory water. Finally, for measurements of ventilation amplitude
(VAMP) and frequency (fV), a third
cannula (PE90) was inserted into the branchial operculum. After surgery the
fish were transferred to an experimental chamber, with a slow water flow
(approx. 1 l min1), and allowed to recover for 24 h before
the experiments started.
Equipment
Branchial and opercular cannulae were connected to pressure transducers
previously calibrated against a static water column. The ventilation and
PVA signals were amplified using a Grass low-level d.c. (model
7P122B, Quincy, USA) and continuously sampled at 20 Hz using a data
acquisition software program (Labview version 5.0, National Instruments,
Austin, USA). VAMP, fV and
fH were calculated from these signals using a
Labview-based calculation program. Mean values were created at 10 s intervals
for both internal and external NaCN exposure, and a representative value from
the resting period and the maximum response, respectively, were chosen for
each animal and plotted as histograms. For the hypoxia exposure experiments,
the mean values were created at 5 s intervals, and to reduce the large set of
data every third value was selected and plotted on the graphs.
Experimental protocols
The experiments started with control injections of seawater (0.8 ml) into
the snout cannula and saline solution (0.9% NaCl, 0.3 ml) into the arterial
cannula to ensure that the vehicle in itself had no effect. The control
injections were followed by stimulating both external oxygen chemoreceptors,
by NaCN injections into the respired water (0.5 ml, 1.0 mg
ml1), and internal receptors, by NaCN injections into the
afferent branchial artery (0.3 ml, 50 µgml1),
respectively. In addition, the fish were also subjected to a 10 min hypoxic
period. The three types of chemoreceptor stimulation were performed randomly
and the fish were allowed to recover to pre-exposure values. Different NaCN
concentrations were assessed to determine the lowest concentration that gave
clear responses for each animal, and then the same concentration was used
after MK801 treatment. The chosen concentrations lie within range of previous
studies (Burleson and Smatresk,
1990; Sundin et al.,
1999
,
2000
). The hypoxic period
started when nitrogen bubbling of the respiratory water was turned on,
resulting in a PO2 drop from 19 kPa to 5 kPa within the 10
min exposure period. The fish were left to recover until the cardiorespiratory
variables had stabilized to pre-hypoxic levels, usually after 1015 min.
The NMDA receptor antagonist MK801 (3.0 mg kg1) was
systemically injected, and after 30 min when the cardiorespiratory parameters
had stabilized, the pre-MK801 protocol was repeated. Prior pilot experiments
showed that the MK801 dose used provided a good blockade for 12 h.
Drugs
Sodium cyanide and Dizocilpine
[5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine,
maleate salt (MK801) were obtained from Sigma (St Louis, USA) and dissolved in
0.9% NaCl solution or seawater.
Statistical analysis
The results are presented as means ± S.E.M. (a value of
P<0.05 was considered significant) and were statistically analysed
by performing paired Student's t-tests comparing the maximum response
for each cardiorespiratory parameter with the resting values directly before
the treatment. The effect of MK801 on the O2 receptor-activated
responses was determined by comparing the change (maximum response value
resting value) for each measured variable in both untreated (control)
and MK801 treated fish.
To establish whether MK801 affected the cardiorespiratory resting values, averages were plotted for each fish as a mean of values from three time points: the pre-hypoxia, pre-internal exposure (NaCN) and pre-external exposure (NaCN) values. These averages were then used to calculate representative mean resting values for each cardiorespiratory parameter. Resting values before and after MK801 treatment were compared with each other and evaluated for statistical significant differences.
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Results |
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Glutamate-like immunoreactivity
Vagal fibres proximal to the ganglion nodosum displayed strong
immunoreactive intensity for glutamate
(Fig. 1A). Accordingly up to
50% of the cell bodies (approx. diameter 3040 µm) and their axons in
the ganglion nodosum stained positive for glutamate
(Fig. 1B). Fig. 1C shows that only the
portion of the vagal nerve trunk containing the sensory fibres, but not motor
fibres, displays glutamate-like IR.
|
NMDAR1-like immunoreactivity
NMDAR1-like IR was found in fibres throughout the vagal sensory column
(Fig. 1D), while small
interneurons (approx. diameter 58 µm) positive for NMDAR1 were only
found rostral to obex (Fig.
1E).
Specificity controls
The absorption and blank tests were negative, confirming the specificity of
the primary and secondary antisera used.
Cardiorespiratory experiments
External and internal vehicle (control) injections occasionally produced
startle reflexes, such as a transient bradycardia and apnea. In such cases the
injections were repeated until the animals were accustomed and no further
responses were observed.
Respiratory responses
Hypoxia significantly increased both fV and
VAMP (Fig.
2A,B). While there was a short delay before the onset of the
frequency response (at PO2 approx. 10 kPa), the amplitude
immediately started to increase upon commencement of the hypoxic period. MK801
treatment abolished the hypoxia-induced fV increase but
did not significantly affect the hypoxia-induced increase in
VAMP.
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Cardiovascular responses
Hypoxia, external and internal NaCN injections decreased
fH (Figs 2,
4). The threshold for the
response was at a water PO2 of approx. 8 kPa (reached
after approx. 5 min) before the heart rate started to drop. The bradycardia
was MK801-insensitive.
|
Before the onset of the hypoxia-induced bradycardia, PVA showed an initial significant increase (at a PO2 of 10 kPa, after approximately 3 min). As soon as the bradycardia commenced PVA declined to baseline values (Fig. 2). The PVA followed the heart rate responses seen during external and internal NaCN injections, and thus decreased significantly (Fig. 4). The only clear effect of MK801 treatment on the PVA responses was the absence of the initial increase during hypoxia (Fig. 2).
Effects of MK801 on resting parameters
MK801 did not significantly change any resting level of the measured
parameters (Table 3), but the
treatment caused major changes in the breathing patterns of the animals. From
being continuous with occasional apneas occurring in conjunction with
spontaneous bradycardia, four types of breathing pattern evolved almost
immediately or up to 60 min after the MK801 treatment before stabilisation;
these were regular continuous breathing but at an increased frequency (two
animals, Fig. 5B), frequency
cycling (two animals, Fig. 5C), episodic breathing (two animals, Fig.
5D) and breathing with decreased ventilation amplitude and
frequency (one animal, not shown).
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Discussion |
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In contrast to the efficient blockade of the respiratory frequency
response, the chemoreceptor elicited bradycardia was MK801-insensitive.
Interestingly, when a bradycardia was evoked instead by microinjection of
glutamate into the Xs of the sculpin
(Sundin et al., 2003), it was
NMDA-receptor dependent. Similarly, a glutamate-induced bradycardia in rats
could also be blocked by NMDA receptor antagonists
(Canesin et al., 2000
;
Colombari et al., 1997
), while
the chemoreceptor-elicited bradycardia has been shown to be both sensitive
(Haibara et al., 1995
) and
insensitive (Ohtake et al.,
1998
) to NMDA receptor blockade. Apparently there might exist
compensatory mechanisms that override the NMDA-receptor-dependent response in
order to maintain the chemoreflex bradycardia (as seen in this study), even
without the presence of functional NMDA receptors.
The hypertension produced by hypoxia in fish is mediated via
-adrenoceptors (Fritsche and
Nilsson, 1990
). In this study the initial hypertension was
abolished when the animals were pre-treated with MK801, which suggests that
the activation of the sympathetic component of the vascular reflex is mediated
by NMDA receptors. Evidence that also supports a role for glutamate in the
transmission of vascular responses during hypoxia in fish is that
microinjection of glutamate into the rostral Xs of the sculpin can produce
pressor effects (Sundin et al.,
2003
).
Corroborating our physiological experiments is the identification of
glutamate in nerve fibres throughout the Xs, the vagal nerve trunk, and in a
large fraction of the cell bodies within the ganglion nodosum. These findings
correspond well with those obtained in mammals
(Saha et al., 1995;
Schaffar et al., 1997
;
Sykes et al., 1997
), thus
showing that glutamate is present in fish vagal sensory pathways. Further
implicating the presence of glutamate in sensory pathways is the finding of
NMDA receptors on interneurons and nerve fibres within the Xs (this study),
NTS in rats (Aicher et al.,
1999
; Lin and Talman,
2000
; Ohtake et al.,
2000
) and cats (Ambalavanar et
al., 1998
). Although we have identified the presence of NMDA
receptors in the sculpin sensory pathways, it should be recognized that these
receptors are found in the majority of glutamatergic synapses in the
vertebrate brain (for a review, see
Colquhoun and Sakmann, 1998
)
and that a systemic administration of MK801 will affect all available
receptors.
There are several reports that fish possess both internal and external
oxygen receptors (Burleson and Milsom,
1993; Milsom and Brill,
1986
) and that they may elicit different cardiorespiratory
responses (Milsom 1996
;
Milsom et al., 2002
;
Sundin et al., 1999
), so we
used injection of NaCN into the respiratory water and intra-arterially to
determine whether the sculpin showed any differences in their
cardiorespiratory reflexes depending on the type of oxygen chemoreceptor group
being stimulated. Overall there was no difference between the external (water)
and internal (blood) NaCN-elicited cardiorespiratory responses other than that
NaCN applied to the respired water significantly increased the ventilation
amplitude and produced a more marked bradycardia. Even though MK801
significantly blocked the externally but not the internally elicited increase
in fV, there was no persuasive evidence for separate
central transmission pathways of cardiorespiratory reflexes for the two oxygen
receptor groups, because in the presence of the antagonist internally applied
NaCN could not significantly increase fV.
Although MK801 treatment did not significantly change any of the
cardiorespiratory mean resting values, it clearly changed the breathing
pattern in the sculpin. From being quite regular and continuous, the animals
displayed more irregular patterns, including frequency cycling and episodic
breathing. Breathing in vertebrates originates from a central respiratory
pattern generator, which is dependent on numerous afferent inputs for
initiation of breathing (Feldman et al.,
1990; Smatresk,
1990
). Removal of the afferent input by selective denervation in
the neotropical fish tambaqui gave rise to similar breathing patterns (as seen
in this study), such as frequency cycling (denervation of the whole
oro-branchial cavity) or episodic breathing (denervation of only the branchial
nerves) (Reid et al., in
press
). Consequently, afferent information to the central
respiratory pattern generator is important for maintaining normal breathing in
fish. As MK801 produced similar irregular breathing patterns in the sculpin
and, in addition, alters breathing patterns in a similar fashion in mammals
(Ling et al., 1994
;
Connelly et al., 1992
;
Harris and Milsom, 2001
), it
is likely that NMDA receptors are involved in regulating the respiratory
rhythms in all vertebrates. The effect of MK801 on breathing patterns in fish
are in keeping with previous findings on isolated lamprey brain preparations,
which showed that ionotropic glutamate receptors participate in the central
respiratory network (Bongianni et al.,
1999
).
In conclusion, this study is the first to show that the excitatory amino acid glutamate, present in the vagal afferent pathway, is involved in central processing of oxygen chemoreceptor information in fish. The most significant observation is that only the increase in fV and initial hypoxic hypertension are NMDA-receptor dependent, while the elevated VAMP and the bradycardic responses are not. Furthermore, NMDA receptors were identified on both nerve fibres and interneurons within the Xs, so the glutamate-NMDA receptor mechanism for regulation of oxygen chemoreceptor reflexes might be present in all vertebrates.
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Acknowledgments |
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