Evidence for glutamatergic mechanisms in the vagal sensory pathway initiating cardiorespiratory reflexes in the shorthorn sculpin Myoxocephalus scorpius
1 Department of Zoology, Göteborg University, Box 463, S-40530
Gothenburg, Sweden
2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham,
B15 2TT, UK
* Author for correspondence (e-mail: lena.sundin{at}zool.gu.se)
Accepted 25 November 2002
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Summary |
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The locations of the vagal sensory and motor (Xm) areas in the medulla were established by the orthograde and retrograde axonal transport of the neural tract tracer Fast Blue following its injection into the ganglion nodosum. Glutamate was then microinjected into identified sites within the Xs in an attempt to mimic chemoreceptor- and baroreceptor-induced reflexes commonly observed in fish. By necessity, the brain injections were performed on anaesthetised animals that were fixed by `eye bars' in a recirculating water system. Blood pressure and heart rate were measured using an arterial cannula positioned in the afferent branchial artery of the 3rd gill arch, and ventilation was measured by impedance probes sutured onto the operculum.
Unilateral injection of glutamate (40-100 nl, 10 mmol l-1) into the Xs caused marked cardiorespiratory changes. Injection (0.1-0.3 mm deep) in different rostrocaudal, medial-lateral positions induced a bradycardia, either increased or decreased blood pressure, ventilation frequency and amplitude and, sometimes, an initial apnea. Often these responses occurred simultaneously in various different combinations but, occasionally, they appeared singly, suggesting specific projections into the Xs for each cardiorespiratory variable and local determination of the modality of the response. Response patterns related to chemoreceptor reflex activation were predominantly located rostral of obex, whereas patterns related to baroreceptor reflex activation were more caudal, around obex.
The glutamate-induced bradycardia was N-methyl-D-aspartate (NMDA) receptor dependent and atropine sensitive. Taken together, our data provide evidence that glutamate is a putative player in the central integration of chemoreceptor and baroreceptor information in fish.
Key words: fish, vagus, reflex control, ventilation, bradycardia, blood pressure, glutamate, NMDA, chemoreceptor, baroreceptor
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Introduction |
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In mammals, the nucleus of the solitary tract (NTS) is the primary synaptic
relay in the brainstem, where afferent information from visceral receptors is
integrated. The NTS has been subdivided into different subnuclei based on its
cytoarchitecture and the afferent and efferent connections of the neurons
within it. The medial and lateral commissural subnucleus of the NTS has been
shown to be the primary site of termination of cardiovascular afferent fibres,
receiving inputs from carotid chemoreceptors, arterial baroreceptors and
cardiopulmonary receptors (Loewy,
1990; Van Giersbergen et al.,
1992
). Glutamate, an excitatory amino acid (EAA), is the strongest
candidate for the neurotransmitter released by these afferents
(Ohtake et al., 1998
;
Talman, 1997
).
Information regarding the location of sensory areas in the medulla
important for control of the cardiorespiratory system in fish is sparse, and
information about the nature of their neurotransmitters and receptors is
essentially lacking. It is documented that the gills are a major site for
chemo- and baroreceptors, with their afferent nerves travelling in cranial
nerves IX and X (Burleson et al.,
1992). In the medulla, the areas of termination of afferent
sensory fibres (Xs) are located dorsally and laterally above the sulcus
limitans of His, whereas the motor area (Xm) is located ventral and lateral to
the sulcus (Meek and Nieuwenhuys,
1998
). Although in most teleosts a clear NTS is absent, the
visceral sensory area forms a continuous column dorso-laterally on either side
of the 4th ventricle in the medulla, into which viscerosensory fibres of
nerves VII, IX and X terminate in a rostrocaudally ordered fashion
(Meek and Nieuwenhuys,
1998
).
Recently, it has been shown that EAAs are the neurotransmitters in taste
pathways in goldfish (Carassius auratus;
Smeraski et al., 1998), and
immunohistochemistry has shown that glutamate is present in the nodose ganglia
and vagal afferents in the shorthorn sculpin Myoxocephalus scorpius
(J. Turesson and L. Sundin, manuscript submitted). Taken together, these
results suggest that EAAs might also be the neurotransmitters in the general
visceral sensory pathways conveying information from chemo- and baroreceptors
via vagal and glossopharyngeal nerves.
A first step on the way to establish if glutamate is a functional neurotransmitter in the central processing of baroreceptor and oxygen chemoreceptor information in fish is to determine whether addition of glutamate into the vagal portion of the visceral sensory column elicits cardiorespiratory responses similar to the reflexes activated by stimulation of peripheral chemo- and baroreceptors. Therefore, the primary aim of this paper was to examine whether microinjection of glutamate, sometimes followed by appropriate antagonists, into different sites of the vagal sensory area (the terminal field of vagal afferent fibres characterised as the NTS in mammals) activates cardiorespiratory responses that mimic chemo- and baroreceptor reflexes. If clear responses were obtained, then glutamate could perhaps be used as a `mapping tool'. Accordingly, a second aim was to elucidate whether there was a distinguishable separation of areas in which different responses were elicited that might reflect a topographical arrangement of the central projection of receptor afferents. As knowledge of the central projections of the vagal afferent and efferent fibres in the medulla of the shorthorn sculpin is a prerequisite for reasonably accurate microinjections into the Xs, the initial aim of this study was to locate the Xs and Xm columns in this species, using a neuroanatomical technique.
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Materials and methods |
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Experimental preparation
On the day of surgery, the fish were anaesthetized in seawater containing
100 mg l-1 MS 222 (ethyl m-amino benzoate; Sigma; 10°C) until
breathing movements ceased. They were transferred to a surgical table where
the gills were continuously irrigated with cooled, recycled water containing
anaesthetic (40-50 mg l-1 MS 222, 10°C).
Topography of the vagal sensory and motor columns
The nodose ganglion was located by tracing the exposed branchial nerves
centrally. Exposure was via a small incision (approximately 1 cm)
made in the epithelium at the dorsal end of the 4th gill arch where it meets
the roof of the opercular cavity, the operculum having been reflected forward.
Using a 25 µl Hamilton syringe equipped with a 27-gauge hypodermic needle,
5-10 µl of Fast Blue (Sigma), as a 2% solution in polyethylene glycol, was
injected through the nerve sheath into the ganglion. When visual observation
confirmed that the ganglion had turned yellowish in appearance, the needle was
withdrawn and the puncture was closed with tissue glue. The incision was
sutured and the fish was tagged, then returned to holding tanks for 7-10 days
to allow axonal transport (orthograde and retrograde) of the tracer into the
projections of the vagus, in the medulla. Each fish was then sacrificed by an
overdose of MS 222 and heparin (0.2 ml, 5000 IU) injected into the caudal
vein. The fish were exanguinated by perfusion with physiological saline (0.9%
NaCl) using a ventral aortic cannula connected to a peristaltic pump. After
10-15 min, when the gills had turned white, the saline was switched to 4%
formaldehyde solution and the fish were perfused for a further 15 min. The
brain was then carefully dissected from the skull and placed in 4%
formaldehyde in 0.1 mol l-1 phosphate-buffered saline (PBS; pH 7.3)
for at least 4-5 h at 4°C. Each brain was then rinsed for 30 min in PBS
and stored in PBS containing 30% sucrose as a cryoprotectant. Finally, it was
quick-frozen in isopentane cooled in liquid nitrogen and mounted on the stage
of a cryostat. Serial, transverse sections, 20 µm thick, were cut,
transferred directly to gelatine-coated glass slides and left to airdry
overnight. The sections were coverslipped with glycerol mounting media and
viewed under a fluorescence microscope (BX60, Olympus) connected to a digital
video camera. Pictures were frozen on a TV monitor and captured by computer
using the Micro Image software (Micro Image, Gothenburg, Sweden). To visualize
the general histology of the labelled sections, some were stained for Nissl
substance.
Microinjection
The day before the experiment, the third afferent branchial artery on the
left side was cannulated (PE 50 tipped with a PE 10) according to the
procedures described for Atlantic cod
(Axelsson and Fritsche, 1994).
This cannula was used to measure ventral aortic blood pressure
(PVA) and heart rate (fH) and for the
administration of drugs. Measurements of ventilation frequency
(fV) and amplitude (VAMP) were made
using impedance probes, fastened with suture thread stitched through each
operculum.
On the day of the experiment, the fish was again anaesthetised (100mg l-1 MS 222) and lowered into a plastic box placed between the steel bars of a modified stereotaxic frame (model SN-2N; Narishige Instruments, Tokyo, Japan). It was fixed in position with eye bars and also, initially, with a mouthpiece through which re-circulating respiratory water (40-50 mg l-1 MS 222) flowed. As the animal started to breath spontaneously, the mouthpiece was withdrawn to deliver water approximately 2 cm in front of the snout. Using a dremel tool and vacuum suction, the skull was carefully opened (incision approximately 1.5 cm long) to expose the whole length of the medulla from the middle portion of the cerebellum to the first pair of the spinal nerves. The fish rested in a horizontal position on a height-adjustable platform inside the box. A standpipe controlled the water level, which was adjusted to cover the gills yet allowed the medulla to be uncovered.
Drugs were delivered into specific locations in the medulla from a
single-barrel microinjection pipette (tip size 10-15 µm). Movements of the
pipette were controlled by a micromanipulator (SM15 equipped with a base
SM-15M). Injection volumes of 40-100 nl were delivered over a period of 1
s by applying a pulse of pressurized N2 using a pressure injector
(model PLI-100; Harvard Medical Systems, Holliston, MA, USA). The volume of
drug delivery was controlled by changing the injection pressure, and the
actual volume of the injection was determined by viewing the movement of the
fluid meniscus in the barrel of the pipette, which was of known internal
diameter, using a microscope (x50 magnification) equipped with a
calibrated eyepiece micrometer.
The cannula was connected to a pressure transducer, the signal was amplified (4Champ; Somedic AB, Sollentuna, Sweden) and the leads from the impedance probes were connected to an impedance converter (model 2991; UFI, Morro Bay, CA, USA). The cardiorespiratory variables were continuously recorded to paper (recorder model 3701, LR 8100; Yokogawa, Tokyo, Japan), and the data were collected online, via data-acquisition software (Labview version 5.0; National Instruments, Solna, Sweden) onto a computer. Sampling frequency was 20 Hz, and mean values were subsequently created at 10s intervals. From the pulsed blood pressure and ventilation signals, fH and fV were derived using a Labview-based calculation program. The injection signal from the PLI-100 pressure injector was also sampled, which allowed exact timing of the injection with the cardiorespiratory responses.
Experimental protocols
In three fish, efforts were made to use a decerebrate and spinalectomized
preparation to avoid any potential influence of anaesthesia on central reflex
mechanisms. However, that approach was abandoned because these fish bled
substantially, displayed low ventral aortic blood pressures (0.9-1.3 kPa) and
never started spontaneous breathing. Instead of decerebration, light
anaesthesia (40-50 mg l-1 MS 222) was used, as it permits a smaller
hole in the skull to be made and leaves the spinal cord intact, maintaining
sympathetic outflow to the vessels. This approach significantly improved the
blood pressure (2.0-3.8 kPa) in the animals, who now also started to breathe
spontaneously. These blood pressures are comparable with those in an
unanaesthetised and free-swimming sculpin
(Fritsche, 1990).
Preliminary trials using 0.1 mmoll-1 and 1.0 mmoll-1
of glutamate were employed to determine a concentration that would give clear
and distinctive responses. The concentration (10 mmoll-1) and
volume range (40-100 nl) chosen are comparable with those commonly used for
microinjections into the medulla of rats
(Canesin et al., 2000;
Dhruva et al., 1998
;
Le Galloudec et al.,
1989
).
General protocol
The general protocol for the experiments was as follows. When stable
cardiorespiratory parameters were established, usually around 40-60 min after
securing the fish in the stereotaxic frame, unilateral microinjections of
glutamate were made sequentially into different discrete areas of the Xs
column along the medulla. When a response was elicited, the following
injection was postponed (between 10 min and 60 min) until stable parameters
were again established. The microinjection pipette was advanced through the
sensory area (as determined by the nerve tracing) in steps of 0.1 mm down to a
depth of 0.3 mm along a rostrocaudal direction from 2.0 mm rostral to -1.0 mm
caudal of obex, in steps of 0.5 mm. Lateral coordinates applied were 0.3 mm,
0.5 mm and 0.7 mm lateral to the midline. Each animal was subjected to 20-50
different injections, although not every coordinate received an injection in
each animal and the order of injection sites varied among animals. When a
clear and concise response was elicited, the pipette was raised, rinsed and
vacuum loaded with the vehicle (0.9% NaCl) then lowered again to the same
depth, and a control injection with the same or a larger injection volume was
performed. To control for tachyphylaxis and possible mechanical damage of an
injection, repetitive injections of glutamate in exactly the same area at 10
min intervals were performed in at least one site in each animal, and
glutamate was sometimes re-injected into the area where a previous vehicle
control had been carried out.
Cardiovascular mechanisms
In five animals, at the end of the above-described general protocol, a site
that had previously elicited a distinct bradycardia was again injected before
an intra-arterial injection of the agonist atropine (1 mg kg-1).
After 20 min, the agonist injection was repeated at the same site.
For experiments using the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, the general protocol was as follows. Having obtained a cardiac response to unilateral microinjection of 40-100 nl of glutamate, the pipette was raised, rinsed then vacuum loaded with the antagonist (3 mg ml-1), which was injected (40-100 nl) at the same depth. The agonist was then reloaded and the injection repeated. The time between the application of the antagonist and the agonist was 5-10 min. To further control for the specificity of the blockade, the pipette was lowered 0.1 mm beyond or moved 0.5 mm in a sagittal direction from the MK-801 saturated area, and the agonist injection was repeated.
Drugs
Monosodium L-glutamate, dizocilpine (5R,
10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5, 10-imine
hydrogen maleate (MK-801) and atropine were obtained from Sigma and dissolved
in 0.9% NaCl.
Statistical analyses
Comparison of the cardiovascular effects before and after glutamate was
made using a paired t-test. The same test was used for the comparison
of the cardiovascular effects of glutamate before versus after
MK-801, and before versus after atropine. Differences were considered
significant at P<0.05. All values are means ± S.E.M.
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Results |
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Retrograde labelling with Fast Blue also identified cells in the nucleus ambiguus (NA) of the vagal motor column. This nucleus consists of a relatively small number of neurones located ventro-laterally with respect to the Xm and separated from it by a tract of nerve fibres (Fig. 1D).
The anterior ends of the two columns are located approximately 1.5-2.0 mm rostral of obex, and the posterior end of both columns stretches to 1 mm caudal of obex. At this caudal extremity, commissural fibres (Fig. 1B) cross above the central canal, to constitute the commissural nucleus of the Xs, while fibres crossing beneath are of motor origin (Fig. 1C).
Motoneurons, probably belonging to the nucleus ambiguus, in a ventrolateral position to the motor column were observed from obex to 0.9 mm rostral of obex. Observations of Fast Blue-filled neurons in this position were not common, probably resulting from incomplete staining and a vague and disperse nucleus in this species.
Microinjections
Control injections
As the dorsal sensory and the ventral motor columns are located rather
close to each other, adjacent at depths of 0.4-0.5 mm from the dorsal surface
of the medulla, the results reported here are restricted to injections made
down to 0.3 mm.
To control for non-specific pressure and volume effects of the injections, the vehicle (0.9% NaCl) alone was delivered into the same sites (equal or larger volume) where glutamate had previously elicited a response. The vehicle produced small insignificant blood pressure increases only in one fish. In addition, two fish displayed bradycardia, a concomitant blood pressure decrease and a short apnea when accidentally large volumes (150-300 nl) were injected. When smaller injection volumes were applied at the same sites no responses were evoked.
Repeated injections of glutamate into the same site at 10 min intervals did not decrease the responsiveness of the animal. Hence, the repetition of a glutamate injection into a vehicle-applied site always produced a response.
Responses to injection of glutamate
Dependent on the injection site in the Xs, glutamate elicited decreases in
heart rate (fH) and either increases or decreases in
ventral aortic blood pressure (PVA), ventilation frequency
(fV) and amplitude (VAMP). A
tachycardia was never observed. Occasionally, an injection elicited a
transient apnea. The coordinates and responses for each injection in all
animals are summarised in Fig.
3. Sometimes, an injection elicited a response in a single
cardiorespiratory parameter (Fig.
4) or, at other times, in two or three or all of them (Figs
5,
6). Blood pressure and the
ventilatory responses could be biphasic, often reflecting the relationship
between fH and PVA on the one hand and
fV and VAMP on the other hand. In clear cases,
such as a bradycardia-induced drop in PVA
(Fig. 7), this depressor event
was excluded from the summarised data in
Fig. 3. Only depressor
responses that were apparently independent of a change in
fH are included (e.g.
Fig. 5). The convention adopted
with respect to ventilatory parameters is that reciprocal changes in frequency
and amplitude are recorded as the appropriate excitatory response. Thus, an
increase in amplitude that led to a decreased frequency or a marked increase
in frequency that resulted in reduced amplitude have been recorded as an
increase in either variable.
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Mapping of the distribution of specific responses
Some salient features of these seemingly complex patterns of response
summarised in Fig. 3 are:
In summary, certain main features emerge with regard to each recorded variable: a bradycardia and specific pressor responses were induced by injections at most reactive sites, both caudal and rostral of obex; depressor responses were obtained at and immediately (0.5 mm) caudal or rostral of obex; fV was increased by injection into some sites at and up to 1.5 mm rostral of obex, while a decrease in fV accompanied injection into sites just caudal (0.5 mm) and 2.0 mm rostral of obex; VAMP was increased by injection into most areas rostral of obex and decreased by injections just caudal (0.5 mm) and rostral (1.0 mm) of obex.
It is clear from these data that, while there is some evidence for rostrocaudal distribution of projections from specific receptor-mediated responses, a reflex bradycardia is induced by injection of glutamate into most sites either side of obex. The induced bradycardia sometimes resulted in cardiac arrest, in one case for up to 4 min (Fig. 7).
Cardiovascular mechanisms
Along the fourth ventricle at the medial (0.3 mm lateral) injection sites,
glutamate always induced a bradycardia that sometimes was very marked, in one
extreme case without a heart beat for up to 4 min
(Fig. 7). In five animals, the
non-competitive antagonist of NMDA receptors, MK-801, was injected into
bradycardia-inductive sites and the glutamate injection was repeated. MK-801
abolished the rapidly glutamate-induced bradycardia (Figs
7,
8). The bradycardia was also
blocked by a systemic injection of atropine
(Fig. 9).
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Discussion |
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Interestingly, the specific location of the terminal field within the NTS
is crucial for the production of respiratory or cardiovascular reflexes in
mammals (Dhruva et al., 1998;
Marchenko and Sapru, 2000
). In
mammals, both chemo- and baroafferents terminate in the commissural nucleus of
the NTS (Loewy, 1990
;
Van Giersbergen et al., 1992
).
Within this restricted area, the baroreflexogenic field is located rostral to
the chemoreflexogenic field (Dhruva et al.,
1998
). With the finding of a depressor area lateral to obex at the
beginning of the commissural segment, our results seem similar to the location
of depressor areas in the commissural nucleus in mammals. However, caudal to
the depressor area, we found no evidence for a chemoreflexogenic zone. In
fact, most of the injections in this region produced no responses at all.
Instead, responses simulating chemoreflexes were elicited rostral of obex.
This is consistent with the fact that most peripheral chemoreceptors have been
described as being located on the gills of fish. The gill arches are
innervated sequentially by the IXth glossopharyngeal nerve then the first four
branches of the vagus. The fifth branch innervates the viscera, including the
heart (Burleson et al., 1992
;
Taylor et al., 1999
). Thus,
chemoreceptor afferents will travel in the more rostral projections into the
Xs from the branchial branches of the vagus nerve, which terminate rostral of
obex (Taylor, 1992
). Caudal of
obex, at the commissural nucleus, the afferents of the most caudal root
terminate. Thus, this structure only receives sensory information from
visceral afferents rather than from the gill arches
(Kanwal and Caprio, 1987
;
Lazar et al., 1992
;
Morita and Finger, 1987
). The
finding of a specific depressor site 0.3 mm lateral to obex substantiates that
the barostatic reflex in fish, implicating changes in the resistance of the
vessels, may project through the area innervating the heart
(Taylor, 1992
).
In addition to the distribution of reaction patterns simulating a specific
reflex, injection of glutamate could sometimes elicit a unitary response such
as an increase in respiratory amplitude or a decrease in blood pressure. This
suggests specific areas in the Xs for reflex control of each cardiorespiratory
variable. Identification of these `single' responses may have been prejudiced
by the extracellular injection technique. Although different response patterns
could be obtained with a pipette movement of just 0.1 mm, the spread of the
injection solution will probably cause stimulation of many neighbouring
neurons. With smaller injection volumes and even smaller steps during mapping,
a better picture of this single response topography may evolve. Nevertheless,
this single response topography may accord with the physiological evidence for
more than one population of oxygen receptors in fish, which elicit different
cardiorespiratory parameters dependent on their peripheral location (specific
gill arch or extrabranchial) or orientation (monitoring respiratory water or
blood oxygen levels) (Burleson and
Smatresk, 1990; Smatresk et
al., 1986
; Sundin et al.,
1999
,
2000
). If glutamate is, as in
mammals (Schaffar et al.,
1997
; Sykes et al.,
1997
; Talman et al.,
1980
), the neurotransmitter released by the afferents of baro- and
chemoreceptors in fish, there should be glutamate receptors on target neurons
binding the EAA. Indeed, our results show that the non-competitive NMDA
receptor antagonist MK-801 effectively blocked the glutamate-induced
bradycardia. Similarly, NMDA receptors mediate a glutamate-induced bradycardia
in rats (Canesin et al., 2000
;
Colombari et al., 1997
). In
addition, the data following systemic injection of atropine show that the
bradycardic responses produced by microinjection of glutamate along the 0.3 mm
lateral coordinates are due to parasympathetic neurotransmission, so that a
glutamatergic mechanism for chemo- and baroreflex activation in fish seems
likely. This is borne out by the demonstration that NMDA receptors in the NTS
are involved in the bradycardic element of both the chemoreflex
(Haibara et al., 1995
) and the
baroreflex in rats (Canesin et al.,
2000
).
In conclusion, glutamate applied to different areas in the Xs of the sculpin evoked responses simulating chemo- and baroreflexes. There was some evidence for a topographic separation of these two areas with a chemoreflexogenic zone rostral to a baroreflexogenic zone. The ubiquitous, glutamate-induced bradycardia depended on NMDA receptors in the sensory pathway and was of muscarinic cholinergic, and therefore vagal, origin. Evidence has thus been presented that glutamate may have been present as a key neurotransmitter in the reflex control of the cardiorespiratory system from early in vertebrate evolution. Thus, this work may provide a first step in establishing the fundamental central mechanisms for the processing of chemo- and baroreceptor signals in all vertebrates.
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Acknowledgments |
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