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1 Department of Physiology and
Biophysics, Cardiovascular
responses to chemoreflex activation by potassium cyanide (KCN, 20 µg/rat iv) were analyzed before and after the blockade of ionotropic
or metabotropic receptors into the nucleus of the solitary tract (NTS)
of awake rats. Microinjection of ionotropic antagonists
[6,7-dinitroquinoxaline-2,3-dione or kynurenic acid
(Kyn)] into the lateral commissural NTS
(NTSlat), the midline
commissural NTS (NTSmid), or
into both (NTSlat+mid), produced
a significant increase in basal mean arterial pressure, and the pressor
response to chemoreflex activation was only partially reduced, whereas
microinjection of Kyn into the
NTSmid produced no changes in the
pressor response to the chemoreflex. The bradycardic response to
chemoreflex activation was abolished by microinjection of Kyn into the
NTSlat or into
NTSlat+mid but not by Kyn microinjection into the NTSmid.
Microinjection of
nucleus of the solitary tract; arterial chemoreceptors; cardiovascular regulation; N-methyl-D-aspartate
receptors; non-N-methyl-D-aspartate
receptors; metabotropic receptors; kynurenic acid; 6,7-dinitroquinoxaline-2,3-dione; THE NUCLEUS OF THE SOLITARY TRACT (NTS) is the most
important synaptic station in the brain stem that integrates afferent information from the cardiovascular system producing the appropriate autonomic and ventilatory adjustments required in different
physiological situations. The medial and lateral commissural subnucleus
of the NTS has been shown to be the primary site of termination of
cardiovascular afferent fibers, receiving inputs from carotid
chemoreceptors, arterial baroreceptors, and cardiopulmonary receptors
(4, 8, 17, 24, 27, 34).
The peripheral chemoreceptors are important in the regulation of
respiratory and cardiovascular functions (8, 25), and the
neurotransmission of the chemoreceptors afferent in the NTS as well as
in other areas of the ventral medulla has been extensively studied (1,
20, 21, 27, 28, 39). Pharmacological studies have indicated that
excitatory amino acid (EAA) receptors in the commissural NTS play an
important role in the neurotransmission of the peripheral chemoreflex
(39-41). However, most of these experiments were performed while
subjects were under anesthesia, which may have a distorting effect on
the cardiovascular and ventilatory responses to chemoreflex activation
(10). In addition, microinjection of
L-glutamate into the NTS of
conscious rats elicited opposite cardiovascular responses when compared
with the responses obtained in the same animals under anesthesia (23),
further confirming that neurotransmission in the NTS is deeply affected
by the anesthesia.
In a previous study, we showed that bradycardic response induced by
chemoreflex activation was mediated by
N-methyl-D-aspartate (NMDA) receptors in the commissural NTS because bilateral
microinjection of
DL-2-amino-5-phosphonovaleric
acid (AP-5), a selective NMDA receptor antagonist, into the lateral
portion of the commissural NTS
(NTSlat) blocked the bradycardic
response in a dose-dependent manner. However, AP-5 produced no effect
on the pressor response to the chemoreflex, suggesting that the
sympathoexcitatory component of the chemoreflex may be mediated by
non-NMDA receptors (16). On the basis of such findings, the present
study aimed to evaluate the role of non-NMDA receptors in the
neurotransmission of the pressor response to chemoreflex activation
(sympathoexcitatory component) in the lateral and medial aspects of the
commissural NTS.
Anatomic studies by Finley and Katz (9) have shown that the projections
of the carotid body afferents occur mainly in the NTSlat. Electrophysiological
studies by Mifflin (27) further confirmed that neurons in this area
received input from carotid chemoreceptors. In contrast, recent studies
by Chitravanshi and colleagues (2, 3) reported that the
carotid chemoreceptor afferents seem to project mainly to the midline
portion of the commissural subnucleus of the NTS
(NTSmid), at the calamus
scriptorium level. In addition, the blockade of EAA receptors in the
NTSmid of anesthetized rats
abolished the pressor and ventilatory responses to chemoreflex
activation (39). Therefore, we also evaluated the role of EAA receptors
(ionotropic and metabotropic) in the different subregions of the
commissural NTS (lateral and midline portions) in the neurotransmission
of the sympathoexcitatory component of the chemoreflex (pressor
response) in conscious rats. A preliminary report of our findings has
been published as an abstract (15).
Guide cannula implantation in direction of NTS.
Male Wistar rats weighing 230-270 g were used in the present
study. Four days before the experiments, the rats were deeply anesthetized with tribromoethanol (250 mg/kg ip; Aldrich, Milwaukee, WI) and placed in a stereotaxic frame (Kopf, Tujunga, CA) for guide
cannula implantation. When the rat reacted to frequent toe pinching
during stereotaxic surgery, additional tribromoethanol was injected.
The technique described by Michelini and Bonagamba (26) was adapted to
implant guide cannulas in the following three experimental protocols:
1) bilateral guide cannulas in the direction of the NTSlat (0.5 mm
lateral to midline and ~0.5 mm rostral to calamus scriptorium),
2) one guide cannula in direction of
the NTSmid (at midline at level of
calamus scriptorium), and 3) three
guide cannulas, with two implanted in the direction of the
NTSlat and one implanted in the
direction of the NTSmid. The implants of all guide cannulas were performed in accordance with the
coordinates of Paxinos and Watson (29). To implant each guide cannula,
we made a small window in the skull caudal to the lambda and a
15-mm-long stainless steel guide cannula (22 gauge; Small Parts) was
introduced perpendicularly through the window at the following
coordinates: 0.5 (NTSlat) or 0.0 mm (NTSmid) lateral to the
bregma, 14.00 (NTSlat) or 14.5 mm (NTSmid) caudal to the
bregma, and 7.9 mm below the skull surface at the bregma (NTSlat and
NTSmid). The tip of the guide
cannula was positioned ~1.0 mm above the dorsal surface of the brain
stem. The guide cannula was fixed to the skull with methacrylate and
watch screws and then closed with an occluder until time of experimentation.
Arterial and venous cannulation.
One day before the experiments, while rats were under tribromoethanol
anesthesia, a catheter (PE-10 connected to PE-50; Clay Adams,
Parsippany, NJ) was inserted into the abdominal aorta through the
femoral artery for measurement of pulsatile arterial pressure (PAP),
mean arterial pressure (MAP), and heart rate (HR). A second catheter
was inserted into the femoral vein for systemic administration of
potassium cyanide (KCN). Both catheters were tunneled subcutaneously and exteriorized through the back of the neck to be connected to the
pressure transducer on the subsequent day. PAP and MAP were measured in
conscious, freely moving rats with a pressure transducer (model CDX
III; Cobe, Lakewood, CO) connected to a Narcotrace 80 physiological
recorder (Narco Bio-Systems, Austin, TX). HR was measured with a Narco
Biotachometer Coupler (model 7302).
Microinjections into NTS.
For microinjections into the NTS, a 33-gauge needle (Small Parts) 1.5 mm longer than the guide cannula was connected by PE-10 tubing to a
1-µl syringe (Hamilton, Reno, NV). After removal of the occluder, the
needle for microinjection was carefully inserted into the guide cannula
and manual injection was started 30 s later. For bilateral
microinjection, the microinjection was initially performed on one side,
the needle was withdrawn and repositioned in the contralateral side,
and the second injection was performed. The same procedure was used in
the protocols with three guide cannulas. Therefore, the time interval
of microinjections into the NTS in the different protocols was ~1
min. The volume for each microinjection was 100 nl in all experimental protocols.
Activation of chemoreflex.
The chemoreflex was activated by intravenous injection of KCN (20 µg/rat; Merck, Darmstadt, Germany) in accordance with the procedures
described by Franchini and Krieger (10) and was previously validated
for our experimental conditions (16). These previous studies
demonstrated that the cardiovascular responses to KCN injection were
reproducible, and no habituation was observed when the same dose of KCN
was systematically injected at intervals of 10 min (16).
Drugs.
The drugs microinjected into the NTS were diluted in artificial
cerebrospinal fluid (aCSF) containing (in mM) 3 KCl, 0.6 MgCl2, 2 CaCl2, 132 NaCl, 24 NaHCO3, and 4 dextrose or 0.9%
saline. The solutions were freshly dissolved in aCSF, except
6,7-dinitroquinoxaline-2,3-dione (DNQX), which was dissolved in 2.5%
DMSO (Sigma, St. Louis, MO). Sodium bicarbonate was added to the
solutions to adjust the pH to a range of 7.0-7.4.
Experimental protocols.
All studies were performed in conscious, freely moving animals. The
experimental protocol for the study of neurotransmission into the NTS
consisted of the activation of the carotid chemoreflex before and after
microinjection of EAA antagonists into the
NTSlat or
NTSmid or into both sites
(NTSlat+mid) by using three guide cannulas. The NTS was functionally identified by previous microinjection of L-glutamate (1 nmol/100 nl), which, in accordance with our previous studies (5, 6,
23), produces pressor and bradycardic responses of short duration. The
peak changes in MAP and HR in response to chemoreflex activation were
evaluated before and 10 min after the microinjections of EAA receptor
antagonists into the NTS. A third injection of KCN was performed 40 min
after antagonist microinjection to evaluate the reversibility of the blockade. The EAA receptor antagonists microinjected into the NTS were
DNQX (Research Biochemicals International, Natick, MA), a selective
non-NMDA receptor antagonist, in three different doses (0.1, 0.5, and
2.0 nmol/100 nl), kynurenic acid (Kyn; Sigma), a nonselective
ionotropic antagonist, in one dose (10 nmol/100 nl), and
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
-methyl-4-carboxyphenylglycine, a metabotropic
receptor antagonist, into the
NTSlat or
NTSmid produced no changes in
baseline mean arterial pressure or heart rate or in the chemoreflex
responses. These results indicate that 1) the processing of the
parasympathetic component (bradycardia) of the chemoreflex seems to be
restricted to the NTSlat and was blocked by ionotropic antagonists and
2) the pressor response of the
chemoreflex was only partially reduced by microinjection of ionotropic
antagonists and not affected by injection of metabotropic antagonists
into the NTSlat or
NTSmid or into
NTSlat+mid in awake rats.
-methyl-4-carboxyphenylglycine
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-methyl-4-carboxyphenylglycine (MCPG; Research Biochemicals International), a selective metabotropic antagonist, in two different doses (2.5 and 5.0 nmol/100 nl). Each rat used in the three different experimental protocols (DNQX, Kyn, or MCPG) received only one dose of
the antagonist. The groups of rats that received microinjection of EAA
receptor antagonists into the NTS also received a control microinjection of saline with DMSO (DNQX group) or aCSF (Kyn and MCPG
groups) in each individual experiment at least 30 min previous to the
microinjection of the respective antagonist.
Histological examination. At the end of the experiments, 100 nl of Evans blue dye (2%) were microinjected for histological identification of the sites of microinjection, and later the animals were submitted to intracardiac perfusion with saline followed by 10% buffered Formalin while they were under ether anesthesia. The brains were removed and stored in buffered Formalin for 2 days. Serial coronal sections (10- 15 µm thick) were obtained and stained by the Nissl method. Only the rats in which the microinjection sites were located in the lateral or midline or in both portions of the commissural NTS, in accordance with the protocol, were considered for data analysis.
Data analysis. All data are expressed as means ± SE. The results were analyzed by one-way ANOVA, and the differences between individual means were determined by Student's t-test, with the level of significance set at 0.05 in all analyses.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effect of bilateral microinjection of DNQX into NTSlat on cardiovascular responses to chemoreflex activation. Figure 1 illustrates the effects of bilateral microinjection of increasing doses of DNQX (0.1, 0.5, and 2.0 nmol/100 nl) into the NTSlat on the cardiovascular responses to chemoreflex activation with KCN (20 µg iv) of three different rats, representative of their respective groups. Figure 1A shows that DNQX (0.1 nmol/100 nl) produced no changes in the pressor or bradycardic responses to chemoreflex activation, whereas the dose of 0.5 nmol/100 nl (Fig. 1B) attenuated the pressor response but produced no change in the bradycardic response induced by chemoreflex activation. Figure 1C shows that the dose of 2.0 nmol/100 nl also attenuated the pressor response to the chemoreflex in the same way as observed with the dose of 0.5 nmol/100 nl and blocked the bradycardic response. Bilateral microinjection of DNQX into the NTS produced a significant increase in basal MAP. Table 1 shows the absolute values of MAP and HR before and 10 min after bilateral microinjection of DNQX into the NTSlat and indicates that DNQX produced a significant increase in basal MAP at all doses, without any significant changes in basal HR.
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173 ± 16 vs. 153 ± 28 and 216 ± 27 vs. 216 ± 27 beats/min). However, at the dose of 2.0 nmol/100 nl, DNQX elicited a
significant reduction in this response (160 ± 25 vs.
50 ± 31 beats/min). The effects of DNQX on the chemoreflex responses were reversible, considering that 40 min after bilateral microinjection of DNQX into the
NTSlat, the chemoreflex responses were back to control values. Bilateral microinjection of vehicle containing 2.5% DMSO into the
NTSlat was associated with
negligible effects on the pressor and bradycardic responses to
chemoreflex activation (Table 2).
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Effect of microinjection of Kyn into
NTSlat or NTSmid
or into
NTSlat+mid on
cardiovascular responses to chemoreflex activation.
The effect of microinjection of Kyn into the
NTSlat or
NTSmid on the cardiovascular
responses to chemoreflex activation is illustrated in Fig.
3. Figure
3A shows a tracing of one rat in which
bilateral microinjection of Kyn (10 nmol/100 nl) into the NTSlat attenuated the pressor
response to the chemoreflex, whereas the bradycardic response was
abolished. Figure 3B shows the
tracings of one rat in which microinjection of Kyn into the
NTSmid induced small changes in
the pressor or bradycardic responses elicited by KCN injection despite
a significant increase in basal MAP. Figure
3C shows the tracings of one rat in
which simultaneous microinjection of Kyn into
NTSlat+mid attenuated the pressor response and abolished the bradycardic response to chemoreflex similar
to the microinjection of Kyn into the
NTSlat (Fig.
3A). These data are summarized in
Fig. 4 and show that Kyn microinjected into
the NTSlat or into the
NTSlat+mid significantly reduced the cardiovascular responses to chemoreflex activation. In these cases,
the pressor responses were significantly reduced
(NTSlat, +50 ± 2 vs. +30 ± 3 mmHg; NTSlat+mid, +48 ± 3 vs. +22 ± 8 mmHg) and the bradycardic responses were almost
abolished (NTSlat, 225 ± 28 vs. 18 ± 7 beats/min;
NTSlat+mid, 213 ± 28 vs. 11 ± 5 beats/min). In contrast, microinjection of Kyn
into the NTSmid did not affect the
pressor (+57 ± 5 vs. 39 ± 8 mmHg) or bradycardic response
(191 ± 37 vs. 163 ± 64 beats/min) to chemoreflex activation. Microinjection of Kyn into the
NTSlat,
NTSmid, or NTSlat+mid induced a significant
increase in baseline MAP but not in baseline HR (Table
5).
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214 ± 26 vs. 216 ± 27 beats/min) responses to chemoreflex activation.
The effects of Kyn on the cardiovascular responses to chemoreflex
activation were restricted to the NTS, because misplaced microinjections into areas adjacent to the NTS did not modify the
pressor or bradycardic responses to chemoreflex activation (Table 3).
Effect of microinjection of MCPG into NTSlat or NTSmid on cardiovascular responses to chemoreflex activation. Neither dose of MCPG (2.5 and 5.0 nmol/100 nl) modified the cardiovascular responses to the chemoreflex activation. Thus only the data for the dose of 2.5 nmol/100 nl are presented in Fig. 5, which illustrates the effects of microinjections of MCPG into the NTSlat or into the NTSmid on the cardiovascular responses to chemoreflex activation. Figure 5A shows the tracings of one rat in which bilateral microinjection of MCPG into the NTSlat induced no changes in the pressor or bradycardic responses to chemoreflex activation, and Fig. 5B shows the tracings of one rat in which microinjection of MCPG into the NTSmid also did not affect the responses to chemoreflex activation.
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241 ± 18 vs.
226 ± 16 beats/min;
NTSmid, 210 ± 45 vs.
212 ± 45 beats/min) to chemoreflex activation.
Microinjection of MCPG into these two subregions of the commissural NTS
did not elicit significant changes in baseline MAP or in baseline HR
(Table 6).
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66 ± 8 vs. 24 ± 9 mmHg) as well as the bradycardic (267 ± 26 vs.
80 ± 3 beats/min) responses induced by
trans-ACPD (250 pmol/100 nl) microinjected into the same site.
Similar blockade was achieved with the dose of 5.0 nmol/100 nl of MCPG
(data not shown).
Histological analyses of microinjection sites. Figure 7 is a photomicrograph of a transverse section of the brain stem of the rat, showing the site of microinjections in the NTS. Figure 7A is a photomicrograph of a coronal section of the brain stem of one rat representative of the group in which the microinjections were performed bilaterally into the NTSlat. Figure 7B is a photomicrograph of a coronal section of the brain stem of one rat representative of the group in which the microinjections were performed into the NTSmid.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Several lines of experimental evidence support the concept that EAA receptors play a major role in the processing of cardiovascular afferents in the NTS (4, 13, 14, 16, 38) and particularly in the neurotransmission of the chemoreflex in the NTS (2, 3, 20, 27, 39, 40). In a recent study on unanesthetized rats, we showed that the bradycardic response to chemoreflex activation was blocked in a dose-dependent manner by AP-5, an NMDA receptor antagonist, whereas the pressor response was not affected (16). However, previous studies performed on anesthetized rats have reported that administration of EAA antagonists into the NTS blocked the pressor response (sympathoexcitatory component) of the chemoreflex activation (39, 40); such blockade was not observed in the present study in the absence of anesthesia. Bilateral microinjection of DNQX (a selective non-NMDA receptor antagonist) at the dose of 0.5 nmol/100 nl, which does not affect NMDA receptors, significantly reduced but did not abolish the pressor response to chemoreflex activation (+64 ± 4 vs. +34 ± 17 mmHg), whereas the highest dose of DNQX used (2.0 nmol/100 nl), which is not selective for non-NMDA receptors, did not elicit any additional reduction in the pressor response to chemoreflex activation (+59 ± 4 vs. +33 ± 10 mmHg).
The partial reduction of the pressor response to chemoreflex activation
observed after DNQX may be related, at least in part, to the
involvement of non-NMDA receptors in the neurotransmission of the
sympathoexcitatory component of the chemoreflex in the NTSlat. However, these results
require careful interpretation because another possibility to explain
why the pressor response to chemoreflex activation was reduced by DNQX
is the significant increase in the baseline MAP observed after the
bilateral microinjection of DNQX into the
NTSlat. With respect to the DNQX
protocol, it is important to note that the additional increase in MAP
in response to chemoreflex activation was smaller than in the control,
probably due to the increase in the baseline sympathetic activity and
consequently in MAP. It is also important to emphasize that the pressor
response to chemoreflex activation is essentially due to sympathetic
activation and not to vasopressin or any other circulating
neurohormone, because in previous studies we verified that the
treatment with prazosin (intravenous), an
1-adrenoceptor antagonist,
almost abolished the pressor response of the chemoreflex (16).
The effects observed after bilateral microinjection of DNQX into the NTSlat on baseline MAP were probably due to the blockade of the sympathoinhibitory pathway of the baroreflex and suggest that non-NMDA receptors located on neurons of this subregion of the NTS are involved in the neurotransmission of this component of the baroreflex. This possibility is supported by a previous study by Gordon and Leone (12) showing that non-NMDA receptors in the NTS play a predominant role in mediating the hypotensive response produced by aortic baroreceptor stimulation. In addition, we verified in a previous study that the blockade of the NMDA receptors in the NTSlat produced no changes in baseline MAP (16).
Another important aspect of the present study relates to the blockade of the bradycardic response to chemoreflex activation observed after the dose of 2.0 nmol/100 nl of DNQX microinjected bilaterally into the NTSlat. In a previous study, we showed that the cardiovagal component of this reflex is mediated by NMDA receptors (16). Therefore, the bradycardic response should not be affected by the non-NMDA receptor antagonist unless the dose used is not selective for this receptor subtype. In this respect, a specific protocol used in the present study showed that DNQX at the dose of 2.0 nmol/100 nl is not selective for non-NMDA receptors because it also blocked the cardiovascular responses to NMDA microinjection into the NTS. The protocol using 0.5 nmol/100 nl of DNQX shows that this dose is in the range of selectivity for non-NMDA receptors, because the cardiovascular responses to NMDA receptors and the bradycardic response to chemoreflex activation were not affected, whereas baseline MAP was increased. Therefore, this evidence supports the concept that the cardiovagal component of the chemoreflex is effectively mediated by NMDA receptors in the NTSlat.
The present data show that DNQX microinjected into the NTSlat did not abolish the pressor response to chemoreflex activation. To explain these findings, at least two possibilities should be considered. First, the EAA receptors in this subregion of the commissural NTS, particularly the non-NMDA receptors, may not be involved in the sympathoexcitatory neurotransmission of the chemoreflex. Second, the blockade of non-NMDA receptors only in the NTSlat may not be sufficient to block all the receptors located in different sites of the NTS probably involved in the processing of the afferent information for the chemoreflex. A study by Zhang and Mifflin (40) showed that the pressor response of the chemoreflex was blocked when Kyn, a broad-spectrum EAA receptor antagonist, was microinjected into the NTSlat. In a study by Vardhan et al. (39), the pressor response to chemoreflex activation was also blocked only after the blockade of NMDA and non-NMDA receptors in the NTSmid. Therefore, in these studies (39, 40), which were performed in anesthetized animals, the pressor response to chemoreflex activation was abolished after a nonselective blockade of ionotropic receptors in the NTSlat or NTSmid. Considering these previous studies and also that isolated blockade of NMDA (16) or non-NMDA receptors (present study) in the NTSlat of unanesthetized rats was not able to block the pressor response to chemoreflex activation, we also evaluated the effects of the Kyn microinjection into the NTSlat or NTSmid on the pressor response to chemoreflex activation in unanesthetized rats.
The reduction in magnitude of the pressor response to chemoreflex activation induced by microinjection of Kyn into the NTSlat was similar to that induced by microinjection of DNQX into the same area. In another group of rats, microinjection of Kyn into the NTSmid did not affect the pressor response to chemoreflex activation. In both cases (NTSlat and NTSmid), the microinjection of Kyn produced a significant increase in basal MAP, similar to the effect of DNQX microinjection into the NTSlat. One possibility that cannot be ruled out to explain the reduction in the pressor response to chemoreflex activation after microinjection of Kyn into NTSlat may be related to the increase in baseline MAP, similar to that observed after microinjection of DNQX into the NTSlat. On the other hand, microinjection of Kyn into NTSmid, despite increasing the baseline MAP, produced no attenuation in the pressor response to chemoreflex activation. Therefore, this observation indicates that the reduction in the pressor response to chemoreflex activation by Kyn microinjected into the NTS probably is not associated with the increase in the baseline MAP. However, it is important to note that the baseline increases in MAP produced by Kyn into the NTSlat or NTSmid were different (+22 vs. +13%, respectively). This difference may explain why the increase in MAP in response to chemoreflex activation after Kyn into the NTSlat was smaller than the increase observed after Kyn into NTSmid. Similar findings were obtained with microinjections of DNQX into NTSlat. The dose of 0.1 nmol/100 nl into the NTSlat increased baseline MAP by 12% and produced no effect on the pressor response to chemoreflex activation, whereas the dose of 0.5 nmol/100 nl increased the baseline MAP by 22% and induced a significant reduction in the pressor response to chemoreflex activation. Alternatively, we might also consider the possibility that the increase in baseline MAP, secondary to the basal increase in sympathetic activity after Kyn or DNQX into the NTSlat, reduces the magnitude of the pressor response to chemoreflex activation because the maximal increase of the sympathetic activity remained the same. The experimental protocol in which we performed blockade of EAA receptors in the NTSlat and NTSmid (NTSlat+mid) with Kyn (10 nmol/100 nl) shows that the pressor response to chemoreflex activation was also only partially reduced, suggesting that the processing of the excitatory component of the chemoreflex in the NTS involves neurotransmitters and receptors other than EAAs and their receptors.
In relation to the parasympathetic component of the chemoreflex, our data show that Kyn microinjected into the NTSlat abolished the bradycardic response, probably due to the blockade of NMDA receptors, as we demonstrated previously with microinjections of AP-5 into the same subregion of the NTS (16). However, Kyn microinjected into the NTSmid produced no change in the bradycardic response to chemoreflex activation, suggesting that this subregion of the commissural NTS is not directly involved in the processing of the parasympathetic component of this reflex. A study by Colombari et al. (7) shows that the integrity of NTSmid is essential for the pressor and bradycardic responses to chemoreflex activation, but their findings do not necessarily imply that the neurotransmission of the parasympathetic component of the chemoreflex occurs in this subregion of the NTS. Studies by Finley and Katz (9) have shown dense labeling in the NTSlat after injection of wheat germ agglutinin-conjugated horseradish peroxidase into the vascularly isolated carotid body, whereas a functional study by Vardhan et al. (39) has shown that the NTSmid plays a key role in the neurotransmission of the sympathoexcitatory component of the chemoreflex in the NTS. No study has evaluated the neurotransmission of the parasympathetic component of the chemoreflex, as we did in the present as well as in a previous investigation from our laboratory (16). Because of this controversy related to the site of termination of the chemoreflex afferents and the processing of the sympathetic and parasympathetic components of the chemoreflex in different subregions of the NTS, in the present study we avoided this problem by performing microinjection into the lateral commissural NTS (NTSlat) and into the midline of commissural NTS (NTSmid), or in both (NTSlat+mid), i.e., in all sites in which the chemoreflex neurotransmission seems to be processed. In this case, we observed that the processing of the parasympathetic component of the chemoreflex seems to be restricted to the NTSlat.
Considering that the antagonism of ionotropic EAA receptors in the NTSlat or NTSmid with DNQX or Kyn (NTSlat+mid) was not able to block the pressor response to chemoreflex activation, and also considering previous evidence that metabotropic receptors mediate excitatory transmission in the NTS (11-13), we also evaluated the possible involvement of this class of EAA receptors in the processing of the neurotransmission of the sympathoexcitatory component of the chemoreflex. The microinjection of MCPG, a metabotropic antagonist, into the NTSlat or NTSmid also did not modify the basal MAP and HR or the pressor and bradycardic responses to chemoreflex activation, indicating that the metabotropic receptors in the NTS play no major role in the neurotransmission of the parasympathetic and sympathetic components of the chemoreflex.
The activation of the chemoreflex with KCN in unanesthetized rats produces, in addition to cardiovascular and ventilatory responses, an important behavioral response (10) which seems to be a consequence of the stimulation of the hypothalamic areas involved with the defense reaction (25). Considering that chemical or electrical stimulation of these hypothalamic defense areas increases sympathetic activity (17, 30-33), we may suggest that, in contrast to studies performed under anesthesia, the pressor response induced by chemoreflex activation in the present study depends at least in part on the hypothalamic defense areas. It is important to note that the behavioral responses to KCN, such as exploration of the cage and alertness, were also eliminated by bilateral ligature of the carotid body artery (16) or by deafferentation of the carotid sinus nerve bifurcation (10). Under anesthesia, the alertness and the defense reaction component of the chemoreflex responses are abolished, a fact that may explain why in the studies by Vardhan et al. (39) and Zhang and Mifflin (40) working with anesthetized rats, the pressor response of the chemoreflex was blocked by EAA receptor antagonists into the NTS. In addition, it is important to note that in the experiments by Vardhan et al. (39) and Zhang and Mifflin (40), no changes in HR were observed in response to chemoreflex activation, whereas in the present study as well as in a previous study from our laboratory (16) we observed an intense bradycardic response. Therefore, when animals are under anesthesia, the behavioral and cardiovascular responses to chemoreflex activation are different, as demonstrated by Franchini and Krieger (10), and the data obtained in conscious and anesthetized animals cannot be easily compared. The degree of involvement of hypothalamic areas in the generation of the pressor response of the chemoreflex in the present study was not experimentally addressed, and therefore further studies on unanesthetized rats are required to explore the possible involvement of hypothalamic projections from and to the NTS on the processing of the sympathoexcitatory component (pressor response) of the chemoreflex.
The involvement of other neurotransmitters in the processing of the chemoreflex afferents in the NTS has been considered. Studies using microdialysis demonstrated that the release of substance P (22, 35) into the NTS was increased during hypoxia. Adenosine may also play a role in the processing of the chemoreflex in the NTS, especially in the projection from the hypothalamic defense areas to the NTS (36, 37). However, the role of these putative neurotransmitters and their different subtypes of receptors in the processing of the pressor response to the chemoreflex in the lateral and medial commissural NTS of unanesthetized rats and particularly in the neurotransmission of the sympathoexcitatory component of the chemoreflex requires additional studies.
The data of the present study indicate that the neurotransmission of the sympathoexcitatory component of the chemoreflex is more complex than the cardiovagal component and suggest that neurotransmitters other than EAAs may play a key role in the processing of the sympathoexcitatory component (pressor response) of the chemoreflex in the lateral and medial commissural NTS.
Perspectives
In a previous study (16), we verified that the bradycardic response to the chemoreflex activation was blocked in a dose-dependent manner by AP-5, a selective NMDA receptor antagonist, whereas the pressor response was not affected. In accordance with those results, we suggested that the sympathoexcitatory component (pressor response) of the chemoreflex could be mediated by non-NMDA receptors at the NTS level, and the experiments presented in the current study were performed to verify this hypothesis. However, the data of this study indicated that different ionotropic (Kyn and DNQX) or metabotropic (MCPG) receptor antagonists were not able to block the pressor response of the chemoreflex, even when Kyn was microinjected simultaneously in three different sites of the NTS. Considering that in a previous study an NMDA receptor antagonist (AP-5) also produced no blockade of the pressor response of the chemoreflex, we may suggest that EAA receptors may not be involved in the processing of the neurotransmission of the sympathoexcitatory component of the chemoreflex in the NTS. Because of this unexpected finding, new and interesting perspectives are open to studies involving the neurotransmission of the sympathoexcitatory component of the chemoreflex in the NTS. One important aspect that must be considered in the present study is related to the fact that the activation of the chemoreflex in unanesthetized rats, in addition to the cardiovascular and respiratory changes, produces behavioral responses, which may contribute to the pressor response. Therefore, studies involving the possible role of the hypothalamic defense area, for example, are required to verify if projections from this area to the NTS participate in the generation of the pressor response to chemoreflex activation. Studies are also required on the possible involvement of other areas, including the parabrachial nucleus and periaqueductal gray in the complex physiological responses to the activation of the chemoreflex in unanesthetized rats, including cardiovascular, respiratory, and behavioral aspects. In addition to the involvement of other areas of the brain in this pressor response, another important aspect to be studied, as a consequence of the present findings, is related to the possibility that neurotransmitters/neuromodulators other than EAAs may play a key role in this neurotransmission. Among these potential neurotransmitters/neuromodulators of the pressor response of the chemoreflex in the NTS, evidence in the literature indicates the involvement of substance P and adenosine in this neurotransmission. Therefore, experiments using the antagonist of the different subtypes of tachycinergic and purinergic receptors in the NTS should be also performed, preferentially in unanesthetized rats, to verify their possible involvement in the neurotransmission of the pressor response to chemoreflex activation.| |
ACKNOWLEDGEMENTS |
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We thank R. F. de Melo for the histological preparations.
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FOOTNOTES |
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This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Programa de Apoio aos Núcleos de Excelência.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: B. H. Machado, Dept. of Physiology, School of Medicine of Ribeirão Preto, Univ. of São Paulo, 14049-900, Ribeirão Preto, São Paulo, Brazil.
Received 20 March 1998; accepted in final form 25 August 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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1 · rat
) in MAP (top) and HR
(bottom) in response to injection of
KCN (20 µg · 0.1 ml
,
n = 7), 0.5 (
,
n = 5), and 2.0 nmol/100 nl (
,
n = 6)] into
NTSlat in 3 groups of rats.
Top: significant differences in
pressor response were observed after doses of 0.5 and 2.0 nmol/100 nl
in comparison with control. Bottom:
changes in HR were significantly reduced only after DNQX at 2.0 nmol/100 nl, a dose that is not selective for non-NMDA receptors (Table
