bradycardia, and tachypnea. In the present study, we activated carotid chemoreceptors with KCN and the neurotransmission of the chemoreceptor reflex into the ...
NMDA receptors in NTS are involved in bradycardic but not in pressor response of chemoreflex ANDRfiA S. HAIBARA, EDUARDO COLOMBARI, DEOCL&CIO A. CHIANCA, LEN1 G. H. BONAGAMBA, AND BENEDITO H. MACHADO Department of Physiology, School of Medicine of Ribeirao Preto, University of Sao Pa&o, 14049-900 Ribeirao Preto, Sao Paulo, Brazil Haibara, And&a S., Eduardo Colombari, Deockio A. Chianca, Jr., Leni G. H. Bonagamba, and Benedito H. Machado. NMDA receptors in NTS are involved in bradycardic but not in pressor response of chemoreflex. Am. J. PhysioZ. 269 (Heart Circ. PhysioZ. 38): H1421-H1427,1995.Activation of carotid chemoreceptors with intravenous potassium cyanide (KCN) produces increases in arterial pressure, bradycardia, and tachypnea. In the present study, we activated carotid chemoreceptors with KCN and the neurotransmission of the chemoreceptor reflex into the commissural nucleus tractus solitarii (NTS) was blocked with phosphonovaleric acid @P-5), an N-methyl-D-aspartate (NMDA)-selective antagonist. The aim of this study was to evaluate the involvement of NMDA receptors in the cardiovascular and respiratory responses produced by chemoreceptor activation in unanesthetized rats. The pressor response to KCN was not changed after microinjection of three different doses of AP-5 into the NTS, whereas the bradycardic response was reduced in a dosedependent manner. The increase in respiratory frequency in response to carotid chemoreceptor activation was also not affected by AP-5 microinjected into the NTS. The data indicate that the activation of the cardiovagal component of the chemoreflex in the commissural NTS is mediated by NMDA receptors, whereas pressor and ventilatory responses are not. excitatory amino acid; phosphonovaleric acid; cardiovascular regulation; heart rate control; autonomic blockade; neurotransmitter; arterial chemoreceptor; respiratory frequency
CHEMORECEPTOR afferents project to the nucleus tractus solitarii (NTS) (6, 7, 11, 17, ZZ), and activation of the chemoreflex produces an increase in arterial pressure, bradycardia, and tachypnea (8,9). The neurotransmission of the afferent information from the carotid chemoreceptors and the processing of the ventilatory and cardiovascular autonomic responses at the NTS level are not completely understood. The neurotransmission of the chemoreflex into the NTS has been studied in anesthetized rats, and several studies (2, 24, 25) have indicated that excitatory amino acid (EAA) receptors participate in the chemoreflex. Considering that the activation of the chemoreflex produces sympathoexcitation (pressor response) as well as cardiovagal excitation (bradycardia), in the present study we evaluated the possibilities that these different autonomic pathways originating at the NTS level may involve not only different postsynaptic neurons but also different subtypes of EAA receptors. Thus, using phosphonovaleric acid (AR-5), an N-methyl-D-aspartate (NMDA)-selective antagonist (5), we evaluated the role of NMDA receptors in the neurotransmission of the chemoreflex into the NTS. THE CAROTID
0363-6135/95
$3.00
Copy-right
o 1995
JR.,
The experiments were performed on conscious rats, because previous studies showed that anesthetics have a distorting effect on the cardiovascular responses to chemoreceptor activation (9) as well as on the hemodynamic responses to microinjection of the EAA Lglutamate into the NTS (14). In the present study, the chemoreflex was activated by intravenous injection of potassium cyanide (KCN), and the changes in mean arterial pressure (MAP), heart rate (HR), and respiratory frequency (RF) were evaluated before and after bilateral microinjection of AR-5 into the NTS. METHODS Male Wistar rats weighing 230-270 g were used in the present study. Four days before the experiments, pentobarbital sodium (40 mg/kg ip; Sigma Chemical, St. Louis, MO)anesthetized rats were placed in a stereotaxic apparatus (David Kopf, Tujunga, CA), and the technique described by Michelini and Bonagamba (16) was used to implant bilateral guide cannulas in the direction of the NTS in accordance with the coordinates of Paxinos and Watson (20). To implant the cannulas, a small window was opened caudal to the lambda, through which a 15-mm-long stainless steel cannula (22 gauge) was introduced perpendicularly 0.5 mm lateral to the midline, 14 mm caudal to the bregma, and 7.9 mm below the skull surface of the bregma. The tip of each guide cannula was placed in the cerebellum 1.0 mm above the dorsal surface of the brain stem. The cannula was fixed to the skull with methacrylate and watch screws and closed with an occluder until the time for the microinjections. The needle (33 gauge) used for microinjection into the NTS was 1.5 mm longer than the guide cannula and was connected by PE-10 tubing to a l-k1 syringe (Hamilton, Reno, NV). One day before the experiments, under ether 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), MAP, and HR and a second catheter was inserted into the femoral vein for KCN injection. Both catheters were tunneled and exteriorized through the back of the neck to be connected to the pressure transducer under conscious freely moving conditions. PAP and MAP were measured with a pressure transducer (model CDX III, Cobe Laboratories, Lakewood, CO) connected to a Narcotrace 80 physiological recorder (Narco Bio-Systems, Austin, TX). HR was measured with a Narco Biotachometer Coupler (model 7302). The efficacy of KCN in activating carotid chemoreceptor was evaluated by intravenous injection of increasing doses of KCN (from 0.4 to 40.0 kg/rat), and dose-response curves for the changes in HR, MAP, and RF were obtained. These experiments were performed on the same animals before and after bilateral surgical ligature of the carotid body artery under ether anesthesia 24 h before the experiments according to the method described by Franchini and Krieger (9). Ventilation was measured by means of whole body plethysmography by use of the technique described by Malan (15), the American
Physiological
Society
H1421
H1422
NMDA
RECEPTORS
AND
which is based on monitoring small pressure changes within a closed animal chamber. A highly sensitive differential pressure transducer (model PM 979, Statham) connected to the recorder was used for these measurements. The catheters from femoral artery and vein were exteriorized through a small hole in the chamber, which was occluded with silicone grease during the recordings. Autonomic evaluation of the cardiovascular responses to carotid chemoreceptor activation was performed in two different groups of rats in which prazosin (1 mg/kg iv) or methylatropine (1 mg/kg iv) was used to block the sympathetic or parasympathetic efferent activity, respectively. In this protocol, KCN (20 pg/rat iv) was injected before and after the selective autonomic blockade, and the changes in MAP and HR were evaluated. The drugs microinjected into the NTS were diluted in artificial cerebral spinal fluid (CSF) containing (in mM) 3 KCl, 0.6 MgC12, 2 CaC12, 132 NaCl, 24 NaHC03, and 4 dextrose, and all microinjections were performed in a volume of 100 nl. The solutions were freshly dissolved in CSF, and sodium bicarbonate was added to adjust the pH to 7.0. The needles for microinjection were carefully inserted sequentially into the guide cannulas, and the manually performed injection was initiated 30 s later. In the protocols for the study of neurotransmission into the NTS, carotid chemoreceptors were stimulated with injection of KCN (20 pg/rat iv), and the involvement of NMDA receptors in this neurotransmission was evaluated by bilateral microinjection of AP-5 (Sigma Chemical). Three doses of AP-5 (0.5, 2.0, and 10.0 nmol/lOO nl) were microinjected into the NTS of three groups of rats [groups A (n = 6), B (n = 9>, and C (n = 6)]. The rats of each group received only one dose of AP-5, and in this protocol KCN was injected intravenously before and 10 min after AP-5 microinjection into the NTS. In this protocol the chemoreflex activation was also performed before and 10 min after bilateral microinjection of artificial CSF into the NTS as a volume control. A third injection of KCN was performed 40 min after AP-5 in order to evaluate the reversibility of the blockade. To determine whether changes in HR after AP-5 were affected by changes in ventilation, a specific protocol was developed for a group of rats (n = 6) in which AP-5 (10 nmol/lOO nl) was injected bilaterally into the NTS with the animals inside the plethysmographic chamber in order to evaluate the possible effect of an NMDA antagonist on the tachypnea produced by activation of carotid chemoreceptors. After the experiments, in conscious animals, 100 nl of Evans blue (2%) were microinjected into the same sites for histological analysis, and later, in ether-anesthetized animals, intracardiac perfusion with saline was followed by 10% buffered Formalin. The brains were removed and stored in buffered Formalin for 2 days, and serial coronal (lo-pm) sections were cut and stained by the Nissl method. Only the rats in which the site of microinjection was the lateral and medial portions of the commissural NTS were used for data analysis. Values are means 2 SE, and the results were analyzed by the paired Student’s t-test. The data related to the dose effect of AP-5 on the cardiovascular responses to chemoreflex activation were analyzed by one-way analysis of variance, and the differences between individual means were determined by Student’s modified t-test with Bonferroni correction for multiple comparisons. The statistical significance was set at the 0.05 level in all procedures. RESULTS
Dose-response curve to KCN. Figure 1 shows the effects of increasing doses of KCN (0.4, 5.0, 10.0, 15.0,
ARTERIAL
CHEMOREFLEX
A
r
80
c
60-
40-
20-
0
B
0 -60
-180
-240 4
L
-300
C
80
rt-
z 6o a u ; 40 cr a 20 0~
g KCN
Fig. 1. Changes in mean arterial pressure (AMAP), heart rate (AHR), and respiratory frequency (ARF) in response to increasing intravenous doses of potassium cyanide (KCN) in conscious rats before (solid line) and 1 day after bilateral ligature of carotid body arteries (dotted line). * Significant difference between control and ligature (P < 0.05).
20.0, and 40.0 pg/rat) on MAP, HR, and RF of intact rats and rats with ligature of the carotid body artery. Increasing doses of KCN produced a dose-dependent change in MAP, HR, and RF. Ligature of the carotid body artery abolished the cardiovascular and respiratory changes in response to KCN injection, and the submaximal dose of 20 pg/rat produced consistent changes for all 3 parameters studied. Therefore, in all other experiments, this dose was used to activate carotid chemoreceptors. Effect of auto lnomic blockade on cardiovascular changes induced by activation of carotid chemoreceptors with KCN. Figure 2, A and C, shows the changes in MAP and HR in response to KCN injection before and after treatment with prazosin (1 mg/kg iv). Blockade of sympathetic efferent tone significantly reduced the pressor response (+ 58 t 4 vs. +17 t 2 mmHg), but
NMDA
80
B 8Ot
60
60-
RECEPTORS
AND
ARTERIAL
7
4020-
* BL
O*
C
50 0 30 I I-
1
-200 I-250 c Fig. 2. A and C: changes
-250
in MAP and HR in response to intravenous KCN before (open bars, n = 8) and after injection of prazosin (1 mg/kg iv; hatched bars, n = 8). B and D: changes in MAP and HR in response to intravenous KCN before (open bars, n = 8) and after injection of methylatropine (1 mg/kg iv; hatched bars, n = 8). *Significant difference from control (P < 0.05).
A before
after
B
H1423
CHEMOREFLEX
the bradycardic response was significantly increased (- 136 t 15 vs. -199 t 22 beats/min) when carotid chemoreceptors were activated. Figure 2, B and D, shows the changes in MAP and HR in response to KCN injection before and after treatment with methylatropine (1 mg/kg iv). The data indicate that the blockade of parasympathetic tone abolished the bradycardic response and elicited a tachycardic response (- 181 t 13 vs. +44 t 5 beats/min) and a significantly greater increase in MAP (+56 t 3 vs. +71 t 3 mmHg) than before methylatropine. Effect of bilateral microinjection of AP-5 into the NTS on cardiovascular changes induced by activation of carotid chemoreceptors with KCN. The effect of increasing doses of AR-5 (0.5,2.0, and 10.0 nmol/lOO nl) on the cardiovascular effects of carotid chemoreceptor activation with KCN on three different rats representative of each of the three groups is illustrated in Fig. 3. Figure 3A shows that AR-5 at 0.5 nmol/lOO nl produced no changes in the pressor or bradycardic response to KCN injection, whereas at 2.0 nmol/lOO nl AR-5 significantly attenuated the bradycardic response but did not change the pressor response (Fig. 3B). Figure 3C shows that the maximal dose of AR-5 (10 nmol/ 100 nl) almost abolished the bradycardic response but did not significantly change the pressor response to carotid chemoreceptor activation with KCN. The blockade of the bradycardic response by AR-5 at 2.0 and 10.0 nmol/lOO nl was reversible, considering that 40 min later the cardiovascular responses to chemoreceptor activation with KCN were similar to those before AR-5 bilateral injection into the NTS. Figure 4 shows the mean changes in MAP and HR in response to activation of carotid chemoreceptors with
before
after
-A-- -J-
C
of ter
before
4+
-+h L1 mm.
OL
t
KCN
t KCN
f KCN
Fig. 3. Changes in HR, pulsatile arterial pressure (PAP), and MAP in response ml-l iv) before and after bilateral microinjection of phosphonovaleric acid nmol/lOO nl (C) into nucleus tractus solitarii of 3 different rats.
t
t
KCN
KCN
to injection of KCN (20 kgratl.O.l (AP-5) at 0.5 (A), 2.0 (B), and
t KCN (2Opg) 10.0
H1424
NMDA
RECEPTORS
AND
ARTERIAL
CHEMOREFLEX
KCN
A
80
r t
60-
KCN (n=6)
KCN (n=91
t!
KCN
(n=6)
4 t
Fig. 4. Changes in MAP and HR in response to injection of KCN (20 kgratl*O.l ml-l iv) before and after bilateral microinjection of increasing doses of AP-5 into nucleus tractus solitarii (NTS) of 3 groups of rats. A: changes in MAP in response to KCN before and after AP-5 was bilaterally injected into NTS at 0.5 (o), 2.0 (a), and 10.0 nmol/lOO nl (0). No statistical differences were observed in pressor responses after the 3 doses of AP-5. B: changes in HR in response to KCN before and after AP-5 was bilaterally injected into NTS as in A. Changes in HR are significantly smaller after AP-5 at 2.0 than at 0.5 nmol/lOO nl but are not different from changes after AP-5 at 10.0 nmol/lOO nl. One-way analysis of variance and differences between individual means were determined by Student’s modified t-test with Bonferroni correction for multiple comparisons.
40-
20-
0-1 CONTROL
1 0.5
1
DOSE
B
OV
-50
I
2.0 AP-5 (nmoles A00 I
1
10.0 nl) 1
+
z oa z I
-100
a -150 1 -200L
KCN in all rats studied in group A (+ 58 t 5 vs. + 53 t 6 mmHgand -183 t 19 vs. -160 t 31 beats/min, n = 6), group B (+55 -+ 2 vs. +53 t 2 mmHgand -181 t 14 vs. -66 t 18 beats/min, n = 9), and group C (+60 t 4 vs. +46 t 6 mmHg and -172 t 15 vs. -138 t 10 beats/min, n = 6). The data summarized in Fig. 4 indicate that AR-5 produced a significant dose-dependent blockade of the bradycardic response and no effect on the pressor response component of carotid chemoreceptor activation. Table 1 shows the absolute values of MAP and HR immediately before and 10 min after bilateral microinjection of AR-5 into the NTS without intravenous KCN injection during this period. The data indicate that Table 1. MU and HR before and 10 min after bilateral microinjections of AP-5 into NTS MAP,
mmHg
HR, beats / min
AP-5, nmol/ 100 nl
Before
After
0.5 2.0 10.0
10326 11123 107+3
10526 112+3 112+4
Before
350 k 26 352 + 13 325 k 12
After
345 + 24 364 t 13 355 2 22
Values are means + SE. MAP, mean arterial pressure; HR, heart rate; AP-5, phosphonovaleric acid; NTS, nucleus tractus solitarii. No statistical difference was observed when MAP and HR were compared before and after AP-5 (paired t-test).
administration of AR-5 bilaterally into the NTS produced no significant changes in the basal MAP and HR. The blockade of the bradycardic response by AR-5 was restricted to the NTS, because misplaced microinjection of AR-5 into dorsal (gracile nucleus, n = 2) or ventral (hypoglossal nucleus, n = 2) structures adjacent to the NTS produced no significant changes in the bradycardic or pressor and tachypneic responses to chemoreflex activation. Bilateral microinjection of AP-5 into the NTS produced no effect on respiratory changes induced by activation of carotid chemoreceptors with KCN. Figure 5 shows the effect of microinjection of AR-5 (10 nmol/ 100 nl) on the changes in MAP, HR, and RF induced by activation of carotid chemoreceptors with KCN (20 kg/kg iv) in a group of six rats. AR-5 at 10 nmol/lOO nl significantly reduced the bradycardic response to carotid chemoreceptor activation (-216 t 5 vs. -63 t 15 beats/min) but did not change the pressor response (+62 t 3 vs. +58 t 3 mmHg) or tachypneic response (+73 t 6~s. +72 + 3 cycles/min). The blockade of the bradycardic response by AR-5 at 10 nmol/lOO nl was reversible, because 40 min later the bradycardic response was similar to control values. In all experimental protocols in which AR-5 was microinjected bilaterally into the NTS, the chemoreflex activation was also performed before and after bilateral
NMDA
A
80
gi E E e
6o
t
AFTER
AP-5
RECEPTORS
AND
1
ARTERIAL
H1425
CHEMOREFLEX
55 km. The dorsoventral distribution restricted to the NTS region.
of the dye was
DISCUSSION
a r a
Recent studies have suggested that the afferents from the carotid chemoreceptors project to the medial and commissural NTS, areas that also play an important role in receiving afferent projections from the arterial baroreceptors (7, 11, 18, 21, 24). In addition to their involvement in ventilatory control, carotid chemoreceptors also play an important role in the autonomic mechanisms related to arterial pressure regulation (10, 18,21,23). In the present study, we show that activation of carotid chemoreceptors with KCN produces an increase in MAP, RF, and bradycardia. The cardiovascular and respiratory effects of intravenous KCN injections are exclusively related to the activation of carotid chemoreceptors, because ligature of the carotid body artery abolished the responses to KCN injection. These results are consistent with those reported by Franchini and Krieger (8,9). In a previous study, we showed that microinjection of L-glutamate into the NTS of conscious freely moving rats produced a dose-dependent pressor and bradycardic response (14). Those findings suggested that the cardiovascular responses to L-glutamate microinjected into the
40
20
0
-250
C
90 75 60 45
Q
0.62 mm rostra1
30 15 0 i
0.50 mm rostra1
CQN
10
40 min
Fig. 5. A, B, and C: changes in MAP, HR, and RF in response to chemoreflex activation with KCN (20 kgrat-l-O.1 ml-l iv) before (KCN CON) and 10 and 40 min (KCN 10 and KCN 40 min, respectively) after bilateral microinjection of AP-5 (10 nmol/ 100 nl) into NTS. * Significant difference from control (P < 0.05).
microinjection of 100 nl of artificial CSF into the NTS; the data show that the vehicle produced no significant changes in the pressor (+59 t 2 vs. +58 t 2 mmHg, n = 27), bradycardic (-188 t 10 vs. -202 t 16 beats/min, n = 27), or tachypneic responses (+74 t 5 vs. +73 t 5 cycles/min, n = 6) to chemoreflex activation. Figure 6 is a schematic representation of the brain stem at the obex level and shows the overlap sites of Evans blue microinjection into the NTS of 27 rats used in the protocols of AP-5 microinjection into the NTS. The center of the microinjection was - 0.5 mm rostra1 to the obex, and the average size of the anteroposterior extension of the area stained by Evans blue was 630 t
0.22
mm rostral
Imm Fig. 6. Line drawing of transverse sections of brain stem from obex to 1 mm rostral to obex [adapted from Paxinos and Watson (20)]. Dark areas, overlap of bilateral distribution of Evans blue dye in NTS of rats used in protocols of AP-5 (n = 27). Most centers of microinjections werelocated- 0.5 mm rostra1 to obex, and average of anteroposterior extension of stained area was 630 + 55 pm. AP, area postrema; Gr, gracile nucleus; 10, dorsal motor nucleus of vagus; 12, hypoglossal nucleus; Sol M, medial NTS; Sol C, commissural NTS; CC, central canal.
H1426
NMDA
RECEPTORS
AND
NTS may be related to the activation of chemoreflex pathway ‘S (4, 14) . The bradycardic response in that case was not reflex in origin, because cxl-adrenergic antagonism with prazosin blocked the pressor response to L-glutamate but did not change bradycardia, which was abolished only after sequential blockade with methylatropine (3) indicating that microinjection of L-glutamate a parasympathetic pathway into the NTS activates related to the bradycardic response and a sympathetic pathway related to the pressor response. Taken together, these data suggest that activation of carotid chemoreceptor afferents with KC N produces a pressor response and bradycardia by mechanisms similar to those observed when L-glutamate was microinjected into the NTS. The neurotransmitters of chemoreceptor afferents into the NTS, as well as the involvement of EAA receptors in this neurotransmission, are controversial. Spyer et al. (22) suggested that serotonin and substance P play an important role in the neurotransmission of chemoreceptors into the NTS, whereas Lindefors et al. (13) showed that h ypoxia increases the release of substance P into the NTS. Several studies have shown that L-glutamate may be involved in this neurotransmission in the NTS (2, 12) and also that EAA receptors participate in this neurotransmission (24, 25), whereas Ohta and Talman (19) suggested that EAA receptors play no role in this neurotransmission. In functional studies, Vardhan et al. (24) showed that the increase in RF and MAP produced by chemoreceptor activation was blocked only when (+)-2-amino-7phosphonoheptanoic acid (AP-7) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) were microinjected into the medial portion of the commissural NTS. The selective blockade of NMDA receptors with AP-7 or non-NMDA receptors with DNQX in the same area produced no changes in the pressor or respiratory responses. In another study, Zhang and Mifflin (25) were able to block the pressor response to chemoreceptor activation when they microinjected kynurenic acid, a nonselective receptor antagonist, into the lateral portion of the commissural NTS. In both studies, the authors were able to block the pressor response when they produced nonselective blockade of EAA receptors in the medial or lateral commissural NTS. In the present study, the selective blockade of NMDA receptors with AP-5 produced no changes in the pressor response to chemoreceptor activation. Our data are similar to those obtained by Vardhan et al. (24), because microinjection of AP-7, an NMDAselective antagonist, into the medial portion of the commissural NTS also produced no blockade of the pressor response to chemoreceptor activation. In relation to the sites of microinjection, Vardhan et al. (24) referred to the midline portion of the commissural NTS as the specific site of chemoreceptor projections to the NTS, whereas Zhang and Mifflin (25) showed that microinjections of kynurenic acid into the lateral portion of the commissural NTS also blocked the pressor response to chemoreceptor activation. In the present study, the areas of distribution of the microinjections were along the medial and lateral portion of the I
ARTERIAL
CHEMOREFLEX
commissural NTS (Fig. 6). Several studies have shown that these subnuclei of the commissural NTS seem to be the projection sites of most chemoreceptor afferents (3, 7, 11). Therefore the areas reached by our microinjections, in accordance with neuroanatomic evidence, correspond to the sites of the NTS in which the processing of chemoreceptor afferents occurs. Misplaced microinjections of AP-5 into structures adjacent to the commissural NTS produced no changes in the bradycardic response to chemoreflex activation, indicating that the effect of the NMDA antagonist on the parasympathetic branch of the chemoreflex is restricted to the sites of neurotransmission of this reflex in the NTS. In a previous study, we observed that anesthesia has distorting effects on the neurotransmission into the NTS, because the pressor response to L-glutamate microinjection into the NTS was changed to a depressor response when the animals were anesthetized with chloralose or urethan (14). With respect to the effect of anesthesia on the chemoreflex, it is important to consider that activation of carotid chemoreceptors in anesthetized animals, in contrast to conscious animals, produces tachycardic responses (1). In addition, studies by Franchini and Krieger (9) and unpublished data from our laboratory demonstrated that activation of the carotid chemoreflex with KCN in conscious rats produces a pressor response, whereas activation of the chemoreflex in the same rat under chloralose anesthesia produces a depressor response. Therefore these data indicate that anesthesia produces important changes in the processing of the chemoreflex pathway, especially in the neurotransmission at the level of the NTS. Studies involving the neurotransmission of the chemoreflex in the NTS usually do not evaluate the changes in HR, probably because the variations of this parameter are blunted by the anesthesia. On the other hand, the magnitude of the bradycardic response observed in unanesthetized rats permits an appropriate evaluation of the cardiovagal component of the chemoreflex. In the present study, AP-5 blocked the bradycardic response but did not affect the tachypneic and pressor response to the chemoreflex activation. These data indicate selectivity at the NTS level in the neurotransmission of the different components of the chemoreflex. To explain why AP-5 produced no changes in pressor or ventilatory responses, we may suggest that the neurotransmission of the chemoreceptor afferents to neurons associated with ventilation and sympathoexcitation in the NTS is not mediated by NMDA receptors. With respect to the possible activation of distinct neurons involved in autonomic neuromodulation in the NTS by chemoreceptor afferents, Mifflin (18) demonstrated that neurons in the NTS do not integrate chemoreceptor afferent inputs in a homogeneous manner and suggested that the multiplicity of NTS unit responses might be related to the specific reflex function of an individual neuron. The present data are in accordance with the suggestions by Mifflin (18), because AP-5 blocked only the cardiovagal component of the chemoreflex, and suggest that the pressor response (sympathetic outflow component) and ventilatory response may be
NMDA
RECEPTORS
AND
mediated not only by different neurons but also by a different subtype of EAA receptors. We conclude that activation of carotid chemoreceptors with KCN produces two independent cardiovascular responses, i.e., an increase pressure and bradycardia. The bradycardic response is not due to baroreflex activation, nor is it dependent on respiratory changes. In addition, the data indicate that the cardiovagal component of the chemoreflex in the NTS is mediated by NMDA receptors, whereas the pressor and ventilatory responses are not. We thank Rubens F. de Melo for histological technical assistance. This work was supported by Fundacao de Amparo a Pesquisa do Estado de S&o Paulo Grants 91/0576-g, 92/2118-O, and 92/3730-l and Conselho National de Desenvolvimento Cientifico e Tecnologico Grant 500864/91-8. A preliminary report of these data was presented at the Society for Neuroscience Meeting, 1993, Washington, DC. Current address: E. Columbari, Dept. of Physiology, UNIFESPEscola Paulista de Medicina, 04023-900, Sao Paulo, SP, Brazil; D.A. Chianca, Jr., Dept. of Biological Sciences, Universidade Federal de Ouro Preto, 35400-000, Ouro Preto, MG, Brazil. Address for reprint requests: B. H. Machado, Dept. of Physiology, School of Medicine of Ribeirao Preto, USP, 14049-900 Ribeirao Preto, SP, Brazil. Received
27 September
1994;
accepted
in final
form
20 March
ARTERIAL
9.
10. 11.
12.
13.
14.
15.
16.
1995. 17.
REFERENCES 1. Amano, M., T. Asari, and T. Kubo. Excitatory amino acid receptors in the rostra1 ventrolateral medulla mediate hypertension induced by carotid body chemoreceptor stimulation. NaunynSchmiedebergs Arch. Pharmacol. 349: 549-554,1994. 2. Brew, S., D. De Castro, G. D. Housley, and J. D. Sinclair. The role of glutamate in transmission of the hypoxic input to respiration through the nucleus tractus solitarius. In: Chemoreceptors and Chemoreceptor Reflexes, edited by H. Acker, A. Trzebski, and D. O’Regan. New York: Plenum, 1990, p. 331-338. 3. Ciriello, J., S. L. Hochstenbach, and S. Roder. Central projections of baroreceptor and chemoreceptor afferent fibers in the rat. In: NucZeus of the Solitary Tract, edited by I. Robin and A. Barraco. Orlando, FL: CRC, 1994, p. 35-50. 4. Colombari, E., L. G. H. Bonagamba, and B. H. Machado. Mechanisms of pressor and bradycardiac responses to L-glutamate microinjected into the nucleus tractus solitarii of conscious rats. Am. J. Physiol. 266 (Regulatory Integrative Comp. Physiol. 35): R730-R738,1994. J. NMDA receptor in synaptic pathways. In: The NM-DA 5. Davies, Receptors, edited by J. C. Watkins and G. L. Collingridge. Oxford, UK: Oxford University Press, 1989, p. 77-91. 6. Donoghue, S., R. B. Felder, D. Jordan, and K. M. Spyer. The central projections of carotid baroreceptors and chemoreceptors in cat: a neurophysiological study. J. PhysioZ. Lond. 347: 397409,1984. 7. Finley, J. C. W., and D. M. Katz. The central organization of carotid body afferent projections to the brainstem of the rat. Brain Res. 572: 108-116,1992. K. G., and E. M. Krieger. Carotid chemoreceptors 8. Franchini, influence arterial pressure in intact and aortic denervated rat.
18*
19.
20. 21.
22 *
23
24.
25.
.
CHEMOREFLEX
H1427
Am. J. Physiol. 262 (Regulatory Integrative Comp. Physiol. 31): R677-R683,1992. Franchini, K. G., and E. M. Krieger. Cardiovascular responses of conscious rats to carotid body chemoreceptor stimulation by intravenous KCN. J. Auton. Nerv. Syst. 42: 63-70,1993. Habeck, J. 0. Peripheral arterial chemoreceptor and hypertension. J. Auton. Nerv. Syst. 34: l-8, 1991. Housley, G. D., R. L. Martin-Brady, M. J. Dawson, and J. D. Sinclair. Brain stem projections of the glossopharyngeal nerve and its carotid sinus nerve branch in the rat. Neuroscience 22: 237-250,1987. Housley, G. D., and J. D. Sinclair. Localization by kainic acid lesion of neurons transmitting the carotid chemoreceptor stimulus for respiration in rat. J. Physiol. Lond. 406: 99-114, 1988. Lindefors, N., Y. Yamamoto, T. Pantaleo, H. Langercrantz, E. Brodin, and U. Ungerstedt. In vivo release of substance P in the nucleus tractus solitarii increases during hypoxia. Neurosci. Lett. 69: 94-97, 1986. Machado, B. H., and L. G. H. Bonagamba. Microinjection of L-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats. Brain Res. 576: 131-138, 1992. Malan, A. Ventilation measured by body plethysmography in hibernating mammals and poikilotherm. Respir. Physiol. 17: 32-44,1973. Michelini, L. C., and L. G. H. Bonagamba. Baroreceptor reflex modulation by vasopressin microinjected into the nucleus tractus solitarii of conscious rats. Hypertension DaZZas 11, Suppl. I: 1-75-I-79, 1988. Mifflin, S. W. Arterial chemoreceptor input to nucleus tractus solitarius. Am. J. Physiol. 263 (ReguZatory Integrative Comp. Physiol. 32): R368-R375,1992. Mifflin, S. W. Inhibition of chemoreceptor inputs to nucleus of tractus solitarius neurons during baroreceptor stimulation. Am. J. Physiol. 265 (ReguZatory Integrative Comp. Physiol. 34): R14R20,1993. Ohta, H., and W. T. Talman. The role of excitatory amino acids in neurotransmission of the chemoreceptor reflex within nucleus tractus solitarii of rats (Abstract). FASEB J. 7: A53 1, 1993. Paxinos, G., and C. Watson. The Rat Brain in Stereotaxic Coordinates. New York: Academic, 1986. Spyer, K. M. The central nervous organization of reflex circulatory control. In: Central Regulation of Autonomic Functions, edited by A. D. Loewy and K. M. Spyer. New York: Oxford University Press, 1990, p. 168-188. Spyer, K. M., P. N. Izzo, R. J. Lin, J. F. R. Paton, L. F. Silva-Carvalho, and D. W. Richter. The central nervous organization of the carotid body chemoreceptor reflex. In: Chemoreceptors and Chemoreceptor Reflex, edited by H. Acker, A. Trzebski, and D. O’Regan. New York: Plenum, 1990, p. 317-321. Sun, M.-K., and K. M. Spyer. Responses of rostroventrolateral medulla spinal vasomotor neurons to chemoreceptor stimulation in rats. J. Auton. Nerv. Syst. 33: 79-84, 1991. Vardhan, A., A. Kachroo, and H. N. Sapru. Excitatory amino acid receptors in commissural nucleus of the NTS mediate carotid chemoreceptor responses. Am. J. Physiol. 264 (Regulatory Integrative Comp. Physiol. 33): R41-R50, 1993. Zhang, W., and S. W. Mifflin. Excitatory amino acid receptors within NTS mediate arterial chemoreceptor reflexes in rats. Am. J. Phvsiol. 265 (Heart Circ. Phvsiol. 34): H770-H773. 1993.