AJP - Regu Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 281: R935-R943, 2001;
0363-6119/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D.-P.
Right arrow Articles by Pan, H.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D.-P.
Right arrow Articles by Pan, H.-L.
Vol. 281, Issue 3, R935-R943, September 2001

Differential roles for glutamate receptor subtypes within commissural NTS in cardiac-sympathetic reflex

De-Pei Li1, David B. Averill2, and Hui-Lin Pan1,3

Departments of 1 Anesthesiology and 3 Neuroscience and Anatomy, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and 2 Hypertension Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ischemic stimulation of cardiac receptors evokes excitatory sympathetic reflexes. Although the nucleus of the solitary tract (NTS) is an important site for integration of visceral afferents, its involvement in the cardiac-renal sympathetic reflex remains to be fully defined. This study examined the role of glutamate receptor subtypes in the commissural NTS in the sympathetic responses to stimulation of cardiac receptors. Renal sympathetic nerve activity (RSNA) was recorded in anesthetized rats. Cardiac receptors were stimulated by epicardial application of bradykinin (BK; 10 µg/ml). Application of BK significantly increased the mean arterial pressure from 78.2 ± 2.2 to 97.5 ± 2.9 mmHg and augmented RSNA by 38.5 ± 2.5% (P < 0.05). Bilateral microinjection of 10 pmol of 6-cyano-7-nitroquinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) antagonist, into the commissural NTS eliminated the pressor and RSNA responses to BK application in 10 rats. However, microinjection of 2-amino-5-phosphonopentanoic acid (0.1 and 1 nmol, n = 8), an NMDA- receptor antagonist, or alpha -methyl-4-carboxyphenylglycine (0.1 and 1 nmol, n = 5), a glutamate metabotropic receptor antagonist, failed to attenuate significantly the pressor and RSNA responses to stimulation of cardiac receptors with BK. Thus this study suggests that non-NMDA, but not NMDA and glutamate metabotropic, receptors in the commissural NTS play an important role in the sympathoexcitatory reflex response to activation of cardiac receptors during myocardial ischemia.

cardiac nociceptors; sympathetic nervous system; cardiovascular reflexes; bradykinin; N-methyl-D-aspartate receptors; myocardial ischemia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION OF CARDIAC RECEPTORS induces chest pain and initiates excitatory cardiovascular reflexes in patients with myocardial ischemia (27, 34). Cardiac primary afferents running in the sympathetic nerve, especially finely myelinated Adelta - and unmyelinated C-fiber afferents, are considered to be the essential pathways for transmission of cardiac nociception to the central nervous system during myocardial ischemia (7, 27, 32). Axonal tracing studies have shown that cardiac sympathetic afferents project to the dorsal horn of the upper thoracic spinal cord through the stellate ganglia and the sympathetic chain (7, 19). Stimulation of cardiac afferents excites neurons in the spinal ascending pathways, such as those located in the spinothalamic tract (5). The vasomotor neurons in the rostral ventrolateral medulla (RVLM) provide critical excitatory output to the preganglionic sympathetic neurons (26, 48). In a recent study, we showed that stimulation of cardiac receptors with bradykinin (BK), an important ischemic metabolite (31), excites barosensitive neurons in the RVLM through cardiac sympathetic afferent pathways (21). However, it remains uncertain how the sensory input from cardiac sympathetic afferents is processed in the supraspinal sites.

The nucleus of the solitary tract (NTS) is an important synaptic station of the cardiopulmonary vagal afferents in the central nervous system and plays a key role in the modulation of the autonomic efferent activity to the cardiovascular system (1, 47, 50). The carotid and aortic baroreceptors (39, 49, 50), carotid chemoreceptors (18, 43), and cardiopulmonary vagal afferents (8, 47) make their first synapse in the NTS. Stimulation of NTS neurons typically modulates sympathetic outflow through excitation of neurons in the caudal ventrolateral medulla, which, in turn, inhibits vasomotor neurons in the RVLM (1, 17, 25). On the other hand, the ascending fibers from the dorsal horn of the cervical and thoracic spinal cord also terminate in the NTS (28). It has been suggested that this spinal-NTS pathway may play a role in certain excitatory somatovisceral and viscerovisceral reflexes (20, 28). The direct sympathoexcitatory pathway from the NTS to the RVLM has been documented by electrophysiology and neuroanatomic techniques (2, 14, 18, 36, 42). In this regard, microinjection of L-glutamate into the NTS produces an increase in blood pressure, which is mediated by the RVLM (42). Also, stimulation of the NTS at the level of the obex (e.g., the commissural NTS) evokes monosynaptic excitatory postsynaptic potential on the RVLM neurons projecting to the spinal cord (14). Furthermore, it has been shown that a portion of the NTS efferents directly projects to the RVLM (2, 36). The functional role of this NTS-RVLM pathway has been studied in sympathoexcitatory reflexes evoked by stimulation of carotid chemoreceptors (15, 43). For example, blockade of N-methyl-D-aspartate (NMDA) and non-NMDA receptors within the commissural NTS abolishes the chemoreflex response evoked by stimulation of the carotid chemoreceptors (43). Thus the NTS, especially its commissural subnucleus, may mediate the excitatory cardiac-sympathetic reflex (10, 43). Although activation of cardiac sympathetic afferents is known to excite RVLM vasomotor neurons and the sympathetic nervous system (21, 24, 30, 32, 41), the role of the commissural NTS and neurotransmitters utilized in this region in the cardiovascular reflex originating from the heart has not been studied. Therefore, in the present study, we tested a hypothesis that glutamate receptors in the commissural NTS mediate the sympathoexcitatory responses to stimulation of cardiac receptors.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical Preparations and Procedures

Experiments were performed on male Sprague-Dawley rats (280-320 g; Harlan Industry, Indianapolis, IN). The experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee and adhered to the Guide for the Care and Use of Laboratory Animals (US Public Health Service). Rats were anesthetized initially with halothane in an induction chamber. The femoral vein was cannulated for intravenous injection of drugs. The halothane was discontinued after alpha -chloralose (50-60 mg/kg iv) and pentobarbital sodium (20 mg/kg iv) were administered. Adequate depth of anesthesia was verified by the absence of responses to noxious pinch of the paw. Supplemental doses of alpha -chloralose (20-25 mg/kg iv) were administered to maintain an appropriate level of anesthesia. The trachea was cannulated, and the rats were ventilated artificially with 100% O2 using a rodent ventilator (model SAR-830/A, IITC/Life Science Instruments, Woodland Hills, CA). The left carotid artery was cannulated, and the arterial blood pressure was measured with a pressure transducer (model PT300, Grass Instruments, Quincy, MA). After an appropriate level of anesthesia was ensured, the rats were paralyzed with pancuronium bromide (1 mg/kg iv) during renal nerve recordings. The adequacy of the anesthesia level during neuromuscular blockade was judged by the stability of the arterial blood pressure and was tested after paralysis wore off and before the next dose of pancuronium was given. Lidocaine (2%) was infiltrated around the surgical wound to minimize the nociceptive afferent input after surgery (21). A limited left lateral thoracotomy was performed to expose the heart. Body temperature was maintained at 37-38°C with a heating lamp. At the end of the experiments, animals were killed by an intravenous injection of an overdose of pentobarbital sodium.

The head of the rat was fixed on a stereotaxic frame (David-Kopf Instruments, Tujunga, CA), and the dorsal surface of the medulla was exposed surgically through the atlantooccipital membrane. The microinjection pipette (tip diameter 20 µm) was placed into the commissural NTS through a hydraulic microdrive (Stoelting, Wood Dale, IL) under direct vision with the use of a surgical microscope (model M900, D. F. Vasconcellos, São Paulo, Brazil). Target stereotaxic coordinates for the commissural NTS were 0.5 mm caudal and 0.5 mm lateral to the caudal tip of the area postrema (calamus scriptorius) and 0.5 mm ventral from the dorsal surface of the medulla (15, 43). Drugs were ejected bilaterally from the pipette in a volume of 50 nl over a 2-s period by application of controlled pulses of pressurized air to the pipette using a microinjection system (Picospritzer II, General Valve, Fairfield, NJ), as described in detail previously (12).

The left kidney was exposed through a left flank incision via a retroperitoneal approach. A small branch of the renal nerve was isolated and carefully dissected free from the renal vessels with the aid of an operating microscope. The renal nerve filament was placed on a stainless steel electrode, and the nerve signal was amplified (20,000-30,000) and filtered (100-3,000 Hz) by an alternating-current amplifier (model P511, Grass Instruments). Renal sympathetic nerve activity (RSNA) was then monitored through an audioamplifier (model AM9, Grass Instruments) and displayed on an oscilloscope (model DSO 450, Gould, Essex, UK). The RSNA and blood pressure were simultaneously monitored and recorded on a thermal-sensitive recorder (model K2G, Astro-Med, West Warwich, RI). In addition, the RSNA was fed into a Pentium computer through an analog-to-digital interface card for subsequent off-line quantitative analysis. Discharge frequency was quantified using a software window discriminator (DataWave Technology, Longmont, CO). The quality of the RSNA was assessed by its pulse synchronous rhythmicity and by examination of the magnitude of decrease in RSNA in response to sinoaortic baroreceptor loading induced by intravenous injection of phenylephrine (1-4 µg). The renal nerve was cut distally to the recording electrode to ensure that the afferent nerve activity was not recorded. The accuracy of the threshold level of the window discriminator was verified by the baroreflex and again at the end of each experiment after the animal was killed. Respective noise levels were subtracted from the nerve recording data before percentage changes from baseline were calculated.

Experimental Protocols

Effect of epicardial application of BK on blood pressure and RSNA. The responses of blood pressure and RSNA to epicardial application of BK were studied in 12 rats. After a good-quality recording of RSNA was obtained, a stabilization period of >= 30 min was allowed. A 60-s control period of nerve activity and blood pressure was recorded. Then, BK (10 µg/ml; Sigma Chemical, St. Louis, MO), dissolved in normal saline, was applied to the anterior surface of the heart with a cotton applicator through an exposed window on the left chest (21, 41). After BK application, blood pressure and RSNA were monitored for 10 min. After each application, the heart and pericardial sac were flushed twice using cotton-tipped applicators soaked with saline to eliminate any residual BK. Responses of blood pressure and RSNA to application of BK were repeated twice, each separated by 15-20 min, to allow the discharge activity of the renal nerve and the blood pressure to return to the control levels. In the same animals, the pressor and RSNA responses to epicardial application of BK were also examined before and 15 min after microinjection of 50 nl of artificial cerebrospinal fluid (CSF; in mM: 124 NaCl, 3 KCl, 1.3 MgSO4, 2.4 CaCl2, 1.4 NaH2PO4, 10 glucose, and 26 NaHCO3, pH 7.4) into the commissural NTS. The artificial CSF was used for preparing different glutamate receptor antagonists (see below) and was delivered in the same manner as drug injection and served as vehicle control.

Effects of blockade of glutamate receptors within NTS on pressor and RSNA responses to epicardial application of BK. The effects of microinjection of 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX), an antagonist for non-NMDA receptors, (±)-2-amino-5-phosphonopentanoic acid (AP5), an antagonist for NMDA receptors, or alpha -methyl-4-carboxyphenylglycine (MCPG), an antagonist for metabotropic receptors, into the commissural NTS on the pressor and RSNA responses to epicardial application of BK were studied in three separate groups of rats. These antagonists were purchased from RBI (Natick, MA) and dissolved in artificial CSF. The initial sympathetic response to epicardial application of BK was examined twice, separated by 15 min, to ensure that the blood pressure and renal nerve responses were reproducible. Subsequently, the selected antagonist was injected into the commissural NTS bilaterally through a glass pipette in a volume of 50 nl over a 2-s period. The pressor and RSNA responses to epicardial application of BK were measured again 15 min after microinjection. The effective concentrations of these glutamate receptor antagonists have been determined in previous studies (12, 43). Additionally, because the effect of MCPG lasts for only 2-3 min after microinjection into the medial NTS (12), separate experiments were performed to examine the effect of 0.1-1 nmol of MCPG 2 min after microinjection into the commissural NTS in five rats.

Histological localization of microinjection sites. Accurate pipette location and spread of injection within the commissural NTS were verified histologically. At the end of the experiment, 50 nl of 2% Chicago blue dye were ejected from the same microinjection pipette at the same site. After euthanasia, the brain stem was removed rapidly and fixed in 10% buffered formalin. Frozen 40-µm coronal sections were made on a freezing microtome, mounted on the slides, and stained with cresyl violet (Sigma Chemical). The dye spot and spread area were identified and plotted on standardized sections according to the atlas of Paxinos and Watson (33). Data were excluded if the microinjection site and spread were not localized in the commissural NTS.

Data Analysis

Values are means ± SE. The RSNA was averaged during control and the entire response period (usually 2.5 min) after BK application. RSNA data are expressed as percentage changes from control values because of the variability in baseline RSNA in each animal. Comparisons between the control and experimental interventions were made by the Student's paired t-test or repeated-measures analysis of variance. Differences were considered to be statistically significant when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A total of 43 rats was used to study the effects of microinjection of glutamate antagonists into the commissural NTS on the sympathetic response to epicardial application of BK. The resting mean arterial pressure in all rats studied was 79.3 ± 2.5 mmHg, and the baseline heart rate was 342 ± 12 beats/min. Figure 1 shows the distribution of microinjection sites into the commissural NTS. The size of the dye spread area was 0.1-0.2 mm around the microinjection site. Four rats were excluded from the study because of misposition of the microinjection pipette. We observed that microinjection of CNQX into the site outside the commissural NTS in these four rats did not change the baseline blood pressure, RSNA, and responses to epicardial application of BK.


View larger version (34K):
[in this window]
[in a new window]
  		Fig. 1.   Distribution of microinjection sites in the commissural nucleus of the solitary tract (NTS) in 43 rats. , Sites within the commissural NTS (ComNTS); open circle , sites not located within the ComNTS. AP, area postrema; Gr, gracile nucleus; Cu, cuneate nucleus; Sol, NTS.

Responses of RSNA and Blood Pressure to Epicardial Application of BK

Epicardial application of BK (10 µg/ml) significantly increased the blood pressure and RSNA in 12 rats. The RSNA evoked by BK increased by 38.5 ± 2.5% (P < 0.05) after a latency of ~10 s. Topical application of BK (10 µg/ml) also significantly increased the mean arterial blood pressure from 78.2 ± 2.2 to 97.5 ± 2.9 mmHg (P < 0.05). Maximal pressor and RSNA responses were usually reached within 40 s and lasted for 2.5-3.0 min. Epicardial application of saline had no effect on the blood pressure and RSNA (data not shown). Microinjection of artificial CSF (50 nl) into the commissural NTS did not alter the pressor and RSNA responses to epicardial application of BK (Fig. 2).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2.   Repeatability of the responses of renal sympathetic nerve activity (RSNA) and mean arterial blood pressure (MAP) to epicardial application of bradykinin (BK) before and after microinjection of artificial cerebrospinal fluid (CSF) into the ComNTS. Values are means ± SE (n = 12). *P < 0.05 compared with respective controls.

Effect of CNQX Microinjection Into the Commissural NTS on the Cardiac-Sympathetic Reflex

The effect of CNQX microinjection into the commissural NTS on the cardiac-sympathetic reflex was studied in 10 rats. Figure 3 is a representative trace showing the effect of microinjection of CNQX, the non-NMDA-receptor antagonist, on the pressor and RSNA responses to epicardial application of BK. Before CNQX microinjection, topical application of BK (10 µg/ml) significantly increased RSNA and blood pressure (Figs. 3 and 4). Microinjection of CNQX (10 pmol) into the commissural NTS increased the baseline discharge activity of RSNA (18.4 ± 2.4%) 3-4 min after microinjection. Pressor and RSNA responses to epicardial application of BK were abolished 15 min after CNQX injection (Figs. 3 and 4). At 30-45 min after CNQX injection, the responses of RSNA (from 102 ± 3.2 to 135 ± 2.7%, P < 0.05) and blood pressure (from 79.2 ± 1.9 to 98.3 ± 2.9 mmHg, P < 0.05) to BK application were restored to the preinjection levels (Figs. 3 and 4).


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 3.   Original traces showing responses of blood pressure (BP) and RSNA to epicardial application of BK before (A) and 15 and 30 min after (B and C, respectively) microinjection of 10 pmol of 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) into the ComNTS in 1 rat.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 4.   Responses of RSNA and BP to epicardial application of BK before and 15 min after CNQX microinjection into the ComNTS. Values are means ± SE (n = 10). *P < 0.05 compared with the respective baseline controls. #P < 0.05 compared with BK-evoked response obtained before CNQX.

Effect of Injection of AP5 Into the Commissural NTS on the Cardiac-Sympathetic Reflex

Neither the pressor nor the RSNA response to epicardial application of BK was affected significantly by microinjection of 100 pmol of AP5 into the commissural NTS. Figure 5 summarizes the responses of blood pressure and RSNA to epicardial application of BK in eight animals before and 15 min after AP5 (100 pmol) microinjection into the commissural NTS. Furthermore, microinjection of a higher concentration of AP5 (1 nmol) into the commissural NTS also failed to alter significantly the responses of RSNA and blood pressure to epicardial application of BK in the same animals (n = 8). Before injection of 1 nmol of AP5, BK application increased blood pressure from 79.1 ± 2.3 to 102.7 ± 2.5 mmHg and excited RSNA by 37.9 ± 2.5%. After AP5 injection, blood pressure increased from 79.5 ± 2.2 to 101.6 ± 2.0 mmHg, and RSNA was augmented by 38.8 ± 2.3% in response to epicardial BK application.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 5.   Responses of RSNA and BP to epicardial application of BK before and 15 min after (±)-2-amino-5-phosphonopentanoic acid (AP5) microinjection into the ComNTS. Values are means ± SE (n = 8). *P < 0.05 compared with respective controls. NS, not significantly different from initial response.

Effect of Microinjection of MCPG Into the Commissural NTS on the Cardiac-Sympathetic Reflex

The pressor and RSNA responses to stimulation of cardiac receptors with BK were not altered 2 min after microinjection of 100 pmol of MCPG into the commissural NTS (Fig. 6; n = 5). Microinjection of 1 nmol of MCPG into the commissural NTS also failed to attenuate significantly the responses of RSNA and blood pressure to epicardial application of BK in the same animals (n = 5). Before microinjection of 1 nmol of MCPG, BK application increased blood pressure from 77.8 ± 2.5 to 104.5 ± 2.1 mmHg and excited RSNA by 35.8 ± 2.5%. After MCPG injection, blood pressure increased from 78.3 ± 1.9 to 103.9 ± 2.2 mmHg, and RSNA was augmented by 36.9 ± 2.1% in response to epicardial BK application. In eight additional animals, BK-elicited pressor and RSNA responses were assessed 15 min after MCPG (100 pmol) was injected into the commissural NTS. The pressor and RSNA responses to epicardial application of BK were not attenuated 15 min after MCPG injection into the commissural NTS (n = 8).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6.   Responses of RSNA and BP to epicardial application of BK before and 2 min after microinjection of 100 pmol of alpha -methyl-4-carboxyphenylglycine (MCPG) into the ComNTS. Values are means ± SE (n = 5). *P < 0.05 compared with respective controls. NS, not significantly different from initial response.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The brain stem nuclei are important for the fine tune of cardiovascular control, including cardiac-sympathetic reflexes (2, 18, 21, 36, 46, 49). The present study focused on the functional role of the commissural NTS in the sympathoexcitatory response initiated by stimulation of cardiac receptors. We found that microinjection of a non-NMDA-receptor antagonist, CNQX, into the commissural NTS eliminated the excitatory responses of RSNA and blood pressure to activation of cardiac receptors. However, these cardiac-sympathetic responses were little affected by microinjection of AP5, an NMDA-receptor antagonist, or MCPG, a glutamate metabotropic receptor antagonist, into the commissural NTS. Therefore, this study provides new information that non-NMDA glutamate receptors in the commissural NTS play an important role in the excitatory cardiac-sympathetic reflex.

Stimulation of cardiac sympathetic afferents during myocardial ischemia typically elicits chest pain and disturbance of the autonomic nervous system in patients with ischemic heart disease (27, 34). Many endogenous ischemic metabolites, including BK, prostaglandins, and lactic acid, are capable of stimulating cardiac sympathetic afferents and contribute to activation of cardiac nociceptors during myocardial ischemia (30, 32, 41). The roles of afferent and efferent limbs of the cardiac-sympathetic reflex have been well established in previous studies (23, 45, 46). However, the integrative mechanisms of the brain stem nuclei in this cardiovascular reflex are complex and remain to be fully defined. The RVLM has been considered to be a major site for generating sympathetic outflow (26, 38). It has been demonstrated that stimulation of cardiac receptors selectively activates RVLM barosensitive neurons through cardiac sympathetic pathways (21). Some studies suggest that stimulation of cardiac vagal afferents could inhibit cardiac-sympathetic reflexes (35, 40). Not all published literature supports the notion that the input from cardiac vagal afferents inhibits the cardiac-sympathetic reflex. For example, a study by Veelken et al. (44) has shown that bilateral cervical vagotomy does not alter the sympathetic and pressor responses to intrapericardial BK in rats. Also, we recently showed that the vagal afferent pathway is not important for the response of RVLM barosensitive neurons to epicardial application of BK (21). It is difficult to reconcile the differences in the above-mentioned studies in the role of cardiac vagal afferents in the cardiac-sympathetic reflex. Future studies are needed to delineate the sites and mechanisms of possible interaction between cardiac vagal and sympathetic afferents in various brain stem nuclei. Because epicardial application of BK induced profound and consistent sympathetic and pressor responses in our preparations, we did not propose to study the interaction between cardiac vagal and sympathetic afferents. It is unlikely that attenuation of RSNA by glutamate antagonist microinjection into the commissural NTS is due to an enhancement of the cardiac vagal input. First, the sympathetic afferent endings in the ventricle are located near the epicardial surface, whereas most of the vagal afferent endings are located deep in the myocardium (4). Thus epicardial application of BK predominantly stimulates cardiac sympathetic afferents. Furthermore, it has been shown that glutamate receptors are involved in the transmission of cardiac vagal input in the NTS mainly through non-NMDA receptors (37). Because microinjection of glutamate receptor antagonists into the NTS can block cardiac vagal input, we would expect to see that the sympathetic response to cardiac afferent activation is augmented (rather than inhibited) after microinjection if there is a significant contribution of cardiac vagal input to the changes in RSNA in the present study.

The NTS has long been known for its role in integration of sensory input from baroreceptors and visceral afferents traveling in the vagal nerve (39, 47, 49). The sympathoinhibitory pathways of the baroreflex and the Bezold-Jarisch reflex involve an excitatory projection from the NTS to the caudal ventrolateral medulla and an inhibitory projection from the caudal ventrolateral medulla to the RVLM (1, 17, 25). However, activation of the peripheral chemoreceptors also excites the sympathetic system, possibly through a direct or indirect pathway from the NTS to the RVLM (14, 16, 42). Using neuroanatomy and electrophysiology techniques, a direct pathway from the NTS to the RVLM neurons projecting to the thoracic spinal cord has been substantiated (2, 18, 36). Also, electrical stimulation of neurons in the NTS at the obex level excites RVLM descending neurons to the thoracic spinal cord (14). Several anatomic and physiological studies have indicated that the commissural NTS plays a unique role in the excitatory cardiovascular reflex (10, 15, 42, 43). For example, microinjection of L-glutamate into the commissural NTS produces a pressor response (9, 22). Electrolytic lesion of the commissural NTS eliminates the pressor response induced by microinjection of L-glutamate into the medial NTS (10). In addition, the excitatory chemoreflex is blocked by glutamate receptor antagonists injected into the commissural NTS (43, 50). Thus it is likely that the commissural NTS is also involved in processing ascending cardiac afferents from the spinal dorsal horns. However, the involvement of the commissural NTS in the sympathoexcitatory response induced by activation of cardiac receptors has not been studied previously.

The cardiac sympathetic afferents are known to project to the deep dorsal horn of the upper thoracic spinal cord (19). The supraspinal sites involved in integration of sensory input from cardiac sympathetic afferents are poorly understood. Although many spinal dorsal horn neurons project to the thalamus and hypothalamus (3, 6, 13), a direct spinal dorsal horn-NTS projection pathway also has been demonstrated (28). Furthermore, electrical stimulation of the stellate ganglia is capable of activating some neurons in the NTS (40). L-Glutamate is a major excitatory neurotransmitter in the central nervous system and is released in the NTS during stimulation of the peripheral chemoreceptors (29). In the present study, we determined the functional significance of this spinal-NTS pathway and the role of glutamate receptors in the excitatory cardiac-sympathetic reflex. We found that microinjection of CNQX into the commissural NTS abolished the sympathetic responses to stimulation of cardiac receptors. These data indicate that non-NMDA receptors in the commissural NTS are important for the integration of cardiac sympathetic afferents.

In the present study, microinjection of an NMDA- receptor antagonist, AP5, or a metabotropic glutamate receptor antagonist, MCPG, into the commissural NTS did not significantly alter the cardiac-sympathetic reflex. These results suggest that NMDA and metabotropic glutamate receptor subtypes are less important than non-NMDA receptors in the commissural NTS in mediating this cardiogenic sympathetic reflex. Because microinjection of a higher concentration of AP5 or MCPG did not significantly alter the cardiac-sympathetic reflex, it is unlikely that the lack of actions of AP5 and MCPG in the commissural NTS was due to inadequate blockade of these two types of glutamate receptors. Our findings are consistent with several studies on the role of glutamate receptors in the NTS in other types of cardiovascular reflexes (15, 47, 49). The pressor response induced by intravenous injection of potassium cyanide is not affected by microinjection of AP5 into the NTS, indicating that the sympathoexcitatory effect is not mediated by NMDA receptors (15). Non-NMDA receptors have been shown to play a major role in the synaptic transmission of baroreceptor afferents in the NTS (49). Although metabotropic receptors can be activated by exogenous L-glutamate injected into the medial NTS, blockade of the metabotropic glutamate receptors with MCPG alone does not attenuate the cardiovascular responses to glutamate injection (12). We observed that neither the pressor nor the RSNA response to stimulation of cardiac receptors was affected by microinjection of MCPG. In fact, we found that microinjection of a metabotropic glutamate receptor agonist, trans-1S,3R-aminocyclopentane-1,3-dicarboxylic acid (12), into the commissural NTS in rats also had no effect on the baseline blood pressure and RSNA (data not shown). It is important to recognize that MCPG does not block all metabotropic glutamate receptors. Eight different metabotropic glutamate receptors have been identified, and they can be categorized into three groups on the basis of their amino acid sequence identity (11). Although groups I and II of metabotropic glutamate receptors can be antagonized by MCPG, no antagonist blocks all types of metabotropic glutamate receptors. However, there is no compelling evidence to implicate a third type of metabotropic glutamate receptor in this reflex response, because CNQX abolished completely the pressor and sympathetic activation elicited by epicardial BK. Furthermore, when we did examine other possibilities (NMDA and the groups I and II of metabotropic glutamate receptors), we did not uncover a contribution to this reflex response.

Microinjection of CNQX into the commissural NTS increased the baseline RSNA. Although the injection site and the dye spread area are within the commissural NTS, this effect still may be due to a spread of the drug solution to other regions of the NTS involved in baroreflex control. Furthermore, the medial and commissural NTS are involved in processing sensory input from peripheral baroreceptors (39), chemoreceptors (43, 50), and cardiopulmonary vagal afferents (47). It is likely that the commissural NTS is not a homogenous structure, and non-NMDA receptors in this region are also important for the baroreflex input (47, 49). Thus, alternatively, an increase in the sympathetic basal tone after CNQX injection could be the result of blockade of non-NMDA receptors and attenuation of sensory input from the baroreceptors and vagal afferents in the commissural NTS.

In summary, we found in the present study that the commissural NTS is involved in integration of the cardiac-sympathetic reflex. Activation of non-NMDA, but not NMDA and glutamate metabotropic, receptors in the commissural NTS is important for the excitatory sympathetic reflex originating from the heart. Thus these data provide new information for our understanding of the regulatory mechanisms in the commissural NTS in the cardiovascular reflex occurring during myocardial ischemia.

Perspectives

Stimulation of cardiac sympathetic afferents during myocardial ischemia is known to elicit an increased sympathetic outflow. Our understanding of the supraspinal mechanisms in the integration of cardiac afferent input and generation of sympathetic output is incomplete. The NTS has a heterogeneous population of neurons and contains neurons that are involved in multiple afferent pathways. The present study demonstrates an important role of the commissural NTS in the sympathoexcitatory response to activation of cardiac receptors. However, the involvement of other subnuclei in the NTS in the cardiac-sympathetic reflex remains unclear. Stimulation of cardiac receptors with BK can excite the RVLM barosensitive neurons (21). Thus it is likely that cardiac afferent input is relayed by the commissural NTS neurons, which may provide excitatory synaptic inputs to the RVLM. In this regard, the response patterns of neurons located in the commissural NTS and other subnuclei in the NTS to stimulation of cardiac sympathetic afferents require further studies. Also, the direct functional link between the commissural NTS and RVLM in the cardiac-sympathetic reflex response needs to be established in future studies.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the secretarial assistance of P. Myers.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-60026 and HL-04419. H.-L. Pan is a recipient of an Independent Scientist Award supported by the National Heart, Lung, and Blood Institute during the course of this study.

Address for reprint requests and other correspondence: H.-L. Pan, Dept. of Anesthesiology, H187, Penn State University College of Medicine, The Milton S. Hershey Medical Center, 500 University Dr., Hershey, PA 17033-0850 (E-mail: hpan{at}psu.edu).

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. Section 1734 solely to indicate this fact.

Received 27 November 2000; accepted in final form 27 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aicher, SA, Kurucz OS, Reis DJ, and Milner TA. Nucleus tractus solitarius efferent terminals synapse on neurons in the caudal ventrolateral medulla that project to the rostral ventrolateral medulla. Brain Res 693: 51-63, 1995[Web of Science][Medline].

2.   Aicher, SA, Saravay RH, Cravo S, Jeske I, Morrison SF, Reis DJ, and Milner TA. Monosynaptic projections from the nucleus tractus solitarii to C1 adrenergic neurons in the rostral ventrolateral medulla: comparison with input from the caudal ventrolateral medulla. J Comp Neurol 373: 62-75, 1996[Web of Science][Medline].

3.   Al-Chaer, ED, Lawand NB, Westlund KN, and Willis WD. Visceral nociceptive input into the ventral posterolateral nucleus of the thalamus: a new function for the dorsal column pathway. J Neurophysiol 76: 2661-2674, 1996[Abstract/Free Full Text].

4.   Barber, MJ, Mueller TM, Davies BG, and Zipes DP. Phenol topically applied to canine left ventricular epicardium interrupts sympathetic but not vagal afferents. Circ Res 55: 532-544, 1984[Abstract/Free Full Text].

5.   Blair, RW, Weber RN, and Foreman RD. Responses of thoracic spinothalamic neurons to intracardiac injection of bradykinin in the monkey. Circ Res 51: 83-94, 1982[Abstract/Free Full Text].

6.   Burstein, R, Falkowsky O, Borsook D, and Strassman A. Distinct lateral and medial projections of the spinohypothalamic tract of the rat. J Comp Neurol 373: 549-574, 1996[Web of Science][Medline].

7.   Cervero, F. Sensory innervation of the viscera: peripheral basis of visceral pain. Physiol Rev 74: 95-138, 1994[Free Full Text].

8.   Chianca, DA, Jr, and Machado BH. Microinjection of NMDA antagonist into the NTS of conscious rats blocks the Bezold-Jarisch reflex. Brain Res 718: 185-188, 1996[Web of Science][Medline].

9.   Colombari, E, Bonagamba LG, and Machado BH. Mechanisms of pressor and bradycardic responses to L-glutamate microinjected into the NTS of conscious rats. Am J Physiol Regulatory Integrative Comp Physiol 266: R730-R738, 1994[Abstract/Free Full Text].

10.   Colombari, E, Menani JV, and Talman WT. Commissural NTS contributes to pressor responses to glutamate injected into the medial NTS of awake rats. Am J Physiol Regulatory Integrative Comp Physiol 270: R1220-R1225, 1996[Abstract/Free Full Text].

11.   Conn, PJ, and Pin JP. Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 37: 205-237, 1997[Web of Science][Medline].

12.   Foley, CM, Moffitt JA, Hay M, and Hasser EM. Glutamate in the nucleus of the solitary tract activates both ionotropic and metabotropic glutamate receptors. Am J Physiol Regulatory Integrative Comp Physiol 275: R1858-R1866, 1998[Abstract/Free Full Text].

13.   Foreman, RD, Hancock MB, and Willis WD. Responses of spinothalamic tract cells in the thoracic spinal cord of the monkey to cutaneous and visceral inputs. Pain 11: 149-162, 1981[Web of Science][Medline].

14.   Granata, AR. Rostral ventrolateral medulla descending neurons excited by nucleus tractus solitarii inputs. Brain Res 648: 299-305, 1994[Web of Science][Medline].

15.   Haibara, AS, Colombari E, Chianca DA, Jr, Bonagamba LG, and Machado BH. NMDA receptors in NTS are involved in bradycardic but not in pressor response of chemoreflex. Am J Physiol Heart Circ Physiol 269: H1421-H1427, 1995[Abstract/Free Full Text].

16.   Hines, T, Toney GM, and Mifflin SW. Responses of neurons in the nucleus tractus solitarius to stimulation of heart and lung receptors in the rat. Circ Res 74: 1188-1196, 1994[Abstract/Free Full Text].

17.   Jeske, I, Reis DJ, and Milner TA. Neurons in the barosensory area of the caudal ventrolateral medulla project monosynaptically on to sympathoexcitatory bulbospinal neurons in the rostral ventrolateral medulla. Neuroscience 65: 343-353, 1995[Web of Science][Medline].

18.   Koshiya, N, and Guyenet PG. NTS neurons with carotid chemoreceptor inputs arborize in the rostral ventrolateral medulla. Am J Physiol Regulatory Integrative Comp Physiol 270: R1273-R1278, 1996[Abstract/Free Full Text].

19.   Kuo, DC, Oravitz JJ, and DeGroat WC. Tracing of afferent and efferent pathways in the left inferior cardiac nerve of the cat using retrograde and transganglionic transport of horseradish peroxidase. Brain Res 321: 111-118, 1984[Web of Science][Medline].

20.   Lan, CT, Wu WC, Ling EA, and Chai CY. Evidence of a direct projection from the cardiovascular-reactive dorsal medulla to the intermediolateral cell column of the spinal cord in cats as revealed by light and electron microscopy. Neuroscience 77: 521-533, 1997[Web of Science][Medline].

21.   Li, DP, and Pan HL. Responses of neurons in rostral ventrolateral medulla to activation of cardiac receptors in rats. Am J Physiol Heart Circ Physiol 279: H2549-H2557, 2000[Abstract/Free Full Text].

22.   Machado, BH, and Bonagamba LG. Microinjection of L-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats. Brain Res 576: 131-138, 1992[Web of Science][Medline].

23.   Malliani, A, Pagani M, Pizzinelli P, Furlan R, and Guzzetti S. Cardiovascular reflexes mediated by sympathetic afferent fibers. J Auton Nerv Syst 7: 295-301, 1983[Web of Science][Medline].

24.   Malliani, A, Schwartz PJ, and Zanchetti A. A sympathetic reflex elicited by experimental coronary occlusion. Am J Physiol 217: 703-709, 1969.

25.   Masuda, N, Terui N, Koshiya N, and Kumada M. Neurons in the caudal ventrolateral medulla mediate the arterial baroreceptor reflex by inhibiting barosensitive reticulospinal neurons in the rostral ventrolateral medulla in rabbits. J Auton Nerv Syst 34: 103-117, 1991[Web of Science][Medline].

26.   McAllen, RM, and Dampney RA. Vasomotor neurons in the rostral ventrolateral medulla are organized topographically with respect to type of vascular bed but not body region. Neurosci Lett 110: 91-96, 1990[Web of Science][Medline].

27.   Meller, ST, and Gebhart GF. A critical review of the afferent pathways and the potential chemical mediators involved in cardiac pain. Neuroscience 48: 501-524, 1992[Web of Science][Medline].

28.   Menetrey, D, and Basbaum AI. Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and viscerovisceral reflex activation. J Comp Neurol 255: 439-450, 1987[Web of Science][Medline].

29.   Mizusawa, A, Ogawa H, Kikuchi Y, Hida W, Kurosawa H, Okabe S, Takishima T, and Shirato K. In vivo release of glutamate in nucleus tractus solitarii of the rat during hypoxia. J Physiol (Lond) 478: 55-66, 1994[Abstract/Free Full Text].

30.   Nerdrum, T, Baker DG, Coleridge HM, and Coleridge JC. Interaction of bradykinin and prostaglandin E1 on cardiac pressor reflex and sympathetic afferents. Am J Physiol Regulatory Integrative Comp Physiol 250: R815-R822, 1986[Abstract/Free Full Text].

31.   Pan, HL, Chen SR, Scicli GM, and Carretero OA. Cardiac interstitial bradykinin release during ischemia is enhanced by ischemic preconditioning. Am J Physiol Heart Circ Physiol 279: H116-H121, 2000[Abstract/Free Full Text].

32.   Pan, HL, Longhurst JC, Eisenach JC, and Chen SR. Role of protons in activation of cardiac sympathetic C-fibre afferents during ischaemia in cats. J Physiol (Lond) 518: 857-866, 1999[Abstract/Free Full Text].

33.   Paxinos, G, and Watson C. The Rat Brain in Stereotaxic Coordinates (4th ed.). San Diego, CA: Academic, 1999.

34.   Perez-Gomez, F, Martin de Dios R, Rey J, and Garcia Aguado A. Prinzmetal's angina: reflex cardiovascular response during episode of pain. Br Heart J 42: 81-87, 1979[Abstract/Free Full Text].

35.   Reimann, KA, and Weaver LC. Contrasting reflexes evoked by chemical activation of cardiac afferent nerves. Am J Physiol Heart Circ Physiol 239: H316-H325, 1980.

36.   Ross, CA, Ruggiero DA, and Reis DJ. Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. J Comp Neurol 242: 511-534, 1985[Web of Science][Medline].

37.   Seagard, JL, Dean C, and Hopp FA. Role of glutamate receptors in transmission of vagal cardiac input to neurones in the nucleus tractus solitarii in dogs. J Physiol (Lond) 520: 243-253, 1999[Abstract/Free Full Text].

38.   Strack, AM, Sawyer WB, Hughes JH, Platt KB, and Loewy AD. A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brain Res 491: 156-162, 1989[Web of Science][Medline].

39.   Talman, WT, Perrone MH, and Reis DJ. Evidence for L-glutamate as the neurotransmitter of baroreceptor afferent nerve fibers. Science 209: 813-815, 1980[Abstract/Free Full Text].

40.   Tjen-A-Looi, SC, Bonham A, and Longhurst JC. Interactions between sympathetic and vagal cardiac afferents in nucleus tractus solitarii. Am J Physiol Heart Circ Physiol 272: H2843-H2851, 1997[Abstract/Free Full Text].

41.   Tjen-A-Looi, SC, Pan HL, and Longhurst JC. Endogenous bradykinin activates ischaemically sensitive cardiac visceral afferents through kinin B2 receptors in cats. J Physiol (Lond) 510: 633-641, 1998[Abstract/Free Full Text].

42.   Urbanski, RW, and Sapru HN. Evidence for a sympathoexcitatory pathway from the nucleus tractus solitarii to the ventrolateral medullary pressor area. J Auton Nerv Syst 23: 161-174, 1988[Web of Science][Medline].

43.   Vardhan, A, Kachroo A, and Sapru HN. Excitatory amino acid receptors in commissural nucleus of the NTS mediate carotid chemoreceptor responses. Am J Physiol Regulatory Integrative Comp Physiol 264: R41-R50, 1993[Abstract/Free Full Text].

44.   Veelken, R, Glabasnia A, Stetter A, Hilgers KF, Mann JF, and Schmieder RE. Epicardial bradykinin B2 receptors elicit a sympathoexcitatory reflex in rats. Hypertension 28: 615-621, 1996[Abstract/Free Full Text].

45.   Weaver, LC, Fry HK, Meckler RL, and Oehl RS. Multisegmental spinal sympathetic reflexes originating from the heart. Am J Physiol Regulatory Integrative Comp Physiol 245: R345-R352, 1983.

46.   Weaver, LC, Meckler RL, Fry HK, and Donoghue S. Widespread neural excitation initiated from cardiac spinal afferent nerves. Am J Physiol Regulatory Integrative Comp Physiol 245: R241-R250, 1983.

47.   Wilson, CG, Zhang Z, and Bonham AC. Non-NMDA receptors transmit cardiopulmonary C fibre input in nucleus tractus solitarii in rats. J Physiol (Lond) 496: 773-785, 1996[Abstract/Free Full Text].

48.   Zagon, A, and Smith AD. Monosynaptic projections from the rostral ventrolateral medulla oblongata to identified sympathetic preganglionic neurons. Neuroscience 54: 729-743, 1993[Web of Science][Medline].

49.   Zhang, J, and Mifflin SW. Differential roles for NMDA and non-NMDA receptor subtypes in baroreceptor afferent integration in the nucleus of the solitary tract of the rat. J Physiol (Lond) 511: 733-745, 1998[Abstract/Free Full Text].

50.   Zhang, W, and Mifflin SW. Excitatory amino acid receptors within NTS mediate arterial chemoreceptor reflexes in rats. Am J Physiol Heart Circ Physiol 265: H770-H773, 1993[Abstract/Free Full Text].

Am J Physiol Regul Integr Comp Physiol 281(3):R935-R943
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
H. Sun, D.-P. Li, S.-R. Chen, W. N. Hittelman, and H.-L. Pan
Sensing of Blood Pressure Increase by Transient Receptor Potential Vanilloid 1 Receptors on Baroreceptors
J. Pharmacol. Exp. Ther., December 1, 2009; 331(3): 851 - 859.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
W.-Z. Wang, L. Gao, H.-J. Wang, I. H. Zucker, and W. Wang
Interaction between cardiac sympathetic afferent reflex and chemoreflex is mediated by the NTS AT1 receptors in heart failure
Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1216 - H1226.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
W.-Z. Wang, L. Gao, Y.-X. Pan, I. H. Zucker, and W. Wang
AT1 receptors in the nucleus tractus solitarii mediate the interaction between the baroreflex and the cardiac sympathetic afferent reflex in anesthetized rats
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1137 - R1145.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. R. Zahner and H.-L. Pan
Role of paraventricular nucleus in the cardiogenic sympathetic reflex in rats
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2005; 288(2): R420 - R426.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
M. R Zahner, D.-P. Li, S.-R. Chen, and H.-L. Pan
Cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats
J. Physiol., September 1, 2003; 551(2): 515 - 523.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
K. L. Barnes, D. M. DeWeese, and M. C. Andresen
Angiotensin potentiates excitatory sensory synaptic transmission to medial solitary tract nucleus neurons
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2003; 284(5): R1340 - R1353.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
H. M. Stauss
Baroreceptor reflex function
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2002; 283(2): R284 - R286.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. A. Sato, E. Colombari, and S. F. Morrison
Inhibition of neurons in commissural nucleus of solitary tract reduces sympathetic nerve activity in SHR
Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1679 - H1684.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D.-P.
Right arrow Articles by Pan, H.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D.-P.
Right arrow Articles by Pan, H.-L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online