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1 Institute of Physiology, National Yang-Ming University, Taipei 11221, Taiwan; and 2 Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Kaohsiung 81346, Taiwan, Republic of China
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We investigated the role of glutamatergic projection from the parabrachial nucleus (PBN) complex to the rostral ventrolateral medulla (RVLM) in the PBN-induced suppression of reflex bradycardia in adult Sprague-Dawley rats that were maintained under pentobarbital anesthesia. Under stimulus conditions that did not appreciably alter the baseline systemic arterial pressure and heart rate, electrical (10-s train of 0.5-ms pulses, at 10-20 µA and 10-20 Hz) or chemical (L-glutamate, 1 nmol) stimulation of the ventrolateral regions and Köelliker-Fuse (KF) subnucleus of the PBN complex significantly suppressed the reflex bradycardia in response to transient hypertension evoked by phenylephrine (5 µg/kg iv). The PBN-induced suppression of reflex bradycardia was appreciably reversed by bilateral microinjection into the RVLM of the N-methyl-D-aspartate (NMDA)-receptor antagonist MK-801 (500 pmol) or the non-NMDA-receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 pmol). Anatomically, most of the retrogradely labeled neurons in the ventrolateral regions and KF subnucleus of the ipsilateral PBN complex after microinjection of fast blue into the RVLM were also immunoreactive to anti-glutamate antiserum. These results suggest that a direct glutamatergic projection to the RVLM from topographically distinct regions of the PBN complex may participate in the suppression of reflex bradycardia via activation of both NMDA and non-NMDA receptors at the RVLM.
rostral ventrolateral medulla; N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors; immunohistochemistry; retrograde tract tracing; rat
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE PARABRACHIAL NUCLEUS (PBN) complex, which consists of 10 spatially segregated groups of cytologically distinct neurons that surround the brachium conjunctivum in the dorsolateral pontine tegmentum, plays an important role in central regulation of cardiovascular functions. Electrical (3) or chemical (3, 26, 31) activation of the PBN complex elicits marked changes in arterial pressure and heart rate. Ablation of this pontine nucleus, on the other hand, reverses the elevation in arterial pressure seen in different forms of experimental hypertension (9, 34). Neuroanatomic results revealed that the PBN complex is reciprocally connected to many brain nuclei that are engaged in circulatory regulation (11, 17, 25, 27, 40). Among these are nucleus tractus solitarii (NTS), the primary terminal site of baroreceptor afferents (6), and rostral ventrolateral medulla (RVLM), the origin of bulbospinal efferents of sympathetic premotor neurons (14). Both NTS and RVLM are well documented as integrative sites for baroreceptor information in the baroreceptor reflex (BRR) circuitry (41).
A wealth of recent evidence further indicates that the PBN complex participates in the modulation of BRR sensitivity. Neuronal excitability in the PBN complex can be evoked by baroreceptor activation or stimulation of the NTS (15, 23). Conversely, dependent on the intensity and timing of the conditioning stimulus, activation of the PBN complex promotes either excitatory or inhibitory modulation of the baroreceptor afferent input to the NTS (8, 30). In addition, the PBN complex participates in BRR-related coronary vasoconstriction (13) and mediates inhibition of BRR responses promoted by forebrain structures (35). More importantly, whereas electrolytic lesion of this pontine structure enhances BRR-mediated cardiovascular responses (20, 39), stimulation of the PBN complex inhibits them (26). These results strongly suggest that the PBN complex exerts an inhibitory modulation on BRR response. However, the neural pathways and chemical mediators that may underlie the PBN-induced suppression of the BRR response are essentially unknown.
Physiological evidence indicates that an increase in the discharge frequency of RVLM cardiovascular neurons induced by PBN accounts for the cardiovascular responses promoted by this pontine nucleus (1, 17). RVLM in turn may participate in BRR control of heart rate (HR) by providing an inhibitory modulation on the excitatory response of vagal preganglionic neurons in the dorsal motor nucleus of the vagus and nucleus ambiguus to NTS activation (46). Anatomically, a glutamatergic descending projection from the PBN complex to the RVLM has also been established (42). The present study was therefore undertaken to investigate whether such a glutamatergic innervation to the RVLM is involved in the suppressive action of the PBN complex on the BRR response. We also elucidated the role of N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic glutamate-receptor subtypes at the RVLM in this process. Our findings suggest that a distinct glutamatergic descending projection to the RVLM from the ventrolateral regions and the Köelliker-Fuse (KF) subnucleus of the PBN complex may participate in the suppression of reflex bradycardia via activation of both NMDA and non-NMDA glutamate receptor subtypes at the RVLM.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The experiments were carried out in compliance with the "Guiding Principles in the Care and Use of Animals" endorsed by the American Physiological Society.
Animal preparation. Adult male Sprague-Dawley rats (280-330 g) obtained from the Experimental Animal Center of the National Science Council (Taiwan, ROC) were used. Animals were initially anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). Preparatory surgery included intubation of the trachea and cannulation of the right femoral artery and both femoral veins. Systemic arterial pressure (SAP) was monitored from the cannulated artery via a pressure transducer (Statham P23 ID) and a pressure processor amplifier (Gould 20-4615-52). HR was determined in a beat-to-beat manner by a cardiotachometer (Gould 20-4615-65) triggered by the arterial pressure pulses. Pulsatile and mean SAP (MSAP), as well as HR, were recorded simultaneously on a polygraph (Gould ES 1000). The head of the animal was thereafter placed horizontally in a stereotaxic head holder (Kopf 1404), with the incisor bar set at 0 mm. The rest of the body was placed on a heating pad and suitably elevated to a position that prevented bending of the tracheal intubation.
During the experiment, animals were artificially ventilated, without paralytic agent, using a rodent respirator (Harvard 683) to maintain end-tidal CO2 within 4-5%, as monitored by a capnograph (Datex Normocap). This was carried out to minimize confounding cardiovascular changes secondary to respiratory perturbations. Anesthetic maintenance was provided by intravenous infusion of pentobarbital sodium at 15-20 mg · kg
1 · h1.
This management scheme was found (48) to provide stable anesthesia while preserving the capability of cardiovascular regulation, including
the BRR response. All data were collected from animals with a
maintained rectal temperature of 37°C, with a steady MSAP >90
mmHg throughout the experiment.
Evaluation of cardiac baroreceptor reflex response.
As in our previous studies (4, 21), the sensitivity of BRR control of
HR was evaluated by measuring the reflex bradycardia in response to
transient hypertension evoked by an intravenous bolus administration of
phenylephrine (5 µg/kg, 250 µl). The quotient calculated from the
peak reflex decrease in HR for a given peak increase in MSAP (in
beats · min1 · mmHg1)
was used as the index for cardiac BRR response. The control value of
this quotient amounted to 0.84 ± 0.06 (mean ± SE,
n = 36). This quotient was further
normalized to a percentage of pretreatment control to compensate for
variations between animals and to allow for comparison between
treatment groups.
Electrical or chemical activation of the parabrachial nucleus complex. We systematically mapped the rostral-caudal extent of the PBN complex and adjacent pontine tegmentum for the distribution of sites at which electrical or chemical activation resulted in a modulation of BRR control of HR. Electrical activation of the PBN complex was achieved using a stainless steel bipolar concentric electrode (Rhodes Medical SNE-100). The stereotaxic coordinates for the PBN complex were 0.8-1.2 mm posterior to the lambda, 2.0-2.7 mm lateral to the midline, and 5.5-7.0 mm below the dural surface. The PBN complex was stimulated with cathodal rectangular current pulses by using a Grass S88 stimulator coupled with a constant current isolation unit (Grass PSIU6).
During each penetration, a locus in the dorsal region of the PBN complex that was capable of inducing an increase in MSAP (>10 mmHg) was first identified by using 10-s trains of 0.5-ms pulses at 25-40 µA and 10-20 Hz. The stimulus parameters, chiefly intensity (to 10-20 µA), were subsequently adjusted to elicit minimal effects on MSAP (
10 mmHg) and HR (10 beats/min) to reduce possible confounding interference with the evaluation of the cardiac BRR response. These parameters were selected on the basis of our preliminary results, in which stimulation of the PBN complex with currents >20 µA invariably induced an increase in MSAP >10 mmHg.
The modulatory effect of PBN complex on BRR control of HR was assessed
by activating the same locus simultaneously with the induction of the
reflex. After MSAP and HR returned to baseline values, the electrode
was advanced to the next locus at 500-µm intervals in a
dorsal-to-ventral direction. The same electrical stimulation and
testing procedures were repeated under the same stimulus parameters.
One to two penetrations were made in either side of the PBN in each
animal to minimize damage or distortion of the brain. Five to seven
sites in each penetration were usually evaluated for their effect on
the BRR response.
Microinjection into the PBN complex of the excitatory amino acid
L-glutamate
(L-Glu; 1 nmol; Sigma) was
carried out in another group of animals to confirm that the effect on
BRR response resulted from activation of perikarya in the PBN complex
rather than fibers of passage (12). Similarly to the procedures for
electrical stimulation, L-Glu
was microinjected unilaterally, at 500-µm intervals, along the
dorsal-ventral axis of the PBN complex. A total volume of 50 nl was
delivered to each locus over 1-2 min to allow for full diffusion
of the chemical using a glass micropipette (50- to 100-µm tip
diameter) that was connected to a 0.5-µl Hamilton microsyringe.
Baroreceptor activation was induced, within 5 min after microinjection
of L-Glu, to evaluate the action
of the PBN locus thus activated on the reflex response. The animal was
allowed to rest for at least 20 min after each evaluation before the
tip of micropipette was positioned downward to the next locus. Again, only one to two penetrations on each side of the PBN were carried out,
with three to five injections of
L-Glu delivered for each penetration.
Microinjection of chemicals into the RVLM. Microinjection of chemicals into the RVLM bilaterally was similarly executed. The stereotaxic coordinates used were 4.5-5.0 mm posterior to the lambda, 1.8-2.1 mm lateral to midline, and 8.0-8.5 mm from the surface of the cerebellum. A total volume of 50 nl was delivered into each side of RVLM over 1-2 min.
To investigate the participation of glutamatergic neurotransmission at the RVLM in the PBN-induced modulation of BRR response, a NMDA-receptor channel blocker, dizocilpine (MK-801; 500 pmol; RBI) (24) or a non-NMDA-receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 pmol; RBI) (19) was microinjected bilaterally into the RVLM to maximize pharmacological blockade. The effect of each chemical treatment on basal BRR response or on modulation of BRR response induced by electrical activation of the PBN was evaluated before and at 5, 10, 20, 40, or 60 min after injection. Microinjection of the same volume of artificial cerebrospinal fluid (aCSF) served as the vehicle control. MK-801 and CNQX were chosen as the antagonists for NMDA and non-NMDA receptors on the basis of their specificity in blocking the respective receptor agonist (19, 24). The dose of MK-801 or CNQX was adopted from studies (4, 36) in which the antagonists were used for the same purpose as in our experiments. At the dose used, both glutamate antagonists were effective in blocking the pressor response to microinjection of L-Glu (1 nmol) at the RVLM in our preliminary study. All solutions were aliquoted, stored frozen, and thawed immediately before use during the experiment. Phenylephrine was freshly prepared with 0.9% saline.Histology. The brain was removed at the conclusion of each experiment and fixed in 30% sucrose in 10% formaldehyde-saline solution for at least 2 days. Frozen 25-µm sections of the pons and medulla oblongata, stained with neutral red, were used for histological verification of the location of stimulation or microinjection sites. For the latter purpose, 1% Evans blue was added to all microinjection solutions.
To create composite drawings of electrical and glutamate stimulation sites, the sections were projected on standardized coronal sections of the brain stem derived from the atlas of Paxinos and Watson (37). Individual stimulation tracts were plotted on the closest standard sections. Composite drawings of all stimulation and microinjection sites were reconstructed and labeled. The nomenclature adopted for the subregions of the PBN complex corresponds to the description of Fulwiler and Saper (11).Retrograde tract tracing in combination with immunohistochemistry. In separate experiments, retrograde tract tracing in combination with immunohistochemistry was carried out to establish a glutamatergic projection from the PBN complex to the RVLM. Rats anesthetized with pentobarbital sodium (50 mg/kg ip) received a single injection of 20-50 nl of 5% fast blue (Sigma) into the RVLM unilaterally. The coordinates of RVLM for fast blue injection were the same as those for microinjection of glutamate-receptor antagonists. Wounds were closed, and rats were allowed to recover in individual cages. After a survival time of 7-14 days, animals were anesthetized and perfused intracardially with warm saline followed by 3.8% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4) at 4°C. The brain stem was removed and postfixed in the latter solution by submersion overnight at 4°C and was cryoprotected by 30% sucrose in 0.1 M PBS.
Frozen 20-µm sections of the brain stem were mounted on plain glass slides and coverslipped with distilled water. They were immediately examined under an Olympus microscope (AX80), equipped with incident-light fluorescence, in conjunction with an Olympus U filter that provided an excitation band pass of 330-385 nm. Bright-field and fluorescence photomicrographs of the injection sites and bilateral PBN complex were taken from the same frame. Slides were then soaked in PBS to remove the coverslips. Sections of the brain stem that contained the PBN complex were further subjected to immunohistochemical processing. They were first washed thoroughly in 0.1 M PBS, preincubated in a 3% normal goat serum in PBS containing 0.75% gelatin and 0.02% Triton X-100, and then incubated in a rabbit anti-glutamate antiserum (1:4,000; Jackson) for 48-72 h. Sections in which anti-glutamate antiserum was omitted served as controls. Thereafter, the sections were processed with avidin-biotin-peroxidase complex (Vectastain ABC Kit, Vector Labs), and the immunoreactive product was visualized with diaminobenzidine. Brain sections were mounted on slides coated with gelatin and chromium potassium sulfate and were counterstained with 1% neutral red. Photomicrographs were taken with an Olympus microscope equipped with an automatic camera system (Olympus AX80). A composite map of the distribution of fast blue-labeled and/or glutamate-immunoreactive neurons in the PBN was constructed by projecting the respective photomicrographs taken from the same brain stem section on standardized coronal sections derived from the atlas of Paxinos and Watson (37). The fast blue labels were mapped with respect to tissue landmarks on bright-field photomicrographs. Cells that contained both retrograde fluorescence and immunoreactivity were detected by overlaying each transparent montage of the PBN complex with tissue landmarks. Both single- and double-labeled neurons in the PBN complex ipsilateral and contralateral to the side of injection were counted.Statistical analysis. Data are presented as means ± SE. The temporal effect of experimental treatments was statistically assessed using two-way ANOVA with repeated measures. This was followed by the Scheffé's multiple-range test for a posteriori comparison of means at comparable time intervals. Difference was considered statistically significant at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Distribution of loci in the PBN complex from which activation
induced suppression of reflex bradycardia.
We systematically activated the rostral-caudal extent of the PBN
complex and adjacent pontine tegmentum to localize loci from which
electrical stimulation induced a suppression of reflex bradycardia. Under a stimulus condition (10-s train of 0.5-ms pulses, at 10-20 µA and 10-20 Hz) that produced no discernible effect on basal MSAP (+8.4 ± 5.1 mmHg, n = 18) and
HR (+7.6 ± 6.5 beats/min, n = 18),
electrical activation of the rostral and middle levels (i.e.,
0.8-1.0 mm posterior to lambda) of the PBN complex elicited a
significant suppression of reflex bradycardia (Fig.
1A).
At these two levels, loci from which electrical stimulation induced >30% inhibition of cardiac BRR response were localized primarily within the ventrolateral regions of the PBN, mainly in the extreme lateral, external medial, and lateral subnuclei, extending to the KF
subnucleus (Fig.
2A).
Activation of the central and dorsal lateral subnuclei of the PBN
complex at these levels was less effective in suppressing the reflex
bradycardiac response (30%). Compared with stimulation of the
rostral and middle levels, electrical stimulation of the ventrolateral
region of the caudal PBN complex elicited much less inhibition (15%)
on the cardiac BRR response (Fig.
2A).
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Time-course effect of MK-801 or CNQX at the RVLM on PBN-induced
suppression of reflex bradycardia.
We next investigated the participation of glutamatergic
neurotransmission at the RVLM in the PBN-induced suppression of reflex bradycardia. In animals that received microinjection of aCSF into the
bilateral RVLM, electrical activation of loci in the ventrolateral regions as well as the KF subnucleus at the rostral or middle level of
PBN complex elicited an overall inhibition in the cardiac BRR response
that amounted to 47.3 ± 3.4% (mean ± SE,
n = 30). Such an inhibitory action of
PBN was significantly reduced after either the NMDA-receptor channel
blocker MK-801 (500 pmol, n = 6) or
the non-NMDA-receptor antagonist CNQX (50 pmol,
n = 7) was microinjected
bilaterally into the RVLM (Figs. 3 and
4). The reduction of PBN-induced BRR
suppression by MK-801 or CNQX peaked at 5-10 min and returned to
pretreatment levels at 60-min postinjection. We also noted that
administration of MK-801 (500 pmol, n = 6) or CNQX (50 pmol, n = 6) into the
RVLM, similar to aCSF, produced no discernible effect on baseline MSAP,
HR (Table 1), and BRR response (Fig.
5).
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Microinjection sites in the RVLM.
Histologically verified microinjection sites indicated that MK-801 or
CNQX was administered randomly into the anatomic confine of RVLM from
1.8 to 2.3 mm rostral to the obex (Fig. 6).
Microinjection of the same chemical agents outside the RVLM, e.g.,
areas dorsal to the RVLM and ventromedial to the nucleus ambiguus, the
lateral reticular nucleus, and the spinal trigeminal nucleus, resulted in no significant change in the BRR response (2.8 ± 2.2%,
n = 10) or PBN-induced suppression of
the same reflex (44.9 ± 7.4%, n = 12).
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Identification of glutamatergic projections from the PBN complex to
the RVLM.
Localized and discrete microinjection (core diameter: 220-300
µm) (Fig.
7B) of
fast blue unilaterally to the RVLM resulted in retrogradely labeled
neurons that were distributed primarily in the ventrolateral division
of the ipsilateral PBN and the KF subnucleus. Some fast blue-labeled
neurons also scattered at the medial as well as central and dorsal
lateral subnuclei. This topographic distribution of the retrogradely
labeled neurons was present mainly in the rostral and middle levels,
with a few in the caudal level, of the PBN complex (Fig.
7A). At the same time, very few
labeled neurons were found in the PBN complex contralateral to the
microinjection site.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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On the basis of combined results from physiological, pharmacological, and immunohistochemical experiments, the present study revealed that the PBN complex, in particular its ventrolateral regions that flank the tip of the brachium conjunctivum as well as the KF subnucleus, may participate in central cardiovascular regulation by suppressing the reflex bradycardia via glutamatergic neurotransmission at the RVLM. We demonstrated that, under an experimental condition that did not discernibly alter baseline SAP and HR, electrical and chemical activation of the ventrolateral regions and the KF subnucleus at the rostral and middle levels of PBN complex significantly inhibited the reflex bradycardia in response to transient hypertension. This PBN-induced cardiac BRR suppression was appreciably attenuated by microinjection into the bilateral RVLM of either MK-801 or CNQX. Of particular interest was the finding that the distribution of glutamatergic projecting neurons to the RVLM within the PBN complex overlapped substantially with loci from which electrical or chemical activation elicited suppression of the cardiac BRR response.
A novel finding in the present study was the engagement of a glutamatergic innervation from the ventral regions and KF subnuclei of the PBN complex to the RVLM in PBN-induced inhibition of reflex bradycardia. The PBN complex is a major relay for transmission of autonomic information from caudal brain stem to forebrain. A significant number of afferent projections to the PBN complex from the caudal brain stem originate in the relay nuclei of BRR arc (17, 18, 27). In particular, afferent innervations from the NTS appear to be topographically organized within the PBN complex. Relevant to the present study, the ventrolateral regions and KF subnucleus of PBN complex are recipients of cardiovascular-related information from the mediocaudal subdivision of the NTS (18), the primary medullary terminal sites of peripheral baro- and chemoreceptor afferents (6). Furthermore, excitatory amino acids, including glutamate, mediate the NTS input to the PBN complex (22, 38). It is therefore intriguing that our present results revealed that neurons within the same regions of the PBN complex in turn inhibited BRR control of HR via a glutamatergic projection to the RVLM, the output end of the BRR loop. Together, these findings suggest that, rather than assuming a passive role as a relay of autonomic information to forebrain structures, the PBN complex may actively participate in cardiovascular homeostasis by providing a negative feedforward on BRR sensitivity via a glutamatergic modulation on RVLM neurons. It has been established that a reciprocal, topographically organized connection between caudal NTS and ventrolateral regions and KF subnucleus of PBN complex (11, 40) is involved in the feedback inhibition of the same reflex response (8, 30). Whether neurons in the ventrolateral regions and KF subnucleus of PBN complex that project to the RVLM send their axon collaterals to the NTS to inhibit the cardiac baroreflex, however, awaits further elucidation. Moreover, the likelihood of indirect connections via intermediate nuclei to the RVLM in the PBN-induced BRR suppression cannot be excluded and warrant further investigation. In addition, because we did not evaluate whether glutamatergic projections from the PBN terminate on glutamatergic or C1 cells in the RVLM, the neuronal mechanisms at the RVLM that underlie the PBN-induced suppression of reflex bradycardia remain to be investigated.
Inhibition of BRR function is one of the means by which centrally elicited pressor response may be sustained under a variety of environmental stressors, including the defense reaction. This is because increases in SAP activate baroreceptors and would normally trigger reflex-mediated decreases in HR and sympathetic activity (41). For example, activation of the paraventricular nucleus of the hypothalamus (5, 7, 21) or the periaqueductal gray area (35) promotes pressor response and inhibition of BRR response. As the respective components of the forebrain defense area and the central nociceptive pathways, these two nuclei possess prominent connections to the lateral region of the PBN complex and the KF subnucleus (25, 32, 40). Thus our demonstration of a descending inhibitory modulation on the BRR regulation of HR by the PBN complex may provide the necessary pathways by which these forebrain nuclei induce pressor responses.
The excitatory amino acid L-Glu, in particular, has been demonstrated to be a major excitatory neurotransmitter in the RVLM in the regulation of cardiovascular functions. Our demonstration of a reversal of PBN-induced BRR suppression when glutamatergic neurotransmission at the RVLM was blocked with either NMDA- or non-NMDA-receptor antagonist suggests an inhibitory role for glutamate at the RVLM in baroreflex control of HR. This suggestion is in line with the conclusion of a previous study (33). The lack of significant alteration in baseline BRR response by MK-801 or CNQX further indicates that this suggested inhibitory modulation by glutamatergic neurotransmission on this reflex machinery may not be tonically active in the RVLM. In support of an inhibitory role for glutamate in baroreflex control of HR, direct application of DL-homocysteic acid to the RVLM inhibits the baroreceptor input to the NTS (46). The excitatory response to NTS stimulation of vagal preganglionic cardiomotor neurons within the dorsal motor nucleus of the vagus and nucleus ambiguus is also inhibited by conditioning activation of the RVLM (46). The impact of this descending glutamatergic projection to the RVLM on the inhibition by PBN of baroreceptor input to the NTS (8) is not immediately clear. Preliminary results from our laboratory indicate that the inhibitory modulation of reflex bradycardia induced by microinjection of L-Glu into the RVLM was attenuated by blockade of GABAergic transmission at the NTS. Together with previous reports (46), these observations suggest that the modulatory effect of PBN on the NTS may be partially mediated via a projection from the PBN to the RVLM. This suggestion, nonetheless, requires further confirmation.
We recognize that our results are at variance with previous studies (28, 49) that suggest that glutamatergic neurotransmission plays a facilitatory role in processing baroreceptive information at the RVLM. Several possibilities may account for this discrepancy. First, the differential results may arise from the difference in concentration of glutamate in the RVLM. By blocking the activity of glutamate with its receptor antagonists, we have depicted the action of endogenously released glutamate in the RVLM. In contrast, receptors at the RVLM are activated in a majority of previous studies (28, 49) by exogenously applied glutamate. Moriguchi et al. (33) reported that angiotensin-induced reduction in BRR sensitivity is accompanied by an increase in concentration of glutamate at the RVLM. Nonetheless, the extracellular concentration of glutamate measured is much lower (pmol vs. nmol) than the reported doses used for microinjection. Furthermore, direct microinfusion of glutamate into the RVLM at doses that match its endogenous levels results in a resetting of baroreflex (33). Second, it is possible that the state of consciousness might also contribute to the difference in results. Instead of conscious rats (49), animals anesthetized with pentobarbital sodium were used in this study. It is of interest that an inhibitory action of glutamate on BRR response was demonstrated in the RVLM of halothane-anesthetized rats (33). Third, glutamate released in different subregions of RVLM may exert different modulatory actions on BRR response. We noted that, compared with those reported in previous studies (28, 49), histologically verified microinjection sites for MK-891 and CNQX in the present study were localized in more ventromedial parts of the RVLM. Activation of the medial and lateral subregions of RVLM induces differential effects on the sympathetic nerve activity (44).
At the receptor level, the present study demonstrated that both NMDA and non-NMDA receptors at the RVLM are involved in the suppression of BRR response by the PBN complex. Because the arithmetic sum of the reversal effects of the two antagonists in the PBN-induced BRR suppression was >100%, and because neither antagonist exerted a tonic effect on BRR response, we speculate that there may be an interaction between NMDA and non-NMDA receptors at the RVLM in the inhibition of BRR response. Interactions of NMDA and non-NMDA receptors have been demonstrated in other regions of the brain (2). We are also aware that, in addition to ionotropic receptors, activation of metabotropic glutamate receptors are involved in cardiovascular responses of glutamate at the RVLM (43). Whether these guanylate nucleotide binding protein-coupled receptors are involved in PBN-induced BRR suppression, however, awaits further investigation.
The functional interpretation of glutamate-immunoreactive neurons in the brain is complicated, because glutamate is a precursor of a variety of other metabolites. As such, instead of a transmitter pool, the glutamate-immunoreactivity may represent the metabolic pool in the PBN complex. Fonnum (10) suggested that the metabolic pool of glutamate in the brain is much larger than its transmitter pool. Because the glutamate-containing neurons we observed in the PBN complex were <30% of the total neuronal population, it is unlikely that the glutamate immunoreactivity detected is involved in a common metabolic compartment. A neurotransmitter role for the immunohistochemically detected glutamate is further suggested by the observations that some of the glutamate-containing neurons in the ipsilateral ventrolateral regions and the KF subnucleus of PBN complex project to the RVLM. More importantly, microinjection of NMDA- or non-NMDA-receptor antagonist into the RVLM resulted in a reversal of the PBN-induced BRR suppression. Glutamate is also a precursor for GABA synthesis in the GABAergic neurons (10, 45). Because most GABAergic neurons are intrinsic to the autonomic nuclei of the brain stem (29), and because no evidence indicates the existence of GABAergic projecting neurons in the PBN complex, it is unlikely that the glutamate-containing neurons in the PBN complex are GABAergic in nature.
Depending on the stimulus intensity (3, 26, 47) and concentration of the excitatory amino acid used (3, 31, 47), activation of the PBN promoted differential effects on SAP and HR that range from no change to an increase in these hemodynamic parameters. To minimize possible confounding interference with the transient hypertension that we used to activate the baroreceptors, the concentration of L-Glu (1 nmol) used in this study was lowered to elicit no significant effect on SAP and HR. We found in our preliminary experiments that, at higher doses (5 or 10 nmol), microinjection of L-Glu into the PBN resulted in an increase in MSAP (+9.6 ± 4.5 or +14.5 ± 6.3 mmHg, respectively) and HR (+9.0 ± 6.3 or +12.4 ± 5.6 beats/min, respectively). These results are consistent with previous reports (3, 31, 47).
We are aware that the pharmacological method we used to evaluate BRR control of HR has limited application on the threshold and maximal capacity of the reflex. Because they could be affected separately, the possibility that glutamatergic inputs from PBN to RVLM may modulate these parameters differentially cannot be ruled out. In addition, because phenylephrine may activate low-pressure receptors in the atria and ventricles, the significance of the glutamatergic projections to the RVLM from the PBN on the reflex mechanism induced by activation of low-pressure receptors may be underestimated. We also recognize that, because the present study did not examine reflex tachycardia in response to a decrease in SAP, the effect of PBN on the other end of the BRR function curve, i.e., BRR control of sympathetic activity, is unknown. In this regard, Hayward and Felder (16) reported recently that PBN neurons reduce the capacity of BRR regulation of sympathetically mediated increases in SAP. Whether the glutamatergic input from the PBN to RVLM is involved in this process, however, awaits further investigation.
In conclusion, the present study demonstrated that glutamatergic descending projections to the RVLM ipsilaterally from the ventrolateral regions and the KF subnucleus of the PBN complex may participate in central regulation of cardiovascular functions by exerting an inhibitory modulation of the cardiac baroreflex response. Furthermore, such an inhibitory modulation on BRR response may take place by activating both NMDA and non-NMDA receptors at the RVLM.
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ACKNOWLEDGEMENTS |
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This work was supported in part by Research Grant VGHKS88-35 from Veterans General Hospital-Kaohsiung, and NSC-88-2314-B075B-016 (J. Y. H. Chan) from the National Science Council, Taiwan, Republic of China.
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FOOTNOTES |
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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 and other correspondence: J. Y. H. Chan, Dept. of Medical Education and Research, Veterans General Hospital-Kaohsiung, Kaohsiung 81346, Taiwan, Republic of China (E-mail: yhwa{at}isca.vghks.gov.tw).
Received 27 April 1998; accepted in final form 11 January 1999.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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), 15-30%
(
), or 0-15% (
) or an increase of 0-15% (
) in
cardiac BRR response; n= 18 animals
per group. For each animal, 1-2 penetrations were made, and
5-7 sites per penetration were tested on BRR control of HR.
