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Am J Physiol Heart Circ Physiol 274: H1284-H1292, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 4, H1284-H1292, April 1998

Noradrenergic neurotransmission at PVN in locus ceruleus-induced baroreflex suppression in rats

Kuo-Rue Hwang1, Samuel H. H. Chan2, and Julie Y. H. Chan1

1 Department of Medical Research, Veterans General Hospital-Taipei, Taipei 11217; and 2 Center for Neuroscience, National Yang-Ming University, Taipei 11221, Taiwan, Republic of China

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We investigated the role of ascending noradrenergic projections from the locus ceruleus (LC) to the paraventricular nucleus (PVN) of the hypothalamus in LC-induced suppression of the baroreceptor reflex (BRR) response in adult Sprague-Dawley rats maintained under pentobarbital anesthesia. On the basis of in vivo microdialysis and high-performance liquid chromatography-electrochemical detection, microinjection of L-glutamate (5 nmol) into the LC resulted in a site-specific increase in norepinephrine (NE) concentration in the dialysate collected from the parvocellular subnucleus of the PVN. The temporal course of this increase in extracellular NE concentration in the PVN coincided with the time course of inhibition elicited by the LC on the BRR response. Microinfusion of NE (10, 50, or 100 nM) into the parvocellular subnucleus of the PVN by reverse microdialysis also promoted a parallel increase in NE at the PVN and a reduction in the BRR response. Inhibition of the BRR response induced by microinjection into the PVN of the alpha 1-adrenoceptor agonist phenylephrine (10 nmol) or chemical activation of the LC was reversed by bilateral PVN microinjection of prazosin (100 pmol). However, local application to the PVN of the alpha 2- or beta -adrenoceptor agonist guanabenz (10 nmol) or isoproterenol (10 nmol) was ineffective. Our results suggest that NE released from the LC-PVN noradrenergic projection may participate in LC-induced suppression of the BRR response by activating the alpha 1-adrenoceptors at the parvocellular subnucleus of the PVN.

paraventricular nucleus of hypothalamus; alpha 1-adrenoceptor; in vivo microdialysis

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE PARAVENTRICULAR NUCLEUS (PVN) of the hypothalamus functions as a site of integration for autonomic and endocrine regulation of cardiovascular functions. Superimposed on its established role in the regulation of neuroendocrine secretions (37, 40), neuroanatomic (10, 23, 34, 37, 40) and electrophysiological (19, 24) findings demonstrated that the PVN is reciprocally connected to areas in the brain stem that are engaged in regulation of blood pressure and modulation of baroreceptor reflex (BRR) sensitivity; for example (10, 35, 37), the PVN receives dense noradrenergic innervations from the A1 cell bodies of the caudal ventrolateral medulla, A2 cell bodies of the nucleus tractus solitarius (NTS), and A6 cell bodies of the locus ceruleus (LC). Efferent fibers from PVN neurons, in turn, descend monosynaptically to the intermediolateral cell column of the spinal cord and the dorsal vagal complex (23, 34, 40), the respective origins of preganglionic sympathetic (14) and parasympathetic (8) neurons. Despite these established neuronal connections between the PVN and brain stem cardiovascular sites, the chemical nature and physiological significance of these circuits in autonomic regulation of cardiovascular performance are not fully understood .

As the nucleus that contains the largest cluster of norepinephrine (NE)-containing neurons in the brain (11), the LC plays an important role in central regulation of cardiovascular functions. Previous studies from our laboratory (3, 4, 38, 39, 44) established that one of these regulatory processes in which the LC participates is the inhibitory modulation of the BRR response. LC-induced suppression of the BRR response is mediated by a direct descending connection to the NTS, the primary terminal site for the baroreceptor afferents (7), and engages NE (3), neuropeptide Y (NPY) (4), and galanin (GAL) (39) as the chemical mediators. We also unveiled the participation of an indirect route between the LC and the NTS via the PVN (38, 44). Pharmacological results suggest that at least noradrenergic (38), NPYergic (38), and GALergic (44) neurotransmission in the parvocellular subnucleus of the PVN participated in this indirect connection that is engaged in LC-induced inhibition of the BRR response. An actual correlation between release of these chemical mediators at the PVN and LC-induced suppression of the BRR response, nonetheless, has not been systematically investigated.

The present study was undertaken to establish the participation of noradrenergic projections from the LC to the PVN in LC-induced BRR suppression. We measured changes in NE concentration in the dialysate collected by in vivo microdialysis in the PVN evoked by activation of LC perikarya and correlated them with LC-elicited inhibition of the BRR response. We also evaluated the effect of altering extracellular NE concentration in the PVN on the BRR response. Pharmacological manipulations were used to identify the subtype(s) of adrenoceptors in the PVN that was involved in LC-induced BRR suppression. Our results suggest that terminal release of NE from the LC-PVN projection may participate in LC-induced suppression of the BRR response by activating the alpha 1-adrenoceptors at the parvocellular subnucleus of the PVN.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The experiments were conducted 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 (250-320 g) were obtained from the Experimental Animal Center of the National Science Council (Taiwan, ROC) and initially anesthetized with pentobarbital sodium (40 mg/kg ip). 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 (model P23 ID, Statham) and a pressure processor amplifier (model 20-4615-52, Gould). Heart rate (HR) was determined by a cardiotachometer (model 20-4615-65, Gould) triggered by the arterial pressure pulses. Pulsatile and mean SAP (MSAP), as well as HR, were recorded simultaneously on a polygraph (model TA11, Gould). The head of the animal was then placed in a stereotaxic head holder (model 1404, Kopf), with the rest of the body positioned on a heating pad and elevated to a suitable position. During the experiment, animals were artificially ventilated using a rodent respirator (model 683, Harvard) to avoid possible confounding cardiovascular changes secondary to respiratory perturbations. Anesthetic maintenance was provided by infusion of pentobarbital sodium at 15-20 mg · kg-1 · h-1. This management scheme (43) was found 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.

Fabrication and calibration of microdialysis probes. To achieve site specificity and enhance detection sensitivity, microdialysis probes with an active exchange area compatible with the PVN were fabricated according to the procedures developed in our laboratory (41). These microdialysis probes had an active exchange area 450-500 µm long and 220 µm diameter. The membrane (Spectra/Por RC; 200-µm ID, 220- µm OD) was made of regenerated cellulose and had a molecular size cutoff of 13,000 Da.

All fabricated microdialysis probes were calibrated for relative recovery rate before the experiment. Each probe was placed in a beaker that contained a modified physiological Ringer solution (in mM: 132 NaCl, 3 KCl, 1.2 CaCl2, 1.2 MgCl2, 0.3 NaH2PO4, 1.2 Na2HPO4, pH 7.2) to which known concentrations of NE (10, 20, 50, or 100 nM) were added. Comparable to that reported previously (5, 42), the relative in vitro recovery for NE thus calculated was 9.8 ± 0.9% (n = 5).

Sampling procedures and measurement of NE content. The microdialysis probe was lowered into the parvocellular subnucleus of the PVN at 1.4-1.6 mm posterior to the bregma, 0.2-0.5 mm from the midline, and 7.4-7.6 mm below the surface of the cortex. Brain tissue was continuously perfused at 1 µl/min during the period of stabilization with a modified physiological Ringer solution by means of a microliter infusion pump (model CMA/102, Carnegie Medicin). Collection of dialysate usually commenced 100-120 min after insertion of the dialysis probe. Samples were collected every 20 min and were stored in Eppendorf tubes kept on ice and shaded with aluminum foil.

The NE concentration in each sample was immediately quantified by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. The sample (15 µl) was injected into the HPLC system using a Rheodyne 9125 injector. The HPLC system consisted of a dual-piston syringe pump (model PM-80, Bioanalytical System) and an electrochemical detector (model LC-4C, Bioanalytical System). The carbon working electrode was held at 0.55 V vs. the reference Ag-AgCl electrode. A mobile phase composed of 0.1 M sodium acetate buffer (pH 6, Merck), 0.1 mM disodium EDTA (Nakarai Chemicals), 2 mM sodium 1-octanesulfonate (Sigma Chemical), and 5% methanol (LC grade, Merck) was pumped, at 0.5 ml/min, through a reverse-phase column (PHASE-II ODS, Bioanalytical System; 3.2 × 100 mm). The concentration of NE was computed by comparing the peak amplitude of each sample in the chromatogram with that for standard NE solutions of known concentration (5 × 10-11-5 × 10-9 M). The minimal detection limit for NE was 1.0 pg. The concentration of NE was normalized to a percentage of pretreatment control to compensate for variations between animals and to allow for comparison between treatment groups.

Evaluation of baroreceptor reflex response. Similar to the method used in our previous studies (3, 4, 39, 44), arterial baroreceptors were stimulated by a transient hypertension induced by administration of phenylephrine (5 µg/kg iv). The quotient that represented unit reflex decrease in HR per unit increase in MSAP (beats · min-1 · mmHg-1) was used as the index for the BRR response. The quotient was normalized to a percentage of pretreatment control to compensate for variations between animals and to allow for comparison between treatment groups.

Microinjection of chemicals into the LC or PVN. To activate the perikarya in the LC, the excitatory amino acid L-glutamate (L-Glu) was microinjected unilaterally into the LC with a glass micropipette that was connected to a 0.5-µl Hamilton microsyringe. The stereotaxic coordinates for the LC were as follows: 0.9-1.0 mm posterior to the lambda, 1.0-1.1 mm lateral to the midline, and 6.0-6.5 mm below the dural surface. A volume of 50 nl was delivered over 1-2 min into one side of the LC to allow for full diffusion of the chemical. Microinjection bilaterally of chemicals into the PVN was similarly executed. The stereotaxic coordinates for the PVN were the same as those for placement of microdialysis probes. An injection volume of 50 nl was delivered consecutively into each side of the PVN.

Experimental protocol. Our first series of experiments examined the concurrent effects of LC activation on extracellular NE concentration in the PVN and the BRR response. The LC was chemically activated by three consecutive injections of L-Glu (5 nmol), at 20-min intervals, to the same site. NE concentration in the dialysate collected from the PVN was determined 5 min after each LC activation, along with determination of the sensitivity of the BRR response. Extracellular NE concentration in the PVN and the BRR response were evaluated for another 60 min after cessation of LC activation.

To ascertain a causative relationship between the increase in NE concentration in the dialysate collected from the PVN and inhibition of the BRR response, we experimentally manipulated the extracellular NE concentration on the basis of the principle of reverse microdialysis and examined the concurrent alterations in the BRR response in our second series of experiments. NE (10, 50, or 100 nM) or a modified physiological Ringer solution was infused into the PVN via the microdialysis probe for 80 min at 1 µl/min. This was followed by the infusion of Ringer solution, at 1 µl/min, for another 60 min. The difference between NE concentrations in the perfusate and dialysate was determined every 20 min during and after infusion of the monoamine into the PVN, together with the sensitivity of the BRR response.

The subtypes of adrenoceptor in the PVN involved in the modulation of the BRR response by LC were studied in our third series of experiments. The alpha 1-, alpha 2-, or beta -adrenoceptor agonist phenylephrine (10 nmol), guanabenz (10 nmol), or isoproterenol (10 nmol) or artificial cerebrospinal fluid (aCSF) was microinjected individually into the bilateral PVN, and the temporal changes in sensitivity of the BRR response were examined for 90 min. Participation of alpha 1-adrenoceptors at the PVN in the modulation of the BRR response by the LC was further ascertained in our last series of experiments. The alpha 1-adrenoceptor antagonist prazosin (100 pmol) or aCSF was administered bilaterally into the PVN, and temporal changes in LC-induced modulation of the BRR response were evaluated for 90 min.

Test agents. NE, phenylephrine, L-Glu, guanabenz, isoproterenol, and prazosin were obtained from Sigma Chemical (St. Louis, MO). All chemical agents were prepared fresh with aCSF (in mM: 149 NaCl, 2.8 KCl, 1.2 CaCl2, 1.2 MgCl2, 5.4 D-glucose, pH 7.4). For microinfusion of NE into the PVN, 0.125 mM ascorbic acid (Nakaraia Chemicals) was added to the Ringer solution to minimize oxidation of the monoamine.

Histology. The brain was removed after each experiment and fixed in 30% sucrose in 10% formaldehyde-saline solution for at least 48 h. The position of the microdialysis probe in the PVN or the tip of the microinjection needle in the PVN or LC was histologically verified on 25-µm frozen sections stained with 1% neutral red. Evans blue (1%) was added to the microinjection solution to aid in subsequent histological identification of the microinjection sites.

Statistical analysis. Values are means ± SE. The temporal effect of treatment(s) on the concentration of NE or the BRR response was statistically assessed using two-way analysis of variance with repeated measures. This was followed by Scheffé's or Dunnett's multiple-range test for a posteriori multiple comparison of individual means at corresponding time intervals. All results were considered statistically significant at P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of LC activation on extracellular NE concentration in the PVN. Repeated activation of the perikarya in the LC by microinjection of L-Glu (5 nmol) resulted in a significant increase in extracellular NE concentration in the ipsilateral parvocellular subnucleus of the PVN (Fig. 1A). Baseline NE concentration in the dialysate collected from the PVN, being the average of three individual samples taken immediately before LC activation, was 59.9 ± 8.6 pg/ml (n = 32). Whereas this value increased to 86.4 ± 12.1 pg/ml (n = 8) on microinjection of L-Glu into the ipsilateral LC, no significant alteration in NE concentration was detected in the PVN (54.6 ± 9.8 pg/ml, n = 5) after local application of aCSF (50 nl) to this pontine nucleus.


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  		Fig. 1.   Time course of simultaneous alterations in norepinephrine concentration ([NE]) in dialysate of paraventricular nucleus (PVN) of hypothalamus and baroreceptor reflex (BRR) response before (C), during (T20, T40, and T60), and after (R20, R40, and R60) microinjection of artificial cerebrospinal fluid (aCSF) or 5 nmol L-Glu into locus ceruleus (LC). A: NE concentration in dialysate collected from PVN. B: temporal alterations in sensitivity of BRR response. Values are means ± SE; n = 5-8 animals/group. * P < 0.05 vs. aCSF by Scheffé's multiple-range test.

Microinjection of L-Glu into the LC also discernibly inhibited the BRR response (Fig. 1B), which manifested a time course that paralleled the increase in extracellular NE concentration in the PVN. Linear regression analysis revealed that these two concurrent changes were highly correlated (y = 0.85x + 2.18, r = 0.97, n = 8). NE concentration in the dialysate from the PVN returned to baseline value (61.5 ± 10.2 pg/ml, n = 8) after the cessation of LC activation, alongside the reversal of the depressed BRR response (Fig. 1B).

Figure 2 depicts the site specificity of the LC-elicited increase in extracellular NE concentration in the PVN and suppression of the BRR response. We found that, under an experimental condition in which chemical activation of the LC discernibly suppressed the BRR response (Fig. 2B), no discernible alteration in NE concentration in the dialysate was detected when the microdialysis probe (n = 10) was placed in the magnocellular subnucleus or in areas immediately outside the PVN (Fig. 2A). Likewise, no significant increase in extracellular NE concentrations was detected in the parvocellular subnucleus of the PVN (Fig. 2A) when L-Glu (5 nmol) was administered into sites adjacent to the LC (n = 8), resulting in minimal alterations in the BRR response (Fig. 2B).


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  		Fig. 2.   Time course of simultaneous alterations in NE concentration in dialysate collected from PVN or areas adjacent to PVN and BRR response before (C), during (T20, T40, and T60), and after (R20, R40, and R60) microinjection of 5 nmol L-Glu into LC or areas outside LC. A: NE concentration in dialysate collected from parvocellular subnucleus of PVN or magnocellular subnucleus of PVN and areas adjacent to PVN (non-PVN). B: temporal alterations in sensitivity of BRR to microinjection of L-Glu into LC or areas outside LC (non-LC). Values are means ± SE; n = 8-10 animals/group. * P < 0.05 vs. non-LC group by Scheffé's multiple-range test.

Figure 3 illustrates the location of sites in the dorsal pontine region on which L-Glu was delivered. Sites in which the excitatory amino acid elicited a significant suppression of the BRR response and a discernible increase in extracellular NE concentration in the PVN were distributed within the anatomic confines of the LC.


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  		Fig. 3.   Diagrammatic representation of dorsal pontine region at 3 rostral-caudal levels showing location of tip of micropipettes through which local application of 5 nmol L-Glu elicited significant suppression (bullet ) or lack of effect (black-triangle) on BRR response and increase in extracellular NE concentration at PVN. Numbers on right denote distance from bregma. DTG, dorsal tegmental nucleus; MeV, mesencephalic trigeminal nucleus; PBN, parabrachial nucleus; V, spinal trigeminal nucleus; 7n, facial nerve; scp, superior cerebellar peduncle.

Temporal changes in extracellular NE concentration in the PVN and the BRR response. To ascertain a causative relationship between the increase in NE concentration in the dialysate collected from the PVN and inhibition of the BRR response, known concentrations of NE (10, 50, or 100 nM) were infused into the parvocellular subnucleus of the PVN via a microdialysis probe. On the basis of our previous work (5, 42), these procedures allowed us to estimate the steady-state extracellular NE concentration in the PVN and the associated changes in the BRR response. This steady-state condition, defined as minimal changes (within 10%) in the difference in NE concentrations between perfusate and dialysate among successive samples collected over 3 h, was reached 60 min after continuous infusion of NE (50 nM, n = 5) into the PVN at 1 µl/min (Fig. 4). An 80-min infusion schedule was therefore adopted in subsequent experiments.


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  		Fig. 4.   Time course changes in difference in NE concentration between perfusate ([NE]IN) and dialysate collected from PVN ([NE]OUT). Samples were collected at 20-min intervals for 3 h after microinfusion of NE (50 nM) at 1 µl/min into PVN. Values are means ± SE; n = 6. 

Figure 5A illustrates that the difference between NE concentrations in the perfusate (10, 50, or 100 nM) and dialysate collected from the PVN followed a time course that rose sharply and significantly during the first 20 min, reaching a peak at 60 min after infusion of the monoamine into the PVN. The microdialysis probe again served as a sampling device after Ringer solution replaced NE as the perfusate. The diffusion gradient, however, now favored the removal of NE from the PVN. Indeed, on replacing NE with Ringer solution as the perfusate, there was a discernible gain of NE in the dialysate during the first 40 min. The subsequent concentration of NE underwent a rapid decrease. We also found that the magnitude and time course of changes in extracellular NE concentrations in the PVN were dependent on the concentration of the monoamine in the perfusate. For example, the highest concentration of NE (100 nM) promoted a greater and more sustained increase in the extracellular NE concentration (Fig. 5A). Microinfusion of Ringer solution at 1 µl/min (n = 5), on the other hand, did not yield discernible changes in NE concentration in the dialysate collected from the PVN.


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  		Fig. 5.   Time course of simultaneous alterations in extracellular NE concentration in PVN and BRR response before (C), during (T20, T40, T60, and T80), and after (R20, R40, and R60) microinfusion of Ringer solution or NE (10, 50, or 100 nM) at 1 µl/min into PVN. A: difference in NE concentration between perfusate and dialysate collected from PVN. B: temporal alterations in sensitivity of BRR. Values are means ± SE; n = 4-6 animals/group. * P < 0.05 vs. Ringer solution group by Scheffé's multiple-range test.

Microinfusion of NE into the PVN also elicited a significant inhibition of the BRR response (Fig. 5B). The inhibitory action of NE was dose dependent and followed a time course that paralleled the elevation in extracellular NE concentration in the PVN. We noted in particular that the sustained rise in extracellular NE concentration in the PVN after microinfusion of 100 nM NE induced a prolonged suppression of the BRR response, which lasted 40 min after NE was replaced with Ringer solution as the perfusate.

We found again that the suppression of the BRR response was dependent on the sites in the hypothalamus that received microinfusion of NE. For example (Fig. 6A), infusion of NE (50 nM), at the same infusion rate, into the magnocellular subnucleus of the PVN (n = 9) or loci adjacent to the confines of the PVN (n = 10) resulted in a temporal change in extracellular NE concentration that was comparable to that in the parvocellular subnucleus (cf. Fig. 5A). Nonetheless, these changes in NE at the PVN were not accompanied by appreciable alterations in the BRR response (Fig. 6B).


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  		Fig. 6.   Time course of simultaneous alterations in extracellular NE concentration in PVN and BRR response before (C), during (T20, T40, T60, and T80), and after (R20, R40, and R60) microinfusion of Ringer solution or NE (10, 50, or 100 nM) at 1 µl/min into PVN. A: difference in NE concentration between perfusate and dialysate collected from magnocellular division of PVN or areas adjacent to PVN (non-PVN). B: temporal alterations in sensitivity of BRR. Values are means ± SE; n = 9-10 animals/group. * P < 0.05 vs. preinfusion control by Dunnett's multiple-range test.

Involvement of alpha 1-adrenoceptors at the PVN in LC-induced BRR suppression. Activation of the alpha 1-adrenoceptors with microinjection of the agonist phenylephrine (10 nmol, n = 7) into the bilateral PVN significantly inhibited the BRR response (Fig. 7A). Such an inhibition was discernibly blocked (Fig. 7A) by a prior application of the alpha 1-adrenoceptor antagonist prazosin (100 pmol, n = 5) into the PVN. Microinjection of the alpha 2- or beta -adrenoceptor agonist guanabenz (10 nmol, n = 5) or isoproterenol (10 nmol, n = 6), respectively, on the other hand, elicited no appreciable alteration in the same reflex response (Fig. 7B).


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  		Fig. 7.   Time course of alterations in BRR response after microinjection into PVN bilaterally of phenylephrine (10 nmol), phenylephrine + prazosin (10 nmol + 100 pmol), or aCSF (A) or guanabenz (10 nmol), isoproterenol (10 nmol), or aCSF (B). Values are means ± SE; n = 5-7 animals/group. * P < 0.05 vs. aCSF group by Scheffé's multiple-range test; # P < 0.05 vs. phenylephrine group by Scheffé's multiple-range test.

The implied participation of alpha 1-adrenoceptors in the PVN on LC-induced suppression of the BRR response (38) was again ascertained. In contrast to aCSF (100 nl, n = 5), blockade of the alpha 1-adrenoceptors with microinjection of prazosin (100 pmol, n = 6) bilaterally into the PVN reversed suppression of the BRR response induced by chemical activation of the LC (Fig. 8). Treatment with prazosin by itself, evaluated at 10 min postinjection, however, elicited no discernible effect on the BRR response (108 ± 3.2%, n = 6).


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  		Fig. 8.   Effect of bilateral microinjection of prazosin (100 pmol) or aCSF into PVN on sensitivity of BRR response and suppression of BRR response by microinjection of 5 nmol L-Glu into LC. Values are means ± SE; n = 5-6 animals/group. * P < 0.05 vs. pretreatment control by Dunnett's analysis; # P < 0.05 vs. LC group by Scheffé's multiple-range test.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study demonstrated that NE released from the LC-PVN noradrenergic projection may participate in LC-induced suppression of the BRR response by activating the alpha 1-adrenoceptors at the PVN. We found that chemical activation of the LC resulted in a site-specific increase in NE concentration in the dialysate collected from the parvocellular subnucleus of the PVN. The temporal elevation in extracellular NE concentration in the PVN, elicited by LC stimulation or by microinfusion of NE via reverse microdialysis, correlated positively with the time course of suppression in the BRR response. At the receptor level, blockade of the alpha 1-adrenoceptors at the PVN reversed the inhibitory action of LC on the BRR response. The latter action, on the other hand, was duplicated by activation of the alpha 1-adrenoceptors in the PVN.

Evidence from a variety of experimental approaches supports a role for noradrenergic neurotransmission at the PVN in the modulation of cardiovascular function. Site-specific changes in NE release in the PVN in response to alterations in SAP have been reported (25, 27, 32). Activation of the LC-PVN ascending noradrenergic pathways that impinge on the PVN reportedly accounts for the increase in NE release in rat PVN induced by systemic hemorrhage (25). An augmented overflow of endogenous NE from the PVN is associated with the development of hypertension in spontaneously hypertensive rats (32, 33), possibly via facilitated neuronal traffic in the noradrenergic projection from the LC (20). Blockade of noradrenergic neurotransmission in the PVN with the neurotoxin 6-hydroxydopamine delays the development of hypertension in young spontaneously hypertensive rats (33).

Few microdialysis studies (25, 28), however, discerned the physiological relevance of the endogenous NE in the PVN released on activation of the LC. Thus a major contribution of the present study was to provide direct evidence that linked the evoked release of NE in the PVN by the LC with the suppression of the BRR response elicited by the same pontine nucleus. By monitoring the simultaneous fluctuations in NE concentration in the dialysate collected from the PVN and the inhibition of the BRR response, we were able to correlate changes in the extracellular NE concentrations with alterations in the sensitivity of the BRR response. We also mimicked the action of LC stimulation by creating a steady-state increase in NE via microinfusion of the monoamine into the PVN. These findings further substantiated the functional significance of the LC-PVN noradrenergic pathways in baroreflex control of blood pressure.

The PVN is a complex nucleus that consists of magnocellular and parvocellular neurons. Whereas the A1 neurons project almost exclusively to the magnocellular neurosecretory neurons (37), projections from the A6 cell bodies in the LC terminate primarily in the parvocellular subnucleus of the PVN (10, 35). Electrical stimulation of the parvocellular subnucleus of the PVN inhibits the BRR response (6, 12) via an action that may take place in the NTS (6, 24). Our present results also pointed to the site-specific engagement of noradrenergic neurotransmission in the parvocellular subnucleus of the PVN in LC-induced BRR suppression. These results were possible because of the dimension of the active exchange area (220 × 450-500 µm) of the microdialysis probe. We found no discernible alteration, in response to activation of the LC, of NE concentration in the dialysate collected from the magnocellular subnucleus of the PVN or sites adjacent to the PVN. Similarly, microinfusion of NE into these sites resulted in an increase in extracellular NE without the concomitant inhibition of the BRR response. On the other hand, activation of areas adjacent to the LC also did not increase extracellular NE concentration in the parvocellular subnucleus of the PVN.

The low basal NE concentration we detected in the PVN is comparable to that reported in previous studies (20, 25, 28-30) in which NE was measured in the same nucleus by in vivo microdialysis or a push-pull technique (32, 33). However, the basal concentration of NE we measured was lower than that detected in the PVN of conscious (28, 29) or urethan-anesthetized rats (20, 25, 30). This discrepancy may be the result of a generalized inhibitory action of pentobarbital sodium on neuronal excitability (31). Nonetheless, our observed magnitude of increase (46.2 ± 9.2%) in NE concentration, evoked in the PVN by LC activation under pentobarbital anesthesia, was actually larger than that (21.8 ± 15.6%) evoked in the hypothalamus of urethan-anesthetized rats (20). As such, pentobarbital anesthesia did not appear to affect significantly the effectiveness of neurons in the LC-PVN circuit.

It is likely that suppression of the BRR response via the LC-PVN noradrenergic pathways is mediated by alpha 1-adrenoceptors in the PVN (38), with minimal involvement of alpha 2- or beta -adrenoceptors. We demonstrated that exogenous application of alpha 1-, but not alpha 2- or beta -, adrenoceptor agonist into the PVN suppressed the BRR response. Furthermore, the LC-induced BRR suppression was attenuated by administration of alpha 1-, but not alpha 2- or beta -, adrenoceptor antagonist into the bilateral PVN. These observations were comparable to the antagonism by alpha 1-, but not alpha 2-, adrenoceptor antagonist of the cardiovascular (2, 13) and neuroendocrine (18, 26) responses to microinjection of NE into the PVN. Furthermore, LC-induced regulation of growth hormone release is relayed in the PVN via a local alpha 1-adrenoceptor population (26). Electrophysiologically, excitatory postsynaptic potentials evoked in the PVN by NE (21, 27) or LC activation (36) are blocked by iontophoretic application of alpha 1-, but not alpha 2- or beta -, adrenoceptor antagonists into the PVN.

We noted that microinjection of prazosin into the PVN did not completely antagonize the LC-induced BRR suppression (cf. Fig. 8). There are two possible, although not mutually exclusive, explanations. First, we previously reported that a direct innervation from the LC to the NTS (3, 4, 39) is also involved in the inhibitory effect elicited by this pontine nucleus on the BRR response. Because it is unaffected by local application of prazosin into the PVN, it is possible that the origin of the remaining BRR suppression was this LC-NTS linkage. Second, other chemical mediators at the PVN may also participate in LC-induced inhibition of the BRR response. In this regard, we previously reported that NPYergic (38) and GALergic (44) neurotransmission in the PVN may also be involved in LC-induced suppression of the BRR response. High concentration of NPY (1) elicits primarily an inhibitory modulation on the discharge rate of PVN neurons. On the other hand, NPY and GAL potentiate NE release in the PVN (22, 30). Because NPY and GAL have been demonstrated to coexist with NE in the LC neurons that project to the PVN (16), it is possible that the combined pre- and postsynaptic actions of these two chemical mediators may account for the superfluous LC-induced BRR suppression after prazosin injection into the PVN.

We are aware that the method used in this study to evaluate the BRR response allowed us to investigate mainly the cardiomotor component of the reflex. Some investigators reported inhibition of the cardiomotor and vasomotor components of the BRR response by the PVN (9, 15). Others reported that whereas the cardiomotor component is suppressed, the vasomotor component of the reflex is unaffected (17). As such, the role of LC-PVN noradrenergic projection in the modulation of the vasomotor component of the BRR response remained to be delineated.

In conclusion, on the basis of in vivo microdialysis in conjunction with pharmacological treatments, the present study demonstrated that NE released from the LC-PVN noradrenergic nerve terminals may function as a chemical mediator in LC-induced suppression of the BRR response by activating the alpha 1-adrenoceptors in the parvocellular subnucleus of the PVN.

    ACKNOWLEDGEMENTS

This work was supported in part by National Science Council (Taiwan, ROC) Grant NSC-87-2312-B075-001 (J. Y. H. Chan).

    FOOTNOTES

Address for reprint requests: J. Y. H. Chan, Dept. of Medical Research, Veterans General Hospital-Taipei, Taipei 11217, Taiwan, ROC.

Received 10 October 1997; accepted in final form 17 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Heart Circ Physiol 274(4):H1284-H1292
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