INTRODUCTION

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THE MAJORITY OF CARDIOVASCULAR and respiratory afferents relay to the central nervous system (CNS) via the vagus and glossopharyngeal nerves and terminate in the nucleus tractus solitarii (NTS) (12, 17). The NTS is a major site of reflex integration, and it is also richly innervated from regions of the CNS concerned directly or indirectly with cardiorespiratory control (17), changing both the activity of the sympathetic nervous system (SNS) and vasopressin (VP) release (27).

The role of GABA in the NTS has been investigated in the last decade. The baroreceptive region of the NTS contains a high density of GABA-containing nerve terminals (2, 11, 16, 20, 24, 25) and a high density of both GABA_A and GABA_B receptors (4, 10, 25). Previous studies (3, 9, 22, 29, 31) demonstrated that stimulation of GABA_B receptors in this region of the NTS elicits a marked pressor response in anesthetized rats. In conscious rats, systemic administration of baclofen causes a sustained hypertension and tachycardia (23).

Anatomic studies showed projections from the NTS to neurons of the paraventricular nucleus of the hypothalamus to and from the NTS to the spinal cord (6, 18, 32). Previous results showed an important participation of VP in the pressor response produced by microinjection of muscimol (GABA_A agonist) and nipecotic acid (inhibitor of GABA uptake) into the NTS of anesthetized rats (7). Moreover, extensive chemical or electrolytic lesions of the NTS demonstrated that, in addition to VP, the SNS is involved in the pressor response (1).

The mechanisms related to the hypertension observed after baclofen injection into the NTS are not yet clear. Several mechanisms can be considered for this hypertension, the possibility of an increase in sympathetic tonus or release of vasopressin, or both. In this study we sought to determine the cardiovascular effects induced by bilateral microinjections of baclofen into the NTS of conscious rats and the mechanisms involved in these effects.

	METHODS

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Male Wistar rats (300-350 g) were used in the present study. Rats were allowed free access to food and water. All experimental protocols were approved by the Institutional Ethical Committee.

Rats were anesthetized with pentobarbital sodium (40 mg/kg ip; Sigma, St. Louis, MO) and placed in a stereotaxic apparatus (model 960; David Kopf, Tujunga, CA). Bilateral guide cannulas were implanted in the direction of the NTS in accordance with a previously described technique (8). In brief, a small window was opened caudal to lambda, through which a pair of 15-mm-long stainless steel guide cannulas (22 gauge) were introduced in a perpendicular way 14.5 mm caudal to the bregma, 0.5 mm lateral to the midline, and 5.7 mm below the skull surface of the bregma. The bottom of the guide cannulas was placed in the cerebellum 1.0 mm above the dorsal surface of the brain stem. Guide cannulas were fixed to the skull with screws and methacrylate and then closed with an occluder until the beginning of the experiment. The needle (33 gauge) used for microinjection into the NTS was 2.5 mm longer than the guide cannulas and was connected with a polyethylene-10 (PE-10) tubing to a 1-µl syringe (Hamilton, Reno, NV). Five days after placement of the NTS cannulas, rats were anesthetized with halothane (Halocarbon), and PE-50 catheters (Clay Adams, Parsippany, NJ) were introduced through the femoral artery and vein to measure pulsatile arterial pressure (PAP) and heart rate (HR) and for drug administration, respectively. Both catheters were tunneled subcutaneously and exteriorized through the back of the neck. PAP and mean arterial pressure (MAP) were determined with a pressure transducer (Statham P23Db) connected to a low-level DC preamplifier in a polygraph (Grass model 7D). HR was derived from arterial pulse wave by a cardiotachometer. Animals were permitted a 24-h period to recover before the experiments.

First protocol. The dose-related effects of baclofen (10, 50, 100, or 1,000 pmol/100 nl; Sigma) on MAP and HR were studied. In this protocol MAP and HR were measured in awake rats immediately before and 15 min after microinjections into the NTS. Different groups of rats were used for each dose. All NTS sites were previously characterized by L-glutamate microinjection (50 mM).

Second protocol. MAP and HR were determined immediately before and 15 min after bilateral microinjections of the GABA_B receptor agonist baclofen (100 or 1,000 pmol/100 nl) into the NTS. Fifteen minutes after baclofen, an ₁-adrenergic antagonist (prazosin, 1 mg/kg iv) and a VP antagonist {1--mercapto-,-cyclopentamethylene propionic acid 2-[0-(methyl) tyrosine] arginine vasopressin; Manning compound, 10 µg/kg iv} that were administrated with a 15-min time interval between drugs. A control group of animals was maintained to verify the effect of the vasopressin antagonist on basal MAP and HR. Bilateral NTS sites were characterized by L-glutamate microinjection (50 mM). After that, vehicle (saline 0.9%) was microinjected bilaterally into the NTS, followed by intravenous injection of the VP antagonist (10 µg/kg).

Third protocol. MAP and HR were determined immediately before and 15 min after bilateral microinjections of the GABA_B receptor agonist baclofen (100 or 1,000 pmol/100 nl) into the NTS. After 15 min, the VP antagonist (Manning compound, 10 µg/kg iv), followed by the ₁-adrenergic antagonist (prazosin, 1 mg/kg iv), were administrated with a 15-min time interval between drugs.

View larger version (66K): [in this window] [in a new window]   Fig. 1.   Photomicrography and schematic representation of a coronal section of the brain stem showing typical bilateral microinjection sites in the nucleus tractus solitarii (NTS). Arrows indicate center of microinjections; DVN, dorsal motor nucleus of vagus; XII, hypoglossal nucleus.

	RESULTS

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Effects of bilateral microinjections of baclofen into NTS on MAP and HR. Bilateral microinjection of baclofen into the NTS of conscious rats (10, 50, or 100 pmol/100 nl) produced dose-dependent increases in MAP [9 ± 5 (n = 5), 18 ± 3 (n = 7), and 37 ± 4 (n = 7) mmHg, respectively; P < 0.05, Fig. 2] but had no effect in HR (Table 1). Bilateral microinjection of 1,000 pmol/100 nl baclofen increased both MAP (66 ± 3 mmHg, n = 8; P < 0.05) and HR (84 ± 5 beats/min, n = 8; P < 0.05) (Table 1; Fig. 2). The pressor response induced by these treatments was sustained for at least 1 h, except in the group microinjected with 1,000 pmol/100 nl baclofen, which animals developed respiratory depression after 30 min.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Changes in mean arterial pressure (MAP) elicited by bilateral microinjection of increasing doses of baclofen (10, 50, 100, and 1,000 pmol/100 nl; n = 5, 7, 7, and 8, respectively) into the NTS of conscious rats. The effect was dose dependent (P < 0.05). Values were measured 15 min after microinjections.

Effects of administration of prazosin and Manning compound on MAP and HR after bilateral microinjections of baclofen into NTS of conscious rats. Bilateral microinjection of baclofen (100 pmol/100 nl) increased MAP from 103 ± 6 to 152 ± 6 mmHg and HR from 345 ± 17 to 388 ± 13 beats/min (n = 6; P < 0.05). Fifteen minutes after NTS microinjection, intravenous injection of the ₁-adrenergic receptor antagonist prazosin (1 mg/kg) lowered MAP from 152 ± 6 to 91 ± 6 mmHg and increased HR to 483 ± 3 beats/min (P < 0.05). Fifteen minutes after prazosin treatment, injection of the vasopressin receptor antagonist Manning compound (10 µg/kg iv) further decreased MAP from 91 ± 6 to 83 ± 8 mmHg (n = 6; P < 0.05). However, no additional change was observed in HR (see Fig. 4A). Figure 3A shows a representative tracing from an animal of this group.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effects of bilateral microinjection of 100 pmol/100 nl (A) and 1,000 pmol/100 nl (B) baclofen into the NTS, followed by intravenous injection of prazosin (1 mg/kg) and vasopressin antagonist (AVP ant; Manning compound, 10 µg/kg) on pulsatile arterial pressure (PAP), MAP, and heart rate [HR, beats/min (bpm)]. Interval of time between treatments was 15 min.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Effects of bilateral microinjection of 100 pmol/100 nl (A; n = 6) and 1,000 pmol/100 nl (B; n = 6) baclofen into the NTS and intravenous injection of prazosin (1 mg/kg) and vasopressin antagonist (Manning compound, 10 µg/kg) on MAP and HR. *Different compared with control (P < 0.05); different from baclofen (P < 0.05).

MAP and HR effects of sequential systemic administration of Manning compound and prazosin after bilateral microinjection of baclofen into NTS of conscious rats. Bilateral microinjection of baclofen (100 pmol/100 nl) increased MAP from 111 ± 3 to 163 ± 7 mmHg (n = 6; P < 0.05); however, HR did not change (358 ± 10 vs. 373 ± 13 beats/min; see Fig. 6A). Fifteen minutes after baclofen microinjection, rats received Manning compound (10 µg/kg iv), which lowered MAP from 163 ± 7 to 148 ± 6 mmHg (P < 0.05). Fifteen minutes after Manning compound treatment, prazosin (1 mg/kg iv) decreased MAP from 148 ± 6 to 68 ± 1 mmHg and elicited a significant increase in HR from 375 ± 13 to 468 ± 10 beats/min (n = 6, P < 0.05; see Fig. 6A). Figure 5A shows a representative tracing from an animal of this group.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effects of bilateral microinjection of 100 pmol/100 nl (A) and 1,000 pmol/100 nl (B) baclofen into the NTS, followed by intravenous injection of prazosin (1 mg/kg) and vasopressin antagonist (Manning compound, 10 µg/kg) on PAP, MAP, and HR. Time between treatments was 15 min.

View larger version (49K): [in this window] [in a new window]   Fig. 6.   Effects of bilateral microinjection of 100 pmol/100 nl (A; n = 6) and 1,000 pmol/100 nl (B; n = 6) baclofen into the NTS and intravenous injection of prazosin (1 mg/kg) and vasopressin antagonist (Manning compound, 10 µg/kg) on MAP and HR. *Different compared with control (P < 0.05); different from baclofen (P < 0.05); #different from baclofen + prazosin (P < 0.05).

	DISCUSSION

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The present study shows that the GABA_B receptor agonist baclofen microinjected into the NTS increases arterial pressure in conscious rats. Other studies (14, 15) showed that GABA microinjections into the NTS increase blood pressure in anesthetized rats. These studies also reported that microinjections of the GABA_A antagonist bicuculine produce hypotension in anesthetized rats (14, 15).

Bilateral microinjection of muscimol (GABA_A agonist) (7), or electrolytic and chemical lesions (1) or bilateral microinjections of baclofen into the NTS (9), elicits tachycardia in anesthetized animals. Our study demonstrated that a GABA_B-selective agonist also elicits a pressor response in conscious rats. Only animals that received microinjections of baclofen at the highest dose (1,000 pmol/100 nl) displayed tachycardia, whereas the other doses did not demonstrate consistent changes in HR. An exception was that, in the second protocol, 100 pmol/100 nl baclofen produced a significant tachycardia. This result could be due to the highest variance observed in HR basal level. We would expect that such an effect could result from baroreflex inhibition in addition to an increased sympathetic tonus elicited by baclofen. The fact that the prazosin-induced fall in blood pressure in rats pretreated with the highest dose of baclofen was not accompanied by tachycardia suggests that impairment of baroreflex activity may be involved in these differences in HR responses.

The dose-related increase in MAP after bilateral microinjection of baclofen into the NTS indicates that the inhibition of this system by microinjection of baclofen results in a pressor response. One of the mechanisms that produce the pressure response is the release of VP (7, 13). Studies in anesthetized rats (7) demonstrated an increase in plasma VP during the increase of arterial pressure elicited by microinjection of muscimol into the NTS. This hypertension elicited by muscimol was reversed by intravenous injection of a vasopressin antagonist (7). Chemical or electrolytic lesions of the NTS produce hypertension that cannot be abolished solely by vasopressin antagonists but can be abolished by lesions or application of glycine into the rostroventrolateral medulla, suggesting that bilateral lesions of the NTS increased sympathetic tonus and also the release of vasopressin (1). In the ongoing study, baclofen-induced hypertension was reversed by peripheral blockade of ₁-adrenoreceptors and additional systemic administration of a vasopressin antagonist further lowered MAP below control levels.

To test the hypothesis that both the release of VP and an increase in sympathetic tonus occur simultaneously when baclofen is microinjected into the NTS rather than the administration of prazosin lowering AP and stimulating VP release, a group of animals received an intravenous injection of VP antagonist first. This produced a significant but small decrease in the baclofen-induced hypertension. The remaining hypertension was then completely reversed by intravenous injection of prazosin. Besides demonstrating an involvement of VP in baclofen-induced hypertension, these data suggest that baclofen-induced hypertension is more dependent on sympathetic tonus activation.

The possible mechanisms for mediation of the hypertension elicited by baclofen within the NTS are not completely understood; however, there is evidence that GABA_B receptors within the NTS modulate arterial baroreflexes (5, 27, 30). A recent study (33) suggested a presynaptic mechanism contributing to the inhibition of aortic depressor nerve inputs by GABA_B receptors within the NTS. This same study also observed that monosynaptic neurons in the NTS were less sensitive to GABA_B-mediated inhibition than polysynaptic neurons (33). However, the aortic depressor nerve-evoked discharge of some NTS neurons was insensitive to baclofen, suggesting the existence of subpopulations of monosynaptic neurons with differing sensitivities to GABA_B inhibition (33). In our present findings, we observed dose-dependent pressor responses to microinjection of baclofen. Doses of 100 pmol/100 nl elicited a pressor response without HR changes. In a subsequent protocol, peripheral blockade of ₁-adrenoreceptors virtually eliminated baclofen (100 pmol/100 nl) induced hypertension and resulted in a possible reflex tachycardia (see Fig. 3). Alternatively, dose-dependent effects of baclofen have been described in in vitro studies of NTS (5). Low doses of baclofen produced presynaptic inhibitory effects, whereas higher doses produced a mixture of pre- and postsynaptic inhibition. In this model, we might speculate that, with a higher dose of baclofen, we would observe enhanced hypertension and significant tachycardia. Treatment in those animals with prazosin and VP receptor antagonist completely reversed hypertension and resulted in no change in HR. Considering that we are using microinjections, we may speculate that different doses of baclofen could be altering the activity of different subpopulations of neurons in the NTS.

These data indicate the involvement of the two systems studied after the inhibition of the NTS by bilateral microinjections of baclofen in conscious rats: 1) the SNS and 2) the release of VP. According to our results, the main system responsible for the hypertension that followed NTS inhibition is the SNS once systemic administration of prazosin was able to completely reverse the hypertension.

In conclusion, our results show that the hypertension induced by baclofen microinjected into the NTS of conscious rats was produced by increases in sympathetic tonus and may involve the release of VP. These data suggest that NTS neurons exert a tonic inhibitory influence on the SNS and possible mechanisms related to VP release.