The nucleus ambiguus region participates in arterial pressure regulation

Neuroscience Letters, 135 (1992) 91-94 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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The nucleus ambiguus region participates in arterial pressure regulation Benedito H. M a c h a d o 2 and Michael J. Brody ~'* IDepartment of Pharmacology and Cardiovascular Center, College of Medicine, University of Iowa, Iowa City, IA 52242 (U.S.A.) and 2Department of Physiology, School of Medicine of Riheirdo Preto, University of Sdo Paulo, SP 14049 (Brazil) (Received 24 August 1991; Revised version received 17 October 1991; Accepted 21 October 1991)

Key words: Heart rate; Lidocaine; Baroreceptor reflex; Parasympathetic tone; Brainstem; Electrical stimulation; Cardiovascular regulation Bilateral microinjections of lidocaine into the nucleus ambiguus produced a significant increase of mean arterial pressure. Electrical stimulation of the nucleus ambiguus produced pressor and bradycardic responses, which are abolished by injection of lidocaine into the rostral ventrolateral medulla. These data indicate that the pressor and bradycardic responses produced by electrical stimulation of the nucleus ambiguus depend on the integrity of the rostral ventrolateral medulla. We conclude that the nucleus ambiguus region is involved in the central neural control of arterial pressure through a possible neuronal inhibitory projection to the rostral ventrolateral medulla.

The nucleus ambiguus (NA) is a secondary projection site of the baroreceptor reflex arch which receives projections from the nucleus tractus solitarii [10]. A ventral division of the NA that extends along the entire length of the medulla contains parasympathetic motoneurons which innervate thoracic and abdominal viscera [1, 2, 9]. In previous studies we showed the important role of the NA in the regulation of heart rate and arterial pressure [3-6]. Chemical and electrolytic lesions of the NA facilitate the development of hypertension in rats with sinoaortic deafferentation [3, 4] while chemical and electrical stimulation of this area produce bradycardia and hypertension, suggesting that the NA may play an inhibitory role on the rostral ventrolateral medulla (RVLM). In addition, electrical stimulation of this area shows a selective change in vascular resistance when compared with stimulation of the RVLM [6]. Evidences obtained in studies from our laboratory suggests a possible role for the NA in the modulation of sympathetic outflow, possibly through inhibitory projections to the neighboring region of the RVLM. The objectives of the present study were (a) to observe the changes in mean arterial pressure after microinjection of lidocaine into the NA to determine the possible * Deceased 3 December 1990.

Correspondence: B.H. Machado, Department of Physiology, School of Medicine of Ribeir~lo Preto, University of S~fo Paulo, SP 14049, Sao Paulo, Brazil. Fax: (55) (16) 633 1586.

inhibitory role of this region on the RVLM and (b) to evaluate the functional interrelationships between N A and RVLM using a combination of electrical stimulation and lidocaine microinjection. Male Sprague-Dawley rats (Biolab, St. Paul, MN) weighing 300-350 g were used in the present study. Direct arterial pressure was measured by means of a cannula (PE-10 connected to PE-50, Clay Adams, Parsippany, N J) inserted into the abdominal aorta through the femoral artery. All of these procedures and the experimental protocols were performed under urethane anesthesia (1 g/kg, i.p.). The arterial line was connected to a Century CP-01 pressure transducer (Century, Inglewood, CA) and to a Beckman recorder (Model R611, Beckman Instruments, Fullerton, CA) and the heart rate was derived from the arterial pulse with a Beckman 9857-B cardiotachometer. The rats were placed in a stereotaxic apparatus (David Kopf, Tujunga, CA) and guide cannula (26 gauge) or electrodes were placed in the N A and RVLM according to the coordinates of Paxinos and Watson [8]. In a first protocol (n=9), microinjections of 100 nl of lidocaine (4%) during 15 s were made bilaterally into NA through a 33 gauge needle connected to a 1/11 microsyringe (Hamilton, Reno, NV). Twenty minutes later when blood pressure returned to control values, the guide cannula was moved ventrally and lidocaine was microinjected into the RVLM. Twenty minutes prior to the first microinjection of lidocaine into NA or RVLM, 100 nl of saline was injected as a volume control and in both areas pro-

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duced negligible effects on mean arterial pressure (MAP) and heart rate (HR). The control values for MAP and HR were those immediately previous to microinjection of lidocaine into NA or RVLM. In a second protocol (n=6), unilateral electrical stimulation of NA or RVLM was performed in the presence of 100 nl of saline (control) or 100 nl of lidocaine (4%) into the other region (1, 5, 10 and 20 min). If the first microinjection was performed into RVLM and NA electrically stimulated, the second microinjection was performed into NA followed by electrical stimulation of RVLM. The second microinjection was performed when the effect of the first microinjection of lidocaine was over, i.e., when the response to electrical stimulation of NA or RVLM was back to control levels. In this protocol, different sequence of microinjection of lidocaine (NA or RVLM first) produced similar responses. Electrical stimulations in NA and RVLM were performed with monopolar tungsten electrodes (tip diameter 50 ¢tm). The duration of each pulse was 0.5 ms, the frequency was fixed at 100 Hz, the intensity at 200 HA and the stimulus was maintained for 10 s. At the end of the experiments+ 100 nl of green-dye was injected uni- (second protocol) or bilaterally (first protocol) into the NA and RVLM and the rats were killed by intracardiac perfusion with saline followed by 10% buffered formalin. The brains were removed and prepared to be stained by the Cresyl violet method. The extent of dye diffusion was mapped and an overlapping analysis was used to determine the common site of microinjection for all rats used. Fig. 1 summarizes the bilateral sites of green-dye microinjections into NA and RVLM of 9 rats used in the first protocol. A two-way analysis of variance with repeated meas-

Irnm Fig. 1. Line drawing of a coronal section of the medulla 3.8 m m posterior to the interaural line shows bilateral overlap sites of green-dye stained areas of the nucleus ambiguus and rostral ventrolateral medulla of 9 rats used in the first protocol (n=9).

urements was used to compare the changes in HR and MAP observed in both protocols. Bilateral microinjection of lidocaine into the NA produced an increase in MAP following the injection (+13%). After the peak increase at the 10th min (116+2 vs 103+4 mmHg), the MAP gradually returned to control values over the following 20 min (104+3 vs 103 +4 mmHg). No significant changes in heart rate were observed after microinjection into the NA while bilateral microinjection of lidocaine into the RVLM, 0.6 mm below the NA, produced a fall in MAP (-30%) immediately after microinjection, with a return to control values within 20 min (Fig. 2). The upper panel in Fig. 3 shows that electrical stimulation in the NA or RVLM under control conditions produced significant pressor responses. When electrical stimulation of the NA was performed after lidocaine injection into the RVLM, the pressor response elicited was significantly reduced (+66+4 vs +10+5 mmHg). However, when the RVLM was electrically stimulated after lidocaine injection into the NA, only a minor reduction in the pressor response was observed (+73+5 vs +55+_7 mmHg). The bottom panel in Fig. 3 shows that electrical stimulation in NA and RYLM produces significant bradycardia (-242+33 and -260+_25 bpm, resp.). When electrical stimulation of the NA was performed after lidocaine injection into the RVLM, bradycardia was abolished (-242+_33 vs 0+_6 bpm). In contrast, when the RVLM was stimulated after lidocaine microinjection into the NA, the bradycardic response was significantly reduced (- 260+_25 vs -40+_3 bpm). Microinjection of the local anesthetic lidocaine was chosen as a method to selectively inhibit small regions of brainstem for a short period of time. In relation to the spread of lidocaine, studies by Sandkuhler and Gebhart [11] showed that injections of 200 nl inactivates a sphere of parenchyma minor than 0.5 mm of radius. In the present study we used a volume of 100 nl and apparently no spreading from RVLM to NA or vice versa occurred considering that microinjection of green-dye in the same volume does not presented overlapping in the spread (Fig. 1). The region of the NA microinjected with lidocaine or electrically stimulated (l 500/lm rostral to the obex) corresponds to the ventrolateral responsive locus described by Nosaka et al. [7]. The area studied also corresponds to that investigated by Stuesse and Fish [10], who showed that cardioinhibitory cells in the ventral medulla were found in the rostral NA. Bilateral microinjection of lidocaine into the NA produces a significant increase in MAP but no changes in HR. When lidocaine was microinjected into the RVLM, an expected significant fall in pressure was observed, while no significant changes in

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Fig. 2. Upper panel: mean arterial pressure (MAP) control (c) and I, 5, 10 and 20 min after bilateral microinjection of lidocaine into the nucleus ambiguus (NA) and rostral ventrolateral medulla (RVLM). Lower panel: heart rate (HR) control and 1, 5, 10 and 20 min after microinjection of lidocaine into the NA and RVLM. "P<0.05 compared to control values (n=9).

HR were observed. In relation to the increase of MAP after injection of lidocaine into the NA, we have shown in previous studies [3, 4] that chemical and electrolytical lesions of the NA facilitate the development of hypertension in rats with sino-aortic deafferentation. The results of this and our previous studies [3, 4] indicate that NA may play an important inhibitory role on the sympathetic efferent tone originating in the RVLM. Electrical stimulation of both the NA and RVLM produces similar pressor responses and bradycardia. When electrical stimulation of the NA was performed after microinjection of lidocaine into the RVLM, the pressor response was reduced from +66_+4 to +10_+5 mmHg and the bradycardia was abolished. On the other hand, when electrical stimulation of the RVLM was performed after microinjection of lidocaine into the NA, the pressor response was reduced from +73_+5 to +55_+7 mmHg, while the bradycardia was significantly attenuated, but not abolished, from -260+25 to -40_+3 bpm. These results indicate that the pressor and bradycardic responses elicited by electrical stimulation of the NA depend on the integrity of the RVLM. In this case lidocaine may be

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Fig. 3. Upper panel: changes in mean arterial pressure (,4 MAP) in response to electrical stimulation of the nucleus ambiguus (NA stim.) or rostral ventrolateral medulla (RVLM stim.) under control conditions (c), i.e., electrical stimulation after microinjection of saline. Electrical stimulation of the NA was repeated l, 5, l0 and 20 rain after microinjection of lidocaine into the RVLM. Electrical stimulation of the RVLM was repeated l, 5, 10 and 20 min after microinjection of lidocaine into the NA. Lower panel: changes in heart rate (A HR) accompanying changes in MAP during electrical stimulation of the NA and RVLM, as described in the upper panel. °P<0.05 comparing the responses observed in the two protocols studied (n=6).

blocking the fibers of passage from NA passing through the RVLM. When NA was blocked with lidocaine, the pressor response elicited by electrical stimulation of the RVLM was maintained with a slight bradycardia, indicating that this reduction in heart rate could be due to activation of cell bodies of preganglionic parasympathetic fibers in the RVLM [12] or fibers of passage from the NA. The present study indicates that the pressor and bradycardic responses produced by electrical stimulation of the NA depend on the integrity of the RVLM. We conclude that the NA has important interrelationships with the RVLM and that NA plays an important role in the central neural control of arterial pressure through a possible inhibitory effect on the RVLM.

94 1 Bieger, D. and Hopkins, D.A., Viscerotopic representation of the upper alimentary tract in the medulla oblongata in the rat: the nucleus ambiguus, J. Comp. Neurol., 211 (1987) 54(~562. 2 Kalia, M. and Sullivan, M.J., Brain stem projections of sensory and motor components of the vagus nerve in the rat, J. Comp. Neurol., 211 (1982) 248 264. 3 Machado, B.H. and Brody, M.J., Effect of nucleus ambiguus lesion on the development of neurogenic hypertension, Hypertension, 11 (1988) 1-135-I-138. 4 Machado, B.H. and Brody, M.J., Role of nucleus ambiguus in regulation of heart rate and arterial pressure, Hypertension, 11 (1988) 602-607. 5 Machado, B.H. and Brody, M.J., Contribution of neurogenic mechanisms to control of intrinsic heart rate, Am. J. Physiol., 256 (Reg. lntegr. Comp. Physiol. 25) (1989) R231 R235. 6 Machado, B.H. and Brody, M.J., Mechanisms of pressor response produced by stimulation of nucleus ambiguus, Am. J. Physiol., 259 (Reg. Integr. Comp. Physiol. 28) (1990) R955-R962. 7 Nosaka, S., Yamamoto, T. and Yasunaga, K., Localization of vagal cardio-inhibitory preganglionic neurons within rat brain stem, J. Comp. Neurol., 186 (1979) 79 92.

8 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 9 Stuesse, S.L., Origins of cardiac vagal preganglionic fibers: a retrograde transport study, Brain Res., 236 (1982) 15-25. 10 Stuesse, S.L. and Fish, S.E., Projections to the cardio-inhibitory region of the nucleus ambiguus of rat, J. Comp. Neurol, 229 (1984) 271-278. 11 Sandkuhler, J. and Gebhart, G.F., Relative contributions of the nucleus raphe magnus and adjacent medullary reticular formation to the inhibition by stimulation in the periaqueductal gray of a spinal nociceptive reflex in the pentobarbithal-anesthetized rat, Brain Res., 305: 77-87, 1984. 12 Varner, K.J., Vasquez, E.C., Lewis, S.J., Machado, B.H., Grosskreutz, C.L., Simon, J.S. and Brody, M.J., Regulation of autonomic cardiovascular function by the rostral ventromedial medulla. In J. Kuno and J. Ciriello (Eds.), Central Neural Mechanisms in Cardiovascular Regulation, Birkhauser Boston~ Inc., Massachusetts, 1991. pp. 29-36.