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Hypotensive effects of lesions of the rostral ventrolateral medulla in rats are anesthetic-dependent
Journal of the Autonomic Nervous System, 22 (1988) 181-187 Elsevier
181
JAN 00804
Research Papers
Hypotensive effects of lesions of the rostral ventrolateral medulla in rats are anesthetic-dependent K a r e n L. C o c h r a n e , R. Allan Buchholz, J o h n W. H u b b a r d , T. K e n t K e e t o n a n d M a r c A. N a t h a n Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78284-7764 (U.S.A.) (Received 30 October 1987) (Accepted 25 January 1988)
Key words: Rostral ventrolateral medulla; Arterial pressure; Vasomotor center; Urethane; a-Chloralose; Sodium pentobarbital
Abstract These studies were designed to determine if the hypotensive effects of bilateral electrolytic lesions of the rostral ventrolateral medulla are dependent on the type of anesthetic agent used. The lesions caused an immediate fall in mean arterial pressure (MAP) in rats anesthetized with urethane, a-chloralose or sodium pentobarbital. At 30 min after placement of the lesions, severe hypotension (MAP = 54 + 5 mm Hg) persisted in the rats anesthetized with urethane. However, 30 min after placement of the lesions, the MAP of rats anesthetized with a-chloralose or sodium pentobarbital was 87 + 9 mm Hg and 99 + 10 mm Hg, respectively. Subsequent transection of the cervical spinal cord produced a much greater decrease in MAP in rats anesthetized with a-chloralose and sodium pentobarbital as compared to rats anesthetized with urethane. Heart rate was significantly lower after placement of the lesions in all 3 groups. We conclude that the magnitude of the hypotensive effect caused by placement of lesions in the rostral ventrolateral medulla is anesthetic-dependent and that the rostral ventrolateral medulla is not the only area of the central nervous system capable of maintaining vasomotor tone.
Introduction A number of investigators have argued that the rostral ventrolateral medulla (RVLM) is the sole area responsible for the maintenance of sympathetic vasomotor tone. This conclusion is based on the observation that placement of electrolytic lesions in the RVLM of anesthetized rabbits or rats caused mean arterial pressure (MAP) to fall
Correspondence: M.A. Nathan, Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.
to a value similar to that observed after spinal transection [5-7]. In a preliminary effort to assess the cardiovascular effects of lesions of the RVLM in rats after recovery from anesthesia, we placed bilateral electrolytic lesions in the RVLM of anesthetized rats. We found that MAP did not fall to the values previously reported [5-7]. In seeking an explanation for these contradictory results, we noted that the previous investigators had selected urethane as their anesthetic agent while we used sodium pentobarbital. The purpose of the present study was to compare the effects of different anesthetic agents on MAP and heart rate (HR) in rats after RVLM lesions.
0165-1838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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Materials and Methods Experiments were performed using male L o n g - E v a n s rats (Blue Spruce Farms) weighing 250-400 g. Three groups of rats were anesthetized with either urethane (n = 12, 1.7 g / k g , i.p.), achloralose (n = 9, 200 m g / k g , i.p.), or sodium pentobarbital (n --9, 50 m g / k g , i.p.). Supplemental doses of the anesthetic agents were given if pressor responses were seen after pinching the tail. Cannulae were inserted into a femoral artery and vein and threaded into the abdominal aorta and vena cava, respectively, for the recording of pulsatile arterial pressure, MAP and HR, and for the administration of drugs [2]. The rats were placed in a stereotaxic apparatus (Kopf), and the head was positioned so that bregma and lambda lay in the same horizontal plane. The rats were paralyzed with o-tubocurarine chloride (0.3 m g / k g , i.v.) and mechanically ventilated (Harvard Rodent Respirator) with 100% 02 (80 times/rain with a 2.5 ml tidal volume). Supplemental doses of o-tubocurarine were given if spontaneous ventilation was observed. A concentric platinum-iridium electrode (outer shaft = 0.25 mm, o.d.; inner shaft = 0.1 mm, o.d.; 0.25 m m tip exposure on both shafts; 0.75 m m intra-tip distance) was lowered into the R V L M at 3 locations caudal to bregma ( - 11.8 nun, - 11.3 mm, - 12.3 mm). Penetrations were made 2.0 m m lateral to each side of the midline at each level, and the tip of the electrode was positioned at 0.2 m m above the ventral surface of the brainstem. The protocol for systematic exploration of cardiovascular responses to stimulation of the R V L M was as follows. First, sympathetic vasomotor tone was evaluated by bolus injection of the short-acting ganglionic blocker trimethaphan camsylate (800 m g / k g , i.v.). Second, the R V L M was electrically stimulated at 11.8 m m caudal to bregma (100 Hz, 0.1 ms pulse width, 50/~A), as a means of eliciting a pressor response and tachycardia, and then the threshold current was determined (7-20 /~A). Third, after M A P returned to the baseline value, a lesion was made by passing a DC anodal current (1.0 m A for 30 s) between the inner shaft of the electrode and a clip
attached to one edge of the skin incision. Fourth, the site was restimulated at 4 × threshold current, and, if a cardiovascular response was still present, additional lesions were made until the pressor response was abolished. These procedures of stimulation and placement of lesions were repeated on the opposite side of the brainstem. Then, the entire protocol was repeated at each of the other two locations. However, at these locations lesions were made only if a pressor response was elicited. Trimethaphan was readministered 10 min after placement of the final lesion. The spinal cord was sectioned by aspiration at the level of the first cervical vertebra 30 min later. A third injection of trimethaphan was then given. The experiment ended with intracardiac perfusion of saline and 10% buffered formalin, and the brain was removed. Frozen sections (40 #m) were cut serially, mounted, and stained with Cresyl violet. Microscopic examination of the sections was performed to localize and determine the extent of the lesions. Coronal sections were selected at 120-/~m intervals through the anterior-posterior extent of the RVLM. The sections were viewed with a projecting microscope. The maximal medial-lateral extent and dorsal-ventral extent of the lesions on each side of the brainstem were measured, and the area of damage at each anteriorposterior level was calculated. A digitizing tablet was used to trace a representative sagittal section taken from the brain atlas of Paxinos and Watson [8]. The anterior-posterior extent of each lesion from each rat was then plotted on the sagittal section. The degree of overlap of each lesion identified the area of damage common to all of the rats. The effect of the lesions on M A P and H R were evaluated for statistical significance using a twoway analysis of variance followed by post hoc analysis of significant main effects using the Newm a n - K e u l ' s procedure [14]. Paired Student's ttests were used to assess the effects of trimethaphan on M A P and HR. The extent of the lesions was also analyzed with a two-way analysis of variance. Differences were considered significant at P < 0.05.
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Results Bilateral destruction of the R V L M s u b s t a n tially decreased M A P i n all groups within 10 m i n after p l a c e m e n t of the last lesion (Table IA). H o w ever, the hypotensive effect of the lesions was largest in the u r e t h a n e group ( - 58 ± 1 m m Hg), a n d the absolute level of M A P i n this group was significantly lower t h a n that observed i n the other two groups. The decrease of M A P i n the s o d i u m p e n t o b a r b i t a l group was also relatively large ( - 51 ± 1 m m Hg), b u t the prelesion M A P was higher i n this group as c o m p a r e d to the other groups. T h e r e d u c t i o n in M A P of the a-chloralose group was less ( - 3 4 ± 1 m m Hg) a n d reached a n absolute level similar to that of the s o d i u m p e n t o b a r b i t a l group. Thus, the prelesion M A P did n o t d e t e r m i n e the m a g n i t u d e of the hypotensive effect of lesions of the R V L M .
T h e i m m e d i a t e h y p o t e n s i v e effect of the lesions i n the a-chloralose a n d s o d i u m p e n t o b a r b i t a l groups d i m i n i s h e d over time (Table IA). By 30 rain after p l a c e m e n t of the lesions, the M A P of the a-chloralose group rose to a level that was n o t significantly less ( - 14 + 3 m m Hg) t h a n the prelesion baseline value, a n d i n the s o d i u m p e n t o b a r b i t a l group, the M A P reached a level that was 22 + 5 m m H g less t h a n the baseline M A P . I n sharp contrast, the effect of the lesions o n the u r e t h a n e g r o u p was m u c h more e n d u r i n g with the M A P r e m a i n i n g at 51 + 1 m m H g below the prelesion baseline value. I n t e r r u p t i o n of d e s c e n d i n g s y m p a t h e t i c p a t h w a y s b y spinal t r a n s e c t i o n caused a further r e d u c t i o n i n the M A P i n all the groups (Table IA). T h e r e d u c t i o n i n M A P was greatest i n the s o d i u m p e n t o b a r b i t a l ( - 51 + 6 m m Hg) a n d a-chloralose ( - 41 + 7 m m Hg) groups b u t consid-
TABLE I Cardiovascular effects of lesions of the rostral ventrolateral medulla Group
Prelesion baseline
A. Mean Arterial Pressure (mm Hg 5: S.E.M.)
Urethane Chloralose Pentobarbital
106 + 4 101 5 : 6 121 5 : 6 *
B. Heart rate (bpm 5: S . E . M . )
Urethane Chloralose Pentobarbital
431 + 8 360 5:20 * 417 + 11
10 mm Postlesion
48 + 3 ..t 67 5 : 5 t 70 5 : 6 t 366 5:13 t 331 5:20 t 353 5 : 9 t
30 min Postlesion
54 5 : 5 *'* 87 5 : 9 t 99 5:10 t,* 366 5:13 * 301 5:17 * *'* 324 -t- 14 t,*
Postspinal transection
36 5 : 2 .,t,* 46 5 : 2 t,* 48 -I- 5 t,* 344 + 13 *'* 300 + 12 * 293 5:15 t,*
* Significantlydifferent from the other two groups; * * significantly different from the urethane group; t significantly different from the preceding recording period; * significantly different from the prelesion baseline recording period; Significance level, P < 0.05. TABLE II Cardiovascular effects of trimethaphan (ram Hg or bpm 5: S.E.M.) Group
A. Prelesion trimethaphan administration
Urethane Chloralose Pentobarbital
B. Postlesion (10 rain) trimethaphan administration
Urethane Chloralose Pentobarbital
Mean arterial pressure
Heart rate
Before
Change
Before
Change
116 + 4 117 + 5 131 + 6
- 5 2 + 5 *,t -32 + 4 * -40 + 5 *
426 + 13 405 -t- 14 439 -I- 12
-43 5- 4 * - 2 2 5- 4 *'* -34 + 5 *
- 12 5:2 * - 15 5:3 * -16 5:4 *
368 5:13 328 5:18 343 + 10
- 31 5- 4 * - 2 2 5:4 * -21 5:7 *
51 -t- 3 * * 75 5:6 82 5:9
* Significantly different from predrug; * * significantly different from chloralose and pentobarbital groups; t significantly different from chloralose group; * significantly different from urethane and pentobarbital groups. Significance level, P < 0.05.
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erably less in the urethane group ( - 1 8 ± 3 mm Hg). However, after spinal transection the absolute level of MAP in the urethane group was
still significantly lower than the MAP of the achloralose or sodium pentobarbital groups. Bradycardia was evident in all the groups 10 min after placement of the lesions, and the magnitude of the bradycardia appeared to be related to the prelesion baseline H R of each group (Table IB). The H R of the a-chloralose group fell the least ( - 29 ___1 bpm) whereas larger decreases were observed in the urethane and sodium pentobarbital groups ( - 6 5 + 5 bpm and - 6 4 ± 2 bpm, respectively). In contrast to MAP, H R showed no signs of recovery in any group at 30 rain after placement of the lesions (Table IB). The H R of the a-chloralose and sodium pentobarbital groups continued to decline ( - 30 ± 2 bpm, - 29 + 5 bpm, respectively), and the H R of the urethane group remained suppressed. Spinal cord transection caused a further reduction in H R in the urethane group ( - 2 2 + 1 bpm) and the sodium pentobarbital group ( - 3 1 ± 1 bpm), but no change was observed in the a-chloralose group (Table IB).
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Fig. 1. Lesion sites In the urethane (top panel), a-chloralose (middle panel) and sodium pentobarbital (bottom panel) group. The hemisections on the right of each figure shows a typical lesion in the R V L M located 11.8 m m caudal to bregma. The line drawings on the left summarize the location of the lesions in the individual rats. The dotted portion of each line drawing indicates the area damaged in all the rats of a particular group. The dashed line shows the boundary of the largest lesions that were made in each group. NTS, nucleus tractus solitarius, Amb, nucleus ambiguus; sp5, spinal trigeminal tract; io, inferior olivary nucleus. Note: the hole near the dorsal surface of the section shown in the top panel is a marking hole.
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P o s t e r i o r to B r e g m o (mr'n) Fig. 2. Sagittal section of lower brainstem and overlying structures showing the lesion sites on the left side of the brain in all rats of the urethane, a-chloralose and sodium pentobarbital groups. The shaded area shows the maximal a m o u n t of overlap of lesion sites (see text for details). The sagittal view is through the center of the R V L M (lateral 1.9 m m from the midline). Amb, nucleus ambiguus; LRt, lateral reticular nucleus; LVe, lateral vestibular nucleus; 7, facial nucleus; NTS, nucleus tractus sol±tar±us.
185
Trimethaphan was used to assess the amount of sympathetic vasomotor tone before and after placement of the RVLM lesions and after spinal cord transection. Trimethaphan significantly decreased the prelesion MAP in all the groups (Table IIA). Trimethaphan caused a small but uniform depression of MAP in all the groups 10 rain after placement of the lesions. At this time, MAP had not recovered in the a-chloralose and sodium pentobarbital groups (Table IIB). Trimethaphan administered after spinal transection caused a slight reduction in the MAP (2-4 mm Hg) of all the groups. Trimethaphan also reduced the prelesion H R in all the groups, (Table IIA). The postlesion bradycardia after trimethaphan was similar in all the 2.000-
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groups (Table IIB). However, trimethaphan variably affected the H R of the groups after spinal cord transection. The reduction in H R was significant in the urethane ( - 3 1 ± 5 bpm) and sodium pentobarbital ( - 1 4 ± 3 bpm) groups, but a bradycardia was inconsistently seen in the ctchloralose group ( - 14 _ 8 bpm). The greatest cross-sectional extent of the lesions in all the groups was found at approximately 11.8 mm posterior to bregma (Fig. 1). The lesions lay between the spinal trigeminal tract and the inferior olive and extended dorsally toward the boundary of the nucleus ambiguus. The RVLM was damaged bilaterally in all of the rats at 11.8 mm posterior to bregma. The area most consistently damaged bilaterally in the majority of the rats (82.7%) lay between 11.3 mm and 12.3 mm posterior to bregma (Fig. 2). The lesions lying between these two levels extended dorsally about 0.8-1 mm from the ventral surface of the RVLM. No statistically significant differences were found in an analysis of the amount of tissue damage on each side of the brainstem within each group (Fig. 3). In addition, no differences in the amount of tissue destroyed were found between the groups.
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Posterior to Bregma (ram) Fig. 3. Line graphs showing the anterior to posterior extent ( m m posterior to Bregma) of damage (ram2-± S.E.M.) to the R V L M on the left (top panel) and right (bottom panel) sides of the brainstem. No statistically significanf differences were found between groups or between sides at any level. The anterior-posterior borders of the lesions varied according to group by 120-240 /~m. However, this a m o u n t of variability was due to a relatively small amount of tissue damage to the posterior R V L M in two rats of the urethane group and to the anterior R V L M in two rats of the sodium pentobarbital group.
The most important finding of our study is that lesions of the RVLM produced only transient hypotension in rats anesthetized with sodium pentobarbital or a-chloralose. This finding is in sharp contrast to the previous reports that severe and persistent hypotension occurs after placement of lesions in the RVLM in animals anesthetized with urethane [5-7]. Although hypotension was observed in all 3 groups 10 min after placement of the lesions, by 30 min postlesion the MAP had recovered in the a-chloralose and sodium pentobarbital groups such that MAP (absolute values of 87 + 9 mm Hg and 99 + 10 mm Hg, respectively) was only 14-18% less than the prelesion values. However, the MAP of the urethane group (absolute value of 54 ± 5 mm Hg) was still 49% below the prelesion value at 30 min after placement of the lesions. Moreover, significant sympathetic vasomotor tone still existed after lesions of the
186 RVLM in the animals anesthetized with a-chloralose and sodium pentobarbital since spinal transection lowered the MAP by 40-50 mm Hg in these two groups. Therefore, the original proposal that the RVLM is the vasomotor center which is responsible for the maintenance of sympathetic vasomotor tone and basal arterial pressure is probably incorrect [6,7,9-12]. The lack of profound and persistent hypotension in the a-chloralose and sodium pentobarbital groups cannot be attributed to differences in the location or size of the lesions. The location of the lesions in all of the groups was similar. The lesions were placed in the correct location since severe hypotension occurred in the urethane group and these results corroborate the findings of others [6,7]. The size of the lesions in the 3 groups was also similar. Although the RVLM does not appear to be the only site of origin of sympathetic vasomotor tone, we suggest that the RVLM can be considered a critical cardiomotor area. We base this suggestion on the finding that the H R in all of the groups was significantly decreased at 10 min after placement of the lesions and that the H R remained depressed or continued to decrease during 30 rain after placement of the lesions. These results raise the question of why lesions of the RVLM produces profound hypotension in rats anesthetized with urethane but not in rats anesthetized with a-chloralose or sodium pentobarbital. One explanation is that, had we observed MAP for a longer time after placement of the lesions in the urethane group, a more complete recovery of MAP may have been seen. The reason that we chose a 30 rain period was because Granata et al. [6,7] had observed their rats for a similar period of time. However, the duration of the postlesion observation period is an unlikely explanation since Dampney and Moon [5] found that arterial pressure remained at a hypotensive level for 3 h after placement of lesions in the RVLM of rabbits anesthetized with urethane. The severe hypotension observed after RVLM lesions in the urethane group, as compared to the other two groups, could have been caused by an initial depression of sympathetic activity followed by a further decrease in activity after placement of
the lesions. Several events could account for this possibility. First, the urethane group could have been more deeply anesthetized than the other groups. However, we attempted to maintain a comparable anesthetic plane in the 3 groups by administering sufficient initial and supplemental doses of each anesthetic agent to prevent the pressor response elicited by pinching the tail. Second, even if all of the groups were at the same anesthetic plane, other pharmacological actions of urethane still could have depressed basal sympathetic activity more than a-chloralose or sodium pentobarbital. However, several studies, in which sympathetic activity was estimated by measurement of plasma norepinephrine concentration in conscious and anesthetized animals, argue against this possibility. For example, Carruba et al. [3] showed that urethane caused a nearly two-fold increase in plasma norepinephrine concentration in rats which was prevented by ganglionic blockade or spinal transection. In contrast, sodium pentobarbital did not alter plasma norepinephrine concentration in rats [4]. In addition, our results are consistent with these observations since the prelesion fall in MAP after administration of trimethaphan was significantly larger in the urethane group as compared to the a-chloralose group and comparable to that observed in the sodium pentobarbital group (Table IIA). The combination of anesthetic agent and the lesion could have reduced sympathetic activity to a greater extent in the urethane group as compared to the other two groups. This explanation is unlikely since sympathetic tone appeared to be comparable after lesions of the RVLM in all of the groups, as indicated by the similar decreases in MAP after trimethaphan (Table IIB). Therefore~ the possibility must be considered that urethane may have peripheral vascular effects which prevented the recovery of MAP after lesions of the RVLM. The results of other studies support this suggestion. For example, urethane, but not sodium pentobarbital or a-chloralose, has been shown to attenuate the pressor responses to angiotensin II [13]. In addition, urethane blunts the pressor response to norepinephrine while a-chloralose and sodium pentobarbital potentiate the pressor response to norepinephrine [1]. Therefore, urethane
187
may have interfered with the actions of endogenous norepinephrine or angiotensin II to a greater extent than did et-chloralose or sodium pentobarbital. In summary, we conclude that the RVLM is not the sole area capable of maintaining vasomotor tone. Previous results leading to this conclusion appear to be due to the use of urethane which potentiates and prolongs the hypotensive effect of lesions of the RVLM. The mechanisms underlying this effect remain to be studied. Whatever the mechanisms, our study shows that the immediate hypotensive effect observed at 10 min after lesion of the RVLM is less severe in rats anesthetized with a-chloralose or sodium pentobarbital. Moreover, the hypotension is transient since, at 30 min postlesion, the MAP had risen to a normotensive value in the a-chloraiose group and was approaching a normotensive value in the sodium pentobarbital group. Therefore, an area, or areas, lying outside the RVLM is (are) capable of maintaining vasomotor tone.
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Acknowledgements The authors wish to thank Drs. Arthur Loewy and Nancy Squires for their valuable suggestions regarding the preparation of the manuscript. The authors also thank Brenda Lemmon for her excellent technical assistance and Joann Faulconer for typing the manuscript. This research was supported by N I H Grant HL33635.
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References 13 1 Brezenoff, H.E., Cardiovascular responses to noradrenaline in the rat before and after administration of various anaesthetics, Br. J. Pharmacol., 49 (1973) 565-572. 2 Buchholz, R.A. and Nathan, M.A., Chronic lability of the
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arterial blood pressure produced by electrolytic lesions of the nucleus tractus solitarii in the rat, Circ. Res., 54 (1984) 227-238. Carruba, M.O., Bondiolotti, G.P., Picotti, G.B., Catteruccia, N. and Da Prada, M., Effects of diethyl ether, halothane, ketamine and urethane on sympathetic activity in the rat, Eur. J. Pharmacol., 134 (1987) 15-24. Carvalho, J.S. and Cherkes, J.K., Renin release by pentobarbital anesthesia in the rat: a role for vascular mechanisms, Life Sci., 30 (1982) 887-897. Dampney, R.A.L. and Moon, E.A., Role of ventrolateral medulla in vasomotor response to cerebral ischemia, Am. J. Physiol., 239 (1980) H349-358. Granata, A.R., Ruggiero, D.A., Park, D.H., Joh, T.H. and Reis, D.J., Lesions of epinephrine neurons in the rostral ventrolateral medulla abolish the vasodepressor components of baroreflex and cardiopulmonary reflex, Hypertension, 5 (Suppl. V) (1983) V80-84. Granata, A.R., Ruggiero, D.A., Park, D.H., Joh, T.H. and Reis, D.J., Brain stem area with C1 epinephrine neurons mediates baroreflex vasodepressor responses, Am. J. Physiol., 248 (1985) H547-567. Paxinos, G. and Watson, C., The Rat Brain, Academic Press, New York, 1982, 79 pp. Reis, D.J., Granata, A.R., Joh, T.H., Ross, C.A., Ruggiero, D.A. and Park, D.H., Brain stem catecholamine mechanisms in tonic and reflex control of blood pressure, Hypertension, 6 (Suppl. II) (1984) II7-15. Reis, D.J., Ross, C.A., Ruggiero, D.A., Granata, A.R. and Joh, T.H., Role of adrenaline neurons of ventrolateral medulla (the C1 group) in the tonic and phasic control of arterial pressure, Clin. Exp. Hypertens., A6 (1984) 221-241. Ross, C.A., Ruggiero, D.A., Joh, T.H., Park, D.H. and Reis, D.J., Adrenaline synthesizing neurons in the rostral ventrolateral medulla: a possible role in tonic vasomotor control, Brain Res., 273 (1983) 356-361. Ross, C.A., Ruggiero, D.J., Park, D.H., Joh, T.H., Sved, A.F., Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor contral by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. Volicer, L. and Loew, C.G., The effect of urethane anesthesia on the cardiovascular action of angiotensin II, Pharmacology, 6 (1971) 193-201. Winer, B.J., Statistical Principles in Experimental Design, McGraw Hill, New York, 1971.
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JAN 00804
Research Papers
Hypotensive effects of lesions of the rostral ventrolateral medulla in rats are anesthetic-dependent K a r e n L. C o c h r a n e , R. Allan Buchholz, J o h n W. H u b b a r d , T. K e n t K e e t o n a n d M a r c A. N a t h a n Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78284-7764 (U.S.A.) (Received 30 October 1987) (Accepted 25 January 1988)
Key words: Rostral ventrolateral medulla; Arterial pressure; Vasomotor center; Urethane; a-Chloralose; Sodium pentobarbital
Abstract These studies were designed to determine if the hypotensive effects of bilateral electrolytic lesions of the rostral ventrolateral medulla are dependent on the type of anesthetic agent used. The lesions caused an immediate fall in mean arterial pressure (MAP) in rats anesthetized with urethane, a-chloralose or sodium pentobarbital. At 30 min after placement of the lesions, severe hypotension (MAP = 54 + 5 mm Hg) persisted in the rats anesthetized with urethane. However, 30 min after placement of the lesions, the MAP of rats anesthetized with a-chloralose or sodium pentobarbital was 87 + 9 mm Hg and 99 + 10 mm Hg, respectively. Subsequent transection of the cervical spinal cord produced a much greater decrease in MAP in rats anesthetized with a-chloralose and sodium pentobarbital as compared to rats anesthetized with urethane. Heart rate was significantly lower after placement of the lesions in all 3 groups. We conclude that the magnitude of the hypotensive effect caused by placement of lesions in the rostral ventrolateral medulla is anesthetic-dependent and that the rostral ventrolateral medulla is not the only area of the central nervous system capable of maintaining vasomotor tone.
Introduction A number of investigators have argued that the rostral ventrolateral medulla (RVLM) is the sole area responsible for the maintenance of sympathetic vasomotor tone. This conclusion is based on the observation that placement of electrolytic lesions in the RVLM of anesthetized rabbits or rats caused mean arterial pressure (MAP) to fall
Correspondence: M.A. Nathan, Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.
to a value similar to that observed after spinal transection [5-7]. In a preliminary effort to assess the cardiovascular effects of lesions of the RVLM in rats after recovery from anesthesia, we placed bilateral electrolytic lesions in the RVLM of anesthetized rats. We found that MAP did not fall to the values previously reported [5-7]. In seeking an explanation for these contradictory results, we noted that the previous investigators had selected urethane as their anesthetic agent while we used sodium pentobarbital. The purpose of the present study was to compare the effects of different anesthetic agents on MAP and heart rate (HR) in rats after RVLM lesions.
0165-1838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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Materials and Methods Experiments were performed using male L o n g - E v a n s rats (Blue Spruce Farms) weighing 250-400 g. Three groups of rats were anesthetized with either urethane (n = 12, 1.7 g / k g , i.p.), achloralose (n = 9, 200 m g / k g , i.p.), or sodium pentobarbital (n --9, 50 m g / k g , i.p.). Supplemental doses of the anesthetic agents were given if pressor responses were seen after pinching the tail. Cannulae were inserted into a femoral artery and vein and threaded into the abdominal aorta and vena cava, respectively, for the recording of pulsatile arterial pressure, MAP and HR, and for the administration of drugs [2]. The rats were placed in a stereotaxic apparatus (Kopf), and the head was positioned so that bregma and lambda lay in the same horizontal plane. The rats were paralyzed with o-tubocurarine chloride (0.3 m g / k g , i.v.) and mechanically ventilated (Harvard Rodent Respirator) with 100% 02 (80 times/rain with a 2.5 ml tidal volume). Supplemental doses of o-tubocurarine were given if spontaneous ventilation was observed. A concentric platinum-iridium electrode (outer shaft = 0.25 mm, o.d.; inner shaft = 0.1 mm, o.d.; 0.25 m m tip exposure on both shafts; 0.75 m m intra-tip distance) was lowered into the R V L M at 3 locations caudal to bregma ( - 11.8 nun, - 11.3 mm, - 12.3 mm). Penetrations were made 2.0 m m lateral to each side of the midline at each level, and the tip of the electrode was positioned at 0.2 m m above the ventral surface of the brainstem. The protocol for systematic exploration of cardiovascular responses to stimulation of the R V L M was as follows. First, sympathetic vasomotor tone was evaluated by bolus injection of the short-acting ganglionic blocker trimethaphan camsylate (800 m g / k g , i.v.). Second, the R V L M was electrically stimulated at 11.8 m m caudal to bregma (100 Hz, 0.1 ms pulse width, 50/~A), as a means of eliciting a pressor response and tachycardia, and then the threshold current was determined (7-20 /~A). Third, after M A P returned to the baseline value, a lesion was made by passing a DC anodal current (1.0 m A for 30 s) between the inner shaft of the electrode and a clip
attached to one edge of the skin incision. Fourth, the site was restimulated at 4 × threshold current, and, if a cardiovascular response was still present, additional lesions were made until the pressor response was abolished. These procedures of stimulation and placement of lesions were repeated on the opposite side of the brainstem. Then, the entire protocol was repeated at each of the other two locations. However, at these locations lesions were made only if a pressor response was elicited. Trimethaphan was readministered 10 min after placement of the final lesion. The spinal cord was sectioned by aspiration at the level of the first cervical vertebra 30 min later. A third injection of trimethaphan was then given. The experiment ended with intracardiac perfusion of saline and 10% buffered formalin, and the brain was removed. Frozen sections (40 #m) were cut serially, mounted, and stained with Cresyl violet. Microscopic examination of the sections was performed to localize and determine the extent of the lesions. Coronal sections were selected at 120-/~m intervals through the anterior-posterior extent of the RVLM. The sections were viewed with a projecting microscope. The maximal medial-lateral extent and dorsal-ventral extent of the lesions on each side of the brainstem were measured, and the area of damage at each anteriorposterior level was calculated. A digitizing tablet was used to trace a representative sagittal section taken from the brain atlas of Paxinos and Watson [8]. The anterior-posterior extent of each lesion from each rat was then plotted on the sagittal section. The degree of overlap of each lesion identified the area of damage common to all of the rats. The effect of the lesions on M A P and H R were evaluated for statistical significance using a twoway analysis of variance followed by post hoc analysis of significant main effects using the Newm a n - K e u l ' s procedure [14]. Paired Student's ttests were used to assess the effects of trimethaphan on M A P and HR. The extent of the lesions was also analyzed with a two-way analysis of variance. Differences were considered significant at P < 0.05.
183
Results Bilateral destruction of the R V L M s u b s t a n tially decreased M A P i n all groups within 10 m i n after p l a c e m e n t of the last lesion (Table IA). H o w ever, the hypotensive effect of the lesions was largest in the u r e t h a n e group ( - 58 ± 1 m m Hg), a n d the absolute level of M A P i n this group was significantly lower t h a n that observed i n the other two groups. The decrease of M A P i n the s o d i u m p e n t o b a r b i t a l group was also relatively large ( - 51 ± 1 m m Hg), b u t the prelesion M A P was higher i n this group as c o m p a r e d to the other groups. T h e r e d u c t i o n in M A P of the a-chloralose group was less ( - 3 4 ± 1 m m Hg) a n d reached a n absolute level similar to that of the s o d i u m p e n t o b a r b i t a l group. Thus, the prelesion M A P did n o t d e t e r m i n e the m a g n i t u d e of the hypotensive effect of lesions of the R V L M .
T h e i m m e d i a t e h y p o t e n s i v e effect of the lesions i n the a-chloralose a n d s o d i u m p e n t o b a r b i t a l groups d i m i n i s h e d over time (Table IA). By 30 rain after p l a c e m e n t of the lesions, the M A P of the a-chloralose group rose to a level that was n o t significantly less ( - 14 + 3 m m Hg) t h a n the prelesion baseline value, a n d i n the s o d i u m p e n t o b a r b i t a l group, the M A P reached a level that was 22 + 5 m m H g less t h a n the baseline M A P . I n sharp contrast, the effect of the lesions o n the u r e t h a n e g r o u p was m u c h more e n d u r i n g with the M A P r e m a i n i n g at 51 + 1 m m H g below the prelesion baseline value. I n t e r r u p t i o n of d e s c e n d i n g s y m p a t h e t i c p a t h w a y s b y spinal t r a n s e c t i o n caused a further r e d u c t i o n i n the M A P i n all the groups (Table IA). T h e r e d u c t i o n i n M A P was greatest i n the s o d i u m p e n t o b a r b i t a l ( - 51 + 6 m m Hg) a n d a-chloralose ( - 41 + 7 m m Hg) groups b u t consid-
TABLE I Cardiovascular effects of lesions of the rostral ventrolateral medulla Group
Prelesion baseline
A. Mean Arterial Pressure (mm Hg 5: S.E.M.)
Urethane Chloralose Pentobarbital
106 + 4 101 5 : 6 121 5 : 6 *
B. Heart rate (bpm 5: S . E . M . )
Urethane Chloralose Pentobarbital
431 + 8 360 5:20 * 417 + 11
10 mm Postlesion
48 + 3 ..t 67 5 : 5 t 70 5 : 6 t 366 5:13 t 331 5:20 t 353 5 : 9 t
30 min Postlesion
54 5 : 5 *'* 87 5 : 9 t 99 5:10 t,* 366 5:13 * 301 5:17 * *'* 324 -t- 14 t,*
Postspinal transection
36 5 : 2 .,t,* 46 5 : 2 t,* 48 -I- 5 t,* 344 + 13 *'* 300 + 12 * 293 5:15 t,*
* Significantlydifferent from the other two groups; * * significantly different from the urethane group; t significantly different from the preceding recording period; * significantly different from the prelesion baseline recording period; Significance level, P < 0.05. TABLE II Cardiovascular effects of trimethaphan (ram Hg or bpm 5: S.E.M.) Group
A. Prelesion trimethaphan administration
Urethane Chloralose Pentobarbital
B. Postlesion (10 rain) trimethaphan administration
Urethane Chloralose Pentobarbital
Mean arterial pressure
Heart rate
Before
Change
Before
Change
116 + 4 117 + 5 131 + 6
- 5 2 + 5 *,t -32 + 4 * -40 + 5 *
426 + 13 405 -t- 14 439 -I- 12
-43 5- 4 * - 2 2 5- 4 *'* -34 + 5 *
- 12 5:2 * - 15 5:3 * -16 5:4 *
368 5:13 328 5:18 343 + 10
- 31 5- 4 * - 2 2 5:4 * -21 5:7 *
51 -t- 3 * * 75 5:6 82 5:9
* Significantly different from predrug; * * significantly different from chloralose and pentobarbital groups; t significantly different from chloralose group; * significantly different from urethane and pentobarbital groups. Significance level, P < 0.05.
184
erably less in the urethane group ( - 1 8 ± 3 mm Hg). However, after spinal transection the absolute level of MAP in the urethane group was
still significantly lower than the MAP of the achloralose or sodium pentobarbital groups. Bradycardia was evident in all the groups 10 min after placement of the lesions, and the magnitude of the bradycardia appeared to be related to the prelesion baseline H R of each group (Table IB). The H R of the a-chloralose group fell the least ( - 29 ___1 bpm) whereas larger decreases were observed in the urethane and sodium pentobarbital groups ( - 6 5 + 5 bpm and - 6 4 ± 2 bpm, respectively). In contrast to MAP, H R showed no signs of recovery in any group at 30 rain after placement of the lesions (Table IB). The H R of the a-chloralose and sodium pentobarbital groups continued to decline ( - 30 ± 2 bpm, - 29 + 5 bpm, respectively), and the H R of the urethane group remained suppressed. Spinal cord transection caused a further reduction in H R in the urethane group ( - 2 2 + 1 bpm) and the sodium pentobarbital group ( - 3 1 ± 1 bpm), but no change was observed in the a-chloralose group (Table IB).
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Fig. 1. Lesion sites In the urethane (top panel), a-chloralose (middle panel) and sodium pentobarbital (bottom panel) group. The hemisections on the right of each figure shows a typical lesion in the R V L M located 11.8 m m caudal to bregma. The line drawings on the left summarize the location of the lesions in the individual rats. The dotted portion of each line drawing indicates the area damaged in all the rats of a particular group. The dashed line shows the boundary of the largest lesions that were made in each group. NTS, nucleus tractus solitarius, Amb, nucleus ambiguus; sp5, spinal trigeminal tract; io, inferior olivary nucleus. Note: the hole near the dorsal surface of the section shown in the top panel is a marking hole.
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P o s t e r i o r to B r e g m o (mr'n) Fig. 2. Sagittal section of lower brainstem and overlying structures showing the lesion sites on the left side of the brain in all rats of the urethane, a-chloralose and sodium pentobarbital groups. The shaded area shows the maximal a m o u n t of overlap of lesion sites (see text for details). The sagittal view is through the center of the R V L M (lateral 1.9 m m from the midline). Amb, nucleus ambiguus; LRt, lateral reticular nucleus; LVe, lateral vestibular nucleus; 7, facial nucleus; NTS, nucleus tractus sol±tar±us.
185
Trimethaphan was used to assess the amount of sympathetic vasomotor tone before and after placement of the RVLM lesions and after spinal cord transection. Trimethaphan significantly decreased the prelesion MAP in all the groups (Table IIA). Trimethaphan caused a small but uniform depression of MAP in all the groups 10 rain after placement of the lesions. At this time, MAP had not recovered in the a-chloralose and sodium pentobarbital groups (Table IIB). Trimethaphan administered after spinal transection caused a slight reduction in the MAP (2-4 mm Hg) of all the groups. Trimethaphan also reduced the prelesion H R in all the groups, (Table IIA). The postlesion bradycardia after trimethaphan was similar in all the 2.000-
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groups (Table IIB). However, trimethaphan variably affected the H R of the groups after spinal cord transection. The reduction in H R was significant in the urethane ( - 3 1 ± 5 bpm) and sodium pentobarbital ( - 1 4 ± 3 bpm) groups, but a bradycardia was inconsistently seen in the ctchloralose group ( - 14 _ 8 bpm). The greatest cross-sectional extent of the lesions in all the groups was found at approximately 11.8 mm posterior to bregma (Fig. 1). The lesions lay between the spinal trigeminal tract and the inferior olive and extended dorsally toward the boundary of the nucleus ambiguus. The RVLM was damaged bilaterally in all of the rats at 11.8 mm posterior to bregma. The area most consistently damaged bilaterally in the majority of the rats (82.7%) lay between 11.3 mm and 12.3 mm posterior to bregma (Fig. 2). The lesions lying between these two levels extended dorsally about 0.8-1 mm from the ventral surface of the RVLM. No statistically significant differences were found in an analysis of the amount of tissue damage on each side of the brainstem within each group (Fig. 3). In addition, no differences in the amount of tissue destroyed were found between the groups.
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Discussion 2,000-
1
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-11.300
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Posterior to Bregma (ram) Fig. 3. Line graphs showing the anterior to posterior extent ( m m posterior to Bregma) of damage (ram2-± S.E.M.) to the R V L M on the left (top panel) and right (bottom panel) sides of the brainstem. No statistically significanf differences were found between groups or between sides at any level. The anterior-posterior borders of the lesions varied according to group by 120-240 /~m. However, this a m o u n t of variability was due to a relatively small amount of tissue damage to the posterior R V L M in two rats of the urethane group and to the anterior R V L M in two rats of the sodium pentobarbital group.
The most important finding of our study is that lesions of the RVLM produced only transient hypotension in rats anesthetized with sodium pentobarbital or a-chloralose. This finding is in sharp contrast to the previous reports that severe and persistent hypotension occurs after placement of lesions in the RVLM in animals anesthetized with urethane [5-7]. Although hypotension was observed in all 3 groups 10 min after placement of the lesions, by 30 min postlesion the MAP had recovered in the a-chloralose and sodium pentobarbital groups such that MAP (absolute values of 87 + 9 mm Hg and 99 + 10 mm Hg, respectively) was only 14-18% less than the prelesion values. However, the MAP of the urethane group (absolute value of 54 ± 5 mm Hg) was still 49% below the prelesion value at 30 min after placement of the lesions. Moreover, significant sympathetic vasomotor tone still existed after lesions of the
186 RVLM in the animals anesthetized with a-chloralose and sodium pentobarbital since spinal transection lowered the MAP by 40-50 mm Hg in these two groups. Therefore, the original proposal that the RVLM is the vasomotor center which is responsible for the maintenance of sympathetic vasomotor tone and basal arterial pressure is probably incorrect [6,7,9-12]. The lack of profound and persistent hypotension in the a-chloralose and sodium pentobarbital groups cannot be attributed to differences in the location or size of the lesions. The location of the lesions in all of the groups was similar. The lesions were placed in the correct location since severe hypotension occurred in the urethane group and these results corroborate the findings of others [6,7]. The size of the lesions in the 3 groups was also similar. Although the RVLM does not appear to be the only site of origin of sympathetic vasomotor tone, we suggest that the RVLM can be considered a critical cardiomotor area. We base this suggestion on the finding that the H R in all of the groups was significantly decreased at 10 min after placement of the lesions and that the H R remained depressed or continued to decrease during 30 rain after placement of the lesions. These results raise the question of why lesions of the RVLM produces profound hypotension in rats anesthetized with urethane but not in rats anesthetized with a-chloralose or sodium pentobarbital. One explanation is that, had we observed MAP for a longer time after placement of the lesions in the urethane group, a more complete recovery of MAP may have been seen. The reason that we chose a 30 rain period was because Granata et al. [6,7] had observed their rats for a similar period of time. However, the duration of the postlesion observation period is an unlikely explanation since Dampney and Moon [5] found that arterial pressure remained at a hypotensive level for 3 h after placement of lesions in the RVLM of rabbits anesthetized with urethane. The severe hypotension observed after RVLM lesions in the urethane group, as compared to the other two groups, could have been caused by an initial depression of sympathetic activity followed by a further decrease in activity after placement of
the lesions. Several events could account for this possibility. First, the urethane group could have been more deeply anesthetized than the other groups. However, we attempted to maintain a comparable anesthetic plane in the 3 groups by administering sufficient initial and supplemental doses of each anesthetic agent to prevent the pressor response elicited by pinching the tail. Second, even if all of the groups were at the same anesthetic plane, other pharmacological actions of urethane still could have depressed basal sympathetic activity more than a-chloralose or sodium pentobarbital. However, several studies, in which sympathetic activity was estimated by measurement of plasma norepinephrine concentration in conscious and anesthetized animals, argue against this possibility. For example, Carruba et al. [3] showed that urethane caused a nearly two-fold increase in plasma norepinephrine concentration in rats which was prevented by ganglionic blockade or spinal transection. In contrast, sodium pentobarbital did not alter plasma norepinephrine concentration in rats [4]. In addition, our results are consistent with these observations since the prelesion fall in MAP after administration of trimethaphan was significantly larger in the urethane group as compared to the a-chloralose group and comparable to that observed in the sodium pentobarbital group (Table IIA). The combination of anesthetic agent and the lesion could have reduced sympathetic activity to a greater extent in the urethane group as compared to the other two groups. This explanation is unlikely since sympathetic tone appeared to be comparable after lesions of the RVLM in all of the groups, as indicated by the similar decreases in MAP after trimethaphan (Table IIB). Therefore~ the possibility must be considered that urethane may have peripheral vascular effects which prevented the recovery of MAP after lesions of the RVLM. The results of other studies support this suggestion. For example, urethane, but not sodium pentobarbital or a-chloralose, has been shown to attenuate the pressor responses to angiotensin II [13]. In addition, urethane blunts the pressor response to norepinephrine while a-chloralose and sodium pentobarbital potentiate the pressor response to norepinephrine [1]. Therefore, urethane
187
may have interfered with the actions of endogenous norepinephrine or angiotensin II to a greater extent than did et-chloralose or sodium pentobarbital. In summary, we conclude that the RVLM is not the sole area capable of maintaining vasomotor tone. Previous results leading to this conclusion appear to be due to the use of urethane which potentiates and prolongs the hypotensive effect of lesions of the RVLM. The mechanisms underlying this effect remain to be studied. Whatever the mechanisms, our study shows that the immediate hypotensive effect observed at 10 min after lesion of the RVLM is less severe in rats anesthetized with a-chloralose or sodium pentobarbital. Moreover, the hypotension is transient since, at 30 min postlesion, the MAP had risen to a normotensive value in the a-chloraiose group and was approaching a normotensive value in the sodium pentobarbital group. Therefore, an area, or areas, lying outside the RVLM is (are) capable of maintaining vasomotor tone.
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Acknowledgements The authors wish to thank Drs. Arthur Loewy and Nancy Squires for their valuable suggestions regarding the preparation of the manuscript. The authors also thank Brenda Lemmon for her excellent technical assistance and Joann Faulconer for typing the manuscript. This research was supported by N I H Grant HL33635.
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References 13 1 Brezenoff, H.E., Cardiovascular responses to noradrenaline in the rat before and after administration of various anaesthetics, Br. J. Pharmacol., 49 (1973) 565-572. 2 Buchholz, R.A. and Nathan, M.A., Chronic lability of the
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arterial blood pressure produced by electrolytic lesions of the nucleus tractus solitarii in the rat, Circ. Res., 54 (1984) 227-238. Carruba, M.O., Bondiolotti, G.P., Picotti, G.B., Catteruccia, N. and Da Prada, M., Effects of diethyl ether, halothane, ketamine and urethane on sympathetic activity in the rat, Eur. J. Pharmacol., 134 (1987) 15-24. Carvalho, J.S. and Cherkes, J.K., Renin release by pentobarbital anesthesia in the rat: a role for vascular mechanisms, Life Sci., 30 (1982) 887-897. Dampney, R.A.L. and Moon, E.A., Role of ventrolateral medulla in vasomotor response to cerebral ischemia, Am. J. Physiol., 239 (1980) H349-358. Granata, A.R., Ruggiero, D.A., Park, D.H., Joh, T.H. and Reis, D.J., Lesions of epinephrine neurons in the rostral ventrolateral medulla abolish the vasodepressor components of baroreflex and cardiopulmonary reflex, Hypertension, 5 (Suppl. V) (1983) V80-84. Granata, A.R., Ruggiero, D.A., Park, D.H., Joh, T.H. and Reis, D.J., Brain stem area with C1 epinephrine neurons mediates baroreflex vasodepressor responses, Am. J. Physiol., 248 (1985) H547-567. Paxinos, G. and Watson, C., The Rat Brain, Academic Press, New York, 1982, 79 pp. Reis, D.J., Granata, A.R., Joh, T.H., Ross, C.A., Ruggiero, D.A. and Park, D.H., Brain stem catecholamine mechanisms in tonic and reflex control of blood pressure, Hypertension, 6 (Suppl. II) (1984) II7-15. Reis, D.J., Ross, C.A., Ruggiero, D.A., Granata, A.R. and Joh, T.H., Role of adrenaline neurons of ventrolateral medulla (the C1 group) in the tonic and phasic control of arterial pressure, Clin. Exp. Hypertens., A6 (1984) 221-241. Ross, C.A., Ruggiero, D.A., Joh, T.H., Park, D.H. and Reis, D.J., Adrenaline synthesizing neurons in the rostral ventrolateral medulla: a possible role in tonic vasomotor control, Brain Res., 273 (1983) 356-361. Ross, C.A., Ruggiero, D.J., Park, D.H., Joh, T.H., Sved, A.F., Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor contral by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. Volicer, L. and Loew, C.G., The effect of urethane anesthesia on the cardiovascular action of angiotensin II, Pharmacology, 6 (1971) 193-201. Winer, B.J., Statistical Principles in Experimental Design, McGraw Hill, New York, 1971.