Brain Research, 530 (1990) 317-320 Elsevier
317
BRES 24330
Spinal cord substance P mediates carbachol-induced cardiovascular responses from the rostral ventrolateral medulla Qing Lin 1, Cong De
Li 2
and Peng Li 1
1Department of Physiology, Shanghai Medical University, Shanghai (People's Republic of China) and 2Departmentof Physiology, Yi Chang Medical College, Hubei (People's Republic of China) (Accepted 3 July 1990)
Key words: Substance P antagonist; Carbachol; Spinal cord; Rostral ventrolateral medulla; Intrathecal infusion; Wistar rat
Intrathecal (i.t.) infusion of substance P (SP) antagonist, (D-Pro2,D-Trp7.9)-substanceP (20/~g), lowered blood pressure profoundly without any significant change of heart rate. The hypertensive and tachycardic responses elicited by microinjection of carbachol (25 ng/site) into the bilateral rostral ventrolateral medulla (rVLM) were blocked by i.t. infusion of SP antagonist. These data provided evidence that an excitatory cardiovascular effect induced by cholinergic system in rVLM may be mediated mainly by the SP receptors in spinal cord. It has been confirmed histologically that substance P (SP)-like neurons of the ventral medulla project to the intermediolateral ceil columns (IML) 9. Functionally, the pressor response elicited by application of kainic acid onto the ventral surface of the medulla was correlated with an increase of substance P (SP) release from the spinal cord and could be blocked by intrathecal (i.t.) infusion of SP antagonist 13"17. Furthermore, Keeler et al. 1°'1~ reported that bicucuiline ( G A B A antagonist)induced activation of cardiovascular responses from the rostral ventrolateral medulla (rVLM) also could be attenuated markedly by spinal application of SP antagonists. It has been well established that cholinergic mechanisms in rVLM are tonically active and involved in the normal maintenance of blood pressure 16'18. This prompted us to investigate whether SP neurons present in the ventral medulla which terminate in IML mediate the cholinergic drug-induced cardiovascular responses from the rVLM. Sixty-four male Wistar rats weighing 250-320 g were anaesthetized with urethane and a-chloralose (800 mg/kg and 60 mg/kg, i.p., respectively). The tail artery was cannulated, blood pressure (BP) and heart rate (HR) were recorded on a polygraph. Body temperature was maintained at 37-38 °C. The head of the animal was mounted on a stereotaxic apparatus in a prone position and flexed with an angle of 45 ° . The dorsal surface of the lower brainstem was exposed by a limited occipital craniotomy. A spinal catheter (PE10 tubing) was inserted through an incision in the atlanto-occipital membrane
and advanced in the spinal subarachnoid space 6.8-7.0 cm caudally to the Tlo-Tll vertebral levels, identified by infusion of 1% pontamine sky blue into the injecting site at the termination of the experiment. A pair of stainlesssteel tubules with external diameter of 0.13 mm were guided into the bilateral rVLM, which extends 2.2-2.5 mm rostral to the obex, 1.5-1.7 m m lateral to the midline and within 0.2-0.5 mm from the ventral surface of the medulla. Drugs were dissolved in normal saline (pH 7.3-7.4) and administered over 0.5-1.0 min in a volume of 10/~1 for i.t. infusion and in an amount of 0.1/~1 within 10 s for rVLM microinjection. At the end of each experiment, 1% pontamine sky blue was infused to the microinjecting sites. The brain was removed and fixed in 10% formaline for 4-10 days. The frozen sections (50 /~m) were made, from which the injecting sites were identified with reference to Fifkova and Marsala's atlas 6. Data are presented as the means + S.E.M. Statistical analysis was performed by the paired and Student t-tests. The effect of i.t. infusion of SP antagonist, (o-Pro2, D-TrpT,9)-SP (20 ~g) was studied on 8 rats and the results were presented in Fig. 1. Prior to SP antagonist administration, the baseline mean blood pressure (MBP) and H R were 103 + 5 m m H g and 410 + 13 beats/min, respectively. The spinal infusion of SP antagonist usually caused an initial depressor and then pressor response which lasted for 3-4 min, followed by a prolonged depressor response. By 15-20 min, the depressor phase had reached its nadir, averaging 59 + 3mmHg (Fig. 1A). A slight but not significant bradycardia was observed
Correspondence: P. Li, Department of Physiology, Shanghai Medical University, Yi Xue Yuan Road 138, Shanghai, 200032, People's Republic of China. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
318
A 12C m
9(3
E ~E
6C
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g
3(3
c 5£ E -~ 4(: ~ 3C T ~6 2C
.,,
g g lC
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6
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C BP(mmHg)
10
15
30
60 (rnin) C
m
i_.~=
I
MBP(mrnHg)
HR(btmin) 480[
AP 9 i
280L'
-
.
5
~
n,,,(~tk
-
280L'
t i.t. SP ant. (20#g)
' 1 min'
Fig. 1. A + B: effect of SP antagonist (20/~g, i.t.) on MBP and HR 15 min after infusion. Open bars: preinfusion. Vertically hatched bars: normal saline infused i.t. (n = 7); solid bars: SP antagonist infused i.t. (n = 8); cross-hatched bars: SP antagonist infused i.v. (n = 6): **P < 0.01, compared to the preinfusion values. C: time course of the cardiovascular effects elicited by i.t. administration of SP antagonist.
(Fig. 1B). Sixty rain later, M B P (86 -+ 4 m m H g ) r e t u r n e d closely to the control level. A r e p r e s e n t a t i v e e x p e r i m e n t
A BP ( mm Hg )
MBP(mrnHg)
HR(b/rain)
B
BP(mmHg) MBP(mmHg)
HR(b/min
t Microinj. car bachol
q1mtn' (25 ng/ site )
Fig. 2. The inhibitory effect of i.t. infusion of SP antagonist on the carbachol-induced cardiovascular responses from the rVLM. A: carbachol responses 15 min after i.t. infusion of normal saline. B: carbachol responses 15 min after i.t. infusion of SP antagonist (20 ~,g).
AP 1
0
.
0
~ IlmmI
Fig. 3. A + B: cardiovascular responses to carbachol (25 rig/site) microinjected into bilateral rVLM 15 rain after normal saline i.t. (open bars, n = 8), SP antagonist i.t. (solid bars, n = 8), SP antagonist i.v. (vertically hatched bars, n = 6) or bleeding (cross-hatched bars, n = 6). **: P < 0.01, comparing normal saline i.t. and SP antagonist i.t.; • and • A : P < 0.05 and P < 0.01, comparing SP antagonist i.t. and SP antagonist i.v.; I l l : P < 0.01, comparing SP antagonist i.t. and bleeding. C: the location of microinjection sites in rVLM. Solid circles: carbachol injecting sites where the cardiovascular responses were elicited; open circles: carbachol injecting sites where no significant response was evoked; nVII, facial nucleus; PGL, nucleus paragigantoceUularis lateralis; RG, nucleus reticularis gigantocellularis; RPG, nucleus paragigantocellularis; V, trigeminal tract.
was s h o w n in Fig. IC. T h e s a m e dose of SP a n t a g o n i s t a d m i n i s t e r e d i n t r a v a n e o u s l y (i.v., n = 6) had n o significant effect o n B P a n d H R , n o r did a c o n t r o l spinal infusion of n o r m a l saline (n = 7). I n 3 a n i m a l s , respiration was c o n t i n u o u s l y m o n i t o r e d by r e c o r d i n g the e l e c t r o m y o g r a m of the d i a p h r a g m to o b s e r v e the possible changes of respiration after the a d m i n i s t r a t i o n of SP a n t a g o n i s t a n d it was s h o w n that t h e r e was n o o b v i o u s change in b r e a t h i n g a l t h o u g h a r e m a r k a b l e i n f l u e n c e o n B P was elicited at the s a m e time. T h e effect of i.t. i n j e c t i o n of SP a n t a g o n i s t o n the cardiovascular r e s p o n s e s elicited by m i c r o i n j e c t i o n of carbachol, A c h agonist, into bilateral r V L M was investigated further. Fig. 2 s h o w e d a typical result of the b l o c k a d e of the c a r b a c h o l - i n d u c e d r e s p o n s e s by SP antagonist. F i f t e e n m i n after i.t. infusion of n o r m a l saline, m i c r o i n j e c t i o n of carbachol (25 ng/site) into r V L M p r o d u c e d a m a r k e d increase in M B P which p e a k e d in 2 - 3 m i n , a n d a short-lasting b r a d y c a r d i a followed by a g r a d u a l increase in H R which r e a c h e d its
319 peak in 6 - 8 min (33 + 3 m m H g and 19 + 6 beat/min, n = 8). The pressor and tachycardic responses lasted for 25-30 min. A control injection of normal saline into the r V L M (n = 7) had no significant effect on the cardiovascular activity. When animals were pretreated spinally with SP antagonist (20 /zg) 15 min before carbachol injection, the cardiovascular responses from r V L M were blocked profoundly (6 + 1 m m H g and - 8 + 4 beat/min, n = 8). In 5 additional animals, the influence of SP antagonist on the cardiovascular responses induced by intra-rVLM injection of carbachol was observed 60 min after i.t. infusion and it was found that the inhibitory effect on pressor response was still obtained (24 + 3 m m H g , P < 0.05) but less than before. However, an i.v. application of SP antagonist with the same dose had no such effect. To exclude the possible influence of severe hypotensive state induced by i.t. infusion of SP antagonist on the responsibility of central nervous system, the resting MBP was lowered to 60-70 m m H g by withdrawing blood of 3-6 ml from the tail artery. In such a case, the carbachol-induced pressor response still existed and an increase in tachycardic response was observed (Fig. 3A,B). The microinjecting sites in the rVLM examined histologically were mostly located in the lateral part of the nucleus paragigantocellularis lateralis with small spread in the facial nucleus, rostral to the obex level from which the pressor and tachycardic response were elicited by carbachol injection (solid circles). No significant response of cardiovascular activities was observed when carbachol was administered in the adjacent sites which were in the facial nucleus and the lateral part of the nucleus reticularis gigantocellularis (open circles, Fig. 3C). Evidence has accumulated that there is a high dense distribtution of SP terminals and receptors in IML of thoracic spinal cord 2"3'12. Iontophoretic or i.t. application of SP increased the firing rate of sympathetic preganglionic neurons and BP accompanied by an increase of plasma epinephrine and norepinephrine 1'4'8'1°'19, while i.t. administration of SP antagonist produced a hypotensive response 11'13, suggesting a tonic sympathoexcitatory role for spinal cord SP. The first part of our present
experiment (Fig. 1) supported this hypothesis. Carbachol, as a cholinergic agonist, has been applied centrally for the studies of neural control of cardiovascular functions both under conscious and anesthetized conditions TM. In our lab, only anesthetized animals were used and the pressor effect of intra-rVLM injection of carbachol was similar as in the conscious one, suggesting that urethane-chloralose anesthesia has no significant effect on the cholinergic cardiovascular responses 2°. Loewy et a1.13 showed that an excitatory cardiovascular effect elicited by application of kainic acid onto r V L M could be blocked by spinal administration of SP antagonist, indicating that SP is probably an important transmitter in bulbospinal sympathoexcitatory pathways. Although it was believed that the r V L M may be under tonic excitatory cholinergic control 16'18, the transmission of its excitatory effect in the bulbospinal pathways still remains a problem. In the present study, it was shown that the pressor and tachycardic responses induced by microinjection of carbachol into bilateral r V L M could be blocked by spinal application of SP antagonist. Such effect did not occur when pretreated with i.v. application of the same dose of SP antagonist, which excluded the possibility of peripheral leakage of the drug and suggested a central blockade action of SP antagonist on SP receptors in the spinal cord. It has been demonstrated that (D-Argl, D-Trp7,9 , Leu11)-SP and (D-Pro2, D-Trp7 ' 9)-SP exerted marked neurotoxic actions when given into the spinal cord, including persistent paralysis and neuronal necrosis 7"15. Such neurotoxicity was not the nature of the antagonism seen in our experiment since the inhibitory effect of SP antagonist was reversible with time and the neurotoxic actions affect mostly the motoneurons in the ventral horns TM. However, the data presented here cannot rule out the possibility of the blockade of transmission from SP interneurons or other SP neuron projections to IML.
1 Backman, S.B. and Henry, J.L., Effects of substance P and neurons on the upper thoracic intermediolateral nucleus of the cat, Can. J. Physiol. Pharmacol., 62 (1984) 248-251. 2 Charlton, C.G. and Helke, C.J., Characterization and segmental distribution of 125I-Bolton-Hunter-labeled substance P binding sites in rat spinal cord, J. Neurosci., 5 (1985) 1293-1299. 3 Charlton, C.G. and Helke, C.J., Autoradiographic localization and charaterization of spinal cord substance P binding sites: high densities in sensory, autonomic, phrenic and Onuf's motor nuclei, J. Neurosci., 5 (1985) 1653-1661. 4 Couture, R., Hassesian, H. and Gupta, A., Studies on the cardiovascular effects produced by the spinal action of substance P in the rat, J. Cardiovasc. Pharmacol., 11 (1988) 270-283. 5 Day, M.D. and Roach, A.G., Cardiovascular effects of carba-
chol and other cholinomimetics administered into the cerebral ventricles of conscious cats, Clin. Exp. Pharmacol. PhysioL, 4 (1977) 431-442. 6 Fifkova, E. and Marsala, J., Stereotaxic atlases for cat, rabbit and rat. In J. Bures, M. Petran and J. Zachar (Eds.), Electrophysiological Methods in Biological Research, Czechoslovak Acad. Sci. Prague, 1967, pp. 656-695. 7 Freedman, J., H0kfelt, T., Post, C., Brodin, E., Sundstrom, E., Jonsson, G., Terenius, L., Leander, S., Fischer, J.A. and Verhofstad, A., Immunohistochemical and behavioral analysis of spinal lesions induced by a substance P antagonist and protection by thyrotropin releasing hormone, Exp. Brain Res., 74 (1989) 279-292. 8 Giibey, M.P., McKenna, K.E. and Schramm, L.P., Effects of
The assistance of Ms Zhu Wen-he is greatly appreciated. This work was in part supported by the Science Fund of the Ministry of Health.
320
9
10
11
12
13
14
substance P on sympathetic preganglionic neurons, Neurosci. Lea., 41 (1983) 157-159. Helke, C.J., Neil, J.J., Massari, V.J. and Loewy, A.D., Substance P neurons project from the ventral medullar to the intermediolateral cell column and ventral horn in the rat, Brain Research, 243 (1982) 147-152. Keeler, J.R., Charlton, C.G. and Helke, C.J., Cardiovascular effects of spinal cord substance P: studies with a stable receptor agonist, J. Pharmacol. Exp. Ther., 233 (1985) 755-760. Keeler, J.R. and Heike, C.J., Spinal cord substance P mediates bicuculline-induced activation of cardiovascular responses from the ventral medulla, J. Auton. Nerv. Syst., 13 (1985) 19-33. Ljungdahl, A., H6kfelt, T. and Niissan, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat - - I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-944. Loewy, A.D. and Sawyer, W.B., Substance P antagonist inhibits vasomotor responses elicited from ventral medulla in rat, Brain Research, 245 (1982) 379-383. Ozawa, H. and Uematsu, T., Centrally mediated cardiovascular effects of intracisternal application of carbachol in anesthetized rats, Jap. J. Pharmacol., 26 (1976) 339-346.
15 Post, C. and Pauisson, I., Antinociceptive and neurotoxic actions of substance P analogues in the rat's spinal cord after intrathecal administration, Neurosci. Lett., 57 (1985) 159-164. 16 Sundaram, K. and Sapru, H., Cholinergic nerve terminals in the ventrolateral medullary area: pharmacological evidence, J. Auton. Nerv. Syst., 22 (1988) 221-228. 17 Takano, Y., Martin, J.E., Leeman, S.E. and Loewy, A.D., Substance P immunoreactivity released from rat spinal cord after kainic acid excitation on the ventral medulla oblongata: a correlation with increases in bood pressure, Brain Research, 291 (1984) 168-172. 18 Willette, R.N., Punnen, S., Krieger, A.J. and Sapru, H.N., Cardiovascular control by cholinergic mechanisms in the rostral ventrolateral medulla, J. Pharmacol. Exp. Ther., 231 (1984) 457-463. 19 Yashpal, K., Gauthier, S. and Henry, J.L., Substance P given intrathecally at the spinal T9 level increases arterial pressure and heart rate in the rat, J. Auton. Nerv. Syst., 18 (1987) 93-103. 20 Zhu, D.N., Guo, X.Q. and Li, P., Relationship between the rostral medullary cholinergic mechanism and the cardiovascular component of the defence reaction, Chin. J. Physiol. Sci., 6 (1990) 86-94.
317
BRES 24330
Spinal cord substance P mediates carbachol-induced cardiovascular responses from the rostral ventrolateral medulla Qing Lin 1, Cong De
Li 2
and Peng Li 1
1Department of Physiology, Shanghai Medical University, Shanghai (People's Republic of China) and 2Departmentof Physiology, Yi Chang Medical College, Hubei (People's Republic of China) (Accepted 3 July 1990)
Key words: Substance P antagonist; Carbachol; Spinal cord; Rostral ventrolateral medulla; Intrathecal infusion; Wistar rat
Intrathecal (i.t.) infusion of substance P (SP) antagonist, (D-Pro2,D-Trp7.9)-substanceP (20/~g), lowered blood pressure profoundly without any significant change of heart rate. The hypertensive and tachycardic responses elicited by microinjection of carbachol (25 ng/site) into the bilateral rostral ventrolateral medulla (rVLM) were blocked by i.t. infusion of SP antagonist. These data provided evidence that an excitatory cardiovascular effect induced by cholinergic system in rVLM may be mediated mainly by the SP receptors in spinal cord. It has been confirmed histologically that substance P (SP)-like neurons of the ventral medulla project to the intermediolateral ceil columns (IML) 9. Functionally, the pressor response elicited by application of kainic acid onto the ventral surface of the medulla was correlated with an increase of substance P (SP) release from the spinal cord and could be blocked by intrathecal (i.t.) infusion of SP antagonist 13"17. Furthermore, Keeler et al. 1°'1~ reported that bicucuiline ( G A B A antagonist)induced activation of cardiovascular responses from the rostral ventrolateral medulla (rVLM) also could be attenuated markedly by spinal application of SP antagonists. It has been well established that cholinergic mechanisms in rVLM are tonically active and involved in the normal maintenance of blood pressure 16'18. This prompted us to investigate whether SP neurons present in the ventral medulla which terminate in IML mediate the cholinergic drug-induced cardiovascular responses from the rVLM. Sixty-four male Wistar rats weighing 250-320 g were anaesthetized with urethane and a-chloralose (800 mg/kg and 60 mg/kg, i.p., respectively). The tail artery was cannulated, blood pressure (BP) and heart rate (HR) were recorded on a polygraph. Body temperature was maintained at 37-38 °C. The head of the animal was mounted on a stereotaxic apparatus in a prone position and flexed with an angle of 45 ° . The dorsal surface of the lower brainstem was exposed by a limited occipital craniotomy. A spinal catheter (PE10 tubing) was inserted through an incision in the atlanto-occipital membrane
and advanced in the spinal subarachnoid space 6.8-7.0 cm caudally to the Tlo-Tll vertebral levels, identified by infusion of 1% pontamine sky blue into the injecting site at the termination of the experiment. A pair of stainlesssteel tubules with external diameter of 0.13 mm were guided into the bilateral rVLM, which extends 2.2-2.5 mm rostral to the obex, 1.5-1.7 m m lateral to the midline and within 0.2-0.5 mm from the ventral surface of the medulla. Drugs were dissolved in normal saline (pH 7.3-7.4) and administered over 0.5-1.0 min in a volume of 10/~1 for i.t. infusion and in an amount of 0.1/~1 within 10 s for rVLM microinjection. At the end of each experiment, 1% pontamine sky blue was infused to the microinjecting sites. The brain was removed and fixed in 10% formaline for 4-10 days. The frozen sections (50 /~m) were made, from which the injecting sites were identified with reference to Fifkova and Marsala's atlas 6. Data are presented as the means + S.E.M. Statistical analysis was performed by the paired and Student t-tests. The effect of i.t. infusion of SP antagonist, (o-Pro2, D-TrpT,9)-SP (20 ~g) was studied on 8 rats and the results were presented in Fig. 1. Prior to SP antagonist administration, the baseline mean blood pressure (MBP) and H R were 103 + 5 m m H g and 410 + 13 beats/min, respectively. The spinal infusion of SP antagonist usually caused an initial depressor and then pressor response which lasted for 3-4 min, followed by a prolonged depressor response. By 15-20 min, the depressor phase had reached its nadir, averaging 59 + 3mmHg (Fig. 1A). A slight but not significant bradycardia was observed
Correspondence: P. Li, Department of Physiology, Shanghai Medical University, Yi Xue Yuan Road 138, Shanghai, 200032, People's Republic of China. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
318
A 12C m
9(3
E ~E
6C
i1_ m E
B
A
B
I 50I" 3 40 E 30 "6 2O
_
g
3(3
c 5£ E -~ 4(: ~ 3C T ~6 2C
.,,
g g lC
lo
6
o
~
5
c
-1C
C BP(mmHg)
10
15
30
60 (rnin) C
m
i_.~=
I
MBP(mrnHg)
HR(btmin) 480[
AP 9 i
280L'
-
.
5
~
n,,,(~tk
-
280L'
t i.t. SP ant. (20#g)
' 1 min'
Fig. 1. A + B: effect of SP antagonist (20/~g, i.t.) on MBP and HR 15 min after infusion. Open bars: preinfusion. Vertically hatched bars: normal saline infused i.t. (n = 7); solid bars: SP antagonist infused i.t. (n = 8); cross-hatched bars: SP antagonist infused i.v. (n = 6): **P < 0.01, compared to the preinfusion values. C: time course of the cardiovascular effects elicited by i.t. administration of SP antagonist.
(Fig. 1B). Sixty rain later, M B P (86 -+ 4 m m H g ) r e t u r n e d closely to the control level. A r e p r e s e n t a t i v e e x p e r i m e n t
A BP ( mm Hg )
MBP(mrnHg)
HR(b/rain)
B
BP(mmHg) MBP(mmHg)
HR(b/min
t Microinj. car bachol
q1mtn' (25 ng/ site )
Fig. 2. The inhibitory effect of i.t. infusion of SP antagonist on the carbachol-induced cardiovascular responses from the rVLM. A: carbachol responses 15 min after i.t. infusion of normal saline. B: carbachol responses 15 min after i.t. infusion of SP antagonist (20 ~,g).
AP 1
0
.
0
~ IlmmI
Fig. 3. A + B: cardiovascular responses to carbachol (25 rig/site) microinjected into bilateral rVLM 15 rain after normal saline i.t. (open bars, n = 8), SP antagonist i.t. (solid bars, n = 8), SP antagonist i.v. (vertically hatched bars, n = 6) or bleeding (cross-hatched bars, n = 6). **: P < 0.01, comparing normal saline i.t. and SP antagonist i.t.; • and • A : P < 0.05 and P < 0.01, comparing SP antagonist i.t. and SP antagonist i.v.; I l l : P < 0.01, comparing SP antagonist i.t. and bleeding. C: the location of microinjection sites in rVLM. Solid circles: carbachol injecting sites where the cardiovascular responses were elicited; open circles: carbachol injecting sites where no significant response was evoked; nVII, facial nucleus; PGL, nucleus paragigantoceUularis lateralis; RG, nucleus reticularis gigantocellularis; RPG, nucleus paragigantocellularis; V, trigeminal tract.
was s h o w n in Fig. IC. T h e s a m e dose of SP a n t a g o n i s t a d m i n i s t e r e d i n t r a v a n e o u s l y (i.v., n = 6) had n o significant effect o n B P a n d H R , n o r did a c o n t r o l spinal infusion of n o r m a l saline (n = 7). I n 3 a n i m a l s , respiration was c o n t i n u o u s l y m o n i t o r e d by r e c o r d i n g the e l e c t r o m y o g r a m of the d i a p h r a g m to o b s e r v e the possible changes of respiration after the a d m i n i s t r a t i o n of SP a n t a g o n i s t a n d it was s h o w n that t h e r e was n o o b v i o u s change in b r e a t h i n g a l t h o u g h a r e m a r k a b l e i n f l u e n c e o n B P was elicited at the s a m e time. T h e effect of i.t. i n j e c t i o n of SP a n t a g o n i s t o n the cardiovascular r e s p o n s e s elicited by m i c r o i n j e c t i o n of carbachol, A c h agonist, into bilateral r V L M was investigated further. Fig. 2 s h o w e d a typical result of the b l o c k a d e of the c a r b a c h o l - i n d u c e d r e s p o n s e s by SP antagonist. F i f t e e n m i n after i.t. infusion of n o r m a l saline, m i c r o i n j e c t i o n of carbachol (25 ng/site) into r V L M p r o d u c e d a m a r k e d increase in M B P which p e a k e d in 2 - 3 m i n , a n d a short-lasting b r a d y c a r d i a followed by a g r a d u a l increase in H R which r e a c h e d its
319 peak in 6 - 8 min (33 + 3 m m H g and 19 + 6 beat/min, n = 8). The pressor and tachycardic responses lasted for 25-30 min. A control injection of normal saline into the r V L M (n = 7) had no significant effect on the cardiovascular activity. When animals were pretreated spinally with SP antagonist (20 /zg) 15 min before carbachol injection, the cardiovascular responses from r V L M were blocked profoundly (6 + 1 m m H g and - 8 + 4 beat/min, n = 8). In 5 additional animals, the influence of SP antagonist on the cardiovascular responses induced by intra-rVLM injection of carbachol was observed 60 min after i.t. infusion and it was found that the inhibitory effect on pressor response was still obtained (24 + 3 m m H g , P < 0.05) but less than before. However, an i.v. application of SP antagonist with the same dose had no such effect. To exclude the possible influence of severe hypotensive state induced by i.t. infusion of SP antagonist on the responsibility of central nervous system, the resting MBP was lowered to 60-70 m m H g by withdrawing blood of 3-6 ml from the tail artery. In such a case, the carbachol-induced pressor response still existed and an increase in tachycardic response was observed (Fig. 3A,B). The microinjecting sites in the rVLM examined histologically were mostly located in the lateral part of the nucleus paragigantocellularis lateralis with small spread in the facial nucleus, rostral to the obex level from which the pressor and tachycardic response were elicited by carbachol injection (solid circles). No significant response of cardiovascular activities was observed when carbachol was administered in the adjacent sites which were in the facial nucleus and the lateral part of the nucleus reticularis gigantocellularis (open circles, Fig. 3C). Evidence has accumulated that there is a high dense distribtution of SP terminals and receptors in IML of thoracic spinal cord 2"3'12. Iontophoretic or i.t. application of SP increased the firing rate of sympathetic preganglionic neurons and BP accompanied by an increase of plasma epinephrine and norepinephrine 1'4'8'1°'19, while i.t. administration of SP antagonist produced a hypotensive response 11'13, suggesting a tonic sympathoexcitatory role for spinal cord SP. The first part of our present
experiment (Fig. 1) supported this hypothesis. Carbachol, as a cholinergic agonist, has been applied centrally for the studies of neural control of cardiovascular functions both under conscious and anesthetized conditions TM. In our lab, only anesthetized animals were used and the pressor effect of intra-rVLM injection of carbachol was similar as in the conscious one, suggesting that urethane-chloralose anesthesia has no significant effect on the cholinergic cardiovascular responses 2°. Loewy et a1.13 showed that an excitatory cardiovascular effect elicited by application of kainic acid onto r V L M could be blocked by spinal administration of SP antagonist, indicating that SP is probably an important transmitter in bulbospinal sympathoexcitatory pathways. Although it was believed that the r V L M may be under tonic excitatory cholinergic control 16'18, the transmission of its excitatory effect in the bulbospinal pathways still remains a problem. In the present study, it was shown that the pressor and tachycardic responses induced by microinjection of carbachol into bilateral r V L M could be blocked by spinal application of SP antagonist. Such effect did not occur when pretreated with i.v. application of the same dose of SP antagonist, which excluded the possibility of peripheral leakage of the drug and suggested a central blockade action of SP antagonist on SP receptors in the spinal cord. It has been demonstrated that (D-Argl, D-Trp7,9 , Leu11)-SP and (D-Pro2, D-Trp7 ' 9)-SP exerted marked neurotoxic actions when given into the spinal cord, including persistent paralysis and neuronal necrosis 7"15. Such neurotoxicity was not the nature of the antagonism seen in our experiment since the inhibitory effect of SP antagonist was reversible with time and the neurotoxic actions affect mostly the motoneurons in the ventral horns TM. However, the data presented here cannot rule out the possibility of the blockade of transmission from SP interneurons or other SP neuron projections to IML.
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The assistance of Ms Zhu Wen-he is greatly appreciated. This work was in part supported by the Science Fund of the Ministry of Health.
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