Brain Research, 520 (1990) 141-150 Elsevier
141
BRES 15597
Selective sensitization by L-glutamate of baroreflex-mediated bradycardia following microinjection into the rostral ventrolateral medulla Xin Zhang, Abdel A. Abdel-Rahman and Wallace R. Wooles Department of Pharmacology, East Carolina University, School of Medicine, Greenville, NC 27858 (U.S.A.) (Accepted 12 December 1989)
Key words: Baroreflex; Blood pressure; Heart rate; Glutamate; Sympathetic efferent discharge; Phenylephrine; Atropine; Propranolol
This study examined the role of L-glutamate receptors in different sites in the rostrai ventrolateral medulla (RVL) on baseline mean arterial pressure (MAP), heart rate (HR), sympathetic efferent discharge (SED) as well as baroreflex control of HR and SED. Depending on the site, the hemodynamic responses varied from an increase to a decrease in MAP which was accompanied by a similar or an opposite change in HR; the change in SED correlated positively with the change in MAP. Because injection of the test dose of glutamate (5 nM) into the deepest portion of the RVL produced the most pronounced increases in MAP, which was accompanied by a brisk bradycardia, we decided to investigate the nature of these responses. Dose-related sympathoexcitatory, pressor and bradycardic effects were obtained in response to 1, 3, 5 and 10 nM glutamate. The glutamate antagonist, glutamate diethylester (GDEE) abolished these responses and decreased baseline MAP, HR and SED. That glutamate-evoked bradycardia was baroreceptor-mediated was supported by: (1) the bradycardia was not only eliminated but was also converted to a small but significant tachycardic response in sinoaortic denervated rats and following cardiac muscarinic blockade; (2) glutamate substantially sensitized the baroreceptor HR and had no effect on SED response when tested by phenylephrine; and (3) GDEE abolished the sensitizing action of glutamate on the baroreflex control of HR. We conclude that glutamatergic pathways in the deepest portion of the RVL are tonically active and subserve sympathoexcitatory, pressor and tachycardic effects. Also, glutamate differentially sensitizes the baroreceptor reflex pathways that control HR by mainly activating the vagal component of the reflex, an effect which masks its tachycardic effect. INTRODUCTION It is widely a c c e p t e d that the m e d u l l a contains structure(s) which control cardiovascular functions 1'9'14. Electrical stimulation of o r g l u t a m a t e injections into the v e n t r o l a t e r a l m e d u l l a ( V L M ) in the a r e a of the paragigantocellularis nucleus e v o k e d p r e s s o r responses 12'21'23. O n the o t h e r h a n d inactivation of the V L M by t e t r o d o toxin causes m a r k e d decreases in systemic b l o o d pressure 2°. Since the V L M contains C1 e p i n e p h r i n e neurons, Reis et al. 2° suggested the C1 a r e a of the V L M is essential for the m a i n t e n a n c e of systemic b l o o d pressure. N e u r o n s in the rostral v e n t r o l a t e r a l m e d u l l a ( R V L ) a r e also involved in the baroreflex control of b l o o d pressure. F i b e r s from the nucleus tractus solitarius ( N T S ) , the first n e u r a l relay of the baroreflex, pass to the R V L 24'25. B r o w n a n d G u y e n e t 5 and Ross et al. 22 have shown that in the rat the most rostral portion of the V L M contains a population of cells which provide a tonic excitatory drive to v a s o m o t o r preganglionic neurons and which function as the descending limb of the sympathetic b a r o r e f l e x . Blessing 3 has shown that bilateral injection of
the 7 - a m i n o b u t y r i c acid ( G A B A ) antagonist bicuculline caused a d o s e - d e p e n d e n t d e c r e a s e in the d e p r e s s o r and s y m p a t h o - i n h i b i t o r y r e s p o n s e to local injection of Lg l u t a m a t e into the caudal m e d u l l a further d e m o n s t r a t i n g a role for the R V L in m o d u l a t i n g p e r i p h e r a l s y m p a t h e t i c v a s o m o t o r activity. A d d i t i o n a l evidence suggesting the R V L is involved in the b a r o r e c e p t o r reflex was p r o v i d e d by the finding that the u p t a k e o f 2-deoxyglucose in the rostral p o r t i o n of the V L M was i n c r e a s e d during activation of arterial b a r o r e c e p t o r afferent fibers in the rat 7. Recently, Chai et al. 6 have shown that in the rat, rabbit and cat pressor responses could b e elicited in all species with either electrical stimulation o r by direct application of g l u t a m a t e into the dorsal m e d i a l and v e n t r o l a t e r a l p o r t i o n s o f the medulla. Chai et al. 6 used microinjections of g l u t a m a t e to d e t e r m i n e if the p r e s s o r responses o b t a i n e d were due to fibers of passage o r to the presence of cell b o d i e s in the areas. G o r d o n and M c C a n n 12 have also used m i c r o i n j e c t i o n s of g l u t a m a t e to m a p the pressor regions of the caudal v e n t r o l a t e r a l medulla. T h e y f o u n d one a r e a which gave consistent p r e s s o r responses but h e a r t rate changes were inconsis-
Correspondence: A.A. Abdel-Rahman, Department of Pharmacology, East Carolina University School of Medicine, Greenville, NC 27858, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
142 using a bipolar platinum electrode under a mineral oil pool as described in our previous studies 36'38. The recorded spikes were amplified by a preamplifier (Grass P511), and visualized on an oscilloscope. The output was also fed to a Spike Processor (Digitimer Model D130) with a loud speaker that measured the frequency of the impulses that exceeded a preset lower window level which was set above the noise level. Digital output (spikes/5 s) was read off the counter and the output was displayed in analog form as a time-frequency histogram on a third channel along with blood pressure and heart rate. For microinjections into the RVL, the rat was placed in a stereotaxic apparatus (David Kopf Instruments) with the bite bar 12 mm below the interaural line. The occipital bone and atlantooccipital membrane were exposed after retraction of the dorsal neck muscles. After separation from the dura the occipital bone was removed. The atlanto-occipital membrane was cut, and the dura overlying the cerebellum was retracted. The floor of the fourth ventricle was exposed as far rostrally as the caudal aspect of the inferior cerebellar peduncle. A single glass pipette, outside tip diameter 50/~m, was carried in a stereotaxic micromanipulator and connected by PE10 polyethylene tubing to a Hamilton microsyringe to make microinjections. The coordinates were approximately 2.0 mm rostral to the calamus scriptorius, 2.0 mm lateral to the midline, and 2.65-3.8 mm below the floor of the fourth ventricle. The drugs were injected into the medulla via the glass micropipettes. The volume of the injections was 0.1 /~1, the interval between two microinjections was 5 min or longer. All microinjected drugs, i.e. L-glutamate monosodium and L-glutamate diethylester were dissolved in the artificial cerebrospinal fluid (ACSF). Microinjection sites were identified at the end of each experiment by injection of the same volume of Fast green dye in the same location. The brain areas of interest were removed and immediately frozen over dry ice. Forty-micron sections were cut on a sliding microtome. Sections were mounted on microscope slides and stained with Cresyl violet or Neutral red. The location of all microinjections was carefully mapped.
t e n t . In t h e p r e s e n t s t u d y w e e v a l u a t e d b l o o d p r e s s u r e , h e a r t r a t e ( H R ) a n d also s y m p a t h e t i c e f f e r e n t d i s c h a r g e ( S E D ) r e s p o n s e s to m i c r o i n j e c t i o n o f g l u t a m a t e i n t o t h e RVL. We found that application of glutamate to areas of the
RVL
medulla
produced
a pressor
or
depressor
response which was associated with a similar or opposite c h a n g e in t h e H R
r e s p o n s e ; t h e c h a n g e in S E D
was
a l w a y s d i r e c t l y r e l a t e d t o t h e c h a n g e in b l o o d p r e s s u r e a n d , in f a c t , p r e c e d e d it. T h e r e is a n a r e a o f t h e R V L , near
the
ventral
pronounced
surface,
which
produced
the
most
i n c r e a s e in S E D a n d b l o o d p r e s s u r e w h i c h
was a s s o c i a t e d w i t h a s e v e r e b r a d y c a r d i a .
We investi-
g a t e d if this s e v e r e b r a d y c a r d i a was r e f l e x l y m e d i a t e d . MATERIALS AND METHODS
Preparation Sprague-Dawley rats (male, 300-400 g) anesthetized with achloralose (150 mg/kg i.v.) were used throughout. The arterial pressure was recorded via a femoral catheter inserted in the aorta, and the HR was computed from the arterial pressure wave. The jugular vein was cannulated for intravenous injection. After the trachea was cannulated, the rats were paralyzed with tubocurarine chloride (0.5 mg/kg i.v.) and artificially ventilated. This dose of tubocurarine had no significant effect on baseline levels of the measured variables, nor on baroreflex responses. We have also reported that arterial blood gases and pH, when routinely monitored, remained within normal range when a very similar experimental protocol was followed 37. The greater splanchnic nerve was exposed through a midline abdominal incision. The nerve was cut at its entrance to the celiac ganglion and approximately 18 mm of the proximal end of the nerve was cleared from surrounding tissues. The abdominal incision was closed and the nerve was then accessed through a retroperitoneal opening which allowed recording of sympathetic nerve activity from the rat after placing it in the stereotaxic head holder. Splanchnic nerve activity was recorded from the central end of the cut nerve
Protocol The baroreceptor HR and SED responses were measured before and after central administration of L-glutamate or its antagonist, glutamate diethylester (GDEE), or the vehicle (ACSF). This was achieved by injecting graded doses of phenylephrine (PE) 0.25-16
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141
BRES 15597
Selective sensitization by L-glutamate of baroreflex-mediated bradycardia following microinjection into the rostral ventrolateral medulla Xin Zhang, Abdel A. Abdel-Rahman and Wallace R. Wooles Department of Pharmacology, East Carolina University, School of Medicine, Greenville, NC 27858 (U.S.A.) (Accepted 12 December 1989)
Key words: Baroreflex; Blood pressure; Heart rate; Glutamate; Sympathetic efferent discharge; Phenylephrine; Atropine; Propranolol
This study examined the role of L-glutamate receptors in different sites in the rostrai ventrolateral medulla (RVL) on baseline mean arterial pressure (MAP), heart rate (HR), sympathetic efferent discharge (SED) as well as baroreflex control of HR and SED. Depending on the site, the hemodynamic responses varied from an increase to a decrease in MAP which was accompanied by a similar or an opposite change in HR; the change in SED correlated positively with the change in MAP. Because injection of the test dose of glutamate (5 nM) into the deepest portion of the RVL produced the most pronounced increases in MAP, which was accompanied by a brisk bradycardia, we decided to investigate the nature of these responses. Dose-related sympathoexcitatory, pressor and bradycardic effects were obtained in response to 1, 3, 5 and 10 nM glutamate. The glutamate antagonist, glutamate diethylester (GDEE) abolished these responses and decreased baseline MAP, HR and SED. That glutamate-evoked bradycardia was baroreceptor-mediated was supported by: (1) the bradycardia was not only eliminated but was also converted to a small but significant tachycardic response in sinoaortic denervated rats and following cardiac muscarinic blockade; (2) glutamate substantially sensitized the baroreceptor HR and had no effect on SED response when tested by phenylephrine; and (3) GDEE abolished the sensitizing action of glutamate on the baroreflex control of HR. We conclude that glutamatergic pathways in the deepest portion of the RVL are tonically active and subserve sympathoexcitatory, pressor and tachycardic effects. Also, glutamate differentially sensitizes the baroreceptor reflex pathways that control HR by mainly activating the vagal component of the reflex, an effect which masks its tachycardic effect. INTRODUCTION It is widely a c c e p t e d that the m e d u l l a contains structure(s) which control cardiovascular functions 1'9'14. Electrical stimulation of o r g l u t a m a t e injections into the v e n t r o l a t e r a l m e d u l l a ( V L M ) in the a r e a of the paragigantocellularis nucleus e v o k e d p r e s s o r responses 12'21'23. O n the o t h e r h a n d inactivation of the V L M by t e t r o d o toxin causes m a r k e d decreases in systemic b l o o d pressure 2°. Since the V L M contains C1 e p i n e p h r i n e neurons, Reis et al. 2° suggested the C1 a r e a of the V L M is essential for the m a i n t e n a n c e of systemic b l o o d pressure. N e u r o n s in the rostral v e n t r o l a t e r a l m e d u l l a ( R V L ) a r e also involved in the baroreflex control of b l o o d pressure. F i b e r s from the nucleus tractus solitarius ( N T S ) , the first n e u r a l relay of the baroreflex, pass to the R V L 24'25. B r o w n a n d G u y e n e t 5 and Ross et al. 22 have shown that in the rat the most rostral portion of the V L M contains a population of cells which provide a tonic excitatory drive to v a s o m o t o r preganglionic neurons and which function as the descending limb of the sympathetic b a r o r e f l e x . Blessing 3 has shown that bilateral injection of
the 7 - a m i n o b u t y r i c acid ( G A B A ) antagonist bicuculline caused a d o s e - d e p e n d e n t d e c r e a s e in the d e p r e s s o r and s y m p a t h o - i n h i b i t o r y r e s p o n s e to local injection of Lg l u t a m a t e into the caudal m e d u l l a further d e m o n s t r a t i n g a role for the R V L in m o d u l a t i n g p e r i p h e r a l s y m p a t h e t i c v a s o m o t o r activity. A d d i t i o n a l evidence suggesting the R V L is involved in the b a r o r e c e p t o r reflex was p r o v i d e d by the finding that the u p t a k e o f 2-deoxyglucose in the rostral p o r t i o n of the V L M was i n c r e a s e d during activation of arterial b a r o r e c e p t o r afferent fibers in the rat 7. Recently, Chai et al. 6 have shown that in the rat, rabbit and cat pressor responses could b e elicited in all species with either electrical stimulation o r by direct application of g l u t a m a t e into the dorsal m e d i a l and v e n t r o l a t e r a l p o r t i o n s o f the medulla. Chai et al. 6 used microinjections of g l u t a m a t e to d e t e r m i n e if the p r e s s o r responses o b t a i n e d were due to fibers of passage o r to the presence of cell b o d i e s in the areas. G o r d o n and M c C a n n 12 have also used m i c r o i n j e c t i o n s of g l u t a m a t e to m a p the pressor regions of the caudal v e n t r o l a t e r a l medulla. T h e y f o u n d one a r e a which gave consistent p r e s s o r responses but h e a r t rate changes were inconsis-
Correspondence: A.A. Abdel-Rahman, Department of Pharmacology, East Carolina University School of Medicine, Greenville, NC 27858, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
142 using a bipolar platinum electrode under a mineral oil pool as described in our previous studies 36'38. The recorded spikes were amplified by a preamplifier (Grass P511), and visualized on an oscilloscope. The output was also fed to a Spike Processor (Digitimer Model D130) with a loud speaker that measured the frequency of the impulses that exceeded a preset lower window level which was set above the noise level. Digital output (spikes/5 s) was read off the counter and the output was displayed in analog form as a time-frequency histogram on a third channel along with blood pressure and heart rate. For microinjections into the RVL, the rat was placed in a stereotaxic apparatus (David Kopf Instruments) with the bite bar 12 mm below the interaural line. The occipital bone and atlantooccipital membrane were exposed after retraction of the dorsal neck muscles. After separation from the dura the occipital bone was removed. The atlanto-occipital membrane was cut, and the dura overlying the cerebellum was retracted. The floor of the fourth ventricle was exposed as far rostrally as the caudal aspect of the inferior cerebellar peduncle. A single glass pipette, outside tip diameter 50/~m, was carried in a stereotaxic micromanipulator and connected by PE10 polyethylene tubing to a Hamilton microsyringe to make microinjections. The coordinates were approximately 2.0 mm rostral to the calamus scriptorius, 2.0 mm lateral to the midline, and 2.65-3.8 mm below the floor of the fourth ventricle. The drugs were injected into the medulla via the glass micropipettes. The volume of the injections was 0.1 /~1, the interval between two microinjections was 5 min or longer. All microinjected drugs, i.e. L-glutamate monosodium and L-glutamate diethylester were dissolved in the artificial cerebrospinal fluid (ACSF). Microinjection sites were identified at the end of each experiment by injection of the same volume of Fast green dye in the same location. The brain areas of interest were removed and immediately frozen over dry ice. Forty-micron sections were cut on a sliding microtome. Sections were mounted on microscope slides and stained with Cresyl violet or Neutral red. The location of all microinjections was carefully mapped.
t e n t . In t h e p r e s e n t s t u d y w e e v a l u a t e d b l o o d p r e s s u r e , h e a r t r a t e ( H R ) a n d also s y m p a t h e t i c e f f e r e n t d i s c h a r g e ( S E D ) r e s p o n s e s to m i c r o i n j e c t i o n o f g l u t a m a t e i n t o t h e RVL. We found that application of glutamate to areas of the
RVL
medulla
produced
a pressor
or
depressor
response which was associated with a similar or opposite c h a n g e in t h e H R
r e s p o n s e ; t h e c h a n g e in S E D
was
a l w a y s d i r e c t l y r e l a t e d t o t h e c h a n g e in b l o o d p r e s s u r e a n d , in f a c t , p r e c e d e d it. T h e r e is a n a r e a o f t h e R V L , near
the
ventral
pronounced
surface,
which
produced
the
most
i n c r e a s e in S E D a n d b l o o d p r e s s u r e w h i c h
was a s s o c i a t e d w i t h a s e v e r e b r a d y c a r d i a .
We investi-
g a t e d if this s e v e r e b r a d y c a r d i a was r e f l e x l y m e d i a t e d . MATERIALS AND METHODS
Preparation Sprague-Dawley rats (male, 300-400 g) anesthetized with achloralose (150 mg/kg i.v.) were used throughout. The arterial pressure was recorded via a femoral catheter inserted in the aorta, and the HR was computed from the arterial pressure wave. The jugular vein was cannulated for intravenous injection. After the trachea was cannulated, the rats were paralyzed with tubocurarine chloride (0.5 mg/kg i.v.) and artificially ventilated. This dose of tubocurarine had no significant effect on baseline levels of the measured variables, nor on baroreflex responses. We have also reported that arterial blood gases and pH, when routinely monitored, remained within normal range when a very similar experimental protocol was followed 37. The greater splanchnic nerve was exposed through a midline abdominal incision. The nerve was cut at its entrance to the celiac ganglion and approximately 18 mm of the proximal end of the nerve was cleared from surrounding tissues. The abdominal incision was closed and the nerve was then accessed through a retroperitoneal opening which allowed recording of sympathetic nerve activity from the rat after placing it in the stereotaxic head holder. Splanchnic nerve activity was recorded from the central end of the cut nerve
Protocol The baroreceptor HR and SED responses were measured before and after central administration of L-glutamate or its antagonist, glutamate diethylester (GDEE), or the vehicle (ACSF). This was achieved by injecting graded doses of phenylephrine (PE) 0.25-16
°tA
-10 -20
"~ 50
50
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o
E
~
g
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2.0
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-20
~-30 ZIZ ~ c>
,o ~ o o
-10
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6
tto z:o 3.ore
