Brain Research, 563 (1991) 203-208 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/911503.50 ADONIS 000689939117121Y
203
BRES 17121
Enkephalins, substance P and acetylcholine microinjected into the nucleus ambiguus elicit vagal bradycardia in rats S.K. Agarwal and F.R. Calaresu Department of Physiology, University of Western Ontario, London, Ont. (Canada) (Accepted 11 June 1991)
Key words: Cardiovascular regulation; Acetylcholine; Peptide; Microinjection
Little is known about putative transmitters in the nucleus ambiguus (NA) mediating parasympathetic control of the heart, although Metenkephalin (m-ENK), Leu-enkephalin (I-ENK), substance P (SP) and acetylcholine (Ach) have been detected in the cell bodies and fibers of this nucleus. The effects of these substances on arterial pressure (AP) and heart rate (HR) were studied by microinjecting them (4-20 nl) into the NA. Experiments were done in 26 spinal (high cervical) rats that were anesthetized with urethane and artificially ventilated. L-Glutamate (GLU) was microinjected into the right NA to identify the location of cell bodies from which decreases in HR and AP could he elicited, m-ENK, 1-ENK, SP or Ach was then microinjected into these sites. Microinjection of 1 nmol of GLU elicited significant decreases in HR (-72.2 +- 9.7 bpm, n = 15) which were not accompanied by significant decreases in mean AE Microinjection of m-ENK (15-200 pmol; n = 7), 1-ENK (15-200 pmol; n = 6), SP (0.9-15 pmol; n = 7) and Ach (2.0-20 pmol; n = 7) into the NA decreased HR in a dose-dependent manner but did not affect AE The magnitudes of HR responses to m-ENK, 1-ENK, SP and Ach were smaller but of longer duration than the changes in HR to microinjection of GLU. These results suggest a physiological role for GLU, enkephalins, SP and Ach in the vagal control of HR mediated by the NA. INTRODUCTION Anatomical and electrophysiological experiments have demonstrated in all mammalian species studied that the ventrolateral division of the loose and external formation of the nucleus ambiguus (NA) is the main site of origin of cardioinhibitory neurons (see reviews 14'18) as well as of axons to other visceral organs ~'18. Neurons in the N A are excited by stimulation of the carotid sinus nerve and by increased sinus pressure 4'17 and can be antidromically activated by electrical stimulation of the cardiac branches of the vagus 4'19'2°. However, although the functional role of the N A in the central control of the cardiovascular system has been established, little is known about neurotransmitters involved in controlling activity of N A neurons. The role of putative neurotransmitters in the function of the central nervous system (CNS) has been investigated by either injections into the ventricular system or by microinjections into discrete CNS sites. Many peptides with a functional role in central cardiovascular regulation have been found (see reviews 8'25) using the first technique. For instance, enkephalins injected into the ventricular system exert potent effects on heart rate (HR), arterial pressure (AP) and respiration. Moreover
enkephalins are present in regions in the brainstem known to be involved in cardiovascular control 13'16. More specifically, the N A has been shown to contain dense binding sites for enkephalins 5'26 and transection of the vagus nerves results in dramatic loss of these binding sites in the N A 26. Finally, microinjection of the morphine-like agents fentanyl and Met-enkephalanamide into the N A of dogs has been shown to elicit decreases in H R 16. Substance P (SP) and acetylcholine (Ach) have also been investigated as putative transmitters in the N A 2A2. SP-like immunoreactivity and choline acetyltransferase immunoreactive cell bodies have been demonstrated in the vicinity of the N A 2'12. Moreover, microintophoretic application of Ach inhibits the firing of cardioinhibitory neurons in the N A 1°'11. However, there is little information about the precise functional role of these putative transmitters (enkephalins, SP and Ach) in the control of the cardiovascular system by the NA. In the present study enkephalins, SP and Ach were microinjected into the N A to study their effects on H R and A P in spinal rats. The spinal transection was done at the C1 level to eliminate the descending pathways that control the sympathetic outflow to the heart, thereby re-
Correspondence: S.K. Agarwal, t)epartment of Physiology, The University of Western Ontario, London, Ont., Canada N6A 5C1. Fax: (1) (519) 661 3827.
204 stricting t h e effects o f m e d u l l a r y s t i m u l a t i o n to a c t i v a t i o n of t h e v a g u s n e r v e s to t h e h e a r t . T h e p e p t i d e s a n d A c h w e r e i n j e c t e d i n t o N A sites d e m o n s t r a t e d
to h a v e car-
dioinhibitory function by microinjections of L-glutamate (GLU).
MATERIALS AND METHODS
General procedure Studies were done in adult male Wistar rats (n = 26, 250-350 g, Charles River, Montreal, Canada), anaesthetized with urethane (Sigma, St. Louis, MO, 1.4 g/kg, i.p. initially and 0.25 g/kg supplements as required). The trachea was cannulated and the animal was artificially ventilated with room air using a small animal ventilator (Harvard Apparatus, model 683). The femoral artery and vein were cannulated. The arterial cannula was connected to a pressure transducer (Century Technology, Inglewood, CA, model CP 01) that was connected to a Grass polygraph (model 79C) for continuous recording of AP. A Grass tachograph (7 P44B) triggered by the arterial pressure pulse was used to monitor HR. The animal was placed in a stereotaxic apparatus, with the bite bar 20 mm below the interaural line. The medulla was exposed by retracting the dorsal neck muscles, incising the atlantooccipital membrane and removing part of the occipital bone and the dura. The spinal cord of the animal was then transected at the C1 level. To maintain AP within the physiological range phenylephrine (PE; Sigma) in physiological saline (2 mg/ml) was infused into the venous cannula, initially at the rate of 700/APa and then at 250/A/h. These amounts of PE were found to maintain mean AP at 96.2 -+ 3.9 mmHg (range 87-105 mmHg; n = 26). Animals that dit not have a steady level of AP over a period of several hours were not included in the results. Rectal temperature was maintained at 37.5 -+ 0.5 °C with a thermostatically controlled heating blanket.
Pressure microin]ection L-Glutamate (Sigma, 0.3-1.25 nmol), Met-enkephalin (Sigma, 15-200 pmol), Leu-enkephalin (Sigma, 15-200 pmol), substance P (Sigma, 0.9-15 pmol) and acetylcholine (Sigma, 2-20 pmol) dissolved in phosphate buffered saline (PBS, pH 7.4) were pressure microinjected (4-20 nl) through multibarrelled glass mieropipettes pulled from Socorex 5 ktl glass capillary tubing (Socorex 851-5, Terochem Laboratories, Mississauga, Ont., Canada). Long shanks were drawn to minimize tissue distortion and the tips were broken to an external diameter of -50/~m. The pipettes were inclined 20 ° from the vertical in the sagittal plane with the tip pointing rostrally. The stereotaxic coordinates used for the placement of the tip in the fight NA were 0.2-0.6 mm caudal to the obex 1.8-2.0 mm lateral and 2.0-2.3 mm below the dorsal surface of the medulla. Volumes of injections were measured directly by monitoring the movement of the fluid meniscus through a 40× microscope fitted with an ocular scale that allowed a resolution of 1 nl. At the start of each experiment the NA was explored for a site where injection of 4-10 nl of 0.15 M GLU consistently elicited decreases in HR. After such a site in the NA was found one of the four substances was microinjected. A third barrel of the micropipette contained PBS used to deliver 20 nl of control saline. Very rarely small (5-10 bpm) changes in HR were elicited by microinjections of saline; in these cases the results were not included in our analysis of the data.
Histology Microinjection sites were marked for histological verification at the end of each experiment. The mieroinjection pipette was retracted from the brain, emptied, filled with 25% India ink solution in saline, reinserted to the same depth and 20 nl of India ink were injected. At the end of the experiments the animals were perfused with 50 ml of PBS followed by 50 ml of a 10% formalin solution in PBS. The brains were removed and stored in formalin for 3-4 days. Frozen transverse sections (50 ~m) were cut and stained with thio-
nin. Injection sites were mapped o n the drawings of transverse sections of the rat brain from an atlas za.
Statistics Mean changes in HR and MAP (i.e., the difference between the peak changes in HR or MAP and the level of these variables before microinjection) were compared by Studenrs t-tests. For comparison of HR responses with different doses of putative transmitters analysis of variance (ANOVA) was performed followed by pairwise comparison by a Least Significant Difference method (LSD method) 21. For dose-response curves percentage changes in HR vs doses were plotted. Linear regression analysis was used to determine the line of best fit for plotted points in the linear portion of the dose-response curve. The probability level taken to indicate a significant change was P < 0.05 for all statistical tests. All data in text and figures are expressed as means + S.E.M.
RESULTS
Microinjection of L-glutamate Microinjection of GLU
(1 n m o l ) i n t o t h e r i g h t N A
e l i c i t e d a m a x i m a l d e c r e a s e in H R o f - 7 2 . 2 -+ 9.7 b p m f r o m a b a s e line o f 389.2 -+ 8.1 b p m (n = 15). A n exa m p l e o f a r e s p o n s e to G L U is s h o w n in Fig. 1A. T h e s e effects w e r e g e n e r a l l y r a p i d in o n s e t ( m e a n 2.3 -+ 0.2 s), p e a k r e s p o n s e w a s r e a c h e d in 12.7 --- 2.9 s a n d t h e dur a t i o n w a s 121 -+ 17.8 s. T h e d o s e - r e s p o n s e
°**I
HR |bpm) 4 0 0 ( / 200 ~ 200 r
GLU
I**I
-
~
A
B
C
M~NK
SALIN!
H
c u r v e for
i
I
0
Stain
6001
D
400 [
~
/ 200L . . . . . . . . . . .
[
E
I i .......................................
L--INK
SP
1oo[
I
0
II
.....
sol
600 r
F
400 [ 200 f
Ach
100 0 Fig. I. Tracings of arterial pressure (AP) and heart rate (HR) during microinjections of L-glutamate, phosphate-buffered saline, Metenkephalin, Leu-enkephalin, substance P and acetylcholine into the right ambiguus of a rat with spinal cord transection. Marks on time scale indicate the time of microinjections. A: L-glutamate (1.2 nmol), B: phosphate-buffered saline (20 nl). C: Met-enkephafin (75 pmol). D: Leu-enkephalin (75 prhol). E: substance P (6.3 pmol). F: acetylcholine (20 pmol).
205 0
n-
15
r - -0.0i540
-10
q;
g..~ t 8 N
-3o!
1D) but had no effect on AP. The dose-response curve for H R responses to I-ENK microinjection is shown in Fig. 3B. The threshold dose for eliciting changes in H R was 15 pmol and the two doses that elicited maximal responses (105 and 200 pmol) elicited decreases in H R that were not significantly different. The regression line calculated for the linear portion of the dose response curve for 1-ENK was significant for H R changes (P < 0.01). The average onset latency of the response was 5.0 ___0.8 s. The peak of the response was reached in 53.4 - 6.6 s and H R gradually returned to preinjection values in 419 - 36.1 s.
Microinjection of substance P mmomloe
Fig. 2. Dose-response curve for microinjection of L-glutamate into the right nucleus ambiguus. Regression lines were calculated for the linear portion of the curve.
H R responses to G L U microinjection is shown in Fig. 2. The threshold dose for eliciting changes in H R was 0.3 nmol and the two maximal doses (1.0 and 1.25 nmol) elicited responses that were not significantly different. The regression line calculated for the linear portion of the dose response curve for G L U was significant for H R changes (P < 0.05). No changes in AP were observed. An example of the lack of response to a microinjection of PBS is shown in Fig. lB. The location of G L U microinjection sites is shown diagrammatically in transverse sections of the medulla (Fig. 5).
Microinjection of Met-enkephalin Microinjection of 105 pmol of m - E N K into the fight NA elicited a maximal decrease in H R of 15.1 --- 2.5 bpm from a baseline of 384.1 - 15.2 bpm (n = 6). An example of a response to m - E N K is shown in Fig. 1C. The dose response curve for H R responses to m-ENK microinjection is shown in Fig. 3A. The threshold dose for eliciting changes in H R was 15 pmol and the two doses that elicited maximal responses (105 and 200 pmol) elicited decreases in H R that were not significantly different. The regression line calculated for the linear portion of the dose response curve for m-ENK was significant for H R changes (P < 0.05)• No changes in AP were observed. The average onset latency of the response was 6.1 --- 1.2 s and the peak response was reached in 25.9 --- 4.2 s. After reaching a maximum, the H R gradually returned to preinjection values in 210 ± 21.2 s.
Microinjection of 6.3 pmol of SP into sites in the right NA elicited a maximal decrease in H R of 21.3 - 4.2 bpm from a baseline of 400.4 _ 13.1 bpm (n = 7; Fig. 1E) but had no effect on AP. The dose-response curve for H R responses to SP is shown in Fig. 4A. The threshold dose for eliciting changes in H R was 0.9 pmol and the two doses that elicited maximal responses (6.3 and 15 pmol) elicited decreases in H R that were not significantly different. The regression line calculated for the linear portion of the dose response curve for SP was significant for H R changes (P < 0.05). H R responses had an onset latency of 6.2 ± 1.1 s, the peak response was reached in 28.5 -z--3.1 s and the H R eventually returned to preinjection values in 376.5 - 39.1 s.
Microinjection of acetylcholine Microinjection of 20 pmol of Ach into the fight N A elicited a maximal decrease in H R of 21.2 _+ 1.0 bpm from a baseline of 392.3 ± 10.2 bpm (n = 7; Fig. 1F)
^
B n-7 r' - - O , m T B
n-6 r -'Ore
J -1(
Microinjection of Leu-enkephalin Microinjection of 105 pmol of I-ENK into the fight NA elicited a maximal decrease in H R of 30.2 ± 6.2 bpm from a baseline of 405.3 -+ 10.1 bpm (n = 7; Fig.
plcNO|eli
plcooole|
Fig. 3. Dose-response curves for microinjection of (A) Met-enkephalin (m-ENK) and (B) Leu-enkephalin (I-ENK) into the right nucleus ambiguus. Regression lines were calculated for the linear portion of the curve.
206 B n-7
p, II
n-7 r' - - 0 , 9 1 9 7
r - -0.9~2
-
,,
\
',~ tl • ,.
Io \
,,
~.~..~..\
i&
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..
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,, L R N /
N_!
,7 5
I0 !ItCOIOlU
t8
20
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5
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20
25
30
plCOIlOlel
Fig. 4. Dose-response curves for microinjection of (A) substance P (SP) and (B) acetylcholine (Ach) into the right nucleus ambiguus. Regression lines were calculated for the linear portion of the curve.
but had no effect on AP. The dose response curve for H R responses to Ach is shown in Fig. 4B. The threshold dose for eliciting changes in H R was 2 pmol and the two doses that elicited maximal responses (20 and 25 pmol) elicited decreases in H R that were not significantly different. The regression line calculated for the linear portion of the dose response curve for Ach was significant for H R changes (P < 0.05). The H R responses had an onset latency of 4.8 + 0.8 s, the peak response was reached in 20.5 -+ 1.9 s and, after reaching a maximum, the H R gradually returned to preinjection values in 243 --- 21.12 s.
203
BRES 17121
Enkephalins, substance P and acetylcholine microinjected into the nucleus ambiguus elicit vagal bradycardia in rats S.K. Agarwal and F.R. Calaresu Department of Physiology, University of Western Ontario, London, Ont. (Canada) (Accepted 11 June 1991)
Key words: Cardiovascular regulation; Acetylcholine; Peptide; Microinjection
Little is known about putative transmitters in the nucleus ambiguus (NA) mediating parasympathetic control of the heart, although Metenkephalin (m-ENK), Leu-enkephalin (I-ENK), substance P (SP) and acetylcholine (Ach) have been detected in the cell bodies and fibers of this nucleus. The effects of these substances on arterial pressure (AP) and heart rate (HR) were studied by microinjecting them (4-20 nl) into the NA. Experiments were done in 26 spinal (high cervical) rats that were anesthetized with urethane and artificially ventilated. L-Glutamate (GLU) was microinjected into the right NA to identify the location of cell bodies from which decreases in HR and AP could he elicited, m-ENK, 1-ENK, SP or Ach was then microinjected into these sites. Microinjection of 1 nmol of GLU elicited significant decreases in HR (-72.2 +- 9.7 bpm, n = 15) which were not accompanied by significant decreases in mean AE Microinjection of m-ENK (15-200 pmol; n = 7), 1-ENK (15-200 pmol; n = 6), SP (0.9-15 pmol; n = 7) and Ach (2.0-20 pmol; n = 7) into the NA decreased HR in a dose-dependent manner but did not affect AE The magnitudes of HR responses to m-ENK, 1-ENK, SP and Ach were smaller but of longer duration than the changes in HR to microinjection of GLU. These results suggest a physiological role for GLU, enkephalins, SP and Ach in the vagal control of HR mediated by the NA. INTRODUCTION Anatomical and electrophysiological experiments have demonstrated in all mammalian species studied that the ventrolateral division of the loose and external formation of the nucleus ambiguus (NA) is the main site of origin of cardioinhibitory neurons (see reviews 14'18) as well as of axons to other visceral organs ~'18. Neurons in the N A are excited by stimulation of the carotid sinus nerve and by increased sinus pressure 4'17 and can be antidromically activated by electrical stimulation of the cardiac branches of the vagus 4'19'2°. However, although the functional role of the N A in the central control of the cardiovascular system has been established, little is known about neurotransmitters involved in controlling activity of N A neurons. The role of putative neurotransmitters in the function of the central nervous system (CNS) has been investigated by either injections into the ventricular system or by microinjections into discrete CNS sites. Many peptides with a functional role in central cardiovascular regulation have been found (see reviews 8'25) using the first technique. For instance, enkephalins injected into the ventricular system exert potent effects on heart rate (HR), arterial pressure (AP) and respiration. Moreover
enkephalins are present in regions in the brainstem known to be involved in cardiovascular control 13'16. More specifically, the N A has been shown to contain dense binding sites for enkephalins 5'26 and transection of the vagus nerves results in dramatic loss of these binding sites in the N A 26. Finally, microinjection of the morphine-like agents fentanyl and Met-enkephalanamide into the N A of dogs has been shown to elicit decreases in H R 16. Substance P (SP) and acetylcholine (Ach) have also been investigated as putative transmitters in the N A 2A2. SP-like immunoreactivity and choline acetyltransferase immunoreactive cell bodies have been demonstrated in the vicinity of the N A 2'12. Moreover, microintophoretic application of Ach inhibits the firing of cardioinhibitory neurons in the N A 1°'11. However, there is little information about the precise functional role of these putative transmitters (enkephalins, SP and Ach) in the control of the cardiovascular system by the NA. In the present study enkephalins, SP and Ach were microinjected into the N A to study their effects on H R and A P in spinal rats. The spinal transection was done at the C1 level to eliminate the descending pathways that control the sympathetic outflow to the heart, thereby re-
Correspondence: S.K. Agarwal, t)epartment of Physiology, The University of Western Ontario, London, Ont., Canada N6A 5C1. Fax: (1) (519) 661 3827.
204 stricting t h e effects o f m e d u l l a r y s t i m u l a t i o n to a c t i v a t i o n of t h e v a g u s n e r v e s to t h e h e a r t . T h e p e p t i d e s a n d A c h w e r e i n j e c t e d i n t o N A sites d e m o n s t r a t e d
to h a v e car-
dioinhibitory function by microinjections of L-glutamate (GLU).
MATERIALS AND METHODS
General procedure Studies were done in adult male Wistar rats (n = 26, 250-350 g, Charles River, Montreal, Canada), anaesthetized with urethane (Sigma, St. Louis, MO, 1.4 g/kg, i.p. initially and 0.25 g/kg supplements as required). The trachea was cannulated and the animal was artificially ventilated with room air using a small animal ventilator (Harvard Apparatus, model 683). The femoral artery and vein were cannulated. The arterial cannula was connected to a pressure transducer (Century Technology, Inglewood, CA, model CP 01) that was connected to a Grass polygraph (model 79C) for continuous recording of AP. A Grass tachograph (7 P44B) triggered by the arterial pressure pulse was used to monitor HR. The animal was placed in a stereotaxic apparatus, with the bite bar 20 mm below the interaural line. The medulla was exposed by retracting the dorsal neck muscles, incising the atlantooccipital membrane and removing part of the occipital bone and the dura. The spinal cord of the animal was then transected at the C1 level. To maintain AP within the physiological range phenylephrine (PE; Sigma) in physiological saline (2 mg/ml) was infused into the venous cannula, initially at the rate of 700/APa and then at 250/A/h. These amounts of PE were found to maintain mean AP at 96.2 -+ 3.9 mmHg (range 87-105 mmHg; n = 26). Animals that dit not have a steady level of AP over a period of several hours were not included in the results. Rectal temperature was maintained at 37.5 -+ 0.5 °C with a thermostatically controlled heating blanket.
Pressure microin]ection L-Glutamate (Sigma, 0.3-1.25 nmol), Met-enkephalin (Sigma, 15-200 pmol), Leu-enkephalin (Sigma, 15-200 pmol), substance P (Sigma, 0.9-15 pmol) and acetylcholine (Sigma, 2-20 pmol) dissolved in phosphate buffered saline (PBS, pH 7.4) were pressure microinjected (4-20 nl) through multibarrelled glass mieropipettes pulled from Socorex 5 ktl glass capillary tubing (Socorex 851-5, Terochem Laboratories, Mississauga, Ont., Canada). Long shanks were drawn to minimize tissue distortion and the tips were broken to an external diameter of -50/~m. The pipettes were inclined 20 ° from the vertical in the sagittal plane with the tip pointing rostrally. The stereotaxic coordinates used for the placement of the tip in the fight NA were 0.2-0.6 mm caudal to the obex 1.8-2.0 mm lateral and 2.0-2.3 mm below the dorsal surface of the medulla. Volumes of injections were measured directly by monitoring the movement of the fluid meniscus through a 40× microscope fitted with an ocular scale that allowed a resolution of 1 nl. At the start of each experiment the NA was explored for a site where injection of 4-10 nl of 0.15 M GLU consistently elicited decreases in HR. After such a site in the NA was found one of the four substances was microinjected. A third barrel of the micropipette contained PBS used to deliver 20 nl of control saline. Very rarely small (5-10 bpm) changes in HR were elicited by microinjections of saline; in these cases the results were not included in our analysis of the data.
Histology Microinjection sites were marked for histological verification at the end of each experiment. The mieroinjection pipette was retracted from the brain, emptied, filled with 25% India ink solution in saline, reinserted to the same depth and 20 nl of India ink were injected. At the end of the experiments the animals were perfused with 50 ml of PBS followed by 50 ml of a 10% formalin solution in PBS. The brains were removed and stored in formalin for 3-4 days. Frozen transverse sections (50 ~m) were cut and stained with thio-
nin. Injection sites were mapped o n the drawings of transverse sections of the rat brain from an atlas za.
Statistics Mean changes in HR and MAP (i.e., the difference between the peak changes in HR or MAP and the level of these variables before microinjection) were compared by Studenrs t-tests. For comparison of HR responses with different doses of putative transmitters analysis of variance (ANOVA) was performed followed by pairwise comparison by a Least Significant Difference method (LSD method) 21. For dose-response curves percentage changes in HR vs doses were plotted. Linear regression analysis was used to determine the line of best fit for plotted points in the linear portion of the dose-response curve. The probability level taken to indicate a significant change was P < 0.05 for all statistical tests. All data in text and figures are expressed as means + S.E.M.
RESULTS
Microinjection of L-glutamate Microinjection of GLU
(1 n m o l ) i n t o t h e r i g h t N A
e l i c i t e d a m a x i m a l d e c r e a s e in H R o f - 7 2 . 2 -+ 9.7 b p m f r o m a b a s e line o f 389.2 -+ 8.1 b p m (n = 15). A n exa m p l e o f a r e s p o n s e to G L U is s h o w n in Fig. 1A. T h e s e effects w e r e g e n e r a l l y r a p i d in o n s e t ( m e a n 2.3 -+ 0.2 s), p e a k r e s p o n s e w a s r e a c h e d in 12.7 --- 2.9 s a n d t h e dur a t i o n w a s 121 -+ 17.8 s. T h e d o s e - r e s p o n s e
°**I
HR |bpm) 4 0 0 ( / 200 ~ 200 r
GLU
I**I
-
~
A
B
C
M~NK
SALIN!
H
c u r v e for
i
I
0
Stain
6001
D
400 [
~
/ 200L . . . . . . . . . . .
[
E
I i .......................................
L--INK
SP
1oo[
I
0
II
.....
sol
600 r
F
400 [ 200 f
Ach
100 0 Fig. I. Tracings of arterial pressure (AP) and heart rate (HR) during microinjections of L-glutamate, phosphate-buffered saline, Metenkephalin, Leu-enkephalin, substance P and acetylcholine into the right ambiguus of a rat with spinal cord transection. Marks on time scale indicate the time of microinjections. A: L-glutamate (1.2 nmol), B: phosphate-buffered saline (20 nl). C: Met-enkephafin (75 pmol). D: Leu-enkephalin (75 prhol). E: substance P (6.3 pmol). F: acetylcholine (20 pmol).
205 0
n-
15
r - -0.0i540
-10
q;
g..~ t 8 N
-3o!
1D) but had no effect on AP. The dose-response curve for H R responses to I-ENK microinjection is shown in Fig. 3B. The threshold dose for eliciting changes in H R was 15 pmol and the two doses that elicited maximal responses (105 and 200 pmol) elicited decreases in H R that were not significantly different. The regression line calculated for the linear portion of the dose response curve for 1-ENK was significant for H R changes (P < 0.01). The average onset latency of the response was 5.0 ___0.8 s. The peak of the response was reached in 53.4 - 6.6 s and H R gradually returned to preinjection values in 419 - 36.1 s.
Microinjection of substance P mmomloe
Fig. 2. Dose-response curve for microinjection of L-glutamate into the right nucleus ambiguus. Regression lines were calculated for the linear portion of the curve.
H R responses to G L U microinjection is shown in Fig. 2. The threshold dose for eliciting changes in H R was 0.3 nmol and the two maximal doses (1.0 and 1.25 nmol) elicited responses that were not significantly different. The regression line calculated for the linear portion of the dose response curve for G L U was significant for H R changes (P < 0.05). No changes in AP were observed. An example of the lack of response to a microinjection of PBS is shown in Fig. lB. The location of G L U microinjection sites is shown diagrammatically in transverse sections of the medulla (Fig. 5).
Microinjection of Met-enkephalin Microinjection of 105 pmol of m - E N K into the fight NA elicited a maximal decrease in H R of 15.1 --- 2.5 bpm from a baseline of 384.1 - 15.2 bpm (n = 6). An example of a response to m - E N K is shown in Fig. 1C. The dose response curve for H R responses to m-ENK microinjection is shown in Fig. 3A. The threshold dose for eliciting changes in H R was 15 pmol and the two doses that elicited maximal responses (105 and 200 pmol) elicited decreases in H R that were not significantly different. The regression line calculated for the linear portion of the dose response curve for m-ENK was significant for H R changes (P < 0.05)• No changes in AP were observed. The average onset latency of the response was 6.1 --- 1.2 s and the peak response was reached in 25.9 --- 4.2 s. After reaching a maximum, the H R gradually returned to preinjection values in 210 ± 21.2 s.
Microinjection of 6.3 pmol of SP into sites in the right NA elicited a maximal decrease in H R of 21.3 - 4.2 bpm from a baseline of 400.4 _ 13.1 bpm (n = 7; Fig. 1E) but had no effect on AP. The dose-response curve for H R responses to SP is shown in Fig. 4A. The threshold dose for eliciting changes in H R was 0.9 pmol and the two doses that elicited maximal responses (6.3 and 15 pmol) elicited decreases in H R that were not significantly different. The regression line calculated for the linear portion of the dose response curve for SP was significant for H R changes (P < 0.05). H R responses had an onset latency of 6.2 ± 1.1 s, the peak response was reached in 28.5 -z--3.1 s and the H R eventually returned to preinjection values in 376.5 - 39.1 s.
Microinjection of acetylcholine Microinjection of 20 pmol of Ach into the fight N A elicited a maximal decrease in H R of 21.2 _+ 1.0 bpm from a baseline of 392.3 ± 10.2 bpm (n = 7; Fig. 1F)
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Microinjection of Leu-enkephalin Microinjection of 105 pmol of I-ENK into the fight NA elicited a maximal decrease in H R of 30.2 ± 6.2 bpm from a baseline of 405.3 -+ 10.1 bpm (n = 7; Fig.
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Fig. 3. Dose-response curves for microinjection of (A) Met-enkephalin (m-ENK) and (B) Leu-enkephalin (I-ENK) into the right nucleus ambiguus. Regression lines were calculated for the linear portion of the curve.
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Fig. 4. Dose-response curves for microinjection of (A) substance P (SP) and (B) acetylcholine (Ach) into the right nucleus ambiguus. Regression lines were calculated for the linear portion of the curve.
but had no effect on AP. The dose response curve for H R responses to Ach is shown in Fig. 4B. The threshold dose for eliciting changes in H R was 2 pmol and the two doses that elicited maximal responses (20 and 25 pmol) elicited decreases in H R that were not significantly different. The regression line calculated for the linear portion of the dose response curve for Ach was significant for H R changes (P < 0.05). The H R responses had an onset latency of 4.8 + 0.8 s, the peak response was reached in 20.5 -+ 1.9 s and, after reaching a maximum, the H R gradually returned to preinjection values in 243 --- 21.12 s.
