Journal of the Autonomic Nervous System, 41 (1992) 121 - 128
121
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01355
Additive effects of dopamine and 8-OH-DPAT microinjected into the nucleus ambiguus in eliciting vagal bradycardia in rats V.C. Chitravanshi 1 and F.R. Calaresu Department of Physiology, University of Western Ontario, London, Ontario, Canada
Key words: Nucleus ambiguus; Vagal bradycardia; Dopamine; 8-OH-DPAT; Microinjection Abstract The effects of combined microinjection into the nucleus ambiguus (NA) of dopamine (DA) and the 5-hydroxytryptamine-lA (5HT-1A) receptor agonist 8-hydroxy-2-[di-n-propylamino]tetralin (8-OH-DPAT) on arterial pressure (AP) and heart rate (HR) were studied in 24 urethane-anaesthetized, artificially ventilated spinal (C1) rats. Sites from which bradycardia was elicited by microinjection of e-glutamate (GLU) were selected for microinjection of DA and 8-OH-DPAT into the NA. Microinjections of 8-OH-DPAT (60-300 pmol in 10 nl) elicited a dose-dependent bradycardia; the H R responses elicited by microinjection of 300 pmol of 8-OH-DPAT were significantly larger ( - 3 4 . 0 + 3.0) than responses elicited by the threshold dose of 60 pmol ( - 3 . 1 _+ 0.1 bpm). The onset latency of the H R responses elicited by the 300 pmol of 8-OH-DPAT was 6.5 + 0.5 s and the peak was reached in 40.0 _+ 8.0 s. The duration of these responses was 615 + 3.5 s. Microinjection of DA (1 nmol in 10 nl) within 3-5 s from the time of 8-OH-DPAT microinjection (60 pmol in 2 nl) into the NA at sites previously shown to elicit decreases in H R following microinjection of GLU, produced significant additive effects in eliciting bradycardia (20.4 +_ 2.9 bpm) when compared with decreases in H R elicited by microinjection of D A (11.1 _+ 1.8 bpm) or of 8-OH-DPAT (3.1 + 0.1 bpm) alone. There were no changes in AP after microinjections of DA, 8-OH-DPAT or of these two substances combined. Microinjections of 10 nl of phosphate buffered saline (PBS) into 18 sites in the NA had no effects on AP and HR. These results demonstrate that microinjections of 8-OH-DPAT into the NA elicit dose-dependent decreases in HR and that DA and 8-OH-DPAT have an additive effect on vagal bradycardia when compared with the bradycardia elicited by the two substances separately.
Introduction
Arterial pressure (AP) and heart rate (HR) are maintained within the physiological range by several mechanisms including the baroreceptor re-
Correspondence to: F.R. Calaresu, Department of Physiology, The University of Western Ontario, London, Ontario, Canada, N6A 5C1. 1 Present address: Section of Neurosurgery, Medical Sciences Building, Room H-586, New Jersey Medical School, 185, S. Orange Avenue, Newark-NJ 07103, USA.
flex. The role of a discrete medullary nucleus, the nucleus ambiguus (NA), in this reflex is well established [14] because cardioinhibitory vagal preganglionic neurons have been located in the ventrolateral part of the NA [12,15]. In addition, several recent studies on the NA have demonstrated the effects on HR of different putative neurotransmitters, such as enkephalins [1], substance P [3], glycine [4] and dopamine (DA) [5] when microinjected into the region of the NA of rats. 5-Hydroxytryptamine (5HT) has been shown to play a physiological role in governing autonomic
122
neural activity and cardiovascular function [16]. More specifically, it has been shown that microinjection of the 5HT-1A receptor agonist 8-hydroxy-2-[di-n-propylamino]tetralin (8-OH-DPAT) into the right NA of cats elicits decreases in HR [13]. Additional information about synaptic transmission in the NA of rats has been provided by the demonstration in the NA of fibers immunoreactive for 5HT [25] and of 5HT-1A binding sites in cats and rats [6,22]. Finally, it has been shown that 8-OH-DPAT elicits hypotension and bradycardia in cats when given systemically [23], probably by its action on central 5HT-1A receptor sites [17]. In another study [8] it has been shown that intravenous administration of 8-OH-DPAT in rats elicited dose-related and sustained decreases in blood pressure and HR. This study has provided direct evidence of involvement of central sites of action as the fall in blood pressure and HR was blocked by the intravenous administration of a putative central receptor antagonist for 5HT-1A. In a recent study [7], it has been reported that in conscious and unrestrained rats intracerebroventricular administration of low doses of 8-OHDPAT elicited significant elevations in AP and HR while higher doses elicited depressor and bradycardic responses. Although binding sites for dopamine (DA) have not been identified in the NA, fibers immunoreactive for tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, have been found in the region of the NA [10,18]. In addition, DA has previously been shown to play a role as a neurotransmitter in the central nervous system [19] and we have recently shown that microinjection of DA into the NA elicits bradycardia in spinal rats [5]. Coexistence of neurotransmitters is well established in the central nervous system. While DA and 5HT have been found co-localized with other peptides in different regions of the rat central nervous system [11], co-localization of DA and 5HT receptors has not been reported. Based on our previous studies showing that microinjection of DA into the NA of spinal rats elicits bradycardia [5] as well as on the studies done with the microinjection of 8-OH-DPAT into the NA of cat [13] it seems likely that these two neurotransmit-
ters coexist in the NA and have a combined physiological involvement in controlling AP and HR. We therefore decided to investigate the effects on AP and HR of simultaneous microinjection of 8-OH-DPAT and DA into the NA of anaesthetized rats. These rats were made spinal by transection of the spinal cord at the level of CI to ensure that responses from chemical stimulation of NA that were due exclusively to vagal mechanisms.
Materials and Methods
General procedure Experiments were done in 24 adult male Wistar rats (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) and artificially ventilated with room air using a small animal ventilator (Harvard Apparatus, model 683). Cannulation of the trachea, left femoral artery and vein was done and AP was continuously measured from the arterial cannula by means of a pressure transducer (Century Technology, Inglewood, CA, model CP 01) which was connected to a Grass polygraph (model 79C). HR was derived from the AP signal through a Grass tachograph (7 P44B). The animal was placed in a Kopf stereotaxic apparatus with the bite bar 20 mm below the interaural line. The medulla was exposed after retracting the dorsal neck muscles, incising the atlantooccipital membrane and removing part of the occipital bone and dura. Spinal cord transection was made at the C1 level in order to eliminate supraspinal inputs to the intermediolaterat cell column, the site of sympathetic preganglionic neurons. AP was maintained within the physiological range (mean AP between 90 and 100 mmHg) by continuously infusing phenylephrine in physiological saline (PE; 2 mg/ml; Sigma) into the venous cannula (initially at a rate of 1 m l / h for 3-5 min and then at 0.25 ml/h). Animals with unstable levels of AP over a period of several hours were not included in the study. Rectal temperature was maintained at 37.5 + 0.5°C with a thermostatically controlled heating blanket.
123
Pressure microinjection
Data analysis
L-Glutamate (GLU; Sigma, 0.15 M), 8-OHD P A T (Research Biochemicals Inc., 0.03 M) and D A (Research Biochemicals Inc.,'0.1 M) were dissolved in phosphate buffered saline (PBS, p H 7.4). The drugs were pressure microinjected through multibarrelled glass micropipettes pulled from glass capillary tubing (Socorex 851-5, T e r o c h e m laboratories, Mississauga, Ontario, Canada) with an external tip diameter of ~ 50 /~m to minimize tissue distortion. The micropipettes were oriented 20 ° from the vertical in the sagittal plane with the tip pointing rostrally. The stereotaxic coordinates used for tip placement in the N A were 0.2-0.6 m m caudal to the obex, 1.6-2.0 m m lateral to the midline and 2.02.4 m m below the dorsal surface of the medulla. Microinjection volumes were observed directly by movement of the fluid meniscus in the micropipette as seen through a 40X microscope fitted with an ocular scale that allowed a resolution of 1 nl.
Mean changes in AP and H R from control values were compared by Student's t-tests. For dose-response relationships, decreases in H R vs. doses were plotted. For changes elicited by co-injection of D A and 8 - O H - D P A T , a one-way analysis of variance ( A N O V A ) was done, followed by the Tukey-Kramer test for comparisons of the changes in H R elicited by D A alone, 8 - O H - D P A T alone and D A plus 8 - O H - D P A T . The probability level taken to indicate a significant difference was P < 0.05 for all statistical tests. All data in text and figures are expressed as means +_ S.E.M.
Selection of doses for microinjection As microinjection of D A has been shown to elicit dose-dependent bradycardia in spinal rats [5] we used the lowest dose of D A (1 nmol in 10 nl) which would elicit a significant decrease in HR. We also chose the minimum dose at which a decrease in H R could consistently be elicited (threshold dose) by 8 - O H - D P A T (60 pmol in 2 nl) to observe the effects elicited by the combination of these two drugs. Amounts between 60 and 300 pmol were chosen for dose-related changes in AP and H R by microinjection of 8 - O H - D P A T into the NA.
Histology At the end of each experiment, microinjection sites were marked for histological verification with India ink using a method previously described [2]. The animals were perfused with 50 ml of phosphate buffered saline (PBS) followed by 50 ml of a 10% formalin solution in PBS. After 3 - 4 days of post-perfusion fixation in formalin, 50 /xm sections were cut and stained with thionin. Injection sites were m a p p e d on drawings of transverse sections of the rat brain from an atlas [21].
Results
Effects of GLU microinjection into the NA G L U (1.5 nmol in 10 nl) was microinjected into the right N A to identify sites which consistently produced bradycardia. G L U microinjection elicited a decrease in H R of 69.7 + 3.0 b p m (n = 24) with an onset latency of 1.7 + 0.3 s from the base line value of 411.6 + 2.1 bpm. The responses reached their peak in 7.8 + 0.9 s and lasted for 66.2 + 3.8 s. A typical response to G L U microinjection is shown in Fig. 1A. There was no effect on AP after microinjection of GLU. Microinjection of 10 nl of PBS had no effect on AP and H R (n = 18; Fig. 1B). The control mean AP was 90.1
]
[]
[]
HR 600 I (bpm) 400 I - ' ~ 200 L 200 AP (mmHg) 100 0
f-GLU
PBS
8-OH-DPAT
Fig. 1. Effects of microinjection of: (A) L-GLU (1.5 nmol in 10 nl); (B) PBS (10 nl); and (C) 8-OH-DPAT (300 pmol in 10 nl) on HR (bpm) into the right NA of a spinal rat. All tracings are from the same rat. Arrows indicate beginning of microinjections.
124
®
®
HR 600[
©
®
®
Ibpm) 400 [-'-~..~ i
200 L
30 s
AP v
I
G LU
1
P BS
I
1
DA
8"OH-DPAT
DA+8"OH'DPAT
Fig. 2. Representative experiment showing the cardiovascular effects of co-administration of DA plus 8-OH-DPAT into the ~ight NA of a spinal rat. Traces show the effects of (A) L-GLU (1.5 nmol in 10 nl); (B) PBS (10 nl); (C) DA (1 nmol in 10 hi); (D) 8-OH-DPAT (60 pmol in 2 nl), and (E) co-microinjection of DA + 8-OH-DPAT (1 nmol and 60 pmol respectively), All recordings are from the same site. Arrows indicate beginning of microinjections.
+ 1.8 mmHg. G L U injection sites are shown in Fig. 3.
Dose-dependent bradycardia elicited by microinjections of 8-OH-DPAT into the NA As there are no data in the literature on the effects on H R and AP of microinjection of 5 H T into the NA we studied these possible effects in 12 rats. The 5HT-1A receptor agonist 8-OHD P A T was microinjected into the right NA at sites where microinjection of G L U elicited decreases in HR. The threshold dose of 8-OHD P A T (60 pmol in 2 nl) elicited a decrease in H R of 3.0 + 0.3 bpm (n = 12; Fig. 2D). Microinjection of 120 pmol in 4 nl (n = 12) did not elicit a significantly greater decrease in H R (4.5 + 0.9 bpm) when compared with the threshold dose. However, a dose of 180 pmol in 6 nl (n = 12) and the largest dose of 300 pmol in 10 nl (n = 12) elicited decreases in H R of 7.0 + 1.1 bpm and 34.0 + 3.0 bpm, respectively, which were significantly greater than responses to the threshold dose of 60 pmol (Figs. 1C and 4). Microinjection of 8 - O H - D P A T at any dose failed to.change AP. Microinjections of 10 nl of PBS did not elicit any effects on AP or H R (n = 6).
Simultaneous microinjection of DA and 8-OHDPA T into the NA D A (1 nmol in 10 nl) was microinjected into the right NA at 12 sites known to elicit bradycardia following G L U microinjection. The decrease
in H R elicited by D A was 11.1 _+ 1.8 b p m which started in 7.3 + 0.4 s, reached the p e a k in 33~8 + 3.5 s and lasted for 228.3 + 15,6 s (Figs, 2 C and 5). Microinjection of 8 , O H . D P A T (60 pmot: in 2 nl) at the same sites elicited a decrease in ~ of 3.1 + 0.2 bpm (threshold dose) with an onset, peak and duration of 5.0 + 0.6 s, 29.9 _+ 2.7 s and 195.8 + 22.5 s, respectively (Figs. 2D and 5). Microinjection of 8 - O H - D P A T (60 pmol in 2 nl) 3-5 s after the microinjection of D A (1 nmol in 10 nl)
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121
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01355
Additive effects of dopamine and 8-OH-DPAT microinjected into the nucleus ambiguus in eliciting vagal bradycardia in rats V.C. Chitravanshi 1 and F.R. Calaresu Department of Physiology, University of Western Ontario, London, Ontario, Canada
Key words: Nucleus ambiguus; Vagal bradycardia; Dopamine; 8-OH-DPAT; Microinjection Abstract The effects of combined microinjection into the nucleus ambiguus (NA) of dopamine (DA) and the 5-hydroxytryptamine-lA (5HT-1A) receptor agonist 8-hydroxy-2-[di-n-propylamino]tetralin (8-OH-DPAT) on arterial pressure (AP) and heart rate (HR) were studied in 24 urethane-anaesthetized, artificially ventilated spinal (C1) rats. Sites from which bradycardia was elicited by microinjection of e-glutamate (GLU) were selected for microinjection of DA and 8-OH-DPAT into the NA. Microinjections of 8-OH-DPAT (60-300 pmol in 10 nl) elicited a dose-dependent bradycardia; the H R responses elicited by microinjection of 300 pmol of 8-OH-DPAT were significantly larger ( - 3 4 . 0 + 3.0) than responses elicited by the threshold dose of 60 pmol ( - 3 . 1 _+ 0.1 bpm). The onset latency of the H R responses elicited by the 300 pmol of 8-OH-DPAT was 6.5 + 0.5 s and the peak was reached in 40.0 _+ 8.0 s. The duration of these responses was 615 + 3.5 s. Microinjection of DA (1 nmol in 10 nl) within 3-5 s from the time of 8-OH-DPAT microinjection (60 pmol in 2 nl) into the NA at sites previously shown to elicit decreases in H R following microinjection of GLU, produced significant additive effects in eliciting bradycardia (20.4 +_ 2.9 bpm) when compared with decreases in H R elicited by microinjection of D A (11.1 _+ 1.8 bpm) or of 8-OH-DPAT (3.1 + 0.1 bpm) alone. There were no changes in AP after microinjections of DA, 8-OH-DPAT or of these two substances combined. Microinjections of 10 nl of phosphate buffered saline (PBS) into 18 sites in the NA had no effects on AP and HR. These results demonstrate that microinjections of 8-OH-DPAT into the NA elicit dose-dependent decreases in HR and that DA and 8-OH-DPAT have an additive effect on vagal bradycardia when compared with the bradycardia elicited by the two substances separately.
Introduction
Arterial pressure (AP) and heart rate (HR) are maintained within the physiological range by several mechanisms including the baroreceptor re-
Correspondence to: F.R. Calaresu, Department of Physiology, The University of Western Ontario, London, Ontario, Canada, N6A 5C1. 1 Present address: Section of Neurosurgery, Medical Sciences Building, Room H-586, New Jersey Medical School, 185, S. Orange Avenue, Newark-NJ 07103, USA.
flex. The role of a discrete medullary nucleus, the nucleus ambiguus (NA), in this reflex is well established [14] because cardioinhibitory vagal preganglionic neurons have been located in the ventrolateral part of the NA [12,15]. In addition, several recent studies on the NA have demonstrated the effects on HR of different putative neurotransmitters, such as enkephalins [1], substance P [3], glycine [4] and dopamine (DA) [5] when microinjected into the region of the NA of rats. 5-Hydroxytryptamine (5HT) has been shown to play a physiological role in governing autonomic
122
neural activity and cardiovascular function [16]. More specifically, it has been shown that microinjection of the 5HT-1A receptor agonist 8-hydroxy-2-[di-n-propylamino]tetralin (8-OH-DPAT) into the right NA of cats elicits decreases in HR [13]. Additional information about synaptic transmission in the NA of rats has been provided by the demonstration in the NA of fibers immunoreactive for 5HT [25] and of 5HT-1A binding sites in cats and rats [6,22]. Finally, it has been shown that 8-OH-DPAT elicits hypotension and bradycardia in cats when given systemically [23], probably by its action on central 5HT-1A receptor sites [17]. In another study [8] it has been shown that intravenous administration of 8-OH-DPAT in rats elicited dose-related and sustained decreases in blood pressure and HR. This study has provided direct evidence of involvement of central sites of action as the fall in blood pressure and HR was blocked by the intravenous administration of a putative central receptor antagonist for 5HT-1A. In a recent study [7], it has been reported that in conscious and unrestrained rats intracerebroventricular administration of low doses of 8-OHDPAT elicited significant elevations in AP and HR while higher doses elicited depressor and bradycardic responses. Although binding sites for dopamine (DA) have not been identified in the NA, fibers immunoreactive for tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, have been found in the region of the NA [10,18]. In addition, DA has previously been shown to play a role as a neurotransmitter in the central nervous system [19] and we have recently shown that microinjection of DA into the NA elicits bradycardia in spinal rats [5]. Coexistence of neurotransmitters is well established in the central nervous system. While DA and 5HT have been found co-localized with other peptides in different regions of the rat central nervous system [11], co-localization of DA and 5HT receptors has not been reported. Based on our previous studies showing that microinjection of DA into the NA of spinal rats elicits bradycardia [5] as well as on the studies done with the microinjection of 8-OH-DPAT into the NA of cat [13] it seems likely that these two neurotransmit-
ters coexist in the NA and have a combined physiological involvement in controlling AP and HR. We therefore decided to investigate the effects on AP and HR of simultaneous microinjection of 8-OH-DPAT and DA into the NA of anaesthetized rats. These rats were made spinal by transection of the spinal cord at the level of CI to ensure that responses from chemical stimulation of NA that were due exclusively to vagal mechanisms.
Materials and Methods
General procedure Experiments were done in 24 adult male Wistar rats (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) and artificially ventilated with room air using a small animal ventilator (Harvard Apparatus, model 683). Cannulation of the trachea, left femoral artery and vein was done and AP was continuously measured from the arterial cannula by means of a pressure transducer (Century Technology, Inglewood, CA, model CP 01) which was connected to a Grass polygraph (model 79C). HR was derived from the AP signal through a Grass tachograph (7 P44B). The animal was placed in a Kopf stereotaxic apparatus with the bite bar 20 mm below the interaural line. The medulla was exposed after retracting the dorsal neck muscles, incising the atlantooccipital membrane and removing part of the occipital bone and dura. Spinal cord transection was made at the C1 level in order to eliminate supraspinal inputs to the intermediolaterat cell column, the site of sympathetic preganglionic neurons. AP was maintained within the physiological range (mean AP between 90 and 100 mmHg) by continuously infusing phenylephrine in physiological saline (PE; 2 mg/ml; Sigma) into the venous cannula (initially at a rate of 1 m l / h for 3-5 min and then at 0.25 ml/h). Animals with unstable levels of AP over a period of several hours were not included in the study. Rectal temperature was maintained at 37.5 + 0.5°C with a thermostatically controlled heating blanket.
123
Pressure microinjection
Data analysis
L-Glutamate (GLU; Sigma, 0.15 M), 8-OHD P A T (Research Biochemicals Inc., 0.03 M) and D A (Research Biochemicals Inc.,'0.1 M) were dissolved in phosphate buffered saline (PBS, p H 7.4). The drugs were pressure microinjected through multibarrelled glass micropipettes pulled from glass capillary tubing (Socorex 851-5, T e r o c h e m laboratories, Mississauga, Ontario, Canada) with an external tip diameter of ~ 50 /~m to minimize tissue distortion. The micropipettes were oriented 20 ° from the vertical in the sagittal plane with the tip pointing rostrally. The stereotaxic coordinates used for tip placement in the N A were 0.2-0.6 m m caudal to the obex, 1.6-2.0 m m lateral to the midline and 2.02.4 m m below the dorsal surface of the medulla. Microinjection volumes were observed directly by movement of the fluid meniscus in the micropipette as seen through a 40X microscope fitted with an ocular scale that allowed a resolution of 1 nl.
Mean changes in AP and H R from control values were compared by Student's t-tests. For dose-response relationships, decreases in H R vs. doses were plotted. For changes elicited by co-injection of D A and 8 - O H - D P A T , a one-way analysis of variance ( A N O V A ) was done, followed by the Tukey-Kramer test for comparisons of the changes in H R elicited by D A alone, 8 - O H - D P A T alone and D A plus 8 - O H - D P A T . The probability level taken to indicate a significant difference was P < 0.05 for all statistical tests. All data in text and figures are expressed as means +_ S.E.M.
Selection of doses for microinjection As microinjection of D A has been shown to elicit dose-dependent bradycardia in spinal rats [5] we used the lowest dose of D A (1 nmol in 10 nl) which would elicit a significant decrease in HR. We also chose the minimum dose at which a decrease in H R could consistently be elicited (threshold dose) by 8 - O H - D P A T (60 pmol in 2 nl) to observe the effects elicited by the combination of these two drugs. Amounts between 60 and 300 pmol were chosen for dose-related changes in AP and H R by microinjection of 8 - O H - D P A T into the NA.
Histology At the end of each experiment, microinjection sites were marked for histological verification with India ink using a method previously described [2]. The animals were perfused with 50 ml of phosphate buffered saline (PBS) followed by 50 ml of a 10% formalin solution in PBS. After 3 - 4 days of post-perfusion fixation in formalin, 50 /xm sections were cut and stained with thionin. Injection sites were m a p p e d on drawings of transverse sections of the rat brain from an atlas [21].
Results
Effects of GLU microinjection into the NA G L U (1.5 nmol in 10 nl) was microinjected into the right N A to identify sites which consistently produced bradycardia. G L U microinjection elicited a decrease in H R of 69.7 + 3.0 b p m (n = 24) with an onset latency of 1.7 + 0.3 s from the base line value of 411.6 + 2.1 bpm. The responses reached their peak in 7.8 + 0.9 s and lasted for 66.2 + 3.8 s. A typical response to G L U microinjection is shown in Fig. 1A. There was no effect on AP after microinjection of GLU. Microinjection of 10 nl of PBS had no effect on AP and H R (n = 18; Fig. 1B). The control mean AP was 90.1
]
[]
[]
HR 600 I (bpm) 400 I - ' ~ 200 L 200 AP (mmHg) 100 0
f-GLU
PBS
8-OH-DPAT
Fig. 1. Effects of microinjection of: (A) L-GLU (1.5 nmol in 10 nl); (B) PBS (10 nl); and (C) 8-OH-DPAT (300 pmol in 10 nl) on HR (bpm) into the right NA of a spinal rat. All tracings are from the same rat. Arrows indicate beginning of microinjections.
124
®
®
HR 600[
©
®
®
Ibpm) 400 [-'-~..~ i
200 L
30 s
AP v
I
G LU
1
P BS
I
1
DA
8"OH-DPAT
DA+8"OH'DPAT
Fig. 2. Representative experiment showing the cardiovascular effects of co-administration of DA plus 8-OH-DPAT into the ~ight NA of a spinal rat. Traces show the effects of (A) L-GLU (1.5 nmol in 10 nl); (B) PBS (10 nl); (C) DA (1 nmol in 10 hi); (D) 8-OH-DPAT (60 pmol in 2 nl), and (E) co-microinjection of DA + 8-OH-DPAT (1 nmol and 60 pmol respectively), All recordings are from the same site. Arrows indicate beginning of microinjections.
+ 1.8 mmHg. G L U injection sites are shown in Fig. 3.
Dose-dependent bradycardia elicited by microinjections of 8-OH-DPAT into the NA As there are no data in the literature on the effects on H R and AP of microinjection of 5 H T into the NA we studied these possible effects in 12 rats. The 5HT-1A receptor agonist 8-OHD P A T was microinjected into the right NA at sites where microinjection of G L U elicited decreases in HR. The threshold dose of 8-OHD P A T (60 pmol in 2 nl) elicited a decrease in H R of 3.0 + 0.3 bpm (n = 12; Fig. 2D). Microinjection of 120 pmol in 4 nl (n = 12) did not elicit a significantly greater decrease in H R (4.5 + 0.9 bpm) when compared with the threshold dose. However, a dose of 180 pmol in 6 nl (n = 12) and the largest dose of 300 pmol in 10 nl (n = 12) elicited decreases in H R of 7.0 + 1.1 bpm and 34.0 + 3.0 bpm, respectively, which were significantly greater than responses to the threshold dose of 60 pmol (Figs. 1C and 4). Microinjection of 8 - O H - D P A T at any dose failed to.change AP. Microinjections of 10 nl of PBS did not elicit any effects on AP or H R (n = 6).
Simultaneous microinjection of DA and 8-OHDPA T into the NA D A (1 nmol in 10 nl) was microinjected into the right NA at 12 sites known to elicit bradycardia following G L U microinjection. The decrease
in H R elicited by D A was 11.1 _+ 1.8 b p m which started in 7.3 + 0.4 s, reached the p e a k in 33~8 + 3.5 s and lasted for 228.3 + 15,6 s (Figs, 2 C and 5). Microinjection of 8 , O H . D P A T (60 pmot: in 2 nl) at the same sites elicited a decrease in ~ of 3.1 + 0.2 bpm (threshold dose) with an onset, peak and duration of 5.0 + 0.6 s, 29.9 _+ 2.7 s and 195.8 + 22.5 s, respectively (Figs. 2D and 5). Microinjection of 8 - O H - D P A T (60 pmol in 2 nl) 3-5 s after the microinjection of D A (1 nmol in 10 nl)
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