308
Brain Research, 583 (1992) 308-311 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25261
Dopamine microinjected into the nucleus ambiguus elicits vagal bradycardia in spinal rats V.c. Chitravanshi and F.R. Calaresu Department of Physiology, University of Western Ontario, London, Onto N6A 5CI (Canada) (Accepted 7 April 1992)
Key words: Cardiovascular regulation ; Dopamine; Microinjection; Nucleus ambiguus; (± )-Sulpiride; SCH-23390
To investigate the effects of dopamine (DA) on vagal efferent activity, DA was microinjected into the right nucleus ambiguus (NA) in rats. Experiments were done in 19 urethane anaesthetized, artificially ventilated spinal (C l ) rats. Sites in the right NA containing cardioinhibitory neurons were identified by observing a marked and reproducible decrease in heart rate (HR; 64.9 + 2.8 bpm; n = 36) elicited by microinjecting t-glutamate (GLU; 1.5. nmol in 10 nl), No decreases in arterial pressure (AP) were obtained at these sites. Microinjection of DA (1-15 nmol in 10 nl) into 24 of these 36 sites caused a dose-dependent decrease in HR . The responses to 1 nmol and 3 nmol DA were blocked by (± )-sulpiride, a specific D z receptor antagonist (0.1 nmol in 10 nl), A higher dose of ( ± l-sulpiride (I nmol in 10 nl) was required to block the responses to 15 nmol of DA. Bradycardia elicited by even the lowest amount of DA (1 nrnol) was not blocked by SCH-23390, a specific D. receptor antagonist. These experiment s demonstrate that the bradycardia caused by microinjection of DA into the NA is due to the excitation of dopamine D z receptors present on vagal preganglionic cardioinhibitory neurons controlling HR .
The baroreceptor reflex is a regulatory mechanism by which the central nervous system (CNS) controls arterial pressure (AP) and heart rate (HR). One of the efferent limbs of this reflex is composed of vagal cardioinhibitory fibers whose cell bodies are located primarily in the ventrolateral division of the nucleus ambiguus (NA)12,14. Although the central pathways of the baroreceptor reflex and the role of NA in this reflex are well established ':' studies investigating the neurochemical mechanisms of synaptic transmission in the NA have begun only recently 1,5.6.9. Dopamine (DA) is one of the most ubiquitous putative transmitters in the central nervous system". For example in the medulla, DA immunoreactive cell bodies have been demonstrated in the dorsal medulla, the dorsal motor nucleus of vagus and the nucleus of the tractus solitarius (NTS)?·11.13 all of which are structures known to be involved in the baroreceptor reflex", Although DA receptors have not been demonstrated in the NA, it has been shown recently that fibers with tyrosine hydroxylase (TH) immunoreactivity are present in the region of the NA10,15. As TH is an enzyme
that converts tyrosine into DOPA in catecholaminergic terminals it is possible that NA neurons may receive dopaminergic innervation. In the present study the effects on AP and HR of microinjection of DA into the NA of spinal rats were investigated. Spinal cord transection was made at the C 1 level to eliminate descending bulbar pathways controlling sympathetic outflow to the heart:' so that any observable effects of DA microinjection were due exclusively to influences on cardioinhibitory vagal fibers . To determine whether the responses to DA were mediated via D 1 or D 2 receptors, D 1 and D 2 antagonists were injected into the NA before microinjection of DA. Experiments were done in 19 adult male Wistar rats anaesthetized with urethane (Sigma, St. Louis, MO, 1.4 g/kg, i.p. initially and 0.25 g/kg supplements as required). The trachea, femoral artery and vein were cannulated and the animal was ventilated artificially with room air using a small animal ventilator (Harvard Apparatus, Model 683). L-Glutamate (GLU; Sigma, 0.15 M), dopamine (Sigma, 0.1-1.5 M), (±) sulpiride, a
Correspondence: V.c. Chitravanshi, Department of Physiology, The University of Western Ontario, London, Ont. , Canada N6A SCI. Fax: (I) (519) 661-3827.
309 O 2 dopamine receptor antagonist (Research Biochemicals Inc., 0.01-0.1 M), and SCH-23390, a 0 1 dopamine receptor antagonist (Research Biochemicals Inc., 5 mM) were dissolved in phosphate buffered saline (PBS, pH 7.4). Each substance was pressure microinjected from a multibarreled. glass micropipette pulled from glass capillary tubing (Socorex 851-5, Terochem Laboratories, Mississauga, Ont., Canada). Medulla and cervical spinal cord were exposed and transection of the spinal cord was made at the C I level. Phenylephrine (Sigma) in physiological saline (2 mgyrnl) was infused into the venous cannula initially at a rate of 1 mljh for 2-3 minutes and then at 0.25 ml /h to maintain AP within the physiological range (90-100 mm Hg), The pipettes were inclined 20° from the vertical in the sagittal plane with the tips pointing rostrally and were positioned in the right NA according to the coordinates of a stereotaxic atlas". Injection volumes were measured by monitoring the movement of the fluid meniscus in the micropipette through a microscope fitted with an ocular scale that allowed a resolution of 1 nl. Microinjection sites were marked for histological verification with India ink using a method previously described/. Injection sites were mapped on drawings of transverse sections of the rat brain from an atlas 18. Mean changes in AP and HR from control values were compared by Student's r-tests, For comparison of HR responses to four different doses of OA analysis of variance was performed followed by Newman-Keuls test. For dose-response relationships, changes in HR vs. doses were plotted. Significance was determined at a confidence level of P < 0.05 for all statistical tests. All data in text and figures are expressed as means ± S.E.M. GLU was microinjected into the right NA to identify sites from which bradycardia could be elicited. The control mean AP was 95.1 ± 3.3 mm Hg in 19 anaesthetized rats. Microinjection of GLU (1.5 nmol in 10 nl) into the NA elicited a decrease in HR (64.9 + 2.8 bpm; n = 36 sites) which began 1.9 ± 0.2 s after injection, reached a peak in 7.4 ± 0.6 s and lasted for 67.2 ± 2.3 s. No changes were observed in AP. A characteristic response to GLU is shown in Fig. 1A. Microinjections of 10 nl of PBS into 10 of these sites had no effect on either AP or HR (Fig. l B), In 12 rats, microinjection of OA into 24 of the 36 sites in the NA that responded to GLU decreased HR without causing changes in AP. A dose as low as 1 nmol in 10 nllowered HR 08.2 ± 2.7 bpm; n = 6). The decrease in HR after microinjection of 3 nmol in 10 nl (30.5 ± 5.0 bpm; n = 6) was not significantly larger than the response to 1 nmol in 10 nl microinjection of OA. However, 10 nmol in 10 nl 05.7 + 6.3 bpm; n = 6)
e
----
HR 600 [ (b....) 400 V
200
AP
2OOt-
(mmH.) 1~
+
otu
+
PBS DA Fig. I. Cardiovascular responses elicited by microinjection of (A) L-glutamate (GLU; 1.5 nmol in 10 nl); (B) phosphate-buffered saline (PBS; 10 nl) ; and (0 dopamine (DA; 15 nmol in 10 nl) into the right nucleus ambiguus (NA) of a spinal rat. Arrows indicate beginning of microinjection.
and 15 nmol in 10 nl (43.3 ± 2.2 bpm; n = 6) of OA elicited significantly greater HR responses (P < 0.05; Figs. lC; 2). Microinjection of the highest dose of OA (15 nmol in 10 nl) elicited HR responses with an onset latency of 9.0 ± 0.7 s which reached a peak at 39.2 ± 5.4 s and gradually returned to preinjection values in 753.3 ± 34.1 s. In 15 rats, microinjections of (± )-sulpiride (0.1-1 nmol in 10 nl) were made into NA (n = 30 sites). Sulpiride injections did not cause changes in AP or
50
n• 6
.: ,!
40
1····· ... c
10
°0
5
10
15
20
0011 (n1lO11) Fig. 2. Dose-dependent changes in heart rate (HR) elicited by DA microinjection into NA. Each dose of DA was injected in 10 nl volumes. The range of doses used was between I and 15 nmol. The points represent mean values (± S.E.M.).
310 c=JBefore 02 antagonist OJAtter 02 antagonist (0.1 nllOl)
50 §After 02 antagonist (1 nllOl)
40
n • 6 sites
Ii
a.
e
30
ex: :J:
....c Ql III
III
Ql
L
20
U Ql
0
10
OA 1 nmol
OA 3 n-al
DA 15 nlIOl
Fig. 3. Significant blocking effect (P < 0.05) of (± )-sulpiride (0.1-1 nmol in 10 nO on the microinjection of DA into the NA in spinal rats. The results are expressed as mean values ± S.E.M.
HR. The responses to 1 nmol in 10 nl and 3 nmol in 10 nl DA were blocked by (± )-sulpiride (0.1 nmol in 10 nl; n = 12; P < 0.05; Fig. 3) microinjected 60-90 s before DA. The decreases in HR elicited by 10 nmol in 10 nl and 15 nmol in 10 nl DA were not blocked by 0.1 nmol in 10 nl of (± )-sulpiride (n = 12). However, the responses to 15 nmol in 10 nl DA were blocked by a higher dose of (± )-sulpiride (l nmol in 10 nl; n = 6; P < 0.05; Fig. 3). In 4 rats microinjection of SCH-23390 (0.05 nmol in 10 nl) into NA did not block the responses to the lowest dose of DA (I nmol in 10 nl) at 6 sites where microinjection of GLU and DA elicited marked bradycardia. SCH-23390 alone did not elicit any effects on HR and AP. The present study shows for the first time that microinjection of DA into the right NA elicits dose-dependent decreases in HR which are mediated by excitation of vagal preganglionic cardioinhibitory neurons which influence HR without affecting AP. Previous studies from our laboratory have also shown that vagal preganglionic cardioinhibitory neurons located in the right NA have effects on HR only 1.6. The bradycardic response elicited by microinjection of DA was blocked by the D 2-specific antagonist (± )-sulpiride and not by the Dj-speciflc antagonist SCH-23390. These results
therefore demonstrate that DA elicits cardiovascular effects in the NA by an action at dopamine D 2 receptors present on vagal cardiac preganglionic neurons. The decreases in HR were elicited by neurons in the caudal and ventrolateral portion of the right NA. The localization of these sites in the main subdivision of the NA corresponds to the location of vagal preganglionic neurons which control HR1. 6•8 •17• Finally, it can be excluded that the changes in HR elicited by microinjection of GLU and DA were the result of mechanical stimulation of nervous tissue because microinjection of 10 nl of PBS into NA did not elicit changes in HR and, in addition, the responses to GLU and DA were site dependent since they could be eliminated by moving the tip of the micropipette as little as 150 JLm. In summary, our experiments have shown that microinjection of DA results in the excitation of vagal preganglionic cardioinhibitory neurons in the right NA causing a dose-dependant decrease in HR in spinal rats. The study also provides evidence for an action of DA on D 2 receptors present in vagal preganglionic neurons in the NA. The authors wish to thank DJ. McKitrick and K. Hayes for critical comments and suggestions. This study was supported by a grant from the Medical Research Council of Canada. 1 Agarwal, S.K. and Calaresu, F.R., Enkephalins, substance-P and acetylcholine microinjected into the nucleus ambiguus elicit vagal bradycardia in rats, Brain Res., 563 (1991) 203-208. 2 Agarwal, S.K., Gelsema, AJ. and Calaresu, F.R., Neurons in the rostral VLM are inhibited by chemical stimulation of caudal VLM in rats, Am. J. Physiol., 257 (1989) R265-R270. 3 Calaresu, F.R., and Yardley, c.P., Medullary basal sympathetic tone, Annu. Rev. Physiol., 50 (1988) 511-524. 4 Calaresu, F.R., Ciriello, J., Caverson, M.M., Cechetto, D.F. and Krukoff, T.L., Functional neuroanatomy of central pathway controlling the circulation, In T.A. Kotchen and c.P. Guthrie (Eds.), Hypertension and Brain, Futura Publications, Mount Kisko, NY, 1984, pp. 3-21. 5 Caverson, M.M. and Zhang, T.x., Substance P (SP) innervation of vagal cardiomotor neurons (VCN) in the nucleus ambiguus, Soc. Neurosci. Abstr., 15 (1989) 966. 6 Chitravanshi, V.c., Agarwal, S.K. and Calaresu, F.R., Microinjection of glycine into the nucleus ambiguus elicits tachycardia in spinal rats, Brain Res., 566 (1991) 290-294. 7 Dahlstrom, A. and Fuxe, K., Evidence for existence of monoamine containing neurons in the central nervous system, I: Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 Suppl, 232 (1964) 1-55. 8 Ermirio, R., Ruggeri, P., Cogo, c.E., Molinari, C. and Calaresu, F.R., Neuronal and cardiovascular responses to ANF microinjected into nucleus ambiguus, Am. 1. Physiol., 260 (1991) R1089R1094. 9 Gilbey, M.P., Jordan, P., Spyer, K.M. and Wood, L.M., The inhibitory action of GABA on cardiac vagal motoneurons in the cat, J. Physiol., 361 (1985) 49P. 10 Halliday, G.M. and McLachlan, E.M., A comparative analysis of neurons containing catecholamine-synthesizing enzymes and neuropeptide Y in the ventrolateral medulla of rats, guinea-pigs and cats, Neuroscience, 43 (1991) 531-550. 11 Hokfelt, T., Fuxe, K., Goldstein, M., Johansson, 0., Ljundgahl, J. and Schultzberg, M., Immunocytochemical studies on cate-
311 cholamine cell systems. In E. Usdin and I. Kopin (Eds.), Catecholamines: Basic and ClinicalFrontiers, Pergamon Press, Oxford, 1979, pp. 1007-1019. 12 Hopkins, D.A, The dorsal motor nucleus of the vagus nerve and the nucleus ambiguus: structures and connections. In R. Hainsworth., P.N. McWilliam and D.A.S.G. Mary (Eds.), CardiogenicReflexes, Oxford University Press, Oxford, 1987, pp. 185-203. 13 Loewy, AD. and Spyer, K.M., Vagal preganglionic neurons. In AD. Loewy and K.M. Spyer (Eds.), Central Regulation of Autonomic Function, Oxford University Press, Oxford, 1990, pp. 68-87. 14 McAllen, M. and Spyer, K.M., The location of cardiac vagal preganglionic motoneurons in the medulla of cat, J. Physiol., 258 (1976) 187-204.
15 Milner, T.A., Pickel, V.M., Giuliano, R. and Reis, OJ., Ultrastructural localization of cholineacetyltransferase in the rat rostroventrolateral medulla: evidence for major synaptic relations with non-catecholaminergic neurons, Brain Res., 500 (1989) 67-89. 16 Moore, R.Y. and Bloom, F.E., Central catecholamine neuron system: anatomy and physiology of the dopamine systems, Annu. Rev. Neurosci., 1 (1978) 129-169. 17 Nosaka, S., Yasunaga, K. and Tarnai, S., Vagal cardiac preganglionic neurons: distribution, cell types, and reflex discharges, Am. 1. Physiol., 243 (1982) R92-R98. 18 Paxinos, G. and Watson, c., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986.
Brain Research, 583 (1992) 308-311 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25261
Dopamine microinjected into the nucleus ambiguus elicits vagal bradycardia in spinal rats V.c. Chitravanshi and F.R. Calaresu Department of Physiology, University of Western Ontario, London, Onto N6A 5CI (Canada) (Accepted 7 April 1992)
Key words: Cardiovascular regulation ; Dopamine; Microinjection; Nucleus ambiguus; (± )-Sulpiride; SCH-23390
To investigate the effects of dopamine (DA) on vagal efferent activity, DA was microinjected into the right nucleus ambiguus (NA) in rats. Experiments were done in 19 urethane anaesthetized, artificially ventilated spinal (C l ) rats. Sites in the right NA containing cardioinhibitory neurons were identified by observing a marked and reproducible decrease in heart rate (HR; 64.9 + 2.8 bpm; n = 36) elicited by microinjecting t-glutamate (GLU; 1.5. nmol in 10 nl), No decreases in arterial pressure (AP) were obtained at these sites. Microinjection of DA (1-15 nmol in 10 nl) into 24 of these 36 sites caused a dose-dependent decrease in HR . The responses to 1 nmol and 3 nmol DA were blocked by (± )-sulpiride, a specific D z receptor antagonist (0.1 nmol in 10 nl), A higher dose of ( ± l-sulpiride (I nmol in 10 nl) was required to block the responses to 15 nmol of DA. Bradycardia elicited by even the lowest amount of DA (1 nrnol) was not blocked by SCH-23390, a specific D. receptor antagonist. These experiment s demonstrate that the bradycardia caused by microinjection of DA into the NA is due to the excitation of dopamine D z receptors present on vagal preganglionic cardioinhibitory neurons controlling HR .
The baroreceptor reflex is a regulatory mechanism by which the central nervous system (CNS) controls arterial pressure (AP) and heart rate (HR). One of the efferent limbs of this reflex is composed of vagal cardioinhibitory fibers whose cell bodies are located primarily in the ventrolateral division of the nucleus ambiguus (NA)12,14. Although the central pathways of the baroreceptor reflex and the role of NA in this reflex are well established ':' studies investigating the neurochemical mechanisms of synaptic transmission in the NA have begun only recently 1,5.6.9. Dopamine (DA) is one of the most ubiquitous putative transmitters in the central nervous system". For example in the medulla, DA immunoreactive cell bodies have been demonstrated in the dorsal medulla, the dorsal motor nucleus of vagus and the nucleus of the tractus solitarius (NTS)?·11.13 all of which are structures known to be involved in the baroreceptor reflex", Although DA receptors have not been demonstrated in the NA, it has been shown recently that fibers with tyrosine hydroxylase (TH) immunoreactivity are present in the region of the NA10,15. As TH is an enzyme
that converts tyrosine into DOPA in catecholaminergic terminals it is possible that NA neurons may receive dopaminergic innervation. In the present study the effects on AP and HR of microinjection of DA into the NA of spinal rats were investigated. Spinal cord transection was made at the C 1 level to eliminate descending bulbar pathways controlling sympathetic outflow to the heart:' so that any observable effects of DA microinjection were due exclusively to influences on cardioinhibitory vagal fibers . To determine whether the responses to DA were mediated via D 1 or D 2 receptors, D 1 and D 2 antagonists were injected into the NA before microinjection of DA. Experiments were done in 19 adult male Wistar rats anaesthetized with urethane (Sigma, St. Louis, MO, 1.4 g/kg, i.p. initially and 0.25 g/kg supplements as required). The trachea, femoral artery and vein were cannulated and the animal was ventilated artificially with room air using a small animal ventilator (Harvard Apparatus, Model 683). L-Glutamate (GLU; Sigma, 0.15 M), dopamine (Sigma, 0.1-1.5 M), (±) sulpiride, a
Correspondence: V.c. Chitravanshi, Department of Physiology, The University of Western Ontario, London, Ont. , Canada N6A SCI. Fax: (I) (519) 661-3827.
309 O 2 dopamine receptor antagonist (Research Biochemicals Inc., 0.01-0.1 M), and SCH-23390, a 0 1 dopamine receptor antagonist (Research Biochemicals Inc., 5 mM) were dissolved in phosphate buffered saline (PBS, pH 7.4). Each substance was pressure microinjected from a multibarreled. glass micropipette pulled from glass capillary tubing (Socorex 851-5, Terochem Laboratories, Mississauga, Ont., Canada). Medulla and cervical spinal cord were exposed and transection of the spinal cord was made at the C I level. Phenylephrine (Sigma) in physiological saline (2 mgyrnl) was infused into the venous cannula initially at a rate of 1 mljh for 2-3 minutes and then at 0.25 ml /h to maintain AP within the physiological range (90-100 mm Hg), The pipettes were inclined 20° from the vertical in the sagittal plane with the tips pointing rostrally and were positioned in the right NA according to the coordinates of a stereotaxic atlas". Injection volumes were measured by monitoring the movement of the fluid meniscus in the micropipette through a microscope fitted with an ocular scale that allowed a resolution of 1 nl. Microinjection sites were marked for histological verification with India ink using a method previously described/. Injection sites were mapped on drawings of transverse sections of the rat brain from an atlas 18. Mean changes in AP and HR from control values were compared by Student's r-tests, For comparison of HR responses to four different doses of OA analysis of variance was performed followed by Newman-Keuls test. For dose-response relationships, changes in HR vs. doses were plotted. Significance was determined at a confidence level of P < 0.05 for all statistical tests. All data in text and figures are expressed as means ± S.E.M. GLU was microinjected into the right NA to identify sites from which bradycardia could be elicited. The control mean AP was 95.1 ± 3.3 mm Hg in 19 anaesthetized rats. Microinjection of GLU (1.5 nmol in 10 nl) into the NA elicited a decrease in HR (64.9 + 2.8 bpm; n = 36 sites) which began 1.9 ± 0.2 s after injection, reached a peak in 7.4 ± 0.6 s and lasted for 67.2 ± 2.3 s. No changes were observed in AP. A characteristic response to GLU is shown in Fig. 1A. Microinjections of 10 nl of PBS into 10 of these sites had no effect on either AP or HR (Fig. l B), In 12 rats, microinjection of OA into 24 of the 36 sites in the NA that responded to GLU decreased HR without causing changes in AP. A dose as low as 1 nmol in 10 nllowered HR 08.2 ± 2.7 bpm; n = 6). The decrease in HR after microinjection of 3 nmol in 10 nl (30.5 ± 5.0 bpm; n = 6) was not significantly larger than the response to 1 nmol in 10 nl microinjection of OA. However, 10 nmol in 10 nl 05.7 + 6.3 bpm; n = 6)
e
----
HR 600 [ (b....) 400 V
200
AP
2OOt-
(mmH.) 1~
+
otu
+
PBS DA Fig. I. Cardiovascular responses elicited by microinjection of (A) L-glutamate (GLU; 1.5 nmol in 10 nl); (B) phosphate-buffered saline (PBS; 10 nl) ; and (0 dopamine (DA; 15 nmol in 10 nl) into the right nucleus ambiguus (NA) of a spinal rat. Arrows indicate beginning of microinjection.
and 15 nmol in 10 nl (43.3 ± 2.2 bpm; n = 6) of OA elicited significantly greater HR responses (P < 0.05; Figs. lC; 2). Microinjection of the highest dose of OA (15 nmol in 10 nl) elicited HR responses with an onset latency of 9.0 ± 0.7 s which reached a peak at 39.2 ± 5.4 s and gradually returned to preinjection values in 753.3 ± 34.1 s. In 15 rats, microinjections of (± )-sulpiride (0.1-1 nmol in 10 nl) were made into NA (n = 30 sites). Sulpiride injections did not cause changes in AP or
50
n• 6
.: ,!
40
1····· ... c
10
°0
5
10
15
20
0011 (n1lO11) Fig. 2. Dose-dependent changes in heart rate (HR) elicited by DA microinjection into NA. Each dose of DA was injected in 10 nl volumes. The range of doses used was between I and 15 nmol. The points represent mean values (± S.E.M.).
310 c=JBefore 02 antagonist OJAtter 02 antagonist (0.1 nllOl)
50 §After 02 antagonist (1 nllOl)
40
n • 6 sites
Ii
a.
e
30
ex: :J:
....c Ql III
III
Ql
L
20
U Ql
0
10
OA 1 nmol
OA 3 n-al
DA 15 nlIOl
Fig. 3. Significant blocking effect (P < 0.05) of (± )-sulpiride (0.1-1 nmol in 10 nO on the microinjection of DA into the NA in spinal rats. The results are expressed as mean values ± S.E.M.
HR. The responses to 1 nmol in 10 nl and 3 nmol in 10 nl DA were blocked by (± )-sulpiride (0.1 nmol in 10 nl; n = 12; P < 0.05; Fig. 3) microinjected 60-90 s before DA. The decreases in HR elicited by 10 nmol in 10 nl and 15 nmol in 10 nl DA were not blocked by 0.1 nmol in 10 nl of (± )-sulpiride (n = 12). However, the responses to 15 nmol in 10 nl DA were blocked by a higher dose of (± )-sulpiride (l nmol in 10 nl; n = 6; P < 0.05; Fig. 3). In 4 rats microinjection of SCH-23390 (0.05 nmol in 10 nl) into NA did not block the responses to the lowest dose of DA (I nmol in 10 nl) at 6 sites where microinjection of GLU and DA elicited marked bradycardia. SCH-23390 alone did not elicit any effects on HR and AP. The present study shows for the first time that microinjection of DA into the right NA elicits dose-dependent decreases in HR which are mediated by excitation of vagal preganglionic cardioinhibitory neurons which influence HR without affecting AP. Previous studies from our laboratory have also shown that vagal preganglionic cardioinhibitory neurons located in the right NA have effects on HR only 1.6. The bradycardic response elicited by microinjection of DA was blocked by the D 2-specific antagonist (± )-sulpiride and not by the Dj-speciflc antagonist SCH-23390. These results
therefore demonstrate that DA elicits cardiovascular effects in the NA by an action at dopamine D 2 receptors present on vagal cardiac preganglionic neurons. The decreases in HR were elicited by neurons in the caudal and ventrolateral portion of the right NA. The localization of these sites in the main subdivision of the NA corresponds to the location of vagal preganglionic neurons which control HR1. 6•8 •17• Finally, it can be excluded that the changes in HR elicited by microinjection of GLU and DA were the result of mechanical stimulation of nervous tissue because microinjection of 10 nl of PBS into NA did not elicit changes in HR and, in addition, the responses to GLU and DA were site dependent since they could be eliminated by moving the tip of the micropipette as little as 150 JLm. In summary, our experiments have shown that microinjection of DA results in the excitation of vagal preganglionic cardioinhibitory neurons in the right NA causing a dose-dependant decrease in HR in spinal rats. The study also provides evidence for an action of DA on D 2 receptors present in vagal preganglionic neurons in the NA. The authors wish to thank DJ. McKitrick and K. Hayes for critical comments and suggestions. This study was supported by a grant from the Medical Research Council of Canada. 1 Agarwal, S.K. and Calaresu, F.R., Enkephalins, substance-P and acetylcholine microinjected into the nucleus ambiguus elicit vagal bradycardia in rats, Brain Res., 563 (1991) 203-208. 2 Agarwal, S.K., Gelsema, AJ. and Calaresu, F.R., Neurons in the rostral VLM are inhibited by chemical stimulation of caudal VLM in rats, Am. J. Physiol., 257 (1989) R265-R270. 3 Calaresu, F.R., and Yardley, c.P., Medullary basal sympathetic tone, Annu. Rev. Physiol., 50 (1988) 511-524. 4 Calaresu, F.R., Ciriello, J., Caverson, M.M., Cechetto, D.F. and Krukoff, T.L., Functional neuroanatomy of central pathway controlling the circulation, In T.A. Kotchen and c.P. Guthrie (Eds.), Hypertension and Brain, Futura Publications, Mount Kisko, NY, 1984, pp. 3-21. 5 Caverson, M.M. and Zhang, T.x., Substance P (SP) innervation of vagal cardiomotor neurons (VCN) in the nucleus ambiguus, Soc. Neurosci. Abstr., 15 (1989) 966. 6 Chitravanshi, V.c., Agarwal, S.K. and Calaresu, F.R., Microinjection of glycine into the nucleus ambiguus elicits tachycardia in spinal rats, Brain Res., 566 (1991) 290-294. 7 Dahlstrom, A. and Fuxe, K., Evidence for existence of monoamine containing neurons in the central nervous system, I: Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 Suppl, 232 (1964) 1-55. 8 Ermirio, R., Ruggeri, P., Cogo, c.E., Molinari, C. and Calaresu, F.R., Neuronal and cardiovascular responses to ANF microinjected into nucleus ambiguus, Am. 1. Physiol., 260 (1991) R1089R1094. 9 Gilbey, M.P., Jordan, P., Spyer, K.M. and Wood, L.M., The inhibitory action of GABA on cardiac vagal motoneurons in the cat, J. Physiol., 361 (1985) 49P. 10 Halliday, G.M. and McLachlan, E.M., A comparative analysis of neurons containing catecholamine-synthesizing enzymes and neuropeptide Y in the ventrolateral medulla of rats, guinea-pigs and cats, Neuroscience, 43 (1991) 531-550. 11 Hokfelt, T., Fuxe, K., Goldstein, M., Johansson, 0., Ljundgahl, J. and Schultzberg, M., Immunocytochemical studies on cate-
311 cholamine cell systems. In E. Usdin and I. Kopin (Eds.), Catecholamines: Basic and ClinicalFrontiers, Pergamon Press, Oxford, 1979, pp. 1007-1019. 12 Hopkins, D.A, The dorsal motor nucleus of the vagus nerve and the nucleus ambiguus: structures and connections. In R. Hainsworth., P.N. McWilliam and D.A.S.G. Mary (Eds.), CardiogenicReflexes, Oxford University Press, Oxford, 1987, pp. 185-203. 13 Loewy, AD. and Spyer, K.M., Vagal preganglionic neurons. In AD. Loewy and K.M. Spyer (Eds.), Central Regulation of Autonomic Function, Oxford University Press, Oxford, 1990, pp. 68-87. 14 McAllen, M. and Spyer, K.M., The location of cardiac vagal preganglionic motoneurons in the medulla of cat, J. Physiol., 258 (1976) 187-204.
15 Milner, T.A., Pickel, V.M., Giuliano, R. and Reis, OJ., Ultrastructural localization of cholineacetyltransferase in the rat rostroventrolateral medulla: evidence for major synaptic relations with non-catecholaminergic neurons, Brain Res., 500 (1989) 67-89. 16 Moore, R.Y. and Bloom, F.E., Central catecholamine neuron system: anatomy and physiology of the dopamine systems, Annu. Rev. Neurosci., 1 (1978) 129-169. 17 Nosaka, S., Yasunaga, K. and Tarnai, S., Vagal cardiac preganglionic neurons: distribution, cell types, and reflex discharges, Am. 1. Physiol., 243 (1982) R92-R98. 18 Paxinos, G. and Watson, c., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986.
