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Spinal cord serotonin release and raised blood pressure after braintem kainic acid injection
354
Brain Refearch, 366 (198b) 354-357
Elsevier BRE 21375
Spinal cord serotonin r ~ u and m ~ b l ~ p ~ brai~ kainic acid in~tion
after
P.M. PILOWSKY,V. KAPOOR, J.B. MINSON, M.J. WEST and J,P. CHALMERS Department of Medicine and Centrefor Neuroscience, Flinders Medical Centre, Adelaide, S.A. 5042 (Australia)
(Accepted October 15th, 1985) Key words: in vivo brain dialysis- - bulbospinal serotonin neuron - - ventrolateral medulla - - sympatheticactivity
The recently developed technique of in vivo dialysis has permitted us to make direct measurements of serotonin release in a specific region of the spinal cord and to relate this to changesin blood pressure elicited by chemicalstimulation of the brainstem. In the present experiments we have shown that chemical stimulation of bulbospinal neurons in the region of the B3 cell group in the ventromedial medulla, causes an increase in the release of serotonin in the thoracic spinal cord and that this release is associated with an increase in blood pressure.
Previous reports on the role of serotonergic bulbospinal neurons in the regulation of blood pressure have variously suggested that they exert a pressor action 6,9,10,1s or that they have a depressor effect4.5,s,14. These diverse results may in part reflect the fact that there are many separate bulbospinal serotonergic pathways 1 and that these may have a wide diversity of functions, whereas few of the studies to date have focussed on a particular defined anatomical tract 2,3. In this study we have examined the effects of stimulating the bulbospinal projection of the ventrolateral component of the B3 serotonin cell group in the medulla oblongatal,6,11. Since we did not wish to stimulate fibres of passage, we used chemical stimulation with microinjections of kainic acid 12. Furthermore, in order to ensure that we were indeed stimulating serotonin neurons descending into the cord, we adapted the technique of in vivo dialysis developed for the study of dopaminergic systems 7A6A9 and measured the efflux of serotonin into a dialysis tubule placed in the lower thoracic spinal cord. Following the injection of kainic acid into the ventrolateral medulla of the rat, the efflux of serotonin is increased and the blood pressure is raised. The results support the hypothesis that activation of specific bulbospinal serotonin neurons can elevate arterial pressure.
All experiments were carried out in male Wistar Kyoto rats (WKY) weighing 250-350 g and anaesthetized with urethane, 1 g/kg i.p. In order to insert the dialysis probe (Fig. 1), the lower thoracic vertebral column was approached by a dorsal incision, the tail was fixed to the operating table, and one of the spinous processes retracted rostratly. The spinal cord was exposed by incision of the overlying dura. A hole was then made over the right or left dorsolateral funiculus with a hypodermic needle, and the dialysis probe inserted by hand. The wound was covered with silicone gel (Wacker Silgel A,B) to provide stability. One limb of the tubule was connected to a Braund infusion pump, and perfusion of the tubule was commenced using Ringers solution (8.6 g NaCI, 0.3 g KC1, 0,32 g CaCI2 per 1000 ml, pH 6.3) containing Zimeldine HC1 (Astra, 100/~g/ml). Samples (50-100 /tl) were collected at 20 min intervals. At the end of the experiment, the spinal cord of some animals was fixed in phosphate-buffered (0.1 M, pH 7.4) formaldehyde (4%) and glutaraldehyde (0.5%), Subsequent histological examination verified that in every case the tubule had been in close proximity to the intermediolateral cell column. Serotonin was measured in 50 ktl samples of the dialysate using high-performance liquid chromatogra-
Correspondence: J.P. Chalmers, Department of Medicine, Hinders Medical Centre, Bedford Park, S.A. 5042, Australia.
0006-8993/86/$03.50© 1986Elsevier Science Publishers B.V. (Biomedical Division)
355
,,-~ 7/0 PROLENE
the spinal cord. A further 3 sets of 5 Wistar Kyoto rats were placed in a stereotaxic apparatus and prepared for microinjection of either kainic acid (5 nmol in 1(30 nl of 10 mM phosphate-buffered saline, pH 7.4, containing 0.01% pontamine sky blue) or vehicle in the region of the lateral elements of the B3 group of serotonin neurons1,6,11 in the ventral medulla (Fig. 1). Injection sites were confirmed histologically in each case. A segment of thoracic spinal cord rostral
~glLASTIC ,
,
DIALYSIS
*ill I[~ II TUBULE~i [~,ARALDIT
E
:
~ KAINIC ACID
120110-
KAINIC A C I D /
100-
~
MAP 90(toreRo) 8o-
7060-
lmm Fig. 1. Upper part: diagram of dialysis probe. Dialysis tubules were obtained from a renal dialyzer (Cfirans TE07 Dialyzer, Terumo). Individual tubules (o.d. = 200/~m, m.w. cut off range = 1500) were threaded with lengths of 7/0 prolene (Ethicon) to prevent occlusion of flow at the tip, and then threaded into 24-gauge hypodermic needles from which the plastic hub had been removed. The tubule was then folded back and glued along the shaft of the needle (5 rain araldite, Ciba-Geigy), so that a 2 mm tip projected from the end of the needle. Only the distal 2 mm of the probe lay within the spinal cord after insertion. Lower part: Drawing of a coronal section through the medulla oblongata showing the sites adjacent to the pyramidal tracts (PYR) at which microinjections were made 1.5 mm rostral to the obex and 1.3 mm lateral to the midline on the ventral surface of the medulla. NA is nucleus ambiguus and NTS is nucleus tractus solitarius.
phy
(HPLC)
with
electrochemical
detection
( E C D ) 13. Chromatography conditions were as fol-
lows: 25 cm, 4.6 mm i.d., 5 #m, O.D.S. II column (Phase Separations) with a mobile phase of KH2PO 4 (13.6 g/l), EDTA (50 mg/l), sodium octyl sulfonate (135 mg/l) and methanol (12.5%), the pH adjusted to 3.5 with glacial acetic acid. Electrochemical detection was carried out using a glassy carbon cell (Bio Analytical Systems), with the working potential set at 0.65 V. An initial experiment showed that inclusion of 100 mM potassium chloride in the dialysate caused a rise of 134 + 46 (S.E.M.) pg/50gl in serotonin efflux (n = 5, P < 0.05, Student's t-test) consistent with the suggestion that the serotonin measured reflected the level of serotonergic nerve activity in
50-
i
6
ff__
8'o
60. 5o
40 5-HT 30
leO
-0- "
0
2;
4; 80 80 100 120 1;0 160 180
Time (rains) Fig. 2. The upper panel shows the effect on mean arterial pressure (mm Hg) of bilateral microinjectionsof kainic acid into the region of the lateral B3 serotonin cells of five normal WKY rats (black circles and thick black fines) and of five WKY rats pretreated with 5,7-dihydroxytryptamme 2 weeks earlier (black circles and thin black lines). Vehicle-injected animals received microinjections of vehicle alone (open circles and dotted line). Microinjections are indicated by the arrow at 80 rain. Values are means and the symbols shown represent standard errors of the mean obtained by analysis of variance using a randomized block design17. In analysing the effect of microinjections the variance was partitioned so as to derive the standard error of the mean for the entire pre-injection period (symbol drawn at 40 min) and also for any one time interval during the post-injection period 15 (symbol drawn at 120 min). The lower panel shows the effects on serotonin efflux (pg per 20 min collection period) of the same bilateral microinjections of kainic acid or vehicle onto the lateral B3 serotonin cells. Notation is as in the upper panel, but as the mean values provided an average for a 20 rain period, results are presented in histogram form.
356 to the site of insertion of the dialysis probe was removed for determination of tissue levels of serotonin13. In the vehicle injected animals the effiux of serotonin gradually settled after measurement began, and was unaffected by the injection of vehicle onto the lateral B3 serotonin cell group (Fig. 2). Vehicle injection had no effect on arterial pressure (Fig. 2). In a second group of 5 animals, the microinjection of kainic acid into the region of the lateral B3 cells caused a marked increase in the efflux of serotonin and in the level of arterial pressure, both of which were sustained for a period of approximately 1 h (Fig. 2). A third group of 5 rats was pretreated 10-14 days earlier with an intracerebroventricular injection of 5,7-dihydroxytryptamine (200/~g in 10/A of normal saline containing 1 mg/ml ascorbic acid). In these animals, spinal cord serotonin concentration measured at the end of the experiments was reduced to less than 10% of the values observed in the vehicle injected controls (P < 0.001, Student's t-test). Microinjection of kainic acid onto the lateral B3 serotonin neurons in these rats had no effect on the efflux of serotonin and did not cause an increase in arterial pressure (Fig. 2). These experiments demonstrate for the first time the feasibility of using in vivo dialysis for studying the release of neurotransmitters in the spinal cord.
1 Bowker, R.M., Westlund, K.N. and Coulter, J.D., Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study, Brain Research, 226 (1981) 187-199. 2 Chalmers, J.P., Brain amines and models of experimental hypertension, Orc. Res,, 36 (1975) 469-480. 3 Chalmers, J.P. and West, M.J., The nervous system in the pathogenesis of essential hypertension. In W.H. Birkenhager and J.L. Reid (Eds.), Handbook of Hypertension. Vol. L Clinical Aspects of Essential Hypertension, Elsevier, Amsterdam, 1983, pp. 64-96. 4 Coote, J.H. and Macleod, V.H., The influence of bulbospinal monoaminergic pathways on sympathetic nerve activity, J. PhysioL (London), 241 (1974)453-475. 5 de Jong, W., Nijkamp, F.P. and Bohus, B., Role of noradrenaline and serotonin in the central control of blood pressure in normotensive and spontaneously hypertensive rats, Arch. Int. Pharmacodyn., 21 (1975) 272-284. 6 Howe, P.R.C., Kuhn, D,M., Minson, J.B,, Stead, B.H. and Chalmers, J.P., Evidence for a b ~ i a a l serotonergic pressor pathway in the rat brain, Brain Research, 270 (1983) 29-36. 7 Imperato, A. and di Chiara, G., Trans-striatal dialysis cou-
Kainic acid was used because it is recognized to stimulate the cell bodies of central neurons rather than fibres of passage 12 and because preliminary experiments had shown that when injected onto the lateral B3 cells, it caused an elevation in arterial pressure lasting for over 1 h, a time span suitable for studying the effiux of serotonin given the sensitivity of the assay available in our laboratory. The results indicate that injection of kainic acid into that part of the medulla that contains the cell bodies of serotonin neurons projecting to the spinal cord TM, elicits an increase in serotonin release in the spinal cord with a temporally related increase in blood pressure. Since pretreatment with the selective neurotoxin 5,7-dihydroxytryptamine prevented both the increase in pressure and the increase in serotonin effiux, it is our hypothesis that stimulation of the B3 serotonin cells evokes an increase in the release of serotonin in the lateral horn of the thoracic spinal cord and that this in turn elevates arterial pressure through stimulation of sympathetic preganglionic neurons. We are grateful to Adriana Caon and Laura Bannister for their excellent technical assistance. The study was supported by grants from the National Health and Medical Research Council, the National Heart Foundation of Australia, and the Clive and Vera Ramaciotti Foundations.
pied to reverse phase high performance liquid chromatography with electrochemical detection: a new method for the study of the in vivo release of endogenous dopamme and metabolites. J, Neurosci., 4 (1984) 966-977. 8 Ito. A. and Schanberg, S.M.. Central nervous system mechanisms responsible for blood pressure elevation induced by p-chlorophenylalanine. J. Pharmacot. Exp. Ther.. 181 (1972) 65-74, 9 Korner. P.I and Head, G.A., Effects of noradrenerg~c and serotonergic neurons on blood pressure, heart rate and baroreceptor-heart rate reflex of the conscious rabbit, J. Auton. Nerv. Syst., 3 (198t)511-523. 10 Kuhn. D.M., Wolf. W.A and Lovenberg, W., Review of the role of the central serotonergic neuronal system in blood pressure regulation, Hypertension, 2 (1980) 243-255. 11 Loewy, A.D. and McKellar. S.. Serotonergic projections from the ventral medulla to the intermediolateral cell column in the rat, Brain Research, 211 (1981) 146-152. 12 McGeer, P.L., MeGeer, E.G. and Hattori, T., Kaiaic Acid as a Tool in Neurobiology. In (McGeer. P.L. et al. (Ed.), Kainic Acid as a Tool in Neurobiology, Raven Press, New York, 1978, pp. 123-138.
357 13 Mefford, I.N., Application of high-performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain, J. Neurosci. Methods, 3 (1981) 207-224. 14 Neumayr, R.J., Hare, B.D. and Franz, D.N., Evidence for bulbospinal control of sympathetic preganglionic neurons by monoaminergic pathways, Life Sci., 14 (1974) 793-806. 15 Shaw, J., Hunyor, S.N. and Korner, P.I., The peripheral circulatory effects of clonidine and their role in the production of arterial hypotension, Eur. J. Pharmacol., 14 (1971) 101-111. 16 Ungerstedt, U., Herrera-Marschitz, M. and Zetterstrom,
T., Doparnine neurotransmission and brain function, Prog. Brain Res., 55 (1982) 41-49. 17 Wallenstein, S., Zuckei, C.L. and Fleiss, J.L., Some statistical methods useful in circulation research, Circ. Res., 47 (1980) 1-9. 18 Wing, L.M.H. and Chalmers, J.P., Participation of central serotonergic neurons in the control of the circulation of the unanaesthetised rabbit, Orc. Res., 35 (1974) 504-513. 19 Zetterstrom, T., Sharp, T., Marsden, C.A. and Ungerstedt, U. In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after D-amphetamine, J. Neurochem., 41 (1983) 1769-1773.
Brain Refearch, 366 (198b) 354-357
Elsevier BRE 21375
Spinal cord serotonin r ~ u and m ~ b l ~ p ~ brai~ kainic acid in~tion
after
P.M. PILOWSKY,V. KAPOOR, J.B. MINSON, M.J. WEST and J,P. CHALMERS Department of Medicine and Centrefor Neuroscience, Flinders Medical Centre, Adelaide, S.A. 5042 (Australia)
(Accepted October 15th, 1985) Key words: in vivo brain dialysis- - bulbospinal serotonin neuron - - ventrolateral medulla - - sympatheticactivity
The recently developed technique of in vivo dialysis has permitted us to make direct measurements of serotonin release in a specific region of the spinal cord and to relate this to changesin blood pressure elicited by chemicalstimulation of the brainstem. In the present experiments we have shown that chemical stimulation of bulbospinal neurons in the region of the B3 cell group in the ventromedial medulla, causes an increase in the release of serotonin in the thoracic spinal cord and that this release is associated with an increase in blood pressure.
Previous reports on the role of serotonergic bulbospinal neurons in the regulation of blood pressure have variously suggested that they exert a pressor action 6,9,10,1s or that they have a depressor effect4.5,s,14. These diverse results may in part reflect the fact that there are many separate bulbospinal serotonergic pathways 1 and that these may have a wide diversity of functions, whereas few of the studies to date have focussed on a particular defined anatomical tract 2,3. In this study we have examined the effects of stimulating the bulbospinal projection of the ventrolateral component of the B3 serotonin cell group in the medulla oblongatal,6,11. Since we did not wish to stimulate fibres of passage, we used chemical stimulation with microinjections of kainic acid 12. Furthermore, in order to ensure that we were indeed stimulating serotonin neurons descending into the cord, we adapted the technique of in vivo dialysis developed for the study of dopaminergic systems 7A6A9 and measured the efflux of serotonin into a dialysis tubule placed in the lower thoracic spinal cord. Following the injection of kainic acid into the ventrolateral medulla of the rat, the efflux of serotonin is increased and the blood pressure is raised. The results support the hypothesis that activation of specific bulbospinal serotonin neurons can elevate arterial pressure.
All experiments were carried out in male Wistar Kyoto rats (WKY) weighing 250-350 g and anaesthetized with urethane, 1 g/kg i.p. In order to insert the dialysis probe (Fig. 1), the lower thoracic vertebral column was approached by a dorsal incision, the tail was fixed to the operating table, and one of the spinous processes retracted rostratly. The spinal cord was exposed by incision of the overlying dura. A hole was then made over the right or left dorsolateral funiculus with a hypodermic needle, and the dialysis probe inserted by hand. The wound was covered with silicone gel (Wacker Silgel A,B) to provide stability. One limb of the tubule was connected to a Braund infusion pump, and perfusion of the tubule was commenced using Ringers solution (8.6 g NaCI, 0.3 g KC1, 0,32 g CaCI2 per 1000 ml, pH 6.3) containing Zimeldine HC1 (Astra, 100/~g/ml). Samples (50-100 /tl) were collected at 20 min intervals. At the end of the experiment, the spinal cord of some animals was fixed in phosphate-buffered (0.1 M, pH 7.4) formaldehyde (4%) and glutaraldehyde (0.5%), Subsequent histological examination verified that in every case the tubule had been in close proximity to the intermediolateral cell column. Serotonin was measured in 50 ktl samples of the dialysate using high-performance liquid chromatogra-
Correspondence: J.P. Chalmers, Department of Medicine, Hinders Medical Centre, Bedford Park, S.A. 5042, Australia.
0006-8993/86/$03.50© 1986Elsevier Science Publishers B.V. (Biomedical Division)
355
,,-~ 7/0 PROLENE
the spinal cord. A further 3 sets of 5 Wistar Kyoto rats were placed in a stereotaxic apparatus and prepared for microinjection of either kainic acid (5 nmol in 1(30 nl of 10 mM phosphate-buffered saline, pH 7.4, containing 0.01% pontamine sky blue) or vehicle in the region of the lateral elements of the B3 group of serotonin neurons1,6,11 in the ventral medulla (Fig. 1). Injection sites were confirmed histologically in each case. A segment of thoracic spinal cord rostral
~glLASTIC ,
,
DIALYSIS
*ill I[~ II TUBULE~i [~,ARALDIT
E
:
~ KAINIC ACID
120110-
KAINIC A C I D /
100-
~
MAP 90(toreRo) 8o-
7060-
lmm Fig. 1. Upper part: diagram of dialysis probe. Dialysis tubules were obtained from a renal dialyzer (Cfirans TE07 Dialyzer, Terumo). Individual tubules (o.d. = 200/~m, m.w. cut off range = 1500) were threaded with lengths of 7/0 prolene (Ethicon) to prevent occlusion of flow at the tip, and then threaded into 24-gauge hypodermic needles from which the plastic hub had been removed. The tubule was then folded back and glued along the shaft of the needle (5 rain araldite, Ciba-Geigy), so that a 2 mm tip projected from the end of the needle. Only the distal 2 mm of the probe lay within the spinal cord after insertion. Lower part: Drawing of a coronal section through the medulla oblongata showing the sites adjacent to the pyramidal tracts (PYR) at which microinjections were made 1.5 mm rostral to the obex and 1.3 mm lateral to the midline on the ventral surface of the medulla. NA is nucleus ambiguus and NTS is nucleus tractus solitarius.
phy
(HPLC)
with
electrochemical
detection
( E C D ) 13. Chromatography conditions were as fol-
lows: 25 cm, 4.6 mm i.d., 5 #m, O.D.S. II column (Phase Separations) with a mobile phase of KH2PO 4 (13.6 g/l), EDTA (50 mg/l), sodium octyl sulfonate (135 mg/l) and methanol (12.5%), the pH adjusted to 3.5 with glacial acetic acid. Electrochemical detection was carried out using a glassy carbon cell (Bio Analytical Systems), with the working potential set at 0.65 V. An initial experiment showed that inclusion of 100 mM potassium chloride in the dialysate caused a rise of 134 + 46 (S.E.M.) pg/50gl in serotonin efflux (n = 5, P < 0.05, Student's t-test) consistent with the suggestion that the serotonin measured reflected the level of serotonergic nerve activity in
50-
i
6
ff__
8'o
60. 5o
40 5-HT 30
leO
-0- "
0
2;
4; 80 80 100 120 1;0 160 180
Time (rains) Fig. 2. The upper panel shows the effect on mean arterial pressure (mm Hg) of bilateral microinjectionsof kainic acid into the region of the lateral B3 serotonin cells of five normal WKY rats (black circles and thick black fines) and of five WKY rats pretreated with 5,7-dihydroxytryptamme 2 weeks earlier (black circles and thin black lines). Vehicle-injected animals received microinjections of vehicle alone (open circles and dotted line). Microinjections are indicated by the arrow at 80 rain. Values are means and the symbols shown represent standard errors of the mean obtained by analysis of variance using a randomized block design17. In analysing the effect of microinjections the variance was partitioned so as to derive the standard error of the mean for the entire pre-injection period (symbol drawn at 40 min) and also for any one time interval during the post-injection period 15 (symbol drawn at 120 min). The lower panel shows the effects on serotonin efflux (pg per 20 min collection period) of the same bilateral microinjections of kainic acid or vehicle onto the lateral B3 serotonin cells. Notation is as in the upper panel, but as the mean values provided an average for a 20 rain period, results are presented in histogram form.
356 to the site of insertion of the dialysis probe was removed for determination of tissue levels of serotonin13. In the vehicle injected animals the effiux of serotonin gradually settled after measurement began, and was unaffected by the injection of vehicle onto the lateral B3 serotonin cell group (Fig. 2). Vehicle injection had no effect on arterial pressure (Fig. 2). In a second group of 5 animals, the microinjection of kainic acid into the region of the lateral B3 cells caused a marked increase in the efflux of serotonin and in the level of arterial pressure, both of which were sustained for a period of approximately 1 h (Fig. 2). A third group of 5 rats was pretreated 10-14 days earlier with an intracerebroventricular injection of 5,7-dihydroxytryptamine (200/~g in 10/A of normal saline containing 1 mg/ml ascorbic acid). In these animals, spinal cord serotonin concentration measured at the end of the experiments was reduced to less than 10% of the values observed in the vehicle injected controls (P < 0.001, Student's t-test). Microinjection of kainic acid onto the lateral B3 serotonin neurons in these rats had no effect on the efflux of serotonin and did not cause an increase in arterial pressure (Fig. 2). These experiments demonstrate for the first time the feasibility of using in vivo dialysis for studying the release of neurotransmitters in the spinal cord.
1 Bowker, R.M., Westlund, K.N. and Coulter, J.D., Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study, Brain Research, 226 (1981) 187-199. 2 Chalmers, J.P., Brain amines and models of experimental hypertension, Orc. Res,, 36 (1975) 469-480. 3 Chalmers, J.P. and West, M.J., The nervous system in the pathogenesis of essential hypertension. In W.H. Birkenhager and J.L. Reid (Eds.), Handbook of Hypertension. Vol. L Clinical Aspects of Essential Hypertension, Elsevier, Amsterdam, 1983, pp. 64-96. 4 Coote, J.H. and Macleod, V.H., The influence of bulbospinal monoaminergic pathways on sympathetic nerve activity, J. PhysioL (London), 241 (1974)453-475. 5 de Jong, W., Nijkamp, F.P. and Bohus, B., Role of noradrenaline and serotonin in the central control of blood pressure in normotensive and spontaneously hypertensive rats, Arch. Int. Pharmacodyn., 21 (1975) 272-284. 6 Howe, P.R.C., Kuhn, D,M., Minson, J.B,, Stead, B.H. and Chalmers, J.P., Evidence for a b ~ i a a l serotonergic pressor pathway in the rat brain, Brain Research, 270 (1983) 29-36. 7 Imperato, A. and di Chiara, G., Trans-striatal dialysis cou-
Kainic acid was used because it is recognized to stimulate the cell bodies of central neurons rather than fibres of passage 12 and because preliminary experiments had shown that when injected onto the lateral B3 cells, it caused an elevation in arterial pressure lasting for over 1 h, a time span suitable for studying the effiux of serotonin given the sensitivity of the assay available in our laboratory. The results indicate that injection of kainic acid into that part of the medulla that contains the cell bodies of serotonin neurons projecting to the spinal cord TM, elicits an increase in serotonin release in the spinal cord with a temporally related increase in blood pressure. Since pretreatment with the selective neurotoxin 5,7-dihydroxytryptamine prevented both the increase in pressure and the increase in serotonin effiux, it is our hypothesis that stimulation of the B3 serotonin cells evokes an increase in the release of serotonin in the lateral horn of the thoracic spinal cord and that this in turn elevates arterial pressure through stimulation of sympathetic preganglionic neurons. We are grateful to Adriana Caon and Laura Bannister for their excellent technical assistance. The study was supported by grants from the National Health and Medical Research Council, the National Heart Foundation of Australia, and the Clive and Vera Ramaciotti Foundations.
pied to reverse phase high performance liquid chromatography with electrochemical detection: a new method for the study of the in vivo release of endogenous dopamme and metabolites. J, Neurosci., 4 (1984) 966-977. 8 Ito. A. and Schanberg, S.M.. Central nervous system mechanisms responsible for blood pressure elevation induced by p-chlorophenylalanine. J. Pharmacot. Exp. Ther.. 181 (1972) 65-74, 9 Korner. P.I and Head, G.A., Effects of noradrenerg~c and serotonergic neurons on blood pressure, heart rate and baroreceptor-heart rate reflex of the conscious rabbit, J. Auton. Nerv. Syst., 3 (198t)511-523. 10 Kuhn. D.M., Wolf. W.A and Lovenberg, W., Review of the role of the central serotonergic neuronal system in blood pressure regulation, Hypertension, 2 (1980) 243-255. 11 Loewy, A.D. and McKellar. S.. Serotonergic projections from the ventral medulla to the intermediolateral cell column in the rat, Brain Research, 211 (1981) 146-152. 12 McGeer, P.L., MeGeer, E.G. and Hattori, T., Kaiaic Acid as a Tool in Neurobiology. In (McGeer. P.L. et al. (Ed.), Kainic Acid as a Tool in Neurobiology, Raven Press, New York, 1978, pp. 123-138.
357 13 Mefford, I.N., Application of high-performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain, J. Neurosci. Methods, 3 (1981) 207-224. 14 Neumayr, R.J., Hare, B.D. and Franz, D.N., Evidence for bulbospinal control of sympathetic preganglionic neurons by monoaminergic pathways, Life Sci., 14 (1974) 793-806. 15 Shaw, J., Hunyor, S.N. and Korner, P.I., The peripheral circulatory effects of clonidine and their role in the production of arterial hypotension, Eur. J. Pharmacol., 14 (1971) 101-111. 16 Ungerstedt, U., Herrera-Marschitz, M. and Zetterstrom,
T., Doparnine neurotransmission and brain function, Prog. Brain Res., 55 (1982) 41-49. 17 Wallenstein, S., Zuckei, C.L. and Fleiss, J.L., Some statistical methods useful in circulation research, Circ. Res., 47 (1980) 1-9. 18 Wing, L.M.H. and Chalmers, J.P., Participation of central serotonergic neurons in the control of the circulation of the unanaesthetised rabbit, Orc. Res., 35 (1974) 504-513. 19 Zetterstrom, T., Sharp, T., Marsden, C.A. and Ungerstedt, U. In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after D-amphetamine, J. Neurochem., 41 (1983) 1769-1773.