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Increased 3,4-dihydroxyphenylacetic acid (DOPAC) levels in spontaneously hypertensive rats (SHR) during the development of hypertension
Neuroscience Letter,~, 86 {1988} 346 35l Elsevier Scientific Publishers Ireland IAd
346
NSL 05219
Increased 3,4-dihydroxyphenylacetic acid (DOPAC) levels in spontaneously hypertensive rats (SHR) during the development of hypertension Ralph D a w s o n Jr. and Michael J. Meldrum Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL 32610 (U.S.A.) (Received 29 June 1987; Revised version received 3 December 1987; Accepted 4 December 1987)
Key words." Spontaneously hypertensive rat; Hypertension; Dopamine; Cerebrospinat fluid; Monoamine metabolite Central dopaminergic neuronal activity was investigated in the spontaneously hypertensive rat (SHR) using an in vivo cerebrospinal fluid (CSF) sampling technique. Increased central dopaminergic activity in the SHR was indicated by a significant (P<0.05) elevation in CSF levels of DOPAC relative to both Wistar-Kyoto and Sprague--Dawley control strains. The increased levels of CSF DOPAC were present at 5, 10 and 16 weeks of age. Homovanitlic acid and 5-hydroxyindoleacetic acid levels were significantly (P<0.05) greater in SHR than WKY at 16 weeks. The possible role of central dopaminergic neurons in the pathogenesis of hypertension in the SHR was discussed.
There is increasing evidence for a role of central dopaminergic neurons in cardiovascular regulation and the pathogenesis of hypertension. A number of studies have specifically implicated central dopaminergic mechanisms as being involved in the development of hypertension in the spontaneously hypertensive rat (SHR), Dopamine (DA) and its major metabolite, 3,4 dihydroxyphenylacetic acid (DOPAC) levels are elevated relative to the normotensive Wistar-Kyoto rat (WKY) in several brain regions [5, 16]. DA receptor numbers have also been shown to be elevated in striatum of SHR [1, 7], while DA uptake is reduced in the striatum and frontal cortex [10]. The depressor actions of bromocriptine, a DA agonist, appears to be centrally mediated in the SHR [11] suggesting that the neurochemical abnormalities present in the SHR may be of pathophysiological relevance. The aim of the present study was to evaluate central DA activity in the SHR by using an in vivo sampling technique to measure DOPAC levels in the cerebrospinal fluid (CSF). Determination of CSF monoamine metabolite levels has been shown to provide a useful index of
Correspondence." R. Dawson Jr., Department of Pharmacodynamics, College of Pharmacy, University o~ Florida, JHMHC, Box J-487, Gainesville, FL 32610, U.S.A. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
347
monoamine turnover [8, 13]. CSF monoamine metabolite levels were assessed at various time points during the developmental progression of hypertension in the SHR. Male SHR and W K Y rats of 4, 9 and 15 weeks of age were obtained from Taconic Farms (Germantown, NY) and housed two per cage under standard temperature and humidity conditions. Animals had access to food and water ad libitum. The light cycle was 14 h light, 10 h dark. Animals were acclimated for 1 week prior to experimentation. CSF samples were withdrawn by a modified method of Haywood et al. [3] from sodium pentobarbital (40 mg/kg) anesthetized animals. Briefly, animals were placed in a stereotaxic holder and the head flexed 45 ° downward. The skin and muscle was separated by a midline incision and the occipital membrane covering the foramen magnum exposed. A 22 gauge needle attached to tygon tubing (15 inches) was attached to a 1 ml syringe. The needle penetrated the occipital membrane and the CSF fluid was withdrawn. Approximately 100-150/tl was removed and 50/tl was added to 50/tl of 0.1 N perchloric acid and quickly frozen for monoamine metabolite assay. The samples were thawed and centrifuged prior to determination of monoamine metabolite levels using high performance liquid chromatography and electrochemical detection as previously described [6]. The values obtained for CSF monoamine metabolite levels using this method are in agreement with values obtained from freely moving rats chronically cannulated in the cisterna magna [2]. Comparisons between SHR and W K Y or Sprague-Dawley (SD) at each age were made using the Student's t-test. The results of our initial observation are presented in Table I. Ten-week-old SHR had significantly (P<0.05) higher CSF levels of DOPAC than either W K Y or SD controls. CSF levels of 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and the amino acid, tryptophan (Trp), did not differ between the groups. A developmental study was then initiatied to confirm our original observation. Age related alterations in CSF D O P A C levels in W K Y and SHR are shown in Fig. 1. SHR had significantly higher DOPAC levels at all ages tested, and the greatest difference between SHR and W K Y occurred at 10 weeks. The DA metabolite, HVA, was significantly elevated in SHR compared to W K Y at 16 weeks, but not at 5 or 10 weeks (Table II). Thus, HVA levels are not consistently elevated in SHR except at week 16. 5-HIAA levels are presented in Fig. 2 and there were no significant differ-
TABLE I CSF M E T A B O L I T E LEVELS IN 10-WEEK-OLD SHR, W K Y A N D SD All values expressed as pg/,ul +__S.E.M. n = 5~5 per group. *P < 0.05 S H R vs W K Y . Group
DOPAC
5-HIAA
HVA
Trp
SD WKY SHR
9.9_+ 1.4 11.8_+0.8 21.1 _+3.7*
113_21 83-t- 14 7 8 + 14
21.4_+2.9 19.6_+2.2 21.0_+2.4
551 _+92 447_+41 493_+47
348
28
[]s..
24 ¸
~20 "5
8S
~
16 ¸
<~ 0,.
12 ¸
"x.l~
x
x
o
t
8
-.x
4
5 week
10 w e e k
16 w e e k
Fig. 1. D O P A C levels in the CSF of S H R and W K Y at various ages during the developmental progression of hypertension. * P < 0 . 0 5 , **P<0.01 S H R vs WKY.
ences between SHR and W K Y except for week 16, at which time 5-HIAA levels in SHR were significantly elevated relative to WKY. Our results clearly show an early and sustained increase in CSF levels of DOPAC in the SHR relative to age-matched WKY. The O-methylated metabolite of DA, HVA, was also slightly increased in SHR, but this effect was small and variable. The increased CSF levels of D O P A C in SHR are consistent with previous studies showing increased tissue levels of DOPAC in SHR [5]. Howes et al. [5] reported increased striatal content of DOPAC in SHR at 6, 14 and 28 weeks of age when compared to WKY. Thus, elevations in CSF and tissue content of DOPAC are present both during the developmental and established phase of hypertension. Studies of DA turnover are difficult to interpret since DA turnover in the neurohypophysis is decreased in 8-week-old SHR [9] but increased in the olfactory tubercle of 7-week-old SHR and unchanged in the striatum [7]. Tyrosine hydroxylase activity has been reported to be significantly increased in the str~atum of 8week-old SHR [12]. DA levels are significantly increased in the frontal cortex of 14TABLE I1 HVA LEVELS IN CSF OF SHR A N D W K Y All values express as pg//A + S.E,M, "HVA levels in 1 W K Y and 2 S H R were below the limits of detection lbr the assay. These animals were assigned a value equal to the detection limit (5 pg//d) for the purposes of data analysis. Number in parentheses represent n. * P < 0 . 0 1 W K Y vs SHR, Age
WKY
SHR
5 Week 10 Week ~ 16 Week
22,04+ 1.24 (9) 12.74 -2:1.46 (8) 16,71 + 1,00 (5)
25.14+3.61 (9) 15.04 _+ 2.47 (8) 2 3 . 0 6 + 0 . 6 2 (5)*
349 140 ]WKY
120
~
*,
100
+l
-'5
80-
~
60-
to
40-
-
20-
0
5 week
10 w e e k
16 w e e k
Fig. 2. CSF levels of 5-HIAA in SHR and WKY at 5, 10 and 16 weeks of age. *P<0.05 SHR vs WKY. week-old SHR [16] and the midbrain of 6-week-old SHR [5]. Therefore, at present it is known that numerous changes occur in DA metabolites, but these changes are age and region dependent. Recent studies suggest that depletion of brain levels of DA and not norepinephrine are responsible for the blood pressure attenuating actions of the neurotoxin 6-hydroxydopamine [14]. Lesions of the substantia nigra but not the ventral tegmental area attenuate the development of hypertension in the SHR, suggesting that the nigrostriatal DA system is involved in cardiovascular control [I 5]. Thus, increased central dopaminergic activity may be involved in the pathogenesis of hypertension in SHR. The specific neuroanatomic locus of the DA neurons involved or the exact neurochemical deficits are unknown. The increase in D O P A C levels in the CSF of S H R suggest several possibilities. These include; increased DA release and/or synthesis, increased intraneuronal or extraneuronal monoamine oxidase activity, increased efflux from the extracellular space to the CSF c o m p a r t m e n t or decreased catechol-O-methyltransferase (COMT) activity. C O M T activity does not appear decreased since HVA levels appear normal or slightly elevated. D O P A C levels are elevated in brain tissue [5] so alterations in blood brain barrier transport are unlikely. Therefore, an increase in D A release appears likely and is consistent with the lesion studies that suggest DA depletion attenuates the development of hypertension [14, 15]. Studies of DA release in the central nervous system of S H R are warranted and the role DA autoreceptors in controlling DA release in S H R are especially of interest as they relate to the antihypertensive potency of DA agonists. DA receptor number has been reported to be elevated in the striatum, olfactory tubercle and frontal cortex of S H R when compared to W K Y [7]. These elevations in spiroperidol binding were evident at 7 weeks of age and maintained in SHR 16 20 weeks old. Thus, the elevation in CSF D O P A C levels parallels the increases in DA receptor number. While the changes in D O P A C levels and DA receptor number in SHR appear related to the pathogenesis of hypertension, the possibility exists that
350
the changes in DA function are related to the alterations in locomotor behavior present in SHR [4]. Further pharmacological characterization of the cardiovascular actions of centrally administered DA receptor subtype selective agonists and antagonists is needed. In conclusion, the present demonstration of elevated CSF DOPAC levels in conjunction with other data suggesting abnormal DA metabolism in SHR are consistent with the involvement of central DA neurons in the pathophysiology of hypertension and/or the expression of behavioral differences. This study was funded in part by grants from the Division of Sponsored Research and College of Pharmacy (BRSG) of the University of Florida, Pharmaceutical Manufacturers Association Research Starter Grant and the Florida Heart Association. 1 Chiu, P., Rajakumar, G,, Chiu, S., Kwan, C.-Y. and Mishra, R.K., Enhanced pH]spiroperidol binding in striatum of spontaneously hypertensive rat (SHR), Eur. J. Pharmacol., 82 (1982) 243-244. 2 De La Riva, C.F. and Yeo, J.A.G., Repeated determination of cerebrospinal fluid amine metabolites by automated direct sampling from an implanted eannula in freely moving rats, J. Neurosci. Meth., 14 (1985) 233-240. 3 Haywood, J.R., Buggy, J., Fink, G.D., DiBona, G.F., Johnson, A.K. and Brody, M.J, Alterations in cerebrospinal fluid sodium and osmolality in rats during one-kidney, one-wrap renal, Hypertens. Clin. Exp. Pharmacol. Physiol,, 11 (1984) 545-549. 4 Hettstrand, K. and Engel, J., Locomotor activity and catecholamine receptor binding in adult normotensive and spontaneously hypertensive rats, J. Neural Transm., 48 (1980) 57-63. 5 Howes, L.G., Rowe, P.R., Summers, R.J. and Louis, W.J., Age related changes of catecholamines and their metabolites in central nervous system regions of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats, Clin: Exp. Hypertens., A6 (1984) 2263-2277. 6 Kontur, P., Dawson, R. and Monjan, A., Manipulation of mobile phase parameters for HPLC separation of endogenous monoamines in rat brain tissue, J. Neurosci. Meth., 11 (I 984) 5-18. 7 Le Fur, G., Guilloux, F., Kabouche, M., Mitrani, N., Ferris, O. and Uzan, A., Central dopaminergic neurons during development of genetic and DOCA-salt hypertension in the rat, Dev. Brain Res., l (1981) 153-163. 8 Mignot, E., Laude, D. and Elghozi, J.L., Kinetics ofdrug-induced changes in dopamine and serotonin metabolite concentrations in the CSF of the rat, J. Neurochem., 42 (1984) 8 ! 9-825. 9 Morris, M. and Sundherg, D.K., Neurohypophyseal dopamine biosynthesis in the spontaneously hypertensive rat, Clin. Exp. Hypertens., 3 (1981) 1165-1181. 10 Myers, M.M., Whittemore, S.R. and Hendley, E.D., Changes in catecholamine neuronal uptake and receptor binding in the brains of spontaneously hypertensive rats (SHR), Brain Res., 220 (1981) 325 338. 11 Nagahama, S., Chen, Y.-F., and Oparil, S., Mechanism of the depressor effect of bromocriptine in the spontaneously hypertensive rat, J. Pharmacol. Exp. Ther., 228 (198,1) 370-375. 12 Nagaoka, A. and Lovenberg, W., Regional changes in the activities of aminergic biosynthetic enzymes in the brains of hypertensive rats, Eur. J. Pharma¢ol., 43 (1977) 297-306. 13 Scheinin, M., Monoamine metabolites in human cerebrospinal fluid: indicators of neuronal activity?, Med. Biol., 63 (1985) 1-17. 14 Van den Buuse, M., Versteeg, D.H.G. and De Jong, W., Role ofdopamine in the development of spontaneous hypertension, Hypertension, 6 (1984) 899-905. 15 Van den Buuse, M., Versteeg, D.H.G. and Jong, W. de, Brain dopamine depletion by lesions in the substantia nigra attenuates the development of hypertension in the spontaneously hypertensive rat, Brain Res., 368 (1986) 69-78. 16 Versteeg, D.H.G., Palkovits, M., Van der Gugten, J., Wijnen, H.L.J.M., Smeets, G.W.M. and Jong, W. de, Catecholamine content of individual brain regions of spontaneously hypertensive rats (SHrats), Brain Res., 112 (1976) 429-434.
346
NSL 05219
Increased 3,4-dihydroxyphenylacetic acid (DOPAC) levels in spontaneously hypertensive rats (SHR) during the development of hypertension Ralph D a w s o n Jr. and Michael J. Meldrum Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL 32610 (U.S.A.) (Received 29 June 1987; Revised version received 3 December 1987; Accepted 4 December 1987)
Key words." Spontaneously hypertensive rat; Hypertension; Dopamine; Cerebrospinat fluid; Monoamine metabolite Central dopaminergic neuronal activity was investigated in the spontaneously hypertensive rat (SHR) using an in vivo cerebrospinal fluid (CSF) sampling technique. Increased central dopaminergic activity in the SHR was indicated by a significant (P<0.05) elevation in CSF levels of DOPAC relative to both Wistar-Kyoto and Sprague--Dawley control strains. The increased levels of CSF DOPAC were present at 5, 10 and 16 weeks of age. Homovanitlic acid and 5-hydroxyindoleacetic acid levels were significantly (P<0.05) greater in SHR than WKY at 16 weeks. The possible role of central dopaminergic neurons in the pathogenesis of hypertension in the SHR was discussed.
There is increasing evidence for a role of central dopaminergic neurons in cardiovascular regulation and the pathogenesis of hypertension. A number of studies have specifically implicated central dopaminergic mechanisms as being involved in the development of hypertension in the spontaneously hypertensive rat (SHR), Dopamine (DA) and its major metabolite, 3,4 dihydroxyphenylacetic acid (DOPAC) levels are elevated relative to the normotensive Wistar-Kyoto rat (WKY) in several brain regions [5, 16]. DA receptor numbers have also been shown to be elevated in striatum of SHR [1, 7], while DA uptake is reduced in the striatum and frontal cortex [10]. The depressor actions of bromocriptine, a DA agonist, appears to be centrally mediated in the SHR [11] suggesting that the neurochemical abnormalities present in the SHR may be of pathophysiological relevance. The aim of the present study was to evaluate central DA activity in the SHR by using an in vivo sampling technique to measure DOPAC levels in the cerebrospinal fluid (CSF). Determination of CSF monoamine metabolite levels has been shown to provide a useful index of
Correspondence." R. Dawson Jr., Department of Pharmacodynamics, College of Pharmacy, University o~ Florida, JHMHC, Box J-487, Gainesville, FL 32610, U.S.A. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
347
monoamine turnover [8, 13]. CSF monoamine metabolite levels were assessed at various time points during the developmental progression of hypertension in the SHR. Male SHR and W K Y rats of 4, 9 and 15 weeks of age were obtained from Taconic Farms (Germantown, NY) and housed two per cage under standard temperature and humidity conditions. Animals had access to food and water ad libitum. The light cycle was 14 h light, 10 h dark. Animals were acclimated for 1 week prior to experimentation. CSF samples were withdrawn by a modified method of Haywood et al. [3] from sodium pentobarbital (40 mg/kg) anesthetized animals. Briefly, animals were placed in a stereotaxic holder and the head flexed 45 ° downward. The skin and muscle was separated by a midline incision and the occipital membrane covering the foramen magnum exposed. A 22 gauge needle attached to tygon tubing (15 inches) was attached to a 1 ml syringe. The needle penetrated the occipital membrane and the CSF fluid was withdrawn. Approximately 100-150/tl was removed and 50/tl was added to 50/tl of 0.1 N perchloric acid and quickly frozen for monoamine metabolite assay. The samples were thawed and centrifuged prior to determination of monoamine metabolite levels using high performance liquid chromatography and electrochemical detection as previously described [6]. The values obtained for CSF monoamine metabolite levels using this method are in agreement with values obtained from freely moving rats chronically cannulated in the cisterna magna [2]. Comparisons between SHR and W K Y or Sprague-Dawley (SD) at each age were made using the Student's t-test. The results of our initial observation are presented in Table I. Ten-week-old SHR had significantly (P<0.05) higher CSF levels of DOPAC than either W K Y or SD controls. CSF levels of 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and the amino acid, tryptophan (Trp), did not differ between the groups. A developmental study was then initiatied to confirm our original observation. Age related alterations in CSF D O P A C levels in W K Y and SHR are shown in Fig. 1. SHR had significantly higher DOPAC levels at all ages tested, and the greatest difference between SHR and W K Y occurred at 10 weeks. The DA metabolite, HVA, was significantly elevated in SHR compared to W K Y at 16 weeks, but not at 5 or 10 weeks (Table II). Thus, HVA levels are not consistently elevated in SHR except at week 16. 5-HIAA levels are presented in Fig. 2 and there were no significant differ-
TABLE I CSF M E T A B O L I T E LEVELS IN 10-WEEK-OLD SHR, W K Y A N D SD All values expressed as pg/,ul +__S.E.M. n = 5~5 per group. *P < 0.05 S H R vs W K Y . Group
DOPAC
5-HIAA
HVA
Trp
SD WKY SHR
9.9_+ 1.4 11.8_+0.8 21.1 _+3.7*
113_21 83-t- 14 7 8 + 14
21.4_+2.9 19.6_+2.2 21.0_+2.4
551 _+92 447_+41 493_+47
348
28
[]s..
24 ¸
~20 "5
8S
~
16 ¸
<~ 0,.
12 ¸
"x.l~
x
x
o
t
8
-.x
4
5 week
10 w e e k
16 w e e k
Fig. 1. D O P A C levels in the CSF of S H R and W K Y at various ages during the developmental progression of hypertension. * P < 0 . 0 5 , **P<0.01 S H R vs WKY.
ences between SHR and W K Y except for week 16, at which time 5-HIAA levels in SHR were significantly elevated relative to WKY. Our results clearly show an early and sustained increase in CSF levels of DOPAC in the SHR relative to age-matched WKY. The O-methylated metabolite of DA, HVA, was also slightly increased in SHR, but this effect was small and variable. The increased CSF levels of D O P A C in SHR are consistent with previous studies showing increased tissue levels of DOPAC in SHR [5]. Howes et al. [5] reported increased striatal content of DOPAC in SHR at 6, 14 and 28 weeks of age when compared to WKY. Thus, elevations in CSF and tissue content of DOPAC are present both during the developmental and established phase of hypertension. Studies of DA turnover are difficult to interpret since DA turnover in the neurohypophysis is decreased in 8-week-old SHR [9] but increased in the olfactory tubercle of 7-week-old SHR and unchanged in the striatum [7]. Tyrosine hydroxylase activity has been reported to be significantly increased in the str~atum of 8week-old SHR [12]. DA levels are significantly increased in the frontal cortex of 14TABLE I1 HVA LEVELS IN CSF OF SHR A N D W K Y All values express as pg//A + S.E,M, "HVA levels in 1 W K Y and 2 S H R were below the limits of detection lbr the assay. These animals were assigned a value equal to the detection limit (5 pg//d) for the purposes of data analysis. Number in parentheses represent n. * P < 0 . 0 1 W K Y vs SHR, Age
WKY
SHR
5 Week 10 Week ~ 16 Week
22,04+ 1.24 (9) 12.74 -2:1.46 (8) 16,71 + 1,00 (5)
25.14+3.61 (9) 15.04 _+ 2.47 (8) 2 3 . 0 6 + 0 . 6 2 (5)*
349 140 ]WKY
120
~
*,
100
+l
-'5
80-
~
60-
to
40-
-
20-
0
5 week
10 w e e k
16 w e e k
Fig. 2. CSF levels of 5-HIAA in SHR and WKY at 5, 10 and 16 weeks of age. *P<0.05 SHR vs WKY. week-old SHR [16] and the midbrain of 6-week-old SHR [5]. Therefore, at present it is known that numerous changes occur in DA metabolites, but these changes are age and region dependent. Recent studies suggest that depletion of brain levels of DA and not norepinephrine are responsible for the blood pressure attenuating actions of the neurotoxin 6-hydroxydopamine [14]. Lesions of the substantia nigra but not the ventral tegmental area attenuate the development of hypertension in the SHR, suggesting that the nigrostriatal DA system is involved in cardiovascular control [I 5]. Thus, increased central dopaminergic activity may be involved in the pathogenesis of hypertension in SHR. The specific neuroanatomic locus of the DA neurons involved or the exact neurochemical deficits are unknown. The increase in D O P A C levels in the CSF of S H R suggest several possibilities. These include; increased DA release and/or synthesis, increased intraneuronal or extraneuronal monoamine oxidase activity, increased efflux from the extracellular space to the CSF c o m p a r t m e n t or decreased catechol-O-methyltransferase (COMT) activity. C O M T activity does not appear decreased since HVA levels appear normal or slightly elevated. D O P A C levels are elevated in brain tissue [5] so alterations in blood brain barrier transport are unlikely. Therefore, an increase in D A release appears likely and is consistent with the lesion studies that suggest DA depletion attenuates the development of hypertension [14, 15]. Studies of DA release in the central nervous system of S H R are warranted and the role DA autoreceptors in controlling DA release in S H R are especially of interest as they relate to the antihypertensive potency of DA agonists. DA receptor number has been reported to be elevated in the striatum, olfactory tubercle and frontal cortex of S H R when compared to W K Y [7]. These elevations in spiroperidol binding were evident at 7 weeks of age and maintained in SHR 16 20 weeks old. Thus, the elevation in CSF D O P A C levels parallels the increases in DA receptor number. While the changes in D O P A C levels and DA receptor number in SHR appear related to the pathogenesis of hypertension, the possibility exists that
350
the changes in DA function are related to the alterations in locomotor behavior present in SHR [4]. Further pharmacological characterization of the cardiovascular actions of centrally administered DA receptor subtype selective agonists and antagonists is needed. In conclusion, the present demonstration of elevated CSF DOPAC levels in conjunction with other data suggesting abnormal DA metabolism in SHR are consistent with the involvement of central DA neurons in the pathophysiology of hypertension and/or the expression of behavioral differences. This study was funded in part by grants from the Division of Sponsored Research and College of Pharmacy (BRSG) of the University of Florida, Pharmaceutical Manufacturers Association Research Starter Grant and the Florida Heart Association. 1 Chiu, P., Rajakumar, G,, Chiu, S., Kwan, C.-Y. and Mishra, R.K., Enhanced pH]spiroperidol binding in striatum of spontaneously hypertensive rat (SHR), Eur. J. Pharmacol., 82 (1982) 243-244. 2 De La Riva, C.F. and Yeo, J.A.G., Repeated determination of cerebrospinal fluid amine metabolites by automated direct sampling from an implanted eannula in freely moving rats, J. Neurosci. Meth., 14 (1985) 233-240. 3 Haywood, J.R., Buggy, J., Fink, G.D., DiBona, G.F., Johnson, A.K. and Brody, M.J, Alterations in cerebrospinal fluid sodium and osmolality in rats during one-kidney, one-wrap renal, Hypertens. Clin. Exp. Pharmacol. Physiol,, 11 (1984) 545-549. 4 Hettstrand, K. and Engel, J., Locomotor activity and catecholamine receptor binding in adult normotensive and spontaneously hypertensive rats, J. Neural Transm., 48 (1980) 57-63. 5 Howes, L.G., Rowe, P.R., Summers, R.J. and Louis, W.J., Age related changes of catecholamines and their metabolites in central nervous system regions of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats, Clin: Exp. Hypertens., A6 (1984) 2263-2277. 6 Kontur, P., Dawson, R. and Monjan, A., Manipulation of mobile phase parameters for HPLC separation of endogenous monoamines in rat brain tissue, J. Neurosci. Meth., 11 (I 984) 5-18. 7 Le Fur, G., Guilloux, F., Kabouche, M., Mitrani, N., Ferris, O. and Uzan, A., Central dopaminergic neurons during development of genetic and DOCA-salt hypertension in the rat, Dev. Brain Res., l (1981) 153-163. 8 Mignot, E., Laude, D. and Elghozi, J.L., Kinetics ofdrug-induced changes in dopamine and serotonin metabolite concentrations in the CSF of the rat, J. Neurochem., 42 (1984) 8 ! 9-825. 9 Morris, M. and Sundherg, D.K., Neurohypophyseal dopamine biosynthesis in the spontaneously hypertensive rat, Clin. Exp. Hypertens., 3 (1981) 1165-1181. 10 Myers, M.M., Whittemore, S.R. and Hendley, E.D., Changes in catecholamine neuronal uptake and receptor binding in the brains of spontaneously hypertensive rats (SHR), Brain Res., 220 (1981) 325 338. 11 Nagahama, S., Chen, Y.-F., and Oparil, S., Mechanism of the depressor effect of bromocriptine in the spontaneously hypertensive rat, J. Pharmacol. Exp. Ther., 228 (198,1) 370-375. 12 Nagaoka, A. and Lovenberg, W., Regional changes in the activities of aminergic biosynthetic enzymes in the brains of hypertensive rats, Eur. J. Pharma¢ol., 43 (1977) 297-306. 13 Scheinin, M., Monoamine metabolites in human cerebrospinal fluid: indicators of neuronal activity?, Med. Biol., 63 (1985) 1-17. 14 Van den Buuse, M., Versteeg, D.H.G. and De Jong, W., Role ofdopamine in the development of spontaneous hypertension, Hypertension, 6 (1984) 899-905. 15 Van den Buuse, M., Versteeg, D.H.G. and Jong, W. de, Brain dopamine depletion by lesions in the substantia nigra attenuates the development of hypertension in the spontaneously hypertensive rat, Brain Res., 368 (1986) 69-78. 16 Versteeg, D.H.G., Palkovits, M., Van der Gugten, J., Wijnen, H.L.J.M., Smeets, G.W.M. and Jong, W. de, Catecholamine content of individual brain regions of spontaneously hypertensive rats (SHrats), Brain Res., 112 (1976) 429-434.