164
Brain Research, 173 (1979) 164-167 © Elsevier/North-Holland Biomedical Press
An abnormal regulation of tyrosine hydroxylase restricted to one catecholamine nucleus in the brain stem of spontaneously hypertensive rats
BERNARD RENAUD*, TONG H. JOH and DONALD J. REIS Laboratory of Neurobiology, Cornell University Medical College, New York, N.Y. 10021 (U.S.A.)
(Accepted May 10th, 1979)
Spontaneously hypertensive (SH) rats of the Okamoto strain contain different amounts of one or several of the catecholamine neurotransmitters dopamine, noradrenaline or adrenaline, regionally in brain than do normotensive controls 12,13,18,17. In some instances the differences may only be evident in young animals 7,13. One molecule through which the genetic variation in central catecholamine metabolism might be expressed is tyrosine hydroxylase (TH), the enzyme catalyzing the initial step in the biosynthesis of catecholamine neurotransmitters from tyrosine. Whether the basal activity and/or the regulation of the enzyme differs in the brain of SH rats is not known. In the present study we have analyzed, in SH rats, the basal activity of T H and one mode of its long-term regulation, the prolonged increase in activity secondary to enhanced accumulation of the enzyme produced in all brain stem noradrenergic groups by reserpine s-l°,ls. T H activity has been analyzed in the locus coeruleus and in two other catecholamine groups of the medulla believed to be of importance in cardiovascular control: the A1 group, which lies ventrolaterally in the medulla2; and the A2 group, which lies in the dorsal medullary tegmentumL The changes in regional T H activity in SH rats elicited by reserpine have been compared in young (4 weeks) and mature rats (14 weeks) with age- and sex-matched normotensive animals of the Sprague-Dawley and Wistar-Kyoto (WKY) strains. Male SH rats of the Okamoto strain and WKY controls were purchased from Taconic Farms (Germantown, N.Y.); male Sprague-Dawley rats from Hilltop Lab Animals (Scottsdale, Pa.). All rats were maintained, 2-3 to a cage, in a thermally controlled light-cycled environment for at least 10 days prior to the experiment. Reserpine (Serpasil, Ciba Pharmaceutical, Summit, N.J. was administered subcutaneously between 09.00 and 11.00 h. The animals were killed by cervical dislocation at various times following the last injection, usually at 96 h, a time at which there is a maximal elevation of enzyme activity in the locus ceruleus, A1 and A2 neurons 5,s-1°. * Present address: D6partement de Physiologic et Pharmacologie, Facult6 de Pharmacie, Universit6 Claude Bernard, Lyon, France.
165 Brains were r a p i d l y r e m o v e d a n d the A1, A 2 a n d locus coeruleus areas r e m o v e d b y microdissection 9,14. Tissues were h o m o g e n i z e d by sonication in fixed vols. o f 5 m M p o t a s s i u m phosp h a t e buffer, p H 6.5, c o n t a i n i n g 0.2 ~ T r i t o n X-100. The h o m o g e n i z a t i o n vols. were 200/~1 p e r p a i r o f locus coeruleus a n d 150 ~1 p e r p a i r o f A1 o r A 2 areas. T h e h o m o g enates were centrifuged for 10 min at 6000 g. T H activity was assayed by m o d i f i c a t i o n o f the m e t h o d o f Coyle 1, as previously described °. There were no significant differences in the b a s a l activity o f T H between A 1, A 2 a n d locus coeruleus areas within the 3 strains o f rats (Table I legend). Reserpine a d m i n i s t e r e d as a single (10 mg/kg, s.c.) o r multiple dose (10 mg/kg, s.c. for 3 consecutive days) increased T H in the A1 a r e a a n d locus coeruleus (Table I). It failed, in the same animals, to alter T H activity in the A 2 region o f a d u l t S H rats. In contrast, reserpine substantially increased T H activity in the A 2 a r e a as well as in the locus coeruleus a n d A1 areas in rats o f the S p r a g u e - D a w l e y a n d W K Y strains (Table I). T h e failure o f reserpine to increase T H activity in the A 2 a r e a o f S H rats was age-dependent. I n y o u n g rats, reserpine elevated T H activity in the A 2 areas o f S H rats with a c o m p a r a b l e m a g n i t u d e to t h a t o b t a i n e d in W K Y a n d S p r a g u e - D a w l e y controls (Table II). T h u s the failure to induce T H in the A 2 a r e a o f S H rats develops, as d o o t h e r differences o f c a t e c h o l a m i n e m e t a b o l i s m l a , 14, p o s t n a t a l l y . T h e present s t u d y d e m o n s t r a t e s an a b n o r m a l i t y in the r e g u l a t i o n o f the catechola m i n e synthesizing enzyme T H in the b r a i n o f a d u l t S H rats. The biochemical defect is TABLE I Tyrosine hydroxylase activity ( % of control) in ,41, `42 and locus coeruleus after single or multiple reserpine injection in adult rats
Sprague-Dawley (SD) male rats (12 weeks okl), Wistar-Kyoto (WKY) male rats (14 weeks old), and male spontaneously hypertensive (SH) rats (14 weeks old) were injected subcutaneously either with saline or reserpine (10 mg/kg) according to two different schedules. Columns 'single' represent the effect of one single injection, animals being sacrificed 4 days later. Columns 'multiple' show the changes in TH activity for animals injected once daily for 3 consecutive days and sacrificed 3 days after the last injection. All statistic calculations were based on n = 6 or 8. Values of TH activity for animals are expressed as percentages of saline injected controls of the same strain. Control values (mean 4- S.E.M.) for SD, WKY, and SH rats, respectively were: (a) A1 area (activity in pmol of DOPA formed per h per mg of protein): 82.1 i 5.6, 87.5 4- 6.1 and 78.7 4- 5.2; (b) A2 area (same units as for A1 area) : 96.7 4- 4.6, 100.2 4- 7.4 and 91.8 i 6.3 ; and (c) locus eoeruleus (activity in pmol DOPA formed per h per pair of locus coeruleus) : 212.6 4- 10.8, 194. 24- 9.3 and 192.9 ± 11.6. Strain
Brain Area and Reserpine Schedule A1
Sprague-Dawley Wistar-Kyoto Spontaneously hypertensive
.42
LC
Single
Multiple
Single
Multiple
Single
160.5±10.1'* 132.84-6"*
190.5±9.4'* 132.1±10.0"*
116.94-1.9'* 114.94-5.3"
133.84-4.4"* 125.94-8.9"
278.34-12.8'* 240.7-4-14.5"*
148.24-8.5"*
140.64-3.9"*
101.1 4-2.2 ns
104.84-3.9 ns
314.74-27.6"*
* P < 0.05; * * P < 0.001 ; ns: not significant compared with saline-injectedcontrols of the same strain.
166 TABLE II Changes in tyrosine hydroxylase activity in A1, .42 and locus coeruleus produced by a single dose o f reserpine in young rats o f W K Y and S H strains
Tyrosine hydroxylase activity has been determined in 4-week-old male Wistar-Kyoto (WKY) rats and spontaneously hypertensive (SH) rats injected subcutaneously either with saline or with reserpine (10 mg/kg) and sacrificed 4 days later. TH activity is expressed as in Table I (mean ± S.E.M.) of 6-8 saline-injected controls of the same strain. WKY AI
Basal activity Reserpine
SH A2
87.1 L3.I 39.2±1.4 116.6~5.3"* 50.6:~2.0" 133 129
LC
AI
A2
1225:5.3 89.7±4.2 37.0il.2 362.3±4.9** 119.3~-2.7"* 47.0_+2.6* 297 133 127
LC
122.3±5.3 333.9±8.6** 273
*P < 0.01; **P < 0.001. anatomically restricted to only one central catecholaminergic system, the A2 group, and is age-dependent, being evident only in mature animals. The biochemical lesion is characterized by a failure to increase T H activity in response to reserpine. This failure probably reflects an inability of the drug to increase the accumulation of T H primarily in noradrenergic neurons of the A2 group of adult SH rats, most likely an inability to further increase the biosynthesis of the enzymO 1. The molecular mechanisms accounting for the failure of reserpine to increase the activity and amount of T H in A2 area of adult SH rats is unknown. The fact that reserpine induces T H in the locus coeruleus and A I regions of SH rats, with a magnitude comparable to that obtained in rats of the W K Y or Sprague-Dawley strains, as well as inducing T H in the adrenal medulla% indicates that the defect cannot be attributed to a strain-dependent difference in the metabolism or pharmacokinetics of the drug. Moreover, the anatomical selectivity and age-dependency of the defect of induction to reserpine in SH rats makes it unlikely to represent a genetic abnormality universally affecting T H regulation in all tissues. The relationship between the biochemical abnormality of T H regulation in SH rats and the appearance of arterial hypertension remains to be established. However, it would appear unlikely that the biochemical defect is itself the cause of hypertension : first, in normal rats, destruction of A2 neurons or of their terminals in the NTS produces only lability of the arterial pressure without hypertension10,14. Thus, abnormal function of A2 neurons is unlikely to express itself as an elevated arterial pressure; and second, the elevation of arterial pressure in developing SH rats appears in advance of the biochemical defect lo. Thus the abnormal regulation of T H in A2 neurons of SH rats may be secondary to, rather than causal for, arterial hypertension. However, since catecholamines appear to facilitate baroreflexes within the NTS 3,4,~° and these catecholamines arise in large measure from A2 neurons 2,15, the results may indicate an adaptive mechanism whereby A2 neurons, by increasing their biosynthetic capacity for the neurotransmitter, seek to facilitate baroreflexes in an attempt to lower the already elevated arterial pressure. Some failure of compensation of baroreflexes,
167 themselves modulated by A2 neurons, might contribute to the cardiovascular abnormalities in this strain. This research was supported by grants from NIH (HL 18974 and NS 03346) and NASA. B.R. was a Fogarty International Fellow from the NIH. 1 Coyle, J. T., Tyrosine hydroxylase in rat brain - - cofactor requirements, regional and subcellular distribution, Biochem. Pharmacol., 21 (1972) 1935-1944. 2 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamine in the cell bodies of brainstem neurons, Actaphysiol. scand., 62: Suppl. 232 (1964) 1-55. 3 DeJong, W., Zandberg, P. and Bohus, B., Central inhibitory noradrenergic cardiovascular control. In W. H. Gispen, Tj. B. van Wimersma Greidanus, B. Bohus and D. de Wied (Eds.), Hormones, Homeostasis and the Brain, Progr. Brain Res., Vol. 42, 1975, pp. 285-298. 4 DeJong, W. and Nijkamp, F. P., Centrally induced hypotension and bradycardia after administration of a-methylnoradrenaline into the area of the nucleus tractus solitarii of the rat, Brit. J. Pharmacol., 58 (1976) 593-598. 5 Renaud, B., Joh., T. H., Snyder, D. W. and Reis, D. J., Induction and delayed activation of tyrosine hydroxylase in noradrenergic neurons of A1 and A2 groups in medulla oblongata of rat, Neurosci. Abstr., 3 (1977) 258. 6 Renaud, B., Joh, T. H. and Reis, D. J., unpublished observation. 7 Renaud, B., Fourni6re, S., Denoroy, L., Vincent, M., Pujol, J. F. and Sassard, J. Early increase in phenylethanolamine-N-methyltransferase activity in a new strain of spontaneously hypertensive rats, Brain Research, 159 (1978) 149-159. 8 Reis, D. J., Joh, T. H., Ross, R. A., and Pickel, V. M., Reserpine selectively increases tyrosine hydroxylase and dopamine-fl-hydroxylase enzyme protein in central noradrenergic neurons, Brain Research, 81 (1974) 380-386. 9 Reis, D. J., Joh, T. H. and Ross, R. A., Effects of reserpine on activities and amounts of tyrosine hydroxylase and dopamine-fl-hydroxylase in catecholaminergic neuronal systems in rat brain, J. Pharmacol. exp. Ther., 193 (1975) 775-784. 10 Reis, D. J., Joh, T. H., Nathan, M. A., Renaud, B., Snyder, D. W., and Talman, W. T., The nucleus tractus solitarii, its catecholaminergic innervation in the normal and abnormal control of arterial pressure. In P. Meyer and H. Schmitt (Eds.), Perspectives in Nephrology and Hypertension, Wiley, New York, 1979, pp. 147-164. 11 Ross, R. A., Joh, T. H. and Reis, D. J., Reduced rate of biosynthesis of dopamine-fl-hydroxylase in the nucleus locus coeruleus during the retrograde reaction, Brain Research, 160 (1979) 174-179. 12 Saavedra, J. M., Grobecker, H. and Axelrod, J., Adrenaline forming enzyme in brainstem: elevation in genetic and experimental hypertension, Science, 191 (1976) 483-484. 13 Saavedra, J. M., Grobecker, H. and Axelrod, I., Changes in central catecholaminergic neurons in the spontaneously (genetic) hypertensive rat, Circulat. Res., 42 (1978) 529-534. 14 Snyder, D. W., Nathan, M. A. and Reis, D. J., Chronic lability of arterial pressure produced by selective denervation of the catecholamine innervation of the nucleus tractus solitarii in rat, Circulat. Res., 43 (1978) 662-671. 15 Torack, R. M., Stranahan, P. and Hartman, B. K., The role of norepinephrine in the function of the area postrema. I. Immunofluorescent localization of dopamine-fl-hydroxylase and electron microscopy, Brain Research, 61 (1973) 235-252. 16 Versteeg, D. H. G., Palkovits, M., Van der Gugten, J., Wijnen, H. L. J. M., Smeets, G. W. M. and DeJong, W., Catecholamine content of individual brain regions of spontaneously hypertensive rats (SH-rats), Brain Research, 112 (1976) 429-434. 17 Wijnen, H. L. J. M., Versteeg, D. H. G., Palkovits, M. and DeJong, W., Increased adrenergic content of individual nuclei of the hypothalamus and the medulla oblongata of genetically hypertensive rats, Brain Research, 135 (1977) 180-185. 18 Zigmond, R. E., Schon, F. and Iversen, L L., Increased tyrosine hydroxylase activity in the locus coeruleus of rat brain stem after reserpine treatment and cold stress, Brain Research, 70 (1974) 547-552.
Brain Research, 173 (1979) 164-167 © Elsevier/North-Holland Biomedical Press
An abnormal regulation of tyrosine hydroxylase restricted to one catecholamine nucleus in the brain stem of spontaneously hypertensive rats
BERNARD RENAUD*, TONG H. JOH and DONALD J. REIS Laboratory of Neurobiology, Cornell University Medical College, New York, N.Y. 10021 (U.S.A.)
(Accepted May 10th, 1979)
Spontaneously hypertensive (SH) rats of the Okamoto strain contain different amounts of one or several of the catecholamine neurotransmitters dopamine, noradrenaline or adrenaline, regionally in brain than do normotensive controls 12,13,18,17. In some instances the differences may only be evident in young animals 7,13. One molecule through which the genetic variation in central catecholamine metabolism might be expressed is tyrosine hydroxylase (TH), the enzyme catalyzing the initial step in the biosynthesis of catecholamine neurotransmitters from tyrosine. Whether the basal activity and/or the regulation of the enzyme differs in the brain of SH rats is not known. In the present study we have analyzed, in SH rats, the basal activity of T H and one mode of its long-term regulation, the prolonged increase in activity secondary to enhanced accumulation of the enzyme produced in all brain stem noradrenergic groups by reserpine s-l°,ls. T H activity has been analyzed in the locus coeruleus and in two other catecholamine groups of the medulla believed to be of importance in cardiovascular control: the A1 group, which lies ventrolaterally in the medulla2; and the A2 group, which lies in the dorsal medullary tegmentumL The changes in regional T H activity in SH rats elicited by reserpine have been compared in young (4 weeks) and mature rats (14 weeks) with age- and sex-matched normotensive animals of the Sprague-Dawley and Wistar-Kyoto (WKY) strains. Male SH rats of the Okamoto strain and WKY controls were purchased from Taconic Farms (Germantown, N.Y.); male Sprague-Dawley rats from Hilltop Lab Animals (Scottsdale, Pa.). All rats were maintained, 2-3 to a cage, in a thermally controlled light-cycled environment for at least 10 days prior to the experiment. Reserpine (Serpasil, Ciba Pharmaceutical, Summit, N.J. was administered subcutaneously between 09.00 and 11.00 h. The animals were killed by cervical dislocation at various times following the last injection, usually at 96 h, a time at which there is a maximal elevation of enzyme activity in the locus ceruleus, A1 and A2 neurons 5,s-1°. * Present address: D6partement de Physiologic et Pharmacologie, Facult6 de Pharmacie, Universit6 Claude Bernard, Lyon, France.
165 Brains were r a p i d l y r e m o v e d a n d the A1, A 2 a n d locus coeruleus areas r e m o v e d b y microdissection 9,14. Tissues were h o m o g e n i z e d by sonication in fixed vols. o f 5 m M p o t a s s i u m phosp h a t e buffer, p H 6.5, c o n t a i n i n g 0.2 ~ T r i t o n X-100. The h o m o g e n i z a t i o n vols. were 200/~1 p e r p a i r o f locus coeruleus a n d 150 ~1 p e r p a i r o f A1 o r A 2 areas. T h e h o m o g enates were centrifuged for 10 min at 6000 g. T H activity was assayed by m o d i f i c a t i o n o f the m e t h o d o f Coyle 1, as previously described °. There were no significant differences in the b a s a l activity o f T H between A 1, A 2 a n d locus coeruleus areas within the 3 strains o f rats (Table I legend). Reserpine a d m i n i s t e r e d as a single (10 mg/kg, s.c.) o r multiple dose (10 mg/kg, s.c. for 3 consecutive days) increased T H in the A1 a r e a a n d locus coeruleus (Table I). It failed, in the same animals, to alter T H activity in the A 2 region o f a d u l t S H rats. In contrast, reserpine substantially increased T H activity in the A 2 a r e a as well as in the locus coeruleus a n d A1 areas in rats o f the S p r a g u e - D a w l e y a n d W K Y strains (Table I). T h e failure o f reserpine to increase T H activity in the A 2 a r e a o f S H rats was age-dependent. I n y o u n g rats, reserpine elevated T H activity in the A 2 areas o f S H rats with a c o m p a r a b l e m a g n i t u d e to t h a t o b t a i n e d in W K Y a n d S p r a g u e - D a w l e y controls (Table II). T h u s the failure to induce T H in the A 2 a r e a o f S H rats develops, as d o o t h e r differences o f c a t e c h o l a m i n e m e t a b o l i s m l a , 14, p o s t n a t a l l y . T h e present s t u d y d e m o n s t r a t e s an a b n o r m a l i t y in the r e g u l a t i o n o f the catechola m i n e synthesizing enzyme T H in the b r a i n o f a d u l t S H rats. The biochemical defect is TABLE I Tyrosine hydroxylase activity ( % of control) in ,41, `42 and locus coeruleus after single or multiple reserpine injection in adult rats
Sprague-Dawley (SD) male rats (12 weeks okl), Wistar-Kyoto (WKY) male rats (14 weeks old), and male spontaneously hypertensive (SH) rats (14 weeks old) were injected subcutaneously either with saline or reserpine (10 mg/kg) according to two different schedules. Columns 'single' represent the effect of one single injection, animals being sacrificed 4 days later. Columns 'multiple' show the changes in TH activity for animals injected once daily for 3 consecutive days and sacrificed 3 days after the last injection. All statistic calculations were based on n = 6 or 8. Values of TH activity for animals are expressed as percentages of saline injected controls of the same strain. Control values (mean 4- S.E.M.) for SD, WKY, and SH rats, respectively were: (a) A1 area (activity in pmol of DOPA formed per h per mg of protein): 82.1 i 5.6, 87.5 4- 6.1 and 78.7 4- 5.2; (b) A2 area (same units as for A1 area) : 96.7 4- 4.6, 100.2 4- 7.4 and 91.8 i 6.3 ; and (c) locus eoeruleus (activity in pmol DOPA formed per h per pair of locus coeruleus) : 212.6 4- 10.8, 194. 24- 9.3 and 192.9 ± 11.6. Strain
Brain Area and Reserpine Schedule A1
Sprague-Dawley Wistar-Kyoto Spontaneously hypertensive
.42
LC
Single
Multiple
Single
Multiple
Single
160.5±10.1'* 132.84-6"*
190.5±9.4'* 132.1±10.0"*
116.94-1.9'* 114.94-5.3"
133.84-4.4"* 125.94-8.9"
278.34-12.8'* 240.7-4-14.5"*
148.24-8.5"*
140.64-3.9"*
101.1 4-2.2 ns
104.84-3.9 ns
314.74-27.6"*
* P < 0.05; * * P < 0.001 ; ns: not significant compared with saline-injectedcontrols of the same strain.
166 TABLE II Changes in tyrosine hydroxylase activity in A1, .42 and locus coeruleus produced by a single dose o f reserpine in young rats o f W K Y and S H strains
Tyrosine hydroxylase activity has been determined in 4-week-old male Wistar-Kyoto (WKY) rats and spontaneously hypertensive (SH) rats injected subcutaneously either with saline or with reserpine (10 mg/kg) and sacrificed 4 days later. TH activity is expressed as in Table I (mean ± S.E.M.) of 6-8 saline-injected controls of the same strain. WKY AI
Basal activity Reserpine
SH A2
87.1 L3.I 39.2±1.4 116.6~5.3"* 50.6:~2.0" 133 129
LC
AI
A2
1225:5.3 89.7±4.2 37.0il.2 362.3±4.9** 119.3~-2.7"* 47.0_+2.6* 297 133 127
LC
122.3±5.3 333.9±8.6** 273
*P < 0.01; **P < 0.001. anatomically restricted to only one central catecholaminergic system, the A2 group, and is age-dependent, being evident only in mature animals. The biochemical lesion is characterized by a failure to increase T H activity in response to reserpine. This failure probably reflects an inability of the drug to increase the accumulation of T H primarily in noradrenergic neurons of the A2 group of adult SH rats, most likely an inability to further increase the biosynthesis of the enzymO 1. The molecular mechanisms accounting for the failure of reserpine to increase the activity and amount of T H in A2 area of adult SH rats is unknown. The fact that reserpine induces T H in the locus coeruleus and A I regions of SH rats, with a magnitude comparable to that obtained in rats of the W K Y or Sprague-Dawley strains, as well as inducing T H in the adrenal medulla% indicates that the defect cannot be attributed to a strain-dependent difference in the metabolism or pharmacokinetics of the drug. Moreover, the anatomical selectivity and age-dependency of the defect of induction to reserpine in SH rats makes it unlikely to represent a genetic abnormality universally affecting T H regulation in all tissues. The relationship between the biochemical abnormality of T H regulation in SH rats and the appearance of arterial hypertension remains to be established. However, it would appear unlikely that the biochemical defect is itself the cause of hypertension : first, in normal rats, destruction of A2 neurons or of their terminals in the NTS produces only lability of the arterial pressure without hypertension10,14. Thus, abnormal function of A2 neurons is unlikely to express itself as an elevated arterial pressure; and second, the elevation of arterial pressure in developing SH rats appears in advance of the biochemical defect lo. Thus the abnormal regulation of T H in A2 neurons of SH rats may be secondary to, rather than causal for, arterial hypertension. However, since catecholamines appear to facilitate baroreflexes within the NTS 3,4,~° and these catecholamines arise in large measure from A2 neurons 2,15, the results may indicate an adaptive mechanism whereby A2 neurons, by increasing their biosynthetic capacity for the neurotransmitter, seek to facilitate baroreflexes in an attempt to lower the already elevated arterial pressure. Some failure of compensation of baroreflexes,
167 themselves modulated by A2 neurons, might contribute to the cardiovascular abnormalities in this strain. This research was supported by grants from NIH (HL 18974 and NS 03346) and NASA. B.R. was a Fogarty International Fellow from the NIH. 1 Coyle, J. T., Tyrosine hydroxylase in rat brain - - cofactor requirements, regional and subcellular distribution, Biochem. Pharmacol., 21 (1972) 1935-1944. 2 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamine in the cell bodies of brainstem neurons, Actaphysiol. scand., 62: Suppl. 232 (1964) 1-55. 3 DeJong, W., Zandberg, P. and Bohus, B., Central inhibitory noradrenergic cardiovascular control. In W. H. Gispen, Tj. B. van Wimersma Greidanus, B. Bohus and D. de Wied (Eds.), Hormones, Homeostasis and the Brain, Progr. Brain Res., Vol. 42, 1975, pp. 285-298. 4 DeJong, W. and Nijkamp, F. P., Centrally induced hypotension and bradycardia after administration of a-methylnoradrenaline into the area of the nucleus tractus solitarii of the rat, Brit. J. Pharmacol., 58 (1976) 593-598. 5 Renaud, B., Joh., T. H., Snyder, D. W. and Reis, D. J., Induction and delayed activation of tyrosine hydroxylase in noradrenergic neurons of A1 and A2 groups in medulla oblongata of rat, Neurosci. Abstr., 3 (1977) 258. 6 Renaud, B., Joh, T. H. and Reis, D. J., unpublished observation. 7 Renaud, B., Fourni6re, S., Denoroy, L., Vincent, M., Pujol, J. F. and Sassard, J. Early increase in phenylethanolamine-N-methyltransferase activity in a new strain of spontaneously hypertensive rats, Brain Research, 159 (1978) 149-159. 8 Reis, D. J., Joh, T. H., Ross, R. A., and Pickel, V. M., Reserpine selectively increases tyrosine hydroxylase and dopamine-fl-hydroxylase enzyme protein in central noradrenergic neurons, Brain Research, 81 (1974) 380-386. 9 Reis, D. J., Joh, T. H. and Ross, R. A., Effects of reserpine on activities and amounts of tyrosine hydroxylase and dopamine-fl-hydroxylase in catecholaminergic neuronal systems in rat brain, J. Pharmacol. exp. Ther., 193 (1975) 775-784. 10 Reis, D. J., Joh, T. H., Nathan, M. A., Renaud, B., Snyder, D. W., and Talman, W. T., The nucleus tractus solitarii, its catecholaminergic innervation in the normal and abnormal control of arterial pressure. In P. Meyer and H. Schmitt (Eds.), Perspectives in Nephrology and Hypertension, Wiley, New York, 1979, pp. 147-164. 11 Ross, R. A., Joh, T. H. and Reis, D. J., Reduced rate of biosynthesis of dopamine-fl-hydroxylase in the nucleus locus coeruleus during the retrograde reaction, Brain Research, 160 (1979) 174-179. 12 Saavedra, J. M., Grobecker, H. and Axelrod, J., Adrenaline forming enzyme in brainstem: elevation in genetic and experimental hypertension, Science, 191 (1976) 483-484. 13 Saavedra, J. M., Grobecker, H. and Axelrod, I., Changes in central catecholaminergic neurons in the spontaneously (genetic) hypertensive rat, Circulat. Res., 42 (1978) 529-534. 14 Snyder, D. W., Nathan, M. A. and Reis, D. J., Chronic lability of arterial pressure produced by selective denervation of the catecholamine innervation of the nucleus tractus solitarii in rat, Circulat. Res., 43 (1978) 662-671. 15 Torack, R. M., Stranahan, P. and Hartman, B. K., The role of norepinephrine in the function of the area postrema. I. Immunofluorescent localization of dopamine-fl-hydroxylase and electron microscopy, Brain Research, 61 (1973) 235-252. 16 Versteeg, D. H. G., Palkovits, M., Van der Gugten, J., Wijnen, H. L. J. M., Smeets, G. W. M. and DeJong, W., Catecholamine content of individual brain regions of spontaneously hypertensive rats (SH-rats), Brain Research, 112 (1976) 429-434. 17 Wijnen, H. L. J. M., Versteeg, D. H. G., Palkovits, M. and DeJong, W., Increased adrenergic content of individual nuclei of the hypothalamus and the medulla oblongata of genetically hypertensive rats, Brain Research, 135 (1977) 180-185. 18 Zigmond, R. E., Schon, F. and Iversen, L L., Increased tyrosine hydroxylase activity in the locus coeruleus of rat brain stem after reserpine treatment and cold stress, Brain Research, 70 (1974) 547-552.
