- Email: [email protected]
Discrete changes in adrenaline-forming enzyme activity in brain stem areas of genetic salt-sensitive hypertensive (Dahl) rats
Brain Research, 193 (1980) 299-303 © Elsevier/North-Holland Biomedical Press
299
Discrete changes in adrenaline-forming enzyme activity in brain stem areas of genetic salt-sensitive hypertensive (Dahl) rats
JUAN M. SAAVEDRA, FERNANDO M. CORREA* and JUNICHI IWAI Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20205 and (J.I.) Brookhaven National Laboratory, Upton, N.Y. 11973 (U.S.A.)
(Accepted February 28th, 1980) Key words: brain adrenaline -- phenylethanolamine N-methyltransferase -- genetic hypertension -salt-sensitive hypertension -- Dahl rats
Dahl and co-workers developed two lines of Sprague-Dawley rats with different, genetically determined susceptibilities to hypertensinogen stimuli, and especially to the the effect of a high salt dietT, 8. Salt-sensitive (S) rats increase their arterial pressure in response to a high salt diet. Salt-resistant (R) rats, on the contrary, manifest no change in blood pressure when submitted to a high salt dietT, s. The coordination of the intake, loss and distribution of fluids in the organism occurs largely as a result of the integrative action of the central nervous system 2. In the S, high salt fed, hypertensive Dahl rats, selective brain lesions prevent and reverse the increase in blood pressure, indicating a role of the central nervous system in this kind of hypertension 5. Central adrenergic mechanisms might play a role in the control of salt and water metabolism and excretion. A sodium-noradrenaline interaction has been documented in the central nervous system 1. Intraseptal or intraventricular administration of noradrenaline or adrenaline results in increased natriuresis 1,3. Sodium depletion in dogs is accompanied by a decrease in the peripheral sympathetic tone, and by increased levels of noradrenaline in the cerebrospinal fluid 6. Central catecholamines, and especially central adrenaline, are involved in the regulation of cardiovascular functions and in several forms of hypertension, including the spontaneously (genetic) hypertension (SHR) 12 and the mineralocorticoid ( D O C A salt) experimental hypertension 15. Increases in the activity of the adrenaline-forming enzyme, phenylethanolaminc N-methyltransferase (PNMT) 4 or noradrenaline Nmethyltransferase (EC 2.1.1.28), are localized to specific brain stem areas, like the A2 * On leave from Department of Pharmacology, Ribcirao Preto, Sao Paulo, Brazil.
300 area, indicating that stimulation of adrenaline synthesis in this area might be related to the production or maintenance of several forms of hypertension 14 lv.21 We report the quantitative estimation of the activity of the adrenaline-forming enzyme in specific areas of the brain stem of S and R Dahl rats, under a low and high salt diet. Hypertensive, sodium-dependent Dahl rats show higher PNMT activity in the A2 area, and lower enzyme activity in the area postrema and nucleus commissuralis than their corresponding controls. Our results suggest the participation of specific adrenaline neurons in the brain stem in the salt-sensitive genetic hypertension (Dahl rats). Mate Dahl salt-sensitive (S) and salt-resistant (R) rats from Brookhaven National Laboratory, Upton, N.Y., were studied. Groups of 16 rats of each S and R lines were fed low sodium chloride chow (0.45 o~ per dry weight) from weaning until 10 weeks of age. Similar groups of each S and R lines were fed high sodium chloride chow (8 o / p e r dry weight) (ICN Pharmaceuticals, Cleveland, Ohio), over the same period. Tap water was provided for drinking ad lib. Systolic blood pressure was measured in unanesthetized rats by tail plethysmography, using a programmed electrosphygmomanometer (Narco Biosystems, Houston, Texas, Model PE-500), as previously described 17. The animals were housed in groups of 4 per cage, under a 0.600-18.00 h light schedule, and were killed by decapitation between 0.900 and 11.00 h, when 1l weeks old. The brains were rapidly removed and frozen on microtome specimen holders on dry-ice. Serial sections of 300 #m thickness were cut in a cryostat at a temperature of --10 °C. Specific brain stem nuclei and areas were dissected by the use of a needle of i.d. of 0.5 ram10,13,19. Phenylethanolamine N-methyltransferase (PNMT) 4 activity was assayed as described earlier ~8. The results were corrected by the use of internal standards of partially purified P N M T 18. Proteins were measured in an aliquot of the tissue homogenate 11. Data were analyzed by a two-way analysis of variance, and a Student's Newman-Keuls test was used for comparisons of individual means 2°. Systolic blood pressures differ significantly between the two lines (FA 250.0, df 2/60, P < 0.01) and after a high salt diet (FB 141.5, df 2/60 P < 0.0t). A high salt diet affects the two lines differently, for there is evidence of a line x salt interaction (FAB 140.3, df 2/60, P <~ 0.0l). S rats under low salt diet have slightly but significantly higher blood pressure than the R rats. After a high salt diet, S rats greatly increase their systolic blood pressure to hypertensive levels, whereas R rats show no change in blood pressure (Table I). Significant changes in P N M T activity are localized to specific brain stem areas. In the A2 area, a high salt diet results in an increase in P N M T activity (FB 6.8, df 2/56, P ~ 0.05) which is more marked in the S rats (Fig. 1). In the area postrema and in the nucleus commissuralis, the high salt diet affects the two lines differently, with evidence of a line x salt interaction (FAB 6.0, and FAB 8.9, respectively; df 2/56, P < 0.05). In these areas, a high salt diet results in significant decreases in P N M T activity only in S rats (Fig. 1). No significant changes in the activity of P N M T occur in other brain stem areas, such as the anterior part of the nucleus tractus solitarius, the A1 area or the locus coeruleus (Fig. 1).
301 TABLE I Systolic blood pressure in Dahl rats
Results are expressed in m m Hg, 4- S.E.M., for groups of 16 rats.
Resistant Sensitive
Low salt diet
High salt diet
113.4 -t- 2.4 123.4 4- 1.8"
113.4 ± 1.7 183.1 ± 3.5**
* P < 0.05, > resistant low salt, > resistant high salt, < sensitive high salt. P < 0.01, > resistant low salt, > resistant high salt, > sensitive low salt.
**
60
~
ResistantLow Salt ResistantHigh Salt
50
SensitiveLow Salt SensitiveHigh Salt
40 3O 2O
AREA POSTREMA .1-
60
NUCLEUS COMMISSURAUS
A.~AREA
At AREA
LOCUSCOERULEUS
!° 40 30
NUCLEUSTRACTUS SOLITARIUS
Fig. 1. P N M T activity in brain stem areas of D a h l rats. Results are expressed as ~ -t- S.E.M. for groups of 15 animals each, assayed individually. Statistical significance, P < 0.05 (Student's N e w m a n - K e u l s test). Area postrema: *,sensitive high salt, < sensitive low salt, < resistant high salt. Nucleus commissuralis: *,sensitive high salt, < sensitive low salt. A2 area: *,sensitive high salt, > sensitive low salt, > resistant low salt.
302 The present results show that R and S Dahl rats respond differently in terms of their brain stem P N M T activity after submission to a high salt diet. Only the S Dahl rats, but not the R rats, show significant changes in brain stem P N M T after a high salt diet. The nature of the role of brain stem adrenaline neurons in the regulation of the genetic, sodium-dependent hypertension is still speculative. The increase in P N M T activity observed in S Dahl rats under a high salt diet is restricted to the A2 area of the brain stem and may represent increased formation of adrenaline by the A2 neurons. This change is similar to the change in P N M T observed in other models of hypertension, such as the SHR and the mineralocorticoid-induced hypertension a5 tT. These adrenaline neurons may be part of a stimulatory system which can be activated by still undetermined central or peripheral mechanisms and result in increased peripheral symphathetic activity and vascular resistance. These mechanisms could be partially dependent on the intracellular or extracellular Na + concentration. Genetic differences, like the ones present in the R and S rats, could modulate the response of A2 adrenaline neurons to a high salt load, and result in consequent blood pressure changes. The possibility remains, however, that the changes in P N M T activity at the A2 area may represent the activation of a vasodepressor system of adrenergic neurons, acting ~s a compensatory mechanism secondary to the rise in blood pressure. Other brain stem areas such as the area postrema and the nucleus commissuralis, in close anatomical relation to the A2 area, show opposite changes in P N M T activity in S Dahl rats under a high salt diet. These results underline the selectivity of the anatomical localization of changes in adrenaline neurons, since the nucleus commissuralis, like the A2 area, is anatomically related to the nucleus tractus solitarius, but shows a decrease, rather than an increase in P N M T activity under a high salt diet (Fig. 1). Thus, the increases in P N M T present in the S Dahl rats under a high salt diet are limited to the A2 area which contains the cell bodies of the adrenaline-forming neurons 9. Our results suggest that brain stem adrenaline neurons form part of complex central mechanisms which play a role in the regulation of blood pressure and sodium metabolism, and indicate that these neurons may be physiologically heterogeneous. The Dahl model of genetic, sodium-sensitive and sodium-resistant rats presents definite advantages for the study of the interaction of genetic factors and a sodium dietary load in the pathogenesis of hypertension. The authors thank Ms. Roberta L. Holcomb for her secretarial assistance.
1 Andersson, B., Leksell, L. G., Lishajko, F. and Rundgren, M., Cerebral sodium-noradrenaline interaction: dipsogenic, antidiuretic and natriuretic effects, Acta physiol, scand., 102 (1978) 254-256. 2 Antunes-Rodriguez, J., Gentil, C. G., Negro Vilar, A. and Covian, M. R.~ Role of adrenals in the changes of sodium chloride intake following lesions in the central nervous system, Physiol. Behav., 5 (1970) 89-93.
303 3 Arruda Camargo, L. A., Saad, W. A., Silva Netto, C. R., Gentil, C. G., Antunes-Rodriquez, J. and Covian, M. R., Effects of catecholamines injected into the septal area of the rat brain on natriuresis, kaliuresis and diuresis, Canad. J. physioL Pharmacol., 54 (1976) 219-228. 4 Axelrod, J., Purification and properties of phenylethanolamine N-methyltransferase, J. bioL Chem., 237 (1962) 1657-1660. 5 Brody, M. J., Fink, G. D., Buggy, J., Haywood, J. R., Gordon, F. J., Knuepfer, M., Mow, M., Mahoney, L. and Johnson, A. K., Critical role of the anteroventral third ventricle (AV3V) region in development and maintenance of experimental hypertension. In P. Meyer and H. Schmitt (Eds.), Nervous System and Hypertension, John Wiley, New York, 1979, pp. 76-84. 6 Brosnihan, K. B., Szilagyi, J. E., Masaki, Z. and Ferrario, C. M., Interaction of sodium (Na ÷) and catecholamines (CA) on the control of blood pressure. In E. Usdin, I. J. Kopin and J. Barchas (Eds.), Catecholamines: Basic and Clinical Frontiers, Vol. 2, Pergamon Press, New York, 1979, pp. 1530-1532. 7 Dahl, L. K., Heine, M. and Tassinari, L., Role of genetic factors in susceptibility to experimental hypertension due to chronic excess salt ingestion, Nature (Lond.), 194 (1962) 480-482. 8 Dahl, L. K., Heine, M. and Tassinari, L., Effects of chronic excess salt ingestion. Evidence that genetic factors play an important role in susceptibility to experimental hypertension, J. exp. Med., 115 (1962) 1173-1190. 9 Hokfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235-251. 10 Konig, J. F. R. and Kiippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams and Wilkins, Baltimore, Md., 1963. 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 12 Okamoto, K. and Aoki, K., Development of a strain of spontaneously hypertensive rats, Jap. Circulat. J. (En.), 27 (1963) 282-290. 13 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 14 Saavedra, J. M., Adrenaline levels in brain stem nuclei and effects of a PNMT inhibitor on spontaneously hypertensive rats, Brain Research, 166 (1979) 283-292. 15 Saavedra, J. M., Brain catecholamines during development of DOCA-salt hypertension in rats, Brain Research, 179 (1979) 121-127. 16 Saavedra, J. M., Grobecker, H. and Axelrod, J., Adrenaline-forming enzyme in brainstem: elevation in genetic and experimental hypertension, Science, 191 (1976) 483-484. 17 Saavedra, J. M., Grobecker, H. and Axelrod, J., Changes in central catecholaminergic neurons in the spontaneously (genetic) hypertensive rat, Circulat. Res., 42 (1978) 529-534. 18 Saavedra, J. M., Palkovits, M., Brownstein, M. J. and Axelrod, J., Localization of phenylethanolamine-N-methyltransferase in the rat brain nuclei, Nature (Lond.), 248 (1974) 695-696. 19 Schlumpf, M., Waser, P., Lichtenstiger, W., Langmann, H. and Schlup, D., Standardized excision of small areas of rat and mouse brain with topographical control, Biochem. PharmacoL, 23 (1974) 2447-2449. 20 Sokal, R. R. and Rohlf, F. J., Biometry, W. H. Freeman, San Francisco, 1969. 21 Wijnen, H. J. L. M., Versteeg, D. H. G., Paikovits, M. and De Jong, W., Increased adrenaline content of individual nuclei of the hypothalamus and the medulla oblongata of genetically hypertensive rats, Brain Research, 135 (1977) 180-185.
299
Discrete changes in adrenaline-forming enzyme activity in brain stem areas of genetic salt-sensitive hypertensive (Dahl) rats
JUAN M. SAAVEDRA, FERNANDO M. CORREA* and JUNICHI IWAI Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20205 and (J.I.) Brookhaven National Laboratory, Upton, N.Y. 11973 (U.S.A.)
(Accepted February 28th, 1980) Key words: brain adrenaline -- phenylethanolamine N-methyltransferase -- genetic hypertension -salt-sensitive hypertension -- Dahl rats
Dahl and co-workers developed two lines of Sprague-Dawley rats with different, genetically determined susceptibilities to hypertensinogen stimuli, and especially to the the effect of a high salt dietT, 8. Salt-sensitive (S) rats increase their arterial pressure in response to a high salt diet. Salt-resistant (R) rats, on the contrary, manifest no change in blood pressure when submitted to a high salt dietT, s. The coordination of the intake, loss and distribution of fluids in the organism occurs largely as a result of the integrative action of the central nervous system 2. In the S, high salt fed, hypertensive Dahl rats, selective brain lesions prevent and reverse the increase in blood pressure, indicating a role of the central nervous system in this kind of hypertension 5. Central adrenergic mechanisms might play a role in the control of salt and water metabolism and excretion. A sodium-noradrenaline interaction has been documented in the central nervous system 1. Intraseptal or intraventricular administration of noradrenaline or adrenaline results in increased natriuresis 1,3. Sodium depletion in dogs is accompanied by a decrease in the peripheral sympathetic tone, and by increased levels of noradrenaline in the cerebrospinal fluid 6. Central catecholamines, and especially central adrenaline, are involved in the regulation of cardiovascular functions and in several forms of hypertension, including the spontaneously (genetic) hypertension (SHR) 12 and the mineralocorticoid ( D O C A salt) experimental hypertension 15. Increases in the activity of the adrenaline-forming enzyme, phenylethanolaminc N-methyltransferase (PNMT) 4 or noradrenaline Nmethyltransferase (EC 2.1.1.28), are localized to specific brain stem areas, like the A2 * On leave from Department of Pharmacology, Ribcirao Preto, Sao Paulo, Brazil.
300 area, indicating that stimulation of adrenaline synthesis in this area might be related to the production or maintenance of several forms of hypertension 14 lv.21 We report the quantitative estimation of the activity of the adrenaline-forming enzyme in specific areas of the brain stem of S and R Dahl rats, under a low and high salt diet. Hypertensive, sodium-dependent Dahl rats show higher PNMT activity in the A2 area, and lower enzyme activity in the area postrema and nucleus commissuralis than their corresponding controls. Our results suggest the participation of specific adrenaline neurons in the brain stem in the salt-sensitive genetic hypertension (Dahl rats). Mate Dahl salt-sensitive (S) and salt-resistant (R) rats from Brookhaven National Laboratory, Upton, N.Y., were studied. Groups of 16 rats of each S and R lines were fed low sodium chloride chow (0.45 o~ per dry weight) from weaning until 10 weeks of age. Similar groups of each S and R lines were fed high sodium chloride chow (8 o / p e r dry weight) (ICN Pharmaceuticals, Cleveland, Ohio), over the same period. Tap water was provided for drinking ad lib. Systolic blood pressure was measured in unanesthetized rats by tail plethysmography, using a programmed electrosphygmomanometer (Narco Biosystems, Houston, Texas, Model PE-500), as previously described 17. The animals were housed in groups of 4 per cage, under a 0.600-18.00 h light schedule, and were killed by decapitation between 0.900 and 11.00 h, when 1l weeks old. The brains were rapidly removed and frozen on microtome specimen holders on dry-ice. Serial sections of 300 #m thickness were cut in a cryostat at a temperature of --10 °C. Specific brain stem nuclei and areas were dissected by the use of a needle of i.d. of 0.5 ram10,13,19. Phenylethanolamine N-methyltransferase (PNMT) 4 activity was assayed as described earlier ~8. The results were corrected by the use of internal standards of partially purified P N M T 18. Proteins were measured in an aliquot of the tissue homogenate 11. Data were analyzed by a two-way analysis of variance, and a Student's Newman-Keuls test was used for comparisons of individual means 2°. Systolic blood pressures differ significantly between the two lines (FA 250.0, df 2/60, P < 0.01) and after a high salt diet (FB 141.5, df 2/60 P < 0.0t). A high salt diet affects the two lines differently, for there is evidence of a line x salt interaction (FAB 140.3, df 2/60, P <~ 0.0l). S rats under low salt diet have slightly but significantly higher blood pressure than the R rats. After a high salt diet, S rats greatly increase their systolic blood pressure to hypertensive levels, whereas R rats show no change in blood pressure (Table I). Significant changes in P N M T activity are localized to specific brain stem areas. In the A2 area, a high salt diet results in an increase in P N M T activity (FB 6.8, df 2/56, P ~ 0.05) which is more marked in the S rats (Fig. 1). In the area postrema and in the nucleus commissuralis, the high salt diet affects the two lines differently, with evidence of a line x salt interaction (FAB 6.0, and FAB 8.9, respectively; df 2/56, P < 0.05). In these areas, a high salt diet results in significant decreases in P N M T activity only in S rats (Fig. 1). No significant changes in the activity of P N M T occur in other brain stem areas, such as the anterior part of the nucleus tractus solitarius, the A1 area or the locus coeruleus (Fig. 1).
301 TABLE I Systolic blood pressure in Dahl rats
Results are expressed in m m Hg, 4- S.E.M., for groups of 16 rats.
Resistant Sensitive
Low salt diet
High salt diet
113.4 -t- 2.4 123.4 4- 1.8"
113.4 ± 1.7 183.1 ± 3.5**
* P < 0.05, > resistant low salt, > resistant high salt, < sensitive high salt. P < 0.01, > resistant low salt, > resistant high salt, > sensitive low salt.
**
60
~
ResistantLow Salt ResistantHigh Salt
50
SensitiveLow Salt SensitiveHigh Salt
40 3O 2O
AREA POSTREMA .1-
60
NUCLEUS COMMISSURAUS
A.~AREA
At AREA
LOCUSCOERULEUS
!° 40 30
NUCLEUSTRACTUS SOLITARIUS
Fig. 1. P N M T activity in brain stem areas of D a h l rats. Results are expressed as ~ -t- S.E.M. for groups of 15 animals each, assayed individually. Statistical significance, P < 0.05 (Student's N e w m a n - K e u l s test). Area postrema: *,sensitive high salt, < sensitive low salt, < resistant high salt. Nucleus commissuralis: *,sensitive high salt, < sensitive low salt. A2 area: *,sensitive high salt, > sensitive low salt, > resistant low salt.
302 The present results show that R and S Dahl rats respond differently in terms of their brain stem P N M T activity after submission to a high salt diet. Only the S Dahl rats, but not the R rats, show significant changes in brain stem P N M T after a high salt diet. The nature of the role of brain stem adrenaline neurons in the regulation of the genetic, sodium-dependent hypertension is still speculative. The increase in P N M T activity observed in S Dahl rats under a high salt diet is restricted to the A2 area of the brain stem and may represent increased formation of adrenaline by the A2 neurons. This change is similar to the change in P N M T observed in other models of hypertension, such as the SHR and the mineralocorticoid-induced hypertension a5 tT. These adrenaline neurons may be part of a stimulatory system which can be activated by still undetermined central or peripheral mechanisms and result in increased peripheral symphathetic activity and vascular resistance. These mechanisms could be partially dependent on the intracellular or extracellular Na + concentration. Genetic differences, like the ones present in the R and S rats, could modulate the response of A2 adrenaline neurons to a high salt load, and result in consequent blood pressure changes. The possibility remains, however, that the changes in P N M T activity at the A2 area may represent the activation of a vasodepressor system of adrenergic neurons, acting ~s a compensatory mechanism secondary to the rise in blood pressure. Other brain stem areas such as the area postrema and the nucleus commissuralis, in close anatomical relation to the A2 area, show opposite changes in P N M T activity in S Dahl rats under a high salt diet. These results underline the selectivity of the anatomical localization of changes in adrenaline neurons, since the nucleus commissuralis, like the A2 area, is anatomically related to the nucleus tractus solitarius, but shows a decrease, rather than an increase in P N M T activity under a high salt diet (Fig. 1). Thus, the increases in P N M T present in the S Dahl rats under a high salt diet are limited to the A2 area which contains the cell bodies of the adrenaline-forming neurons 9. Our results suggest that brain stem adrenaline neurons form part of complex central mechanisms which play a role in the regulation of blood pressure and sodium metabolism, and indicate that these neurons may be physiologically heterogeneous. The Dahl model of genetic, sodium-sensitive and sodium-resistant rats presents definite advantages for the study of the interaction of genetic factors and a sodium dietary load in the pathogenesis of hypertension. The authors thank Ms. Roberta L. Holcomb for her secretarial assistance.
1 Andersson, B., Leksell, L. G., Lishajko, F. and Rundgren, M., Cerebral sodium-noradrenaline interaction: dipsogenic, antidiuretic and natriuretic effects, Acta physiol, scand., 102 (1978) 254-256. 2 Antunes-Rodriguez, J., Gentil, C. G., Negro Vilar, A. and Covian, M. R.~ Role of adrenals in the changes of sodium chloride intake following lesions in the central nervous system, Physiol. Behav., 5 (1970) 89-93.
303 3 Arruda Camargo, L. A., Saad, W. A., Silva Netto, C. R., Gentil, C. G., Antunes-Rodriquez, J. and Covian, M. R., Effects of catecholamines injected into the septal area of the rat brain on natriuresis, kaliuresis and diuresis, Canad. J. physioL Pharmacol., 54 (1976) 219-228. 4 Axelrod, J., Purification and properties of phenylethanolamine N-methyltransferase, J. bioL Chem., 237 (1962) 1657-1660. 5 Brody, M. J., Fink, G. D., Buggy, J., Haywood, J. R., Gordon, F. J., Knuepfer, M., Mow, M., Mahoney, L. and Johnson, A. K., Critical role of the anteroventral third ventricle (AV3V) region in development and maintenance of experimental hypertension. In P. Meyer and H. Schmitt (Eds.), Nervous System and Hypertension, John Wiley, New York, 1979, pp. 76-84. 6 Brosnihan, K. B., Szilagyi, J. E., Masaki, Z. and Ferrario, C. M., Interaction of sodium (Na ÷) and catecholamines (CA) on the control of blood pressure. In E. Usdin, I. J. Kopin and J. Barchas (Eds.), Catecholamines: Basic and Clinical Frontiers, Vol. 2, Pergamon Press, New York, 1979, pp. 1530-1532. 7 Dahl, L. K., Heine, M. and Tassinari, L., Role of genetic factors in susceptibility to experimental hypertension due to chronic excess salt ingestion, Nature (Lond.), 194 (1962) 480-482. 8 Dahl, L. K., Heine, M. and Tassinari, L., Effects of chronic excess salt ingestion. Evidence that genetic factors play an important role in susceptibility to experimental hypertension, J. exp. Med., 115 (1962) 1173-1190. 9 Hokfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235-251. 10 Konig, J. F. R. and Kiippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams and Wilkins, Baltimore, Md., 1963. 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 12 Okamoto, K. and Aoki, K., Development of a strain of spontaneously hypertensive rats, Jap. Circulat. J. (En.), 27 (1963) 282-290. 13 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 14 Saavedra, J. M., Adrenaline levels in brain stem nuclei and effects of a PNMT inhibitor on spontaneously hypertensive rats, Brain Research, 166 (1979) 283-292. 15 Saavedra, J. M., Brain catecholamines during development of DOCA-salt hypertension in rats, Brain Research, 179 (1979) 121-127. 16 Saavedra, J. M., Grobecker, H. and Axelrod, J., Adrenaline-forming enzyme in brainstem: elevation in genetic and experimental hypertension, Science, 191 (1976) 483-484. 17 Saavedra, J. M., Grobecker, H. and Axelrod, J., Changes in central catecholaminergic neurons in the spontaneously (genetic) hypertensive rat, Circulat. Res., 42 (1978) 529-534. 18 Saavedra, J. M., Palkovits, M., Brownstein, M. J. and Axelrod, J., Localization of phenylethanolamine-N-methyltransferase in the rat brain nuclei, Nature (Lond.), 248 (1974) 695-696. 19 Schlumpf, M., Waser, P., Lichtenstiger, W., Langmann, H. and Schlup, D., Standardized excision of small areas of rat and mouse brain with topographical control, Biochem. PharmacoL, 23 (1974) 2447-2449. 20 Sokal, R. R. and Rohlf, F. J., Biometry, W. H. Freeman, San Francisco, 1969. 21 Wijnen, H. J. L. M., Versteeg, D. H. G., Paikovits, M. and De Jong, W., Increased adrenaline content of individual nuclei of the hypothalamus and the medulla oblongata of genetically hypertensive rats, Brain Research, 135 (1977) 180-185.