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Central and peripheral contributions to hypothalamic epinephrine
Brain Research, 224 (1981) 175 179
175
Elsevier/North-Holland Biomedical Press
Central and peripheral contributions to hypothalamic epinephrine
IVAN N. MEFFORD, KEVIN A. ROTH, GEORGE PAXINOS and JACK D. BARCHAS Nancy Pritzker Laboratory of Behavioral Neurochemist~T, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U,S.A.) and (G.P.) Department of P~ychology, The University of New South Wales, P.O. Box 1, Kensington, N.S.W. 2033 (Australia)
(Accepted July 16th, 1981) Key words: epinephrine - - hypothalamus - - rat brain - - adrenal demedullation - - brain stem
hemisection
Hypothalamic epinephrine concentrations were examined following adrenal demedullation or after surgical hemisection of ascending projections to the hypothalamus. Following surgical transection epinephrine in the ipsilateral hypothalamus was depleted by approximately 60~. Adrenal demedullation had no effect on hypothalamic epinephrine concentrations, it was concluded that hypothalamic epinephrine was central in origin. The presence o f epinephrine (E) in the central nervous system of mammals was first observed some 30 years agoa,6,10A8,19. Initially, it was thought that the E detected in brain tissue was primarily found as a result o f E presence in blood. Tritium-labeled E is only sparingly taken up by brain from plasma, apparently crossing the blood-brain barrier only at the hypothalamus and, to a lesser extent, in the brain stem area of the floor of the fourth ventricle2, 20. More recently phenylethanolamine-N-methyltransferase (PNMT), the enzyme which converts norepinephrine (NE) to E, has been localized 5 in mammalian brain. The observation of the presence o f this enzyme in brain 1,2,8,14,15 and its immunohistochemical detection in neurons ~ suggested that most if not all the E in brain was derived from C N S synthesis. Both stress 17 and glucocorticoid administration 11 have been shown to increase central P N M T activity as well as increasing levels of E 11. If any uptake of peripheral E does occur, it must be derived from adrenal E as nearly all peripheral E is synthesized in the adrenal medulla. Further, since the cell bodies for the central E neurons are found in the brain stem areas C1 and Ce, transection of these ascending axons should eliminate dendritic hypothalamic E associated with these cell bodies. In the present study, the possible peripheral contribution to hypothalamic E was examined by comparing hypothalamic E levels of adrenal demedullated rats, 3 weeks following surgery, to sham-operated controls. F o r determination o f the contribution of the brain stem cell body projections, 3 separate knife cuts were made in the left hemisphere. After 7 days the rats were 0006 8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
176 sacrificed and the hypothalami dissected into left and right hemispheres. Catecholamine levels were determined in the two hemispheres and compared, with the hypothalamic dissection contralateral to the hemisection serving as the control. Male Sprague-Dawley rats (Simonsen Labs, Gilroy, CA) 200-250 g were used throughout. Adrenal demedullated and sham-operated rats were obtained from Zivic Miller. All rats were maintained on a 12 h light/dark cycle and on ad libitum food and and water. The water for adrenal demedullated animals was replaced with saline (0.9 oi NaCI). Cuts of the ascending brain stem projections were accomplished with the use of a retractable knifO 6. With the skull horizontal, the guide cannula was inserted 2 mm anterior to the interaural line and at a position that placed the tip of the knife at 0.0,
100 -
~k uJ u
¢/3 I-"
75
¢J
z I.-z
5O
IJJ n" LU 0,.
25
0 A
B
C
Fig. 1. Effects of transection of ascending n e u r o n s on hypothalamic epinephrine. **P -:: 0.01, ***P < 0,001 by 2-tailed Student's t-test, n == 4 or 5 in each case.
177 1.0 and 2.0 mm lateral to the midline. After sectioning the brains in a horizontal plane, the cuts were interpreted with the use of an atlas of horizontal sections 13 and reconstructed on coronal plates of the K6nig and Klippel atlas v. Following decapitation, brains were dissected and tissues immediately frozen at --80 °C until analyzed for catecholamines. Catecholamines were determined by high performance liquid chromatography with electrochemical detection as previously described 9. Fig. 1 shows the location and dimensions of the lateralized knife cuts as made. The bar graphs above each section show the ratios of E concentrations in the hypothalami from the cut side to the uncut side. Approximately 30~o~ depletion was observed with each of the two more medial cuts. The most lateral cut was ineffective in lowering hypothalamic E. The depletion of hypothalamic E following hemisection of the ascending brain stem neurons was slightly more than 6 0 ~ . Hypothalamic NE content was lowered by 12~, 3 7 ~ and 6 ~ following hemisections A, B and C, respectively, a sum of 55 % depletion. Table I shows the catecholamine concentrations in hypothalamus following adrenal demedullation. There were no observable effects on hypothalamic E concentrations. Norepinephrine was unaffected while dopamine was significantly elevated (P < 0.02 by 2 tailed Student's t-test). Palkovits et al. lz have previously reported on the effects of lesions of the medullary cell body areas C1 and C2 on hypothalamic epinephrine concentrations. Using either partial transection or electrolytic lesioning, decreases of 59-78 o~ were observed in bypothalamic E levels. The hemisections made in our work were at A900 #m 7 compared to the transections made by Palkovits, P 13,500 #m. These hemisections were at the level of the dorsal tegmental decussation and were intended to transect the dorsal and ventral bundles ipsilateral to the knife cuts. The contralateral hypothalamus was used as the control in each case. The degree of depletion observed ( ~ 60'~,~,)is comparable to that observed by Palkovits. Others have shown that following surgical isolation of the hypothalamus, P N M T activity is lowered by 58-66 °/oZ. These data support anatomical evidence for the origin of hypothalamic E from medullary projections. None of these studies, however, show complete elimination of hypothalamic E, leaving open the possibility of an as yet undetermined source of hypothalamic E.
TABLE l
Hypotha&mic catecholamOte levels following adrenal demedullation All values are ng/g wet weight ~z S.E.M. (n).
Sham-operated controls Adrenal demedullated
Norepinephrine
Ephtephrine
Dopamine
1524 ± 76 (7) 1744 5__ 114 (8)
29.0 5z 1.4 (7) 33.0 Jz 2.2 (8)
253 ± 14 (7) 363 ± 36 (8)
178 A l t h o u g h earlier studies have shown E to cross the b l o o d - b r a i n barrier only to a small extent 20, we chose to observe what, if any, effect the e l i m i n a t i o n of the source of peripheral E had on h y p o t h a l a m i c E. The results from a d r e n a l medullated a n i m a l s d e m o n s t r a t e d that indeed peripheral E does n o t c o n t r i b u t e to the c o n c e n t r a t i o n of h y p o t h a l a m i c E. O u r data offer further evidence that h y p o t h a l a m i c E is central in origin, in part from ascending projections from the perikarya in the medullary C~ a n d Cz cell groups a n d perhaps from other as yet unidentified sources in the h y p o t h a l a m u s itself. This work was supported by N I M H Program-Project G r a n t M H 23861 a n d an a w a r d from the O N R , SRO-001.
1 Axelrod, J., Purification and properties of phenytethanolamine N-methyltransferase, J. biol. Chem., 237 (1962) 1657-1660. 2 Barchas, J. D., Ciaranello, R. D. and Steinman, A. M., Epinephrine formation and metabolism in mammalian brain, Biol. Psychiat., l (1969) 31-48. 3 Brownstein, M. J., Palkovits, M., Tappaz, M. L., Saavedra, J. M. and Kizer, J. S., Effect of surgical isolation of the hypothalamus on its neurotransmitter content, Brain Research, 117 (1976) 287-295. 4 Gunne, L.-M., Relative adrenaline content in brain tissue, Actaphysiol. seand., 56 (1962) 324-333. 5 H6kfelt, 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. 6 Holtz, P., Uber die sympathicommitsehe Wirksamkeit von Gehirnextraken, Acta physiol, stand., 20 (1950) 354-362. 7 Kt~nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas, Robert E. Krieger, Huntington, NY, 1974. 8 McGeer, P. L. and McGeer, E. G., Formation of adrenaline by brain tissue, Biochem. bi~)phys. Res. Commun., 17 (1964) 502-507. 9 Mefford, I. N., Gilberg, M. and Barchas, J. D., Simultaneous determination of catecholamines and unconjugated 3, 4-dihydroxyphenylacetic acid in brain tissue by ion-pairing reverse-phase high-performance liquid chromatography with electrochemical detection, Analyt. Bioehem., 104 (1980) 46%472. 10 Montagu, K. A., Adrenaline and noradrenaline concentrations in rat tissues, Biochem. J., 63 (1956) 559-565. 11 Moore, K. E. and Phillipson, O. T., Effects of dexamethasone on phenylethanolamineN-methyltransferase and adrenaline in the brains and superior cervical ganglion of adult and neonatal rats, J. Neurochem., 25 (1975) 289-294. 12 Palkovits, M., Mezey, E., Zaborozky, L., Feminger, A., Versteeg, D. H. G., Wijner, H. J. L. M., DeJong, W., Fekete, M. I. K., Herman, J. P. and Kanyicska, B., Adrenergic innervation of the rat hypothalamus, Neurosci. Lett., 18 (1980) 237-243. 13 Paxinos, G., Watson, C. R. R. and Emson, P., AChE-stained horizontal sections of the rat brain in stereotaxic coordinates, J. Neurosci. Meth., 3 (1980) 129-149. 14 Pohorecky, L. A., Zigmond, M., Karten H. and Wurtman, R. J., Enzymatic conversion of norepinephrine to epinephrine by the brain, J. Pharmacol. exp. Ther., 165 (1969) 190--195. 15 Saavedra, J. M., Palkovits, M., Brownstein, M. J. and Axelrod, J., Localization of phenylethanolamine-N-methyltransferase in rat brain nuclei, Nature (Lond.), 248 (1974) 695-696. 16 Sclafani, A. and Grossman, S. P., Hyperphagia produced by knife cuts between the medial and lateral hypothalamus in the rat, Physiol. Behav., 4 (1969) 533-537. 17 Turner, B. B., Katz, R. J., Roth, K. A. and Carroll, B. J., Central elevation of pheny!ethanolamine-N-methyltransferase activity following stress, Brain Research, 153 (1978) 419-422. 18 Vogt, M., The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs, J. Physiol. (Lond.), 123 (1954) 451481.
179 19 Von Euler, U. S., A specific sympathomimetic ergone in adrenergic nerve fibers (sympathin) and its relation to adrenaline and noradrenaline, Acta physiol, scand., 12 (1946) 73-97. 20 Weil-Malherbe, H., Axelrod, J. and Tomchick, R., Blood brain barrier for adrenaline, Science, 129 (1958) 1226-1227.
175
Elsevier/North-Holland Biomedical Press
Central and peripheral contributions to hypothalamic epinephrine
IVAN N. MEFFORD, KEVIN A. ROTH, GEORGE PAXINOS and JACK D. BARCHAS Nancy Pritzker Laboratory of Behavioral Neurochemist~T, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U,S.A.) and (G.P.) Department of P~ychology, The University of New South Wales, P.O. Box 1, Kensington, N.S.W. 2033 (Australia)
(Accepted July 16th, 1981) Key words: epinephrine - - hypothalamus - - rat brain - - adrenal demedullation - - brain stem
hemisection
Hypothalamic epinephrine concentrations were examined following adrenal demedullation or after surgical hemisection of ascending projections to the hypothalamus. Following surgical transection epinephrine in the ipsilateral hypothalamus was depleted by approximately 60~. Adrenal demedullation had no effect on hypothalamic epinephrine concentrations, it was concluded that hypothalamic epinephrine was central in origin. The presence o f epinephrine (E) in the central nervous system of mammals was first observed some 30 years agoa,6,10A8,19. Initially, it was thought that the E detected in brain tissue was primarily found as a result o f E presence in blood. Tritium-labeled E is only sparingly taken up by brain from plasma, apparently crossing the blood-brain barrier only at the hypothalamus and, to a lesser extent, in the brain stem area of the floor of the fourth ventricle2, 20. More recently phenylethanolamine-N-methyltransferase (PNMT), the enzyme which converts norepinephrine (NE) to E, has been localized 5 in mammalian brain. The observation of the presence o f this enzyme in brain 1,2,8,14,15 and its immunohistochemical detection in neurons ~ suggested that most if not all the E in brain was derived from C N S synthesis. Both stress 17 and glucocorticoid administration 11 have been shown to increase central P N M T activity as well as increasing levels of E 11. If any uptake of peripheral E does occur, it must be derived from adrenal E as nearly all peripheral E is synthesized in the adrenal medulla. Further, since the cell bodies for the central E neurons are found in the brain stem areas C1 and Ce, transection of these ascending axons should eliminate dendritic hypothalamic E associated with these cell bodies. In the present study, the possible peripheral contribution to hypothalamic E was examined by comparing hypothalamic E levels of adrenal demedullated rats, 3 weeks following surgery, to sham-operated controls. F o r determination o f the contribution of the brain stem cell body projections, 3 separate knife cuts were made in the left hemisphere. After 7 days the rats were 0006 8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
176 sacrificed and the hypothalami dissected into left and right hemispheres. Catecholamine levels were determined in the two hemispheres and compared, with the hypothalamic dissection contralateral to the hemisection serving as the control. Male Sprague-Dawley rats (Simonsen Labs, Gilroy, CA) 200-250 g were used throughout. Adrenal demedullated and sham-operated rats were obtained from Zivic Miller. All rats were maintained on a 12 h light/dark cycle and on ad libitum food and and water. The water for adrenal demedullated animals was replaced with saline (0.9 oi NaCI). Cuts of the ascending brain stem projections were accomplished with the use of a retractable knifO 6. With the skull horizontal, the guide cannula was inserted 2 mm anterior to the interaural line and at a position that placed the tip of the knife at 0.0,
100 -
~k uJ u
¢/3 I-"
75
¢J
z I.-z
5O
IJJ n" LU 0,.
25
0 A
B
C
Fig. 1. Effects of transection of ascending n e u r o n s on hypothalamic epinephrine. **P -:: 0.01, ***P < 0,001 by 2-tailed Student's t-test, n == 4 or 5 in each case.
177 1.0 and 2.0 mm lateral to the midline. After sectioning the brains in a horizontal plane, the cuts were interpreted with the use of an atlas of horizontal sections 13 and reconstructed on coronal plates of the K6nig and Klippel atlas v. Following decapitation, brains were dissected and tissues immediately frozen at --80 °C until analyzed for catecholamines. Catecholamines were determined by high performance liquid chromatography with electrochemical detection as previously described 9. Fig. 1 shows the location and dimensions of the lateralized knife cuts as made. The bar graphs above each section show the ratios of E concentrations in the hypothalami from the cut side to the uncut side. Approximately 30~o~ depletion was observed with each of the two more medial cuts. The most lateral cut was ineffective in lowering hypothalamic E. The depletion of hypothalamic E following hemisection of the ascending brain stem neurons was slightly more than 6 0 ~ . Hypothalamic NE content was lowered by 12~, 3 7 ~ and 6 ~ following hemisections A, B and C, respectively, a sum of 55 % depletion. Table I shows the catecholamine concentrations in hypothalamus following adrenal demedullation. There were no observable effects on hypothalamic E concentrations. Norepinephrine was unaffected while dopamine was significantly elevated (P < 0.02 by 2 tailed Student's t-test). Palkovits et al. lz have previously reported on the effects of lesions of the medullary cell body areas C1 and C2 on hypothalamic epinephrine concentrations. Using either partial transection or electrolytic lesioning, decreases of 59-78 o~ were observed in bypothalamic E levels. The hemisections made in our work were at A900 #m 7 compared to the transections made by Palkovits, P 13,500 #m. These hemisections were at the level of the dorsal tegmental decussation and were intended to transect the dorsal and ventral bundles ipsilateral to the knife cuts. The contralateral hypothalamus was used as the control in each case. The degree of depletion observed ( ~ 60'~,~,)is comparable to that observed by Palkovits. Others have shown that following surgical isolation of the hypothalamus, P N M T activity is lowered by 58-66 °/oZ. These data support anatomical evidence for the origin of hypothalamic E from medullary projections. None of these studies, however, show complete elimination of hypothalamic E, leaving open the possibility of an as yet undetermined source of hypothalamic E.
TABLE l
Hypotha&mic catecholamOte levels following adrenal demedullation All values are ng/g wet weight ~z S.E.M. (n).
Sham-operated controls Adrenal demedullated
Norepinephrine
Ephtephrine
Dopamine
1524 ± 76 (7) 1744 5__ 114 (8)
29.0 5z 1.4 (7) 33.0 Jz 2.2 (8)
253 ± 14 (7) 363 ± 36 (8)
178 A l t h o u g h earlier studies have shown E to cross the b l o o d - b r a i n barrier only to a small extent 20, we chose to observe what, if any, effect the e l i m i n a t i o n of the source of peripheral E had on h y p o t h a l a m i c E. The results from a d r e n a l medullated a n i m a l s d e m o n s t r a t e d that indeed peripheral E does n o t c o n t r i b u t e to the c o n c e n t r a t i o n of h y p o t h a l a m i c E. O u r data offer further evidence that h y p o t h a l a m i c E is central in origin, in part from ascending projections from the perikarya in the medullary C~ a n d Cz cell groups a n d perhaps from other as yet unidentified sources in the h y p o t h a l a m u s itself. This work was supported by N I M H Program-Project G r a n t M H 23861 a n d an a w a r d from the O N R , SRO-001.
1 Axelrod, J., Purification and properties of phenytethanolamine N-methyltransferase, J. biol. Chem., 237 (1962) 1657-1660. 2 Barchas, J. D., Ciaranello, R. D. and Steinman, A. M., Epinephrine formation and metabolism in mammalian brain, Biol. Psychiat., l (1969) 31-48. 3 Brownstein, M. J., Palkovits, M., Tappaz, M. L., Saavedra, J. M. and Kizer, J. S., Effect of surgical isolation of the hypothalamus on its neurotransmitter content, Brain Research, 117 (1976) 287-295. 4 Gunne, L.-M., Relative adrenaline content in brain tissue, Actaphysiol. seand., 56 (1962) 324-333. 5 H6kfelt, 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. 6 Holtz, P., Uber die sympathicommitsehe Wirksamkeit von Gehirnextraken, Acta physiol, stand., 20 (1950) 354-362. 7 Kt~nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas, Robert E. Krieger, Huntington, NY, 1974. 8 McGeer, P. L. and McGeer, E. G., Formation of adrenaline by brain tissue, Biochem. bi~)phys. Res. Commun., 17 (1964) 502-507. 9 Mefford, I. N., Gilberg, M. and Barchas, J. D., Simultaneous determination of catecholamines and unconjugated 3, 4-dihydroxyphenylacetic acid in brain tissue by ion-pairing reverse-phase high-performance liquid chromatography with electrochemical detection, Analyt. Bioehem., 104 (1980) 46%472. 10 Montagu, K. A., Adrenaline and noradrenaline concentrations in rat tissues, Biochem. J., 63 (1956) 559-565. 11 Moore, K. E. and Phillipson, O. T., Effects of dexamethasone on phenylethanolamineN-methyltransferase and adrenaline in the brains and superior cervical ganglion of adult and neonatal rats, J. Neurochem., 25 (1975) 289-294. 12 Palkovits, M., Mezey, E., Zaborozky, L., Feminger, A., Versteeg, D. H. G., Wijner, H. J. L. M., DeJong, W., Fekete, M. I. K., Herman, J. P. and Kanyicska, B., Adrenergic innervation of the rat hypothalamus, Neurosci. Lett., 18 (1980) 237-243. 13 Paxinos, G., Watson, C. R. R. and Emson, P., AChE-stained horizontal sections of the rat brain in stereotaxic coordinates, J. Neurosci. Meth., 3 (1980) 129-149. 14 Pohorecky, L. A., Zigmond, M., Karten H. and Wurtman, R. J., Enzymatic conversion of norepinephrine to epinephrine by the brain, J. Pharmacol. exp. Ther., 165 (1969) 190--195. 15 Saavedra, J. M., Palkovits, M., Brownstein, M. J. and Axelrod, J., Localization of phenylethanolamine-N-methyltransferase in rat brain nuclei, Nature (Lond.), 248 (1974) 695-696. 16 Sclafani, A. and Grossman, S. P., Hyperphagia produced by knife cuts between the medial and lateral hypothalamus in the rat, Physiol. Behav., 4 (1969) 533-537. 17 Turner, B. B., Katz, R. J., Roth, K. A. and Carroll, B. J., Central elevation of pheny!ethanolamine-N-methyltransferase activity following stress, Brain Research, 153 (1978) 419-422. 18 Vogt, M., The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs, J. Physiol. (Lond.), 123 (1954) 451481.
179 19 Von Euler, U. S., A specific sympathomimetic ergone in adrenergic nerve fibers (sympathin) and its relation to adrenaline and noradrenaline, Acta physiol, scand., 12 (1946) 73-97. 20 Weil-Malherbe, H., Axelrod, J. and Tomchick, R., Blood brain barrier for adrenaline, Science, 129 (1958) 1226-1227.