410
Brain Research, 99 (1975) 410 414 ~(~ Elsevier Scientific Publishing Company, Amsterdam -Printed in The Netherlands
Regional distribution of DOPA decarboxylase activity in hypothalamus of the rat
MASASHI SAITO, MAKOTO HIRANO AND HIDEYUKI UCHIMURA
Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga 842-01 (Japan) (Accepted August 5th, 1975)
The enzyme, DOPA decarboxylase (3,4-dihydroxy-L-phenylalaninedecarboxylase; D-DC; EC 4.1.1.26) catalyzes the formation of dopamine (DA)from DOPA, whereas 5-HTP decarboxylase (5-hydroxy-L-tryptophan decarboxylase; 5-HTP-DC; EC 4.1.1.28) catalyzes the formation of serotonin (5-hydroxytryptamine; 5-HT) from 5-HTP. These enzymes are commonly called aromatic L-amino acid decarboxylase (A-DC) as a single enzymel,S,9,16. In mammalian central nervous system, A-DC is mainly distributed in caudate nucleus, corpus striatum, hypothatamus and mesencephalon3,6,10,12,14,ts. Concerning the subcellular localization of A-DC in rat brain, the enzyme is known to appear predominantly not only in the high speed supernatant fraction but also in the nerve ending (or synaptosomal) fraction4015,19,~2. Broch e t al. 4 reported that D-DC was more soluble in regions with a high content of monoaminergic cell bodies, whereas it was more particulate m regions with a high concentration of monoaminergic terminals. Moreover, Htkfelt et a l p demonstrated by the immunohistochemical method that A-DC in mesencephalon was located mainly in the DA and 5-HT nerve cell systems and not in other neuronal or extraneuronal systems. In the brain tissue, therefore, A-DC is localized mainly in monoaminergic neurons, while there are some findings that A-DC occurs also in brain capillaries2, la. Since monoamines seem to play important roles on the hypothalamic functions as neurotransmitters, it is essential to study the quantitative distributions of monoamines and their related enzymes in the hypothalamic nuclei for understanding the hypothalamic functions and the roles of monoamines in hypothalamus. In fact, the levels of monoamines and their related enzymes have been studied recently in the individual hypothalamic nucleiS,tT,20,23~ However, the experiment for D-DC activities in the hypothalamic nuclei is not yet undertaken. In the present study, D-DC activities in the hypothalamic nuclei of the rat were examined, as a necessary supplement for the investigation of the hypothalamic functions. Seventeen-week-old Wistar-King male rats which were kept in a group were used for the experiment. The animals were killed by decapitation at 4:00 p.m. The
411 TABLE I REGIONAL DISTRIBUTION OF D - D E
IN RAT HYPOTHALAMUS
The values of D-DC activity represent means ± S.E.M. The numbers of animals are in parentheses. D-DC activity is expressed as/~moles 14COzformed/g dry wt./h. Nueh, us o f hypothalamus
D-DC activiO,
Pars anterior Nucleus anterior
25.46 ± 2.15 (5)
Area lateralis Nucleus paraventricularis Area retrochiasmaticus
21.68 j= 2.12 (5) 40.30 ± 5.58 (4) 39.56 4 3.95 (3)
Pars medialis Nucleus ventromedialis Nucleus dorsomedialis
29.52 -- 4.41 (4) 27.31 -- 1.79 (4)
Area lateralis Nucleus periventricularis Nucleus arcuatus
20.38 : 1.99 (5) 30.04 - 3.91 (5) 36.54 ± 3.08 (4)
Pars posterior Nucleus posterior
25.34 r__ 2.13 (4)
Area lateralis Nucleus premammillaris dorsalis Nucleus premammillaris ventralis Nucleus arcuatus
16.73 ± 12.49 ± 32.80 -. 32.48 --
0.61 (4) 1.48 (4) 2.68 (4) 3.73 (4)
brain was immediately removed and placed on ice. The tissue block which contained hypothalamus was isolated and frozen in liquid nitrogen. A series of frontal sections of the tissue block was made at 55 #m or 85 # m thickness in a cryostat (--15 °C). The sections were freeze-dried overnight at - - 3 0 °C and 10 3 mm Hg and stored in evacuated tubes at - - 2 0 °C until use. The individual hypothalamic nuclei were carefully dissected freehand under a stereomicroscope not to include brain vessels. Each sample was weighed by an electronic microbalance (Type 4152, Sartorius Co.) with a digital voltmeter (Type EO-12, Eto Co.), and was transferred into a mictrotube and crushed with a stainless steel needle. D O P A decarboxylase was assayed by a modification of the method of Giacobini and Nor~ 7. Five #1 of cold buffer substrate was added to the dry sample in a pointed microtube (final concentrations; 1.5 m M DL-[1-14C]DOPA, 69.4 m M phosphate buffer, pH 7.4, 0.042 m M pyridoxal-5-phosphate, 4.2 m M L-cysteine chloride, 0.17 m M BSA). The incubation tube was connected by rubber tubing to another microtube containing 20 #1 of 1 M hyamine solution in methanol. The two tubes connected to each other were incubated for 15 min at 38 °C. The reaction was stopped by injecting 16 #1 of 2.5 M H2SO4 into the reaction mixture through the rubber tubing. The tubes were replaced in the water bath for 30 min at 38 °C for the complete diffusion of CO2. The tube containing hyamine was separated from the connection and transferred into a counting vial containing 10 ml of toluene scintillator. After shaking the
412
....~ -2
!J_ .....
MT
LD pE L"O
0 0
a~
"
2
3 Fig. 1. Distribution of D-DC in the rat hypothalarnic nuclei. Drawings of frontal sections through the hypothalamus (1-3). 1, pars anterior: 2, pars medialis: 3, pars posterior. Abbreviations: A, nucleus anterior; AR, nucleus arcuatus; DM. nucleus dorsomedialis; F, fornix; L, area lateralis; MT. tractus mammillothalamicus; OT, tractus opticus; P. nucleus posterior; PA, nucleus paraventricularis; PD, nucleus premammillaris dorsalis; PE, nucleus periventricularis: PV, nucleus premam-
millaris ventralis; RC. area retrochiasmaticus; VM. nucleus ventromedialis. Concentrations of D-DC: solid area, high: at least 20% above the average of the hypothalarnic nuclei; crosshatched area, relatively high: within +20% of the average; hatched area, relatively low: within--20 % of the average; stippled area. low: at least 20% below the average. counting vial for 30 min, the radioactivity was measured in a Packard Tricarb model 3320 liquid scintillation spectrometer, The counting efficiency was 75 %. The results are summarized in Table I and Fig. l. The highest D - D C activities were found in the nucleus paraventricularis and area retrochiasmaticus. Relatively high activities were found in the arcuate, ventromedial and periventricular nuclei, and nucleus premammillaris ventralis. The lateral hypothalamic area and the nucleus premammiltaris dorsalis had low D - D C activities. Within the hypothalamic nuclei, the highest activity of D - D C was about 3-fold greater than the lowest activity. Kuntzman et al. i2 reported that D - D C activity was evenly distributed throughout the cat hypothalamus. In their study, D - D C activity was determined in the several parts of hypothalamus such as anterior, posterior and middle thirds, a n d ventral and dorsal halves, which might include several hypothalamic nuclei and their surrounding tissues. In the individual hypothalamic nuclei of rat, however, D - D C activities were found to be distributed widely and unevenly as shown in the present study. The gross regional distribution of D - D C activities in mammalian brain has been
413 indicated to correlate with those of monoamine levels by several authors t,14,16. Recently, Palkovits et al. 17 and Saavedra et al. 2° reported the distribution of N E, DA and of 5-HT in the discrete hypothalamic nuclei. Comparing our data with their results, the nucleus paraventricularis and area retrochiasmaticus, which showed high D-DC activities in our study, contained high levels of NE and DA, while the lateral hypothalamic area and nucleus premammillaris dorsalis, which showed low activities, contained low levels of NE and DA. On the contrary, nucleus paraventricularis and area retrochiasmaticus had low contents of 5-HT, whereas the lateral hypothalamic area and nucleus premammillaris dorsalis had high contents of 5-HT. It is of interest that, in the individual hypothalamic nuclei, the distribution of D-DC activities correlates directly with those of NE and DA, and yet correlates inversely with that of 5-HT. These relationships between the distribution of D-DC and those of monoamine levels suggest that the levels of D-DC obtained in the present study mainly reflect the activities of D-DC in the neuronal elements. Furthermore, these findings bring the possibility that D-DC may be different from 5-HTP-DC. It is generally supported that A-DC is involved in the decarboxylation steps both in DA neurons and in 5-HT neurons as a single enzyme1,5, 9,16. However, in the recent study in which the differences in assay characteristics, distribution and the effect of 6-hydroxydopamine, of D-DC and 5-HTP-DC were examined, Sims et al. ~1,'-2 suggested that the two different proteins catalyzed the D-DC and 5-HTP-DC reactions in brain. Furthermore, Korf et al. 11 also reported the existence of specific 5-HTP-DC enzyme in 5-HT containing nerve endings through the study of the decarboxylation of exogenous L-5-HTP after destruction of the rat cerebral raphe system. Our present data may also support the suggestion that D-DC is different from 5-HTP-DC. It is necessary, therefore, to examine the distribution of 5-HTP-DC activities in hypothalamic nuclei. This work was supported by a grant from the Science and Technology Agency of Japan, and by a grant from the Ministry of Pubic Welfare of Japan.
1 AURES, D., HA,KANSON, R., AND CLARK, W. G., Histidine decarboxylase and dopa decarboxylase. In A. LAJTHA(Ed.), Control Mechanisms in the Nervous System, Handbook o f Neurochemistry, Vol. 4, Plenum Press, New York, 1970, pp. 165-196. 2 BERTLER,A., FALCK,B., OWMAN,CH., AND ROSENGREN,E., The localization of monoaminergic blood-brain barrier mechanisms, Pharmacol. Rev., 18 (1966) 369-385. 3 BOGDANSKI,D. F., WEISSBACH,H., ANDUDENFRIEND,S., The distribution of serotonin, 5-hydroxytryptophan-decarboxylase, and monoamine oxidase in brain, J. Neurochem., 1 (1957) 272-278. 4 BROCH,JR., O. J., AND FONNUM,F., The regional and subcellular distribution of catechoI-Omethyltransferase in rat brain, J. Neurochem., 19 (1972) 2049 2055. 5 CHRISTENSON,J. G., DIARMAN,W., ANDUDENFRIEND,S., On the identity of DOPA decarboxylase and 5-hydroxytryptophan decarboxylase, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 343-347. 6 DE RoPP, R. S., AND FURST, A., Effect of analogs of phenylalanine and tryptophan on kinetics of DOPA decarboxylase in rat brain, Brain Research, 2 (1966) 323 332. 7 G1ACOBIN[,E., AND NORI~, B., Dopa-decarboxylase in autonomic and sensory ganglia of the cat, Acta physiol, stand., 82 (1971) 209-217.
414 8 HIRANO,M., UCHIMURA,H., ANDSAITO, M., Regional distribution ofmonoamine oxidase activity for 5-hydroxytryptamine and tyramine in hypothalamus of the rat, Brain Research, 93 (1975) 558-565. 9 H6KFELT, T., FUXE, K., AND GOLDSTEIN, M., Immunohistochemical localization of aromatic L-amino acid decarboxylase (DOPA-decarboxylase) in central dopamine and 5-hydroxytryptamine nerve celt bodies of the rat, Brain Research, 53 (1973) 175-180. 10 HOLTZ, P., AND WEST•RMANN, E., Uber die Dopadecarboxylase und Histidindecarboxylase des Nervengewebes, Nauyn-Schmiedeberg's Arch. exp. Path. Pharmak., 227 (1956) 538-546. 11 KORF, J., VENEMA,K., AND POSTEMA,F., Decarboxylation of exogenous L-5-hydroxytryptophan after destruction of the cerebral raphe system, J. Neurochem., 23 (1974) 249-252. 12 KUNTZMAN,R., SHORE, P. A., BOGDANSKI,D., AND BRODIE,B. B., Microanalytical procedures for fluorometric assay of brain DOPA-5HTP decarboxylase, norepinephrine and serotonin, and a detailed mapping of decarboxylase activity in brain, J. Neurochem., 6 (1961) 226-232. 13 LANGELIER, P., PARENT, A., AND POIRIER, L. J., Decarboxytase activity of the brain capillary walls and parenchyma in the rat, cat and monkey, Brain Research, 45 (1972) 622-629. 14 LLOYD, K. G., AND HORNYKIEWICZ,O., Occurrence and distribution of aromatic L-amino acid (L-DOPA) decarboxylase in the human brain, J. Neurochem., t9 (1972) 1549-1559. 15 MCGEER, P. L., BAGCHI, S. P., AND MCGEER, E. G., Subcellular localization of tyrosine hydroxylase in beef caudate nucleus, Life Sei., 4 (1965) 1859-1867. 16 MCGEER, P. L., AND McGEER, E. G., Neurotransmitter synthetic enzymes. In G. A. KERKUTAND .l.W. PHILLIS(Eds.), Progress in Neurobiology, VoL 2, Pergamon Press, Oxford, 1973, pp. 69-117. 17 PALKOVITS,M., BROWNSTEIN,M., SAAVEDRA,J. M., AND AXELROD,J., Norepinephrine and dopamine content of hypothalamic nuclei of the rat, Brain Research, 77 (1974) 134-149. 18 ROBERGE, A. G., AND POIRIER, L. J., Effect of chronically administered L-DOPA on Dopa/ 5HTP decarboxylase and tyrosine and tryptophan hydroxylases in cat brain, J. Neural Transmission, 34 (1973) 171-185. 19 RODRIGUEZ DE LORES ARNAIZ, G., AND DEROaERTIS, E., 5-Hydroxytryptophan decarboxylase activity in nerve endings of the rat brain, J. Neurochem., 11 (1964) 213-219. 20 SAAVEDRA,J. M., PALKOVITS,M., BROWNSTEIN,M. J., AND AXELROD,J., Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region, Brain Research, 77 (1974) 157-165. 21 SIMS, K. L., AND BLOOM, F. E., Rat brain L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan decarboxylase activities: differential effect of 6-hydroxydopamine, Brain Research, 49 (1973) 165-175. 22 SIMS, K. L., DAWS, G. A , AND BLOOM, F. E., Activities of 3,4-dihydroxy-L-phenylalanine and 5hydroxy-L-tryptophan decarboxylases in rat brain: assay characteristics and distribution, J. Neurochem., 20 (1973) 449~164. 23 UCmMURA, H., SAXTO,M., AND HIRANO, M., Regional distribution of choline acetyltransferase in hypothalamus of the rat, Brain Research, 91 (1975) 161-164.
Brain Research, 99 (1975) 410 414 ~(~ Elsevier Scientific Publishing Company, Amsterdam -Printed in The Netherlands
Regional distribution of DOPA decarboxylase activity in hypothalamus of the rat
MASASHI SAITO, MAKOTO HIRANO AND HIDEYUKI UCHIMURA
Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga 842-01 (Japan) (Accepted August 5th, 1975)
The enzyme, DOPA decarboxylase (3,4-dihydroxy-L-phenylalaninedecarboxylase; D-DC; EC 4.1.1.26) catalyzes the formation of dopamine (DA)from DOPA, whereas 5-HTP decarboxylase (5-hydroxy-L-tryptophan decarboxylase; 5-HTP-DC; EC 4.1.1.28) catalyzes the formation of serotonin (5-hydroxytryptamine; 5-HT) from 5-HTP. These enzymes are commonly called aromatic L-amino acid decarboxylase (A-DC) as a single enzymel,S,9,16. In mammalian central nervous system, A-DC is mainly distributed in caudate nucleus, corpus striatum, hypothatamus and mesencephalon3,6,10,12,14,ts. Concerning the subcellular localization of A-DC in rat brain, the enzyme is known to appear predominantly not only in the high speed supernatant fraction but also in the nerve ending (or synaptosomal) fraction4015,19,~2. Broch e t al. 4 reported that D-DC was more soluble in regions with a high content of monoaminergic cell bodies, whereas it was more particulate m regions with a high concentration of monoaminergic terminals. Moreover, Htkfelt et a l p demonstrated by the immunohistochemical method that A-DC in mesencephalon was located mainly in the DA and 5-HT nerve cell systems and not in other neuronal or extraneuronal systems. In the brain tissue, therefore, A-DC is localized mainly in monoaminergic neurons, while there are some findings that A-DC occurs also in brain capillaries2, la. Since monoamines seem to play important roles on the hypothalamic functions as neurotransmitters, it is essential to study the quantitative distributions of monoamines and their related enzymes in the hypothalamic nuclei for understanding the hypothalamic functions and the roles of monoamines in hypothalamus. In fact, the levels of monoamines and their related enzymes have been studied recently in the individual hypothalamic nucleiS,tT,20,23~ However, the experiment for D-DC activities in the hypothalamic nuclei is not yet undertaken. In the present study, D-DC activities in the hypothalamic nuclei of the rat were examined, as a necessary supplement for the investigation of the hypothalamic functions. Seventeen-week-old Wistar-King male rats which were kept in a group were used for the experiment. The animals were killed by decapitation at 4:00 p.m. The
411 TABLE I REGIONAL DISTRIBUTION OF D - D E
IN RAT HYPOTHALAMUS
The values of D-DC activity represent means ± S.E.M. The numbers of animals are in parentheses. D-DC activity is expressed as/~moles 14COzformed/g dry wt./h. Nueh, us o f hypothalamus
D-DC activiO,
Pars anterior Nucleus anterior
25.46 ± 2.15 (5)
Area lateralis Nucleus paraventricularis Area retrochiasmaticus
21.68 j= 2.12 (5) 40.30 ± 5.58 (4) 39.56 4 3.95 (3)
Pars medialis Nucleus ventromedialis Nucleus dorsomedialis
29.52 -- 4.41 (4) 27.31 -- 1.79 (4)
Area lateralis Nucleus periventricularis Nucleus arcuatus
20.38 : 1.99 (5) 30.04 - 3.91 (5) 36.54 ± 3.08 (4)
Pars posterior Nucleus posterior
25.34 r__ 2.13 (4)
Area lateralis Nucleus premammillaris dorsalis Nucleus premammillaris ventralis Nucleus arcuatus
16.73 ± 12.49 ± 32.80 -. 32.48 --
0.61 (4) 1.48 (4) 2.68 (4) 3.73 (4)
brain was immediately removed and placed on ice. The tissue block which contained hypothalamus was isolated and frozen in liquid nitrogen. A series of frontal sections of the tissue block was made at 55 #m or 85 # m thickness in a cryostat (--15 °C). The sections were freeze-dried overnight at - - 3 0 °C and 10 3 mm Hg and stored in evacuated tubes at - - 2 0 °C until use. The individual hypothalamic nuclei were carefully dissected freehand under a stereomicroscope not to include brain vessels. Each sample was weighed by an electronic microbalance (Type 4152, Sartorius Co.) with a digital voltmeter (Type EO-12, Eto Co.), and was transferred into a mictrotube and crushed with a stainless steel needle. D O P A decarboxylase was assayed by a modification of the method of Giacobini and Nor~ 7. Five #1 of cold buffer substrate was added to the dry sample in a pointed microtube (final concentrations; 1.5 m M DL-[1-14C]DOPA, 69.4 m M phosphate buffer, pH 7.4, 0.042 m M pyridoxal-5-phosphate, 4.2 m M L-cysteine chloride, 0.17 m M BSA). The incubation tube was connected by rubber tubing to another microtube containing 20 #1 of 1 M hyamine solution in methanol. The two tubes connected to each other were incubated for 15 min at 38 °C. The reaction was stopped by injecting 16 #1 of 2.5 M H2SO4 into the reaction mixture through the rubber tubing. The tubes were replaced in the water bath for 30 min at 38 °C for the complete diffusion of CO2. The tube containing hyamine was separated from the connection and transferred into a counting vial containing 10 ml of toluene scintillator. After shaking the
412
....~ -2
!J_ .....
MT
LD pE L"O
0 0
a~
"
2
3 Fig. 1. Distribution of D-DC in the rat hypothalarnic nuclei. Drawings of frontal sections through the hypothalamus (1-3). 1, pars anterior: 2, pars medialis: 3, pars posterior. Abbreviations: A, nucleus anterior; AR, nucleus arcuatus; DM. nucleus dorsomedialis; F, fornix; L, area lateralis; MT. tractus mammillothalamicus; OT, tractus opticus; P. nucleus posterior; PA, nucleus paraventricularis; PD, nucleus premammillaris dorsalis; PE, nucleus periventricularis: PV, nucleus premam-
millaris ventralis; RC. area retrochiasmaticus; VM. nucleus ventromedialis. Concentrations of D-DC: solid area, high: at least 20% above the average of the hypothalarnic nuclei; crosshatched area, relatively high: within +20% of the average; hatched area, relatively low: within--20 % of the average; stippled area. low: at least 20% below the average. counting vial for 30 min, the radioactivity was measured in a Packard Tricarb model 3320 liquid scintillation spectrometer, The counting efficiency was 75 %. The results are summarized in Table I and Fig. l. The highest D - D C activities were found in the nucleus paraventricularis and area retrochiasmaticus. Relatively high activities were found in the arcuate, ventromedial and periventricular nuclei, and nucleus premammillaris ventralis. The lateral hypothalamic area and the nucleus premammiltaris dorsalis had low D - D C activities. Within the hypothalamic nuclei, the highest activity of D - D C was about 3-fold greater than the lowest activity. Kuntzman et al. i2 reported that D - D C activity was evenly distributed throughout the cat hypothalamus. In their study, D - D C activity was determined in the several parts of hypothalamus such as anterior, posterior and middle thirds, a n d ventral and dorsal halves, which might include several hypothalamic nuclei and their surrounding tissues. In the individual hypothalamic nuclei of rat, however, D - D C activities were found to be distributed widely and unevenly as shown in the present study. The gross regional distribution of D - D C activities in mammalian brain has been
413 indicated to correlate with those of monoamine levels by several authors t,14,16. Recently, Palkovits et al. 17 and Saavedra et al. 2° reported the distribution of N E, DA and of 5-HT in the discrete hypothalamic nuclei. Comparing our data with their results, the nucleus paraventricularis and area retrochiasmaticus, which showed high D-DC activities in our study, contained high levels of NE and DA, while the lateral hypothalamic area and nucleus premammillaris dorsalis, which showed low activities, contained low levels of NE and DA. On the contrary, nucleus paraventricularis and area retrochiasmaticus had low contents of 5-HT, whereas the lateral hypothalamic area and nucleus premammillaris dorsalis had high contents of 5-HT. It is of interest that, in the individual hypothalamic nuclei, the distribution of D-DC activities correlates directly with those of NE and DA, and yet correlates inversely with that of 5-HT. These relationships between the distribution of D-DC and those of monoamine levels suggest that the levels of D-DC obtained in the present study mainly reflect the activities of D-DC in the neuronal elements. Furthermore, these findings bring the possibility that D-DC may be different from 5-HTP-DC. It is generally supported that A-DC is involved in the decarboxylation steps both in DA neurons and in 5-HT neurons as a single enzyme1,5, 9,16. However, in the recent study in which the differences in assay characteristics, distribution and the effect of 6-hydroxydopamine, of D-DC and 5-HTP-DC were examined, Sims et al. ~1,'-2 suggested that the two different proteins catalyzed the D-DC and 5-HTP-DC reactions in brain. Furthermore, Korf et al. 11 also reported the existence of specific 5-HTP-DC enzyme in 5-HT containing nerve endings through the study of the decarboxylation of exogenous L-5-HTP after destruction of the rat cerebral raphe system. Our present data may also support the suggestion that D-DC is different from 5-HTP-DC. It is necessary, therefore, to examine the distribution of 5-HTP-DC activities in hypothalamic nuclei. This work was supported by a grant from the Science and Technology Agency of Japan, and by a grant from the Ministry of Pubic Welfare of Japan.
1 AURES, D., HA,KANSON, R., AND CLARK, W. G., Histidine decarboxylase and dopa decarboxylase. In A. LAJTHA(Ed.), Control Mechanisms in the Nervous System, Handbook o f Neurochemistry, Vol. 4, Plenum Press, New York, 1970, pp. 165-196. 2 BERTLER,A., FALCK,B., OWMAN,CH., AND ROSENGREN,E., The localization of monoaminergic blood-brain barrier mechanisms, Pharmacol. Rev., 18 (1966) 369-385. 3 BOGDANSKI,D. F., WEISSBACH,H., ANDUDENFRIEND,S., The distribution of serotonin, 5-hydroxytryptophan-decarboxylase, and monoamine oxidase in brain, J. Neurochem., 1 (1957) 272-278. 4 BROCH,JR., O. J., AND FONNUM,F., The regional and subcellular distribution of catechoI-Omethyltransferase in rat brain, J. Neurochem., 19 (1972) 2049 2055. 5 CHRISTENSON,J. G., DIARMAN,W., ANDUDENFRIEND,S., On the identity of DOPA decarboxylase and 5-hydroxytryptophan decarboxylase, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 343-347. 6 DE RoPP, R. S., AND FURST, A., Effect of analogs of phenylalanine and tryptophan on kinetics of DOPA decarboxylase in rat brain, Brain Research, 2 (1966) 323 332. 7 G1ACOBIN[,E., AND NORI~, B., Dopa-decarboxylase in autonomic and sensory ganglia of the cat, Acta physiol, stand., 82 (1971) 209-217.
414 8 HIRANO,M., UCHIMURA,H., ANDSAITO, M., Regional distribution ofmonoamine oxidase activity for 5-hydroxytryptamine and tyramine in hypothalamus of the rat, Brain Research, 93 (1975) 558-565. 9 H6KFELT, T., FUXE, K., AND GOLDSTEIN, M., Immunohistochemical localization of aromatic L-amino acid decarboxylase (DOPA-decarboxylase) in central dopamine and 5-hydroxytryptamine nerve celt bodies of the rat, Brain Research, 53 (1973) 175-180. 10 HOLTZ, P., AND WEST•RMANN, E., Uber die Dopadecarboxylase und Histidindecarboxylase des Nervengewebes, Nauyn-Schmiedeberg's Arch. exp. Path. Pharmak., 227 (1956) 538-546. 11 KORF, J., VENEMA,K., AND POSTEMA,F., Decarboxylation of exogenous L-5-hydroxytryptophan after destruction of the cerebral raphe system, J. Neurochem., 23 (1974) 249-252. 12 KUNTZMAN,R., SHORE, P. A., BOGDANSKI,D., AND BRODIE,B. B., Microanalytical procedures for fluorometric assay of brain DOPA-5HTP decarboxylase, norepinephrine and serotonin, and a detailed mapping of decarboxylase activity in brain, J. Neurochem., 6 (1961) 226-232. 13 LANGELIER, P., PARENT, A., AND POIRIER, L. J., Decarboxytase activity of the brain capillary walls and parenchyma in the rat, cat and monkey, Brain Research, 45 (1972) 622-629. 14 LLOYD, K. G., AND HORNYKIEWICZ,O., Occurrence and distribution of aromatic L-amino acid (L-DOPA) decarboxylase in the human brain, J. Neurochem., t9 (1972) 1549-1559. 15 MCGEER, P. L., BAGCHI, S. P., AND MCGEER, E. G., Subcellular localization of tyrosine hydroxylase in beef caudate nucleus, Life Sei., 4 (1965) 1859-1867. 16 MCGEER, P. L., AND McGEER, E. G., Neurotransmitter synthetic enzymes. In G. A. KERKUTAND .l.W. PHILLIS(Eds.), Progress in Neurobiology, VoL 2, Pergamon Press, Oxford, 1973, pp. 69-117. 17 PALKOVITS,M., BROWNSTEIN,M., SAAVEDRA,J. M., AND AXELROD,J., Norepinephrine and dopamine content of hypothalamic nuclei of the rat, Brain Research, 77 (1974) 134-149. 18 ROBERGE, A. G., AND POIRIER, L. J., Effect of chronically administered L-DOPA on Dopa/ 5HTP decarboxylase and tyrosine and tryptophan hydroxylases in cat brain, J. Neural Transmission, 34 (1973) 171-185. 19 RODRIGUEZ DE LORES ARNAIZ, G., AND DEROaERTIS, E., 5-Hydroxytryptophan decarboxylase activity in nerve endings of the rat brain, J. Neurochem., 11 (1964) 213-219. 20 SAAVEDRA,J. M., PALKOVITS,M., BROWNSTEIN,M. J., AND AXELROD,J., Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region, Brain Research, 77 (1974) 157-165. 21 SIMS, K. L., AND BLOOM, F. E., Rat brain L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan decarboxylase activities: differential effect of 6-hydroxydopamine, Brain Research, 49 (1973) 165-175. 22 SIMS, K. L., DAWS, G. A , AND BLOOM, F. E., Activities of 3,4-dihydroxy-L-phenylalanine and 5hydroxy-L-tryptophan decarboxylases in rat brain: assay characteristics and distribution, J. Neurochem., 20 (1973) 449~164. 23 UCmMURA, H., SAXTO,M., AND HIRANO, M., Regional distribution of choline acetyltransferase in hypothalamus of the rat, Brain Research, 91 (1975) 161-164.
