558
Brain Research, 93 (1975) 558-563 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Regional distribution of monoamine oxidase activity for 5-hydroxytryptamine and tyramine in hypothalamus of the rat
MAKOTO HIRANO, HIDEYUK1 UCHIMURA AND MASASHI SAITO Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga 842-01 (Japan)
(Accepted May 7th, 1975)
Monoamine oxidase (MAO) (EC 1.4.3.4) catalyzes the oxidative deamination of the putative neurotransmitter substances norepinephrine, dopamine and 5-hydroxytryptamine (5-HT). MAO in the brain, particularly in the hypothalamus, has been reported to play some important roles in the regulation of emotional behavior 6,17 and neuroendocrine mechanisms11,16. It is important to estimate MAO levels in discrete hypothalamic nuclei since the hypothalamus consists of heterogeneous structures with different functions. The presence of a high content of MAO in the hypothalamus has been shown by histochemical a°,13, biochemical z, spectrofluorimetric 9 and manometric 23 techniques. The histochemical staining methods, however, were not quantitative. Other previous techniques also were not sufficiently sensitive for the estimation of MAO in individual hypothalamic nuclei because a large amount of tissue was required. In recent years, the multiplicity of MAO has been shown in various species and organs 19,26. Johnston 12 reported that two forms of MAO could be distinguished in rat brain and designated the enzymes type A and B with the MAO inhibitor clorgyline. Norepinephrine and 5-HT are specific substrates for type A MAO, while benzylamine and fl-phenylethylamine25 are specific substrates for type B MAO. Tyramine is metabolized by both forms of the enzymes. In the present paper, the regional distribution of MAO in the hypothalamus, using 5-HT and tyramine as substrates, was studied according to quantitative micromethods14,20,21. Seventeen-week-old, male Wistar-King rats, housed in groups of 5 per cage, were used. The animals were killed at 4 p.m. by decapitation and the brains were immediately removed and placed on ice. The parts of the hypothalamus were isolated and frozen in liquid nitrogen. Frontal sections of 55/~m or 85 /zm thickness were made in a cryostat at --15 °C. The sections were freeze-dried overnight at --30 °C and 10-3 mm Hg and stored in evacuated tubes at --20 °C until use. The individual hypothalamic nuclei were dissected freehand under a stereomicroscope. Each sample was weighed using an electronic microbalance (Type 4125, Sartorius Co.) with a digital voltmeter (Type EO-12, Eto Co.). The sensitivity of this balance is 0.1 #g. The weight of each sample was 2-6 #g.
559 TABLE I MONOAMINE OXIDASE LEVELS AND THE RATIOS BETWEEN
5-HT-MAO
AND T Y R A M I N E - - M A O IN t t Y P O -
THALAMIC NUCLEI
The results are expressed as mean values 4- S.E.M. (number of animals). 5-HT-MAO, /~moles 5-HT oxidized/g dry wt./h; tyramine-MAO, #moles tyramine oxidized/g dry wt./h. Using same rats, both 5-HT-MAO and tyramine-MAO were determined. Nucleus ofhypothalamus
5-HT-MA 0
Pars anterior Nucleus anterior Arealateralis Nucleus paraventricularis Arearetrochiasmaticus
69.32 ± 4.71 41.11 4- 3.75 85.07 4- 6.75 66.59 ± 5.55
Pars medialis Nucleus ventromedialis Nucleus dorsomedialis Arealateralis Nucleus periventricularis Nucleus arcuatus Pars posterior Nucleus posterior Arealateralis Nucleuspremammillaris dorsalis Nucleus premammillarisventralis Nucleus arcuatus
Tyramine-MA 0
(5) (5) (5) (4)
54.07 ± 4.65 41.13 ± 4.07 76.47 4- 8.31 70.00 4- 4.95
Ratio *
(5) (5) (4) (4)
1.28 1.00 1.11 0.95
91.90 ± 10.67 (4) 79.75 ± 9.57 (4) 54.96 4- 3.32 (4) 72.76 4- 9.28 (4) 53.43 4- 6.58 (5)
78.53 4- 5.89 (4) 65.60 4- 10.39 (4) 45.62 i 2.78 (4) 201.35 4- 47.49 (4) 158.65 4- 18.14 (4)
1.17 1.22 1.20 0.36 0.34
68.20 4- 5.94 (4) 39.48 -- 3.63 (5) 47.16 4- 4.50 (4) 88.08 4- 12.05 (4) 74.12 4- 6.22 (5)
46.72 ± 5.20 (5) 37.25 4- 2.06 (5) 79.58 ± 6.22 (5) 82.10 4- 1.93 (4) 221.51 4- 24.79 (5)
1.46 1.06 0.59 1.07 0.33
* 5-HT-MAO/tyramine-MAO. M A O was determined by a modification o f the radiochemical assay o f McC a m a n et al. 14. Two microliters o f 0.05 ~ Triton X-100 containing 0.05 ~ BSA were added to the sample in a pointed microtube. After preincubation for 15 min at 0 °C, 2/zl ice-cold buffer substrate were added (final concentrations: 0.1 M phosphate buffer, p H 7.2; 1.0 m M [2-14C]5-hydroxytryptamine binoxalate, 27.5 mCi/mmole, New England Nuclear Co.). All tubes were mixed without warming and incubated at 38 °C for 15 min. The reaction was stopped by the addition o f 1/A o f 3 N H C 1 and 50 #1 o f ethyl acetate were added. The preparation was thoroughly mixed and centrifuged to separate the phases. Thirty-five microliters o f the ethyl acetate layer was removed and transferred to a clean tube containing 30 #1 o f 0.3 N HC1. After thorough mixing and centrifugation, 25/~1 o f the ethyl acetate layer were transferred to a counting vial and mixed with 1 ml o f absolute methanol followed by 15 ml o f scintillator toluene solution. Radioactivity was determined in a Packard Tricarb model 3320 liquid scintillation spectrometer. The counting efficiency was 78 ~ . The same procedure was used with tyramine as the substrate (final concentration: 1.5 m M [14C]tyramine hydrochloride, 10 mCi/mmole, New England Nuclear Co., in 0.1 M phosphate buffer, p H 7.95; incubation time, 30 min at 38 °C). Our results are summarized in Table I and Fig. 1. M A O activities towards 5-HT ( 5 - H T - M A O ) and towards tyramine ( t y r a m i n e - M A O ) were detected in all
560
MT
AR
2
3 Fig. 1. Distribution of 5-HT-MAO in the rat hypothalamic 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; PE, nucleus periventricularis; PD, nucleus premammillaris dorsalis; PV, nucleus premammillaris ventralis; RC, area retrochiasmaticus; VM, nucleus ventromedialis. Concentrations of 5-HT-MAO: solid area, high, at least 20 ~ above the average of the hypothalamic nuclei; hatched area, medium, ± 2 0 ~ of the average; stippled area, low, at least 2 0 ~ below the average.
561 nuclei and found to be unevenly distributed in the hypothalamus; tyramine-MAO and 5-HT-MAO had a 6-fold and 2.5-fold difference, respectively, between the nuclei with the lowest and highest MAO level. The highest 5 - H T - M A O activities were found in the nuclei of ventromedialis, premammillaris ventralis, paraventricularis and dorsomedialis. The nuclei of the medial part of arcuatus, periventricularis, anterior and posterior, had relatively high activities, while the lateral hypothalamic area and the nucleus premammillaris dorsalis had low activities of 5-HT-MAO. On the other hand, extremely high concentrations of tyramine-MAO were found in the nucleus periventricularis, and in the nuclei of medial and posterior parts of arcuatus. The nuclei of ventromedialis, paraventricularis and premammillaris had relatively high levels, while the lateral hypothalamic area and the nucleus posterior had low levels of tyramine-MAO. In the several hypothalamic nuclei, the distribution of 5 - H T - M A O is strikingly different from that of 5-HT levels 12. High 5-HT contents are found in the nucleus premammillaris dorsalis and the area lateralis, while these nuclei have low 5-HTMAO activities. In contrast, low 5-HT contents are found in the nuclei of ventromedialis, paraventricularis and dorsomedialis where high 5-HT-MAO activities exist as described above. This inverse correlation between 5-HT-MAO levels and 5-HT contents in the hypothalamic nuclei may reflect the functional activity of each nucleus. Compared with the regional distribution of norepinephrine and dopamine in the hypothalamus la, 5-HT-MAO levels did not correlate with those biogenic monoamine levels in various hypothalamic nuclei. It seems to be difficult to interprete this point. Further examination of MAO distribution using norepinephrine and dopamine as substrates will be needed. The ratios of 5 H T - M A O to tyramine-MAO varied from 0.33 to 1.46 (Table I). Concerning the ratios of 5-HT-MAO to tyramine-MAO, Harada e t al 9 reported that no difference in substrate specificity had been observed in several regions of rat brain. Furthermore, McCaman et a114 found that in various layers and areas of cortex, cerebellum and spinal cord, there were similar distribution patterns of MAO activity with either 5-HT or tyramine as substrates. By contrast, Goridis and coworkers7, z4 reported that the ratios of 5-HT to tyramine metabolized by various tissues were different from each other in superior cervical ganglion, pineal gland and cerebral hemispheres. They suggested the existence of several MAO forms in these tissues. In the present study, marked differences of the ratios between 5-HT-MAO and tyramine-MAO were found in hypothalamic nuclei. This result suggests that multiple forms of MAO exist in individual hypothalarnic nuclei and also that the relative distribution of 5-HT MAO and tyramine-MAO is different in the hypothalamus. The lowest values of the ratio (0.33 - 0.36) were strictly found in the nucleus periventricularis and in the nuclei of the medial and posterior parts of the arcuatus. This finding indicates that these nuclei contain mainly type B MAO since 5-HT is metabolized by type A MAO, and tyramine is a substrate for both type A and type B MAO. It is of interest that these nuclei contain dopaminergic cell bodies 1,5,2z. The low levels of the ratio in these nuclei, therefore, would be in part due to a high content
562 of dopaminergic n e u r o n s , since d o p a m i n e as well as t y r a m i n e is metabolized by both type A a n d type B M A O s. Moreover, all these nuclei are k n o w n to be located close to the third ventricle. Several workers have suggested that the ependymal cells of the third ventricle have characteristic structures such as 'bleb-like surface p r o t r u s i o n s ' a n d ' d o m e - s h a p e d structure' which were tentatively identified as small neurons3, 4. O n the other h a n d , Goridis et al v d e m o n s t r a t e d that type B M A O in the pineal gland was associated with the pineal cells whereas type A M A O was f o u n d within sympathetic nerve endings. It is possible to assume, therefore, that some n e u r o n a l or n o n - n e u r o n a l structures which have a high c o n t e n t of type B M A O might be involved in those nuclei neighboring with the third ventricle. I n conclusion, M A O activities in discrete h y p o t h a l a m i c nuclei of the rat were estimated quantitatively, a n d the different ratios of 5 - H T - M A O to t y r a m i n e - M A O were found. The findings indicate that the relative levels of type A a n d type B M A O are different from one a n o t h e r in h y p o t h a l a m i c nuclei. This work was supported by a g r a n t from the Science a n d Technology Agency of Japan.
1 BJORKLUND, A., AND NomY, A., Fluorescence histochemical and microspectrofluorometric mapping of dopamine and noradrenaline cell groups in the rat diencephalon, Brain Research, 5 l (1973) 193-205. 2 BOGDANSKI,D. F., WEISSBACH,H., AND UDENFRIEND,S., The distribution of serotonin, 5-hydroxytryptophan decarboxylase, and monoamine oxidase in brain, J. Neurochem., 1 (1957) 272-278. 3 BRUNI, J. E., MONTEMURRO,n . G., CLATTENBURG,R. E., AND SINGH,R. P., A scanning electron microscopic study of the ependymal surface of the third ventricle of the rabbit, rat, mouse and human brain, Anat. Rec., 174 (1972) 407-420. 4 CLEMENTI,F., AND MARINI, O., The surface fine structure of the walls of cerebral ventricles and choroid plexus in cat, Z. Zellforsch., 123 (1972) 82-95. 5 CUELLO,A. C., WEINER,R. I., AND GANONG,W. F., Effect of lateral deafferentation on the morphology and catecholamine content of the mediobasal hypothalamus, Brain Research, 59 (1973) 191-200. 6 ELEFTHERIOU,B. E., AND BOEHLKE, K. W., Brain monoamine oxidase in mice after exposure to aggression and defeat, Science, 55 (1967) 1693-1694. 7 GORIDIS,C., AND NEFF, N. H., Evidence for a specific monoamine oxidase associated with sympathetic nerves, Neuropharmacology, 10 (1971) 557-564. 8 HALL,D. W. R., LOGAN,B. W., ANDPARSONS,G. H., Further studies on the inhibition of monoamine oxidase by M + B 9302 (clorgyline). I. Substrate specificity in various mammalian species, Biochem. Pharmacol., 18 (1969) 1447-1454. 9 HARADA,M., KONDO,Y., KUZUYA,H., ANDNAGATSU,T., Monoamine oxidase activities towards biogenic monoamines in several regions of rat brain, J. Neurochem., 24 (1975) 193-195. l0 HASHIMOTO,P. H., MAEDA,T., TORn, K., AND SHIMIZU, N., Histochemical demonstration of autonomic regions in the central nervous system of the rabbit by means of a monoamine oxidase staining, Med. J. Osaka Univ., 12 (1962) 425-439. 11 HOLZBAUER,M., AND YOUDIM, M. B. H., The oestrous cycle and monoamine oxidase activity, Brit. J. Pharmacol., 20 (1973) 600-608. 12 JOHNSTON,J. P., Some observations upon a new inhibitor of monoamine oxidase in brain tissues, Biochem. Pharmacol., 17 (1968) 1285-1297. 13 MANOCHA,S. L., SHANYA,T. R., ANDBOURNE,G. H., Histochemical mapping of the distribution of monoamine oxidase in the diencephalon and basal telencephalic centers of the brain of squirrel monkey (Saimiri sciureus), Brain Research, 6 (1967) 570-586.
563 ~4 McCAMAN, R. E., MCCAMAN, M. W., HUNT, J. M., AND SMITH, M. S., Microdetermination of monoamine oxidase and 5-hydroxytryptophan decarboxylase activities in nervous tissues, J. Neurochem., 12 (1965) 15-23. 15 PALKOVITS,M., BROWNSTEIN,M., SAAVEDRA,J. M., AND AXELROD,J., Norepinephrine and dopamine content of hypothalamic nuclei of the rat, Brain Research, 77 (1974) 137-149. 16 PARVEZ, H., AND PARVEZ, S., The effects of metopirone and adrenalectomy on the regulation of the enzymes monoamine oxidase and catechol-O-methyltransferase in different brain regions, J. Neurochern., 20 (1973) 1011-1020. 17 PRYOR, G. T., SCOTT, M. K., AND PEACHE, S., Increase monoamine oxidase activity following repeated electroshock seizures, J. Neurochern., 19 (1972) 891-893. 18 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. 19 SQUIRES,R. F., Multiple forms of monoamine oxidase in intact mitochondria as characterized by selective inhibitors and thermal stability: a comparison of eight mammalian species. In E. COSTA AND M. SANDLER (Eds.), Advances in Biochemical Psychopharrnacology, Vol. 5, Raven Press, New York, 1972, pp. 355-370. 20 UCHIMURA,H., HIRANO, M., SAITO, M., MUKAI, A., AND HAZAMA,H., The quantitative histochemistry of monoamine oxidase in the central nervous tissues, Brain and Nerve (Tokyo), 26 (1974) 341-345. 21 UCHIMURA,H., SAITO, M., AND HmANO, M., Regional distribution of choline acetyltransferase in hypothalamus of the rat, Brain Research, 91 (1975) 161-164. 22 UNGERSTEDT,U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., 82, Suppl. 367 (1971) 1-48. 23 WEINER,N., The distribution of monoamine oxidase and succinic oxidase in brain, J. Neurochern., 6 (1960) 79-86. 24 YANG, H. Y. T., GORIDIS,C., AND NEFF, N. H., Properties of monoamine oxidase in sympathetic nerve and pineal gland, J. Neurochern., 19 (1972) 1241-1250. 25 YANG, H. Y. T., ANDNEFF, N. H., fl-Phenylethylamine : a specific substance for type B monoamine oxidase of brain, J. Pharrnacol. exp. Ther., 187 (1973) 365-371. 26 YOUDIM, M. B. H., Multiple forms of monoamine oxidase and their properties. In E. COSTAAND M. SANDLER (Eds.), Advances in Biochemical Psychopharrnacology, Vol. 5, Raven Press, New York, 1972, pp. 67-77.
Brain Research, 93 (1975) 558-563 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Regional distribution of monoamine oxidase activity for 5-hydroxytryptamine and tyramine in hypothalamus of the rat
MAKOTO HIRANO, HIDEYUK1 UCHIMURA AND MASASHI SAITO Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga 842-01 (Japan)
(Accepted May 7th, 1975)
Monoamine oxidase (MAO) (EC 1.4.3.4) catalyzes the oxidative deamination of the putative neurotransmitter substances norepinephrine, dopamine and 5-hydroxytryptamine (5-HT). MAO in the brain, particularly in the hypothalamus, has been reported to play some important roles in the regulation of emotional behavior 6,17 and neuroendocrine mechanisms11,16. It is important to estimate MAO levels in discrete hypothalamic nuclei since the hypothalamus consists of heterogeneous structures with different functions. The presence of a high content of MAO in the hypothalamus has been shown by histochemical a°,13, biochemical z, spectrofluorimetric 9 and manometric 23 techniques. The histochemical staining methods, however, were not quantitative. Other previous techniques also were not sufficiently sensitive for the estimation of MAO in individual hypothalamic nuclei because a large amount of tissue was required. In recent years, the multiplicity of MAO has been shown in various species and organs 19,26. Johnston 12 reported that two forms of MAO could be distinguished in rat brain and designated the enzymes type A and B with the MAO inhibitor clorgyline. Norepinephrine and 5-HT are specific substrates for type A MAO, while benzylamine and fl-phenylethylamine25 are specific substrates for type B MAO. Tyramine is metabolized by both forms of the enzymes. In the present paper, the regional distribution of MAO in the hypothalamus, using 5-HT and tyramine as substrates, was studied according to quantitative micromethods14,20,21. Seventeen-week-old, male Wistar-King rats, housed in groups of 5 per cage, were used. The animals were killed at 4 p.m. by decapitation and the brains were immediately removed and placed on ice. The parts of the hypothalamus were isolated and frozen in liquid nitrogen. Frontal sections of 55/~m or 85 /zm thickness were made in a cryostat at --15 °C. The sections were freeze-dried overnight at --30 °C and 10-3 mm Hg and stored in evacuated tubes at --20 °C until use. The individual hypothalamic nuclei were dissected freehand under a stereomicroscope. Each sample was weighed using an electronic microbalance (Type 4125, Sartorius Co.) with a digital voltmeter (Type EO-12, Eto Co.). The sensitivity of this balance is 0.1 #g. The weight of each sample was 2-6 #g.
559 TABLE I MONOAMINE OXIDASE LEVELS AND THE RATIOS BETWEEN
5-HT-MAO
AND T Y R A M I N E - - M A O IN t t Y P O -
THALAMIC NUCLEI
The results are expressed as mean values 4- S.E.M. (number of animals). 5-HT-MAO, /~moles 5-HT oxidized/g dry wt./h; tyramine-MAO, #moles tyramine oxidized/g dry wt./h. Using same rats, both 5-HT-MAO and tyramine-MAO were determined. Nucleus ofhypothalamus
5-HT-MA 0
Pars anterior Nucleus anterior Arealateralis Nucleus paraventricularis Arearetrochiasmaticus
69.32 ± 4.71 41.11 4- 3.75 85.07 4- 6.75 66.59 ± 5.55
Pars medialis Nucleus ventromedialis Nucleus dorsomedialis Arealateralis Nucleus periventricularis Nucleus arcuatus Pars posterior Nucleus posterior Arealateralis Nucleuspremammillaris dorsalis Nucleus premammillarisventralis Nucleus arcuatus
Tyramine-MA 0
(5) (5) (5) (4)
54.07 ± 4.65 41.13 ± 4.07 76.47 4- 8.31 70.00 4- 4.95
Ratio *
(5) (5) (4) (4)
1.28 1.00 1.11 0.95
91.90 ± 10.67 (4) 79.75 ± 9.57 (4) 54.96 4- 3.32 (4) 72.76 4- 9.28 (4) 53.43 4- 6.58 (5)
78.53 4- 5.89 (4) 65.60 4- 10.39 (4) 45.62 i 2.78 (4) 201.35 4- 47.49 (4) 158.65 4- 18.14 (4)
1.17 1.22 1.20 0.36 0.34
68.20 4- 5.94 (4) 39.48 -- 3.63 (5) 47.16 4- 4.50 (4) 88.08 4- 12.05 (4) 74.12 4- 6.22 (5)
46.72 ± 5.20 (5) 37.25 4- 2.06 (5) 79.58 ± 6.22 (5) 82.10 4- 1.93 (4) 221.51 4- 24.79 (5)
1.46 1.06 0.59 1.07 0.33
* 5-HT-MAO/tyramine-MAO. M A O was determined by a modification o f the radiochemical assay o f McC a m a n et al. 14. Two microliters o f 0.05 ~ Triton X-100 containing 0.05 ~ BSA were added to the sample in a pointed microtube. After preincubation for 15 min at 0 °C, 2/zl ice-cold buffer substrate were added (final concentrations: 0.1 M phosphate buffer, p H 7.2; 1.0 m M [2-14C]5-hydroxytryptamine binoxalate, 27.5 mCi/mmole, New England Nuclear Co.). All tubes were mixed without warming and incubated at 38 °C for 15 min. The reaction was stopped by the addition o f 1/A o f 3 N H C 1 and 50 #1 o f ethyl acetate were added. The preparation was thoroughly mixed and centrifuged to separate the phases. Thirty-five microliters o f the ethyl acetate layer was removed and transferred to a clean tube containing 30 #1 o f 0.3 N HC1. After thorough mixing and centrifugation, 25/~1 o f the ethyl acetate layer were transferred to a counting vial and mixed with 1 ml o f absolute methanol followed by 15 ml o f scintillator toluene solution. Radioactivity was determined in a Packard Tricarb model 3320 liquid scintillation spectrometer. The counting efficiency was 78 ~ . The same procedure was used with tyramine as the substrate (final concentration: 1.5 m M [14C]tyramine hydrochloride, 10 mCi/mmole, New England Nuclear Co., in 0.1 M phosphate buffer, p H 7.95; incubation time, 30 min at 38 °C). Our results are summarized in Table I and Fig. 1. M A O activities towards 5-HT ( 5 - H T - M A O ) and towards tyramine ( t y r a m i n e - M A O ) were detected in all
560
MT
AR
2
3 Fig. 1. Distribution of 5-HT-MAO in the rat hypothalamic 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; PE, nucleus periventricularis; PD, nucleus premammillaris dorsalis; PV, nucleus premammillaris ventralis; RC, area retrochiasmaticus; VM, nucleus ventromedialis. Concentrations of 5-HT-MAO: solid area, high, at least 20 ~ above the average of the hypothalamic nuclei; hatched area, medium, ± 2 0 ~ of the average; stippled area, low, at least 2 0 ~ below the average.
561 nuclei and found to be unevenly distributed in the hypothalamus; tyramine-MAO and 5-HT-MAO had a 6-fold and 2.5-fold difference, respectively, between the nuclei with the lowest and highest MAO level. The highest 5 - H T - M A O activities were found in the nuclei of ventromedialis, premammillaris ventralis, paraventricularis and dorsomedialis. The nuclei of the medial part of arcuatus, periventricularis, anterior and posterior, had relatively high activities, while the lateral hypothalamic area and the nucleus premammillaris dorsalis had low activities of 5-HT-MAO. On the other hand, extremely high concentrations of tyramine-MAO were found in the nucleus periventricularis, and in the nuclei of medial and posterior parts of arcuatus. The nuclei of ventromedialis, paraventricularis and premammillaris had relatively high levels, while the lateral hypothalamic area and the nucleus posterior had low levels of tyramine-MAO. In the several hypothalamic nuclei, the distribution of 5 - H T - M A O is strikingly different from that of 5-HT levels 12. High 5-HT contents are found in the nucleus premammillaris dorsalis and the area lateralis, while these nuclei have low 5-HTMAO activities. In contrast, low 5-HT contents are found in the nuclei of ventromedialis, paraventricularis and dorsomedialis where high 5-HT-MAO activities exist as described above. This inverse correlation between 5-HT-MAO levels and 5-HT contents in the hypothalamic nuclei may reflect the functional activity of each nucleus. Compared with the regional distribution of norepinephrine and dopamine in the hypothalamus la, 5-HT-MAO levels did not correlate with those biogenic monoamine levels in various hypothalamic nuclei. It seems to be difficult to interprete this point. Further examination of MAO distribution using norepinephrine and dopamine as substrates will be needed. The ratios of 5 H T - M A O to tyramine-MAO varied from 0.33 to 1.46 (Table I). Concerning the ratios of 5-HT-MAO to tyramine-MAO, Harada e t al 9 reported that no difference in substrate specificity had been observed in several regions of rat brain. Furthermore, McCaman et a114 found that in various layers and areas of cortex, cerebellum and spinal cord, there were similar distribution patterns of MAO activity with either 5-HT or tyramine as substrates. By contrast, Goridis and coworkers7, z4 reported that the ratios of 5-HT to tyramine metabolized by various tissues were different from each other in superior cervical ganglion, pineal gland and cerebral hemispheres. They suggested the existence of several MAO forms in these tissues. In the present study, marked differences of the ratios between 5-HT-MAO and tyramine-MAO were found in hypothalamic nuclei. This result suggests that multiple forms of MAO exist in individual hypothalarnic nuclei and also that the relative distribution of 5-HT MAO and tyramine-MAO is different in the hypothalamus. The lowest values of the ratio (0.33 - 0.36) were strictly found in the nucleus periventricularis and in the nuclei of the medial and posterior parts of the arcuatus. This finding indicates that these nuclei contain mainly type B MAO since 5-HT is metabolized by type A MAO, and tyramine is a substrate for both type A and type B MAO. It is of interest that these nuclei contain dopaminergic cell bodies 1,5,2z. The low levels of the ratio in these nuclei, therefore, would be in part due to a high content
562 of dopaminergic n e u r o n s , since d o p a m i n e as well as t y r a m i n e is metabolized by both type A a n d type B M A O s. Moreover, all these nuclei are k n o w n to be located close to the third ventricle. Several workers have suggested that the ependymal cells of the third ventricle have characteristic structures such as 'bleb-like surface p r o t r u s i o n s ' a n d ' d o m e - s h a p e d structure' which were tentatively identified as small neurons3, 4. O n the other h a n d , Goridis et al v d e m o n s t r a t e d that type B M A O in the pineal gland was associated with the pineal cells whereas type A M A O was f o u n d within sympathetic nerve endings. It is possible to assume, therefore, that some n e u r o n a l or n o n - n e u r o n a l structures which have a high c o n t e n t of type B M A O might be involved in those nuclei neighboring with the third ventricle. I n conclusion, M A O activities in discrete h y p o t h a l a m i c nuclei of the rat were estimated quantitatively, a n d the different ratios of 5 - H T - M A O to t y r a m i n e - M A O were found. The findings indicate that the relative levels of type A a n d type B M A O are different from one a n o t h e r in h y p o t h a l a m i c nuclei. This work was supported by a g r a n t from the Science a n d Technology Agency of Japan.
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