Folia Psychiatrica et Neurologica Japonica, Vol. 33, No. 1, 1979
Distribution of Type A and Type B Monoamine Oxidase Activity in Hypothalamic Nuclei of the Rat Norifumi Kunimoto, M.D. and Hidebumi Hazama, M.D. Department of Neuro-psychiatry, Tottori University School of Medicine, Yonago
INTRODUCTION Type A and type B monoamine oxidase (MAO) [EC 1.4.3.4.1 have been differentiated by their substrate specificity and inhibitor sensitivity in homogenates of the rat brain.X Whereas the distribution of type A M A 0 was reported to be similar to that of type B M A 0 in the central nervous system of the rat,"' the relative levels of type A and type B M A 0 were suggested to differ in the hypothalamic nuclei." In the present paper, the regional distribution of type A, type A and B and type B M A 0 activity in individual nuclei of the rat hypothalamus was studied according to quantitative micromethods with clorgyline as a specific inhibitor of type A MAO. MATERIALS AND METHODS Male Wistar rats (200-260 g) kept in a group were used. The rats were killed by decapitation at 9 a.m. and the brains were immediately taken out and placed on ice. The blocks including the hypothalamus were isolated and frozen in liquid nitrogen. Frontal serial sections of 60 pm thickness were made in a cryostat at - 15°C and freeze-dried. The individual hypothalamic nuclei were dissected from the freeze-dried sections with a microknife under a stereoReceived for publication July 31, 1978.
microscope. After being weighed with an electronic microbalance (Type 4125, Sartorius Co.), each sample was placed into a microtube. The sensitivity of this balance was 0.1 pg. The weight of each sample was 1 .O to 7.0 pg. M A 0 activity was measured by the radiochemical assay method.4 Two pl of 0.1 M phosphate buffer, pH 7.2, containing BSA and Triton X-100in each final concentration 0.05% was added to the weighed sample in the microtube. After a 2 0 min preincubation at OOC, 2 pl of icecold substrate buffer solution was added. 5-Hydroxytryptamine (5-HT) and dopamine (DA) were used as a substrate for type A and type A and B MAO, respectively (the final concentration of the substrate: 1.1 mM [2-14C] 5-hydroxytryptamine binoxalate, 45 mCi/mM; 1.0 mM [ 12-I4C] dopamineethylamine, 60 mCi/mM, New England Nuclear Co.). When the assay of type B MAO, 1 pl of the buffer and 1 pl of M clorgyline solution were added into a microtube, and after a preincubation at 0°C for 20 min, 2 pI of ice-cold DA-buffer solution (the same solution that was used for the assay of type A and B MAO) was added. All of these procedures were made in an ice-cold bath. After an incubation at 38OC for 45 min, the reaction products were extracted in ethyl acetate and the radioactivity was measured with a liquid scintillation spectrometer. The inhibiting effect of the deamination of 5-HT and DA by clorgyline is shown in Fig. 1.
N. Kunimoto and H. Hazama
106
0
a I
Clorgyline -log final conc. M Fig. 1: Inhibiting effect of the deamination of 5-HT and DA by clorgyline. Assays were performed with homogenates of the freeze-dried sample of the rat.
The inhibition of DA deamination with clorgyline showed a plateau at the final concentration of clorgyline between 2 X lo-' M and 8 X 10-"M. In case of 5-HT, a small plateau was also recognized but the detail of the cause is unknown. When 1 pI of M clorgyline was used (the final concentration of clorgyline, 2.5 X lo-" M ) , the inhibition of 5-HT and DA deamination was found to be about 100% and 5376, respectively. Accordingly M A 0 activity for DA in this condition was considered to possibly mean type B M A 0 activity (denoted as DA MAO-type B) , even though the comparison with the activity shown with typical substrate for types such as P-phenylethylamine is necessary due to the indirect measurement of type B M A 0 in the present study. Moreover, it was ascertained in this experiment that the enzyme reaction towards
DA and 5-HT as the substrate proceeded linearly with or without the inhibitor with the use of the freeze-dried rat cerebral cortex as the enzyme sample at least that between 0.5 pg and 8 pg in weight. RESULTS
As shown in Table 1, type A, type A and B and type B M A 0 activities were distributed unevenly in each hypothalamic nucleus. Type A M A 0 activity was obviously higher in medial parts than in lateral parts of the hypothalamus. The highest activities of type A and B M A 0 for DA existed in the nuclei of suprachiasmaticus in the anterior part and arcuatus in the medial and posterior part of the hypothalamus. The activities of type B M A 0 were highest in the nucleus arcuatus of the
Type A and Type B MA0 in Rat Hypothalamus
Table 1 :
Distribution of M A 0 Activity in the Hypothalamic Nuclei of the Rat
~
~
para anterior. N; anterior A. lateralir N. paraventricularir N . aupraopticua N. ruprachianmatiaur
55.7 t 7.0 52.4 f 1.9 05.1 f 7.2 54.1 2 9.7 75.9 i 5.6
para medialia N . ventromrdialii N. dorromrdialii A. l a t e r a l i r N. periventricularii N. arcuatua
81.9 89.1 47.6 96.3 96.6
t 3.9 t 4.1
par. porterior N . poaterior A. l a t e r r l i a N . premmillrrir dorralir N . premunillarir ventrrlir N. arcuatua
14.6 54.3 99.5 72.3 79.1
f
I h e rerultr
clorgylina)
2.4 t 2,l t 9.7
f
3.8 t 1.6 210.7 t 6.1 tll.1
eo.1 ti6.i 44.5 f 5.0 64.2 f10.0 84.6 f 5.1 133.6 f12.7 70.5 87.3 65.4 86.0 124.0
t 8.8
fl2.Z k 8.2 t13.6 t 8.7
, /J
p
mOl.8
19.1 f 12.9 f 36.3 f 53.4 f 39 $ 6 f
1.5 0.9 6.6 7.3 3.9
0.32 0.41 1.29 1.71 0.42
20.0 f 21.9 f 17.0 f 37,O t
1.9 2.8 0.9 2.8
0.40
8 8 . 3 t16.2
88.8 t 8.8
6 3 , 3 t 6.9
72.0 213.5 94.0 t 9.3 181.4 f u . 3
are rxprrried am mew valuerfS,E.M.. q y p r A
wt./hr lype A and B N O , with
107
20.2 f 1.5 16.7 f 1'.9 29.3 f 2.3 57.6 f 8.2 106.5 214 - 2
0.33 0.35
0.76 2.52 0.29 0.35 0.69 1.58
1.42
N O l )I molar 5-HT oxidized/g dry
molra DA oxidized/g dry w t . / h r lype B MA0 (DA an nubatratel treated DA oxidiard/g dry w t . / h . Number of animal8 i a f i v e ,
*DA MO-type A wai calculated according t o deduction
of
DA MO-typ.
B from DA MA0 (typa A
and 6).
medial part and the posterior part of the hypothalamus, relatively high in the nuclei of paraventricularis, supraopticus, and premamillaris ventralis and low in the lateral hypothalamic area. The ratio of DA MAOtype B to DA MAO-type A ranged from 0.29 to 2.52. DISCUSSION
Recently it has been revealed that M A 0 activities are unevenly distributed in the hypothalamus as well as in the limbic and the lower brain stem n ~ c l e i . ~Whether -~ or not regional distribution of the two M A 0 types, A and B, in the brain is different from one another is not exactly clear. The
present study describes the relative proportions of the two types of enzyme within the hypothalamus, assessing M A 0 activity with the use of a common substrate (DA) for the two types and a specific inhibitor (clorgyline) against type A. The ratio of DA MAO-type B to DA MAO-type A ranged from 0.29 to 2.52 in the present study. This indicates that the distribution pattern of type B M A 0 differs from type A M A 0 within the rat hypothalamus. The highest values of the ratio were found in the circumventricular areas particularly in the nucleus arcuatus. This implies that remarkably high activities of type B M A 0 exist in these areas. In this context, type A M A 0 is absolutely domi-
N. Kunimoto and H. Hazama
108
nant within the rat hippocanipus.4 While the existence of clorgyline-insensitive 5-HT M A 0 was postulated in circumventicular structures by histochemical technique,I7 we could not detect clorgyline-insensitive M A 0 activity in the circumventricular areas when using lo-" M to M clorgyline and 5HT as a substrate by the method described in this report." The regional distribution of DA M A 0 activity in this study is not exactly the same as that of tyramine MAO,(' although tyramine is also considered as a substrate for both type A and type B. This difference might be explained by some regional difference in substrate specificity of individual nuclei. Harada et ~ 1 observed . ~ a difference in substrate specificity of M A 0 in the cerebellum whereas not so distinct in several roughly dissected regions of the rat brain. It is known that the nucleus of paraventricularis and supraopticus produces oxytocin and vasopressin, respectively, and the nucleus arcuatus contains dopaminergic neurons and regulates the secretion of hypophysial hormones. It is of interest that high activities of type B M A 0 exist in these nuclei. Among the biogenic amines in the central nervous system, specifically catalysed amines by type B M A 0 are less understood in their detailed distributions and physiological roles as compared with those by type A MAO. It might be tentatively assumed that not only DA but the biogenic amines which are substrates for type B M A 0 such as P-phenylethylamine are actively concerned in neurosecretion. Recently P-phenylethylamine has been shown to exert a direct, stimulating effect on postsynaptic DA receptors in the nigrostriatal pathway.1 SUMMARY The regional distribution of type A, type A and B and type B activity of monoamine oxidase (MAO) in individual nuclei of the rat hypothalamus was studied according to quantitative micromethods with clorgyline as
a specific inhibitor of type A MAO. As results, type A and type B M A 0 were distributed differently in the rat hypothalamus. It is suggested that type B M A 0 acts differently from type A M A 0 in regard to the function of the hypothalamus. REFERENCES 1 Antelman, S. M., Edwards, D. J. and Lin,
M.: Phenylethylamine: evidence for a direct, postsynaptic dopamine-receptor stimulating action, Brain Res, 127: 317322, 1977. 2 Harada, M., Kondo, Y., Kuzuya, H. and Nagatsu, T. : Monoamine oxidase activities towards biogenic monoamines in several regions of rat brain, J Neurochem, 24: 193-1 95, 1975. 3 Hazama, H. and Ito, M.: Effects of psy-
chotropic drugs on monoamine oxidase activity in individual nuclei of hypothalamus and amygdala in rat, Yonago Acta med, 20: 221-224, 1976. 4 Hazama, H. and Kunimoto, N.: Distrihution of monoamine oxidase in hippocampal region of the rat, Experientia, 34: 424425, 1978. 5 Hazama, H., Kunimoto, N. and Takeshita,
H.: Clorgyline inhibition of monoamine oxidase activities in circumventricular areas of rat hypothalamus, Yonago Acta med, 21: 140-144, 1978. 6 Hirano, M., Uchimura, H. and Saito, M.: Regional distribution of monoamine oxidase activity for 5-Hydroxytryptamine and tyramine in hypothalamus of the rat, Brain Res, 93: 555-563, 1975. 7 Hirano, M., Kim, J. S., Saito, M., Uchimura, H., Ito, M. and Nakahara, T.: Monoamine oxidase activities for serotonin and tyramine in individual limbic and lower brain stem nuclei of the rat, J Neurochem, 30: 263-267, 1978. 8 Johnston, J. P.: Some observation upon a new inhibitor of monoamine oxidase in brain tissues, Biochem Pharmacol, 17: 1285-1297, 1968. 9 McCaman, R.E., McCaman, M. W., Hunt, J. M. and Smith, M. S.: Microdetermination of monoamine oxidase and 5-hydroxytryptophan decarhoxylase activities in nervous tissues, J Neurochem, 12: 15-23, 1965. 10 Suzuki, 0. and Yagi, K.: Distribution of type A and B monoamine oxidase activities
Type A and Type B M A 0 in Rat Hypothalamus in the central nervous system of rat and chick, Experientia, 32: 13-14, 1976. I I Williams, D., Gascoigne, J. E. and Williams,
109
E. D.: A specific form of rat brain monoamine oxidase in circumventricular structures, Brain Res, 100: 231-235, 1975.
Distribution of Type A and Type B Monoamine Oxidase Activity in Hypothalamic Nuclei of the Rat Norifumi Kunimoto, M.D. and Hidebumi Hazama, M.D. Department of Neuro-psychiatry, Tottori University School of Medicine, Yonago
INTRODUCTION Type A and type B monoamine oxidase (MAO) [EC 1.4.3.4.1 have been differentiated by their substrate specificity and inhibitor sensitivity in homogenates of the rat brain.X Whereas the distribution of type A M A 0 was reported to be similar to that of type B M A 0 in the central nervous system of the rat,"' the relative levels of type A and type B M A 0 were suggested to differ in the hypothalamic nuclei." In the present paper, the regional distribution of type A, type A and B and type B M A 0 activity in individual nuclei of the rat hypothalamus was studied according to quantitative micromethods with clorgyline as a specific inhibitor of type A MAO. MATERIALS AND METHODS Male Wistar rats (200-260 g) kept in a group were used. The rats were killed by decapitation at 9 a.m. and the brains were immediately taken out and placed on ice. The blocks including the hypothalamus were isolated and frozen in liquid nitrogen. Frontal serial sections of 60 pm thickness were made in a cryostat at - 15°C and freeze-dried. The individual hypothalamic nuclei were dissected from the freeze-dried sections with a microknife under a stereoReceived for publication July 31, 1978.
microscope. After being weighed with an electronic microbalance (Type 4125, Sartorius Co.), each sample was placed into a microtube. The sensitivity of this balance was 0.1 pg. The weight of each sample was 1 .O to 7.0 pg. M A 0 activity was measured by the radiochemical assay method.4 Two pl of 0.1 M phosphate buffer, pH 7.2, containing BSA and Triton X-100in each final concentration 0.05% was added to the weighed sample in the microtube. After a 2 0 min preincubation at OOC, 2 pl of icecold substrate buffer solution was added. 5-Hydroxytryptamine (5-HT) and dopamine (DA) were used as a substrate for type A and type A and B MAO, respectively (the final concentration of the substrate: 1.1 mM [2-14C] 5-hydroxytryptamine binoxalate, 45 mCi/mM; 1.0 mM [ 12-I4C] dopamineethylamine, 60 mCi/mM, New England Nuclear Co.). When the assay of type B MAO, 1 pl of the buffer and 1 pl of M clorgyline solution were added into a microtube, and after a preincubation at 0°C for 20 min, 2 pI of ice-cold DA-buffer solution (the same solution that was used for the assay of type A and B MAO) was added. All of these procedures were made in an ice-cold bath. After an incubation at 38OC for 45 min, the reaction products were extracted in ethyl acetate and the radioactivity was measured with a liquid scintillation spectrometer. The inhibiting effect of the deamination of 5-HT and DA by clorgyline is shown in Fig. 1.
N. Kunimoto and H. Hazama
106
0
a I
Clorgyline -log final conc. M Fig. 1: Inhibiting effect of the deamination of 5-HT and DA by clorgyline. Assays were performed with homogenates of the freeze-dried sample of the rat.
The inhibition of DA deamination with clorgyline showed a plateau at the final concentration of clorgyline between 2 X lo-' M and 8 X 10-"M. In case of 5-HT, a small plateau was also recognized but the detail of the cause is unknown. When 1 pI of M clorgyline was used (the final concentration of clorgyline, 2.5 X lo-" M ) , the inhibition of 5-HT and DA deamination was found to be about 100% and 5376, respectively. Accordingly M A 0 activity for DA in this condition was considered to possibly mean type B M A 0 activity (denoted as DA MAO-type B) , even though the comparison with the activity shown with typical substrate for types such as P-phenylethylamine is necessary due to the indirect measurement of type B M A 0 in the present study. Moreover, it was ascertained in this experiment that the enzyme reaction towards
DA and 5-HT as the substrate proceeded linearly with or without the inhibitor with the use of the freeze-dried rat cerebral cortex as the enzyme sample at least that between 0.5 pg and 8 pg in weight. RESULTS
As shown in Table 1, type A, type A and B and type B M A 0 activities were distributed unevenly in each hypothalamic nucleus. Type A M A 0 activity was obviously higher in medial parts than in lateral parts of the hypothalamus. The highest activities of type A and B M A 0 for DA existed in the nuclei of suprachiasmaticus in the anterior part and arcuatus in the medial and posterior part of the hypothalamus. The activities of type B M A 0 were highest in the nucleus arcuatus of the
Type A and Type B MA0 in Rat Hypothalamus
Table 1 :
Distribution of M A 0 Activity in the Hypothalamic Nuclei of the Rat
~
~
para anterior. N; anterior A. lateralir N. paraventricularir N . aupraopticua N. ruprachianmatiaur
55.7 t 7.0 52.4 f 1.9 05.1 f 7.2 54.1 2 9.7 75.9 i 5.6
para medialia N . ventromrdialii N. dorromrdialii A. l a t e r a l i r N. periventricularii N. arcuatua
81.9 89.1 47.6 96.3 96.6
t 3.9 t 4.1
par. porterior N . poaterior A. l a t e r r l i a N . premmillrrir dorralir N . premunillarir ventrrlir N. arcuatua
14.6 54.3 99.5 72.3 79.1
f
I h e rerultr
clorgylina)
2.4 t 2,l t 9.7
f
3.8 t 1.6 210.7 t 6.1 tll.1
eo.1 ti6.i 44.5 f 5.0 64.2 f10.0 84.6 f 5.1 133.6 f12.7 70.5 87.3 65.4 86.0 124.0
t 8.8
fl2.Z k 8.2 t13.6 t 8.7
, /J
p
mOl.8
19.1 f 12.9 f 36.3 f 53.4 f 39 $ 6 f
1.5 0.9 6.6 7.3 3.9
0.32 0.41 1.29 1.71 0.42
20.0 f 21.9 f 17.0 f 37,O t
1.9 2.8 0.9 2.8
0.40
8 8 . 3 t16.2
88.8 t 8.8
6 3 , 3 t 6.9
72.0 213.5 94.0 t 9.3 181.4 f u . 3
are rxprrried am mew valuerfS,E.M.. q y p r A
wt./hr lype A and B N O , with
107
20.2 f 1.5 16.7 f 1'.9 29.3 f 2.3 57.6 f 8.2 106.5 214 - 2
0.33 0.35
0.76 2.52 0.29 0.35 0.69 1.58
1.42
N O l )I molar 5-HT oxidized/g dry
molra DA oxidized/g dry w t . / h r lype B MA0 (DA an nubatratel treated DA oxidiard/g dry w t . / h . Number of animal8 i a f i v e ,
*DA MO-type A wai calculated according t o deduction
of
DA MO-typ.
B from DA MA0 (typa A
and 6).
medial part and the posterior part of the hypothalamus, relatively high in the nuclei of paraventricularis, supraopticus, and premamillaris ventralis and low in the lateral hypothalamic area. The ratio of DA MAOtype B to DA MAO-type A ranged from 0.29 to 2.52. DISCUSSION
Recently it has been revealed that M A 0 activities are unevenly distributed in the hypothalamus as well as in the limbic and the lower brain stem n ~ c l e i . ~Whether -~ or not regional distribution of the two M A 0 types, A and B, in the brain is different from one another is not exactly clear. The
present study describes the relative proportions of the two types of enzyme within the hypothalamus, assessing M A 0 activity with the use of a common substrate (DA) for the two types and a specific inhibitor (clorgyline) against type A. The ratio of DA MAO-type B to DA MAO-type A ranged from 0.29 to 2.52 in the present study. This indicates that the distribution pattern of type B M A 0 differs from type A M A 0 within the rat hypothalamus. The highest values of the ratio were found in the circumventricular areas particularly in the nucleus arcuatus. This implies that remarkably high activities of type B M A 0 exist in these areas. In this context, type A M A 0 is absolutely domi-
N. Kunimoto and H. Hazama
108
nant within the rat hippocanipus.4 While the existence of clorgyline-insensitive 5-HT M A 0 was postulated in circumventicular structures by histochemical technique,I7 we could not detect clorgyline-insensitive M A 0 activity in the circumventricular areas when using lo-" M to M clorgyline and 5HT as a substrate by the method described in this report." The regional distribution of DA M A 0 activity in this study is not exactly the same as that of tyramine MAO,(' although tyramine is also considered as a substrate for both type A and type B. This difference might be explained by some regional difference in substrate specificity of individual nuclei. Harada et ~ 1 observed . ~ a difference in substrate specificity of M A 0 in the cerebellum whereas not so distinct in several roughly dissected regions of the rat brain. It is known that the nucleus of paraventricularis and supraopticus produces oxytocin and vasopressin, respectively, and the nucleus arcuatus contains dopaminergic neurons and regulates the secretion of hypophysial hormones. It is of interest that high activities of type B M A 0 exist in these nuclei. Among the biogenic amines in the central nervous system, specifically catalysed amines by type B M A 0 are less understood in their detailed distributions and physiological roles as compared with those by type A MAO. It might be tentatively assumed that not only DA but the biogenic amines which are substrates for type B M A 0 such as P-phenylethylamine are actively concerned in neurosecretion. Recently P-phenylethylamine has been shown to exert a direct, stimulating effect on postsynaptic DA receptors in the nigrostriatal pathway.1 SUMMARY The regional distribution of type A, type A and B and type B activity of monoamine oxidase (MAO) in individual nuclei of the rat hypothalamus was studied according to quantitative micromethods with clorgyline as
a specific inhibitor of type A MAO. As results, type A and type B M A 0 were distributed differently in the rat hypothalamus. It is suggested that type B M A 0 acts differently from type A M A 0 in regard to the function of the hypothalamus. REFERENCES 1 Antelman, S. M., Edwards, D. J. and Lin,
M.: Phenylethylamine: evidence for a direct, postsynaptic dopamine-receptor stimulating action, Brain Res, 127: 317322, 1977. 2 Harada, M., Kondo, Y., Kuzuya, H. and Nagatsu, T. : Monoamine oxidase activities towards biogenic monoamines in several regions of rat brain, J Neurochem, 24: 193-1 95, 1975. 3 Hazama, H. and Ito, M.: Effects of psy-
chotropic drugs on monoamine oxidase activity in individual nuclei of hypothalamus and amygdala in rat, Yonago Acta med, 20: 221-224, 1976. 4 Hazama, H. and Kunimoto, N.: Distrihution of monoamine oxidase in hippocampal region of the rat, Experientia, 34: 424425, 1978. 5 Hazama, H., Kunimoto, N. and Takeshita,
H.: Clorgyline inhibition of monoamine oxidase activities in circumventricular areas of rat hypothalamus, Yonago Acta med, 21: 140-144, 1978. 6 Hirano, M., Uchimura, H. and Saito, M.: Regional distribution of monoamine oxidase activity for 5-Hydroxytryptamine and tyramine in hypothalamus of the rat, Brain Res, 93: 555-563, 1975. 7 Hirano, M., Kim, J. S., Saito, M., Uchimura, H., Ito, M. and Nakahara, T.: Monoamine oxidase activities for serotonin and tyramine in individual limbic and lower brain stem nuclei of the rat, J Neurochem, 30: 263-267, 1978. 8 Johnston, J. P.: Some observation upon a new inhibitor of monoamine oxidase in brain tissues, Biochem Pharmacol, 17: 1285-1297, 1968. 9 McCaman, R.E., McCaman, M. W., Hunt, J. M. and Smith, M. S.: Microdetermination of monoamine oxidase and 5-hydroxytryptophan decarhoxylase activities in nervous tissues, J Neurochem, 12: 15-23, 1965. 10 Suzuki, 0. and Yagi, K.: Distribution of type A and B monoamine oxidase activities
Type A and Type B M A 0 in Rat Hypothalamus in the central nervous system of rat and chick, Experientia, 32: 13-14, 1976. I I Williams, D., Gascoigne, J. E. and Williams,
109
E. D.: A specific form of rat brain monoamine oxidase in circumventricular structures, Brain Res, 100: 231-235, 1975.
