Department of Anatomy (Prof. H. MASAI), Yokohama City University School of Medicine, Urafunecho, Yokohama, Japan. Results

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1 Department of Anatomy (Prof. H. MASAI), Yokohama City University School of Medicine, Urafunecho, Yokohama, Japan Throughout all classes of vertebrates, the telencephalon of bony fishes has developed in a peculiar direction on the evolutionary chain, that is, the telencephalon shows the eversion of the medial wall towards dorsolateral side. In another respect, bony fishes show the most different patterns in those parts of central nervous system related to their life habits, as LISSNER (1932), EVANS (1940), UCHIHASHI (1953) and SVETOVIDOV (1958) have reported. On the other hand, it was recently noticed by BRODIE and co-workers (1957) that brain amines i. e, catecholamines and serotonin have an important significance in carrying out the visceral functions of the brain. Since a few years ago, the present authors have been investigating the histochemical distribution of monoamine oxidase metabolizing catecholamines and serotonin of the central nervous system of frog and toad (KUSUNOKI, in press), Uroloncha (KUSUNOKI et al. 1964) and chick embryos (KUSUNOKI et al. 1965), in order to reveal the relationship of brain amines to the visceral areas of the central nervous system. In the brain of chick embryos on the 9th day of incubation, it was found that the activity of monoamine oxidase is relatively intense in the visceral areas i. e. along the sulcus limitans, the hypothalamus, the preoptic area and the septal area. In addition, as far as I know, no histochemical studies on the distribution of monoamine oxidase have been systematically performed in the central nervous system of fishes. Concerning the neuronal activity, it is recognized that there is a resting metabolism participating in the energy production and a functional metabolism related to the chemical transmitters. The authors intended to study the histochemical distribution of monoamine oxidase in the central nervous system of goldfish, comparing with the distribution patterns of succinic dehydrogenase. Material and Method As the experimental material, use was made of 10 individuals of goldfish which is called "Wakin" (trilobed caudal fin) in Japan. They were collected in March, August and September. The brains were removed as quickly as possible after decapitation, and cut into frontal and GLENNER's method to demonstrate the activity of monoamine oxidase and with the method of NACHIAS to demonstrate the activity of succinic dehydrogenase. Results Monoamine oxidase The gray matter exhibits positive reaction, and white matter also is partly positive. The This work was supported by a grant for scientific research from the Ministry of Education of Japan. 363

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3 positive granules present in the gray matter are located mainly in the neuropil. But, it is difficult to detect whether there are reactive granules within the perikaryon with the poor cytoplasm. The epiphysis, the dorsal sack and chorioid plexus are positive, while the ependyma is negative (Fig. 4, 5, 17). In general, the forebrain is abundant in positive granules, compared to other parts. The reaction of monoamine oxidase appears highly in the following areas: the olfactory bulb (Fig. 1, 16) in particular, glomerulus and anterior olfactory nucleus, pars dorsalis and pars lateralis of the area dorsalis (Fig. 2, 3), supracommissural part of the area ventralis (Fig. 3, 4) (these nomenclature are after NIEUWENHUYS, 1963 b) the preoptic area (Fig. 3, 4), the hypothalamus (Fig. 5, 6, 7, 16), in particular the nucleus periventricularis. A fairly positive reaction is found in the tectum opticum (Fig. 5, 6, 7, 8, 9), the nucleus interpeduncularis (Fig. 9, 16), the central gray of the transitional part from the mesencephalon to the mesencephalon (Fig. 10), the area postrema (Fig. 14) and the substantia gelatinosa of the spinal cord (Fig. 15). The fasciculus retroflexus, the commissura habenularum (Fig. 17), the commissura posterior (Fig. 6) and some other fibers show a strong activity among the white matter. Faint reactions are found in the nucleus habenulae (Fig. 5, 16, 17), the cerebellum (corpus and valvula) (Fig. 7, 9, 10, 11, 12, 16), crista cerebellaris, lobus vagus (Fig. 12, 13, 14, 16) and lobus facialis (Fig. 12, 13, 16). Succinic dehydrogenase The activity of succinic dehydrogenase is revealed in both the neuropil and the perikaryon in the gray matter, but not in the white matter. A strong reaction of succinic dehydrogenase is shown in the following areas, comparing with other portions: the dorsal sack (Fig. 18, 19, 27, 28), the epiphysis (Fig. 28), the tectum opticum (Fig. 19, 20, 21, 22), the nucleus pretectalis, the nucleus geniculatus lateralis (Fig. 20), the nucleus interpeduncularis (Fig. 22), the rotundus complex, the nucleus isthmi, the torus semicircularis (Fig. 22), the torus longitudinalis (Fig. 21), corpus et valvula cerebelli (Fig. 22, 27), the lobus vagus (Fig. 24, 25, 27), the lobus facialis (Fig. 24), the crista cerebellaris (Fig. 23) and the substantia gelatinosa of the spinal cord (Fig. 26). Contrary to the distribution patterns of monoamine oxidase, each part of the telencephalon, the nucleus preopticus and the hypothalamus shows a slight reaction of succinic dehydrogenase. In the hypothalamus, however, considerably positive reaction of the succinic dehydrogenase is found in some individuals. Discussion Regarding distribution of monoamine oxidase, it was noticed that in goldfish the olfactory bulb, supracommissural part of the area ventralis, preoptic area, hypothalamus and nucleus interpeduncularis show an intense reaction. Similar locality of positive reaction was reported in the amphibia (Rana and Bufo), in birds (Uroloncha and chick embryo) and in the Mammals (rabbit) (HASHIMOTO, 1962). From these data, it can be said that the visceral areas show high activity of monoamine oxidase also in goldfish. The pars lateralis of the area dorsalis and the area ventralis of the everted telencephalon of bony fishes show relatively strong reaction of monoamine oxidase, as do the primordium hippocampi and pars septalis of other vertebrates. Moreover, these parts in the fish correspond to the medial wall of the evaginated telencephalon, which is occupied by the primordium hippocampi and pars septalis, from the standpoints of phylogeny and ontogeny as reported by NIEUWENHUYS (1962, 1963a, b). From

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5 these results, the pars lateralis of the area dorsalis and the area ventralis are supposed to be homologous to the primordium hippocampi and pars septalis, respectively. The monoamine oxidase activity present in the central gray of the transitional part from the mesencephalon to the metencephalon seems to coincide with that occurring in the area along the sulcus limitans in chick embryo. The activity of succinic dehydrogenase is shown in the nucleus geniculatus lateralis, nucleus pretectalis, rotundus complex, tectum opticum, nucleus isthmi, torus semicircularis, corpus et valvula cerebelli, crista cerebellaris, lobus vagus and lobus facialis. The distribution of succinic dehydrogenase activity is similar to the previous results obtained by MASAI and MATANO (1961) using Seligman-Rutenburg's method, but the present reaction is finer and more delicate than the previous one, and thus the parts presenting a strong activity are found to greater extent. The somatic areas reveal a relatively intense reaction of succinic dehydrogenase and a faint reaction of monoamine oxidase, while the visceral areas presenting a strong reaction of monoamine oxidase show a weak reaction of succinic dehydrogenase. This seems to be similar to the positive and negative figures of photography. Refering to the results in other vertebrates investigated by the present authors, the fundamental metabolism of the brain of goldfish is quite similar to that of other classes. However, goldfishes have a stronger activity of monoamine oxidase and succinic dehydrogenase than the amphibia. Judging from this, the amphibia represent the most primitive stage among the vertebrates, as far as the evolution of the enzymic system is concerned. Differing from a reaction of mono- the species of the other classes, the tectum opticum of goldfish exhibits amine oxidase. There is a question why the lobus vagus and the lobus facialis are faintly reactive in monoamine oxidase and highly positive in succinic dehydrogenase, which are thought to be homologous to the nucleus solitarius and the dorsal vagus nucleus. Concerning the life manner of goldfish, it is supposed that they seek for food mainly by taste and, moreover, the lobus vagus and lobus facialis are much developed, especially forming the lamination of the lobus vagus. These lobi are thought to play a higher integration, and this fact is presumed to influence the enzymic patterns. This phenomenon must be analyzed in the future. The fiber bundles are, as a rule, stained by this method of staining for monoamine oxidase in goldfish. The stainable fiber bundles are found in amphibia also. The fact that the fiber bundles are positive in the reaction of monoamine oxidase should not be simply thought to be an artifact, considering the fact that the fiber bundles of other poikilothermal animals tend to be stainable. Finally, we shall discuss the relationship between the distribution patterns of these both enzymes and the main fiber-connections in the telencephalon and diencephalon. The septal parts, the preoptic area and the hypothalamus, which are connected to each other by the medial forebrain bundle, are highly positive in the reaction of monoamine oxidase, while the rotundus complex and nucleus geniculatus lateralis, which are contributed mainly by the lateral forebrain bundle and optic tract respectively, show a relatively strong reaction of succinic

6 Fig. 15. Frontal section of the spinal cord. The substantia gelatinosa is distinguishable in monoamine

7 dehydrogenase. This seems to be analogous to the fact that the characters of the function of the thalamocortical connections reflect the intensity gradient of succinic dehydrogenase in the cortical areas and the thalamic nuclei related to the corresponding connection, as found by FRIEDE (1961). Summary The histochemical distribution of monoamine oxidase and succinic dehydrogenase in the central nervous system of goldfish was studied by GLENNER's and NACHLAS' methods. The reaction of monoamine oxidase is higher in the septal area, the preoptic area, the hypothalamus and the area postrema which belong to the visceral areas. On the other hand, the reaction of succinic dehydrogenase is stronger in the nucleus geniculatus lateralis, the rotundus complex and the corpus et valvula cerebelli, which belong originally to the somatic areas. Moreover, the homology of each part of the everted telencephalon characteristic to bony fishes was discussed on the basis of the distribution patterns of monoamine oxidase. Abbreviation ap area postrema ca commissura anterior cc canalis centralis ce cerebellum ch commissura habenularum co chiasma opticum

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9 Literatures Brodie, B. B. and P. A. Shore: A concept for a role of serotonin and norepinephrine as chemical mediators in the brain. Ann. New York Acad. Sci. 66: (1957) Evans, H. M.: Brain and body of fish. London, Technical Press, 164 pp, Friede, R. L.: Thalamocortical relations reflected by local gradation of oxidative enzymes; with some notes on patterns of enzyme distribution in nerve cells. Regional Neurochemistry, Proc. IVth. Int. Neurochem. Sympos. Varenna, Italy. London, Pergamon Press P Hashimoto, P. H. et al.: 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) Kusunoki, T., H. Ishibashi and H. Masai: A histochemical study on the fundamental plan of the central nervous system. Experientia 21: 572, (1965) ---: The distribution of monoamine oxidase and melanin pigment in the central nervous system of amphibia. J. Hirnforschung (in press). and succinic dehydrogenase in the central nervous system. (in Japanese.) Acta anat. nippon. 39:73 (1964) Lissner, H.: Das Gehirn der Knochenfische. Wissenschaftliche Meeresuntersuchungen herausgegeben von der Kommission zur wissenschaftlichen Untersuchung der deutschen Meere in Kiel und der Biologischen Anstalt auf Helgoland. 2 (1932) Masai, H. and S. Matano: Comparative neurological studies in respiratory enzymic activity in the central nervous system of submammals. II. Fishes and amphibia. Yokohama med. Bull., 12: (1961) Nieuwenhuys, R.: The morphogenesis and the general structure of the actinopterygian forebrain. Acta morphol. neerl.-scand. 5: (1962) -: Further studies on the general structure of the actinopterygian forebrain. Ibid. 6: (1963 a) -: The comparative anatomy of the actinopteygian forebrain. J. Hirnforshung 6: (1963 b). Svetovidov, A. N.: The structure of the brain of fishes in relation to classification and habits. Proc. XVth Int. Congr. Zool., Sect. V, Paper 4 (1958) Uchihashi, K.: Ecological study of the Japanese teleosts in relation to the brain morphology. (in Japanese.) Bull. Japan Sea Regional Fisheries Res. Lab. 2: (1953).