The Morphological Changes of Oligodendroglia During the Formation of Myelin Sheaths

Okajimas Fol. anat. jap., 50: 327-344, 1973

-Golgi Study and Electron Microscopy-

Yoshiro Inoue, Yoshihiro Sugihara, Shoji Nakagawa and Kazuyo Shimai

Department of Anatomy, School of Medicine, Keio University Shinano-machi, Shinjuku-ku, Tokyo, Japan

-Received for Publication, July 27, 1973-

Morphological studies of oligodendroglia in the adult central nervous system have been performed chiefly with metal impregnation methods at the light microscopic level. Rio-Hortega (1928) investigated this type of glial cells in detail with the Golgi method and classified it into four types. According to Rio-Hortega's observations three types of oligodendroglia (II, III and IV Type) occurred mainly in the white matter. These oligodendroglia possessed spiral or ring structures at the tip of their processes, which appeared to be closely associated with the neuronal axons. From these findings, it has been suggested that the myelin-forming cell might be oligodendroglia in the central nervous system. At the electron microscopic level, however, direct continuity between myelin sheaths and processes from oligodendroglia cell bodies has never been demonstrated in the adult animal brain (Peters 1968, Peters, Palay and Webster 1970). On the other hand, electron microscopic findings in the young animal brain or under experimental con- ditions, and observations in tissue cultures have supported the concept that oligodendroglia might be myelin-forming cells in the central nervous system (Yonezawa 1961, Yonezawa, Bornstein, Peterson and Murray 1962, Bunge, Bunge and Pappas 1962, Peters 1964, Bunge and .Glass 1965, Yonezawa and Iwanami 1966, Calay 1967, Peters 1968, Hirano 1968, Hirano, Livine and Zimmermann 1968, Peters, Palay and Webster 1970). Since attempts to follow the progress of myelin formation in young animal brains have been performed only with electron microscopy, there was the avoidable limitation that three-dimen- * This study was partly supported by a Scientific Research Grant 877013 from the Japanese Ministry of Education. 327 328 Y. Inoue, Y. Sugihara, S. Nakagawa and K. Shimai sional changes during development had to be inferred from numerous plane figures of ultra-thin sections (Bunge 1968, Peters 1968). Thus, the authors designed this study to observe the young chick brain chiefly with the Golgi method and partially with electron microscopy, to investigate the three-dimensional changes of oligodendroglia, and to examine the relationship between myelin formation and the morphological changes of oligodendroglia, comparing the results with previous reports based on electron microscopic data.

Materials and Methods The medulla oblongata, diencephalon, optic tectum, optic tract and optic nerve of chick embryos (Hamburger-Hamilton stage 36-45) (Hamburger and Hamilton 1951) and young chicks just after hatching were used.

1. Light microscopic studies a. Golgi method Materials were fixed and silver-impregnated according to the Colonnier's modification (1961), embedded in celloidin and cut in HO p thick serial sections. b. Hematoxylin-eosin and Kliiver-Barrera staining For confirmation of the cell architecture of the brain, materials were fixed with Bouin's solution and cut in 10 p thick serial paraffin sections, followed by staining. c. Toluidine blue staining For observation of the aspects of myelin formation, materials were embedded with Epon 812 after 1% osmic acid fixation, and cut in about 1 p thick sections, followed by 1% toluidine blue staining.

2. Electron microscopic studies a. The tissue blocks stained with the Golgi method were cut in 100 p thick sections without embedding, using a sliding microtome. The glial cells, one object of this study, were confirmed in these sections under a light microscope, and the portions where glial cells were observed very frequently were then trimmed out. Then the pieces of the sections were refixed with 1% osmic acid in Millonig's solution and embedded in Epon 812 with the usual method. The ultra-thin sections were stained with 3% uranyl acetate aqueous solution. b. The young chicks just after hatching were fixed 1% glutaraldehyde in Millonig's buffer solution (pH. 7.2) or a mixed solution of 1% glutaraldehyde and 1% paraformaldehyde in Millonig's solution (pH. 7.2) by vascular perfusion through the left ventricle. After perfusion, the brain was removed from the skull and refixed with 2% Morphological Changes of Oligodendroglia During Development, 329 osmic acid in the above buffer solution or Dalton's solution, followed by embedding in Epon 812. The ultra-thin sections for electron microscopic observation were stained with 3% uranyl acetate aqueous solution, followed by staining with a mixed lead solution (Sato 1967).

Observation and Discussion All glial cells observed for this study followed approximately the same pattern of morphological changes in each part of the brain except for the optic nerve and tract, although some differences were observed in the stages of development of the glial cells. Therefore, the chick medulla oblongata just after hatching was first selected for observation in this study. In the medulla oblongata of the young chick just after hatching, the portions in which myelin sheaths were being formed were confirmed by observations of the paraffin sections stained by the Kliiver- Barrera method or of the epon sections stained with toluidine blue, and then the confirmed portions were investigated with the Golgi method. Various types of immature cells were observed, which had not been found in the adult chicken brain. Among these cells was found an immature type of astrocyte which was identified by the characteristically rough surface of the processes and by the observation of its attachment to the vascular walls with perivascular feet (Fig. 1), and another immature type presumed to be immature microglia, because they had angularly tortuous processes with a few, spine-like, small protrusions on the surface (Fig. 2). Besides these immature cells, there were two other types of cells which possessed very numerous, thin processes (Fig. 3, 4, 5). One type had a polygonal cell body about 10 p in longer diameter with numerous, relatively smooth-surfaced processes, branching several times and extending approximately 50 if in overall length (Fig. 3, 5), and the other type had slightly wider, membraneous cytoplasnlic protrusions from the cell body, with numerous processes from the somal protrusions, extending approximately 50 p in overall length (Fig. 4). Both of those types of cells will be referred to as "multi-process-cells" in this paper. The tips of the processes of both types varied : some tapered away freely, some were nodularly enlarged (Fig. 6), and some terminated in a short tubular structure (Fig. 5, 6). Frequently the end of the process touching the tubular structure was nodularly enlarged (Fig. 5, 6). RamOn-Moliner (1958) investigated the young cat brain with the Golgi method, and observed an "undifferentiated type" of glial cells (cells with "mossy or seaweed-like processes") and "transitional forms" which seemed to be transformed to oligodendroglia. He proposed that the "undifferentiated type cells" were transformed to oligodendroglia 330 Y. Inoue, Y. Sugihara, S. Nakagawa and K. Shimai through the "transitional forms". These "undifferentiated type cells" and "transitional forms" at some stages were similar to the above "multi -process-cells" found in the chick brain . Oligodendroglia in the adult chicken brain had a round or oval cell body, from which three to ten, smooth or sometimes bead-like processes extended. The ends of the processes, as a rule, tapered away, but some were in contact with a short tubular-structure (Inoue 1970, Inoue, Nakagawa, Sugihara and Shimai 1971). Glial cells resembling mature oligodendroglia were also observed in the chick brain just after hatching, showing a round or oval cell body with a few to ten processes like a mature oligodendroglia (Fig. 7, 8, 21). The processes were smooth-surfaced, and often enlarged nodularly especially at the branching points, producing a bead-like shape. The tips of the processes, however, differed from those of adult processes. This type of cells will be referred to as "oligo-like-cells" in this report. The "oligo-like-cells" found in the chick brain just after hatching possessed tubular structures of various lengths (longer ones exceeded 200 a) attached to the ends of the processes or the cell bodies (Fig. 7, 8, 21). The number of these tubular structures varied, but those cells with more than a few usually had approximately ten or so structures per cell. The contact points of the processes with the tubular structures were frequently nodularly enlarged and silver granules precipitated more densely at the tubular sides of these points (Fig. 8). The electron microscopic investigation of the Golgi stained materials embedded in epon resin and cut into ultra-thin sections clearly demonstrated that the silver granules, which constructed the tubular structures at the light microscopic level, were precipitated at the portions corresponding mainly with the inner cytoplasmic tongue between the axon and myelin lamellae and partly with the outer cytoplasmic tongue outside the myelin lamellae (Fig. 9, 10). The cytoplasm, however, could not be clearly identified because of the predominant precipitation of silver granules. On the other hand, the electron microscopy of the medulla oblongata of chicks just after hatching showed clearly that the lamellae of the myelin sheaths were much looser (Fig. 12) than those in the adult brain (Fig. 11). Furthermore, a greater volume of the inner cytoplasmic tongue was observed, considered to be a part of the cytoplasm of oligodendroglia (Fig. 12). Since the tubular structures of the "oligo-like-cells" seemed to be silver-impregnated due to the precipitation of the silver granules in the inner and outer cytoplasmic tongues, these cells were considered to be an immature type of olip- dendroglia at a somewhat advanced stage of myelin lamellae formation. More detailed observation of glial cells with such tubular struc- Morphological Changes of Oligodendroglia During Development 331 senting the different stages of the transition from "multi-process-cells" with numerous processes to "oligo-like-cells" with long tubular structures (Fig. 13). That is, as the number of processes of "multi-process- cells" decreased during development, the tubular structures on the tips of the processes tended to increase in length, and the shape of the cell bodies changed from an irregularly polygonal shape to a tri- angular or pear-like shape (Fig. 17). The cell bodies finally became oval or round and smooth-surfaced, resembling the characteristic form of the "oligo-like-cell" soma with approximately ten processes and elongated tubular structures (Fig. 8, 21).

Fig. A. The morphologicalchanges of oligodendrogliafrom the immature type, the "multi-process-cell", to the mature type through transitional forms, the "oligo-like-cells" with the tubular structures varying in length (arrows).

These findings led to the following conclusions (see Fig. A) : An immature type of oligodendroglia has numerous processes, some of which are attached to neuronal axons running nearby. The axons are enclosed by the processes, eventually becoming part of the outer and inner cytoplasmic processes. This is revealed by the formation of nodules or short tubular structures at the tips of processes in the Golgi stained materials. Then as the inner and outer cytoplasmic tongue extends along the axon, the free processes decrease in number to about three to ten (probably due to the movement of their cyto- 332 Y. Iuoue, Y. Sugihara, S. Nakagawa and K. Shimai

plasm to surround the axon), and finally the cell body and processes begin to take a shape similar to that of mature oligodendroglia. The layer of the inner cytoplasmic tongue, which appears as a tubular structure as a result of silver impregnation, gradually becomes thinner with the formation of myelin lamellae, as the whole cytoplasmic amount may decrease and the cytoplasm still continues to envelope the axon. At the same time, the layer of the myelin lamellae become much denser. Therefore, at the final stage of myelin formation a clear precipitation of silver granules into the inner cytoplasmic process is extremely difficult probably because of the small volume of the cytoplasmic layer and the remarkably limited penetration of the fixa- tive and silver solution through the dense myelin lamellae. These facts may give rise to a complete disappearance of the tubular structures or remnants of short tubular-reticulate structures in the Golgi stained material. Consequently, the whole silver-impregnated images may seem to take the characteristic shape of the mature type of oligodendroglia with a round or oval cell body and free cytopiati*c processes. Next, after confirming the stages of myelin sheath formation in the medulla oblongata of chick embryos under a light microscope using the materials fixed with osmic acid, embedded in epon resin, and stained with toluidine blue, the same stages of myelin sheath formation were investigated using the Golgi stained materials. In the medulla oblongata at Hamburger-Hamilton stage 36, myelin sheaths seldom occurred, and the immature type of oligodendroglia were almost all "multi-process-cells" (Fig. 14). Some of the cells appeared to be somewhat differentiated since they possessed cytoplasmic processes with short tubular structures on their tips (Fig. 14). No "oligo- like-cells" with long tubular structures, however, were ever observed. At stage 36, the immature cell types which might be transformed to astrocytes (Fig. 15) and microglia (Fig. 16) already existed. This fact suggested that at the latest the glial cells might begin differentiation at this stage to those type of cells identified in the adult brain. From this stage on, "multi-process-cells" and "oligo-like-cells" showed similar transformations to those in the chick brain just after hatching, base upon the stages of myelin. formation (Fig. 17). In the optic nerve and tract which consisted only of neuronal axons, myelin formation began at stage 42 (about 16 days chick embryo). At that time, cells possessing a round or oval cell body (about 10 p in longer diameter) with numerous short and thin processes (extending in all directions about 20 p in overall width) (Fig. 18) were observed together with the immature type of astrocytes. Since some of the former cells also had nodules or short tubular structures on the tips of their processes (Fig. 19), this type seemed to belong to MorphologicalChanges of Oligodendroglia During Development 333

the "multi-process-cell", although they seemed somewhat different in shape from those found in other parts of the brain. Since "oligo- like-oells" with long tubular structures were found in the optic tract and nerve of the chick just after hatching (Fig. 20), however, it was assumed that the same process of differentiation of oligodendroglia as observed in the medulla oblongata might also occur during myelin formation. In 1958, RamOn-Moliner first described the immature type of oligodendroglia with the silver impregnation method. In his report he noted two types of glial cells, "undifferentiated cells" and "transitional forms to oligodendroglia". The former had numerous seaweed-like processes and the latter had processes whose peripheral parts ran parallel with the nerve fibers. Bunge et al (1962) then proposed from electron microscopic data that the parallel parts of the processes men- tioned above might correspond to the outer cytoplasmic process sur- rounding the myelin sheaths, which consisted of a part of cytoplasm of oligodendroglia. On the other hand, in our materials stained with the Colonnier's modification, silver granules were precipitated in all of the oligodendroglia cytoplasm ; in the cell body, cytoplasmic processes and the outer and inner cytoplasmic tongues around the axon. Therefore, it was postulated that the various silver-impregnated figures described above represent the morphological characteristics of oligodendroglia at each of the developmental stages from the immature to adult type. Until the present development of myelin sheath formation in the central nervous system has been investigated chiefly by electron microscopy (Bunge, Bunge and Pappas 1962, Peters 1964, Peters 1968, Bunge 1968) and by tissue cultures (Hild 1957, Yonezawa 1961). Peters (1968) diagrammatically portrayed the progress of myelin sheath development from electron microscopy, explaining it as follows : "In the first stage, an axon becomes encircled by a process of a myelin-forming, neuroglial cell. Next the outer surfaces of the lips of the enveloping process come together to form a mesaxon. The mesaxon then elongates in a spiral manner around the enclosed axon until many turns are completed. Then compact myelin results and eventually cytoplasm per- sists only at the outer and inner ends of the spiral. However, the unanswered problems arise when an attempt is made to explain how the internodal length increases during development and the Ranvier's node is formed completely." Hild (1957), however, described the stages in the formation of internodal segments in tissue cultures. First , short collars of myelin sheaths were formed around an axon. These collars gradually elongated and finally, at the contiguous portion, each of the adjacent collars became thinner to complete the Ranvier's node . In the Golgi stained materials of the chick medulla oblongata just 334 Y. Inoue, Y. Sugihara, S. Nakagawa and K. Shimai after hatching, two tubular structures were often found closely adjacent to each other, separated by a narrow gap (Fig. 8, 17, 21). Electron microscopy of the same developmental stage at the portions of the Ranvier's node where both end-structures of the adjacent internodal segments (so-called "pockets of the paranodal cytoplasm") were simultaneously observed, frequently showed the length of the node to be somewhat wide (about 2 to 3 p in length) (Fig. 22) and the myelin lamellae to be less dense than the completed ones. From these findings it was concluded that myelination in the central nervous system might first develop through the formation of the spiral lamellae and the simultaneous elongation of the internodal segments to form the Ranvier's node, and then the incomplete myelin lamellae became denser until compact myelin lamellae were completely formed. Detailed observation of the immature type of oligodendroglia with the tubular structures showed ten or so of these tubules running parallel to one another (Fig. 20) or in randam directions (Fig. 7, 8) according to the directions of the associated axons. From thes6, findings it was inferred that an oligodendroglia might extend their processes to more than one axon (generally ten or so) and be associated with myelin formation. This agreed with observations from both electron microscopy and tissue culture studies (Bunge, Bunge and Pappas 1962, Peters 1964, Peters 1968, Yonezawa 1961). In these cases in which tubular structures were, by chance, silver- impregnated closely adjacent to each other, appearing to enclose the same axon, two arrangements were formed one in which each tubular structure arose from two different " oligo-like-cells " (Fig. 8), and one in which both tubular structures originated from the same cell (Fig.

Fig. B. One oligodendroglia (A) may be associated with myelination of more than one axon (a, b, c, d). One oligodendroglia (A) may form more than one internodal segment along one axon (a). Internodal segments of one axon (a) may be formed by the processes of more than one oligodendroglia (A, B, C). MorphologicalChanges of Oligodendroglia During Development 335

17, 21). This showed that the internodal segments of one myelinated nerve fiber in the central nervous system were associated with more than one oligodendroglia, as was demonstrated by electron microscopy (Bunge, Bunge and Pappas 1962, Peters 1964, Bunge 1968, Peters, Palay and Webster 1970) and by tissue culture findings (Yonezawa 1961). In addition it was demonstrated in the Golgi stained materials that one oligodendroglia might form two or more internodal segments along an axon. These findings are diagramatically shown in Fig. B.

Summary In the brain of chick embryos and young chicks just after hatching the morphological changes of oligodendroglia during myelin formation were investigated with the Golgi method and the relationship between their changes in shape and myelin formation was discussed. It was inferred that an immature type of oligodendroglia sent out numerous thin processes, some tips of which might attach nearby neuronal axons, enclose them and elongate along them. The portions corresponding to the enclosing tongues was considered to be demonstrated as the various length of the tubular structures on the tips of the cytoplasmic processes by the Golgi method, since silver granules were precipitated as well in the outer and inner cytoplasmic tongue around the axon as in a cell body and cytoplasmic processes of oligodendroglia, which was demostrated by the electron microscopy of the Golgi stained materials. As the tubular structures became more elongated, the processes decreased in number, and as the myelin lamellae developed more densely, the tubular structures might be probably no longer silver-impregnated, to result in the characteristic silver-impregnated images of the mature oligodendroglia. From the findings of the arrangement of the tubular structures along an axon, internodal segments of a myelinated nerve fiber were apparently made by the plural oligodendroglia, and furthermore, single oligodendroglia seemed to be able to form two or more internodal segments along an axon. On the other hand, it was clear that an oligodendroglia was simultaneously associated with the formation of myelin sheaths of the plural axons (probably ten or so axons). It was presumed that the myelin lamellae developed in a spiral manner simultaneously with the elongation of the internodal length to reach the Ranvier's node, and then the spiral lamellae became much denser to complete the compact myelin sheaths. 336 Y. Inoue, Y. Sugihara, S. Nakagawa and K. Shimai

Acknowledgement

The authors wish to express thanks to Miss Sumiko Hashimoto and Mr. Hidehiko Okushige for their expert technical assistance.

References

Bunge, M. B., R. P. Bunge and G. D. Pappas. Electron microscopic demonstration of connections between glia and myelin sheaths in the developing mammalian central nervous system. J. Cell. Biol., 12 : 448-453, 1962. Bunge, R. P. and P. M. Glass. Some observations on myelin-glial relationships and on the etiology of the cerebrospinal fluid exchange lesion. Ann. N. Y. Acad. Sci., 122 : 15-28, 1965. Bunge, R. P. Glial cells and the central myelin sheath. Physiol. Rev., 48 : 197-251, 1968. Caley, D. W. Ultrastructural differences between central and peripheral myelin sheath formation in the rat. Anat. Rec., 157 : 223, 1967. Colonnier, M. The tangential organization of the visual cortex. J. Anat., 98 327- 344, 1961. Hamburger, V. and H. Hamilton. A series of normal stages in the development of the chick embryo. J. Morphology, 88 : 4 -92, 1951. Mid, W. Myelogenesis in cultures of mammalian central nervous tissue. Z. Zell- forsch., 46 : 71-95, 1957. Hirano, A. A confirmation of the oligodendroglial origin of myelin in the adult rat. J. Cell Biol., 38: 637-640, 1968. Hirano, A., S. Levine and H. M. Zimmermann. Remyelination in the central nervous system after cyanide intoxication. J. Neuropathol. Exptl. Neurol., 27 : 234-245, 1968. Inoue, Y. The glioarchitectonics of the chicken brain. I. The glial cells in the optic tract. Okajimas Fol. anat. jap., 41: 229-265, 1970. Inoue, Y., S. Nakagawa, Y. Sugihara and K. Shimai. The glioarchitectonics of the chicken brain. III. Astrocytes, oligodendroglia and other neuroglial cells. Okajimas Fol. anat. jap., 48: 237-270, 1971. Peters, A. Observation on the connections between myelin sheaths and glial cells in the optic nerves of young rats. J. Anat., 98: 125-134, 1964. Peters, A. The morphology of axons of the central nervous system. The structure and function of nervous tissue. (edited by Bourne, G. H.) Vol. 1: 141-204, 1963. Peters, A., S. L. Palay and H. de F. Webster. The cellular sheaths of neurons. The fine structure of the nervous system. The cells and their processes. (Hoeber), pp. 70- 104, 1970. RamOn-Moliner, E. A study on neuroglia : The problem of transitional forms. J. Comp. Neurol., 110: 157-167, 1958. Rio-Hortega, P. Del. Tercera aportacion al conocimiento morfologico e interpretacion functional de la oligodendroglia. Mem. Real. Soc. esp. Hist. nat., 14: 5-122, 1928. Sato, T. A modified method for lead staining of thin sections. J. Electronmicro- scopy, 16: 193, 1967. Yonezawa, T. Studies on myelin formation of central and peripheral nervous tissue Morphological Changes of Oligodendroglia During Development 337

Yonezawa, T., M. B. Bornstein, E R. Peterson and M R. Murray. A histochemical study of oxidative enzymes in myelinating cultures of central and peripheral nervous tissue. J. Neuropath. exp. Neurol ., 21 : 479-487, 1962. Yonezawa, T. and H. Iwanami. An experimental study of thiamine defficiency in nervous tissue, using tissue culture technics . J. Neuropath. exp. Neurol., 25: 362-372, 1966. 338 Y. Inoee, Y. Sugihara, S. Nakagawa and K. Shimai

Explanation of figures

Plate I Fig. 1. Astrocyte (as) in the medulla oblongata of the chick just after hatching. vf : vascular feet, by : blood vessel. Golgi method 600x Fig. 2. Microglia (arrows) in the medulla oblongata of the chick just after hatching. Golgi method 600x Fig. 3 and 4. Two types of the "multi-process-cells" in the medulla oblongata of the chick just after hatching. Golgi method 600x Fig. 5. The "multi-process-cell" with very short tubular structures (arrows) on tips of the processes. Golgi method 600x Fig. 6. High magnification figure of 'a' in the Fig. 5. An arrow : the short tubular structure and nodule on the tip of the process. An arrow head : the nodular enlargement of the tip of the process. Golgi method 1500x Fig. 7. "Oligo-like-cell" with long tubular structures (arrows) on the tip of the processes. Golgi method 600x Fig. 8. Two "oligo-like-cells" with tubular structures apparently adjacent with each other. Rn : the intimate contact point of the adjacent tubular structures, probably corresponding to the Ranvier's node. Arrows : nodularly enlarged tips of the processes. Golgi method 600x 339

Plate I

Y.Inoue et al 340 Y. Inoue, Y. Sugihara, S. Nakagawa and K. Shimai

Plate II

Fig. 9. Electron microscopy of tubular staucture stained with the Golgi method. M: myelin lamellae, an arrow : the precipitation of silver granules in the portion corresponding to the inner cytoplasmic tongue. 12500x Fig. 10. Electron microscopy of the "oligo-like-cell" stained with the Golgi method. N: the nucleus, S: the cell body, P: the process, 0: the outer cytoplasmic tongue, I: the inner cytoplasmic tongue. 12500 x Fig. 11. Electron microscopy of the compact myelin lamellae of the adult chicken ectostriatum. 12500 x Fig. 12. Electron microscopy of the myelin lamellae in the chick medulla oblongata just after hatching. 12500x 341

Plate 11

Y. Inoue et al 342 Y- Inoue, Y. Sugihara, S. Nakagawa and K- Shimai

Plate III

Fig. 13. Transitional form between the "multi-process-cells" and "oligo-like-cells". Arrows : relatively elongated tubular structures. Golgi method 600 x Fig. 14. Two "multi-process-cells" in the medulla oblongata of the chick embryo at stage 36. Arrows : short tubular structures on the ends of the processes. Golgi method 400 x Fig. 15. Probable immature type of astrocytes (arrows) in the medulla oblongata of the chick embryo at stage 36. Golgi method 600 x Fig. 16. Probable immature type of microglia (an arrow) in the medulla oblongata of the chick embryo at stage 36. Golgi method 600 x Fig. 17. The "multi-process-cell" with the more elongated tubular structures (vrows) in the medulla oblongata of the chick embryo at stage 40. Two tubular structures (arrow heads) of this cell were apparently adjacent with each other at the point ‘Rn', probably corresponding to the Ranvier's node. Golgi method 600 x • Fig. 18. The "multi-process-cells" (arrows) in the optic nerve of the chick embryo at stage 42. Golgi method 600 x Fig, 19. The "multi-process-cell" in the optic nerve of the chick embryo at stage 42. Arrows : the short tubular structures, an arrow head : a nodular enlargement on the tip of the process. Golgi method 600 x Fig. 20. The "oligo-like-cell" in the optic tract of the chick just after hatching. Arrows : the tubular structures on the ends of the processes. Golgi method 600 x Fig. 21. The "oligo-like-cell" with two tubular structures adjacent closely with each other at the point `Rn' probably corresponding to the Ranvier's node. An 'x' and 'y' showed two processes associated with the tubular structures . Golgi method 1000 x Fig. 22. Electron microscopy of the developing Ranvier's node (Rn). The length of the node estimated to be about 2. 5 p long. 15000 x 343

Plate III

Y. Inoue et al