The myelination of the cerebellar cortex in the cat

Cell Tiss, Res. 188, 509-520 (1978) Cell and Tissue Research

by Springer-Verlag 1978

The Myelination of the Cerebellar Cortex in the Cat* **

W. Lange

Department of Anatomy Rheinisch-Westf'~ilische Technische Hochschule Aachen

Summary. The myelination of the cerebellar cortex of the cat was investigated in 61 cats aged from 3 hrs post partum to two and a half years. The first myelinated fibers appear at the time of birth in the central medullary ray. Before the onset of myelination, all fibers reach a critical diameter of about 1 gin. About the 14th day of life the number of oligodendrocytes in the prospective white matter increases markedly. Thereafter, the oligodendrocytes invade the inner granular layer. It therefore seems that the myelination of the cerebellar cortex proceeds from the central medullary ray towards the granular layer. At the 60th day of postnatal life, most of the afferent and efferent fiber systems are myelinated. These findings are discussed in relation to the development of function and the maturation of the electrical activity of the cerebellar circuit.

Key words: Cerebellar cortex (Cat) - Postnatal development - Myelination of efferent and afferent fiber systems - Electron microscopy.

Introduction

During the last years, many investigations on the cyto-, synapto- and histogenesis of the cerebellar cortex have demonstrated (Altman, 1966, 1969, 1972a, 1972b, 1972c, 1975, 1977; Fujita, 1969; Mugnaini, 1969; Laramendi, 1969; Korneliussen, 1972) that the cerebellum can be subdivided in different longitudinal zones, which reach their final developmental stage at different times. The phylogenetically older parts are situated in the vermis or immediately paravermal, while the phylogenetically younger parts are localized in the hemispheres (Korneliussen, 1972). Each longitudinal zone again can be subdivided into three compartments, which correspond to the transversal organization of the cerebellum (Larsell, 1952; Jansen

Send offprint requests to." Professor Dr. W. Lange, Department of Anatomy, Rhein.-Westf. Techn. Hochschule Aachen, Melatenerstr. 211, 5100 Aachen, Federal Republic of Germany

* Dedicated to Prof. Dr. H. Leonhardt in honour of his 60th birthday ** Supported by the Deutsche Forschungsgemeinschaft (La 184/3)

0302-766X/78/0188/0509/$02.40

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and Brodal, 1958; Jansen, 1969). In spite of the fact that there is a large temporal phase-displacement in the maturation of the different parts (Jakob, 1928; Korneliussen, 1972) the cyto-, histo- and corticogenesis within all parts of the cerebellum follows the same pattern.

In contrast to this, our knowledge about the myelogenesis in the cerebellar cortex is very poor, although meanwhile investigations on the myeloarchitecture of the adult cat cerebellum have been published by Lange (1972) and Voogd (1977). Besides this, there are recent publications available dealing with the onset of function and the maturation of the cerebellar circuit (Puro and Woodward, 1977a, b). These findings show that the functional maturation of the cerebellar cortex may be correlated with the myelination of the afferent and efferent fiber systems, which according to Jakob (1928) make up three myelinated plexus in the adult cerebellar cortex.

The aim of the present study is to determine the onset of myelination in the cerebellar cortex and to study the time sequence in the myelination of the different afferent and efferent systems. The results obtained will be correlated to physiological data. The investigation is restricted to the vermal part of lobule II because in this region the three myelinated plexus mentioned above are well developed.

Materials and Methods

61 cats aged from 3 hrs post partum to two and a half years were perfused with glutaraldehyde (6 % in 0.05 M Millonig phosphate-buffer, pH 7.2). The cerebella were removed 2 h after fixation and pieces of the vermal and hemispherical parts of the lobule II, IX and X were postfixed for 1 h in a 1%OsO4 solution and embedded in Araldite. Semithin sections were stained by the method of Ito and Winchester (1963), and thin sections were stained with lead citrate and uranyl acetate.

Results

In the three-hour-old cat the cerebellar cortex consists of the external granular layer, the molecular layer, the Purkinje cell layer and the inner granular layer (Fig. 1 a). The external granular layer comprises 8 to 9 cell rows, the migration of cells from this layer into the internal granular layer being completed. The central medullary ray lying under the internal granular layer is very small and by light microscopy is not recognizable at this time (Fig. 2a). Only few oligodendrocytes are visible. They can be easily distinguished by the dark staining of their cytoplasm and their ellipsoid shape.

Electron microscopically, myelinated fibers are very rarely seen (Fig. 3 a, b). The majority of fibers are unmyelinated. The extracellular space is very wide. Cross- and longitudinally-sectioned fibers are arranged in an irregular pattern. Just prior to myelination, the diameter of the fibers increases (Fig. 3a, b).

This increase in diameter appears to be a prerequisite for myelination, because all the unmyelinated fibers in the prospective medullary ray of one folium have only half the size compared to those in which myelination starts (Figs. 2b, 3a, b). Concomitantly to the thickening of the unmyelinated fibers the content of organelles in the axoplasm changes, in particular the number of neurofilaments increases. A

Myelination of Cerebellar Cortex 511

Fig. la-d. Cerebellar cortex of the cat at different times of postnatal development, a three hours post partum, b 27th day of postnatal life, e 60th day of postnatal life d adult cat 756 days of age. Note the increase in thickness of the cerebellar cortex and the poorly developed intragranular and supraganglionic plexus in the 60-day-old cat. At this developmental stage the outer granular layer exhibits only one layer. In the adult cat, all plexus in the cerebellar cortex are well developed. Vermal part of lob. II. x 230

dis t inc t ion between c l imbing and mossy fibers c anno t be made at this deve lopmenta l s tage (Figs. 2 b, 3 a, b). However , also in the adu l t cat mossy and c l imbing fibers are ha rd to dis t inguish in the centra l medu l l a ry ray.

The inner g ranu la r layer, unti l the ten th pos tna ta l day, is free o f o l igoden- drocytes . Mossy and c l imbing fibers wi thout a myel in sheath are seen runn ing

512 W. Lange

Fig. 2a-d. Central medullary ray of one folium in the vermal part of lob. II a At three hours post par tum only glial cells are visible, which cannot be classified as astrocytes or oligodendrocytes, b between the 10th and 14th days post partum, the unmyelinated fibers begin to orientate, e the oligodendrocytes at this time are arranged in rows, d between the 30th and 40th postnatal days most of the fibers in the central medullary ray are myelinated and exhibit a distinct lamination of the white matter. Fig. a, c, d • 560, b x 5550


Fig. 3a. Axon in the vermal part of lob. II, which having reached the critical diameter of about 1 lam is enveloped by only some myelin lamellae. Cat, 3 hrs p.p. • 27,500 b Myelinated axon of about 1 Ixm in diameter in an oligodendrocyte. In the inset, smaller unmyelinated axons are visible. Cat, 3 hrs p.p. Vermal part of lob. II, • 25,600

through this layer (Fig. 4a), although the synaptic contacts with the different cell types are only partly established.

At the 14th postnatal day the number of oligodendrocytes in the central medullary ray increases markedly (Fig. 2c). This process occurs very rapidly, the time course of which being very distinct and showing only a minor time. The oligodendrocytes are arranged in rows and show a pearl-string pattern. Under the electron microscope it can be seen that at this time the majority of the unmyelinated fibers begin to organize (Fig. 2b). During the first postnatal days the fibers do not show any kind of alignment. With the beginning of the multiplication of the oligodendrocytes a parallel orientation with only a small intercellular space becomes evident.

Shortly afterwards, about the 16th postnatal day of life, the first oligodendrocytes appear in the internal granular layer. They are first visible in the lower third, just above the central medullary ray, in which the myelogenesis meanwhile proceeds. Step by step the oligodendrocytes invade other parts of the internal granular layer, followed by the myelination of the Purkinje cell axons.

The first myelinated Purkinje cell axons (Fig. 4b) can be easily recognized by reason of their dark axoplasm, which contains some longitudinally oriented neurofilaments. An even better distinguishing feature is the presence of endoplasmic reticulum arranged in a tubular fashion. The myelin sheath of the

Fig. 4a. Unmyelinated fiber, running through the inner granular layer. Vermal part of lob. II. Cat, 3 hrs p.p. x 4840 b . Myelinated Purkinje cell axon at 14 days post partum. Vermal part of lob. II, x 7150 c. Adult cerebellar cortex; all fiber systems are myelinated. A mossy fiber is seen entering a glomerulus cerebellosus. Vermal part of lob. II, x 2800


Fig. 5. At the 55th day of postnatal life no myelinated parallel fiber or recurrent collateral in the lower third of the molecular layer is visible. Vermal part of lob, II, x 7500

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Fig. 6a. Lower third of the molecular layer in a 60-day-old cat. Compare with Fig. lc. Only few myelinated fibers in the supraganglionic plexus are visible, x 820. b. Lower third of the molecular layer in the adult cat. Compare with Fig. 1 d. The supraganglionic plexus is fully myelinated. • 820

Purkinje cell axon, at this time, is relatively thin, the number of myelin lamellae increasing during the further development.

Meanwhile the myelination in the central medullary ray is almost completed. The orientation of the myelinated fibers further proceeded and the axons of the Purkinje cells form distinct bundles (about 30th postnatal day), which reach the cerebellar nuclei at the shortest distance. On their way a lamellation in the white matter becomes visible (Fig. 2 d), which apparently is established very early during postnatal life.

At this time, about the 35th day of postnatal life, the myelination in the internal granular layer has proceeded. All Purkinje cell axons are myelinated and also most of the climbing fibers (Fig. lc). Even the majority of the mossy fibers are by now myelinated and loose their myelin sheath, when they penetrate a glomerulus cerebellosus (Fig. 4c).

The external granular layer in the cat disappears about the 60th day of postnatal life (Fig. 1 c). Until this time the supraganglionic plexus in the lower third of the molecular layer cannot be identified (Figs. 5, 6 a). The formation of the plexus initiates with an augmentation of the oligodendrocytes in the Purkinje cell layer. The oligodendrocytes during this phase are frequently found lying in pairs, a feature which may be interpreted as evidence for mitosis in loco. Few myelinated fibers are visible at this time, but the plexus is still poorly developed (Fig. 6a). This


can be clearly demonstrated, when one compares the supraganglionic plexus in a 55 days old cat with that of a 2.5-years-old cat. In the 55-day-old cat (Fig. 6 a) the lower third of the molecular layer comprises only very few myelinated fibers, which are of a fairly thick caliber. In the adult cat, (Fig. 6b), the supraganglionic plexus is very well developed and occupies the lower third of the molecular layer. The majority of fibers are very thin and of the same caliber; the small number of thicker fibers are apparently recurrent collaterals of the Purkinje cell axons. By means of the electron microscope, the different fiber types in the supraganglionic plexus are better distinguishable.

Beyond the 90th day of life no other fibers become myelinated. The increase of the volume-fraction of myelinated fibers in the further development of the cerebellar cortex until the 90th to 160th day of postnatal life is due to a thickening of the individual myelin sheaths. Quantitative data on this aspect will be published separately.

Discussion

De Sanctis (1898), Vogt (1905), Naito (1923), Riese (1925), and Jakob (1928) investigated the myelination of the cerebellar cortex in man. According to de Sanctis, the first myelinated fibers are visible in the central medullary ray and the cortex of some parts of the vestibulo-cerebellum in fetuses aged seven to eight months. Vogt, Naito and Riese report the onset of myelination somewhat later in the intrauterine or extrauterine life. However, all investigators agree that the first myelinated fibers are visible in the central medullary ray of a folium.

As in other parts of the central nervous system, e.g. the corpus callosum (Fleischhauer and Wartenberg, 1967) or in the optic nerve (More et al., 1976), the onset of myelination is associated with two events: an increase in the diameter of the unmyelinated fibers and an increase in the number of glial cells (Fleischhauer and Hillebrand, 1965; Fleischhauer, 1968). The increase in diameter seems to come first, because at the first day of life there are only a few fibers with two or three myelin lamellae, which have already reached the critical diameter of 1 Ixm (Fig. 3 a). All the other fibers lacking a thin myelin sheath, are much thiner at this time. Even when the unmyelinated fibers start to arrange themselves in a parallel direction, the diameter has not markedly increased.

However, at this time, about the 12th to 24th day of postnatal life, mitoses of the glial cells in the central medullary ray are frequent. The glial cells become distinguishable in astrocytes and oligodendrocytes, and start to arrange themselves in rows; the latter being a typical feature during myelogenesis in the central nervous system (Fleischhauer, 1968; Moore et al., 1976). But the results obtained by Fleischhauer and Moore et al. cannot be directly compared with those obtained in the cerebellum, because in the corpus callosum and the optic tract already at the time of birth the unmyelinated fibers appear to be arranged parallel. In the cerebellum the unmyelinated fibers are mixed up and are later on organized in a preferential direction. Whether the parallel arrangement of the fibers induces the augmentation of glial cells or vice versa cannot be decided at this time. It also seems

518 w. Lange

impossible to determine from the fine structure of the fibers in the medullary ray, whether they are mossy or climbing fibers.

In the internal granular layer, oligodendrocytes and myelinated fibers appear later than in the central medullary ray. Furthermore, there is a gradient between the inner and the outer zone in the granular layer. In the inner zone of the internal granular layer myelinated Purkinje cell axons are first visible, very seldom in the outer zone. A little later, myelinated mossy and climbing fibers also appear in the inner zone. From this point of view it seems possible, that the findings reported by de Sanctis (1898), Vogt (1905), Naito (1923) and Riese (1925) are correct, who all state that myelination proceeds from the medullary ray into the inner granular layer. This standpoint is confirmed by the fact that the oligodendrocytes migrate from the white matter into the granular layer.

Also Moore et al. (1976) found that there are differences in the time sequence of myelination between the optic nerve and the optic tract. All these results seem to indicate that myelination does not take place at the same time over all the length of one axon.

Even more difficult is it to decide this problem for the supraganglionic plexus, where myelination takes place about the 50th day of postnatal life. Only a quantitative study will shed light on this question.

In spite of the fact that in the newborn kitten there are only a few myelinated fibers, Crepel (1971, 1972a, b, 1974) Crepel et al. (1976); Delhaye-Bouchaud (1976); Martinez et al. (1971); and Puro and Woodward (1977a, b) were able to demonstrate an electrical activity in the cerebellar cortex. At this time the cerebellar circuit already functions, but the latencies are very long. This indicates that both the pathways and the synaptic contacts are established very early after birth (Larramendi, 1969; Mugnaini, 1969). In the fourth week after birth the latencies in the rat change to the adult values. This means a decrease about the factor 10. Puro and Woodward (1977a, b) explain these shorter latencies with a maturation in the time scale of synapses. An aspect, which has not been considered by these authors, is the myelination of the ascending and descending fiber systems. Unmyelinated fibers with a diameter of about 0.3-1 ktm have a conduction velocity of some 0.5-2 m p.s., while myelinated fibers with a thin myelin sheath like those in the adult cerebellar cortex (1-3 lam in diameter) have a conduction velocity of about 3-15 m p.s. These fiber spectra are similar to those found in the immature and mature cerebellar cortex. Therefore, a decrease in the latencies in the cerebellar circuit, even by a factor of 10 could be explained by the myelination of the ascending and descending fiber systems.

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Accepted November 19, 1977

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The myelination of the cerebellar cortex in the cat