Cell Tiss. Res. 166, 489496 (1976) Cell and Tissue Research �9 by Springer-Verlag 1976

The Myelinated Parallel Fibers of the Cerebellar Cortex and Their Regional Distribution* **

W. Lange

Anatomisches Institut der Universit/it Kiel (Direktor: Prof. Dr. med. H. Leonhardt)

Summary. In the cerebellar cortex of the Rhesus monkey and the cat, the supraganglionic plexus in the molecular layer exhibits regional differences. The plexus is very well developed in the vermal parts of the anterior lobe, but only poorly developed in the nodulofloccular lobe. Most of the fibers of this plexus are myelinated parallel fibers, which synapse in the typical manner with dendritic thorns of Purkinje cells. Only very few fibers of this plexus are recurrent collaterals of Purkinje cells. Their distribution throughout the cerebellar cortex does not display regional differences. These findings agree with physiological data on the disinhibition of Purkinje cells in different parts of the cerebellar cortex.

Key words: Cerebellar cortex (Rhesus monkey, cat) - Supraganglionic plexus - Myelinated parallel fibers - Regional distribution - Electron micro- scopy.

Introduction

The supraganglionic plexus of the cerebellar cortex, which is situated in the lower third of the molecular layer just above the Purkinje cells, has previously been thought to consist exclusively of recurrent collaterals of Purkinje cells (Fox et al., 1964; Eccles et al., 1967). This plexus displays differences in various species (very sparse in the rat, Palay and Chan Palay, 1974; very extensive in the Rhesus monkey and cat, Lange, 1972). Furthermore, regional differences in the cerebellar cortex have been demonstrated in the cat, Rhesus monkey

Send offprint requests to: Prof. Dr. W. Lange, Abteilung Anatomie der Medizinischen Fakult~it an der Rhein.-Westf. Techn. Hochschule, 5100 Aachen, Melatener Stral3e 211, Federal Republic of Germany

* Dedicated to Professor Dr. med. Drs. h .c .W. Bargrnann in honour of his 70th birthday. ** Supported by the Deutsche Forschungsgemeinschaft (La 184/2).

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Fig, 1. (a) Cerebellar cortex of the Rhesus monkey, nodulus. The supraganglionic plexus in the lower third of the molecular layer is very sparse. Kltiver-Barrera (1953). x480. (b) Cerebellar cortex of the Rhesus monkey, vermal part of lobule VIII. The supraganglionic plexus is well- developed. Note the ascending myelinated fibers, which traverse the plexus to the molecular layer. Kltiver-Barrera (1953). x480

The Myelinated Parallel Fibers of the Cerebellar Cortex 491

and man (Lange, 1972). In the phylogenetically eldest part of the cerebellum, the nodulofloccular lobe, the plexus is poorly developed, but very well-developed in the anterior lobe, especially in its vermal parts.

If this plexus is composed only of recurrent collaterals of Purkinje cells, the regional differences in its distribution throughout the cerebellar cortex would suggest a stronger disinhibition of Purkinje cells by recurrent collaterals in the anterior lobe than in the nodulofloccular lobe. Recent physiological experi- ments failed to demonstrate these differences (Leicht and Oppermann, personal communication). For this reason it appears necessary to reexamine the supra- ganglionic plexus and to re-evaluate its fiber constituents.

Material and Methods

Two Rhesus monkeys (Macaca rhesus) and two cats (Felis domestica) were perfused with glutaralde- hyde (6% in 0.05 M Millonig-phosphate-buffer, pH 7.2). The cerebella were removed and pieces of the vermal part of the anterior lobe were postfixed for one hour in a 1% OsO4 solution and embedded in Epon 812. Semithin sections were stained by the method of Ito and Winchester (1963), thin sections with lead citrate and uranyl acetate.

For paraffin sections some parts of the anterior lobe were postfixed in Bouin's fluid and 8 ~tm sections were stained with Luxol fast blue according to Kliiver-Barrera (1953).

Results

In paraffin sections from perfused brains stained with Luxol fast blue, the supraganglionic plexus can be easily recognized. It is situated in the lower third of the molecular layer just above the Purkinje cells. The blue-stained myelinated fibers of this plexus are cut in cross section because these fibers run parallel to the axis of the folium (Fig. 1 b). Between these fibers, other myelinated fibers are visible, running directly from the granular layer to the molecular layer. These fibers, however, can only be recognized in those parts of the cerebellar cortex in which the supraganglionic plexus is well- developed (Fig. l b). In the nodulus where the supraganglionic plexus is very sparse myelinated fibers coming from the granular layer and running per- pendicularly to the molecular layer are difficult to identify or may be totally absent (Fig. 1 a). The myelinated fibers in the supraganglionic plexus almost all have the same diameter. However, some fibers do differ distinctly from the majority of the other myelinated fibers. They do not possess a round outline and are twice as thick as other fibers of the plexus (Fig. 2). The thicker fibers are found equally distributed in all parts of the cerebellar cortex.

With the electron microscope, ascending myelinated fibers can be easily demonstrated. They contain microtubules and occasionally filaments. They are

Fig. 2. (a) Purkinje cell from the vermal part of lobule VIII, Rhesus monkey. The rectangle indicates the area magnified in Fig. 2b. x 840. (b) The supraganglionic plexus consists of fibers of equal diameter. Note the ascending myelinated fiber and the cross-sectioned larger fibers in this plexus, x 2,000

492 W. Lange

Fig. 3. Cerebellar cortex of the Rhesus monkey. Ascending fiber from the granular layer, which bifurcates in the lower third of the molecular layer, x 8,000

generally accompanied by bundles of unmyelinated granule cell axons. In the lower third of the molecular layer the myelin sheath ends and the axons bifurcate in a T-like fashion (Fig. 3). After the bifurcation, the fiber branches again and is enveloped by a myelin sheath (Fig. 3) which encloses the axon along its course through the molecular layer, where the parallel fibers meet.

Nodes of Ranvier are often seen in myelinated fibers of the molecular layer. Here, the axons are enlarged, like a varicosity, in unmyelinated parallel fibers and are also filled with vesicles. These vesicles are round and have a mean diameter of about 380 A (Fig. 4a). Elongated mitochondria frequently surround the synaptic vesicles in these varicosities (Fig. 4a). Synaptic junctions are mainly detectable when myelinated fibers traverse thorns of Purkinje cells (Fig. 4b). At the synapse the axon is enlarged. The vesicles are round and of the same diameter as in the nodes of Ranvier. The typical membrane thickening of a synaptic contact can also be observed. The myelin sheath ends before the axon swelling and does not reappear after the synaptic junction. The unmye- linated fiber accompanies the other parallel fibers and forms varicosities for a distance of about 2 p.m from the first synaptic contact. From these observations it appears as though some of the myelinated fibers in the lower third of the molecular layer loose their myelin sheath very soon after the bifurcation of the axon, which originates from the granule cells in the depth of the granular layer. But most of the myelinated fibers within the supraganglionic plexus are

The Myelinated Parallel Fibers of the Cerebellar Cortex 493

Fig. 4. (a) Myelinated fiber of the supraganglionic plexus, which runs parallel to other fibers. The part of the axon which is not enveloped by a myelin sheath resembles a varicosity of parallel fibers and contains synaptic vesicles, x 17,800. (b) Varicosity o f a myelinated fiber of the supragang- lionic plexus, which synapses with a dendritic thorn of a Purkinje cell. Cerebellar cortex of the Rhesus monkey, x 28,500

enveloped throughout the molecular layer, and are filled with synaptic vesicles at the nodes of Ranvier. However, the diameter of these varicosities is not as large as in those fibers in which the myelin sheath ends very soon after its appearence.

In the Rhesus monkey and cat, myelinated fibers of the molecular layer are numerous in the vermal parts of the anterior lobe. These same fibers are very seldom in identical areas in the rat. In marsupials, e.g. the opossum (unpub- lished data), the myelinated fibers coming from the depths of the granular

The Myelinated Parallel Fibers of the Cerebellar Cortex 495

layer ascend in bundles to the molecular layer, in contrast to the cat and Rhesus monkey where these fibers mostly ascend individually.

Discussion

In 1911, Ram6n y Cajal denied the existence of myelinated parallel fibers in the cerebellar cortex in spite of the fact that K611iker (1900) had already demon- strated myelinated fibers in the molecular layer of some marsupials. Most investi- gators (Fox et al., 1964; Eccles et al., 1967) believed that the myelinated fibers in the molecular layer were only recurrent collaterals of Purkinje cells, building up the supraganglionic plexus. This plexus exhibits regional differences in density in man (Jakob, 1928), Rhesus monkey and cat (Lange, 1972). In the vermal parts of the anterior lobe, the plexus is very well-developed, but poorly developed in the nodulofloccular lobe. Most of the fibers have approximately the same diameter (1.5/am), but in all parts of the cerebellar cortex there are some mye- linated fibers, the diameter of which is twice that of most of the plexal fibers. These thicker myelinated fibers in the lower third of the molecular layer are short and do not end on Purkinje cell dendrites, but rather on secondary and tertiary dendrites. Thus, it appears that the larger myelinated fibers in the molecular layer are branches of recurrent collaterals of Purkinje cells, which synapse in the typical manner as previously described by Larramendi and Lem- key-Johnston (1970) and Palay and Chan-Palay (1974).

The smaller fibers of the supraganglionic plexus have the same direction as the parallel fibers and make synaptic junctions with dendritic thorns of Purkinje cells. At the nodes of Ranvier they form swellings filled with synaptic vesicles, similar to parallel fibers. All these findings indicate, in agreement with Mugnaini (1972), that the smaller fibers are myelinated parallel fibers, arising from myelinated granule cell axons. However, Mugnaini did not mention their regional distribution. The present results point out that more than 90% of the myelinated fibers of the supraganglionic plexus are parallel fibers. In those parts in which the supraganglionic plexus is well-developed as in the anterior lobe (Lange, 1972), the plexus is mainly composed of myelinated parallel fibers, while in the nodulus the number of recurrent collaterals and myelinated parallel fibers is the same. These findings are in good agreement with the physiological results of Leicht and Oppermann (personal communication), who could not find any difference with regard to the disinhibition of Purkinje cells by recurrent collaterals in the vermal parts of the anterior lobe and in the nodulofloccular lobe. Additionally, these physiological data seem to confirm our morphological findings, which indicate that the distribution of recurrent collaterals in all parts of the cerebellar cortex is the same.

Fig. 5. (a) Myelinated fiber of the supraganglionic plexus with a vesicle-containing varicosity. Note the preterminal fiber (arrow) at the opposite side of the vesicle-containing part. • 33,000. (b) Magnification of Fig. 5 a. The membrane thickening at the varicosity (arrow) indicates a possible axo-axonal synapse on a myelinated parallel fiber, x 65,000

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There is another point of interest. The myelinated parallel fibers exhibit varicosities and synapses with Purkinje cell dendrites most frequently at the nodes of Ranvier. Occasionally at such swellings, another fiber can be observed which synapses at the opposite side of the synaptic junction with a parallel fiber (Fig. 5 a, b).

These axo-axonal synapses in our material are rare, and we could not recog- nize the source of this preterminal fiber. Interestingly, Anderson (1975) reported similar findings in the hippocampus and discussed the question whether the excitation in such a fiber is cut off at this axo-axonal junction. Considering the rarity of such synaptic junctions, another question seems more relevant: It is open to discussion whether the higher conducting velocity from the mossy fibers to the Purkinje cells, observed in those parts of the cerebellar cortex in which the myelinated parallel fibers are very numerous, is of any significance for the cerebellar circuit.

References

Anderson, L. : Paper presented at the Institute of Anatomy, University of Kiel 1975 Eccles, J., lto, M., Szent/tgothai, J. : The cerebellum as a neuronal machine. Berlin-Heidelberg-New

York: Springer 1967 Fox, C.A., Siegesmund, K.A., Dutta, C.R. : The Purkinje cell dendritic branchlets and their relation

with the parallel fibers: Light and electron microscopic observations. In: Morphological and biochemical correlates of neural activity (eds. : M.M. Cohen and R.S. Snider), p. 112-141. New York: Harper & Row 1964

Jakob, A. : Das Kleinhirn. In: Handbuch der mikroskopischen Anatomie des Menschen (ed. : W. von M611endorff), Bd. 4, Teil 1. S. 674-916. Berlin: Springer 1928

Kliiver, H., Barrera, E. : A method for combined staining of cells and fibres in the nervous system. J. Neuropath. exp. Neurol. 12, 40ff403 (1953)

K611iker, A. : Sulla presenza di un gran numero di fibre nervose a mielina nello strato moleculare del cervelletto dei Monotremi e di un Marsupiale. 1900 (Quoted by Ramon y Cajal 1911)

Lange, W. : Ober regionale Unterschiede in der Myeloarchitektonik der Kleinhirnrinde. I. Der Plexus supraganglionaris. Z. Zellforsch. 134, 12%142 (1972)

Larramendi, L.M.H., Lemkey-Johnston, N.J.: The distribution of recurrent Purkinje collateral synapses in the mouse cerebellar cortex: An electron microscopic study. J. comp. Neurol. 138, 451482 (1970)

Leicht, R., Oppermann, J. : Personal communication, 1975 Mugnaini, E. : The histology and cytology of the cerebellar cortex. In : The comparative anatomy

and histology of the cerebellum. The human cerebellum, cerebellar connections and cerebellar cortex, (eds. : O. Larsell and J. Jansen), p. 201-264. Minneapolis: The University of Minnesota Press 1972

Palay, S., Chan-Palay, V. : Cerebellar cortex. Cytology and organization. Berlin-Heidelberg-New York: Springer 1974

Ram6n y Cajal, S.: Histologie du syst~me nerveux de l'homme et des vert6br6s. Paris: Maloine 1911

Received August 18, 1975 / in revisedJbrm September 30, 1975