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Further Electron Microscopical Investigations of the Inferior Olive of the Cat F R E D WALBERG Anatomical Institute, University of Oslo, Oslo

Most electron microscopical studies of the central nervous system have so far been made on tissue taken at the surface. Relatively little is known of the ultrastructure of subcortical regions. Below some findings made in the feline inferior olive with the electron microscope will be reported. Some of the blocks were selected from a cat, where following anaesthesia with nembutal the brainstem was dissected free. Thin horizontal slices were cut from the medulla and immediately immersed in chilled 2 % osmium tetroxide fixative with 4.5 % sucrose buffered with veronal acetate to p H 7.2-7.5. The last slice was isolated within 6 min after cessation of the circulation. Fixation was continued for about 2 h. After this the tissue was dehydrated in acetone and embedded in Araldite. During fixation in osmium pieces of the olive were isolated under a dissection microscope. Isolation for electron microscopy of parts of the central nervous system not immediately accessible from the surface presents considerable difficulties. First it is important to make a dissection of the region without compressing or destroying the part to be isolated, and the material has to be fixed as soon as possible after cessation of the blood circulation. Therefore, although it turned out to be possible to obtain suitable material from the inferior olive in animals not perfused, this procedure is far from ideal, and can be largely avoided by intravital fixation. Various fixatives have been tried for intravital perfusion. The best appears to be osmium tetroxide. Palay et al. (1962) have recently described a technique by which intravital perfusion with this fixative is made. The authors have used rats and fish, and the structures of the cells in the central nervous system are excellently preserved. The high cost of osmium fixatives restricts their use in the investigation of larger animals, e.g. adult cats. Good formalin fixation of the fine structures of the cells has so far not been obtained. Recently, however, Holt and Hicks (1961) have introduced a new formaldehyde fixative which has been shown to preserve the cellular components well in their excised pieces of various tissues. The fixative used is a solution of 4 % formaldehyde buffered at p H 7.2 with 0.067 Mphosphate and containing 7.5 % sucrose. This fixative has been used in some of the adult cats employed in the present study of the inferior olive. Following anaesthetization with nembutal, the cats were first perfused intravitally with 100 ml Ringer solution at 37". Then perfusion followed with References p . 74/75

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approximately 750 ml chilled fixative. The bottle was placed 120 cm above the animal, and the perfusion lasted approximately 30 min. After this, the medulla was isolated, transferred to chilled fixative, and cut in thin slices with a razor blade. From each slice, pieces of the inferior olive were isolated under the dissection microscope. After isolation, the pieces were transferred to osmium tetroxide fixative, fixed for 1.5 h, dehydrated in ethyl alcohol or acetone and embedded in Araldite. The micrographs show that the components of the cells are well preserved. Furthermore, the extracellular spaces are usually about 200 A in the cats perfused with formalin, i.e. of the same sizes as in cats from which tissue is fixed directly in osmium. These observations make it likely that intravital perfusion with formaldehyde will be valuable when in larger animals a rapid fixation of regions not immediately accessible from the surface is wanted. The findings show that the fine structure of the inferior olive in many respects is similar to that found in other regions of the central nervous system examined with the electron microscope. On some points, the structure of the nuclear complex differs from what has been observed in other regions of the central nervous system. In the micrographs boutons, dendrites, perikaryon, axons and glial cells are identified. Since the detailed structure of these elemerits has been considered in a recent publication (Walberg, 1963) only certain patterns found in the olive will be dealt with. Terminal boutons

The terminal boutons, like those described in other regions of the central nervous system, contain synaptic vesicles. These are usually not concentrated towards the presynaptic membrane. Characteristic of the boutons is the high content of mitochondrial profiles. As many as eight in a single bouton are not unusual. According to Gray (1961a) axo-dendritic synapses may be divided into two types, type 1 and 2, according to their morphological picture. While in the first there is no thickening of the presynaptic membrane, the density of the postsynaptic membrane is very marked. In addition, dense material is present in the synaptic cleft. Also the cleft between the pre- and postsynaptic membranes is enlarged. In type 2 synapse there is a moderate thickening of the pre- as well as of the postsynaptic membrane. The thickenings are of the same size. No dense material is present in the synaptic cleft, and this is not enlarged. In the olive the majority of the axo-dendritic synapses are similar to Gray’s type 2. Relatively few are of type 1. In addition to these two categories there is also a third type, which is found relatively often. Like type 1 this synapse is characterized by thickening only of the postsynaptic membrane. This thickening is, however, not very marked, and the synaptic cleft is not enlarged. Furthermore, no dense material is present in the cleft. This synapse, therefore, appears to represent an intermediate type. Figs. la-c show schematical drawings of the three synapse types. For micrographs of the synapses the reader is referred to the paper mentioned above (Walberg, 1963). At present no correlation can be established between structure and function of the

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synapses in the central nervous system. However, it should be noted that in their extensive study of the stratum radiatum in the rat hippocampus, Westrum and Blackstad (1962) found only type 1 synapse. Furthermore, all axo-somatic synapses in the central nervous system in regions hitherto examined have been shown to be of type 2. The same finding has also been made in the olive. On the other hand, a relatively

Fig. la-c. Drawing illustratingthe three types of axo-dendritic synapses found in the inferior olive. Fig. Id shows the dendro-dendritic contact present in the same nucleus. Abbreviations for all figures: a, axon; b, bouton; bm, basement membrane;c, Golgi complex; ca, capillary; cv, compound vesicle; d, dendrite; e, granular endoplasmic reticulum; en, endothelial cell; f, filaments; g, part of fibrous astrocyte; n, nucleus; m, mitochondrium. Unless otherwise indicated, the scale line represents 1 p.

great number of the axo-dendritic synapses in the olive appears to be of an intermediate type. This is an indication that a subdivision of synapses into different groups may not be fortunate, since this may give a too schematical picture of the possible properties of these structures. Axons In the few places in the olive where a bouton is found connected with an unmyelinated axon, the latter, which here will be called a terminal axon, is seen to contain filaments, not tubuli (Fig. 9). As regards small unmyelinated axons which are not terminals, these contain tubuli. Small myelinated axons display both filaments and tubuli. The micrographs from the olive show that both organelles are present in myelinated axons up to about 2 p References p. 74/75

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Fig. 2. Region of the olive showing glial processes (g) filled with filaments. At arrows glial sheets which are continuous with processes. To the left, part of nerve cell (n) at the level of the perikaryon, to the upper right myelinated axon (a) cut longitudinally. In this, filaments (f) as well as tubuli (t) are present. At (e) granular endoplasmic reticulum. Formalin perfusion and osmium fixation. Rostra1 part of medial accessory olive. Abbreviations, see legend to Fig. 1.

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(Fig. 2). On the other hand, in myelinated axons from the olive larger than this, only filaments have been seen. So far tubuli have been described only in small, unmyelinated axons (Gray, 1959b; Blackstad and Kjaerheim, 1961). Myelinated axons have been shown to have filaments only. More observations are needed to reveal whether small myelinated axons in addition to filaments display tubuli also in other regions. A special type of small myelinated axons has been found in the medial accessory olive and in the ventral lamella. These are shown in Figs. 3-5. As is evident two or

Fig. 3. Serial sections through two myelinated axons surrounded by a common myelin sheath. The myelin sheath in axon a, is made up of 14, in a2 of 10 larnellae, respectively. Note that division into two myelinated axons, each with a double myelin sheath, is almost completed in section d. Asterisks in section c indicate three glial sheets embracing myeiinated axon a2. Outer sheet ends at double arrow. Single arrow points to sheet shown at higher magnification in inset in section d. The glial sheet between arrows in inset is only about 200 A wide. At the two arrows in section (a) the plasma membranes of the glial cells are in close contact. Rostra1 part of medial accessory olive. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1.

three axons are surrounded by a common myelin sheath. Only speculations can be made concerning the origin of these types of axons. A structure like that shown in Fig. 3 might be assumed to appear when an axon making a bend in an acute angle is References p . 74/75

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Fig. 4. Two myelinated axons included within a common myelin sheath. Only one myelin lamella appears to surround each axon. Arrow points to fusion of the lamellae. Ventral lamella of principal olive. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1. Fig. 5. Three myelinated axons included within a common myelin sheath. Ventral lamella. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1.

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sectioned close to the vertex. However, since the number of myelin lamellae in axon a1 and a2 is not the same (14 and 10, respectively), the possibility that they represent parts of the same axon can be excluded. In keeping with this is also the finding that even three myelinated axons can be included within a common myelin sheath (Fig. 5). The structures shown in Figs. 3-5 probably occur in regions of division of axons. Further evidence that they are due to a division of myelinated axons is provided by the following observation. In Fig. 3 two axons are followed in serial sections, and a final separation of these axons is indicated in the last micrograph. One might object that if this explanation is correct, a node of Ranvier should be present at the site of division of the axon. This has not been found in any of the micrographs of the axons here presented. However, although it is well known that nodes of Ranvier are present at the site of division of peripheral nerve fibres, very little is known concerning this point as regards central axons. Actually, although studies with the electron microscope have shown that nodes of Ranvier are present in axons also within the central nervous system (see e.g. Bunge, Bunge and Ris, 1960; Metuzals, 1960, 1962), they appear to be far less frequent than peripherally. Thus, although they have been found in the olive, only one node of Ranvier has so far been observed in the micrographs. The absence of nodes of Ranvier, therefore, does not militate against the assumption that close to the site of division of myelinated axons structures like those shown in Figs. 3-5 may be formed. Although it cannot be excluded that each of the fibres are individual axons lying very close and for some unknown reason included in a common myelin sheath, this explanation appears to be the most probable. As regards the small myelinated axons, i.e., axons below 2-3 p, there is apparently no relation between the total width of the fibres and the thickness of the myelin sheath. Large fibres may be surrounded by only a thin myelin sheath composed of a few lamellae, in width like that surrounding adjacent, much smaller, fibres. Examples of such fibres are shown in Fig. 8. The micrograph shows that of fibres a i and az, both having approximately the same total diameter, one has a large amount of axoplasm, the other very little. Whether the ratio of the axon diameter to the total diameter is inconstant also for larger fibres within the central nervous system, is not known.

Den& ites In a few places specialization of opposed membranes of dendrites has been found. The membrane thickenings appear to be the same on the two sides, and dense material between the contacting membranes is present (Fig. Id, for micrographs the reader is referred to Walberg, 1963). The same observation has been made by Gray (1961b) in the cerebellum between adjacent dendrites of granule cells. At present it is not known whether they correspond to desmosomes described in other tissues and belong to dendrites of the same or different cells. Glial cells As mentioned elsewhere (Walberg, 1963) glial sheets and processes are found in close approximation to all neural elements. These sheets may either form narrow References p. 74/75

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profiles in which no structures are present (Figs. 2 and 3), or they can be seen to be wider at one or both ends. Here filaments are present (Figs. 2,6 and 10). Also, sheets may be continuous with processes. The latter, which show densely packed bundles of filaments, are very characteristic of the inferior olive (Figs. 2, 3, 6, 7, 11 and 13-15).

Fig. 6. Part of neuropil showing relation between expansions from fibrous astrocytes and other elements. Processes filled with filaments (f) as well as ‘empty’ profiles (g) are seen. Arrows point to close contact between plasma membranes of fibrous astrocytes. Ventral lamella. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1. Fig. 7. Micrograph from other region of neuropil showing profiles of fibrous astrocytes. At arrow close contact between process filled with filaments (f) and glial sheet (g). Ventral lamella. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1 .

Depending upon the plane of section, the filaments are cut transversely, obliquely or longitudinally. The sheets as well as the processes can be followed to their parent cell. This has an oval nucleus and is relatively rich in organelles, a finding of interest when the classification of glial cells is considered, a problem recently discussed by Hartmann (1961). Typical glial cells are shown in Figs. 11 and 12. As is evident, filaments are also present in the perikaryon, either single or packed in bundles. Furthermore, there is a double nuclear membrane. Although the cytoplasm is relatively dense, the presence of filaments justifies that this type of cell is interpreted as a fibrous astrocyte. Golgi studies by Cajal(l909-11) and especially by Scheibel and Scheibel(l955) have revealed that fibrous astrocytes are abundant in the inferior olive, and that they are interspersed between the various elements in the neuropil. The micrographs in Figs. 2, 3, 6, 7, 10 and 11 show details. Also the myelinated axons are surrounded by the glial sheets, which in some regions lie very closely packed. The illustration in Fig. 3 shows an example of this. The asterisks indicate three sheets lying immediately adjacent to the outer myelin lamella of axon a2. At the lower end of the axon the distance between the outside of the plasma membranes of the inner sheet (arrow Fig. 3c, inset Fig. 3d) is only about 200 A.

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Another example of the relation between the glial sheets and the other structures

of the neuropil is given in Fig. 10. Here the part of the bouton visible in the micrograph is entirely embraced by two glial sheets. The organelles of the perikaryon of the fibrous astrocytes in the inferior olive are shown in Figs. 11 and 12. As is evident, the cytoplasm contains ribosomes with and

without connection with endoplasmic reticulum, Golgi complex, mitochondria and filaments. Also compound vacuoles are found. In some regions there is only a narrow strand of cytoplasm outside the nucleus. This strand may be filled with filaments (Fig. 12). Furthermore, the filaments in the perikaryon are single or grouped in relatively loose bundles (Fig. 1l), contrasting to the filaments in the processes. Here they are packed very densely (see e.g. Figs. 2, 6, 7 and 11). The filaments measure about 80 A in diameter and show signs of beading in some regions.

Fig. 8. Small myelinated axons in the neuropil. In spite of the finding that axon a1 and as have almost the same total diameter there is only a narrow myelin sheath around the former. At arrow small myelinated axon with sheath of almost same width as axon al. Medial accessory olive. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1. Fig. 9. Axon terminating in bouton. Note that at arrow only filaments are present in the terminal axon. Medial accessory olive. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1. Fig. 10. Bouton (b) which in this section is entirely surrounded by glial elements (g). Note that profile gl and g2 are parts of same process. Ventral lamella. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1.

Relatively empty glial profiles are also found. These profiles are wider than the sheets, and are sometimes continuous with these (Figs. 6 and 7). They are poor in organelles and contain only a few cisternae and vesicles, the latter in some profiles References p. 74/75

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being as small as about 500 A. These profiles are cross sections of peripheral parts of glial-cell cytoplasm and may be mistaken for empty sections of boutons. The fibrous astrocytes of the olive in the adult cat have some resemblance to those found in the white matter of the spinal cord in the same animal by Bunge, Bunge and Ris (1960). When their Fig. 3 is compared with Figs. 11 and 12 shown here, it is apparent that individual filaments and grouping of filaments in bundles are found in the perikaryon in both types of cells. Furthermore, the cisternae of the endoplasmic reticulum associated with ribosomes show no orientation. Also free ribosomes, Golgi complex and mitochondriae are present. In the glial cells of the olive the latter mostly appear to have a higher electron density than those found in adjacent structures. However, the bodies of varying densities present in the fibrous astrocytes in the spinal cord have not been identified in astrocytes from the olive. On the other hand, compound vacuoles are present in the latter (Fig. 11). On other points the astrocytes in the olive differ more from those found in the spinal cord. Thus the processes with densely packed filaments are very characteristic of the astrocytes in the olive, they are actually present in almost all micrographs. Typical examples are shown in Figs. 2, 6, 7 and 11. As mentioned previously, the processes are often in continuity with thin glial sheets, in which no filaments are found (see e.g. Fig. 2). The same type of processes entirely filled with filaments has been shown to be present only in reactive astrocytes at the margin of healing cortical wounds in the cerebral cortex of the rat (Palay, 1958, Fig. ll), and in cells termed reactive macroglia in the spinal cord of adult cats 460 days after operation (Bunge, Bunge and Ris, 1961, Fig. 23). In, the fibrous astroglial cells of the cerebral cortex of the normal rat (Schultz el al., 1957; Maynard et al., 1957; Farquhar and Hartmann, 1957; Gray, 1959a, 1961a) and cat (Pappas and Purpura, 1961) such processes have not been described. Also, although filaments are present in the processes of the glial cells in the optic nerve of the mouse (Peters, 1962, see e.g. his Figs. 6-9), they are not studded with these structures. Furthermore, the attenuated glial sheets which extend from the perikaryon or from the cell processes and intrude between all other neuron elements are very characteristic of the fibrous astrocytes of the olive. Whether this difference is due to species differences or to variations in the sites of the cells in the central nervous system, is not known. However, it should be noted that Peters (1962) has found that in the toad fibrous astrocytes differ from those present in the same region in mouse and rat. Also it is not known whether the fine structure of the fibrous astrocytes is the same in newborn and adult animals in the same species. Referring to what is said here it is obvious that the cell classified by Bunge, Bunge and Ris (1961) as reactive macroglia and shown by them to be related to remyelination of axons in the spinal cord of adult cats is almost identical to that found in the inferior olive in adult normal cats. Only speculations can be made concerning the role played by the fibrous astrocytes in myelination of axons in the olive of normal cats. Although the thin sheets of fibrous astrocytes in many regions of the olive embrace and almost surround myelinated axons in the same manner as the processes of the reactive macroglial cells do in remyelination in the spinal cord (see Bunge, Bunge and Ris, 1961, Fig. 4), a continuity

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between the glial cell and the myelin sheath has not been observed in the olive. On the other hand, oligodendrocytes like those shown by Farquhar and Hartmann (1957),

Fig. 11. Part of perikaryon of fibrous astrocyte. The nucleus (n) is surrounded by a double membrane. Filaments in bundles (f) or single are abundantly present. Granular endoplasmic reticulum (e) and

Golgi complex (c) is seen, and arrows at (cv) point to compound vesicles. Arrow above (f) in upper right corner points to sheet intruding between bouton (b) and dendrite (d). In dendrite (d) in lower right comer a compound vacuole (cv) is present. The glial processes adjacent to the perikaryon (gl-gl) or penetrating into this (g5) are parts of the same cell or neighbouring fibrous astrocytes. Ventral lamella. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1. Fig. 12. Region of perikaryon from another fibrous astrocyte. Arrow points to double nuclear membrane. Note that at filaments (f) the cytoplasm forms only a narrow strand which at arrow in upper right corner appears as a thin sheet. Ventral lamella. Formalin perfusion and osmium fixation. Abbreviations, see legend to Fig. 1. References p. 74/75

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Schultz et al. (1957), Hartmann (1958), Bunge, Bunge and Ris (1961) and Peters (1962) (for classification of glial cells, see also Luse, 1958), have not been observed. Although oligodendrocytes are present in the olive (concerning the oligodendrocyte see especially the Golgi studies by Scheibel and Scheibel, 1955, 1958) processes and sheets of fibrous astrocytes are included in almost all micrographs from the olive hitherto examined. Whether the same distribution of fibrous astrocytes is found in the olive also in newborn kittens is not known. Referring to the fact that electron microscopical studies have shown that glial-cell types intermediate in structure between astrocytes and oligodendroglia are present in the central nervous system (see Hartmann, 1961 for references), and that Bunge, Bunge and Pappas (1962) have shown that the oligodendrocyte is the myelin-forming cell in the spinal cord, the possibility exists that in young cats the oligodendrocyte, in adult the fibrous astrocyte is the common glial cell in the olive. However, more studies are needed to reveal the nature of the myelin-forming cell in various parts of the central nervous system.

Fig. 13. Micrograph showing part of capillary (ca). Only glial feet of fibrous astrocytes (g) are in contact with the basement membrane (bm). At arrows close contact between plasma membranes of glial processes. Medial accessory olive. Formalin perfusion and osmium fixation. Abbreviations,see legend to Fig. 1.

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The specialization present at the contact region between adjacent glial-cell processes first described by Gray (1961a), and also found by Peters (1962) and Westrum and Blackstad (1962), is likewise present in the inferior olive of the cat. This has been briefly mentioned elsewhere (Walberg, 1963). At the site of contact the plasma membranes of the glial processes show an increased density, and between the membranes a third line is present. This is only seen at high magnifications. As demonstrated by Gray (1961a) the distance between the membranes is reduced to 150 8, at the place

Fig. 14. Other part of same capillary.Note the processes of fibrous astrocytes (g) adjacent to basement membrane. Abbreviations, see legend to Fig. 1. References p. 74/75

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of contact. The micrographs in Figs. 3,6,7 and 13 show examples of such membranes forming close contacts. As is obvious they are present between glial sheets (Figs. 3 and 6) as well as between sheets and processes filled with filaments (Fig. 7). Referring to the finding by Peters (1962) that in the optic nerve the processes containing filaments usually do not form close contacts with other glial membranes, the latter observation is of some interest. In the olive processes filled with filaments appear to join

Fig. IS. Section through endothelial cell (en) of a capillary. Along basement membrane (bm) five ‘empty’ profiles of fibrous astrocytes. These are indicated by asterisks. Note that the first of the profiles is interposed between basement membrane and glial process 81. The second profile begins at arrow to the left of axon al, narrows between the axons a1 and a2 at the two arrows to a thin sheet, and widens to the right of the axon. This region is indicated by an open triangle. The profile then again narrows in the region between the two arrows to the right of the open triangle and ends with a wider part indicated by a filled triangle. Medial accessory olive. Formalin Ferfusion and osmium fixation. Abbreviations, see legend to Fig. 1.

in contact with dial sheets rather frequently. On the other hand, close contacts have only occasionally been observed between membranes of opposing processes filled with filaments. Although being found relatively frequently the contacts are not very numerous, especially when the high number of glial sheets and processes in the olive are considered. The contacts appear mostly to be present where glial membranes meet end to end (Fig. 6). They are more seldom found where glial membranes lie parallel to each other over a longer distance. The functional importance of the glial contacts is not known. Gray (1961a) has

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suggested that they may seal off the extracellular spaces, so that metabolites are forced to traverse glial cytoplasm, a view also advocated by Peters (1962). However, more studies are needed to clarify whether this suggestion is correct. In the olive the vascular glial feet to a large extent are formed by glial processes filled with filaments(Figs. 13-14, see also Fig. 14 in Walberg, 1963). In micrographs of perivascular tissue from various regions published by previous authors, the glial feet in contact with basement membranes of capillaries mostly appear to be of the clear type (see e.g. Farquhar and Hartmann, 1957; Maynard et al., 1957; Hartmann, 1958; Gray, 1961a; Pappas and Purpura, 1961; Peters, 1962). Only a few places in the olive clear glial processes and thin sheets from such processes have been found adjacent to the basement membrane (Fig. 15). The membrane measures about 800 A at the narrowest place. In adult cats Donahue and Pappas (1961) found the membranes to be 1000 A. Expansions from fibrous astrocytes appear to be the only elements in the neuropil in contact with capillaries in the olive. Although in some regions other structures lie apparently very close to the basement membrane they are obviously always separated from this by glial cells. The micrograph in Fig. 15 is particularly illustrating. Here a very narrow glial sheet lies between two myelinated axons and the basement membrane. The sheet is part of a clear glial process. Furthermore, the capillary endothelium in the olive appears to form a continuous layer and not to be fenestrated, a finding in agreement with that made in the cerebral cortex by Maynard et al. (1957). As pointed out by Gray (1961a) and more fully discussed by Peters (1962) the finding that only astrocytes are found in direct apposition to the basement membrane, raises the question regarding the neural elements engaged in the blood-brain barrier. Referring to the comments given on this point especially by the latter author, it should be stressed that as in the optic nerve (Peters, 1962) also in the olive processes of fibrous astrocytes form a layer between capillaries and the other nervous structures. In the olive, however, most of the processes are filled with filaments. The same relation is probably present also in other regions. The data presented here, together with those given in a previous publication (Walberg, 1963) show that a certain area of the central nervous system, in addition to similarities with regions previously described, also has features characteristic of this area. ACKNOWLEDGEMENT

This study was supported by grant NB 0221544 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. The aid is gratefully acknowledged. SUMMARY

Perfusion with formalin has been used in this study in which the fine structure of the olive of the cat is considered. Since in a previous paper the various elements in the neuropil of the olive have been described (Walberg, 1963) special attention is here given to certain findings. Of these the structure of small myelinated axons and that of the glial cells should especially be mentioned. References p . 74/75

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SCHEIBEL, M. E..ANDSCHEIBEL, A. B., (1958); Neurons and neuroglia cells as seen with the light microscope. Biology of Neuroglia. W. F. Windle, Editor. Springfield, Illinois. Charles C. Thomas (p. 5-23). SCHULTZ, R. L., MAYNARD, E. A.,ANDPEASE,D. C., (1957); Electron microscopy of neurons and neuroglia of cerebral cortex and corpus callosum. Amer. J . Anaf., 100, 369-388. WALBERG, F., (1963); An electron microscopic study of the inferior olive of the cat. J. comp. Neurol., 120, 1-15. L. E.,ANDBLACKSTAD, T~.,(1962);An electron microscopic study of the stratum radiatum WESTRUM, of the rat hippocampus (regio superior, CA 1) with particular emphasis on synaptology. J. comp. Neurol., 119, 281-309. DISCUSSION

GLEES:Dr. Walberg’s use of formalin and fixation by perfusion is very important, because now classical neurohistology and electron microscopic histology appear possible on the same material. His findings of two medullated axons ensheathed by a common myelin is extraordinary and has not been seen in light microscopy. WALBERG: An obvious advantage when formalin fixatives are used, is that pieces can be taken of the same material for light microscopy. So far, however, in our laboratories the Glees’ sections made of material fixed with the formalin solution introduced by Holt and Hicks (1961) have not been usable. At present, nothing can be said whether this is due to the special formalin solution used for perfusion. Further work is necessary before any statement can be made on this point. VERHAART: Haggqvist’s method shows the axon and the myelin sheath in different colours and allows to distinguish myelinated fibres not surpassing 1 p in diameter, the sheath included. Still I never saw a common myelin sheath around 2 myelinated axons. WALBERG: One of the reasons why axons like those shown in the present communication are not seen with the light microscope, is probably that they are very small. Whether they are present in other regions than the inferior olive, is not known. VANDER Loos: In regard to the postsynaptic ‘membrane thickenings’ Dr. Walberg described, I would like to ask whether with higher resolution he was able to separate a membranous component (directly continuous with the non-synaptic membrane of the postsynaptic element) from a submembranous accumulation of electrodense material? Was it possible to make this distinction in very lightly stained and unstained preparations? Concerning the apparent non-existence of a constant ratio axon diameter/myelin sheath thickness in the inferior olive I can confirm this point for myelinated axons in the neocortex cerebri. WALBERG: The sections from the inferior olive have all been stained with uranyl acetate, and it has not been possible to separate between a membranous and a submembranous part on the postsynaptic side. Probably a decisive answer to the question could be given if instead lead monoxide was used as a staining agent. Studies from other regions of the central nervous system probably will reveal that also here there is no constant ratio for the axon diameter/myelin sheath thickness for small myelinated fibres.