Does silver impregnate normal and degenerating boutons? A study based on light and electron microscopical observations of the inferior olive

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BRAIN RESEARCH

DOES SILVER I M P R E G N A T E N O R M A L A N D D E G E N E R A T I N G BOUTONS? A STUDY BASED ON L I G H T A N D E L E C T R O N MICROSCOPICAL OBSERVATIONS OF T H E I N F E R I O R OLIVE

FRED WALBERG Anatomical Institute, University of Oslo, Oslo (Norway) (Accepted February 10th, 1971)

INTRODUCTION

The argyrophilia of boutons and axons depends upon factors poorly understood. Although several efforts have been made to clarify whether axons as well as boutons are impregnated in silver-stained sections in normal and operated animals2,5,0,8,10-15, 18,23,z9, no general conclusions have yet been reached. The present study deals with the problem whether the rings and clubs (so-called boutons) present in silver-stained sections of the inferior olive are the boutons of the central nervous system. The observations on operated adult cats show that the rings and clubs in Glees' sections in the inferior olive are cross-sectioned normal or degenerating terminal myelinated axons, and not boutons. Degenerating boutons were not impregnated in the Nauta-Laidlaw stained sections used in the present study, but degenerating boutons were responsible for the smallest light-microscopically visible argyrophilic particles seen in the Fink-Heimer sections. MATERIALAND METHODS Twelve cats were anesthetized with Nembutal. Lesions were made with a Horsley-Clarke stereotaxic instrument in the left mesencephalic reticular formation rostral to the inferior olive. A diagram of one of the lesions is shown in Fig. 1. The survival time of the animals was 6, 7 or 11 days. Eleven cats were perfused with a rinsing solution followed by aldehydes either through the left ventricle (2 cats) or the abdominal aorta (9 cats). The rinsing solution consisted of 200 ml 0.9% NaC1 buffered with 0.16 M sodium cacodylate (10 ml/liter) and with CaC12 • H20 (230 mg/liter) added. The first aldehyde solution consisted of 4% formaldehyde (freshly made from paraformaldehyde) in 500 ml 0.16 M sodium cacodylate. After dissolution of the paraformaldehyde the pH was adjusted to 7.4 with a few drops of 1 M HC1. CaC12 • H20 (230 mg/liter) was then added. This and the first solution were perfused at 38°C. The second aldehyde solution consisted of 2 liters of 2.5% glutaraldehyde in 0.16 M Brain Research, 31 (1971) 47-65

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sodium cacodylate with 10 ml 1 ~ CaCl2/liter. This solution was perfused at 3°C. The procedure was similar to that used by Sousa-Pinto and Vaaland z6. The entire brain with the rostral part of the cervical spinal cord was dissected out, and stored in glutaraldehyde for some days or several weeks. The brain stem was then isolated. The mesencephalon was separated and further processed for investigation of the lesions. Histological examination revealed that the lesions were all of the type shown in Fig. 1. The medulla was cut horizontally either on a kryotome after storing in DMSO (for details see ref. 32) or on a TC-2 tissue sectioner. In the former, sections were cut 20 or 30/zm thick; in the latter, 100-200 #m. The sections and slices were stained according to the methods of Glees 4, Nauta-Laidlaw 22 and Fink-Heimer 3. Drawings of the sections and slices were made before staining. After staining, small pieces were isolated from the left and right inferior olive in distilled water under the dissecting microscope. The pieces were taken from the rostral part of the medial accessory olive (see Fig. 1), and were postfixed in 1~o OsO4 solution, buffered with Millonig's 19 solution. The pieces were dehydrated in graded solutions of ethanol or acetone, and embedded in Araldite according to the method of Holl~inder 16. The ultrathin sections were stained with lead citrate 27. Some cryostate sections were only postfixed in 170 OsO4 solution, and not stained with silver. These sections were processed for electron microscopy, and served as control for the preservation of the tissue before staining with silver. In some animals blocks from the right inferior olive (unoperated side) were selected for the same purpose. One operated cat was treated as described by Guillery 9, i.e., the animal was perfused with a buffered Ringer solution followed by the aldehyde mixture introduced by Karnowsky 17. The blocks isolated from the rostral part of the left medial accessory olive in this animal were postfixed in 1.3 ~ OsO4 in a 0.067 M s.-collidine buffer. The blocks were stained with 1 70 phosphotungstic acid in absolute ethanol and embedded in Araldite. The ultrathin sections from these blocks were carefully studied to show whether, in PTA-treated material, neurofilaments arranged in the form of a ring were present in the normal and degenerating boutons of the inferior olive. One operated cat needs special comment. Cat B.St.L. 124 (described in a previous publication 28) was operated on 4 December 1956 and killed on 12 December. This cat was included in the present study because the Glees sections were extraordinarily good and showed a great many open rings and compact clubs (Fig. 8a). One of the Glees sections that had been stored for 14 years was therefore selected for electron microscopy. The section was isolated by placing the object-glass in ajar with xylol for 24 h, thus permitting the cover glass to float away. The section was then transferred to 10070 ethanol, and gradually hydrated to water. Postfixation in 1 ~o OsO4 lasted for 1.5 h, and the section was thereafter dehydrated through another graded series of ethanol. Dissection of the pieces to be examined in the electron microscope was then performed in 10070 ethanol under the dissecting microscope. The areas isolated corresponded to the region of the rostral part of the medial accessory olive indicated in Fig. 1. The pieces were transferred to Araldite and polymerized. The ultrathin sections obtained from the 20 #m thick slices were stained with uranyl acetate and lead citrate, and examined in the electron microscope. Two series in Brain Research, 31 (1971) 47-65

ROSTRAL

R

L

dm.c.c. d.I.

L

DORSAL

VENTRAL

Fig. 1. Diagram showing the lesion (above) and horizontal sections through the left inferior olive (below) in cat B.St.L.124 with a survival time of 6 days. The dots indicate the regions of the inferior olive that receive fibers from the destroyed mesencephalic area. The rectangle indicates the part of the medial accessory olive studied in all animals. All the illustratioas of Figs. 2-11 are taken from this area. Abbreviations: fl, nucleus fl; d.l., dorsal lamella; dm.c.c., dorsomedial cell column; D, dorsal accessory olive; F.l.m., fasciculus longitudinalis medialis; F.r., fasciculus retroflexus; L, left; M, medial accessory olive; N.c.p., nucleus of the posterior commissure; N.D., nucleus of Darkschewitsch; N.int., nucleus interstitialis (of Cajal); N.n.III, nucleus of the Ilrd nerve; N.r.l., nucleus reticularis lateralis; R, right; v.l., ventral lamella; v.l.o., ventrolateral outgrowth.

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Fig. 2. Electron micrographs of Glees sections from cats 1367 (a and b) and B.St.L. 546 (c and d) with a survival time of 7 days. a and b, normal boutons b, bl and b2 show only unspecific silver precipitate; d, dendrite; a, al and a2, small myelinated axons showing a moderate silver precipitate; arrow in a points to the synaptic region; c and d, degenerating boutons b2; bl and b3 are normal boutons; d, dendrite; arrow points to postsynaptic thickening at the degenerating bouton. × 30,000. addition to single sections were studied in this animal. The first series consisted o f 60 sections (20 grids), the second o f 130 sections (28 grids). The findings in the series will be described below. OBSERVATIONS

Light microscopy of Glees sections The periaqueductal region o f the mesencephalon and its surroundings is one o f the main areas giving origin to efferent fibers to the inferior olive. Lesions like those shown in Fig. 1 were followed by an intense degeneration in certain parts o f the nuclear complex, especially the rostral part o f the medial accessory olive and the principal olive (for details see ref. 28 and Fig. 1). The degeneration appeared after 6 and 7 days

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in Glees sections as a great increase in 'boutons', rings as well as compact clubs. In addition, some degenerating fine-beaded fibers were present. The photomicrograph of Fig. 8a, when compared with that from the unoperated side (Fig. 3g) shows the difference in 'bouton' density.

Fig. 3. Electron micrographs of Glees sections (g is light micrograph) from cat B.St.L. 546 (see above). a-c, cross-sectioned normal small myelinated fibers; d-f, cross-sectioned degenerating small myelinated fibers; g, Glees section from medial accessory olive on unoperated (right) side in cat B.St.L. 124. Note that no rings ('boutons') are present (cf. text and Fig. 8a); h, one degenerating (a3) and three normal (al, as and a4) small myelinated fibers, a - f and h, × 30,000; g, × 1560.

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This characteristic finding makes the rostral part of the medial accessory olive a favorable area for an electron microscopical study of rings and clubs present in Glees sections. Sections from this region were therefore used in the present study.

Electron microscopy of Glees sections The ultrastructural preservation of the tissue was not satisfactory, In most instances, however, the various nervous components could be identified electron microscopically. Control sections from the contralateral inferior olive or from the olive on the operated side which had not been processed for silver staining, showed that the tissue was well preserved after perfusion with aldehydes. The impaired quality of the Glees sections was therefore due to the staining procedure. Silver grains varying from 3 to 35 nm were present in the electron micrographs from these sections. The smallest particles lay scattered on most structures, and occurred as a fine precipitate. Larger grains were related to nuclei of the various types of cells and to myelinated fibers of all sizes. In the normalfibers the myelin sheaths and usually the mitochondria were free from large silver particles which showed great affinity to the axoplasm. The silver particles could sometimes be seen on tubuli or filaments in the axons, but it was usually impossible to relate the grains to specific organelles. A typical silver precipitate on normal fine-myelinated fibers is shown in Fig. 3a-c. In cross-sectioned fibers where mitochondria were present (Fig. 3a), the silver was precipitated so as to form more or less complete rings. Cross-sectioned small normal myelinated fibers without mitochondria appear as compact structures (Fig. 3b, c). The degenerating myelinated axons (Fig. 3d-f, h) also showed precipitates in the axoplasm. The particles were of the same size as those present in normal axons. At the 7th day after operation a certain number of the mitochondria were enlarged and sometimes filled almost the entire axoplasm. Others were disintegrating, and their fragments could not be clearly distinguished from the degenerating axoplasm. Many of the degenerating mitochondria had acquired a greater affinity for silver. Hence, cross-sectioned degenerating fine-myelinated fibers showed either silver precipitate only in the matrix, as do normal fibers, or also on the mitochondria. In the latter event, such axons appeared as compact clubs. The normal boutons showed no, or very few, silver particles. The particles were mostly small, and lay scattered in the matrix (Fig. 2a, b). The degenerating boutons showed the same relation to silver as did the normal ones. A few scattered particles were found on some, but most degenerating boutons were free from silver (Fig. 2c, d). No precipitate occurred, which was reminiscent of the rings and compact clubs of the light microscopical Glees sections. A heavy, localized silver precipitate was found in some dendrites. The majority of the particles appeared to lie in the matrix (Fig. 4a, b). Silver particles occasionally showed affinity for astrocytic filaments.

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Fig. 4. a and b, Electron micrographs of Glees sections from cat B.St.L. 546, to show conspicuous local silver precipitate in dendrites, d. A small normal myelinated axon with silver precipitate in a is present at the upper right side of the dendrite. The dendrite in b is probably sectioned close to the perikaryon. Arrow points to endoplasmic reticulum. × 30,000.

Electron microscopy of Nauta-Laidlaw sections The N a u t a - L a i d l a w sections had small silver particles occurring as a fine precipitate on all structures in the tissue. In addition, larger silver particles were concentrated on degenerating myelinated fibers. The particles were present in the degenerating axoplasm, and especially on degenerating mitochondria, but at the 7 day stage it was difficult to identify axoplasmic organelles. The myelin was usually free from silver and the sections investigated did not show the staining artifact sometimes present in N a u t a - L a i d l a w sections 14. Cross-sections o f degenerating small myelinated fibers are shown in Fig. 5b, c. N o r m a l myelinated fibers have very little silver precipitate.

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Fig. 5. Electron micrographs of Nauta-Laidlaw sections from cat B.St.L. 547. a, the degenerating bouton; b, shows only little silver precipitate; d, dendrite; arrows point to synaptic regions, b and c, cross-sectioned degenerating small myelinated fibers with conspicuous silver precipitate, x 30,000.

Fig. 6. Electron micrographs of Fink-Heimer (II)-stained sections from cat B.St.L. 548. a and b, degenerating boutons with conspicuous silver precipitate. The number of coarse silver particles varies in the sections, but in all degenerating boutons it is clearly higher than that found on the surrounding structures; d, dendrites; arrows point to synaptic regions, c shows a cross-sectioned degenerating small myelinated fiber with a conspicuous silver precipitate. × 30,000. The n o r m a l a n d degenerating b o u t o n s showed only few and scattered silver particles (Fig. 5a). T h e few particles in degenerating b o u t o n s a p p e a r e d mostly related to the disintegrating m i t o c h o n d r i a .

Electron microscopy of Fink-Heimer sections T h e sections showed a fine diffusely distributed silver precipitate on all tissue structures. The precipitate consisted o f needle-like and small g l o b u l a r particles. In addition, coarser silver particles were c o n c e n t r a t e d on degenerating m y e l i n a t e d fibers and on degenerating b o u t o n s (Fig. 6a-c).

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The silver granules in the degenerating fibers were aggregated in smaller or larger groups, or filled the axon entirely. Ring-like figures were encountered, as well as c o m p a c t clubs. The myelin showed only unspecific precipitates of silver. The argyrophilia o f the degenerating b o u t o n s was less intense t h a n that o f the degenerating fibers. The individual silver particles could be identified b u t the c o n c e n t r a t i o n o f silver o n the degenerating b o u t o n s was clearly greater t h a n o n the s u r r o u n d i n g tissue structures.

Fig. 7. Electron micrographs of normal and degenerating boutons in 3 cats with a survival time of 7 days. a, normal bouton; b, with filaments, synaptic vesicles and a mitochondrion. Boutons of this type with filaments are extremely rare in the rostral part of the medial accessory olive (cat 1368, section stained with lead citrate); b and c, degenerating boutons; b and bl, as such structures appear when lead citrate is used as a stain; b2, normal bouton; arrow points to synaptic region (cat 1367); d and e, normal, bl 3, and degenerating, b, boutons, as such structures appear when PTA is used as a block stain; d, dendrites; arrow points to synaptic region (cat B.St.L. 553). × 30,000. Brain Research, 31 (1971) 47-65

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Fig. 8. Light and electron micrographs of a 14-year-old Glees section, a, the micrograph shows the great increase in the number of open rings and compact clubs occurring in the rostral part of the medial accessory olive after a lesion of the mesencephalon and after a survival of 6 days (cf. Figs. 1 and 3g). × 1520. b-j, Electron micrographs of the light micrograph shown in a; f, normal bouton; b, showing only unspecific silver precipitate; d, dendrite; arrow points to synaptic region; b, c and e, degenerating boutons, b. Note that these, like the normal ones, have only unspecific precipitate. Arrows point to synaptic regions, d and g-j, cross-sectioned small impregnated normal and degenerating fibers showing heavy silver precipitate. The silver accumulations form rings (d, g, i, j) or compact clubs (h) (cat B.St.L. 124). x 30,000.

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Electron microscopy of PTA-stained blocks Gray and Guillery6, 9 have emphasized that neuro-fibrillar rings are clearly visualized in boutons only when PTA is used as a stain. The inferior olive of one operated cat was therefore treated with this stain (see Material and Methods), and examined in the electron microscope. Examination of the PTA-treated material from the inferior olive showed that filaments, tubuli and synaptic contacts were well impregnated when PTA was used as a stain. However, the findings are essentially the same as those made in sections stained with lead (cf. especially Fig. 7b, c, with Fig. 7e). Note that neither normal nor degenerating boutons of the PTA-stained blocks examined had filaments arranged in the form of a ring (Fig. 7d, e). The filamentcontaining bouton shown in Fig. 7a was one of the rare exceptions of this type found in the inferior olive. This particular bouton was found in a section stained with lead citrate.

Electron microscopy of a 14-year-old Glees section During the present study it became clear that neither normal nor degenerating boutons were responsible for the light microscopically visible rings and clubs of the Glees sections. Hence it was essential to study, in the electron microscope, one of the author's old favorite Glees sections, which had been processed in the 'classical' manner. The section had been optimally stained, and showed a large number of rings and clubs in the rostral part of the medial accessory olive 28. Electron microscopy of single sections gave essentially the same results as the Glees sections from the above-described animals. The tissue was not well preserved, but neurons and glial cells could easily be seen. The neuropile was partly destroyed, but normal and degenerating boutons were clearly identified. None of these boutons showed a concentrated silver precipitate (Fig. 8b, c, e, f). Such precipitate was present only in axons. The myelin sheath was free from impregnation. The silver particles in the small myelinated axons were concentrated in the axoplasm, but it was impossible to relate them to particular organelles. The mitochondria were not stained. A distinction between normal and degenerating fibers could not always be made. Typical impregnated axons are shown in Fig. 8d, g-j; Fig. 8d is a degenerating fiber. In the first series of 60 sections, one particular visual field was photographed. This field was identified from the location of the surrounding vessels and cells. The magnification of the electron micrographs was 10,000. An overlay of acetate foil (0.2 mm) was placed above each micrograph. The single argyrophilic particles or aggregates of particles in the axons were then indicated with ink on each foil. The foils were placed above each other and orientation was maintained with the vessels and cells as points of reference. None of the normal and degenerating boutons of this series showed a concentrated precipitate of silver, but the micrographs showed an unspecific precipitate which also occurred on boutons. The other series consisted of 130 sections (a few sections were lost). An identifiable visual field was also selected in this series and photographed. The argyrophilic

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Fig. 9. Two rings ('boutons') reconstructed from a series taken from a Glees section in cat B.St.L. 124. The silver particles in each of 26 successive electron micrographs were drawn with ink on acetate foil overlying each micrograph. The foils were then placed above each other and photographed. The rings represent cross-sectioned small myelinated fibers. The diameter of the rings corresponds to that of the previeusly described rings in the medial accessory olive1. None of the electron micrographs of this and another series showed concentrated silver precipitate in normal and degenerating boutons (for details see text). Scale line 1/~m.

axons were indicated as in the first series. Two o f the rings that could be followed through all the sections were studied in detail in the first 46 sections (grids 1-12) at a higher magnification (primary magnification 10,000). The electron micrographs o f these sections had a magnification o f 30,000. The rings were indicated on foil overlying 26 successive electron micrographs. The rings had a diameter o f approximately 1.3 # m , and are shown in Fig. 9. N o n e o f the normal and degenerating boutons o f this series showed a precipitate o f the type found in the axons. In this respect the findings were similar to those made in the first series. DISCUSSION The neurofibrillar rings and clubs o f silver sections are usually considered by light microscopists to be boutons o f the central nervous system. The electron microscope made it possible to identify organelles related to argyrophilia and to decide whether only normal or also degenerating boutons were stained with silver. The first step towards a solution o f this problem was made by G r a y and Guillery 5. The gray matter o f the spinal cord o f unoperated cats and rats was examined in silverstained sections, and Bielschowsky-stained blocks f r o m the same areas were studied in the electron microscope. The authors found that some boutons had filaments arranged in the f o r m o f a ring in their matrix, and concluded that neurofilaments were the basis of the argyrophilic material o f boutons. They suggested that the classical rings and clubs o f light microscopy are boutons containing neurofilamentous rings. Brain Research, 31 (1971) 47-65

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Glees sections

There is no reason to doubt that Gray and Guillery's conclusions are correct as regards their observations in the spinal cord. However, in a previous publication, the present author expressed doubt that their statement was valid generally 29. His observations on the inferior olive did not agree with those on the spinal cord. Experimental light microscopical studies with the Glees method had shown that, after lesions of the part of the mesencephalon giving origin to fibers to the inferior olive, and after 6-7 days of survival, a great increase in the number o f ' b o u t o n s ' occurred in certain parts of the inferior olive. The 'boutons' were especially abundant in the rostral part of the medial accessory olive, where they appeared as rings or solid clubs 28. Here more than 200 such 'boutons' were present per microscopical field ( × 1200). In unoperated animals only 0-2-3 'boutons' were found in the same region. Experimental electron microscopical studies with the same type of lesion, with identical survival times and from the same region, showed degenerating boutons of the dark type with no identifiable filaments in lead-citrate-stained sections 30. It was concluded that in the inferior olive, degenerating dark boutons devoid of filaments could show argyrophilia. It was tacitly surmised that the rings and clubs present in the Glees sections from the inferior olive and studied in the light microscope were boutons. This conclusion was later criticized in a review by Gray and Guillery 8, who maintained that the rings and clubs present in Glees sections from the inferior olive, and described by the present author, like those found in the spinal cord, were boutons with filaments (pp. 142-143). The discrepancies between their findings and those made in our laboratory were explained as being due to methodological differences, and it was claimed that only when PTA is used as a stain can filaments in boutons be clearly visualized. Gray and Guillery used this stain in the spinal cord, and in the inferior olive we had used lead citrate. In the present study the rostral part of the medial accessory olive was examined after 7 days survival in material fixed and stained according to the technique used by Guillery 9. The findings were essentially the same as those made in lead-citratestained sections. Normal boutons with neurofilaments arranged in the form of a ring were not found in the PTA-treated blocks hitherto examined. It is more important, however, that none of the degenerating boutons showed neurofilamentous rings. Such boutons cannot therefore be responsible for the great increase in open rings and compact clubs seen in light microscopically studied Glees sections from the inferior olive of operated cats with a survival of 6-7 days. Observations made in other nuclei in our laboratory support the view that methodological differences cannot explain the discrepancy between Guillery's findings and ours. Filaments were clearly visualized in normal as well as degenerating boutons in the vestibular nuclear complex when lead citrate was used as a stain (see, e.g., ref. 20, Figs. 5-9, and ref. 21, Fig. 16b). In our Glees sections neither normal nor degenerating boutons showed a silver precipitate heavy enough to make them visible as rings or clubs in the light microscope. The cross-sectioned normal and degenerating small myelinated axons were, on the Brain Research, 31 (1971) 47-65

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other hand, like the larger ones, heavily stained in the electron micrographs. They had diameters down to about 0.5/~m. Most of the light microscopically visible rings and clubs present in the medial accessory olive and measured in Glees sections, ranged from 0.8 to 2/~m (ref. 1). The electron micrographs of the Glees-stained material showed furthermore that myelin and mitochondria were usually unstained in normal fibers (see also ref. 5). The silver particles were aggregated in the axoplasm, but apparently were not related only to one organelle. Small normal myelinated axons in the rostral part of the medial accessory olive had, in addition to mitochondria, tubuli and a few scattered filaments. The latter were so few that they alone cannot be responsible for the argyrophilia making the axons visible in the light microscope. Furthermore, the filaments were usually not arranged in the form of a ring. The degenerating small myelinated fibers at the 6-7-day stage were swollen, and had a beaded appearance. The swollen parts usually included large disintegrating mitochondria. Unlike normal mitochondria, the degenerating mitochondria were often stained in the Glees sections taken at the 6-7th day of survival. Except for unspecific precipitates, the myelin was unstained, as in normal fibers. The 14-year-old Glees section showed the same electron microscopical findings as did the other animals used in this study. None of the boutons showed a heavy argyrophilia (Fig. 8b, c, e, f). The normal and degenerating fine-myelinated fibers had, on the other hand, a conspicuous silver precipitate (Fig. 8d, g-j). The serial sections confirmed this observation and made it possible to make a reconstruction of rings. Two of these are shown in Fig. 9. In the light microscope these same sections showed a concentration of rings and compact clubs in the inferior olive (Fig. 8a). Great difficulties arise when one selects material for electron microscopy. Furthermore, the present findings can be met with the critical comment that silver grains in some (or several) structures can be lost during preparation of the sections for electron microscopy. This comment is obviously relevant for all the previous studies of this type. The present findings are, however, based on observations on a large number of blocks and on serial sections. The following conclusion is therefore considered permissible. The ring-like, so-called boutons, present in Glees sections of the inferior olive in unoperated cats and seen in the light microscope, were cross-sectioned small myelinated fibers with one or more centrally placed mitochondria. These mitochondria were unstained but surrounded by silver precipitate. The compact 'boutons' (clubs) of unoperated animals were cross-sectioned myelinated axons of the same size in regions where mitochondria are absent. The ring-like 'boutons' of operated cats at the 6-7day stage, were cross-sectioned parts of degenerating small myelinated axons in which the degenerating (?) mitochondria were not yet stained. The compact 'boutons' were cross-sectioned parts of the same type of degenerating axons including stained degenerating mitochondria, or without mitochondria. This conclusion needs the following comment. Students familiar with the light microscopical appearance of the open rings ('boutons') present in Glees sections will observe that the rings are clearly focused only through a small part of a section

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15-20 #m thick. They are absent above or below this focus, i.e., they present themselves clearly as 'rings'. This could only occur if the terminal myelinated fiber had a local deposit of silver in its axoplasm. Electron micrographs show that this is so. The bouton 'tail', occasionally observed in the inferior olive, probably represents closely related longitudinally sectioned small fibers or outpocketings of axoplasm in the crosssectioned fiber. These statements are based on observations made in the series in this study, and do not allow for general conclusions as regards the nature of rings and clubs seen when the Glees method or other non-suppressive silver stains are used in light microscopy. However, it is appropriate to mention the observations by Pecci Saavedra et al. 23, who studied degeneration in the optic pathways in monkeys and examined with the electron microscope Glees-stained sections from the lateral geniculate nucleus 4 and 5 days after enucleation. The findings were compared with those made under the light microscope. These authors found that normal and degenerating boutons showed very little silver deposit, but that degenerating myelinated optic fibers were heavily impregnated. Ring-like 'boutons' occurred where mitochondria were present in their center. Compact 'boutons' appeared where mitochondria were absent. The degenerating optic tract terminals reacted with an intense neurofilamentous hypertrophia, and such boutons would be expected to stain heavily with silver. No such correlation was found and the authors concluded that argyrophilia is slight in synaptic terminals where neurofilaments are abundant. However, these findings, like those in other regions, do not permit general conclusions. Thus, it is possible and even probable, that degenerating filament-filled boutons in other nuclei are argyrophilic. In our laboratory, filament-filled degenerating Purkinje cell axons in Glees-stained sections showed great affinity for silver 31. Studies now in progress reveal that this is also so for the filament-filled degenerating boutons. Guillery9 has recently presented an alternative explanation of the nature of the so-called boutons (rings) from the lateral geniculate body of the cat seen in the light microscope in Glees sections. He was unable to find boutons with neurofilamentous rings in this region in the electron microscope. He observed, however, that some dendrites showed appendages in which neurofilaments were arranged in the form of a ring. He therefore concluded that the light microscopically visible rings (boutons) in the Glees-stained lateral geniculate nucleus are filament-filled dendritic appendages. However, decisive evidence for the suggestion that ring-like structures in the lateral geniculate nucleus are silver-stained filaments in dendritic appendages can only be obtained from a study of Glees sections in the electron microscope. Such a study is necessary to decide whether the situation in the lateral geniculate nucleus is different from that in the inferior olive. Ralston and Herman 25 have arrived at the same conclusion as Guillery concerning the nature of 'neurofibrillar rings' present in Glees-stained sections from the ventral basal thalamie nucleus of the cat studied in the light microscope. These rings are said to be silver-stained neurofilaments in dendrites. However, in a subsequent experimental study of the lemniscal afferents to the same region in animals with a Brain Research, 31 (1971) 47-65

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survival time of 3-4 days, Ralston 24 finds a great increase in the 'neurofibrillar rings' in the sections studied in the light microscope. Contrary to those present in unoperated animals, these rings are considered to be degenerating filament-filled boutons. However, electron microscopy of Glees-stained sections of this nucleus must also be done before decisive conclusions can be drawn. Although, as stressed above, the findings made in the inferior olive do not allow general conclusions, the Glees method generally fails to stain 'boutons' in the cerebral and cerebellar cortices. It follows, from the observation made in the present study, that one could not expect to find 'boutons' in regions where the majority of the terminal fibers are unmyelinated. Nauta-Laidlaw sections

Problems occur when one tries to make general conclusions from observations based on sections where suppressive silver stains are used. The Nauta-Laidlaw sections showed that degenerating boutons had only very little silver precipitate. Other students have, however, shown degenerating boutons with heavy silver deposits in NautaGygax sections 11. The degree of suppression will therefore be one of the factors that decides whether degenerating boutons can be visualized in the light microscope. Electron microscopy of Nauta-Gygax and Nauta-Laidlaw sections is therefore necessary if a student wants to make decisive conclusions concerning the nature of the light microscopically visible silver particles. Fink-Heimer sections

The claim, made by Heimer and Peters 15 that boutons are stained when the Fink-Heimer method is used, has been met with the argument that the published electron micrographs show degnerating boutons too faintly stained to be visible as silver particles in the light microscope 1°. However, the authors had purposely used diluted silver solutions, and this criticism is therefore not appropriate 13. Heimer's previous detailed light microscopical study, showing clear regional differences in the distribution of degeneration when the Fink-Heimer or other silver methods are used 12, also strongly indicates that the fine dust of the Fink-Heimer sections represents degenerating boutons and their terminal unmyelinated stalks. The observations made in the inferior olive support Heimer's conclusions. The degenerating boutons shown in Fig. 6a, b are clearly visible in the light microscope. Such boutons would be responsible for the fine dust present in Fink-Heimer sections from the medial accessory olive. A shorter suppression of the sections would probably have resulted in an even better impregnation of the degenerating boutons. However, although the present findings are clear, the pitfalls can be great even when the Fink-Heimer method is used. For instance, unspecific precipitates may complicate the picture. The presence of fine dust in light microscopical sections only at certain limited stages of survival would therefore be a valuable support for the interpretation of the dust as representing bouton degeneration. The disappearance of the dust after a slightly longer survival time than that when the dust is found would indicate that Brain Research, 31 (1971) 47-65

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the degenerating boutons had been phagocytosed and digested by glial cells. To what extent unmyelinated degenerating terminal fibers participate in the formation of the fine dust remains to be shown. The findings made in the substantia nigra by Grofovh and Rinvik7 indicate that such fibers do not swell during degeneration, but that they show fragmentation at early survival stages. Their diameter ranges from 0.2 to 0.5 #m, and they would therefore be the smallest visible particles in the light microscope. Dendrites

A brief comment is necessary as regards stainability of dendrites in Glees sections. Several sections in the present study showed intense local impregnation in some dendrites in the electron microscope. Most of these were small (Fig. 4a), but a few were probably dendritic stems (Fig. 4b). The sections gave no clear information as to the nature of the organelles responsible for the argyrophilia, but it is clear that no rings of filaments occurred in the argyrophilic portions of dendrites hitherto observed. Such local impregnation of dendrites would be clearly visible in the light microscope, and could obviously be interpreted as 'boutons'. The present study, together with those made by Guillery9, Ralston~5, Ralston and Herman 24 and Pecci Saavedra et al. 23, shows that one should be extremely careful with conclusions as to the nature of rings and clubs present in Glees sections studied in the light microscope. Silver precipitation in axons and dendrites, as well as in boutons, can obviously lead to the formation of structures which in the light microscope would give the student the impression that he deals with the boutons of the central nervous system. Electron microscopical studies are necessary to show whether a similar situation exists when other non-suppressive silver stains are used. The present findings also give a warning against general statements when suppressive silver methods are used. SUMMARY

The problem of argyrophilia in normal and degenerating axons and boutons has been studied in experimental material from the inferior olive. The conclusionsare based on single and serial electron micrographs of Glees, Nauta-Laidlaw and FinkHeimer sections. The rings seen when the Glees method is used are not filament-filled boutons of the type described by Gray and Guillery5. They are cross-sectioned small myelinated normal or degenerating axons, in which the axoplasm, but not the centrally located mitochondria, are stained. Compact 'boutons' in the same sections are crosssectioned normal fibers without mitochondria or degenerating fibers of the same type, where, in addition to axoplasm, degenerating mitochondria are stained. In Nauta-Laidlaw sections of the present study, degenerating boutons have very little precipitate, and are not visible in the light microscope. The fine dust present in the Fink-Heimer sections is degenerating boutons and the coarser argyrophilic particles are fragments of degenerating fibers. The observations made in the inferior olive show that one should be extremely Brain Research, 31 (1971) 47-65

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careful with statements concerning the nature of open rings and clubs ('boutons') seen when silver stains are used. Studies from various regions have so far shown that it is impossible to reach general conclusions. The capriciousness of the silver methods should always be considered when such techniques are used. ACKNOWLEDGEMENTS

The author is greatly indebted to Mrs. J. Line Vaaland for expert technical assistance, especially with the serial reconstructions.

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