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Further studies on silver impregnation of normal and degenerating boutons. A light and electron microscopical investigation of a filamentous degenerating system
BRAIN RESEARCH
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FURTHER STUDIES ON SILVER IMPREGNATION OF NORMAL AND DEGENERATING BOUTONS. A LIGHT AND ELECTRON MICROSCOPICAL INVESTIGATION OF A FILAMENTOUS DEGENERATING SYSTEM
F R E D WALBERG
Anatomical Institute, University of Oslo, Oslo (Norway) (Accepted July 25th, 1971)
INTRODUCTION
Experimental electron microscopical studies have revealed that most axons and boutons of the central nervous system when they degenerate show one of two types of reaction. The first of these is the dark type of degeneration. This is from the onset of degeneration characterized by a darkening of the axoplasm, in which the disintegrating organelles gradually become unidentifiable. This type of reaction has been described in detail by a variety of authors, and is the one most commonly found in fiber tracts of the central nervous system of vertebrates. The other type of degeneration is the filamentous reaction, which is characterized by an initial hypertrophy of axons and boutons. Their swollen parts become filled with filaments, resulting in a displacement and clumping of the other organelles. This type is not as common as the dark type of degeneration, but has been described in several pathways. The filamentous degeneration is characteristic for the Purkinje cell axons and boutons of the cat1, 20. The reaction is very prominent, and is from the 3rd-4th day followed by a rapid shrinkage and darkening of the degenerating fibers and boutons (see also ref. 20). To the author's knowledge no systematical combined light and electron microscopical study has been done on a filamentous degenerating fiber system, in order to reveal to what extent the various silver methods demonstrate degeneration of this type. The present paper deals with the filamentous degeneration of the Purkinje cell axons and boutons of the cat, as this appears in Glees, Nauta-Laidlaw, Fink-Heimer (I) and Eager sections. The findings show that the 4 methods differ markedly in their ability to impregnate filamentous degenerating structures. MATERIAL AND METHODS
Altogether 9 adult cats have been used. The cats were anesthetized with Nembutal, and lesions were made with a knife and sucker in various parts of the Brain Research, 36 (1972) 353-369
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cerebellar cortex. A diagram of one of the lesions is shown in Fig. I. The survival time of the animals was 3-8 days. All cats were perfused with a rinsing solution followed by aldehyde through the abdominal aorta. The rinsing solution consisted of 200 ml 0 . 9 ~ NaCI buffered with 10 ml/l of 0.16 M sodium cacodylate and with 10 ml 1 ~o CaCI2/I added. The first aldehyde solution consisted of 4 ~ formaldehyde (freshly made of 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 I N HC1. 230 mg CaCI2 was then added. This and the first solution was perfused at 38°C. The second aldehyde solution consisted of 2 liters of 2.5 To glutaraldehyde in the same cacodylate buffer, with 10 ml I ~ CaC12/liter. This solution was perfused at 3°C. The procedure was similar to that used by Sousa-Pinto and Vaaland 25. The brain with the rostral part of the cervical spinal cord was dissected out and stored in 4 ~ formaldehyde from some days to several weeks. The cerebellum was isolated and the extent of the lesion indicated on a diagram of the cerebellar surface. A frontal section was then made with a razor blade through the central part of the lesion, so that the cerebellum was divided into two parts. The depth of the lesion was in this way controlled, and further control of the depth was made during the subsequent treatment of the two halves. Some of them were cut on a kryotome after storing in DMSO 31, others were cut on a TC-2 tissue sectioner, and the rest on an ordinary freezing microtome. In the first and latter cases, 20 or 30/~m sections were cut. The chopper gave slices of 100-200 /zm. The sections and slices were stained according to the method of Glees 5, Nauta-Laidlaw '21, Fink-Heimer 4 and Eager 2.
Q
L
1450
R
Fig. 1. Diagram showing the extent of the lesion (hatching) in the cerebellum of cat 1450. The same type of lesion was made in the other cats used in this study. The degenerating Purkinje cell axons and boutons in the nuclei fastigii and interpositus were in all cats studied in the light and electron microscope after staining of sections and slices with the Glees, Nauta-Laidtaw, Fink-I-/eimer (I) and Eager methods. The findings made in the various animals are shown in Figs. 2-7. L, left; R, right. Brain Research, 36 (1972) 353-369
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Small pieces were isolated from the sections and slices containing the nuclei fastigii and interpositus underlying the lesions. The isolation was made in distilled water under the dissecting microscope after the impregnation. The pieces were postfixed in 1% OsO4 solution buffered with Millonig's19 solution. The pieces were then dehydrated in graded solutions of alcohol or acetone, and transferred to section embedding in Araldite according to the method of Hollfinder14. Some of the pieces were very thick, and therefore embedded directly in Araldite blocks. The ultrathin sections were stained with uranyl acetate and lead citrate 27, and studied in the electron microscope. Some of the sections cut on the kryotome and freezing microtome were, after staining with the various methods, mounted in the ordinary way for light microscopy. Such sections were compared with sections from the same animals studied in the electron microscope. OBSERVATIONS The findings were essentially the same in all animals studied. Some ultrathin sections showed a heavy silver impregnation, others had only a scanty precipitate. However, only the central part of an argyrophilic region of the used ultrathin section was photographed. Degenerating axons and boutons without silver precipitate described in the present study, were therefore always present directly adjacent to structures showing impregnation. The conclusions drawn concerning argyrophilia or lack of argyrophilia of degenerating axons and boutons are therefore considered to be valid. The following description is based on light and electron microscopical observations on sections from the nucleus fastigii as well as the nucleus interpositus underlying the lesions. Corresponding observations were made in the two nuclei, and no specification will therefore be made in the description of the findings. In the ultrathin sections degenerating fibers and boutons in various stages of degeneration were present. The majority of these were in most sections of the hypertrophic filamentous type. Some degenerating fibers and boutons had, however, reached a state intermediate between the hypertrophic and dark degeneration. Such structures were hypertrophic and dark, and the filaments could be difficult to identify. Other degenerating fibers and boutons were of the dark, collapsed type (cf. ref. 20). Fibers and boutons in all these 3 stages of degeneration were in several ultrathin sections located close to each other, and this made it possible to show variations as regards argyrophilia of fibers and boutons in the different stages of degeneration. Details will be described below.
Glees sections Light microscopy. The sections showed argyrophilic fibers of various sizes. The shape of the fibers varied. Some were straight, others had swollen parts, but fragmentation of fibers was only occasionally seen. Rings and clubs similar in size and Brain Research, 36 (1972) 353-369
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structure to those c o n v e n t i o n a l l y described as ' b o u t o n s ' were also present, either close to nerve cells or at some distance from these (Fig. 2a). Electron microscopy. The sections showed silver grains varying in size from 3 to 35 nm. The smallest particles were present o n most structures, a n d occurred as a fine precipitate. The larger grains were present o n nuclei of the various types of cells, o n myelinated fibers a n d o n b o u t o n s . The n o r m a l fibers showed large silver particles with affinity to the axoplasm. The myelin sheaths a n d m i t o c h o n d r i a were free from such precipitate. The silver particles could in some instances be seen o n tubuli or filaments, b u t it was usually
Fig. 2. Light microscopical sections from nucleus fastigii of cat 1450, survival time 3 days. a, Glees section showing open rings and clubs in the neuropil. Arrows point to some of these structures. Such structures can be normal or degenerating boutons, but the observations made in this and other publications clearly show that they also can be impregnated part of dendrites or cross-sectioned terminal myelinated axons, b, Nauta-Laidlaw section showing degenerating fibers, c, Fink-Heimer (I) section showing degenerating fibers, d, Eager section from nucleus fastigii, showing many degenerating fibers. Degenerating filamentous as well as dark fibers are impregnated. The number of degenerating fibers present in Eager sections is therefore much higher than that present in NautaLaidlaw and Fink-Heimer sections (cf. text). × 1680.
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Fig. 3. Electron micrographs of Glees sections from nucleus interpositus in cat 1416. a, Normal bouton, b, with heavy silver precipitate. Synaptic vesicles, v, can be seen in the central unimpregnated part of the bouton, and peripherally at the lower left. b, Impregnated normal bouton, b. The centrally located mitochondria are unstained. Arrow points to synaptic region, c, Silver impregnated hypertrophic filamentous degenerating bouton, b. Some synaptic vesicles can be seen in the lower part of the bouton, d, Another filamentous degenerating bouton. Arrow points to synaptic region, e, Silver impregnated hypertrophic filamentous degenerating myelinated axon. Some normal small unimpregnated and impregnated myelinated axons are also seen. a-d, x 30,000; e, x 10,000.
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impossible to relate the grains to specific organelles. M a n y o f the n o r m a l b o u t o n s h a d only a fine precipitate, others were heavily i m p r e g n a t e d with greater silver particles. B o u t o n s with centrally located m i t o c h o n d r i a h a d a precipitate in the form o f a ring outside the m i t o c h o n d r i a (Fig. 3a,b). O t h e r n o r m a l b o u t o n s a p p e a r e d as
Fig. 4. Electron micrographs of silver impregnated dendrites in nucleus interpositus from cats 549 and 1416. a, A centrally located dendrite is surrounded by several normal boutons, b. Arrows point to synaptic regions, b, Prominent silver precipitate in a dendrite. Note that silver particles in this and the preceding electron micrograph usually avoid the normal mitochondria. ,~ 30,000. Brain Research, 36 (1972) 353-369
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compact bulbs (not illustrated). The silver particles could in some instances be seen on filaments in the boutons. In others it was impossible to relate the grains to specific organelles. Heavy silver precipitate was present also in many dendrites (Fig. 4a,b). Such dendrites could be surrounded by unimpregnated boutons (Fig. 4a). Some of the silver grains in the dendrites were related to organelles, but it was difficult to make decisive statements on this point. The degenerating filamentous axons were heavily impregnated with silver (Fig. 3e). The silver grains were precipitated on the filaments, but particles appeared to be located also in the matrix. The degenerating hypertrophic boutons were likewise heavily impregnated. Also here most of the silver grains were related to the filaments. Two typical degenerating filamentous boutons are illustrated in Fig. 3c, d. The boutons presented in these figures were only moderately impregnated. Many of the degenerating filamentous boutons were so heavily impregnated that they could be identified with certainty only where a postsynaptic specialization was present on an apposed dendrite. A certain number of the degenerating dark myelinated fibers and boutons were also impregnated with silver. Electron micrographs of such structures are not included in the present study. "
Nauta-Laidlaw sections Light microscopy. Only a few degenerating fibers were impregnated (Fig. 2b). Electron microscopy. No impregnation was found of normal fibers and boutons (Fig. 5a). Neither did hypertrophic filamentous degenerating fibers and boutons show silver precipitate, except for the very small particles present also on other structures (Fig. 5a,f). Darkened filamentous degenerating fibers showed, however, some precipitation (Fig. 5c), and the dark degenerating fibers were heavily impregnated (Fig. 5b,e). Myelin was usually free from silver, and the sections did not show the staining artefact sometimes present in Nauta-Laidlaw sections11. Larger silver granules could very occasionally be found on filamentous darkened degenerating boutons, and the same was the case for degenerating boutons of the dark type. Fink-Heimer sections Light microscopy. The sections showed a moderate number of impregnated degenerating fibers (Fig. 2c), and in addition some isolated small argyrophilic particles. Electron microscopy. The sections showed a fine silver precipitate diffusely distributed on all tissue structures. The precipitate consisted of needle-like and small globular particles. The degenerating filamentous fibers and boutons were unimpregnated (Fig. 6a,d), but impregnation with silver was obvious in filamentous degenerating fibers and boutons that had started to darken (Fig. 6b,e). Many degenerating dark fibers Brain Research, 36 (1972) 353-369
Fig. 5. Electron micrographs from Nauta-Laidlaw sections of cat 1450. a, The normal (bl), the light filamentous degenerating (b2) and the dark filamentous degenerating (b3) boutons show only unspecific silver precipitate, b, The dark degenerating myelinated fiber shows a heavy silver precipitate. c, The darkened filamentous degenerating fiber shows a light silver precipitate, which consists of particles of the same size and larger than those seen in a. d, The dark degenerating bouton shows only unspecific silver precipitate. Arrow points to synaptic region, e, Dark degenerating small myelinated fiber (like 5 b) showing a heavy silver precipitate, f, Filamentous degenerating hypertrophic myelinated fiber in an early stage showing only unspecific silver precipitate. A comparison of this degenerating fiber with those in b, c and e, clearly shows that argyrophilia of the hypertrophic filamentous degenerating fibers increases when they darken. Argyrophilia is very prominent when the degenerating fibers have collapsed and have been transformed to the dark type. ~" 30,000.
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Fig. 6. Electron micrographs of Fink-Heimer (I) sections from nuclei fastigii and interpositus in cats 1450 and 1418. a, The degenerating hypertrophic filamentous bouton shows only unspecific silver precipitate. Arrow points to synaptic region, b, The darkened degenerating bouton shows an incipient silver impregnation. Arrow points to synaptic region, c, The dark degenerating bouton is heavily impregnated with silver particles. Arrows point to synaptic vesicles, d, The degenerating filamentous myelinated axon shows only unspecific silver precipitate, e, The darkening hypertrophic degenerating myelinated fiber shows an incipient silver impregnation. The silver particles are concentrated in the periphery of the degenerating fiber, f and g, Cross- and longitudinally-sectioned dark degenerating small myelinated fibers with a heavy silver precipitate, a-b and d-g, Cat 1450, survival time 3 days, nucleus fastigii, c, Cat 1418, survival time 4 days, nucleus interpositus. × 30,000. Brain Research, 36 (1972) 353-369
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Fig. 7. Electron micrographs of Eager-stained sections from nucleus fastigii in cat 1450. a, Normal (bl and b2) and filamentous degenerating (b3) boutons with only very little silver precipitate. Arrows point to synaptic regions, b, Degenerating hypertrophic filamentous myelinated axon with heavy silver precipitate. Some normal small unimpregnated myelinated axons are also present, c, Horizontally sectioned small myelinated dark degenerating fiber with heavy silver precipitate, d, Dark degenerating bouton with only unspecific silver precipitate. Arrow points to synaptic region, e, Degenerating dark bouton, showing so~-ne silver particles. This bouton was showing the heaviest silver impregnation in the series studied. Arrow points to synaptic region, a and c-e, :, 30,000; b, ~ 15,000.
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and boutons showed a conspicuous silver precipitate (Fig. 6c,f,g). The myelin showed only the fine precipitate.
Eager sections Light microscopy. The sections showed a more intense degeneration than did the Nauta-Laidlaw and Fink-Heimer sections. The degenerating fibers were very characteristic, and contrasted well with the pale background (Fig. 2d). The degeneration was much more pronounced than appears from the light micrograph of Fig. 2d, which only shows degenerating fibers from one focus. Electron microscopy. The sections showed a fine diffusely distributed silver precipitate, almost similar to that found in Fink-Heimer sections. The coarser silver particles were present on all types of degenerating fibers, and filamentous as well as dark degenerating fibers were heavily impregnated (Fig. 7b,c). The degenerating filamentous and dark boutons were, however, usually unimpregnated (Fig. 7d). The degenerating filamentous and dark boutons shown in Fig. 7a,e were the ones having the heaviest silver precipitate. The illustrations reveal that the precipitate was moderate. DISCUSSION
The capriciousness of the silver methods makes it always difficult to reach decisive conclusions in studies of the type made here. Such studies can therefore be met with the criticism that negative conclusions concerning argyrophilia are of little value. Lack of argyrophilia of certain structures can also be a result of loss of silver particles in ultrathin sections during their processing. Because of this, it is essential that conclusions concerning argyrophilia in ultrathin sections are based on observations made only in parts of the sections where there is a prominent silver precipitate. Valuable additional information can, however, be obtained from regions of ultrathin sections showing moderate argyrophilia, but such areas should not be used for decisive conclusions. Furthermore, observations of a large number of ultrathin sections with prominent silver precipitate are necessary before conclusions are drawn. Only in this way is it possible to decide whether observations made in a single ultrathin section are generally valid. However, even with these qualifications it is necessary to stress that conclusions concerning argyrophilia can be drawn only for the fiber system studied specifically. General conclusions should be avoided, since they may often be misleading.
Glees sections The findings made in the present study show clearly that normal as well as filamentous degenerating fibers and boutons are stained in the cerebellar nuclei of the cat, and that argyrophilic particles in degenerating fibers and boutons are related to filaments. This finding is in agreement with previous observations in our labora-
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tory '~9. it cannot be excluded that the same is the case for normal boutons. A certain number of the normal boutons in the cerebellar nuclei have filaments, but a heavy impregnation like that found of the normal boutons of Fig. 3a,b, prevents decisive conclusions concerning a relation between filaments and silver particles. However, even if there seems to be a relation between filaments and silver particles in electron micrographs, such particles could be precipitated on proteins in the matrix close to the filaments. Such a possibility was already mentioned by Gray and Guillery 6. Credence to this view has recently been given by Kiernan 17, who investigated subcellular fractions of nervous tissue and their relation to silver. He concluded that argyrophilia is a property of a component of the axoplasm, which can be dissolved and reprecipitated, and that it therefore is likely that a substance or group of substances rather than a formed organelJe is responsible for argyrophilia. He found that the properties of this protein component differed markedly from those of known membranous and fibrous intracellular proteins. Even if Kiernan applied only a rarely used silver method (Ungewitter's Urea Silver Nitrate Method), and even if his silver staining was made on precipitates pretreated with various chemical agents, his observations make it clear that detailed discussions on this point at present are of doubtful value. Only studies of the type made by Kiernan can lead to important and valuable conclusions concerning the chemical and physical factors responsible for precipitation of silver particles and their possible relation to various organelles of normal and degenerating axons and boutons. The findings made in the present study are only partly in agreement with those made by Pecci Savedra et al. 2a with the Glees method in another filamentous degenerating system. They found that degenerating filamentous myelinated optic fibers in the monkey were heavily impregnated with silver, but that the degenerating filamentous boutons showed only very little silver deposit. They concluded, therefore, that argyrophilia is slight in synaptic terminals with neurofilaments. The present study, on the other hand, shows clearly that degenerating filamentous fibers, as well as boutons in the cerebellar nuclei, are heavily stained with silver (Fig. 3c-e). One might suspect that the negative observation made by Pecci Savedra et al. was caused by the fact that these authors looked for degenerating boutons in areas of the ultrathin sections with only a moderate silver precipitate. However, the present author has made almost similar findings with the Eager method in the intracerebellar nuclei (vide infra), and his observations were made in regions with heavy silver precipitate. The findings by Pecci Savedra et al. therefore probably are another reflection of the capriciousness of the silver methods, and another warning against general conclusions. The open rings and clubs found in the Glees sections studied in the light microscope could be boutons (Fig. 2a). However, since dendrites also are impregnated in the cerebellar nuclei (Fig. 4a,b), it is clear that such structures also could mimic 'boutons'. Previous studies have shown that dendrites are impregnated also in the inferior olive 2s. Guillery 7 has furthermore suggested that rings in the lateral geniculate body of the cat are filament-filled dendritic appendages, and Ralston 24 has arrived at almost the same conclusion in the ventral basal thalamic nucleus of the same animal. Brain Research, 36 (1972) 353-369
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In addition, it has been shown that the rings and clubs present in Glees sections from the medial accessory olive of the cat studied in the light microscope, are crosssectioned normal and degenerating small myelinated axons 2s. All these studies, therefore, together with the observations by Pecci Savedra et al. clearly show that the rings and clubs present in Glees sections studied in the light microscolre, can be myelinated terminal axons and stained parts of dendrites as well as boutons. No conclusions concerning synaptology or distribution of boutons can therefore be made from Glees sections. The method can, however, in suitable regions be used for a study of fibro-architectonics and for the location of degenerating fibers which pass intermingled with normal ones. An illustrating example of this is given in a recent study on the distribution of the VIIIth nerve fibers within the primary cochlear nuclei2L The fallacies of the Nauta modifications and the Fink-Heimer and Eager techniques will be discussed below. Nauta-Laidlaw sections
Degenerating filamentous Purkinje cell axons and boutons are not stained when the Nauta-Laidlaw method is used. It is interesting, however, that argyrophilia of degenerating fibers begins when these are transformed to the dark type of degeneration. The argyrophilia starts when the degenerating filamentous fibers begin to darken. Such darkened filamentous lightly stained degenerating fibers can be found side by side with unstained light filamentous degenerating fibers (Fig. 5c,f), and degenerating heavily stained dark collapsed fibers (Fig. 5b,e). It is important, however, that only occasionally was silver precipitate (except for the fine type) found on degenerating boutons. These were, regardless of the type of degeneration, usually unstained in the sections of the series studied here. Thus, it is clear that it is hazardous only from the light microscopical Nauta-Laidlaw sections, to make conclusions as to the nature of the smallest argyrophilic particles present. F i n k - H e i m e r sections
The Fink-Heimer method (I) likewise fails to stain the degenerating filamentous cerebellofugal fibers and their boutons. An insipient impregnation is, however, evident in the darkened filamentous fibers and boutons (Fig. 6b,e). The silver particles are in such fibers mostly precipitated in the peripheral part (Fig. 6e). Whether this only is an occasional observation made in the cerebellar nuclei, or whether it is an indication that the degeneration in the peripheral part of the axoplasm proceeds more quickly, is unknown. When the degenerating fibers and boutons have been transformed to the dark collapsed type, argyrophilia becomes very prominent (Fig. 6c,f,g). An important difference from Nauta-Laidlaw sections is, however, that dark degenerating boutons usually are heavily stained in the Fink-Heimer sections. This corresponds to what has been described previously by Heimer and Peters 12, Heimer 1° and Walberg 2s. Brain Research, 36 (1972) 353-369
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Eager sections Degenerating hypertrophic filamentous fibers are heavily impregnated when the Eager technique is used (Fig. 7b). Darkened filamentous fibers, and dark collapsed degenerating fibers are likewise heavily stained (Fig. 7c). This method, therefore, impregnates both types of degenerating fibers, and will, therefore, probably prove to be very valuable for a study of the distribution of filamentous as well as dark degenerating fiber tracts. The present author has had little success with staining of degenerating boutons with Eager's method. In this respect the method in his hands appears to be similar to the Nauta-Laidlaw modification. Great efforts have been made to find impregnated degenerating boutons in the ultrathin sections. The most heavily impregnated boutons, found in the present study, are shown in Fig. 7a,e. Eager 3 has suggested that his method is comparable to the Fink-Heimer technique. Since his conclusions were based only on light microscopical observations, decisive conclusions can scarcely be drawn. Further electron microscopical studies of unmyelinated as well as myelinated hypertrophic and dark degenerating fiber systems will show whether we now in addition to the Fink-Heimer method have another method, which can impregnate degenerating boutons equally well. If this will turn out to be the case, we would thus for the first time have a method capable of impregnating selectively degenerating axons and terminals of filamentous as well as dark degenerating fiber tracts.
Concluding remarks Several silver staining methods, in addition to those considered above, have been described in recent years (see especially 16,1s,2a,3°), but none of these methods will be considered here. The present author has until lately routinely made Nauta-Laidlaw and FinkHeimer (I or II) sections of the series to be studied in the light microscope. After introduction of the Eager method, this has been included in the routine. The observations made so far on material treated with that method are restricted to the cerebellum of the cat. In the brain stem of the cat the author has hitherto had greater success with the Nauta-Laidlaw method than with the Fink-Heimer stain. Although probably degenerating boutons only occasionally are impregnated in Nauta-Laidlaw sections, the impregnation of degenerating terminal myelinated fibers (their axoplasm) makes it possible in the brain stem to map in great detail in the light microscope thefield of termination of a degenerating fiber tract. The Eager method may turn out to be superior to the Nauta-Laidlaw (or -Gygax) technique in this respect. The method is quick, no 'suppression' of normal fibers is necessary, and the counterstaining with cresyl violet gives a good background contrast. The problem is a different one in regions where fiber bundles are unmyelinated. Heimer s and Heimer and Wall la have clearly shown that the Fink-Heimer method in such regions is superior to the Nauta modifications, especially where there is a
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selective distribution of terminal unmyelinated fibers to certain layers. Although Eager's a studies from the olfactory glomeruli show that his method is capable of staining unmyelinated fibers, it remains to be clarified with the electron microscope whether also the degenerating boutons of such fibers are equally well impregnated. Furthermore, species differences will probably be as important for the successful application of this method as for the other techniques considered here. Interesting problems arise when a student wants to make a detailed study of the termination of a fiber tract in the central nervous system. Such a study obviously calls for a trial of several methods. The present author wants again to stress, that regional differences can be so great in the central nervous system, that general conclusions seldom can be drawn from studies of a single fiber system. Experimental light microscopical studies with a careful mapping of the terminalfieM of the fiber tract, should always precede experimental electron microscopy. A detailed trimming of the blocks, chosen for electron microscopy, should furthermore be made after staining of semithin sections from such blocks. Only in this way is it possible to select the area with terminal degeneration to be studied with the electron microscope. The staining procedure of semithin sections introduced by Holl~inder and Vaaland 15 is quick and reliable in regions where the terminal fibers are myelinated. The Heimer method a should obviously be preferred in areas with unmyelinated fiber bundles and tracts. Future studies will show whether the Eager method in this respect is equal or superior to the other methods. SUMMARY The filamentous degenerating Purkinje cell axons and boutons have been studied in the light and electron microscope after staining with the Glees, NautaLaidlaw, Fink-Heimer (I) and Eager methods. Degenerating filamentous fibers and boutons are stained when the Glees method is used, but local silver deposit is present also in dendrites. Previous observations have shown that dendritic appendages and terminal myelinated axons can be impregnated in Glees sections. No conclusions as regards distribution of boutons or concerning synaptic details can therefore be drawn from light microscopical observations of Glees sections. Degenerating filamentous fibers and boutons are not stained with the NautaLaidlaw and Fink-Heimer techniques. However, the degenerating filamentous fibers start to show argyrophilia when they darken, and are heavily stained with both techniques when they have been transformed to the dark type. The degenerating darkened boutons show affinity to silver in Fink-Heimer sections, and are heavily impregnated when they have reached the dark stage. Degenerating boutons are only occasionally impregnated when the Nauta-Laidlaw method is used. Degenerating filamentous, as well as degenerating dark fibers, are impregnated in Eager sections. Degenerating boutons are, however, only occasionally stained. In the series examined in thepresent study, this method therefore appears to be similar to the Nauta-Laidlaw technique as regards impregnation of degenerating boutons.
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v. WALBERG H o w e v e r , it is i m p o r t a n t to n o t e t h a t f i l a m e n t o u s as well as d a r k d e g e n e r a t i n g
fibers are s t a i n e d in E a g e r sections. ACKNOWLEDGEMENTS T h e a u t h o r is g r e a t l y i n d e b t e d to M r s . J. L i n e V a a l a n d f o r e x c e l l e n t t e c h n i c a l assistance.
REFERENCES 1 EAGER, R. P., Modes of termination of Purkinje cell axons in the cerebellum of the cat. In R. LLINLS (Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. Med. Ass., Chicago, II1., 1969, pp. 585-601. 2 EAGER, R. P., Selective staining of degenerating axons in the central nervous system by a simplified silver method: spinal cord projections to external cuneate and inferior otivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 3 EAGER,R. P., Olfactory nerve projections to the olfactory bulb in rabbit: demonstration by means of a simplified ammoniacal silver degeneration method, Brain Research, 23 (1970) 250-254. 4 FXNK, R. P., AND HEIMER, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 5 GLEES, P., Terminal degeneration within the central nervous system as studied by a new silver method, J. Neuropath. exp. Neurol., 5 (1946) 54-59. 6 GRAY, E. G., AND GUILLERY,R. W., The basis for silver staining of synapses of the mammalian spinal cord: a light and electron microscope study, J. Physiol. (Lond.), 157 (1961) 581-588. 7 GUILLERY,R. W., A light and electron microscopical study of neurofibrils and neurofilaments at neuro-neuronal junctions in the dorsal lateral geniculate nucleus of the cat, J. Anat. (Lond:), 120 (1967) 583-604. 8 HEIMER, L., Silver impregnation of terminal degeneration in some forebrain fiber systems: a comparative evaluation of current methods, Brain Research, 5 (1967) 86-108. 9 HEIMER, L., Silver impregnation of degenerating axons and their terminals on Epon-Araldite sections, Brain Research, 12 (1969) 246-249. 10 HEIMER, L., Bridging the gap between light and electron microscopy in the experimental tracing of fiber connections. In W. J. H. NAUTA AND S. O. E. EnnESSON (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 162-172. 11 HEIMER, L., ANO EgnOLM, R., Neuronal argyrophilia in early degenerative states: a light and electron microscopic study of the Glees and Nauta techniques, Experientia (Basel), 23 (1967) 237-239. 12 HEIMER, L., AND PETERS, A., An electron microscope study of a silver stain for degenerating boutons, Brain Research, 8 (1968) 337-346. 13 HEIMER,L., AND WALL,P. O., The dorsal root distribution to the substantia gelatinosa of the rat with a note on the distribution in the cat, Exp. Brain Res., 6 (1968) 89-99. 14 HOLL.~NDER,H., The section embedding (SE) technique. A new method for the combined light microscopic and electron microscopic examination of central nervous tissue, Brain Research, 20 (1970) 39-47. 15 HOLL~,NDER,H., AND VAALAND,J. L., A reliable staining method for semi-thin sections in experimental neuroanatomy, Brain Research, 10 (1968) 120--126. 10 JOHNSTONE,G., AND BOWSHER,D., A new method for the selective impregnation of degenerating axon terminals, Brain Research, 12 (1969) 47-53. 17 KIERNAN, J. A., Silver staining of axons in subcellular fractions of nervous tissue, Experientia (Basel}, 26 (1970) 1352. 18 LOEwY, A. D., Ammoniacal silver staining of degenerating axons, Acta neuropath. (Berl.), 14 (1969) 226-236. 19 MILLONIG,G., Further observations on a phosphate buffer for osmium solutions in fixation. In
S. S. BREESE (Ed.), Proceedings Fifth International Congress for Electron Microscopy, Vol. 2, Academic Press, New York, 1962, p. P-8.
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20 MUGNAIN1,E., AND WALBERG,F., An experimental electron microscopical study on the mode of termination of cerebellar corticovestibular fibres in the cat lateral vestibular nucleus (Deiters' nucleus), Lap. Brain Res., 4 (1967) 212-236. 21 NAUTA, W. J. H., Silver impregnation of degenerating axons. In W. F. WINDLE (Ed.), New Research Techniques of Neuroanatomy, Thomas, Springfield, Ill., 1957, pp. 17-26. 22 OSEN, K. K., Course and termination of the primary afferents in the cochlear nuclei of the cat, Arch. ital. Biol., 108 (1970) 21-51. 23 PECCI SAVEDRA, J., MASCI'VI'I, T. A., VACCAREZZA, O. L., AND READER, T. A., Non-synaptic ring images in silver-impregnated lateral geniculate nucleus, Brain Research, 14 (1969) 733-738. 24 RALSTON, H. J., The synaptic organization of lemniscal projections to the ventrobasal thalamus of the cat, Brain Research, 14 (1969) 99-115. 25 SOUSA-PINTO,A., AND VAALAND,J. L., Personal communication. 26 STERLING,P., AND KUYPERS, H. G. J. M., Simultaneous demonstration of normal boutons and degenerating nerve fibres and their terminals in the spinal cord, J. Anat. (Lond.), 100 (1966) 723-732. 27 VENABLE,J. H., AND COGGESI-IALL,R., A simplified lead citrate stain for use in electron microscopy, J. Cell Biol., 25 (1965) 407-408. 28 WALBERG,F., Does silver impregnate normal and degenerating boutons? A study based on light and electron microscopical observations of the inferior olive, Brain Research, 31 (1971) 47-65. 29 WALBERG, F., AND MUGNAINI, E., Distinction of degenerating fibres and boutons of cerebellar and peripheral origin in the Deiters' nucleus of the same animal, Brain Research, 14 (1969) 57-75. 30 WIITANEN, J. T., Selective silver impregnation of degenerating axons and axon terminals in the central nervous system of the monkey, Brain Research, 14 (1969) 546-548. 31 ZAGURY,D., MODEL, P. G., AND PAPPAS, G. O., The preservation of the fine structure of cryostatsectioned tissue with dimethylsulfoxide for combined light and electron microscopy, J. Histochem. Cytochem., 16 (1968) 40-48.
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353
FURTHER STUDIES ON SILVER IMPREGNATION OF NORMAL AND DEGENERATING BOUTONS. A LIGHT AND ELECTRON MICROSCOPICAL INVESTIGATION OF A FILAMENTOUS DEGENERATING SYSTEM
F R E D WALBERG
Anatomical Institute, University of Oslo, Oslo (Norway) (Accepted July 25th, 1971)
INTRODUCTION
Experimental electron microscopical studies have revealed that most axons and boutons of the central nervous system when they degenerate show one of two types of reaction. The first of these is the dark type of degeneration. This is from the onset of degeneration characterized by a darkening of the axoplasm, in which the disintegrating organelles gradually become unidentifiable. This type of reaction has been described in detail by a variety of authors, and is the one most commonly found in fiber tracts of the central nervous system of vertebrates. The other type of degeneration is the filamentous reaction, which is characterized by an initial hypertrophy of axons and boutons. Their swollen parts become filled with filaments, resulting in a displacement and clumping of the other organelles. This type is not as common as the dark type of degeneration, but has been described in several pathways. The filamentous degeneration is characteristic for the Purkinje cell axons and boutons of the cat1, 20. The reaction is very prominent, and is from the 3rd-4th day followed by a rapid shrinkage and darkening of the degenerating fibers and boutons (see also ref. 20). To the author's knowledge no systematical combined light and electron microscopical study has been done on a filamentous degenerating fiber system, in order to reveal to what extent the various silver methods demonstrate degeneration of this type. The present paper deals with the filamentous degeneration of the Purkinje cell axons and boutons of the cat, as this appears in Glees, Nauta-Laidlaw, Fink-Heimer (I) and Eager sections. The findings show that the 4 methods differ markedly in their ability to impregnate filamentous degenerating structures. MATERIAL AND METHODS
Altogether 9 adult cats have been used. The cats were anesthetized with Nembutal, and lesions were made with a knife and sucker in various parts of the Brain Research, 36 (1972) 353-369
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cerebellar cortex. A diagram of one of the lesions is shown in Fig. I. The survival time of the animals was 3-8 days. All cats were perfused with a rinsing solution followed by aldehyde through the abdominal aorta. The rinsing solution consisted of 200 ml 0 . 9 ~ NaCI buffered with 10 ml/l of 0.16 M sodium cacodylate and with 10 ml 1 ~o CaCI2/I added. The first aldehyde solution consisted of 4 ~ formaldehyde (freshly made of 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 I N HC1. 230 mg CaCI2 was then added. This and the first solution was perfused at 38°C. The second aldehyde solution consisted of 2 liters of 2.5 To glutaraldehyde in the same cacodylate buffer, with 10 ml I ~ CaC12/liter. This solution was perfused at 3°C. The procedure was similar to that used by Sousa-Pinto and Vaaland 25. The brain with the rostral part of the cervical spinal cord was dissected out and stored in 4 ~ formaldehyde from some days to several weeks. The cerebellum was isolated and the extent of the lesion indicated on a diagram of the cerebellar surface. A frontal section was then made with a razor blade through the central part of the lesion, so that the cerebellum was divided into two parts. The depth of the lesion was in this way controlled, and further control of the depth was made during the subsequent treatment of the two halves. Some of them were cut on a kryotome after storing in DMSO 31, others were cut on a TC-2 tissue sectioner, and the rest on an ordinary freezing microtome. In the first and latter cases, 20 or 30/~m sections were cut. The chopper gave slices of 100-200 /zm. The sections and slices were stained according to the method of Glees 5, Nauta-Laidlaw '21, Fink-Heimer 4 and Eager 2.
Q
L
1450
R
Fig. 1. Diagram showing the extent of the lesion (hatching) in the cerebellum of cat 1450. The same type of lesion was made in the other cats used in this study. The degenerating Purkinje cell axons and boutons in the nuclei fastigii and interpositus were in all cats studied in the light and electron microscope after staining of sections and slices with the Glees, Nauta-Laidtaw, Fink-I-/eimer (I) and Eager methods. The findings made in the various animals are shown in Figs. 2-7. L, left; R, right. Brain Research, 36 (1972) 353-369
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Small pieces were isolated from the sections and slices containing the nuclei fastigii and interpositus underlying the lesions. The isolation was made in distilled water under the dissecting microscope after the impregnation. The pieces were postfixed in 1% OsO4 solution buffered with Millonig's19 solution. The pieces were then dehydrated in graded solutions of alcohol or acetone, and transferred to section embedding in Araldite according to the method of Hollfinder14. Some of the pieces were very thick, and therefore embedded directly in Araldite blocks. The ultrathin sections were stained with uranyl acetate and lead citrate 27, and studied in the electron microscope. Some of the sections cut on the kryotome and freezing microtome were, after staining with the various methods, mounted in the ordinary way for light microscopy. Such sections were compared with sections from the same animals studied in the electron microscope. OBSERVATIONS The findings were essentially the same in all animals studied. Some ultrathin sections showed a heavy silver impregnation, others had only a scanty precipitate. However, only the central part of an argyrophilic region of the used ultrathin section was photographed. Degenerating axons and boutons without silver precipitate described in the present study, were therefore always present directly adjacent to structures showing impregnation. The conclusions drawn concerning argyrophilia or lack of argyrophilia of degenerating axons and boutons are therefore considered to be valid. The following description is based on light and electron microscopical observations on sections from the nucleus fastigii as well as the nucleus interpositus underlying the lesions. Corresponding observations were made in the two nuclei, and no specification will therefore be made in the description of the findings. In the ultrathin sections degenerating fibers and boutons in various stages of degeneration were present. The majority of these were in most sections of the hypertrophic filamentous type. Some degenerating fibers and boutons had, however, reached a state intermediate between the hypertrophic and dark degeneration. Such structures were hypertrophic and dark, and the filaments could be difficult to identify. Other degenerating fibers and boutons were of the dark, collapsed type (cf. ref. 20). Fibers and boutons in all these 3 stages of degeneration were in several ultrathin sections located close to each other, and this made it possible to show variations as regards argyrophilia of fibers and boutons in the different stages of degeneration. Details will be described below.
Glees sections Light microscopy. The sections showed argyrophilic fibers of various sizes. The shape of the fibers varied. Some were straight, others had swollen parts, but fragmentation of fibers was only occasionally seen. Rings and clubs similar in size and Brain Research, 36 (1972) 353-369
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structure to those c o n v e n t i o n a l l y described as ' b o u t o n s ' were also present, either close to nerve cells or at some distance from these (Fig. 2a). Electron microscopy. The sections showed silver grains varying in size from 3 to 35 nm. The smallest particles were present o n most structures, a n d occurred as a fine precipitate. The larger grains were present o n nuclei of the various types of cells, o n myelinated fibers a n d o n b o u t o n s . The n o r m a l fibers showed large silver particles with affinity to the axoplasm. The myelin sheaths a n d m i t o c h o n d r i a were free from such precipitate. The silver particles could in some instances be seen o n tubuli or filaments, b u t it was usually
Fig. 2. Light microscopical sections from nucleus fastigii of cat 1450, survival time 3 days. a, Glees section showing open rings and clubs in the neuropil. Arrows point to some of these structures. Such structures can be normal or degenerating boutons, but the observations made in this and other publications clearly show that they also can be impregnated part of dendrites or cross-sectioned terminal myelinated axons, b, Nauta-Laidlaw section showing degenerating fibers, c, Fink-Heimer (I) section showing degenerating fibers, d, Eager section from nucleus fastigii, showing many degenerating fibers. Degenerating filamentous as well as dark fibers are impregnated. The number of degenerating fibers present in Eager sections is therefore much higher than that present in NautaLaidlaw and Fink-Heimer sections (cf. text). × 1680.
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Fig. 3. Electron micrographs of Glees sections from nucleus interpositus in cat 1416. a, Normal bouton, b, with heavy silver precipitate. Synaptic vesicles, v, can be seen in the central unimpregnated part of the bouton, and peripherally at the lower left. b, Impregnated normal bouton, b. The centrally located mitochondria are unstained. Arrow points to synaptic region, c, Silver impregnated hypertrophic filamentous degenerating bouton, b. Some synaptic vesicles can be seen in the lower part of the bouton, d, Another filamentous degenerating bouton. Arrow points to synaptic region, e, Silver impregnated hypertrophic filamentous degenerating myelinated axon. Some normal small unimpregnated and impregnated myelinated axons are also seen. a-d, x 30,000; e, x 10,000.
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impossible to relate the grains to specific organelles. M a n y o f the n o r m a l b o u t o n s h a d only a fine precipitate, others were heavily i m p r e g n a t e d with greater silver particles. B o u t o n s with centrally located m i t o c h o n d r i a h a d a precipitate in the form o f a ring outside the m i t o c h o n d r i a (Fig. 3a,b). O t h e r n o r m a l b o u t o n s a p p e a r e d as
Fig. 4. Electron micrographs of silver impregnated dendrites in nucleus interpositus from cats 549 and 1416. a, A centrally located dendrite is surrounded by several normal boutons, b. Arrows point to synaptic regions, b, Prominent silver precipitate in a dendrite. Note that silver particles in this and the preceding electron micrograph usually avoid the normal mitochondria. ,~ 30,000. Brain Research, 36 (1972) 353-369
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compact bulbs (not illustrated). The silver particles could in some instances be seen on filaments in the boutons. In others it was impossible to relate the grains to specific organelles. Heavy silver precipitate was present also in many dendrites (Fig. 4a,b). Such dendrites could be surrounded by unimpregnated boutons (Fig. 4a). Some of the silver grains in the dendrites were related to organelles, but it was difficult to make decisive statements on this point. The degenerating filamentous axons were heavily impregnated with silver (Fig. 3e). The silver grains were precipitated on the filaments, but particles appeared to be located also in the matrix. The degenerating hypertrophic boutons were likewise heavily impregnated. Also here most of the silver grains were related to the filaments. Two typical degenerating filamentous boutons are illustrated in Fig. 3c, d. The boutons presented in these figures were only moderately impregnated. Many of the degenerating filamentous boutons were so heavily impregnated that they could be identified with certainty only where a postsynaptic specialization was present on an apposed dendrite. A certain number of the degenerating dark myelinated fibers and boutons were also impregnated with silver. Electron micrographs of such structures are not included in the present study. "
Nauta-Laidlaw sections Light microscopy. Only a few degenerating fibers were impregnated (Fig. 2b). Electron microscopy. No impregnation was found of normal fibers and boutons (Fig. 5a). Neither did hypertrophic filamentous degenerating fibers and boutons show silver precipitate, except for the very small particles present also on other structures (Fig. 5a,f). Darkened filamentous degenerating fibers showed, however, some precipitation (Fig. 5c), and the dark degenerating fibers were heavily impregnated (Fig. 5b,e). Myelin was usually free from silver, and the sections did not show the staining artefact sometimes present in Nauta-Laidlaw sections11. Larger silver granules could very occasionally be found on filamentous darkened degenerating boutons, and the same was the case for degenerating boutons of the dark type. Fink-Heimer sections Light microscopy. The sections showed a moderate number of impregnated degenerating fibers (Fig. 2c), and in addition some isolated small argyrophilic particles. Electron microscopy. The sections showed a fine silver precipitate diffusely distributed on all tissue structures. The precipitate consisted of needle-like and small globular particles. The degenerating filamentous fibers and boutons were unimpregnated (Fig. 6a,d), but impregnation with silver was obvious in filamentous degenerating fibers and boutons that had started to darken (Fig. 6b,e). Many degenerating dark fibers Brain Research, 36 (1972) 353-369
Fig. 5. Electron micrographs from Nauta-Laidlaw sections of cat 1450. a, The normal (bl), the light filamentous degenerating (b2) and the dark filamentous degenerating (b3) boutons show only unspecific silver precipitate, b, The dark degenerating myelinated fiber shows a heavy silver precipitate. c, The darkened filamentous degenerating fiber shows a light silver precipitate, which consists of particles of the same size and larger than those seen in a. d, The dark degenerating bouton shows only unspecific silver precipitate. Arrow points to synaptic region, e, Dark degenerating small myelinated fiber (like 5 b) showing a heavy silver precipitate, f, Filamentous degenerating hypertrophic myelinated fiber in an early stage showing only unspecific silver precipitate. A comparison of this degenerating fiber with those in b, c and e, clearly shows that argyrophilia of the hypertrophic filamentous degenerating fibers increases when they darken. Argyrophilia is very prominent when the degenerating fibers have collapsed and have been transformed to the dark type. ~" 30,000.
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Fig. 6. Electron micrographs of Fink-Heimer (I) sections from nuclei fastigii and interpositus in cats 1450 and 1418. a, The degenerating hypertrophic filamentous bouton shows only unspecific silver precipitate. Arrow points to synaptic region, b, The darkened degenerating bouton shows an incipient silver impregnation. Arrow points to synaptic region, c, The dark degenerating bouton is heavily impregnated with silver particles. Arrows point to synaptic vesicles, d, The degenerating filamentous myelinated axon shows only unspecific silver precipitate, e, The darkening hypertrophic degenerating myelinated fiber shows an incipient silver impregnation. The silver particles are concentrated in the periphery of the degenerating fiber, f and g, Cross- and longitudinally-sectioned dark degenerating small myelinated fibers with a heavy silver precipitate, a-b and d-g, Cat 1450, survival time 3 days, nucleus fastigii, c, Cat 1418, survival time 4 days, nucleus interpositus. × 30,000. Brain Research, 36 (1972) 353-369
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Fig. 7. Electron micrographs of Eager-stained sections from nucleus fastigii in cat 1450. a, Normal (bl and b2) and filamentous degenerating (b3) boutons with only very little silver precipitate. Arrows point to synaptic regions, b, Degenerating hypertrophic filamentous myelinated axon with heavy silver precipitate. Some normal small unimpregnated myelinated axons are also present, c, Horizontally sectioned small myelinated dark degenerating fiber with heavy silver precipitate, d, Dark degenerating bouton with only unspecific silver precipitate. Arrow points to synaptic region, e, Degenerating dark bouton, showing so~-ne silver particles. This bouton was showing the heaviest silver impregnation in the series studied. Arrow points to synaptic region, a and c-e, :, 30,000; b, ~ 15,000.
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and boutons showed a conspicuous silver precipitate (Fig. 6c,f,g). The myelin showed only the fine precipitate.
Eager sections Light microscopy. The sections showed a more intense degeneration than did the Nauta-Laidlaw and Fink-Heimer sections. The degenerating fibers were very characteristic, and contrasted well with the pale background (Fig. 2d). The degeneration was much more pronounced than appears from the light micrograph of Fig. 2d, which only shows degenerating fibers from one focus. Electron microscopy. The sections showed a fine diffusely distributed silver precipitate, almost similar to that found in Fink-Heimer sections. The coarser silver particles were present on all types of degenerating fibers, and filamentous as well as dark degenerating fibers were heavily impregnated (Fig. 7b,c). The degenerating filamentous and dark boutons were, however, usually unimpregnated (Fig. 7d). The degenerating filamentous and dark boutons shown in Fig. 7a,e were the ones having the heaviest silver precipitate. The illustrations reveal that the precipitate was moderate. DISCUSSION
The capriciousness of the silver methods makes it always difficult to reach decisive conclusions in studies of the type made here. Such studies can therefore be met with the criticism that negative conclusions concerning argyrophilia are of little value. Lack of argyrophilia of certain structures can also be a result of loss of silver particles in ultrathin sections during their processing. Because of this, it is essential that conclusions concerning argyrophilia in ultrathin sections are based on observations made only in parts of the sections where there is a prominent silver precipitate. Valuable additional information can, however, be obtained from regions of ultrathin sections showing moderate argyrophilia, but such areas should not be used for decisive conclusions. Furthermore, observations of a large number of ultrathin sections with prominent silver precipitate are necessary before conclusions are drawn. Only in this way is it possible to decide whether observations made in a single ultrathin section are generally valid. However, even with these qualifications it is necessary to stress that conclusions concerning argyrophilia can be drawn only for the fiber system studied specifically. General conclusions should be avoided, since they may often be misleading.
Glees sections The findings made in the present study show clearly that normal as well as filamentous degenerating fibers and boutons are stained in the cerebellar nuclei of the cat, and that argyrophilic particles in degenerating fibers and boutons are related to filaments. This finding is in agreement with previous observations in our labora-
Brain Research, 36 (1972) 353-369
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v. WALB[RG
tory '~9. it cannot be excluded that the same is the case for normal boutons. A certain number of the normal boutons in the cerebellar nuclei have filaments, but a heavy impregnation like that found of the normal boutons of Fig. 3a,b, prevents decisive conclusions concerning a relation between filaments and silver particles. However, even if there seems to be a relation between filaments and silver particles in electron micrographs, such particles could be precipitated on proteins in the matrix close to the filaments. Such a possibility was already mentioned by Gray and Guillery 6. Credence to this view has recently been given by Kiernan 17, who investigated subcellular fractions of nervous tissue and their relation to silver. He concluded that argyrophilia is a property of a component of the axoplasm, which can be dissolved and reprecipitated, and that it therefore is likely that a substance or group of substances rather than a formed organelJe is responsible for argyrophilia. He found that the properties of this protein component differed markedly from those of known membranous and fibrous intracellular proteins. Even if Kiernan applied only a rarely used silver method (Ungewitter's Urea Silver Nitrate Method), and even if his silver staining was made on precipitates pretreated with various chemical agents, his observations make it clear that detailed discussions on this point at present are of doubtful value. Only studies of the type made by Kiernan can lead to important and valuable conclusions concerning the chemical and physical factors responsible for precipitation of silver particles and their possible relation to various organelles of normal and degenerating axons and boutons. The findings made in the present study are only partly in agreement with those made by Pecci Savedra et al. 2a with the Glees method in another filamentous degenerating system. They found that degenerating filamentous myelinated optic fibers in the monkey were heavily impregnated with silver, but that the degenerating filamentous boutons showed only very little silver deposit. They concluded, therefore, that argyrophilia is slight in synaptic terminals with neurofilaments. The present study, on the other hand, shows clearly that degenerating filamentous fibers, as well as boutons in the cerebellar nuclei, are heavily stained with silver (Fig. 3c-e). One might suspect that the negative observation made by Pecci Savedra et al. was caused by the fact that these authors looked for degenerating boutons in areas of the ultrathin sections with only a moderate silver precipitate. However, the present author has made almost similar findings with the Eager method in the intracerebellar nuclei (vide infra), and his observations were made in regions with heavy silver precipitate. The findings by Pecci Savedra et al. therefore probably are another reflection of the capriciousness of the silver methods, and another warning against general conclusions. The open rings and clubs found in the Glees sections studied in the light microscope could be boutons (Fig. 2a). However, since dendrites also are impregnated in the cerebellar nuclei (Fig. 4a,b), it is clear that such structures also could mimic 'boutons'. Previous studies have shown that dendrites are impregnated also in the inferior olive 2s. Guillery 7 has furthermore suggested that rings in the lateral geniculate body of the cat are filament-filled dendritic appendages, and Ralston 24 has arrived at almost the same conclusion in the ventral basal thalamic nucleus of the same animal. Brain Research, 36 (1972) 353-369
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In addition, it has been shown that the rings and clubs present in Glees sections from the medial accessory olive of the cat studied in the light microscope, are crosssectioned normal and degenerating small myelinated axons 2s. All these studies, therefore, together with the observations by Pecci Savedra et al. clearly show that the rings and clubs present in Glees sections studied in the light microscolre, can be myelinated terminal axons and stained parts of dendrites as well as boutons. No conclusions concerning synaptology or distribution of boutons can therefore be made from Glees sections. The method can, however, in suitable regions be used for a study of fibro-architectonics and for the location of degenerating fibers which pass intermingled with normal ones. An illustrating example of this is given in a recent study on the distribution of the VIIIth nerve fibers within the primary cochlear nuclei2L The fallacies of the Nauta modifications and the Fink-Heimer and Eager techniques will be discussed below. Nauta-Laidlaw sections
Degenerating filamentous Purkinje cell axons and boutons are not stained when the Nauta-Laidlaw method is used. It is interesting, however, that argyrophilia of degenerating fibers begins when these are transformed to the dark type of degeneration. The argyrophilia starts when the degenerating filamentous fibers begin to darken. Such darkened filamentous lightly stained degenerating fibers can be found side by side with unstained light filamentous degenerating fibers (Fig. 5c,f), and degenerating heavily stained dark collapsed fibers (Fig. 5b,e). It is important, however, that only occasionally was silver precipitate (except for the fine type) found on degenerating boutons. These were, regardless of the type of degeneration, usually unstained in the sections of the series studied here. Thus, it is clear that it is hazardous only from the light microscopical Nauta-Laidlaw sections, to make conclusions as to the nature of the smallest argyrophilic particles present. F i n k - H e i m e r sections
The Fink-Heimer method (I) likewise fails to stain the degenerating filamentous cerebellofugal fibers and their boutons. An insipient impregnation is, however, evident in the darkened filamentous fibers and boutons (Fig. 6b,e). The silver particles are in such fibers mostly precipitated in the peripheral part (Fig. 6e). Whether this only is an occasional observation made in the cerebellar nuclei, or whether it is an indication that the degeneration in the peripheral part of the axoplasm proceeds more quickly, is unknown. When the degenerating fibers and boutons have been transformed to the dark collapsed type, argyrophilia becomes very prominent (Fig. 6c,f,g). An important difference from Nauta-Laidlaw sections is, however, that dark degenerating boutons usually are heavily stained in the Fink-Heimer sections. This corresponds to what has been described previously by Heimer and Peters 12, Heimer 1° and Walberg 2s. Brain Research, 36 (1972) 353-369
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v. WAtBER(I
Eager sections Degenerating hypertrophic filamentous fibers are heavily impregnated when the Eager technique is used (Fig. 7b). Darkened filamentous fibers, and dark collapsed degenerating fibers are likewise heavily stained (Fig. 7c). This method, therefore, impregnates both types of degenerating fibers, and will, therefore, probably prove to be very valuable for a study of the distribution of filamentous as well as dark degenerating fiber tracts. The present author has had little success with staining of degenerating boutons with Eager's method. In this respect the method in his hands appears to be similar to the Nauta-Laidlaw modification. Great efforts have been made to find impregnated degenerating boutons in the ultrathin sections. The most heavily impregnated boutons, found in the present study, are shown in Fig. 7a,e. Eager 3 has suggested that his method is comparable to the Fink-Heimer technique. Since his conclusions were based only on light microscopical observations, decisive conclusions can scarcely be drawn. Further electron microscopical studies of unmyelinated as well as myelinated hypertrophic and dark degenerating fiber systems will show whether we now in addition to the Fink-Heimer method have another method, which can impregnate degenerating boutons equally well. If this will turn out to be the case, we would thus for the first time have a method capable of impregnating selectively degenerating axons and terminals of filamentous as well as dark degenerating fiber tracts.
Concluding remarks Several silver staining methods, in addition to those considered above, have been described in recent years (see especially 16,1s,2a,3°), but none of these methods will be considered here. The present author has until lately routinely made Nauta-Laidlaw and FinkHeimer (I or II) sections of the series to be studied in the light microscope. After introduction of the Eager method, this has been included in the routine. The observations made so far on material treated with that method are restricted to the cerebellum of the cat. In the brain stem of the cat the author has hitherto had greater success with the Nauta-Laidlaw method than with the Fink-Heimer stain. Although probably degenerating boutons only occasionally are impregnated in Nauta-Laidlaw sections, the impregnation of degenerating terminal myelinated fibers (their axoplasm) makes it possible in the brain stem to map in great detail in the light microscope thefield of termination of a degenerating fiber tract. The Eager method may turn out to be superior to the Nauta-Laidlaw (or -Gygax) technique in this respect. The method is quick, no 'suppression' of normal fibers is necessary, and the counterstaining with cresyl violet gives a good background contrast. The problem is a different one in regions where fiber bundles are unmyelinated. Heimer s and Heimer and Wall la have clearly shown that the Fink-Heimer method in such regions is superior to the Nauta modifications, especially where there is a
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selective distribution of terminal unmyelinated fibers to certain layers. Although Eager's a studies from the olfactory glomeruli show that his method is capable of staining unmyelinated fibers, it remains to be clarified with the electron microscope whether also the degenerating boutons of such fibers are equally well impregnated. Furthermore, species differences will probably be as important for the successful application of this method as for the other techniques considered here. Interesting problems arise when a student wants to make a detailed study of the termination of a fiber tract in the central nervous system. Such a study obviously calls for a trial of several methods. The present author wants again to stress, that regional differences can be so great in the central nervous system, that general conclusions seldom can be drawn from studies of a single fiber system. Experimental light microscopical studies with a careful mapping of the terminalfieM of the fiber tract, should always precede experimental electron microscopy. A detailed trimming of the blocks, chosen for electron microscopy, should furthermore be made after staining of semithin sections from such blocks. Only in this way is it possible to select the area with terminal degeneration to be studied with the electron microscope. The staining procedure of semithin sections introduced by Holl~inder and Vaaland 15 is quick and reliable in regions where the terminal fibers are myelinated. The Heimer method a should obviously be preferred in areas with unmyelinated fiber bundles and tracts. Future studies will show whether the Eager method in this respect is equal or superior to the other methods. SUMMARY The filamentous degenerating Purkinje cell axons and boutons have been studied in the light and electron microscope after staining with the Glees, NautaLaidlaw, Fink-Heimer (I) and Eager methods. Degenerating filamentous fibers and boutons are stained when the Glees method is used, but local silver deposit is present also in dendrites. Previous observations have shown that dendritic appendages and terminal myelinated axons can be impregnated in Glees sections. No conclusions as regards distribution of boutons or concerning synaptic details can therefore be drawn from light microscopical observations of Glees sections. Degenerating filamentous fibers and boutons are not stained with the NautaLaidlaw and Fink-Heimer techniques. However, the degenerating filamentous fibers start to show argyrophilia when they darken, and are heavily stained with both techniques when they have been transformed to the dark type. The degenerating darkened boutons show affinity to silver in Fink-Heimer sections, and are heavily impregnated when they have reached the dark stage. Degenerating boutons are only occasionally impregnated when the Nauta-Laidlaw method is used. Degenerating filamentous, as well as degenerating dark fibers, are impregnated in Eager sections. Degenerating boutons are, however, only occasionally stained. In the series examined in thepresent study, this method therefore appears to be similar to the Nauta-Laidlaw technique as regards impregnation of degenerating boutons.
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v. WALBERG H o w e v e r , it is i m p o r t a n t to n o t e t h a t f i l a m e n t o u s as well as d a r k d e g e n e r a t i n g
fibers are s t a i n e d in E a g e r sections. ACKNOWLEDGEMENTS T h e a u t h o r is g r e a t l y i n d e b t e d to M r s . J. L i n e V a a l a n d f o r e x c e l l e n t t e c h n i c a l assistance.
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