Silver impregnation of terminal degeneration in some forebrain fiber systems: a comparative evaluation of current methods

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SILVER I M P R E G N A T I O N OF T E R M I N A L D E G E N E R A T I O N IN SOME F O R E B R A I N FIBER SYSTEMS: A C O M P A R A T I V E E V A L U A T I O N OF CURRENT METHODS

LENNART HEIMER* Department of Anatomy, University of G6teborg, G6teborg (Sweden) and Department of Psychology, Massachusetts Institute of Technology, Cambridge, Mass. (U.S.A.)

(Received November 14th, 1966)

INTRODUCTION Ever since formulation of the neuron doctrine at the end of the nineteenth century the histological demonstration of synaptic structures has been of major interest to neurohistologists. The first description of an axon terminal was given by Cajal in 1888, and since then many studies on the structure of synapses, both normal and degenerative, have been published (for a recent review see Gray and Guillery16). The dichromate-silver method of Golgi, introduced in the late nineteenth century, has been of particular value for the morphological corroboration of the synapse concept. Although uniquely suitable for revealing normal synaptic articulations, the Golgi method has so far been inadequate for studies of degenerated material. The reduced silver techniques of Cajal 4° and of Bielschowsky4 were developed at the beginning of this century. For a long time these techniques--sometimes described as 'neurofibrillar methods'--were used only for visualizing normal nerve fibers. Hoff 21 appears to have been the first to employ the Cajal impregnation principle to identify boutons terminaux degenerating as a result of transection of their parent axons. Since then, several techniques better suited to this experimentalanatomical purpose have been developed; it is remarkable, however, that those most commonly employed are based upon the Bielschowsky principle of frozen section impregnation rather than Cajal's block impregnation method**. * Present address: Department of Psychology, Massachusetts Institute of Technology, Cambridge, Mass. (U.S.A.) ** Many attempts have been made to clarify the chemical and physical mechanisms involved in silver impregnation. The first theory regarding the mechanism of silver impregnation25 postulated that nuclei of reduced silver are formed during the initial impregnation in the silver solution, and deposition of silver occurs on these nuclei during the treatment in an ammoniacal silver solution. This theory has been extended by many later workers2~,23,3z--37,42--44,46,~L Peters33 further suggested that the initial reducible silver is taken up by the amino acid histidine. Seki45 drew attention to the importance of the degree of structural compactness of the material being impregnated. (continued on next page) Brain Research, 5 (1967) 86-108

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Fig. 1. The effect of varying different factors in the Glees silver impregnation method. A, impregnation in a diluted (0.005~) silver nitrate solution of pH 7.6 results in heavily impregnated nuclei; B, by lowering the pH to 5.1 and keeping other factors constant, a selective fiber impregnation is obtained; and C, further lowering of pH to 3.6 again results in heavily impregnated nuclei, provided the silver ion concentration is increased (10 % silver nitrate solution). It was not until 1946 that a relatively simple Bielschowsky modification for the impregnation of degenerated axons was developed by Glees 14. This technique has proved to be of great value for the identification of certain types of degenerating synaptic endstructures, and thus, of the actual termination sites of degenerating axons. It has been employed most successfully in studies of brain stem connections, but appears to have been of limited usefulness for the impregnation of degenerating synaptic endstructures in the forebrain. Research by the Glees method has, moreover, been hampered by a heavy concomitant impregnation of normal fibers. Nauta and Gygax further developed the silver impregnation technique and their work resulted in two valuable methods, the original non-suppressive Nauta method2S, a0 in which no attempt was made to suppress the impregnation of normal fibers, and the suppressive Nauta-Gygax method 31 in which such suppression was achieved by the introduction of a phosphomolybdic acid-potassium permanganate pretreatment. The suppressive N a u t a - G y g a x method was later modified by replacing the ammoniated silver nitrate solution with the ammoniated silver carbonate solution of Laidlawa, 29. The introduction of Laidlaw's solution seems to have been generally considered an important modification. It is not always realized that the original non-suppressive Nauta method and the suppressive N a u t a - G y g a x method are two fundamentally different techniques. The original N a u t a method was introduced as a technique for the impregnation of The importance of the physical and chemical properties of the tissues and solutions used in techniques of silver impregnation has been stressed repeatedly (for references see WilliamsSO,51). A clear illustration of how different, interdependent factors influence the impregnation is given by Fig. 1. The pictures are taken from sections of the rat brain obtained during experiments with the Glees modification 14 of the Bielschowsky technique. Brain Research, 5 (1967) 86-108

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degenerating axon terminals in frozen sections. The pretreatment used in the suppressive Nauta-Gygax method reduces the uptake of metallic silver by normal axons and thereby considerably facilitates the identification of degenerating fibers. As a result, the suppressive Nauta-Gygax method has become widely used as a convenient technique for the tracing of axon trajectories. Depending on the handling of the technique, as well as on the fiber system studied, the method can give a more or less detailed picture of the terminal axon arborization, including a variable proportion of the degenerating axon terminals19,zo, 47. As will be documented below, however, there is good reason to believe that the suppression of normal fiber impregnation is often obtained at the expense of a considerable reduction in the impregnation of the terminal axon arbor. Following the introduction of the attractive suppressive method of NautaGygax, the original Nauta method has been used only occasionally; in fact, few neuroanatomists appear to be familiar with the characteristics of the latter technique. Nevertheless, Blackstad's 5 and Raisman's et al. ~s,~9 analyses of hippocampal fiber systems, as well as White's study of the olfactory connections 49 of the rat, have shown that the synaptic fields of these fiber systems can be more adequately outlined with the original Nauta method than with either the Glees technique or the suppressive Nauta-Gygax method. By the use of the original Nauta method, White recently showed that the projection field of the fibers from the olfactory bulb in the rat is wider than can be demonstrated with the more commonly used suppressive Nauta-Gygax method. In White's study with the original Nauta method a considerable number of olfactory bulb fibers were shown to extend as far caudally as the ventral entorhinal area, well beyond the caudal limit of the distribution field usually outlined by the fiber degeneration observed in material impregnated by the more commonly used Nauta-Gygax technique. As is the case with the Glees method, however, the study of original Nauta sections is often complicated by the heavy impregnation of normal fibers. The terminal distribution of a fiber system can be satisfactorily outlined only when the method used permits identification of the synaptic endstructures. It is therefore unfortunate that the two most commonly used silver techniques, viz. the Glees and the suppressive Nauta-Gygax methods, are of limited value for the demonstration of degenerating axon terminals, a fact that is reflected to some extent by the existing controversies concerning the terminal distribution of several central nervous fiber connections. The present paper represents an attempt to evaluate the relative merits of different reduced silver methods as a means of outlining the terminal distribution of several fiber systems in the forebrain of the rat. MATERIALS AND METHODS

Surgical lesions were placed, under direct vision, in the olfactory bulb, olfactory peduncle or lateral olfactory tract of male albino rats anesthetized with Nembutal. In other animals, lesions were made in the neocortex, in the fimbria hippocampi or in the optic nerve. After survival times varying from 1 to 10 days, the animals were Brain Research, 5 (1967) 86-108

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Fig. 2. Terminal degeneration in the plexiform layer of the olfactory tubercle impregnated with the original Nauta method 2s. The ipsilateral olfactory bulb was removed 2 days before sacrifice. The high power photograph (E) was taken from the most posteromedial part of the tubercle as demonstrated by the successive steps in A-C. Note the dorsolaterally directed extension of massive degeneration in the medial part of the tubercle. killed by an overdose of Nembutal and perfused transcardially with physiological saline followed by 10 % formalin. The brains were removed and subjected to further fixation in 10 % formalin for periods varying between 1 and 3 weeks. The whole forebrain was subsequently sectioned frontally, horizontally or sagittally, either on the freezing microtome at 15-40/~, or after previous paraffin embedding at 5-10/~. O f the specimens sectioned frozen, two parallel series, each consisting of about 6 sections/ Brain Research, 5 (1967) 86-108

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mm were stained by, respectively, the original Nauta '~8 and the suppressive NautaGygax z9 techniques. Selected neighboring sections were impregnated according to the Glees method la or a modified original Nauta method lz. The paraffin sections were impregnated according to the original Nauta method. For demonstrating normal synaptic endings, some of the paraffin sections were treated in potassium dichromate before Nauta impregnation, as described in the appendix. RESULTS

(1) Terminal degeneration in the olfactory cortex (A) The original non-suppressive Nauta method. Fig. 2 shows the brain of an animal sacrificed 2 days after a lesion of the left olfactory bulb. The lesion did not extend into the accessory olfactory bulb or the anterior olfactory nucleus. Fig. 2E shows a massive accumulation of heavily impregnated, generally spherical structures in the plexiform layer of the caudomedial part of the ipsilateral olfactory tubercle of this brain. Similar concentrations of argyrophilic structures were found in the plexiform layer throughout the whole ipsilateral olfactory tubercle; none, however, were present contralateral to the lesion. Pictures comparable to those shown in Fig. 2 were observed following longer survival times of 4 and 5 days. It is a fundamental question whether the argyrophilic bodies in this figure really represent degenerated axon terminals*. Light microscopic studies have not been able to provide a conclusive answer to this question. An electron microscopic study of sections previously impregnated with the original Nauta method was therefore performed, and a comparison was made between the light microscopic and the electron microscopic appearance of the degeneration. The photographs shown in Figs. 3 and 4 were taken from the prepiriform cortex of a rat in which an extensive lesion of the ipsilateral olfactory bulb was made 3 days prior to sacrifice. Fig. 3A shows a dense accumulation of argyrophilic structures especially in the superficial zone of the plexiform layer (the lower part of the photograph), whereas Fig. 3B shows more sparsely distributed similar structures within the pyramidal cell layer. For the purpose of comparison, Fig. 4 shows electron micrographs of ultrathin sections prepared from a neighboring 50 ,u thick frozen section previously impregnated by the original Nauta method. Despite the inevitably poor quality of electron microscopic pictures obtained by this procedure, boutons terminaux are clearly identifiable, by shape as well as by characteristic postsynaptic densities. The figures show the presence of massive and apparently random silver accumulations within the boutonal axoplasm. It seems likely that the corresponding light microscopic appearance would be that of more or less spherical, almost solid black, corpuscles such as those shown in Figs. 2 and 3. It is therefore suggested that many of the diffusely distributed black * The terms axon terminal, synaptic endstructure, synaptic thickening and boutons terminaux are used interchangeably for the reason that the techniques used in this study as a rule do not allow distinction to be made between various forms of synaptic endstructures. The interested reader is referred to Gray and Guillery's recent review of this subject16.

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Fig. 3. Terminal degeneration in the prepiriform cortex 3 days after an ipsilateral olfactory bulb lesion. Impregnation according to the original Nauta method. A dense accumulation of argyrophilic bodies is demonstrated in the superficial part of the plexiform layer (lower part of A). B shows more sparsely distributed similar structures in the pyramidal cell layer.

Fig. 4. Electron micrographs of ultrathin sections prepared from 50/~ thick frozen sections adjacent to the original Nauta sections shown in Fig. 3. The axon terminals, with an apparently erratic silver deposition, can be identified by the postsynaptic thickening (st).

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Fig. 5. A, terminal degeneration in the posteromedial part of the olfactory tubercle impregnated with t he original Nauta method on paraffin embedded material. The ipsilateral olfactory bulb was removed 2 days before sacrifice. Compare the corresponding pictures in frozen sections (Figs. 2 and 6). B, contralateral normal side.

!i i ii!i~!i~i!!!?ii~!i Fig. 6. Massive terminal degeneration in the posteromedial part of the olfactory tubercle 2 days after an ipsilateral bulb lesion. Impregnation after Glees. Compare with the adjacent original Nauta section shown in Fig. 2.

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Fig. 7, Terminal degeneration in the lateral part of the olfactory tubercle impregnated with the suppressive Nauta-Gygax method29. Compare this relatively sparse degeneration with the massive degeneration demonstrated by the original Nauta method in the same animal (Fig. 2). bodies seen under the light microscope represent degenerating boutons. Others are undoubtedly part of fine degenerating fibers, often recognizable only under oil immersion. It may be of importance to know, especially in the study of small brains and in brains with large lesions, that degenerated boutons can be impregnated also on paraffin sections with the original Nauta method 6. The degeneration pattern obtained in the olfactory tubercle on paraffin embedded material (Fig. 5) corresponds to that observed in frozen sections of comparable cases (Figs. 2 and 6). In the olfactory cortex degenerated boutons can be impregnated after a survival time of only 24 h. (B) The Glees method. Fig. 6 shows terminal degeneration in the posteromedial olfactory tubercle in a Glees stained section adjacent to the original Nauta section shown in Fig. 2. The Glees method, slightly modified as described in the appendix, produces the same picture of diffusely distributed, solid black particles as does the original Nauta technique. Also the distribution of the degeneration in the tubercle is the same with the two methods. Pictures of terminal degeneration, similar to those seen with the original Nauta method (Fig. 3A), have also been obtained with the

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Fig. 8. A, sparse degenerating callosal fibers in layer I1 of the rat's frontal cortex 5 days after contralateral hemidecortication. Impregnation according to the suppressive Nauta-Gygax method. Compare with B. B, field corresponding to A as appearing in a section impregnated following a modified original Nauta method (Fink and Heimer12, Procedure I1) and counterstained with cresylechtviolet. The solid black spherules on and between neuronal perikarya are interpreted as representing degenerating fine fibers and synaptic endstructures.

Glees method in prepiriform cortex. It is therefore concluded that the Glees method, like the original Nauta method, can reveal degenerated synaptic terminals in this part of the brain. It must be noted, however, that in these forebrain regions more consistent results are obtained with the original Nauta method than with the Glees technique. (C) The suppressive Nauta-Gygax method. With a similar combination of light and electron microscopy as described above it has been shown that the suppressive N a u t a - G y g a x method can impregnate degenerated synaptic endings in the spinal cord and the mammillary body19, ~0. An example of the capacity of this method to show terminal degeneration in the olfactory cortex of the rat is given in Fig. 7, which shows the lateral part of the olfactory tubercle of the same animal from which the photograph of Fig. 2 was taken. A comparison of the two photographs, however, shows that the quantity of impregnated synaptic structures in sections stained by the suppressive N a u t a - G y g a x method is considerably smaller than in sections prepared by the original N a u t a technique. Furthermore, using the suppressive N a u t a - G y g a x method, terminal degeneration can be detected only in the lateral part of the olfactory tubercle near the lateral olfactory tract, where it is still quite conspicuous in animals having survived surgery for 5 days. In no case were similar pictures of terminal degeneration or degenerating axons observed in more medial regions of the tubercle; this negative result was obtained irrespective of the duration of the potassium permanganate treatment which, when unduly prolonged, causes a progressively increasing refractoriness of degenerating axons to silver impregnation. It thus appears justified to conclude that, unlike the original Nauta method, the suppressive N a u t a - G y g a x

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Fig. 9. A, degenerating hippocampal commissural fibers in sector CA3 of a rat whose contralateral fimbria hippocampi was destroyed 5 days before sacrifice. Impregnation according to the suppressive Nauta-Gygax method. Compare with B. B, field corresponding to A as appearing in a section impregnated according to Fink and Heimer (Procedure II) and counterstained with cresylechtviolet. A relatively massive terminal degeneration is seen in the stratum radiatum above the layer of the mossy fibers. Degenerating fibers are seen penetrating the pyramidal cell layer at the bottom of the figure. technique fails to impregnate degenerating boutons as well as degenerating fibers in a major extent of the olfactory tubercle of rats subjected to ipsilateral olfactory bulb removal.

(2) Terminal degeneration in neocortex, hippocampus and tectum mesencephali While the original Nauta method as well as the Glees method were found useful for showing degenerated boutons in several regions of the brain, their application in other areas met with difficulties. More consistent and more easily interpretable results were obtained by either one of two recent modifications of the original Nauta technique (Fink and Heimer12). Figs. 8-10 show results obtained by one of these modifications as compared to the effects of the suppressive N a u t a - G y g a x method. Fig. 8 shows, in adjacent N a u t a - G y g a x (A) and Fink-Heimer (B) sections respectively, degeneration in the second layer of the rat's frontal cortex 5 days after contralateral frontal decortication. In the suppressive N a u t a - G y g a x section (Fig. 8A) sparse degenerating callosal fibers can be followed up into the second layer and the density of degenerating fibers gradually diminishes as one moves from the deeper to the more superficial layers of the cortex. The new method (Fig. 8B), by contrast, often

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~ i i ,¸

.....i~iiiiiii~i~i~7

Fig. 10. A, sparse degeneration in the stratum griseum superficiale of the superior colliculus following a contralateral enucleation 5 days before sacrifice. Impregnation according to the suppressive methe,d of Nauta-Gygax. B, field corresponding to A as appearing in a section impregnated according to Fink and Heimer (Procedure II). A heavy terminal degeneration is now revealed throughout the extent of the stratum griseum superficiale. shows degeneration throughout all layers; sometimes the most massive terminal degeneration is seen in the superficial layers of the cortex. Fig. 9 shows degeneration in the hippocampal sector CA3 of a rat sacrificed 5 days after a lesion in the contralateral fimbria hippocampi. In both the suppressive N a u t a - G y g a x (Fig. 9A) and the Fink-Heimer (Fig. 9B) sections many degenerating axons are seen to penetrate the pyramidal cell layer at the bottom of the figure. While in the suppressive N a u t a - G y g a x material it is difficult to determine the sites of termination of these fibers, the new modification demonstrates terminal degeneration in the stratum radiatum above the layer of the mossy fibers, a distribution which agrees well with Blackstad's light 5 and electron microscopic findings (personal communication) concerning the termination of commissural afferents to the hippocampus. The last example of terminal degeneration (Fig. 10) is from the superior colliculus of a rat whose contralateral eye was removed 5 days before sacrifice. Fig. 10A shows the resulting degeneration in the stratum griseum superficiale obtained by the suppressive N a u t a - G y g a x method whereas Fig. 10B shows the corresponding region in a nearby section impregnated according to the Fink-Heimer modification.

(3) Disappearance of normal synaptic endings after experimental lesions In normal brain tissue the usual reduced silver methods reveal only sporadic synaptic endings. The same techniques, however, impregnate a multitude of normal

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Fig. 11. Synaptic endings in the thalamus of an unoperated rat. The section shown in A is prepared according to the method of Armstrong and Stephens a, whereas the section shown in B was treated in potassium dichromate after dewaxing and then impregnated as described in the appendix. C, synaptic endings on a pyramidal cell of the hippocampus of an unoperated rat. In addition to boutons covering the dendrite, there are many other argyrophilic particles whose identity remains uncertain. Impregnation according to the original Nauta method after pretreatment of the single paraffin section in potassium dichromate.

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Fig. 12. Electron micrograph showing silver granules within mitochondria. The micrograph was prepared from a paraffin section impregnated with the method for normal boutons as described in the appendix.

Fig. 13. Prepiriform cortex of a rat sacrificed 10 days after unilateral section of the lateral olfactory tract. Impregnation according to the method for normal boutons on paraffÉn sections as described in the appendix. A, the superficial part of the plexiform layer contralateral to the lesion. Lateral olfactory tract fibers are seen at the bottom of the figure. B, the corresponding region on the side of the lesion, where a marked reduction in the number of impregnated synaptic endstructures has taken place.

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Fig. 14. Prepiriform cortex of a rat sacrificed 3 days after resection of the ipsilateral olfactory bulb. The lighter region dorsal to the lateral olfactory tract at the bottom of the figure indicates the reduced number of impregnated boutons in t he superficialpart of t he plexiform layer. Impregnation according to the original Nauta method after pretreatment of the paraffin section in potassium dichromate. boutons terminaux in brain and spinal cord tissue that has been pretreated with potassium dichromate2,3, 41. This is demonstrated in Fig. 11A, which shows a part of the thalamus in a normal rat brain prepared according to the method of Armstrong and Stephens 3. Electron micrographs of sections treated in potassium dichromate before silver impregnation show considerable metallic silver deposits in the mitochondria (Fig. 12). Thus, the efficacy of the Armstrong and Rasmussen techniques in demonstrating normal boutons could well be related to the large number of mitochondria present in synaptic endstructures. Similar results can be obtained when, instead of whole brain tissue, deparaffinized sections are chromated prior to silver impregnation. Examples are given by Fig. 11B which, like Fig. 11A, shows synaptic endformations in the thalamus of the normal rat, and Fig. 11C showing boutons on a pyramidal cell in hippocampus. If the termination of a fiber system in a certain region is massive enough, this method of staining normal synaptic endings shows the disappearance of boutons after Brain Research, 5 (1967) 86-108

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destruction of their cell bodies or axons 41. That this method, applied to appropriate fiber systems, can indeed supply indirect evidence of synaptic fields is further demonstrated by Fig. 13, which shows a marked depletion of the normally closely spaced boutons in the superficial zone of the plexiform layer of the ipsilateral prepiriform cortex 10 days after section of the olfactory tract. The reduction in the number of impregnated synaptic endstructures is particularly striking when comparing the operated (B) with the unoperated (A) side. A further example is given by the low power photograph in Fig. 14, which shows the prepiriform cortex of a rat whose ipsilateral olfactory bulb was resected 3 days before sacrifice. The light zone above the lateral olfactory tract at the bottom of the figure corresponds to the superficial part of the plexiform layer where a marked reduction in the number of impregnated normal synaptic endstructures has taken place. DISCUSSION

The observations reported in the foregoing account emphasize once more the importance of an adequate impregnation of terminal degeneration in the experimental study of central nervous fiber connections. Despite its obvious value in tracing degenerating axons, the much used suppressive Nauta-Gygax method in many cases supplies no more than a general indication of axonal termination sites, even though, according to Guillery and Ralston's electron microscopical findings 19, it can impregnate, depending on the fiber system studied, a smaller or larger percentage of the degenerating synaptic endstructures. This limitation of the Nauta technique was evident in all of the four fiber systems used as test models in the present study. Thus, the distribution of the lateral olfactory tract as shown by the suppressive Nauta-Gygax method would appear to involve no more than the lateral half of the olfactory tubercle, and not extend to the entorhinal area. By contrast, alternate series of the same specimens impregnated by the original non-suppressive Nauta method reveal abundant terminal degeneration in the plexiform layer of the olfactory tubercle throughout the latter's extent, as well as in the ventrolateral zone of the entorhinal area. These findings are in complete accord with White's recent report 49 on the efferent connections of the rat's olfactory bulb as demonstrated by the original Nauta method. The limitation of the suppressive Nauta-Gygax technique was no less striking in the other fiber systems studied. Following hemidecortication, a modified original Nauta method produced pictures of dense terminal degeneration, not only in the deep layers, but also in the superficial layers of several contralateral cortical areas and in the stratum radiatum of the contralateral hippocampal sector CA3, regions in which the suppressive Nauta-Gygax technique failed to impregnate any degeneration at all or only demonstrated an occasional fiber of passage. Following unilateral eye enucleation, the modified original Nauta method stains a multitude of degenerating boutons terminaux in the superficial grey layer of the contralateral superior colliculus, whereas in the same layer the suppressive Nauta-Gygax method impregnates degenerating fine axons but reveals only few degenerating synaptic endstructures. While the suppressive Nauta-Gygax method in general fails to impregnate a large percentage Brain Research, 5 (1967) 86-108

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of degenerating boutons, the possibilities of revealing degenerating fine axon arborizations is less predictable: in some fiber systems, such as the sensory lemnisci, clear pictures of the terminal axon plexus seem to be obtained, whereas in others, notably the lateral olfactory tract, little more than relatively rectilinear stem fibers are impregnated, with the result that even the general region of distribution may remain in doubt. Such intersystemic differences are probably determined in part by differences in fiber caliber, but the involvement of other factors such as chemical specificities cannot be excluded. Recognizing the limitations of the suppressive Nauta-Gygax method, many have used the Glees technique to obtain more accurate pictures of synaptic fieldsT,1L Although this combination of techniques has proven useful in some regions, the Glees method has so far been of limited value for the demonstration of degenerated boutons in the forebrain, notably the cerebral cortex. In the present experiments on the projections of the olfactory bulb, however, the Glees technique provided pictures of terminal degeneration in the olfactory cortex similar to those obtained with the original Nauta method. The degenerated boutons came out solid black and in large number. Ring-like structures suggesting the impregnation of rings of neurofilaments in the boutons 17,1s were not seen. In accordance with Walberg4s we therefore do not believe that the presence of neurofilaments is a prerequisite for positive Glees impregnation. The Glees method, appropriately applied, may prove valuable for the demonstration of synaptic fields in the forebrain, especially if they are dense and well-defined. In more diffusely distributed fiber systems, however, the massive impregnation of normal fibers will probably restrict the usefulness of the Glees technique. Cowan and PowelP by using the Glees method on non-operated monkeys, obtained pictures of 'terminal degeneration' in some hypothalamic nuclei, and on the basis of this observation questioned the results of earlier Glees studies of degeneration in hypothalamus. Adey et al. 1, however, who studied a large number of normal monkeys with the Glees method, failed to reveal the pseudo-degeneration described by Cowan and Powell. The debate on pseudo-degeneration shows the difficulties which may arise when metallic impregnation is used to evaluate changes in synaptic endings, structures which are more in the domain of the electron microscope than of the light microscope. This weakness, inherent in light microscopic studies of terminal degeneration, can be offset only by examining a large number of animals under different experimental conditions and if possible with different techniques. Although both the Glees and the suppressive Nauta-Gygax method have been widely used for tracing fiber systems in the central nervous system, little is known about their mechanisms. Some studies10,11 suggest that the Glees and the NautaGygax methods stain different constituents of the fiber. Evans and Hamlyn11 found that sections prepared with the Glees method were unaltered or even improved by lipid extraction in alcohol-chloroform before silver impregnation. Similar treatment of sections prepared according to the Nauta-Gygax method completely abolished the impregnation of degenerated fibers and the removed lipid was therefore thought to be the Nauta positive material. The failure of the suppressive Nauta-Gygax method to demonstrate the finest unmyelinated axon arborizations seemed to give Brain Research, 5 (1967) 86--108

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this hypothesis additional support. Eager and Barrnett 10 further suggested that the permanganate oxidizes unsaturated lipids into an argyrophilic compound. Also Giolli 13 suggests that the argyrophilia in the suppressive Nauta-Gygax technique is mainly related to unsaturated lipids. It is interesting to note, however, that electron microscopic studies of degenerated Nauta-Gygax material usually show very little silver deposition in the lipid rich myelin sheaths10,19,z0, zT. Other chemical mechanisms must also be considered. The oxidation ofhistidine and related argyrophilic protein components 33, for instance, may well be a significant factor in the production of the 'argyrophilia suppressing' effect of permanganateZL It seems reasonable to assume that the water soluble permanganate ions have a more easy access to the argyrophilic components after lipid extraction of the tissue. The unsuccessful attempt to impregnate degenerated axons with the suppressive NautaGygax method after lipid extraction may therefore be due to a more intense 'suppressive' effect~6 rather than to the extraction of argyrophilic material as was suggested by Evans and Hamlyn. The potassium permanganate may also have additional functions. Oxidation of remaining formalin, for instance, may initially enhance the impregnation. With further permanganate treatment the suppressive effect, first apparent in the less argyrophilic normal fibers, may give the illusion of a 'selective' stain. Excessively long exposure eliminates argyrophilia of all fibers. This well-known progression of effect indeed suggests a quantitative rather than a qualitative difference between normal and degenerating fibers with respect to the organic substratum of argyrophilia. Electron microscopic studies of silver impregnated sections demonstrate a striking variability in the form and site of metallic deposition 10,19,~°,27. Many physical as well as chemical factors probably contribute to the well-known capriciousness of the silver impregnation mechanism. The optimal postsurgical survival time is another important variable, which may vary, not only between different species, but also between different fiber systems within the same brain. Furthermore, a survival time suitable for the demonstration of degenerating axons of passage may be less appropriate or even inadequate for the impregnation of terminal degeneration.

'Terminal' and 'preterminal' degeneration While Glees 14 suggested the term 'terminal degeneration' for degenerative changes seen in synaptic endings, Nauta 2a adopted this term to include degeneration in both the axon arborizations and their synaptic endings. With increased use of the Glees and Nauta methods the opinion developed that only the Glees method could show degenerated boutons and the finest degenerating fibers, whereas the suppressive Nauta-Gygax method impregnated 'preterminal' fibers of somewhat coarser caliber, usually in the form of fine droplets arranged around the cell bodies ('pericellular arborizations'). Additional support for this view was given by Evans and Hamlyn 11 who suggested that the Nauta positive material was lipid which appeared in too small amounts in the synaptic endings and the finest degenerating fibers to be detected by the suppressive Nauta-Gygax method. The term 'preterminal degeneration' was there-

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fore proposed for the picture of 'pericellular arborizations' seen in the suppressive Nauta-Gygax method; the fine 'tail' connected to a degenerated terminal, sometimes seen with the Glees technique, was included in the term 'terminal degeneration'7. The difficult task of evaluating the nature of the argyrophilic bodies was thus circumvented by assuming that the silver particles seen with the suppressive Nauta-Gygax method represented 'preterminal degeneration' whereas degenerated synaptic endstructures ('terminal degeneration') could be demonstrated only with the Glees method. It has now been shown, however, that all commonly used reduced silver methods can impregnate synaptic endings as well as fibers. Since we are usually uncertain of the nature of the single argyrophilic body, and the method used does not permit a categorical identification, the unqualified terms 'preterminal' and 'terminal' degeneration in relation to silver techniques seem unjustified4s. The existence of'bouton en passage' verified with the electron microscope in several regions of the central nervous system, makes this distinction between 'terminal' and 'preterminal' still more dubious. It therefore seems appropriate to use the term 'terminal degeneration' for the degeneration in both the axon arborizations and their synaptic endings as suggested by Nauta 2s and more recently by Sprague and Ha 47.

Impregnation of normal synaptic endings for studying sites of termination In the Armstrong and Rasmussen methods for the impregnation of normal synaptic endings2, 41 whole tissue blocks are mordant in potassium dichromate before embedded in paraffin. Similar results can also be obtained when single paraffin sections are pretreated in dichromate before silver impregnation, thereby permitting the preparation of alternate series of sections stained by several techniques. Electron microscopic studies indicate that the potassium dichromate, widely used as a fixative for mitochondria, increases the argyrophilia of the mitochondria. The successful impregnation of normal boutons after postchroming may therefore be related to an increased silver deposition in the mitochondria as suggested by Armstrong et aLL Since mitochondria are found throughout the neuron, however, the silver deposits do not invariably indicate the presence of boutons, and a differential diagnosis of single argyrophilic particles is often impossible. The dichromate-silver method has recently been used for studying changes in synaptic endings after injury to the spinal cord 24. Silver techniques, however, require the aid of the electron microscope in evaluating minute changes in synaptic endings. Although the dichromate-silver method may give quantitative information about specific synaptic fields, its value as an experimental method appears to be limited to fiber systems having circumscript and dense synaptic fields. The disappearance of argyrophilic boutons must be quite extensive to be appreciated, and the fact that inferences must be drawn from the absence of stainable material is in itself a sign of inadequacy, especially with respect to silver techniques. CONCLUDING REMARKS

The foregoing account emphasizes the need for a thorough familiarity with

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the limitations and vagaries of experimental silver methods. Although all of the techniques currently used for the identification and tracing of axon degeneration have their unquestionable merits, all have their peculiar shortcomings, and hence, none should be relied upon exclusively. This point should be stressed in particular with respect to the suppressive Nauta-Gygax technique, the most widely used of the experimental silver methods. This technique is an excellent one for the study of fiber trajectories, and with proper application affords, in some fiber systems at least, fairly comprehensive identification of terminal distribution areas. Nevertheless, in the present comparative study of four different fiber systems, it consistently failed to disclose the quantity and mode of termination demonstrated with the original Nauta method or an improved recent modification of the latter. In one case, viz. that of the lateral olfactory tract, even the general region of distribution remained in doubt. These observations emphasize the necessity to supplement observations in suppressive Nauta-Gygax material by the study of sections stained by methods providing more detailed pictures of the terminal segments of degenerating axons. The latter methods, by contrast, may present difficulties in interpreting the often extremely rich pictures of terminal axon degeneration and thus require experience and cautious evaluation, particularly because the exact binding sites of metallic silver in degenerating neurons and the glial environment have not yet been identified satisfactorily. It must be emphasized, that even the most effective light microscopic methods can do little more than identify the synaptic fields of degenerating fiber systems. While such methods occasionally allow distinctions to be made between axosomatic and axodendritic synapses, they are inadequate for the identification of axoaxonic contacts, the presumable substratum of presynaptic inhibition. Details of this finesse can be obtained only by experimental studies with the electron microscope, which, however, in turn requires the guidance provided by light-microscopic observations. The innumerable modifications of the reduced silver technique reflect the difficulties encountered when metallic impregnation is applied to biological material. In commenting on silver techniques in particular, Wolman expressed this state of affairs by writing: 'Their practical application represents more often an art than a science'. It seems that one way out of this dilemma is to be aware of the dynamic character of the impregnation mechanism. Another one must be the perpetual search for the physicochemical principles involved in silver impregnation. SUMMARY

The relative value of current silver methods for the impregnation of terminal degeneration in the forebrain was reviewed. Thelateral olfactory tract, the commissural system in neocortex and hippocampus, and the optic tract were used as test models. The widely used suppressive Nauta-Gygax methodzg,al failed to show the quantity and mode of termination in all these fiber systems. In the lateral olfactory tract, as well as in the two commissural systems, even the region of distribution of axons was often doubtful. The original non-suppressive Nauta method2s, on the other hand, allowed an adequate impregnation of terminal degeneration, especially Brain Research, 5 (1967) 86-108

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in the lateral olfactory tract. However, more consistent impregnation of fine terminal fibers and synaptic thickenings are obtained with a recently developed modification of the original Nauta method (Fink and Heimer12), which in addition permits a differential impregnation of degenerating axons and synaptic endings. Although the Glees method 14 was found to yield good pictures of terminal degeneration in the olfactory cortex, it proved more capricious than the original Nauta method, and also less successful in the other fiber systems studied, The Armstrong2,8 and Rasmussen41 methods for the impregnation of normal synaptic endings may give quantitative information about specific synaptic fields. The value of these techniques for the experimental demonstration of fiber connections appears to be limited, however, to fiber systems of circumscript distribution and high synaptic density. APPENDIX METHODS AND MODIFICATIONS USED IN THE PRESENT STUDY

(1) Impregnation of degenerated synaptic endings (A) Frozen sections. Four methods were used: (1) the original Nauta method, (2) the Glees method, (3) the suppressive Nauta-Gygax method and (4) a modified original Nauta method. Even if the procedure as described originally by Nauta z8 was found useful for demonstration of degenerated axon terminals in many regions of the brain, its application in other areas was less successful and more consistent results were obtained by either one of two recent modifications of the original Nauta method (Fink and Heimer12). The results with the Glees method 14 seemed to be optimal when a diluted ammoniacal silver solution was used according to Lundberg26. It also proved valuable to reduce the formalin concentration to 0.5% (ref. 1). As already pointed out by Glees, the rapid transfer of the sections through several dishes of formalin was found to be of utmost importance both in the step following the initial silver nitrate bath and in the final reduction process. As suggested by Chambers, Liu and Lius the ammoniacal silver nitrate solution was replaced by the Laidlaw's ammoniacal silver carbonate solution in the suppressive Nauta-Gygax methodSL In some cases uranyl nitrate was used instead ofphosphomolybdic acid prior to oxidation with potassium permanganate (Nauta, personal communication). For electron microscopical study pieces were excised from some of the silver impregnated frozen sections, fixed in an osmic acid solution, dehydrated in ethanol and embedded in epon. (B) Paraffin sections. Most of the paraffin sections were impregnated with the original Nauta method 2s. The impregnation time (varying from 1 h to 2 days) in the initial silver-pyridine solution was found to be of secondary importance. No times Brain Research, 5 (1967) 86-108

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can be fixed for the development in the ammoniacal silver solution and in the reducing fluid. Treatment of the sections in ammoniated alcohol before silver impregnation was found to be unnecessary. To prevent the sections separating from the slides, it is helpful to coat the slides with a film of chrome-alum prior to mounting the sections. The chrome-alum is prepared by dissolving 300 mg of chrome-alum in 600 ml of a 0.5 ~o aqueous gelatin solution. The slides are dipped in this solution and permitted to dry. The sections are then placed on the slide.

(2) Impregnation of normal synaptic endings in paraffin sections After dewaxing, singly mounted sections were soaked in a 3 ~o potassium dichromate solution for periods varying between 2 and 14 days. They were then impregnated in a silver-pyridine solution (5 parts pyridine plus 95 parts 1.5~ silver nitrate) for 1-2 h, transferred to the Laidlaw solution, and reduced in the formalincitric acid-alcohol reducer of Nauta. Good results were also obtained when the dichromate treated sections were impregnated with the original non-suppressive Nauta method. For electron microscopical study some of the sections were treated as described in section 1A. ACKNOWLEDGEMENTS

Part of this work was done while the author was supported by a Training Fellowship from the Foundations Fund for Research in Psychiatry, New Haven, Conn. This work was supported by the U.S. Public Health Service, National Institute of Child Health and Human Development (HD 00344-04) and U.S. Department of Health, Education and Welfare (NB 06542-01), and by a grant from the Medical Faculty at the University of GSteborg. Grateful appreciation is expressed to Drs. T. Blackstad, K.-A. Karlsson, H. Karten, K. Larsson and W. J. H. Nauta for valuable assistance and criticism. The author is indebted to the late Mrs. Dorah Guttmark and to Miss Janet Aronson for technical support. ADDENDUM

After this article had gone to press Sterling and Kuypers [J. Anat. (Lond.), 100 (1967) 723-732] described a method for the silver impregnation of boutons terminaux in the spinal cord. The characteristic of this technique appears to lie in simultaneous but differential impregnation of normal and degenerating boutons. REFERENCES 1 ADEY,W. R., RUDOLPH,A. F., HINE,I. F., AND HARRITH,N. J., Glees staining of the monkey hypothalamus: A critical appraisal of normal and experimental material, J. Anat. (Lond.), 92 (1958) 219-235.

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2 ARMSTRONG, J., RICHARDSON, K. C., AND YOtmG, J. Z., Staining neural end-feet and mitochondria after postchroming and carbowax embedding, Stain TechnoL, 31 (1956) 263-270. 3 ARMSTRONG, J., AND STEPHENS, P. R., A modified chrome-silver paraffin wax technique for staining neural end-feet, Stain Technol., 35 (1960) 71-75. 4 BIELSCHOWSKY,M., Die Silberimpregnation der Neurofibrillen, J. Psychol. Neurol. (Ipz.), 3 (1904) 169-188. 5 BLACKSTAO,T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. NeuroL, 105 (1956) 417-537. 6 BLACKSTAD,T. W., On the termination of some afferents to the hippoeampus and fascia dentata. An experimental study in the rat, Acta anat. (Basel), 35 (1958) 202-214. 7 BOWSHER,n., BROOAL,A., AND WALBERG,F., The relative values of the Marchi method and some silver impregnation techniques, Brain, 83 (1960) 150-160. 8 CHAMBERS,W. W., LIU, CHANG-YN,AND LIU, CHAN-NAO,A modification of the Nauta technique for staining of degenerating axons in the central nervous system, Anat. Rec., 124 (1956)391-392. 9 COWAN, W. M., AND POWELL, T. P. S., A note on terminal degeneration in the hypothalamus, J. Anat. (Lond.), 90 (1956) 188-192. 10 EAGER, R. P., AND BARRNETT, R. J., Morphological and chemical studies of Nauta-stained degenerating cerebellar and hypothalamic fibers, J. comp. Neurol., 126 (1966) 487-510. 11 EVANS, D. H. L., AND HAMLYN, L. H., A study of silver impregnation methods in the central nervous system, J. Anat. (Lond.), 90 (1956) 193-202. 12 FINK, 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. 13 GIOLLI, R. A., A note on the chemical mechanism of the Nauta-Gygax technique, J. Histochem. Cytochem., 13 (1965) 206-210. 14 GLEES, P., Terminal degeneration within the central nervous system as studied by a new silver method, J. Neuropath. exp. Neurol., 5 (1946) 54-59. 15 GLEES,P., AND NAUTA,W. J. H., A critical review of studies on axonal and terminal degeneration, Mschr. Psychiat. Neurol., 129 (1955) 74-91. 16 GRAY, E. G., AND GUILLERY,R. W., Synaptic morphology in the normal and degenerating nervous system, Int. Rev. Cytol., 19 (1966) 111-182. 17 GRAY, E. G., AND HAMLYN,L. H., Electron microscopy of experimental degeneration in the avian optic tectum, d. Anat. (Lond.), 96 (1962) 309-316. 18 GUILLERY, R. W., Some electron microscopical observations of degenerative changes in the central nervous synapses. In W. BARGMANNAND J. e. SCHAOE(Eds.), Degeneration Patterns in the Nervous System, Progress in Brain Research, Vol. 14, Elsevier, Amsterdam, 1965, pp. 57-76. 19 GUILLERY, R. W., AND RALSTON, H. J., Nerve fibers and terminals: electron microscopy after Nauta staining, Science, 143 (1964) 1331-1332. 20 HEIMER, L., AND EKHOLM, R., Neuronal argyrophilia in early degenerative states: A light and electron microscopical study of the Glees and Nauta techniques, Experientia (Basel), 23 (1967) (in press). 21 HOFF, E. C., The distribution of spinal terminals (boutons) of the pyramidal tract determined by experimental degeneration, Proc. roy. Soc. B, 111 (1932) 226-237. 22 HOLMES,W., Silverstaining of nerve axons in paraffin sections, Anat. Rec., 86 (1943) 157. 23 HOLMES,W., Peripheral nerve biopsy. In S. C. DYKE (Ed.), Recent Advances in ClinicalPathology, Churchill, London, 1947, pp. 402-417. 24 ILLIS, L., Changes in spinal cord synapses and a possible explanation for spinal shock, Exp. Neurol., 8 (1963) 328-335. 25 LIESEGANG,R. E., Die Kolloid-Chemie der histologischen Silberf~irbung, KolloidBeitr., 3 (1911) 1. 26 LUNDBERO, P. O., Cortico-Hypothalamic Connexions in the Rabbit. An Experimental NeuroAnatomical Study. Acta physiol, scand., Suppl. 171, 49 (1960). 27 LUND, R. D., AND WESTRUM, L. E., Neurofibrils and the Nauta method, Science, 151 (1966) 1397-1399. 28 NAUTA, W. J. H., Ober die sogenannte terminale Degeneration im Zentralnervensystem und ihre Darstellung durch Silberimpriignation, Schweiz. Arch. Neurol. Psychiat., 66 (1950) 353-376. 29 NAUTA, W. J. H., Silver impregnation of degenerating axons. In W. F. WINDLE (Ed.), New Research Techniques of Neuroanatomy, Thomas, Springfield, Ill., 1957, p. 1% 30 NAUTA, W. J. H., AND GYGAX, P, A., Silver impregnation of degenerating axon terminals in the central nervous system: (1) Technic, (2) Chemical notes, Stain Technol., 26 (1951) 5-11.

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