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An improved cupric-silver method for impregnation of axonal and terminal degeneration
SHORT COMMUNICATIONS
523
An improved cupric-silver method for impregnation of axonal and terminal degeneration In a previous report, one of the authors ~ introduced a cupric-silver procedure which produces a highly selective demonstration of degenerating axoplasm in frozen sections of brains from experimental animals. However, as was pointed out in that report, the experimental identification of neural pathways by this method may become complicated by the coincidental appearance of granular varicosities in an impregnated normal neuropil, especially in certain cerebral areas. This particular impregnated neuropil appears to be formed by the processes of granular types of argyrophilic neurons which have been described by Knoche s and Bliimcke 1. These cells are loosely grouped in certain areas of the hypothalamus and related structures, as previously discussed2. Since the use of the cupric-silver method has proved to be valuable in tracing fiber connections3,4, efforts have been made to eliminate this complication as well as some capriciousness due to occasional variations in the preparation of the original ammoniacal silver nitrate solution. A background granularity, possibly resulting from the use of phosphate buffered fixatives, has also been brought under control. The present communication is therefore concerned with modifications of the original impregnation procedUre which not only assure more consistent results and a 'cleaner' background, but also provide means of controlling, within certain limits, the stainability of the granular argyrophilic neuropil. The latter purpose is accomplished by a treatment of the sections with pure acetone immediately after they have been exposed to the copper-silver mixture. This effect of acetone, probably largely physical; depends also upon other variables which will be discussed further on. The procedure described below is now routinely used in ~this laboratory, with consistent and satisfactory results, for the demonstration of degenerating axons and terminals in experimental material from rats and cats. Preparation of the tissues. (1) Fix the brain material by perfusion with 4 ~ paraformaldehyde in 0.067 M cacodylate buffer (pH 7.2-7.4) containing 4 ~ sucrose, following exsanguination by an aqueous solution containing sodium chloride 0.8 ~, glucose 0.4 ~ , and sucrose 0.8 ~o (w/v) with pH adjusted to 7.2-7.4 with sodium bicarbonate. (2) Following removal and further fixation in the perfusion fluid for 2-8 days (a time span which can vary according to species and agee), immerse the brains in - 30 ~o sucrose in 0.067 M cacodylate buffer until the infiltrated tissue sinks. (3) Cut frozen sections at 25-30 #m and store them for 2-4 days in the fixative solution. Silver impregnation. (1) Rinse the sections in 3 changes of distilled water. (2) Transfer them to the copper-silver mixture (rat, 3-4 days; cat, 4-6 days). The stock solution is prepared as follows: 1000 ml 2.5 ~ silver nitrate, 15 ml 0.5 ~ cupric nitrate, 40 ml pyridine, 80 ml 95 ~o ethyl alcohol. (3) Place sections in pure acetone for 3-5 min. (4) Transfer them for 7-15 min to an ammoniacal silver nitrate solution freshly prepared as follows: 7.4 ml of a stock solution consisting of 200 ml 0.36 ~ sodium hydroxide and 90 ml concentrated ammonium hydroxide are added to 10 ml of 20 ~o silver nitrate. This mixture is then diluted 1:1 with distilled water. (5) Place directly
Brain Research, 33 (1971) 523-529
524
SHORq COMMUNI( A]IONS
for 2-4 rain in a reducer composed of 24 ml 1 0 ~ non-neutralized lbrmalin, 14 ml (for rat) or 12 ml (for cat) 1 ~o citric acid, 200 ml 100~ ethyl alcohol, and distilled water to make up 2000 ml. The sections now become dark brown. (6) Rinse in distilled water (time non-critical). (7) Bleach in 0.5 ~ potassium ferricyanide (timing determined by examination). The sections should grossly become golden-yellowish in color. (8) Rinse thoroughly in distilled water. (9) Place in 1 ~o sodium thiosulfate for 1 rain. (10) Rinse in distilled water. Brain Research, 33 (1971) 523-529
SHORT COMMUNICATIONS
525
Fig. 1. Parasagittal section through the rat cerebellum 2 days after production of a lesion in the dorsal aspect of lobule V and its medullary core; cupric-silver method, a, The degenerative changes appear in black. Degenerating fibers descend from the lesion (L) to reach and distribute topically in the nucleus interpositus (I). x 40. b, Higher magnification ( x 500) of the area in and near (I) in a to show axonal degeneration (above) and degeneration of terminals (below).
Brain Research, 33 (1971) 523-529
526
SHORT ('OMMUNICATIONS
The sections are now rapidly dehydrated in 95 ~,, ethyl alcohol followed by pure acetone and cleared in a mixture of xylene 70 ml, creosote 20 ml, phenol 10 g, and mounted in Permount. For best results, room temperature should not be allowed to fluctuate beyond 22-25°C. The experimental results obtainable with this procedure compare favorably with those of the original version. Degenerating axons and terminals stand out sharply Brain Research, 33 (1971) 523-529
SHORT COMMUNICATIONS
527
Fig. 2. Transverse section through the rat cerebral cortex 2 days after production of a lesion in the internal capsule, a, 'Pre-terminal'-like degeneration in the deep cortical layers. Note also the spot-like degeneration in the striatum (Str) and the axonal degeneration in the corona radiata (CR). b, Terminal degeneration in cortical layer II. Cupric-silver method. × 500. in dark brown or black against a light golden-yellow background, while impregnation o f n o r m a l fibers is well suppressed. As with several comparable techniquesS,~, i°, this staining process belongs to the category o f silver methods which demonstrate degenerating axon terminals in the f o r m o f more or less globular black corpuscles (Figs. Brain Research, 33 (1971) 523-529
528
SHORT COMMUNICATIONS
l b, 2b). The method described here does this very effectively. A most important advantage over its prototype lies in the possibility of reducing the impregnation of normal granular neuropil as has been discussed. It also appears that the present version produces a more intensive impregnation of non-terminal axon segments, in particular 'pre-terminal' axon arborizations (Fig. 2a). As with other silver procedures, problems of interpretation may arise because of the occurrence of variable amounts of irregular, mainly extracellular, silver deposits, particularly in the glomerular layer of the olfactory bulbsT,L For an accounting of some common sources of error in interpreting the results of silver techniques of this general category, reference is made to a recent article by Heimer 7. The procedure suggested here may help to reduce such sources of error. The staining schedule may vary somewhat for different species or even for animals of the same species but different age. The length of time during which the tissues are stored in the fixative is important. These and other factors have been discussed at length elsewhere 2. Furthermore, in dealing with species other than those used here, consideration might, if necessary, be given to altering the concentration of citric acid in the reducer. On the other hand, experience indicates that within the time factors established in the above schedule, use of long periods of fixation (up to 8 days) plus thorough acetone treatment and brief immersion of the sections in the ammoniacal silver nitrate solution and in the reducer are associated with a reduction in the staining of the granular argyrophilic neuropil. This action is probably related to facilitation of the bleaching action of the ferricyanide solution. This work was supported by Grants 5ROI NS08166 and 5TO1 NS05249 from the NINDS. Department of Anatomy, The University of Iowa, Iowa City, Iowa 52240 (U.S.A.)
JOSI~ S. DE OLMOS WALTER R. I N G R A M
1 BLOMCKE, S., Vergleichend experimentell-morphologische Untersuchungen zur Frage einer retino-hypothalamischen Bahn bei Huhn, Meerschweinchen und Katze, Z. mikr.-anat. Forsch., 67 (1962) 469-513. 2 DE OLMOS, J. S., A cupric-silver method for impregnation of terminal axon degeneration and its further use in staining granular argyrophilic neurons, Brain Behav. Evol., 2 (1969) 213-237. 3 DE OLMOS,J. S., The amygdaloid projection field in the rat brain as studied with different silver procedures, Anat. Rec., 166 (1970) 298. 4 DE OLMOS,J. S., The projection field of the stria terminalis in the rat brain. An experimental study with silver techniques. Thesis, The University of Iowa, Iowa City, Iowa, 1971. 5 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 olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 6 F~NK, R. P., AND HEMMER,L., Two methods for selective impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 7 HEIMER, L., Selective silver-impregnation of degenerating axoplasm. In W. J. H. NAUTA AND S. O. E, EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, New York, 1970, pp. 106-131. 8 KNOCHE, H., Clber die Ausbreitung und Herkunft der nerv6sen Nodulusfasern in Hypothalamus und Retina, Z. Zellforsch., 48 (1958) 602-616. (Also Morph. Jb., 105 (1964) 477--494.)
Brain Research, 33 (1971) 523-529
SHORT COMMUNICATIONS
529
9 LAND, L. J., EAGER, R. P., AND SHEPHERD,G. M., 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. 10 NAUTA,W. J. H., AND GYGAX,P. A., Silver impregnation of degenerating axons in the central nervous system: 1. Technic, 2. Chemical notes, Stain Technol., 26 (1951) 5-11. (Accepted July 28th, 1971)
Brain Research, 33 (1971) 523-529
523
An improved cupric-silver method for impregnation of axonal and terminal degeneration In a previous report, one of the authors ~ introduced a cupric-silver procedure which produces a highly selective demonstration of degenerating axoplasm in frozen sections of brains from experimental animals. However, as was pointed out in that report, the experimental identification of neural pathways by this method may become complicated by the coincidental appearance of granular varicosities in an impregnated normal neuropil, especially in certain cerebral areas. This particular impregnated neuropil appears to be formed by the processes of granular types of argyrophilic neurons which have been described by Knoche s and Bliimcke 1. These cells are loosely grouped in certain areas of the hypothalamus and related structures, as previously discussed2. Since the use of the cupric-silver method has proved to be valuable in tracing fiber connections3,4, efforts have been made to eliminate this complication as well as some capriciousness due to occasional variations in the preparation of the original ammoniacal silver nitrate solution. A background granularity, possibly resulting from the use of phosphate buffered fixatives, has also been brought under control. The present communication is therefore concerned with modifications of the original impregnation procedUre which not only assure more consistent results and a 'cleaner' background, but also provide means of controlling, within certain limits, the stainability of the granular argyrophilic neuropil. The latter purpose is accomplished by a treatment of the sections with pure acetone immediately after they have been exposed to the copper-silver mixture. This effect of acetone, probably largely physical; depends also upon other variables which will be discussed further on. The procedure described below is now routinely used in ~this laboratory, with consistent and satisfactory results, for the demonstration of degenerating axons and terminals in experimental material from rats and cats. Preparation of the tissues. (1) Fix the brain material by perfusion with 4 ~ paraformaldehyde in 0.067 M cacodylate buffer (pH 7.2-7.4) containing 4 ~ sucrose, following exsanguination by an aqueous solution containing sodium chloride 0.8 ~, glucose 0.4 ~ , and sucrose 0.8 ~o (w/v) with pH adjusted to 7.2-7.4 with sodium bicarbonate. (2) Following removal and further fixation in the perfusion fluid for 2-8 days (a time span which can vary according to species and agee), immerse the brains in - 30 ~o sucrose in 0.067 M cacodylate buffer until the infiltrated tissue sinks. (3) Cut frozen sections at 25-30 #m and store them for 2-4 days in the fixative solution. Silver impregnation. (1) Rinse the sections in 3 changes of distilled water. (2) Transfer them to the copper-silver mixture (rat, 3-4 days; cat, 4-6 days). The stock solution is prepared as follows: 1000 ml 2.5 ~ silver nitrate, 15 ml 0.5 ~ cupric nitrate, 40 ml pyridine, 80 ml 95 ~o ethyl alcohol. (3) Place sections in pure acetone for 3-5 min. (4) Transfer them for 7-15 min to an ammoniacal silver nitrate solution freshly prepared as follows: 7.4 ml of a stock solution consisting of 200 ml 0.36 ~ sodium hydroxide and 90 ml concentrated ammonium hydroxide are added to 10 ml of 20 ~o silver nitrate. This mixture is then diluted 1:1 with distilled water. (5) Place directly
Brain Research, 33 (1971) 523-529
524
SHORq COMMUNI( A]IONS
for 2-4 rain in a reducer composed of 24 ml 1 0 ~ non-neutralized lbrmalin, 14 ml (for rat) or 12 ml (for cat) 1 ~o citric acid, 200 ml 100~ ethyl alcohol, and distilled water to make up 2000 ml. The sections now become dark brown. (6) Rinse in distilled water (time non-critical). (7) Bleach in 0.5 ~ potassium ferricyanide (timing determined by examination). The sections should grossly become golden-yellowish in color. (8) Rinse thoroughly in distilled water. (9) Place in 1 ~o sodium thiosulfate for 1 rain. (10) Rinse in distilled water. Brain Research, 33 (1971) 523-529
SHORT COMMUNICATIONS
525
Fig. 1. Parasagittal section through the rat cerebellum 2 days after production of a lesion in the dorsal aspect of lobule V and its medullary core; cupric-silver method, a, The degenerative changes appear in black. Degenerating fibers descend from the lesion (L) to reach and distribute topically in the nucleus interpositus (I). x 40. b, Higher magnification ( x 500) of the area in and near (I) in a to show axonal degeneration (above) and degeneration of terminals (below).
Brain Research, 33 (1971) 523-529
526
SHORT ('OMMUNICATIONS
The sections are now rapidly dehydrated in 95 ~,, ethyl alcohol followed by pure acetone and cleared in a mixture of xylene 70 ml, creosote 20 ml, phenol 10 g, and mounted in Permount. For best results, room temperature should not be allowed to fluctuate beyond 22-25°C. The experimental results obtainable with this procedure compare favorably with those of the original version. Degenerating axons and terminals stand out sharply Brain Research, 33 (1971) 523-529
SHORT COMMUNICATIONS
527
Fig. 2. Transverse section through the rat cerebral cortex 2 days after production of a lesion in the internal capsule, a, 'Pre-terminal'-like degeneration in the deep cortical layers. Note also the spot-like degeneration in the striatum (Str) and the axonal degeneration in the corona radiata (CR). b, Terminal degeneration in cortical layer II. Cupric-silver method. × 500. in dark brown or black against a light golden-yellow background, while impregnation o f n o r m a l fibers is well suppressed. As with several comparable techniquesS,~, i°, this staining process belongs to the category o f silver methods which demonstrate degenerating axon terminals in the f o r m o f more or less globular black corpuscles (Figs. Brain Research, 33 (1971) 523-529
528
SHORT COMMUNICATIONS
l b, 2b). The method described here does this very effectively. A most important advantage over its prototype lies in the possibility of reducing the impregnation of normal granular neuropil as has been discussed. It also appears that the present version produces a more intensive impregnation of non-terminal axon segments, in particular 'pre-terminal' axon arborizations (Fig. 2a). As with other silver procedures, problems of interpretation may arise because of the occurrence of variable amounts of irregular, mainly extracellular, silver deposits, particularly in the glomerular layer of the olfactory bulbsT,L For an accounting of some common sources of error in interpreting the results of silver techniques of this general category, reference is made to a recent article by Heimer 7. The procedure suggested here may help to reduce such sources of error. The staining schedule may vary somewhat for different species or even for animals of the same species but different age. The length of time during which the tissues are stored in the fixative is important. These and other factors have been discussed at length elsewhere 2. Furthermore, in dealing with species other than those used here, consideration might, if necessary, be given to altering the concentration of citric acid in the reducer. On the other hand, experience indicates that within the time factors established in the above schedule, use of long periods of fixation (up to 8 days) plus thorough acetone treatment and brief immersion of the sections in the ammoniacal silver nitrate solution and in the reducer are associated with a reduction in the staining of the granular argyrophilic neuropil. This action is probably related to facilitation of the bleaching action of the ferricyanide solution. This work was supported by Grants 5ROI NS08166 and 5TO1 NS05249 from the NINDS. Department of Anatomy, The University of Iowa, Iowa City, Iowa 52240 (U.S.A.)
JOSI~ S. DE OLMOS WALTER R. I N G R A M
1 BLOMCKE, S., Vergleichend experimentell-morphologische Untersuchungen zur Frage einer retino-hypothalamischen Bahn bei Huhn, Meerschweinchen und Katze, Z. mikr.-anat. Forsch., 67 (1962) 469-513. 2 DE OLMOS, J. S., A cupric-silver method for impregnation of terminal axon degeneration and its further use in staining granular argyrophilic neurons, Brain Behav. Evol., 2 (1969) 213-237. 3 DE OLMOS,J. S., The amygdaloid projection field in the rat brain as studied with different silver procedures, Anat. Rec., 166 (1970) 298. 4 DE OLMOS,J. S., The projection field of the stria terminalis in the rat brain. An experimental study with silver techniques. Thesis, The University of Iowa, Iowa City, Iowa, 1971. 5 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 olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 6 F~NK, R. P., AND HEMMER,L., Two methods for selective impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 7 HEIMER, L., Selective silver-impregnation of degenerating axoplasm. In W. J. H. NAUTA AND S. O. E, EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, New York, 1970, pp. 106-131. 8 KNOCHE, H., Clber die Ausbreitung und Herkunft der nerv6sen Nodulusfasern in Hypothalamus und Retina, Z. Zellforsch., 48 (1958) 602-616. (Also Morph. Jb., 105 (1964) 477--494.)
Brain Research, 33 (1971) 523-529
SHORT COMMUNICATIONS
529
9 LAND, L. J., EAGER, R. P., AND SHEPHERD,G. M., 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. 10 NAUTA,W. J. H., AND GYGAX,P. A., Silver impregnation of degenerating axons in the central nervous system: 1. Technic, 2. Chemical notes, Stain Technol., 26 (1951) 5-11. (Accepted July 28th, 1971)
Brain Research, 33 (1971) 523-529