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Argyrophilic interconnexions of brain blood vessels
548
SHORT COMMUNICATIONS
Argyrophilic interconnexions of brain blood vessels Mammalian brain tissue stained by the common reduced silver methods frequently shows argyrophilic connexions between vessels. These were referred to by Cajal 1, who wrote when describing the vessels of the spinal cord: ' D a n s les capillaires un peu plus gros il existe, en dehors de l'endoth61ium, une membrane adventice tr~s mince, form6e par l'entrelacement compliqu6 de fibres conjonctives. Cette adventice 6met souvent des faisceaux qui traversent la substance grise pour aller s'ins6rer sur l'adventice d'autres capillaires.' This was observed by Cajal in sections of the spinal cord as well as in other areas of the brain stained by his silver method. Similar connexions can be seen in the microphotographs in papers where the Nauta method and some of its modifications are usedZ,3, 5. In cat brain tissue fixed by vascular perfusion of formaldehyde and stained by the Wiitanen 11 modification of the Nauta method, the vessels are often outlined by an argyrophilic network (Fig. 1) and connexions between them are frequently found (Figs. 1-8). The argyrophilic perivascular network and its intervascular connexions may appear solid (Figs. 1-3 and 5-8) but they may also have a somewhat granular appearance (Fig. 4). The perivascular network is well defined around the larger vessels (Fig. I). Capillaries are outlined only by a faint and powdery silver precipitate with no evident structure and with scattered dark endothelial nuclei (Figs. 7 and 8). At the places where connexion with the intervascular argyrophilic bridges is established the capillary outline shows a filamentous structure (Figs. I and 6). The intervascular argyrophilic bridges extend between vessels of all calibres and frequently have a ' Y ' shape connecting 3 vessels (Fig. 2). These bridges have a thickness ranging from 1.5
Fig. 1. Argyrophilic intervascular connexions (arrows) between two vessels larger than capillaries (on the left) and from one of these with a capillary (on the right). The larger vessels are clearly outlined by an argyrophilic perivascutar network. This is incomplete in the capillary. Cat parietal cortex: 30/~m thick section stained by the Wiitanen 11 method. × 1,600. Abbreviations used in this and the following figures: as, astrocyte nucleus; c, capillary vessel; f, astroglial microfilaments; Iv, vessel larger than capillaries; n, glial nucleus; and pv, artifactual (?) perivascular space. Brain Research, 33 (1971) 548-553
SHORT COMMUNICATIONS
549
Fig. 2. Argyrophilic branching intervascular connexion (arrows). Note the solid appearance of the impregnation in the intervascular connexion and the granular pattern in the capillary wall. Tissue treatment and magnification as in Fig. 1.
Fig. 3. Silver impregnation of the vascular network in a 100 #m thick section of cat parietal cortex stained by the Wiitanen 11 method. The larger vessels are evidently outlined by a silver-impregnated network, while capillaries are shown by a powdery silver precipitate with scattered endothelial nuclei. Two argyrophilic bridges are visible (arrows). × 100. /zm (Fig. 2) to 0.4 # m (Fig. 4) and at high magnification their fibrillar appearance is often discerned. W h e n thick sections are used, the argyrophilic perivascular network and bridges are more a b u n d a n t (Figs. 3 and 6-8). In 100/zm sections, the vessels are clearly and more completely outlined (Fig. 3). In these sections a perivascular network is clearly seen a r o u n d the large vessels, while capillaries a p p e a r as an argyrophilic veil with scattered and heavily impregnated endothelial nuclei. At high magnification the connexion o f the bridges with the large vessels (Fig. 6) and capillaries (Figs. 6-8) is Brain Research, 33 (1971) 548-553
550
SHORT COMMUNICATIONS
clearly recognized. O c c a s i o n a l l y these bridges are seen in close relation to d a r k nuclei p r e s u m e d to be glial (Figs. 6 and 8). N o structure in the central nervous system has been clearly d e m o n s t r a t e d t h a t m a y a c c o u n t for these a r g y r o p h i l i c i n t e r v a s c u l a r connexions. Cajal's suggestion t h a t they represent connective tissue strands between the a d v e n t i t i a l sheaths o f capillaries is u n t e n a b l e t o d a y , since such c o n n e x i o n s have never been f o u n d in electron microscopic studies o f the nervous system. F u r t h e r m o r e , such studies have failed to disclose a connective tissue a d v e n t i t i a a r o u n d the capillaries o f the central nervous system. The a r g y r o p h i l i c n e t w o r k a r o u n d larger vessels could represent the well-known connective tissue a d v e n t i t i a . However, this i n t e r p r e t a t i o n does n o t a p p e a r the most likely. The p e r i v a s c u l a r n e t w o r k o f larger vessels, as seen in this material, is m o r p h o logically similar a n d a p p a r e n t l y c o n t i n u o u s with t h a t o f smaller vessels where no connective tissue is f o u n d (Fig. 3). F u r t h e r m o r e , it is also c o n t i n u o u s with a n d similar to the i n t e r v a s c u l a r bridges. M o r i a n d L e b l o n d 6 suggested t h a t bundles o f filaments can pass in astrocytic processes between capillaries and establish such bridges. This suggestion agrees with classical views c o n c e r n i n g astrocytic vascular 'feet'. A s t r o c y t e s have processes a t t a c h e d to the v a s c u l a r walls a n d each astrocyte m a y have several such processes. C o n s e q u e n t l y , there is considerable o p p o r t u n i t y for an astrocyte to establish a bridge between different sites o f the vascular tree. The a u t h o r s m e n t i o n e d a b o v e suggest, in a d d i t i o n , t h a t the filamentous astroglial bridges m a y p r o v i d e s u p p o r t for the n e r v o u s tissue by reinforcing the vascular n e t w o r k . However, these suggestions rest only on indirect evidence.
Fig. 4. Four argyrophilic intervascular connexions. Note the granular appearance of the impregnation in the intervascular connexions and capillary walls. Tissue treatment as in Fig. 1. x 900. Fig. 5. Argyrophilic bridges in 100/~m thick sections of cat parietal cortex. Two bridges are visible (arrows), connecting the same capillary with two other vessels. Tissue treatment as in Fig. 3. x 900. Fig. 6. Argyrophilic connexion (arrow) between a large vessel and a capillary. The larger vessel is clearly shown by a perivascular network, and the capillary by an irregular silver precipitate with a net-like structure at the site of insertion of the intervascular connexion. Tissue treatment as in Fig. 3. x 600. Fig. 7. Argyrophilic connexion (arrow) between two capillaries. Tissue treatment as in Fig. 3. x 900. Fig. 8. Argyrophilic connexion (arrows) between two capillaries, closely related to a glial nucleus. Tissue treatment as in Fig. 3. × 900. Fig. 9. Layers of filament bundles around a large vessel. Six different layers of microfilament-filled astroglial processes are visible around the wall of a large vessel. Cat parietal cortex fixed by perfusion with formaldehyde and glutaraldehyde, and postfixed with osmium tetroxide and uranyl acetate8. Ethanol dehydration and embedding in Spurr's 9 resin. Grid staining with lead10. × 30,000. Fig. 10. Filament bundles in astroglial profiles around a capillary wall. Tissue treatment and magnification as in Fig. 9. Fig. 11. Filament bundle approaching and encircling a capillary. Tissue treatment as in Fig. 9. × 3,300. Fig. 12. Photomontage showing a filament bundle connecting the wall of a vessel with the perikaryon of an astrocyte. The filament bundle is interrupted (arrows) but in the next section it has been possible to confirm the continuity of the two segments. Tissue treatment as in Fig. 8. z 5,000. Brain Research, 33 (1971) 548-553
mm
552
SHORT COMMUNICAI-IONS
Additional indirect evidence in support of this interpretation was provided by the electron microscopic study of cat cerebral cortex fixed by perfusion with phosphatebuffered formaldehyde and glutaraldehyde, followed by osmium postfixation, in some blocks uranyl acetate was used as a postfixative 8, in addition to osmium, a procedure which helped in making astrocytic filaments prominent. Under the electron microscope the larger vessels often appear surrounded by astroglial processes filled with bundles of filaments. These bundles frequently form several layers around the vessel (Fig. 9). Astroglial filaments are also frequent around capillaries (Fig. 10), but large areas of these vessels have no such filaments around them. The filamentous astroglial processes appear to be confined to some segments of the capillaries (Fig. 10) and in favourable sections it is possible to see a filamentous bundle approaching and encircling a capillary wall (Fig. 11). This perivascular distribution of filament-filled astroglial processes, as well as the range of thickness of the microfilament bundles, agree well with the dimensions and distribution of the argyrophilic structures seen in the light microscope. It appears very likely that most of the perivascular network and the intervascular connexions shown by light microscopy may be composed of silver-impregnated bundles of astroglial filaments. This interpretation agrees with Mori and Leblond's 6 suggestion that astrocytes establish microfilament filled bridges between the central nervous system vessels. Strong support for this view is provided by the finding of a bundle of astrocytic filaments encircling a vessel and passing through the astrocyte perikaryon towards another vessel as shown in Fig. 12. Here a bundle of filaments close to the wall of a large vessel is seen to pass through the neuropil to the perikaryon of an astrocyte. Gray and Guillery 4 have adduced evidence that the filaments in nerve cells of the spinal cord can be impregnated with silver by the methods of Cajal and Bielschowsky. In many studies of filaments it is apparently assumed that these structures are basically similar in every situation and in different animal species. However, Wuerker 12 has recently shown that astroglial filaments have structural subunits which are smaller than those of neurofilaments, and has suggested on the basis of this finding that the two types of filament may be composed of different proteins. Cajal's original description, together with the observations made in the present study, suggest that glial filaments can be impregnated along with neurofilaments by a variety of more or less routine silver techniques. This suggestion emphasizes once more the need for great care in the interpretation of silver-impregnated sections of brain tissue. Intracellular support, intracellular transport and mechanochemical transduction have been suggested as possible functions of filaments 7. If the argyrophilic bridges observed in the present study are indeed composed of astroglial filaments, their concentration around the large vessels and the fact that they connect different, but nearby, points of the vascular network would be in agreement with Mori and Leblond's 6 suggestion that such bridges contribute to the support of nervous tissue. Studies of serial EM sections of silver-stained material are necessary to decide if the present interpretation of the argyrophilic intervascular bridges is tenable.
Brain Research, 33 (1971) 548-553
SHORT COMMUNICATIONS
553
H e l p f u l discussions with Profs. A . C o i m b r a a n d M. M. Magalh[les, L a b o r a t o r y o f H i s t o l o g y , M e d i c a l S c h o o l o f O p o r t o , P o r t u g a l are gratefully a c k n o w l e d g e d . This w o r k received financial s u p p o r t f r o m the I n s t i t u t o p a r a a A l t a C u l t u r a as well as f r o m the C a l o u s t e G u l b e n k i a n F o u n d a t i o n , Lisbon, P o r t u g a l . N o t e added in proof. A f t e r this c o m m u n i c a t i o n was sent to press a p a p e r by A b a d i a - F e n o l l et al. (Rev. Roum. Neurol., 7 (1970) 19-25) c a m e to the a t t e n t i o n o f the a u t h o r . In t h a t p a p e r , similar a r g y r o p h i l i c i n t e r v a s c u l a r bridges are shown by a different silver m e t h o d . T h o s e a u t h o r s suggest a different i n t e r p r e t a t i o n o f these structures. Anatomical Institute, Medical School University of Oporto, Oporto (Portugal)
A. SOUSA-PINTO
1 CAJAL, S. RAMtSNY, Histologie du Syst~me Nerveux de l'Homme et des Vertebras, Cons. Sup.
Inv. Cient., Madrid, 1952, 520. pp. 2 EAGER,R. P., Selective staining of degenerating axons in the central nervous system by a simplified
3 4
5 6 7 8 9 10
11
12
silver method: spinal cord projection to external cuneate and inferior olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 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. 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. LOEWY,A. D., Ammoniacal silver staining of degenerating axons, Acta Neuropath. (Berl.), 14 (1969) 226-236. MORI, S., AND LEBLOND, C. P., Electron microscopic features and proliferation of astrocytes in the corpus callosum of the rat, J. comp. Neurol., 137 (1969) 197-226. SCHMITT, F. O., Fibrous proteins - - neuronal organelles, Proc. nat. Acad. Sci. (Wash.), 60 (1968) 1092-1101. SILVA, M. T., CARVALHOGUERRA, F., AND MAGALHAES,M. M., The fixative action of uranyl acetate in electron microscopy, Experientia (Basel), 24 (1968) 1074. SPURR,A. R., A low viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res., 26 (1969) 31-43. VENABLE,J. H., AND COGGESHALL,R., A simplified lead citrate stained for use in electron microscopy, J. Cell Biol., 25 (1965) 407-408. 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. WUERKER,B. B., Neurofilaments and glial filaments, Tissue and Cell, 2 (1970) 1-10.
(Accepted July 23rd, 1971)
Brain Research, 33 (1971) 548-553
SHORT COMMUNICATIONS
Argyrophilic interconnexions of brain blood vessels Mammalian brain tissue stained by the common reduced silver methods frequently shows argyrophilic connexions between vessels. These were referred to by Cajal 1, who wrote when describing the vessels of the spinal cord: ' D a n s les capillaires un peu plus gros il existe, en dehors de l'endoth61ium, une membrane adventice tr~s mince, form6e par l'entrelacement compliqu6 de fibres conjonctives. Cette adventice 6met souvent des faisceaux qui traversent la substance grise pour aller s'ins6rer sur l'adventice d'autres capillaires.' This was observed by Cajal in sections of the spinal cord as well as in other areas of the brain stained by his silver method. Similar connexions can be seen in the microphotographs in papers where the Nauta method and some of its modifications are usedZ,3, 5. In cat brain tissue fixed by vascular perfusion of formaldehyde and stained by the Wiitanen 11 modification of the Nauta method, the vessels are often outlined by an argyrophilic network (Fig. 1) and connexions between them are frequently found (Figs. 1-8). The argyrophilic perivascular network and its intervascular connexions may appear solid (Figs. 1-3 and 5-8) but they may also have a somewhat granular appearance (Fig. 4). The perivascular network is well defined around the larger vessels (Fig. I). Capillaries are outlined only by a faint and powdery silver precipitate with no evident structure and with scattered dark endothelial nuclei (Figs. 7 and 8). At the places where connexion with the intervascular argyrophilic bridges is established the capillary outline shows a filamentous structure (Figs. I and 6). The intervascular argyrophilic bridges extend between vessels of all calibres and frequently have a ' Y ' shape connecting 3 vessels (Fig. 2). These bridges have a thickness ranging from 1.5
Fig. 1. Argyrophilic intervascular connexions (arrows) between two vessels larger than capillaries (on the left) and from one of these with a capillary (on the right). The larger vessels are clearly outlined by an argyrophilic perivascutar network. This is incomplete in the capillary. Cat parietal cortex: 30/~m thick section stained by the Wiitanen 11 method. × 1,600. Abbreviations used in this and the following figures: as, astrocyte nucleus; c, capillary vessel; f, astroglial microfilaments; Iv, vessel larger than capillaries; n, glial nucleus; and pv, artifactual (?) perivascular space. Brain Research, 33 (1971) 548-553
SHORT COMMUNICATIONS
549
Fig. 2. Argyrophilic branching intervascular connexion (arrows). Note the solid appearance of the impregnation in the intervascular connexion and the granular pattern in the capillary wall. Tissue treatment and magnification as in Fig. 1.
Fig. 3. Silver impregnation of the vascular network in a 100 #m thick section of cat parietal cortex stained by the Wiitanen 11 method. The larger vessels are evidently outlined by a silver-impregnated network, while capillaries are shown by a powdery silver precipitate with scattered endothelial nuclei. Two argyrophilic bridges are visible (arrows). × 100. /zm (Fig. 2) to 0.4 # m (Fig. 4) and at high magnification their fibrillar appearance is often discerned. W h e n thick sections are used, the argyrophilic perivascular network and bridges are more a b u n d a n t (Figs. 3 and 6-8). In 100/zm sections, the vessels are clearly and more completely outlined (Fig. 3). In these sections a perivascular network is clearly seen a r o u n d the large vessels, while capillaries a p p e a r as an argyrophilic veil with scattered and heavily impregnated endothelial nuclei. At high magnification the connexion o f the bridges with the large vessels (Fig. 6) and capillaries (Figs. 6-8) is Brain Research, 33 (1971) 548-553
550
SHORT COMMUNICATIONS
clearly recognized. O c c a s i o n a l l y these bridges are seen in close relation to d a r k nuclei p r e s u m e d to be glial (Figs. 6 and 8). N o structure in the central nervous system has been clearly d e m o n s t r a t e d t h a t m a y a c c o u n t for these a r g y r o p h i l i c i n t e r v a s c u l a r connexions. Cajal's suggestion t h a t they represent connective tissue strands between the a d v e n t i t i a l sheaths o f capillaries is u n t e n a b l e t o d a y , since such c o n n e x i o n s have never been f o u n d in electron microscopic studies o f the nervous system. F u r t h e r m o r e , such studies have failed to disclose a connective tissue a d v e n t i t i a a r o u n d the capillaries o f the central nervous system. The a r g y r o p h i l i c n e t w o r k a r o u n d larger vessels could represent the well-known connective tissue a d v e n t i t i a . However, this i n t e r p r e t a t i o n does n o t a p p e a r the most likely. The p e r i v a s c u l a r n e t w o r k o f larger vessels, as seen in this material, is m o r p h o logically similar a n d a p p a r e n t l y c o n t i n u o u s with t h a t o f smaller vessels where no connective tissue is f o u n d (Fig. 3). F u r t h e r m o r e , it is also c o n t i n u o u s with a n d similar to the i n t e r v a s c u l a r bridges. M o r i a n d L e b l o n d 6 suggested t h a t bundles o f filaments can pass in astrocytic processes between capillaries and establish such bridges. This suggestion agrees with classical views c o n c e r n i n g astrocytic vascular 'feet'. A s t r o c y t e s have processes a t t a c h e d to the v a s c u l a r walls a n d each astrocyte m a y have several such processes. C o n s e q u e n t l y , there is considerable o p p o r t u n i t y for an astrocyte to establish a bridge between different sites o f the vascular tree. The a u t h o r s m e n t i o n e d a b o v e suggest, in a d d i t i o n , t h a t the filamentous astroglial bridges m a y p r o v i d e s u p p o r t for the n e r v o u s tissue by reinforcing the vascular n e t w o r k . However, these suggestions rest only on indirect evidence.
Fig. 4. Four argyrophilic intervascular connexions. Note the granular appearance of the impregnation in the intervascular connexions and capillary walls. Tissue treatment as in Fig. 1. x 900. Fig. 5. Argyrophilic bridges in 100/~m thick sections of cat parietal cortex. Two bridges are visible (arrows), connecting the same capillary with two other vessels. Tissue treatment as in Fig. 3. x 900. Fig. 6. Argyrophilic connexion (arrow) between a large vessel and a capillary. The larger vessel is clearly shown by a perivascular network, and the capillary by an irregular silver precipitate with a net-like structure at the site of insertion of the intervascular connexion. Tissue treatment as in Fig. 3. x 600. Fig. 7. Argyrophilic connexion (arrow) between two capillaries. Tissue treatment as in Fig. 3. x 900. Fig. 8. Argyrophilic connexion (arrows) between two capillaries, closely related to a glial nucleus. Tissue treatment as in Fig. 3. × 900. Fig. 9. Layers of filament bundles around a large vessel. Six different layers of microfilament-filled astroglial processes are visible around the wall of a large vessel. Cat parietal cortex fixed by perfusion with formaldehyde and glutaraldehyde, and postfixed with osmium tetroxide and uranyl acetate8. Ethanol dehydration and embedding in Spurr's 9 resin. Grid staining with lead10. × 30,000. Fig. 10. Filament bundles in astroglial profiles around a capillary wall. Tissue treatment and magnification as in Fig. 9. Fig. 11. Filament bundle approaching and encircling a capillary. Tissue treatment as in Fig. 9. × 3,300. Fig. 12. Photomontage showing a filament bundle connecting the wall of a vessel with the perikaryon of an astrocyte. The filament bundle is interrupted (arrows) but in the next section it has been possible to confirm the continuity of the two segments. Tissue treatment as in Fig. 8. z 5,000. Brain Research, 33 (1971) 548-553
mm
552
SHORT COMMUNICAI-IONS
Additional indirect evidence in support of this interpretation was provided by the electron microscopic study of cat cerebral cortex fixed by perfusion with phosphatebuffered formaldehyde and glutaraldehyde, followed by osmium postfixation, in some blocks uranyl acetate was used as a postfixative 8, in addition to osmium, a procedure which helped in making astrocytic filaments prominent. Under the electron microscope the larger vessels often appear surrounded by astroglial processes filled with bundles of filaments. These bundles frequently form several layers around the vessel (Fig. 9). Astroglial filaments are also frequent around capillaries (Fig. 10), but large areas of these vessels have no such filaments around them. The filamentous astroglial processes appear to be confined to some segments of the capillaries (Fig. 10) and in favourable sections it is possible to see a filamentous bundle approaching and encircling a capillary wall (Fig. 11). This perivascular distribution of filament-filled astroglial processes, as well as the range of thickness of the microfilament bundles, agree well with the dimensions and distribution of the argyrophilic structures seen in the light microscope. It appears very likely that most of the perivascular network and the intervascular connexions shown by light microscopy may be composed of silver-impregnated bundles of astroglial filaments. This interpretation agrees with Mori and Leblond's 6 suggestion that astrocytes establish microfilament filled bridges between the central nervous system vessels. Strong support for this view is provided by the finding of a bundle of astrocytic filaments encircling a vessel and passing through the astrocyte perikaryon towards another vessel as shown in Fig. 12. Here a bundle of filaments close to the wall of a large vessel is seen to pass through the neuropil to the perikaryon of an astrocyte. Gray and Guillery 4 have adduced evidence that the filaments in nerve cells of the spinal cord can be impregnated with silver by the methods of Cajal and Bielschowsky. In many studies of filaments it is apparently assumed that these structures are basically similar in every situation and in different animal species. However, Wuerker 12 has recently shown that astroglial filaments have structural subunits which are smaller than those of neurofilaments, and has suggested on the basis of this finding that the two types of filament may be composed of different proteins. Cajal's original description, together with the observations made in the present study, suggest that glial filaments can be impregnated along with neurofilaments by a variety of more or less routine silver techniques. This suggestion emphasizes once more the need for great care in the interpretation of silver-impregnated sections of brain tissue. Intracellular support, intracellular transport and mechanochemical transduction have been suggested as possible functions of filaments 7. If the argyrophilic bridges observed in the present study are indeed composed of astroglial filaments, their concentration around the large vessels and the fact that they connect different, but nearby, points of the vascular network would be in agreement with Mori and Leblond's 6 suggestion that such bridges contribute to the support of nervous tissue. Studies of serial EM sections of silver-stained material are necessary to decide if the present interpretation of the argyrophilic intervascular bridges is tenable.
Brain Research, 33 (1971) 548-553
SHORT COMMUNICATIONS
553
H e l p f u l discussions with Profs. A . C o i m b r a a n d M. M. Magalh[les, L a b o r a t o r y o f H i s t o l o g y , M e d i c a l S c h o o l o f O p o r t o , P o r t u g a l are gratefully a c k n o w l e d g e d . This w o r k received financial s u p p o r t f r o m the I n s t i t u t o p a r a a A l t a C u l t u r a as well as f r o m the C a l o u s t e G u l b e n k i a n F o u n d a t i o n , Lisbon, P o r t u g a l . N o t e added in proof. A f t e r this c o m m u n i c a t i o n was sent to press a p a p e r by A b a d i a - F e n o l l et al. (Rev. Roum. Neurol., 7 (1970) 19-25) c a m e to the a t t e n t i o n o f the a u t h o r . In t h a t p a p e r , similar a r g y r o p h i l i c i n t e r v a s c u l a r bridges are shown by a different silver m e t h o d . T h o s e a u t h o r s suggest a different i n t e r p r e t a t i o n o f these structures. Anatomical Institute, Medical School University of Oporto, Oporto (Portugal)
A. SOUSA-PINTO
1 CAJAL, S. RAMtSNY, Histologie du Syst~me Nerveux de l'Homme et des Vertebras, Cons. Sup.
Inv. Cient., Madrid, 1952, 520. pp. 2 EAGER,R. P., Selective staining of degenerating axons in the central nervous system by a simplified
3 4
5 6 7 8 9 10
11
12
silver method: spinal cord projection to external cuneate and inferior olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 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. 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. LOEWY,A. D., Ammoniacal silver staining of degenerating axons, Acta Neuropath. (Berl.), 14 (1969) 226-236. MORI, S., AND LEBLOND, C. P., Electron microscopic features and proliferation of astrocytes in the corpus callosum of the rat, J. comp. Neurol., 137 (1969) 197-226. SCHMITT, F. O., Fibrous proteins - - neuronal organelles, Proc. nat. Acad. Sci. (Wash.), 60 (1968) 1092-1101. SILVA, M. T., CARVALHOGUERRA, F., AND MAGALHAES,M. M., The fixative action of uranyl acetate in electron microscopy, Experientia (Basel), 24 (1968) 1074. SPURR,A. R., A low viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res., 26 (1969) 31-43. VENABLE,J. H., AND COGGESHALL,R., A simplified lead citrate stained for use in electron microscopy, J. Cell Biol., 25 (1965) 407-408. 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. WUERKER,B. B., Neurofilaments and glial filaments, Tissue and Cell, 2 (1970) 1-10.
(Accepted July 23rd, 1971)
Brain Research, 33 (1971) 548-553