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Is there a cortico-nigral tract? A comment based on experimental electron microscopic observations in the cat
742
SHORT COMMUNICATIONS
Is there a cortico-nigral tract? A comment based on experimental electron microscopic observations in the cat The existence of a cortico-nigral projection appears to be widely accepted and a large number of investigators have claimed that the cerebral cortex is in fact the main source of afferents to the substantia nigra (for references see ref. 6). This notion was seriously challenged by one of us (E.R.) in a previous paper where the cortico-nigral projection in the ca: was reinvestigated with the silver impregnation methods of Nauta 5 and of GleesL Although Rinvik 6 found that in operated animals bundles of degenerating fibers traversed the substantia nigra and ultimately ended in more dorsally situated central nervous structures, he could only find very few scattered axons which did not follow the main bundles. These scattered axons, however, are obviously too few to represent a cortico-nigral projection. Rinvik 6 emphasized that electron microscopical investigations were needed to establish whether the fibers passing through the substantia nigra had b~utons en p a s s a g e in synaptic contact with its cells. The aim of the present study was to investigate with the electron microscope whether degenerating boutons are present in the substantia nigra following cortical lesions. Large cortical lesions were made in 7 adult cats. All of the lesions involved the coronal and pre- and postcruciate gyri, and often in addition affected parts of the gyrus proreus, the anterior ectosylvian gyrus and the gyrus lateralis. The lesions were made deep so that they also would interrupt fibers originating on the medial aspect of the hemisphere. The basal ganglia were intact. Following a survival time of 2 to 13 days the animals were briefly perfused intravitally with a warmed physiological saline solution, followed by 0.5 l of 4 ~ paraformaldehyde in Millonig's buffer solution of the same temperature ~ucceeded by 2 1 of 2 . 5 ~ glutaraldehyde in the same buffer at r o o m temperature. The brains were dissected free and thin coronal slices through the entire mesencephalon were postfixed in chilled 1 ~ osmiumtetroxide for 1.5-2 h. From every transverse slice small blocks were cut out from the entire mediolateral extent of the substantia nigra thus providing pieces from all levels of the nucleus and from the pars compacta as well as the pars reticulata. The blocks were dehydrated in increasing gradients of alcohol, embedded in Araldite and cut on an LKB Ultrotome. Semithin sections from every block were stained with paraphenylenediamine 4 to select the blocks which included degenerating fibers (Fig. la). Ultrathin sections from these blocks were stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop I B. No detailed description will be given here of the ultrastructure of the normal substantia nigra. Concerning this the reader is referred to the recent study by Schwyn and Fox v. It is of relevance to the present problem that the nucleus is traversed by a great number of myelinated as well as unmyelinated fibers. The smallest among the former have a diameter of 0.5-1 #. The latter are thinner and are usually arranged in bundles. These unmyelinated fibers represent a characteristic element of the nucleus. Another very typical element is a glomerulus-like structure. This is built up by a central dendrite which is entirely surrounded by boutons and the whole complex is Brain Research, 14 (1969) 742-744
SHORT COMMUNICATIONS
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Fig. 1. a, Semithin section through substantia nigra in a cat with a lesion of the sensorimotor cortex. Single arrows point to cross sections of some degenerating fibers, the two arrows point to a degenerating fiber leaving the pes pedunculi (Pp) and entering substantia nigra. Lm, medial lemniscus. Cat 1331, survival time 3 days. Paraphenylenediamine stain. A 370. b, Electron micrograph showing one small myelinated degenerating fiber (al) and parts of 4 myelinated normal fibers (a2-as) in substantia nigra in the same cat. z 30,000. c, Electron micrograph showing small myelinated degenerating fibers (a~ and aa) and one myelinated normal fiber (al) in substantia nigra in the same animal as shown in b. g Astroglial process with glycogen granules. × 30,000.
Brain Research, 14 (1969) 742-744
744
SHORT COMMUNICATIONS
ensheathed by glial lamellae. Other dendritic profiles are, however, contacted by processes and sheets of glial cells which intrude between the boutons. This appears especially to be the case for the proximal parts of the dendrites. A varying number of degenerating myelinated nerve fibers of different caliber, single or in bundles, were found in the ultrathin sections. In all animals they showed the characteristic features of the electron dense type of degeneration (Fig. lb, c). In none of the blocks, however, were degenerating thin unmyelinated axons or degenerating dark or filamentous boutons observed (for references see refs. 1 and 3). Degenerating boutons can, of course, have escaped our recognition despite the meticulous care which was taken in sampling of the blocks. Our negative finding, therefore, does not exclude the possibility that a few corticofugal fibers may terminate in the substantia nigra. On the other hand, the presence in all operated animals of an enormous number of apparently normal boutons literally enveloping most of the nigral cells shows that an afferent connection of functional importance for the substantia nigra does not originate in the cerebral cortex. Experiments are in progress in our laboratory to disclose the probable subcortical origin of this large number of normal boutons. The present experimental study indicates that a cortico-nigral projection is absent in the cat. Furthermore, our findings make it questionable whether the projection is present in other mammals. Anatomical Institute, University of Oslo, Oslo 1 (Norway)
ERIC RINVIK FRED WALBERG
1 ALKSNE,J. F., BLACKSTAD, T. W., WALBERG,F., AND WHITE, JR., L. E., Electron microscopy of
2 3 4 5
6 7
axon degeneration: A valuable tool in experimental neuroanatomy, Ergebn. Anat. Entwickl.Gesch., 39 (1966) 1-32. GLEES,P., Terminal degeneration within the central nervous system as studied by a new silvermethod, J. Neuropath. exp. Neurol., 5 (1946) 54-59. GRAY, E. G., AND GUILLERY,R. W., Synaptic morphology in the normal and degenerating nervous system, Int. Rev. Cytol., 19 (1966) 111-182. HOLL~.NDER, H., AND VAALAND, J. L., A reliable staining method for semi-thin sections in experimental neuroanatomy, Brain Research, 10 (1968) 120-126. NAUTA,W. J. H., Silver impregnation of degenerating axons. In W. F. W~NDLE(Ed.), New Research Techniques of Neuroanatomy, Thomas, Springfield, Ill., 1957, pp. 17-26. RINVIK,E., The cortico-nigral projection in the cat. An experimental study with silver impregnation methods, J. comp. Neurol., 126 (1966) 241-254. SCHWVN,R. C., AND FOX, C. A., A Golgi and electron microscopic study of the substantia nigra in Macaca mulatta and Saimiri sciureus, Anat. Rec., 163 (1969) 342.
(Accepted May 22nd, 1969)
Brain Research, 14 (1969) 742-744
SHORT COMMUNICATIONS
Is there a cortico-nigral tract? A comment based on experimental electron microscopic observations in the cat The existence of a cortico-nigral projection appears to be widely accepted and a large number of investigators have claimed that the cerebral cortex is in fact the main source of afferents to the substantia nigra (for references see ref. 6). This notion was seriously challenged by one of us (E.R.) in a previous paper where the cortico-nigral projection in the ca: was reinvestigated with the silver impregnation methods of Nauta 5 and of GleesL Although Rinvik 6 found that in operated animals bundles of degenerating fibers traversed the substantia nigra and ultimately ended in more dorsally situated central nervous structures, he could only find very few scattered axons which did not follow the main bundles. These scattered axons, however, are obviously too few to represent a cortico-nigral projection. Rinvik 6 emphasized that electron microscopical investigations were needed to establish whether the fibers passing through the substantia nigra had b~utons en p a s s a g e in synaptic contact with its cells. The aim of the present study was to investigate with the electron microscope whether degenerating boutons are present in the substantia nigra following cortical lesions. Large cortical lesions were made in 7 adult cats. All of the lesions involved the coronal and pre- and postcruciate gyri, and often in addition affected parts of the gyrus proreus, the anterior ectosylvian gyrus and the gyrus lateralis. The lesions were made deep so that they also would interrupt fibers originating on the medial aspect of the hemisphere. The basal ganglia were intact. Following a survival time of 2 to 13 days the animals were briefly perfused intravitally with a warmed physiological saline solution, followed by 0.5 l of 4 ~ paraformaldehyde in Millonig's buffer solution of the same temperature ~ucceeded by 2 1 of 2 . 5 ~ glutaraldehyde in the same buffer at r o o m temperature. The brains were dissected free and thin coronal slices through the entire mesencephalon were postfixed in chilled 1 ~ osmiumtetroxide for 1.5-2 h. From every transverse slice small blocks were cut out from the entire mediolateral extent of the substantia nigra thus providing pieces from all levels of the nucleus and from the pars compacta as well as the pars reticulata. The blocks were dehydrated in increasing gradients of alcohol, embedded in Araldite and cut on an LKB Ultrotome. Semithin sections from every block were stained with paraphenylenediamine 4 to select the blocks which included degenerating fibers (Fig. la). Ultrathin sections from these blocks were stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop I B. No detailed description will be given here of the ultrastructure of the normal substantia nigra. Concerning this the reader is referred to the recent study by Schwyn and Fox v. It is of relevance to the present problem that the nucleus is traversed by a great number of myelinated as well as unmyelinated fibers. The smallest among the former have a diameter of 0.5-1 #. The latter are thinner and are usually arranged in bundles. These unmyelinated fibers represent a characteristic element of the nucleus. Another very typical element is a glomerulus-like structure. This is built up by a central dendrite which is entirely surrounded by boutons and the whole complex is Brain Research, 14 (1969) 742-744
SHORT COMMUNICATIONS
743
Fig. 1. a, Semithin section through substantia nigra in a cat with a lesion of the sensorimotor cortex. Single arrows point to cross sections of some degenerating fibers, the two arrows point to a degenerating fiber leaving the pes pedunculi (Pp) and entering substantia nigra. Lm, medial lemniscus. Cat 1331, survival time 3 days. Paraphenylenediamine stain. A 370. b, Electron micrograph showing one small myelinated degenerating fiber (al) and parts of 4 myelinated normal fibers (a2-as) in substantia nigra in the same cat. z 30,000. c, Electron micrograph showing small myelinated degenerating fibers (a~ and aa) and one myelinated normal fiber (al) in substantia nigra in the same animal as shown in b. g Astroglial process with glycogen granules. × 30,000.
Brain Research, 14 (1969) 742-744
744
SHORT COMMUNICATIONS
ensheathed by glial lamellae. Other dendritic profiles are, however, contacted by processes and sheets of glial cells which intrude between the boutons. This appears especially to be the case for the proximal parts of the dendrites. A varying number of degenerating myelinated nerve fibers of different caliber, single or in bundles, were found in the ultrathin sections. In all animals they showed the characteristic features of the electron dense type of degeneration (Fig. lb, c). In none of the blocks, however, were degenerating thin unmyelinated axons or degenerating dark or filamentous boutons observed (for references see refs. 1 and 3). Degenerating boutons can, of course, have escaped our recognition despite the meticulous care which was taken in sampling of the blocks. Our negative finding, therefore, does not exclude the possibility that a few corticofugal fibers may terminate in the substantia nigra. On the other hand, the presence in all operated animals of an enormous number of apparently normal boutons literally enveloping most of the nigral cells shows that an afferent connection of functional importance for the substantia nigra does not originate in the cerebral cortex. Experiments are in progress in our laboratory to disclose the probable subcortical origin of this large number of normal boutons. The present experimental study indicates that a cortico-nigral projection is absent in the cat. Furthermore, our findings make it questionable whether the projection is present in other mammals. Anatomical Institute, University of Oslo, Oslo 1 (Norway)
ERIC RINVIK FRED WALBERG
1 ALKSNE,J. F., BLACKSTAD, T. W., WALBERG,F., AND WHITE, JR., L. E., Electron microscopy of
2 3 4 5
6 7
axon degeneration: A valuable tool in experimental neuroanatomy, Ergebn. Anat. Entwickl.Gesch., 39 (1966) 1-32. GLEES,P., Terminal degeneration within the central nervous system as studied by a new silvermethod, J. Neuropath. exp. Neurol., 5 (1946) 54-59. GRAY, E. G., AND GUILLERY,R. W., Synaptic morphology in the normal and degenerating nervous system, Int. Rev. Cytol., 19 (1966) 111-182. HOLL~.NDER, H., AND VAALAND, J. L., A reliable staining method for semi-thin sections in experimental neuroanatomy, Brain Research, 10 (1968) 120-126. NAUTA,W. J. H., Silver impregnation of degenerating axons. In W. F. W~NDLE(Ed.), New Research Techniques of Neuroanatomy, Thomas, Springfield, Ill., 1957, pp. 17-26. RINVIK,E., The cortico-nigral projection in the cat. An experimental study with silver impregnation methods, J. comp. Neurol., 126 (1966) 241-254. SCHWVN,R. C., AND FOX, C. A., A Golgi and electron microscopic study of the substantia nigra in Macaca mulatta and Saimiri sciureus, Anat. Rec., 163 (1969) 342.
(Accepted May 22nd, 1969)
Brain Research, 14 (1969) 742-744