Brain Research, 83 (1975) 469473
469
© Elsevier ScientificPublishing Company, Amsterdam- Printed in The Netherlands
Short Communications
Peroxidase labeling of motor cortex neurons projecting to the ventrolateral nucleus in the cat
MARYANN A. ROMAGNANO AND RAYMOND J. MACIEWICZ Department of Neuroscience and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, The Albert Einstein College of Medicine, Bronx, iV. Y. 10461 (U.S.A.)
(Accepted October 9th, 1974)
Anterograde degeneration studies 1°,t7-22 have demonstrated a projection from motor cortex (MC) onto the ventrolateral nucleus (VL) of the thalamus. Although the exact cells of origin of this pathway have remained obscure, previous authors have suggested that corticofugal efferents in general originate in the deeper layers (V and VI) of the cerebral cortexS,9,23 and electrophysiological studies in the MC of the cat I have confirmed these observations. To determine the precise cells of origin of the projection of MC onto VL we have used the method of retrograde transport of horseradish peroxidase (HRP) 11,13-16. Single injections of 0.05 #1 of a 50 ~ HRP (Sigma type VI) solution were made stereotaxically in both VL nuclei in each of 4 cats. The stereotaxic coordinates for the injections were A11.0, H + 1.5 and L4.0 (see ref. 8). The animals were anesthetized with pentobarbital sodium during surgery and were kept anesthetized for forty-eight postoperative hours. They were then killed by aortal perfusion with cold 1 ~ formaldehyde and 1.25% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.6). The brains were blocked in frontal stereotaxic planes and allowed to sink in a cold, buffered 30 ~ sucrose solution. The brains were sectioned at 30-35/,m on a freezing microtome, and reacted with 3,Y-diaminobenzidine and hydrogen peroxide according to the method of Graham and Karnovsky6. Sections were mounted and counterstained with cresyl violet. The size of the injection site in the thalamus was defined as the extent of the extracellular HRP reaction product at the injection site. The limits of MC were determined using the cytoarchitectural criteria of Hassler and MuhsClement 7. In 4 of the 8 injection sites, the spread of HRP was largely confined to VL. Some spread of the injected HRP also occurred along the path of the injecting cannula, in the central lateral nucleus and the anterior ventral nucleus (Fig. 1). Neurons that had retrogradely transported the injected HRP were identified by the
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Fig. I. A: diagram of 3 of the injection sites. Blackened areas represent the maximal extent of the extracellular HRP reaction product in the ventrolateral nucleus. AV, anterior ventral nucleus; CL, central lateral nucleus; VA, ventral anterior nucleus; VL, ventrolateral nucleus; VPE, ventrat posterior lateral nucleus. B: photomicrograph of the fourth injection site. HRP reaction product can be seen in the ventrolateral nucleus. Staining along the path of the injecting cannula is due largely to tissue damage, although some spread of HRP occurs along the cannula track as noted in text.
appearance of the granular H R P reaction product in the cell body. Such cells could be found in several of the nuclei known to project onto VL. In agreement with the findings reported from anterograde degeneration studies, HRP-positive cells were found in the globus pallidus 3,24, the pars reticularis of the substantia nigra 2,a, and the dentate, interpositus and fastigial nuclei of the cerebellum 12. In the MC, HRP-positive cells were localized to the deeper cortical layers. Granules of the H R P reaction product were found only in the medium-sized pyramidal cells of this region. Such cells were located subjacent to the Betz cells of layer V in layer Vb and the dorsal part of layer VI. Within the band of labeled cells, however, many medium-sized pyramids did not contain HRP, even in the regions of MC containing the highest density of HRP-positive cells. Presumably such cells either failed to transport the H R P or do not project to the injection site. The demonstration of HRP-positive cells in the globus pallidus, pars reticularis of the substantia nigra, and all three of the deep cerebellar nuclei provide further evidence that these neuronal groups are afferent to the VL. Consistent with the observations of others 1°,17-22 we have also found HRP-positive cells in the MC. Their localization in layers Vb and VI suggest that the medium-sized pyramidal cells
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Fig. 2. A and B: HRP-positive pyramidal neurons in layer Vb of motor cortex. Granules of the H R P reaction product can be seen in the cell body and proximal dendrites. Bar represents 20 #m. C: representative section through motor cortex. Black dots in layers Vb and VI identify location of HRP-positive cells. Bar represents 2 mm.
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o f these layers m a y be the origin o f the c o r t i c o - t h a l a m i c p a t h w a y from M C o n t o VL. This conclusion is consistent with the available a n a t o m i c a l a n d physiological evidence on the origin o f M C efferents 1,5. The electrophysiological studies o f T o y a m a et al. 2r' show t h a t the visual cortex efferents to the t h a l a m u s a n d m e s e n c e p h a l o n originate in layer V, so t h a t o u r results m a y well reflect a general feature o f cortical o r g a n i z a t i o n a n d n o t a peculiarity specific to MC. The d e m o n s t r a t i o n t h a t m e d i u m - s i z e d p y r a m i d s o f layers Vb a n d VI o f M C project to VL is evidence that these cells m a y be the only M C efferents t e r m i n a t i n g in VL. As the p y r a m i d a l tract is k n o w n to c o n t r i b u t e collaterals to VL 4 these results suggest t h a t such fibers arise f r o m m e d i u m - s i z e d p y r a m i d a l t r a c t neurons and n o t f r o m the giant Betz ceils. However, it a p p e a r s to be a general experience t h a t n o t all neurons p r o j e c t i n g to a given region are clearly labeled with the r e t r o g r a d e t r a n s p o r t m e t h o d used 16, a n d hence the conclusions d r a w n f r o m o u r present findings can only be considered tentative. A t the very least, however, it is clear that layers Vb and VI o f the M C are a m a j o r source o f the c o r t i c o - t h a l a m i c p a t h w a y to VL. The a u t h o r s wish to t h a n k Drs. G e o r g e D. P a p p a s and D o m i n i c k P. P u r p u r a for their c o n t i n u e d e n c o u r a g e m e n t and support. M a r i e Buschke is t h a n k e d for her valuable technical advice. W e are grateful to M a r k F l o m e n b a u m for his help in p r e p a r i n g the figures. This study was s u p p o r t e d in parts by G r a n t s NS-11431, NS-07512, and 5T5-GM1674 f r o m the N a t i o n a l Institutes o f Health.
1 ASANUMA, H., AND ROSEN, L, Electrical cytoarchitectural study of the cat's motor cortex. In T. L. FRIGYES/,E. RINVIKAND M. D. YAHR(Eds.), Corticothalamic Projections and Sensorimotor Activities, Raven Press, New York, N.Y., 1972, pp. 375-377. 2 CARPENTER, M. B., AND PETER, P., Nigrostriatal and nigrothalamic fibers in the rhesus monkey, J. comp. Neurol., 144 (1972) 93-116. 3 CARPENTER, M. B., AND STROMINGER, N. L., Efferent fibers of the subthalamic nucleus in the
4 5 6 7 8 9
10 ll
monkey. A comparison of the efferent projections of the subthalamic nucleus, substantia nigra and globus paUidus, Amer. J. Anat., 121 (1967) 41-71. CLARE, M. H., LANDAU, W. M., AND BISHOP,G. H., Electrophysiological evidence of a collateral pathway from the pyramidal tract to the thalamus in the cat, Exp. Neurol., 9 (1969) 262-267. FULTON,J. F., Physiology of the Nervous System, 3rd ed., Oxford University Press, New York, 1949. GRAHAM,R. C., AND KARNOVSKY,M. J., Glomerular permeability. Ultrastructural cytochemical studies using peroxidases as protein tracers, J. exp. Med., 124 (1966) 1123-1133. HASSLER, R., AND MUnS-CLEMENT, K., Architektonischer Aufbau des sensomotorischen und parietalen Cortex der Katze, J. Hirnforsch., 6 (1964) 377-420. JASPER, H. H., AND AJMONE MARSAN,C., A Stereotaxic Atlas of the Diencephalon of the Cat, National Research Council of Canada, Ottawa, 1954. KRUGER, L., AND MALIS, L. ]., Distribution of afferent and efferent fibers in the cerebral cortex of the rabbit revealed by heavy ionizing particles, Exp. Neurol., 10 (1964) 509-524. KUSAMA,Z., OTANI, K., AND KAWANA,E., Projections of the motor, somatic sensory, auditory and visual cortices in cats. In T. TOKIZANEAND J. P. SCHADI~(Eds.), Correlative Neurosciences, Progress in Brain Research, Vol. 21A, Elsevier, Amsterdam, 1966, pp. 292-322. KUYPERS, H. G. J. M., KIEVlT, J., AND GROEN-KLEVANT,A. C., Retrograde axonal transport of horseradish peroxidase in rat's forebrain, Brain Research, 67 (1974) 211-218.
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12 LARSELL, O., AND JANSEN, J., The Comparative Anatomy and Histology of the Cerebellum: The Human Cerebellum, Cerebellar Connections, and Cerebellar Cortex, University of Minnesota Press, Minneapolis, Minn., 1972, pp. 151-153. 13 LAVAIL, J. H., AND LAVAIL, M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 14 LAVAIL,J. H., WINSTON,K. R., ANDTISH, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. 15 MACIEWICZ,R. J., Afferents to the lateral suprasylvian gyrus of the cat traced with horseradish peroxidase, Brain Research, 78 (1974) 139-143. 16 NAUTA, H. J. W., PRITZ, M. B., AND LASEK, R. J., Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. 17 PETRAS, J. M., Some fiber connections of the precentral cortex (areas 4 and 6) with the diencephalon in the monkey (Macaca mulatta), Anat. Rec., 148 (1964) 322. 18 PETRAS, J. M., Some fiber connections of the precentral and postcentral cortex with the basal ganglia, thalamus and subthalamus, Trans. Amer. neurol. Ass., 90 (1965) 274-275. 19 PETRAS,J. M., Some fiber connections of the motor and somatosensory cortex of simian primates and felid, canid, procyonid carnivores, Ann. N.Y. Acad. Sci., 167 (1969) 469-505. 20 PETRAS, J. M., Corticostriate and corticothalamic connections in the chimpanzee. In T. L. FRIGYESI, E. RINVIK AND M. D. YAHR (Eds.), Corticothalamic Projections and Sensorimotor Activities, Raven Press, New York, N.Y., 1972, pp. 201-220. 21 RINVIK, E., The corticothalamic projection from the pericruciate and coronal gyri in the cat. An experimental study with silver impregnation methods, Brain Research, 10 (1968) 79-119. 22 RINVIK, E., Origin of thalamic connections from motor and somatosensory cortical areas in the cat. In T. L. FRIGYESI, E. RtNVIK AND M. D. YAHR (Eds.), Corticothalamic Projections and Sensorimotor Activities, Raven Press, New York, N.Y., 1972, pp. 57-90. 23 SnOLL, D. D., The Organization of the Cerebral Cortex, Methuen, London and Wiley, New York, 1956. 24 SZABO,J., AND RENAUD, L., Cerebello- and pallidothalamic projections in the cat, Proc. Canad. Fed. biol. Soc., 7 (1964) 50-51. 25 TOYAMA,K., MATSUNAMI,K., AND OHNo, T., Antidromicidentification of association, commissural and corticofugal efferents in cat visual cortex, Brain Research, 14 (1959) 513-517.
469
© Elsevier ScientificPublishing Company, Amsterdam- Printed in The Netherlands
Short Communications
Peroxidase labeling of motor cortex neurons projecting to the ventrolateral nucleus in the cat
MARYANN A. ROMAGNANO AND RAYMOND J. MACIEWICZ Department of Neuroscience and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, The Albert Einstein College of Medicine, Bronx, iV. Y. 10461 (U.S.A.)
(Accepted October 9th, 1974)
Anterograde degeneration studies 1°,t7-22 have demonstrated a projection from motor cortex (MC) onto the ventrolateral nucleus (VL) of the thalamus. Although the exact cells of origin of this pathway have remained obscure, previous authors have suggested that corticofugal efferents in general originate in the deeper layers (V and VI) of the cerebral cortexS,9,23 and electrophysiological studies in the MC of the cat I have confirmed these observations. To determine the precise cells of origin of the projection of MC onto VL we have used the method of retrograde transport of horseradish peroxidase (HRP) 11,13-16. Single injections of 0.05 #1 of a 50 ~ HRP (Sigma type VI) solution were made stereotaxically in both VL nuclei in each of 4 cats. The stereotaxic coordinates for the injections were A11.0, H + 1.5 and L4.0 (see ref. 8). The animals were anesthetized with pentobarbital sodium during surgery and were kept anesthetized for forty-eight postoperative hours. They were then killed by aortal perfusion with cold 1 ~ formaldehyde and 1.25% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.6). The brains were blocked in frontal stereotaxic planes and allowed to sink in a cold, buffered 30 ~ sucrose solution. The brains were sectioned at 30-35/,m on a freezing microtome, and reacted with 3,Y-diaminobenzidine and hydrogen peroxide according to the method of Graham and Karnovsky6. Sections were mounted and counterstained with cresyl violet. The size of the injection site in the thalamus was defined as the extent of the extracellular HRP reaction product at the injection site. The limits of MC were determined using the cytoarchitectural criteria of Hassler and MuhsClement 7. In 4 of the 8 injection sites, the spread of HRP was largely confined to VL. Some spread of the injected HRP also occurred along the path of the injecting cannula, in the central lateral nucleus and the anterior ventral nucleus (Fig. 1). Neurons that had retrogradely transported the injected HRP were identified by the
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Fig. I. A: diagram of 3 of the injection sites. Blackened areas represent the maximal extent of the extracellular HRP reaction product in the ventrolateral nucleus. AV, anterior ventral nucleus; CL, central lateral nucleus; VA, ventral anterior nucleus; VL, ventrolateral nucleus; VPE, ventrat posterior lateral nucleus. B: photomicrograph of the fourth injection site. HRP reaction product can be seen in the ventrolateral nucleus. Staining along the path of the injecting cannula is due largely to tissue damage, although some spread of HRP occurs along the cannula track as noted in text.
appearance of the granular H R P reaction product in the cell body. Such cells could be found in several of the nuclei known to project onto VL. In agreement with the findings reported from anterograde degeneration studies, HRP-positive cells were found in the globus pallidus 3,24, the pars reticularis of the substantia nigra 2,a, and the dentate, interpositus and fastigial nuclei of the cerebellum 12. In the MC, HRP-positive cells were localized to the deeper cortical layers. Granules of the H R P reaction product were found only in the medium-sized pyramidal cells of this region. Such cells were located subjacent to the Betz cells of layer V in layer Vb and the dorsal part of layer VI. Within the band of labeled cells, however, many medium-sized pyramids did not contain HRP, even in the regions of MC containing the highest density of HRP-positive cells. Presumably such cells either failed to transport the H R P or do not project to the injection site. The demonstration of HRP-positive cells in the globus pallidus, pars reticularis of the substantia nigra, and all three of the deep cerebellar nuclei provide further evidence that these neuronal groups are afferent to the VL. Consistent with the observations of others 1°,17-22 we have also found HRP-positive cells in the MC. Their localization in layers Vb and VI suggest that the medium-sized pyramidal cells
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Fig. 2. A and B: HRP-positive pyramidal neurons in layer Vb of motor cortex. Granules of the H R P reaction product can be seen in the cell body and proximal dendrites. Bar represents 20 #m. C: representative section through motor cortex. Black dots in layers Vb and VI identify location of HRP-positive cells. Bar represents 2 mm.
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o f these layers m a y be the origin o f the c o r t i c o - t h a l a m i c p a t h w a y from M C o n t o VL. This conclusion is consistent with the available a n a t o m i c a l a n d physiological evidence on the origin o f M C efferents 1,5. The electrophysiological studies o f T o y a m a et al. 2r' show t h a t the visual cortex efferents to the t h a l a m u s a n d m e s e n c e p h a l o n originate in layer V, so t h a t o u r results m a y well reflect a general feature o f cortical o r g a n i z a t i o n a n d n o t a peculiarity specific to MC. The d e m o n s t r a t i o n t h a t m e d i u m - s i z e d p y r a m i d s o f layers Vb a n d VI o f M C project to VL is evidence that these cells m a y be the only M C efferents t e r m i n a t i n g in VL. As the p y r a m i d a l tract is k n o w n to c o n t r i b u t e collaterals to VL 4 these results suggest t h a t such fibers arise f r o m m e d i u m - s i z e d p y r a m i d a l t r a c t neurons and n o t f r o m the giant Betz ceils. However, it a p p e a r s to be a general experience t h a t n o t all neurons p r o j e c t i n g to a given region are clearly labeled with the r e t r o g r a d e t r a n s p o r t m e t h o d used 16, a n d hence the conclusions d r a w n f r o m o u r present findings can only be considered tentative. A t the very least, however, it is clear that layers Vb and VI o f the M C are a m a j o r source o f the c o r t i c o - t h a l a m i c p a t h w a y to VL. The a u t h o r s wish to t h a n k Drs. G e o r g e D. P a p p a s and D o m i n i c k P. P u r p u r a for their c o n t i n u e d e n c o u r a g e m e n t and support. M a r i e Buschke is t h a n k e d for her valuable technical advice. W e are grateful to M a r k F l o m e n b a u m for his help in p r e p a r i n g the figures. This study was s u p p o r t e d in parts by G r a n t s NS-11431, NS-07512, and 5T5-GM1674 f r o m the N a t i o n a l Institutes o f Health.
1 ASANUMA, H., AND ROSEN, L, Electrical cytoarchitectural study of the cat's motor cortex. In T. L. FRIGYES/,E. RINVIKAND M. D. YAHR(Eds.), Corticothalamic Projections and Sensorimotor Activities, Raven Press, New York, N.Y., 1972, pp. 375-377. 2 CARPENTER, M. B., AND PETER, P., Nigrostriatal and nigrothalamic fibers in the rhesus monkey, J. comp. Neurol., 144 (1972) 93-116. 3 CARPENTER, M. B., AND STROMINGER, N. L., Efferent fibers of the subthalamic nucleus in the
4 5 6 7 8 9
10 ll
monkey. A comparison of the efferent projections of the subthalamic nucleus, substantia nigra and globus paUidus, Amer. J. Anat., 121 (1967) 41-71. CLARE, M. H., LANDAU, W. M., AND BISHOP,G. H., Electrophysiological evidence of a collateral pathway from the pyramidal tract to the thalamus in the cat, Exp. Neurol., 9 (1969) 262-267. FULTON,J. F., Physiology of the Nervous System, 3rd ed., Oxford University Press, New York, 1949. GRAHAM,R. C., AND KARNOVSKY,M. J., Glomerular permeability. Ultrastructural cytochemical studies using peroxidases as protein tracers, J. exp. Med., 124 (1966) 1123-1133. HASSLER, R., AND MUnS-CLEMENT, K., Architektonischer Aufbau des sensomotorischen und parietalen Cortex der Katze, J. Hirnforsch., 6 (1964) 377-420. JASPER, H. H., AND AJMONE MARSAN,C., A Stereotaxic Atlas of the Diencephalon of the Cat, National Research Council of Canada, Ottawa, 1954. KRUGER, L., AND MALIS, L. ]., Distribution of afferent and efferent fibers in the cerebral cortex of the rabbit revealed by heavy ionizing particles, Exp. Neurol., 10 (1964) 509-524. KUSAMA,Z., OTANI, K., AND KAWANA,E., Projections of the motor, somatic sensory, auditory and visual cortices in cats. In T. TOKIZANEAND J. P. SCHADI~(Eds.), Correlative Neurosciences, Progress in Brain Research, Vol. 21A, Elsevier, Amsterdam, 1966, pp. 292-322. KUYPERS, H. G. J. M., KIEVlT, J., AND GROEN-KLEVANT,A. C., Retrograde axonal transport of horseradish peroxidase in rat's forebrain, Brain Research, 67 (1974) 211-218.
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12 LARSELL, O., AND JANSEN, J., The Comparative Anatomy and Histology of the Cerebellum: The Human Cerebellum, Cerebellar Connections, and Cerebellar Cortex, University of Minnesota Press, Minneapolis, Minn., 1972, pp. 151-153. 13 LAVAIL, J. H., AND LAVAIL, M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 14 LAVAIL,J. H., WINSTON,K. R., ANDTISH, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. 15 MACIEWICZ,R. J., Afferents to the lateral suprasylvian gyrus of the cat traced with horseradish peroxidase, Brain Research, 78 (1974) 139-143. 16 NAUTA, H. J. W., PRITZ, M. B., AND LASEK, R. J., Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. 17 PETRAS, J. M., Some fiber connections of the precentral cortex (areas 4 and 6) with the diencephalon in the monkey (Macaca mulatta), Anat. Rec., 148 (1964) 322. 18 PETRAS, J. M., Some fiber connections of the precentral and postcentral cortex with the basal ganglia, thalamus and subthalamus, Trans. Amer. neurol. Ass., 90 (1965) 274-275. 19 PETRAS,J. M., Some fiber connections of the motor and somatosensory cortex of simian primates and felid, canid, procyonid carnivores, Ann. N.Y. Acad. Sci., 167 (1969) 469-505. 20 PETRAS, J. M., Corticostriate and corticothalamic connections in the chimpanzee. In T. L. FRIGYESI, E. RINVIK AND M. D. YAHR (Eds.), Corticothalamic Projections and Sensorimotor Activities, Raven Press, New York, N.Y., 1972, pp. 201-220. 21 RINVIK, E., The corticothalamic projection from the pericruciate and coronal gyri in the cat. An experimental study with silver impregnation methods, Brain Research, 10 (1968) 79-119. 22 RINVIK, E., Origin of thalamic connections from motor and somatosensory cortical areas in the cat. In T. L. FRIGYESI, E. RtNVIK AND M. D. YAHR (Eds.), Corticothalamic Projections and Sensorimotor Activities, Raven Press, New York, N.Y., 1972, pp. 57-90. 23 SnOLL, D. D., The Organization of the Cerebral Cortex, Methuen, London and Wiley, New York, 1956. 24 SZABO,J., AND RENAUD, L., Cerebello- and pallidothalamic projections in the cat, Proc. Canad. Fed. biol. Soc., 7 (1964) 50-51. 25 TOYAMA,K., MATSUNAMI,K., AND OHNo, T., Antidromicidentification of association, commissural and corticofugal efferents in cat visual cortex, Brain Research, 14 (1959) 513-517.
