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An ultrastructural study of the anomalous corticorubral projection following neonatal lesions in the albino rat
162
Brain Research, 1 II (1976) 162 1(~6 ;~' Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
An ultrastructural study of the anomalous corticorubral projection following neonatal lesions in the albino rat
S. H. N A H and S. K. L E O N G
Department of Anatomy, Faculty of Medicine, University of Malaya, Kuala Lumpur (Malaysia) (Accepted April 12th, 1976)
Nah and Leong11 have shown that unilateral ablation of the sensorimotor and adjacent cortex (SMC) in the newborn albino rat results in the sprouting of a crossed corticorubral projection from the opposite intact cortex. The aim of our present experiment was to determine whether the anomalous corticorubral fibre to the contralateral red nucleus establishes any synaptic contacts and if so, whether these contacts are comparable to those formed by the normal ipsilateral corticorubral projection. Eight newborn albino rats received an aspiration lesion of the left SMC. After the lesion the animals were returned to the mother for nursing and were allowed to survive for a period of 2-4 months. Two of the animals were sacrificed at the end of two months to test if the degenerative products resulting from the initial lesion were cleared. The remaining animals received a second lesion at a corresponding region in the opposite intact cortex, and were sacrificed at days 3, 5 and 9 following the second lesion. Ether anaesthesia was used during all the operative procedures. The perfusion fluid was made up of 4 ~ paraformaldehyde and 1 ~ glutaraldehyde in phosphate buffer2L To every 100 ml of solution prepared, 0.54 g of dextrose was added and the pH was adjusted to 7.2-7.4 with 10 N sodium hydroxide. At least 150 ml of perfusate was used for each animal. After perfusion the brain was dissected out carefully and kept in the perfusate overnight at 4 °C. Tissue blocks were then sampled from the rostrocaudal extent of the right and left red nuclei, treated with buffered osmium tetroxide, dehydrated and embedded in Epon. Semi-thin sections were cut from each tissue block and stained with Richardson's stain (ref. 17) for the purpose of orientation, trimming and sectioning for electron microscopy. The sections obtained were stained with lead citrate and uranyl acetate, studied and photographed in an Hitachi HS-8 electron microscope. In addition to the experimental animals 3 adult control animals were used for the study of the normal corticorubral projections from the SMC. In the normal adult rats ultrastructural studies of the red nucleus revealed neurones of 4 size categories as described by Reid et al. 15,16. In the caudal onethird of the nucleus the cells were predominantly giant and large neurones. Small and medium sized neurones predominated in the rostral parvocellular part of the nu-
163 cleus, whereas the middle third of the nucleus contained a mixture of large, medium and small neurones. Following ablation of the SMC in the normal adult control rats, degeneration was mainly observed in the rostral two-thirds of the ipsilateral red nucleus, with a definite predominance in the rostral one-third of the nucleus. Degeneration was rarely encountered in a corresponding region of the contralateral red nucleus. At most 1-2 degenerated axons and one degenerated terminal could be detected in some sections, and in most other sections no degeneration was observed. In contrast in the rostral part of the ipsilateral red nucleus it was not uncommon to find 2-3 degenerated axons and 1-2 degenerated terminals in a grid square for every 2-3 squares examined. In the middle third of the ipsilateral red nucleus fewer degenerated axons and terminals could be observed; about 1-2 degenerated axons and 0-2 degenerated terminals in a grid square for every 4--5 squares examined. The majority of the fibres showing degeneration were myelinated with only a few being non-myelinated. The degenerated myelinated fibres measured from 0.2 to 3/tm and the non-myelinated fibres about 0.2 #m. The affected fibres showed various degrees of degenerative changes. In some the axoplasm appeared electron-dense without any distinct neurotubules and filaments, although remnants of mitochondria were occasionally encountered. In others the axons looked almost empty. Still others showed a complete darkening of the axoplasm masking all organelles and such dark axons were commonly found at day 9 postoperative. The periodicity of the myelin sheath did not appear altered at 3 and 5 days postoperative though out-pocketing of the myelin was occasionally seen. At 9 days, while many degenerated axons still demonstrated a normal myelin sheath pattern, others showed much out-pocketing of the sheath and the periodicity of the sheath had become indistinct. Also at 9 days postoperative, more non-myelinated axons showed degenerative changes. Electron-dense degenerating synaptic endings were present throughout the range of postoperative survival periods. The degenerating profiles measured 0.2-2 /~m and were in contact with dendrites measuring 0.2-3 #m. No degenerated axosomatic terminals were seen. In most degenerating terminals the synaptic vesicles were not recognizable but synaptic vesicles still visible in some of the degenerating terminals appeared primarily round in shape, although some flattened or ellipsoid vesicles might also be present. The mitochondria seen in the degenerating terminals were generally swollen or distorted with fragmented cristae. Scattered glycogen particles were quite often seen in the degenerating terminals. Although terminals in their various stages of degeneration were seen throughout the survival period studied, the more advanced stages of degeneration with the terminals completely distorted and showing no recognizable organelles were more often seen in animals allowed a longer period of survival, viz., 5 and 9 days postoperative. The postsynaptic membranes showed an increase in electron density suggestive of Gray's type I asymmetrical contacts 4,5 and the degenerating terminals formed synaptic relations mostly with one dendrite, although contacts with two dendrites were occasionally seen. Some astrocytic processes were seen to be hypertrophied and to contain scattered glycogen particles, various dense bodies, membranous formations, abundant
164 cisternae of rough endoplasmic reticulum and much increased Golgi apparatus. Degenerating axons and terminals engulfed by astrocytes and oligodendrocytes were more frequently seen in animals allowed 5 and 9 days postoperative survival. The present findings with respect to the normal corticorubral projection are in general agreement with previous reports1, 3. In addition to the features described above, growth cones consisting of swellings filled with numerous small mitochondria were often seen. Occasionally a glial cell or a myelin sheath was seen to contact a dendrite exhibiting a sub-surface density, suggesting the taking over of a vacated synaptic site. This feature has also been reported by previous workers6,7,14. Degenerative products resulting from an SMC lesion at birth were cleared when the animal was sacrificed two months later. Following a recent SMC lesion in the experimental animals in which the contralateral SMC had been destroyed at birth, degeneration was observed in the rostral two-thirds of both the ipsilateral and the contralateral red nucleus (Fig. 1), with the anomalous fibres running across the mid-line to the contralateral red nucleus as demonstrated in the light microscopic studies of Nah and Leong 11. The pattern of degeneration and the synaptic relationships in the ipsilateral and the contralateral red nucleus were similar to those of the normal corticorubral projection described above. Degeneration in the ipsilateral red nucleus was slightly more than that in the normal control animal. About 3-4 degenerated axons and 1-3 degenerated terminals were found in a grid square for e v e r y 2-3 squares examined. The density of degeneration in the contralateral red nucleus in these experimental animals was only slightly less than that in the ipsilateral r e d nucleus, about 2-3 degenerated axons and 1-2 degenerated terminals in a grid square for every 2-3 square examined. Glial cells or myelin sheaths in contact with a dendrite exhibiting a sub-surface density were also observed in these cases. The glial
Fig. 1. A degenerating terminal forming axodendritic asymmetrical relationships. The postsynaptic density is indicated by arrows.|No organnetles are visible in, the terminal except for a few degenerating m~tochondrla. Degeneration seen m the contralateral red,nucleus after a recent large SMC lesion m the remaining intact cerebral hemisphere in an animal with a large neonatal SMC lesion. × 30.000,
165 reaction seen in the experimental animals in both the ipsilateral and contralateral red nucleus was similar to that found in the control animals. Our light microscopic observations11 suggest that the corticorubral fibres terminate in the neuropil of the anterior two-thirds of the red nucleus. This impression is confirmed in the present electron microscopic preparations which show that the normal as well as the anomalous corticorubral fibres establish synapses with small and medium sized dendrites. The present studies could not, however, establish whether the dendrites upon which the degenerating terminals were located were dendrites projecting from the giant and large cells in the caudal magnocellular portion of the red nucleus, or dendrites from cells situated in the more rostral portion of the nucleus. Our findings suggest that both the normal and anomalous corticorubral fibres terminate in asymmetric synaptic contact with dendrites, and contain mostly spherical vesicles. Shortening or prolonging the postoperative survival time in our experiments has not resulted in degeneration of a second type of terminal. The terminals undergoing degeneration in this study have been shown to be excitatory12,19,21. However, these corticorubral fibres are slow-conducting and the synapses are not efficient2° when compared with the rapid and efficient cerebellorubral afferentsis. Padel et al. 13 have demonstrated physiologically that subpopulations of rubral neurons receive afferents from those regions of the motor cortex which control, respectively, proximal and distal limb muscles, an observation suggesting that the red nucleus likewise affects the motor mechanisms of both proximal and distal muscles. The convergence pattern of the efferent from the motor cortex to single red nucleus cells seems highly complex and could be the anatomical substrate for coordinated activation of the proximal and distal muscles of the limbs. Whatever the functions of the corticorubral fibres in normal adult animals may be, it might not seem unreasonable to assume a similar function for the anomalous corticorubral fibres, as both the normal and anomalous fibres form the same type of synapses. However, as previous studies have shown that sprouting may occur without equivalent electrophysiological2 and behavioural s-l° signs of change, electrophysiological and behavioural studies would be necessary to determine the functional significance of the anomalous corticorubral afferents. The present study, coupled with a similar study6 demonstrating the formation of anomalous corticofugal connections with the pons, superior colliculus and the spinal cord after neonatal SMC lesion, suggests that the process of reinnervation following injury to the sensorimotor cortex follows a precise and rigidly determined pattern. In all 4 regions studied: the spinal cord, pons, superior colliculus and red nucleus, the newly established synapses are not visibly different from the normal ones, and all form Gray's type I asymmetrical contacts. We wish to thank the staff of the Electron Microscopic laboratory, Faculty of Medicine, University of Malaya, for preparing the sections for the present study.
166 1 Brown, L. T., Corticorubral projections in the rat, J. comp. Neurol., 154 (1974) 149-168. 2 Chow, K. L , Mathers, H. and Spear, D., Spreading of uncrossed retinal projection in superior colliculus of neonatally enucleated rabbits, J. eomp. Neurol., 151 (1973) 307 322. 3 Flumerfelt, B. A. and Gwyn, D. G., Synaptology and afferent connections of the red nucleus in the rat, Anat. Rec., 175 (1973) 321. 4 Gray, E. G., Electron microscopy of synaptic contacts on dendritic spines of the cerebral cortex, Nature (Lond.), 183 (1959) 1592-1593. 5 Gray, E. G., Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study, J. Anat. (Lond.), 93 (1959) 420-433. 6 Leong, S. K., A qualitative electron microscopic investigation of the anomalous corticofugal projections following neonatal lesions in the albino rats, Brain Research, In Print. 7 Lurid, R. D. and Lund, J. S., Synaptic adjustment after deafferentation of the superior colliculus of the rat, Science, 171 (1971) 804-807. 8 Marotte, L. R. and Mark, R. F., The mechanism of selective reinnervation of fish eye muscle. I. Evidence from muscle function during recovery, Brain Research, 19 (1970) 41-51. 9 Marotte, L. R. and Mark, R. F., The mechanism of selective reinnervation offish eye. I(. Evidence from electron-microscopy of nerve endings, Brain Research, 19 (1970) 53-62. 10 Mollgaard, K., Diamond, M. C., Bennett, E. L., Rosenzweig, M. R. and Linder, B., Qualitative synaptic changes with differential experience in rat brain, Int. J. Neurosci., 2 (1971) 113-128. 11 Nab, S. H. and Leong, S. K., Bilateral corticofugal projection to the red nucleus after neonatal lesions in the albino rats, Brain Research, 107 (1976) 433436. 12 Nakajima, Y., Fine structure of the medial nucleus of the trapezoid body of the bat with special reference to two types of synaptic endings, J. Cell Biol., 50 (1971) 121-134. 13 Padel, Y., Smith, A. M. and Armand, J., Topography of projections from the motor cortex to rubrospinal units in the cat, Exp. Brain Res., 17 (1973) 315-332. 14 Raisman, G. and Field, P. M., A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei, Brain Research, 50 (1973) 241-264. 15 Reid, J. M., Gwyn, D. G. and Flumerfelt, B. A., A cytocarchitecture and Golgi study of the red nucleus in the rat, J. eomp. Neurol., 162 (1975) 337-362. 16 Reid, J. M., Flumerfelt, B. A. and Gwyn, D. G., An ultrastructural study of the red nucleus in the rat, J. comp. Neurol., 162 (1975) 363-386. 17 Richardson, K. C., Jarett, L. and Finke, E. H., Embedding in epoxy resins for ultrathin sectioning in electron microscopy, Stabt Technol., 35 (1960) 313-323. 18 Toyama, K., Tsukahara, N. and Udo, M., Nature of the cerebellar influences upon the red nucleus neurons, Exp. Brain Res., 4 (1967) 292-309. 19 Tsukahara, N., Fuller, D. R. G. and Brooks, V. B., Collateral pyramidal influences induced post-synaptic potentials in red nucleus neurons, J. NeurophysioL, 32 (1969) 35-42. 20 Tsukahara, N. and Kosaka, K., The mode of cerebral excitation of red nucleus neurons, Exp. Brain Res., 5 (1968) 102 117. 21 Uchizono, K., Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat, Nature (Lond.), 207 (1965) 642-643. 22 Vaughn, J. E. and Peters, A., Aldehyde fixation of nerve fibres, J. Anat. (Loml.), 100 (1966) 687.
Brain Research, 1 II (1976) 162 1(~6 ;~' Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
An ultrastructural study of the anomalous corticorubral projection following neonatal lesions in the albino rat
S. H. N A H and S. K. L E O N G
Department of Anatomy, Faculty of Medicine, University of Malaya, Kuala Lumpur (Malaysia) (Accepted April 12th, 1976)
Nah and Leong11 have shown that unilateral ablation of the sensorimotor and adjacent cortex (SMC) in the newborn albino rat results in the sprouting of a crossed corticorubral projection from the opposite intact cortex. The aim of our present experiment was to determine whether the anomalous corticorubral fibre to the contralateral red nucleus establishes any synaptic contacts and if so, whether these contacts are comparable to those formed by the normal ipsilateral corticorubral projection. Eight newborn albino rats received an aspiration lesion of the left SMC. After the lesion the animals were returned to the mother for nursing and were allowed to survive for a period of 2-4 months. Two of the animals were sacrificed at the end of two months to test if the degenerative products resulting from the initial lesion were cleared. The remaining animals received a second lesion at a corresponding region in the opposite intact cortex, and were sacrificed at days 3, 5 and 9 following the second lesion. Ether anaesthesia was used during all the operative procedures. The perfusion fluid was made up of 4 ~ paraformaldehyde and 1 ~ glutaraldehyde in phosphate buffer2L To every 100 ml of solution prepared, 0.54 g of dextrose was added and the pH was adjusted to 7.2-7.4 with 10 N sodium hydroxide. At least 150 ml of perfusate was used for each animal. After perfusion the brain was dissected out carefully and kept in the perfusate overnight at 4 °C. Tissue blocks were then sampled from the rostrocaudal extent of the right and left red nuclei, treated with buffered osmium tetroxide, dehydrated and embedded in Epon. Semi-thin sections were cut from each tissue block and stained with Richardson's stain (ref. 17) for the purpose of orientation, trimming and sectioning for electron microscopy. The sections obtained were stained with lead citrate and uranyl acetate, studied and photographed in an Hitachi HS-8 electron microscope. In addition to the experimental animals 3 adult control animals were used for the study of the normal corticorubral projections from the SMC. In the normal adult rats ultrastructural studies of the red nucleus revealed neurones of 4 size categories as described by Reid et al. 15,16. In the caudal onethird of the nucleus the cells were predominantly giant and large neurones. Small and medium sized neurones predominated in the rostral parvocellular part of the nu-
163 cleus, whereas the middle third of the nucleus contained a mixture of large, medium and small neurones. Following ablation of the SMC in the normal adult control rats, degeneration was mainly observed in the rostral two-thirds of the ipsilateral red nucleus, with a definite predominance in the rostral one-third of the nucleus. Degeneration was rarely encountered in a corresponding region of the contralateral red nucleus. At most 1-2 degenerated axons and one degenerated terminal could be detected in some sections, and in most other sections no degeneration was observed. In contrast in the rostral part of the ipsilateral red nucleus it was not uncommon to find 2-3 degenerated axons and 1-2 degenerated terminals in a grid square for every 2-3 squares examined. In the middle third of the ipsilateral red nucleus fewer degenerated axons and terminals could be observed; about 1-2 degenerated axons and 0-2 degenerated terminals in a grid square for every 4--5 squares examined. The majority of the fibres showing degeneration were myelinated with only a few being non-myelinated. The degenerated myelinated fibres measured from 0.2 to 3/tm and the non-myelinated fibres about 0.2 #m. The affected fibres showed various degrees of degenerative changes. In some the axoplasm appeared electron-dense without any distinct neurotubules and filaments, although remnants of mitochondria were occasionally encountered. In others the axons looked almost empty. Still others showed a complete darkening of the axoplasm masking all organelles and such dark axons were commonly found at day 9 postoperative. The periodicity of the myelin sheath did not appear altered at 3 and 5 days postoperative though out-pocketing of the myelin was occasionally seen. At 9 days, while many degenerated axons still demonstrated a normal myelin sheath pattern, others showed much out-pocketing of the sheath and the periodicity of the sheath had become indistinct. Also at 9 days postoperative, more non-myelinated axons showed degenerative changes. Electron-dense degenerating synaptic endings were present throughout the range of postoperative survival periods. The degenerating profiles measured 0.2-2 /~m and were in contact with dendrites measuring 0.2-3 #m. No degenerated axosomatic terminals were seen. In most degenerating terminals the synaptic vesicles were not recognizable but synaptic vesicles still visible in some of the degenerating terminals appeared primarily round in shape, although some flattened or ellipsoid vesicles might also be present. The mitochondria seen in the degenerating terminals were generally swollen or distorted with fragmented cristae. Scattered glycogen particles were quite often seen in the degenerating terminals. Although terminals in their various stages of degeneration were seen throughout the survival period studied, the more advanced stages of degeneration with the terminals completely distorted and showing no recognizable organelles were more often seen in animals allowed a longer period of survival, viz., 5 and 9 days postoperative. The postsynaptic membranes showed an increase in electron density suggestive of Gray's type I asymmetrical contacts 4,5 and the degenerating terminals formed synaptic relations mostly with one dendrite, although contacts with two dendrites were occasionally seen. Some astrocytic processes were seen to be hypertrophied and to contain scattered glycogen particles, various dense bodies, membranous formations, abundant
164 cisternae of rough endoplasmic reticulum and much increased Golgi apparatus. Degenerating axons and terminals engulfed by astrocytes and oligodendrocytes were more frequently seen in animals allowed 5 and 9 days postoperative survival. The present findings with respect to the normal corticorubral projection are in general agreement with previous reports1, 3. In addition to the features described above, growth cones consisting of swellings filled with numerous small mitochondria were often seen. Occasionally a glial cell or a myelin sheath was seen to contact a dendrite exhibiting a sub-surface density, suggesting the taking over of a vacated synaptic site. This feature has also been reported by previous workers6,7,14. Degenerative products resulting from an SMC lesion at birth were cleared when the animal was sacrificed two months later. Following a recent SMC lesion in the experimental animals in which the contralateral SMC had been destroyed at birth, degeneration was observed in the rostral two-thirds of both the ipsilateral and the contralateral red nucleus (Fig. 1), with the anomalous fibres running across the mid-line to the contralateral red nucleus as demonstrated in the light microscopic studies of Nah and Leong 11. The pattern of degeneration and the synaptic relationships in the ipsilateral and the contralateral red nucleus were similar to those of the normal corticorubral projection described above. Degeneration in the ipsilateral red nucleus was slightly more than that in the normal control animal. About 3-4 degenerated axons and 1-3 degenerated terminals were found in a grid square for e v e r y 2-3 squares examined. The density of degeneration in the contralateral red nucleus in these experimental animals was only slightly less than that in the ipsilateral r e d nucleus, about 2-3 degenerated axons and 1-2 degenerated terminals in a grid square for every 2-3 square examined. Glial cells or myelin sheaths in contact with a dendrite exhibiting a sub-surface density were also observed in these cases. The glial
Fig. 1. A degenerating terminal forming axodendritic asymmetrical relationships. The postsynaptic density is indicated by arrows.|No organnetles are visible in, the terminal except for a few degenerating m~tochondrla. Degeneration seen m the contralateral red,nucleus after a recent large SMC lesion m the remaining intact cerebral hemisphere in an animal with a large neonatal SMC lesion. × 30.000,
165 reaction seen in the experimental animals in both the ipsilateral and contralateral red nucleus was similar to that found in the control animals. Our light microscopic observations11 suggest that the corticorubral fibres terminate in the neuropil of the anterior two-thirds of the red nucleus. This impression is confirmed in the present electron microscopic preparations which show that the normal as well as the anomalous corticorubral fibres establish synapses with small and medium sized dendrites. The present studies could not, however, establish whether the dendrites upon which the degenerating terminals were located were dendrites projecting from the giant and large cells in the caudal magnocellular portion of the red nucleus, or dendrites from cells situated in the more rostral portion of the nucleus. Our findings suggest that both the normal and anomalous corticorubral fibres terminate in asymmetric synaptic contact with dendrites, and contain mostly spherical vesicles. Shortening or prolonging the postoperative survival time in our experiments has not resulted in degeneration of a second type of terminal. The terminals undergoing degeneration in this study have been shown to be excitatory12,19,21. However, these corticorubral fibres are slow-conducting and the synapses are not efficient2° when compared with the rapid and efficient cerebellorubral afferentsis. Padel et al. 13 have demonstrated physiologically that subpopulations of rubral neurons receive afferents from those regions of the motor cortex which control, respectively, proximal and distal limb muscles, an observation suggesting that the red nucleus likewise affects the motor mechanisms of both proximal and distal muscles. The convergence pattern of the efferent from the motor cortex to single red nucleus cells seems highly complex and could be the anatomical substrate for coordinated activation of the proximal and distal muscles of the limbs. Whatever the functions of the corticorubral fibres in normal adult animals may be, it might not seem unreasonable to assume a similar function for the anomalous corticorubral fibres, as both the normal and anomalous fibres form the same type of synapses. However, as previous studies have shown that sprouting may occur without equivalent electrophysiological2 and behavioural s-l° signs of change, electrophysiological and behavioural studies would be necessary to determine the functional significance of the anomalous corticorubral afferents. The present study, coupled with a similar study6 demonstrating the formation of anomalous corticofugal connections with the pons, superior colliculus and the spinal cord after neonatal SMC lesion, suggests that the process of reinnervation following injury to the sensorimotor cortex follows a precise and rigidly determined pattern. In all 4 regions studied: the spinal cord, pons, superior colliculus and red nucleus, the newly established synapses are not visibly different from the normal ones, and all form Gray's type I asymmetrical contacts. We wish to thank the staff of the Electron Microscopic laboratory, Faculty of Medicine, University of Malaya, for preparing the sections for the present study.
166 1 Brown, L. T., Corticorubral projections in the rat, J. comp. Neurol., 154 (1974) 149-168. 2 Chow, K. L , Mathers, H. and Spear, D., Spreading of uncrossed retinal projection in superior colliculus of neonatally enucleated rabbits, J. eomp. Neurol., 151 (1973) 307 322. 3 Flumerfelt, B. A. and Gwyn, D. G., Synaptology and afferent connections of the red nucleus in the rat, Anat. Rec., 175 (1973) 321. 4 Gray, E. G., Electron microscopy of synaptic contacts on dendritic spines of the cerebral cortex, Nature (Lond.), 183 (1959) 1592-1593. 5 Gray, E. G., Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study, J. Anat. (Lond.), 93 (1959) 420-433. 6 Leong, S. K., A qualitative electron microscopic investigation of the anomalous corticofugal projections following neonatal lesions in the albino rats, Brain Research, In Print. 7 Lurid, R. D. and Lund, J. S., Synaptic adjustment after deafferentation of the superior colliculus of the rat, Science, 171 (1971) 804-807. 8 Marotte, L. R. and Mark, R. F., The mechanism of selective reinnervation of fish eye muscle. I. Evidence from muscle function during recovery, Brain Research, 19 (1970) 41-51. 9 Marotte, L. R. and Mark, R. F., The mechanism of selective reinnervation offish eye. I(. Evidence from electron-microscopy of nerve endings, Brain Research, 19 (1970) 53-62. 10 Mollgaard, K., Diamond, M. C., Bennett, E. L., Rosenzweig, M. R. and Linder, B., Qualitative synaptic changes with differential experience in rat brain, Int. J. Neurosci., 2 (1971) 113-128. 11 Nab, S. H. and Leong, S. K., Bilateral corticofugal projection to the red nucleus after neonatal lesions in the albino rats, Brain Research, 107 (1976) 433436. 12 Nakajima, Y., Fine structure of the medial nucleus of the trapezoid body of the bat with special reference to two types of synaptic endings, J. Cell Biol., 50 (1971) 121-134. 13 Padel, Y., Smith, A. M. and Armand, J., Topography of projections from the motor cortex to rubrospinal units in the cat, Exp. Brain Res., 17 (1973) 315-332. 14 Raisman, G. and Field, P. M., A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei, Brain Research, 50 (1973) 241-264. 15 Reid, J. M., Gwyn, D. G. and Flumerfelt, B. A., A cytocarchitecture and Golgi study of the red nucleus in the rat, J. eomp. Neurol., 162 (1975) 337-362. 16 Reid, J. M., Flumerfelt, B. A. and Gwyn, D. G., An ultrastructural study of the red nucleus in the rat, J. comp. Neurol., 162 (1975) 363-386. 17 Richardson, K. C., Jarett, L. and Finke, E. H., Embedding in epoxy resins for ultrathin sectioning in electron microscopy, Stabt Technol., 35 (1960) 313-323. 18 Toyama, K., Tsukahara, N. and Udo, M., Nature of the cerebellar influences upon the red nucleus neurons, Exp. Brain Res., 4 (1967) 292-309. 19 Tsukahara, N., Fuller, D. R. G. and Brooks, V. B., Collateral pyramidal influences induced post-synaptic potentials in red nucleus neurons, J. NeurophysioL, 32 (1969) 35-42. 20 Tsukahara, N. and Kosaka, K., The mode of cerebral excitation of red nucleus neurons, Exp. Brain Res., 5 (1968) 102 117. 21 Uchizono, K., Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat, Nature (Lond.), 207 (1965) 642-643. 22 Vaughn, J. E. and Peters, A., Aldehyde fixation of nerve fibres, J. Anat. (Loml.), 100 (1966) 687.