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Neocortical transplants in the cerebellum of the rat: their afferents and efferents
228
Brain Resea,ch, 189 (.1980) 228 232 ( Elsevier/North-Holland Biomedical Press
Neocortical transplants in the cerebellum of the rat: their afferents and efferents
MONICA M. OBLINGER, BRIAN H. HALLAS and GOPAL D. DAS* Department of Biological Sciences, Purdue University, West La]ayette, Ind. 47907 (U.S.A.)
(Accepted January 3rd, 1980) Key words: neocortical transplants - - cerebellum - - embryonic neural tissue
Successful transplantation of embryonic neural tissues in the brains of neonate or adult host mammals has been achieved by several investigators 2-5,s,1°,~3. In these investigations neural tissues were transplanted homo- or heterotopically and the transplants were seen to have differentiated and survived. Das 2,3 and Das and Hallas 5 observed that the neural transplants in the parenchyma of the host brain grew and differentiated, became integrated with the host tissue, and shared neuropil containing dendrites as well as axons at the interface between the transplant and the host brain. Other investigators transplanted embryonic brain tissue into different regions of the host CNS and observed that such transplants established afferent as well as efferent connections with the host brain1, 9,11. The present report is addressed to the afferent and efferent connections of neocortical tissue heterotopically transplanted in the cerebellum of host animals. Using the technique of transplantation described previously 2,6, 16- or 17-dayold embryonic neocortical tissue was transplanted into the right cerebellar hemisphere of neonatal Long-Evans rats. Ninety days after transplantation, at which time the transplants were fully grown and differentiated, lesions were made in 16 host animals in either the inferior olivary nuclei, pons, left cerebellar hemisphere, ventral thalamus or transplant. Three animals sustained hemisections of the cervical spinal cord. These animals were sacrificed 3-7 days after the lesion by transcardial perfusion with 1 0 ~ formalin. Frozen serial sections were cut at 33/~m in a coronal plane and processed using the Fink-Heimer metho&. In 12 other animals 0.2.-0.6 #1 of H R P (Sigma type VI, 30 ~ solution) was injected either directly into the transplant or into the left cerebellar hemisphere. These animals were sacrificed 20-24 h after the injection by perfusion with 2 ~ glutaraldehyde, 2 ~ paraformaldehyde, in a phosphate buffer. Frozen serial sections, 33/~m thick, were cut in a coronal plane and every other section was reacted according to the H R P blue reaction product histochemical procedure of Mesulam 12. Alternate sections were stained with the Bodian fiber stain. * To whom correspondence and reprint requests should be addressed.
229
Fig. 1. a: coronal view of a 17 day neocortical transplant (Tr) in the right hemisphere of host cerebellum (Cb). Arrows indicate some regions of interface. Fink-Heimer stain, ( x 10). b: interface of the transplant and host cerebellum. Fibers from the medullary layer (mdl) of the cerebellum are seen penetrating the transplant. Bodian fiber stain, (× 250). c: degenerating fibers in the transplant as a result of a contralateral lesion of the pontine gray. Degenerating axons from the rr,edullary layer are indicated by arrows. Fink-Heimer stain, ( x 400). d : labelled neurons in the contralateral inferior olivary nucleus following an injection of HRP into the transplant. Mesulam's blue reaction product H R P procedure, ( x 400).
230 In all host animals the transplants were seen to have survived and grown. The cortical transplants had replaced nearly the entire right hemisphere of the host cerebellum (Fig. la), and in a few cases had extended into the vermal region of the cerebellum. All transplants were anatomically integrated with the host cerebellum in the sense that normal neuropil was present at the interface and no pia matter or glia scar formation separated the transplant from the host brain. The Bodian stained material revealed numerous axons crossing the interface between the host brain and the transplant (Fig. l b). A common observation in all preparations was axons from the pyramidal cells of the transplants, axons of cerebellar neurons, as well as axonal bundles from the cerebellar medullary regions crossing the interface. The sources of the afferent fibers to, as well as the destination of efferent fibers from, the transplants were analyzed in the Fink-Heimer and H R P material. A summary of these findings is presented in Table I. In general, the inferior olivary nuclei, pons, spinal cord and the left cerebellar hemisphere provided afferent fibers to the transplant, After lesions in these regions, degenerating axons and axonal terminals were seen in various regions of the transplant. Degenerating fibers could be observed extending from the interface to deep areas of the transplants (Fig. 1c). The course and pattern of degenerating fibers within the transplants varied to some extent from animal to animal, and this was related to the internal cytoarchitectural organization of the transplants. In animals that received thalamic lesions, no degenerating fibers in the transplant were seen. When H R P was injected into the transplants, labeled cells were observed in the contralateral inferior olivary nuclei, pontine nuclei, reticular formation of the brain stem, cerebellar cortex and in regions of the transplant distant to the injection site (Fig. TABLE I
Afferents to the transplants Condition
Number of anhnals
Connections with the transplant
Inferior olive lesion Pontine lesion Cerebellar lesion Spinal cord lesion Thalamic lesion H R P injected into transplant
3 3 3 3 3 6
Yes Yes Yes Yes No Yes*
* Labelled cells observed in contralateral olivary nuclei, pontine nuclei, reticular formation and host cerebellum.
Efferents of the transplants Condition
Number of animals
Connections
Transplant lesion
4
Deep cerebellar nuclei, reticular formation, host cerebellum Labelled neurons in transplant
HRP injected into host cerebellum 6
231 ld). Only those preparations in which there was no diffusion of HRP outside the transplant were analyzed. Efferents of transplants were analyzed in Fink-Heimer preparations following lesions of the transplant. Degenerating fibers were commonly seen in medullary cerebellar regions and in the deep cerebellar nuclei unilaterally. In two cases aberrant degenerating bundles were observed in the region of the dorsal longitudinal fasciculus of Schfitz and in the reticular formation. Since Iesions were generally restricted to only parts of transplants, degenerating fiber bundles ramifying within the transplants were also commonly observed. It was noted that these intratransplant bundles were quite prevalent. This study demonstrated that embryonic cortical transplants in the cerebellum of neonate host animals received afferent fibers from the host brain as well as providing efferent fibers that ramified both within the host brain and the transplant itself. Our observations show that afferents to the transplants were provided by the fibers immediately available around it, i.e. fibers normally present in the intact cerebellar hemisphere. These findings support the suggestion of Jaeger and Lund 9 and Lund tl that the 'proximity of transplant and host brain' is an important factor in the formation of connections between the two. Fibers far removed from the transplant such as thalamic fibers that normally would have projected to the cortex did not project to the cortical tissue when it was heterotopically transplanted. Efferent fibers were seen to prefer terminal sites that would normally receive fibers from the region of cerebellum that was replaced by the transplant. In conclusion, the afferents and efferents of transplants seem to be non-specific. The milieu in which the transplants are located, their growth characteristics, and the presence or absence of glial scar formation at the interface may be the major factors determining the nature and growth of the afferents and efferents of the transplants. This research was supported by NIH Research Grant NS-08817.
1 Bj6rklund, A., Segal, M. and Stenevi, U., Functional reinnervation of rat hippocampus by locus coeruleus implants, Brain Research, 170 (1979) 409-426. 2 Das, G. D., Transplantation of embryonic neural tissue in the mammalian brain. I. Growth and differentiation of neuroblasts from various regions of the embryonic brain in the cerebellum of neonate rats, T.I.T.J. Life Sci., 4 (1974) 93-124. 3 Das, G. D., Differentiation of dendrites in the transplanted neuroblasts in the mammalian brain. In G. W. Kreutzberg (Ed.), Advances in Neurology, VoL I2, Raven Press, New York, 1975, pp. 181-199. 4 Das, G. D. and Altman, J., Studies on the transplantation of developing neural tissue in the mammalian brain. I. Transplantation of cerebellar slabs into the cerebellum of neonate rats, Brain Research, 38 (1972) 233-249. 5 Das, G. D. and Hallas, B. H., Transplantation of brain tissue in the brain of adult rats, Experientia (Basel), 34 (1978) 1304-1305. 6 Das, G. D., Hallas, B. H. and Das, K. G., Transplantation of neural tissues in the brains of laborataory mammals: technical details and comments, Experientia (Basel), 35 (1979) 143-153. 7 Fink, R. P. and Helmet, 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.
232 8 Greene, H. S. N. and Arnold, H., The homologous and heterologous transplantation of brain and brain tumors, J. Neurosurg., 2 (1945) 315-331. 9 Jaeger, C. B. and Lund, R. D., Efferent fibers from transplanted cerebral cortex of rats, Brain Research, 165 (1979) 338-342. 10 LeGros Clark, W. E., Neuronal differentiation in implanted foetal cortical tissue, J. Neurol. Psychiat., 3 (1940) 263-272. 11 Lurid, R. D. and Hauschka, S. D., Transplanted neural tissue develops connections with host rat brain, Science, 193 (1976) 582-584. 12 Mesulam, M. M,, Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 13 Stenevi, U., Bj6rklund, A. and Svengaard, N.-A., Transplantation of central and peripheral monoamine neurons to the adult rat brain: techniques and conditions for survival, Brain Research, 114 (1976) 1 -20.
Brain Resea,ch, 189 (.1980) 228 232 ( Elsevier/North-Holland Biomedical Press
Neocortical transplants in the cerebellum of the rat: their afferents and efferents
MONICA M. OBLINGER, BRIAN H. HALLAS and GOPAL D. DAS* Department of Biological Sciences, Purdue University, West La]ayette, Ind. 47907 (U.S.A.)
(Accepted January 3rd, 1980) Key words: neocortical transplants - - cerebellum - - embryonic neural tissue
Successful transplantation of embryonic neural tissues in the brains of neonate or adult host mammals has been achieved by several investigators 2-5,s,1°,~3. In these investigations neural tissues were transplanted homo- or heterotopically and the transplants were seen to have differentiated and survived. Das 2,3 and Das and Hallas 5 observed that the neural transplants in the parenchyma of the host brain grew and differentiated, became integrated with the host tissue, and shared neuropil containing dendrites as well as axons at the interface between the transplant and the host brain. Other investigators transplanted embryonic brain tissue into different regions of the host CNS and observed that such transplants established afferent as well as efferent connections with the host brain1, 9,11. The present report is addressed to the afferent and efferent connections of neocortical tissue heterotopically transplanted in the cerebellum of host animals. Using the technique of transplantation described previously 2,6, 16- or 17-dayold embryonic neocortical tissue was transplanted into the right cerebellar hemisphere of neonatal Long-Evans rats. Ninety days after transplantation, at which time the transplants were fully grown and differentiated, lesions were made in 16 host animals in either the inferior olivary nuclei, pons, left cerebellar hemisphere, ventral thalamus or transplant. Three animals sustained hemisections of the cervical spinal cord. These animals were sacrificed 3-7 days after the lesion by transcardial perfusion with 1 0 ~ formalin. Frozen serial sections were cut at 33/~m in a coronal plane and processed using the Fink-Heimer metho&. In 12 other animals 0.2.-0.6 #1 of H R P (Sigma type VI, 30 ~ solution) was injected either directly into the transplant or into the left cerebellar hemisphere. These animals were sacrificed 20-24 h after the injection by perfusion with 2 ~ glutaraldehyde, 2 ~ paraformaldehyde, in a phosphate buffer. Frozen serial sections, 33/~m thick, were cut in a coronal plane and every other section was reacted according to the H R P blue reaction product histochemical procedure of Mesulam 12. Alternate sections were stained with the Bodian fiber stain. * To whom correspondence and reprint requests should be addressed.
229
Fig. 1. a: coronal view of a 17 day neocortical transplant (Tr) in the right hemisphere of host cerebellum (Cb). Arrows indicate some regions of interface. Fink-Heimer stain, ( x 10). b: interface of the transplant and host cerebellum. Fibers from the medullary layer (mdl) of the cerebellum are seen penetrating the transplant. Bodian fiber stain, (× 250). c: degenerating fibers in the transplant as a result of a contralateral lesion of the pontine gray. Degenerating axons from the rr,edullary layer are indicated by arrows. Fink-Heimer stain, ( x 400). d : labelled neurons in the contralateral inferior olivary nucleus following an injection of HRP into the transplant. Mesulam's blue reaction product H R P procedure, ( x 400).
230 In all host animals the transplants were seen to have survived and grown. The cortical transplants had replaced nearly the entire right hemisphere of the host cerebellum (Fig. la), and in a few cases had extended into the vermal region of the cerebellum. All transplants were anatomically integrated with the host cerebellum in the sense that normal neuropil was present at the interface and no pia matter or glia scar formation separated the transplant from the host brain. The Bodian stained material revealed numerous axons crossing the interface between the host brain and the transplant (Fig. l b). A common observation in all preparations was axons from the pyramidal cells of the transplants, axons of cerebellar neurons, as well as axonal bundles from the cerebellar medullary regions crossing the interface. The sources of the afferent fibers to, as well as the destination of efferent fibers from, the transplants were analyzed in the Fink-Heimer and H R P material. A summary of these findings is presented in Table I. In general, the inferior olivary nuclei, pons, spinal cord and the left cerebellar hemisphere provided afferent fibers to the transplant, After lesions in these regions, degenerating axons and axonal terminals were seen in various regions of the transplant. Degenerating fibers could be observed extending from the interface to deep areas of the transplants (Fig. 1c). The course and pattern of degenerating fibers within the transplants varied to some extent from animal to animal, and this was related to the internal cytoarchitectural organization of the transplants. In animals that received thalamic lesions, no degenerating fibers in the transplant were seen. When H R P was injected into the transplants, labeled cells were observed in the contralateral inferior olivary nuclei, pontine nuclei, reticular formation of the brain stem, cerebellar cortex and in regions of the transplant distant to the injection site (Fig. TABLE I
Afferents to the transplants Condition
Number of anhnals
Connections with the transplant
Inferior olive lesion Pontine lesion Cerebellar lesion Spinal cord lesion Thalamic lesion H R P injected into transplant
3 3 3 3 3 6
Yes Yes Yes Yes No Yes*
* Labelled cells observed in contralateral olivary nuclei, pontine nuclei, reticular formation and host cerebellum.
Efferents of the transplants Condition
Number of animals
Connections
Transplant lesion
4
Deep cerebellar nuclei, reticular formation, host cerebellum Labelled neurons in transplant
HRP injected into host cerebellum 6
231 ld). Only those preparations in which there was no diffusion of HRP outside the transplant were analyzed. Efferents of transplants were analyzed in Fink-Heimer preparations following lesions of the transplant. Degenerating fibers were commonly seen in medullary cerebellar regions and in the deep cerebellar nuclei unilaterally. In two cases aberrant degenerating bundles were observed in the region of the dorsal longitudinal fasciculus of Schfitz and in the reticular formation. Since Iesions were generally restricted to only parts of transplants, degenerating fiber bundles ramifying within the transplants were also commonly observed. It was noted that these intratransplant bundles were quite prevalent. This study demonstrated that embryonic cortical transplants in the cerebellum of neonate host animals received afferent fibers from the host brain as well as providing efferent fibers that ramified both within the host brain and the transplant itself. Our observations show that afferents to the transplants were provided by the fibers immediately available around it, i.e. fibers normally present in the intact cerebellar hemisphere. These findings support the suggestion of Jaeger and Lund 9 and Lund tl that the 'proximity of transplant and host brain' is an important factor in the formation of connections between the two. Fibers far removed from the transplant such as thalamic fibers that normally would have projected to the cortex did not project to the cortical tissue when it was heterotopically transplanted. Efferent fibers were seen to prefer terminal sites that would normally receive fibers from the region of cerebellum that was replaced by the transplant. In conclusion, the afferents and efferents of transplants seem to be non-specific. The milieu in which the transplants are located, their growth characteristics, and the presence or absence of glial scar formation at the interface may be the major factors determining the nature and growth of the afferents and efferents of the transplants. This research was supported by NIH Research Grant NS-08817.
1 Bj6rklund, A., Segal, M. and Stenevi, U., Functional reinnervation of rat hippocampus by locus coeruleus implants, Brain Research, 170 (1979) 409-426. 2 Das, G. D., Transplantation of embryonic neural tissue in the mammalian brain. I. Growth and differentiation of neuroblasts from various regions of the embryonic brain in the cerebellum of neonate rats, T.I.T.J. Life Sci., 4 (1974) 93-124. 3 Das, G. D., Differentiation of dendrites in the transplanted neuroblasts in the mammalian brain. In G. W. Kreutzberg (Ed.), Advances in Neurology, VoL I2, Raven Press, New York, 1975, pp. 181-199. 4 Das, G. D. and Altman, J., Studies on the transplantation of developing neural tissue in the mammalian brain. I. Transplantation of cerebellar slabs into the cerebellum of neonate rats, Brain Research, 38 (1972) 233-249. 5 Das, G. D. and Hallas, B. H., Transplantation of brain tissue in the brain of adult rats, Experientia (Basel), 34 (1978) 1304-1305. 6 Das, G. D., Hallas, B. H. and Das, K. G., Transplantation of neural tissues in the brains of laborataory mammals: technical details and comments, Experientia (Basel), 35 (1979) 143-153. 7 Fink, R. P. and Helmet, 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.
232 8 Greene, H. S. N. and Arnold, H., The homologous and heterologous transplantation of brain and brain tumors, J. Neurosurg., 2 (1945) 315-331. 9 Jaeger, C. B. and Lund, R. D., Efferent fibers from transplanted cerebral cortex of rats, Brain Research, 165 (1979) 338-342. 10 LeGros Clark, W. E., Neuronal differentiation in implanted foetal cortical tissue, J. Neurol. Psychiat., 3 (1940) 263-272. 11 Lurid, R. D. and Hauschka, S. D., Transplanted neural tissue develops connections with host rat brain, Science, 193 (1976) 582-584. 12 Mesulam, M. M,, Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 13 Stenevi, U., Bj6rklund, A. and Svengaard, N.-A., Transplantation of central and peripheral monoamine neurons to the adult rat brain: techniques and conditions for survival, Brain Research, 114 (1976) 1 -20.