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Abnormal organization of the cerebellar cortex in the mutant Japanese quail,Coturnix coturnix japonica
Brain Research, 177 (1979) 183-188 © Elsevier/North-Holland Biomedical Press
183
Abnormal organization of the cerebellar cortex in the mutant Japanese quail,
Cofurnix cofurnix japonica SHINSUKE UEDA, HIRONOBU ITO, HIDEO MASAI and TAKATADA KAWAHARA Department o/ Anatomy, Osaka University Medical School, Osaka, 530 and ( T. K.) National Institute of Genetics, Mishima, 411 (Japan)
(Accepted July 19th, 1979)
A number of mutant cerebellar organizations in mice have been described and have provided valuable information for understanding the genetics and the development of the central nervous system6-8,11,14,16,17. In recent years, the Mexican axolotl was studied electropbysiologically4 and anatomically .5 because of its cerebellar deficiency. A new mutant Japanese quail described here was obtained from the National Institute of Genetics (Mishima, Japan) where domestication of normal quail has been carried out for the purpose of providing new experimental animals. Morphological analyses on mutant 'shaker fowl' birds were studied by hematoxylin-eosin staining showing Purkinje cell degeneration and sex-linked semi-lethal nervous disorders 13. This new mutant quail is characterized by cerebellar functional disorders of fine tremor of the extremities and trunk, gait disturbance, and tumbling when the animal is active or excited. They sometimes dislocate the hip joint. However, there is a large variation in severity and onset-times of the cerebellar disorders. In addition to clinical signs described above, this mutant has dark feathers, is lighter in body weight than the normal, and tends to die at an early age. We have, therefore, named the new mutant 'dark feather nervous disorder' (gene symbol dn). It is a new autosomal recessive mutation (Kawahara, in preparation). This study describes the structural organization of normal and mutant cells of the cerebellum of dn mutant quail at light microscopic level. In this study 6 mature normal and 7 mature dn mutant quails between postnatal weeks 16 and 20 were examined (Japanese quail becomes mature 8 weeks after hatching). All animals were anesthetized by i.p. injection of sodium pentobarbital (Nembutal). Two brains of each group were fixed by perfusion through the heart with 10 ~ neutral formalin and removed from the skull. One cerebellum of each group was postfixed for 24 h in the same fresh solution. After being embedded in celloidin, serial sagittal sections were cut at 25 ,um and stained with 0.1 ~ cresyl violet. Another cerebellum from each group was postfixed in Bodian II solution, embedded in paraffin, cut into 15 #m sagittal sections and stained by a modification of the Bodian method after Otsuka et al. 9. Three mutant brains and two normal brains were fixed and stained in Golgi-Cox solution for 4 weeks, embedded in celloidin and cut into serial sagittal
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185 sections of 100 #m. In order to visualize dendritic spines of Purkinje cells one cerebellum of each group was impregnated with the rapid Golgi method. In addition one cerebellum of each group was fixed by immersion in a formalinammonium bromide solution, and sagittal frozen sections 25/~m thick were impregnated with Cajal's gold sublimate method 16 for astrocytes. In the midsagittal sections of the Nissl and Bodian stained preparations, the mutant shows smaller profiles than the normal (Figs. 1 and 2). Both the molecular and granule cell layers are thinner in the mutant, but the difference in thickness of molecular layer between the two types is more noticeable than granule cell layer (Figs. 3 and 4). In order to compare the mean thickness between the mutant and the normal the areas of the molecular and granule cell layers were measured by a planimeter and the mean lengths of each layer were also measured by a string on the magnified photographs of the Nissl stained preparations. The ratio of the mean thickness of the molecular layer and the granule cell layer in the normal to those in the mutant are 1:0.86 and 1:0.93 respectively. The boundary between the Purkinje cell layer and the granule cell layer is obscure in the mutant because the Purkinje cells are not aligned in a single row. Some of the Purkinje cells are embedded in the granule cell layer (Fig. 4), and the granule ceils remain in the molecular layer (Fig. 5). In the mutant cerebellum, the Purkinje cell soma is small and fusiform in shape independent of its location (Fig. 4), while in the normal the Purkinje soma is flaskshaped. Purkinje cells in the mutant were more intensely stained in the Bodian and Nissl preparations. Counting of Purkinje cells was carried out, but no difference was found in mean cell number per unit area. The number of the granule cells, which appears morphologically normal, was also counted per unit area, but mean density was almost the same as in the normal. However, the total number of Purkinje cells and granule cells must be reduced because of the smaller size of the granule and molecular cell layers in the mutant cerebellum. Golgi-Cox preparations showed hypoplasia of the dendritic trees of some Purkinje cells. Their branches are restricted to a small zone in a middle portion of the molecular layer, while the Purkinje cell dendrites in the normal cerebellum have a wide range of arborization in the molecular layer (Fig. 6). Some abnormal Purkinje cells have long primary dendrites from which poorly developed branches arise (Fig. 7), some have fusiform somata without primary dendrites (Fig. 8), and some others are embedded in the granule cell layer having relatively well-developed dendrites compared to the two types described above (Fig. 9). Although the number of spines per unit length on the peripheral dendrites is not significantly different between the mutant and Fig. 1. Midsagittal view of normal type cerebellum. Cresyl violet. ~ 10. Fig. 2. Midsagittal view of mutant type cerebellum. Cresyl violet. ~. 10. Fig. 3. Midsagittal section of normal type cerebellar cortex. G, granule cell layer; M, molecular layer; P, Purkinje layer. Bodian staining. × 300. Fig. 4. Midsagittal section of mutant ty.,zecerebellar cortex. Note that Purkinje cell somata are fusiform and some of them are embedded in granule cell layer. G, granule cell layer; M, molecular layer. Bodian staining, x 300. Fig. 5. Parasagittal section of mutant type cerebellum. Arrows indicate the granule cell aggregation in molecular layer. G, granule cell layer; M, molecular layer. Cresyl violet. ~ 300.
186
Fig. 6. Two Purkinje cells in normal type cerebellum. Molecular layer is indicated by an arrow. GolgiCox method. Y, 200. Figs. 7--9. A b n o r m a l Purkinje cells in mutant type cerebellum. Molecular layer is indicated by an arrow. G o l g i - C o x method. ,-, 200. Fig. 10. Bergmann glia in mutant type cerebellum. Molecular layer is indicated by an arrow. GolgiCox method. ~ 200.
187 normal in the rapid Golgi preparation, the total number of spines must be reduced because of the hypoplasia of the Purkinje cell dendrites. Basket, stellate, granule, Bergmann glia (Fig. t0), and other glial cells appear morphologically normal. The different thickness of the molecular layer appears to be due to the hypoplasia of dendritic trees of Purkinje cells. An additional factor may be the reduced number of total parallel fibers, because the size of the whole granule cell layer of this mutant is smaller than that of the normal. The granule cell aggregation in molecular layer in the present mutant suggests incomplete migration of granule cells. However, the morphology of Bergmann glia was normal in the Golgi-Cox preparations. Reduced rate of granule cell migration in Weaver mouse has been reported by Rezai and Yoon a2. In addition, Sidmanl0,15 has suggested the close interrelationship between granule cell and Bergmann fiber during migration. The development of Bergmann glia in a few mutant mice was studied by immunofluorescence with glial fibrillary acidic (GFA) protein antiserum 1. However, much agreement cannot be discovered on the point of fine details such as Weaver mouse ~,1'). Ultrastructural and quantitative analyses in the Bergmann glia of this mutant will be needed to discuss the interrelationship between granule cell and Bergmann glia. Although the dendritic trees of Purkinje cell are obviously diminished, they develop numerous spines. Yet, it is uncertain whether the normal synapses are formed between the spines and parallel fibers, because some workers have reached the consensus that the dendritic spines of Purkinje cells arise independently of any inductive action by parallel fibers3,10,11. This new mutant is phenotypically similar to Reeler mutant mouse 2 in histopathology and in clinical features. However, in this mutant there were no Golgi 11 neurons in the white matter of the cerebellum which were observed in Reeler mutant mouseL The abnormality of Reeler mutant cerebellum is thought to be the result of the failure of neuronal migration in spite of its normal arising at the embryonic stages. We are now carrying out a study of the development of this mutant quail cerebellum. The present study shows that there exists a mutant type in avian species similar to the Reeler mutant mouse. Further investgation of this new mutant quail cerebellum will bring useful information about normal and abnormal cerebellum development in all vertebrates. This research was supported by a Grant 212113 for scientific research from the Ministry of Education of Japan. I Bignami, A. and Dahl, D., The development of Bergmann glia in mutant mice with cerebellar malformation: Reeler, Staggerer and Weaver. lmmunofluorescence study with antibodies to glial fibrillary acidic protein, J. comp. Neurol., 155 (1974) 219-230. 2 Hamburgh, M., Analysis of the postnatal developmental effects of 'Reeler', a neurological mutation in mice. A study in developmental genetics, Develop. Biol., 8 (1963) 165-185. 3 Hirano, A., Dembitzer, H. M. and Yoon, C. H., Development of Purkinje cell somatic spines in the weaver mouse, Acta neuropath. (BerL), 40 (1977) 85-90. 4 Ide, C. F., Neurophysiology of spastic, a behavior mutant of the Mexican axolotl: altered vestibular projection to cerebellar auricle and area acousticolateralis, J. comp. Neurol., 176 (1977) 359-372.
188 5 lde, C. F., Tompkins, R. and Miszkowski. N.. Neuroanatomy of spastic, a behavior mutant of the Mexican axolotl: Purkinje celt distribution in the adult cerebellum. J. comp. NeuroL. 176 (1977~ 373-386. 6 Landis, S. C., Ultrastructural changes in the mitochondria of cerebellar Purkinje cells of nervous mutant mice, J. Cell Biol., 57 (t973) 782-797. 7 Landis, D. M. D. and Reese. T. S., Structure of the Purkinje cell membrane in staggerer and weaver mutant mice, J. comp. Neurol., 171 (1977) 247-260. 8 Landis, S. C. and Mullen, R. J., The development and degeneration of Purkinje cells m pcd mutant mice, J. comp. Neurol.. 177 (1977) 125-143. 9 Otsuka, N.. Miyanaga, A.. Tanaka, F. and Kimura. A.. Neue Silberimpr~ignationsversuche zur Darstellung der Neurofibrillen an Paraffinshnitten. J. Kyoto P r e f Univ. Med., 68 (1960) 1125-1128. 10 Rakic, P. and Sidman, R. L., Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice, J. comp. Neurol., 152 (1973) 103-132. 11 Rakic, P. and Sidman, R. L.. Organization of cerebellar cortex secondary deficit of granule cells in weaver mutant mouse, J. eomp. Neurol.. 152 (1973) 133-162. 12 Rezai, Z. and Yoon, C H., Abnormal rate of granule cell migration in the cerebellunl of 'weaver' mutant mice, Develop. BioL, 29 (1972l 17-26. 13 Scott, H. M., Morril, C. C.. Alberts, J. O. and Roberts, E.. The 'shaker' fowl. J. Hered,, 41 (19501 255-257. 14 Sidman. R. L., Green, M. C. and Appel, S. H., Catalog o f the Neurological Mutants o/'the Mouae, Harvard University Press, Cambridge, Massachusetts, 1965. 15 Sidman. R L. and Rakic, P., Neuronal migration, with special reference to develo ping human brain : a review, Brain Research, 62 (1973) 1-35. 16 Sotelo. C. and Changeux. J.-P., Bergmann fibers and granular cell migration in the cerebellum of homozygous weaver mutant mouse, Brain Research, 77 (1974) 48~491. 17 Yoon, C. H. and Frouhar. Z. R.. Interaction of cerebellar mutant genes. I. Mice doubly affected by 'staggerer' and 'weaver' conditions, J. comp. Neurol., 150 (1973) 137-146.
183
Abnormal organization of the cerebellar cortex in the mutant Japanese quail,
Cofurnix cofurnix japonica SHINSUKE UEDA, HIRONOBU ITO, HIDEO MASAI and TAKATADA KAWAHARA Department o/ Anatomy, Osaka University Medical School, Osaka, 530 and ( T. K.) National Institute of Genetics, Mishima, 411 (Japan)
(Accepted July 19th, 1979)
A number of mutant cerebellar organizations in mice have been described and have provided valuable information for understanding the genetics and the development of the central nervous system6-8,11,14,16,17. In recent years, the Mexican axolotl was studied electropbysiologically4 and anatomically .5 because of its cerebellar deficiency. A new mutant Japanese quail described here was obtained from the National Institute of Genetics (Mishima, Japan) where domestication of normal quail has been carried out for the purpose of providing new experimental animals. Morphological analyses on mutant 'shaker fowl' birds were studied by hematoxylin-eosin staining showing Purkinje cell degeneration and sex-linked semi-lethal nervous disorders 13. This new mutant quail is characterized by cerebellar functional disorders of fine tremor of the extremities and trunk, gait disturbance, and tumbling when the animal is active or excited. They sometimes dislocate the hip joint. However, there is a large variation in severity and onset-times of the cerebellar disorders. In addition to clinical signs described above, this mutant has dark feathers, is lighter in body weight than the normal, and tends to die at an early age. We have, therefore, named the new mutant 'dark feather nervous disorder' (gene symbol dn). It is a new autosomal recessive mutation (Kawahara, in preparation). This study describes the structural organization of normal and mutant cells of the cerebellum of dn mutant quail at light microscopic level. In this study 6 mature normal and 7 mature dn mutant quails between postnatal weeks 16 and 20 were examined (Japanese quail becomes mature 8 weeks after hatching). All animals were anesthetized by i.p. injection of sodium pentobarbital (Nembutal). Two brains of each group were fixed by perfusion through the heart with 10 ~ neutral formalin and removed from the skull. One cerebellum of each group was postfixed for 24 h in the same fresh solution. After being embedded in celloidin, serial sagittal sections were cut at 25 ,um and stained with 0.1 ~ cresyl violet. Another cerebellum from each group was postfixed in Bodian II solution, embedded in paraffin, cut into 15 #m sagittal sections and stained by a modification of the Bodian method after Otsuka et al. 9. Three mutant brains and two normal brains were fixed and stained in Golgi-Cox solution for 4 weeks, embedded in celloidin and cut into serial sagittal
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185 sections of 100 #m. In order to visualize dendritic spines of Purkinje cells one cerebellum of each group was impregnated with the rapid Golgi method. In addition one cerebellum of each group was fixed by immersion in a formalinammonium bromide solution, and sagittal frozen sections 25/~m thick were impregnated with Cajal's gold sublimate method 16 for astrocytes. In the midsagittal sections of the Nissl and Bodian stained preparations, the mutant shows smaller profiles than the normal (Figs. 1 and 2). Both the molecular and granule cell layers are thinner in the mutant, but the difference in thickness of molecular layer between the two types is more noticeable than granule cell layer (Figs. 3 and 4). In order to compare the mean thickness between the mutant and the normal the areas of the molecular and granule cell layers were measured by a planimeter and the mean lengths of each layer were also measured by a string on the magnified photographs of the Nissl stained preparations. The ratio of the mean thickness of the molecular layer and the granule cell layer in the normal to those in the mutant are 1:0.86 and 1:0.93 respectively. The boundary between the Purkinje cell layer and the granule cell layer is obscure in the mutant because the Purkinje cells are not aligned in a single row. Some of the Purkinje cells are embedded in the granule cell layer (Fig. 4), and the granule ceils remain in the molecular layer (Fig. 5). In the mutant cerebellum, the Purkinje cell soma is small and fusiform in shape independent of its location (Fig. 4), while in the normal the Purkinje soma is flaskshaped. Purkinje cells in the mutant were more intensely stained in the Bodian and Nissl preparations. Counting of Purkinje cells was carried out, but no difference was found in mean cell number per unit area. The number of the granule cells, which appears morphologically normal, was also counted per unit area, but mean density was almost the same as in the normal. However, the total number of Purkinje cells and granule cells must be reduced because of the smaller size of the granule and molecular cell layers in the mutant cerebellum. Golgi-Cox preparations showed hypoplasia of the dendritic trees of some Purkinje cells. Their branches are restricted to a small zone in a middle portion of the molecular layer, while the Purkinje cell dendrites in the normal cerebellum have a wide range of arborization in the molecular layer (Fig. 6). Some abnormal Purkinje cells have long primary dendrites from which poorly developed branches arise (Fig. 7), some have fusiform somata without primary dendrites (Fig. 8), and some others are embedded in the granule cell layer having relatively well-developed dendrites compared to the two types described above (Fig. 9). Although the number of spines per unit length on the peripheral dendrites is not significantly different between the mutant and Fig. 1. Midsagittal view of normal type cerebellum. Cresyl violet. ~ 10. Fig. 2. Midsagittal view of mutant type cerebellum. Cresyl violet. ~. 10. Fig. 3. Midsagittal section of normal type cerebellar cortex. G, granule cell layer; M, molecular layer; P, Purkinje layer. Bodian staining. × 300. Fig. 4. Midsagittal section of mutant ty.,zecerebellar cortex. Note that Purkinje cell somata are fusiform and some of them are embedded in granule cell layer. G, granule cell layer; M, molecular layer. Bodian staining, x 300. Fig. 5. Parasagittal section of mutant type cerebellum. Arrows indicate the granule cell aggregation in molecular layer. G, granule cell layer; M, molecular layer. Cresyl violet. ~ 300.
186
Fig. 6. Two Purkinje cells in normal type cerebellum. Molecular layer is indicated by an arrow. GolgiCox method. Y, 200. Figs. 7--9. A b n o r m a l Purkinje cells in mutant type cerebellum. Molecular layer is indicated by an arrow. G o l g i - C o x method. ,-, 200. Fig. 10. Bergmann glia in mutant type cerebellum. Molecular layer is indicated by an arrow. GolgiCox method. ~ 200.
187 normal in the rapid Golgi preparation, the total number of spines must be reduced because of the hypoplasia of the Purkinje cell dendrites. Basket, stellate, granule, Bergmann glia (Fig. t0), and other glial cells appear morphologically normal. The different thickness of the molecular layer appears to be due to the hypoplasia of dendritic trees of Purkinje cells. An additional factor may be the reduced number of total parallel fibers, because the size of the whole granule cell layer of this mutant is smaller than that of the normal. The granule cell aggregation in molecular layer in the present mutant suggests incomplete migration of granule cells. However, the morphology of Bergmann glia was normal in the Golgi-Cox preparations. Reduced rate of granule cell migration in Weaver mouse has been reported by Rezai and Yoon a2. In addition, Sidmanl0,15 has suggested the close interrelationship between granule cell and Bergmann fiber during migration. The development of Bergmann glia in a few mutant mice was studied by immunofluorescence with glial fibrillary acidic (GFA) protein antiserum 1. However, much agreement cannot be discovered on the point of fine details such as Weaver mouse ~,1'). Ultrastructural and quantitative analyses in the Bergmann glia of this mutant will be needed to discuss the interrelationship between granule cell and Bergmann glia. Although the dendritic trees of Purkinje cell are obviously diminished, they develop numerous spines. Yet, it is uncertain whether the normal synapses are formed between the spines and parallel fibers, because some workers have reached the consensus that the dendritic spines of Purkinje cells arise independently of any inductive action by parallel fibers3,10,11. This new mutant is phenotypically similar to Reeler mutant mouse 2 in histopathology and in clinical features. However, in this mutant there were no Golgi 11 neurons in the white matter of the cerebellum which were observed in Reeler mutant mouseL The abnormality of Reeler mutant cerebellum is thought to be the result of the failure of neuronal migration in spite of its normal arising at the embryonic stages. We are now carrying out a study of the development of this mutant quail cerebellum. The present study shows that there exists a mutant type in avian species similar to the Reeler mutant mouse. Further investgation of this new mutant quail cerebellum will bring useful information about normal and abnormal cerebellum development in all vertebrates. This research was supported by a Grant 212113 for scientific research from the Ministry of Education of Japan. I Bignami, A. and Dahl, D., The development of Bergmann glia in mutant mice with cerebellar malformation: Reeler, Staggerer and Weaver. lmmunofluorescence study with antibodies to glial fibrillary acidic protein, J. comp. Neurol., 155 (1974) 219-230. 2 Hamburgh, M., Analysis of the postnatal developmental effects of 'Reeler', a neurological mutation in mice. A study in developmental genetics, Develop. Biol., 8 (1963) 165-185. 3 Hirano, A., Dembitzer, H. M. and Yoon, C. H., Development of Purkinje cell somatic spines in the weaver mouse, Acta neuropath. (BerL), 40 (1977) 85-90. 4 Ide, C. F., Neurophysiology of spastic, a behavior mutant of the Mexican axolotl: altered vestibular projection to cerebellar auricle and area acousticolateralis, J. comp. Neurol., 176 (1977) 359-372.
188 5 lde, C. F., Tompkins, R. and Miszkowski. N.. Neuroanatomy of spastic, a behavior mutant of the Mexican axolotl: Purkinje celt distribution in the adult cerebellum. J. comp. NeuroL. 176 (1977~ 373-386. 6 Landis, S. C., Ultrastructural changes in the mitochondria of cerebellar Purkinje cells of nervous mutant mice, J. Cell Biol., 57 (t973) 782-797. 7 Landis, D. M. D. and Reese. T. S., Structure of the Purkinje cell membrane in staggerer and weaver mutant mice, J. comp. Neurol., 171 (1977) 247-260. 8 Landis, S. C. and Mullen, R. J., The development and degeneration of Purkinje cells m pcd mutant mice, J. comp. Neurol.. 177 (1977) 125-143. 9 Otsuka, N.. Miyanaga, A.. Tanaka, F. and Kimura. A.. Neue Silberimpr~ignationsversuche zur Darstellung der Neurofibrillen an Paraffinshnitten. J. Kyoto P r e f Univ. Med., 68 (1960) 1125-1128. 10 Rakic, P. and Sidman, R. L., Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice, J. comp. Neurol., 152 (1973) 103-132. 11 Rakic, P. and Sidman, R. L.. Organization of cerebellar cortex secondary deficit of granule cells in weaver mutant mouse, J. eomp. Neurol.. 152 (1973) 133-162. 12 Rezai, Z. and Yoon, C H., Abnormal rate of granule cell migration in the cerebellunl of 'weaver' mutant mice, Develop. BioL, 29 (1972l 17-26. 13 Scott, H. M., Morril, C. C.. Alberts, J. O. and Roberts, E.. The 'shaker' fowl. J. Hered,, 41 (19501 255-257. 14 Sidman. R. L., Green, M. C. and Appel, S. H., Catalog o f the Neurological Mutants o/'the Mouae, Harvard University Press, Cambridge, Massachusetts, 1965. 15 Sidman. R L. and Rakic, P., Neuronal migration, with special reference to develo ping human brain : a review, Brain Research, 62 (1973) 1-35. 16 Sotelo. C. and Changeux. J.-P., Bergmann fibers and granular cell migration in the cerebellum of homozygous weaver mutant mouse, Brain Research, 77 (1974) 48~491. 17 Yoon, C. H. and Frouhar. Z. R.. Interaction of cerebellar mutant genes. I. Mice doubly affected by 'staggerer' and 'weaver' conditions, J. comp. Neurol., 150 (1973) 137-146.