Transient accumulation of Gallyas-Braak-positive and phosphorylated tau-immunopositive substances in neuronal lipofuscin granules in the amygdala, hippocampus and entorhinal cortex of rats during long-term chloroquine intoxication.

Long-term chloroquine (CQ) intoxication of normal and groggy mutant rats resulted in transient accumulation of Gallyas-Braak (G-B) -positive and phosphorylated tau (AT8) -immunopositive substances in neuronal lipofuscin granules in the amygdala, hippocampus and entorhinal cortex. In the facial nuclei of normal rats, the neuronal lipofuscin granules were only AT8-immunopositive but G-B-negative, throughout CQ intoxication, while in groggy rats, the granules were positive by both staining methods irrespective of CQ intoxication. The results indicate that there are different mechanisms in the production of G-B-positive substances in neuronal lipofuscin granules between CQ-intoxicated rats and untreated groggy mutant rats.

Age-related changes in number of phosphorylated tau-immunopositive particles in neurons of facial and lateral cerebellar nuclei of normal and groggy mutant rats.

The numbers of cytoplasmic particles immunopositive to an anti-phosphorylated tau antibody (AT8) were counted in the neurons of facial and lateral cerebellar nuclei of Slc:Wistar and groggy mutant rats aged from 20 to 360 days. These particles greatly increased in number from 20 to 30 days of age in the Slc:Wistar rats, whereas in the groggy rats, they increased at such a slower rate than in the Slc:Wistar rats as to reach the peak at 60 days of age. The particles decreased to lower levels at 120 days of age in both rat strains, and increased again from 180 days of age. The results indicate that the numbers of AT8-immunopositive particles in neuronal cytoplasm change dynamically according to the physiological state associated with the growing and aging processes.

Accumulation of argyrophilic substances in neuronal cytoplasm of aging groggy mutant rat.

A groggy rat is a single autosomal recessive mutant displaying a movement disorder. Using Gallyas-Braak (G-B) staining of sections of brain and spinal cord from groggy and Slc:Wistar rats, argyrophilic neurons were seen in some regions of the 180-day-old groggy rats. The numbers of these neurons and of the regions exhibiting these neurons in groggy rats increased with age. In 730-day-old groggy rats, these neurons were especially numerous in the red nucleus, reticulotegmental nucleus of pons, intertrigeminal nucleus, facial nucleus, all the reticular nuclei of medulla, hypoglossal nucleus, and spinal cord layers 7-9. Using electron microscopy, silver grains after G-B staining and immunodeposits after staining with an anti-phosphorylated tau antibody, AT8, showed the same localization in the lipofuscin granules in the neurons of facial and hypoglossal nuclei of 730-day-old groggy rats. However, AT8 immunoreactivity was found not only in the lipofuscin granules of the facial and hypoglossal nuclei of the aged-matched Slc:Wistar rat, but also in those of the G- B-negative cerebellar nuclei of groggy and Slc:Wistar rats. These facts suggest that the AT8-immunopositive tau in the argyrophilic neurons of aged groggy rats is modified to react with the G-B staining.

Age-related appearance of dystrophic axon terminals in cerebellar and vestibular nuclei of Mongolian gerbils.

In the brains of 360-day-old Mongolian gerbils, numerous swellings immunoreactive to anti-neurofilament antibody were observed in cerebellar and vestibular nuclei. The number of these swellings was the same in two gerbil strains with different susceptibility to spontaneous motor seizures by various stimuli, but much more numerous in gerbils as compared with the 360-day-old Slc:Wistar rats. Such swellings were only occasionally found before 60 days of age in gerbils, but they increased in number about fivefold from 60 to 180 days of age and about quadruple from 180 to 360 days of age. Electron microscopic observation showed that these swellings were dystrophic axon terminals (DATs) whose cytoplasms were occupied with large bundles of neurofilaments, numerous vesicular structures containing membranous and/or granular materials, and many rod-shaped mitochondria. Additionally, other types of DATs displaying degenerative changes of cytoplasmic organelles were observed. ACPase cytochemistry showed that the vesicular structures in the DATs contained ACPase and released it into the cytoplasm.

Altered axon terminals containing concentric lamellar bodies of cerebellar Purkinje cells in Mongolian gerbil.

Altered axon terminals containing concentric lamellar bodies were observed in cerebellar and vestibular nuclei of the Mongolian gerbil. The terminals increased in number from 30 days of age onward, and reached about tenfold at 360 days. The numbers were the same in two gerbil strains with different susceptibility to spontaneous motor seizures by various stimuli, but about threefold those in Slc:Wistar rat.

Concentric lamellar bodies in axon terminals and preterminal axons of cerebellar Purkinje cells in groggy mutant rat: acid phosphatase cytochemistry.

The process of formation and degeneration of concentric lamellar bodies (CLBs) in axon terminals and preterminal axons of Purkinje cells were examined by acid phosphatase (ACPase) cytochemistry. In myelinated axons, tubular structures measuring 20-50 nm in diameter contained ACPase reaction products. In altered axon terminals and preterminal axons of Purkinje cells, elongated saccular structures contained numerous reaction products. These saccular structures were arranged concentrically and enclosed cytoplasmic organelles. In the CLBs displaying degenerative profiles, ACPase reaction products were dispersed in the degenerating materials. The nature of the tubular structures and their roles in the transport of ACPase and the formation of CLBs are discussed.

Axonal swellings in cerebellar white matter of groggy mutant rat.

In the groggy mutant rat, a number of axonal swellings appeared in the cerebellar white matter from 180 days of age onward. Since these axonal swellings were immunostained with an antibody against calbindin D28k, the axons forming these swellings were considered to belong to Purkinje cells. They were also immunostained with an anti-neurofilament antibody, and ultrastructurally characterized by the presence of myelin sheaths around them and abnormal accumulations of filamentous structures, mitochondria and smooth endoplasmic reticula (SER) in their axoplasm. The SER were considered to convey acid phosphatase (ACPase) to the swelling's axoplasm, where ACPase was set free from the SER throughout the axoplasm and engaged in the digestion of cytoplasmic organelles. The groggy rat is useful model model for the study of the mechanism of the age-related formation of axonal swellings.

Electron microscopic cytochemistry of acid phosphatase activity in dystrophic axons of rat gracile nuclei.

Dystrophic axons (DAs) in rat gracile nuclei were largely classified into two types. One type had a focal swelling measuring about 2-8 microns in diameter, and contained greatly increased numbers of various cellular organelles. Numerous reaction products of acid phosphatase (ACPase) cytochemistry were deposited in the cytoplasmic matrix and the multilaminated bodies. The second type ranged from 8 microns to 50 microns in diameter, and was characterized by the presence of a large number of fine tubular structures in the cytoplasm. Only a few reaction products of ACPase cytochemistry were localized in the small vesicular structures. The functional role of ACPase in the degradation and accumulation of cellular organelles in DAs is discussed.

Age-related and region-specific increase in number of concentric lamellar bodies in axon terminals and presynaptic axons in central nervous system of groggy mutant rat.

In some regions of the central nervous system (CNS) of the groggy rat, a mutant with a movement disorder, concentric lamellar bodies (CLBs) were formed in numerous axon terminals and presynaptic axons. These bodies were counted electron microscopically in the lateral cerebellar nuclei of Slc:Wistar and groggy mutant rats at 20 to 180 days of age. In the Slc:Wistar rat groups, the mean numbers of axonal CLBs were mostly under 1.0 per 100 microns2, except for the 30-day-old rat group which showed a value of 1.7. In the groggy rat groups, the numbers of axonal CLBs greatly increased from 40 days of age onward, reaching the maximum mean number of 23.7 per 100 microns2 in the 90-day-old rat group and subsequently decreasing; however, significant numbers were still present in the 180-day-old rat group. Since these bodies have been reported to contain an acid phosphatase (ACPase), the regional specificity of their appearance in the CNS was examined by light and electron microscopic ACPase histochemistry. In the 90- and 180-day-old groggy rats, numerous particulate deposits of ACPase reaction products were found in the neuropil of the lateral, interposed and medial cerebellar nuclei, the superior, lateral and spinal vestibular nuclei, and the spinal gracile nuclei. By electron microscopy, the particulate deposits in the cerebellar and vestibular nuclei were confirmed as the CLBs in axon terminals, while those in the gracile nuclei were revealed to be the dystrophic axons. Thus, it was suggested that the axon terminals and presynaptic axons, having a high capacity to form the CLBs in the groggy rats from 40 days of age onward, belong to the Purkinje cells.

Neuronal degeneration in the striatum of the groggy rat: a new mutant with a movement disorder.

A new mutation displaying abnormal movement was obtained in the progeny of a female Wistar rat which had been given 10 mg/kg methylnitrosourea at an early stage of the gestational period. Genetic studies revealed that the character is inherited by an autosomal single recessive gene, and we designated this mutation groggy (gene symbol gr). The abnormal movement of the groggy rat was first apparent around postnatal day 15, while the histological studies revealed the appearance of numerous necrotic neurons in the striatum of the groggy rat on postnatal days 60 and 120.

Critical period for induction of congenital hydrocephalus and dysplasia of subcommissural organ by prenatal X-irradiation in rats.

A single whole-body X-irradiation of pregnant Wistar rats at a dose of 1.05 Gy at 10.30, 12.30 and 14.30 h respectively, of gestational day 10 resulted in significantly high incidences of hydrocephalic offspring. No hydrocephalic offspring resulted from X-irradiation of pregnant rats with 1.05 Gy at 16.30 h, whereas a dose of 1.22 Gy at 16.30 h resulted in a low but statistically significant incidence of hydrocephalus. Neither 1.05 Gy nor 1.22 Gy X-irradiation of pregnant rats at 18.30 h resulted in any hydrocephalic offspring. Dysplasia of the subcommissural organ was noticed in all the hydrocephalic brains histologically examined.

Ethanol-phosphotungstic acid and bismuth staining of spermatid nucleoli in mouse spermiogenesis.

The nucleoli of developing mouse spermatids were examined with ethanol-phosphotungstic acid (E-PTA) staining, and also with bismuth staining following formaldehyde fixation (FA-Bi staining) and glutaraldehyde fixation (GA-Bi staining). Only the cortical zone of the nucleolar dense fibrillar component (DFC) in the round spermatids was stained with E-PTA, while the inner area remained either faintly (early Golgi-phase spermatids) or completely unstained (cap-phase spermatids). Incubation of the fixed testis with dithiothreitol before E-PTA staining resulted in homogeneously intense staining of the DFC. The facts suggest that numerous E-PTA-positive basic proteins were present in the DFC, but disulfide crosslinks formed in the DFC proteins prevent penetration of PTA into the DFC interior. The DFC was stained with bismuth after FA-Bi and GA-Bi staining until the disappearance of the nucleoli occurring in acrosome-phase spermatids. The fibrillar center, homogeneously stained using E-PTA, FA-Bi, and GA-Bi methods was present in the nucleoli of Golgi-phase and early cap-phase spermatids, but disappeared in the nucleoli of late cap-phase spermatids. These results are discussed based on the previous studies dealing with the ribosomal RNA synthesis in mouse spermiogenesis.

Perichromatin granule-like granular structures in the nuclei of antral-follicular oocytes of the Chinese hamster.

In the nuclei of antral-follicular oocytes of the Chinese hamster (Cricetulus griseus), numerous granular structures ranging from 60 nm to 250 nm in diameter were present in the nucleolar area, and a few were observed on the surface of heterochromatin materials and within the interchromatin space. When treated with bismuth staining en bloc after glutaraldehyde fixation (GA-Bi staining), these granular structures were shown to be composed of fine fibrils intensely contrasted with bismuth, indicating that these may be regarded as one type of perichromatin granule.

Appearance of perichromatin granules in decondensing sperm nuclei in fertilized mouse eggs.

A number of granular structures measuring about 35-45 nm in diameter appeared in the dispersing chromatin materials of the decondensing sperm nuclei in fertilized mouse eggs. These granules were contrasted by the bismuth staining en bloc following either formaldehyde or glutaraldehyde fixation, and were encircled by an electron-lucent "halo." The facts indicate that these are perichromatin granules.

Genetical and histological studies on a mouse displaying microphthalmia and cataract.

A male mouse displaying bilateral microphthalmia and cataract was found among the offspring of pregnant Slc: ICR mouse treated intraperitoneally with 10 mg/kg methylnitrosourea on gestational day 4. This mutant has been maintained by brother-sister mating. By the mating test with normal Slc: ICR mice, this character was revealed to be inherited by an autosomal single recessive gene. Linkage test with the brown locus showed that this mutant gene is linked with the B gene and mapped on chromosome 4. The histological study of the eyes of adult mutant mice revealed various abnormalities all over the eyes, especially in the lens and neural retina. Embryologically, the mutant mice showed persistent connection between the lens vesicle and the surface ectoderm by a cellular stalk, and also the formation of retinal infolding, in the early stages of eye development. Both were considered to be responsible for the consequent abnormal development and degradation of the lens. These results suggest that the mutant mouse we found may be an allele of the dysgenetic lens (dyl) reported by Sanyal and Hawkins.

Congenital hydrocephalus following X-irradiation of pregnant rats on an early gestational day.

When pregnant rats were X-irradiated at a dose of 100 R on gestational day 9.5, a considerable number of postnatally-viable hydrocephalic offspring resulted, all of which were accompanied with bilateral micro- or anophthalmia. Histological studies revealed that the cerebral aqueduct of the congenital hydrocephalic brain was severely stenosed, and the subcommissural organ was reduced in size and displaced at some distance from the anterior end of the cerebral aqueduct. From embryological studies, it was considered that the maldevelopment of the subcommissural organ in the X-irradiated fetus might cause a reduction in the amount of its secretions which function as a cushion preventing complete closure of the cerebral aqueduct during fetal life, resulting in stenosis of the cerebral aqueduct.

5-Azacytidine-induced exencephaly in mice.

Maternal intraperitoneal administration of 1 mg/kg 5-azacytidine to pregnant S1c:ICR mice on gestational Day 7.5 resulted in a high incidence of exencephalic offspring. Histological examination of untreated 7.5 days mouse embryos revealed that the head folds were formed in the anterior halves of the embryos, whereas the primitive streaks still remained in the posterior halves. At 12 hours after 5-azacytidine administration (8.0 days embryos), numerous pyknotic cells were observed in the neurectoderm of the head folds, in the embryonic ectoderm and the migrating mesoderm of the primitive streak region. These pyknotic cells had almost disappeared from the embryonic tissues, and few abnormalities were encountered, in embryos 24 hours after 5-azacytidine administration (8.5 days embryos), except for the slight reduction in thickness of the neurectoderm of the head folds compared with that in untreated 8.5 days embryos. In untreated 9.5 days embryos, the head folds had entirely closed along the anterior neuroaxes, whereas those treated with 5-azacytidine 48 hours earlier displayed head folds that were open in various degrees along the neuroaxes anterior to the fourth ventricle. The primary cause of 5-azacytidine-induced exencephaly is considered to be attributable to a powerful cell-killing action of 5-azacytidine and the subsequent loss of germinal cells in the neurectoderm of the head folds. The precise mechanisms by which this damage results in the failure of neural tube closure in the cephalic region remains unclear.