Effects of methylazoxymethanol given at different stages of postnatal life on development of the rat brain. Comparison with those of thyroid deficiency

Newborn rats were treated at different stages of their development with low doses of methylazoxymethanol acetate. The postnatal increase of the DNA content of the cerebrum did not differ from that of controls. In the cerebellum, the DNA content was transitorily reduced, but later, the external granular layer became thicker and DNA deposition increased in comparison with controls; finally, the cerebellar DNA returned to a normal value. Morphological abnormalities of the cerebellum, abnormal orientation of migrating cells, scattering of Purkinje cell bodies within the internal granule cells and specially striking abnormalities of the morphology and orientation of Purkinje cell dendrites were noted in rats treated with MAM from birth to day 3. The effects on the Purkinje cell morphogenesis persisted but were much less marked when MAM was given from 4 to 7 or from 8 to 11 days. Neonatal thyroid deficiency, as MAM-treatment between days 0 and 3, leads to an abnormal position of Purkinje cell bodies within the cerebellar cortex; it also leads to morphological abnormalities of their dendritic arborization which closely resemble those observed after MAM-treatment during the second postnatal week. It also alters the cell formation in the cerebellum. Thyroid deficiency probably exerts its effect on cell formation earlier than previous biochemical studies have shown. On another hand, the morphological abnormalities of Purkinje cell arborizations in the thyroid-deficient animals may be partly due to the perturbations of cell formation which persist later in the cerebellum.     

Intra- and interpopulation heterogeneity of the cerebellar nerve cells in topography and ribosomal gene content

Using the method of in situ hybridization of ribosomal DNA with DNA of isolated nuclei, a study was made of localization and relative content of ribosomal genes in the Purkinje cells and other cells of the rat cerebellum. It has been shown that various cells of cerebellum have, on the average, the same number of ribosomal genes. A subpopulation with amplified ribosomal genes have been found among the nuclei of Purkinje's cells.     

Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei

Summary A cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of DNA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats.Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region.     

Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei. A cytophotometric study.

A cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of NDA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats. Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region.     

Cytophotometric comparisons of DNA levels in neuronal and glial cells of the cerebellum: A comparative study

Several cytochemical studies of the DNA content and ploidy status of neuronal cell nuclei in the central nervous system have reported the occurrence of hyperdiploid amounts of DNA in Purkinje cells and suggest the existence of some type of ‘extra’ DNA, the biological significance of which is, as yet, unknown. To explore this phenomenon further, the DNA content of glial and Purkinje cell nuclei was determined in several vertebrate species, using the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole (DAPI) to stain isolated cerebellar nuclei for analysis with a single parameter flow cytometer. The Feulgen reaction for DNA was used to stain liver and cerebellar tissue imprints for the measurement of individual nuclei with a Vickers M86 integrating microdensitometer. In both types of analyses, chicken erythrocyte nuclei served as an internal reference standard of 2.5 pg DNA per cell. The mean DNA content of Purkinje cells and glial or granule cells was essentially the same as that found for diploid (2C) non-neuronal cells, such as hepatocytes, in rainbow trout, Amazon molly fish, salamander (Plethodon), mouse, rat, rabbit, cat, dog, monkey and human. Although Purkinje cell nuclei with 4C DNA levels were found in all of these species, except salamander and rabbit, the frequency of such cells was low (1–7%) and varied with the species. There was a low incidence of Purkinje cell nuclei with interclass DNA amounts in all species examined. Our data show that most neuronal cell nuclei in the cerebellum contain 2C levels of DNA.     

Basic factors in the polyploidization of cerebellar Purkinfe cells in chick embryogenesis. III. Kinetics of RNA and DNA in Purkinje cell nuclei]

The content and concentration of RNA and DNA in the nuclei of the Purkinje cells of cerebellum of chick embryos (10-21 day-old) were determined by cytospectrophotometry. The RNA concentration shows a dayly rhythm, its content significantly increasing. The periods of the increase of RNA synthesis coincide with the periodicity of protein synthesis, the morphogenesis of the Purkinje cells and with the embryo's development as a whole. The DNA concentration remaining constant, its content increases sharply with the onset of the specific functional activity of cells and of the intensive growth of axons and dendrites. The variability of the mean values of this growth increases which is conditioned by the appearance of cells with hyperdiploid, tetraploid and hypertetraploid DNA contents in their nuclei. The Purkinje cells do not divide, their increased DNA content may be regarded as the expression of polyploidization or polytenization (which are functionaly equivalent) of a part of the nuclei. It is not excluded that in this case a differential gene amplification may take place. The increase of DNA content is associated with the onset of morphofunctional maturity of cells and may be due to a continuously increasing intensity of protein synthesis secured by the matrix material.     

DNA CONTENTS IN PURKINJE CELLS AND INNER GRANULE NEURONS IN THE DEVELOPING RAT CEREBELLUM

Cytophotometric studies revealed that the content of Feulgen DNA in Purkinje cells in the developing rat cerebellum remains at the diploid level throughout the postnatal life. No evidence was found to suggest resumption of DNA synthesis in the neurons. This finding is in accord with autoradiographic observations that neuroblasts once differentiated from matrix cells do not synthesize DNA nor divide again.     

Congenital cerebellar hypoplasia in newborn calves

A sporadic outbreak of congential cerebellar ataxia (CCA) was seen in calves. The abnormality of this disorder of the central nervous system characteristic was cerebellar hypoplasia, which was observed in eight calves. The cerebellum was almost completely absent in some of these calves and approximately one-fifth of the normal size in some others. Six of the eight calves were also affected with hydrocephaly. The significant changes of the cerebellum were complete or partial destruction of the cortex, deficiency of granules and/or Purkinje cells, clumping of the remaining granule cells, and an irregular cavity formation in the terminal portion of the folium. The clinicopathological changes of CCA quite closely resembled those of the bovine viral diarrhea-mucosal disease virus infection.     

Cytophotometric Re-investigation of DNA content in Purkinje cells of the rat cerebellum

Summary Stage scanning cytophotometry of Feulgen-stained cell nuclei of the cerebellum of young adult rats revealed that the absorbance values of the majority of the Purkinje cells show a Gaussian distribution with a low coefficient of variance. The peak absorbance of this population is the same as that of the granule cells. About 1% of the Purkinje cells measured, were found to have a stain content which indicates a 4C amount of DNA. For both the granular and the Purkinje cell population, a very small number of nuclei possesses absorbance values intermediate between 2C and 4C. The present data suggest prevalent diploidy of the Purkinje cells, and are at variance with those postulating a tetraploid and/or hyperdiploid status of this population.     

The Murine Stem Cell Virus Promoter Drives Correlated Transgene Expression in the Leukocytes and Cerebellar Purkinje Cells of Transgenic Mice

The murine stem cell virus (MSCV) promoter exhibits activity in mouse hematopoietic cells and embryonic stem cells. We generated transgenic mice that expressed enhanced green fluorescent protein (GFP) under the control of the MSCV promoter. We obtained 12 transgenic founder mice through 2 independent experiments and found that the bodies of 9 of the founder neonates emitted different levels of GFP fluorescence. Flow cytometric analysis of circulating leukocytes revealed that the frequency of GFP-labeled leukocytes among white blood cells ranged from 1.6% to 47.5% across the 12 transgenic mice. The bodies of 9 founder transgenic mice showed various levels of GFP expression. GFP fluorescence was consistently observed in the cerebellum, with faint or almost no fluorescence in other brain regions. In the cerebellum, 10 founders exhibited GFP expression in Purkinje cells at frequencies of 3% to 76%. Of these, 4 mice showed Purkinje cell-specific expression, while 4 and 2 mice expressed GFP in the Bergmann glia and endothelial cells, respectively. The intensity of the GFP fluorescence in the body was relative to the proportion of GFP-positive leukocytes. Moreover, the frequency of the GFP-expressing leukocytes was significantly correlated with the frequency of GFP-expressing Purkinje cells. These results suggest that the MSCV promoter is useful for preferentially expressing a transgene in Purkinje cells. In addition, the proportion of transduced leukocytes in the peripheral circulation reflects the expression level of the transgene in Purkinje cells, which can be used as a way to monitor transgene expression properties in the cerebellum without invasive techniques.     

Dynamics of quantitative RNA changes in the Purkinje cells of the rat cerebellum in various functional states

UV cytophotometry was used to study over time the effect of vestibular stimulation on RNA content in Purkinje's cells of the rat cerebellum nodulus. The animals placed in narrow boxes were rotated horizontally (60 r.p.m.) for an hour. To evaluate partially the effects of stress and hypoxia, the content of RNA was examined in the immobilized animals placed in the same boxes without rotation. Rhythmic quantitative changes in RNA in the cytoplasm and nucleus of Purkinje cells were observed upon vestibular stimulation of cerebellum function. In the nucleolus, the changes in the content of RNA were less pronounced. The periodic quantitative changes in the content of RNA in the cytoplasm, nucleus and nucleolus of Purkinje cells were statistically significant in the majority of the animals, but weakly pronounced.     

Misregulation of poly(ADP-ribose) polymerase-1 activity and cell type-specific loss of poly(ADP-ribose) synthesis in the cerebellum of aged rats

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.     

Change in the staining properties of pyriform neurocytes in the mouse cerebellum during protein-calorie deficiency and subsequent rehabilitation

Quantitative analysis has been carried out on semithin sections of cerebellum cortex to investigate the relation between Purkinje cells with different dyeing properties. The number of dark Purkinje cells was found to increase after a month-long food rehabilitation of ill-fed mice. At the same time addition of carnitine to the mouse food has resulted in a significant decline in the number of dark Purkinje cells, as compared to control animals. The data obtained suggest that the rising number of dark Purkinje cells in the cerebellum cortex under conditions of malnutrition is probably due to the increased intracellular accumulation of free fatty acids.     

Loss of Sulfoglucuronyl and Other Neolactoglycolipids in Purkinje Cell Abnormality Murine Mutants

A significant reduction in the content of two members of the sulfoglucuronyl-neolacto series of glycolipids (SGGLs), 3-sulfoglucuronyl-lacto-N-neotetraosylceramide (SGGL-1) and 3-sulfoglucuronyl lacto-N-norhexaosylceramide (SGGL-2), in the cerebellum of the Purkinje cell abnormality mutants, Purkinje cell degeneration (pcd/pcd), lurcher (Lc/+), and staggerer (sg/sg), was also confirmed in the mildly affected nervous (nr/nr) mutant. The expression of SGGLs was studied during development of the pcd/pcd mutant cerebellum, and it was shown that the rate of decline in the level of SGGLs practically coincided with the loss of Purkinje cell perikarya. This indicated that SGGLs are primarily localized in Purkinje cells and that initially, at least, there is no genetic defect in the biosynthesis of SGGLs in the mutant. The precursors of SGGLs, viz., lacto-N-neotetraosylceramide (paragloboside) and lacto-N-norhexaosylceramide, as well as other glycolipids derived from these precursors, such as X-determinant fucoglycolipids and disialosyllacto-N-neotetraosylceramide, were also present in normal cerebellum. Levels of paragloboside and its other derivatives, similar to SGGLs, were also significantly reduced in the Purkinje cell abnormality mutants pcd/pcd, sg/sg, Lc/+, and nr/nr but were normal in other cerebellar mutants, such as quaking (qk/qk), weaver (wv/wv), and reeler (rl/rl), where Purkinje cells are not involved. Thus, the entire paragloboside family of glycolipids is primarily associated with Purkinje cells in the cerebellum. Although levels of monoclonal antibody HNK-1-reactive glycolipids were reduced in the Purkinje cell abnormality mutants, HNK-1-reactive glycoproteins were not affected in these mutants.     

Zebrin II / Aldolase C Expression in the Cerebellum of the Western Diamondback Rattlesnake (Crotalus atrox)

Aldolase C, also known as Zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+), alternating with stripes of Purkinje cells with little or no expression (ZII-). The patterns of ZII+ and ZII- stripes in the cerebellum of birds and mammals are strikingly similar, suggesting that it may have first evolved in the stem reptiles. In this study, we examined the expression of ZII in the cerebellum of the western diamondback rattlesnake (Crotalus atrox). In contrast to birds and mammals, the cerebellum of the rattlesnake is much smaller and simpler, consisting of a small, unfoliated dome of cells. A pattern of alternating ZII+ and ZII- sagittal stripes cells was not observed: rather all Purkinje cells were ZII+. This suggests that ZII stripes have either been lost in snakes or that they evolved convergently in birds and mammals.     

RGS8 expression in developing cerebellar Purkinje cells

The regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 belongs to B/R4 subfamily of RGS proteins and is specifically expressed in Purkinje cells of adult cerebellum. Here, to examine the expression of RGS8 mRNA in developing cerebellum, we performed in situ hybridization. Apparent signals for expression of RGS8 mRNA were first detected on day 9 after birth, then RGS8 mRNA expression in Purkinje cells increased up to day 21, and its levels decreased to some extent in adult Purkinje cells. We also studied the expression of RGS7, which is expressed in Golgi cells in the granule cell layer of adult cerebellum. The expression of RGS7 mRNA was recognized in 7 day neonatal cerebellum. When examined with anti-RGS8 antibody, the RGS8 protein was already excluded from nucleus on day 9, and was distributed in cell body and dendrites in differentiating Purkinje cells of 14 day neonates.     

Postnatal development of the cerebellar Purkinje cell shape in guinea pig ontogenesis

In sagittal cerebellum sections, morphometrical study of cerebellum of mature-born animals - guinea pigs - was performed using Nissl's procedure. A change of shape and volume of Purkinje cells and their nuclei in the course of the guinea pig postnatal ontogenesis was studied. It has been shown that both the growth process itself and the rate of formation of the definite form of Purkinje cells and of their nuclei in the course of ontogenesis proceeds non-uniformly. The most intensive growth of vertical and horizontal diameters of Purkinje cells and of their nuclei is observed during the 1st and 4th weeks of postnatal life. Especially rapid is an increase of horizontal diameters of Purkinje cells and of their nuclei, which impairs the ovoid-bear-like shape to the cerebellar Purkinje cells of adult guinea pigs.     

Disturbed Purkinje cell migration due to reduced expression of Reelin by X-irradiation in developing rat cerebellum.

The major histogenetic events of the rat cerebellum take place in the early postnatal days. During this period, precursors of microneurons, such as granule cells, form the external granular layer (EGL), extend over the surface of the primordial cerebellum, and actively proliferate. Postmitotic granule cells leave the EOL and migrate to the internal granular layer (IGL). On the other hand, guided by radial glial fibers, immature Purkinje cells migrate from the ventricular zone of the fourth ventricle and settle in the Purkinje cell plate with thickness of several cells. Various cell adhesion molecules are involved in the interaction between the migratory immature Purkinje cells and processes of the radial glia as the basis for contact guidance. The second process is the formation of immature Purkinje cells to the monolayer. This process takes place at the first week after birth of the rat and cell adhesion molecules such as neural cell adhesion molecule (NCAM), fibronectin, tenascin and Reelin are also suggested to play an important role for the cell patterning. When rat fetuses are exposed to X-radiation in the last gestation period, abnormal foliation of the cerebellum develops with ectopic Purkinje cells. The molecular mechanism that contributes to abnormal migration of Purkinje cells and foliar malformation induced by X-irradiation in the cerebellum are not yet clear. This study was undertaken to elucidate the mechanisms of ectopic Purkinje cell formation by examining the expression of cell adhesion molecules.     

A quantitative cytochemical study of the DNA content of neurons of rat cerebellar cortex

Abstract— Measurements of nuclear DNA content with quantitative cytochemical methods for determining amounts in single nuclei reveal tetraploid quantities of DNA in cerebellar Purkinje neurons of the rat, or twice the amount of nuclear DNA of rat somatic cells in general. The findings suggest that tetraploidy is probably a universal phenomenon among rat Purkinje cells. Granule and basket cells have a diploid DNA content.     

Postnatal development of the shape of cerebellar Purkinje cells in guinea pig ontogenesis

In sagittal cerebellum sections, morphometrical study of cerebellum of mature-born animals—guinea pigs—was performed using Nissl’s procedure. A change of shape and volume of Purkinje cells and their nuclei in the course of the guinea pig postnatal ontogenesis was studied. It has been shown that both the growth process itself and the rate of formation of the definite form of Purkinje cells and of their nuclei in the course of ontogenesis proceeds non-uniformly. The most intensive growth of vertical and horizontal diameters of Purkinje cells and of their nuclei is observed during the 1st and 4th weeks of postnatal life. Especially rapid is an increase of horizontal diameters of Purkinje cells and of their nuclei, which impairs the ovoid-bear-like shape to the cerebellar Purkinje cells of adult guinea pigs.