Differentiation of astroglial cells of the cerebellar cortex in the postnatal development of the rat: a morphological and histochemical study

The morphological autoradiographic and cytospectrophotometric analysis of proliferation and differentation of the cerebellar cortex astroglial cells has been carried out during the rat early postnatal development. The proliferating astroglial cells constitute a major part of the whole cell population of the internal granular layer during the first week. It was proved by means of double labelling (3H- and 14C-thymidine) that these cells synthesize DNA and divide repeatedly, their division proceeding without preliminary morphological dedifferentiation, i. e. with the preservation of plasmatic processes. A suggestion is put forward that the precursors of the cerebellar cortex astroglial cells under study take their origin from the subependymal zone during the prenatal development. The results obtained allow to identify the proliferating glial cells as the Bergman's glia.     

The polypeptide PEP-19 is a marker for Purkinje neurons in cerebellar cortex and cartwheel neurons in the dorsal cochlear nucleus

This light and electron microscopic immunocytochemical study shows that the polypeptide PEP-19, a presumptive calcium binding protein specific to the nervous system, represents an excellent marker for cerebellar Purkinje cells and dorsal cochlear nucleus (DCoN) cartwheel cells. The polypeptide clearly reveals the entire populations of both types of neurons, including their complete dendritic and axonal arborizations. Other PEP-19 containing neurons in the two regions display weak immunoreactivity restricted to the cell body or to cell body and principal dendrites. Electron microscopic localization of PEP-19-like immunoreactivity reveals similarities between this polypeptide, parvalbumin, and a 28K vitamin D-dependent calcium binding protein. However, calmodulin, which is expressed in both Purkinje and granule cells, may differ from PEP-19. Similarities between the organization of the cerebellar cortex and the DCoN superficial layers have been known for some time, with several types of neurons in one system having their presumed homologue in the other. These data provide further support for the proposed structural and functional homology between Purkinje and cartwheel neurons, and establishes PEP-19 as a useful marker for examining degeneration of these two neuronal populations in murine cerebellar mutants.     

Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice.

SV40 T antigen (Tag) expression directed to cerebellar Purkinje cells resulted in the generation of three transgenic mouse lines that displayed ataxia, a neurological phenotype characteristic of cerebellar dysfunction. Onset of symptoms and cerebellar pathology, characterized by specific Purkinje cell degeneration, appeared to be directly dependent upon transgene copy number. The SV5 line (containing > 30 transgene copies), exhibited embryonic transgene expression that caused selective death of immature Purkinje cells and a subsequent block in cerebellar development and ataxia at 2 weeks. The developmental effect of the disruption of Purkinje cells in SV5 mice suggests that a normal complement of these cells is required for early development of the cerebellar cortex, especially granule cell proliferation and migration from external to internal layers. Transgene expression in a second line, SV4 (10 copies), was detectable during the second postnatal week. Death of mature Purkinje cells in the SV4 line resulted in onset of ataxia at 9 weeks. Ataxia in a third line, SV6 (2 copies), was detected after 15 weeks. The distinct cerebellar phenotypes of the SV4-6 lines correlate with specific Tag-induced Purkinje cell ablation as opposed to tumorigenesis.     

Histological changes during development of the cerebellum in the chick embryo exposed to a static magnetic field

Few studies have been performed to evaluate the ultrastructural changes that exposure to static magnetic fields (SMF) can cause to the processes of cell migration and differentiation in the cerebellum during development. Thus, we have studied the development of the cerebellum in the chick embryo (n = 144) under a uniform SMF (20 mT). All of our observations were done on folium VIc of Larsell's classification. The cerebella of chick embryos, which were exposed solely on day 6 of incubation and sacrificed at day 13 of incubation [short exposure (S)1; n = 24], showed an external granular layer (EGL) that was less dense than the EGL in the control group (n = 24). The molecular layer (ML) exhibited a low number of migratory neuroblastic elements. Moreover, the internal granular layer (IGL) was immature, with the cellular elements less abundant and more dispersed than in controls. In chick embryos exposed on day 6 of incubation and sacrificed at day 17 (S2; n = 24), the outstanding feature was the regeneration of the different layers of the cerebellar cortex. The cerebellar cortex of chick embryos exposed continuously to an identical field from the beginning of the incubation up to day 13 [long exposure (L)1; n = 24] or day 17 (L2; n = 24) of incubation showed a higher number of alterations than that of group S1. Electron microscopy confirmed the findings from light microscopy and, at the same time, showed clear signs of cell degeneration and delay in the process of neuronal differentiation. This was more apparent in groups L1 (100%) and L2 (100%) than in groups S1 (95.4%) and S2 (65.2%). In conclusion, the present study showed that SMF can induce irreversible developmental effects on the processes of cell migration and differentiation of the chick cerebellar cortex. Bioelectromagnetics 18:36–46, 1997. © 1997 Wiley-Liss, Inc.     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.     

Retrograde degeneration of the neurons of the cerebellar nuclei after partial removal of the associative cerebral cortex

Retrograde degeneration of the cerebellar nuclei cells has been studied after partial ablation of the associative parietal cerebral cortex in the cat. The material is stained after Nissl. Retrogradely degenerated and normal cells are counted. The "ghost-cells" in the cerebellar nuclei indicate that a direct axonal connection exists between some neurons and the cerebral cortex operated, while the cells that are at other stages of degeneration are, perhaps, connected with this part of the cortex by means of axonal collaterals.     

Histological and ultrastructural features in the early stage of Purkinje cell degeneration in the cerebellar calcification (CC) rat.

The cerebellar calcification (CC) rat is a new neurodegenerative mutant with severe Purkinje cell loss and symmetrical calcifications in the cerebellar cortex manifesting ataxia: lack of coordination in body movements. In the present study, histopathological features were examined in the Purkinje cell degeneration in postnatal homozygous suckling rats without clinical signs, which were genotyped by microsatellite markers. In addition, the calcified Purkinje cells were investigated ultrastructurally and elemental analysis was performed on the deposits. Body weight of the homozygous (cc/cc) rats was already slightly lower compared with the heterozygotes (cc/+) in the neonatal stage. The degeneration of the Purkinje cells in the cc/cc rats was recognized obviously in lobules VI, VII, VIII and IX from 14 days after birth, a few days before the appearance of the ataxic behavior. The Purkinje cells in the region along the fissure between the VIII and IX lobule areas were intensely positive for periodic acid-Schiff reaction specific to glycoconjugates, and in this region, calcium depositions were weakly positive for von Kossa's stain. Electron microscopy also revealed that the calcified Purkinje cells possessed numerous electron-dense bodies containing inclusions with cystic structures such as vesicles, mitochondria and lysosomes, and these bodies were mainly composed of calcium and phosphorous. These findings suggest abnormal storage of glycoconjugates might be a trigger of Purkinje cell degeneration and serves as a matrix for accumulation of calcium phosphate in the cerebellum of CC rats.     

Effect of ethanol on the cerebellar cortex of the chick embryo

The effect of ethanol on the cerebellar cortex of chick embryos was studied in semi-thin sections of material prepared for electron microscopy. The embryos were injected with ethanol on the 3rd or 6th day of incubation and observed until days 13, 15, 17 and 21 of development. A decrease was seen in the number of germinal cells generated, together with defects in neuronal migration and the existence of a lower quantity of cells due to a generalised process of cell death. At the same time, a progressive neuronal degeneration was observed until the 15th day of incubation, the tissue recovering progressively on days 17 and 21. On the other hand, the embryos treated with ethanol on the 3rd day were less affected than those injected on the 6th day.     

猫小脑皮质GABA能神经元年龄相关性变化

为探讨青年猫和老年猫小脑皮质GABA能神经元及其表达的年龄相关性变化,利用Nissl染色显示小脑皮质结构及神经元,免疫组织化学ABC法标记GABA免疫阳性神经元。光镜下观察,采集图像,并利用图像分析软件对分子层、蒲肯野细胞层和颗粒层神经元及GABA免疫阳性神经元及其灰度值进行分析统计。结果显示,GABA免疫阳性神经元、阳性纤维及终末在青年猫和老年猫小脑皮质各层均有分布。与青年猫相比,老年猫分子层、蒲肯野细胞层神经元和GABA免疫阳性神经元密度及其GABA免疫阳性反应强度均显著下降(P<0.01),颗粒层神经元密度和GABA免疫阳性强度也显著下降(P<0.01),但其GABA免疫阳性神经元密度无显著变化(P>0.05);蒲肯野细胞的胞体萎缩,阳性树突分枝减少。因此认为,衰老过程中猫小脑皮质GABA能神经元的丢失和GABA表达的下降,可能是老年个体运动协调、精确调速和运动学习等能力下降的重要原因之一。     

Unequal patterns of development of succinate-dehydrogenase and acetylcholinesterase in Purkinje cell bodies and granule cells isolated in bulk from the cerebellar cortex of the immature rat

Abstract— –A preparative procedure for the isolation in bulk of two cellular populations of the cerebellar cortex of the immature rat, the granule cells and the Purkinje cell bodies, is described. The procedure is used to delineate the developmental pattern of succinate-INT-reduclase (EC 1.3.99.1) and acetylcholinesterase (EC 3.1.1.7) in the crucial period of cerebellar maturation, i.e. between 12 and 19 days postnatally. Although the overall yield of neuronal RNA diminished with age, the proportion of RNA in the Purkinje cell body fraction increased while that in the granule cells decreased and microscopic examination of the fractions confirmed this result. The yields of succinate-INT-reductase and of acetylcholinesterase in the fractions paralleled the yields of RNA. A significant finding was the trend toward diminishing specific activities (units/μg of RNA) with age of both enzymes in the Purkinje cell bodies as against the opposite, upward trend of their specific activities in the granule cells. An additional finding of interest was the different ratio of true acetylcholinesterase/total cholinesterase activity in the two cell types, with the granule cells consistently exhibiting higher true acetylcholinesterase values than the Purkinje cell bodies. The present report thus supplements the histoenzymological data on the developing rat cerebellum in that it reveals specific differences in the enzymatic development of two different cerebellar types, a finding which was greatly facilitated by the availability of the procedure for their bulk isolation.     

小熊猫小脑皮层的组织结构及RT-97、KGF和Bax蛋白在小脑皮层的表达

为了解小熊猫(Ailurus fulgens)小脑皮层的结构特征,观察神经丝蛋白抗体RT-97、角质细胞生长因子(KGF)及Bax蛋白在小脑皮层中的表达,利用组织学方法和免疫组织化学方法观察了小熊猫小脑皮层的显微结构,检测了RT-97、KGF和Bax蛋白的表达.结果表明,小脑皮层从外向内依次可分为分子层、Purkinje细胞层、颗粒层3层.RT-97在小熊猫小脑皮层Purkinje细胞层、颗粒层中神经细胞的轴突、分子层中颗粒细胞的轴突及小脑髓质中有阳性表达;KGF在小脑皮层分子层、Purkinje细胞层和颗粒细胞层及髓质中均有阳性表达;Bax蛋白在小脑皮层分子层、Purkinje细胞层和颗粒细胞层中有阳性表达.RT-97、KGF和Bax蛋白在小脑皮层神经结构的构筑中可能发挥着不同的功能.     

A new neurological mutant rat with symmetrical calcification of Purkinje cells in cerebellum.

A new neurological mutant has been found in the inbred F344 strain of rats. The mutation is inherited as an autosomal recessive trait and is manifest clinically by a hesitant and wobbling gait with asynergic limbs and slight tremor. These symptoms begin at 16-18 days of age and remain essentially constant thereafter. Histologic examination revealed severe degeneration of the Purkinje cells and symmetrical calcification in these and in their dendritic branches in the cerebellar cortex. Such calcified Purkinje cells were intensely stained with the periodic acid-Schiff (PAS) method. PAS-positive substances in the Purkinje cells and extending diffusely over the lesioned sites in the molecular layer were also evident before calcification took place. We have named this neurological mutant the Cerebellar Calcification (CC) rat with the gene symbol cc. This offers a new animal model for the study of the Purkinje cell degeneration and intracranial calcification.     

Aspects of biochemical differentiation in the central nervous system.

A demonstration of cell-specific patterns of development in the immature CNS is provided by examples of characteristic, cell-specific time-courses of enzyme development in different classes of brain cells isolated in highly purified form by bulk-separation from the cerebral and cerebellar cortex of the growing rat. The enzymatic analysis was carried out at the level of the nerve and glial cell lysosomes and mitochondria, two subcellular organelles crucial to the economy of all cells. The findings reveal rather similar developmental patterns for the lysosomal hydrolase N-acetyl-beta-D-glucosaminidase in neurons and glial cells of the cerebral cortex as well as in two different cerebellar nerve cell types, the Purkinje and the granule cell. However, significant differences in the post-natal chronology of development of the mitochondrial enzyme alpha-glycerophosphate dehydrogenase were noted between cortical nerve and glial cells, the glial enzyme exhibiting 6-fold higher levels of activity than the neuronal one throughout the first month of postnatal life. The findings emphasize the feasibility as well as the necessity of studies aimed at the elucidation of the cell-specific aspects of the biochemistry of developing nerve and glial cells.     

Aging of cerebellar Purkinje cells

Cerebellar Purkinje cells (PCs), the sole output neurons in the cerebellar cortex, play an important role in the cerebellar circuit. PCs appear to be rather sensitive to aging, exhibiting significant changes in both morphology and function during senescence. This article reviews such changes during the normal aging process, including a decrease in the quantity of cells, atrophy in the soma, retraction in the dendritic arborizations, degeneration in the subcellular organelles, a decline in synapse density, disorder in the neurotransmitter system, and alterations in electrophysiological properties. Although these deteriorative changes occur during aging, compensatory mechanisms exist to counteract the impairments in the aging PCs. The possible neural mechanisms underlying these changes and potential preventive treatments are discussed.     

Molecular morphology of neuronal apoptosis: Analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex

We have used the mammalian post-natal cerebellar cortex as a model to dissect out the molecular morphology of neuronal apoptosis in a well-defined population of central neurons: the cerebellar granule cells. By immunocytochemistry, in situ labeling of apoptotic cells, and analysis of cerebellar slices following particle-mediated gene transfer (biolistics), we have studied the relationship of cell death and cleavage of caspase 3, a key molecule in the execution of apoptosis, and monitored caspase 3 activation in living cells. Our results demonstrate the existence of caspase dependent and independent apoptotic pathways affecting the cerebellar granule cells at different stages of their life. Apoptosis of proliferating precursors and young pre-migratory cells occurs in the absence of caspase 3 cleavage, whereas cell death of post-mitotic post-migratory neurons is directly linked to caspase 3 activation. Data obtained from cerebellar cortex can be generalized to outline a more comprehensive picture of the cellular and molecular mechanisms of neuronal death not only in development, but also in a number of pathological conditions leading to neuronal loss.     

Cell reassociation behavior and lectin-induced agglutination of embryonic mouse cells from different brain regions

Cells from dissociated embryonic mouse cerebellum, when plated at high cell density in a 10 μl microwell, assembled into a reproducible three-dimensional pattern of reaggregates containing 2–10000 cells each and interconnecting cables of processes. The details of this pattern were modifiable by coating the culture substratum with poly-d-lysine and by the use of different types of serum in the growth medium. Formation of interconnecting cables was inhibited reversibly by the dimeric lectin Succinyl Concanavalin A (Succ-ConA). Cells from cerebellum, midbrain, medulla and cerebral cortex, cultured separately in microwells, formed distinguishable patterns that appeared to be more region-specific than developmental stage-specific.With lectins as probes of carbohydrate-containing surface macromolecules, cells harvested from different tissue regions had different cell surface properties. In addition the concentration of concanavalin A (ConA) or wheat germ agglutinin (WGA) required for half maximal agglutination was greatly increased for cerebellar cells harvested later than embryonic day 16. These observations suggest both regional and age-specific alterations in cell surface elements required for lectin-induced agglutination during mouse brain development.     

Proliferation of piriform neurons in the rat cerebellar cortex in the pre- and postnatal periods of development]

The method of histoautoradiography with the use of H3-thymidine was applied to the study; there were established the periods of appearance in the cerebellar anlage of albino rats of neuroblasts differentiated into the piriform neurones of the cerebellar cortex; dynamics of the proliferative activity of these cellular elements in the course of the pre- and postnatal periods of development of the experimental animals was investigated. On the basis of the material obtained a conclusion was drawn that the last cell divisions (the result of cell differentiation were Purkinje's cells of the cerebellar cortex) stopped by the 13th--15th day of the embryonic development. No incorporation of the labeled precursor into the DNA of the nuclei of the differentiating piriform neurons occurred later.     

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.     

Besides Purkinje cells and granule neurons: an appraisal of the cell biology of the interneurons of the cerebellar cortex

Ever since the groundbreaking work of Ramon y Cajal, the cerebellar cortex has been recognized as one of the most regularly structured and wired parts of the brain formed by a rather limited set of distinct cells. Its rather protracted course of development, which persists well into postnatal life, the availability of multiple natural mutants, and, more recently, the availability of distinct molecular genetic tools to identify and manipulate discrete cell types have suggested the cerebellar cortex as an excellent model to understand the formation and working of the central nervous system. However, the formulation of a unifying model of cerebellar function has so far proven to be a most cantankerous problem, not least because our understanding of the internal cerebellar cortical circuitry is clearly spotty. Recent research has highlighted the fact that cerebellar cortical interneurons are a quite more diverse and heterogeneous class of cells than generally appreciated, and have provided novel insights into the mechanisms that underpin the development and histogenetic integration of these cells. Here, we provide a short overview of cerebellar cortical interneuron diversity, and we summarize some recent results that are hoped to provide a primer on current understanding of cerebellar biology.     

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea pig, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.