Zonal distribution of Purkinje cells in the zebrafish cerebellum: analysis by means of a specific monoclonal antibody

We have isolated a monoclonal antibody that recognizes a 42-kDa protein from adult zebrafish brain. The antibody stains the typical drop-shaped perikaryon of Purkinje cells and their dendrites. The cerebellum of teleosts has complex features. It is composed of three parts; the valvula cerebelli (Va), the corpus cerebelli (CCe), and the crista cerebellaris (CC). In higher vertebrates, the molecular layer is always found as the most outer layer of the cerebellum, but in teleosts, some of the granular cells are located on the surface of the Va. In higher vertebrates, the boundary between the granular and molecular layers always contains Purkinje cells, but this does not occur in teleosts. The Purkinje cells are found only in a part of the boundary in Va. We have found that the layer containing Purkinje cells forms a continuous zone in the cerebellum in the zebrafish. The complex structure of the cerebellum is more easily understood with the aid of the concept of a "Purkinje zone". The Purkinje zone starts at the caudal end of Val (lateral division of Va), turns at the edge of Va toward Vam (medial division of Va), connects to CCe, and ends at the bottom of CCe. The dendrites are found only on one side of the zone. The dendrites of the Purkinje cells in Vam are planar and are packed regularly, similar to those of higher vertebrates. However, the dendrites in Val and the posterior part of CCe are not planar and are irregularly packed.     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.     

Immunohistochemical Localization of α-Mannosidase During Postnatal Development of the Rat Cerebellum

The localization of alpha-D-mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At bith an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.     

CEREBELLAR GRANULE CELLS IN VITRO : A Light and Electron Microscope Study

The behavior of granule cells in mature cerebellar cultures derived from newborn mice was studied by light and electron microscopy. Many granule cells remained in the explants as an external granular layer. These cells were differentiated, as evidenced by formation of bundles of parallel fibers and by development of synapses between granule cell axons and Purkinje cell branchlet spines, and between Golgi cell axons and granule cell dendrites. Although the over-all architecture of the cerebellar explants after 18–33 days in vitro was similar to that of the newborn mouse, the evident differentiation of the granule cells suggested that interneuronal relationships resemble those of the mature cerebellum in vivo.     

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.     

生长休止蛋白7在大鼠小脑皮质中的免疫细胞化学研究

目的探讨生长休止特定蛋白7(Gas7)在大鼠小脑中的表达定位。方法应用Gas7抗血清,对大鼠小脑组织切片进行免疫组织化学染色。结果在小脑皮质分子层可见大量的Gas7阳性神经纤维;蒲氏细胞层中,Gas7主要表达在神经元胞膜和部分胞质处;颗粒层中可见Gas7阳性神经纤维。结论Gas7主要在小脑神经元的定位特征可能与Gas7促进神经元和神经突起发育的调节功能有关。     

Species variation in the localisation of esterases in the cerebellar cortex of mouse and bat

A comparative study of the distribution of a simple esterase and acetylcholinesterase in the cerebellar cortex of mouse and bat has been made. The Purkinje layer is intensely positive for simple esterase in both species. The granular and molecular layers showed mild to moderate activity in mouse and intense activity in bat. Acetylcholinesterase in cerebellar layers of bat is more intense than in mouse. In bat cerebellum, acetylcholinesterase is observed in the dendrites of Purkinje cells, but not in their cell bodies. Acetylcholinesterase was not found in Purkinje cells of mouse.     

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.     

Distribution of Kiaa0319-like immunoreactivity in the adult mouse brain--a novel protein encoded by the putative dyslexia susceptibility gene KIAA0319-like

Kiaa0319L is a novel protein encoded by a recently discovered gene KIAA0319-like(L) that may be associated with reading disability. Little is known about the characteristics of this protein and its distribution in the brain. We investigated here expression of this protein in adult mice, using an antibody specific for human and rodent Kiaa0319L. In the brain, Kiaa0319L was localized strongly in the olfactory bulb, and strong expression was found in other regions, including hippocampus, cerebellum, diencephalon and the cerebral cortex. Immunohistochemistry confirmed expression in these brain regions, and showed further that the protein was expressed preferentially in neurons in layer IV and VI of the neocortex, CA1 and CA2 subfields of the hippocampus and a subpopulation of neurons in CA3 and dentate gyrus. Furthermore, the protein was confined to dendrites of CA1 neurons in the stratum radiatum, but not those in the stratum oriens, and in astrocytes within the hippocampus. In the cerebellum, the protein was observed in the molecular layer and a fraction of Purkinje neurons. These findings confirmed expression of Kiaa0319L in brain regions that are involved in reading performance, supporting its possible involvement in reading disability. The specific patterns of localization in the neocortex, hippocampus and cerebellum suggest further that this protein may be related to other biological processes in a subpopulation of neurons within these regions, eg. formation and maintenance of polarity in the neuron.     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.     

Transient accumulations of glycosaminoglycans in rat and chicken cerebellar cortex during development

Modifications of glycosaminoglycans at neuropile of rat and chicken cerebellum during development were histochemically studied. The application of Alcian Blue staining techniques and enzymatic degradations permitted to reveal in both species that in earlier stages of cerebellar development hyaluronic acid is present throughout neuropile of entire cerebellum but it accumulated preferentially at the medullary region and around precursory Purkinje cells where it showed a mucoid-like appearance. This substance was related with cell migration and aligning processes. At the middle of cerebellar development, around 2nd postnatal week in rat and 12-16 embryonary days in chick, a new polyanionic transient accumulation, presumably chondroitinsulphate, became present at the medullary region following the longitudinal axis of folium and limiting the forming granular layer, being this substance mainly related with polarity processes by controlling or guiding the growing cones of afferent fibers, which enter massively to cerebellar cortex. It disappeared as myelination progressed. Also from the middle stage of development onward, beside glycosaminoglycans, other polyanionic substances were present at the molecular and granular layer neuropile and at the cytoplasm of some nerve cells. These macromolecules were rather related with nerve cell differentiation and maturation.     

An immunohistochemical analysis of rat hippocampal antigens in early postnatal ontogeny by using monoclonal antibodies]

In order to study the molecular mechanisms of neurogenesis, monoclonal antibodies (MAbs) were produced against antigens of the developing rat hippocampus. MAb 3G7-F8 was used for immunohistochemical localization of the corresponding antigen of paraffin sections of the rat brain at days 0, 5, 14, and 21 of the postnatal development. In the hippocampus of newborn and 5-day-old rats, positive immunostaining was observed in the cytoplasm and proximal segments of processes of neurons located in granular, polymorph, and pyramidal layers, as well as in entorhinal cortex. In granule cell bodies and neurons of entorhinal cortex specific staining decreased by day 14 and disappeared by day 21 after birth, whereas neurons of pyramidal and polymorph layers remained immunopositive. Diffuse specific staining in the cerebellum was observed beginning from day 5 after birth in the Purkinje cell layer. On days 14-21 positive reaction was observed in Purkinje cell bodies and in the layer containing dendrites of Purkinje cells and parallel fibers. External and internal granular layers remained immunonegative. No specific staining was observed in other regions of the brain, as well as in the control slices. These data suggest that the antigen detected by the 3G7-F8 antibody is involved in the formation of the neuronal connections.     

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.     

Neurotransmitters, neuropeptides and calcium binding proteins in developing human cerebellum: a review

Many endogenous neurochemicals that are known to have important functions in the mature central nervous system have also been found in the developing human cerebellum. Cholinergic neurons, as revealed by immunoreactivities towards choline acetyltransferase or acetylcholinesterase, appear early at 23 weeks of gestation in the cerebellar cortex and deep nuclei. Immunoreactivities gradually increase until the first postnatal month. Enkephalin is localized in the developing cerebellum, initially in the fibers of the cortex and deep nuclei at 16–20 weeks and then also in the Purkinje cells, granule cells, basket cells and Golgi cells at 23 weeks onward. Another neuropeptide, substance P, is localized mainly in the fibers of the dentate nucleus from 9 to 24 weeks but substance P immunoreactivity declines thereafter. GABA, an inhibitory neurotransmitter of the central nervous system, starts to appear at 16 weeks in the Purkinje cells, stellate cells, basket cells, mossy fibers and neurons of deep nuclei. GABA expression is gradually upregulated toward term forming networks of GABA-positive fibers and neurons. Catecholaminergic fibers and neurons are also detected in the cortex and deep nuclei at as early as 16 weeks. Calcium binding proteins, calbindin D28K and parvalbumin, make their first appearance in the cortex and deep nuclei at 14 weeks and then their expression decreases toward term, while calretinin appears later at 21 weeks but its expression increases with fetal age. The above findings suggest that many neurotransmitters, neuropeptides and calcium binding proteins (1) appear early during development of the cerebellum; (2) have specific temporal and spatial expression patterns; (3) may have functions other than those found in the mature neural systems; and (4) may be able to interact with each other during early development.     

Distribution and developmental expression of lamin-like immunoreactivity in the central nervous system.

Lamins are the major proteic constituents of the nuclear lamina, the innermost layer of the nuclear membrane. The immunolocalization of lamins in the rat central nervous system was studied using polyclonal antibodies. Besides an ubiquitarious localization in the nuclear membranes of neurons and glial cells, an intense lamin-like immunoreactivity was found in the soma and dendrites of cerebellar Purkinje cells. The same specific reaction was also observed in the human cerebellum. Experiments performed in newborn animals demonstrated that the cytoplasmic expression of lamins in Purkinje cells begins during postnatal development.     

Comparative aspects of cerebellar cytoarchitecture in the European eel life cycle

The major anatomical divisions of the cerebellum of the European eel, i.e., corpus cerebelli, lobus vestibulolateralis, and valvula, were studied morphologically and morphometrically. There were differences in cerebellar cytoarchitecture and gross morphology in two stages of the eel life cycle, the trophic stage (yellow eel), and the reproductive stage (silver eel), which are characterized by different degrees of swimming activity. The principal differences between silver and yellow eels in the cytoarchitecture of the corpus cerebelli and the lobus vestibulolateralis were in distribution of Purkinje or Purkinje-like cells in the molecular layer, which is wider in silver eels, in part because of a decreased thickness of the granular cell layer. In the silver eel, the scattering of Purkinje cells was more evident in the lobus vestibulolateralis where the molecular layer is also thicker. The data indicate the transition from the yellow eel to the silver eel is characterized by a migration of granule cells from the ganglionic cell layer to the internal granular layer and by a further development of molecular layer components, e.g., parallel fibers, Purkinje-cell dendrites, etc. In contrast, the thickness of the granular layer and of the Purkinje cell layer, limited to the lower part of the valvula, decreased. There is also a slight increase of cerebellar volume in the silver eel. The volume of the lobus vestibulolateralis was constant. Hypertrophy of the valvula and eminentiae granulares is observed and is due to the migration of cells from the granular layer of the corpus cerebelli whose volume slightly decreases. Perhaps the lobus vestibulolateralis also contributes to the increased volume of eminentiae granulares. Our findings suggest that the cerebellum continues to develop during the passage from the trophic to the reproductive stage of the eel. The appearance of new afferents from the lateral line which becomes more visible in the silver eel probably completes cerebellar ontogeny.     

Differential neuronal loss following early postnatal alcohol exposure

Neonatal rats were exposed to 6.6 g/kg of alcohol each day between postnatal days 4 and 10 while artificial-rearing procedures were used, in a manner which produced high peak and low trough blood alcohol concentrations each day. Gastrostomy controls were reared artificially with maltose/dextrin isocalorically substituted for alcohol in the milk formula, and suckle controls were reared normally by dams. The pups were sacrificed on day 10 and tissue sections (2 microns thick) were obtained in the sagittal plane through the cerebellum and in the horizontal plane through the hippocampal formation. Overall area measures were obtained for the hippocampus proper, area dentata, and cerebellum, along with areas of the cell layers of these regions. In the hippocampal formation, cell counts were made of the pyramidal cells of the hippocampus proper, the multiple cell types of the hilus, and the granule cells of the area dentata. In the cerebellum, cell counts of Purkinje cells, granule cells of the granular layer, granule cells of the external granular layer, and mitotic cells of the external granular layer were obtained from lobules I, V, VII, VIII, and IX. Alcohol selectively reduced areas and neuronal numbers in the cerebellum but had no significant effects on neuronal numbers in the hippocampal formation. Purkinje cells exhibited the greatest percent reductions, and cerebellar granule cells were significantly reduced in the granular layer but not in the external granular layer. All lobules showed these effects, but lobule I was significantly more affected than the other four lobules that were analyzed. The results demonstrate the differential vulnerability of selected neuronal populations to the developmental toxicity of alcohol exposure during the brain growth spurt.     

Immunological studies on the Purkinje cells from rat and mouse cerebella. II. Immunohistochemical specificity of the antiserum to purkinje cells.

An antiserum raised against an enriched preparation of isolated rat cerebellar Purkinje cells has been studied with the indirect immunofluorescence technique to establish its specificity and localisation. On cryostat sections, the unabsorbed IgG fraction stained large and small neurons in all brain regions. This staining was greatly reduced in the forebrain after the serum was absorbed on heart and liver membranes, and abolished after additional absorption on cerebral membranes. In the cerebellum, these absorptions also removed background staining in the internal granular layer, while the perikarya and dendrites of the Purkinje cells remained positive. Large neurons in the deep cerebellar nuclei and the brain stem were also stained, but further absorption on membranes prepared from the brain stem removed staining in both these areas without affecting that of the Purkinje cells. Thus, using immunohistochemical screening, it was possible through a series of absorptions to obtain a serum that is specific to cerebellar Purkinje cells.