LOCALIZATION OF THE THY-1 ANTIGEN IN THE CEREBELLAR CORTEX OF RAT BRAIN BY IMMUNOFLUORESCENCE DURING POSTNATAL DEVELOPMENT

Abstract— At birth in the rat brain the Thy-1 antigen was present at 10% of the adult level and increased rapidly to reach near adult levels after 3 weeks. Localization studies by immunofluorescence on sections of rat cerebellar cortex during this period showed that at day 5 there was weak fluorescence associated mainly with the molecular layer and some fibre-like structures in the centre of the folium; no fluorescence was found around the cells of external granular layer. From 5 to 16 days there was a rapid increase in Thy-1 immunofluorescence with noticeably higher levels associated with the white matter than the molecular layer. However, by 21 days the reverse was found' with lower levels in white matter than in the molecular layer with a similar distribution to that observed previously in adult rat cerebellum. Small rings and patches of fluorescence were observed in the molecular and granular layers. The results indicated that Thy-1 was present on axons, mature neurons and their processes. In addition, Thy-1 immunofluorescence was found in the pia-arachnoid until around day 16.     

Light and electron microscopical immunocytochemistry of 5'-nucleotidase in rat cerebellum

5'-Nucleotidase in nervous tissue has so far not been localised at the ultrastructural level using immunocytochemical techniques. We have now applied monoclonal antibodies and a polyclonal antiserum raised against this ecto-enzyme and describe the distribution of 5'-nucleotidase antigenicity in rat cerebellum both at the light and electron microscopic levels. Within all cerebellar layers, 5'-nucleotidase immunoreactivity was found on plasma membranes of glial elements, i.e. Bergmann glial cell processes crossing the molecular layer, astrocytic end-feet around blood vessels and glial cell extensions surrounding single Purkinje cells. In the granular layer, 5'-nucleotidase immunoreactivity was present on glial membranes interposed between granule cells. Neuronal cells or processes were devoid of immunoreactivity. The immunocytochemical results were compared with conventional 5'-nucleotidase histochemistry. Both techniques showed the same ecto-localisation of the enzyme and favour the view of 5'-nucleotidase being predominantly situated at glial plasma membranes.     

Immunohistochemical demonstration of serotonin-containing nerve fibers in the cerebellum

Summary The localization of serotonin (5-HT)-immunoreactive nerve fibers in the cerebellum of the rat and cat was investigated by means of the peroxidase-anti-peroxidase (PAP) method using highly specific antibodies to 5-HT.Serotonin-containing nerve fibers were distributed throughout the entire cerebellum including the deep cerebellar nuclei, while 5-HT-positive neuronal somata were not detected in the cerebellum of either species. A different pattern of 5-HT innervation was found among the three layers of the cerebellar cortex. There were also interspecific differences in the pattern of distribution of 5-HT. In the rat, the pool of 5-HT nerve fibers mainly consisted of tangential elements, which were predominant in the molecular layer, while in the cat only a few 5-HT fibers were found in the molecular layer of the cerebellar cortex; dense networks of 5-HT nerve fibers were present in the granular layer. Some differences are evident in the pattern of distribution of 5-HT fibers in cerebellar regions classified on an anatomical and functional basis.This work was supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan     

THE DEVELOPMENT OF D-AMINO ACID OXIDASE IN RAT CEREBELLUM

D-Amino acid oxidase (D-amino acid: O₂ oxidoreductase (deaminating), EC 1.4.3.3; D-AAO) activity is biochemically undetected in rat brain stem, cerebellum and forebrain until 14 days after birth. Adult levels are attained by day 30 in the brain stem, and by day 36 in the cerebellum. At adulthood, forebrain D-AAO activity per g wet weight of tissue is less than 2% that of the cerebellum. In contrast to the pattern in the CNS, substantial D-AAO activity is present in both liver and kidney 2 days before birth and adult levels are approached within 2 weeks of birth. Nonetheless, D-AAO activities in rat liver, kidney, brain stem and cerebellum are likely to be due to a single enzyme which has properties very similar to the purified hog D-AAO. The late ontogenesis of D-AAO activity in cerebellum and brain stem relative to that in liver and kidney parallels reported phylogenetic data. Histochemical staining for D-AAO in rat cerebellar cortex is absent until 15 days after birth when activity is first observed in some cells of the external germinal zone and adjacent molecular layer. These cells appear to migrate to a final destination around the Purkinje cell soma and leave processes at the pial surface. By 21 days of age an adult pattern of staining is manifest throughout the cerebellum but it is of weak intensity. The adult pattern includes some staining in the granular layer which seems to be associated with mossy fibers and certain cerebellar glomeruli, and strong staining at the pial surface, in the molecular layer, and in cells surrounding, but not within, the Purkinje cell soma. The data suggest that the biochemical appearance of D-AAO in developing cerebellum derives from two sources: one associated with differentiation of one of the last cell types to form from the external germinal zone, and the other with maturation of mossy fibers and their synapses (cerebellar glomeruli).     

Expression of FSH and its co-localization with FSH receptor and GnRH receptor in rat cerebellar cortex

The expression of follicle-stimulating hormone (FSH) and its receptor in extrapituitary and non-HPG axis tissues has been demonstrated and their non-reproductive functions in these tissues have been found. However, there have been no reports concerning the expression and function of FSH and its receptor in the cerebellum. In our study, immunofluorescence staining and in situ hybridization were used to detect the expression of FSH, double-labeled immunofluorescence staining was used to detect co-localization of FSH and its receptor and co-localization of FSH and gonadotropin-releasing hormone (GnRH) receptor in the rat cerebellar cortex. Results showed that some cells of the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex showed both FSH immunoreactivity and FSH mRNA positive signals; not only for FSH and FSH receptor, but also for FSH and GnRH receptor co-localized in some cells throughout the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex. These suggested that rat cerebellum could express FSH; cerebellum is a target tissue of FSH; FSH may exert certain functions through FSH receptor in a paracrine or autocrine manner; GnRH may regulate FSH positive cells through GnRH receptor in the cerebellum. Our study provides morphological evidence for further functional research on FSH and related hormones in the cerebellum.     

Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex

Abstract: The monoclonal antibody 4C5 recognizes a neuron-specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age-dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8-day-old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8-day-old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron-neuron interactions.     

Production of an antiserum against cyclic nucleotide phosphodiesterase and its use for the immunocytochemical demonstration of this enzyme in rat cerebellum

A procedure for the separation of cyclic AMP phosphodiesterase from a commercially available preparation and for raising antibodies against this enzyme in rabbits is described. An antiserum thus obtained was used for the immunocytochemical detection of cyclic nucleotide phosphodiesterase in rat cerebellum. The molecular layer, the granular layer and the cerebellar white matter exhibited different degrees of immunoreactivity. Only a few cell bodies (possibly glial cells) were stained. Most of the antigenic sites were present in the neuropil of the molecular layer and around Purkinje cells. Cerebellar glomeruli, sites of synaptic interactions between mossy fibres, Golgi cells and granule cells, were also stained by this antiserum. Control reactions using preimmune serum were consistently negative.     

Neuronal migration independent of glial guidance: light and electron microscopic studies in the cerebellar cortex of neonatal rats

Migrating cells from the external germinal layer of the newborn rat cerebellum were studied by light and electron microscopy. Each migrating cell possessed a single, broad, leading process oriented perpendicular to the pial surface. These cell processes were sometimes associated with profiles of other granule cells, but were not necessarily associated with the electron-lucent Bergmann fibers present at these early developmental stages. Migrating granule cells could be observed circumventing both blood vessels and the perikarya/processes of other cells present in the developing molecular layer. Thus, during the early stages of cerebellar ontogeny, when the migration pathway through the molecular layer is sparsely populated with cells and processes, the vertical process of a granule cell may seek actively a path of least resistance, utilizing 'contacts' with surrounding objects for avoidance, rather than as guideposts imperative for directing migration. Cellular associations observed at this stage of cerebellar development may thus be more fortuitous than requisite.     

The topographical localization of acetylcholinesterase in the adult rat cerebellum: A reappraisal

Summary The adult rat cerebellum has been investigated histochemically for acetylcholinesterase activity by the direct-coloring thiocholine technique. Results obtained are as follows:Staining indicative of sites of acetylcholinesterase (AChe) activity are predominantly delimited to the granular layer of the cerebellar cortex. The white matter directly adjacent to the granular layer of any lobule exhibit stronger activity than the medullary core. Often the molecular layer stain, diffusely and weakly.A great proportion of the enzyme staining is attributed to afferent mossy terminals. Golgi cells are also considered to possess intracellular AChe.Topographically, the vermian lobules of the cerebellum stain stronger relative to the hemispherical lobules except for the flocculus and paraflocculus. Mediolateral gradation of activity if present is not convincing. In the vermis, the lingula stains moderately. A distinctive feature of the present study is the enzyme activity in sub-lobuli VIb and VIc of the declive and the anterior portion of lobule VII. These areas stain densely and strongly. The nodule and lower part of the uvula exhibit dense and intense staining for AChe. All other lobules of the cerebellum stain weakly, more so those of the anterior lobe (besides the lingula).A rich core of AChe staining fibres radiate from the white matter adjacent to lobule X to reach lobules I, II, III, IV and V. A similarly intense core of AChe is found subjacent to the granular layer of lobules VI and VII.The three pairs of cerebellar peduncles stain differentially. Staining in the intracerebellar nuclei are however uniformly weak.The present findings are discussed as they relate to previous studies and in the light of current thoughts in cerebellar anatomy and function. Attention is drawn particularly to the functional implication of the dense and strong enzyme activity herein reported for the declive-tuber vermis complex of the cerebellum as these areas are believed to be sites of termination of cerebellar teleceptive inputs.     

Meningeal cells are involved in foliation, lamination, and neurogenesis of the cerebellum: evidence from 6-hydroxydopamine-induced destruction of meningeal cells

In the present paper we report on experiments conducted to find out if there is a positive correlation between the destruction of meningeal cells over the newborn rat cerebellum by 6-hydroxydopamine (6-OHDA) and the subsequent development of abnormalities in cerebellar fissuration, lamination, and granule cell number. Both destruction of meningeal cells and quality and magnitude of 6-OHDA-induced cerebellar defects show the same threshold sensitivity without further dose responsiveness. Blockade of neuronal uptake 1 for catecholamines with nomifensine prevents neither destruction of meningeal cells nor the development of abnormalities in cerebellar structure after 6-OHDA treatment. Blockade of extraneuronal uptake 2 for catecholamines with normetanephrine prevents both destruction of meningeal cells and the development of typical cerebellar abnormalities after 6-OHDA treatment. All three parallel experiments suggest that there is a positive correlation between the destruction of meningeal cells and the development of abnormal cerebellar structure, indicating that meningeal cells are involved in these defective morphogenetic processes, i.e., fissuration, lamination, and cell proliferation in the external granular layer. The preferential localization of defects in cerebellar fissures indicates that, in analogy to the mesenchyme surrounding other epithelia with a branching morphogenesis, the role of meningeal cells could be the production of interstitial collagen which is necessary to stabilize the epithelial basal lamina in the fissures.     

Secretogranin II and its Derivative Peptide,Manserin, are Differentially Localized in Purkinje Cells and Unipolar Brush Cells in the Rat Cerebellum

The cerebellum has long been recognized as the primary center of motor coordination in the central nervous system. Cerebellar neuropeptides have been postulated to be involved in such motor coordination, though this role is not fully understood. We herein investigated the localization of novel neuropeptide, “manserin” in the adult rat cerebellum. Punctate signals of manserin immunoreactivity were observed in the granular layer of the rat cerebellum. Manserin signals were also observed in the fibers and fiber terminals in the granular layer as well as the molecular layer. Manserin did not localize in Purkinje cells. Interestingly, cerebellar manserin was preferentially colocalized with unipolar brush cells, a class of excitatory granular layer interneuron, which are known to be involved in vestibullocerebellar functions. These results indicate that manserin plays pivotal roles in the cerebellar functions.     

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.     

Production of an antiserum against cyclic nucleotide phosphodiesterase and its use for the immunocytochemical demonstration of this enzyme in rat cerebellum

Summary A procedure for the separation of cyclic AMP phosphodiesterase from a commercially available preparation and for raising antibodies against this enzyme in rabbits is described. An antiserum thus obtained was used for the immunocytochemical detection of cyclic nucleotide phosphodiesterase in rat cerebellum. The molecular layer, the granular layer and the cerebellar white matter exhibited different degrees of immunoreactivity. Only a few cell bodies (possibly glial cells) were stained. Most of the antigenic sites were present in the neuropil of the molecular layer and around Purkinje cells. Cerebellar glomeruli, sites of synaptic interactions between mossy fibres, Golgi cells and granule cells, were also stained by this antiserum. Control reactions using preimmune serum were consistently negative.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday.     

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO₂. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.     

Ontogenetic development of the brain of the platyhelminth Fasciola hepatica

During ontogenetic development in the definitive host, the cerebral ganglia of the parasitic flatworm Fasciola hepatica lose their cell rind integrity and develop specialized nerve processes. The organization and cytological features of the central nervous system were examined during three developmental stages in the parasitic life cycle of F. hepatica to determine when the changes occur. The cerebral ganglion cell bodies of migrating juvenile worms (5 days post-infection) are organized into a one-cell-thick rind that surrounds a central neuropile composed of small unmyelinated nerve processes (less than 3 microns in diameter). In young, sexually-immature adult worms (30 days post-infection), the cell bodies of the ganglia are no longer organized into a complete or tight cell rind around the ganglia. In addition, large diameter ('giant') unmyelinated nerve processes (greater than 12 microns) are found in the neuropile area. These giant nerve processes are also found in the transverse commissure and the longitudinal nerve cords. In mature adult worms (4-6 months post-infection), the rind of nerve cell bodies has completely disappeared and cell bodies are scattered around and within the neuropile. More than half of the volume of the mature adult neuropile is composed of giant nerve processes. The three developmental stages of the parasite that were used in this study differ significantly in their sizes, behaviours and microhabitat locations in the host. The results suggest that the organizational and morphological changes in the ganglia reflect selective adaptations to changes in the parasitic microenvironment.     

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。     

pp60c-src in the developing cerebellum.

pp60c-src was localized in the cerebellum of developing chicken embryos by immunoperoxidase staining with antisera raised against bacterially expressed pp60v-src. Immunoreactivity (IR) appeared in the cerebellum of the chicken embryos at the time of neuronal differentiation. pp60c-src IR was detected in regions of the developing cerebellum where processes of developing neurons and glia are located. In the early embryo (stage 17), pp60c-src IR was localized in the marginal zone of the cerebellar plate. By stage 40, pp60c-src IR was localized in the process-rich molecular layer of the cerebellum and between the cells of the developing internal granular layer. Cell bodies of cerebellar neurons did not show pp60c-src IR at any stage of development. Mitotically active neuroepithelial cells of the metencephalon did not express pp60c-src before the onset of differentiation in the early embryo, nor did proliferating cells of the external granular layer express pp60c-src at later stages. Although it is not possible to ascertain whether pp60c-src is localized in developing neurons or glia at the light microscope level, the time of its appearance and pattern of distribution in the molecular layer is suggestive of a localization within the developing neuronal processes which compose the bulk of this layer. Biochemical analyses of pp60c-src in the developing cerebellum by the immune complex protein kinase activity and sensitivity of the kinase to inhibition by P1,P4-di(adenosine-5')tetraphosphate confirmed that the expression of pp60c-src coincided with the time of neuronal differentiation. We conclude from these results that in the central nervous systems, pp60c-src may be more important in an aspect of cell differentiation or a mature neuronal function than in the proliferation of neuronal or glial precursors.     

Alterations in Actin-Binding β-Thymosin Expression Accompany Neuronal Differentiation and Migration in Rat Cerebellum

The β₄-and β₁₀-thymosins, recently identified as actin monomer-sequestering proteins, are developmentally regulated in brain. Using specific mRNA and protein probes, we have used in situ hybridization and immunohis-tochemical techniques to investigate the distribution of the β-thymosin mRNAs and their proteins in developing rat cerebellum. Early in postnatal development, both β-thymosin mRNAs were expressed at highest levels in the postmitotic, premigratory granule cells of the external granular layer; expression diminished as granule cells migrated to and differentiated within the developing internal granular layer. In addition, both β-thymosin proteins were present in bundles of cerebellar afferent fibers in the white matter at this time. Throughout the maturation period, both proteins were present in elongating parallel fibers in the upper portion of the molecular layer. Later in cerebellar development, thymosin β₄, but not thymosin β₁₀, was expressed in Golgi epithelial cells and Bergmann processes. Thymosin β₄ was expressed in a small population of cells with microglial morphology scattered throughout the gray and white matter. Thymosin β₁₀ was detected in an even smaller population of glia. Expression of thymosin β₄ and thymosin β₁₀ in premigratory granule cells and in growing neuronal processes is consistent with the possibility that both β-thymosins are involved in the dynamics of actin polymerization during migration and process extension of neurons.     

Developmental Expression of HNK-1-Reactive Antigens in the Rat Cerebellum and Localization of Sulfoglucuronyl Glycolipids in Molecular Layer and Deep Cerebellar Nuclei

Monoclonal antibody HNK-1-reactive carbohydrate epitope is expressed on proteins, proteoglycans, and sulfoglucuronyl glycolipids (SGGLs). The developmental expression of these HNK-1-reactive antigens was studied in rat cerebellum. The expression of sulfoglucuronyl lacto-N-neotetraosylceramide (SGGL-1) was biphasic with an initial maximum at postnatal day one (PD 1), followed by a second rise in the level at PD 20. The level of sulfoglucuronyl lacto-N-norhexaosyl ceramide (SGGL-2) in cerebellum was low until PD 15 and then increased to a plateau at PD 20. The levels of SGGLs increased during postnatal development of the cerebellum, contrary to their diminishing expression in the cerebral cortex. The expression of HNK-1-reactive glycoproteins decreased with development of the rat cerebellum from PD 1. Several HNK-1-reactive glycoproteins with apparent molecular masses between 150 and 325 kDa were visualized between PD 1 and PD 10. However, beyond PD 10, only two HNK-1-reactive bands at 160 and 180 kDa remained. The latter appeared to be neural cell adhesion molecule, N-CAM-180. A diffuse HNK-1-reactive band seen at the top of polyacrylamide electrophoretic gels was due mostly to proteoglycans. This band increased in its reactivity to HNK-1 between PD 15 and PD 25 and then decreased in the adult cerebellum. The lipid antigens were shown by two complementary methodologies to be localized primarily in the molecular layer and deep cerebellar nuclei as opposed to the granular layer and white matter. A fixation procedure which eliminates HNK-1-reactive epitope on glycoproteins and proteoglycans, but does not affect glycolipids, allowed selective immunoreactivity in the molecular layer and deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)