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.     

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。     

Immunolocalization of the acid-sensing ion channel 2a in the rat cerebellum

The acid-sensing ion channels (ASICs) are members of the DEG/ENaC superfamily of Na+ channels. Acid-gated cation currents have been detected in neurons from multiple regions of the brain including the cerebellum, but little is known about their molecular identity and function. Recently, one of ASICs (ASIC1a) was implicated in synaptic plasticity. In this study we examined the subcellular distribution of ASIC2a in rat cerebellum by immunostaining and confocal microscopy. Monoclonal antibodies for labeling of defined brain structures, for example, astroglia, Purkinje cell dendrites, nuclei, and presynaptic terminals were used for colocalization analyses. In the gray matter, the anti-ASIC2a antibody intensively stained dendrite branches of Purkinje cells evenly distributed throughout the entire molecular layer (ML). In the granule cell layer (GL), anti-ASIC2a antibody stained synaptic glomeruli. Neuronal localization of ASIC2a was confirmed by lack of co-staining with glial fibrillary acidic protein. Anti-ASIC2a staining in the ML colocalized with metabotropic glutamate receptor 1alpha (mGluR1alpha) in Purkinje cell dendrites and dendritic spines. Both proteins, mGluR1alpha and ASIC2a, were enriched in a crude synaptic membrane fraction prepared from cerebellum, suggesting synaptic expression of these proteins. Dual staining with anti-syntaxin 1A and anti-ASIC2a antibodies demonstrates characteristic complementary distribution of two proteins in both ML and GL. Because syntaxin 1A localized in presynaptic membranes and synaptic vesicles, complementary distribution with ASIC2a suggests postsynaptic localization of ASIC2a in these structures. This study shows specific localization of ASIC2a in both Purkinje and granule cell dendrites of the cerebellum and enrichment of ASIC2a in a crude cerebellar synaptic membrane fraction. The study is the first report of synaptic localization of ASIC2a in the CNS. The synaptic localization of ASIC2a in the cerebellum makes this channel a candidate for a role in motor coordination and learning.     

ATM, the Mre11/Rad50/Nbs1 complex, and topoisomerase I are concentrated in the nucleus of Purkinje neurons in the juvenile human brain

The genetic disease ataxia telangiectasia (AT) results from mutations in the ataxia telangiectasia mutated (ATM) gene. AT patients develop a progressive degeneration of cerebellar Purkinje neurons. Surprisingly, while ATM plays a criticial role in the cellular reponse to DNA damage, previous studies have localized ATM to the cytoplasm of rodent and human Purkinje neurons. Here we show that ATM is primarily localized to the nucleus in cerebellar Purkinje neurons in postmortem human brain tissue samples, although some light cytoplasmic ATM staining was also observed. No ATM staining was observed in brain tissue samples from AT patients, verifying the specificity of the antibody. We also found that antibodies against components of the Mre11/Rad50/Nbs1 (MRN) complex showed strong staining in Purkinje cell nuclei. However, while ATM is present in both the nucleoplasm and nucleolus, MRN proteins are excluded from the nucleolus. We also observed very high levels of topoisomerase 1 (TOP1) in the nucleus, and specifically the nucleolus, of human Purkinje neurons. Our results have direct implications for understanding the mechanisms of neurodegeneration in AT and AT-like disorder.     

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.     

Localization of Hexokinase in Neural Tissue: Electron Microscopic Studies of Rat Cerebellar Cortex

: The distribution of hexokinase (ATP:d -hexose 6-phosphotransferase, EC 2.7.1.1) in the rat cerebellar cortex has been studied at the electron microscopic level using the peroxidase-antiperoxidase procedure. Extensive staining of cytoplasmic regions, with some increased staining at mitochondrial profiles, was seen in the cell bodies of both neurons (basket, stellate, Lugaro, Golgi, and granule cells) and astrocytes. Oligodendrocytes showed little or no detectable staining. Purkinje cell perikarya were much less intensely stained than were the perikarya of other neurons. The initial portion of the Purkinje dendrite was, like the perikaryon from which it emerged, lightly stained. More intense staining was seen in the secondary and tertiary branches of the Purkinje dendrite, but the terminal branches were devoid of stain. Granule cell dendrites were well stained in their initial portions but devoid of stain in their terminal dendritic digits which form part of the cerebellar glomeruli. In contrast to the unstained granule cell dendritic digits, the central mossy fiber nerve terminal of the glomerulus exhibited intense staining of the mitochondrial profiles and of synaptic vesicles adjacent to the mitochondria. Axons of basket cells showed intense staining in the segments adjacent to the Purkinje cell soma, while terminal twigs of the basket axons in the pinceau surrounding the (unstained) initial segment of the Purkinje axon showed markedly decreased staining intensity. These results indicate that there may be substantial variation in hexokinase levels between the various regions of neuronal processes. Hexokinase was seen at both cytoplasmic and mitochondrial locations in a variety of cells. It does not appear likely that location of hexokinase can be directly correlated with cell type, i.e., with neurons versus glia.     

Actin as a cytoskeletal basis for cell architecture and a protein essential for ecdysis in Prorocentrum minimum (Dinophyceae,Prorocentrales)

The specific cell architecture of prorocentroid dinoflagellates is reflected in the internal cell structure, particularly, in cytoskeleton organization. Cytoskeleton arrangement in a Prorocentrum minimum cell was investigated using fluorescent labeling approaches, electron‐microscopy and immunocytochemical methods. The absence of cortical microtubules was confirmed. Phalloidin – tetramethylrhodamine isothiocyanate conjugate staining demonstrated that F‐actin forms a dense layer in the cortical region of the cell; besides, it was detected in the ‘archoplasmic sphere’ adjacent to the nucleus. In some cells the rest of the cytoplasm and the nucleus were also slightly stained. In dividing cells, F‐actin was mainly distributed in the cortical region and in the cleavage furrow. Fluorescent deoxyribonuclease I staining demonstrated more evenly distributed cytoplasmic non‐polymerized actin; the basis of the nuclear actin pool is monomeric actin. It concentrates in the nucleoplasm and forms a meshwork around chromosomes. The significant amount of G‐actin is apparently localized in the P. minimum nucleolus. Assumed involvement of F‐actin in the process of stress‐induced ecdysis – cell cover shedding – was examined. A sharp decrease in the level of ecdysis was observed after treatment with actin‐depolymerizing agent latrunculin B. The fluorescent staining of treated cells demonstrated disturbance of the actin cytoskeleton and disappearance of the cortical F‐actin layer. Our results support the recent data on the actin involvement in fundamental nuclear processes: cytoplasmic F‐actin appears to participate in cell shape determination, cell cover rearrangement and development. Actin may play a substitute role in the absence of cortical microtubules, representing the cytoskeletal basis of P. minimum cell architecture.     

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.     

Immunoelectron microscopy of fodrin in the rat uriniferous and collecting tubular epithelium

We examined the localization of fodrin in epithelial cells of rat uriniferous and collecting tubules by immunofluorescence and immunoelectron microscopy of frozen sections. In the uriniferous tubule, fodrin was found along the cell membrane and in the well-developed terminal web, as previously reported in other epithelial cells: in the terminal web and along the basolateral cell membrane in the proximal tubule; all around the cell surface in the thin limb of Henle; along the basolateral surface in the thick limb of Henle's thick segment and the distal tubule. In the intercalated cells of the collecting tubule, fodrin was found not only along the basolateral cell membrane but also in the apical cytoplasm. The most peculiar labeling was obtained in the principal cells of the collecting tubule. In addition to labeling in the basolateral cell membrane, fodrin was found diffusely in the cytoplasmic matrix. Association of fodrin with any particular structure could not be identified, but the Golgi area was apparently free of labeling. Cytoplasmic labeling was more conspicuous in the principal cells of the medulla than in those of the cortex. The present results show that fodrin need not always exist in association with the cell membrane or the cytoskeleton but can occur in the cytoplasmic matrix, at least in epithelial cells. We discuss the possible physiological significance of the latter distribution.     

Immunocytochemical Characterization of Glutamate Dehydrogenase in the Cerebellum of the Rat

The immunocytochemical distribution of glutamate dehydrogenase was studied in the cerebellum of the rat using antibodies made in rabbit and guinea pig against antigen purified from bovine liver. Antiserum was found to block partially enzymatic activity both of the purified enzyme and of extracts of the rat cerebellum. Using immunoblots of proteins of rat cerebellum, a major immunoreactive protein and several minor immunoreactive proteins were detected with antiserum. Only a single immunoreactive protein was detected using affinity-purified antibody preparations. This protein migrates with a molecular weight identical to that of the subunit of glutamate dehydrogenase. Further evidence that the antibodies were selective for glutamate dehydrogenase in rat cerebellum was obtained through peptide mapping. Purified glutamate dehydrogenase and the immunoreactive protein from rat cerebellum generated similar patterns of immunoreactive peptides. No significant cross-reaction was observed with glutamine synthetase. Immunocytochemistry was done on cryostat- and Vibratome-cut sections of the cerebellum of rats that had been perfused with cold 4% paraformaldehyde. Glial cells were found to be the most immunoreactive structures throughout the cerebellum. Most apparent was the intense labeling of Bergmann glial cell bodies and fibers. In the granule cell layer, heavy labeling of astrocytes was seen. Purkinje and granule cell bodies were only lightly immunoreactive, whereas stellate, basket, and Golgi cells were unlabeled. Labeling of presynaptic terminals was not apparent. These findings suggest that glutamate dehydrogenase, like glutamine synthetase, is enriched in glia relative to neurons.     

Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.     

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.     

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.     

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Muller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans.     

Alpha-fodrin in the adrenal gland: localization by immunoelectron microscopy

Localization of fodrin, the brain equivalent of spectrin (a protein constituent of the erythrocyte membrane cytoskeleton), was investigated at the ultrastructural level in rat adrenal gland. By use of an affinity purified antibody directed against the alpha-fodrin subunit, all chromaffin cells, cortical cells, nerve fibers, and their surrounding Schwann cells were found to be labeled close to the cytoplasmic side of their plasma membranes. The labeling appeared more intense for chromaffin cells, and secretory granules and mitochondria were frequently found to be associated with the zone containing alpha-fodrin in these cells. The immunostained zone was estimated to extend 230 +/- 70 nm into the cytoplasm. This localization is discussed in terms of what is known of the properties of spectrin, and possible roles of the molecule in the chromaffin cell are suggested.     

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.     

MIT-23: A mitochondrial marker for terminal neuronal differentiation defined by a monoclonal antibody

We describe a monoclonal antibody directed against a neuron-specific mitochondrial protein from rat brain. On protein blots the antibody recognizes a single polypeptide of apparent molecular weight 23,000. By solid-phase immunoassay the antigen was detected in all brain regions tested but was not detected in nonneural tissues. Within neurons, the antibody stains cytoplasmic granules that immunoelectron microscopy shows are mitochondria, hence the designation MIT-23. Immunocytochemical staining of the cerebellar cortex showed that MIT-23 occurs in all the neuronal types but is absent from glial and other nonneuronal cells. During neonatal development of the cerebellum, MIT-23 appears in neurons after their final cell division or migration is completed, suggesting that specific proteins associated with mitochondria participate in neuronal maturation.     

Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons.

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.     

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.