Mutually exclusive expression of fibronectin and glial fibrillary acidic protein in cultured brain cells

The reported expression of the cell surface-associated, mainly mesenchymal glycoprotein fibronectin by cultured glial cells is in discrepancy with recent work on brain tissue failing to demonstrate any glial or neuronal fibronectin. We have investigated the expression of fibronectin in relation to glial fibrillary acidic protein in cultured human glial and glioma cell lines as well as in cultures derived from newborn rat brain. Using double immunofluorescence technique we found that cells containing glial fibrillary acidic protein do not express fibronectin, and vice versa. The only exception to this rule was the occasional finding of fibronectin at points of cell-to-cell adhesion also in relation to cells containing glial fibrillary acidic protein. The results were also tested by polyacrylamide gel electrophoresis of the culture media of the human cell lines, and by subcultures from the brain of newborn rat, cultures stimulated with dibutyryl cyclic AMP (db-cAMP), and by vinblastine treatment of the cells. The lack of expression of fibronectin in cells containing glial fibrillary acidic protein, a gliospecific cytoskeletal protein, is discussed with reference to glio-mesenchymal interactions and glial markers in vitro.     

Udp-galactose: glycoprotein galactosyltransferase activity in a clonal line of rat brain.

1. UDPgalactose:glycoprotein galactosyltransferase (EC 2.4.1.-) activity was demonstrated in homogenates from whole rat brain, isolated neuromal perikarya, enriched glial cell fractions, and cultured rat glial tumor cells (clone C6). 2. Galactosyltransferase activity was enriched 3-9-fold in neuronal perikarya and 1.4--1.8-fold in the glial cell fraction over the activity in whole brains from 19- and 40-day-old rats. The activity of galactosyltransferase in neuronal perikarya decreased with age. Extensive contamination of the glial cell fraction with membranous fragments appeared to obscure the precise specific activity of this fraction. 3. The specific activity of the enzyme in glial tumor cells was 4--8-fold higher than in brain tissue when the enzyme was assayed under identical conditions using endogenous and different exogenous acceptors. 4. Galactosyltransferase activities from adult brain and glial tumor cells had similar properties. They both required Mn-2 plus and Triton, and exhibited pH optima between 5 and 7. The apparent Km of the enzyme for UDPgalactose was 1.3-10-minus 4 M for brain tissue and 2.2-10-minus 4 M for glial tumor cells. 5. The high galactosyltransferase activity in glial tumor cells and in neuronal perikarya of younger rats is compatible with the possibility of a role of this enzyme in developing brain.     

Glial patterning during postembryonic development of central neuropiles in the brain of the honeybee

 Glial cells are involved in several functions during the development of the nervous system. To understand potential glial contributions to neuropile formation, we examined the cellular pattern of glia during the development of the mushroom body, antennal lobe and central complex in the brain of the honeybee. Using an antibody against the glial-specific repo-protein of Drosophila, the location of the glial somata was detected in the larval and pupal brain of the bee. In the early larva, a continuous layer of glial cell bodies defines the boundaries of all growing neuropiles. Initially, the neuropiles develop in the absence of any intrinsic glial somata. In a secondary process, glial cells migrate into defined locations in the neuropiles. The corresponding increase in the number of neuropile-associated glial cells is most likely due to massive immigrations of glial cells from the cell body rind using neuronal fibres as guidance cues. The combined data from the three brain regions suggest that glial cells can prepattern the neuropilar boundaries. Received: 3 November 1996 / Accepted: 7 February 1997     

Morphogenesis and proliferation of the larval brain glia in Drosophila

Glial cells subserve a number of essential functions during development and function of the Drosophila brain, including the control of neuroblast proliferation, neuronal positioning and axonal pathfinding. Three major classes of glial cells have been identified. Surface glia surround the brain externally. Neuropile glia ensheath the neuropile and form septa within the neuropile that define distinct neuropile compartments. Cortex glia form a scaffold around neuronal cell bodies in the cortex. In this paper we have used global glial markers and GFP-labeled clones to describe the morphology, development and proliferation pattern of the three types of glial cells in the larval brain. We show that both surface glia and cortex glia contribute to the glial layer surrounding the brain. Cortex glia also form a significant part of the glial layer surrounding the neuropile. Glial cell numbers increase slowly during the first half of larval development but show a rapid incline in the third larval instar. This increase results from mitosis of differentiated glia, but, more significantly, from the proliferation of neuroblasts.     

Organization of a midline proliferative cluster in the embryonic brain of the grasshopper

We have studied a group of midline cells in the embryonic brain of the grasshopper by using immunocytochemical and intracellular dye injection techniques. This cluster of midline cells differentiates between the pars intercerebralis lobes of the protocerebrum during early embryogenesis, and is composed of putative midline progenitors as well as neuronal and glial cells. Annulin immunoreactive glial processes surround the borders of the midline cell cluster and also form a network of processes extending from there to the borders of proliferative clusters in the brain hemispheres. Among the cells that derive from the midline cluster are two bilaterally symmetrical pairs of identified primary commissure pioneer neurons. By navigating along the glial bound borders of the midline proliferative cluster, the axons of these pioneers establish an initial axonal bridge across the brain midline. This analysis identifies a glial-bound midline proliferative cluster in the brain and shows that neuronal and glial cells of this cluster are closely associated with neurons pioneering the primary brain commissure. Comparable features of midline cells in the ventral ganglia and similarities to other proliferative clusters in the brain hemispheres are discussed.     

Astrocyte dysfunction in neurological disorders: a molecular perspective

Recent work on glial cell physiology has revealed that glial cells, and astrocytes in particular, are much more actively involved in brain information processing than previously thought. This finding has stimulated the view that the active brain should no longer be regarded solely as a network of neuronal contacts, but instead as a circuit of integrated, interactive neurons and glial cells. Consequently, glial cells could also have as yet unexpected roles in the diseased brain. An improved understanding of astrocyte biology and heterogeneity and the involvement of these cells in pathogenesis offers the potential for developing novel strategies to treat neurological disorders.     

中华蜜蜂工蜂蕈形体胚后发育中神经胶质的形成模式

本研究通过形态解剖、免疫组织化学等技术,对中华蜜蜂Apis cerana cerana工蜂蕈形体胚后发育中神经胶质的形成过程进行了比较研究。结果表明：蕈形体中神经胶质增殖的高峰期集中在幼虫发育末期到蛹发育早期;在工蜂蕈形体的蕈体冠、蕈体柄以及小叶的发育过程中,神经胶质细胞往往先于神经纤维网出现在特定的区域,引导神经纤维网的形成。它们一方面规定了神经纤维网的边界和区域,为神经纤维网提供内部的分隔;另一方面也为神经纤维的移动提供特定的“路标"和靶向。与神经纤维网相关联的神经胶质的数量的持续增加,除了神经胶质的分裂增殖外,还有一部分来自于外部细胞体层的神经胶质的迁入。     

Neuroglia in ageing and disease

The proper operation of the mammalian brain requires dynamic interactions between neurones and glial cells. Various types of glial cells are susceptible to morpho-functional changes in a variety of brain pathological states, including toxicity, neurodevelopmental, neurodegenerative and psychiatric disorders. Morphological modifications include a change in the glial cell size and shape; the latter is evident by changes of the appearance and number of peripheral processes. The most blatant morphological change is associated with the alteration of the sheer number of neuroglia cells in the brain. Functionally, glial cells can undergo various metabolic and biochemical changes, the majority of which reflect upon homeostasis of neurotransmitters, in particular that of glutamate, as well as on defence mechanisms provided by neuroglia. Not only glial cells exhibit changes associated with the pathology of the brain but they also change with brain aging.     

Biosynthesis of Galactocerebrosides and Glucocerebrosides in Glial Cell Lines

UDP-galactose:ceramide galactosyltransferase (CGalT, EC 2.4.1.45) and UDP-glucose:ceramide glucosyltransferase (CGlcT, EC 2.4.1.80) were determined in the glial cell lines G26-20, G26-24, C6, and C6TK-. The enzymatic assay for CGalT in cultured glial cells was complicated by a rapid conversion of UDP-galactose to UDP-glucose, due to the elevated UDP-galactose-4'-epimerase activity in certain glial cell clones. It seems that mechanisms regulating UDP-galactose-4'-epimerase activity and levels of UDP sugars in the glial cell lines differ from those in brain tissue. Compared with the maximum activity of CGalT in the myelinating rat brain, the enzyme activities in the oligodendroglioma clonal cell lines G26-20 and G26-24 were 16-30 times lower. On the other hand, CGalT levels in G26-20 and G26-24 cells were comparable to the values found in young rat brain before myelination starts. No CGalT activity could be detected in C6 or C6TK- cells by the method used in this study, whereas CGlcT activity was found in all glial cell lines tested and its levels were close to the values observed in the young rat brain.     

Fibronectin associated with the glial component of embryonic brain cell cultures

In a basic approach to investigations of neuronal–glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti-GFA serum. When unfractionated cerebrum cells dissociated from 10-day chick or 14-day mouse embryos were plated as monolayers and cultured for 1-14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane-associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal–glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti-fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti-GFA or anti-fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal–glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal–glial interactions is discussed.     

Osmotic swelling characteristics of glial cells in the murine hippocampus, cerebellum, and retina in situ

Glial cells are proposed to play a major role in the ionic and osmotic homeostasis in the CNS. Swelling of glial cells contributes to the development of edema in neural tissue under pathological conditions such as trauma and ischemia. In this study, we compared the osmotic swelling characteristics of murine hippocampal astrocytes, cerebellar Bergmann glial cells, and retinal Müller glial cells in acutely isolated tissue slices in response to hypoosmotic stress and pharmacological blockade of Kir channels. Hypoosmotic challenge induced an immediate swelling of somata in the majority of Bergmann glial cells and hippocampal astrocytes investigated, whereas Müller cell bodies displayed a substantial delay in the onset of swelling and hippocampal astroglial processes remained unaffected. Blockade of Kir channels under isoosmotic conditions had no swelling-inducing effect in Müller cell somata but caused a swelling in brain astrocytic somata and processes. Blockade of Kir channels under hypoosmotic conditions induced an immediate and strong swelling in Müller cell somata, but had no cumulative effect to brain astroglial somata. No regulatory volume decrease could be observed in all cell types. The data suggest that Kir channels are differently implicated in cell volume homeostasis of retinal Müller cells and brain astrocytes and that Müller cells and brain astrocytes differ in their osmotic swelling properties.     

Shiga toxin-2 enhances heat-shock-induced apoptotic cell death in cultured and primary glial cells

The blood–brain barrier (BBB) selectively controls the homeostasis of the central nervous system (CNS) environment using specific structural and biochemical features of the endothelial cells, pericytes, and glial limitans. Glial cells, which represent the cellular components of the mature BBB, are the most numerous cells in the brain and are indispensable for neuronal functioning. We investigated the effects of Shiga toxin on glial cells in vitro. Shiga toxin failed to inhibit cell proliferation but attenuated expression of heat shock protein 70, which is one of the chaperone proteins, in cultured and primary glial cells. Furthermore, the combination of Shiga toxin and a heat shock procedure induced cell apoptosis and decreased cell proliferation in both cells. Thus, we speculate that glial cell death in response to the combination of Shiga toxin and heat shock might weaken the BBB and induce central nervous system complications.     

A light and electron microscopic study of the iron transporter protein DMT-1 in the monkey cerebral neocortex and hippocampus

We have studied by immunocytochemistry, the distribution of DMT-1, a cellular iron transporter responsible for transport of metal irons from the plasma membrane to endosomes, in the normal monkey cerebral neocortex and hippocampus. Light to moderate DMT-1 staining was observed in glial cell bodies in the neocortex, the subcortical white matter, and the hippocampus. Despite light labeling of cell bodies, glial end feet around cortical and subcortical blood vessels were heavily labeled. In the neocortex, the glial cell bodies displayed the morphological features of protoplasmic astrocytes. Labeled glial cells in the subcortical white matter contained dense bundles of glial filaments and were identified as fibrous astrocytes. The observation that DMT-1 was present on astrocytic endfeet suggests that these cells are involved in uptake of iron from endothelial cells. It is possible that the iron could then be redistributed into the extracellular space in the brain parenchyma.     

Laminin and fibronectin in normal and malignant neuroectodermal cells

Laminin and fibronectin, the major noncollagenous matrix glycoproteins, were studied in connection with normal brain cells and neuroectodermal cell lines. Laminin, a Mr 900,000 dalton matrix glycoprotein and an essential component of basement membranes, was found to be produced by cultured cells of several malignant cell lines of neuroectodermal origin. In cultured mouse C1300 neuroblastoma line cells laminin was localized, by immunoelectron microscopy, to the rough endoplasmic reticulum and, to sites of cell-to-cell and cell-to-substratum adhesion. Further experiments on the intracellular transport of this glycoprotein in C1300 cells confirmed that laminin is, at least partially, transported through the Golgi pathway. These results favor a role for laminin in attachment and cellular interactions of malignant neuronal cells. Laminin was also found in connection with neurons and glial cells from mammalian brain. In primary cultures from developing rat brain the vast majority of non-neuronal cells (80%) expressed immunoreactivity for the glial fibrillary acidic protein, a cytoskeletal protein specific for astrocytes. During the first week in culture all the glial fibrillary acidic protein-positive cells, with the exception of mature-looking star-shaped astrocytes, exhibited immunoreactivity for laminin. The intracellular laminin disappeared gradually after a few weeks in culture, but an extensive laminin matrix persisted and seemed to be localized on the upper surface of the non-neuronal cells. The neurofilament-positive neurons were negative for laminin. Pretreatment of the cultures with the ionophore monensin, caused accumulation of laminin-immunoreactivity within the Golgi region, which confirmed that laminin is, indeed, produced by cultured astrocytes and secreted through the Golgi complex. No fibronectin immunoreactivity was found in the majority of glial cells. However, under culture conditions where fibronectin was omitted from the culture medium there was, in the primary cultures, a minor population of glial fibrillary acidic protein-positive flat glial cells that exhibited intracytoplasmic immunofluorescence for fibronectin. In the presence of fibronectin in culture medium no fibronectin-positive glial cells could be detected. It thus appears that laminin, and to a minor extent fibronectin, are proteins that normal glial cells are capable of producing under specific conditions. Laminin and fibronectin were localized in adult rat brain in capillary and meningeal structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

The appearance of neural and glial cell markers during early development of the nervous system in the amphibian embryo

Cell-type-specific antibodies have been used to follow the appearance of neurones and glia in the developing nervous system of the amphibian embryo. Differentiated neurones were recognized with antibodies against neurofilament protein while glial cells were identified with antibodies against glial fibrillary acidic protein (GFAP). The appearance of neurones containing the neurotransmitters 5-hydroxytryptamine and dopamine has been charted also. In Xenopus, neurofilament protein in developing neurones was observed occasionally at NF stage 21 and was present reliably in the neural tube and in caudal regions of the brain at stage 23. Antibodies to the low molecular weight fragment of the neurofilament triplet recognized early neurones most reliably. Radial glial cells, identified with GFAP antibody, were identified from stage 23 onwards in the neural tube and caudal regions of the brain. In the developing spinal cord, GFAP staining was apparent throughout the cytoplasm of each radial glial cell. In the brain, the peripheral region only of each glial cell contained GFAP. By stage 36, immunohistochemically recognizable neurones and glia were present throughout the nervous system. In the axolotl, by stage 36 the pattern of neural and glial staining was identical to that observed in Xenopus. GFAP staining of glial cells was obvious at stage 23, although neuronal staining was clearly absent. This implies that glial cells differentiate before neurones. 5-HT-containing cell bodies were first observed in caudal regions of the developing brain on either side of the midline at stage 26. An extensive network of 5-HT neurones appeared gradually, with a substantial subset crossing to the opposite side of the brain through the developing optic chiasma. 5,7-dihydroxytryptamine prevented the appearance of 5-HT. Depletion of 5-HT had little effect on development or swimming behaviour. Dopamine-containing neurones in the brain first differentiated at stage 35-36 and gradually increased in number up to stage 45-47, the latest stage examined. The functional role of 5-HT- or dopamine-containing neurones remains to be elucidated. We conclude that cell-type-specific antibodies can be used to identify neurones and glial cells at early times during neural development and may be useful tools in circumstances where functional identification is difficult.     

Fine structure of the neural sheath,glia and neurons in the brain of the housefly,Musca domestica

Summary The fine structure of the neural sheath, glial cells and nerve cells in the brain of adult male houseflies is described. The neural sheath is composed of neural lamella and perineurium. The neural lamella consists of an external lamina and collagen-like fibrils which are embedded in an amorphous matrix. The perineurial cells form a continuous layer around the brain. On their inner surface, perineurial cells form junctional complexes with glial cell processes. A cortical cellular layer composed of neurons and glial cells surrounds the centrally located neuropil. Three types of glial cells are identified. Glial cells differ in size and in relative development and distribution of organelles. Thin processes of glioplasm completely surround the cell bodies of the neurons. Five types of neurons are described. Most of the neurons are monopolar, a few are bipolar.Supported by a grant from the National Science Foundation     

Immunohistochemistry of Atrial Natriuretic Peptide in Brain Infarction

Atrial natriuretic peptide (ANP) was originally isolated from cardiac atria, and has potent natriuretic, diuretic, and vasorelaxant properties. It has been localized in neurons and astrocytes in the cerebral cortex and the white matter. We hypothesize that glial ANP may contribute to the regulation of cerebral blood flow in brain infarction. In order to elucidate this possible role, the immunohistochemistry of ANP was studied in cases of brain infarction and in other cases of brain trauma for comparison. A statistically significant increase in the number of ANP-immunoreactive glial cells (mainly astrocytes) was observed in the white matter surrounding the brain infarction compared with the intact area. No statistically significant increase in ANP-immunoreactive glial cell number was observed in the cerebral white matter from brain haemorrhage, contusion and control cases. Our results indicate that glial ANP may increase in number in brain infarction, and that it may be involved in the regulation of the cerebral blood flow in the infarcted area.     

Glial cells phagocytose neuronal debris during the metamorphosis of the central nervous system in Drosophila melanogaster

 Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes and other remains from neuronal degeneration in phagosomes. Received: 23 January 1996 / Accepted in revised form: 21 May 1996     

Cell volume regulation in the nervous system

There is good evidence that the three main compartments of the brain, i.e. extracellular space, neurones and glial cells, change their volume during physiological and pathophysiological neuronal activity. However, there is strikingly little knowledge about the mechanisms underlying such alterations in cell volume. For this purpose, a better understanding of the electrophysiological behavior of the neurones and glial cells during volume changes is necessary. Examples are discussed for which changes in cell volume can be derived from the underlying changes in membrane permeabilities. Volume regulatory mechanisms in the brain have not been described under isotonic conditions. However, a rapid volume regulatory decrease is occurring in cultured glial cells during exposure to hypotonic solutions. In contrast, in these cells no volume regulatory increase was found during superfusion with hypertonic media. On the other hand, the entire brain is able to compensate chronic hypertonic perturbations within hours to days. Interestingly, not only ion fluxes induce cellular volume changes but, in turn, water movements can also influence ion fluxes in both neurones and glial cells. With respect to this it should be considered that volume regulatory membrane processes might not exclusively be activated by changes in transmembranal ion gradient, but also by changes of membrane surface shape. Future studies on cellular mechanisms of volume regulation in the brain should imply a combined use of recent techniques such as computerized video-imaging, radiotracer flux measurements and ion-sensitive microelectrodes in defined cell cultures. Optical monitoring and ion-sensitive microelectrodes should enable measurements of volume changes in identified cellular elements of intact nervous structures such as brain slices.