Presence Of Glial Cells Research Articles

Chapter 6 Molecular organization of cerebellar glutamate synapses

The organization of key molecules at glutamatergic synapses in the rat cerebellar cortex as analyzed by high resolution immunocytochemical techniques using gold particles as markers. The distinct compartmentation of glutamate and glutamine was consistent with biochemical data indicating an active role of glia in the removal of released glutamate and in the supply of glutamine for de novo synthesis of transmitter glutamate. The presence in glial cells of two different glutamate transporters, GLT1 and GLAST, provided further support of this concept. Both transporters were selectively expressed in glial membranes and occurred at higher densities in glial processes surrounding parallel fiber synapses with spines than in glial processes associated with parallel fiber synapses with dendritic shafts. At the former type of synapse, gold particles signalling GLT1 and GLAST could be found within a few nanometers of the postsynaptic density. The rat cerebellum also contains a homologue (rEAAC1) of the glutamate transporter EAAC1, originally cloned from rabbit, mRNA encoding this transporter was restricted to neurons. The exact localization of the rEAAC1 transporter molecules at cerebellar synapses remains to be determined but immunocytochemical and physiological data from other laboratories suggest that they may be preferentially expressed in postsynaptic membranes. Gold particles representing immunoreactivity for the AMPA receptor subunits GluR2/3 were found along the entire mediolateral extent of the postsynaptic specialization of parallel fiber synapses and were rarely encountered at non-synaptic membranes. The present data show that molecules engaged in signalling at cerebellar glutamatergic synapses are precisely organized, consistent with the requirements for rapid signal transmission and efficient removal and recycling of transmitter.

Hypoxic and Excitotoxic Damage to Cultured Rat Retinal Ganglion Cells

The cytotoxic effects of hypoxia and excitatory amino acids on cultured retinal ganglion cells were studied. The influence of coculture with retinal Müller glia and cortical astrocytes on cell survival was evaluated, as were the effects of the N-methyl- dl-aspartate inhibitor, MK-801. Dissociated retinal neurons from 7-day-old Sprague–Dawley rats were plated on a laminin substrate, neocortex-derived glial monolayers, or Müller cell monolayers. Ganglion cells were labeled by injection of DiI into the superior colliculus 2 days prior to dissociation. Exposure of cultured ganglion cells to glutamate and N-methyl- dl-aspartate showed a time- and concentration-dependent survival rate. Exposure of cells to hypoxia demonstrated a survival rate that was dependent on time and O 2concentration. Excitotoxic and hypoxic damage was entirely blocked by the specific non-competitive inhibitor of N-methyl- dl-aspartate, MK-801. Retinal ganglion cells cultured on cortical astrocytes and retina-derived Müller glia showed significantly better survival rates ( P<0.001) than cells cultured on laminin-coated dishes under control conditions, in hypoxia (9% to 15% O 2), and after exposure to 200μ mglutamate. Retinal ganglion cells cultured on Müller glia showed significantly better survival rates ( P<0.01) than those cultured on cortical astrocytes under conditions of hypoxia (9% to 15% O 2) and exposure to 200μ mglutamate. The results demonstrate that excitotoxic and hypoxic damage to cultured retinal ganglion cells is moderated by NMDA receptor blockade and by the presence of glial cells, especially retinal Müller cells. This system may provide a useful model for studying the pathophysiology of excitotoxicity and hypoxia on cultured retinal ganglion cells, and may be used to help identify potentially clinically useful therapeutic agents.

Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes.

Effects of increased levels of arachidonic acid (AA) were analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in a physiological medium added with arachidonic acid (AA) in a concentration range from 0.01 to 0.5 mM. The concentration profiles of the fatty acid and AA-metabolites were subsequently followed for 90 min. AA was measured by gas chromatography, whereas the AA-metabolites PGF2 alpha and LTB4 by radioimmunoassay (RIA). Following administration of AA at 0.05 or 0.1 mM the medium was completely cleared from the fatty acid within 10 to 15 min. However, when 0.5 mM were added, AA concentrations of 0.36 +/- 0.055 mM were found at 20 min, while 0.275 +/- 0.045 mM at 90 min. Addition of AA (0.1 mM) to cell-free medium was also associated with a steady decline of its concentration, although the decrease was markedly delayed as compared to the clearance in the presence of glial cells. AA was subjected to dose-dependent metabolisation in the cell suspension as demonstrated by the production of PGF2 alpha and LTB4. Following addition of 0.01 or 0.5 mM, concentrations of PGF2 alpha increased to a 1.9- or 4.9-fold level within 10 min, whereas those of LTB4 rose to a 1.3- or 33.7-fold level. This was attenuated or completely blocked, respectively, by the cyclo- and lipoxygenase inhibitor BW 755C. Formation of both metabolites from AA was also observed when studying astrocytes from primary culture. The current findings demonstrate an impressive efficacy of C6 glioma cells and astrocytes to clear arachidonic acid from the suspension medium and to convert the lipid compound into prostaglandins and leukotrienes. Uptake and metabolisation of AA by the glial elements may play an important role in vivo, for example in cerebral ischemia.

Immunohistochemistry of cellular proliferation in eyes with longstanding retinal detachment.

To further characterize the content and nature of cyclitic membranes (n = 4), ringschwielen (n = 3) and a subretinal strand on sections of complete globes with long-standing retinal detachment after trauma (n = 7) or retinal reattachment surgery (n = 1), we applied a panel of cellular and extracellular antibodies using the APAAP (alkaline phosphatase and monoclonal anti-alkaline phosphatase) technique. Only one cyclitic membrane from an eye that was enucleated two months after trauma was positive for glial fibrillary acidic protein, demonstrating the presence of glial cells. All cyclitic membranes contained macrophages and expressed intracellular contractile proteins. Ringschwielen stained consistently for cells of epithelial origin (RPE) with cytokeratin and for macrophages. The subretinal strand stained for cytokeratin, macrophages and alpha-smooth muscle actin, supporting cellular contraction in the genesis of this form of proliferative vitreoretinopathy.

Locust Medial Neurosecretory Cells in vitro: Morphology, Electrophysiological Properties and Effects of Temperature

ABSTRACT The medial neurosecretory cells of the pars intercerebralis in the protocerebrum of larval and adult locusts (Locusta migratoria) were cultured in a chemically defined serum-free culture medium. The morphology of the cells was investigated by light microscopy and the electrophysiological properties were studied using the patch-clamp technique in the whole-cell configuration. The dissociated neurosecretory cells grew new processes under these conditions and were maintained in culture for up to 2 months. The percentage of cells showing outgrowth was significantly higher in third-instar larvae than in instars 4 and 5 and adults. A primary axonal stump promoted a unipolar cell morphology; in other cases, most neurosecretory cells became multipolar. The presence of glial cells in undissociated groups of neurosecretory cells improved outgrowth and the formation of neurite bundles. A considerable number of the recorded cells showed spiking activity in response to depolarization. The influences of temperature on spike frequency, duration and amplitude as well as on membrane potential and ionic currents were investigated. The results suggest that temperature may directly affect the function of neurosecretory cells.

Early nerve growth factor-induced events in developing rat septal neurons

A culture system enriched for nerve growth factor (NGF) receptor bearing cells was developed to investigate signal transduction events activated by NGF in postmitotic central nervous system neurons. Cells from the septal region of embryonic rats at 16 days of gestation were grown on glass coverslips above a glial cell layer established from postnatal rat cortex. The separation of glial and neuronal planes in this “bilaminar” system permits the diffusion of glial-derived factors required by septal neurons for survival yet allows the investigation of NGF responses in a pure neuronal population. Approximately 15% of the neurons in this culture system were immunoreactive for the low affinity NGF receptor. NGF rapidly increased MAP kinase activity (2–5 min) and transiently induced expression of c- fos in septal neurons. NGF treatment also increased choline acetyltransferase activity, while the number of cholinergic neurons remained constant. Septal neuron survival depended on the presence of glial cells, but neuronal viability in the bilaminar system was unaffected by anti-NGF antiserum, indicating that glial-derived neurotrophic support is not mediated by NGF alone. These data suggest that the bilaminar culture system is a useful system for the study of early events in NGF-activated signal transduction and the nature of glial-derived trophic support of developing basal forebrain neurons.

Presence of glial cells in the rat pineal gland: a light and electron microscopic immunohistochemical study.

Immunoperoxidase methods for the demonstration of three glial antigens, vimentin, glial fibrillary acidic protein, and S-100 protein, were applied to routine-fixed paraffin sections of rat pineal gland. A pre-embedding electron microscope immunoperoxidase method was also used to study the ultrastructural localization of S-100 protein in pineal cells. Light and electron microscopic results showed the presence of these antigenic glial markers in the second pineal cell type. The term glial cell is proposed for the second of parenchymatous cell in rat pineal gland.

Quinolinic acid phosphoribosyltransferase: preferential glial localization in the rat brain visualized by immunocytochemistry.

The excitotoxic brain metabolite quinolinic acid has been hypothetically linked to the pathogenesis of neurodegenerative disorders. By using antibodies prepared against a homogeneous preparation of its catabolic enzyme, quinolinic acid phosphoribosyltransferase [QPRTase; nicotinate-nucleotide:pyrophosphate phosphoribosyltransferase (carboxylating), EC 2.4.2.19], immunocytochemical methods were applied to assess the cellular and subcellular localization of quinolinic acid in the rat brain. On the light-microscopic level, the enzyme was found to be preferentially associated with glial elements of variable morphology. In addition to its presence in glial cells, QPRTase was contained in tanycytes and ependymal cells of the cerebral ventricles and, sporadically, in neurons. Overall, QPRTase immunoreactivity was noted in every brain region studied, the histological pattern being in good accordance with the regional variation of enzyme activity established in biochemical studies. As judged on the ultrastructural level, QPRTase, in all cell types examined so far, was often noted in densely stained roundish cytoplasmic bodies (0.1-0.8 micron in diameter), which were bounded by a single membrane. In functional terms, these structures may represent early lysosomes, secretory granules, or residual bodies. The particular anatomical arrangement of the quinolinic acid system may reflect the brain's defense strategy against detrimental effects of the endogenous excitotoxin.

Specific stimulation of in vitro maturation of mesencephalic dopaminergic neurones by striatal membranes.

We have previously established that dissociated mesencephalic dopaminergic (DA) neurones from embryonic mouse differentiate in primary cultures and that their development is enhanced when they are grown in the presence of their target cells from the striatum1–3. The capacity of individual DA neurones to take up and synthesize 3H-DA was increased in co-culture. This phenomenon does not depend on the presence of glial cells because it also occurred in serum-free conditions in which glial development was largely impaired4. Therefore, striatal target neurones favour the maturation of afferent DA neurones. Diffusible or membrane-bound factors could be involved in this process. To test one of these possibilities, we have now examined the in vitro development of embryonic mesencephalic DA neurones exposed to striatal membranes isolated from postnatal mice at various ages. 3H-DA uptake was used as an index of maturation of the DA neurones5. We show that striatal membranes from 2- and 3-week-old animals stimulate the development of DA neurones. The finding that this effect was not seen with membranes from 1-week-old or adult animals suggests that a developmentally regulated membrane-bound ‘factor’is probably involved. This factor seems to be specific for the DA neurones.

Behaviour of neuroblasts in the presence of glial cells, fibroblasts and meningeal cells in culture

Dissociated nerve cells from 7-day-old chick embryo cerebral hemispheres were cultivated on plastic surfaces, astroblast layers, fibroblast layers and meningeal cell layers. The cell suspensions obtained by mechanical dissociation and plated on these layers contained primarily neuroblasts. The neuroblasts cultured on astroblast layers behaved differently from those cultured on fibroblast or on meningeal cell layers. They adhered within 2 h to the preformed astroblast monolayers and remained scattered over it. In contrast, in the two other cases, the neuroblasts formed floating aggregates which adhered to the layers only after 24 h. Neuroblasts behaved on monolayers made of fibroblasts or meningeal cells as on plastic surfaces. The neuronal cells grown on astroblast layers were much more differentiated than those plated on plastic or on the two other layers studied. After 2–3 weeks of culture the neurons were large and the fibres were longer, thicker and more ramified. However, the fibroblast and the meningeal cells enhanced slightly the growth of the neuroblasts relative to plastic surfaces. These results support the possibility of specific interactions between astroblasts and neuroblasts.