Nikos Sakellaridis

Early and late passage C-6 glial cell growth: similarities with primary glial cells in culture.

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell numer and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into “astrocytic phenotype”.Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.

Effects of opiates on the growth of neuron-enriched cultures from chick embryonic brain.

Neuron‐enriched cultures derived from 6‐day‐old chick embryo cerebral hemispheres were treated with morphine or methadone, 10−5 M or 10−6 M, on days 4–6 or 6–8 in culture and were evaluated morphologically and biochemically at day 9 using phase contrast microscopy and choline acetyltransferase activity (ChAT) as a cholinergic marker. The treatment of the cultures with morphine markedly affected their growth pattern; specifically, we observed an increased number of flat cells presumptively glia, and aggregates sided by flat cells and devoid of thick bundles of neuritic processes that normally characterize neuron‐enriched cultures. These morphologic changes were reflected in a drastic decrease of ChAT activity in cultures treated from day 4 to day 6 but not from 6 to 8. In contrast to morphine, exposure to 10−6 M methadone from day 4 to day 6 resulted in reduced ChAT activity but the growth pattern of the cultures remained morphologically intact. We suggest that morphine exerts a general neurotoxic effect whereas methadone may affect some specific cholinergic function.

Factors influencing neuronal growth in primary cultures derived from 3-day-old chick embryos

We compared neuronal growth patterns in primary cultures prepared by dissociating 3‐day‐old chick embryos, either whole embryo (E3WE) or head only (E3H) and plating the dispersed cells onto Petri dishes coated with either poly‐l‐lysine, collagen or laminin. The culture medium was Dulbeccos Modified Eagles Medium (DMEM), supplemented with either 5 or 10% fetal bovine calf serum (FCS). As we have previously described, in E3WE cultures on poly‐l‐lysine the neuronal primary growth patterns were aggregation with neuritic fasciculation, presence of growth cones with microspikes and very few flat cells. In contrast with cultures grown on poly‐l‐lysine, in cultures grown on collagen or laminin the distinct growth pattern was extensive networks of isolated and differentiated neurons lying on acquired monolayers of flat cells. When 5% PCS was used, as compared to 10% PCS, neuronal aggregates were fewer and smaller on poly‐l‐lysine; on collagen or laminin a tendency to aggregate was observed. Several differences were observed in the E3H cultures when compared to E3WE: (a) aggregates were less numerous with the prevailing pattern being a web‐like, self‐contained aggregate; (b) aggregates connected with other aggregates or flat cells were rare and the aggregate adhesivity was minimized; (c) neurons on collagen or laminin formed networks with the exception of a few, small aggregates displaying no fasciculation; (d) flat cells did not form a monolayer but islets which hosted the neuronal meshy networks. We attribute these differences in the growth patterns between the various types of cultures to be the combined result of a variety of environmental signals, derived from the provided substrata, the serum and the nonneuronal cell factors and cell surface, all primarily regulating neuronal adhesivity.

C-6 Glioma Cells of Early Passage Have Progenitor Properties in Culture

Although considerable progress has been made in the last decade in our understanding of the role of glial cells in neuronal development and function, the factors which regulate glia cell growth and function are only recently being investigated (see refs in review Vernadakis, 1988). C-6 glioma cells have provided a useful model to study glial cell properties, glial factors and sensitivity of glial cells to various substances and conditions. In an early study, we reported (Parker et al, 1980) that C-6 glioma cells, 2B clone, exhibited differential enzyme expression with cell passage: the activity of cyclic nucleotide phosphohydrolase (CNP) an enzyme marker for oligodendrocytes (Poduslo and Norton, 1972; Poduslo, 1975) was markedly high and that of glutamine synthetase (GS), an enzyme marker for astrocytes (Martinez-Hernandez et al, 1977; Norenberg and Martinez-Hernandez, 1979) was low in early passages (up to passage 26) and this relationship was reversed in the late passages (beyond passage 70)

Evidence for plasticity in neurotransmitter expression in neuronal cultures derived from 3-day-old chick embryo

We have previously reported the developmental profiles of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT) bio- and immunocytochemically, assessing GABAergic and cholinergic neuronal phenotypes respectively, in neuroblast-enriched cultures from 3-day-old chick embryo, plated on poly-L-lysine. We have also reported that collagen as culture substrate inhibits neuronal aggregation and neuritic fasciculation in this culture system. In this study we assessed the same parameters for cultures on collagen. In addition, we evaluated the effects of nerve growth factors (NGF) on cholinergic and GABAergic expression on neurons plated either on polylysine or collagen. We found that non-neuronal cells and NGF prolonged the survival of cholinergic and GABAergic neuronal populations and that both markedly stimulated GABAergic expression. In contrast, cholinergic expression was only enhanced by NGF. Immunostaining for GABA and ChAT reflected the biochemical findings. Glutamine synthetase and cyclic nucleotide phosphohydrolase, used as markers for astrocytes and oligodendrocytes respectively, showed very low activity in both substrata and were not related to GAD or ChAT peak activities. Our findings suggest that humoral factors and cell-cell contacts markedly influence neuronal phenotypic expression in culture. Moreover, it appears that during early neuronal differentiation GABAergic neurons are more responsive to microenvironmental regulation compared to cholinergic neurons.

Glial cell growth in culture: Influence of living cell substrata

The role of the microenvironment in the growth of glial cells in culture has been the topic of ongoing research in this laboratory. Recently, we reported a study on the contribution of fibroblast cell substratum and extracellular matrix in glial cell growth. In the present study we report data concerning a) the influence of a neuronal-enriched living substratum from chick embryo on the growth of glial cells derived from chick embryonic brain and plated onto the substratum; b) the influence of dissociated cells derived from chick embryonic brain on the growth of established glial cells in culture, and c) the influence of dissociated cells derived from adult rat spinal cord on the growth of established glial cells from newborn rat in culture. The activities of glutamine synthetase (GS) and 2′, 3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were the biochemical probes determined for astrocytes and oligodendrocytes, respectively. We found that glial growth as assessed by both enzyme activities, was enhanced when a nervous tissue derived cell population was plated onto a glial-enriched substratum, whereas glial growth was inhibited when the neuronal-enriched population was the cell substratum.

Cholinergic neurotoxicity induced by ethylcholine aziridinium (AF64A) in neuron-enriched cultures.

The sequence of events in neuronal changes induced by the cholinotoxin ethylcholine aziridinium (AF64A) was studied. Neuron-enriched cultures derived from 8-day-embryonic chick cerebra were treated with AF64A at concentrations of 10(-5), 10(-4) and 10(-3) M. Choline acetyltransferase (ChAT) was used as an index of cholinergic neurons. Changes in cell morphology, the immunocytochemical and biochemical presence of ChAT, and DNA and protein content were assessed. Neuron-enriched cultures exposed to AF64A showed a dose-dependent response; after 24 h of exposure to 10(-3) M toxin all cells were dead, whereas a concentration of 10(-5) M did not alter culture morphology or DNA and protein contents. Despite the lack of cytological changes and the presence of ChAT immunoreactivity, biochemically assessed ChAT activity was reduced 36% in 10(-5) M treated cultures. Thus, the implicated decrease in acetylcholine synthesis in these cells cannot entirely account for the neuronal degeneration. Simultaneous exposure of cultures to both AF64A and 10 times higher concentrations of choline chloride delayed or diminished the neurotoxic changes. The protective effect of high choline concentrations was interpreted as evidence of competition between choline and AF64A for the high affinity choline transport system and as constituents in the cell membrane. Examination of the temporal sequence of cytotoxic changes in 10(-4) M exposed cultures revealed that disruption of neuronal aggregates and fragmentation of neurites occurred between 4 and 8 hours of exposure. After 24 h, some neurons survived but with attenuated arbors; in contrast, astrocytes appeared intact, suggesting that glial cells are more resistant than neurons to the toxic effects of AF64A. These findings suggest this culture model may be useful to further elucidate the mechanisms of AF64A drug action and study differentiation of cultured neuronal populations in the absence of cholinergic cells.

Developmental profiles of glial enzymes in the chick embryo: In vivo and in culture.

The activities of two glial cell enzymes, glutamine synthetase (a marker for astrocytes) and 2?,3?-cyclic nucleotide 3?-phosphohydrolase (a marker for oligodendrocytes and myelination) were studied in the developing chick embryo brain in vivo and in cultures derived from chick embryos. The in vivo findings showed that the activities of both enzymes parallel the patterns of gliogenesis and myelination. Glutamine synthetase follows similar patterns in culture and in vivo, whereas the developmental profile of 2?,3?-cyclic nucleotide 3?-phosphohydrolase appears to be affected by the culture conditions.

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of μ and δ opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [3H]dihyromorphine was used as a μ ligand and with 5×10−7 M levorphanol for non-specific binding, and [3H](d-Ala2-d-Leu5)-enkephalin was used as a δ with 5×10−7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both μ and δ opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity μ sites during early embryogenesis but only one δ site. By 18 days of embryonic age, only one μ site remained. This developmental change is interpreted as a transitory state of the receptor to the adult μ pattern. The presence of only one δ site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the μ receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.

Comparisons of Glial Cells from Newborn and Aged Mouse in Culture

Only in the last decade has consideration been given to glial cells in the aging brain, and the role of glial cells in the neuronal aging process is far from being understood. Morphological changes in number and volume of astroglia cells have been reported in various brain areas in the aging rat (Geinisman et al., 1978; Landfield et al., 1977; Lindsey et al., 1979). The phenomenon of “gliosis” with aging has been interpreted to reflect a compensatory response of astrocytes to their microenvironment and specifically to neuronal loss. On the other hand, the decline in synaptic function with aging (see review by Vernadakis, 1985) may partially be attributed to the lack of responsiveness of glial cells to synaptic plasticity including “reactive synaptogenesis,” a phenomenon observed in neuronal regeneration (Cotman and Nadler, 1978; Cotman and Scheff, 1979). Studies using the hippocampus as a model system to compare reactive synaptogenesis in the mature and aged rat brain have shown that reactive synaptogenesis is slower in the aged animals. Reactive synaptogenesis observed after partial denervation in brain-lesion studies appears to depend upon trophic signals provided by hormones and other growth-regulating factors, and the regulatory role of glial cells, i.e., the ability of glial cells to prepare the neuropil for the growth of new synapses. It is likely that, in the aging brain, glial cells may not supply the required growth factors for new synapse formation. Thus the proposal has been put forth that glial cells change with aging and may ultimately contribute to neuronal aging through changes in the microenvironment.