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Biomedical subjects

A Vernadakis

Publications and source records attributed to A Vernadakis.

At least 73 records · Page 4Linked to original sources
PubMed1990

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.

Animals↗
PubMed1990

Opioids influence neurotransmitter phenotypic expression in chick embryonic neuronal cultures.

There is considerable interest in the role of endogenous opioid peptides in neural growth and differentiation. In this study we used neuron-enriched cultures derived from 3-day-old chick embryos to test the effects of endogenous enkephalins on neurotransmitter phenotypic expression. Cultures were grown in serum-free chemically defined medium and were treated with either Met-enkephalin antiserum (anti-Met) to immunoneutralize enkephalins, or with naloxone, a universal opioid receptor antagonist, to block receptor-mediated actions of released endogenous opioids. The enzyme activities of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) were used as markers for cholinergic and catecholaminergic phenotypic expression, respectively. We found that cultures treated with anti-Met or naloxone exhibited strikingly different neuronal growth patterns as compared to controls. In addition, ChAT activity was enhanced by anti-Met, and TH activity by both anti-Met and naloxone. These findings lend support to the possibility that neuropeptides may be co-localized with neurotransmitters and that peptides released into the microenvironment affect neuronal phenotypic expression by differential receptor subtypes.

Animals↗
PubMed1989

Growth hormone-releasing hormone influences neuronal expression in the developing chick brain. I. Catecholaminergic neurons.

We have examined catecholaminergic expression during development in the chick embryonic brain using tyrosine hydroxylase (TH) activity as a biochemical marker for catecholaminergic neurons. TH activity was detectable as early as after 4 days of incubation in whole brain homogenates and increased throughout embryonic development. The greatest increase in enzyme activity was observed between embryonic days 8 and 15, a period of active neuronal maturation and synaptogenesis. Growth hormone-releasing hormone (GHRH) was tested for its influence on TH activity during embryonic development. Eight-day-old embryos that received GHRH (50 ng/50 microliters) in ovo on days 1, 3, 5 and 7 exhibited a significant (P less than 0.001) increase in TH activity. Similar results were obtained when GHRH was administered in a single 50 ng/50 microliter dose on day 1 or day 3 of development. However, embryos receiving the same dose of GHRH on day 5 exhibited no significant difference in TH activity as compared to controls. When growth hormone (GH, 100 ng/50 microliters) was administered during the same critical period (day 3) no difference was observed in TH activity as compared to controls. Thus, the effects of GHRH on TH activity do not appear to be mediated through GH. We interpret these data to mean that GHRH can enhance catecholaminergic phenotypic expression in the chick embryonic brain when administered during a discrete critical period of development from days 1 to 3 of embryonic age.

Animals↗
PubMed1989

Developmental profile of glutamine synthetase in lines of mice bred for ethanol sensitivity.

Glutamine synthetase (GS) activity was used as a marker to examine differences in astrocyte development in mice selectively bred for ethanol sensitivity: long sleep (LS), short sleep (SS), mild ethanol withdrawal (MEW), severe ethanol withdrawal (SEW) and control ethanol withdrawal (CEW). We found that 1) GS activity in MEW and SEW was higher than in LS and SS during the first 2 weeks of postnatal development, in the forebrain but not in the cerebellum; 2) lower GS activity was observed consistently in all areas examined with the SS mice as compared to the LS; 3) glutamine synthetase activity in MEW and SEW differed significantly from their controls (CEW) during the early developmental period regardless of the brain region examined; however, after 30 days of maturation, GS activity in SEW was higher than that in MEW and CEW in the forebrain. Astrocytes are known to contribute in the regulation of the neuronal microenvironment. Therefore, we interpret the differences we found in astrocytic function during early brain development among these lines of mice to account in part for the neuronal predisposition to ethanol sensitivity.

Aging↗
PubMed1989

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 number 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.

Animals↗
PubMed1988

GABAergic neurons in cultures derived from three-, six- or eight-day-old chick embryo: a biochemical and immunocytochemical study.

Cultures were prepared from 3-, 6- and 8-day-old chick embryos. Whole chick embryos were dissociated by sieving through a nylon mesh for E3 cultures and cerebral hemispheres for E6 and E8 cultures. The dispersed cells were plated onto poly-L-lysine coated culture dishes in Dulbecco's modified Eagle's medium, containing 10% fetal bovine calf serum. Growth patterns in these cultures have been previously described. Biochemical and immunocytochemical markers were used to identify GABAergic neurons in culture. Neurons exhibiting GABA-like immunoreactivity were present in all 3 types of cultures as early as 4-6 days in culture. The developmental profile of glutamic acid decarboxylase (GAD) derived from 3-day-old whole chick embryo cultures showed low activities whereas the enzyme activity markedly rose in cultures derived from 6- or 8-day-old chick embryo cerebral hemispheres during the first two weeks. The changes in GAD activity observed in these cultures are interpreted to reflect the maturational state of GABAergic neurons and also their responsiveness to microenvironmental factors.

Animals↗
PubMed1988

Cholinergic neurons in cultures derived from three-, six- or eight-day-old chick embryo: a biochemical and immunocytochemical study.

Cultures were prepared by dissociating 3-day-old whole chick embryos, or 6- or 8-day-old chick embryo cerebral hemispheres. The dispersed cells were plated onto poly-L-lysine coated dishes in Dulbecco's modified Eagle's medium fortified with 10% fetal bovine calf serum. Immunocytochemistry, histochemistry and biochemistry were used to characterize the cholinergic neurons in these cultures. Double staining for acetylcholinesterase (AChE) and neurofilament revealed that a high percentage of neurons also contained AChE; fewer neurons though were positive for choline acetyltransferase (ChAT). The presence of AChE in non-cholinergic neurons was interpreted to be indicative of 'cholinoceptive' neurons in culture. The developmental profile for ChAT activity in cultures derived from 3-day-old whole chick embryos was previously described; we have reported that activity reached high levels by 7 days in culture. In this study we found that in cultures derived from 6-day-old chick embryos ChAT activity increased slowly up to 6 days in culture and rose markedly thereafter; in contrast, in cultures derived from 8-day-old chick embryos activity was low until day 9 and rose moderately thereafter. The differences in ChAT expression between cultures derived from chick embryos of different developmental stages may reflect a loss of plasticity of the cholinergic population with age.

Acetylcholinesterase↗
PubMed1988

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 Dulbecco's Modified Eagle's 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% FCS was used, as compared to 10% FCS, 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.

Animals↗
PubMed1988

Neuron-glia interrelations.

Considerable progress in our understanding of neuron and glial cell interrelationships has emerged during the last decade from in vitro and in vivo studies. Neural culture systems have provided powerful tools to delineate cellular and molecular events. Moreover, the advances in development of immunocytochemical and biochemical specific cell markers has made possible the characterization of complex cell behaviors. Glial cells actively participate in several aspects of neuronal growth and differentiation both by providing cell-cell contact interactions and by secreting neuronal growth-promoting factors. In turn, neurons influence the cellular behavior of both astrocytes and oligodendrocytes, primarily by secreting substances into the microenvironment. Such substances as neurohormones and neurotransmitters have been shown to affect several glial functions including electrophysiological responses, energy metabolism, and ionic homeostasis. In several instances these effects appear to be mediated through receptors on glial cells. Astrocytes actively participate in the regulation of the ionic environment. They take up and release several neurotransmitter substances and can modulate the concentration of a neurotransmitter substance at the synaptic cleft and thus monitor neuronal activity. The evidence of neuron-astroglia synaptic contacts supports the view that such contacts are present during early neuroembryogenesis and thus may provide contact signals for neuronal growth. The process of myelination in the CNS appears to be regulated by both neuronal signals to the oligodendrocyte and also intrinsic programming in the oligodendrocytes to produce myelin components. The prevailing view that astrocytes impede regeneration appears to be shifting towards a more favorable notion of the role of these cells in promoting this process. Of interest is the concept that there is a critical period in the ability of astrocytes either to enhance regeneration or to form a gliotic scar and impede this process. The role of glial cells in the aging process of the neuron is only beginning to be appreciated. If glial cells are actively involved in the regulation of the microenvironment, then it follows that any changes in the behavior of glial cells with aging will ultimately affect neuronal function. It is abundantly clear from in vitro studies that glial cells are pluripotential cells with several functional capabilities. Their responsiveness to an environment in which neurons are maturing as compared to an environment where neurons are injured or aging clearly portrays the multifunctional role of the astrocyte.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Differential maturation of mu and delta opioid receptors in the chick embryonic brain.

The developmental profiles of the binding of mu and delta 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 mu ligand and with 5 X 10(-7) M levorphanol for non-specific binding, and [3H](D-Ala2-D-Leu5)-enkephalin was used as a delta with 5 X 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 mu and delta opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity mu sites during early embryogenesis but only one delta site. By 18 days of embryonic age, only one mu site remained. This developmental change is interpreted as a transitory state of the receptor to the adult mu pattern. The presence of only one delta 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 mu receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Growth patterns of glial cells dissociated from newborn and aged mouse brain with cell passage.

Glial cell cultures derived from newborn and aged (18-month-old) mouse cerebral hemispheres and maintained up to cell passage 11 were characterized immunocytochemically by using glial fibrillary acidic protein (GFA), and biochemically by using glutamine synthetase (GS), for astrocytes, and 2',3' cyclic nucleotide 3' phosphohydrolase (CNP) for oligodendrocytes. We report here the changes occurring during passages 5-11. GS and CNP activities did not significantly change with cell passage in cultures from newborn mouse. In cultures derived from aged mouse, CNP activity did not change significantly whereas GS activity increased severalfold. A characteristic finding in higher cell passages (passage #7) was the loss of GFA-positive stained cells and the appearance of multinucleated cells. We interpret these changes in culture to represent possible signs of cellular senescence.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1986

Growth patterns of primary cultures dissociated from 3-day-old chick embryos: morphological and biochemical comparisons.

Cultures were prepared by dissociating 3-day-old whole chick embryos and plating the dispersed cells on poly-L-lysine-coated dishes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum. By 48 hr in culture, aggregates and neuritic sprouting were observed. Long neuritic bundles connecting cell aggregates were evident by 4 days in culture. Consistent patterns throughout the lifespan of the cultures were contacts between neurites, and flat isolated cells, presumptively glial, emerged. Throughout the lifespan of the cultures, the cholinergic cell population was characterized histochemically by the method of Karnovsky and Roots and biochemically by assaying choline acetyltransferase. By 4 days in culture, all aggregates showed light cholinesterase-positive staining; however, with days in culture, several aggregates had no staining, and some positive-stained aggregates were interconnected with other aggregates showing only spotted positive staining. Choline acetyltransferase activity showed a developmental profile in agreement with the histological findings. The early presence of choline acetyltransferase activity is taken as indication of the early commitment of cholinergic neurons.

Acetylcholinesterase↗
PubMed1986

Responses in astrocytic C6 glioma cells to ethanol and dibutyryl cyclic AMP.

Late passage C-6 glioma cells exhibit astrocytic properties as shown by a characteristic cell morphology and by high levels of the astrocytic cell maker glutamine synthetase (GS). In this study the effects of ethanol (0.2%-1.0% w/v) on the pattern of dibutyryl cyclic AMP (dBcAMP, 1 mM)-induced differentiation were examined using cell number and DNA content as indices for proliferation and cell morphology and GS activity to evaluate differentiation. Differences were observed in the susceptibility of cells to dBcAMP alone, ethanol alone, or simultaneous exposure to both drugs, when cultures were compared at logarithmic and postconfluent phases of growth. Exposure to dBcAMP decreased cell proliferation, induced a characteristic change in cell shape and increased GS activity. In logarithmic phase, simultaneous exposure of cells to ethanol and dBcAMP delayed the dBcAMP-induced change in cell shape and attenuated the mitosis-restricting properties of exposure to dBcAMP. Furthermore, GS activity was greater in dually treated cultures than in cultures treated with dBcAMP alone. We interpret this higher enzyme activity to be the consequence of increased cell-cell contact resulting from larger numbers of cells in the dually treated cultures, coupled with a subsequent dBcAMP-induction of this cytosolic enzyme. In postconfluent cultures, ethanol-exposure did not statistically alter DNA content; whereas GS activity was lower, suggesting that synthesis of GS may be impaired by cellular exposure to ethanol. Furthermore, enzyme activity was also lower in cultures treated with dBcAMP in concert with ethanol than in those treated with dBcAMP alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Effects of neuron-conditioned medium and fetal calf serum content on glial growth in dissociated cultures.

The influence of the microenvironment as assessed by medium conditioned by 6-day-old chick embryo neurons in culture and of the nutrients derived from fetal bovine serum was evaluated in cultures of primary chick embryo glial cells. Glia-enriched cultures from 15-day-old chick embryo were incubated from culture days 3-9 with various concentrations of neuron-conditioned medium, with or without 10% fetal bovine serum in the final culture medium. Also, glial growth was studied in cultures with 5%, 10% or 20% fetal bovine serum in the medium. Glutamine synthetase and 2',3',-cyclic nucleotide 3'-phosphohydrolase were used as astrocytic and oligodendrocytic markers, respectively. Cultures were harvested at day 9. The presence of neuron-conditioned medium in the cultures was associated with persistence of immature glioblast-like cells. This persistence of glial immature cells was also reflected by the lower glutamine synthetase activity in the cultures with neuron-conditioned medium as compared to cultures with neuron-conditioned medium and fetal calf serum. In cultures with 5% neuron-conditioned medium without fetal bovine serum, cyclic nucleotide phosphohydrolase activity was increased. We are assuming that the input of neurons to the microenvironment is partially mediated through the neuron-conditioned medium. Thus, the present findings show that neurons influence the growth and differentiation of glial cells in culture.

Animals↗