共查询到20条相似文献,搜索用时 31 毫秒
1.
Glutamine synthetase and energy metabolism enzymes in cultivated chick neurons and astrocytes: modulation by serum and hydrocortisone 总被引:1,自引:0,他引:1
Primary cultures of astroglial cells and of neurons obtained from chick embryos were grown in culture medium with and without serum added. The expression of glutamine synthetase (GS) in the cultured nerve cells was investigated immunocytochemically and biochemically. The cellular localization of GS in cerebellar tissue sections and in cerebral cortex of chick embryos was investigated by immunohistochemical staining. In tissue sections the enzyme is only present in astrocytes and their processes; neurons and their structures do not express the enzyme. In contrast, in pure neuronal primary cultures, a high level of GS was detected by biochemical and immunochemical methods. Thus, our results clearly indicate the presence of GS in pure neuronal cell cultures and its absence in this type of cells in vivo. Removal of serum from the culture medium enhanced GS levels in primary astrocyte cultures, but was without effect on GS activity in neurons. Addition of calf serum to the culture medium induces a two-fold increase of cellular lactate dehydrogenase (LDH) activity in neurons by increasing specifically the M subunit containing isoenzymes. The sensitivity of chick astroglial cells and neurons toward the GS inducing effect of hydrocortisone and modulation of its effect by serum was also investigated. Differences in the sensitivity of the two types of nerve cells in culture toward the GS inducing effect of hydrocortisone, and the effect of serum could be demonstrated. 相似文献
2.
Modifications in astrocyte morphology and calcium signaling induced by a brain capillary endothelial cell line 总被引:4,自引:0,他引:4
Astrocytes extend specialized endfoot processes to perisynaptic and perivascular regions, and thus are positioned to mediate the bidirectional flow of metabolic, ionic, and other transmissive substances between neurons and the blood stream. While mutual structural and functional interactions between neurons and astrocytes have been documented, less is known about the interactions between astrocytes and cerebrovascular cells. For example, although the ability of astrocytes to induce structural and functional changes in endothelial cells is established, the reciprocity of brain endothelial cells to induce changes in astrocytes is undetermined. This issue is addressed in the present study. Changes in primary cultures of neonatal mouse cortical astrocytes were investigated following their coculture with mouse brain capillary endothelial (bEnd3) cells. The presence of bEnd3 cells altered the morphology of astrocytes by transforming them from confluent monolayers into networks of elongated multicellular columns. These columns did not occur when either bEnd3 cells or astrocytes were cocultured with other cell types, suggesting that astrocytes undergo specific morphological consequences when placed in close proximity to brain endothelial cells. In addition to these structural changes, the pharmacological profile of astrocytes was modified by coculture with bEnd3 cells. Astrocytes in the cocultures showed an increased Ca2+ responsiveness to bradykinin and glutamate, but no change in responsiveness to ATP, as compared to controls. Coculturing the astrocytes with a neuronal cell line resulted in increased responsiveness of the glial responses to glutamate but not to bradykinin. These studies indicate that brain endothelial cells induce changes in astrocyte morphology and pharmacology. 相似文献
3.
《International journal of developmental neuroscience》1994,12(4):363-372
We have investigated the effects of cell passaging and time in culture on astrocyte morphology, transferrin expression and the expression of two main astrocyte markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS; EC 6.3.1.2). When primary astrocytes were subcultured, giving rise to secondary and tertiary cultures, their morphology changed, regardless of the split ratio used to passage the cells. Correlating with this morphological change, a dramatic increase in the accumulation of GFAP and GS mRNAs was observed after cells had been passaged. This effect was in marked contrast to the moderate increase in the levels of GFAP and GS mRNAs observed over several weeks in primary culture. Hydrocortisone induction of GS gene expression was not affected by cell passage. Transferrin mRNA, which is not normally found in astrocytes in vivo, was expressed at a high level in primary cultures of astrocytes. However, transferring mRNA almost completely disappeared after the second passage. Astrocyte-conditioned media, or co-cultures with oligodendrocytes, modified transferrin gene expression. Taken together, these results show that subculturing of primary rat astrocytes leads to a dramatic change in the genetic expression of several proteins and provides a new approach to modify astrocyte differentiation in vitro. 相似文献
4.
In this study we examined the effects of prolonged l-trans-pyrrolidine-2,4-dicarboxylate (PDC)-induced glutamate reuptake blockade on the viability of glial cells in cerebellar granule cell cultures. Immunofluorescence staining for the glial-specific intermediate filament protein, GFAP, revealed that the PDC- induced increase of extracellular glutamate concentration was accompanied by increased astrocyte death, while neurons and oligodendrocytes remained intact and viable. Astrocytic cell death was manifested as fragmentation of processes and cell bodies. The selective astrocyte death was completely prevented by the competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, NBQX (10 microM), whereas MK-801 (10 microM), a noncompetitive blocker of N-methyl-D-aspartate receptors, gave only partial protection. Double staining for GFAP and the AMPA receptor subunits GluR2/3 showed that astrocytes had much higher immunoreactivity for GluR2/3 than neurons or oligodendrocytes, suggesting that the number of AMPA receptors is likely to be higher on astrocytes. Furthermore, we employed real-time RT-PCR to measure GluR1-4 subunit mRNA expression in control and PDC-exposed cultures. Following treatment with PDC, GluR1 and GluR4 mRNAs were reduced by 40% and GluR3 was reduced by 70% relative to control levels. In contrast, GluR2 expression was not affected by the PDC treatment, indicating that GluR3 was the dominant type of AMPA receptor subunit expressed on astrocytes. Our results show that astrocytes appear to be more vulnerable than neurons or oligodendrocytes to a gradual increase in the extracellular glutamate concentration, suggesting that astrocytes may be critically involved in the pathophysiology of slowly developing chronic neurodegenerative disorders. 相似文献
5.
Glutamine and glutamate transport in cultured neuronal and glial cells 总被引:15,自引:0,他引:15
The uptake of L-glutamine in neuronal and glial cultures derived from rat cerebral hemispheres was found to be mediated by a low affinity-high capacity mechanism which was concentrative in both cell types; the calculated Km and Vmax were twice as high in glial than in neuronal cultures. In contrast L-glutamate was taken up by a high affinity system which was particularly efficient and concentrative in the glial cells. Different transport mechanisms for L-glutamine appeared to operate in the two cell types: L-glutamine uptake in neurons was sodium-dependent, specifically inhibited by L-glutamine but not affected by high potassium concentrations in the external medium; on the other hand, glial glutamine transport was decreased when potassium concentration increased, was sodium-independent and significantly inhibited by 3 structurally related amino acids. No significant contribution of homoexchange could be detected in either cell type. After [14C]glutamine preincubation, the radioactivity released into the superfusion medium by neuronal cells was increased in the presence of a high potassium concentration; no such effect could be seen in the case of glial cultures. A regulatory mechanism is suggested where astrocyte depolarization and repolarization would channel a flux of glutamine toward the neurons, subsequent to a glutamate flux in the opposite direction. 相似文献
6.
Marina Pizzi Alessandra Valerio Virginia Arrighi Paola Galli Marco Belloni Marina Ribola Antonella Alberici PierFranco Spano Maurizio Memo 《The European journal of neuroscience》1995,7(7):1603-1613
Short-term exposure of primary cultures of cerebellar granule cells from neonatal rat brain to high concentrations of glutamate resulted in a significant increase of both immunoreactivity to and mRNA levels of tau protein. Time-course experiments revealed the increases of tau immunoreactivity and mRNA levels to be maximal 2 h after the glutamate pulse. To investigate the relationship between newly synthesized tau protein and glutamate-induced neurotoxicity, neurons were preincubated with a specific tau antisense oligonucleotide. This treatment resulted in (i) inhibition of the glutamate-induced increase of tau immunoreactivity and (ii) a decrease in the sensitivity of the neurons to neurotoxic concentrations of glutamate. These data indicate that induction of the cytoskeleton-associated tau protein participates in the cascade of events promoted by glutamate leading to neurodegeneration. 相似文献
7.
The effect of selective injury to dopaminergic neurons on the expression of glial cell line-derived neurotrophic factor (GDNF) was examined in substantia nigra cell cultures. H(2)O(2), mimicking increased oxidative stress, or l-DOPA, the main symptomatic treatment for Parkinson's disease, increased GDNF mRNA and protein levels in a time-dependent mode in neuron-glia mixed cultures. The concentration dependence indicated that mild, but not extensive, injury induced GDNF up-regulation. GDNF neutralization with an antibody decreased dopaminergic cell viability in H(2)O(2)-treated cultures, showing that up-regulation of GDNF was protecting dopaminergic neurons. Neither H(2)O(2) nor l-DOPA directly affected GDNF expression in astrocyte cultures, but conditioned media from challenged mixed cultures increased GDNF mRNA and protein levels in astrocyte cultures, indicating that GDNF up-regulation was mediated by neuronal factors. Since pretreatment with 6-OHDA completely abolished H(2)O(2)-induced GDNF up-regulation, we propose that GDNF up-regulation is triggered by failing dopaminergic neurons that signal astrocytes to increase GDNF expression. 相似文献
8.
Increased matrix metalloproteinase (MMP) proteolytic activity contributes to the pathogenesis of many neuroinflammatory and neurodegenerative conditions in the CNS. To fully understand this process, it is important to define the MMP expression profile of specific cell types, including the CNS-resident cells astrocytes and microglia. While previous studies have characterized astrocyte MMP expression by using mixed glial cultures, these results are likely complicated by the presence of contaminating microglia within these cultures. In the current study, we sought to clarify this complexity, by taking a novel approach to prepare pure astrocyte cultures entirely devoid of microglia, by promoting neural stem cell (NSC) differentiation into astrocytes. The MMP expression profile of mixed glial cultures, neurosphere-derived astrocytes, and pure microglia was characterized by RNase protection assay. This revealed that MMP gene expression is largely cell-type specific. Astrocytes constitutively expressed MMP-11, MMP-14, and MMP-2 and showed induction of MMP-3 in response to IL-1beta but did not respond to lipopolysaccharide (LPS). In contrast, microglia constitutively expressed high levels of MMP-12 and showed strong induction of MMP-9 and MMP-14 in response to LPS. Gelatin zymography confirmed that LPS and TNF-alpha induced strong expression of MMP-9 in microglia but not astrocytes. In summary, these studies demonstrate that neurosphere-derived astrocytes represent an attractive alternative system in which to study astrocyte behavior in vitro. Using this system, we have shown that astrocytes and microglia express distinct sets of MMP genes and that microglia, not astrocytes, are the major source of MMP-9 in response to LPS or TNF-alpha. 相似文献
9.
10.
We have previously shown that in hypothalamic mixed neuronal-glial cultures both astrocytic shape and distribution of glial fibrillary acidic protein (GFAP) are modified by estradiol. In the present study, we have investigated whether or not the presence of neurons is necessary for these hormonal effects. In mixed neuronal-glial hypothalamic cultures the proportion of process-bearing GFAP-immunoreactive cells was significantly increased after treatment for 30 min with 10(-12) M 17 beta estradiol. This effect was present for at least 1 day and was reverted by incubating the cells in estradiol-free medium. Estradiol incubation resulted in a progressive differentiation of GFAP-immunoreactive cells from a flattened epithelioid morphology to bipolar, radial, and stellate shapes. This effect was not observed in pure hypothalamic glial cultures. Furthermore, incubation of hypothalamic glial cells with medium conditioned by estradiol-treated mixed hypothalamic cultures did not affect the shape of GFAP-immunoreactive astrocytes. In contrast, addition of hypothalamic neurons, but not cerebellar neurons or fibroblasts, to established hypothalamic glial cultures affected the development of estradiol sensitivity in astrocytes. These results indicate that estradiol induction of shape changes in hypothalamic astrocytes is not only dependent on the presence of hypothalamic neurons, but that physical contact between astrocytes and neurons is necessary for the manifestation of the effect of this hormone. 相似文献
11.
Glutamate is the most abundant excitatory amino acid in the central nervous system. It has also been described as a potent toxin when present in high concentrations because excessive stimulation of its receptors leads to neuronal death. Glial influence on neuronal survival has already been shown in the central nervous system, but the mechanisms underlying glial neuroprotection are only partly known. When cells isolated from newborn rat retina were maintained in culture as enriched neuronal populations, 80% of the cells were destroyed by application of excitotoxic concentrations of glutamate. Massive neuronal death was also observed in newborn retinal cultures containing large numbers of glia, or when neurons were seeded onto feeder layers of purified cells prepared from immature (postnatal 8 day) rat retina. When newborn retinal neurons were seeded onto feeder layers of purified glial cells prepared from adult retinas, application of excitotoxic amino acids no longer led to neuronal death. Furthermore, neuronal death was not observed in mixed neuron/glial cultures prepared from adult retina. However, in all cases (newborn and adult) application of kainate led to amacrine cell‐specific death. Activity of glutamine synthetase, a key glial enzyme involved in glutamate detoxification, was assayed in these cultures in the presence or absence of exogenous glutamate. Whereas pure glial cultures alone (from young or adult retina) showed low activity that was not stimulated by glutamate addition, mixed or co‐cultured neurons and adult glia exhibited up to threefold higher levels of activity following glutamate treatment. These data indicate that two conditions must be satisfied to observe glial neuroprotection: maturation of glutamine synthetase expression, and neuron‐glial signalling through glutamate‐elicited responses. GLIA 25:229–239, 1999. © 1999 Wiley‐Liss, Inc. 相似文献
12.
Mercuric chloride, but not methylmercury, inhibits glutamine synthetase activity in primary cultures of cortical astrocytes 总被引:4,自引:0,他引:4
Methylmercury (MeHg) is highly neurotoxic with an apparent dose-related latency period between time of exposure and the appearance of symptoms. Astrocytes are known targets for MeHg toxicity and a site of mercury localization within the central nervous system (CNS). Glutamine synthetase (GS) is an enzyme localized predominately within astrocytes. GS converts two potentially toxic molecules, glutamate and ammonia, to the relatively non-toxic amino acid, glutamine. During prolonged exposure to MeHg, inorganic mercury (I-Hg) accumulates within the brain, suggesting in situ demethylation of MeHg to I-Hg. To determine if speciation of mercurials would differentially alter GS activity and expression, neonatal rat primary astrocyte cultures were exposed to MeHg or mercuric chloride (HgCl2) for 1 or 6 h. MeHg produced no changes in GS activity, protein, or mRNA at any time or dose tested. In contrast, HgCl2 produced a dose dependent decrease in astrocytic GS activity at both 1 and 6 h. There were no changes in GS protein or mRNA levels following HgCl2 exposure. Additional studies were carried out to determine GS activity in cell lysates incubated with HgCl2 or MeHg. In cell lysates, HgCl2 was three-times more potent than MeHg in inhibiting GS activity. The inhibition of GS activity in cell lysates by HgCl2 was reversed by the addition of dithiothreitol (DTT), while DTT did not restore GS activity following MeHg. These data suggest that astrocytic GS activity is not inhibited by physiologically relevant concentrations of MeHg, but is inhibited by I-Hg, which is present in CNS following chronic MeHg exposure. 相似文献
13.
F. Maher 《Journal of neuroscience research》1995,42(4):459-469
Immunofluorescence analysis was used to study the cellular localization of glucose transporters 1 and 3 (GLUT1 and GLUT3) in primary rat neuronal and glial cultures. In primary cultured cerebellar granule neurons and cortical neurons, GLUT3 was detected in a pattern consistent with a generalized cell surface distribution. GLUT3 distribution corresponded most closely with the neural cell adhesion molecule (NCAM), and showed overlapping but distinct distributions compared to synaptophysin, microtubule-associated protein 2 (MAP2), neurofilament protein, and growth-associated protein (GAP43). Culture of neurons in the presence of glia did not alter the cellular localization of GLUT3. GLUT1 was detectable in primary cerebellar granule neurons both at the cell surface and in the cytoplasm, and appeared decreased in neurons cocultured with glia. GLUT1, but not GLUT3, was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes present in mixed neuronal-glial cultures derived from cerebellum and cerebral cortex, as well as in cortical astrocyte cultures. GLUT1, but not GLUT3, was also detected in microglia and oligodendrocytes present in these cultures. This study indicates a generalized cell surface expression of the glucose transporters expressed in neurons and glia, rather than selective targeting to different cellular domains or subcellular locations. © 1995 Wiley-Liss, Inc. 1 This article is a US Government work and, as such, is in the public domain in the United States of America. 相似文献
14.
A Espinosa de los Monteros S Kumar S Scully R Cole J de Vellis 《Journal of neuroscience research》1990,25(4):576-580
We have previously shown by immunocytochemistry in rat primary glial cultures that transferrin (Tf) is an early developmental marker for oligodendrocytes. The present work addresses the issue of Tf gene expression and synthesis by neural cells in vitro. For this purpose, we used rat embryonic neuronal cultures and newborn glial cultures of astrocytes and oligodendrocytes. Cultured fibroblasts and C6 glioma cells were used as negative controls. We found that Tf mRNA is present in oligodendrocytes, astrocytes, and neurons. However, oligodendrocytes and astrocytes, but not neurons, were shown to synthesize and secrete Tf. Neither fibroblasts nor C6 glioma cells expressed detectable amounts of Tf mRNA. Tf mRNA levels in astrocyte cultures appeared to be under hormonal control since hydrocortisone markedly reduced message levels. These results show that both astrocytes and oligodendrocytes can synthesize and secrete Tf under cell culture conditions. However, epigenetic factors, such as hydrocortisone, may repress the expression of Tf in astrocytes in vivo. 相似文献
15.
16.
Glial cells dissociated from newborn and aged mouse brain 总被引:3,自引:0,他引:3
A Vernadakis D Mangoura N Sakellaridis S Linderholm 《Journal of neuroscience research》1984,11(3):253-262
Changes occurring with days in culture and cell passage in cultured glial cells derived from newborn vs aged (18-mo) mouse cerebral hemispheres were compared. The activities of the enzymes glutamine synthetase (GS), an astrocyte marker, and 2',3'-cyclic nucleotide 3'- phosphohydrolase (CNP), an oligodendrocyte marker, were determined. In addition, glial fibrillary acidic protein (GFA) and glycerol phosphate dehydrogenase (GPDH) immunoreactivity was used to morphologically identify astrocytes and oligodendrocytes, respectively. In cultures derived from newborn mouse cerebral hemispheres, both GS and CNP activity and GFA-positive and GPDH-rhodamine-positive cells were present with cell passage. In general, GS activity did not change in early cell passage in cultures from either newborn or aged mouse; in passage 5, GS was high in both sources of cell populations. CNP activity increased with cell passage in cultures derived from newborn mouse; in cultures derived from aged mouse CNP was low in the primary cultures, increased with cell passages 2 and 3, and declined with passages 4 and 5. The survival of astrocytes as shown by GS and the decline in oligodendrocytes as shown by CNP was also supported by an increase in the proportions of GFA and GPDH immunoreactive cells. We interpret the increase in GS activity to parallel the astrogliosis observed in vivo in the aging brain. Moreover, the decline in oligodendrocytes in culture may represent a shift of balance between glial cell types that appears to be influenced by the age of brain tissue and time in culture. 相似文献
17.
Using an antiserum against calretinin, a calcium-binding protein, we discovered two distinct neuronal cell types that stain intensely in enriched cerebellar granule cells. One neuronal cell type resembles unipolar brush cells, whereas the other resembles Lugaro cells. During early culture times, these calretinin-positive neurons are most numerous but represent less than one percent of the total neuronal population. In cultured cells, calretinin mRNA levels peak at day three in vitro, followed by a rapid decline to undetectable levels by day six in vitro. However, calretinin-immunoreactive neurons are observed up to 29 days in vitro. Excitotoxic concentrations of glutamate receptor agonists failed to elicit an excitotoxic response on the intensely staining calretinin-positive neurons, whereas greater than 95% of the cerebellar granule cells were susceptible to the excitotoxic actions of the glutamate receptor agonists. To distinguish between the two possibilities that calretinin-positive neurons either do not express glutamate receptors or they are not susceptible to the excitotoxic effects of glutamate receptor agonists, we performed immunocytochemistry using glutamate receptor antibodies to detect the presence of receptor protein. We found that the AMPA/kainate glutamate receptor (GluR2R3) colocalized with calretinin, suggesting that calretinin-immunoreactive neurons express the AMPA/kainate receptor; cerebellar granule cells, which are known to express this receptor, were also immunoreactive for the GluR2R3 receptor. 相似文献
18.
Pyruvate recycling is a pathway for complete oxidation of glutamate. The cellular location and the physiological significance of such recycling has been debated during the last decade. The present study was aimed at elucidating whether recycling takes place in neuron-enriched cultures of dissociated cerebella, consisting mainly of glutamatergic granule cells, some GABAergic neurons, and few astrocytes. These cultures and cultures of astrocytes from cerebellum were incubated in medium containing [U-(13)C]glutamate, and cell extracts were analyzed by gas chromatography and mass spectrometry. Additionally, in the case of the neuron-enriched cultures, a magnetic resonance (MR) spectrum was obtained. It could be shown that the atom percentage excess of the isotopomer representing pyruvate recycling in glutamate (M + 4) was similar for astrocytes and neuron-enriched cultures. However, the latter showed more recycling in glutamine (synthesized in the small fraction of astrocytes) than the pure astrocyte cultures, whereas the reverse was the case for aspartate. In fact, the atom percentage excess of the isotopomer representing pyruvate recycling in glutamine was slightly but significantly higher than that in glutamate in the neuron-enriched cultures. It can be concluded that pyruvate recycling is clearly present in neurons, and this was verified by MR spectroscopy. 相似文献
19.
Annie Lannuzel Marc Tardieu Christiane Hery Jean Vianney Barnier Jean Didier Vincent Bernard Guibert Huynh Van Tan Francoise Gray 《Annals of neurology》1997,42(6):847-856
Detection of apoptotic neurons and microglial cells in the brains of human immunodeficiency virus type 1 (HIV-1)-infected patients has suggested that programmed cell death may be implicated in the physiolpathology of HIV-1 encephalopathy. To analyze in vitro the intracellular signals induced by HIV-1 in human neurons and the associated neuronal death, we tested cultured human central nervous system (CNS) cells for apoptosis induced by HIV-1 and gp120 and for signaling pathways activated by gp120. HIV-1 and gp120 induced apoptosis of neurons and microglial cells but not of astrocytes or transformed microglial cells. Gp120 activated c-Jun N-terminal kinase (JNK) and p42 extracellular regulated kinase (ERK) in primary CNS cells, with an early peak of activation at 2 to 5 minutes that was not present when pure microglial or astrocyte cultures were tested, followed by a late and sustained activation (10 and 60 minutes) in primary and enriched glial cell cultures as well as in transformed microglial cells. This demonstrates that gp120 could be an effector of HIV-1-induced apoptosis in the CNS and act directly on neuronal and glial cells. 相似文献
20.
《Brain research. Molecular brain research》1994,21(1-2):150-156
The high-affinity GABA transporter in neurons and glial cells is the primary means of inactivating synaptic GABA. In the present study, a rat GABA transporter (GAT-1)-specific probe was used to quantitate GAT-1 mRNA in cultured neurons and glial cells from rat brain. GAT-1 mRNA is expressed in neurons but not in pure cultures of astrocytes. Incubation of neurons with forskolin led to concentration- and time-dependent decreases in GAT-1 mRNA. This effect could be also achieved by chronic exposure of neurons to 8-Br-cAMP and dib-cAMP but not with 1,9-dideoxyforskolin. This effect on the levels of GAT-1 mRNA correlates with a decrease in the Na+-dependent GABA transport activity in neurons. Treatment with agents that increase cellular levels of cAMP did not affect GABA transport or GAT-1 mRNA expression in glial cells. 相似文献