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D F Condorelli

Publications and source records attributed to D F Condorelli.

At least 55 records · Page 3Linked to original sources
PubMed1995

Growth conditions differentially affect the constitutive expression of primary response genes in cultured cerebellar granule cells.

Cultured cerebellar granule cells underwent apoptotic degeneration when grown in medium containing 10 instead of 25 mM K+. Knowing that apoptosis is associated with changes in the expression of primary response genes, we have measured c-fos, zif/268, and c-jun mRNA levels during maturation of cultured granule cells grown in 10 or 25 mM K+. The constitutive expression of c-fos and zif/268 was differentially regulated by extracellular K+ concentration at 5 days of maturation in vitro (DIV), when cells grown under suboptimal conditions (i.e. in 10 mM K+) are committed to degenerate. At this stage, c-fos mRNA levels were detectable only in cultures grown in 25 mM K+, whereas zif/268 mRNA levels were dramatically elevated in cultures grown in 10 mM K+. This provides one of the few conditions in which c-fos and zif/268 are differentially regulated in nerve cells. Substantial changes in c-jun, or beta-actin mRNA levels were detectable only at 7 DIV, when the percentage of apoptotic cells had already reached a plateau in cultures grown in 10 mM K+. We speculate that changes in the expression of zif/268 are important in the gene program associated with the induction of apoptosis by trophic deprivation in cultured neurons.

Animals↗
PubMed1995

Seizures increase trkC mRNA expression in the dentate gyrus of rat hippocampus. Role of glutamate receptor activation.

In this study we have shown, by in situ hybridization and RNase protection assay, a significant trkC mRNA increase confined to the dentate gyrus of hippocampus, both after seizures induced by intracerebroventricular injection of kainic acid and bicuculline. Moreover, after bicuculline treatment we observed an earlier increase of trkC mRNA level, which peaked after 3 h and returned back to normal levels by 12 h. In contrast, the kainic acid treatment produced a delayed increase of trkC mRNA, which initiated after 6 h, peaked at 12 h, and returned to normal levels at 24 h. This increase, which involves also trkC mRNA receptor with tyrosine kinase activity, was mediated by non-NMDA receptors and counteracted by GABA potentiating agent diazepam. Using embryonic neuronal cultures from cerebral hemispheres, including hippocampus, we found that glutamate receptor agonists, including glutamate, kainate, NMDA, and t-ACPD, increase trkC mRNA levels with the following rank order of efficacy: NMDA > t-ACPD > kainic acid > glutamate. In conclusion, our data show that trkC mRNA expression in granule cells of the hippocampus dentate gyrus is increased during seizure activity and that it is mediated by non-NMDA receptors.

Animals↗
PubMed1995

Neurotrophins and their trk receptors in cultured cells of the glial lineage and in white matter of the central nervous system.

Previous studies have analyzed the expression of different members of the neurotrophin family and their trk receptors in glial cultures composed mainly or exclusively of type-1 astrocytes, whereas only partial data have been published on other cultured glial types. In this article we compare the mRNA levels for neurotrophins (NGF, BDNF, NT-3, NT-4) and their high-affinity receptors (trkA, trkB, trkC) in cultures enriched in specific glial types, such as microglia, type-1 astroglia, and cells of the O/2A lineage (type-2 astroglia and oligodendroglia). Relatively high levels of NGF mRNA (comparable to those observed in adult rat cerebral cortex) are present in all types of cultured glial cells, except for a low level of expression in cultures enriched in microglial cells. In contrast, BDNF mRNA is undetectable in all cultures examined. NT-3 and NT-4 mRNA molecules, at a level equal to that observed in adult rat cerebral cortex, are easily detected in type-1 astrocyte cultures, whereas their hybridization signals are undetectable in cells of the O/2A lineage and in microglial cultures. The analysis of neurotrophin receptor mRNAs confirms the absence of trkA mRNA, the presence of relatively high levels of trkB mRNA (70-100% of cerebral cortex values), and low levels of trkC mRNA (10-18% of cerebral cortex values) in both cultured astroglial and oligodendroglial cells. Only very low levels of trkB and trkC mRNAs are observed in microglial cultures. Although cultured glial cells express mainly mRNAs encoding for the truncated form of trkB and trkC, a low level of mRNA encoding for the full-length catalytic form of these receptors is detected by the sensitive ribonuclease protection assay.

Animals↗
PubMed1995

Activation of metabotropic glutamate receptors coupled to inositol phospholipid hydrolysis amplifies NMDA-induced neuronal degeneration in cultured cortical cells.

We have studied the influence of class I metabotropic glutamate receptors (mGluRs) on excitotoxic neuronal degeneration in cultured murine cortical neurons grown on a monolayer of astrocytes. These cultures expressed high levels of mGluR5 mRNA, which were comparable to those found in RNA extracts from cerebral cortex. Cortical neurons in mixed cultures were heavily stained with antibodies raised against mGluR5 and were also stained--albeit to a much lower extent--with mGluR1a but not with mGluR1b or c antibodies. Preferential agonists of class I mGluRs, such as quisqualate, 3,5-dihydroxyphenylglycine (DHPG), and trans-azetidine-2,4-dicarboxylic acid (t-ADA), as well as the mixed mGluR agonist, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) all stimulated PPI hydrolysis in cultured cortical cells. The potency of N-methyl-D-aspartate (NMDA) in inducing neuronal degeneration was substantially enhanced when the drug was coincubated with quisqualate, DHPG or t-ADA during a 10-min pulse (paradigm of "fast" toxicity). None of the mGluR agonists influenced neuronal viability by itself. The amplification of NMDA toxicity by quisqualate or DHPG was attenuated by a series of protein kinase C (PKC) inhibitors, suggesting that class I mGluRs operate, at least in part, through activation of PKC. Quisqualate and, in particular, DHPG enhanced excitoxic neuronal degeneration even when applied after the toxic pulse with NMDA. This action is likely to occur early in the maturation of excitotoxic damage, because the functional activity of class I mGluRs was substantially reduced at 2 or 3 hr after the NMDA pulse. These results suggest that activation of class I mGluRs enhances NMDA-receptor mediated neuronal toxicity and encourage the search for selective antagonists for the experimental therapy of acute or chronic neurodegenerative diseases.

Amino Acid Sequence↗
PubMed1995

Inducible and constitutive transcription factor NF-kappa B-like DNA binding activities in rat brain cells cultured in vitro.

Description of constitutive and inducible transcription factors in brain cells is a prerequisite for understanding phenomena of long-term neuronal plasticity. In this report we report that basal levels of NF-kappa B-like transcription factor DNA binding activity (measured by electrophoretic mobility shift assay) are observed in two kinds of cultured rat brain cells, namely neurons of fetal cerebral hemispheres and astroglia derived from newborn brains. In the latter culture, phorbol ester induces additional forms of DNA binding activity, whereas L-glutamate fails to do so. In neurons, neither treatment is effective in inducing NF-kappa B-like DNA binding activity over the basal level. These results are in contrast to the fact that L-glutamate in both neurons and glia elevates DNA binding activity of AP-1, another transcription factor. These data, besides describing behavior of NF-kappa B-like DNA binding activities, also provide some evidence that L-glutamate exerts its modulatory functions on neurons and glia through specific transcription factors like AP-1.

Animals↗
PubMed1995

NMDA receptor-dependent and -independent immediate early gene expression induced by focal mechanical brain injury.

In the present study we analysed, by in situ hybridization, the effects of an extremely localized mechanical brain injury, obtained by the simple needle insertion (30 g) in rat hippocampus or cortex, on the expression of several immediate early genes (c-fos, fosB, c-jun, junB, junD, zif/268). When the needle is deepened into the hippocampus through the cortex, a simultaneous ipsilateral activation of all examined IEGs is observed in both the cerebral cortex and in the dentate gyrus of hippocampus. Maximal effects are detected between 30 and 60 min with the following rank order of induction: zif/268 > c-fos- > junB > fosB > c-jun > junD. On the other hand, when the penetration of the needle is limited to the cerebral cortex the activation of the IEGs (c-fos, fosB, junB and zif/268) spreads throughout the ipsilateral cortex but does not involve the hippocampal region. Systemic administration of ketamine, a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors, blocks IEG expression induced by brain injury in the cerebral cortex and in the hippocampal dentate gyrus. Pretreatment with the anticonvulsant diazepam, the anaesthetic urethane, or the muscarinic receptor antagonist scopolamine do not affect the injury-induced genomic response. An important regional difference in the sensitivity to the blocking effect of ketamine can be observed analysing the results regarding the zif/268 gene expression in the hippocampus. A clear induction of this gene by needle insertion can be detected both in the dentate gyrus and in the hippocampal layers. However, the dentate gyrus induction is completely blocked by the ketamine pretreatment, while the induction in the hippocampal layers is not affected by this NMDA antagonist. The zif/268 induction in the hippocampal layers is not blocked even if the intracerebroventricular administration of a non-NMDA glutamate receptor antagonist is associated to the systemic pretreatment with ketamine. This result represents the first observation of injury-induced neuronal genomic responses that are not critically dependent on the NMDA receptor activity.

Animals↗
PubMed1995

Activation of metabotropic glutamate receptors prevents neuronal apoptosis in culture.

Cultured granule cells grown in serum-containing medium with a "low K+" concentration (10 mM) underwent apoptosis after maturation for 5 days in vitro (5 DIV), a time that coincides with the developmental decline in the activity of metabotropic glutamate receptors (mGluRs) coupled to polyphosphoinositide hydrolysis. The mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) prevented the development of low K(+)-induced apoptosis and the presence of the drug was critical at 6 and 7 DIV, i.e., after the drop of mGluR activity. The neuroprotective action of 1S,3R-ACPD was prevented by the mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) and was mimicked by N-methyl-D-aspartate or carbamylcholine but not by agonists of the mGluR subtypes negatively linked to adenylyl cyclase. In cultures treated either with Li(+)-which reduced polyphosphoinositide response to concentrations of glutamate (5 microM) that approximate those physiologically present in the incubation medium--or MCPG, the development of low K(+)-induced apoptosis already occurred at 4 DIV. Thus, the activation of mGluRs coupled to polyphosphoinositide hydrolysis by endogenous glutamate could contribute to protect cultured granule cells against apoptosis during early stages of maturation.

Animals↗
PubMed1994

Tissue-specific DNA methylation patterns of the rat glial fibrillary acidic protein gene.

The glial fibrillary acidic protein (GFAP) is an intermediate filament protein, specific of the cytoskeleton of astrocytes in the central nervous system. In the present work, as a preliminary step to the study of glial-specific gene expression, we cloned the rat GFAP gene, and we report the sequence of 1.9 kb of the 5' flanking region, exon 1, and the majority of the first intron. By digestion with methylation-sensitive restriction enzymes followed by Southern blot analysis, the methylation status of various CpG sites was examined in this genomic segment. We tested whether structural modification of the GFAP gene, such as DNA methylation, could be related to its tissue-specific transcriptional activity. Therefore, we compared a GFAP-expressing cell population (primary culture of astroglial cells), a mixed population of GFAP-expressing and -nonexpressing cells (adult rat cerebral hemispheres), and a GFAP-nonexpressing tissue (liver). In the 5' flanking region we identified a CpG site at position -1176 whose level of methylation is inversely correlated to GFAP expression. In primary cultured astrocytes, 75% of the GFAP gene alleles were demethylated at this site, while the corresponding value obtained for the cerebral hemispheres was 45%, and for liver only 9%. On the basis of the sequence data, a CpG-rich region (putative CpG island) was identified extending from -38 to +347 and overlapping 80% of the first exon. HhaI and HpaII sites located in the putative CpG island showed a relatively high level of methylation in all the cell populations examined, and did not show any clear correlation with the level of GFAP gene expression or with the methylation status of the -1176 site. Further in vivo developmental studies and in vitro differentiation studies are necessary to better understand the functional differences of the various methylatable CpG sites in the 5' end of the GFAP gene.

Animals↗
PubMed1994

Glutamate receptor-driven activation of transcription factors in primary neuronal cultures.

We have used primary neuronal cultures prepared from fetal cerebral hemispheres to investigate the effects of different glutamate receptor agonists and antagonists on the expression of transcription factor encoding genes, such as c-fos, fosB, c-jun, junB, junD, c-myc, and zif/268. The addition of glutamate (100 microM) to the culture medium rapidly activated c-fos, fosB, c-jun, junB and zif/268 gene expression, reaching the maximal level at 30-60 minutes for zif/268 and at 60 minutes for the other genes. The onset of fosB mRNA accumulation was slightly delayed in comparison to the other genes. No clear induction was found for junD and c-myc. Different glutamate receptor agonists, such as NMDA, kainate, quisqualate, trans-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) were able to increase c-fos, c-jun, and zif-268 mRNA levels with rapid and transient kinetics similar to those observed after glutamate treatment. Similar results were obtained for junB and fosB after kainate and quisqualate stimulation. Pretreatment with MK-801, a non competitive NMDA antagonist, produced an almost complete inhibition of glutamate-driven expression of transcription factor genes, thus suggesting that NMDA receptor plays a major role in glutamate induced-gene expression. On the contrary the kainate/AMPA receptor antagonist, DNQX, did not influence glutamate induced-gene expression. Under the conditions used in the present study, NMDA was effective in inducing the simultaneous activation of several IEGs even when added to the culture medium containing millimolar concentration of magnesium. When experiments were performed in Krebs solution, NMDA was effective in stimulating zif/268 and c-fos mRNAs only in the absence of Mg2+, while glutamate activated c-fos and zif/268 both in the presence and absence of magnesium ions. As expected, NMDA effect was fully inhibited by MK-801. The level of AP-1 DNA binding activity, as measured by electrophoretic mobility shift assay, increased after addition of glutamate and NMDA to cultured neurons and such increase was antagonized by the pretreatment with MK-801.

Animals↗
PubMed1994

Changes in gene expression of AMPA-selective glutamate receptor subunits induced by status epilepticus in rat brain.

In the present investigation we address the question of whether one of the responses to increased neuronal activity is a modification of the expression of the different subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptors (GluR-1, GluR-2, GluR-3). Thus, we used two different models of generalized status epilepticus, as widespread elevated neuronal activity, to study in vivo responses of the AMPA receptor mRNA expression in rat forebrain. By Northern blot analysis and in situ hybridization, we show that one of the delayed responses to LiCl/pilocarpine-induced status epilepticus is a dramatic change in the mRNA level of some subunits of AMPA-selective glutamate receptors. These effects, which appear between 6 and 12 h after the drug treatment, are subunit and brain region specific. The most striking example of differential expression of the three examined GluR mRNAs can be observed in the dentate gyrus of the hippocampus. In this specific brain subregion an increase of GluR-3 mRNA level is induced 12 h after LiCl/pilocarpine treatment, while a clear decrease in GluR-1 mRNA level and no significant change in GluR-2 mRNA level can be observed in the same area under these experimental conditions. Both the GluR-1 decrease and the GluR-3 increase are transient effects and a return to basal level can be observed after 48-72 h. In the CA1 layer of the hippocampus, a parallel decrease of both GluR-1 and GluR-3 expression is found 12-24 h after drug treatment, followed by a recovery of the expression to control values at 48 h. In kainate-induced epilepsy we could reproduce the late increase (12-24 h) in GluR-3 mRNA in the dentate gyrus; however, under this experimental condition, no clear decrease of GluR-1 expression can be observed in this area. A general decrease in mRNA level for the AMPA receptor subunits (GluR-1-3) in the hippocampal layers, in particular in CA3 and CA4 subfields, was also observed. In conclusion the results reported in the present paper reveal a specific regulation of GluR gene expression in the granule cells of the hippocampal dentate gyrus and stimulate further investigation on the functional role of the GluR-3 subunit in the receptor-channel complex.

Animals↗
PubMed1994

Expression of neurotrophins and their receptors in primary astroglial cultures: induction by cyclic AMP-elevating agents.

By northern blot analysis and ribonuclease protection assay, we observed the presence of a high level of trkB mRNA in primary brain cultures devoid of neuronal cells and highly enriched in glial fibrillary acidic protein-positive astroglial cells prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocampus, and striatum. In primary astroglial cultures, the more abundant trkB transcripts code for the truncated receptor without tyrosine kinase activity; probes specific for the full-length trkB mRNA did not detect any signal in northern blot analysis. By the sensitive ribonuclease protection assay, we could show the presence of trkC mRNA in cultured astrocytes, whereas no trkA mRNA was detected. We confirmed the presence of relatively high levels of nerve growth factor and neurotrophin-3 mRNA, and very low basal level of brain-derived neurotrophic factor mRNA. Moreover, we demonstrated that another member of the neurotrophin family, neurotrophin-4, is also expressed in cultured astroglial cells. In view of the fact that many functional receptors for conventional neurotransmitters or neuropeptides present on astroglial cells may act via the adenylate cyclase system, we studied also the effect of agents able to increase the intracellular cyclic AMP concentration. A sharp increase in the trkB mRNA level was observed after treatment of primary astroglial cultures with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. On the contrary, trkC mRNA levels were unaffected by treatment with cyclic AMP-elevating agents. All the neurotrophin mRNAs examined, except neutrophin-4, were increased by 3-isobutyl-1-methylxanthine treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1993

AMPA-selective glutamate receptor subunits in astroglial cultures.

We analysed AMPA ionotropic receptor subunits at the mRNA level (GluR-1 to -4) and at the protein level (GluR-1 and GluR-2/3/4c) in "primary astroglial cultures" (non-neuronal cell cultures highly enriched in glial fibrillary acidic protein [GFAP] positive cells) prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocampus, and striatum and in "brain non-neuronal cell cultures" (low percentage of GFAP positive cells) prepared from cerebellum, brainstem, mesencephalon, and hypothalamus. For comparison, we also determined AMPA subunit mRNA and protein levels in different brain regions. By Northern blot analysis mRNAs for the AMPA receptor subunits (GluR-1,-2,-3,-4) were detected in primary rat cerebral hemispheres astroglial cultures. Immunoblotting analysis with anti-GluR-1 and anti-GluR-2/3/4c polyclonal antibodies confirmed the presence of low level of immunoreactive proteins of the same size of those identified in vivo as GluR subunits. Expression of GluR genes varied depending on the brain area used as starting material for the preparation of the cultures: GluR-1, -2, and -3 were mainly expressed in cortical cultures, while GluR-4 expression predominated in brainstem derived cultures. Interestingly this pattern of expression correlates with that observed in the intact brain, where high levels of GluR-4 mRNA and low levels of the other GluR subunits were found in the brainstem. In conclusion our results confirm the existence of glutamate ionotropic receptors of the AMPA type in primary astroglial cultures and suggest that GluR-4 is the main AMPA receptor subunit expressed in non-neuronal cells of the central nervous system.

Animals↗
PubMed1993

Characterization of metabotropic glutamate receptors negatively linked to adenylyl cyclase in brain slices.

We have characterized the pharmacological profile of activation of metabotropic glutamate receptors negatively linked to adenylyl cyclase (mGluR decreases cAMP) in brain slices. Among the putative mGluR agonists, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), were the most potent inhibitors of forskolin-stimulated cAMP formation in hippocampal slices, followed by ibotenate, L-2-amino-3-phosphonopropionate (AP3), quisqualate, L-glutamate and beta-N-methylamino-L-alanine (BMAA). Inhibition of forskolin-stimulated cAMP formation by DL-2-amino-4-phosphonobutanoate (AP4) was biphasic, suggesting that the drug interacts with more than one mGluR decreases cAMP subtype. Both L-AP4 and L-serine-O-phosphate (a restricted analogue of AP4) were much more effective in inhibiting forskolin-stimulated cAMP formation than their D-isomers, indicating that interaction of these drugs with the mGluR decreases cAMP is stereoselective. Despite the fact that DCG-IV and ibotenate behave as NMDA receptor agonists, their effect was insensitive to MK-801. The regional pattern of expression of mGluR decreases cAMPS, as estimated by using 1S,3R-ACPD as an agonist, did not correlate with the steady-state levels of mGluR2 mRNA. Thus, 1S,3R-ACPD inhibited forskolin-stimulated cAMP in slices from hippocampus, cerebral cortex, corpus striatum, olfactory tubercle or hypothalamus, but not in slices from olfactory bulb or cerebellum; in contrast, mGluR2 mRNA levels were high in the olfactory bulb and very low in the corpus striatum. 1S,3R-ACPD also inhibited forskolin-stimulated cAMP formation in cortical membranes, excluding the involvement of trans-synaptic mechanisms in the activity of mGluR decreases cAMPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1993

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells.

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu > quisqualate (quis) = ibotenate (ibo) > (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) > beta-methyl-amino-L-alanine (BMAA) and in terms of potency, quis > ACPD > Glu > ibo = BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg2+). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1S,3R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg2+, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg(2+)-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms).(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1993

Platelet-activating factor and its methoxy-analogue ET-18-OCH3 stimulate immediate early gene expression in rat astroglial cultures.

In the present paper we analyzed c-fos and zif/268 expression in rat primary astroglial cell cultures after treatment with Platelet-activating Factor (PAF) and its 2-O-methyl-analogue, 1-O-octadecyl-2-O-methoxy-glycero-3-phosphocholine (ET-18-OCH3). Both compounds, at a dose (2 microM) that did not produce toxic effects on astroglial cells, induced a rapid and transient increase of c-fos and zif/268 mRNA level. Pretreatment of astroglial cells with the PAF antagonist BN50730 (5 microM) 10 min prior to the addition of alkyl-phospholipids almost completely prevented the activation of the immediate early genes. On the contrary triazolam, another PAF inhibitor, did not block PAF induced gene expression when added to the medium at 5 microM concentration. ET-18-OCH3 effect on gene expression is blocked by the same antagonist (BN50730) which is effective in inhibiting PAF effect on astrocytes, suggesting that both substances act through the same binding site. Results obtained support the view that astroglial cells are a cellular target for this lipid mediator, and, like macrophages, respond to its methoxy-analogue.

Animals↗
PubMed1993

Metabotropic glutamate receptors in cultured cerebellar granule cells: developmental profile.

Excitatory amino acid (EAA)-induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR) agonists, including L-Glu, quisqualate, and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid: The stimulation of [3H]inositol monophosphate ([3H]-InsP) formation was low at 2 days in vitro (DIV), but the response increased steeply, reaching a peak at 4 DIV, followed by a progressive decline. In contrast, carbamylcholine-induced PPI hydrolysis exhibited a plateau after a pronounced increase during the first week in vitro. At 6 DIV, but not at 4 DIV, when the activity peaked, PPI hydrolysis elicited by Glu was reduced by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, indicating that in cultured granule cells, NMDA receptors contribute to [3H]-InsP formation and that this component of the response develops relatively late. Accordingly, NMDA-induced [3H]-InsP formation, estimated under Mg(2+)-free conditions, increased markedly from very low values at 2 DIV to a plateau at 8-10 DIV. The developmental pattern of EAA-induced PPI hydrolysis was paralleled by changes in the level of an mRNA for a specific mGluR subtype (mGluR1 mRNA). RNA blot analysis performed with the pmGR1 cDNA probe revealed that the hybridization signal in RNA extracts from cultures at 1 DIV was very weak, but mGluR mRNA levels increased dramatically between 1 and 3 DIV, followed by a progressive decrease, so that by 15 DIV the mRNA levels were only approximately 10% of the values at 3 DIV.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

Induction of primary response genes by excitatory amino acid receptor agonists in primary astroglial cultures.

We have characterized the genomic response of astroglial cells to excitatory amino acids by using selective agonists and antagonists for the various receptor subtypes and by analyzing different primary response genes, such as members of the Fos (c-fos and fosB) and Jun (c-jun, junB, and junD) families, zif/268, and c-myc. A rapid and transient elevation of mRNA levels for c-fos, fosB, c-jun, junB, and zif/268 was observed after addition of glutamate to cultured astrocytes, whereas junD and c-myc expression was not affected. The level of AP-1 DNA binding activity, as measured by the electrophoretic mobility shift assay, also increased after addition of glutamate to cultured astrocytes. Glutamate-induced c-fos expression was not affected by the N-methyl-D-aspartate receptor antagonists MK-801 and D-2-amino-5-phosphonopentanoate, by the kainate/alpha-amino-3-hydroxy-5- methylisoxazole-4-propionate (AMPA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or by the broad-spectrum antagonist kynurenate. Kainate and AMPA were also effective in inducing primary response gene expression, and their actions were antagonized by kynurenate and DNQX but not by MK-801. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid, a selective agonist for the metabotropic glutamate receptor, induced primary response gene expression, but its action was not antagonized by different glutamate antagonists, including L-2-amino-3-phosphonopropionate. In conclusion, our data suggest that cultured astrocytes express both kainate/AMPA ionotropic receptors and metabotropic receptors coupled to the rapid and coordinated activation of different classes of transcriptional factor genes.

Animals↗