Biomedical subjects

R Balázs

Publications and source records attributed to R Balázs.

At least 19 recordsLinked to original sources

PubMed1995

Agonist-induced down-regulation of NMDA receptors in cerebellar granule cells in culture.

In contrast to the acute toxic effect of NMDA on mature cerebellar granule cells, chronic treatment with NMDA (140 microM from 1 to 9 days in vitro) did not compromise cell survival. Such treatment markedly suppressed NMDA receptor activity: at 8 days in vitro NMDA-induced 45Ca2+ influx was reduced by approximately 60% and acute exposure to NMDA (highest concentration tested, 1 mM) at 9 days in vitro did not cause detectable toxicity. The reduction in NMDA receptor activity was accompanied by a significant decrease (approximately 80% at 9 days in vitro) in the level of the NR1 and the NR2A NMDA receptor subunit protein, detected using the selective photoaffinity ligand [125I]CGP55802A. It seems, therefore, that the agonist-induced decrease in NMDA receptor activity is due to receptor down-regulation. In contrast to the marked influence of chronic NMDA exposure on the cellular content of the NMDA receptor subunit proteins, mRNA levels of the different subunits (NR1, NR2A, NR2B and NR2C) were not significantly affected. It seems, therefore, that agonist-induced down-regulation of the NMDA receptor involves critically mRNA translation and/or post-translational regulation.

Animals↗

PubMed1995

NMDA and kainate induce internucleosomal DNA cleavage associated with both apoptotic and necrotic cell death in the neonatal rat brain.

Injection of N-methyl-D-aspartate (NMDA) or kainate in the striatum of 7-day-old rats induced massive cell loss in the ipsilateral striatum, hippocampus and inner cortical layers. In order to examine whether apoptosis contributes to cell death in this model of excitotoxic injury we examined the progression of internucleosomal DNA fragmentation and changes in cellular ultrastructure. Agarose gel electrophoresis of DNA extracted from the ipsilateral striatum, cerebral cortex and hippocampus clearly showed breakdown of DNA into oligonucleosome-sized fragments, indicative of apoptosis, 12 h post-NMDA injection. In addition, an increase between 12 and 24 h was observed as well as a continuous presence 5 days later. Kainate induced a similar time course of oligonucleosomal DNA fragmentation, but the intensity of the ethidium bromide stained bands was less compared with that observed for NMDA. DNA fragmentation was not detected in animals intrastriatally injected with Tris-HCl or in animals treated with MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate, 1 mg/kg] 30 min after NMDA injection. MK-801 had no effect on DNA fragmentation induced by kainate. In addition to agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) was used for detection of DNA fragmentation in sections. A gradual increase in the density of both apoptotic and non-apoptotic TUNEL nuclei was found in the anterior cingulate (ACC) and retrosplenial (RSC) areas of the cortex, the striatum, and the CA1 area and dentate gyrus of the hippocampus over the first 24 h post-NMDA or kainate injection. In the contralateral hemisphere hardly any TUNEL nuclei were present and their density was comparable with that in animals injected with vehicle only. In the ipsilateral mammillary nucleus (MN), which showed no signs of acute cell swelling after intrastriatal injection with NMDA, internucleosomal DNA fragmentation was found 24 and 48 h after intrastriatal NMDA injection. Here, the density of TUNEL cells with apoptotic morphology was high at 12 and 24 h post-NMDA injection but returned to control levels by 5 days. Electron microscopy showed cells with a clearly apoptotic morphology in the ACC and RSC and in the MN 24 h after NMDA injection. In the CA1 area of the hippocampus a necrotic, rather than an apoptotic, ultrastructure prevailed, indicating that the TUNEL method stained both apoptotic and necrotic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

AMPA receptors in cerebellar granule cells during development in culture.

The survival and maturation of differentiating cerebellar granule cells in culture are known to be promoted by excitatory amino acids (EAAs) which, however, compromise the survival of mature cells. In contrast to the trophic effect, the toxic effect of alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropiate (AMPA) could only be elicited when the desensitisation of AMPA receptors was blocked, cyclothiazide being used in this study. Nevertheless, even under these conditions, toxicity induced by AMPA in contrast to kainate was, at 9 DIV, only half of the maximal toxicity attained by 13-16 DIV. Since cellular responses to AMPA depend so dramatically on the maturational stage of granule cells, we examined here whether this characteristic is related to developmental changes in AMPA receptor properties, which may result from changes in the subunit composition of the receptor. In contrast to toxicity, AMPA-induced 45Ca2+ influx (determined in the presence of cyclothiazide and the NMDA receptor blocker MK-801) reached a maximum already at 9 DIV. This also applied to a fraction of the 45Ca2+ uptake which persisted either after Cd2+ application or under Na(+)-free conditions and therefore presumably was mediated directly through AMPA receptor channels. Quantitative analysis of Western blots showed that the amounts of GluR4 and to a lesser extent GluR2/3/4c are substantial already at 2 DIV, remaining fairly constant until 9 DIV, followed by an increase by 16 DIV. However GluR1, which is hardly detectable in granule cells in vivo and is also low early in vitro, increased almost linearly with cultivation time.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Regulation of the expression of NMDA receptor subunits in rat cerebellar granule cells: effect of chronic K(+)-induced depolarization and NMDA exposure.

The influence of K(+)-induced membrane depolarization and NMDA treatment on the regulation of NMDA receptor subunit (NR) expression was investigated during the development of granule cells in culture, as a follow-up of previous work on NMDA receptor activity. In spite of the increase in NMDA receptor activity elicited by these treatments (K25 or K10 + NMDA cultures), the main developmental changes in receptor mRNA levels were similar to those in untreated cells (K10) (a threefold increase in total NMDA receptor mRNA, quantitative dominance of NR1 mRNA, late expression of NR2C, and virtual absence of NR2D. However, high K+ and NMDA treatment resulted in a greater increase of NR2A mRNA levels and a retardation in the developmental changes in the relative amounts of NR2B and NR2C mRNAs. The correspondence between NMDA receptor activity and the amount of NR1 and NR2A subunit proteins was excellent, the rank order being K25 > K10 + NMDA > K10 at 9 days in vitro. Because the increase in subunit mRNA was not always paralleled by an increase in subunit protein, the control of NMDA receptor expression involves critically, in addition to gene transcription, regulation of translational and/or posttranslational events.

Affinity Labels↗

PubMed1995

Properties of AMPA receptors expressed in rat cerebellar granule cell cultures: Ca2+ influx studies.

Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or 45Ca2+ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45Ca2+ accumulation. 45Ca2+ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall 45Ca2+ influx. These included Ca2+ uptake through NMDA-receptor channels, voltage-sensitive Ca2+ channels, and via Na+/Ca2+ exchange. However, nearly one-fifth of the total 45Ca2+ influx remained unaccounted for and this estimate was similar to 45Ca2+ influx observed under Na(+)-free conditions. This observation suggested that a significant proportion of the Ca2+ flux passes through the AMPA-receptor channel proper, a view supported by Co2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro.

Animals↗

PubMed1995

Development of voltage-activated potassium currents in cultured cerebellar granule neurons under different growth conditions.

1. The functional expression of two potassium currents in cultured cerebellar granule cells was investigated with the whole cell patch-clamp technique in relation to development and growth condition. Cells were grown in medium containing different concentrations of potassium: 25 mM (K25) and 40 mM (K40), together referred to as "high K+"; 10 mM (K10) or "low K+"; and K10 with 100 microM N-methyl-D-aspartate (KNMDA). All conditions are known to influence maturation and survival of granule cells in culture. 2. At 2 days in vitro (DIV) the membrane capacitance, taken as index of membrane surface area, was the same for cells grown in each growth condition. At 7-9 DIV it had increased in each condition, but to a substantially larger extent in cells grown in KNMDA, K25, and K40 than in cells grown in K10. During development the input resistance only decreased in cells grown in KNMDA and high K+. 3. A delayed potassium current (IK) and a fast transient potassium current (IA) could both be recorded at 2 DIV in each growth condition, although a few neurons only expressed the IK. The IK was partially suppressed by tetraethylammonium (5 mM), whereas IA was predominantly sensitive to 4-aminopyridine (5 mM). 4. Normalized for cell capacitance, the specific IA conductance hardly changed during development in cells grown in high K+ and KNMDA. Cells in K10, however, displayed an IA with totally different properties in 23 of 24 cells; the specific IA conductance in these cells was considerably smaller at 7-9 DIV, suggesting a deletion of these channels during development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Growth conditions differentially modulate the vulnerability of developing cerebellar granule cells to excitatory amino acids.

The survival of immature nerve cells in a cerebellar culture, predominantly excitatory granule cells, is known to be promoted by chronic exposure to high K+ (> 20 mM) or glutamate (Glu) receptor agonists. These treatments are believed to mimic the in vivo effect of the incoming glutamatergic afferents, the mossy fibres. Here we report that with maturation the cells become vulnerable to excitatory amino acids (EAAs) and that the characteristics of EAA sensitivity are dependent on the environmental influences being either "trophic" (25 mM K+ or 140 microM NMDA, K25 or K10 + NMDA) or "non-trophic" (10 mM K+, K10). Toxicity was assayed routinely at 9 days in vitro (DIV) after 24 h exposure to EAAs. Under all the tested conditions, the effect of Glu was mediated exclusively through NMDA receptors. However, the efficacy and potency of Glu were high in K25- and K10 + NMDA-grown cells compared with K10-grown cells. Growth conditions had the same influence on NMDA as on Glu-induced toxicity, but with the following special features: (1) in comparison with K25 cells, the potency of NMDA was significantly lower in K10 + NMDA cells. The K10 + NMDA cultures behaved as if they were completely insensitive to the NMDA which is present in their growth medium. (2) The K10-grown cells were not vulnerable to NMDA, unless the cell membrane was depolarised by shifting the cells into K25 medium. The efficacy of NMDA became then similar to that in K25 cultures, although the potency was about 7-fold less. Thus NMDA receptors can be activated by the depolarisation of K10 cells, implying the operation of Mg2+ blockade of the channel at normal resting membrane potential. Although non-NMDA receptors did not seem to be involved in Glu toxicity, cells were vulnerable to kainate, which killed significantly more cells than Glu (about 80% vs 70%). This was partly due to the resistance of GABAergic interneurons present in the cultures to Glu- or NMDA-induced toxicity. In contrast to the effects of Glu or NMDA, KA vulnerability was lower in cells grown in K25 or K40 than K10 medium (rank order K10 > K25 > K40). Under our experimental conditions, cultured cells were resistant to AMPA, quisqualate and the selective metabotropic Glu receptor agonist 1S,3R-ACPD. Collectively, the observations indicated that EAA sensitivity of cultured cerebellar interneurons is significantly and differentially influenced by environmental factors, believed to mimic in vivo trophic influences on these cells.

Animals↗

PubMed1994

Comparison of diffusion-weighted MRI with changes in cell volume in a rat model of brain injury.

The precise mechanisms that underlie acute changes in tissue water diffusion following cerebral ischemia or related insults such as glutamate exposure remain unexplained, but it has been suggested that these may be caused by cell swelling due to water uptake. This study was undertaken to compare the changes observed in diffusion-weighted MR images with changes in the cellular volume measured by electrical impedance in a model of N-methyl-D-aspartate-induced brain injury in perinatal rats. The results show that the temporal course of the intensity changes in the diffusion-weighted images parallelled the progressive shrinkage of the extracellular space measured from the electrical impedance. After administration of the N-methyl-D-aspartate antagonist MK-801 the signal enhancement in the images was reversed, which paralleled the normalization of the extracellular space observed by the impedance measurements. It was estimated that the extracellular space decreased from 24 to 12% while the apparent diffusion coefficient of water decreased from 0.89 x 10(-9) in normal tissue to 0.42 x 10(-9) m2/s in tissue exposed to N-methyl-D-aspartate. These data indicate that changes in tissue water diffusion are related to changes in cell volume.

Animals↗

PubMed1994

Selective stimulation of excitatory amino acid receptor subtypes and the survival of granule cells in culture: effect of quisqualate and AMPA.

Differentiating granule cells develop survival requirements in vitro which can be met by treatment with high K+ or excitatory amino acids. Promotion of cell survival by N-methyl-D-aspartate (NMDA) or kainate has already been established and here we report that treatment of the cells with alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) or quisqualate (QA) also leads to cell rescue. In comparison with the effect of NMDA, the influence of AMPA/QA is small, resulting in a 20-30% increase in cell survival, with a peak at a very narrow concentration range (0.5-2.0 microM QA and 5-10 microM AMPA). The effect is exclusive to AMPA receptor stimulation, since stimulation of metabotropic glutamate receptors with (1S3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) has no effect. Furthermore, AMPA/QA rescue of cells is blocked by ionotropic non-NMDA receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline (NBQX). In addition, both nifedipine and dizolcipline (MK-801) interfered with the cell survival promoting effect of AMPA, suggesting that the influence of AMPA is mediated via calcium influx involving both depolarization-activated voltage sensitive calcium channels and NMDA receptors stimulated as a result of AMPA-induced release of glutamate. Possible reasons for the small cell survival promoting effect of AMPA/QA compared with the influence of high K+ or NMDA are discussed.

Animals↗

PubMed1994

Structural maturation, cell proliferation and bioelectric activity in long-term slice-cultures of immature rat hippocampus.

Explants of transverse slices of the 6-day-old rat hippocampus were grown in a serum-free medium for 2-14 days. Histology performed after various culturing periods demonstrated that these slices maintain a high degree of 3-dimensional organotypy, while undergoing growth and differentiation of the main cellular elements similar to that seen in vivo. Histological indications of continuing cell proliferation were verified by autoradiography showing a labelling of neuroblasts in the dentate gyrus and of glioblasts at the sites of gliogenesis observed in vivo. Spontaneous bioelectric activity and evoked potentials were recorded, both indicating the development of impulse generation and neuronal connectivity within the explant. Silver impregnation and electron microscopic studies lent further support for the presence of neuronal networks intrinsic to the hippocampus. These findings suggest that within the period studied the hippocampal slice cultures mature in a fashion similar to that seen in situ.

Animals↗

PubMed1994

The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration.

The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stained viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K+ in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing 'low' K+ (5 or 10 mM), as well as in the presence of 'high' K+ (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in 'high' as compared with 'low' K(+)-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-D-glucose accumulation and increased N-methyl-D-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in 'high' K+ promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K(+)-induced membrane depolarization.

Animals↗

PubMed1994

Early evolution and recovery from excitotoxic injury in the neonatal rat brain: a study combining magnetic resonance imaging, electrical impedance, and histology.

We explored the therapeutic potentials of two N-methyl-D-aspartate (NMDA) receptor antagonists in vivo using different techniques. NMDA injected into the striatum of neonatal rats (20 nmol/0.5 microliters) induced a rapid increase in the diffusion-weighted (DW) image intensity, spreading over a large part of the ipsilateral hemisphere. Subcutaneous injection of the NMDA receptor antagonist MK-801 (1 mg/kg) or D-(E)-4-(3-phosphono-2-prop-enyl)-2-piperazine-carboxylic acid (D-CPPene; 1.5 mg/kg) reversed both the volume and the grading of the NMDA-induced hyperintensity of DW images, the reversal by MK-801 being more rapid than that by D-CPPene. In the cerebral cortex, there was an inverse relationship between changes in DW image intensity and the size of the extracellular space, assessed by electrical impedance measurements. The reduction of the hyperintense volume in DW images 1 or 2 h after MK-801 or D-CPPene treatment of NMDA-injected animals depended on the type of antagonist used and on the interval between intrastriatal NMDA injection and antagonist treatment. The reduction was 95% when MK-801 was given with a delay of 90 min and decreased to 20% when it was given at 360 min. With D-CPPene, the reduction was 80% after a delay of 30 min and virtually absent when it was administered at 360 min. Quantitative analysis showed significant correlations between the residual hyperintense volume 1 or 2 h after MK-801 or D-CPPene treatment and the final lesion volume, assessed from either T2-weighted images (R = 0.89, p < 0.001) or histology (R = 0.80, p < 0.001) 5 days after the insult. This study illustrates the sensitivity of DW magnetic resonance imaging to monitor in vivo early events after an excitotoxic insult and the effect of putative protective drugs that may counteract the resulting damage.

Animals↗

PubMed1993

Temporal evolution of NMDA-induced excitoxicity in the neonatal rat brain measured with 1H nuclear magnetic resonance imaging.

The aim of this study is to characterize the evolution of excitotoxic damage in neonatal rat brain by diffusion-weighted and T2-weighted magnetic resonance imaging. Results are compared with histological findings. Magnetic resonance imaging was performed at various times (15 min, 24 h, 3 days and 5 days) after intrastriatal microinjection of N-methyl-D-aspartate (NMDA) at postnatal day 8. The transverse relaxation time (T2) and apparent diffusion coefficient of water were determined. The results show an acute reduction of the apparent diffusion coefficient, reflected by an ipsilateral hyperintensity in the diffusion-weighted images, within 15 min after intrastriatal NMDA injection. At this time no changes in the T2-weighted images were apparent. The volume of the hyperintensity was relatively large with a radius of approximately 2 mm and coincided with histological signs of pronounced karyo-dendritic swelling. Subcutaneous administration of MK-801 25 min after the intracerebral NMDA injection readily reversed the hyperintensity and resulted in complete protection as verified by histology. Areas with increased T2 values were observed 1 day after NMDA microinjection and corresponded to regions with obvious cell necrosis. Five days after NMDA injection the lesion was evident using both diffusion- and T2-weighted images and coincided with an overt lesion comprising areas of cell loss and dilatation of the ipsilateral ventricle. In conclusion, this study illustrates the possibility of using diffusion-weighted imaging as a tool to monitor efficacy of treatment strategies at an early stage of excitotoxic injury.

Animals↗

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

Promotion of granule cell survival by high K+ or excitatory amino acid treatment and Ca2+/calmodulin-dependent protein kinase activity.

Cerebellar granule cells in culture develop survival requirements which can be met either by chronic membrane depolarization (25 mM K+) or by stimulation of ionotropic excitatory amino acid receptors. We observed previously that this trophic effect is mediated via Ca2+ influx, either through dihydropyridine-sensitive, voltage-dependent calcium channels (activated directly by high K+ or indirectly by kainate) or through N-methyl-D-aspartate receptor-linked ion channels. Steps after Ca2+ entry in the transduction cascade mediating the survival-supporting effect of high K+ and excitatory amino acids have now been examined. Using protein kinase inhibitors (H-7, polymixin B and gangliosides), and modulating protein kinase C activity by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, we obtained evidence against the involvement of protein kinase C and cyclic nucleotide-dependent protein kinases in the transduction cascade. On the other hand, calmidazolium (employed as a calmodulin inhibitor) counteracted the trophic effect of elevated K+ with high potency (IC50 0.3 microM), which exceeded by approximately 10-fold the potency for the blockade by the drug of voltage-sensitive calcium channels. The potency of calmidazolium in interfering with the N-methyl-D-aspartate rescue of cells was also much higher in comparison with the inhibition of 45Ca2+ influx through N-methyl-D-aspartate receptor-linked channels. Our results indicated that after calmodulin the next step in the trophic effects involves Ca2+/calmodulin-dependent protein kinase II activity. KN-62, a fairly specific antagonist of this enzyme, compromised elevated K+ or excitatory amino acid-supported cell survival with high potency (IC50 2.5 microM). In the relevant concentration range, KN-62 had little or no effect on Ca2+ entry through either voltage- or N-methyl-D-aspartate receptor-gated channels. Combining information on the toxic action of glutamate in "mature" granule cells with the trophic effect of either excitatory amino acids or high K+ treatment on "young" cells, we conclude that after the initial steps involving calcium in both cases the respective transduction pathways diverge. The toxic action of glutamate seems to be mediated through protein kinase C [Favaron et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1983-1987 whereas a Ca2+/calmodulin-dependent protein kinase, which can be inhibited by KN-62 (but is resistant to gangliosides and to inhibitors whose potency is higher for protein kinase C than for Ca2+ calmodulin-dependent protein kinases, such as H-7 and polymixin B), is involved critically in the trophic effect.

Amino Acids↗

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

Growth conditions differentially regulate the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured neurons.

We have studied the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K+ (10, 25, or 40 mM) with or without 100 microM N-methyl-D-aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K+ (K10) and increased dramatically in cultures grown in 25 mM K+ (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 + NMDA). In cultures grown in 40 mM K+ (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and -3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA-stimulated 45Ca2+ influx and 4 beta-[3H]phorbol 12,13-dibutyrate binding in intact cells. Both functional parameters increased along with the K+ concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Mechanisms underlying developmental changes in the expression of metabotropic glutamate receptors in cultured cerebellar granule cells: homologous desensitization and interactive effects involving N-methyl-D-aspartate receptors.

Glutamate receptors coupled to polyphosphoinositide (PPI) hydrolysis (metabotropic glutamate receptors, mGluR), are highly efficient during the early stages of postnatal life and are thought to be involved in developmental plasticity. The dramatic decrease with age in mGluR activity suggests the existence of mechanisms that down-regulate this receptor after a certain stage of neuronal maturation. In cultured cerebellar granule neurons grown under conditions that promote the survival and maturation of cells (serum-containing medium with 25 mM K+), enzymatic depletion of extracellular glutamate prevented the age-dependent decrease in mGluR agonist-stimulated PPI hydrolysis that normally occurs after 4 days of maturation in vitro, suggesting that mGluR activity declines as a result of developmental changes affecting homologous desensitization. This was borne out by the observation that glutamate at low concentrations (1-10 microM) readily desensitized mGluR at 7 days but not at 4 days in culture. Furthermore, the critical period during which the high sensitivity to agonist-induced desensitization of mGluR developed coincided with the period when phorbol ester-activated protein kinase C acquired the ability to suppress mGluR activity. The developmental pattern of mGluR agonist-induced PPI hydrolysis was similar in granule cells grown under "trophic" and "nontrophic" conditions (in cultures in 25 mM K+ and in a medium containing "low" K+, in this study, 10 mM, respectively). However, the developmental decline in the response to mGluR stimulation after 4 days in vitro was not prevented in cells grown in 10 mM K+ by the removal of extracellular glutamate; rather, it could be counteracted by treatment with N-methyl-D-aspartate (NMDA) (EC50, approximately 4 microM), which blocked the development of mGluR desensitization. The effect was NMDA receptor mediated and required DNA transcription and protein synthesis. However, NMDA exerted a different effect in cells grown in 25 mM K+, inducing a substantial decrease rather than an increase in mGluR activity. The effect of growth conditions was also examined on mGluR mRNA levels, which were not always correlated with mGluR activity. In general, either increases in the medium K+ concentrations or NMDA supplementation of the cultures resulted in a decrease in mGluR mRNA levels. It is noteworthy that NMDA could also restore mGluR activity after the metabotropic response had reached its peak. This implies that NMDA receptor activation may be involved in the increase in mGluR activity in adult life under conditions that elicit plastic changes in the nervous system.

Aging↗