Expression and Agonist-Induced Down-Regulation of mRNAs of m2- and m3-Muscarinic Acetylcholine Receptors in Cultured Cerebellar Granule Cells

The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization, mRNA species for m2- and m3-mAChRs but not m1- and m4-mAChRs were detected in these cells. The expression of mRNAs of both m2- and m3-mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 microM carbachol led to differential down-regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 microM) and pirenzepine (10 microM), prevented carbachol-induced m3-mAChR mRNA down-regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up-regulation of m3-mAChR mRNA. Thus, the mRNA species for both m2- and m3-mAChR subtypes are differentially expressed in culture and down-regulated by agonist stimulation. The loss of these mRNA species may play a role in the down-regulation of mAChR binding sites that occurs after desensitization.     

Ethanol Promotes Apoptosis in Cerebellar Granule Cells by Inhibiting the Trophic Effect of NMDA

Abstract: When primary cultures of cerebellar granule neurons are grown in a physiological concentration of KCl (5 m M ) they undergo apoptosis, which can be prevented by growing the cells in the presence of N -methyl- d -aspartate (NMDA). We now show that ethanol inhibits this trophic effect of NMDA, i.e., promotes apoptosis, and also inhibits the NMDA-induced increase in intracellular Ca²⁺ concentration in cells grown in 5 m M KCl. Both effects of ethanol show a similar concentration dependence and are reversed by a high concentration of glycine, the co-agonist at the NMDA receptor. The data suggest that the effect of ethanol on apoptosis is mediated, at least in part, by inhibition of NMDA receptor function. This effect of ethanol to increase apoptosis could contribute to the previously described in vivo sensitivity of the developing cerebellum to ethanol-induced damage.     

Chronic Dizocilpine (MK-801) Reversibly Delays GABAA Receptor Maturation in Cerebellar Granule Neurons In Vitro

Abstract: We investigated the effect of chronically blocking NMDA receptor stimulation to examine changes in GABA_A receptor expression and pharmacology in cerebellar granule cells at different stages of maturation. We have previously shown that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunits. When NMDA receptor stimulation is blocked with MK-801 during the first week in vitro, a decrease in the α1, γ2S, and γ2L receptor subunit mRNAs occurred. When present only during the second week, changes were limited to the α1 and γ2L mRNAs. Finally, if MK-801 was present during the first week and removed during the second week, these changes reversed. Whole-cell voltage-clamp recordings showed that treatment with MK-801 during either the first or second week increased the EC₅₀ of the receptors for GABA and attenuated the potentiation mediated by flunitrazepam. Last, these properties were reversed if MK-801 was removed after the first week in vitro. Our results suggest that MK-801 reversibly inhibits GABA_A receptor maturation by modulating receptor subunit expression and that the altered pharmacological responses appear to be dominated by changes in the levels of allosteric modulation mediated by the γ2 receptor subunit.     

Effects of Depolarization and NMDA Antagonists on the Survival of Cerebellar Granule Cells: A Pivotal Role for Protein Kinase C Isoforms

Abstract: Primary cultures of cerebellar granule cells (CGCs) grown in high-K⁺ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3–4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K⁺ concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC α, γ, μ, λ, and ι from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC ε and βII, respectively, whereas no significant change was observed for βI. The isoforms of δ, θ, η, and ζ were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC ι and ε were decreased, those of PKC α, βI, βII, γ, and λ were unchanged, whereas that of PKC μ was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2–4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K⁺ cultures, levels of PKC α, μ, λ, and ι, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC γ, λ, ι, and μ at 10 and 14 DIV. In addition, the PKC ε level was increased at 14 DIV but decreased at early stages, whereas PKC α, βI, and βII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up-regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK-801.     

Protein Kinase C Activation Attenuates N-Methyl-d-Aspartate-Induced Increases in Intracellular Calcium in Cerebellar Granule Cells

Abstract: Activation of the N-methyl-d -aspartate (NMDA) subtype of glutamate receptor increases levels of intracellular calcium and can lead to stimulation of protein kinase C activity. Several reports have demonstrated that stimulation of protein kinase C can, in turn, increase electrophysiological responses to NMDA in certain cells or in oocytes expressing certain NMDA receptor subunits. In the present study, the effects of protein kinase C activation on NMDA receptor-mediated increases in intracellular Ca²⁺ levels were investigated in primary cultures of rat cerebellar granule cells using fura-2 fluorescence spectroscopy. Pretreatment of the cells with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not the inactive analogue 4α-phorbol 12-myristate 13-acetate, inhibited NMDA-induced increases in intracellular Ca²⁺ levels. Coincubation of cells with PMA and the kinase inhibitor staurosporine or calphostin C blocked the PMA effect. The potency of NMDA was reduced twofold, and the potency of the NMDA receptor coagonist, glycine, to enhance the response to NMDA was decreased fourfold by pretreatment of cells with PMA. The effect on glycine was mimicked by pretreatment with okadaic acid, a protein phosphatase inhibitor. PMA treatment did not significantly alter Mg²⁺ inhibition of the NMDA response but decreased the potency of the competitive antagonist CGS-19755. These data suggest that, in cerebellar granule cells, the function of the NMDA receptor may be subject to feedback inhibition by protein kinase C stimulation. Under physiological conditions, this inhibition may result from a decreased effectiveness of the endogenous coagonists, glutamate and glycine.     

Complexity of Depolarization-mediated ERK Phosphorylation in Cerebellar Granule Cells in Primary Cultures

We previously showed that cultured mouse cerebellar granule cells during incubation in glutamine-replete medium respond to 45 mM [K⁺]_e after 20 and 60 min incubation with extracellular-signal regulated kinase 1 and 2 (ERK_1/2) phosphorylation which is mainly, but probably not exclusively, secondary to glutamate release and transactivation of epidermal growth factor (EGF) receptors. In the present study the response after 20 min was shown to be abolished by protein kinase C (PKC) inhibition, whereas that at 60 min was PKC-independent. Addition of 50 μM glutamate to the cells caused ERK_1/2 phosphorylation already after 5 min most of which was sensitive to PKC inhibition although a minor part was PKC inhibition-resistant. Exposure to [K⁺]_e during incubation in glutamine-depleted medium caused no stimulated release of glutamate but a transactivation-independent ERK_1/2 phosphorylation at 20 and 60 min. The response at 20 min was insensitive to PKC inhibition. The potential importance of these complex responses for synaptic plasticity is discussed. Special issue article in honor of Dr. Frode Fonnum.     

Metabotropic Glutamate Receptors in Cultured Cerebellar Granule Cells: Developmental Profile

Abstract: 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 Mg²⁺-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 ∼10% of the values at 3 DIV. These observations indicate that the functional expression of the mGluR is subject to developmental regulation, which critically involves receptor mRNA levels.     

γ-Aminobutyric Acid Affects the Developmental Expression of Neuron-Associated Proteins in Cerebellar Granule Cell Cultures

The effect of gamma-aminobutyric acid (GABA) on the expression of the neuron-associated D2 and neuron-specific enolase (NSE) was studied during development in culture of cerebellar granule cells. It was found that the presence of GABA during culture development increased the overall protein content. D2 content was also increased but not above the general increase in protein whereas NSE increased above the general level of protein. The presence of GABA in the growth medium also appeared to accelerate the changes in molecular forms of D2 and NSE seen during neuronal development. This suggests that GABA promotes or accelerates the general maturation of neurons, as these two neuron-associated proteins otherwise differ from each other with respect to their subcellular localization and their physiological and biochemical properties.     

Ethanol Specifically Inhibits NMDA Receptors with Affinity for Ifenprodil in the Low Micromolar Range in Cultured Cerebellar Granule Cells

Abstract: The effect of ethanol on the intracellular Ca²⁺ concentration response to NMDA in rat cerebellar granule cells grown in low or high KCI concentrations has been studied using image analysis. The cells grown in low KCI displayed high sensitivity for glycine. The subtype-selective antagonist ifenprodil inhibited the response with high (in the low micromolar range) and low (in the high micromolar range) potency. Ethanol affected the high-potency component in these cultures. In cells grown in high KCI the glycine sensitivity was lower, and a low potency for ifenprodil (high micromolar) dominated. These cells were not significantly sensitive to ethanol. The results indicate that the component displaying potency for ifenprodil in the low micromolar range with properties of the NR2B subunit is the target for ethanol action on the NMDA receptor.     

Identification of an Ectokinase Activity in Cerebellar Granule Primary Neuronal Cultures

Abstract: Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 n M [γ-³²P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for ∼20 min. Unlike what was seen with [γ-³²P]ATP, in the presence of [³²P] orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80–90% in the presence of 1 µ M unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 m M PO₄³⁻. Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 m M KCl or 100 µ M glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl₂ but inhibited in the presence of MnCl₂ or NaF and in the absence of CaCl₂. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80–90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.     

Homologous Desensitization of Muscarinic Cholinergic, Histaminergic, Adrenergic, and Serotonergic Receptors Coupled to Phospholipase C in Cerebellar Granule Cells

Cultured cerebellar granule cells express phospholipase C-coupled muscarinic cholinergic, histaminergic, alpha 1-adrenergic, and serotonergic receptors. In an attempt to study desensitization of these neurotransmitter receptors, cells were prestimulated with saturating concentrations of carbachol, histamine, norepinephrine, or serotonin during the labeling of cells with myo-[3H]inositol and then rechallenged with various receptor agonists for their ability to elicit accumulation of [3H]inositol monophosphate in the presence of lithium. Prestimulation with each of these receptor agonists was found to cause a time-dependent desensitization to subsequent stimulation with the desensitizing agonist. Thus, prestimulation for 0.5, 4, and 18 h decreased carbachol response to 87 +/- 4, 52 +/- 2, and 40 +/- 1% of the control, respectively; histamine response to 37 +/- 2, 24 +/- 2, and 18 +/- 2%, respectively; norepinephrine response to 55 +/- 5, 14 +/- 1, and 10 +/- 1%, respectively; and serotonin response to 36 +/- 1, 18 +/- 1, and 9 +/- 2%, respectively. In all cases, the responses mediated by receptors which were not prestimulated remained virtually unchanged, thus indicating homologous desensitization. Dose-response studies indicate that the desensitization was associated with a major reduction in the maximal extent of agonist-induced responses. The basal accumulation was markedly enhanced following 0.5- and 4-h prestimulation, but returned to near normal after 18-h pretreatment. Biologically active phorbol ester, 4 beta-phorbol 12-myristate 13-acetate, rapidly attenuated basal phospholipase C activity, as well as the responses mediated by carbachol, histamine, norepinephrine, and serotonin, suggesting that activation and translocation of protein kinase C might play a role in the desensitization of phospholipase C-coupled receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA Induces Functionally Active Low-Affinity GABA Receptors on Cultured Cerebellar Granule Cells

The effect of γ-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low-affinity GABA receptors (K_D 546 ± 117 nM) in addition to the high-affinity receptors (K_D 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED₅₀ 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABA_A receptors.     

Properties of AMPA Receptors Expressed in Rat Cerebellar Granule Cell Cultures: Ca2+ Influx Studies

Abstract: 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)-α-amino-3-hydroxy-5-methylisoxazole-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.     

Sialidase in Cerebellar Granule Cells Differentiating in Culture

The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using [3H]GD1a and 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUB-NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3H]GD1a and 0.1 M for MUB-NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB-NeuNAc and 0.1 mM for [3H]GD1a; enzyme activity linear with time up to 30 min with MUB-NeuNAc and up to 90 min with [3H]GD1a; and enzyme activity linear with enzyme protein content up to 80 micrograms with MUB-NeuNAc and up to 20 micrograms with [3H]GD1a. The assay with [3H]GD1a required the presence of Triton X-100 in a molar ratio to GD1a of 15:1. Poly-L-lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GD1a/Triton X-100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB-NeuNAc. Using no more than 20 micrograms of cellular protein, the contamination, if any, by poly-L-lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7-8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB-NeuNAc and from 1 to 100 nmol of product released/h/mg of protein with [3H]GD1a.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic Cholinoceptor-Stimulated Synthesis and Degradation of Inositol 1,4,5-Trisphosphate in the Rat Cerebellar Granule Cell

Abstract: A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [³H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP₅) and inositol hexakisphosphate (InsP₆), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 m M ) caused an immediate increase in level of Ins(1,4,5)P₃ (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP₅ and InsP₆ was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P₃ and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P₃ metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P₃ 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidylinositol 4-phosphate cannot be excluded.     

Disruption by Lithium of Phosphoinositide Signalling in Cerebellar Granule Cells in Primary Culture

Abstract: Mild depolarisation (20 m M KCI) synergistically enhances the ability of a muscarinic agonist to activate phosphoinositide turnover and to elevate inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] in cerebellar granule cells in primary culture. The effects of lithium on this intense stimulation of phosphoinositide turnover was studied. Lithium causes depletion of cytoplasmic inositol and phosphoinositides, which results in the inhibition of phosphoinositide turnover within 15 min and the return of Ins(1,4,5)P₃ to basal levels at this time. This inhibition could not be reversed by culturing and preincubating cerebellar granule cells in concentrations of inositol similar to those detected in the CSF. Inositol concentrations substantially in excess of those in the CSF not only reversed the effects of lithium on stimulated Ins(1,4,5)P₃ levels, but significantly enhanced this level in comparison with stimulation in the absence of lithium. sn -1,2-Diacylglycerol elevation during stimulated phosphoinositide turnover was also disrupted by lithium, but in contrast to Ins(1,4,5)₃, the presence of lithium resulted in a transient enhancement of the elevation evoked by carbachol plus mild KCI depolarisation, which was reversed by 500 µ M inositol, but not by 200 µ M inositol. The implications of these phenomena in relation to the mechanism of action of lithium in the treatment of manic depression are discussed.     

Modulation by Ionotropic Excitatory Amino Acids and Potassium of (±)-1-Aminocyclopentane-trans-1,3-Dicarboxylic Acid-Stimulated Phosphoinositide Hydrolysis in Mouse Cerebellar Granule Cells

Abstract: The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was studied in mouse cerebellar granule cells. In Mg²⁺-containing buffers, NMDA (50–100 µM), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 10–1,000 µM), and high potassium (10–30 mM) enhanced synergistically the response to a maximally effective concentration of 500 µMtrans-ACPD. Potentiation of the trans-ACPD response was blocked by higher concentrations of NMDA (>500 µM) and potassium (>35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans-ACPD-stimulated phosphoinositide hydrolysis was blocked by d,l -2-amino-5-phosphonopentanoic acid (APV), a competitive NMDA-receptor antagonist. Under Mg²⁺-free conditions, the accumulation of inositol phosphates in the presence of trans-ACPD alone was equal to that attained by trans-ACPD in Mg²⁺-containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 µM). trans-ACPD potentiated synergistically the NMDA-evoked increases in cytosolic free-Ca²⁺ levels in Mg²⁺-containing but not in Mg²⁺-free solutions, and moreover did not enhance the AMPA-evoked increases in cytosolic free-Ca²⁺ levels. The calcium ionophore A23187 caused a dose-dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans-ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein-coupled phospholipase C activation and regulated elevation of cytosolic free-Ca²⁺ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.