Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.     

Kainate-induced apoptosis correlates with c-Jun activation in cultured cerebellar granule cells

We have investigated the involvement of c-Jun in cell death induced by exposure of primary cultures of murine cerebellar granule cells to the glutamate receptor agonist kainate (KA) and evaluated its possible use as a marker for apoptosis. Using cerebellar granule cell neurones from postnatal day 7 mice, we found that 1 hr exposure to KA (1–1000 μM) induced a concentration-dependent neuronal cell death with characteristic apoptotic morphology, including cell shrinkage, neurite blebbing and DNA fragmentation. In addition KA-induced a concentration-dependent expression of c-Jun mRNA and protein as determined by in situ hybridization and immunocytochemistry respectively. DNA fragmentation was detected using terminal transferase-mediated nick-end (TUNEL) labelling and agarose gel electrophoresis. KA-induced cell death was significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM), which shifted the concentration-response curve significantly rightward. The number of apoptotic cell bodies, determined by TUNEL, was also reduced by CNQX (50 μM), with only 15–20% of neurones staining positive after exposure to 1mM KA. In addition, the number of positively stained cells for c-Jun protein and mRNA was substantially reduced by CNQX (50 μM) as determined by random and representative cell counts. These results show for the first time that KA induced apoptotic neuronal death in cultured murine cerebellar granule cells involves the induction of c-Jun mRNA and protein, suggesting the involvement of this immediate early gene in excitotoxic receptor-mediated apoptosis and its potential use as a marker for apoptotic cell death. J. Neurosci. Res. 52:69–82, 1998. © 1998 Wiley-Liss, Inc.     

AMPA/kainate receptor-mediated up-regulation of GABAA receptor delta subunit mRNA expression in cultured rat cerebellar granule cells is dependent on NMDA receptor activation

We have studied the effects of AMPA/kainate receptor agonists on GABA(A) receptor subunit mRNA expression in vitro in cultured rat cerebellar granule cells (CGCs). Kainate (KA) (100 microM) and high K(+) (25 mM) dramatically up-regulated delta subunit mRNA expression to 500-700% of that in control cells grown in low K(+) (5 mM). KA or high K(+) had no effect on the expression of the other major GABA(A) receptor subunits alpha1, alpha6, beta2, beta3 or gamma2. Up-regulation of delta mRNA was also detected with the AMPA receptor-selective agonist CPW-399 and to a lesser extent with the KA receptor-selective agonist ATPA. AMPA/kainate receptor-selective antagonist DNQX completely inhibited KA-, CPW-399- and ATPA-induced delta mRNA up-regulation indicating that the effects were mediated via AMPA and KA receptor activation. NMDA receptor-selective antagonist MK-801 inhibited 76% of the KA- and 57% of the CPW-399-induced delta up-regulation suggesting that KA and CPW-399 treatments may induce glutamate release resulting in NMDA receptor activation, and subsequently to delta mRNA up-regulation. In CGCs, delta subunit is a component of extrasynaptic alpha6betadelta receptors that mediate tonic inhibition. Up-regulation of delta during prolonged glutamate receptor activation or cell membrane depolarization may be a mechanism to increase tonic inhibition to counteract excessive excitation.     

Adenosine and ADP prevent apoptosis in cultured rat cerebellar granule cells

Cerebellar granule cells (CGCs) explanted in vitro undergo death via apoptosis when the concentration of potassium is shifted from 25 mM to 5 mM. We report that adenosine and ADP, which act as neurotransmitters and neuromodulators in the brain, exert in cultured cerebellar granule cells a specific and marked antiapoptotic action with half-maximal effect in the 10–100 μM range. The action of adenosine is partly inhibited by the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and is mimicked by the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA), while ADP effect, that is completely blocked by the P2x, P2y receptors noncompetitive antagonist suramine, is restored in the presence of the selective P2x purinoceptors agonist β,γ-methylene- -ATP. These findings demonstrate that adenosine and ADP markedly inhibit the program of cell death in cerebellar granule cells and suggest that such an action is mediated via interaction with, respectively, A1 and P2x receptors.     

The neurotoxicity of 1-methyl-4-phenylpyridinium in cultured cerebellar granule cells

Cerebellar granule cells in enriched primary culture are susceptible to the neurotoxic effects of 1-methyl-4-phenylpyridinium (MPP+). Relatively high MPP+ concentrations are required to elicit neurotoxic effects at early culture times, but lower concentrations of MPP+ produce comparable neurotoxic effects at later culture times. Under identical culture conditions 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is not neurotoxic. Preincubation with the glutamate uptake blockers, DL-threo-3-hydroxyaspartic acid or dihydrokainate, or the dopaminergic uptake blocker mazindol, protects the granule cells from the cytotoxic effects of MPP+. Although MPTP is not neurotoxic in an enriched granule cell culture, in coculture with cerebellar astrocytes MPTP is toxic to granule cells, presumably because it is converted in astrocytes to MPP+. Cerebellar astrocytes remain confluent and viable. The addition of pargyline to the coculture abolishes the neurotoxicity consistent with a role of MAO B in bioactivation of MPTP. The concentration of MPP+ in the coculture medium (13 microM) was less than that required for the toxic effect in enriched neuronal cultures at earlier culture times, suggesting that an astroglial-neuronal interaction, perhaps by proximity, enhances the neurotoxicity of MPP+. These results might explain reported effects of MPTP on some cerebellar cells in mice.     

Developmental profile of excitatory GABA(A) responses in cultured rat cerebellar granule cells

GABA induced a transient increase in cytosolic free Ca2+ in cerebellar granule cells, which decreased from 3 to 8 days in vitro (DIV). Cytosolic Ca2+ changes induced by glutamate/glycine were comparable at 3 and 7 DIV. The GABA response was ascribed to GABA(A)-receptor mediated depolarization activating L-type Ca2+ channels since the response was inhibited by bicuculline or nifedipine. GABA-mediated Ca2+ rise at 4 DIV was potentiated by pentobarbital or by the neurosteroid 5beta-pregnan-3alpha-ol-20-one, or by decreasing the extracellular Cl- concentration. Neurons cultured for > 7 DIV showed no rise in intracellular Ca2+ in response to GABA regardless of the Cl- gradient. GABA(A) receptor-mediated cytosolic Ca2+ rise suggests an important role for the excitatory activity of GABA in developing cerebellar granule neurons.     

Kainate down-regulates a subset of GABA<Subscript>A</Subscript> receptor subunits expressed in cultured mouse cerebellar granule cells

The effect of kainate, an agonist selective for ionotropic AMPA/kainate type of glutamate receptors, on GABAA receptor subunit expression in cultured mouse cerebellar granule cells was studied using quantitative RT-PCR, ligand binding and electrophysiology. Chronic kainate treatment, without producing excitotoxicity, resulted in preferential, dose- and time-dependent down-regulation of alpha1, alpha6 and beta2 subunit mRNA expression, the expression of beta3, gamma2 and delta subunit mRNAs being less affected. The down-regulation was reversed by DNQX, an AMPA/kainate-selective glutamate receptor antagonist. A 14-day kainate treatment resulted in 46% decrease of total [3H]Ro 15-4513 binding to the benzodiazepine sites. Diazepam-insensitive [3H]Ro 15-4513 binding was decreased by 89% in accordance with very low amount of alpha6 subunit mRNA present. Diazepam-sensitive [3H]Ro 154513 binding was decreased only by 40%, contrasting >90% decrease in alpha1 subunit mRNA expression. However, this was consistent with lower potentiation of GABA-evoked currents in kainate-treated than control cells by the alpha1-selective benzodiazepine site ligand zolpidem, suggesting compensatory expression of alpha5 (and/or alpha2 or alpha3) subunits producing diazepam-sensitive but zolpidem-insensitive receptor subtypes. In conclusion, chronic kainate treatment of cerebellar granule cells selectively down-regulates oil, alpha6 and beta2 subunits resulting in altered GABAA receptor pharmacology.     

Brain-derived neurotrophic factor mitigates chronic ethanol-induced attenuation of gamma-aminobutyric acid responses in cultured cerebellar granule cells

This study examined the effect of chronic exposure to ethanol and brain-derived neurotrophic factor (BDNF) on the responsiveness of cerebellar granule cells to gamma-aminobutyric acid (GABA). Cerebellar granule cell cultures were chronically exposed to ethanol (100 mM), BDNF (20 ng/ml), or the combination of ethanol and BDNF. Whole-cell current responses of granule cells to exogenously applied GABA were monitored following at least 5 days of chronic exposure. In the ethanol-treated cultures, granule cell responsiveness to GABA was attenuated. Concomitant exposure of cultures to ethanol and BDNF mitigated the ethanol-induced attenuation of GABA response, although BDNF, by itself, did not affect responsiveness to GABA. BDNF increased the expression of the GABA(A) receptor alpha6 subunit, whereas ethanol had no effect, in chronically treated granule cell cultures. In addition, concomitant treatment with BDNF and ethanol did not increase the expression of the GABA(A) receptor alpha6 subunit, so the subunit expression alone could not account for the mitigating effect of BDNF. We propose that different mechanisms regulating responsiveness to GABA underlie the effects induced by ethanol and BDNF, with the former influencing the expression of functional GABA(A) receptors and the latter involving the activation of the TrkB receptor and its downstream signaling pathways.     

Effects of chronic ethanol exposure on cultured cerebellar granule cells

The aim of this study was to investigate the lipid content and composition of rat cerebellar granule cells grown in the presence of ethanol (40, 55, or 80 mM) during in vitro differentiation. Quantitative analyses showed no effects of 40 mM ethanol, whereas a significant increase of total cholesterol was observed at 55 mM. Cells exposed to the highest ethanol dose (80 mM) were characterized by a higher sialidase activity, and by the modification of the ganglioside pattern and phospholipid fatty acid composition. The observed modifications were accompanied by changes of membrane anisotropy fluorescence assessed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene.     

Transport of 5-formyltetrahydrofolate into primary cultured cerebellar granule cells

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured cerebellar granule cells (CGC) was studied. Uptake of 5-FTHF into CGC was saturable with K(m)=2.86 microM and V(max)=40.8 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF in the astrocytes has a similar style in the time curve. Uptake of 5-FTHF is characterized by countertransport because adding unlabeled 5-FTHF in the medium resulted in the efflux of labeled 5-FTHF. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methotrexate and folic acid (K(i)=6.64, 7.69, and 19.38 microM, respectively). Uptake was significantly decreased by high concentrations of sodium azide and sodium arsenate but not by sodium cyanide. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Acute exposure of the cells to ethanol (100 mM) did not affect the uptake. It is concluded that CGC have a carrier-mediated system for the uptake of 5-FTHF and other folates.     

Single-channel and whole-cell currents in rat cerebellar granule cells

The patch-clamp technique was used to study both whole-cell and single-channel currents in cultured rat cerebellar granule cells. In whole-cell recordings under voltage-clamp conditions 3 types of current were found; a transient inward sodium current and a transient and a sustained outward potassium current. Single-channel currents were recorded from both inside-out and outside-out membrane patches. Three types of potassium channel were identified; two non-inactivating channels with unit conductances of 160 and 60 pS and one inactivating channel of 20 pS. No calcium currents were detected.     

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis

Summary. In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage. Received December 17, 1997; accepted May 19, 1998     

Two-photon imaging of calcium accumulation in rat cerebellar granule cells

Topical accumulation of calcium ions in neurites and cell bodies of rat cerebellar granule cells was studied by two-photon microscopy in neurons loaded with the Ca-sensitive fluorescent indicator Oregon Green 488 Bapta. High potassium caused a rapid surge of internal calcium ([Ca2+]i) in the cell body, followed by a plateau. In neurites, [Ca2+]i reached a peak and then decreased back to the control level. In contrast, in neurons stimulated by NMDA, [Ca2+]i reached a steady level and remained constant as long as the agonist was present in the bath, either in the cell bodies or in neurites. In the latter, the response to NMDA treatment was smaller and heterogeneous, and [Ca2+]i increased in certain segments of the neurite, but not in others.     

高钾可诱导培养的小脑颗粒神经元凋亡

采用四唑盐比色、琼脂糖凝胶电泳、乙二酸荧光素染色和Hoechst332 58染色等方法研究高钾对原代培养的大鼠小脑颗粒神经元的毒性作用及其机制。结果发现 :①高钾诱导神经元死亡呈剂量 ( 50～ 10 0mmol/L)和时间依赖性 ;②神经元死亡呈现明显的凋亡特征 :胞体缩小 ,染色质浓缩 ,DNA“梯形”条带形成和蛋白质合成抑制剂 (cycloheximide ,1.0mg/L)可阻断其毒性等 ;③MK 80 1( 2 μmol/L)、尼莫地平 ( 10 μmol/L)、硫酸镁 ( 2 0mmol/L)可阻断高钾的大部分毒性作用。结果提示 :高钾可能通过刺激内源性谷氨酸释放从而诱导小脑颗粒神经元凋亡     

Sodium-dependent high-affinity uptake of taurine in cultured cerebellar granule cells and astrocytes

Taurine uptake in cultured cerebellar granule cells and astrocytes consisted of a saturable high-affinity component and nonsaturable diffusion. The transport constant (Km) was significantly lower and the maximal velocity (V) higher in granule cells than in astrocytes. The uptakes were strictly sodium dependent and also moderately decreased in potassium-free medium. The specificity profile of taurine uptake was similar in both cell types, hypotaurine, beta-alanine, and guanidinoethanesulphonic acid being the most potent inhibitors, followed by GABA and homotaurine. Glutamate inhibited taurine uptake more in astrocytes than in granule cells. In principle, the uptake systems were similar in granule cells and astrocytes, exhibiting features characteristic of uptake of a neurotransmitter or -modulator.