Stimulation of the N-methyl-d-aspartate receptor promotes the biochemical differentiation of cerebellar granule neurons and not astrocytes

Cerebellar granule cells are believed to be glutamatergic, but, as they receive excitatory amino acidergic input from mossy fibers, they also possess N-methyl-d-aspartate (NMDA) receptors. The possible involvement of these NMDA receptors in the biochemical differentiation of cultured granule neurons was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 3 days (i.e. between 2 and 5 days in vitro), it elevated specific activity of glutaminase in the dose- and time-dependent manners. The half-maximal effect was obtained at about 10 μM NMDA, whereas the maximum concentration, which produced about a 2.7-fold increase in 5-day-old cultures, was about 50 μM NMDA. This increase in glutaminase was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca²⁺ ion channel blockers, MK 801 and Mg²⁺. The effect of NMDA was not related to the survival of the granule cells, as the experiments were carried out before the dependence on high K⁺ for the survival of granule cells develops in culture, and during the period of investigation none of the compounds used compromised the survival of these cells. The enhancement of glutaminase activity was due to an induction in enzyme protein, since it was completely blocked by cycloheximide and actinomycin D. In contrast to granule neurons, the treatment with NMDA had no significant effect on the activity of glutaminase and glutamine synthetase in cultured cerebellar astroglial cells. Our present results on glutaminase enzyme would indicate that an increase in the cellular concentration of free Ca²⁺ mediated through the NMDA induced increase in Ca²⁺ conductance, leads to long term changes in differentiating cerebellar granule neurons, and it is possible that this kind of physiological stimulation of granule cells is normally provided in vivo by the presynaptic glutamatergic mossy fibers.     

尼莫地平诱导小脑颗粒神经元凋亡及机制研究

目的探讨尼莫地平能否诱导小脑颗粒神经元凋亡及可能机制。方法取Sprague-Dawley(SD)大鼠小脑颗粒神经元,体外培养7d,用含10μM尼莫地平培养基处理神经元24h,对照组为含1‰二甲亚砜(DMSO)培养基处理细胞。Hoechst33258染胞核检测凋亡率。Westernblot检测c-Fos和c-Jun蛋白的表达水平;腺病毒作载体过表达c-Fos和负显性c-Jun突变体阻断c-Jun功能,检测能否抑制尼莫地平诱导的神经元凋亡,免疫细胞化学方法检测腺病毒感染率。结果对照组神经元的凋亡率为(10±4)%,尼莫地平处理6、12、24h后神经元的凋亡率分别为(13±4)%、(28±5)%、(45±3)%。尼莫地平处理使c-Fos表达下调,但c-Jun表达水平上调。腺病毒Ad-c-Fos和Ad-c-JunDN(负显性c-Jun突变体)对神经元感染率为85%以上,过表达c-Fos或负显性c-Jun突变体使尼莫地平诱导的神经元凋亡率从(42±6)%减少到(28±6)%或(20±3)%。结论尼莫地平通过下调c-Fos和上调c-Jun表达诱导小脑颗粒神经元凋亡。     

The chemokine receptor CXCR4 and not the N-methyl-D-aspartate receptor mediates gp120 neurotoxicity in cerebellar granule cells

The human immunodeficiency virus type 1 (HIV-1) glycoprotein gp120 causes neuronal cell death; however, the molecular mechanisms of the neurotoxic effect remain largely unresolved. It has been suggested that gp120 evokes cell death by inducing the release of neurotoxins, including glutamate. The objective of this work was to examine the role of glutamate in gp120-mediated neurotoxicity. We used as an experimental tool cerebellar granule cells prepared from 8-day-old rat cerebella, in which both glutamate and gp120 cause cell death. Cerebellar granule neurons were exposed to gp120 or glutamate alone or in combination with the glutamate receptor antagonist MK801 as well as other antiglutamatergic compounds. Cell viability was measured at various times by using several markers of cell death and apoptosis. MK801, at a concentration that blocked glutamate-induced neuronal cell death, failed to prevent gp120-mediated apoptotic cell death. Moreover, interleukin-10, which has previously been shown to block glutamate toxicity in these neurons, was not neuroprotective against gp120. Because gp120 toxicity is mediated by activation of the chemokine receptor CXCR4, neurons were incubated with the CXCR4 inhibitor AMD3100. This compound prevented gp120- but not glutamate-mediated cell death. These findings suggest that gp120 is toxic to neurons even in the absence of the virus and that the toxic mechanism involves primarily activation of CXCR4 receptor. Therefore, antagonists to the CXCR4 receptor may be more suitable compounds for inhibiting HIV-1 neurotoxicity.     

Ephrin-B1 promotes dendrite outgrowth on cerebellar granule neurons

Ephrins are developmentally regulated molecules that may contribute to axonal pathfinding through their binding to Eph receptor tyrosine kinases. In many cases, ephrins act as negative molecules that stimulate growth cone collapse, although some forms may promote axonal growth. Here, we have addressed the role played by ephrin-B1 during rat postnatal cerebellar development. Ephrin-B1 is expressed by both granule and Purkinje neurons whereas EphB is present in granule neurons in early postnatal cerebellum at a time coincident with axonal and dendrite outgrowth. Stably transfected 3T3 cells overexpressing ephrin-B1 enhance survival and neurite growth from cultured cerebellar granule neurons, an effect that is inhibited by the presence of an excess of a soluble EphB protein. Ephrin-B1-induced neuritogenesis is correlated with an increased expression of certain neuronal-specific microtubule-associated proteins (MAPs). Cerebellar granule neurons plated on stably transfected 3T3 cells overexpressing ephrin-B1 show an up-regulation of the expression of axonal MAPs such as Tau and phosphorylated MAP2C compared with neurons cultured on control 3T3 cells. The level of expression of these axonal MAPs is similar to that found in neurons plated on poly-L-lysine. Interestingly, there is a noteworthy up-regulation of somatodendritic MAPs such as high-molecular-weight MAP2 and mode II-phosphorylated MAP1B in neurons cultured on stably transfected 3T3 cells overexpressing ephrin-B1 compared with neurons plated on either control 3T3 cells or poly-L-lysine. In view of these data, we suggest that ephrin-B1 favors dendritogenesis of granule neurons during cerebellum development.     

Tri-iodothyronine regulates survival and differentiation of rat cerebellar granule neurons.

The effects of tri-iodothyronine (T3), which are known to affect cerebellar development, were tested on neuronal survival and differentiation of cultured cerebellar granule neurons. T3 in physiological concentrations increased both granule neuron survival after three days in culture and synaptic vesicle protein formation, as shown by immunostaining with antibodies against synaptophysin. Likewise, T3 increased the mRNA level for synapsin(I), but not that for GAP43 in granule neurons. Antibodies against microtubule associated protein Tau, which is expressed in developing neurites, showed that T3 also enhanced neurite formation.     

Laminin promotes cerebellar granule cells migration in vitro and is synthesized by cultured astrocytes

Newborn rat cerebellum microexplants have been used as a model to study neuronal migration. Laminin in a substrate-bound form modifies extensively the migratory behavior of the neurones, an effect which is blocked if antilaminin antibodies are present during the assay. 35S methionine incorporation followed by SDS-PAGE electrophoresis, fluorography and immunoprecipitation with antilaminin antibodies allowed to demonstrate that laminin is synthesized and secreted by cultured newborn rat cerebellum as well as by cultured newborn rat cerebral cortex astrocytes.     

Vitronectin promotes the progress of the initial differentiation stage in cerebellar granule cells

Vitronectin (VN), which is an extracellular matrix protein, is known to be involved in the proliferation and differentiation of primary cultured cerebellar granule cell precursors (CGCPs); however, the effect of VN is not fully understood. In this study, we analyzed the effects of VN loss on the proliferation and differentiation of CGCPs in VN knockout (VNKO) mice in vivo. First, immunohistochemistry showed that VN was distributed in the region from the inner external granule layer (iEGL) through the internal granule layer (IGL) in wild-type (WT) mice. Next, we observed the formation of the cerebellar cortex using sagittal sections of VNKO mice at postnatal days (P) 5, 8 and 11. Loss of VN suppressed the ratio of NeuN, a neuronal differentiation marker, to positive cerebellar granule cells (CGCs) in the external granule layer (EGL) and the ratio of CGCs in the IGL at P8, indicating that the loss of VN suppresses the differentiation into CGCs. However, the loss of VN did not significantly affect the proliferation of CGCPs. Next, the effect of VN loss on the initial differentiation stage of CGCPs was examined. The loss of VN increased the expression levels of Transient axonal glycoprotein 1 (TAG1), a marker of neurons in the initial differentiation stage, in the cerebella of VNKO mice at P5 and 8 and increased the ratio of TAG1-positive cells in the primary culture of VNKO-derived CGCPs, indicating that the loss of VN accumulates the CGCPs in the initial differentiation stage. Taken together, these results demonstrate that VN promotes the progress of the initial differentiation stage of CGCPs.     

Taurine-deficient cultured cerebellar astrocytes and granule neurons obtained by treatment with guanidinoethane sulfonate.

Mouse cerebellar granule neurons and astrocytes grown in the presence of 2 mM guanidinoethane sulfonate (GES) exhibited a progressive and rapid decrease in taurine concentration. A reduction of 20% was observed as early as 1 hr after exposure to GES and the loss of cell taurine continued until the taurine pool was reduced by about 90%. This remaining taurine persisted without further decrease even after 3 weeks of exposure to GES. Taurine reduction caused by GES was similar in both types of cells. The effect of GES was dose-dependent, with significant decreases in taurine levels already detected at 100 microM. It was selective for taurine, since none of the other free amino acids were affected. Taurine depletion induced by GES was totally reversible. Intracellular taurine was not mobilized by GES. Taurine uptake in both astrocytes and granule neurons, examined at the taurine concentration present in the culture medium, was practically abolished by 2 mM GES. This approach represents an in vitro model of taurine depletion that may be useful to investigate the cell abnormalities responsible for the failure of differentiation and migration of granule cells and astrocytes observed in taurine-deficient cats.     

Nitric oxide promotes survival of cerebellar granule neurons cultured in vitro through the Akt pathway

In this study,cerebellar granule neurons were used to examine the role of nitric oxide on cell survival.The N-methyl-D-aspartic acid receptor antagonist,MK-801,and the soluble guanylate cyclase antagonist,1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one,decreased cell viability,induced caspase-3,and decreased phosphorylated-Akt levels,suggesting that blockade of nitric oxide production promotes apoptosis of differentiating cerebellar granule neurons.After administration of sodium nitroprusside,an endogenous nitric oxide donor,cell viability recovered,caspase-3 expression was decreased,and phosphorylated-Akt levels increased.This study provides direct evidence that nitric oxide can sustain the survival of developing cerebellar granule neurons in vitro through the nitric oxide-Akt pathway.Moreover,endogenous nitric oxide exerts these effects in a cyclic guanosine monophosphate-dependent manner while exogenous nitric oxide does so in a cyclic guanosine monophosphate-independent manner.     

Cell-type specific effects of N-methyl-D-aspartate on biochemical differentiation of subcortical neurons in culture

The possible involvement of N-methyl-D-aspartate (NMDA) receptors in the biochemical differentiation of cultured neurons derived from the medial frontal part of the forebrain containing the septum-diagonal band region was studied in terms of the activities of enzymes important in the synthesis of neurotransmitter compounds. The activity of choline acetyltransferase (ChAT) was used as a marker for cholinergic neurons, glutamate decarboxylase (GAD) for GABAergic neurons and phosphate-activated glutaminase (GLNase) and aspartate aminotransferase (ASP-AT) for glutamatergic neurons, while lactate dehydrogenase (LDH) was included as an ubiquitous enzyme. The exposure of cultures to a depolarizing concentration of K+ (40 mM) for the last 3 days (i.e. between 2 and 5 days in vitro) significantly enhanced the expression of ChAT, GAD and GLNase activities, but high K+ caused little alteration in the activities of ASP-AT and LDH. On the other hand, treatment with NMDA markedly elevated the specific activities of GAD and GLNase only, and the compound had no significant effects on the activities of ChAT, ASP-AT and LDH enzymes. The enhancements of the specific activities of GAD and GLNase were completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca2+ ion channel blocker, MK-801. On the basis of the present findings it is concluded that, (a) contrary to an earlier proposal, ASP-AT does not appear to be a good marker for the glutamatergic neurons, (b) the failure of the subcortical cholinergic neurons to respond by an increase in ChAT activity to NMDA may indicate that these nerve cells lack NMDA subtype excitatory amino acid receptors, and (c) as the septal GABAergic input in the hippocampus is involved in the modulation of long-term potentiation, the presence of NMDA receptors on these neurons would now suggest that NMDA receptors are linked to both the initiation and the modulation of hippocampal plasticity in the mammalian brain.     

Thyroid hormone promotes BCL-2 expression and prevents apoptosis of early differentiating cerebellar granule neurons

Programmed cell death is a basic cellular process that has aroused much interest in recent years. Like immune cells, cultures of cerebellar granule neurons are very homogeneous and provide a unique opportunity for quantifying by flow cytometry one form of programmed cell death in the CNS, the apoptosis, and for studying its regulation by neurotrophic factors. We found that thyroid hormone promoted postmitotic survival by preventing the apoptosis of newly formed and early differentiated granule neurons in a dose-dependent manner. This regulation could be through the protein bcl-2, which is known to prevent cell death. This protein was present at all stages of granule neuron differentiation and appeared to be developmentally regulated. It was underexpressed in apoptotic granule neurons. The protein content of the cerebellum in hypothyroid rats was drastically reduced. In contrast, thyroid hormone caused a marked dose-dependent increase in the amounts of this protein in granule neuron cultures. The possibility that thyroid hormone may be directly or indirectly required to promote cell survival is discussed, in terms of the hormone control of the local delivery of neurotrophins, such as NGF and NT-3, as well as the expression of their low affinity receptors, gp75. We suggest that thyroid hormone has a permissive action on the developing CNS.     

Apoptotic morphology does not always require caspase activity in rat cerebellar granule neurons

The death of a cell via apoptosis is characterized by morphological changes including cell shrinkage and nuclear condensation. Intracellularly, proteases, including caspases, are activated. In the present article we have compared the ability of three different neurotoxic agents to induce caspase activity in cerebellar granule cells (CGC). These compounds are the micro-tubule-disrupting agent colchicine and the oxidative stress-inducing agents hydrogen peroxide and meth-ylmercury (MeHg). We have previously shown that each of these agents causes nuclear changes that are consistent with apoptosis, i.e., induction of chromatin condensation and DNA cleavage into fragments of regular size (700, 300 and 50 kbp). However, only colchicine causes a large increase in caspase activity, as monitored by the ability of whole cell extracts to cleave the synthetic caspase substrate DEVD-MCA. In contrast, MeHg and hydrogen peroxide do not induce any significant increase of DEVDase activity as compared to control cells. Immunocytochemistry confirms that active caspase-3 is abundant only in colchicine-exposed cells. In agreement with these findings, the pan-caspase inhibitor, z-VAD-fmk, is efficient in protecting CGC against colchicine, but not against hydrogen peroxide or MeHg. These data suggest that in CGC the activation of caspases is not always required to induce morphological changes and pattern of DNA fragmentation consistent with apoptosis.     

Increased sensitivity to N-methyl-D-aspartate receptor-induced excitotoxicity in cerebellar granule cells from interleukin-1 receptor type I-deficient mice

The effects of chronic exposure to excitatory amino acids (EAAs) were examined in cultured cerebellar granule cells (CGCs) from wild type (WT) and interleukin-1 receptor type I (IL-1RI)-deficient mice. After 8 days in culture, the cells were exposed to 100 microM glutamate or 300 microM N-methyl-D-aspartate (NMDA) for 24 h. Analysis of cell viability, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and phase-contrast microscopy revealed that CGCs from IL-1RI-deficient mice were more vulnerable to EAAs as compared to the WT controls. The results indicate that IL-1RI signalling is important for neuronal survival. The effect of glutamate on the CGCs from IL-1RI-deficient mice was decreased by the non-competitive NMDA-receptor antagonist MK-801, supporting the involvement of NMDA receptors in the glutamate-induced excitotoxicity.     

Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons

Cerebellar granule neurons can be readily maintained in culture if depolarized with high concentrations of K⁺ or subtoxic concentrations of various excitatory amino acids. We now report that these depolarizing stimuli promote cerebellar granule neuron survival by blocking their programmed death via apoptosis. Cerebellar granule neurons maintained in depolarizing conditions and then changed to non-depolarizing conditions, exhibit the morphological and biochemical features of apoptosis, including cytoplasmic blebbing, condensation and aggregation of nuclear chromatin internucleosomal DNA fragmentation. Inhibitors of RNA or protein synthesis greatly attenuate cell death induced by non-depolarizing culture conditions. In contrast, cerebellar granule neurons, when exposed to fresh serum-containing medium or to high concentrations of glutamate, exhibit a delayed-type of neurotoxicity which is non-apoptotic in nature. Given the actions of excitatory amino acid receptor agonists in preventing apoptosis of cultured cerebellar granule neurons, we hypothesize that the functional innervation of postmigratory granule neurons during cerebellar development may prevent further elimination of these neurons by blocking their programmed death.     

Acute neuregulin-1 signaling influences AMPA receptor mediated responses in cultured cerebellar granule neurons

Neuregulin-1 (NRG1) is a trophic and differentiation factor that signals through ErbB receptor tyrosine kinases to regulate nervous system development. Previous studies have demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons of the hippocampus and frontal cortex; however, immunohistochemical and genetic analyses strongly suggest these effects are indirect and mediated via ErbB4 receptors on GABAergic interneurons. Here, we used cultured cerebellar granule cells (CGCs) that express ErbB4 to analyze the cell-autonomous effects of NRG1 stimulation on glutamatergic function. These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We show that acute NRG1 treatment does not affect whole-cell AMPA or NMDA receptor (NMDAR) mediated currents in CGCs at 10-12 days in vitro. NRG1 also does not affect the frequency or amplitude of spontaneous AMPAR or NMDAR mediated miniature excitatory post-synaptic currents (mEPSCs). To further investigate the effects of NRG1 on activity-dependent plasticity of glutamatergic synapses in CGCs, we characterized the effects of high-glyine/0 Mg²⁺ (which activates synaptic NMDARs) on AMPAR-mEPSC frequency and amplitude. We show that high-glycine induces a form of chemical long-term potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency but not amplitude. Moreover, NRG1 induces a decrease in AMPAR-mEPSC frequency following chemLTP, but does not affect AMPAR-mEPSC amplitude. CGCs in our cultures conditions express low levels of GluR1, in contrast to dissociated hippocampal cultures, but do express the long isoform of GluR4. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and (2) that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar to Schaeffer collateral to CA1 synapses, NRG1 effects are activity dependent and mediated via modulation of synaptic AMPARs.     

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.     

Characterization of the activation of glutaminase induced by N-methyl-D-aspartate and potassium in cerebellar granule cells

Chronic stimulation of cerebellar granule cells with N-methyl-D-aspartate (NMDA) or KCl induces a specific activation of the enzymes directly involved in glutamate neurotransmitter synthesis. Phosphate-activated glutaminase (PAG) activity is enhanced in cultured granule neurons incubated with 150 μM NMDA or 25 mM KCl. Other enzymes are not affected by this treatment like lactate dehydrogenase (LDH) and glutamate dehydrogenase (GLDH), which is also a mitochondrial enzyme but not directly involved in neurotransmitter synthesis. This effect is dependent on protein synthesis and is induced after 12 hr of NMDA or KCl stimulation. Kinetics of PAG activity showed that K_m values were unaffected, in contrast to V_max values that were increased approximately 70% and 215% over control by NMDA and KCl treatment, respectively. For GLDH, we found two isoforms that were affected differentially by the experimental conditions. Western blot analysis clearly evidenced an increase of approximately 120–180% in the amount of PAG in NMDA- and KCl-treated cells, whereas GLDH was not significantly modified. These results demonstrate that the NMDA- and KCl-induced activation of PAG are not due to the modification of the preexisting enzyme, but to an increase in the synthesis of this enzyme. This suggests that NMDA receptor stimulation during critical periods of the cerebellar granule cell development leads to the activation of gene expression involved in the process of cell differentiation. © 1996 Wiley-Liss, Inc.     

[U-13C]aspartate metabolism in cultured cortical astrocytes and cerebellar granule neurons studied by NMR spectroscopy

The metabolism of [U-¹³C]aspartate was studied in cultured cortical astrocytes and cerebellar granule neurons in the presence of glucose and during inhibition of glycolysis. Redissolved, lyophilized cell extracts and incubation media were analyzed by ¹³C nuclear magnetic resonance spectroscopy for the determination of metabolites labeled from aspartate. Uniformly labeled lactate was prominent in control media of astrocytes and cerebellar granule neurons. In both cell types, aspartate entered the tricarboxylic acid (TCA) cycle, as shown by labeling patterns in glutamate and, in astrocytes, in glutamine. From the complex labeling patterns in aspartate in astrocytic perchloric acid extracts it was clear that acetylcoenzyme A (acetyl-CoA) derived from aspartate via oxaloacetate and pyruvate could enter the TCA cycle. Such “recycling,” however, could not be detected in cerebellar granule neurons. Inhibition of glycolysis reduced aspartate uptake and metabolism in both cell types. Most notably, lactate derived from aspartate showed a large reduction, and in astrocytes, incorporation of labeled acetyl-CoA into the TCA cycle was significantly reduced. Thus, astrocytes and cerebellar granule neurons differ in their handling of aspartate. Furthermore, inhibition of glycolysis clearly affected aspartate metabolism by such cells. GLIA 23:271–277, 1998. © 1998 Wiley-Liss, Inc.     

Role of brain-derived neurotrophic factor in the protective action of N-methyl-D-aspartate in the apoptotic death of cerebellar granule neurons induced by low potassium

Several neurotrophic factors, including brain-derived neurotrophic factor (BDNF), and neurotransmitters, such as glutamate, may influence neuronal apoptotic death. Rat cerebellar granule neurons (CGN) cultured in low potassium (5 or 10 mM KCl) for more than 5 days in vitro (DIV) die apoptotically. These cells survive in the presence of high potassium (25 mM KCl, K25) or N-methyl-D-aspartate (NMDA), an agonist of glutamatergic receptors. CGN transferred from high to low potassium die apoptotically. Here, we characterized the effect of BDNF and NMDA on the apoptotic death induced by low potassium in CGN. Cell death of CGN by culturing in low potassium for 6 DIV was inhibited by BDNF and NMDA. When CGN were cultured in K25 and transferred to a low-potassium medium, 65% of neurons died after 48 hr. Under these conditions, BDNF, NMDA, or BDNF + NMDA increased CGN survival. Both BDNF and NMDA decreased caspase-9 activity and mRNA caspase-3 levels and activity induced by low potassium. CGN survival induced by BDNF is mediated by TrkB activation, whereas that induced by NMDA is mediated by NMDA receptor and TrkB activation. NMDA, but not BDNF, raised [Ca(2+)](i), which was reduced by low-potassium treatment. These results suggest that NMDA receptor stimulation induces CGN survival through the influx of extracellular Ca(2+) that may evoke the release of BDNF and the activation of TrkB. Complementary mechanisms induced by depolarization and changes in Ca(2+) levels would also contribute to the neuroprotection exerted by NMDA and potassium.