Involvement of NMDA Receptor in the Modulation of Excitatory and Inhibitory Amino Acid Neurotransmitters Release in Cortical Neurons

The purpose of this paper was to examine the function of N-methyl-D-aspartate (NMDA) glutamate receptor in cortical neurons on amino acid neurotransmitters release as well as the fraction of neurons implicated in the response of this receptor. Local stimulation of these cells at different concentrations of NMDA, agonist of this ionotropic glutamate receptor, produced a dose dependent release of aspartate, glutamate, glycine and GABA. These effects were blocked by DAP5, an antagonist of the NMDA receptor. The amino acid Ca²⁺ dependent release mediated by the NMDA receptor, is induced by the opening of voltage-dependent Ca²⁺ channels that this receptor promotes. Ca⁺⁺ movements were explored in single cells loaded with fura-2. When single cells were stimulated with 100 μM NMDA, the calcium recording performed showed that 82% of the cells responded to this agonist increasing the intracellular calcium concentration, although the amplitude of these increments was variable. The results suggest that NMDA-elicited neurotransmitter release from cortical neurons involves Ca²⁺-dependent and Ca²⁺-independent components, as well as neuron depolarisation, and different VDCC subtypes of N, P/Q or L depending of the amino acid neurotransmitter release elicited by this receptor.     

Excitatory Amino Acid-Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons

Abstract: A large body of evidence suggests that disturbances of Ca²⁺ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca²⁺]_i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage-sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca²⁺]_i but via different mechanisms. By using the drug dantrolene, it has been shown that 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) apparently exclusively stimulates Ca²⁺ influx through agonist-operated calcium channels and voltage-operated calcium channels. Increased [Ca²⁺]_i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca²⁺]_i may be attributed to a release of Ca²⁺ from intracellular stores. Quisqualate (QA) stimulates Ca²⁺ release from an intracellular store that is independent of Ca²⁺ influx; presumably this store is activated by inositol phosphates. The increase in [Ca²⁺]_i due to exposure to glutamate or N-methyl-d -aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca²⁺ release from internal stores. Only one of the latter stores is dependent on Ca²⁺ influx with regard to release of Ca²⁺, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca²⁺ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca²⁺ pool(s). By using this drug it has been possible to show to what extent these Ca²⁺ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca²⁺ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca²⁺ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex.     

Mechanisms of heterogeneity of calcium response to kainate and neuronal types in rat cortical primary culture

The dynamics of intracellular Ca²⁺ signal in response to NMDA (N-methyl-D-aspartate, 30 μM) or KA (kainite, 30 μM), its dependence on extracellular Ca²⁺ and the mechanisms of KA-triggered Ca²⁺ entry into neurons have been tested in neurons of rat cortical primary cultures. The level of intracellular free Ca²⁺ concentrations ([Ca²⁺] _i ) was evaluated on Leica SP5 MF confocal microscope using Fluo-3 fluorescent dye, which resolves changes in [Ca²⁺] _i in the micromolar range. The dynamics of [Ca²⁺] _i increase in response to NMDA and KA was different but in both cases the [Ca²⁺] _i increase required the presence of Ca²⁺ in the extracellular solution. The neuronal population was found to be heterogeneous, based on the response to KA applied together with either L-type calcium channel blocker nifedipine (3 μM) or IEM-1460 (3 μM), a blocker of Ca²⁺-permeable AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) lacking GluR2 subunit. Experiments exhibited three types of calcium responses, characteristically belonging to interneurons (expressing Ca²⁺-permeable AMPAR), pyramidal neurons (with AMPAR containing GluR2, making them impermeable to Ca²⁺), and intermediate type of cells expressing both AMPAR types. Thus, we have demonstrated the role of AMPAR and L-type calcium channels in KA-triggered Ca²⁺ entry into neurons. The dynamics of [Ca²⁺] _i during the KA treatment was shown to depend on subunit composition of particular AMPAR subtype expressed in neurons. The data suggest that neuronal types existing in adult cortical tissue are probably presented in primary culture, too.     

Epileptiform curents in rat cortical neurons increase over time of the in vitro culture period

Here we show that in primary culture of rat cortical neurons the number of episodes of epileptiform curents (EC) provoked by extracellular magnesium removal increases over time. We demonstrate that NMDA receptor agonists in low concentrations induce an elevation of frequency of miniature postsynaptic currents followed by their synchronization resulting in EC. Ifenprodil did not block EC but strongly inhibited NMDA-evoked whole-cell currents, which say for a key role of the ifenprodil-resistant synaptic GluN2A-containing NMDA receptors in the generation of EC. We suppose that in cultured neurons the onset of EC and a gradual increase of the EC amplitude over the time of culture period is directed by an increase of synaptic connections density and displacement of the GluN2B subunit by GluN2A in synapses.     

Knockdown of the aryl hydrocarbon receptor attenuates excitotoxicity and enhances NMDA-induced BDNF expression in cortical neurons

NMDA receptors play dual and opposing roles in neuronal survival by mediating the activity-dependent neurotrophic signaling and excitotoxic cell death via synaptic and extrasynaptic receptors, respectively. In this study, we demonstrate that the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is involved in the expression and the opposing activities of NMDA receptors. In primary cultured cortical neurons, we found that NMDA excitotoxicity is significantly enhanced by an AhR agonist 2,3,7,8-tetrachlorodibenzo- p -dioxin, and AhR knockdown with small interfering RNA significantly reduces NMDA excitotoxicity. AhR knockdown also significantly reduces NMDA-increases intracellular calcium concentration, NMDA receptor expression and surface presentation, and moderately decreases the NMDA receptor-mediated spontaneous as well as miniature excitatory post-synaptic currents. However, AhR knockdown significantly enhances the bath NMDA application– but not synaptic NMDA receptor-induced brain-derived neurotrophic factor (BDNF) gene expression, and activating AhR reduces the bath NMDA-induced BDNF expression. Furthermore, AhR knockdown reveals the calcium dependency of NMDA-induced BDNF expression and the binding activity of cAMP-responsive element binding protein (CREB) and its calcium-dependent coactivator CREB binding protein (CBP) to the BDNF promoter upon NMDA treatment. Together, our results suggest that AhR opposingly regulates NMDA receptor-mediated excitotoxicity and neurotrophism possibly by differentially regulating the expression of synaptic and extrasynaptic NMDA receptors.     

Psoralidin Stimulates Expression of Immediate-Early Genes and Synapse Development in Primary Cortical Neurons

Upon synaptic stimulation and glutamate release, glutamate receptors are activated to regulate several downstream effectors and signaling pathways resulting in synaptic modification. One downstream intracellular effect, in particular, is the expression of immediate-early genes (IEGs), which have been proposed to be important in synaptic plasticity because of their rapid expression following synaptic activation and key role in memory formation. In this study, we screened a natural compound library in order to find a compound that could induce the expression of IEGs in primary cortical neurons and discovered that psoralidin, a natural compound isolated from the seeds of Psoralea corylifolia, stimulated synaptic modulation. Psoralidin activated mitogen-activated protein kinase (MAPK) signaling, which in turn induced the expression of neuronal IEGs, particularly Arc, Egr-1, and c-fos. N-methyl-d-aspartate (NMDA) receptors activation and extracellular calcium influx were implicated in the psoralidin-induced intracellular changes. In glutamate dose–response curve, psoralidin shifted glutamate EC₅₀ to lower values without enhancing maximum activity. Interestingly, psoralidin increased the density, area, and intensity of excitatory synapses in primary hippocampal neurons, which were mediated by NMDA receptor activation and MAPK signaling. These results suggest that psoralidin triggers synaptic remodeling through activating NMDA receptor and subsequent MAPK signaling cascades and therefore could possibly serve as an NMDA receptor modulator.

     

Over-expression of X-Linked Inhibitor of Apoptosis Protein Modulates Multiple Aspects of Neuronal Ca2+ Signaling

X-linked inhibitor of apoptosis (XIAP) protects and preserves the function of neurons in both in vitro and in vivo models of excitotoxicity. Since calcium (Ca²⁺) overload is a pivotal event in excitotoxic neuronal cell death, we have determined whether XIAP over-expression influences Ca²⁺-signaling in primary cultures of mouse cortical neurons. Using cortical neuron cultures derived from wild-type (Wt) mice transiently transfected with XIAP or from transgenic mice that over-express XIAP, we show that XIAP opposes the rise in intracellular Ca²⁺ concentration by a variety of triggers. Relative to control neurons, XIAP over-expression produced a slight, but significant, elevation of resting Ca²⁺ concentrations. By contrast, the rise in intracellular Ca²⁺ concentrations produced by N-methyl-d-aspartate receptor stimulation and voltage gated Ca²⁺ channel activation were markedly attenuated by XIAP over-expression. The release of Ca²⁺ from intracellular stores induced by the sarco/endoplasmic reticulum Ca²⁺ ATPase inhibitor thapsigargin was also inhibited in neurons transiently transfected with XIAP. The pan-caspase inhibitor zVAD did not, however, diminish the rise in intracellular Ca²⁺ concentrations elicited by l-glutamate suggesting that XIAP influences Ca²⁺ signaling in a caspase-independent manner. Taken together, these findings demonstrate that the ability of XIAP to block excessive rises in intracellular Ca²⁺ by a variety of triggers may contribute to the neuroprotective effects of this anti-apoptotic protein.     

Neuron-derived factors negatively modulate ryanodine receptor-mediated calcium release in cultured mouse astrocytes

Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) produced by ryanodine receptor (RyR) agonist, caffeine (caf), and ionotropic agonists: N-methyl-d-aspartate (NMDA) receptor (NMDAR) agonist, NMDA and P2X7 receptor (P2X7R) agonist, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP) were measured in cultured mouse cortical astrocytes loaded with the fluorescent calcium indicator Fluo3-AM in a confocal laser scanning microscope. In mouse astrocytes cultured in standard medium (SM), treatment with caf increased [Ca²⁺]_i, with a peak response occurring about 10 min after stimulus application. Peak responses to NMDA or BzATP were observed about <1 min and 4.5 min post stimulus, respectively. Co-treatment with NMDA or BzATP did not alter the peak response to caf in astrocytes cultured in SM, the absence of the effects being most likely due to asynchrony between the response to caf, NMDA and BzATP. Incubation of astrocytes with neuron-condition medium (NCM) for 24 h totally abolished the caf-evoked [Ca²⁺]_i increase. In NCM-treated astrocytes, peak of [Ca²⁺]_i rise evoked by NMDA was delayed to about 3.5 min, and that induced by BzATP occurred about three minutes earlier than in SM. The results show that neurons secrete factors that negatively modulate RyR-mediated Ca²⁺-induced Ca²⁺ release (CICR) in astrocytes and alter the time course of Ca²⁺ responses to ionotropic stimuli.     

Neuroprotective effects of vitexin by inhibition of NMDA receptors in primary cultures of mouse cerebral cortical neurons

The accumulation of glutamate can excessively activate the N-methyl-d-aspartate (NMDA) receptors and cause excitotoxicity. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside, Vit) is a c-glycosylated flavone which was found in the several herbs, exhibiting potent hypotensive, anti-inflammatory, and neuroprotective properties. However, little is known about the neuroprotective effects of Vit on glutamate-induced excitotoxicity. In present study, primary cultured cortical neurons were treated with NMDA to induce the excitotoxicity. Pretreatment with Vit significantly prevented NMDA-induced neuronal cell loss and reduced the number of apoptotic neurons. Vit significantly inhibited the neuronal apoptosis induced by NMDA exposure by regulating balance of Bcl-2 and Bax expression and the cleavages of poly (ADP-ribose) polymerase and pro-caspase 3. Furthermore, pretreatment of Vit reversed the up-regulation of NR2B-containing NMDA receptors and the intracellular Ca²⁺ overload induced by NMDA exposure. The neuroprotective effects of Vit are related to inhibiting the activities of NR2B-containing NMDA receptors and reducing the calcium influx in cultured cortical neurons.     

N-Methyl-D-aspartate receptor-dependent and -independent cytotoxic effects of Dictyostelium discoideum differentiation-inducing factor-1 on rat cortical neurons

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (small lipophilic hormone) that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. Recent studies have revealed that DIF-1 inhibits growth and induces the differentiation of mammalian tumor cells. The present study examines the effects of DIF-1 on rat cortical neurons in primary culture. We found that DIF-1 induced rapid neuronal cell death. The release of lactate dehydrogenase (LDH), as an indicator of cell death, increased dose-dependently with DIF-1. The release of LDH was inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists MK801 and AP5, suggesting that the NMDA receptor is involved in the induction of cell death by DIF-1. However, glutamate cytotoxicity could not explain the entire action of DIF-1 on neurons because the estimated concentration of glutamate around DIF-1-treated neurons was below 50 microM and DIF-1 caused more severe cell death than 500 microM glutamate. We discovered that another portion of DIF-1 cytotoxicity is independent of the NMDA receptor; that is, coaddition of DIF-1 and MK801 induced dendritic beading and increased expression of the immediate early genes c-fos and zif/268. These results indicate that DIF-1 induces rapid cell death via both NMDA receptor-dependent and -independent pathways in rat cortical neurons.     

Calcium transients in the model of rapidly induced anoxic tolerance in rat cortical slices: involvement of NMDA receptors

In this study, we investigated the effects of NMDA receptor antagonists on calcium transients induced by a single 2-min preconditioning anoxia (PA) in rat olfactory cortical slices, and on the ability of PA to prevent pathological calcium overload induced by subsequent 10-min test anoxia (TA). Relative changes in the intracellular Ca(2+) concentration (Ca(i)) and in the level of Ca(2+) bound to intracellular hydrophobic domains (Ca(b)) were monitored using fura-2 and chlortetracycline, respectively. Our results confirmed that TA induces prominent long-lasting increases in Ca(i) and Ca(b), reflecting cellular calcium overload. It was found that PA produces moderate increases in both Ca(2+) pools and prevents Ca(2+) overload induced by TA carried out 90 min later. Calcium transients and the protective effects of PA were significantly suppressed in slices treated with NMDA receptor antagonists during and 30 min after PA. These results indicate that moderate activation of the NMDA receptors participates in the mechanism of the PA-induced anoxic tolerance of cortical neurons.     

芝麻素抑制NMDA诱导的小鼠皮层神经元损伤

目的:研究芝麻素对NMDA所致原代培养小鼠皮层神经元损伤的保护作用及其机制。方法:体外培养原代皮层神经元;免疫荧光染色鉴定细胞纯度;MTT法测定细胞存活率;Hoechst/PI双染色观察细胞凋亡的形态学变化;激光共聚焦显微镜技术观察钙成像,检测细胞内钙离子浓度变化;Western blot检测各组细胞中Bcl-2、Bax、NR2A、NR2B蛋白的表达。结果:NMDA(200μM)60分钟能使神经元细胞存活率显著下降,细胞凋亡百分比明显增加(P0.01),芝麻素(0.1μM)能提高细胞存活率,减少细胞凋亡(P0.01)。与NMDA组相比,芝麻素能抑制钙超载;降低Bax和NR2B蛋白表达;增加和Bcl-2蛋白表达(P0.01)。结论:芝麻素具有神经保护作用,这种作用可能与抑制钙超载、下调NMDA受体亚型NR2B的表达以及调节Bcl-2家族蛋白有关。     

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl-d-aspartate receptors in cultured rat hippocampal neurons

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl₂ similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons.     

Modulation of NMDA Receptor Subunits Expression by Concanavalin A

The processes of N-methyl-d-aspartate (NMDA) receptor subunits expression were examined in cortical neurons and rat brain in order to investigate how the concanavalin A (Con A) modulates neuronal cells. Con A modulated the expression of NMDA receptor subunits in cultured cortical cells. Con A augmented the level of intracellular Ca²⁺ by α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA). We determined whether activation of AMPA receptors was involved in the regulation of NMDA receptor expression with Con A by blocking the desensitization of AMPA receptors. The results showed that AMPA receptor antagonists suppressed NMDA receptor subunits expression in Con A-treated cortical neuronal cells. PMA elevated the expression of NMDA receptor subunits, while PKC inhibitor and tyrosine kinases inhibitor suppressed the expression of NMDA receptor subunits. Furthermore, it was shown that NMDA receptor subunits expression was modulated in a region-specific manner after the sustained microinfusion of Con A into the cerebroventricle of the rat brain. Collectively, it could be presumed that the AMPA receptor activation was involved in Con A-induced modulation of NMDA receptor subunits expression.     

Increased Na+/Ca2+ Exchanger Activity Promotes Resistance to Excitotoxicity in Cortical Neurons of the Ground Squirrel (a Hibernator)

Ground squirrel, a hibernating mammalian species, is more resistant to ischemic brain stress than rat. Gaining insight into the adaptive mechanisms of ground squirrels may help us design treatment strategies to reduce brain damage in patients suffering ischemic stroke. To understand the anti-stress mechanisms in ground squirrel neurons, we studied glutamate toxicity in primary cultured neurons of the Daurian ground squirrel (Spermophilus dauricus). At the neuronal level, for the first time, we found that ground squirrel was more resistant to glutamate excitotoxicity than rat. Mechanistically, ground squirrel neurons displayed a similar calcium influx to the rat neurons in response to glutamate or N-methyl-D-aspartate (NMDA) perfusion. However, the rate of calcium removal in ground squirrel neurons was markedly faster than in rat neurons. This allows ground squirrel neurons to maintain lower level of intracellular calcium concentration ([Ca²⁺]_i) upon glutamate insult. Moreover, we found that Na⁺/Ca²⁺ exchanger (NCX) activity was higher in ground squirrel neurons than in rat neurons. We also proved that overexpression of ground squirrel NCX2, rather than NCX1 or NCX3, in rat neurons promoted neuron survival against glutamate toxicity. Taken together, our results indicate that ground squirrel neurons are better at maintaining calcium homeostasis than rat neurons and this is likely achieved through the activity of ground squirrel NCX2. Our findings not only reveal an adaptive mechanism of mammalian hibernators at the cellular level, but also suggest that NCX2 of ground squirrel may have therapeutic value for suppressing brain ischemic damage.     

Neurotrophic effect of isoquinoline derivatives in primary cortical culture.

Recent studies indicate that the N-methyl-D-aspartate (NMDA) antagonist, (+)-1-methyl-1-phenyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (FR 115427), enhanced neuronal survival in primary culture of cortical neurons from mouse embryos. In the present study isoquinoline derivatives were examined for the neurotrophic activity in primary culture of cortical neurons and were also examined for anti-NMDA activity. In spite of varying level of anti-NMDA activity, isoquinoline derivatives enhanced neuronal survival at the concentration of 10 microM. To elucidate of the mechanisms of neurotrophic activity in primary cortical culture, nicardipine and flunarizine, known calcium channel blockers, were also tested. Neither nicardipine nor flunarizine showed neurotrophic activity up to the doses causing toxicity in cultured neurons. NBQX, an AMPA receptor antagonist, was also tested for neurotrophic activity. However no enhancement of neuronal survival was observed. These data suggest that one of the mechanisms to promote neuronal survival may depend on the structure of isoquinoline ring. Moreover neurotrophic activity observed in our culture systems might not relate on anti-NMDA activity, blockade of voltage dependent L-type calcium channels and antagonization of AMPA receptor.     

NMDA受体的活化调节原代皮层神经元的Wnt/β-catenin信号通路

经典的Wnt/β-catenin信号通路在中枢神经系统突触形成和功能中发挥重要的调节作用。作为兴奋性神经递质的谷氨酸,与其受体结合,参与许多信号调节活动。为了探讨NMDA受体活化对Wnt/β-catenin信号通路的作用,该文利用18 d的C57小鼠胚胎培养皮层神经元（离体10 d）,用10μmol/L谷氨酸钠（monosodium glutamate,MSG）和50μmol/L N-甲基-D-天冬氨酸（NMDA）处理细胞,通过蛋白免疫印迹技术或者细胞免疫荧光染色分析Wnt/β-catenin信号通路关键成员。结果发现,NMDA受体的活化能使GSK-3β的Ser9位磷酸化水平增加,活性被抑制,胞浆内β-catenin蛋白降解减少,入核增加,激活下游基因表达。这些结果提示,NMDA受体激活能够上调Wnt/β-catenin信号通路。     

Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-D-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP.     

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP⁺ induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP⁺ injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca^2 + concentration ([Ca^2 +]_cyt) and Ca^2 + in endoplasmic reticulum (ER) ([Ca^2 +]_ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca^2 + release up to 120 min after MPP⁺ injury. Furthermore, decrease of [Ca^2 +]_cyt induced by Homer1 knockdown in MPP⁺ treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP⁺ insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.     

Mechanisms for regulating synaptic efficiency in the visual cortex

Brief epochs of pairing of low frequency synaptic activation and postsynaptic depolarization, in vitro, in supragranular neurons of mature guinea-pig visual cortex lead to a transient (20–60 min) synaptic potentiation. This process is due to a true up-regulation of excitatory synapse efficiency onto the activated neuron. The potentiation requires NMDA receptor activation and a postsynaptic calcium signal for induction and it is modifiable by endogenous nitric oxide (NO) production in the mature cortex. In the cortex of young animals (< PND 21), the pairing-induced potentiation is robust and depends on a postsynaptic calcium signal but it is independent of NMDA receptor activation and NO production. The ability of cortical synaptosomes to release endogenous glutamate is enhanced by NMDA receptor activation and this enhancement is NO-dependent. The NO signal, however, does not amplify the glutamate release of all synapses but only those that have activated voltage-gated calcium channels and were presumably more active at the time of the NO signal. Electrophysiological recordings from visual cortical neurons in anesthetized cats with local iontophoresis of compounds that inhibit or facilitate endogenous cortical NO production reveal the capacity for NO to modulate visual responses in vivo. NO appears to act in the intact cortex by amplifying signals of visual inputs that were co-active at the time of the NO production. The adult visual cortex is capable of dramatic alterations in synaptic efficiency over brief periods suggesting a dynamic cortical network. NMDA receptors and nitric oxide contribute to these processes.