Microwave Exposure Impairs Synaptic Plasticity in the Rat Hippocampus and PC12 Cells through Over-activation of the NMDA Receptor Signaling Pathway

Objective The aim of this study is to investigate whether microwave exposure would affect the N-methyI-D-aspartate receptor （NMDAR） signaling pathway to establish whether this plays a role in synaptic plasticity impairment. Methods 48 male Wistar rats were exposed to 30 mW/cm^2 microwave for 10 min every other day for three times. Hippocampal structure was observed through H＆E staining and transmission electron microscope. PC12 cells were exposed to 30 mW/cm^2 microwave for 5 min and the synapse morphology was visualized with scanning electron microscope and atomic force microscope. The release of amino acid neurotransmitters and calcium influx were detected. The expressions of several key NMDAR signaling molecules were evaluated. Results Microwave exposure caused injury in rat hippocampal structure and PC12 cells, especially the structure and quantity of synapses. The ratio of glutamic acid and gamma-aminobutyric acid neurotransmitters was increased and the intracellular calcium level was elevated in PC12 cells. A significant change in NMDAR subunits （NR1, NR2A, and NR2B） and related signaling molecules （CaZ＋/calmodulin-dependent kinase II gamma and phosphorylated cAMP-response element binding protein） were examined. Conclusion 30 mW/cm^2 microwave exposure resulted in alterations of synaptic structure, amino acid neurotransmitter release and calcium influx. NMDAR signaling molecules were closely associated with impaired synaptic plasticity.

Influences of Microwave on the Cognitive Function of Chickling and the Gene Expression of NMDA Receptor Subunit

[Objective] The research aimed to study the effects of microwave on the chick embryo development and the cognitive function of chickling. [Method] The microwave which was transmitted by the permatron and was 2 450 MHz was used to simulate the microwave radiation source to radiate the hatching eggs until the chickling was hatched out. The disposable passive avoidance learning and RT-PCR were respectively used to detect the influences of microwave on the cognitive function of chickling and the expression amounts of NMDA receptor NR1 and NR2 subunits. [Result] After the microwave radiation,the avoidance rate of exposed group was significantly lower than that in the control group. Especially the avoidance rate of highest radiation intensity group was extremely significantly lower than that in the control group. Meanwhile,the body weights of two groups of chickling in the exposed group increased,and the hatching time in one group increased. Via RT-PCR analysis,the expression amount of NR2 subunit increased on the 10th day and the 15th day. The expression amount of NR1 subunit only decreased on the 15th day. [Conclusion] The microwave had the certain influence on the individual development. By changing the structure composition and function of NMDA receptor in the endbrain,the microwave made the self-regulation ability of chickling decline,which had the certain damage on the cognitive function.

Isoflurane Enhances the Expression of Cytochrome C by Facilitation of NMDA Receptor in Developing Rat Hippocampal Neurons In Vitro

This study examined the effects of clinically relevant concentrations of isoflurane on the amplitude of NMDA receptor current （INMDA） and the expression of cytochrome C in cultured developing rat hippocampal neurons. The hippocampi were dissected from newborn Sprague-Dawley rats. Hippocampal neurons were primarily cultured for 5 days and then treated with different concentrations of isoflurane [（0.25, 0.5, 0.75, 1 minimum alveolar concentration （MAC））]. The peak of INMDA was re- corded by means of the whole cell patch clamp technique. The cytochrome C level was detected by Western blotting and quantitative real-time PCR. Our results showed that isoflurane （0.25, 0.5, 0.75 and 1 MAC） potentiated the amplitude of INMDA by （116±8.8）%, （122±11.7）%, （135±14.3）% and （132~14.6）%, respectively, and isoflurane increased the mRNA expression of cytochrome C in a concentration-dependent manner. The cytochrome C mRNA expression reached a maximum after 0.5 MAC isoflurane stimulation for 6 h （P〈0.05）. It was concluded that isoflurane enhances the expression of cytochrome C in cultured rat hippocampal neurons, which may be mediated by facilitation of NMDA receptor.

Effect of glycine site/NMDA receptor antagonist MRZ2/576 on the conditioned place preference and locomotor activity induced by morphine in mice

Objective: To study the effect of glycine site/NMDA (N-methyl-D-aspartate) receptor antagonist MRZ2/576 on the conditioned place preference (CPP) and locomotor activity induced by morphine in mice. Methods: Different doses (1.25, 2.5 and 5 mg/kg, i.p.) of MRZ2/576 were used to evaluate the effect of MRZ2/576 on the acquisition and expression of CPP induced by morphine (5 mg/kg) in mice. In addition, we examined the locomotor activity of mice in conditioning and testing phase of CPP paradigm. Results: MRZ2/576 alone could not establish place preference, but a 5 mg/kg dose of MRZ2/576 could block both acquisition and expression of morphine-induced CPP. In testing phase of CPP, there was no statistical difference for locomotor activity between the groups; injection of MRZ2/576 showed a dose-dependent decrease of locomotor activity on both control and morphine-treated mice, especially 5 mg/kg of MRZ2/576 significantly suppressed the locomotor activity of mice. Conclusion: Based on the present results, we assume that MRZ2/576 can antagonize the rewarding effect of morphine, suggesting that this glycine site/NMDA receptor antagonist could be used to treat addictions due to its light side effect profile.

The Effect of Coriaria Lactone on NMDA Receptor Mediated Currents in Rat Hippocampal CA1 Neurons

Summary: To investigate the exact mechanism of epileptogenesis induced by coriaria lactone (CL), the effect of CL on NMDA receptor mediated current (IAsp) in rat hippocampal CA1 neu- rons was investigated by using nystatin perforated whole-cell patch clamp. 10-6-10-4 mol/L Asp acted on NMDA receptors and elicited an inward current (IAsp) at a holding potential (VH) of -40 mV in presence of 10-6 mol/L glycine and absence of Mg2+ extracellularly. CL enhanced NMDA receptor mediated current induced by Asp, but had no effect on threshold concentration, EC50, Hill coefficient as well as maximal-effect concentration and reversal potential of IAsp. The effect had no relationship with holding potential. These results showed that CL could enhance NMDA receptor mediated current to increase [Ca2+]i of neurons by acting on Gly site, thereby inducing epilepsy.

Salicylate enhances expression and function of NMDA receptors in cochlear spiral ganglion neurons

Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons （SGNs）. Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by Real time fluorescence quantitative PCR （FQ-PCR）. NMDA receptor whole-cell currents were recorded using patch clamp in acute isolated SGNs. Results Compared with the control group, salicylate significantly increased the mRNA level of NR1 subunit in SGNs. NMDA of concentrations ranging from 0.1 mM to 10 mM evoked no current in SGNs. NMDA （0. 1mM and 0.5 mM） applied with salicylate （5 mM）, however, induced inward currents （212.6±15.2pA, n=5; 607.9±44.3pA, n=5） in a dose-dependent manner, which could be inhibited by APV. Salicylate alone did not produce any current in SGNs. Conclusion Salicylate increases the expression of NMDA receptors and facilitates the currents mediated by NMDA receptors in SGNs.

Influences of NR2B-containing NMDA Receptors Knockdown on Neural Activity in Hippocampal Newborn Neurons

Summary： Adult-bom neurons undergo a transient period of plasticity during their integration into the neural circuit. This transient plasticity may involve NMDA receptors containing NR2B, the major sub unit expressed at early developmental stages. The main objective of the present study was to investigate the effects of NR2B gene knockdown on the functional integration of the adult-born granule cells gen- erated from the subgranule zone （SGZ） in the hippocampus. The small interfering RNA （siRNA） was used to knock down the NR2B gene in the adult-born hippocampal neurons. In the functional integration test, the mice were exposed to a novel environment （open field arena）, and the expression of c-fos was immunohistochemically detected in the hippocampus. After exposure to the novel environment, siRNA-NR2B mice were significantly different from control mice in either the number of squares or the number of rears they crossed, showing decreased horizontal and vertical activity （P〈0.05）. Moreover, the c-fos expression was increased in both control and siRNA-NR2B mice after open field test. But, it was significantly lower in siRNA-NR2B neurons than in control neurons. It was concluded that the neu- ral activity of newborn neurons is regulated by their own NR2B-containing NMDA glutamate receptors during a short, critical period after neuronal birth.

K2P channels and NMDA receptor as new targets of fast onset antidepressants

Major depressive disorder(MDD)is a common neuropsychiatric disorder characterized by diverse symptoms.There are big limitations of clinic medicine which highlighted an urgent and clear need for more efficacious and faster-acting therapeutic agents to treat patients with MDD,especially those who are refractory to the traditional antidepressants.In the present study,we assessed a novel compound,YY-21,from timosaponin B-Ⅲ derived from sarsasapogenin of Anemarrhenae Rhizoma.We found that YY-21 obviously increased presynaptic glutamate release and enhanced long-term synaptic activity within 10 min as determined by excitatory postsynaptic current(EPSC) and field excitatory postsynaptic potential(fEPSP) in medial prefrontal cortex(mPFC) slices.YY-21 demonstrated anxiolytic-like effects following acute administration in animals and reversed the depressivelike and anxiety phenotypes induced by chronic unpredictable mild stress(CMS) with a relatively fast therapeutic onset.Our mechanism research reveals that NMDA receptors and two-pore domain potassium(K2P)(TREK1) channels emerged as new drug targets for faster acting antidepressants.K2 P channels generate leak currents that are responsible the maintenance of resting membrane potential.They are potential targets for the treatment of multiple diseases.Here we identify TKDC,an inhibitor of the TREK subfamily,including TREK1,TREK2 and TRAAK channels.Using TKDC as a chemical probe,a combined study of computations,mutagenesis,and electrophysiology reveal an allosteric ligand-binding site in the extracellular cap of the channels.The molecular dynamics simulations suggest that ligand-induced allosteric conformational transitions cause a blockage of the ion conductive pathway.The identification of the extracellular ligand-binding site is confirmed by the discovery of new inhibitors targeting this site using virtual screening.These results suggest that the extracellular cap of a K2P channel can act as a new allosteric site and may serve as a direct drug target.

Preparation of ^(99m)Tc-PQQE and preliminary biological evaluation for the NMDA receptor

The 4,5-dioxo-4,5-dihydro-1H-pyrrolo(2,3-f)quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester (PQQE) was synthesized on the basis of Pyrroloquinoline quinine (PQQ). 99m Tc-PQQE was prepared using stannous fluoride (SnF 2 ) as reducing agent. Biological characteristics of 99m Tc-PQQE include lipophilic and the charge properties were compared to 99m Tc-PQQ. The biodistributions of 99m Tc-PQQE in mice and brain regional distribution were performed. In vivo distribution of 99m Tc-PQQE in mice indicates that the concentration ratio of drug and blood increases steadily over time. The major radioactivity may be metabolized by the hepatic and renal system. The elimination-phase half-time (t1/2β) results indicate that the residence time of 99m Tc-PQQE (203.92) in the body is twice as long as 99m Tc-PQQ (100.45). The uptake of 99m Tc-PQQE in brain was improved due to the ameliorating of charge and lipophilicity. The highest total regional brain uptake of 99m Tc-PQQE was in the frontal lobe and hippocampus, where the NMDA receptor is very abundant. 99m Tc-PQQE had a good target to nontarget ratio (hippocampus/cerebellum) which preserved a higher value (peak 4.0 at 120 min) from 60 min to 180 min after injection. In vitro autoradiographic results are in close agreement with the regional brain map. The enrichment can be blocked by N-methyl-D-aspartate receptor (NMDAR) redox modulatory site antagonists-ebselen (EB). This work suggests that 99m Tc-PQQE has some specific targeting to the NMDA receptor.

Radio-ligand receptor binding assav in vitro and animal biodistribution in vivo of ^(99)Tc^m-N-ethyl-N_2S_2-memantine as a potential NMDA receptor imaging agent

The pharmacologic characteristics of ^(99)Tc^m-N-ethyl-N_2S_2-memantine,an NMDA receptor imaging agent,was investigated.It was prepared by a one-step reaction from N-ethyl-N_2S_2-memantine.The affinity and specificity were determined by radio-ligand receptor binding assay(RRA).Biodistribution in vivo in mice was performed.The results showed that ^(99)Tc^m-N-ethyl-N_2S_2-memantine bound to a single site on NMDA receptor with a K_d of 584.32 nmol/L and a B_(max)of 267.05 nmol/mg.A competitive analysis showed that such specific binding could be inhibited by specific inhibitors of NMDA receptor,such as ketamine and(+)-MK-801.The biodistribution exhibited rapid uptake and favorable retention in mice brains.The major radioactivity was metabolized by the hepatic system.A two-compartment model of C=4.49e^(-0.083t)+ 1.42e^(-0.0016t)was established,and the half life was 8.35 min in blood.In conclusion,the new radio-ligand ^(99)Tc^m-N-ethyl-N_2S_2-Memantine has a moderate affinity and specific binding to NMDA receptor,and can easily cross the blood-brain barrier(BBB).Therefore,it may be a potential NMDA receptor imaging agent.

"Smart fats", healthy brain and function of lipid-sensing NMDA receptors

NMDA receptor channels play a significant role in learning and memory and their dysfunction can cause neuronal cell death leading to dementia. Research had shown that lipids change the risk for dementia, especially some omega-3 lipids appear to lower Alz-heimer’s risk, yet only limited research exists on the modulation of NMDA receptor channels by lipids. Here we review recent literature concerning molecular determinants that influence the NMDA receptor channel gating via membrane lipids and fatty acids with profound significance for understanding how altered NMDA signalling leads to neuronal cell death linked to age-related dementia’s. Future discovery of lipid-like modulators of NMDA receptor function offer the potential for the development of new bioceu-ticals and affordable nutritional supplements to combat neuronal degeneration as well as to promote well being and healthy aging.

Myosin Va‑dependent Transport of NMDA Receptors in Hippocampal Neurons

N-methyl-D-aspartate receptor(NMDAR)trafficking is a key process in the regulation of synaptic efficacy and brain function.However,the molecular mechanism underlying the surface transport of NMDARs is largely unknown.Here we identified myosin Va(MyoVa)as the specific motor protein that traffics NMDARs in hippocampal neurons.We found that MyoVa associates with NMDARs through its cargo binding domain.This association was increased during NMDAR surface transport.Knockdown of MyoVa suppressed NMDAR transport.We further demonstrated that Ca^(2+)/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)regulates NMDAR transport through its direct interaction with MyoVa.Furthermore,MyoVa employed Rab11 family-interacting protein 3(Rab11/FIP3)as the adaptor proteins to couple themselves with NMDARs during their transport.Accordingly,the knockdown of FIP3 impairs hippocampal memory.Together,we conclude that in hippocampal neurons,MyoVa conducts active transport of NMDARs in a CaMKII-dependent manner.

氟中毒大鼠脑组织中NMDA受体及内质网应激相关通路蛋白的表达

背景:前期研究发现N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid receptor,NMDA)受体与氟相关,但其在氟诱导的内质网应激中的作用尚不明确。目的:观察实验性氟中毒大鼠脑组织中兴奋性神经递质NMDA受体及内质网应激IRE1α-ASK1-JNK通路蛋白表达变化,并应用NMDA受体抑制剂干预SH-SY5Y细胞,探讨氟中毒神经系统损伤的发病机制。方法:(1)动物模型:18只1月龄SD大鼠随机分为对照组(饮水含氟量<0.5 mg/L)、低氟组(饮水含氟量为10.0 mg/L)、高氟组(饮水含氟量为100.0 mg/L),每组6只,雌雄各半。饮水摄氟饲养6个月后,观察大鼠氟斑牙发生情况,测定24 h尿氟含量;大鼠麻醉处死后取脑组织观察组织病理变化,蛋白印迹法检测脑组织中NMDA受体及IRE1α、ASK1、JNK蛋白表达。(2)细胞模型:体外培养SH-SY5Y细胞,选择终浓度为0.3 mmol/L和3 mmol/L的氟化钠染氟处理,并用10μmol/L的NMDA受体拮抗剂Ifenprodil、MK-801对染氟细胞进行干预,观察相关蛋白改变。结果与结论:(1)高氟组大鼠氟斑牙发生率、尿氟水平显著高于对照组和低氟组(P<0.05);(2)与对照组相比,低氟组大鼠海马CA3区神经细胞胞质嗜碱性略增加,高氟组CA3区神经细胞排列紊乱,嗜碱性增加,部分细胞核固缩;(3)大鼠脑组织内,高氟组NR2A与低氟组NR2B的蛋白表达明显高于对照组(P<0.05),且高氟组NR2B、IRE1、ASK1、p-JNK蛋白表达明显高于对照组和低氟组(P<0.05);(4)SH-SY5Y细胞内,高氟组NR1、NR2A、NR2B蛋白表达明显高于对照组(P<0.05),且高氟+Ifenprodil组、高氟+MK-801组NR1、NR2A蛋白表达均明显低于高氟组(P<0.05),高氟+Ifenprodil组NR2B蛋白表达也明显低于高氟组(P<0.05);(5)SH-SY5Y细胞内,高氟组IRE1、ASK1、p-JNK蛋白表达明显高于对照组(P<0.05),且高氟+Ifenprodil组、高氟+MK-801组ASK1、p-JNK蛋白表达均明显低于高氟组(P<0.05),高氟+Ifenprodil组IRE1蛋白水平也明显低于高氟组(P<0.05);(6)提示:过量氟摄入可激活中枢神经系统NMDA受体,引起内质网应激IRE1α、ASK1、p-JNK蛋白表达增加,使用NMDA受体抑制剂对氟中毒所致内质网应激具有一定缓解作用。

Clustering of surface NMDA receptors is mainly mediated by the C-terminus of GluN2A in cultured rat hippocampal neurons

N-methyI-D-aspartate receptors （NMDARs） containing different GluN2 subunits play distinct roles in synaptic plasticity. Such differences may not only be determined by the channel properties, but also by differential surface distribution and synaptic localization. In the present study, using a Cy3-conjugated Fab fragment of the GFP antibody to label surface-located GluN2 subunits tagged with GFP at the N-terminus, we observed the membrane distribution patterns of GluN2A- or GluN2B-containing NMDARs in cultured rat hippocampal neurons. We found that surface NMDARs containing GluN2A, but not those containing GluN2B, were inclined to cluster at DIV7. Swapping the carboxyl termini of the GluN2 subunits completely reversed these distribution patterns. In addition, surface NMDARs containing GluN2A were preferentially associated with PSD-95. Taken together, the results of our study suggest that the clustering distribution of GluN2A- containing NMDARs is determined by the GluN2A C-terminus, and its interaction with PSD-95 plays an important role in this process.

Formaldehyde increases intracellular calcium concentration in primary cultured hippocampal neurons partly through NMDA receptors and T-type calcium channels

Objective Formaldehyde at high concentrations is a contributor to air pollution. It is also an endogenous metabolic product in cells, and when beyond physiological concentrations, has pathological effects on neurons. Formaldehyde induces mis-folding and aggregation of neuronal tau protein, hippocampal neuronal apoptosis, cognitive impairment and loss of memory functions, as well as excitation of peripheral nociceptive neurons in cancer pain models. Intracellular calcium （[Ca2＋]i） is an important intracellular messenger, and plays a key role in many pathological processes. The present study aimed to investigate the effect of formaldehyde on [Ca2＋]i and the possible involvement of N-methyl-D-aspartate receptors （NMDARs） and T-type Ca2＋ channels on the cell membrane. Methods Using primary cultured hippocampal neurons as a model, changes of [Ca2＋]i in the presence of formaldehyde at a low concentration were detected by confocal laser scanning microscopy. Results Formaldehyde at 1 mmol/L approximately doubled [Ca2＋]i. （2R）-amino-5-phosphonopentanoate （AP5, 25 μmol/L, an NMDAR antagonist） and mibefradil （MIB, 1 μmol/L, a T-type Ca2＋ channel blocker）, given 5 min after formaldehyde perfusion, each partly inhibited the formaldehyde-induced increase of [Ca：＋]i, and this inhibitory effect was reinforced by combined application of AP5 and MIB. When applied 3 min before formaldehyde perfusion, AP5 （even at 50μmol/L） did not inhibit the formaldehyde-induced increase of [Ca2＋]i, but MIB （1 μmol/L） significantly inhibited this increase by 70%. Conclusion These results suggest that formaldehyde at a low concentration increases [Ca2＋]i in cultured hippocampal neurons; NMDARs and T-type Ca2＋ channels may be involved in this process.

Recent development in NMDA receptors

Purpose To identify the structure and the function of NMDA receptors, to understand the modulatory mechanism of some endogenous and exogenous compounds on NMDA receptors, and to provide theoretical basis for developing new drugs that modulate NMDA receptors Data sources A total of 24 originally identified articles were selected Study selection A total of 24 articles were selected from several hundred original articles or reviews. The content of selected articles are in accordance with our purpose and the authors are authorized scientists in the study on NMDA receptors.Data extraction After careful review of the selected papers, the meaningful results and conclusions were extracted using scientific criteria and our experience in the research of NMDA receptors.Results NMDA receptor contains at least five subunits. They were designated as the NR1 (ζ1), NR2A (ε1), NR2B (ε2), NR2C (ε3), and NR2D (ε4). A unique feature of NMDA receptor is the requirement for both glutamate and the co agnist glycine for the efficient gating. NMDA receptor is modulated by a number of endogenous and exogenous compounds. Mg 2+ not only blocks the NMDA channel in a voltage dependent manner but also potentiates NMDA induced responses at positive membrane potentials. Na +, K + and Ca 2+ not only pass through the NMDA receptor channel but also modulate the activity of NMDA receptors. Zn 2+ blocks the NMDA current in a noncompetitive and a voltage independent manner. It has been demonstrated that polyamines do not directly activate NMDA receptors, but instead act to potentiate or inhibit glutamate mediated responses. The activity of NMDA receptors is also strikingly sensitive to the changes in H + concentration, and partially inhibited by the ambient concentration of H + under physiological conditions.Conclusions NMDA receptors are glutamate regulated by ion channels that are permeable to Ca 2+ , Na +, K + and are sensitive to voltage dependent Mg 2+ block This channel complex contributes to excitatory synaptic transmission at sites throughout the brain and the spinal cord,and is modulated by a number of endogenous and exogenous compounds NMDA receptors play a key role in wide range of physiologic and pathologic processes Five NMDA receptor subunits have now been characterized in both rat and mouse brain

TRPV4 Regulates Soman-Induced Status Epilepticus and Secondary Brain Injury via NMDA Receptor and NLRP3 Inflammasome

Nerve agents are used in civil wars and terrorist attacks,posing a threat to public safety.Acute exposure to nerve agents such as soman(GD)causes serious brain damage,leading to death due to intense seizures induced by acetylcholinesterase inhibition and neuronal injury resulting from increased excitatory amino-acid levels and neuroinflammation.However,data on the anticonvulsant and neuroprotective efficacies of currently-used countermeasures are limited.Here,we evaluated the potential effects of transient receptor vanilloid 4(TRPV4)in the treatment of soman-induced status epilepticus(SE)and secondary brain injury.We demonstrated that TRPV4 expression was markedly up-regulated in rat hippocampus after soman-induced seizures.Administration of the TRPV4 antagonist GSK2193874 prior to soman exposure significantly decreased the mortality rate in rats and reduced SE intensity.TRPV4-knockout mice also showed lower incidence of seizures and higher survival rates than wild-type mice following soman exposure.Further in vivo and in vitro experiments demonstrated that blocking TRPV4 prevented NMDA receptor-mediated glutamate excitotoxicity.The protein levels of the NLRP3 inflammasome complex and its downstream cytokines IL-1βand IL-18 increased in soman-exposed rat hippocampus.However,TRPV4 inhibition or deletion markedly reversed the activation of the NLRP3 inflammasome pathway.In conclusion,our study suggests that the blockade of TRPV4 protects against soman exposure and reduces brain injury following SE by decreasing NMDA receptor-mediated excitotoxicity and NLRP3-mediated neuroinflammation.To our knowledge,this is the first study regarding the“dual-switch”function of TRPV4 in the treatment of soman intoxication.

Serine 707 of APPL1 is Critical for the Synaptic NMDA Receptor-Mediated Akt Phosphorylation Signaling Pathway

Accumulating evidence indicates that the synaptic activation of N-methyl-o-aspartate receptors （NMDARs） has a neuroprotective effect on neurons. Our previous study demonstrated that APPL1 （adaptor protein containing pleckstrin homology domain, phosphotyrosine- binding domain, and leucine zipper motif） mediates the synaptic activity-dependent activation of PI3K-Akt signaling via coupling this pathway with NMDAR-PSD95 （postsynaptic density protein 95） complexes. However, the molecular mechanism underlying this process is still unknown. In the present study, we investigated the inter- action of APPL1 with PSD95 using co-immunocyto- chemical staining and western blotting. We found that the PDZ2 domain of PSD95 is a binding partner of APPL1. Furthermore, we identified serine 707 of APPL1, a pre- dicted phosphorylation site within the PDZ-binding motif at the C-terminus, as critical for the binding of APPL1 to PSD95, as well as for activation of the Akt signaling pathway during synaptic activity. This suggests that serine 707 of APPL1 is a potential phosphorylation site and may be involved in regulating the neuroprotective Akt signaling pathway that depends on synaptic NMDAR activity.

Induction of Increased Intracellular Calcium in Astrocytes by Glutamate through Activating NMDA and AMPA Receptors

To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of NMDA and AMPA receptors in the procedure, the change of calcium signal was investigated by monitoring the fluctuation of intracellular Ca 2+ concentration ([Ca 2+ ] i) on the basis of Fura-2 single cell fluorescent ratio (F345/F380). The changes in the effect of glutamate on the intracellular calcium signal were observed after blockage of NMDA and(or) AMPA receptors. It was found that L-glutamate could induce an increased [Ca 2+ ] i in most of the cells in concentration- and time-dependent manner. D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5, a selective antagonist of the NMDA receptor) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of the AMPA receptor) could abolish the effects of NMDA and AMPA respectively. The treatment of D-AP-5 and CNQX simultaneously or respectively could attenuate the effect of L-glutamate at varying degrees. All these indicated that glutamate could modulate intracellular Ca 2+ of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.

Synaptic non-GluN2B-containing NMDA receptors regulate tyrosine phosphorylation of GluN2B 1472 tyrosine site in rat brain slices

Activation of N-methyl-D-aspartate receptors（NMDARs）mediates changes in the phosphorylation status of the glutamate receptors themselves.Previous studies have indicated that during synaptic activity,tyrosine kinases（Src and Fyn）or phosphatases（PTPαand STEP）are involved in regulating the phosphorylation of NMDARs.In this study,we used immunoblotting to investigate the role of an NMDAR subpopulation on the phosphorylation level of the GluN2B subunit at the Y1336 and Y1472sites in rat brain slices after NMDA treatment.We found that NMDA stimulation dramatically decreased the phosphorylation level of GluN2B at Y1472 in a dose-and time-dependent manner,but not at Y1336.Extrasynaptic NMDAR activation did not reduce the phosphorylation of GluN2B at Y1472.In addition,ifenprodil,a selective antagonist of GluN2Bcontaining NMDARs,did not abolish the decreased phosphorylation of GluN2B at Y1472 triggered by NMDA.These results suggest that the activation of synaptic GluN2A-containing NMDARs is required for the decreased phosphorylation of GluN2B at Y1472that is induced by NMDA treatment in rat brain slices.