Effects of pien-tze-huang on expression of NMDAR1 and GluR2 in cortex of MCAO rats / 中国药理学通报

Aim: To investigate the effects of Pien-Tze-Huang (PZH) on mRNA and protein expression of NMDAR1 and GluR2 in cortex of MCAO rats. Methods: Forty healthy adult male Sprague-Dawley rats were randomly divided into three groups: sham, MCAO, MCAO + PZH groups. The rats were subjected to focal cerebral ischemia/reperfusion with suture-occluded method. Neurological deficit testing was performed with Zea Longa scale. The volume of cerebral infarction was evaluated by TTC staining. The mRNA and protein expression of NMDAR1 and GluR2 in cortex of side cerebral ischemic tissues were determined using qPCR and Western blot analysis. Results: Compared with MCAO group, PZH significantly improved the neurological deficit, decreased the volume of cerebral infarction, and up-regulated the mRNA and protein expression of NMDAR1 and GluR2. Conclusions: PZH attenuates the down-regulation of mRNA and protein expression of NMDAR1 and GluR2 after focal cerebral ischemic injury in rats, which may be associated with the cerebral protective effect of PZH.

Protective effects and mechanism of Zuogui Jiangtang Jieyu Formulation on hippocampal neuron of NVU induced by autophagy in diabetes mellitus with depression / 中草药

Objective: To investigate the protective effects and mechanism of Zuogui Jiangtang Jieyu Formulation (ZGF) on hippocampal neuron of neurovascular unit (NVU) induced by autophagy in diabetes mellitus with depression (DD). Methods Hippocampal neurons, astrocytes and brain microvascular endothelial cells from SD rats were primitively isolated, purified and cultured, and then immunocytochemistry staining was employed to identify primitive cells. The DD model of NVU was established by applying intervention of high glucose (150 mmol/L) and cortisone (200 μmol/L) after co-culture system was built in vitro. The cultured cells were randomly divided into normal group, blank serum group, model group, and medicated serum of ZGF group. Intracellular calcium levels (Ca2+) on hippocampal neuron of NVU was detected by Furo-3/AM staining. The expression of autophagy marker Beclin-1, LC3-II, and GluR2/Ca2+/mTOR signaling pathway key proteins was detected by high content analysis. The apoptosis of hippocampal neurons was observed by Tunel staining. Results:Compared with the normal group, Ca2+ levels was remarkably increased, expression of autophagy marker Beclin-1 and LC3-II was dramatically up-regulated, proteins expression of GluR2 and mTOR was obviously down-regulated while AMPK up-regulated, and the apoptosis in hippocampal neuron of NVU was significantly increased in model and blank serum group. Compared with the model group, intracellular calcium levels was obviously decreased, expression of autophagy marker Beclin-1 and LC3-II was down-regulated, GluR2/Ca2+/mTOR signaling pathway proteins expression of GluR2, mTOR was up-regulated while AMPK down-regulated, furthermore the apoptosis of hippocampal neurons was significantly decreased in ZGF group. Conclusion: ZGF has significant protective effects on hippocampal neuron of NVU induced by autophagy in DD, and its mechanism is related to the GluR2/Ca2+/mTOR signaling pathway and apoptosis.

Effect of Catgut Implantation on Spatial Learning-memory Ability, Expression of Hippocampal Protein Kinase C Interacting Protein 1 and GluR 2 and Ca2+ Content in Rats with Chronic Ischemic Cognitive Impairment / 针刺研究

OBJECTIVE: To observe the effect of catgut embedment at "Baihui" (GV 20), "Dazhui" (GV 14), etc. on learning-memory ability, expression of hippocampal protein kinase C interacting protein 1 (PICK 1) and glutamate receptor 2 (GluR 2) proteins and level of calcium ions, so as to explore its mechanism underlying improvement of vascular cognitive impairment. METHODS: A total of 56 male SD rats were randomly divided into sham operation, model, catgut embedment and medication groups (n=14 in each). The chronic ischemic cognitive impairment model was established by permanent occlusion of bilate-ral common carotid arteries. The catgut embedment was applied to GV 20, GV 14, "Shenshu" (BL 23) and "Xuanzhong" (GB 39), once a week, for 4 weeks. Rats of the medication group received intraperitoneal injection of monosialate tetrahexose ganglioside sodium (GM-1, 0.33 mg/kg), once daily for 4 weeks. The rats' learning-memory ability was detected by Morris water maze tasks, pathological changes of hippocampal Nissl's bodies were tested by Nissl staining. The expression levels of PICK 1 and GluR 2 proteins in the hippocampus were detected by Western bolt (WB), and the concentration of calcium ions in the hippocampus tissue was measured by Bicinchoninic acid (BCA) assay. RESULTS: After modeling, the mean escape latencies of place navigation test were significantly increased while the crossing times of target platform quadrant of space probing test notably decreased in the model, catgut embedment and medication groups compared with their own individual pre-modeling (P0.05). CONCLUSION: Catgut implantation at GV 20 etc. can effectively improve the learning-memory ability in rats with chronic ischemic cognitive impairment, which may be related to its effects in down-regulating the expression of PICK 1 and calcium ion concentration and up-regulating the expression of AMPA receptor subunit GluR 2 protein in the hippocampus.

MiR-124 involves in methamphetamineaddiction in PC12 cells by inhibiting GluR2 / 中国药理学通报

Aim To investigate the change of miR-124 expression in methamphetamine-induced addiction in PC12 cells and the possible regulatory mechanism that it involves.Methods PC12 cells were randomly divided into 6 groups as follows: control group, methamphetamine group, agomir Negative Control group, miR-124 agomir group, agomir Negative Control+methamphetamine group and miR-124 agomir+methamphetamine group.After the treatment, the total RNA and protein were extracted in PC12 cells.The expression of miR-124 was measured by Real-time PCR and the expression of GluR2 was determined by Western blot in PC12 cells.Results Compared with those in the control group, the expression of miR-124 was remarkably decreased and the expression of GluR2 was significantly increased in the methamphetamine group in PC12 cells.Compared with those in the agomir Negative Control+methamphetamine group, the expression of miR-124 was remarkably increased and the expression of GluR2 was significantly decreased in the miR-124 agomir+methamphetamine group in PC12 cells.Conclusion MiR-124 might involve in methamphetamine-induced addiction in PC12 cells by inhibiting GluR2.

Protective effects of SFE-CO2 of Notoginseng against glutamate-induced apoptosis in PC12 cells / 中国药理学通报

Aim To investigate the protective effects of supercritical CO2 fluid extract(SFE)of Notoginseng a-gainst glutamate-induced PC12 cells damage and the underlying mechanism. Methods PC12 cells were dealt with glutamate to establish cell models. MTT as-say,LDH method,Hoschst 33342 staining,Fluo-3 /AM fluorescence staining and Western blot were used to observe the changes of cell viability,intracellular Ca2 + concentration and the expression of protein that interacted with C kinase l(PICK1)and glutamate re-ceptors 2 (GluR2),respectively. Results Glutamate was cytotoxic to PC12 cells with an inhibitory concen-tration 50(IC 50 )of 25 mmol·L - 1 . Pretreatment with SFE(25,50,100 mg·L-1)and FSC231(100 μmol ·L-1 )and SFE(100 mg·L-1 )+FSC231(100μmol ·L-1 )remarkablely improved cell viability,reduced LDH leakage,decreased apoptosis rate,debased intra-cellular calcium concentration,decreased the expres-sion of PICK1 ,and increased the expression of GluR2 . Conclusions SFE of Notoginseng shows protective effects against glutamate-induced PC12 cell damage, and its mechanism may be related to the inhibition of PICK1 and the increase of GluR2 protein expression.

Influences of levodopa and cytidine diphosphate choline on the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid GluR2 in visual cortex of monocular deprivation rats / 中华实验眼科杂志

Background Research demonstrated that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid GluR2 (AMPA-GluR2) is associated with amblyopia.It has been shown that levodopa and cytidine diphosphate choline can improve visual function of amblyopic children,but the mechanism is unclear.Objective This study was to explore the possible effects of levodopa and cytidine diphosphate choline on amblyopia.Methods Monocular deprivation (MD) animal models were created in 60 2-week-old SD rats by monolateral eyelid suturing and observed for 31 days and reared in natural light together with 15 other matched normal healthy SD rats.The models were randomly divided into the MD group,levodopa group,cytidine diphosphate choline group and normal saline control group,with 15 rats for each group.40 mg/kg of levodopa,80 mg/kg of cytidine diphosphate choline,I ml normal saline were given to the rats,respectively,for 28 consecutive days.Expressions of the AMPA-CluR2 protein and AMPA-CluR2 mRNA in the rat visual cortex were detected by immunohistochemistry,Western blot and real-time fluorescence quantitative PCR.Use of the animals followed the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results The expression values of the AMPA-GluR2 protein (AMPA-GluR2/β-actin) and AMPA-GluR2 mRNA (2-△△Ct) were significantly lower in the MD group than those of the normal control group (protein:0.32 ± 0.02 vs.0.64 ± 0.05,t =13.287,P＜0.05 ;mRNA:0.30±0.01 vs.0.84±0.03,t=38.184,P＜0.05).Those in the levodopa group were significantly increased in comparison with the normal saline solution group (protein:0.59 ±0.04 vs.0.33 ±0.03,t =11.628,P＜0.05 ; mRNA:0.71±0.06 vs.0.33 ±0.02,t =13.435,P＜0.05).The expression values of the AMPA-GluR2 protein and AMPA-GluR2 mRNA were significantly increased in the cytidine diphosphate choline group compared with the normal saline solution group (protein:0.52 ± 0.04 vs.0.33 ± 0.03,t =8.497,P ＜ 0.05 ; mRNA:0.48± 0.04 vs.0.33 ± 0.02,t =7.500,P＜0.05).Conclusions AMPA-GluR2 is associated with the plasticity of visual development.Levodopa and cytidine diphosphate choline may improve visual function by down-regulating the expression of AMPA-GluR2 in the visual cortex.

El DHEAS incrementa la expresión de GluR2/3 y GLUR2 del receptor AMPA en el hipocampo de ratones C57/BL6 / DHEAS increases levels of GluR2/3 and GluR2, AMPA receptor subunits, in C57BL/6 mice hippocampus

El DHEAS es un neuroesteroide con efecto neuromodulador de la transmisión sináptica y en la neuroprotección, sin embargo las vías moleculares a través de las cuales se inducen estos cambiosno están completamente claras. Como varios de los neuroesteroides actúan a través de los recetores ionotrópicos de glutamato, se evaluó el efecto del DHEAS en las subunidades GluR2 y GluR3 del receptor AMPA para esclarecer sus efectos. Con este fin se administró DHEAS o una sustancia control durante 7 días a ratones C57/BL6. La expresión de las subunidades se evaluó por Westernblotting.Los resultados presentados muestran que la administración prolongada de 40mg/kg/día de DHEAS a ratones C57/BL6 produce un incremento en los niveles de proteína de las subunidades GluR2/3 yGluR2 del receptor AMPA en el hipocampo. Dado el papel específico que juega la subunidad GluR2 del receptor AMPA en el control de la entrada de calcio durante los procesos de muerte celular y de plasticidad sináptica, este hallazgo contribuye al estudio de los neuroesteroides como una estrategia terapéutica relevante en enfermedades neurodegenerativas y eventos cerebrovasculares.

Dehydroepiandrosterone sulfate (DHEA-S) is a neurosteroid that has effects such as neuromodulator of synaptic transmission and neuroprotection. The specific signaling pathways for these effects are not elucidated yet. Given that, some neurosteroids act through the activation of ionotropic glutamate receptors, therefore the effect of DHEA-S on the subunits GluR2 and GluR3of the AMPA receptor was evaluated. Either DHEA-S or a control substance was administered to C57/BL6 mice. Subunit expression of the AMPA receptor was analyzed by Western blotting. Results show that long-term DHEA-S administration toC57/BL6 mice, increases the protein levels of the subunits GluR2 and GluR2/3 of the AMPA receptors located in the hippocampus. Due to the role of AMPA receptor, specifically GluR2subunit in the regulation of intracellular calcium levels, cellular apoptosis, and synaptic plasticity, the study of neurosteroids as a therapeutic strategy in neurodegenerative diseases and cerebrovascular events is very relevant.

Role of GluR2-lacking AMPAR in synaptic plasticity: Recent progress / 第二军医大学学报

AMPARs are excitatory glutamate receptors that mediate the fast synaptic transmission. AMPARs are homo- or hetero-tetramers composed of selective combinations of four subunits: GluR1, GluR2, GluR3 and GluR4. AMPARs containing GluR2 are mainly in the central nervous system and are Ca2+ impermeable. MPARs-lacking GluR2 are Ca2+ permeable, and GluR2-lacking AMPARs are confined to certain neurons or certain physiological or pathological conditions. Recent research showed that GluR2-lacking AMPARs play special roles in the synaptic function and plasticity and transduction of local signal transduction. This paper reviews the GluR2-lacking AMPARs and their roles in the synaptic function and plasticity.

GluR2 expression in developing rat cochlear nucleus and relationship with development of synapse / 上海第二医科大学学报

Objective To investigate the trend of developmental expression of GluR2,subtype of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA) receptor and synaptophysin(SYP) in the rat cochlear nucleus(CN) in different developmental stages,and explore the association of GluR2 expression with the development of synapse. Methods SD rats of 2,3,4,6,8 and 10 weeks old were selected,the expression of GluR2 and SYP in CN was detected with immunofluorescence histochemical method,and the association of them was explored. Results GluR2 expression was observed in all the neurons of CN in each postnatal groups.The expression was relatively weaker in the second and third week,became denser in the fourth week,reached the peak in the sixth week and then sharply decreased to the weakest in the tenth week.The expression of GluR2 was denser at granular cell layer,while weaker at molecular layer and multipolar cell layer in the dorsal CN.SYP expression was detected in all the neurons of CN in each postnatal groups.The expression was weakest in the second week,significantly denser in the fourth week,reached the peak in the sixth week,was then sharply decreased and stably maintained. Conclusion The expressions of GluR2 and SYP in the postnatal rat CN exhit an equally age-dependent tendency.The expression of GluR2 in the CN may be associated with the the maturation and function development of the CN.The different expression and distribution of GluR2 and SYP in the rat CN of different developmental stages may be involved in the development and plasticity of auditory center.

Inhibitory and Excitatory Postsynaptic Currents of Medial Vestibular Nucleus Neurons of Rats

The medial vestibular nucleus (MVN) neurons are controlled by excitatory synaptic transmission from the vestibular afferent and commissural projections, and by inhibitory transmission from interneurons. Spontaneous synaptic currents of MVN neurons were studied using whole cell patch clamp recording in slices prepared from 13- to 17-day-old rats. The spontaneous inhibitory postsynaptic currents (sIPSCs) were significantly reduced by the GABAA antagonist bicuculline (20micrometer), but were not affected by the glycine antagonist strychnine (1micrometer). The frequency, amplitude, and decay time constant of sIPSCs were 4.3 0.9 Hz, 18.1 2.0 pA, and 8.9 0.4 ms, respectively. Spontaneous excitatory postsynaptic currents (sEPSCs) were mediated by non-NMDA and NMDA receptors. The specific AMPA receptor antagonist GYKI-52466 (50micrometer) completely blocked the non-NMDA mediated sEPSCs, indicating that they are mediated by an AMPA-preferring receptor. The AMPA mediated sEPSCs were characterized by low frequency (1.5 0.4 Hz), small amplitude (13.9 1.9 pA), and rapid decay kinetics (2.8 0.2 ms). The majority (15/21) displayed linear I-V relationships, suggesting the presence of GluR2-containing AMPA receptors. Only 35% of recorded MVN neurons showed NMDA mediated currents, which were characterized by small amplitude and low frequency. These results suggest that the MVN neurons receive excitatory inputs mediated by AMPA, but not kainate, and NMDA receptors, and inhibitory transmission mediated by GABAA receptors in neonatal rats.

AMPA Receptor-Induced Neuronal Cell Death in Rat Hippocampus Following Transient Global Ischemia: Relationship to Calpain and Caspase-3 Expression / 대한해부학회지

A central challenge in ischemia-induced neuronal death research is understanding the mechanisms by which apoptotic or necrotic cascades are initiated and affected. We tested potential roles for AMPA and NMDA receptor protein levels and activation of calpain, caspase-3 in the hippocampus at times after transient global ischemia when detectable necrotic or apoptotic cell damage was observed by neurofilament 200 (NF200) degradation, TUNEL, and H & E. We determined that the decrease in the AMPA receptor subunit, GluR2, in response to the transient global ischemia plays a major role in triggering the neuronal cell death in hippocampus. We also examined potential roles for calpain and caspase-3 in ischemic cell death and found that (1) calpain is activated at a time following caspase-3 activation and paralleled degradation of NR2A, NR2B, and GluR2 and irreversible necrotic neuronal changes, (2) caspase-3 is has their maximal expression at the time of highest apoptosis, (3) the NF200 degradation, one of the neuronal deathinducing factors was correlated well with the calpain activation and necrotic changes in the hippocampal CA1 neurons. These results suggest that the significant degradation of GluR2 subunits of AMPA receptor and calpain activation are possibly involved in NF 200 degradation-mediated necrotic hippocampal cell death after transient global ischemia.

The Expression of GluR2/3 in Cochlear Nucleus of Different Age SD Rat / 听力学及言语疾病杂志

Objective To investigate the expression of ?-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid(AMPA) receptor subunit proteins GluR2/3 in the postnatal rat cochlear nucleus(CN), and the relationship with the auditory brainstem response(ABR).Methods The thresholds of ABR of different age SD rats were recorded respectively,and the expression of GluR2/3 in CN was detected with FITC-immunocytochemistry.Results ①P1w SD rats did not show ABR wave, and there was no significant difference in the ABR threshold among P4w,P9w and P15w SD rats. ②GluR2/3 was expressed in all sorts of neurons of CN of every postnatal groups, the expression of P4w rats was denser than that of P1w, P9w and P15w rats. The immunostaining of P4w rats was located at the cell membrance, while P9w and P15w rats in the endochylema majoritily. Conclusion The different expression and distribution of GluR2/3 in different age rat CN may be involved in the development of auditory centre.