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Aim To investigate the improving effect of transcranial direct current stimulation (tDCS) on endogenous hippocampal neurogenesis in mice with cerebral ischemiaand the possible mechanism. Methods The model of acute cerebral ischemia in mice was established by bilateral common carotid artery occlision (BCCAO). The pathological changes of mice were detected by hippocampal HE staining. The learning and memory function of mice was assessed by Morris water maze. The number of BrdU, DCX and BrdU/NeuN-positive cells was observed through immunofluorescence staining for detecting hippocampal neurogenesis. The mRNA and protein expressions of NMDAR subunits NR2a and NR2b in hippocampus were detected by qRT-PCR and Western blot. Results The neuronal damage in the hippocampal CA1 region was marked (P <0. 01), and the learning and memory function significantly decreased (P<0. 01) in cerebral ischemia mice, suggesting the successful establishment of cerebral ischemia model. At the same time, the number of BrdU, DCX and BrdU/NeuN positive cells was up-reg-ulated significantly (P < 0. 01 ) , indicating the occurrence of neurogenesis in hippocampus after cefebral ischemia. Treatment with tDCS significantly ameliorated the pathological damage in CA1 region of mice, improved learning and memory, and promoted hippocam-pal neurogenesis. Meanwhile, the mRNA and protein expression levels of NR2a and NR2b in hippocampus were also up-regulated (P < 0. 05 or P < 0.01). Conclusions tDCS can promote hippocampal neurogenesis and improve learning and memory function in cerebral ischemia mice, which may be related to theup-regula-tion ofNR2a and NR2b expression.
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Objective@#To investigate the effect of Lactobacillus casei on depressive behavior and the expression of NR1, NR2A receptor in postpartum depression rats induced by maternal separation.@*Methods@#Twenty-four Sprague-Dawley female rats were randomly divided into control group(CON group) (n=8), postpartum depression group(PPD group) (n=8), postpartum depression with lactobacillus casei group(PPD+ Lactobacillus casei group) (n=8). Rats of the control group were given no interventions.Rats in PPD group and PPD+ Lactobacillus casei group were given maternal separation to establish depression model.And then the rats in PPD+ Lactobacillus casei group were treated with Lactobacillus casei(8×108CFU/(kg·d)) for 4 weeks after 14 days of maternal separation.The forced swimming test (FST) was employed to detect the depressive behaviors.Lactobacillus, Bifidobacterium, Enterococcus faecalis and Escherichia coli in cecum of rats and the expression of NR1 and NR2A mRNA in hippocampus of rats were detected by real-time fluorescence quantitative PCR.@*Results@#Compared with CON group ((157.50±8.13) s), the immobility time of PPD group((200.00±10.35) s) was significantly longer (t=-3.23, P<0.05). Compared with PPD group, the immobility time of PPD+ Lactobacillus casei group ((153.25±7.41) s) was significantly shortened (t=3.67, P<0.05), and the depressive behavior was improved.Compared with CON group, the mRNA expression of Lactobacillus ((1.47±0.08), t=-5.87, P<0.01), Enterococcus faecalis ((1.23±0.08), t=-2.85, P<0.05) and Escherichia coli( (1.33±0.07), t=-4.96, P<0.01) in caecum were significantly increased in PPD group, while that of Bifidobacterium decreased significantly((0.65±0.07), t=5.18, P<0.01). Compared with PPD group, the mRNA expression of Lactobacillus( (1.05±0.05), t=3.67, P<0.01), Enterococcus faecalis ((0.97±0.05), t=2.74, P<0.05) and Escherichia coli ((1.06±0.05), t=3.31, P<0.01) were significantly decreased in PPD+ Lactobacillus casei group, while that of Bifidobacterium increased significantly ((0.98±0.04), t=-4.26, P<0.01). Compared with CON group, the mRNA expression of NR1 receptor ((0.57±0.04), t=9.65, P<0.01) and NR2A receptor ((0.60±0.05), t=8.64, P<0.05) in hippocampus of rats in PPD group were significantly decreased.Compared with PPD group, the expression of NR1 receptor ((1.01±0.05), t=-5.39, P<0.01) and NR2A receptor ((0.98±0.07), t=-3.91, P<0.05) in hippocampus were increased significantly in PPD+ Lactobacillus casei group.@*Conclusion@#Lactobacillus casei can improve the depressive behavior of postpartum depression in rats, and improve the intestinal flora, which affect the expression of NR1 and NR2A in hippocampus.
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Objective To investigate the effect of Lactobacillus casei on depressive behavior and the expression of NR1, NR2A receptor in postpartum depression rats induced by maternal separation. Methods Twenty-four Sprague-Dawley female rats were randomly divided into control group( CON group) (n=8),postpartum depression group(PPD group) (n=8),postpartum depression with lactobacillus casei group(PPD+ Lactobacillus casei group) (n=8). Rats of the control group were given no interventions. Rats in PPD group and PPD+Lactobacillus casei group were given maternal separation to establish depression model. And then the rats in PPD+Lactobacillus casei group were treated with Lactobacillus casei ( 8× 108CFU/(kg·d)) for 4 weeks after 14 days of maternal separation. The forced swimming test ( FST) was employed to detect the depressive behaviors. Lactobacillus,Bifidobacterium,Enterococcus faecalis and Esche-richia coli in cecum of rats and the expression of NR1 and NR2A mRNA in hippocampus of rats were detec-ted by real-time fluorescence quantitative PCR. Results Compared with CON group ((157. 50±8. 13) s), the immobility time of PPD group((200. 00±10. 35) s) was significantly longer (t=-3. 23,P<0. 05). Com-pared with PPD group,the immobility time of PPD+ Lactobacillus casei group ((153. 25±7. 41) s) was sig-nificantly shortened (t=3. 67,P<0. 05),and the depressive behavior was improved. Compared with CON group,the mRNA expression of Lactobacillus ((1. 47± 0. 08),t=-5. 87,P<0. 01),Enterococcus faecalis ((1. 23±0. 08),t=-2. 85,P<0. 05) and Escherichia coli( (1. 33±0. 07),t=-4. 96,P<0. 01) in caecum were significantly increased in PPD group, while that of Bifidobacterium decreased significantly (( 0. 65 ± 0. 07),t=5. 18,P<0. 01). Compared with PPD group,the mRNA expression of Lactobacillus ( ( 1. 05 ± 0. 05),t=3. 67,P<0. 01),Enterococcus faecalis ((0. 97±0. 05),t=2. 74,P<0. 05) and Escherichia coli ((1. 06±0. 05),t=3. 31,P<0. 01) were significantly decreased in PPD+ Lactobacillus casei group,while that of Bifidobacterium increased significantly ((0. 98± 0. 04),t=-4. 26,P<0. 01). Compared with CON group,the mRNA expression of NR1 receptor (( 0. 57 ± 0. 04), t=9. 65, P<0. 01) and NR2A receptor ((0. 60±0. 05),t=8. 64,P<0. 05) in hippocampus of rats in PPD group were significantly decreased. Com-pared with PPD group,the expression of NR1 receptor ((1. 01±0. 05),t=-5. 39,P<0. 01) and NR2A re-ceptor ((0. 98±0. 07),t=-3. 91,P<0. 05) in hippocampus were increased significantly in PPD+ Lactoba-cillus casei group. Conclusion Lactobacillus casei can improve the depressive behavior of postpartum depression in rats,and improve the intestinal flora,which affect the expression of NR1 and NR2A in hippocampus.
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Objective To observe the effect of Kidney-nourishing, Blood-activating, Phlegm-resolving and Resuscitation-inducing Decoction(KBPRD)on the expression of N-methyl-D-aspartate(NMDA)receptor subtype 1,2A,2B(NR1,NR2A,NR2B)in the cochlear spiral ganglion neurons(SGN)of tinnitus rats and to explore its mechanism, thus to provide experimental evidence for the treatment of tinnitus with KBPRD. Methods Sixty rats were randomly divided into normal group,model group,western medicine group,and low-,middle-,and high-dose Chinese medicine groups, 10 rats in each group. Rats were given intraperitoneal injection of sodium salicylate combined with water deprivation to induce tinnitus model. After successful establishment of the model, the rats in low-,middle-,and high-dose Chinese medicine groups were given gastric administration of KBPRD in the dosage of 5.5, 11, 22 g·kg-1·d-1 respectively, the rats in western medicine group were given gastric administration of 5 mg·kg-1·d-1 of carbamazepine,and rats in the model group and normal group were given gastric administration of 2 mL of normal saline,once every day,treatment time covering 8 weeks. The expression levels of NR1,NR2A,and NR2B in the cochlear SGN was detected by immunoblotting and real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)after 8 weeks of treatment. Results Compared with the normal group,the expression levels of NR1, NR2A and NR2B in the model group were increased, the difference being significant (P < 0.05). Compared with the model group,the expression of NR1,NR2A and NR2B in low-,middle-,and high-dose Chinese medicine groups were significantly decreased(P<0.05).Conclusion KBPRD is effective on relieving tinnitus of rats, and its mechanism is correlated with lowering the increased expression of NR1,NR2A and NR2B in SGN of tinnitus rats.
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This study was aimed to observe the effect ofZi-Bu Pi-Yin Recipe (ZBPYR) on the mRNA expressions of NMDA receptor subunits NR1, NR2A, NR2B in different brain regions of spleen-yin deficiency Alzheimer's Disease (AD) model rats. The levels of NR1, NR2A, NR2B mRNA expressions were detected by using RT-PCR method. The results showed that the levels of NR1, NR2A, NR2B mRNA expressions of AD group and spleen-yin deficiency AD group decreased significantly (P < 0.05). The levels of NR1, NR2A, NR2B mRNA expressions of ZBPYR treatment group increased significantly (P < 0.05). It was concluded that the expression levels of NMDAR mRNA in different brain regions of the ZBPYR treatment group increased significantly, which indicated that ZBPYR may up-regulate the protein expressions of NMDAR by increasing the expression levels of NMDAR mRNA, thereby to play the anti-dementia effect.
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ObjectiveTo discuss the correlation between kidney-deficiency constitution and brain function decline, and material basis of kidney-storing-spirit theory and neurobiology mechanism. Methods By employing the method of “cats scare rats”, composite offspring rat models with deficiency and acquired dystrophy were built, then they were divided into model group,ZuoguiPill group and Yougui Pill group. Blank group was composed from normal pregnant rats. When the intimidation of offspring began,Zuogui Pill andYougui Pill groups received gavage with corresponding doses of medicine, 1 times per day for 2 consecutive months. Model and blank groups received the same amount of normal saline. Exercise capacity was detected by suspension test. Learning and memory capacity was detected by Y maze test. Expressions of NMDA receptor subunits NR2A and NR2B were detected by immunohistochemical method.Results In suspension test, the duration of model group was shorter than blank group (P<0.05), while duration ofZuogui Pill andYougui Pill groups was longer than the model group (P<0.05). In Y maze test, the correct number of model group was less than blank group, and increased significantly after treatment (P<0.05). The optical density of NR2A and NR2B was lower in model group than blank group (P<0.05) and higher inZuogui Pill andYougui Pill groups than model group (P<0.05).ConclusionThe neurobehavioral is abnormal and NMDA receptor expression depresses in rats with kidney- deficiency constitution.ZuoguiPill andYouguiPill can rise NMDA receptor expression and improve brain function of rats, which reveal the correlation between kidney-deficiency constitution and brain function decline, and material basis of kidney-storing-spirit theory and neurobiology mechanism.
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ObjectiveTo observe the effect ofZuogui Jiangtang Jieyu Formula on the expressions of glutamate, NR2A and NR2B in hippocampus of diabetic rats with depression;To explore the mechanism of protective effect. Methods Diabetes with depression rat models were established and then were randomly divided into the model group, positive medicine group, high-, medium- and low-doseZuogui Jiangtang Jieyu Formula groups. Normal rats acted as normal group, 16 rats per group. After 28 days of administration, Open-field test was used to detect the behavior of the rats;glutamate content of hippocampus was detected by ELISA;the expressions of NR2A and NR2B in rat hippocampus were detected by immunofluorescence.Results Compared with normal group, automatic activity times of rats in model group decreased significantly (P<0.01);both glutamate content (P<0.01) and expressions of NR2A, NR2B (P<0.01) increased significantly. Compared with the model group, automatic activity times of rats in positive medicine group and high-doseZuogui Jiangtang Jieyu Formula group significantly increased (P<0.01);glutamate content dropped (P<0.01);expressions of NR2A and NR2B decreased (P<0.05).ConclusionZuogui Jiangtang Jieyu Formula can improve depressive behavior of diabetic rats with depression, which may be related to the regulation of glutamate content and expressions of NR2A and NR2B in hippocampus.
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Background: This study aims to determine the association between neuroinflammation and oxidative stress with prognosis of brain injury patients and the association between neurosurgical procedure with neuroinflammation and oxidative stress condititons. Methods: The study design is a prospective observation of 40 brain injury patients who underwent surgery. IL-6, uric acid, MDA, NR2A antibodies and GSH serum level of pre- and 1 day post-operation on brain injury patients were measured, and their association with GCS, GOS and neurosurgical procedures were analyzed. Results: The post-operative IL-6 serum level showed a downward trend compared to pre-operative value (mean decrease: -190.61 pg/mL). The post-operative IL-6 level was significantly associated with GCS 7 days post-operation (p = 0.006), with OR 24. The post-operative IL-6 serum level was significantly associated with GOS 3 months post-trauma (p = 0.016) with OR 11.6. The post-operative uric acid serum level showed a downward trend compared to pre-operative value (mean decrease: -0.26 mg/dL). There was a significant difference between the mean value of post-operative uric acid serum level in patients with 7 days post-trauma with GCS ≤ 8 (mean: 4.16 mg/dL) and GCS > 8 (mean: 2.71 mg/dL), (p = 0.042). The post-operative MDA serum level showed a downward trend compared to pre-operative value (mean decrease: -0.08 nmol/mL). There is no significant association between MDA serum level, GCS and GOS and no significant association of NR2A antibody and GSH serum level with GCS, GOS and neurosurgical procedure. From the multivariate analysis, the most important neuroinflammatory variable associated with GCS and GOS is IL-6. Conclusion: Neuroinflammation and oxidative stress may have prognostic values in brain-injured patients, in particular IL-6. Neurosurgical procedures may decrease the neuroinflammation process
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Brain InjuriesABSTRACT
Objetivo. Identificar la influencia de los cambios respecto a la estructura secundaria y a la relación evolutiva de los receptores NMDA, AMPA Y KAINATO en las especies Homo sapiens, Pan troglodytes, Pongo pygmaeus, y Macaca mulata. Materiales y métodos. Se recopilaron 91 secuencias correspondientes a los receptores NMDA, AMPA y Kainato y se sometieron a los programas de predicción de estructura secundaria, sitios de fosforilación, alineamientos múltiples, selección del modelo de evolución y predicción filogenética. Resultados. Se encontró que las subunidades GLUR5, NR2A, NR2C y NR3A presentaron cambios de estructura en la región C-terminal y aparición o pérdida de sitios de fosforilación en esta zona. Adicionalmente la predicción filogenética nos propone que las subunidades NR2 de NMDA son las más cercanas al nodo ancestral que da origen a los demás subunidades. Conclusiones. Los cambios de estructura y sitios de fosforilación en las subunidades GLUR5, NR2A, NR2C y NR3A nos sugieren variaciones en la interacción de la región C-terminal con proteínas quinasas y con proteínas con dominios PDZ lo cual podría afectar el tráfico y anclaje de las subunidades. Por otra parte la predicción filogenética nos propone que los cambios que se presentaron en las subunidades NR2 dieron origen a las demás subunidades de los receptores ionotrópicos de glutamato, básicamente porque son las subunidades de NMDA y en particular NR2D las que se encuentran más estrechamente relacionadas con el nodo ancestral que posiblemente dio origen a los iGluRs.
Objective. To identify the influence of changes on the secondary structure and evolutionary relationship of NMDA, AMPA and kainate receptors in Homo sapiens, Pan troglodytes, Pongo pygmaeus and Macaca mulatta. Materials and methods. We identified 91 sequences for NMDA, AMPA and kainate receptors and analyzed with software for predicting secondary structure, phosphorylation sites, multiple alignments, selection of protein evolution models and phylogenetic prediction. Results. We found that subunits GLUR5, NR2A, NR2C and NR3A showed structural changes in the C-terminal region and formation or loss of phosphorylation sites in this zone. Additionally the phylogenetic prediction suggests that the NMDA NR2 subunits are the closest to the ancestral node that gives rise to the other subunits. Conclusions. Changes in structure and phosphorylation sites in GLUR5, NR2A, NR2C and NR3A subunits suggest variations in the interaction of the C-terminal region with kinase proteins and with proteins with PDZ domains, which could affect the trafficking and anchoring of the subunits. On the other hand, the phylogenetic prediction suggests that the changes that occurred in the NR2 subunits gave rise to the other subunits of glutamate ionotropic receptors, primarily because the NMDA and particularly the NR2D subunits are the most closely related to the ancestral node that possibly gave rise to the iGluRs.
Objetivo. Identificar a influência das mudanças da estrutura secundária e da relação evolutiva dos receptores NMDA, AMPA e cainato nas espécies Homo sapiens, Pan troglodytes, Pongo pygmaeus e Macaca mulatta. Materiais e métodos. Foram recopiladas 91 seqüências correspondentes aos receptores NMDA, AMPA e cainato e foram submetidos a programas de predição de estrutura secundária, sítios de fosforilação, alinhamentos múltiplos, seleção do modelo de evolução e predição da filogenia. Resultados. Descobrimos que as subunidades GLUR5, NR2A, NR2C e NR3A apresentaram alterações estruturais na região C-terminal e aparecimento ou perda de sítios de fosforilação nesta área. Além disso, a predição filogenética sugere ainda que as subunidades NR2 de NMDA são as mais próximas ao nó ancestral que dá origem às demais subunidades. Conclusões. As mudanças na estrutura e nos sítios de fosforilação nas subunidades GLUR5, NR2A, NR2C e NR3A sugerem variações na interação da região C-terminal com proteínas quinase e com proteínas de domínios PDZ que poderia afetar o tráfego e fixação das subunidades. Além disso, a predição filogenética sugere que as mudanças ocorridas nas subunidades NR2 deram origem às outras subunidades de receptores ionotrópicos de glutamato, principalmente porque são subunidade NMDA e particularmente NR2D aquelas que são mais estreitamente relacionadas com o nó ancestral que provavelmente deu origem aos iGluRs.
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ObjectiveTo study the distribution and expression of NMDA receptor subunit 2A in the spinal cord of morphine tolerant rats. MethodsTwelve Sprague-Dawley rats were randomly divided into two groups with 6 rats each: control group (C) were intrathecally administrated 0.9% NaCl 10μl and morphine group(M) received 10μg morphine (i.t.). Drugs were administrated twice daily for 7 consecutive days. Tail flick latency (TFL) in the hot water immersion test was used to evaluate changes of thermal hyperalgesia latency of each group before and 30min after administration every morning. Rats were killed the day after last administration and L4～5 segment of the spinal cord was removed for determination the expression of NR2A by immunofluorescence method. ResultsTFL of group M was decreased gradually after chronic administration of morphine intrathecally. There was no significant difference between group M[(3.25±0.93)s] and group C[(2.66±0.27)s] on the 7th day (P＞0.05). A morphine tolerant model was established successfully. NR2A was distributed throughout the rat spinal cord. The expression of NR2A in group M(OD:9617±1233) was increased compared with group C(OD:2.66±0.93) (t=3.133,P＜0.05).ConclusionThe expression of NR2A was upregulated after repeated administration of morphine intrathecally in the superficial dorsal horn of spinal cord of morphine tolerant rats,which may be part of the mechanisms of morphine tolerance.
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Aim To investigate the effect of curcumin on the expressions of NR2A, NR2B and apoptotic neurons in hippocampus after global brain ischemia-reperfusion in SD rats. Methods Global ischemic model induced by 4-vessel occlusion was adopted. Rats were randomly divided into 5 groups: sham group (SH), ischemia-reperfusion group (I/R), curcumin 30(Cur 30), 100 (Cur 100) and 300 mg?kg-1 group (Cur 300). At different time points after ischemia, animals were designated as subgroups 2 h, 6 h, 1 d, 3 d, 7 d. Cell morphology was observed on HE stain slides. The apoptotic neurons were detected in CA1 region by TUNEL method. Expressions of NR2A and NR2B in hippocampal CA1 and CA3/DG regions were detected by Immunohistochemical technique. Results At each time point the number of apoptotic neurons was much more in I/R group than that in SH group and Cur 100 group,and in Cur 300 group than that in Cur 100 group. The expression of NR2A was higher in the hippocampal CA1 and CA3/DG regions in Cur 100,Cur 300, Cur 30 and SH groups than that in I/R group at each time point(P
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Objective To investigate whether the partially C-terminal deletion of NR2A subunit alters the surface expression and channel function of NMDA receptors in both HEK293 cells and cultured hippocampal neurons of rats. Methods Four plasmids for NR2A mutants with N-terminally GFP-tagged and C-terminal deletion NR2A?C1(?897L-1017S),NR2A?C2(?1024D-1142P),NR2A?C3(?1149D-1347G),and NR2A?C4(?1354S-1464V) were generated,and transfected into HEK293 cells and hippocampal neurons in culture.Surface staining was performed using anti-GFP antibody and Cy3 conjugated secondary antibody.Glutamate evoked currents were also detected using whole-cell recording. Results Positive surface staining was found for all the HEK293 cell co-transfected NR1-1a/NR2A?C1,NR1-1a/NR2A?C2,NR1-1a/NR2A?C3 or NR1-1a/NR2A?C4,and quantitative analysis showed no significant decrease in surface expression level when compared to that from NR1-1a/NR2A transfection.Glutamate-evoked currents were recorded in HEK293 cells co-transfected with NR1-1a/NR2A?C2 or NR1-1a/NR2A?C4.Surface expression level of NMDA receptor clusters on dendrites was significantly decreased in the neurons transfected with NR2A?C1,NR2A?C2 or NR2A?C3 than in those transfected with NR2A.Conclusion C-terminal deletion of NR2A subunit differentially effects surface expression of NMDA receptors in HEK293 cells and in hippocampal neurons in culture.
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PURPOSE: The developing brain has been reported to be extremely susceptible to toxicity of ischemia and/or hypoxia during a restricted developmental period. Hippocampal neuronal cell death is a typical type of perinatal hypoxic brain lesion and often coexists with other forms of cerebral hypoxic injuries. In the present study, we examined whether transcriptional changes of NR2A and NR2B subunits of the N-Methyl-D-aspartate(NMDA) receptors related to the neuronal cell death to hypoxic toxicity are involved in developing neurons in the hippocampus. METHODS: We examined the lesion produced by in vivo direct exposure of 92% N2 and 8% O2 for 2 hours at postnatal day 7. Hippocampal sections from the 7th and 14th days after hypoxia were obtained, and the amount of the NR2A and NR2B mRNA subunits were measured by in situ hybridization using the antisense probe to the NR2A and NR2B subunits. To determine the effects of molecular changes of NMDA receptor subunits, morphological changes of neurons and/or accompanying astrocytosis were evaluated by H&E and immunohistochemical stain. RESULTS: Fourteen days after hypoxia, the expression of NR2B significantly increased whereas NR2A showed distinct reduction compared with that of control rat pups. At this time, unexpectedly, neurons in CA3 region showed prominant reduction of the actual numbers and accompanied reactive astrocytosis. CONCLUSION: Alteration of NR2A and NR2B expression to hypoxic insults, suggest the possibility that changes of the NR2 subunits which can alter the function of the NMDA receptor play a crucial role in the occurrence of developmentally specific hippocampal neuronal injury.