Distribution of PSD95 and GluA2 subunit during maturation of primary hippocampal neurons / 中国药理学通报

Aim To investigate the distribution of postsynaptic density protein 95 (PSD95) and AMPA receptor GluA2 subunit during the maturation of rat hippocampal neurons in vitro. Methods Cultured rat hippocampal neurons at 4(days in vitro, DIV), 7DIV, 14DIV and 20DIV were used in an immunofluorescence assay to test co-localization of PSD95 and GluA2 by laser confocal microscope at a magnification of 60 ×. Results PSD95 puncta were gradually distributed densely in dendrites and dendritic spines during neuron maturation progresses. GluA2 subunits were uniformly distributed in the cell body, axons, dendrites and dendritic spines. The Pearson' s correlation of PSD95 and GluA2 was 0. 830 ±0. 033 and 0. 734 ±0. 019 respectively at the dendrites of 4DIV and 7DIV neurons, which showed strong co-localization. While at the dendrites of 14DIV and 20DIV neurons, a moderate correlation was demonstrated by the Pearson correlation 0. 547 ± 0. 021 and 0. 574 ± 0. 024. Conclusions GluA2 has no specific intracellular distribution during neuron maturation, while the functional localization of PSD95 depends on the appearance of dendritic spines. The co-localization of GluA2 with PSD95 in dendritic spines suggests that there are stable synapses containing AMPA receptors.

Effect of PSD-95 on long-term learning and memory impairment in neonatal rats induced by sevoflurane anesthesia / 中华行为医学与脑科学杂志

Objective:To investigate the effect of postsynaptic density-95(PSD-95)on long-term learning and memory impairment in neonatal rats induced by sevoflurane anesthesia.Methods:A total of 54 SD rats aged 7 days of SPF grade were randomly divided into 3 groups: control group (exposed to air), model group (exposed to 2.1% sevoflurane, 4 h/d, consecutive 3 days) and PSD-95 inhibitor group (inhaled sevoflurane+ intraperitoneal injection NA-1, consecutive 5 days), with 18 rats in each group.Morris water maze test and new object recognition test were used to detect the ability of visuospatial learning and memory and recognition memory of rats in each group.RT-qPCR was used to detect the mRNA levels of kalirin, Rac1 and PSD-95 in rat hippocampus.The expressions of kalirin, Rac1, PSD-95 and apoptosis related proteins Caspase-3, Bcl-2 and Bax in rat hippocampus were detected by Western blot.The expression levels of kalirin and Rac1 in hippocampus were detected by immunohistochemistry.SPSS 23.0 software was used for statistical analysis.Repeated measurement ANOVA and one-way ANOVA was used for comparing among groups.Results:Repeated measurement ANOVA showed that in the water maze test, the interaction between time and group of platform seeking latency and swimming distance of the three groups were significant ( Ftime×group=36.539, 41.548, both P<0.01). Simple effect analysis showed that the platform latency and swimming distance in the model group from day 2 to 6 were longer than those in the control group (platform latency from day 2 to 6: t=14.039, 17.147, 13.155, 13.831, 27.247, all P<0.01; swimming distance from day 2 to 6: t=10.122, 20.987, 7.267, 10.011, 8.121, all P<0.01). Compared with the model group, from day 2 to 6, the platform latencies of PSD-95 inhibitor group were prolonged( t=7.948, 14.768, 11.582, 12.832, 24.346, all P<0.01) and the swimming distances were increased( t=8.235, 24.325, 11.234, 12.031, 7.036, all P<0.01). The new object recognition test found that the new object exploration time in the model group was significantly longer than that in the control group ((21.30±2.27)s, (19.21±1.42)s, t=1.843, P<0.01), and the new object exploration time in the PSD-95 inhibitor group was significantly longer than that in the model group ((26.83±2.13)s, t=4.844, P<0.01). The difference index of novel objects in the model group was significantly lower than that in the control group ((0.41±0.12), (0.59±0.10), t=3.416, P<0.01), and the difference index of novel objects in the PSD-95 inhibitor group was significantly lower than that in the model group ((0.37±0.08), t=0.696, P<0.05). The qRT-PCR results showed that the expressions of Rac1, kalirin and PSD-95 mRNA in the model group were significantly lower than those in the control group ( t=9.969, 3.954, 6.561, P<0.05), and the expressions of Rac1, kalirin and PSD-95 mRNA in the PSD-95 inhibitor group were significantly lower than those of the model group ( t=2.132, 2.251, 3.502, all P<0.05). Immunohistochemistry results showed that the kalirin in the hippocampus CA1 area of the model group was significantly lower than that in the control group((8.18±1.94) vs (15.47±3.35), t=11.47, P<0.01), and kalirin in the PSD-95 inhibitor group was significantly lower than that in the model group((4.98±1.53), t=10.28, P<0.01); Rac1 in the model group was significantly lower than that in the control group ((3.72±1.53), (8.17±2.91), t=6.76, P<0.01), and the Rac1 in the PSD-95 inhibitor group(2.73±0.37) was significantly lower than the model group ( t=4.72, P<0.05). Western blot results showed that Caspase-3((1.37±0.16) vs (0.54±0.01), t=5.71, P<0.01) and Bax((1.87±0.31) vs (1.23±0.25), t=12.01, P<0.01) protein levels in the model group were significantly higher than those in the control group.Caspase-3 and Bax protein levels in the PSD-95 inhibitor group were significantly higher than those in model group (Caspase-3: (1.79±0.17), t=9.87, P<0.01; Bax: (2.19±0.21), t=16.19, P<0.01). The Bcl-2 protein level in the model group was significantly lower than that of the control group ((1.22±0.21) vs (1.96±0.38), t=11.92, P<0.01). And the Bcl-2 protein level in the PSD-95 inhibitor group (1.01±0.19) was significantly lower than that in the model group ( t=10.73, P<0.01). Conclusion:Sevoflurane anesthesia can damage the long-term learning and memory function and reduce the expression of PSD95 protein in neonatal rats.Inhibiting the expression of PSD95 can aggravate this damage, which may be related to the synaptic plasticity and apoptosis of neurons involved in PSD95.

Effects of electroacupuncture at "Baihui" and "Yongquan" on the levels of synaptic plasticity related proteins postsynaptic density-95 and synaptophysin in hippocampus of APP/PS1 mice / 针刺研究

OBJECTIVE: From the perspective of β-amyloid (Aβ) toxicity and synaptic plasticity, the mechanism of electroacupuncture to improve learning and memory ability in the early pathological stages of Alzheimer's disease was explored. METHODS: Twelve male amyloid-protein precursor (APP)/γ-secretase (PS1) double transgenic AD mice were randomly and equally divided into electroacupuncture (EA) group and model group, and other 6 male C57BL/6 mice were used as the normal group. EA (1 Hz/50 Hz, 0.5 mA) was applied to "Baihui" (GV20) and bilateral "Yongquan"(KI1) for 15 min, once every other day for 6 weeks. Immunofluorescence was used to observe the positive expression of Aβ in the left hippocampus. Immunohistochemistry was used to observe the positive expression of postsynaptic density-95 (PSD-95) in the left hippocampus. Western blot was used to detect the expression of PSD-95 and synaptophysin (SYN)in the right hippocampus. RESULTS: Immunofluorescence results showed that extracellular Aβ was seen in the model group and electroacupuncture group, but no senile plaques were seen. Compared with the normal group, the expression level of Aβ in the hippocampus of the model group increased significantly (P<0.01). Compared with the model group, the expression of Aβ in the hippocampus of the EA group decreased (P<0.05). Immunohistochemical results showed that compared with the normal group, the PSD-95 positive expression in the model group was decreased(P<0.05). Compared with the model group, the expression of PSD-95 in the EA group was increased (P<0.05). Western blot results showed that compared with the normal group, the expression levels of PSD-95 and SYN in the hippocampus of the model group were decreased (P<0.05, P<0.01). Compared with the model group, the expression levels of PSD-95 and SYN in the EA group were increased (P<0.05，P <0.01). CONCLUSION: EA can reduce the expression of Aβ in the hippocampus of APP/PS1 mice and increase the expression of PSD-95 and SYN, which may contribute to its effect in improving the synaptic plasticity.

The Memory-Enhancing Effects of Liquiritigenin by Activation of NMDA Receptors and the CREB Signaling Pathway in Mice

Liquiritigenin (LQ) is a flavonoid that can be isolated from Glycyrrhiza radix. It is frequently used as a tranditional oriental medicine herbal treatment for swelling and injury and for detoxification. However, the effects of LQ on cognitive function have not been fully explored. In this study, we evaluated the memory-enhancing effects of LQ and the underlying mechanisms with a focus on the N-methyl-D-aspartic acid receptor (NMDAR) in mice. Learning and memory ability were evaluated with the Y-maze and passive avoidance tests following administration of LQ. In addition, the expression of NMDAR subunits 1, 2A, and 2B; postsynaptic density-95 (PSD-95); phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII); phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2); and phosphorylation of cAMP response element binding (CREB) proteins were examined by Western blot. In vivo, we found that treatment with LQ significantly improved memory performance in both behavioral tests. In vitro, LQ significantly increased NMDARs in the hippocampus. Furthermore, LQ significantly increased PSD-95 expression as well as CaMKII, ERK, and CREB phosphorylation in the hippocampus. Taken together, our results suggest that LQ has cognition enhancing activities and that these effects are mediated, in part, by activation of the NMDAR and CREB signaling pathways.

Change of Caspase-3 and PSD-95 Expression in Hippocampus of Mice with Chronic Ketamine Addiction / 中国医科大学学报

Objective To study the changes of hippocampal caspase-3 and PSD-95 expression levels in the mice exposed to ketamine 30 mg/(kg·d)for three months. Methods Forty C57BL/6 mice were randomly divided into two groups,and the chronic ketamine addiction model was established by giving mice a three month course of daily intraperitoneal injections of ketamine. Immunohistochemical study and Western blot-ting were applied to observe the expression of caspase-3 and PSD-95 protein. Results There were more expression of caspase-3 and less of PSD-95 in ketamine group as detected by immuohistochemistry. Western blotting results showed caspase-3 active fragment level significantly increased com-pared to saline group,but PSD-95 protein level was decreased. Conclusion The increased level of caspase-3 protein and reduced expression of PSD-95 are observed after long-term ketamine administration. These findings may provide an evidence for the neurotoxicity in mouse hippocampus of chronic ketamine addition as a recreational drug.

In vitro study of bone marrow mesenchymal stem cells differentiated into dopaminergic neuron-like calls / 国际生物医学工程杂志

Objective Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate to the special histological types of neurons in vitro.The morphological change of cells and positive expression of specific antigen on membrane were studied,and the function of connection between the induced BMSCs was also detected.The feasibility of BMSCs differentiate to the special histological types of neurons was investigated.Methods BMSCs were divided into group Ⅰ (induced with bFGF+GDNF),group Ⅱ (induced with bFGF+GDNF+WHI-P131 +Shh),and control group (no revulsive).The morphologic change of cells was observed,and the positive rate of neuron specific surface antigen and the content of dopamine were detected.Formation of mature synaptic structure was detected by immunohistochemical assay of postsynaptic density protein 95 (PSD-95) expression,and synaptic loop was shown by FM1-43 stain synaptic vesicles.Results By immunohistochemical staining,the positive rates of dopamine transporter (DAT) and tyrosine hydroxylase (TH) in group Ⅱ were significantly higher than those in group Ⅰ,and dopamine can been detected in cell culture supematant of group Ⅱ.After BMSCs was induced into dopamine neuron-like cells,number and length of cell protrusions,positive rate of PSD-95 and fluorescence intensity of FM1-43 in group Ⅱ were significantly higher than those of group Ⅰ.Conclusions There were no significant change in positive rate of neuron-specific surface markers,rate of cell survival and differentiation rate after BMSCs differentiated to dopaminergic neuron-like cells.The number and length of cell protrusions,content of dopamine in cell culture supematant,positive rate of dopaminergic neuron-specific surface antigen (DAT and TH),synaptic function index (positive rate of PSD-95 and fluorescence intensity of synaptic loop) of group Ⅱ were all significantly higher than that of group Ⅰ.

Relationship between Hippocampal NMDA Receptor together with Associated Synaptic Proteins and Development of Vascular Dementia in Rats / 华中科技大学学报(医学版)

Objective To investigate the influence of Memantine on changes of N-methyl-D-aspartic acid receptor 2B (NMDAR-2B) level, the expression of postsynaptic density 95 ( PSD-95) and calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ)activities in the hippocampus of vascular dementia(VD)rats and their recognition. Methods The VD rat model was established by permanent,bilateral occlusion of the common carotid arteries. One hundred and forty-four Wistar rats were randomly divided into sham-operated group, VD model group and Memantine-treated group. The water maze test was performed to detect the ability of learning and memory of the rats,the changes of NMDAR-2B level were measured by RT-PCR,the changes in the expression of PSD-95 were detected by immunohistochemical staining,and the changes in the CaMK Ⅱ activities were determined by incorporation of ~(32)P into histone. Results Compared with the sham-operated group, the ability of learning and memory of VD rats was decreased significantly(P<0. 01); the level of NMDAR-2B and the expression of PSD-95 increased significantly at the 4th week after operation(F<0. 05 or 0. 01)and decreased at the 8th to 16th week after operation (P<0. 01). Total CaMK Ⅱ activity was increased significantly at the 4th week after operation(P<0. 01) ,then dropped at the 8th week after operation(P>0. 05) ,and decreased significantly at the 12th,16th week after operation(P<0. 01). As for Memantine-treated group,at the 4th week after operation,there was no statistically significant difference in the parameters mentioned above in contrast with the sham-operated group. At the 8th,and 12th week after operation,the ability of learning and memory,the level of NMDAR-2B and the expression of PSD-95 were significantly increased as compared with VD rats(P<0. 01 or 0. 05) ,but total CaMK Ⅱ activity was similar to that of VD rats. Conclusion With the development of VD,NMDAR-2B level,the expression of PSD-95 and CaMK Ⅱ activities were firstly over-activated,and then decreased greatly. Memantine can improve VD rats' recognition by modulating the expression of NMDAR-2B,but its effect on CaMK Ⅱactivity was limited.

Effect of electroacupuncture combined with repetitive transcranial magnetic stimulation on postsynaptic density 95 in the hippocampus of rats after cerebral ischemia / 中华物理医学与康复杂志

Objective To investigate the effect of electroacupuncture (EA) combined with repetitive transcranial magnetic stimulation ( rTMS) on postsynaptic density 95 ( PSD- 95) in the hippocampus of rats after cerebral ischemia. Methods Sixty healthy male Wistar rats were divided into normal, model, EA, rTMS and EA + rTMS groups randomly, and the rats in each group were further divided into 7 d, 14 d and 28 d subgroups. A model of middle cerebral artery occlusion ( MCAO) was established, followed by EA, rTMS or EA + rTMS treat-ment. The expression of PSD-95 in the dentate gyros and area CA3 of the hippocampus of the ischemic cerebral hemisphere were investigated. Results The expression of PSD-95 in the dentate gyrus and area CA3 of the ische-mic cerebral hemisphere had decreased 7 d after cerebral ischemia. The expression of PSD-95 in the three treat-ment groups increased after 14 d of treatment and was significantly greater compared with the model group after 28 d of treatment. The EA + rTMS group was especially superior to the EA and rTMS groups in terms of the expression of PSD-95 in area CA3 by the 28th day. Conclusions EA + rTMS can significantly increase the expression of PSD-95 in the hippocampus after cerebral ischemia. This may help explain how EA + rTMS improves learning and mem-ory after cerebral ischemia.

EFFECTS OF NMDA RECEPTORS IN SYNAPSE AND EXTRASYNAPSE OF RAT HIPPOCAMPAL NEURONS ON THE EXPRESSION OF POSTSYNAPTIC DENSITY-95 INDUCED BY AMYLOID-BETA / 解剖学报

Objective To investigate the mechanism of the soluble A? oligomers-induced alteration of synaptic proteins. Methods This study applied immunocytochemistry technique to investigate the changes of the expression of postsynaptic density-95(PSD-95) in primary hippocampal neurons, which was exposed to A?_ 25-35 after NMDAR antagonist or agonist treatment. Results The results showed that A?_ 25-35 downregulated PSD-95 protein in a dose- and time-dependent manner. Treatment of cells with MK801 (a general NMDA receptor antagonist) prevented A?-induced PSD-95 degradation. Moreover, when extrasynaptic NMDA receptors were blocked by ifenprodil (a NR2B subunit specific antagonist), the A?-induced downregulation of PSD-95 was significantly attenuated. Whereas, when synaptic NMDA receptors were blocked by bicuculline (a GABA receptor antagonist) in combination with MK801, the PSD-95 degradation did not change significantly.Conclusion The results suggest that A?-induced downregulation of PSD-95 depends on NMDAR activity, and extrasynaptic NMDA receptors may be involved in A?-induced synaptic protein degradation.