脑源性神经营养因子与不含GLuR2的AMPA受体在增强AMPA受体功能及活化突触囊泡中的关系

Relationship of brain-derived neurotrophic factor and AMPA receptor without GLuR2 in enhancing the function of AMPA and activating synaptic vesicle

摘要:

目的：研究脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）与α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid，AMPA）受体尤其不含GluR2的AMPA受体的相互关系，为进一步针对性调控BDNF及AMPA受体，保护海马神经元提供理论依据。方法：孕17～18 d SD大鼠8只，取胎鼠体外培养海马神经元细胞爬片，同批细胞爬片随机分为空白对照组（N组）、BDNF实验组（BDNF组）、特异性阻断不含GLuR2 的AMPA 受体的阻断剂1-萘乙酰精胺三盐酸（1-naphthylacetyl spermine Trihydrochloride，NASPM）对照组（N+NASPM组）、BDNF+NASPM实验组（BD－NF+NASPM组）4组，各组随机选择爬片2张，共3批细胞爬片应用于实验。各组应用Synapto GreenTM C4（FM1-43）结合激光共聚焦标记突触囊泡，活体追踪同一视野给予相应处理前后2次染色突触囊泡变化；应用膜片钳记录AMPA受体mEPSCs频率及幅度变化。结果：BDNF组与N组第2次染色比较，荧光强度BDNF组（750.23±137.81）明显高于N组（554.41±16.42）（χ2=7.22，P=0.030）；而BDNF组添加NASPM后，荧光强度（525.93±72.64）较BDNF组有明显减少（χ2=13.18，P=0.000），表明BDNF对不含GLuR2的AMPA受体调控可能是BDNF使功能突触囊泡增多的机制之一。BDNF组较N组AMPA受体微兴奋性突触后电流的频率从（1.730±0.217）增长到（3.340±0.280）（P=0.000），幅度从（-16.30±1.98）增加到（-25.00±2.57）（P=0.000）。BDNF组使用NASPM后，频率（1.740±0.207）（P=0.000）及幅度（-15.50±2.52）（P=0.000）均减小，表明BDNF能通过对不含GLuR2的AMPA受体调控来增加AMPA受体的功能。结论：BDNF可能部分通过调节不含GLuR2的AMPA受体，来参与BDNF增强AMPA受体功能及活化突触囊泡的作用。

Abstract:

Objective：To study the relationship of brain-derived neurotrophic factor（BDNF） and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor（AMPA） receptor without GLuR2，to further regulate BDNF and the receptor of AMPA and to pro－vide theoretical basis for protecting hippocampal neuron. Methods：Eight pregnant SD rats of 17-18 d were selected and their fetal rats’ hippocampal cells were separated and cultured. The cells were randomly divided into normal group（N group），BDNF group，N+NASPM group and BDNF+NASPM group. All groups were treated with Synapto GreenTM C4（FM1-43） and patch clamp. Quantity of synaptic vesicles and the function of synapse were recorded. Results：Results showed that the second fluorescene intensity of BDNF group（750.23±137.81） was obvious higher than that of N group（554.41±16.42）（ χ2=7.22，P=0.030）；while fluorescene intensity of BDNF+NASPM group（525.93±72.64） was lower than that of BDNF group（ χ2=13.18，P=0.000），indicating that BDNF regulating AMPA receptor without GluR2 may be one of the mechanisms of BDNF increasing the function of synaptic vesicle. What’s more，BDNF group had higher frequency（3.34±0.280） and amplitude（-25.00±2.57） of AMPA receptor micro excitatory postsynaptic currents compared with those of N group（1.730±0.217）（P=0.000） and （-16.3±1.98）（P=0.000）. After BDNF group being adding NASPM，frequency（1.740±0.207）（P=0.000） and amplitude（-15.50±2.52）（P=0.000） were reduced，indicating that BDNF can increase AMPA receptor mEPSCs by regulating the AMPA receptor without GluR2. Conclusions：BDNF may partially adjust the AMPA receptor without GluR2 subunit to enhance the function of AMPA and activate the synaptic vesicles.