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

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（BDNF＋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。

Objective：To study the relationship of brain-derived neurotrophic factor（BDNF） and a-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）（X^2=7.22,P=0.030） ;while fluorescene intensity of BDNF＋NASPM group （525.93 ±72.64） was lower than that of BDNF group （X^2=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 postsynaptie 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.