Objective To explore the role of the extracellular signal-regulated kinase （ERK）/cAMP response element binding protein （CREB） pathway in the induction of long-term potentiation （LTP） in the anterior cingulate co...Objective To explore the role of the extracellular signal-regulated kinase （ERK）/cAMP response element binding protein （CREB） pathway in the induction of long-term potentiation （LTP） in the anterior cingulate cortex （ACC） that may be implicated in pain-related negative emotion. Methods LTP of field potential was recorded in ACC slice and the expressions of phospho-ERK （pERK） and phospho-CREB （pCREB） were examined using immunohistochemistry method. Results LTP could be induced stably in ACC slice by high frequency stimulation （2-train, 100 Hz, 1 s）, while APv （an antagonist of NMDA receptor） could block the induction of LTP in the ACC, indicating that LTP in this experiment was NMDA receptor-dependent. Bath application of PD98059 （50 μmol/L）, a selective MEK inhibitor, at 30 min before tetanic stimulation could completely block the induction of LTP. Moreover, the protein level of pERK in the ACC was transiently increased after LTP induction, starting at 5 rain and returning to basal at 1 h after tetanic stimulation. The protein level of pCREB was also increased after LTP induction. The up-regulation in pERK and pCREB expressions could be blocked by pretreatment of PD98059. Double immunostaining showed that after LTP induction, most pERK was co-localized with pCREB. Conclusion NMDA receptor and ERK-CREB pathway are necessary for the induction of LTP in rat ACC and may play important roles in pain emotion.展开更多
原代培养的海马神经元是研究神经细胞中蛋白运输与亚细胞空间定位的有效研究工具。本文旨在建立海马神经元与皮层胶质细胞混合培养的方法,以期提供状态良好的神经元供研究用。新生2～3 d Sprague-Dawley(SD)大鼠断头处死,从大脑半球表...原代培养的海马神经元是研究神经细胞中蛋白运输与亚细胞空间定位的有效研究工具。本文旨在建立海马神经元与皮层胶质细胞混合培养的方法,以期提供状态良好的神经元供研究用。新生2～3 d Sprague-Dawley(SD)大鼠断头处死,从大脑半球表面片取2～3片皮层组织,切碎后用胰蛋白酶消化成单细胞悬液,种植于25cm2培养瓶内。种植后第四天,采用剧烈敲击培养瓶的方法去除非星形胶质细胞的杂质细胞,继续培养贴壁细胞直至细胞接近铺满瓶底,改用含阿糖胞苷的神经细胞培养液。原代培养的大鼠海马神经元种植在这种预先培养好的皮层星形胶质细胞层上。镜下观察结果显示,本方法培养的原代星形胶质细胞纯度较高,共培养的神经元状态较佳,存活时间长,突触前结构发育良好,可以耐受转染操作。以上结果提示,本文所建立的原代神经元与星形胶质细胞的混合培养方法操作简单,能够可靠地培养出健康的神经元。展开更多
基金supported by Shanghai Municipal Science and Technology Major Project(No.2018SHZDZX01)Shanghai Center for Brain Science and Brain-Inspired Technology。
基金supported by National Natural Science Fundation of China (No.30870835,30821002,and 30900444)National Basic Research Program of China (No. 2007CB512303,2007CB512502,and 2006CB500807)Postdoctoral Fundation of China (No.20080440578)
文摘Objective To explore the role of the extracellular signal-regulated kinase （ERK）/cAMP response element binding protein （CREB） pathway in the induction of long-term potentiation （LTP） in the anterior cingulate cortex （ACC） that may be implicated in pain-related negative emotion. Methods LTP of field potential was recorded in ACC slice and the expressions of phospho-ERK （pERK） and phospho-CREB （pCREB） were examined using immunohistochemistry method. Results LTP could be induced stably in ACC slice by high frequency stimulation （2-train, 100 Hz, 1 s）, while APv （an antagonist of NMDA receptor） could block the induction of LTP in the ACC, indicating that LTP in this experiment was NMDA receptor-dependent. Bath application of PD98059 （50 μmol/L）, a selective MEK inhibitor, at 30 min before tetanic stimulation could completely block the induction of LTP. Moreover, the protein level of pERK in the ACC was transiently increased after LTP induction, starting at 5 rain and returning to basal at 1 h after tetanic stimulation. The protein level of pCREB was also increased after LTP induction. The up-regulation in pERK and pCREB expressions could be blocked by pretreatment of PD98059. Double immunostaining showed that after LTP induction, most pERK was co-localized with pCREB. Conclusion NMDA receptor and ERK-CREB pathway are necessary for the induction of LTP in rat ACC and may play important roles in pain emotion.
基金supported by the National Natural Science Foundation of China(No.31571098,32071026)Shanghai Municipal Science and Technology Major Project(No.2018SHZDZX01)+1 种基金ZJ LabShanghai Center for Brain Science and Brain-Inspired Technology。
基金supported by the National Basic Research Program of China(No.2013CB531905)the National Natural Science Foundation of China(No.81571064)the National Basic Science Talent Cultivation Foundation of China(No.J1210041)
基金supported by the Nationa l985 Program from the Ministry of Education of China
文摘原代培养的海马神经元是研究神经细胞中蛋白运输与亚细胞空间定位的有效研究工具。本文旨在建立海马神经元与皮层胶质细胞混合培养的方法,以期提供状态良好的神经元供研究用。新生2～3 d Sprague-Dawley(SD)大鼠断头处死,从大脑半球表面片取2～3片皮层组织,切碎后用胰蛋白酶消化成单细胞悬液,种植于25cm2培养瓶内。种植后第四天,采用剧烈敲击培养瓶的方法去除非星形胶质细胞的杂质细胞,继续培养贴壁细胞直至细胞接近铺满瓶底,改用含阿糖胞苷的神经细胞培养液。原代培养的大鼠海马神经元种植在这种预先培养好的皮层星形胶质细胞层上。镜下观察结果显示,本方法培养的原代星形胶质细胞纯度较高,共培养的神经元状态较佳,存活时间长,突触前结构发育良好,可以耐受转染操作。以上结果提示,本文所建立的原代神经元与星形胶质细胞的混合培养方法操作简单,能够可靠地培养出健康的神经元。
