论文部分内容阅读
目的探讨细胞外信号激酶(ERK)/cAMP反应原件蛋白(CREB)信号通路对大鼠前扣带皮层神经元长时程增强(LTP)诱导的影响。方法采用离体脑片场电位记录方法观察ERK激酶抑制剂对大鼠前扣带皮层(ACC)LTP诱导的影响,采用免疫组织化学方法观察ACC脑片在强直刺激后不同时间点磷酸化ERK(pERK)和磷酸化CREB(pCREB)的表达情况。结果在大鼠ACC脑薄片上,高频刺激(2-train,100Hz,1s)能诱导出稳定的场电位(fEPSP)长时程增强。预先给予NMDA受体竞争性拮抗剂APv(50μmol/L)可完全阻断LTP的产生,提示本实验中ACC神经元LTP是NMDA受体依赖性的。灌流液中预先给予MEK抑制剂PD98059(50μmol/L)能完全阻断LTP的产生。取高频刺激后不同时间点的脑片进行免疫组化检测,结果显示,高频刺激后5min时,pERK表达显著升高,在10min达到最高峰,1h后回复到基础表达水平。同样,高频刺激后ACC脑片中pCREB的表达也显著增加。预先灌流液中给予MEK抑制剂PD98059能够阻断高频刺激引起的pERK和pCREB表达上调。免疫双标结果显示几乎所有的pERK都能与pCREB共定位于同一个神经细胞。结论大鼠前扣带皮层中NMDA受体和ERK/CREB信号通路是长时程增强诱导所必需的。
Objective To investigate the effect of extracellular signal kinase (ERK) / cAMP response protein (CREB) signaling pathway on the long-term potentiation (LTP) induced by anterior cingulate cortical neurons in rats. Methods The effects of ERK kinase inhibitors on the induction of LTP in rat anterior cingulate cortex were observed by in vitro brain field potential recording. The expression of phosphorylated ERK (pERK) was detected by immunohistochemical method at different time point after anotraphic stimulation ) And phosphorylated CREB (pCREB) expression. Results Long-term potentiation of stable field potential (fEPSP) was induced by high-frequency stimulation (2-train, 100 Hz, 1 s) in rat brain slices of ACC. Pretreatment with APv (50μmol / L), a competitive antagonist of NMDA receptor, completely blocked the production of LTP, suggesting that LTP in ACC neurons is NMDA receptor-dependent in this experiment. Pretreatment with MEK inhibitor PD98059 (50μmol / L) in perfusate solution completely blocked the production of LTP. The hippocampal slices were taken for immunohistochemistry at different time points after stimulation. The results showed that pERK expression increased significantly at 5 min after high-frequency stimulation, peaked at 10 min, and returned to basal level after 1 h. Similarly, the expression of pCREB in ACC brain slices after high-frequency stimulation was also significantly increased. PD98059, a MEK inhibitor pre-perfusion fluid, blocked the up-regulation of pERK and pCREB induced by high-frequency stimulation. Immunoblotting showed that almost all pERKs co-localized with pCREB in the same neuron. Conclusion NMDA receptor and ERK / CREB signaling pathway in the anterior cingulate cortex of rats is necessary for long-term potentiation induction.