The Study Of Machanism On CAMP Response Element Binding Protein Participates In The Phosphorylated Extracellular Signal Regulate Protein Kinase Mediated Epilepsy Mossy Fiber Sprouting

Objective:To observe the effects of the inhibitor of extracellular signal-regulated protein kinase 1/2 on the expression of phosphorylated cAMP response-element binding protein, growth associated prorein and synaptophysin after epileptiform discharge of hippocampal neurons, and investigate the effects of ERK/CREB on epilepsy mossy fiber sproutingMethods:Neonatal Wistar(<24 hours) rats were adopted. Hippocampal neurons were cultured in Neurobasal medium supplemented with 10% fetal bovine serum, and placed into a 37oC, 5% CO2 cell culture incubator. The neurons were devided into:1.Normal control group: On day 9 after beginning the incubation, we replaced the maintenance medium with normal extracellular fluid for three hours, then recorded the discharge using whole-cell patch clamp techniques. 2.Model group: On day 9 after beginning the incubation, we replaced the maintenance medium with magnesium-free extracellular fluid for three hours, then recorded the epileptiform activity using whole-cell patch clamp techniques. 3.U0126 group: On day 9 after beginning the incubation, we replaced the maintenance medium with magnesium-free extracellular fluid and 10μm/l U0126 for three hours. We evaluated two groups, model and U0126, and six time points, 0 minutes, 30 minutes, 2 hours, 6 hours, 12 hours, and 24 hours following epileptiform discharge. We investigated changes in pERK1/2 and pCREB expression by double-label immunofluorescence, for each sample, photos were collected using a laser scanning confocal microscope. Using western blotting, we observed changes in expression of GAP-43 and synaptophysin.Results:1.Hippocampal neurons epileptiform discharge was recorded by EPC-10 system in whole-cell patch clamp. Action potential could be recorded in neurons, who had been treated with normal extracellular fluid for 3 hours. Epileptiform discharges of neurons recordings from cultured neurons that were exposed to magnesium-free extracellular fluid for 3 hours manifested larger, longer duration synaptic potentials and multiple action potentials. These evolved into continuous tonic high-frequency burst discharges that resolved into recurrent epileptiform discharges after return to normal medium. 2.Following immunofluorescence double labeling, differentially expressed products were detected. In the normal control group, pCREB were observed in the nucleus, and pERK1/2 were detected in the cytoplasm predominantly. pERK1/2 could be observed at 0 min, and the expression peaked at 30 min. The expression pattern of pCREB was the same as pERK1/2 in the model group. In the U0126 group, pERK1/2 expression was inhibited completely. Moreover, pCREB expression also was partially inhibited, and the intensity of its expression was similar at each time point.3. We measured the levels of SYP and GAP-43 by western blotting.In normal neurons, both SYP and GAP-43 were observed. In model group, both proteins were detected at the first time point, and changes in expression followed the same pattern as pERK1/2. On the other hand, in U0126 group, neurons expression of both proteins were reduced across all six time points.Conclusions:1.Neurons cultured in Neurobasal medium with fetal bovine serum was fit to be treated with magnesium-free extracellular fluid for three hours. The neuronal epileptiform discharge model was stabile for next study.2.ERK/CREB signal transduction pathway is functionally regulated by phosphorylation, which was activated after neuronal epileptiform discharge, and accompanied by increased levels of GAP-43 and SYP, which were considered to be markers of MFS. Moreover, bouth the activation of the signal transduction pathway was inhibited, and the expression of the markers were decreased by U0126.3.In cellular level, these findings indicate that MFS after epileptic seizure are related to activation of the ERK/CREB signal transduction pathway.