The Effect And Potential Mechanism Of GPR40 Involved In Epileptic Seizures

Part one: The expression of GPR40 in epileptic tissues of patients and animal modelObjective: We determined the expression location and level of GPR40 in tissues of patients with temporal lobe epilepsy(TLE)and epilepsy mouse modelMethods: 1.From our previous established human brain tissues containing over 300 neocortical tissues from pharmacoresistent TLE patients and 100 histological normal neocortical tissues from head trauma patients,we randomly selected 20 epileptic tissues as experimental group and 10 normal tissues as control group.2.The adult male C57BL/6 mice were unilateral intrahippocampal injected of 200 ng kainic acid(KA)to establish TLE model.Hippocampus and cortex from mice with spontaneous recurrent seizures(SRS,n=5)group and without SRS(non-SRS,n=5)were obtained.3.Western-blot was used to detect the expression level of GPR40 and immunofluorescent staining was used to determine the expression location.Results: 1.Immunofluorescent staining found that GPR40 was highly expressed in the pyramidal cell layer of the CA1 and CA3 regions of the hippocampus.In temporal neocortices from patients with TLE and the hippocampus from mice model,GPR40 is expressed mainly in neurons but not in astrocytes.Furthermore,GPR40 is distributed in the postsynaptic area of excitatory neurons.2.In the human tissues,The expression of GPR40 was significantly increased in temporal neocortex of TLE patients compared with that in controls.3.In the animal model,The expression of GPR40 in both hippocampus and cortex was significantly increased in SRS group compared with that in non-SRS group.Conclusion: In brain tissues from epileptic patients and mice model,the expression of GPR40 is significantly increased and mainly expressed in excitatory neurons,which indicating GPR40 may participate in the development of epilepsy.Part two: GPR40 regulates epileptic seizures activity in two animal modelsObjective: To investigated the effect of GPR40 in the development of epilepsy,the GPR40 selective agonist GW9508 and the selective antagonist GW1100 were intracerebroventricularly administrated during epileptogenesis in the KA-induced TLE model and the pentylenetetrazole(PTZ)kindling model,and the epileptic activity was assessed by behavioral observation and local field potential(LFP)recording.Methods: 1.KA model: Adult male C57BL/6 mice were induced status epilepticus(SE)by unilateral intrahippocampal injected of 200 ng KA.Three days later,mice was randomly divided into 3 groups: GW9508 group,GW1100 group and DMSO group(n=6 in each group).Mice was daily treated for consecutive 7 days through the intracerebroventricular guide cannula.The SRS behavior was continuously monitored,and 30 days later,the LFPs was recorded.2.PTZ kindling model: Adult male C57BL/6 mice were equipped with guide cannula and treated with GW9508,GW1100 or DMSO(n=12 in each group)through the intracerebroventricular guide cannula prior to an injection of a subthreshold dose of PTZ(35mg/kg)every other day for total 15 injections.The seizure score was assessed during the 5 injections.Results: 1.In the KA model,behavior observation showed that the seizure frequency was reduced in GW9508 group and increased in GW1100 group compared with control group(p <0.05).LFPs recording showed,compared with control group,GW9508 reduced the seizure-like events(SLEs)frequency and the total time spent in SLEs during 30 minutes,and GW1100 showed the opposite effect(p< 0.05).2.In the PTZ kindling model,GW9508 lead to a significant decrease in the seizure severity scores in all assessed points and a significant increase in the survival rate over time compared with that in the control group.GW1100 showed the opposite effect.Conclusion: Through two animal models,it was confirmed that,during the development of epilepsy,GPR40 activation alleviate the result epileptic activity and GPR40 inhibition exacerbate the result epileptic activity.Part three: GPR40 regulates the synaptic transmissionObjective: Using the whole-cell patch-clamp recordings in mice brain slices,to explore the effect of GPR40 on the synaptic transmission.Methods: Whole-cell patch-clamp recordings were performed on thehippocampus CA1 pyramidal neurons in mice brain slices at baseline and after GW9508 or GW1100 perfusion.The action potential(AP),miniature excitatory postsynaptic currents(m EPSCs),miniature inhibitory postsynaptic currents(m IPSCs),AMPA and NMDA receptor mediated EPSC currents,and paired-pulse ratio(PPR)were recorded to determine the influenced postsynaptic receptors.Results: Whole-cell patch-clamp recordings showed that GW9508 reduced the frequency of AP and GW1100 increased it,compared with the baseline.The frequency and amplitude of m EPSCs was reduced by GW9508 and increased by GW1100.However,neither the frequency nor the amplitude of the m IPSCs was affected by either treatment.Compared with the baseline condition,the amplitude of the NMDAR-mediated EPSCs was reduced by GW9508 and increased by GW1100.The AMPAR-mediated EPSCs was unchanged by either treatment.The PPR for NMDA-mediated EPSCs showed no significant changes were found following either treatment.Conclusion: GPR40 affects NMDA receptor-mediated excitatory postsynaptic transmission.Part four: The potential mechanism of GPR40 involved in epilepsyObjective: To explore underlying mechanisms of GPR40 involved in epilepsy by understanding the mechanisms of GPR40 in regulating NMDA receptors.Methods: 1.In the brain tissue after animal experiment,the expression of total protein and membrane protein of these affected NR2 A and NR2 B was detected by Western blotting.2.Immunofluorescent staining was used to detect the colocalization of GPR40 and NR2 A and NR2 B.Immunoprecipitation was used to detect the interaction of GPR40 with NR2 A and NR2 B.Quantitative immunoprecipitation was used to detect the effect of GPR40 on this interaction.Results: 1.Western blotting showed that the ratio of membrane/total protein of NR2 A and NR2 B in GW9508 group was significantly decreased,and that in GW1100 group was significantly increased.in NRBA The total protein expression was not significant changed in either treatment.2.Immunofluorescent staining showed the colocalization of GPR40 with NR2 A and NR2 B.Immunoprecipitation showed that GPR40 interacted with NR2 A and NR2 B.Quantitative immunoprecipitation showed that,compared with the control,GW9508 reduced this interactionbetween GPR40 and NR2 A as well as NR2 B,and GW1100 enhanced this interaction.Conclusion: GPR40 affects the surface expression of NR2 A and NR2 B.This effect may be through regulating the interaction of GPR40 with NR2 A and NR2 B.