Study On The Effect And Mechanism Of Decreased Excitability In Dentate Gyrus Neurons In The Epileptogenesis Of Temporal Lobe Epilepsy

Objective: Epilepsy is a chronic brain disease with different etiology and different clinical manifestations,but it was characterized by recurrent epileptic seizures.Epilepsy affects more than 70 million people worldwide,although the seizures of most patients can be well controlled with administration of antiepileptic drug treatment.However,there are still 20%-30% of patients who still experience uncontrolled seizures despite adequate treatment with existing anti-epileptic drugs.Therefore,exploring the underlying mechanisms of epilepsy has become a top priority in central nervous system disease research.DREADD is one of the most commonly used chemical genetics.G protein-coupled receptors were modified,so that those receptors can only bind to special synthetic compounds and then the signaling pathway of corresponding G protein-coupled receptor was activated to mediate neuronal excitability.The excitability of neurons in hippocampal dentate gyrus has attracted much attention in the study about epileptogenesis.Therefore,this study explore the role of neuronal excitability of hippocampal dentate gyrus in epileptogenesis and underlying mechanism in pentylenetetrazole kindling rat model by using DREADD.Method:(1)Four-week-old healthy male SD rats were randomly divided into three groups: DREADD+CNO group,DREADD+NS group,and non-DREADD+CNO group.Rats in the DREADD+CNO and DREADD+NS groups were injected with 0.5μl of h M4Di-containing virus into the bilateral hippocampal dentate gyrus through stereotaxic surgery,while rats in the non-DREADD+CNO group were injected with0.5μl of control virus.Four weeks later,rats were injected intraperitoneally with 35mg/kg pentylenetetrazol(PTZ)daily.Subsequently,we observed epilepsy-related behavioral performance such as mortality rate,kindling rate,kindling latency and daily seizure severity.After continuously observing behavioral performance for thirty minutes,rats in the DREADD+CNO and non-DREADD+CNO groups were injected with 10mg/kg CNO,while rats in the DREADD+NS group were injected with an equal volume of saline.On day 28,after the behavioral observation,brain tissue was collected from all rats to evaluate mossy fiber sprouting using Timm staining.(2)Different doses of CNO(1mg/kg,5mg/kg,10mg/kg)were used to compare the effects of different CNO doses on epilepsy-related behavioral performance and mossy fiber sprouting in the PTZ kindling rat model of epilepsy.The expression levels of MOB1 and NDR2,axon growth-related proteins,in the hippocampus of each group of rats under different CNO doses were detected by western blotting and immunofluorescence.(3)Four-week-old healthy male SD rats were used to establish the DREADD+CNO+NDR2 group.A mixture of h M4Di-containing virus and NDR2-expressing virus was injected into the bilateral hippocampal dentate gyrus through stereotaxic surgery.Four weeks later,rats were injected with 35mg/kg PTZ intraperitoneally daily.After continuously observing behavioral performance for thirty minutes,the rats were injected with 10mg/kg CNO intraperitoneally.On day 28,after the behavioral observation,brain tissue was collected from all rats to evaluate the expression levels of NDR2 in the hippocampus using western blotting and immunofluorescence,and to evaluate mossy fiber sprouting using Timm staining.Results:1.h M4 Di receptor required for DREADD technique was selectively expressed in neurons of dentate gyrus in pentylenetetrazole kindling rat model.2.Silencing of dentate gyrus significantly prolonged the latency of kindling,delayed the tendency of kindling,relieved the degree of seizure severity and reduced rate of kindling,meanwhile,moss fiber sprouting was suppressed.3.Compared with DREADD rats receiving 1mg/kg CNO dose,the rats receiving 10mg/kg CNO dose had a lower degree of seizure severity,and there were no significant differences in mortality,rate of kindling,latency and tendency of kindling,severe epileptic seizure latency and moss fiber sprouting among rats treated with different CNO doses.4.When silencing of dentate gyrus inhibited the epileptogenesis of TLE,MOB1 expression was significantly increased and NDR2 expression was significantly decreased in hippocampus;5.The NDR2 expression in hippocampus was up-regulated in rat pentylenetetrazole kindling with viral transfection;6.The inhibiting effect of silencing of dentate gyrus on epileptogenesis of TLE and mossy fiber sprouting was blocked by the up-regulation of NDR2 expression in hippocampus.Conclusion1.Decreased excitability of neurons in the dentate gyrus inhibit the epileptogenesis of temporal lobe epilepsy;2.The inhibiting effect of silencing of dentate gyrus on mossy fiber sprouting and epileptogenesis of TLE may be related to MOB1 protein in hippocampus.3.Silencing of dentate gyrus neurons may inhibit mossy fiber sprouting and prevents epileptogenesis of TLE through NDR2 kinase.