TNF? Promotes Neuronal Excitability In Low-Mg²⁺ Epilepsy Model Via Astrocyte Gap Junction Pathway

Background Epilepsy,as a common CNS functional disorder,is characterized with by epileptic seizures activity in one brain region,usually paralleling with hippocampal damage.In the brain tissues from epilepsy patient and epilepsy disease animal models,many researchers observed that the expression of the pro-inflammatory factor TNF ? increases and the activity of astrocytic gap junction is abnormal.There are abundant gap junctions between adjacent astrocytes in hippocampal tissue,which make astrocytes as a functional syncytium.This structure plays an important role in maintaining the balance of internal environment in brain and an appropriate condition for neurons.However,whether the increasing TNF? influences the function of gap junction,and how it promotes the development of epilepsy,are not determined.Objective and Method To induce high excitability of neuronal network in vivo,brain slice from mouse postnatal 21 days was incubated in Low-Mg2+ a CSF.Brain slice field potential recording and whole cell recording are used in the current study,combined with quantitative PCR of single astrocyte,western blotting and immunofluorescence staining.In addition,the application of recombinant TNF?,TNF? antagonist and gap junction antagonist help us to observe the effect of TNF? on neuronal networks and figure out whether gap junction involves in the mechanisms.Results 1)The m RNA level of both TNFR1 and TNFR2 are high in single hippocampal astrocyte,and TNFR1 is the main type of TNF receptor;2)Recurrent epileptiform charges was induced in CA1 region of hippocampal via Low-Mg2+ a CSF incubation added with PTX(Picrotoxin);3)TNF? increase the interictal-like events,promoting the excitability of neuronal networks.And the application of TNF? could remove the effect;4)While conducting the dual electrodes recording,high synchronization is observed between the response of membrane current of astrocyte and the neuronal network field potential;5)Gap junction antagonist CBX and MFA decrease epileptiform in Low-Mg2+ epilepsy model,which reverse the enhanced discharge induced by TNF ? bath application;6)The gap junction Cx43 expresses abundantly in astrocyte and the expressionis age dependent;7)The gap junction Cx43 is distributed almost over hippocampal region,mainly the astrocytic endfeets near vascular wall and the fine process of astrocytes.Conclusion In the epilepsy model in brain slices induced by low-Mg2+ incubation,TNF? promotes the high excitability of neuronal network.And gap junction protein Cx43 in astrocyte may involve in the epileptiform induced by TNF?.Our research would contribute to clarify the mechanism of epilepsy generation and epilepsy development,providing more information for epilepsy therapy and potential molecular target for pharmacological treatments.