The Role Of KCC2 In Epileptogenesis And Potential Of Anti-convulsasnt Target Study

Epilepsy is one of the most frequent central nervous system (CNS) disorder, multiple factors feed into epilepsy causation, the underline mechanism of epileptogenesis has not yet fully understood. However, the imbalance between excitation and inhibition in CNS is the basic inductive factor. This imbalance causes a group of neurons to make a sudden, excessive synchronized discharges, and exhibited as an abnormal behavior. GABA and GABA receptors is one of the main inhibitory system in adult CNS, and play an important role in epileptogenesis. Previous studies mostly focus on the changes of the GABA transmission and GABAA receptor composition during epileptogenesis, however, more and more evidence showing the alteration of Cl- homeostasis also makes a very important role to the epileptogenesis The intracellular Cl- homeostasis is mainly under cation-chororide cotransporter KCC2 activity control in adult. To explore the relationships between KCC2 and epileptogenesis, CTZ epilepsy model, both in vitro and in vivo preparation, was used.The first part of this thesis demonstrated, by using western-blot and immunohistochemistry techniques, that CTZ induced cell membrane KCC2 down regulation in both cultured, brain slice and anaesthetized rat hippocampal neurons after seizure induction. Further, by using patch-clamp electrophysiological techniques, we found that cell membrane KCC2 down regulation caused the reducation of intracellular Cl" exclusion probability and a positive shift in EGABA.Blockade of KCC2 function by using furosemide, a KCC2 inhibitor, facilitated CTZ induced epileptiform activity in hippocampal slice. By using overexpression KCC2 plasmid, we further demonstrated that upregulation of KCC2, a expected, significantly suppressed the probability of neurons into epilepsy after CTZ treatment in cultured hippocampal neurons. In conclusion, our results demonstrated that cell membrane KCC2 down regulation is one of the causes for reduced GABA inhibitory function which contributes to the CTZ-induced epileptogenesis.In the second part of this thesis, we found that furosemide, as a blockade for KCC2, has protect function against, at least demonstrated in this study, CTZ induced seizure activity related KCC2 down-regulation. And this protection property was likely not related to the anti-epilepsy effects of furosemide. This conclusion was based on the finding that, co-incubation with CTZ, furosemide significantly reversed CTZ seizure induced membrane KCC2 down regulation (western-blot and immunostaining) and the positive shift of EGABA(electrophysiology). By protection of KCC2 from seizure activity induced down regulation, furosemide also reversed seizure induced GAB A current impairment and GAB A receptor down regulation. This was evidenced with the observation that furosemide inhibited CTZ induced both phasic mIPSC amplitude and tonic IPSCs amplitude reducetion, and GABA receptor β2/β3 subunit downregulation. The underline mechanism of this protection is probably due to one of the other KCC2 function that it could act as membrane GABA receptor immobolizer to stabilize receptors. This hyposis is based on the evidence that down-regulation of KCC2 with specific shRNA in cultured hippocampal neurons decreased in amplitude of miPSCs and an increase in tonic GABA current density without affect the total GABA current density. The further study showing the increased tonic GABA current density was not mediated by the δ-containing extra-synaptic GABAA receptors to leave the probability of an increased α5-containing extra-synaptic GABAA receptors. In summary, furosemide could protect KCC2 from the down regulation by seizure like stimulation, and down stream to protect GABA recertor distribution and function which might be important in seizure control.In conclusion, our results demonstrated that neuronal membrane KCC2 down regulation is one of the important facts in seizure induction and development, while KCC2 down regulation also influences of GABA receptor distribution and functional impaiment, which might be the underline mechanism for epilepsy. In addition, we also demonstrated that blockade of KCC2 by its inhibitor furosemide rescued the KCC2 protein to remain on cell membrane during seizure stimulation, which indicates that binding site of the KCC2 protein for its inhibitor might be an important target for anti-convulasant drug development.