Objective:Epilepsy is a multi-etiological brain dysfunction syndrome characterized by synchronously repeated spontaneous discharges from neuronal cells.Its pathogenesis involves excitatory/inhibitory imbalance,ion cha...Objective:Epilepsy is a multi-etiological brain dysfunction syndrome characterized by synchronously repeated spontaneous discharges from neuronal cells.Its pathogenesis involves excitatory/inhibitory imbalance,ion channel abnormalities and may be related to the abnormal structure and function of neurotransmitter receptors.The research was to observe the effects of rapid increase inhbitory neurotransmitter GABA on the excitatory/inhibitory imbalance,ion channel abnormalities and abnormal neurotransmitter receptors in living epileptic mice from the level of free moving animal,cell,sub-cell and receptor.Methods:The techniques of fiber photometry and laser uncage of Rub-GABA for epileptic free moving mice induced by administration of Kainic acid(KA)or 4-AP,local field potentials(LFP),multicell bolus lording of calcium(OGB-1AM),virus transfection of calcium-sensitive protein GCaMP6f,transfection of fluorescent labeled AMPA receptors in cortical neurons by in utero electroporation,two-photon uncage of Rubi-GABA,two-photon imaging,two-photon calcium imaging,twophoton cell attach,two-photon targeted patch clamp and two-photon shadow patch clamp in living epileptic mice induced by 4-AP were used.Results:Laser photolysis of Rubi-GABA in hippocampal CA1 could immediately alleviate KA induced acute epileptic seizures in living mice.In cellular level,two-photon uncage Rub-GABA or rapid release of GABA significantly decreased the number of neurons releasing the calcium signals by multicell bolus lording of calcium,inhibited spikes recording by LFP and immediately inhibited both calcium signals and spikes using two-photon cell attaching technique in the living epileptic mice induced by 4-AP.Two-photon uncage Rub-GABA significantly decreased spikes induced by 4-AP recording with two-photon shadow patch clamp in vivo.In sub-cellular level,two-photon Rubi-GABA uncage of dendritic spines of cortical neurons transfected with GCaMP6f obviously decreased the frequency and amplitude of calcium signal on those dendritic spines in 4-AP induced epileptic living mice.Two-photon targeted patch clamp and shadow patch clamp were performed separately to both the cortical neurons transfected fluorescent labeled AMPA receptors in the mice by in utero electroporation and the cortical neurons with no fluorescent labeled AMPA receptors in control mice.The results showed that there was no difference in the electrophysiological indices between the two kinds of cortical neurons,meaning that there was no obvious change of electrophysiological characteristics in the cortical neurons transfected fluorescent labeled AMPA receptors.Further study showed that rapid disappearance of the AMPA receptors was found before disappearance of dendritic spines in epileptic living mice induced by high concentration of 4-AP,and the disappearance of the AMPA receptors was significantly delayed or the AMPA receptors even reappeared after rapid release of GABA with the different concentration of 4-AP in the living mice.Conlusion:Rapid release of GABA can rectify the excitatory/inhibitory imbalance and ion channel abnormalities of epilepsy,and rapidly inhibits epilepsy in free moving mice,and from cellular and sub-cellular level in vivo.It also protects the structure and function of neurotransmitter receptors during epilepsy in vivo.展开更多
文摘Objective:Epilepsy is a multi-etiological brain dysfunction syndrome characterized by synchronously repeated spontaneous discharges from neuronal cells.Its pathogenesis involves excitatory/inhibitory imbalance,ion channel abnormalities and may be related to the abnormal structure and function of neurotransmitter receptors.The research was to observe the effects of rapid increase inhbitory neurotransmitter GABA on the excitatory/inhibitory imbalance,ion channel abnormalities and abnormal neurotransmitter receptors in living epileptic mice from the level of free moving animal,cell,sub-cell and receptor.Methods:The techniques of fiber photometry and laser uncage of Rub-GABA for epileptic free moving mice induced by administration of Kainic acid(KA)or 4-AP,local field potentials(LFP),multicell bolus lording of calcium(OGB-1AM),virus transfection of calcium-sensitive protein GCaMP6f,transfection of fluorescent labeled AMPA receptors in cortical neurons by in utero electroporation,two-photon uncage of Rubi-GABA,two-photon imaging,two-photon calcium imaging,twophoton cell attach,two-photon targeted patch clamp and two-photon shadow patch clamp in living epileptic mice induced by 4-AP were used.Results:Laser photolysis of Rubi-GABA in hippocampal CA1 could immediately alleviate KA induced acute epileptic seizures in living mice.In cellular level,two-photon uncage Rub-GABA or rapid release of GABA significantly decreased the number of neurons releasing the calcium signals by multicell bolus lording of calcium,inhibited spikes recording by LFP and immediately inhibited both calcium signals and spikes using two-photon cell attaching technique in the living epileptic mice induced by 4-AP.Two-photon uncage Rub-GABA significantly decreased spikes induced by 4-AP recording with two-photon shadow patch clamp in vivo.In sub-cellular level,two-photon Rubi-GABA uncage of dendritic spines of cortical neurons transfected with GCaMP6f obviously decreased the frequency and amplitude of calcium signal on those dendritic spines in 4-AP induced epileptic living mice.Two-photon targeted patch clamp and shadow patch clamp were performed separately to both the cortical neurons transfected fluorescent labeled AMPA receptors in the mice by in utero electroporation and the cortical neurons with no fluorescent labeled AMPA receptors in control mice.The results showed that there was no difference in the electrophysiological indices between the two kinds of cortical neurons,meaning that there was no obvious change of electrophysiological characteristics in the cortical neurons transfected fluorescent labeled AMPA receptors.Further study showed that rapid disappearance of the AMPA receptors was found before disappearance of dendritic spines in epileptic living mice induced by high concentration of 4-AP,and the disappearance of the AMPA receptors was significantly delayed or the AMPA receptors even reappeared after rapid release of GABA with the different concentration of 4-AP in the living mice.Conlusion:Rapid release of GABA can rectify the excitatory/inhibitory imbalance and ion channel abnormalities of epilepsy,and rapidly inhibits epilepsy in free moving mice,and from cellular and sub-cellular level in vivo.It also protects the structure and function of neurotransmitter receptors during epilepsy in vivo.
