The Establishment Of4-AP Epileptic Models For Neuronal Network Ex Vivo And Investigation For Characteristics Of Energy Metabolism

Part I The establishment of4-AP epileptic models for rats fetal hippocampal neurons ex vivoObjective To investigate the properties of primary cultured hippocampal neurons from embryo rats ex vivo and observe the features of Seizure-like-events (SLEs) induced by Mg2+free media or4-AP (50μM) in the cultures, and thereby establishing a new cultured hippocampal neuron SLEs model for further study on epileptogenesis.Methods After isolated from the18-19embryonic rats, the hippocampal neurons were digested and plated on poly-L-lysine-coated covers lips and cultured. The morphological characteristics were observed through phase-contrast microscope at different time point after seeding. The cultured neurons were treated with zero-Mg2+medium or4-AP and the epileptiform activities were immediately detected with whole cell patch clamp recording techniques for30minutes.Results The hippocampal neurons almost adhere to the plates24h after planting, and were found to form synapses since the second day and make thriving connection with each other. The neurons were well-developed8days after culturing, and then be treated with zero-Mg2+medium or4-AP for whole cell patch clamp recording. Immediately after being processed, the neurons developed continuous, spontaneous SLEs. The average amplitude of both SLEs increased in the first10minutes and decreased10minutes later.Conclusions Continuos SLEs could be immediately induced in cultured hippocampal neurons of18-19embryonic rats ex vivo via treatment of zero Mg2+medium or4-AP, which could sustain at least30minutes. The average amplitude and frequency of SLEs induced by4-AP were higher than the zero Mg2+model, exhibiting a similar SLEs model which could be used for the investigation of the molecular mechanisms underlying the induction, propagation and termination of epileptic activities ex vivo. Part Ⅱ The investigation of high energy burst of SLEs ex vivo and its relationship with chemical transmissionObjective To investigate the changes of mitochondrial membrane potentials (Γψm), intracellular ATP, ADP and the extracellular glutamate and GABA of the SLEs network of cultured hippocampal neurons. Thereby illuminating the energy burst of ex vivo SLEs and its relationship with chemical transmission.Methods After being treated with zero-Mg2+medium or4-AP (50μM) for Omin,5min,10min,15min,20min,25min,30min, the mitochondrial membrane potentials of cultured hippocampal neurons were measured by JC-1kits with fluorescence images made on a fluorescence spectrophotometer. Measurements of intracellular ATP, ADP levels with corresponding kits were performed on a Spectrofluorophotometer, while the measurements of extracellular glutamate and GABA levels were performed via ELISA.Results These two SLEs models could both be used for the investigation of epileptic activities ex vivo. During the progress of SLEs, the mitochondrial membrane potentials and glutamate concentration increased at first and turned to decrease, while ATP and GABA decreased during early period and gradually increased afterward. The changes suggested that the epileptic activities are bursts of energy. However, the chemical transmission started slowly compared with SLEs.Conclusions SLEs are energy burst of neuronal high potential, companied with high biological metabolism. The chemical transmission may not be the main power for the epleptic activity during the early period.