The Change Of Slient Synapse And AMPA Receptor Subunits In Vitro Model Of Epilepsy

Part I the establishment of epileptic discharge co-culture cellmodelObjective To obtain a simple, efficient and stable method co-culturedwith neurons and astrocytes, then to establish a stable cell model ofepilepsy.Method the pregnant16-17d of SD rats and borned within24h of SDneonatal rats, respectively, using a mixed culture method (mixed culturegroup)、improved Banker culture method (modified Banker co-culturegroup) and plug-dish method (plug-dish group) producte of hippocampalneurons and astrocytes in co-culture model, and then three groups ofcultured cells were applied magnesium-free extracellular fluid and normalextracellular fluid induced3h, making epilepsy model and normal controls,each method is repeated three times. Using immunofluorescence neuronNSE and astrocyte GFAP expression, identification of comparative culturesystem neurons and astrocytes purity; whole-cell patch clamp mode torecord different groups of neurons’ seizure discharge conditions andcompare the success rate of discharge. Results (1) groups of neurons insert dish purity [94.3%-100%] purityof astrocytes [93%-100%] compared to the co-culture group to giveimproved Banker neurons [87.6%-95%] and astrocytes [88.2%-93%].(2)about95percent plug-dish hippocampal neurons (n=10) can record theparoxysmal spike-like eruptions and Paroxysmal depolarizing shifts(PDSs);improved Banker of hippocampal neurons cultured group of about93%(n=10) can record the paroxysmal spike-like eruptions and PDSs; mixedculture group the proportion was about90%.(3) after treatment of externalliquid magnesium cells72h, plug-dish is still85%of the group of neurons(n=10) can record the paroxysmal spike-like eruptions and PDSs.improved Banker group has about82%of the (n=10) paroxysmalspike-like eruptions and PDSs; mixed culture group this proportion is about80%.Conclusion The three methods all can be successfully established cellsin vitro co-culture model of epilepsy, but the plug-in co-culture dish modelestablished method easier and has higher cell culture purity. Part II: the change of silent synapse and AMPA receptorsubunits in vitro model of epilrpsyObjective to study the change of silent synapse and AMPA receptorsubunits in vitro model of epilrpsy.Methods the plug-dish method to establish in vitro model of epilrpsyare divided into two groups, the experimental group withoutmagnesium,which magnesium-free extracellular fluid culture3h inducedspontaneous recurrent epileptic seizure-like discharge. Normal controlgroup using normal extracellular magnesium instead of magnesium-freeextracellular fluid culture3h, the experiment was repeated three times.application of Synapto GreenTM C4(FM1-43) and synaptophysin(Synaptophysin, SYN) to test whether the presynaptic silent synapses hassome changes; micro excitatory postsynaptic currents(mEPSCs) ofα-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(a-mino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, AMPA)recorded by patch clamp; NR1and GLuR1subunit doubleimmunofluorescent labeling reflect whether the postsynaptic silentsynapses has changed; SYN and GLuR1, GLuR2, GLuR3, GLuR4subunitdouble immunofluorescence to detect the expression of AMPA receptorsubunits.Results FM1-43and SYN double-labeled test showedmagnesium-treated group compared with the control group decreased withpresynaptic silent synapses (P <0.05); AMPA receptor’s mEPSCs frequencyand amplitude showed a significant increase than the normal group (P <0.05); postsynaptic silent synapses decrease compared with the controlgroup occurred (P <0.05); the number of fluorescence GLuR2, GLuR4subunit decreased in normal control group (P <0.05), GLuR1, GLuR3subunit increased compared with the control group (P <0.05).Conclusion No magnesium-induced seizures can lead to reduction inpresynaptic and postsynaptic silent synapses, reduced silent synapses arelikely to translated into functional synapses and becomed pathological basisof epilepsy;no magnesium-induced co-cultured neurons after epilepticdischarge can induce GLuR2、GLuR4subunit decrease and GLuR1、GLuR3subunits increase.