Study On The Origin Of Ictogenesis Site,mechanisms And Pharmacological Intervention Of Epileptiform Discharges In Neonatal Mouse Hippocampus

Objective Neonatal seizures are catastrophic conditions occurring in neonates,and the related treatment is closely associated with ictogenesis mechanisms.However,the mechanisms underlying neonatal seizures vary from initiation sites.To explore the initiation sites of neonatal seizures,the initiations and propagation patterns of epileptiform discharges in neonatal mouse hippocampal slices were analyzed by mult-electrode recording techniques;the pharmacological experiment and Western blotting analysis were applied to investigate the mechanisms underlying ictogenesis in different sub-regions;the effect of traditional anti-epileptic drug,phenobarbital sodium（PB）,PB combined with Na⁺-K⁺-2Cl^-cotransporter isoform 1（NKCC1）blocker,bumetanide（BMT）,upon the epileptiform discharges in different sub-regions were explored to seek the efficient treatment for neonatal seizures.Methods Part 1:The multi-electrode array（MEA）was used to record epileptiform discharges induced by low-Mg²⁺artificial cerebrospinal fluid（ACSF）in hippocampal slices.The initiations,propagation patterns and parameters（frequency,amplitude,duration）of epileptiform discharges were analyzed.Part 2:（1）In low-Mg²⁺model,the effect of 10μM BMT,10μM gamma-aminobutyric Acid A receptor（GABA_AR）antagonist,bicuculline（BIC）,upon epileptiform discharges in the hippocampus proper and the subiculum were investigated.（2）In high-K⁺model,the antagonist of N-methyl-D-aspartatereceptor（NMDAR）,D-（-）-2-Amino-5-phosphonopentanoic acid（D-AP5）（50μM）,was applied to test its effect upon epileptiform discharges in the hippocampus proper and the subiculum.（3）The levels of NKCC1,NR2A/B expression in the hippocampus proper and the subiculum were measured by Western blotting.Part 3:In low-Mg²⁺model,the effect of 100μM PB,100μM PB combined with 10μM BMT,upon epileptiform discharges in the hippocampus proper and the subiculum were also tested.Results Part 1:（1）Interictal-like discharges（IIDs）and ictal-like discharges（IDs）were both induced in hippocampal slices after perfusion of low-Mg²⁺ACSF.（2）CA3-and Sub-origin IIDs/IDs were found in hippocampal slices,CA3-origin IIDs/IDs initiated in CA3a/b,propagated bi-directionally to CA3c,dentate gyrus（DG）（retrograde）,to CA1 and subiculum（anterograde）.Sub-origin IIDs/IDs did not propagate backward to CA1.（3）The latencies to onset of the first CA3-and Sub-origin IID/ID were not significantly different（P>0.05）.（4）The frequencies of CA3-and Sub-origin IIDs/IDs were not significantly different（P>0.05）,the amplitudes of CA3-origin IIDs/IDs were significantly lower than that of Sub-origin IIDs/IDs（**P<0.01）,and the duration of CA3-origin IDs was significantly longer than that of Sub-origin IDs（*P<0.05）.Part 2:（1）BMT（10μM）reversely abolished CA3-origin IDs,and depressed the CA3-origin IIDs and Sub-origin IDs（**P<0.01）,while the Sub-origin IIDs were not affected（P>0.05）.（2）BIC（10μM）reversely abolished the CA3-origin IDs,and depressed the Sub-origin IDs（**P<0.01）,while the CA3-and Sub-origin IIDs were unaffected（P>0.05）.（3）In high-K⁺model,D-AP5 irreversely abolished the Sub-origin IDs,but the parameters of Sub-origin IIDs and CA3-origin IDs/IIDs were not significantly affected（P>0.05）.（3）BMT,BIC or D-AP5 had no significant effect upon the propagation patterns of two original IIDs.（4）The level of NKCC1 expression was significantly higher in the hippocampus proper than that in the subiculum（**P<0.01）.The levels of NR2A/B expression were significantly lower in the hippocampus proper than that in the subiculum（**P<0.01）.Part 3:（1）PB blocked the Sub-origin IDs and depressed the Sub-origin IIDs（**P<0.01）,but it had no significant effect upon the parameters of CA3-origin IIDs/IDs（P>0.05）.（2）PB had no effect on the propagation patterns of the two original IIDs.（3）PB combined with BMT abolished the CA3-and Sub-origin ID/IIDs in hippocampal slices.Conclusions（1）As well as CA3,the subiculum was a potential site of ictogenesis in a neonatal seizure model,and possessed the similar seizure susceptibility to the hippocampus proper.（2）Gamma-aminobutyric acid（GABA）excitation resulting from NKCC1played a dominant role during ictogenesis in the hippocampus proper;glutamate excitation mediated by NMDARs might play a dominant role during ictogenesis in the subiculum.（3）PB was efficient in suppressing seizures initiated in the subiculum,but it was invalid in treating with seizures initiated from the hippocampus proper.（4）BMT enhanced PB efficiency in suppressing seizures initiated from the hippocampus proper and the subiculum.