Variations Of Ionotropic Glutamate Receptors Mediating Epileptiform Activity In Rat Hippocampal CA1 Region

ObjectiveThe epileptiform activity was induced by perfusing GABA_A receptor antagonist bicuculline (Bic, 30μmol/L) in normal hippocampal slices and temporal lobe epilepsy (TLE) model was established by kainic acid (KA) injected into the center site of right hippocampus CA3 region with the stereotaxic technology. To investigate the variations of ionotropic glutamate receptors mediating epileptiform activity in rat hippocampal CA1 region by using extracellular recording in hippocampal slices, and to explore the mechanism of epileptogenesis.Methods1 Model establishment.1.1 TLE model. Adult male Wistar rats (200²40 g and clean stage) were used in the experiments. Under chloral hydrate (350 mg/kg) anesthesia, rats were placed on the stereotaxic apparatus and 2.5³Î¼l KA (0.4μg/μl) was slowly injected (about 15 min) to the CA3 region of right hippocampus (4.0 mm posterior to bregma, 4.4 mm lateral to the midline, 3.8 mm below dura). The microsyringe was removed 10 min later and the rat scalp was sutured. The behavioral progression of KA-induced seizures was scored according to Racine's standard classification. Only those rats that could reach at least the classâ…£seizures were used in subsequent studies. The equivalent volume of normal saline to the same site was injected in the control rats.1.2 Acute epileptiform activity. The normal hippocampal slices were perfused with GABA_A receptor antagonist Bic (30μmol/L) to induce epileptiform activity. 2 Slice preparation. Rats were anesthetized with 2% pentobarbital sodium and decapitated. Their brains were then rapidly removed and placed in oxygenated (95% O2/5% CO₂) 4°C artificial cerebrospinal fluid (ACSF). Transverse hippocampal slices (400μm thick) were cut and transferred to a storage chamber for 1² h. The ACSF was gassed continuously with 95% O2/5% CO₂ at 30±2°C.3 Field-potential recording. After a recovery period of 1² h, an individual slice was transferred to the recording chamber, in which it was continuously superfused with oxygenated ACSF at a rate of 2 ml/min at 32°C. Population spike (PS) was recorded in CA1 pyramidal cell layer by stimulating the Shaffer collaterals in stratum radiatum.4 Statistical Analysis. All statistical analysis was performed using SPSS 13.0 software. Values are expressed as mean±SEM. Comparisons between means were performed using Student's t-test. The level of significance was set at P < 0.05.Results1 Variations of epileptiform activity PS in hippocampal CA1 region. Single PS was usually recorded in normal pyramidal cell layer of hippocampal CA1 region. Perfusing normal hippocampal slices with Bic (30μmol/L) could induce multiple epileptiform discharges, in which the PS number was (5.07±0.30, n = 11), the amplitude of the first PS was increased significantly by (150.86±22.56)% (n = 11, P< 0.01) compared with the control. Epileptiform discharges could recorded in slices of TLE model rats, the PS number was (6.12±0.33, n = 9). The PS number was increased significantly in slices of epileptiform activity compared with the control (P< 0.01).2 Variations of ionotropic glutamate receptors mediating PS in hippocampal CA1 region. The PS recorded in normal pyramidal cell layer of hippocampal CA1 mainly mediated by non-N-methyl-D-aspartate (non-NMDA) receptors. The PS recorded in normal slices after perfusing with Bic was partially mediated by NMDA receptor in addition to non-NMDA receptors. After perfusing with NMDA receptor antagonist, DL-2-amino-5-phosphonovaleric acid (APV, 50μmol/L), the amplitude of the first PS was not significantly affected (n = 11, P>0.05), however, the amplitude of the fourth and fifth PS was decreased obviously (n = 11 , P<0.05; n = 9, P<0.05). After perfusing APV in slices of TLE model rats, the amplitude of the first PS was not significantly affected (n = 9, P>0.05), but the amplitude of the fourth and fifth PS was decreased obviously (n = 8, P<0.05; n = 8, P<0.01), moreover, APV could inhibit the residual potential after perfusing non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 10μmol/L).Conclusion1 Both slices of normal rats perfused with Bic and slices of TLE model rats could induce epileptiform activity, and increased the PS number in CA1 region.2 In addition to non-NMDA receptors, the activation of NMDA receptor contributes to epileptiform activity in hippocampal CA1 region.