| Changes in hippocampal connexin32, connexin36and connexin43levels in the model of epilepsy induced by4-aminopyridine in vivoObjective:To observe the changes of hippocampal Cx32, Cx36and Cx43mRNA and protein levels after seizures induced by4-aminopyridine(4-AP) and to investigate the relationship between gap junction and seizure.Method:55adult S-D male rats (weighing200-230g) were divided randomly into control group and experimental group. Seizure was induced by intraperitoneal injection of low-dose4-AP in experimental group, while the controls were injected with equal dose of saline. Experimental group were divided into five subset groups:electrode,2h,8h,24h and48h group. Behavior was observed and the latent period of generalized tonic-clonic seizure(GTCS) were recorded in each experimental group.Electroencephalogram(EEG) was used to record seizure-induced EEG signals of each rat in electrode group. The expressions of Cx32, Cx36and Cx43protein in normal and epileptogenic hippocampus were detected by immunohistochemical staining and western blot, while the expressions of Cx32, Cx36and Cx43mRNA were examined by RT-PCR.Results:1. Behavior and EEG results:In experimental group, the successful rate of the model was88.9%, the average latent period of GTCS was32.2±4.8min.There were multiform epileptic waves in EEG after4-AP injection, including sharp wave, spike wave and sharp-slow wave.2. Immunohistochemical staining showed that Cx32, Cx36and Cx43are wildly expressed in the brain.In experimental group, Upregulation in Cx32, Cx36and Cx43immunoreactive cell has been detected in the hippocampal CA3regions2h following intraperitoneal injection of4-AP(P<0.05).The number of Cx32immunoreactive cell reached the peak at8h (P<0.05), and declined gradually over the following few hours, until48h their number is still higher than control group (P<0.05);The number of Cx36and Cx43immunoreactive cell reached the peak at24h after intraperitoneal injection of4-AP (P<0.05).3.Western blot analysis showed that the expression of hippocampal Cx32, Cx36and Cx43protein in experimental group increased at2h following intraperitoneal injection of4-AP (P<0.05).The expression of Cx32protein reached the peak at8h (P<0.05), and declined gradually over the following hours, until48h its expression level is still higher than control group (P<0.05);The expression of Cx36and Cx43protein reached the peak at24h after intraperitoneal injection of4-AP (P<0.05).4. Results of RT-PCR showed that the expression of hippocampal Cx32, Cx36and Cx43mRNA in experimental group increased at2h following intraperitoneal injection of4-AP (P<0.05).The expression of Cx32mRNA reached the peak at8h after intraperitoneal injection of4-AP (P<0.05), and declined gradually over the following hours, until48h its expression level is still higher than control group (P<0.05);The expression of Cx36and Cx43mRNA reached the peak at8h and maintained at a high level until the end of this study.Conclusion:Hippocampal Cx32, Cx36and Cx43mRNA and protein levels were upregulated after seizures induced by4-AP, indicating that they may involved in seizure. The anticonvulsant effect of gap junction blockers in hippocampal slice in vitro and its mechanismObjective:To investigate the effect of gap junction blocker carbenoxolone (CBX) and Cx36blocker quinine on neuronic epileptiform discharges in hippocampal CA3region and the mechanism.Method:Brain tissues of S-D male rats (weighing50-100g) were divided and hippocampal slices(300μm) were prepared.The blind patch clamp whole-cell technique was used to record the effect of CBX and quinine on neuronic local field potential activity in hippocampal CA3region induced by bicuculline (BMI) and high-K+. Long-term potentiation (LTP) was induced by high frequency stimulus in hippocampal CA3region, which may mimic epileptiform discharges.Using voltage clamp technique to record the effect of quinine on glumatic acid-mediated LTP(LTPGlu).Results:1. CBX inhibits neuronic epileptiform discharges induced by BMI and high-K+in hippocampal CA3region:The number of interictal burstsper min induced by BMI in hippocampal CA3region was significantly reduced after bath application of CBX (from12.5±3.3to5.7±1.4/min,n=8)(P<0.05), after washout CBX and return to normal ACSF with BMI, the number of interictal bursts per min (8.3±2.7/min) partly return to the original level; The number of interictal bursts per min induced by high-K+was significantly reduced after bath application of CBX (from18.3±3.6to7.9±2.7/min,n=8)(P<0.05), after washout CBX and return to normal ACSF with high-K+, the number of interictal bursts per min (15.8±3.3/min) return to the original level.2. Quinine inhibits neuronic epileptiform discharges induced by BMI and high-K+in hippocampal CA3region:The number of interictal bursts per min induced by BMI in hippocampal CA3region was significantly reduced after bath application of quinine (from7.2±1.6to2.35±1.2/min,n=8)(P<0.05), after washout quinine and return to normal ACSF with BMI, the number of interictal bursts per min (5.5±1.8/min) partly return to the original level; The number of interictal bursts per min induced by high-K+was significantly reduced after bath application of quinine (from27.4±4.1to13.8±2.6/min,n=8)(P<0.05), after washout quinine and return to normal ACSF with high-K+,the number of interictal bursts per min (25.8±3.7/min) return to the original level.3. Repeat high frequency stimulus induced LTPGlu of neuron in normal hippocampal CA3region. When brain slices were perfusied with quinine, repeat high frequency stimulus can’t induce neuronic LTPGlu in hippocampal CA3region.Conclusions:1. Both CBX and quinine suppress the interictal epileptiform discharges induced by BMI and high-K+in hippocampal CA3region, the inhibition effects of CBX and quinine on epileptiform discharges induced by BMI might be long-lasting.2. Quinine may exert its anticonvulsant effects partially through decreasing glutamate release induced by Cx36gap junction inhibition. |
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