Electrophysiological Studies Of The Antagonistic Effect Of Bexarotene On A?_25-35 Induced Toxic Effects In Hippocampal CA1 Pyramidal Neurons

Objective:The excessive accumulation of amyloid beta-protein(A?)in the brain is the pathogenesis of Alzheimer's disease.Bexarotene,a retinoid X receptor agonist,has recently been demonstrated to promote the removal of A? in the mouse model of Alzheimer's disease by improving the expression of apolipoprotein E(ApoE).The results of this research provides a new hope for the Alzheimer's disease drug research,but the synapses function and mechanism of bexarotene is unclear.The research purpose of this paper is to explore the possible antagonistic effect of bexarotene on A?25-35 induced toxic effect in synaptic transmission,potassium channels and neuronal excitability with electrophysiology techniques and to provide the theoretical basis for potential AD clinical treatment drug.Methods1.Groups of experimentExperiments were performed in hippocampal slices from postnatal 7-14-day-old Wistar rats.This study was included two parts,the first part is the effect of bexarotene to the hippocampus CA1 pyramidal neurons with A?25-35 application,the experiment was divided into three groups: control group,A?25-35 group,A?25-35+Bexarotene group.The second part is the effect of bexarotene to the normal hippocampus CA1 pyramidal neurons,and the experiment was divided into two groups: control group,Bexarotene group.2.Preparation of hippocampal slicesThe rats were decapitated under diethyl ether anesthesia,then the brain was rapidly removed and immersed in ice-cold(0-4°C)artificial cerebrospinal fluid(ACSF)with 95%O2+5%CO2 for 3 min.Then the hippocampal slices were prepared(350?m in thickness)with a vibratome tissue slicer,incubated with ice-cold ACSF with 95%O2+5%CO2.The hippocampal slices were incubated in ACSF solution constantly oxygenated with 95%O2+5%CO2 for one hour at 32°C in a storage chamber and then incubated at 28°C after one hour.3.Preparation of glass patch electrodesPatch electrodes were pulled from borosilicate glass capillaries with a horizontal puller to yield tip openings of 1-2?m.When filled with the internal solution,the resistance of the pipettes was 3-5M?.4.Date record and drug applicationThe whole-cell patch clamp technique was applied to record spontaneous excitatory postsynaptic currents(sEPSCs),miniature excitatory postsynaptic currents(mEPSCs),potassium currents,evoked action potential of CA1 pyramidal neurons in wistar rat hippocampal slices.Final concentration of A?25-35 or bexarotene were added into extracellular solution.5.Date analysis and statisticsThe Clampfit 10.4 software was used to analyze dates.Application of One-way ANOVA to calculate the difference between the dates of control group,A?25-35 group and A?25-35+Bexarotene group.Application of paired samples t-test to calculate the difference between the dates of control group and Bexarotene group.Result:1.The amplitude and frequency changes of sEPSCs and mEPSCs in hippocampus CA1 pyramidal neurons between groupsAfter A?25–35(1 ?mol/L)application,the average amplitude and frequency of sEPSCs and mEPSCs were significantly reduced compared with the control group(n=7,P< 0.05).Application bexarotene(1 ?mol/L)to A?25-35 group,the average amplitude and frequency of sEPSCs and mEPSCs were not significantly difference compared with the A?25–35 group(n = 7,P > 0.05).Application bexarotene(5 ?mol/L?10 ?mol/L)to A?25-35 group,the average amplitude and frequency of sEPSCs and mEPSCs were significantly increased compared with the A?25–35 group(n = 7,P < 0.05).Application bexarotene(1 ?mol/L?5 ?mol/L?10 ?mol/L)to hippocampal slices that without any processing before,record sEPSCs and mEPSCs.The results show that the average amplitude and frequency of sEPSCs and mEPSCs were not significantly difference between Bexarotene group and control group(n = 7,P > 0.05).2.The amplitude changes of potassium currents in hippocampus CA1 pyramidal neurons between groupsCampared with control group,the A?25-35 group average amplitude of potassium currents were reduced from 2713.83 ± 203.15 pA in control group to 1919.67 ± 162.40 pA(n = 6,P < 0.05),the current-voltage curve of potassium currents was decreased.Following application of bexarotene(5 ?mol/L)to A?25-35 group,the A?25-35+Bexarotene group amplitude of potassium currents were increased form 1919.67± 162.40 pA to 2436.51 ± 217.27 pA(n = 6,P < 0.05),the current-voltage curve of potassium currents was increased,and compared with the control group,the difference was not statistically significant(n = 6,P > 0.05).Application bexarotene(5 ?mol/L)to hippocampal slices that without any processing before,record potassium currents.The results show that the control group average amplitude of potassium currents were 2780.50 ± 187.85 pA;the Bexarotene group average amplitude of potassium currents were 2813.92 ± 175.74 p A;There is not significantly difference between two guroups(n = 6,P > 0.05).3.The frequency changes of evoked action potential in hippocampus CA1 pyramidal neurons between groupsCampared with control group,the A?25-35 group average frequency of evoked action potential were reduced from 12.85 ± 0.70 in control group to 10.56 ± 0.80(n = 6,P < 0.05).Following application of bexarotene(5 ?mol/L)to A?25-35 group,the A?25-35+Bexarotene group average frequency of evoked action potential were increased from 10.56 ± 0.80 to 12.42 ± 0.54(n = 6,P < 0.05),and compared with the control group,the difference was not statistically significant(n = 6,P > 0.05).Application bexarotene(5 ?mol/L)to hippocampal slices that without any processing before,record the evoked action potential of hippocampus CA1 pyramidal neurons.The results show that the Bexarotene group average frequency of evoked action potential were 12.69 ± 0.78,the con group average frequency of evoked action potential were 12.75 ± 0.82,there were not significantly difference(n = 6,P > 0.05).Conclusions:1.A?25-35 can significantly reduce the amplitude and frequency of sEPSCs and mEPSCs in hippocampus CA1 pyramidal neurons,results in the decrease of excitatory postsynaptic potential and synaptic function damage.While bexarotene can increase the amplitude and frequency of sEPSCs and mEPSCs in hippocampus CA1 pyramidal neurons to normal levels.This suggests that bexarotene has a presynaptic and postsynaptic site of action to antagonize A?25-35-induced synaptic dysfunction in hippocampal CA1 pyramidal neurons.2.A?25-35 can reduce the amplitude of potassium currents,this suggest that A?25-35 has a toxic effect on potassium channel of hippocampus CA1 pyramidal neurons.While bexarotene has an antagonistic effect to A?25-35-induced dysfunction in potassium channel.3.A?25-35 can reduce the excitability of neurons through reduced the frequency of evoked action potential in hippocampus CA1 pyramidal neurons.While bexarotene can improve the excitability of hippocampus neurons to normal levels through the antagonistic effect to A?25-35-induced dysfunction in potassium channel.4.Bexarotene have no significant influence to sEPSCs,mEPSCs,potassium currents and evoked action potential in normal hippocampus CA1 pyramidal neurons.