| ObjectiveWe investigate whether Isoliquiritigenin(ISL)can improve the anxiety-like behavior of young adults in Presenilin 1/2 conditional double knockout(PS cDKO)mice with Alzheimer disease(AD).At the same time,we further study whether ISL can restore the balance between excitatory synaptic transmission and inhibitory synaptic transmission(E/I)in hippocampal CA1 pyramidal neurons of PS cDKO mice.We investigated how ISL regulates potassium current mediated by Kv1.1 potassium channel in the parvalbumin positive(PV+)interneurons of hippocampal CA1 region of PS cDKO mice,and ultimately improves the anxiety-like behavior of PS cDKO mice in young adultsMethordBased on the observation of abnormal mental behavior in PS cDKO mice,we used whole-cell patch clamp technique,behavioral experimental technique and immunofluorescence technique to determinate the anxiety-like behavior of PS cDKO mice.(1)Reproduction and genotyping of PS cDKO mice(2)2.5-month-old PS cDKO mice and their littermate wild-type mice were used in this experiment.These mice consisted of half male and half female,and their weigh were about 20 g.They were randomly divided into four groups:wildtype group(WT,n=16),wildtype+ISL group(WT+ISL,n=16),PS cDKO group(cDKO,n=16)and PS cDKO+ISL group(cDKO+ISL,n=16).WT+ISL group and cDKO+ISL group were intraperitoneally injected with ISL(30 mg/kg,diluted with 0.2%DMSO)once daily for 30 days,and WT group and cDKO group were injected with the same dose of 0.2%DMSO dilution.At 3.5 months old,behavioral experiments were performed for a total of 7 days.Medication was given continuously during this period.The change of anxiety-like behavior between PS cDKO mice and WT mice and the effect of ISL treatment were observed by open field test,bright black box experiment,sucrose preference test.(3)Whole-cell patch-clamp technique was used to observe the E/I balance of synapses in the hippocampal CA1 pyramidal cells of WT and PS cDKO mice,including spontaneous excitatory postsynaptic current(sEPSC)and spontaneous inhibitory postsynaptic current(sIPSC)and the effect of ISL on mini inhibitory postsynaptic currents(mIPSC).(4)PS cDKO mice and WT mice were mated with GAD67-GFP mice that specifically labeled interneuron green fluorescence,respectively,and cDKO-GAD mice were bred.The 2.5 month-old mice,including half male and half female,and were randomly divided into four groups,10 mice in each group:including WT:GAD group,WT:GAD+ISL group,cDKO:GAD group,cDKO:GAD+ISL group.WT:GAD+ISL group and cDKO:GAD+ISL group mice were injected intraperitoneally with ISL at a dose of 30 mg/kg,diluted with 0.2%DMSO.WT:GAD group and cDKO:GAD group were given intraperitoneal injection of the same dose of 0.2%DMSO dilution once a day for 30 days,and then electrophysiological experiments were performed after treatment.(5)Whole-cell patch clamp technique was used to detect the action potential release,membrane resistance,threshold potential,resting potential,time constant,potassium channel and other interneuron excitability indexes of interneurons in the hippocampal CA1 region of PS cDKO miceResults(1)Behavioral test results showed that there were no difference in the percentage of time among the mice in each group in the open field.However,the moving distance and moving speed of PS cDKO mice were significantly higher than those in WT mice,indicating PS cDKO mice may have anxiety-like behavior and improved significantly after ISL treatment(P<0.05).In the bright black box experiment,the activity time and moving distance of PS cDKO mice in the bright black box and the number of shuttles between the bright black boxes were significantly lower than those of WT mice,and the improvement was achieved by ISL treatment(P<0.05).In the sucrose preference test,there were no significant difference in the amount of sucrose water consumed by each group of mice,suggesting that young adults PS cDKO mice did not have depression-like behavior(P>0.05).(2)The whole cell patch clamp technique was used to detect the E/I balance of hippocampal CA1 pyramidal cells in each group.The frequency of sIPSC in PS cDKO mice were significantly lower than that in WT mice(P<0.01),and there were no significant differences in amplitude.The sEPSC results showed that there were no significant differences in the frequency or amplitude of the mice in each group.In the comparison of the charge of sEPSC and sIPSC transfer,the inhibitory charge transfer in the hippocampal CA1 region of PS cDKO mice were lower than that in WT mice.There were no significant differences in excitatory charge transfer.Therefore,the E/I balance ratio in hippocampal CA1 region of PS cDKO mice were significantly higher than that in WT mice.However,ISL treatment improved the transfer balance of charge in PS cDKO mice,thereby reducing the increase in E/I balance in PS cDKO mice.The results of mIPSC recording of pyramidal cells in hippocampal CA1 area showed that the frequency of mIPSC in PS cDKO mice were significantly lower than that in WT mice,there were no significant differences in amplitude,but the mIPSC frequency of PS cDKO mice could not be changed after ISL treatment.(3)After mating PS cDKO mice with GAD67-GFP mice,the action potentials of interneurons in the hippocampal CA1 region were recorded by whole-cell patch clamp technique.The results showed that the non fast spiking interneuron action potential threshold,resting membrane potential,time constant,input resistance,action potential amplitude,and action potential duration were not significantly differences among four groups of mice;while the results of recording fast-spiking interneurons in hippocampal CA1 of cDKO:GAD mice showed that the action potential thresholds were significantly higher than that of WT:GAD mice.The action potential threshold of cDKO:GAD mice were decreased after ISL treatment(P<0.001),and there were no significant differences among other indicators.(4)The results of whole-cell patch clamp technique showed that the potassium ion current of PV+interneurons in hippocampal CA1 region of cDKO:GAD mice were significantly higher than that of WT:GAD mice,and ISL could attenuate higher potassium currents in cDKO:GAD mice(P<0.001).Further,the broad-spectrum potassium channel blocker 4-AP blocked the attenuate effects of ISL.However,the specific Kv1.1 potassium channel blocker DTx-K was used to verify Kv1.1 current change.The Kv1.1 currents in the hippocampal CA1 region of cDKO:GAD mice were significantly higher than that in other groups,and ISL could decrease the Kv1.1 current of cDKO:GAD mice(P<0.01).Conclusion(1)Young adult PS cDKO mice have anxiety-like behavior but no depression-like behavior,due to the inhibitory functional damage to neuronal synapses which leads to an increase in E/I balance.ISL has an effect on improving anxiety-like behavior and restoring synaptic E/I balance in PS cDKO mice.(2)ISL can improve the excitability by decreasing the threshold of action potential of PV+interneurons in hippocampal CA1 region of PS cDKO mice,thus improving the anxiety-like behavior of PS cDKO mice.(3)ISL can reduce the threshold of action potential in PV+interneurons by inhibiting the Kv1.1 channel-mediated potassium current in the hippocampal CA1 region of PS cDKO mice,and finally improve the anxiety-like behavior of PS cDKO mice. |
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