| Objective:In this study,the synaptic vesicle recycling was taken as the breakthrough point,and the "Yin and Yang" theory of traditional Chinese medicine was applied to the regulation of synaptic transmission,and the possible biological relationship between the "Yin and Yang balance" theory and the nervous system was discussed.Based on this theory,the specific action forms of quercetin on the regulation of synaptic function in central nervous system were clarified,and the regulatory mechanisms of quercetin on synaptic transmission dynamics and synaptic plasticity were further explored.This study provides a theoretical basis and mechanism for the clinical use of quercetin in the field of neurology.Methods:1.Study on the effect of quercetin on postsynaptic response in central nervous system: Acute brainstem slices containing MNTB brain regions in rat brain stem were prepared and divided into the control group and quercetin-treated groups with different concentrations.Subsequently,the whole-cell patch clamp technique was used to record mini-excitatory postsynaptic currents(m EPSCs)on postsynaptic neurons at the calyx of Held synapse,measuring basic electrophysiological parameters such as m EPSCs frequency,amplitude,rise time,decay time,and comparing and analyzing between groups.The purpose of this part of the study is to elucidate the possible postsynaptic mechanism of quercetin.2.Study on the presynaptic effect and mechanism of quercetin in central nervous system: A lock-in amplifier was incorporated directly into the resistance and capacitance compensation circuitry of a patch clamp set-up,to allow exocytosis and endocytosis to be monitored in the whole-cell mode by measuring changes in cell membrane capacitance.We used membrane capacitance measurement using patch clamp to stimulate nerve endings of the calyx-type synapses with different intensities and frequencies,and measured key electrophysiological parameters in the process of presynaptic calcium influx,vesicle release,vesicle retrieve,and the recovery and replenishment of the readily releasable pool(RRP).The purpose of this part of the study is to clarify the regulation and mechanism of quercetin in the process of presynaptic vesicle release.3.Study on the effect of quercetin on short-term plasticity of central mammalian synapses: A combination of nerve Fiber stimulation and whole-cell patch-clamp technique was used in this part of the study.We performed fiber stimulation at 5 Hz,50 Hz,100 Hz,and 200 Hz at the axons of calyx-type synapses,respectively.The inhibitory level(EPSC40/EPSC1ratio)of postsynaptic short-term depression(STD)and the EPSC successful rate were recorded in control group and the quercetin-treated group,to clarify the regulation effect of quercetin on short-term synaptic plasticity in central nervous system.Results:1.There were no significant differences in m EPSCs frequency,amplitude,or key parameters,rise time and decay time,between the control group and quercetin-treated groups with different concentrations(10 μM,100 μM,250 μM)(frequency: p = 0.99;amplitude: p = 0.84;rise time: p = 0.53;decay time: p = 0.24).Our results show that quercetin has no effect on postsynaptic spontaneous release,suggesting that quercetin has no significant regulatory effect on presynaptic vesicle release probability,quantum size and postsynaptic receptor characteristics,that is,it does not participate in the regulation of spontaneous release of vesicles.2.Under depol20 msstimulation,quercetin significantly slowed down the clathrin-dependent slow endocytosis compared to the control group(control group: 40 ± 2%,quercetin-treated group: 56 ± 4%,p = 0.01),while it did not affect the initial dynamic rate of vesicle retrieve(control group: 42 ± 4 f F/s,quercetin-treated group: 37 ± 5 f F/s,p= 0.56).Furthermore,quercetin did not affect the calcium currents(control group: 2.0 ± 0.2 n A,quercetin-treated group: 2.1 ± 0.1 n A,p = 0.60)and the presynaptic vesicle release(control group: 511 ± 27 f F,quercetin-treated group: 573 ± 37 f F,p = 0.24).Based on the above experimental results,we believe that quercetin is directly involved in the regulation of presynaptic vesicle release/retrieve.Although quercetin does not affect the calcium inflow and vesicle release stimulated by depol20 ms,quercetin significantly inhibits vesicle retrieve kinetics.Due to the limited number of vesicles directly involved in presynaptic transmission,inhibition of vesicle recycling will directly affect the recovery of the readily releasable pool before the recycling of vesicles to the synapse,helping to maintain the frequency of synaptic transmission in an appropriate range.3.Under depol20msx10 stimulation,quercetin significantly slowed down the clathrin-dependent overall vesicle retrieval rate compared to the control group(control group: 16 ± 3%,quercetin-treated group: 32 ±3%,p = 0.002).The initial the dynamic rate of vesicle retrieve in the quercetin-treated group did not significantly differ from that of the control group(control group: 238 ± 24 f F/s,quercetin-treated group:222 ± 13 f F/s,p = 0.61).Furthermore,quercetin did not affect the amount of calcium(control group: 315 ± 19 p C,quercetin-treated group: 325 ± 27 p C,p = 0.77)and the vesicle release(control group: 1461 ± 97 f F,quercetin-treated group: 1526 ± 101 f F,p = 0.67).Based on the above experimental results,we found that quercetin under either depol20 msor depol20msx10stimulation did not affect the amount of calcium inflow and the vesicle release presynaptic nerve terminals,but significantly inhibited vesicle retrieve,especially clathrin-dependent vesicle recycling kinetics.4.We tested the replenishment of RRP by administering 10 x 1 Hz depol20 mssquare wave stimuli in calyx-type synapses.Within the 10 s square wave stimulation,starting from the second 20 ms stimulation,RRP supplementation in quercetin-treated group slowed down compared with the control group,and there was a statistical difference between the 2,4,5,8,9,and 10 stimuli(the second stimulus: control group,79.3 ± 2.4%;quercetin-treated group,64.3 ± 5.0%;p = 0.047;Third stimulation:control group,63.6 ± 5.0%;quercetin-treated group,46.6 ± 6.1%;p =0.09;Fourth stimulation: control group,54.3 ± 2.8%;quercetin-treated group,39.8 ± 3.8%;p = 0.03;The fifth stimulation: control group,50.5± 4.2%;quercetin-treated group,36.6 ± 3.2%;p = 0.04;The sixth stimulus: control group,42.8 ± 5.8%;quercetin-treated group,37.6 ±6.6%;p = 0.61;The seventh stimulation: control group,51.1 ± 5.1%;quercetin-treated group,36.0 ± 5.6%;p = 0.11;The eighth stimulus:control group,44.1 ± 3.9%;quercetin-treated group,26.3 ± 4.6%;p =0.03;Ninth stimulus: control group,44.4 ± 3.2%;quercetin-treated group,26.2 ± 5.2%;p = 0.03;Tenth stimulation: control group,42.8 ±4.9%;quercetin-treated group,26.8 ± 4.1%;p = 0.048).5.In order to further verify the regulatory effect of quercetin on RRP,we plotted the RRP recovery kinetics curve of presynaptic nerve.After emptying the RRP with depol20 ms square wave stimulation(ΔCm1),the vesicle release was induced again with depol20 ms square wave stimulation at different intervals to calculate the ratio of ΔCm2/ ΔCm1and plot the RRP recovery curve.Our results showed that after different time intervals of stimulation,the number of vesicle recovery in quercetin group was smaller than that in control group,suggesting that quercetin can significantly slow down the recovery rate of RRP(0.2 s: control group,40.2 ± 1.1%,quercetin-treated group,31.8 ± 2.7%,p = 0.03;0.5 s:control group: 51.6 ± 1.5%,quercetin-treated group: 43.2 ± 1.9%,p =0.02;1 s: control group: 63.3 ± 2.6%,quercetin-treated group: 53.5 ±2.4%,p = 0.04;2 s: control group: 69.8 ± 2.0%,quercetin-treated group:57.9 ± 3.1%,p = 0.02;5 s: control group: 83.2 ± 2.2%,quercetin-treated group: 70.4 ± 3.8%,p = 0.04;10 s: control group: 89.5 ± 2.5%,quercetin-treated group: 85.7 ± 1.3%,p = 0.02).Quercetin mainly inhibits clathrin-dependent vesicle retrieve,which is a slower component of vesicle retrieve kinetics,but has no significant inhibitory effect on clathrin-independent vesicle retrieve.6.EPSCs were induced by giving 40 stimuli with frequencies of 5 Hz,50 Hz,100 Hz,and 200 Hz at the calyx of Held axon terminal.The degree of short-term depression(STD)was characterized by the ratio of the average EPSC amplitude from the 30 th and 40 th stimulation to that of the first stimulus(EPSC40/EPSC1).There was no significant difference in STD between the control group(15.3 ± 1.2%,n = 6)and the quercetin group(13.8 ± 1.5%,n = 6)when the axons were given 40 stimuli at 5 Hz.As the stimulation frequency increased,the degree of STD was enhanced in the quercetin group compared to the control group.At 50 Hz,100 Hz,and200 Hz,the ratios of EPSC40/EPSC1 were 1.4 ± 0.5%(p = 0.03),1.4 ± 0.5%(p = 0.02),and 1.0 ± 0.4%(p = 0.049)in the quercetin group,respectively,while in the control group,the ratios were 4.5 ± 1.1%,3.4 ± 0.5%,and 3.0 ± 0.6%,respectively.In addition,we measured the success rate of inducing EPSCs between the control group and the quercetin group.Under the condition of 5 Hz stimulation,the success rate of inducing EPSCs in quercetin group and control group was 100%.However,the success rate of inducing EPSCs in quercetin group decreased significantly compared with control group under higher frequency stimulation conditions.(50 Hz: control group,98.6 ± 1.0%,quercetin group,73.9 ± 14.7%,p = 0.15.100 Hz: control group: 90.4 ± 7.4%,quercetin group: 27.1 ± 6.5%,p < 0.001;200 Hz: control group: 55.0 ±3.1%,quercetin group: 23.1 ± 10.7%,p = 0.048).These results suggest that quercetin significantly enhances synaptic STD under strong stimulation and has a regulatory effect on short-term plasticity.Conclusion:1.Quercetin did not affect the amplitude and frequency of synaptic after m EPSCs in calyx-type synapses in the brainstem,indicating that it does not affect the characteristics of postsynaptic receptors and the frequency of presynaptic vesicle release.2.Quercetin reduced the rate of endocytosis in the calyx-type synaptic terminals,regardless of whether the stimulation was strong or weak.Because the amount of presynaptic vesicles available for release is limited,inhibition of vesicle recycling during continuous stimulation can affect the maintenance of synaptic transmission.3.Quercetin inhibits the replenishment and recovery of the readily releasable pool(RRP)of vesicles.4.Quercetin enhances short-term depression(STD)in calyx-type synapses under strong stimulation,indicating its regulatory effect on short-term plasticity of central nervous system synapses. |
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