Effects Of Etomidate On Sensory Information Transmission In The Molecular Layer Of Mouse Cerebellar Cortex

Background:Etomidate is widely used as an intravenous anesthetic with short duration.The inhibitory effect of etomidate on the activity of Purkinje cells(PC)in vivo is regulated by GABA_A(γ-aminobutyric acid A)and glycine receptor.However,the mechanism of etomidate on sensory information processing in the molecular layer of cerebellar cortex in vivo is still unclear.Therefore,it is necessary to further study the effect of etomidate on the response of sensory stimulation in the cerebellar molecular layer of mice under urethane anesthesia,Objective:To explore the effect of etomidate on the sensory information transmission function in the cerebellar molecular layer of mice,so as to provide experimental basis for elucidating its mechanism.Methods:Thirty male or female adult(6-8 weeks old)HA/ICR mice were anesthetized with urethane(1.3g/kg body weight,i.p.)and underwent craniotomy through a circular hole with a diameter of 1-1.5 mm.The peristaltic pump was used to inject oxygen-containing artificial cerebrospinal fluid(ACSF)into the cerebellum at a rate of 0.4ml/min.Rectal temperature was monitored with a thermometer and maintained at 37±0.2℃.The local field potential of the molecular layer was recorded by the axopatch200B amplifier.The electrophysiological data were obtained through digidata1440 series analog-to-digital interface on the computer by using clappex10.3software.A thick wall borosilicate glass puller was used to make a patch pipette.Through the pipette,the recording electrode was filled with ACSF,and the resistance of the electrode was3–5mΩ.Etomidate and gabazine were purchased from sigma Shanghai company.The drugs were dissolved in ACSF and perfused in the exposed area by peristaltic pump.The ipsilateral palp pad was stimulated by blowing air(30 ms,60 psi)connected to a special blowing device.The stimulation was controlled by a computer and synchronized with the electrophysiological recording,and transmitted at 0.05Hz by Master8 controller and clappex10.3 software.All the chemicals were dissolved into solutions and frozen as aliquots;finally,they were infused into the cerebellar surface at the rate of 0.4 ml/min in ACSF.The electrophysiological data were analyzed by clapfit 10.3 software.Result:(1)10 min after etomidate(50μM/L)was perfused to the cerebellar surface,the response latency did not change significantly(P>0.05;n=10;not shown),but the P1amplitude decreased significantly(P<0.001;n=7)(Fig.2A,B and C).The decrease of P1 amplitude induced by etomidate was concentration dependent.The lowest effective dose was 10μm,which was 2.19%lower than baseline(P<0.05;n=5),and IC50 was64.57μM.When etomidate concentration was 500μm,P1 amplitude was 98.68%lower than baseline(P<0.0001;n=5).(2)When etomidate(50μM/L)was infused,the normalized value of P1 half width was lower than that of baseline(ACSF:100.26%)(P=0.032;n=7)and121.4±7.8%(Fig.3A).The normalized area under the P1 curve(AUC)was significantly lower than baseline(P<0.001;n=7)(ACSF:100.1 8%)was 47.6±8.4%of the baseline.In addition,the rise time of etomidate increased significantly(P<0.05).(3)Perfusion of GABA_A receptor blocker(SR95531)20μM/L on the cerebellar surface blocked the GABA ergic component P1 induced by sensory stimulation,and showed the excitatory component N2 induced by sensory stimulation,which represented the excitatory component of PCs induced by sensory stimulation.In the presence of gabazine,etomidate had no significant effect on the amplitude of N2(gabazine:52.5±4.5%,gabazine+etomidate:54.12±5.4%;n=7).There was no significant difference in the AUC of N2 between the two groups(gabazine:50.82±6.8%,gabazine+etomidate:52.79±7.5%;n=7;P>0.05).In the presence of strychnine(10μm),a glycine receptor antagonist,sensory stimulation had no significant effect on P1 amplitude.The standardized amplitude of P1 was 98.20±4.2%of baseline(100.06±3.5%;P>0.05;n=7).In the presence of strychnine(10μm),etomidate(50μm)reduced P1 amplitude to62.19±5.8%of baseline(100.06±3.5%;P<0.001;n=7),similar to etomidate alone(50μm;60.56±6.4%of baseline;P>0.05;n=7).(4)The peak potential of cerebellar MLI(molecular layer interneuron)could be evoked by the stimulation of ipsilateral whisker pad.Etomidate(50μm)for 10 min significantly inhibited spontaneous and sensory evoked spikes,and the standardized number of spontaneous spikes decreased by 1.64±1.6%(100.0±1.35%;P<0.0001;n=6)compared with baseline.The standardized number of sensory evoked potentials decreased by 21.0±6.4%(102.0±10.07%;P<0.001;n=6)compared with baseline.Conclusion:Etomidate modulates the evoked field potential response of cerebellar cortex molecular layer by sensory stimulation through GABA_Areceptor.Glycine receptor blockers could not block the inhibitory response induced by etomidate.Etomidate perfusion on the cerebellar surface can inhibit spontaneous and sensory evoked spike activities in cerebellar MLI,resulting in the weakening of inhibitory effect of molecular layer on sensory stimulation.