Effective Mechanisms Of Etomidate On Cerebellar Purkinje Cell Spontaneous Activity And Sensory Information Transmission In Mouse Granule Cell Layer

[Purpose]Etomidate,an ultrashort-acting intravenous anesthetic,is used for the induction and maintenance of general anesthesia.Etomidate is believed to produce sedation by binding on the y-aminobutyric acid type A(GABAA)receptors.Electrophysiological studies showed that etomidate enhanced the amplitude of Cl-currents,increased GABAergic inhibitory transmission and potentiated the glycine-induced current in the rat spinal dorsal horn neurons,indicating that etomidate potentiated both GABAA and glycine receptors activity.However,the effects of etomidate on the spontaneous simple spike(SS)activity of cerebellar Purkinje cells(PCs)and sensory information processing in cerebellar GCL in living mouse have not been fully understood.In this study,we investigated the effects of etomidate on the spontaneous simple spike activity of purkinje cells and tactual stimulation-evoked responses in the cerebellar GCL by electrophysiological and pharmacological methods.We aim to know the mechanisms of etomidate inhibit the spontaneous simple spike firing rate of cerebellar PCs and tactual stimulation-evoked responses in the cerebellar GCL in vivo in mice.[Methods]The ICR mice(6-8-week-old)were anesthetized with urethane.A 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Vermis Ⅵ-Ⅶor Crus II.Cell-attached recordings from PCs and local field potential recordings from GC layer were obtained with an Axopatch-200B amplifier under current-clamp mode(I=0).The signals of electrophysiology were acquired through a Digidata 1440 series analog-to-digital interface on a computer using Clampex 10.3 software.All chemicals were dissolved to form solutions and kept frozen;they were applied to the cerebellar surface at 0.4 mL/min in ACSF.In the experiments involving KT5720,the application of KT5720 was started at least 30 min before recording and continuing throughout the experiments.All data are expressed as the mean ± SEM.Differences between the mean values recorded under control and test conditions were evaluated with the Student paired t test or one-way ANOVA using SPSS 21.0 software.P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results]Part I:(1)Cerebellar purkinje cells express regular spontaneous simple spike firing accompanied with irregular CS firing under current-clamp mode.Cerebellar surface application of etomidate reduced the PC simple spike(SS)firing rate in a concentration-dependent manner.The IC50 was 43.4 μM.(2)Application of either a y-aminobutyric acid type A(GABAA)receptor antagonist,SR95531 or a glycine receptor antagonist strychnine failed to significantly affect the simple spike firing rate of PCs,but significantly attenuated the etomidate-induced decrease in PC SS firing rate.(3)Co-application of SR95531 and strychnine abolished the etomidate-induced decrease in PC SS firing rate.(4)Intraperitoneal injection of etomidate(3 mg/kg body weight)also induced a significant depression in PC SS firing rate,which was blocked by the co-application of GABAA receptor antagonist and glycine receptor antagonist on the cerebellar surface.Part Ⅱ:(1)Cerebellar surface perfusion of etomidate significantly depressed the air-puff stimulation on ipsilateral whisker pad evoked field potential responses in GCL,which exhibited depresses in the amplitude and area under the curve(AUC)of stimulus onset responses(N1).(2)Application of GABAA receptor antagonist,SR95531 significantly attenuated,but not completely prevented the etomidate-induced decrease in amplitude and AUC of N1.(3)Application of CB1 receptor antagonist,AM-251 completely prevented the etomidate-induced decrease in amplitude and AUC of N1 in the absence of GABAA receptors activity.(4)Application of CB1 receptor agonist,WIN55212-2 induced a decrease in amplitude and AUC of N1,as well occludes the etomidate-induced depression of N1 in the absence of GABAA receptors activity.(5)In addition,application of a specific PKA inhibitor,KT5720 completely abolished the effects of etomidate on amplitude and AUC of N1 in the absence of GABAA receptors activity.[Conclusions](1)Etomidate inhibited PC SS firing via facilitation of GABAA and glycine receptors activity in cerebellar PCs in vivo in mice,significantly decreased firing rate of PCs.(2)Etomidate facilitates CB1 receptors in the absence of GABAA receptors activity,resulting in a depression of the sensory stimulation-evoked MF-GC synaptic transmission via PKA signaling pathway in mouse cerebellar granule cell layer.