Mechanism Of Serotonin Modulates Simple Spike Activity Of Cerebellar Purkinje Cells In Mice

[Objective]Serotonin（5-HT）nerve fibers from the dorsal raphe nucleus can project to the wide areas of the cerebellar cortex,regulating the functional activity of the cerebellar cortical neural circuit through the 5-HT receptor,and modulating the command output of Purkinje cells（PC）of the deep cerebellar nucleus.However,the regulatory mechanisms of the 5-HT on the spontaneous discharge activity of PC in mouse cerebellar cortex are not clear.In this study,we used the electrophysiological recording technology in vivo to study the effect of the 5-HT on PC spontaneous discharge activity of the vermis area of mouse cerebellar cortex in urethane anesthetized mice and to explore the pharmacological mechanisms of the effect of the5-HT on PC spontaneous simple peak potential（SS）discharge activity.[Methods]Adult Kunming mice were aged 6-8 weeks and anesthetized by intraperitoneal injection of urethane（1.3 g/kg）.After endotracheal intubation,the mice were fixed on the brain stereotactic platform.Cerebellar Vermis areas were exposed by the surgical approach.The temperature was maintained at 37±0.2℃,using the temperature maintenance instrument.The surface of the cerebellum was continuously perfused with oxygenated artificial cerebrospinal fluid（ACSF）through the peristaltic pump.PC and molecular layer interneurons（MLI）in the vermis area of the mouse cerebellar cortex were recorded by cell-attached recording technique.The internal solution was used to fill recorded electrodes with resistances of 3-5 M（?）.The types of recorded cells were identified based on the depth and discharge characteristics.The PC and MLI were characterized by the regular simple spike（SS）and irregular complex spikes（CS）on the depth of 150-250μm,and the single and irregular discharge activity,respectively.All recording data were analyzed by the clampfit 10.3 software.The data were represented by Mean±SEM.SPSS 17.0 software for statistical analysis.The mean discharge frequency before and after administration was compared by one-way ANOVA.P<0.05,and was considered to be statistically different.[Results]1.Under cell-attached recording mode,the average spontaneous SS discharge frequency of PC was 31.80±2.9 Hz,and the average discharge frequency of CS was0.75±0.2 Hz in the Vermis area of the cerebellar cortex of mice.2.The perfusion of different concentrations of the 5-HT resulted in a time-dependent and concentration-dependent decrease in the frequency of spontaneous SS discharge of PC.The inhibition reached the peak value 10 minutes after administration（P<0.05）of 100μM of the 5-HT,accompanied by a significant increase in dispersion coefficient（CV）（P<0.05）.The maximum concentration of the5-HT for reducing the discharge frequency of PC spontaneous SS was 500μM.However,the minimum concentration was 1μM,and the median effective inhibitory concentration(IC₅₀)was 37.55μM。3.Administration of the 5-HT1A receptor blocker had no significant effect on spontaneous SS discharge frequency and CV of PC（P>0.05）.In the presence of the receptor blocker,administration of the 5-HT could significantly reduce the spontaneous SS discharge frequency of PC（P<0.05）and widely increase the CV（P<0.05）.These results indicated that the 5-HT1A receptor antagonist could not block the inhibitory effect of the 5-HT on spontaneous SS discharge of PC.4.Perfusion of 5-HT7 receptor blocker had no significant effect on spontaneous SS discharge frequency and CV of PC（P>0.05）.In the presence of a 5-HT7 receptor blocker,5-HT could deeply decrease the spontaneous SS discharge frequency of PC（P<0.05）and significantly enhance the CV（P<0.05）.These results demonstrated that the 5-HT7 receptor antagonist could not block the inhibitory effect of 5-HT on spontaneous SS discharge of PC.5.Administration of the 5-HT2B receptor blocker had no significant effect on spontaneous SS discharge frequency and CV of PC（P>0.05）.However,the presence of a 5-HT2B receptor blocker and the inhibitory effect of 5-HT on PC discharge activity disappeared.There were no differences in spontaneous SS discharge frequency of PC before and after administration of 5-HT（P>0.05）,and the CV also did not change significantly（P>0.05）.The results showed that the 5-HT2B receptor antagonist can block the inhibitory effect of the 5-HT on PC spontaneous SS discharge,suggesting that 5-HT inhibits PC spontaneous SS were discharged by activating the 5-HT2B receptor.6.Blocking AMPA receptors had no significant effect on spontaneous SS discharge activity of PC,and there were no significant changes in SS frequency and CV before and after administration（P>0.05）.In the presence of an AMPA receptor blocker,after administration of 5-HT,the spontaneous SS discharge frequency of PC was decreased（P<0.05）,and CV was increased significantly（P<0.05）.These results showed that the 5-HT can inhibit the discharge frequency of PC spontaneous SS by blocking excitatory glutamatergic synaptic transmission,indicating that the inhibitory effect of 5-HT on PC spontaneous SS was not related to excitatory glutamatergic synaptic transmission.7.Blocking the GABA_Areceptor had no significant effect on the spontaneous SS discharge frequency of PC,resulting in a significant decrease in CV（P<0.05）.In the presence of a GABA_Areceptor blocker,the inhibitory effect of 5-HT on spontaneous SS discharge activity of PC was abolished.Meanwhile,the discharge frequency did not change（P>0.05）,and there was no significant difference in CV before and after administration（P>0.05）.These results demonstrated that blocking the GABA_Areceptor could eliminate the inhibitory effect of 5-HT on PC spontaneous SS discharge,showing that 5-HT may lead to the inhibition of PC spontaneous SS discharge by exciting MLI.8.Under the cell-attached recording mode,the perfusion of the 5-HT could enhance the spontaneous discharge activity of MLI,and the discharge frequency was increased after administration（P<0.05）,indicating that the 5-HT could reinforce the spontaneous activity of MLI.Furthermore,the administration of the 5-HT2B receptor blocker had no effect on the spontaneous discharge frequency of MLI,blocking the excitatory effect of 5-HT on MLI.In the presence of 5-HT2B receptor blockade,the discharge frequency of MLI did not increase after administration of 5-HT（P>0.05）.These results suggested that the 5-HT increases the spontaneous discharge activity of MLI by activating the 5-HT2B receptor and then enhances the inhibitory effect of MLI on PC.[Conclusion]1.The 5-HT increases the spontaneous discharge frequency of MLI by activating the 5-HT2B receptor,resulting in the inhibition of spontaneous SS discharge activity of PC.2.The 5-HT projection may enhance the inhibitory synaptic transmission of MLI-PC by exciting MLI in the cerebellar cortex to participate in the regulation of motor behavior.