Effect Of CRF On Spontaneous Glutamatergic Synaptic Transmission Of Mouse Cerebellar Purkinje Cell In Vivo

Objective:The cerebellum of mammals is an important central organ related to motor function External information comes from the climbing fiber(CF),moss fiber(MF)into the cerebellar cortex.After integration and calculation by the neuronal network,Purkinje cell(PC)sends out motion adjustment instructions to complete the relevant functions of motor regulation.Corticotrophin releasing factor(CRF)is an important neuropeptide that participates in the regulation of stress response and visceral function of mammals.CRF nerve fibers are projected into the cerebellar cortex,which affects the spontaneous discharge activity of the mammalian cerebellar cortical PCs,but the mechanism of its influence have not yet been fully understood.In this study,we studied the effect of CRF on the spontaneous excitatory glutamic acid synaptic transmission of Purkinje cells in cerebellar cortex of urethane-enesthatized mice,by using electrophysiological recording technique and neuropharmacology methods.We also investigated the mechanism of CRH affectd on the spontaneous excitatory glutamic acid synaptic transmission of the mouse cerebellar PCs.Methods:Adult ICR mice of 6-8 weeks old were used in this study.After intraperitoneal injection of 1.3 g/kg urethane for anesthesia,a hole of 1-1.5 mm diameter is opened at the corresponding position in the Vermis region of the cerebellum.After the dura mater was gently removed,the exposed area was continuous perfused with artificial cerebrospinal fluid(ACSF).The PC spontaneous discharge activity was recorded by cell-attached methods,and the PC spontaneous activity of the cerebellar cortex is recorded by the Axopatch-200B patch clamp amplifier with data acquisition software.CRH and receptor blockers were treated with cerebellar surface perfusion.Clampfit 10.3 software was used for the analysis of electrophysiological experimental data.Data are expressed as the mean ± S.E.M.The number of recorded cells was expressd with n.The SPSS(Version 21)software was used to analyze the data,and the one way ANOVA was used to compare the average values.P<0.05 thought there was a statistical significanceResults:(1)Cerebellar surface perfusion of CRF(100 nM)significantly increased the spontaneous discharge frequency of PC SS,the average frequency of SS discharge was 118.2 + 3.7%of baseline,and the non selective CRF-Rs blocker could completely prevented the CRF-induced increase of the spontaneous SS discharge frequency of PCs.(2)CRF-R1 selective receptor antagonist,BMS-763534 partly blocked the effect of CRF-R1 on the increase of CRF induced PC spontaneous SS.(3)Application antisauvagine-30,a CRF-R2 selective receptor blocker,also significantly prevented the CRF-induced increased in the SS frequency of PCs,and CRF-R1 is greater than CRF-R2.(4)In the presence of antisauvagine-30 and BMS-763534,the CRF-induced increase in SS firing rate of PCs was completle blocked.(5)CRF significantly shortened the mEPSCs interval,but has no effect on the amplitude of mEPSCs.In the presence of CRF-R2 blocker,CRF no longer shortened the mEPSCs interval.(6)PKA inhibitor,H-89 significantly increased the interval of mEPSCs and significantly reduced the amplitude of mEPSCs,and completely blocked the effect of CRF on the frequency of mEPSCs.ConclusionsThe results of this study showed that CRF could increase the release of glutamic acid and increase the excitatory afferent of PC by activating CRF-R2,which could increase the spontaneous SS discharge frequency of PC,suggesting that stress stimulation could regulate the spontaneous activity of PC in the cerebellar cortex by presynaptic CRF-Rs,and then affect the instruction output and motor regulation function of PCs.