Effect Of Corticotropin Release Hormone On Spontaneous Simple Spike Activity Of Mouse Cerebellar Purkinje Cell In Vivo

[Objective]Cerebellum is a major motor center in mamalians.The outside information transferred to cerebellar cortex via cliumbing fibers and mossy fibers,which calculated and integrated by cerebellar cortical neuronal network,and finally the commands sent by cerebellar Purkinje cells(PC)to complete various physiological events.Corticotropin releasing hormone(CRH)is a critical neuropeptide that involves in regulation of stress response and internal organs activities.CRH distributes in cerebellar circuitry in mamalians,and modulates the spontaneous discharge and command outputs of PCs.However,its mechanisms are currently unclear.In the present study,we investigated the the effects of different concentrations of CRH on the spontaneous SS activity in cerebellar PC in urethane-anesthetized mice by cell-attached recording and pharmacological methods.To study the possible mechanisms of CRH modulates the spontaneous SS activity.The results will contribute to understand the effective mechanisms of stress stimulus on the activity of cerebellar neuronal circuitry and motor learning.[Methods]Adult(6-8 weeks old)ICR mice were anesthetized with urethane(1.3g/kg Body weight,i.p.),and craniotomy with opening a 1-1.5mm in diameter was performed to expose the cerebellar surface corresponding to Vermis.After carefully removed the dura,the surface of cerebellum was continually perfused by oxygenated artificial cerebrospinal fluid.The ACSF consist of(mM)NaCl 118;KCl 3;MgCl2 1;NaH2P04.2H20 1,NaHC03 25;D-Glucose 10,CaCl2 2。pH was 7.25-7.35,osmolarity was 295-300 mOsm.In vivo cell-attached recording was used to record the spontaneous spike firing activity in the study.Recording electrodes were filled with 10μ l ACSF,with resistances of 4-6MΩ.The PC spontaneous firing were acquired through a Patch Clamp Amplifier System(Axopatch-200B)and data acquisition software.The electrophysiological data were analyzed using Clampfit 10.3 software.Values are expressed as the mean ± S.E.M.Differences were evaluated with the One-way AN OVA using SPSS software.P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results]Cerebellar surface perfusion of CRH induced a significant decrease in instant frequency which expressed the peak value shift to left.The mean frequency of SS firing was decreased from 24.3 ± 1.6 Hz to 19.4 ± 2.1 Hz(P<0.05).However,CRH did not significantly change the CV value of SS.In the presence of GABAA receptor antagonist,CRH induced a significant increase in instant frequency which expressed the peak value shift to right.The mean frequency of SS firing was increased from 25.5 ± 1.8 Hz to 30.7 ± 2.4 Hz(P<0.05).These results indicated that CRH excited cerebellar cortical MLIs resulting in a decrease in SS firing rate of PCs.Blocking the MLls-mediated inhibitory inputs,CRH might excite PCs resulting in an increase in SS firing rate.Blockade of MLIs-mediated GABAergic synaptic transmission,CRH increased the spontaneous SS firing rate in a concentration-dependent mannar.The half effective concentration(EC50)was 53.8 nM;and the maxmal concentration was 1 μ M.Blockade of GABAA and AMPA receptors,CRH induced a significant increase in instant frequency which expressed the peak value shift to right,but without effect CV value.These results indicated that CRH excited PCs accompanied with an increase in SS firing rate was not through increasing the CF and PF excitatory inputs.In the presence of non-selective CRH receptors blocker,CRH failed to induce increases in the instant frequency and SS firing rate.These results indicated that CRH excited cerebellar PCs accompanied with an increase in SS firing rate through CRH receptors.In the presence of a selective CRH-R2 antagonist,the effect of CRH on instant frequency of SS firing was insistent,and the mean SS rate was still significantly increased.These results indicated that CRH excited cerebellar PCs was not dependent CRH-R2,suggesting that CRH directly combined with CRH-R1 and excited PCs resulting in an increase in SS firing rate.[Conclusions]The present results indicated that CRH excited cerebellar MLIs resulting in a decrease in SS frimng rate of PCs.Blockade of GABAergic synaptic transmission between MLI-PC syanpses,CRH excited PCs resulting in an increase in the spontaneous SS firing rate via CRH-R1.These results suggested that CRH could act as cerebellar PCs and affet the PC command output,as well as the motor regulation via CRH receptors.