Mechanisms Of Crf-r2 In Excitatory Synaptic Transmission And Long-term Plasticity Of Purkinje Cells In Mouse Cerebellar Cortex

[Purpose]The cerebellar Purkinje cell(PC)mainly receives external excitatory information from the mossy fiber(MF)-granule cell(GrC)-parallel fiber(PF)pathway originating from a wide range of brain regions and the climbing fiber(CF)pathway originating from the neurons of the inferior olivary nuclus(IO).Corticotropin releasing factor(CRF),as the main stress hormone,can be released to cerebellar cortex through CF terminals.CRF receptor plays an important regulatory role in synaptic transmission and long-term plasticity of cerebellar neural circuit.CRF receptors belong to G protein coupled receptors,including CRF receptor 1(CRF-R1)and receptor 2(CRF-R2),which are expressed in cerebellum.Previous studies have found that CRF mainly regulates the discharge activities of simple spike(SS)and complex spikes(CSs)of PC,and modulates the synaptic transmission of molecular layer interneuron(MLI)-PC mainly through CRF-R1.However,the mechanism of CRF-R2 on PF-PC and CF-PC synaptic transmission and long-term plasticity in mouse cerebellar cortex is not clear.Therefore,the present study using in vivo cell-attachment recording,in vitrowhole-cellrecording,immunohistochemicaltechniqueand neuropharmacological methods to study the roles of CRF-R2 in PF-PC synaptic transmission in mouse cerebellar cortex induced by facial air-puff stimulation,and to explore the effect of CRF-R2 on CF-PC synaptic transmission and synaptic plasticity.I aimed to clarify the mechanism of CRF-R2 in PC information transmission and long-term plasticity in cerebellar cortex.[Methods]1.In vivo experimentC57BL/6 male mice(6-8 weeks)were anesthetized by intraperitoneal injection of urethane(1.3g/kg body weight).In Crus II or vermis VI-VII area,the skull was drilled,and a hole of about 1-1.5 mm was opened to expose the cerebellar surface.The oxygen-containing artificial cerebrospinal fluid(ACSF)was continuously perfused to the exposed part of the operation.In vivo electrophysiological experiments were performed by PC-attached recording.The depth of the recording electrode into the brain tissue was about 160μm to 210μm.the presence of spontaneous SS and CSs was used to determine whether it was PC.The spontaneous SS and CSs activities and electrical activities caused by sensory stimulation were recorded.The sensory stimulation is the air-puff stimulation of the ipsilateral whisker pad.The system consists of a Picospritzer,a Master 8 and a computer.The duration of each recording is 25 sec,and the stimulation interval is set to 30 sec.All drugs were dissolved in ACSF and administered by brain surface perfusion.Excitatory glutamatergic synaptic transmission was isolated by addingγ-amino butyric acid receptor A(GABA_A)blocker to ACSF.2.In vitro experimentAfter inhalation anesthesia with isoflurane,the cerebellum was cut into 250μM thick sagittal section.The whole-cell recordings were performed on PCs,and biocytin was added to the filling solution in the electrode.After the experiment,formalin was used to fix the brain slices,and then biocytin staining was carried out.In order to record the synaptic transmission of CF-PC,we placed a stimulation electrode in the granular layer(GL),and electrically stimulated CF to induce postsynaptic current response of PCs.The test stimulation was paired-pulses with an interval of 50 ms,and the pulse width was 0.2 ms.The stimulation could induce a pair of excitatory postsynaptic currents(EPSCs),N1 and N2,and the N2/N1 ratio was calculated.In the CF-PC long-term synaptic plasticity experiment,the stable response was recorded with the test stimulus for 10 minutes before induction.The stimulus to induce long-term plasticity was 5 Hz,continuous stimulation for 30 seconds,Then,continuous recording was obtained for 50 minutes after the end of the induction stimulus.In addition,the distribution of CRF-R2 in cerebellar cortex was determined by immunohistochemical staining with the cerebellum from the mouse of the same age.[Results]Part Ⅰ:Mechanism of CRF-R2 in PF-PC synaptic transmission in mouse cerebellar cortex(1)The results of PC-attached recording in the cerebellar cortex showed that a selective CRF-R2 agonist,UrocortinⅢ(UCNⅢ)perfused on the cerebellar surface significantly increased the mean discharge frequency and instantaneous frequency of SS,but significantly reduced the interspike interval(ISI).Perfusion of CRF-R2receptor antagonist,antisauvagine-30,did not affect the discharge frequency of SS,but eliminated the effect of UCNⅢon the discharge activity of PCs.(2)Air-puff stimulation of the ipsilateral tentacle pad in mice caused inhibitory postsynaptic potentials in Crus II area of cerebellar cortex,inhibited PC discharge activities,and caused a certain duration of discharge pause.In the presence of GABA_Areceptor blockers,sensory stimulation induced PC to produce evoked simple spike(e SS)discharges,indicating that sensory stimulation induced excitatory response of PF-PC synapse after blocking inhibitory afferent of molecular layer interneurons.(3)In the presence of UCNⅢ,the number of e SS discharges caused by sensory stimulation increased significantly,and this increase could be eliminated by CRF-R2antagonist,antisauvagine-30.The enhanced effect of UCNⅢon sensory stimulation response can be blocked by protein kinase A(PKA)inhibitors,H-89 and KT5720,or by an adenylate cyclase(AC)inhibitor SQ22536,but can not be blocked by protein kinase C(PKC)inhibitor,chelerythrine,indicating that activated CRF-R2 up-regulates sensory stimulation-evoked PF-PC synaptic transmission through AC-PKA pathway.Part Ⅱ:Mechanism of CRF-R2 in CF-PC synaptic transmission in mouse cerebellar cortex(1)Perfusion of UCNⅢon the cerebellar surface of anesthetized mice can significantly enhance the spontaneous discharge activity of CSs,resulting in the increase of the number of spikelets and the increase of discharge pause duration of CSs.This enhancement can be completely blocked by CRF-R2 antagonist,antisauvagine-30.(2)In the presence of CRF-R1 antagonist,BMS-763534,CRF significantly enhanced the spikelet number and pause duration of CSs.The enhancement of CSs activity could be completely blocked by CRF-R2 antagonist,antisauvagine-30.(3)Under in vitro conditions,CF was stimulated with a paired electrical stimulation to produce excitatory postsynaptic currents N1 and N2 in PC.UCNⅢsignificantly increased the synaptic transmission of CF-PC,showing a significant increase in the amplitude of N1 and a significant decrease in the N2/N1 paired-pulse ratio(PPR),indicating that UCNⅢenhanced the synaptic transmission of CF-PC through presynaptic action.Administration of antisauvagine-30 did not affect the amplitude and PPR of N1,but completely eliminated the enhancement of UCNⅢon the CF-PC synaptic transmission.(4)Perfused CRF-R1 blocker BMS-763534 had no effect on N1 amplitude and PPR,but in the presence of BMS-763534,perfused CRF could cause N1 amplitude to increase and PPR to decrease,which could be blocked by CRF-R2 antagonist.(5)Perfusion of PKA inhibitor KT5720 could eliminate the enhancement of N1induced by UCNⅢ,but the addition of membrane impermeable PKA blocker,PKI14-22 to the internal solution could not block the enhancement of N1 induced by UCNⅢ,indicating that UCNⅢenhanced CF-PC synaptic transmission occurred in presynaptic rather than postsynaptic sites.(6)Fluorescence immunostaining of CRF-R2 in cerebellar cortex showed that there were abundant CRF-R2 distribution in PC and molecular layer(ML)of cerebellar cortex.Part Ⅲ:Mechanisms of CRF-R2 in CF-PC long-term plasticity in mouse cerebellar cortex(1)Electrical stimulation of CF with frequency of 5 Hz for 30 seconds could significantly reduce the amplitude of N1 for a long-term,without significant changes in PPR,indicating that tetanic stimulation with this frequency could induce long-term depression(LTD)in mouse cerebellar cortex CF-PC.(2)In the presence of UCNⅢ,CF-PC LTD could not be induced by electrical stimulation with CF frequency of 5 Hz for 30 sec.However,when CRF-R2 was blocked,the stimulation could induce CF-PC LTD,but did not change PPR,indicating that the postsynaptic CF-PC LTD induced by tetanic stimulation in mouse cerebellar cortex was blocked by the activation of CRF-R2.(3)Blocking the activity of metabotropic glutamate receptor 1(m Glu R1),5 Hz,30 sec CF electrical stimulation did not change the amplitude of N1 and PPR.However,in the presence of m Glu R1 blocker and UCNⅢ,5 Hz,30 sec CF tetanic stimulation increased the amplitude of N1,accompanied by a decrease in PPR,indicating that blocking m Glu R1 dependent CF-PC LTD and activating CRF-R2 can stimulate CF-PC to produce presynaptic long-term potentiation(LTP).(4)Using PKI 14-22 to inhibit postsynaptic PKA,tetanic stimulation of CF did not change the amplitudes of N1 and PPR,but after perfusion of UCNⅢ,tetanic stimulation increased the amplitudes of N1 and decreased PPR,indicating that activated CRF-R2 stimulated CF-PC LTP,which was independent of postsynaptic PKA signal pathway.However,CF-PC LTP stimulated by activated CRF-R2 can be eliminated by perfusion PKA inhibitor KT5720.When KT5720 and UCNⅢexist simultaneously,tetanic stimulation of CF will not change N1 amplitude and PPR,indicating that CF-PC LTP stimulated by activated CRF-R2 depends on presynaptic PKA signal pathway.[Conclusions](1)Activated CRF-R2 can facilitate the sensory information transmission of PF-PC synapse through AC-PKA signaling pathway rather than PKC signaling pathway,and increase the sensitivity of cerebellar cortex to sensory information.(2)The activation of CRF-R2 augments CF-PC synaptic transmission through a presynaptic but not postsynaptic PKA signaling pathway in the mouse cerebellar cortex.The information transmission from the IO to the cerebellar cortex is augmented by the activation of CRF-R2.(3)Activation of CRF-R2 can eliminate postsynaptic PKA mediated CF-PC LTD,and its mechanism may be related to the activation of CRF-R2 to induce presynaptic PKA mediated CF-PC LTP.