Mechanism Of NMDA Receptor On Cerebellar Mossy Fiber-Granule Cell Synaptio Function And Motor Coordination In Mice

[Purpose]N-methyl-D-aspartate receptor(NMDAR)is involved in important physiological processes of cerebellar cortical neuronal circuitry,such as synaptic plasticity,motor learning and memory,neuropathy and cerebellar related dyskinesia.Cerebellar cortex receives various sensory inputs from the outside through mossy fiber(MF)-granule cell(GrC)and climbing fiber(CF),and Purkinje cells(PC)produce motor related command outputs,which participates in the sensory perception,fine regulation of motor coordination,motor learning and autonomous movement.The granular layer of cerebellum mainly includes two kinds of neurons,GrC and Golgi cells.GrC is the relay neuron of cerebellar cortex and the only excitatory neuron,which receives excitatory input from MFs and transmits it to PCs and molecular layer interneurons(MLI)through parallel fibers.In cerebellar cortex,NMDAR expresses in cerebellar GrCs and involves in synaptic transmission of MF-GrC in cerebellar cortex.These findings suggest that NMDAR may play an important role in the functional activity of MF-GrC synaspses and motor coordination in cerebellar cortex,but its mechanism is not clear.Therefore,I used in vivo electrophysiological recording,immunohistochemistry,pharmacology,microinjection and ethology methods to investigate the following contents: The mechanism of NMDAR in MF-GrC synaptic transmission induced by facial stimulation in urethane anesthetized mice;the role of NMDAR in motor coordination of mice;the mechanism of NMDAR in the facial stimulation-evoked long-term plasticity of MF-GrC synapse.This study aims to clarify the influence mechanism of NMDAR on the synaptic transmission and long-term plasticity of MF GrC induced by sensory stimulation in cerebellar cortex,and to clarify its role in motor coordination.[Methods](1)Animal preperation.Adult mice(6-8 weeks old)were anesthetized with urethane(1.3 g/kg body weight)by intraperitoneal injection.A 1-1.5 mm hole was drilled to expose the corresponding cerebellar surface of Crus I/II area.The brain surface was perfused continuously with artificial cerebrospinal fluid(ACSF)containing oxygen at the rate of 0.5 ml/min.The rectal temperature was monitored and maintained at 37 ± 0.2 °C.(2)Electrophysiological recording.Axopatch-200 B amplifier was used for cell-attached recording of cerebellar GrC.Using Clappex10.4 software to acquire MF-GrC synaptic response signal through digidata 1440 digital to analog converter.The recording electrode is filled with ACSF,and the resistance is 3-5 MΩ.The glass electrode was slowly inserted under the pia mater by a micromanipulator about300-400 μm.After reaching the granular layer,the field potential response induced by sensory stimulation was recorded in the current-clamp mode.The GABAA receptor blocker,picrotoxin was used to eliminate the inhibitory components of Golgi cells,and then the MF-GrC excitatory synaptic transmission induced by sensory stimulation was isolated and recorded.(3)Sensory stimulatiomn.The effects of sensory stimulation on MF-GrC synaptic transmission were studied by air-puff stimulation(duration: 10 ms,pressure:60 psi).Paired stimulation was used as a test stimulus for induction of synaptic plasticity.The long-term plasticity of MF-GrC synapses was induced by 10 ms,20 Hz and 240 pulses,and then the effect of NMDAR on the long-term plasticity of MF-GrC synaptic transmission was studied.The drugs were dissolved in ACSF and applied directly to the cerebellar surface by peristaltic pump at a speed of 0.5 ml/min.(4)Immunohistochemistry and imaging.Mice were intraperitoneally injected with 7% chloral hydrate(5ml/kg)for deep anaesthesia,followed by heart perfusion with PBS and 4% paraformaldehyde PBS solution.At 4 ° C,the brain was fixed for48 hours,and the cerebellum and brain were separated with a blade.After embedding the cerebellum,the cerebellum was sliced into 8 μm sections in the sagittal plane.The sections were stored at 4 °C for immunohistochemistry.They were cultured in rabbit anti-GluN2 A,Alexa Fluor 555 donkey anti-rabbit and 4’,6-diamidino-2-phenylindole(DAPI),respectively.Fluorescence images were obtained by confocal laser scanning microscope(Nikon C2,Tokyo,Japan).On the Nikon C2 laser confocal system,a large area image containing the required area(Crus II)is obtained with a 10 x objective lens.(5)Microinjection into cerebellar cortex and motor behavior experiments.Microinjection into Crus I / II area of cerebellar cortex: After 4-5-week-old mice were anesthetized by 10% chloral hydrate anesthesia,a craniotomy was performed under brain stereotactic instrument and a dental drill.The prepared cannula for injection was implanted into the Crus I / II area of the mouse cerebellum.After the operation, antibacterial ointment was applied and antibiotics were injected intraperitoneally.After 7 days of recovery,the animals were divided into control group and administration group.At 9 a.m.on the 8th day after operation,100 nl ACSF(control group)or receptor blocker(administration group)were microinjected into the surface of cerebellar cortex by microinjection pump respectively,and then walking disorder analysis and rotarod test were carried out.(6)The electrophysiological data were analyzed by Clampfit 10.4 software.The values are expressed as mean ± S.E.M.SPSS software was used for one-way ANOVA and two-way ANOVA to determine the statistical significance level differences among the groups.P-values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results]Part I.Effects of NMDARs on sensory information MF-GrC synaptic transmission and motor coordination(1)In the presence of GABAA receptor antagonist picrotoxin,the MF-GrC synaptic transmission in mouse cerebellar cortex evoked by facial air-puff stimulation could be recorded.(2)Activation of NMDARs enhanced facial stimulation-evoked MF-GrC synaptic transmission in mouse cerebellar cortex.However,blocking NMDARs significantly reduced facial stimulation-evoked MF-GrC synaptic transmission.It is suggested that the activation of NMDARs is involved in the sensory information processing in the mouse cerebellar cortex.(3)Blocking NMDARs containing GluN2 A subunit significantly reduced facial stimulation-evoked MF-GrC synaptic transmission in cerebellar granular layer,and completely blocked the enhancement of MF-GrC induced by NMDA.However,blocking NMDARs containing GluN2 B subunit or GluN2C/D subunit did not affect facial stimulation-evoked MF-GrC synaptic transmission in cerebellar granular layer,nor did it affect the enhancement of MF-GrC induced by NMDA.It is suggested that NMDARs containing GluN2 A subunit,but not GluN2 B subunit and GluN2C/D subunit is involved in the sensory information transmission in the mouse cerebellar granular layer.(4)The selective GluN2A-containing NMDAR positive regulator,GNE-0723 enhanced the MF-GrC synaptic transmission.The application of NMDARs containing GluN2 A subunit blocker weakened the MF-GrC synaptic transmission of cerebellar granular layer induced by sensory stimulation,and completely blocked the GNC-induced enhancement of MF-GrC transmission.These results further suggested that NMDARs enhances facial stimulation-evoked MF-GrC synaptic transmission in mouse cerebellar cortex through NMDARs containing GluN2 A subunit.(5)Microinjection of D-APV or PEAQX onto cerebellar surface and blocking NMDARs or NMDARs containing GluN2 A subunit in Crus II region of cerebellar cortex significantly increased the number of error number and time of walking steps in walking disorder and significantly reduced the latency and speed of falling from the rotating rod in the rotating rod experiment.It is suggested that blockade of NMDARs or NMDARs containing GluN2 A subunit in Crus II impairs the walking coordination ability of mice.Part II.Mechanism of NMDARs in sensory stimulation-evoked MF-GrC long-term plasticity(1)When GABAA receptor was blocked,20 Hz facial stimulation could induce a LTP of MF-GrC in cerebellar granular layer.However,blocking NMDARs,20 Hz facial stimulation failed to induce MF-GrC LTP in cerebellar granular layer.The results show that MF-GrC LTP induced by 20 Hz facial blowing stimulation is dependent on NMDARs.(2)Under the condition of blocking NMDARs containing GluN2 A subunit,20 Hz facial stimulation could not induce MF-GrC LTP in cerebellar granular layer.However,blocking NMDARs containing GluN 2B or GluN 2C/D subunit had no effect on 20 Hz facial stimulation-induced MF-GrC LTP in the cerebellar granular layer.In addition,activation of NMDARs containing GluN2 A subunit could induce MF-GrC LTP,and lead to sensory stimulation can not further induce MF-GrC LTP.The result suggests that the LTP of cerebellar MF-GrC induced by facial stimulation is mediated by NMDARs containing GluN2 A subunit.(3)The application of nitric oxide synthase(NOS)inhibitor could eliminate the MF-GrC LTP induced by facial stimulation in mouse cerebellar cortex.The application of NO donor,SNOP could not only induce the production of MF-GrC LTP,but also block the 20 Hz stimulation to further induce MF-GrC LTP.The results indicate that the MF-GrC LTP induced by 20 Hz facial stimulation is mediated by NO signal.(4)Intracellular signaling mechanism of MF-GrC LTP in cerebellar granular layer induced by 20 Hz facial stimulation.Inhibiting protein kinase A(PKA)or blocking PKC signaling pathway could lead to the inability of facial stimulation to induce MF-GrC LTP in the mouse cerebellar cortex.In addition,the depletion of intracellular Ca2+ led to the failure of facial stimulation to induce MF-GrC LTP in the mouse cerebellar cortex.These results show that the induction of MF-GrC LTP requires to be mediated by PKA and PKC signaling pathways,and also needs the participation of intracellular calcium.[Conclusions](1)Activation or inhibition of NMDAR,especially NMDAR containing GluN2 A subunit,can significantly enhance or weaken MF GRC synaptic transmission in mouse cerebellar cortex induced by sensory stimulation.(2)Blocking NMDAR containing GluN2 A subunit can lead to impairment of motor coordination in mice,suggesting that NMDAR containing GluN2 A subunit play an important role in sensory information synaptic transmission and motor coordination of MF-GrC pathway in mouse cerebellar cortex.(3)NMDAR containing GluN2 A subunit exists in GrCs of mouse cerebellar cortex.MF-GrC LTP induced by facial sensory stimulation depends on the NMDAR containing GluN2 A subunit / NO cascade,which is co-mediated by PKA and PKC signaling pathways and requires the participation of intracellular calcium ions.(4)The present results suggest that NMDAR containing GluN2 A subunit,control the long-term plasticity of MF-GrC synaptic transmission,which may play a key role in motor learning of animals.