Role Of NMDA Receptors During The Sensory Stimulation-evoked Field Potential Response In Mouse Cerebellar Cortex In Vivo

[Purpose]N-methyl-D-aspartate receptors(NMDARs)are a group of glutamate ion channels,which are involved in the regulation of presynaptic neurotransmitter release and the postsynaptic response.In mammalian cerebellar cortex,NMDARs are found existed on the membrane of granule cells(GC),terminals of parallel fibers(PF),molecular layer intemeurons(MLI)and climbing fiber-Purkinje cell(PC)synapses,but the functional NMDARs are absence in the membrane of the adult PCs.We previously found that the air-puff stimulation on ipsilateral whisker pad evoked a sequence of transient tiny excitation response followed by strong inhibitory response in cerebellar molecular layer via mossy fiber pathway.However,whether the NMDARs are involved in the sensory-evoked responses in cerebellar molecular layer under in vivo conditions are currently unknown.In the present study,we investigated the role of NMDARs during the air-puff stimulation evoked field potential responses in cerebellar molecular layer of urethane-anesthetized mice by electrophysiological recording technique and pharmacological methods.[Methods]In this part,we used 37 adult(6-8 weeks)ICR mice.These mice were anesthetized by intraperitoneal injection of urethane(1.3 g/kg body weight),and then tracheotomized for avoiding respiratory obstruction.Further,the mice were fixed on a custom-made stereotaxic devises.Following a watertight chamber created,a 1-1.5 mm craniotomy hole was drilled to expose the cerebellar surface corresponding to cerebellar lobule Crus II.The duramater was carefully removed using a needle,and the surface of cerebellum was constantly superfused with oxygenated artificial cerebrospinal fluid(ACSF)at 0.4 ml/min with a peristaltic pump.Body temperature was monitored using a temperature monitor and maintained at 37.0±0.2? using a heating instrument(Chengdu instrument,China).Local field potential recordings from the cerebellar molecular layer were performed with an Axopatch-200B amplifier(Molecular Devices,Foster City,CA).The field potentials were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer by Clampex 10.3 software.Recording electrodes were filled with ACSF and with resistances of 3-5 M?.NMDARs agonist(NMDA)and NMDARs antagonist(D-APV)were bought from Sigma-Aldrich(Shanghai,China).All chemicals were applied to the cerebellar surface at 0.4 ml/min in ACSF.Electrophysiological data were analyzed using Clampfit 10.3 software.All values are expressed as the mean±SEM.Differences between the mean values recorded under control and test conditions were evaluated with the Student's paired t-test or one-way ANOVA using SPSS 21.0 software.P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results](1)Cerebellar surface administration of NMDA(10-200 ?M)induced a dose-dependent decrease in amplitude of the facial stimulation-evoked inhibitory responses(PI)in cerebellar molecular layer.The IC50 of NMDA induced inhibition of P1 was 46.5 ?M.High concentration(100-200 ?M)NMDA completely inhibited the sensory-evoked P1.(2)Administration of NMDA affected the dynamic properties of sensory stimulation-evoked P1 in mouse cerebellar molecular layer,which expressed decreases in decay time,half-width and area under curve(AUC)of P1.(3)Application of NMDA induced significant increases in the decay time,half-width and AUC values of the facial stimulation-evoked excitatory responses(N1)in cerebellar molecular layer.However,NMDA did not significantly affect the amplitude,half-amplitude of N1 and rise time of N1..(4)Application an NMDAR blocker,D-APV(250 ?M)abolished the facial stimulation-evoked GABAergic inhibitory components(P1)in mouse cerebellar molecular layer.(5)Blockade of NMDARs did not significantly change the amplitude and half-amplitude of N1,but significantly increased the half-width and AUC value of N1.[Conclusions](1)Either pharmacological activation or inhibition of NMDARs abolished the facial stimulation evoked GABAergic inhibitory components,but enhanced the sensory stimulation-evoked PF excitatory inputs in cerebellar molecular layer.(2)The functional NMDARs play a critical role during the sensory stimulation-evoked field potential responses in the mouse cerebellar molecular layer.[Purpose]Sensory information transferred to cerebellar PCs through MF-GC-PF pathway,and involves in generation of motor-related outputs,such as sensory perception,motor coordination,motor learning and fine adjustment of voluntary movement.GCs express spontaneous spike firing at low frequency,but they are extremely sensitive to different sensory stimulation inputs.We previously found that cerebellar surface administration of NMDA enhanced the sensory stimulation-evoked PF excitatory inputs in cerebellar molecular layer,suggesting that activation of NMDARs in GC layer might contribute to sensory information processing under in vitro conditions.However,the role of NMDARs during the sensory information processing in cerebellar granule cell layer in living mouse is currently unclear.We here studied the function of MF-GC NMDARs during the facial stimulation evoked field potential responses in cerebellar GC layer in urethane-anesthetized mice by electrophysiological recording technique and pharmacological methods.We aimed to understand the role of NMDARs during the sensory information processing in cerebellar granule cell layer in vivo in mice.[Methods]A total of 37 adult(6-8 weeks)ICR mice were used in this study.The mice were anesthetized by intraperitoneal injection of urethane(1.3 g/kg body weight),and then tracheotomized for avoiding respiratory obstruction.Further,the mice were fixed on a custom-made stereotaxic devises.Following a watertight chamber created,a 1-1.5 mm craniotomy hole was drilled to expose the cerebellar surface corresponding to cerebellar lobule Crus ?.The dura mater was carefully removed using a needle,and the surface of cerebellum was constantly superfused with oxygenated artificial cerebrospinal fluid(ACSF)at 0.4 ml/min with a peristaltic pump.Body temperature was monitored using a temperature monitor and maintained at 37.0±0.2? using a heating instrument(Chengdu instrument,China).Local field potential recordings from the cerebellar GC layer were performed with an Axopatch-200B amplifier(Molecular Devices,Foster City,CA).The field potentials were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer by Clampex 10.3 software.Recording electrodes were filled with ACSF and with resistances of 3-5 M?.NMDARs agonist(NMDA)and NMDARs antagonist(D-APV)were bought from Sigma-Aldrich(Shanghai,China).All chemicals were applied to the cerebellar surface at 0.4 ml/min in ACSF.Electrophysiological data were analyzed using Clampfit 10.3 software.All values are expressed as the mean ± SEM.Differences between the mean values recorded under control and test conditions were evaluated with the Student's paired t-test or one-way ANOVA using SPSS 21.0 software.P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results](1)Facial stimulation evoked a couple of excitatory components in the GC layer in mouse cerebellar cortical folium Crus ?,which identified as stimulus-onset response(Ron)and stimulus-offset response(Roff).(2)Application of NMDA induced a significant decrease in the half-width and AUC value of the facial stimulation-evoked Ron,without changing the amplitude and half-amplitude of Ron.(3)NMDA significantly decreased the amplitude,AUC value,half-width of the facial stimulation evoked Roff,and decreased Roff/Ron.ratio in cerebellar cortical GC layer.(4)Application of NMDARs antagonist,D-APV(250 ?M)induced significant decreases in the half-width and AUC value of the facial stimulation evoked Ron,without changing the amplitude and half-amplitude of Ron in mouse cerebellar GC layer.(5)Blockade of NMDARs significantly decreased the amplitude,half-width,and AUC value of the facial stimulation evoked Roff,and decreased Roff/Ron ratio in cerebellar GC layer.[Conclusion]Functional NMDARs contribute to the facial stimulation-evoked field potential responses in cerebellar cortical GC layer.Either pharmacological activation or blockade of NMDARs impacts the high-fidelity properties of the cerebellar GCs for transferring the sensory information.