Roles Of NMDA Receptor During The Spontaneous Activities Of Cerebellar Purkinje Cells In Vivo In Mice

[Purpose]N-methyl-D-aspartate receptors (NMDARs) are glutamate ion channels involved in the postsynaptic neuronal response and the regulation of presynaptic neurotransmitter release, and play critical roles in the modulation of synaptic transmission in the central nervous system (CNS). It is well known that NMDARs are widely expressed on the membrane of granule cells (GCs), parallel fibers (PFs), and molecular layer interneurons (MLI) in the cerebellar cortex of mammals. Activation of NMDARs increases inhibitory postsynaptic currents (IPSCs) of Purkinje cells (PCs) in cerebellar slices. However, the effects of NMDA on the cerebellar neuronal network under in living mouse are currently unclear. In our present study, we explored the effects of NMDA on the spontaneous activity of PCs in urethane-anesthetized mice by electrophysiological recording and pharmacological methods. We aim to understand the mechanisms of NMDA affect the spontaneous simple spike (SS) activities of cerebellar PCs in vivo in mice.[Methods]Twenty-five adult (6-8-week-old) ICR mice were anesthetized with urethane (1.3 g/kg body weight intraperitoneal injection), then racheotomized to avoid respiratory obstruction. The mice were fixed on a custom-made stereotaxic apparatus. After a watertight chamber was created, a 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Vermis Ⅵ. The dura mater was carefully removed, and the brain surface was constantly superfused (0.4 ml/min) with oxygenated artificial cerebrospinal fluid (ACSF) with a peristaltic pump. Body temperature was monitored using a rectal temperature probe and maintained at 37.0 ±0.2℃ using a heating pad. We used an Axopatch-200B amplifier (Molecular Devices, Foster City, CA) under cell-attached recordings conditions, recording the PCs. The PC spontaneous action potentials were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.3 software. Recording electrodes were filled with inteacellular fluid and the resistances of 4-6 MΩ. PCs were identified by the both simple spikes and complex spikes under cell-attached recording conditions at the same time. Spontaneous activity was calculated from a train of interspike intervals recorded for 100 seconds. We used clampfit 10.3 software to analyze electrophysiological data. All values are expressed as the mean±SEM. Differences between the mean values recored 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 perfusion of NMD A (5-200μM) reduced the PC simple spike (SS) firing rate in a dose-dependent manner. The IC50 of NMD A for inhibiting SS firing of PCs was 50.3μM.(2) Application of GABAA receptor (GABAAR) antagonist, SR95531 (20μM) abolished NMDA-induced inhibition of PCs spontaneous activity, and revealed NMDA-induced excitation of cerebellar PCs.(3) NMDA receptor antagonist, D-APV (250μM) did not affect the mean frequency of SS firing, but the SS firing rate of PCs became more regular than the control.(4) In addition, NMDA increased the spike firing of both basket-type and stellate-type MLIs.[Conclusions]NMDA-induced excitation of MLIs at the cerebellar surface may inhibit PC activity. NMDARs of MLIs may play an important role in regulating the spontaneous activity of PCs, and in information transmission and integration in cerebellar cortex.[Purpose]NMDARs are postsynaptically expressed at climbing fiber-Purkinje cell (CF-PC) synapses in cerebellar cortex in adult mice, which contribute to CF-PC synaptic transmission under in vitro conditions. However, the role of CF-PC NMDARs in intact cerebellar cortex of living mouse is currently unclear. We here studied the function of CF-PC NMDARs in cerebellar cortex in urethane-anesthetized mice by whole-cell patch-clamp recording technique and pharmacological methods. Our purpose is to understand the roles of NMDA receptors in spontaneous complex spike activity in cerebellar cortex.[Methods]Twenty nine adult (6-8-week-old) ICR mice were anesthetized with urethane (1.3 g/kg body weight intraperitoneal injection), then racheotomized to avoid respiratory obstruction. The mouse was fixed on a custom-made stereotaxic apparatus. After a watertight chamber was created, a 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Vermis VI. The dura mater was carefully removed, and the brain surface was constantly superfused (0.4 ml/min) with oxygenated ACSF with a peristaltic pump. Body temperature was monitored using a rectal temperature probe and maintained at 37.0±0.2℃ using a heating pad. The whole-cell patch-clamp recordings from cerebellar PCs were performed from an Axopatch-200B amplifier (Molecular Devices, Foster City, CA). The PC spontaneous action potentials were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.3 software. Recording electrodes were filled with inteacellular fluid and the resistances of 4-6 MΩ. PCs were identified by the both simple spikes and complex spikes under whole-cell recording conditions at the same time. Spontaneous activity was calculated from a train of interspike intervals recorded for 100 seconds. We used clampfit 10.3 software to analyze electrophysiological data. All values are expressed as the mean±SEM. Differences between the mean values recored 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) Under current-clamp conditions, cerebellar surface application of NMDA (50μM) induced an increase in the CS-evoked pause of SS firing accompanied with a decrease in the frequency of SS firing rate.(2) Under voltage-clamp conditions, application of NMDA enhanced the waveform of CS-evoked inward currents, which expressed increases in the area under curve (AUC) and spikelets of CS. NMDA increased the AUC of spontaneous CS in a concentration-dependent manner. The EC50 of NMDA for increasing AUC of spontaneous CS was 33.4μM.(3) NMDA significantly increasing the amplitude, half width and decay time of CS-evoked after-hyperpolarization (AHP) currents.(4) Application of NMDARs antagonist, D-APV (250μM) attenuated the waveform of spontaneous CS which decreased their AUC, number of spkelets and amplitude of AHP potential.[Conclusions]Postsynaptic NMDARs of CF-PC synapses contribute to the enhancement of CS activity, and play an important role in CF information transmission and integration in the cerebellar cortex.