Effects Of Ethanol On Complex Spike Activity Of Cerebellar Purkinje Cells In Vivo In Mice

OBJECTIVEThe cerebellum is an important target of the ethanol. Excessive drinking impairs cerebellar function, even results in brain atrophy. Purkinje cells constitute the single output of the cerebellar cortex to the deep cerebellar nuclei and it is highly sensitive to ethanol. Alcoholic intoxication lead to PC denaturation and dendritic reduction, then it produced ataxia. Ethanol had little effect on spontaneous firing of Purkinje cells, the mechanisms of ethanol affects cerebellar PC spontaneous CSs in vivo are currently unclear. Here, thestudyusing electrophysiological technique and pharmacological methods to study the effect of ethanol on complex spike activity of Purkinje cells (PC) in the cerebellar cortex in vivo in mice. To examine the pharmacological mechanisms that ethanol affects the characteristic and current property of complex spikes.METHODSAdult (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. In vivo whole-cell patch-clamp recording accompanied biocytinstaining technique was used as electrophysiological recording in the study. Recording electrodes were filled with 10μl internal solution, 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 ± SEM. Differences were evaluated with the One-way ANOVA using SPSS software. P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.RESULTS1. Under urethane anesthesia, PCs expressed regular SS firing with irregular complex spikes in cerebellar cortex folium Vermis, for which the mean frequency were 19 ± 4.2 Hz and 0.31 ± 0.12 Hz, respectively.2. Under current-clamp recording, ethanol(300mM) depressed the pause time and the amplitude of after-hyperpolarization.3. Under voltage-clamp recording, ethanol significantly reduced the number of spikelets and the area under curve of CS, but it had no significantly effect on the frequency of inter-spikelet interval.4. Ethanol depressed the area under curve of CSs in a dose-dependent manner, the IC50=168.5mM5. Blocking of NMDA and mGluRl receptors activity failed to prevent the ethanol-induced inhibition of CSs, whereas blockade of CB1 receptors activity abolished the ethanol-induced inhibition of CSs.CONCLUSIONOur present results indicate that ethanol inhibits the CS activity of Purkinje cell via activity of CBIin vivoin mice, suggesting that excessive ethanol intake inhibits periphery afferent information transferring to cerebellar PC via the activation of presynaptic CB1 receptor ofCF-PC synapse.