| [Purpose]The cerebellum is a target of the actions of EtOH.EtOH consumption causes alterations of motor coordination,balance,behavior,speech,and certain cognitive functions are considered to be caused partly by impairment of cerebellar circuits function and modulation of synaptic transmission.The molecular layer interneuron-Purkinje cell synapses(MLI-PC)are critical for sensory information integration and transmission in cerebellar cortex,which are sensitive to acute and chronic ethanol consumption.The aim of this study is to investigate the effect of chronic ethanol consumption on the tactile-evoked MLI-PC synaptic transmission and long-term plasticity by electrophysiological recording and pharmacological methods.[Methods]A total of 125(5-week-old)ICR mice were used in this study.The first part the mice were divided into ethanol consumption group(33 mice)and control group(33 mice).The second part the mice were divided into ethanol consumption group(29 mice)and control group(27 mice).Mice in EtOH consumption group were intraperitoneal(i.p.)injection of EtOH(0.8 g/kg;15%in saline),while mice in control group were i.p.injection same volume saline.The electrophysiological recordings were performed after i.p.injection of EtOH for 28 days.After the mice were anesthetized with urethane(1.3 g/kg body weight i.p.),the craniotomy was drilled to expose the cerebellar surface corresponding to Crus II.The cerebellar surface was constantly superfused with oxygenated ACSF with a peristaltic pump at 0.5 ml/min.Rectal temperature was monitored and maintained at 37.0±0.2? using body temperature equipment.Cell-attached recordings from cerebellar PCs were performed with an Axopatch-200B amplifier.The signals of PC cell-attached recordings were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.3 software.Facial stimulation was performed by air-puff(10 ms,60 psi)of the ipsilateral whisker pad through a 12-gauge stainless steel tube connected with a pressurized injection system.For the induction of MLI-PC synaptic plasticity,air-puff stimulus at 1Hz(240pulses)was delivered 10 min after the recording became stable.The amplitude of P1 before and after 1 Hz facial stimulation was normalized by the mean value of baseline.All values are expressed as the mean ± S.E.M,and differences were evaluated by the Student's paired t-test,one-way ANOVA and Tow-way ANOVA using SPSS 17.0 software.P<0.05 was considered to indicate a statistically significant difference between experimental groups.[Results](1)Facial stimulation evoked an inhibitory component(P1)followed by a pause of SS firing in cerebellar PCs.Chronic ethanol consumption did less affect the latency of the facial stimulation-evoked responses of cerebellar PCs,but induced significant enhancement of MLI-PC synaptic transmission,which expressed increases in amplitude of P1 and pause of SS firing.The mean amplitude of P1 and pause of SS in ethanol consumption group were significant higher than control group.However,the mean amplitude of N1 and number of the evoked SS in ethan ol consumption group were no significant different than control group,indicating that ethanol consumption did less effect on the parallel fiber volley and parallel fiber-PC synaptic transmission.(2)Perfusion of NOS inhibitor,L-NNA(5 mM)significantly decreased the amplitude of P1,accompanied with a decrease of pause of SS firing in ethanol consumption mice,but no effect on control group,indicating that inhibition of NOS significantly decreased the facial stimulation-evoked responses in ethanol consumption mice.Perfusion of NO donor,SNAP(100 ?M)significantly increased the amplitude of P1,accompanied with an increase of pause of SS firing in control mice,indicating that NO donor significantly increased the facial stimulation-evoked responses in control group.Chronic ethanol consumption did less effect on the spontaneous SS and CSs activity of cerebellar PCs.(3)The repetitive facial stimulation at 1 Hz(240 pulses)produced a MLI-PC LTD,which was expressed as decreases in P1 amplitude and pause of SS for over 50 min in control mice.However,the repetitive facial stimulation fails to induce MLI-PC LTD in chronic EtOH consumption mice.(4)Blockade of CB1 receptors activity,facial stimulation fails to induce MLI-PC LTD in control mice,but induces MLI-PC LTP in EtOH consumption mice.Perfusion of an NOS inhibitor,L-NNA,the repetitive stimulation induced MLI-PC LTD in EtOH consumption mice,indicating that facial stimulation induces MLI-PC LTD in EtOH consumption mice when NOS is inhibited.However,perfusion of NO donor,SNAP,delivery of repetitive stimulation failed to induce MLI-PC LTD in control mice,indicating that application of NO donor prevents the MLI-PC LTD in control mice.(5)Co-application of NO donor and CB1 receptors blocker,1 Hz facial stimulation induces MLI-PC LTP rather than MLI-PC LTD in control mice.Inhibition of NOS and CB1 receptors activity,facial stimulation fails to induce neither MLI-PC LTD nor MLI-PC LTP in EtOH consumption mice.In addition,blocking CB1 receptors activity neither MLI-PC LTD nor MLI-PC LTP was induced in acute EtOH consumption mice.[Conclusions](1)Chronic ethanol consumption did less effect on the spontaneous SS and CSs activity of cerebellar PC,but induced significant increases in amplitude of the facial stimulation-evoked P1 and pause of SS firing through activation of NO signaling pathway.(2)Chronic EtOH consumption can impair the sensory stimulation-induced LTD at MLI-PC synapses via activation of NO signalling pathway in cerebellar cortical Crus II in vivo in mice,suggesting that the EtOH consumption attenuates motor coordination and motor learning may be involve in its impairing MLI-PC synaptic plasticity in vivo in mice. |
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