| [Objective]Cerebellum,as a center of motor regulation,is mainly used to regulate muscle tension,maintain body balance and coordinate random movement.It is generally believed that external information is transmitted to cerebellar cortex through bryophyte granule cell parallel fiber pathway,integrated by cortical neural network,output from Purkinje cell(PC)to cerebellar nucleus,and finally participate in motor coordination and motor learning.Long term alcohol intake can affect the transmission of sensory information in the cerebellar cortex,even lead to cerebellar ataxia and atrophy,but the mechanism of long-term alcohol intake on the transmission of sensory information from bryophyte fibers to granulosa cells in the cerebellar cortex is not clear.Therefore,this study used electrophysiological and pharmacological methods to study the mechanism of long-term alcohol intake on the transmission of information synapses from bryophyte fibers to cerebellar granule cells in sensory stimulation mice,and to explore the damage of long-term alcohol intake on motor coordination and motor learning function.[Methods]20 ICR male mice(6-8 weeks old)were divided into control group and alcohol group.The control group was given the same amount of 0.9%sodium chloride solution,and the alcohol group was given 1.6g/kg alcohol.The electrophysiological record was made 28 days after injection of ethanol into the abdominal cavity.Urethane(1.3g/kg)was used for anesthesia and tracheotomy.In order to avoid airway obstruction,the mice were fixed on the self-made stereotactic frame by mouth and ear stick.After that,craniotomy was performed on the head of mice to remove the scalp and muscle tissue to be recorded and fully expose the skull.Remove the skull with a dental drill,drill 1-1.5mm area,peel off the dura mater,expose the crus Ⅱ area corresponding to the surface of cerebellum,and use peristaltic pump(0.4ml/min)to perfuse the surface of the record site with the artificial cerebrospinal fluid oxygenated with oxygen containing artificial cerebrospinal fluid.The rectal temperature was maintained at 37.0±0.2℃.Electrophysiological recording and stimulation the granulosa cells were recorded with axopatch-ld amplifier.Using Clampex 10.3 software,the potential is obtained through 1440 series analog-digital interface on personal computer.The recording electrode is made of borosilicate glass,filled with ACSF,with a resistance of 3-5m.Tactile stimulation of the ipsilateral whisker pads was performed by inflating(60 ms,60 psi)through 12 stainless steel tubes connected to the pressurized injection system.Synchronous electrophysiological recording was performed by using marster 8 controller(a.m.p.i.)and Clampex 10.3 software.The recording electrode is used to record the field potential induced by sensory stimulation through patch clamp amplifier,the recording part of cerebellum is searched by microscope,the recording glass electrode is placed in the recording part,the recording electrode is carefully punctured under the pia mater by the micromanipulator,after reaching the granular cell layer of cerebellum cortex,the sensory stimulation is given by blowing,and the field potential changes are recorded after finding the largest response recording part,perfusion of PTX、JNJ16259685 and D-APV in the cerebellar surface.The electrophysiological data were analyzed by using the software of clampfit 10.3.The experimental data were expressed by means of mean ± standard deviation,and the differences were evaluated by standard paired t-test or single ANOVA using SPSS analysis software.When P<0.05,it was considered that there was a difference between the mean values.[Results]1.N1 is the excitatory glutamatergic synaptic transmission of mossy fiber granulosa cells(MF-GC),while P1 is the GABA synaptic transmission of Golgi cells granulosa cells,while blocking the inhibitory afferent of Golgi cells significantly enhances the mf-gc synaptic transmission induced by sensory stimulation.2.Sensory stimulation induces high fidelity MF-GC excitatory glutamatergic synaptic transmission,accompanied by GABA energy feedback synaptic transmission(P1)of Golgi cells granulosa cells,while blocking the inhibitory afferent of Golgi cells can significantly enhance MF-GC excitatory synaptic transmission,suggesting that P1 plays an important role in the modulation of MF-GC synaptic transmission.3.Alcohol can prolong the latency of field potential response,decrease the N1 amplitude and increase the P1 rising time,suggesting that long-term alcohol intake can enhance the inhibitory component P1 and decrease the excitatory synaptic transmission.4.The attenuation time of P1,the ratio of AUC,N2/N1 and the inhibition time of the inhibitory component of the field potential response in the alcohol group increased,suggesting that the inhibitory component of synaptic transmission in the granular layer induced by sensory stimulation in the alcohol group increased significantly.5.Long term alcohol intake had no significant effect on Roff in granular layer induced by sensory stimulation.6.It was found that blocking mGluRl had no significant effect on P1,the inhibitory component of field potential response in granular layer of long-term alcohol intake mice induced by sensory stimulation.7.Block NMDA receptor,significantly inhibit the long-term alcohol intake mice’s sensory stimulation induced granular layer potential response,suppress the inhibitory component P1,suggesting that the long-term alcohol intake may enhance the inhibitory component of sensory stimulation induced granular cell layer potential response in the cerebellar cortex of alcohol rats through NMDA receptor.8.Activation of NMDA receptor in the control group significantly enhanced the field potential response of granular cells induced by sensory stimulation and increased the inhibitory component P1,suggesting that activation of NMDA receptor can enhance the inhibitory component of the field potential response of granular cells induced by sensory stimulation.[Conclusions]1.Facial sensory stimulation induces MF-GC to produce a high fidelity synaptic transmission response,which is manifested in glutamate mediated excitatory response and GABAA receptor-mediated inhibitory response.2.Long term ethanol intake enhanced GABAA receptor-mediated inhibitory response and significantly suppressed MF-GC excitatory glutamatergic synaptic transmission induced by sensory stimulation.3.The enhancement of inhibitory components of MF-GC synaptic transmission caused by long-term ethanol intake is dependent on NMDA receptor,but is not dependent on type 1 metabotropic glutamate receptor,suggesting that long-term heavy drinking may affect the fine motor regulation and motor learning function of cerebellum through NMDA receptor enhancing the inhibitory synaptic transmission of sensory information in granular layer. |
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