| ObjectiveThe external information is transmitted to cerebellar cortex through mossy fiber granule cell parallel fiber pathway,and then transmitted and integrated through molecular layer interneuron(MLIs),which is output from Purkinje cell(PC)to cerebellar deep nucleus,and finally participates in motor coordination and motor learning.Alzheimer’s disease(AD)is a degenerative disease of central nervous system,which usually involves the external information synaptic transmission in cerebellar cortex,which in turn impairs motor coordination and motor learning ability,but the mechanism of the effect of AD on information synaptic transmission in the cerebellar cortex and motor coordination and motor learning is not clear.Therefore,we employed 5XFAD model mice to study the effects of AD on the sensory information synaptic transmission and plasticity in MLI-PC synapses in the molecular layer of the mouse cerebellar cortex by electrophysiological recording,pharmacology and ethology methods,and to explore the damage of ad on motor coordination and motor learning function.Methods1.In this study,PCR amplification technology was first used to identify the genotype of 5 × FAD transgenic mice,and 5 × FAD positive mice were selected as the model(AD)group.The purpose was to screen out the successfully transformed 5 ×FAD genes.The pups that failed to transfer the gene were the wild-type negative control group.2.In the electrophysiological experiment,5 × FAD mice aged 9-12 months were used,with 10 mice in each group.The weight of 5 × FAD mice was about 30-40g.Intraperitoneal injection of urethane(1.3 g/kg)of 5 × FAD anesthesia in mice,row after endotracheal intubation through the ears and mouth holder will be 5 × FAD mice on the homemade record on the shelf in the body of experimental animals,cut open the mice skin and muscles of the head,skull exposure,in preparation for recording area around making perfusion slot,line open-brain surgery,after carefully remove the dura mater with oxygen filling of the surface of the artificial cerebrospinal fluid(ACSF)epidemic cerebrospinal meningitis,at the same time,with temperature to maintain the instrument to control the temperature of 37+0.2℃.The ipsilateral whisker pad was used for tactile stimulation(the duration of stimulation was 10ms and the intensity of stimulation was 60 psi).Through Marster 8 controller and a simultaneous Clampex 10.3 software tactile stimuli and electrophysiological recording,recording electrodes with ACSF,impedance is about 3-5 M Ω.Use record electrode by patch clamp amplifier sensory stimulation induced potential,find the cerebellum corresponding record part with a microscope,using micro meter will record carefully Pierce pia mater,reaching the position of the Purkinje fibers of the cerebellum,the sensory stimulation of the wind is given,looking for parts after the biggest reaction,record the change of the field potential,observe the AD mice synaptic transmission of the cerebellum change caused by the molecular layer of potential change;1 Hz(10 ms,60 psi)stimulation was administered to investigate the synaptic transmission of mli-pc in AD mice before and after stimulation.Clampfit 10.3 software was used for electrophysiological data analysis,and all data were presented as mean ± standard error.SPSS 17.0 software was used for one-way variance analysis and two-factor variance analysis.P<0.05 was considered to be statistically different between the experimental groups.3.Behavioral tests were conducted on 5 × FAD mice and wild type(WT)mice aged 3 months,6 months,9 months and 12 months.The movement coordination and motor learning ability of 5 × FAD mice and wild type mice aged 5 × FAD mice and 12 months were respectively observed through the movement learning function testing experiment of walking disorder analyzer and stick rotating instrument.After the data were sorted out,SPSS 17.0 analysis software was used for statistical analysis of the data.One-way analysis of variance and two-factor analysis of variance were used to compare the mean values of each group.P<0.05 was considered to be statistically different.Results1.Given facial blow stimulation(10ms,60psi),the incubation period of PC response induced by facial blow stimulation in AD mice was significantly longer than that in WT mice(P<0.05),the amplitude of P1 in AD mice and the Pause time of PC discharge were significantly lower than that in WT mice(P<0.05),and the amplitude CV of P1 in AD mice was significantly higher than that in WT mice(P<0.05).2.Give a continuous facial hair drier stimulation(1 hz)in mice,found the AD mice inhibitory response amplitude and discharge the Pause time,(Pause)were significantly lower than that of WT mice showed the AD to MLI-PC sensory information synaptic transmission is an obvious damage(P<0.05),caused the MLIPC synaptic transmission latency period longer,the amplitude decreased,SS discharge Pause time shortened,and the amplitude variation coefficient increased,prompt AD damage MLI-PC synaptic transmission.3.Compared with WT group,there was no significant difference in the amplitude of molecular layer field potential N1 in AD mice induced by facial hair dryer stimulation(P>0.05),indicating that AD had little effect on the excitatory afferent of parallel fibers.4.1Hz sensory stimulation can induce significant mli-pc synaptic transmission LTD in WT mice PC,but the expression of LTD in AD mice PC is not obvious,suggesting that AD damages the induction and maintenance of mli-pc synaptic plasticity.5.AD has obvious damage effect on mli-pc sensory information synaptic transmission,which affects external information transmission and processing,and may damage cerebellum motor coordination function.6.Compared with the WT group,the error rate and error time of the limbs of AD mice during walking were significantly increased,which were gradually aggravated with the increase of the month age of AD mice(P<0.05),indicating the impairment of AD motor coordination function.7.Mice in the WT group and AD group were trained for 5 days to observe the duration of the stick rotating instrument.The duration of the stick rotating instrument in AD mice did not increase with the number of times of training(P<0.05),and the learning ability of AD mice was low and increased with the increase of month age.8.Compared with the WT group,the duration of the stick rotation in mice was significantly shortened(P<0.05),and the rotation speed requirement of the stick rotation instrument was significantly reduced.Alzheimer’s disease had a significant impairment on the motor learning and motor coordination function of mice,which was aggravated with the increase of the age of months.Conclusion1.AD has an obvious damaging effect on the synaptic transmission of MLI-PC sensory information,resulting in the prolonged latency,decreased amplitude and increased coefficient of variation of MLI-PC synaptic transmission,but it has no obvious effect on the synaptic transmission of PF-PC.2.The induction and maintenance of MLI-PC synaptic plasticity induced by AD injury sensory stimulation significantly impaired the motor learning and motor coordination functions of mice,which were aggravated with the increase of the age of months. |
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