Regulation Of LiCl On LFPs In Hippocampal CA1 Of APP/PS1 Mice

Background and objectiveAlzheimer's disease(AD)is one of neurodegenerative diseases that is common in the elderly.With the rapid aging of the population,around 115 million people worldwide will be living AD by 2050.Currently,there are large numbers of pathological and clinical researches on AD,but no effective treatment has been found.Recent studies have found that there is a lowering of network activity in the brain of AD patients and AD model mice,which has become hot spot in AD research.In the pathogenesis of AD,the A? cascade hypothesis has been widely accepted.Abnormally high amount of A? in the brain of patients with AD is thought to be neurotoxic,leading to synaptic damage,which is highly correlated with early cognitive defects of AD.In recent years,it has been proved that A?1-42 can damage the function of neural network which is important to cognitive function,resulting in the decreased energy of local field potentials(LFPs).Especially,it was discovered that theta oscillations(4-12Hz)and gamma oscillations(30-80Hz)in the hippocampus of AD patients are reduced.LFPs reflect the firing activity of neuron population and is the linear sum of postsynaptic potential of neurons in local brain regions.Neurons interact with each other through excitatory and inhibitory synapses,generating rhythmic oscillations to process information in the brain.Especially,theta and gamma oscillations in the hippocampus are believed to be the basis of cognitive processes such as attention,sensory perception and learning and memory.Studies have found a close relation between LFPs and synaptic plasticity.Changes in neurotransmitters and the inhibition and activation of corresponding receptors affect the generation of rhythmic oscillations in LFPs.Recent studies have found that the Wnt/?-catenin signaling pathway is closely related to the occurrence and development of AD,and this pathway is involved in the synaptic plasticity of the brain and the regulation of memory processes.Abnormal Wnt/?-catenin signals were found in the brain tissues of AD patients and various AD mouse models.Lithium chloride(LiCl),a drug used to treat bidirectional mental disorders,is glycogen synthase kinase-3?(GSK-3?)inhibitor.Studies have found that LiCl can reduce the damage of A? neurotoxicity to brain tissue.The molecular mechanism is that LiCl inhibits GSK-3? activity and thus activates Wnt/?-catenin signal pathway.However,most studies on the neuroprotective effects of LiCl focus on the changes of molecular and biochemical indicators such as A? and Tau protein,but there are few reports on whether it can effectively reverse the abnormal neural network activity in AD brains caused by A?.Therefore,we use the APP/PS1 transgenic mice as the AD model to test the LiCl effect on the LFPs in hippocampal CA1 region.With administration of LiCl,i.p,for continuous 2 months,LFPs were recorded using the technology of multi-channel in vivo recording in order to observe theta,gamma oscillations,and corresponding behavior change and molecular biological technology were tested as well.Meanwhile protein expression of synaptophysin(SYN)and post synaptic density-95(PSD95),and those related to Wnt/?-catenin signaling pathway was also tested to illustrate the potential underlying mechanism.Methods1.Genotype identification of APP/PS1 mice.APP/PS1 transgenic mice were paired with C57BL/6 wild type(WT)mice in a ratio of 1:2 for reproduction,and the offsprings were included in the following experiment.At the age of one month,DNA was extracted from the mouse tail,and the target gene APP was amplified by PCR and seperated inagarose gel by electrophoresis.2.Grouping of animals.Male APP/PSl positive transgenic mice and WT mice were divided into 4 groups,10 in each group,coding as WT+NS group,WT+LiCl group,AD+NS group and AD+LiCl group.LiCl was dissolved in 0.9% normal saline(NS),which was given a dose of 6 mmol/Kg.Mice in each group were intraperitoneally injected with LiCl solution or NS of the same volume at the age of 6 months,once a day,for 2 months.3.Morris water maze was used to detect the spatial learning and memory ability.The Morris water maze was tested for 6 days,the first 5 days were for the place navigation test,in which the mice were trained to find a hidden platform.Escape latency was collected from each mice in all 4 groups.On the 6 day mice were for the spatial probe test,in which the number of crossing times that the animals passed by the target platform and the percentage of time the mice spent in the target quadrant were recorded and calculated.4.Multi-channel LFPs in hippocampus CA1 were recorded in vivo.After the Morris water maze was completed,mice in each group were implanted with four-channel electrodes in the hippocampus CA1.Then the LFPs in hippocampus CA1 was recorded 5 days after recovery.The LFPs data were imported into software Matlab to analyze.5.The expression of A?1-42 in hippocampal CA1 was detected by immunofluorescence.After the Morris water maze and electrophysiological test were completed,the mice were perfused with 4% PFA and brain tissue was taken.After frozen sectioning,immunofluorescence staining was performed using antibody to detect the deposition of A?1-42 in hippocampal CA1.The percentage of A?1-42 positive area in the observed area was analyzed by software IPP6.0.6.Western blotting was used to detect the expression of Wnt-related proteins.After the Morris water maze and electrophysiological test were completed,the hippocampal tissues were rapidly isolated from each group of mice,and the protein quantification of GSK-3?,P-GSK-3?-ser9,?-catenin and CyclinD1 were performed by western blotting.7.Western blotting was used to detect the expression of synaptic proteins.After the Morris water maze and electrophysiological test were completed,the hippocampal tissues of each group were rapidly isolated,and the protein quantification of SYN,PSD95 were performed by western blotting.8.Statistical analysis.The experimental results were expressed as Mean±S.E.M,the LFPs data were imported into software Matlab 2015 b to analyze,and software SPSS 21.0 was used for statistical analysis.The data of place navigation test of Morris water maze adopted repeated measurement ANOVA,and the rest data adopted one-way ANOVA.The difference was statistically significant at P<0.05.Results1.Through genotype identification,the DNA of the offspring mice can be amplified to an APP band of about 350 bp,which can be considered as APP /PS1 transgenic mice.2.During the first 5 days of the place navigation test,the time it took for each group of mice to find the hidden platform gradually decreased with the increase of training days.Compared with WT+NS group,the escape latency of AD+NS group was significantly increased on the days of 3,4 and 5(P<0.001,P<0.01,P<0.01).Compared with the AD+LiCl group,the escape latency was significantly shortened on the days of 4 and 5(P<0.05,P<0.05).On the 6th day of spatial probe test,compared with the WT+NS group,the times of crossing the platform and the percentage of time spent in the target quadrant in AD+NS group were significantly reduced(P<0.001,P<0.001).Compared with the AD+NS group,The times of crossing the platform and the percentage of time spent in the target quadrant in the AD+LiCl group were significantly increased(P<0.05,P<0.01).3.The results of multi-channel in vivo recording showed that compared with WT+NS group,the power of the theta and gamma oscillations in the LFPs in AD+NS group mice were significantly decreased(P<0.001,P<0.001).Compared with AD+NS group,the power of the theta and gamma oscillations in AD+LiCl group were up-regulated(P<0.05,P<0.01).4.The results of immunofluorescence staining showed that compared with the WT+NS group,the percentage of A?1-42 positive area in the hippocampal CA1 of mice in AD+NS group was significantly increased(P<0.001).Compared with AD+NS group,the deposition of A?1-42 in the hippocampal CA1 was significantly reduced in AD+LiCl group(P < 0.01).5.Western blotting analysis of results showed that there is no significant difference in the expression of GSK-3? between the groups(P > 0.05).Compared with the WT+NS group,The expression level of P-GSK-3?-ser9(P<0.01),?-catenin(P<0.01)and CyclinD1(P<0.001)in the AD+NS group is significantly lowered.Compared with the AD+NS group,the expression levels of P-GSK-3?-ser9(P<0.05),?-catenin(P<0.05)and CyclinD1(P<0.01)were up-regulated in the AD+LiCl group.6.Western blotting results showed that the expression of SYN(P<0.001)and PSD95(P<0.001)in AD+NS group was significantly lower than that in WT+NS group,and the expression of SYN(P<0.05)and PSD95(P<0.01)in AD+LiCl group was significantly higher than that in AD+NS group.ConclusionThe administration with LiCl alleviated the lowering of power of theta and gamma oscillations in hippocampal CA1 of APP/PS1 mice.Its possible molecular mechanism is activation of Wnt/?-catenin pathway,reduction of the neurotoxicity of A?1-42,and enhance of the expression of hippocampal synapse related proteins SYN and PSD95,thus improving the electrical activity of the neural network and learning and memory function.