The Molecular And Pathological Mechanisms Of Chronic Sleep Deprivation Exacerbates Cognitive Impairments In APP/PS1 Alzheimer’s Disease Model Mice

Objective:To observe the effects of chronic sleep deprivation(SD)on short-term working memory,fear conditioning memory,spatial memory and synaptic plasticity in 8-monthold APP/PS1 double transgenic Alzheimer’s disease(AD)model mice,and to investigate the possible molecular and pathological mechanisms.Methods:1.Animal grouping and treatment8-month-old male APP/PS1 mice and wild type(WT)littermates were randomly divided into 4 groups(n=10-12/group): platform control WT mice(WT-PC),sleepdeprived WT mice(WT-SD),platform control APP/PS1 mice(APP/PS1-PC)and sleepdeprived APP/PS1 mice(APP/PS1-SD).The modified multiple platform method was utilized to establish a chronic SD model of APP/PS1 mice and WT mice.SD was given from 12:00 PM to 8:00 AM of the next day,and all mice were placed to their home cages from 08:00 AM to 12:00 PM.The SD process lasted for 21 days,then behavioral tests were performed to evaluate learning and memory abilities.2.Y maze spontaneous alternation testThe mice were put into the laboratory for 24 hours and the experiment began.Each mouse was placed successively in the triangle center region of the maze,and then the mice were allowed to explore freely in the maze for 8 min.The movement trajectory,the number of arm entries and order of exploration of the arms in the maze within 8minutes were recorded,and the percentage of correct spontaneous alteration spontaneous alternating was calculated.3.Morris water maze testThis experiment is divided into three parts: the hidden platform test,the probe test and the visible platform test.The hidden platform test was from the 1st to the 5th day,during which each mouse learned 4 times a day.The mice were put into the water from the four quadrants respectively,and the time was 60 s.If the mice found the platform within 60 s and stayed on the platform for 5 s,it was deemed to have successfully stepped on the platform.If the mice did not find the platform within 60 s,the experimenter should induce the mice to the platform.The hidden platform test recorded each mouse’s movement trajectory,the time mice took to find the platform(the escape latency)and its swimming speed.After the hidden platform test,the probe test was started(the 6th day of the experiment).Each mouse was tested twice.Mice were put into water from two quadrants except the target quadrant,and allowed to swim for 60 s in the maze.The swimming track,swimming speed,the number of platform crossing and swimming time in the target quadrant were recorded,and the swimming time percentage spent in the target quadrant was calculated.After the hidden platform test,the visible platform test was conducted,and each mouse was tested twice.The mice were put into water from two quadrants except the target quadrant,and the swimming track,swimming speed and the time each mouse arrived at the target platform were recorded.4.Fear conditioning testThis experiment includes two parts: training session and testing session.At the training session,each mouse was placed in the chamber and moved freely for 90s(Exploration),then underwent 5 cycles of 30 s sound stimulation(90 dB,2900HZ)accompanied by electrical shock(0.3 m A)at last 2 s,within a 90 s time span.The testing session was performed 24 h after training session.Each mouse was put into the conditioned fear box with different background,and 150 s of conditioning stimulus was given after 150 s of adaptation.The percentage of freezing in the period of Exploration,Pre-END,Pre-CS and CS were recorded and calculated.5.In vivo hippocampal electrophysiological recordingAfter the mice were anesthetized(5% chloral hydride,0.007 ml/g,i.p.),the head of the mice was positioned in a stereotaxic device.After the skull was removed,the stimulating electrode and recording electrode were lowered slowly into the hippocampus of the mice(the Schaffer collateral-CA1 synaptic pathway),and the field excitatory postsynaptic potentials(f EPSPs)could be observed.At this point,the inputoutput(I/O)was firstly drawn by giving the gradually increasing stimuli intensity,and then the 50% maximum stimulus intensity was selected to induce and record the stable f EPSP for 30 min,namely BASIC.Then the mice were given paired pulse stimulation,and the paired-pulse facilitation(PPF)of mice was recorded.Finally,long-term potentiation(LTP)in the hippocampus was induced by high frequency stimulation(HFS)and recorded for 60 min continuously.6.Western blotAfter in vivo hippocampal electrophysiological recording,mice were randomly selected from each group for Western blot.The mice were anesthetized(5% chloral hydrate,0.007 ml/g,i.p.)and the brain tissues of the mice were perfused with PBS and the hippocampal tissues on both sides were isolated.After the hippocampal tissue was ground and the protein was extracted,the total protein concentration was measured,the sample loading quantity was selected and the sample loading ran electrophoresis.After the proteins escaped,they were transferred to the membrane,and the primary antibody(SYP and PSD-95)were incubated at 4°C overnight after being sealed with 5% skim milk at room temperature for 2 h.On the second day,the secondary antibodies were incubated at room temperature for 2 h after washing the membrane for 3 times with TBST,and the membrane was washed again for 3 times.The bands were detected with an ECL kit,scanned with detection system,and the expression levels of SYP and PSD-95 were analyzed by software.7.ImmunohistochemistryAfter electrophysiological recording,the mice were randomly selected from each group.The brains of the mice were dissected,dehydrated,embedded and sliced with a thickness of 2 μm.The primary antibody(6E10 or Iba-1)was incubated overnight at4°C.The second antibody was incubated at room temperature for 2 h the next day.protein localization was visualized using diaminobenzidine(DAB)for 2 min,and the sections were lightly counterstained in Harris’ hematoxylin solution.After dehydration and vitrification,the sections were imaged with an optical microscope to observe the area of Aβ(6E10)plaques and microglia(Iab-1)in hippocampus of mice.Results:1.Chronic SD exacerbated short-term working memory impairment of APP/PS1 mice.In the Y maze test,compared with the WT-PC group,the percentage of correct spontaneous alternation of APP/PS1-PC group was significantly decreased(P<0.001),indicating that the short-term working memory of APP/PS1 mice was impaired.After chronic sleep deprivation,the percentage of correct spontaneous alternation of APP/PS1 mice was further decreased(P=0.002),indicating that chronic sleep deprivation aggravated the short-term working memory impairment of APP/PS1 mice.At the same time,there was no difference in the total arm entries representing the motor ability of mice among each group,indicating that the motor ability had no effect on the percentage of correct spontaneous alternation of mice.2.Chronic SD exacerbated long-term spatial reference memory impairment of APP/PS1 mice.Compared with WT-PC group,APP/PS1-PC group had a significantly longer the escape latency during the hidden platform test of the Morris water maze(day 3:P=0.024;day 4:P=0.005;day 5:P<0.001),indicating impaired long-term spatial learning in APP/PS1 mice.After chronic sleep deprivation,the escape latency of APP/PS1 mice decreased(day 3:P<0.001;day 4:P=0.027;day 5:P=0.034).In the probe test,the percentage of swimming time(P=0.001)and cross-platform times(P=0.044)in the target quadrant of mice in the WT-PC group were higher than those in the APP/PS1-PC group,indicating that APP/PS1-PC had impaired long-term spatial learning ability.After chronic sleep deprivation,the percentage of swimming time in the target quadrant(P=0.002)and the number of crossings(P=0.044)of APP/PS1 mice were further decreased.These results indicated that chronic sleep deprivation aggravates the impairment of long-term spatial learning ability in APP/PS1 mice.In the visible platform test,there was no significant difference the swimming time to arrive the platform,indicating that the swimming speed of the mice had no effect on the experimental results.3.Chronic SD exacerbated fear conditioning memory impairment of APP/PS1 mice.In the Pre-END period of training session,the freezing percentage of mice in APP/PS1-PC group was significantly lower than that of mice in WT-PC group(P<0.001),and chronic SD further decreased the freezing percentage of APP/PS1 mice(P=0.018).In the CS period of testing session,the freezing percentage of mice in APP/PS1-PC group was significantly lower than that of mice in the WT-PC group(P<0.001),and chronic SD further decreased the freezing percentage of APP/PS1 mice(P=0.025).4.Chronic SD aggravated the LTP depression in the hippocampus of APP/PS1 mice.The in vivo hippocampal electrophysiological recording was performed after behavioral tests.The input-output(I/O)curves in the hippocampus of the four groups increased with the increase of the stimulus intensity,and there was no significant difference in f EPSP induced by the same stimulus intensity,indicating that the basic synaptic transmission ability of the four groups was normal.After 30 min stable f EPSP was recorded in all the four groups,mice were given HFS,and LTP was successfully induced in all the other three groups except the APP/PS1-SD group.At 60 min after HFS,the f EPSP slope of APP/PS1-PC group was significantly lower than that of WTPC group(P=0.023),and at 0 min and 30 min after HFS,the f EPSP slope of APP/PS1-SD group was significantly lower than that of APP/PS1-PC group(0 min: P<0.001,30min: P=0.003).These results indicated that APP/PS1 mice had suppressed in vivo hippocampal LTP compared with WT mice.Chronic sleep deprivation further aggravated hippocampal synaptic plasticity suppression in APP/PS1 mice.Meanwhile,there was no significant difference in PPF(f EPSP2/f EPSP1)between the four groups(P>0.05),indicating that APP/PS1 gene mutation and chronic sleep deprivation did not affect the release of presynaptic neurotransmitters.5.Chronic SD downregulated the expression level of PSD-95 in the hippocampus of APP/PS1 mice.The expression level of SYP in WT-PC group was significantly higher than that in APP/PS1-PC group(P<0.001),and the expression level of PSD-95 in WT-PC group was also significantly higher than that in APP/PS1-PC group(P<0.001).After chronic sleep deprivation,the expression level of PSD-95 in the hippocampus of APP/PS1 mice was further decreased(P<0.001),but the expression level of SYP was not affected.6.Chronic SD increased amyloid β(Aβ)deposition and activation of microglia in the hippocampus of APP/PS1 mice.The deposition of Aβ plaques was observed in the hippocampus of APP/PS1 mice,and the Aβ plaques were significantly increased after chronic SD(P<0.001).At the same time,the Iba-1 positive area of APP/PS1-PC group was significantly higher than that of the WT-PC group(P<0.001),and chronic SD caused further increase of Iba-1positive area in APP/PS1mice(P<0.001).Conclusion:Chronic SD exacerbates the cognitive deficits in 8-month-old APP/PS1 mice by increasing Aβ deposition and microglia activation,reducing the expression of PSD-95 and aggravating the LTP suppression in hippocampus.