| Objective:To investigate whether suvorexant,a dual orexin receptor antagonist,could improve behavior circadian rhythm disturbance and alleviate cognitive impairment occurred in9-month-old APP/PS1 mice,and to investigate the possible neuroprotective mechanisms of suvorexant exerted in Alzheimer’s disease(AD).Methods:1.Animal grouping and drug administration8-month-old male APP/PS1 and wild type(WT)C57BL/6 mice with same genetic background were randomly divided into four groups:APP/PS1-Vehicle(APP/PS1-Veh),APP/PS1-Suvorexant(APP/PS1-Suv),WT-Vehicle(WT-Veh)and WT-Suvorexant(WT-Suv),n=15-20 for each group.Suvorexant(30 mg/kg)or equal volume of vehicle(0.5%methyl cellulose 400)was oral gavage administrated daily in the beginning of the light phase(ZT0)for 28 days.2.Recording of wheel-running activityFirstly,each mouse was maintained under a 12 h:12 h light dark(LD)cycle(light on at 06:00,off at 18:00),and the light intensity was 360 Lx during the light-on phase.All mice were acclimated for 1 week and then continuously recorded for 28 days.Then mice were transformed into a constant dark(DD)environment and recorded for another 14days.After the wheel-running activity recording was completed,locomotor activities of mice were calculated.The period,activity duration,robustness,amplitude,and intradaily variability(IV)under LD and DD conditions were performed using Actiview and Clocklab analysis software to measure the locomotor circadian rhythm and its stability.3.Cognitive behavioral tests(1)Y-maze spontaneous alternation test:Each mouse was placed at the intersection point of the three arms and allowed to explore the maze freely for 8 min.The total arm entries and the order of exploration of the arms were recorded.The spontaneous alternation percentage,an index of spatial working memory,was measured.(2)Morris water maze test:The experiment was performed 24 h after the Y maze test,including the hidden platform test,the probe test and the visible platform test.In the hidden platform test,each mouse was trained four times per day for 5 consecutive days,with the escape latency and swim path being recorded.On the sixth day,each mouse underwent a 60 s probe trial,and the searching behavior of the mouse in the target quadrant was measured.After the probe test,the visible platform test was performed to record the time each mouse arrived at the target platform.In addition,the swimming speed of each mouse during hidden platform test and probe test was recorded.4.In vivo hippocampal LTP recordingThe same mice used in the behavioral tests were used for the in vivo electrophysiological study.Mice were anesthetized with 5%chloral hydrate(0.007 ml/g,i.p.)and placed in a stereotaxic device for LTP recording.According to brain stereotaxic map,bound stimulating/recording electrode was inserted into the hippocampal Schaffer collateral-CA1 synaptic pathway.Baseline field excitatory postsynaptic potentials(fEPSPs)evoked by test stimulation were monitored for 30 min.Paired-pulse facilitation(PPF)was observed by using paired-pulse stimulation.LTP was induced by a high-frequency stimulation(HFS)protocol,and test stimuli were used again after HFS for a period of at least 1 h to record any change in slope of the fEPSPs.5.Immunohistochemistry experimentsThree mice were randomly selected from each group,perfused with phosphate-buffered saline(PBS)and 4%paraformaldehyde in order.The brains of the mice were rapidly dissected and post-fixed for 24 h at room temperature.The brain blocks were sliced into 10-?m-thick coronal sections by frozen sectioning.The sections were incubated overnight with anti-Aβ1_-16(6E10)primary antibody at 4°C,followed by incubation with secondary antibody for 2 h and DAPI for 30 min.The nuclei were stained with hematoxylin,gradient alcohol dehydration,xylene transparency,neutral gum seal.A light microscope was used to obtain images of Aβplaques in the SCN,hippocampus and cortex slices,with the area of Aβdeposits in each slice section being quantified.6.Western blot experiment5-6 mice in each group were anesthetized by 5%chloral hydrate in ZT2(light phase)and ZT14(dark phase)respectively.The brain was decapitated and the tissues of SCN and hippocampus were isolated to extract the total protein.Then,SDS-PAGE electrophoresis was carried,and the protein was transferred to the PVDF membrane.After5%BSA treatment 2 h,the primary antibody,second antibody and ECL were added in proper order.The relative gray values of target protein(PER1,CREB,pCREB,β-actin)were analyzed by using Alpha SA software.Results:1.Suvorexant treatment improved the abnormal circadian pattern of APP/PS1 mice.In the 12 h light/12 h dark cycle(LD)environment,the total daily activity of APP/PS1-Veh group was significantly lower than that of WT-Veh group(P=0.002),especially the average activity in ZT12-ZT24(dark phase)was significantly reduced(P=0.003).After suvorexant treatment,the total daily activity of APP/PS1 mice was increased,yet there was no significant difference compared with APP/PS1-Veh group(P=0.071).In addition,compared with the WT-Veh group,the relative amplitude(P<0.001)and robustness(P<0.001)of the APP/PS1-Veh group were significantly decreased,while the intradaily variability(IV)was increased(P<0.001).After suvorexant treatment,the relative amplitude(P=0.001)and robustness(P=0.003)of APP/PS1 mice were significantly increased,and IV was decreased(P<0.001).These results indicated that the locomotor circadian rhythm stability of APP/PS1 mice in the LD environment was impaired,while suvorexant treatment improved circadian rhythm stability and alleviated the circadian rhythm disturbance in APP/PS1 mice.After transformed into constant darkness(DD)environment,mice in the APP/PS1-Veh group showed scattered activity during the subjective day,and the free running period was significantly longer than that of the WT-Veh group(P<0.001).After suvorexant treatment,the free running period of APP/PS1 mice was shortened(P=0.021).Meanwhile,the total daily activity of mice in the APP/PS1-Veh group was significantly decreased(P=0.003).Compared with WT-Veh group,the average activity of mice in the APP/PS1-Veh group in CT0-CT12 was significantly higher(P=0.031),and the activity in CT12-CT24 was much lower(P=0.002).After suvorexant treatment,the total daily activity(P=0.073)and average activity in CT12-CT24(P=0.061)of APP/PS1 mice increased,but there was no significant difference between APP/PS1-Veh and APP/PS1-Suv group.In addition,compared with WT-Veh group,the activity duration of APP/PS1-Veh group was significantly prolonged(P<0.001),the relative amplitude(P=0.004)and robustness(P=0.005)significantly decreased,and IV significantly increased(P<0.001).The suvorexant treatment shortened the activity duration of APP/PS1 mice(P<0.001),increased the relative amplitude(P=0.004),decreased IV(P<0.001),and relatively increased the robustness but without statistical difference(P=0.051).These results indicated that the circadian rhythm stability of APP/PS1 mice in DD environment was disturbed,and suvorexant treatment increased the circadian rhythm stability and partially improved the circadian rhythm disturbance of APP/PS1 mice in DD environment.2.Suvorexant treatment ameliorated the cognitive impairments in APP/PS1 mice.In Y maze spontaneous alternation test,the percentage of right spontaneous alternation in APP/PS1-Veh group was significantly lower than that in WT-Veh group(P=0.001)and APP/PS1-Suv group(P<0.001).In the hidden platform test of Morris water maze,the APP/PS1-Suv mice exhibited longer escape latencies than WT-Veh group mice on day 2-5(P<0.001),while suvorexant treatment shortened the escape latency of APP/PS1 mice(day 2:P<0.01;day 3-5:P<0.001).On the 6th day of probe trial,suvorexant treatment significantly prolonged the swimming time percentage of APP/PS1mice in the target quadrant(P=0.005).At the same time,there was no significant difference of swimming speed during 6 days and the swimming time in visible platform among four groups(P>0.05).These results indicated that suvorexant treatment effectively rescued the working memory and long-term spatial memory impairments in APP/PS1mice.3.Suvorexant treatment reversed the LTP depression in the hippocampus ofAPP/PS1 mice.After behavioral tests,in vivo hippocampal fEPSPs and LTP in the CA1 region were recorded.There was no significant difference of baseline fEPSP slope 30 min before HFS and PPF induced by paired-pulse stimulation among four groups,indicating that APP/PS1gene mutation and suvorexant treatment did not affect baseline synaptic transmission and presynaptic neurotransmitter release.Immediately after HFS,the LTP was successfully induced in the four groups.However,the fEPSP slope in the APP/PS1-Veh group was gradually decreased with the prolongation of recording time.At 30 min(P<0.001)and 60min(P<0.001)post-HFS,the fEPSP slope in APP/PS1-Veh group was significantly lower than that in WT-Veh group,which was rescued by suvorexant pretreatment(30 min:P=0.015;60 min:P<0.001).4.Suvorexant treatment reduced the Aβpathology in the hippocampus and cortexof APP/PS1 mice.There was no 6E10 positive plaques detected in the SCN region of APP/PS1 mice.However,the Aβplaques were observed in the hippocampus and cortex of 9-month-old APP/PS1 mice.After suvorexant pretreatment,the Aβimmunopositive area in the hippocampus(P<0.001)and cortex(P<0.001)was significantly decreased.5.Suvorexant treatment restored the circadian rhythm of PER1 and pCREBexpression in the SCN and hippocampus of APP/PS1 mice.The expression of PER1 and pCREB in the SCN and hippocampus of WT mice showed obvious circadian rhythm.The expression of PER1 in both SCN(P<0.001)and hippocampus(P=0.008)for WT-Veh mice was higher in ZT2 and lower in ZT14,while the expression of pCREB in the hippocampus was lower in ZT2 and higher in ZT14(P=0.002).However,the expression of PER1 in both the SCN(P=0.737)and hippocampus(P=0.225)of APP/PS1-Veh mice lost the circadian rhythm.In addition,the expression of pCREB,the downstream protein of PER1,in the hippocampus of APP/PS1mice was also lost circadian rhythm(P=0.052).After suvorexant treatment,the circadian rhythmic expression of PER1 protein in SCN region was partially restored(P=0.054),and the circadian rhythmic expression of PER1(P=0.036)and pCREB(P=0.001)in hippocampus was restored.Conclusion:Suvorexant effectively alleviates the behavioral circadian rhythm disturbance and cognitive deficits observed in 9-month-old APP/PS1 mice by decreasing Aβdeposition in the hippocampus and cortex,improving hippocampal synaptic plasticity and restoring the circadian rhythm of PER1 and pCREB expression both in SCN and hippocampus,suggesting that suvorexant could be significant in the prevention and treatment of AD. |
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