| Objective:To investigate the effects of citalopram on behavioral performance and synaptic plasticity in female 3xTgAD mice and illuminate the possible mechanism.Methods:Experiments were performed using 5-month-old female 3xTgAD mice.The ageand sex-matched wild-type(WT)mice were used as control.Citalopram was given to the mice through drinking water at a concentration of 0.06 mg/ml(approximately 10 mg/kg/day).Mice were treated with citalopram or water from 5 months of age for 3 months and divided into 4 groups: WT + water,WT + citalopram,3xTgAD + water and 3xTgAD + citalopram.Behavioral tests were performed since 7 months old,including the following tests.(1)The open feld test(OFT)was used to measure locomotion,exploration,and anxiety-like behavior.Each mouse was placed in the apparatus for a 10 min period.The total distance travelled in the open field and time spent in inner square were measured.(2)Y maze test was used to assess working memory.Each mouse was put into the intersection of the arms,and allowed to move freely through the maze during an 8-minute session.The total number and the order of arm-entry were recorded.The correct rate of spontanious alternation behavior was calculated.(3)Morris water maze(MWM)test were used to evaluate the spatial learning and memory ability of mice.The test was carried out in a circular tank containing tap water.The swimming traces of mice were recorded and analyzed.For the hidden platform task,a platform was set 1 cm below the water surface and each animal was trained four times per day for 5 consecutive days to find the platform.Te escape latency was used to assess learning ability.On day 6,a probe trial was conducted with no platform in the pool for each animal to evaluate the ability of memory retention by measuring the number of platform crossings.(4)The elevated plus maze(EPM)test was used to evaluate anxiety-like behavior in mice.The metal apparatus was raised 50 cm above ground and consisted of two opposite open arms and two opposite closed arms which extended from a common central platform.The mice were allowed to move freely in the maze for 5 min.Total distance travelled in the maze and time spent in the open arms were recorded.(5)The tail suspension test(TST)was used to assess depression-like behavior in mice.The mouse was suspended by the tail from a hook in a white-painted box for 5 min.Movements were recorded by a camera.Immobility time was measured.The effect of citalopram on synaptic plasticity was tested by in vivo hippocampal long-term potentiation(LTP)recording.The mice were anesthetized with chloral hydrate(350 mg/kg,i.p.)and placed in a stereotaxic device for acute surgery and LTP recording.A hole with an approximate 2.0 mm diameter was drilled on the skull.A concentric stimulating electrode was placed at the Schaffer collateral/commissural pathway.A recording electrode was inserted into the CA1 region of the hippocampus to record feld excitatory postsynaptic potentials(fEPSPs)in stratum radiatum.An electronic stimulator and a coupled isolator were used to generate pulse stimulation.The signals from the recording electrode were amplifed by a multichannel biological signal acquisition/processing system.Test stimulation was given for at least 15 min to establish a stable baseline fEPSPs.High-frequency stimulation(HFS)was applied to induce LTP of fEPSP.After the HFS,fEPSPs were recorded for 1 h.Te slope of fEPSPs was normalized to basal fEPSPs and averaged.Area under the curve(AUC)that showed time-course response was measured from 0 to 10 min and 21 to 60 min afer HFS.Paired-pulse facilitation(PPF)was also measured before HFS to analyze presynaptic functions.Afer LTP recording,the mice were euthanized at once.Brains were rapidly removed and hippocampi and cortex were dissected from the brains and immediately frozen and stored.Hippocampal and cortical tissues were homogenized.The concentrations of Aβ were measured using a commercial ELISA kit according to the manufacturer’s protocol.Te extracts for detergent soluble Aβ ELISA from hippocampal tissues were also used to perform western blot analysis.Proteins were separated by electrophoresis with the 12% SDS-PAGE and transferred onto PVDF membranes.Te blot was probed with A8717(Sigma,USA)to detect APP and CTF and rabbit anti-β-actin to control for loading differences,followed by peroxidase-conjugated goat anti-rabbit IgG.The protein bands were visualized by ECL detection reagents.Western blot images were quantitated using ImageJ.Results:(1)In OFT,the total running distance by 3xTgAD mice was decreased compared to that in WT mice(P < 0.001).Citalopram treatment signifcantly increased total distance in WT mice(P = 0.017),but not in 3xTgAD mice(P = 0.123).3xTgAD mice spent more time in inner square than WT mice(P = 0.009),while Citalopram treatment had no signifcant effect on it in WT mice(P = 0.308)and 3xAD mice(P = 0.290).(2)In EPM test,the total running distance by 3xTgAD mice was less than that in WT mice(P = 0.003),and citalopram treatment did not affect the distance in WT mice(P = 0.106)and 3xTgAD mice(P = 0.827).Time spent in open arms was signifcantly increased in 3xTgAD mice when compared to WT mice(P = 0.012),but citalopram treatment did not affect the time spent in open arms in WT mice(P = 0.387)and 3xTgAD mice(P = 0.184).(3)In TST,no signifcant difference in immobility time was found between 3xTgAD and WT mice,and citalopram treatment had no effect on the immobility time in WT mice or 3xTgAD mice(P > 0.05).(4)In Y maze test,the total arm entries of 3xTgAD mice were significantly less than those of WT mice(P<0.001),and citalopram treatment did not affect arm enties of WT mice(P = 0.395)or 3xTgAD mice(P = 0.925).There was no significant difference in spontaneous alternation between 3xTgAD mice and WT mice,and citalopram treatment had no effect on spontaneous alternation in WT mice or 3xTgAD mice(P > 0.05).(5)In Morris water maze test,the escape latency of all mice decreased over 5-day training period(P < 0.001).3xTgAD mice spent longer time to fnd the hidden platform than WT mice over the course of the 5 days(P < 0.05).Citalopram treatment resulted in a significantly greater decrease in escape latency in 3xTgAD mice(P < 0.05).For the probe test,the number of platform crossings of 3xTgAD mice was signifcantly fewer than that of WT mice(P = 0.004),and citalopram reversed the effect(P = 0.003).(6)In hippicampal LTP recording,genotype and citalopram treatment did not affect the AUC from 0 to 10 min(P > 0.05).AUC from 21 to 60 min was signifcantly smaller in 3xTgAD mice(P = 0.010),and citalopram reversed the effect(P = 0.047).Genotype and citalopram had no signifcant effects on the PPF(P > 0.05).(7)In ELISA,levels of detergent soluble Aβ40 and Aβ42 were below the limit of detection in hippocampus and cerebral cortex samples in 3xTgAD mice.The insoluble Aβ40 concentrations in the hippocampus and cerebral cortex samples from citalopram-treated 3xTgAD mice were signifcantly lower than the concentrations in water-treated 3xTgAD mice(P = 0.018 and P = 0.0348).However,citalopram did not have significant effect on the levels of insoluble Aβ42 in the hippocampus and cerebral cortex(P > 0.05).(8)In western blot,the levels of APP and CTFβ in 3xTgAD mice were higher than those in WT mice(P = 0.001 and P < 0.001),and citalopram treatment signifcantly decreased the amount of APP and CTFβ in 3xTgAD mice(P = 0.018 and P < 0.023).Conclusions:(1)At the age of 7-8 months,female 3xTgAD mice showed spatial memory impairment,and chronic administration of citalopram signifcantly ameliorate memory deficit.The working memory of female 3xTgAD mice was intact at this age.(2)7-8 month-old female 3xTgAD mice exhibited apathy and disinhibition compared to WT mice,and citalopram did not work on it.At this age,female 3xTgAD mice did not present depression-like behavior.(3)Chronic administration of citalopram signifcantly improved synaptic plasticity in female 3xTgAD mice.(4)Chronic administration of citalopram signifcantly decreased insoluble Aβ in female 3xTgAD mice by inhibiting APP expression or increasing α-secretase enzymatic activity. |
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