Effects Of Fluoxetine On The Neuronal And Synaptic Pathological Changes In The Medial Prefrontal Cortex Of The Transgenic AD Mice

Part oneEffects of fluoxetine on the behaviors and medial prefrontal cortex in the AD transgenic miceObjective:Studies have shown that fluoxetine(FLX)can improve the cognitive function of AD,but the mechanism remains unclear.The medial prefrontal cortex(MPFC)is one of the major brain regions involved in AD.Multiple studies have shown that in the early stages of Alzheimer’s disease(AD)disease,a certain proportion of AD patients had significant executive dysfunction,and AD patients had significant executive dysfunction and associated prefrontal cortical atrophy.Therefore,this study focused on the effects of fluoxetine on the behaviors,MPFC volume,Aβ protein deposition and Tau protein phosphorylation in AD transgenic mice.Methods:Forty 8-month-old male APP/PS1 double transgenic AD mice and forty 8-month-old male wild-type mice of the littermate were selected.APP/PS1 transgenic mice of the littermate were randomly divided into normal saline control group(AD+NS)and fluoxetine intervention group(AD+FIX),and the wild-type mice were used as wild-type saline group(WT+NS)and wild-type fluoxetine intervention group(WT+FLX).Fluoxetine(10 mg/kg/d)was intraperitoneally injected into AD+FLX group and WT+FLX group for 2 months.At the same time,the littermate AD+NS group and WT+NS group mice were intraperitoneally injected with the same dose of normal saline.Each group mice received an intraperitoneal injection of BrdU(50 mg/kg/d)for 7 days at the 5th week of the fluoxetine intervention.The body mass of 4 group mice were tested weekly.After 2 months of intervention,the mice in each group were tested in the open field to test the anxiety-like behavior of the mice.The Morris water maze was used to test the spatial learning and memory ability of the mice.The Y-maze was used to test the execution ability and working memory of the mice.At the end of the behavioral test,6 mice were randomly selected from each group.After anesthesia,4cr%paraformaldehyde was administered through cardiac perfusion.Each mouse brain was divided into left and right hemispheres,dehydrated in a gradient of sucrose water,embedded in an OCT embedding agent,and then cut into 60 μm thick serial sections along the coronal plane in a cryostat.From the sections containing area MPFC,every fourth section was sampled in a systematic-random manner and four sets of sampled sections were acquired in the end.One sets of sampled sections was randomly selected and placed on glass slides to dry and then stained with toluidine blue nissl’ s staining.Under the optical microscope,the cell structure was used to delineate the MPFC boundary,and the Cavalieri’s principle was applied to each group.The MPFC volume of the mice was measured.The remaining frozen tissue sections were observed by immunohistochemcal staining and immunofluorescence double labeling to observe Aβ plaques in MPFC.After the behavioral test,another 6 mice randomly selected from each group were anesthetized.After direct neck dissection,the mouse brain was dissected and the MPFC was isolated.The MPFC was homogenized and the protein was detected to detect the toxicity AP40,the toxicity Aβ42 and phosphorylated Tau protein(p-Tau)in MPFC.Results:1.Body mass:There was no significant difference in body weight between the 4 groups of mice before FLX intervention.However,FLX intervention from the first week of FLX intervention to 2 months ended,the body mass before intervention of each group mice was no significant difference compared with post-intervention.There was no significant change in body mass of the 4 groups from the beginning of FLX intervention to the end of the intervention.2.Open field test:In this study,after FLX intervention,the parameters of the open field test including the total distance of activities in the open field,the central distance of the open field,the percentage of the central distance of the open field,the activity time of the central area of the open field,the percentage of the activity time of the open field and the average speed were not significantly different among the four group mice.3.Water maze test:There was no significant difference in the mean swim speed among the 4 group mice.The escape latency of the AD+NS mice was significantly longer than that of the WT+NS mice(p<0.01).The escape latency of the AD+FLX group mice was significantly shorter than that of the AD+NS group mice(p<0.05).The escape latency of the AD+FLX group mice was not significantly different from the escape latency of the WT+FLX mice.The number of crossing platforms of the AD+NS group mice was significantly lower than that of the WT+NS group mice.The number of crossing platforms of the AD+FLX group mice was significantly higher than that of the AD+NS group mice(p<0.05),and the percentage of swimming time in target quadrant in the 4 group mice was not significantly different.4.Y-maze test:Compared with the WT+NS group mice,the percentage of correct alternating times in the AD+NS group mice was significantly reduced(p<0.01).Compared with the AD+NS group mice,the percentage of correct alternating in the AD+FLX group mice was significantly increased(p<0.01).5.Stereological results of MPFC volume;The MPFC volume of the AD+NS group mice was significantly smaller than that of the WT+NS group mice(p<0.01),while after fluoxetine intervention,the MPFC volume of the AD+FLX group mice was significantly larger than that of the AD+NS group mice(p<0.05),and there was no significant difference in MPFC volume between the AD+FLX group mice and the WT+FLX group mice.6.There were significant differences in the expressions of insoluble toxic Ap40 and Aβ42 in MPFC among the WT+NS group mice,the WT+FLX group mice,the AD+NS group mice and the AD+FLX group mice.The expressions of insoluble toxic Aβ40 and Aβ42 in the MPFC of the 10-month-old AD+NS group mice were significantly lower than those of the WT+NS group mice(p<0.01),while there was no significant difference in the insoluble toxic Aβ40 between the AD+NS group mice and the AD+FLX group mice.The expression of insoluble toxic Aβ42 in the MPFC of the AD+FLX group mice was significantly lower than that of the AD+NS group mice(p<0.01).7.There was no senile plaque deposition in the MPFC of the 10-month-old WT+NS group mice and the WT+FLX group mice,but there was a small amount of A[B plaque deposition in the MPFC of the littermate AD+NS group mice and AD+FLX group mice.The number of Aβ plaques deposited in the MPFC of the AD+FLX group mice was less than that of the AD+NS group mice.8.After quantifying the number of p-Tau+(ser396)cells per unit area,the density of p-Tau+(ser396)cells in MPFC of the AD+NS group mice was significantly higher than that of the littermate WT+NS group mice(p<0.01),and the density of p-Tau+(ser396)cells in MPFC of the AD+FLX group mice was significantly lower than that of the AD+NS group mice(p<0.05).ELISA results indicated that the expression of p-Tau protein in MPFC of 10 month-old AD+NS group mice was significantly higher than that of the WT+NS group mice(p<0.01),and the expression of p-Tau in MPFC of the AD+FLX group mice was significantly lower than that of the AD+NS group mice(p<0.01).Conclusions:1.The fluoxetine intervention could significantly improve the spatial learning and memory ability of the transgenic AD mice.2.10-month-old APP/PS1 transgenic AD mice had working memory impairment,and two month intervention of fluoxetine could significantly improve the executive ability and working memory of the transgenic AD mice.3.At 10-month-old,there was senile plaque deposition in the MPFC of APP/PS1 transgenic AD mice,and the expressions of insoluble toxic Aβ40 and Aβ42 were significantly increased,while FLX treatment could reduce the formation of senile plaques and reduce the expression of Aβ424.Accurate stereological results showed that the MPFC volume of APP/PS1 transgenic AD mice at 10-month-old was significantly atrophy,and the two month fluoxetine intervention significantly improved the atrophy of MPFC in the transgenic AD mice.Part twoEffects of fluoxetine on the neurons in the medial prefrontal cortex of transgenic AD mice and the possible mechanism of the effectsObjective:To study the effects of fluoxetine on the neurons in the medial prefrontal cortex of transgenic AD mice and the possible mechanism of the effects in order to provide experimented basis for searching the new intervention methods for the prevention and treatment of AD.Methods:After the end of the behavioral tests,the two sets of sampled sections were randomly selected from the eight set of the sampled sections containing area MPFC for toluidine blue staining and anti-DCX immunohistochemcal staining.The number of neurons in the MPFC and the number of DCX+neurons were calculated with the stereological method.The remaining frozen tissue sections was stained with immunofluorescence double labeling technique to examine the 5HT4R+/NeuN+neurons and the new neurons in the MPFC of the four group mice.Results:1.The stereological results of the number of the neurons in MPFC:The total number of the neurons in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.05),and the total number of the neurons in the MPFC of the AD+FLX group mice was significantly more than t that of he AD+NS group mice(p<0.05).The total number of the neurons in the MPFC was not significantly different between the AD+FLX group mice and the WT+FLX group mice.2.The stereological results of DCX+(newborn neuron)in MPFC:The total number of the DCX+neurons in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.01),and the total number of the DCX+neurons in the MPFC of the AD+FLX group mice was significantly more than that of the AD+NS group mice(p<0.01).The total number of the DCX+neurons in the MPFC of the AD+FLX group mice was significantly less than that of the WT+FLX group mice(p<0.05).3.Immunofluorescence staining of the BrdU+/NeuN+neurons(mature newborn neurons):The number of the BrdU+/NeuN+neurons per unit area indicated that there were significant differences in the BrdU+/NeuN+neuron density among the WT+NS group mice,the WT+FLX group mice,the AD+NS group mice and the AD+FLX group Pice.The density of the BrdU+neurons in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.01),while the density of the BrdU+/NeuN+neurons in the MPFC of the AD+FLX group mice was significantly more than that of the AD+NS group mice(p<0.05).4.Immunofluorescence staining results of 5HT4R+/NeuN+neurons:The number of the 5HT4R+/NeuN+neurons per unit area indicated that there were significant differences in the density of the 5HT4R+/NeuN+neurons in the MPFC of the WT+NS group mice,the WT+FLX group mice,the AD+NS group mice and the AD+FLX group mice.The density of the 5HT4R+/NeuN+neurons in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.01),while the density of the 5HT4R+/NeuN neurons in the MPFC of the AD+FLX group mice was significantly more than that of the AD+NS group mice(p<0.05).Conclusions:1.At 10-month age,the neurons in the MPFC of APP/PS1 transgenic AD mice were significantly lost.FLX intervention could delay the loss of the neurons in the MPFC of transgenic AD mice.2.At 10-month age,the ability of neurogenesis and survival of the new neurons in APP/PS1 transgenic AD mice was decreased.FLX intervention could promote the neurogenesis and survival of the newborn neurons in the MPFC of transgenic AD mice.3.At 10-month age,the expression of 5HT4R in MPFC of the transgenic AD mice was significantly decreased,and FLX intervention could increase the expression of 5HT4R in the neurons of the MPFC of transgenic AD mice,which suggested that FLX may protect the loss of neurons and improve the neurogenesis and survival of the newborn neurons in the MPFC of transgenic AD mice through increasing or activating the expression of 5HT4R.Part three Effects of fluoxetine on the synapses in the medial prefrontal cortex of the AD transgenic mice and the possible mechanism of the effectsObjective:To study the effect of fluoxetine intervention on the synapses in the MPFC of the early transgenic AD mice and the possible mechanism of the effects in order to provide an experimental basis for seeking the new interventions for the prevention and treatment of AD.Methods:After the end of the behavioral tests,the two sets of the sampled sections were randomly selected from the eight sets of the sampled sections containing area MPFC for anti-spinophilin immunogold staining.The number of spinophilin+spines(synapses)were calculated using the stereological method.The remaining frozen tissue sections were stained with inlunofluorescence double labeling technique to examine the 5HT1AR+/NeuN+neurons and anti-PSD95+synapses in the MPFC of the four group mice.After behavior tests,another 6 mice were randomly selected from each group were anesthetized.After direct neck dissection,the mouse brain was dissected and MPFC was isolated.The protein was homogenized and the β-catenin、GSKβ3、p-GSK3β、SYP、BDNF and c-fos protein in MPFC were detected with ELISA.Results:1.The stereological results of the spinophilin+ spines(synapses)in the MPFC:The total number of the spinophilim+ spines in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.05),and the total number of the spinophilin+spines(synapses)in the MPFC of the AD+FLX group mice was significantly more than that of the AD+NS group mice(p<0.05).The total number of the spinophilin+spines in MPFC was not significantly different between the AD+FLX group mice and the WT+FLX group mice.2.Immunofluorescence staining results of the 5HT1AR+/NeuN+neurons:The number of the 5HT1AR+/NeuN+neurons per unit area indicated that there were significant differences in the density of the 5HT1AR+/NeuN+neurons in the MPFC among the WT+NS group mice,the WT+FLX group mice,the AD+NS group mice and the AD+FLX group mice.The density of the 5HT1AR+/NeuN+neurons in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.01),while the density of the 5HT1AR+/NeuN+neurons in the MPFC of the AD+FLX group mice was significantly more than that of the AD+NS group mice(p<0.05).3.Immunofluorescence staining results of the PSD95+synapses:The fluorescence intensity of the PSD95+synapses per unit area indicated that the expression of PSD95 in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.01).There was not significantly different on the expression of PSD95 in the MPFC between the AD+FLX group mice and the AD+NS group mice(p>0.05).4.The expression of SYP in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.01),and the expression of SYP in the MPFC of the AD+FLX group mice was significantly more than that of the AD+NS group mice(p<0.01).5.The expression of GSK3B protein in MPFC of the AD+NS group mice was signifieantly more than that of the WT+NS group mice(p<0.01),the expression of GSK3B protein in the MPFC of the AD+FLX group mice was significantly less than that of the AD+NS group mice(p<0.01).However,the expressions of β-catenin and p-ser9-GSK3β protein in the MPFC of the AD+NS group mice were significantly less than those of the WT+NS group mice(p<0.01,p<0.01);the expressions of β-catenin and β-ser9-GSK3βprotein in the MPFC of the AD+FLX group mice were significantly more than those of the AD+NS group mice(p<0.01,p<0.01).6.The expression of c-fos in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.01),and the expression of c-fos in the MPFC of the AD+FLX group mice was significantly more than that of the AD+NS group mice(p<0.01).7.The expression of BDNF in the MPFC of the AD+NS group mice was significantly less than that of the WT+NS group mice(p<0.01),the expression of BDNF in the MPFC of the AD+FLX group mice was significantly more than that of the AD+NS group mice(p<0.01).Conclusions:1.At 10-month age,the synapse numbers of the MPFC in the APP/PS1 transgenic AD mice were significantly decreased.FLX intervention could protect the synapses in the MPFC of the early transgenic AD mice.2.At 10-month age,the expression of 5HT1AR in the MPFC of the APP/PS1 transgenic AD mice was significantly decreased.FLXintervention could promote the expression of 5HT1 AR in the MPFC of the transgenic AD mice.3.FLX could activate the key molecules in the classical Wnt pathway through activating or increasing 5HT1AR,which might be one of possible mechanisms for the protective effects of FLX on the synapses in MPFC.4.At 10-month age,the expressions of the BDNF and c-fos protein in the MPFC of the APP/PS 1 transgenic AD mice were significantly decreased,while FLX treatment could increased the expressions of BDNF and c-fos protein.5.FLX could enhance the expressions of BDNF and synapse protein and reduce synaptic loss by activating or increasing 5HT1AR in the MPFC of the transgenic AD mice,thereby improving cognitive dysfunction in the transgenic AD mice.