Effects And The Mechanism Of The Effects Of Fluoxetine On The Neurons And Synapses In The Hippocampus Of Early Alzheimer's Disease Through 5-HT System

PART ONE THE EFFECTS AND THE MECHANISM OF THE EFFECTS OF FLUOXETINE ON BEHAVIOR CHANGES AND A?AND PHOSPHORYLATED TAU PROTEIN OF THE HIPPOCAMPUS IN TRANSGENIC MOUSE MODEL OF EARLY ALZHEIMER'S DISEASEObjective:To investigate the effects and the mechanism of the effects of fluoxetine?FLX?on behavioral changes in early AD mice and pathological changes in the hippocampus of early AD mice in order to further provide the scientific foundation for further studies on the mechanism for the fact that fluoxetine delays the cognitive function decline in early AD.Methods:Forty male 8 months old APP/PS1 mice were selected and randomly distributed into an APP/PS1+NS group and an APP/PS1+FLX group.Forty male 8-month-old wild-type?WT?littermates were selected and randomly distributed into a WT+NS group and a WT+FLX group.The mice in the WT+FLX group and APP/PS1+FLX group were intraperitoneally injected daily with FLX?10 mg/kg i.p.dissolved in0.9%NS?regime for 10 weeks.The mice in the WT+NS group and APP/PS1+NS group were intraperitoneally injected daily with NS?equivalent 0.9%NS i.p.?for 10 weeks.At late two weeks injection,the autonomous activity of the mice was detected with the open field test,and the spatial learning and memory ability of the mice was detected with Moriss water maze and the working memory ability was detected with Y-maze.Immunofluorescence was used to detect the changes of amyloid plaques and 5HT1A receptor in the neurons of hippocampus subregions in each group mice.The changes of phosphorylated Tau protein,and levels of GSK3?and p-ser9-GSK3?in four group mice were detected with ELISA technique.Results:Compared with that of the WT+NS group,the parameters of the open field test including total route,the time spent in center area,the route spent in center area,percentage of time in center area,percentage of route in center area and the mean speed of the APP/PS1+NS group has no significant differences?p?29?0.05;p?29?0.05;p?29?0.05;p?29?0.05;p?29?0.05;p?29?0.05?.After 10 week of chronic FLX treatment,the time spent in center area,the route spent in center area,percentage of time in center area,percentage of route in center area and the mean speed of the APP/PS1+FLX group were not significantly different from those of the APP/PS1+NS group?p?29?0.05;p?29?0.05;p?29?0.05;p?29?0.05;p?29?0.05;p?29?0.05?.In Morris water maze,the mean escape latencies of the WT+NS group was significantly decreased compared with that of the APP/PS1+NS group?p<0.01?.Compared with that of the APP/PS1+FLX group,the mean escape latencies of the APP/PS1+NS group were significantly increased?p<0.01?.For the probe test used for evaluating the spatial memory ability,the numbers of platform location crosses of the WT+NS group were significantly increased compared with those of the APP/PS1+NS group?p?27?0.05?.Compared with those of the APP/PS1+NS group,the numbers of platform location crosses of the APP/PS1+FLX group were significantly increased?p?27?0.05?.The results of the Y maze showed a significantly decrease in alternation performance in the APP/PS1+NS group compared with the WT+NS group?p<0.01?.The alternation behavior in the APP/PS1+FLX group was significantly increased compared with the APP/PS1+NS group?p<0.01?.The amyloid plaques of the APP/PS1+FLX group were fewer than those of the APP/PS1+NS group in DG and CA1/2 of the hippocampus?p<0.01;p<0.01?,whereas compared with the WT+NS group,the APP/PS1+NS group exhibited numerous plaques in DG and CA1/2 of the hippocampus?p<0.01;p<0.01?.The immunofluorescence showed that the 5HT1A⁺/NeuN⁺cells in the DG and CA1/2 regions of hippocampus of the APP/PS1+NS group were significantly decreased compared with the WT+NS group?p<0.01;p<0.01?.The5HT1A⁺/NeuN⁺cells in the DG,CA1/2,and CA3 regions of the hippocampus of the APP/PS1+FLX group were significantly increased compared with the APP/PS1+NS group?p<0.05;p<0.01;p<0.01?.ELISA results showed that the levels of GSK3?and phosphorylated Tau protein were significantly decreased?p<0.05?,and p-ser9-GSK3??p<0.05?was significantly increased,A?₄₀ and A?₄₂?p<0.01;p<0.01?were significantly decreased in APP/PS1+FLX group compared with APP/PS1+NS group.Conclusions:1.10 week Fluoxetine treatment could delay the decline progress in the learning and memory ability and working memory ability of early AD mice,and 10 week fluoxetine treatment had no effect on the autonomic activity of four group mice.2.Fluoxetine treatment could not only significantly decrease the amyloid plaques and A?₄₀ and A?₄₂ in the hippocampus of early AD,but also reduce the expression levels of GSK3?and phosphorylated Tau protein and inhibit the activity of GSK3?.At the same time,fluoxetine treatment could also increase the expression of 5HT1A receptors in the neurons of hippocampus.3.We speculated that the effects of fluoxetine on the A?₄₀,A?₄₂ and GSK3?in the hippocampus of early AD mice might be associated with the increased expression of the 5HT1A receptor in the hippocampus neurons.PART TWO THE EFFECTS AND THE MECHANISM OF THE EFFECTS OF FLUOXETINE ON NEURONS AND NEWBORN NEURONS OF THE HIPPOCAPUS IN EARLY ALZHEIMER'S DISEASEObjective:To investigate the effects of fluoxetine on the volume of hippocampus and the changes of 5HT4 receptors in neurons,newborn neurons and neurons in early AD and the effects of fluoxetine on immature neurons of dentate gyrus in early AD in order to provide an effective scientific basis for studying the effect and the mechanism of the effect of fluoxetine on the cognition function in early AD.Methods: Eight months old male APP/PS1 double transgenic AD mice were given 10 week of fluoxetine treatment,and one week of continuous injection of Brd U was started from the fifth week.After 10 weeks,6 mice were randomly selected from WT + NS group mice,WT + FLX group mice,APP/PS1 + NS group mice and APP/PS1 + FLX group mice for perfusion.Serial coronal sections with a thickness of 50 ?m were cut with a cryo-ultramicrotome.The sections were randomly sampled according to the stereological sampling principle.The sections from four groups were stained with toluidine blue,and the related structural parameters in the dentate gyrus,CA1/2 and CA3 regions of hippocampus were estimated with the stereological methods.The immature neurons in the dentate gyrus of the hippocampus from four groups were stained with immunohistochemistry and cresol purple,and the immature neurons of the dentate gyrus from four groups were accurately quantified using stereological methods.Newborn neurons and 5HT4R+/Neu N+ cells in the hippocampal DG,CA1/2 and CA3 regions in the four groups were counted with immunofluorescence technique.Results: Compared with the WT + NS group,the volume and neurons in DG and CA1/2 of hippocampal showed a significantly decrease in APP/PS1 + NS group at the 10 month old?p < 0.05;p < 0.01??p < 0.01;p < 0.01?.Meanwhile,the newborn neurons and 5HT4+/Neu N+ cells in DG,CA1/2,and CA3 regions of hippocampus in APP/PS1 + NS group also showed significantly decrease compared with those of WT + NS group?p < 0.01;p < 0.01;p < 0.01??p < 0.01;p < 0.05;p < 0.01?.After 10 week fluoxetine treatment,the volumes of hippocampal DG and CA1/2 in the APP/PS1+FLX group were significantly increased compared with those in the APP/PS1 + NS group?p < 0.01;p < 0.05?.Compared with the APP/PS1 + NS group,the numbers of neurons,newborn neurons and 5HT4+/Neu N+ cells in DG,CA1/2 the and CA3 regions of hippocampus were significantly increased?p < 0.05;p < 0.01;p < 0.05?.The immature neurons in the DG of hippocampal in the APP/PS1+FLX group were significantly increased compared with the APP/PS1 + NS group?p < 0.05?.Conclusions: 1.10 week fluoxetine treatment could delay the volume shrinkage of DG and CA1/2 of hippocampus in early AD mice.2.Fluoxetine treatment could delay the loss of neurons in DG and CA1/2 regions of hippocampus in early AD mice,and fluoxetine treatment could significantly increase the number of newborn neurons in DG,CA1/2 and CA3 regions of hippocampus in early AD mice.3.Fluoxetine treatment could significantly increase the number of immature neurons in DG region of hippocampus in early AD mice.4.Fluoxetine treatment could significantly increase the number of 5HT4+/Neu N+ cells in DG,CA1/2 and CA3 regions of hippocampus in the early AD.5.The results of this experiment further confirmed that neurogenesis in hippocampus might be associated with the early pathogenesis of AD,while the protective effects of fluoxetine on neurons,newborn neurons and the promotion of immature neurons in hippocampus might be the one of the structure mechanisms for the positive effect of fluoxetine on AD.We speculated that the effects of fluoxetine on the hippocampual neurons in early AD mice with the increased expression of the 5HT4 receptor in the hippocampual neurons.PART THREE THE EFFECTS AND THE MECHANISM OF THE EFFECTS OF FLUOXETINE ON THE SYNAPSES IN SUB-REGIONS OF HIPPOCAMPUS IN EARLY ALZHEIMER'S DISEASEObjective:To investigate the effects of fluoxetine treatment on the dendritic spines and PSD95 in the sub-regions of hippocampus and the levels of the SYP and BDNF in the hippocampus of the early AD in order to provide a effective scientific basis for studing the mechanism for the fact that FLX improves the behaviors of early AD.Methods: Eight months old male APP/PS1 mice were injected with fluoxetine for 10 weeks,and one week of continuous injection of Brd U was started from the fourth week.After 10 weeks,6 mice were randomly selected from WT + NS group mice,WT + FLX group mice,APP/PS1 + NS group mice and APP/PS1 + FLX group mice for perfusion.Serial coronal sections with a thickness of 50 ?m were cut with a cryo-ultramicrotome.The sections were randomly sampled according to the stereological sampling principle.The dendritic spines in DG,CA1/2 and CA3 regions of the hippocampus in four groups were quantified with the immunohistochemical techniques and the stereological methods.The changes on levels of SYP and BDNF in hippocampus of four group mice were detected with ELISA technique.The density of PSD95 in the DG?CA1/2 and CA3 regions of the hippocampus in four groups was quantified using immunofluorescence technique.Results: Compared with the WT + NS group,APP/PS1 + NS group at 10 months of age showed significant decrease in the number of dendritic spines in DG,CA1/2 and CA3 regions of the hippocampus?p < 0.01;p < 0.05;p < 0.01?,as well as the density of PSD95 in DG,CA1/2 and CA3 regions of the hippocampus was reduced.At the same time,the expression levels of SYP and BDNF of APP/PS1 + NS group in the hippocampus also showed significantly decreased comparing with WT + NS group?p < 0.05;p < 0.05?.After 10 week fluoxetine treatment,compared with the APP/PS1 + NS group,the number of dendritic spines in the DG,CA1/2 and CA3 regions of the hippocampus were significantly increased in the APP/PS1+FLX group?p < 0.01;p < 0.01;p < 0.05?.Compared with the APP/PS1 + NS group,the density of PSD95 of the APP/PS1+FLX group in the DG,CA1/2 and CA3 regions of the hippocampus was increased.Meanwhile,the expression levels of SYP and BDNF in hippocampus of APP/PS1+FLX group were significantly increased comparing with the APP/PS1 + NS group?p < 0.05;p < 0.05?.Conclusions: 1.There were a large number of dendritic spine loss in the DG,CA1/2 and CA3 regions in the hippocampus at 10 months APP/PS1 mice,suggesting that the dendritic spines change in the hippocampus may be related to cognitive function decline of early AD.2.10-week fluoxetine treatment could delay the loss of dendritic spines in the DG,CA1/2 and CA3 regions of the hippocampus in early AD and increase the density of PSD95 in the DG,CA1/2 and CA3 regions of the hippocampus.10-week fluoxetine treatment could delay the expression level decline of SYP in the hippocampus of early AD.3.10-week fluoxetine treatment could delay the expression level decline of BDNF in the hippocampus of early AD.4.The above results further demonstrated that dendritic spines in the hippocampus were involved in the structural changes of hippocampus in early AD,and fluoxetine could delay delay the loss of dendritic spines in early AD.These results might provide a scientific foundation for the further studies on the mechanism for the effect of fluoxetine on early AD.