| Objective: Taking the primary cultured hippocampal neurons as observed objective,aiming to observe the rapid effects of testosterone(T)?dihydrotestosterone(DHT)and Testosterone-bovine serum albumin(T-BSA)on the density and morphology of dendritic spines and Synaptic proteins PSD95 and SYN in primary hippocampal neurons of rats.To explore the non-genomic effect of androgen on the synaptic plasticity of hippocampal neuronsMethods:1 The optimization method of hippocampal neurons of rats GFP transfection based on the dendritic spines morphological observation,Chose embryonic day 18 SD rats,decapitated fetal rat and picked up the brains,dissected hippocampus and culture the hippocampal neurons.Lipofectamine 2000 and Lentivirus were transfected into the 8 days in vitro(DIV)neurons with green fluorescence protein(GFP)plasmid,respectively.Then observed the transfection efficiency and fluorescent expression,the neuronal survival rate was detected with trypan blue staining.After that,choosing the optimization method to transfect hippocampal neurons in DIV 8,then observing the growth and development of dendritic spine on DIV 10,15,20 and 25.Finally,we observed the effect on the morphology of hippocampal neuron dendritic spines when transfected in DIV 8,12 and 16 different times.2 To observe the rapid effects of androgen on the morphology and density and surface area of dendritic spines in primary hippocampal neurons of rats.Culture of rat primary hippocampal neurons,choosing the optimization transfection method of Lipofectamine 2000 to transfect GFP in hippocampal neurons,On DIV 20,dendritic spines which are mature and successful transfection were imaged in living cells by confocal microscope and living cells workstation.The experiment was divided into 4 groups randomly: control group(Con group),treated with testosterone group(T group),treated with dihydrotestosterone(DHT group)and treated with T-BSA group(T-BSA group).According to the reference and previous experimental results,the concentration of T group and DHT group is 10 nM and T-BSA group is 0.36 nM,control group is treated with the same amount of dimethyl sulfoxide,Using laser confocal microscope records the change of density and morphology of dendritic spines before and after drug intervention.Every 30 min scan pictures,a total of 120 min.3 The rapid effects of androgen on synaptic proteins PSD95 and SYN in primary hippocampal neurons of rats.Culture of rat primary hippocampal neurons,conducting experiment on DIV 20,the experimental grouping and drug concentration were the same as above.Based on previous experimental results,the action time is 60 min and then using immunofluorescence cytochemical staining to observe the synaptic proteins PSD95 and SYN.Results:1 The optimization method of hippocampal neurons of rats GFP transfection based on the dendritic spines morphological observation.The transfection effect of Lipofectamine 2000 is better than Lentivirus based on morphological observation of dendritic spines.The transfection efficiency of Lipofectamine 2000 is 5.21% lower than 82.53% of Lentivirus group.But according to the survival rate of before and after transfection,Lipofectamine 2000 group is 93.78% better than 83.37% of Lentivirus group.One week after,the survival rate of Lipofectamine 2000 group is 91.59% better than 72.92% of Lentivirus group obviously.At the same time,neurons which transfected by Lipofectamine 2000 are in good state,the development of dendritic spines is more mature on DIV 20,filiform pseudopodia decreased significantly,forming dense short or mushroom of dendritic spines and DIV12 for transfection is the best time for dendritic spines morphological observation.Cell culture grows well to 20 days when the fluorescence expression dendritic spines shape is clearly visible,suitable for living cells dendritic spines morphological observation for a long time.2 The rapid effects of androgen on the morphology and density and surface area of dendritic spines in primary hippocampal neurons of rats.The morphology of dendritic spines: the dendritic spines are various in diverse groups,mushroom,stubby,thin and so on can be found on the same dendrites at the same time,and the shape of dendritic spines is not fixed.Compared with each group,the dendritic spine morphology of Con group was relatively stable,and there was no obvious change.The other three groups after administration,some dendritic spine morphology has been more obvious changes,more mature.The performances are from the thin into a stubby or from stubby into a mushroom.The density of dendritic spines: In 120 min period,the density of dendritic spines in the Con group,T group,DHT and T-BSA group has no statistical significance(P > 0.05).The surface area of dendritic spines: the surface area of dendritic spines in Con group changes with time,relatively stable and little change.The surface area of dendritic spine in T group,DHT group and T-BSA group changed with time.At 60 min,compared with Con group(1.26 ± 0.41),there was a significant difference in the surface area of dendritic spines between group T(2.47 ± 0.64),DHT group(2.68 ± 1.08)and T-BSA group(2.64 ± 0.67)(P < 0.05).At 90 min,compared with Con group(1.41 ± 0.31)there was a significant difference in the surface area of dendritic spines between group T(2.41 ± 0.52),DHT group(2.57 ± 0.80)and T-BSA group(2.38 ± 0.60)(P <0.05).The difference was most significant at 60 min and the other time periods were no statistically significant difference(P > 0.05).There was no significant difference in the surface area of dendritic spines between T group,DHT group and T-BSA group(P > 0.05).3 Androgen rapid effect on Synaptic proteins PSD95 and SYN in primary hippocampal neurons of rats.PSD95 immunofluorescence staining showed: compared with Con group(63.60±2.06),fluorescence intensity of PSD95 are increased in T group(90.78±2.83)?DHT group(85.48±2.41)and T-BSA group(88.42±3.40),the difference was statistically significant(P < 0.05).There was no significant difference in the fluorescence intensity of PSD95 between T group,DHT group and T-BSA group(P > 0.05).SYN immunofluorescence staining showed: compared with Con group(52.96±0.97),fluorescence intensity of SYN are increased in T group(63.02±0.66)?DHT group(65.00±1.46)and T-BSA group(63.47±1.05).The difference was statistically significant(P < 0.05).There was no significant difference in the fluorescence intensity of SYN between T group,DHT group and T-BSA group(P > 0.05).Conclusion:1 Based on morphological observation of dendritic spines,the method of Lipofectamine 2000 transfect GFP in hippocampal neurons on DIV 12 and then observe the morphology of dendritic spines on DIV 20 has good results.2 Androgen failed to increase the density of dendritic spine in primary hippocampal neurons of rats in a short time,but the impact of the dendritic spine morphology,so that the dendritic spine more mature,and increase the dendritic spine surface area.3 Androgen increases the expression of synaptic proteins PSD95 and SYN in primary hippocampal neurons of rats in a short time... |
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