Rapid Effects Of Androgen On Dendritic Spines And Synaptic Proteins In Neurons Of Acute Hippocampal Slices

Objective: To explore the effect of androgen non-genomic effect on synaptic morphological plasticity of hippocampal neurons,acute hippocampal slices were observed in this study for the effects of testosterone(T),dihydrotestosterone(DHT)and testosterone-bovine serum albumin(T-BSA)on dendritic spine density and synaptic proteins SYN and PSD95.Methods:1 Preparation of acute hippocampal slices and identification of their activities.SD rats,after anesthesia,were decapitated brain and placed in artificial cerebrospinal fluid.The hippocampal tissue was cut off and the 300?m coronal hippocampal slices were prepared by vibration slicer and then placed in artificial cerebrospinal fluid.Five acute were prepared from each rat.The hippocampal slices were incubated in and then moved into artificial cerebrospinal fluid containing DAPI and PI at 0 min,15 min,30 min,60 min and 120 min,respectively.The excess fluorescent dyes were swilled out.The morphology of hippocampal neurons was observed by laser scanning confocal microscopy,and the activity of hippocampal neurons in acute brain slices was measured by staining of fluorescent dyes.2 Effects of androgen on dendritic spine density of neurons in acute hippocampal slices.The acute hippocampal slices were randomly divided into control group(CON group),T group,DHT group and T-BSA group.According to the preliminary test results,the drug concentration of T group and DHT group is 10 nM,T-BSA group was 0.36 nM,and CON group given the same amount of DMSO.According to the preliminary test results,the drug action time is 1 h.After completion of the administration,the acute hippocampal slices was fixed with 4% paraformaldehyde and subjected to Golgi-Cox staining.Laser confocal microscopy was used to calculate the dendritic spines of the level 2 or 3 dendrites in the hippocampal CA1 neurons.The dendritic spine density of the selected dendrites was calculated by Imaris software.Density of spine was compared between groups.3 Effects of androgen on synaptic protein SYN and PSD95 in neurons of acute hippocampal slices.Preparation of acute hippocampal slices.The experimental grouping and drug concentration were the same as above.According to the preliminary test results,the drug action time is 1 h.After the end of the administration,the acute hippocampal slices was fixed with 4% paraformaldehyde and was dehydrated by 30% sucrose solution.After embedding,three slices were taken by frozen sections,and the depth of each slice was fixed.The slice thickness was 10?m.The synaptic protein SYN and PSD95 in hippocampal CA1 region were observed by immunofluorescence histochemical staining and confocal laser scanning microscopy.The relative mean fluorescence intensity(RMFI)of each group was compared.Then we analyses the relative mean fluorescence intensity.Results:1 Preparation of acute hippocampal slices and identification of their activities.Prepare of acute brain slices,which is pale yellow,stretch and flat in naked eye.The cells below the surface layer had a clear boundary,good refraction and a strong sense of three-dimensional and depth.The nucleus and particles are not visible.Double-staining of DAPI and PI showed that the percentages of viable cells were 79.92±1.66% 80.06±0.94% 80.60±0.97%,79.48±0.92% and 79.87±0.88% at the time of 0 min,15 min,30 min,60 min and 120 min,respectively.There was no significant difference in neuronal activity between the every two groups(P > 0.05).2 Effects of androgen on dendritic spine density of neurons in acute hippocampal slices.The results of Golgi-Cox staining showed that compared with CON group(2.10±0.15 thorns/?m),T group(2.89±0.19 thorns/?m),DHT group(3.01±0.16 thorns/?m)and T-BSA group(2.90±0.22 thorns/?m)were increased in dendritic spine density(P < 0.05).But there was no significant difference between T group,DHT group and T-BSA group(P > 0.05).3 Effects of androgen on Synaptic protein SYN and PSD95 in neurons of acute hippocampal slices.The immunofluorescence assay of SYN showed that the mean relative fluorescence intensity of T group(54.22±4.10),DHT group(56.01±4.90)and T-BSA group(55.34±6.12)were significantly higher than that of CON group(42.26±3.66)(P < 0.05).There was no significant difference between T group,DHT group and T-BSA group(P > 0.05).The immunofluorescence assay of PSD95 showed that the mean relative fluorescence of T group(84.12±5.34),DHT group(83.98±3.37)and T-BSA group(85.84±4.45)was significantly higher than that of CON group(70.26±4.55)(P < 0.05).There was no significant difference between T group,DHT group and T-BSA group(P > 0.05).Conclusion:1 The viability of acute hippocampal slices of neurons prepared under experimental conditions was stable within 2 hours.2 Androgen can quickly increase the dendritic spine density of neurons in acute hippocampal slices in a short time.3 Androgen can rapidly increase the expression level of synaptic protein SYN and PSD95 in a short time.