Study On Androgen Membrane Binding Sites Of Hippocampal Neurons And The Effect Of CaM/CaMK? Signaling Pathway On The Synaptic Protein PSD95

Objective:The objects of study were the primary cultured hippocampal neurons of SD rats.Seeking direct evidences for the presence of androgen cell membrane binding sites,vefifying similarities and differences between cell membrane binding sites and intracellular receptors and types of neurons expressing membrane receptors,in order to understand the way how androgens play a physiological role.Afterwards,the effect of the non-genomic pathway of androgen on the synaptic protein PSD95 in hippocampal neurons was clarified,and the influencing mechanism of CaM/CaMKII signaling pathway was elucidated to fully understand the ways in which androgen exerts its physiological effects,in order to fully understand the physiological role of androgen play mode,provide the basis for synaptic plasticity study to further promote the neuroprotective mechanism of androgens.Methods:1 Study on androgen membrane receptors of hippocampal neurons(1)Verify the presence of androgen membrane binding sites(membrane receptors)in hippocampal neuronsChose embryonic day 18 SD rats and remove the fetus after anesthesia.The brains were aseptically removed and the hippocampus was removed.The rats were digested,pipetted,resuspended and filtered,and inoculated in confocal dishes.They were cultured to 15~20 days and were randomly divided into BSA-FITC group and T-BSA-FITC group.BSA-FITC and T-BSA-FITC were incubated respetively for 15 min.Confocal laser scanning microscopy was used to observe the binding of T-BSA-FITC to hippocampal neurons and to verify the existence of androgen membrane binding sites(membrane receptors)in hippocampal neurons.(2)Study on the difference of androgen membrane receptors and intracellular receptors in hippocampal neuronsChose embryonic day 18 SD rats and remove the fetus after anesthesia.The brains were aseptically removed and the hippocampus was removed.The rats were digested,pipetted,resuspended and filtered,and inoculated in confocal dishes.After 15~20 days of culture,flutamide was given in advance for 15 min and T-BSA-FITC was given for 15 min.Laser confocal microscopy was used to observe whether the classical androgen receptor antagonist flutamide can inhibit the binding of androgen to membrane receptors.In addition,competitive experiments were conducted to incubate neurons with excess free T for 15 min before adding T-BSA-FITC for 15 min to observe the binding of T-BSA-FITC to hippocampal neuronal cell membranes.(3)Study on the type of hippocampal neurons expressing androgen membrane receptors(excitatory / inhibitory)Chose embryonic day 18 SD rats and remove the fetus after anesthesia.The brains were aseptically removed and the hippocampus was removed.The rats were digested,pipetted,resuspended and filtered,and inoculated in confocal dishes.It was cultured to 15~20 day,adding T-BSA-FITC to incubate15 min,and then fixed with 4% polyoxymethylene.The immunofluorescent cytochemical technique was used to mark the glutamatergic neurons(excitability)with vGLUT1 antibody.GABA antibody was used to mark gamma aminobutyric acid neurons(inhibitory).Laser confocal microscopy was used to observe the co-localization of T-BSA-FITC and antibody expression,and to analyze excitatory and inhibitory neuronal subsets in neurons with androgen receptor.2 The effect of androgen membrane receptor mediated CaM/Ca MKII signaling pathway on synaptic protein PSD95 in hippocampal neurons(1)The effect of androgen on the synaptic protein PSD95 in the primary hippocampal neuronsChose embryonic day 18 SD rats and remove the fetus after anesthesia.The brains were aseptically removed and the hippocampus was removed.Therats were digested,pipetted,resuspended and filtered,and inoculated in 6 hole plate.It was cultured to 15~20 day to extract cell protein.They were randomly divided into control group(Con group),T group,T-BSA group.The time of drug action was 1 h.Western blotting was used to detect the expression of PSD 95 in hippocampal neurons.(2)The effect of androgen on the CaM/CaMKII signaling pathway in primary hippocampal neuronsChose embryonic day 18 SD rats and remove the fetus after anesthesia.The brains were aseptically removed and the hippocampus was removed.The rats were digested,pipetted,resuspended and filtered,and inoculated in 6 hole plate.It was cultured to 15~20 day to extract cell protein.They were randomly divided into control group(Con group),T group,T-BSA group.The time of drug action was 1 h.Western blotting was used to detect the expression of CaM,CaMKII and P-CaMKII in hippocampal neurons.(3)The effect of androgen membrane receptor mediated CaM/CaMK II signaling pathway on the synaptic protein PSD 95 of hippocampal neuronsChose embryonic day 18 SD rats and remove the fetus after anesthesia.The brains were aseptically removed and the hippocampus was removed.The rats were digested,pipetted,resuspended and filtered,and inoculated in 6 hole plate.It was cultured to 15~20 day to extract cell protein.They were randomly divided into Con group,T group,T-BSA group,and CaMKII inhibitor KN93+T group(KN93+T group).The time of drug action was 1 h.The KN93+T group was pretreated with KN93 for 30 min before giving T.Western blotting was used to detect the expression of PSD 95 in hippocampal neurons.Result:1.Verify the presence of androgen membrane receptors in hippocampal neuronsWhen the hippocampal neurons were cultured for 15~20 days,the cell bodies were plump and varied in shape,with cones,triangles,spindles,ellipses and so on.The protrusions are thickened and lengthened,branches of the dendrites increased,and the interweaves became reticulate.The specificlocation of T-BSA-FITC and hippocampal neurons showed green fluorescence.In group T-BSA-FITC,there was extensive green fluorescence labeling on the root and distal part of hippocampal neurons and neurites,while no green fluorescence labeling was found in the cytoplasm and nucleus of neurons,which verified the existence of androgen receptor in hippocampal neurons.No green fluorescent binding sites were observed in the hippocampal neurons in BSA-FITC group,and the possible effects of the interaction of BSA and cell membrane on the results were excluded.(Fig.1)2.Study on the difference of androgen membrane receptors and intracellular receptors in hippocampal neuronsPre-administration of flutamide and T-BSA-FITC resulted in green fluorescence at the root and distal ends of hippocampal neuronal membranes and processes,but the number of labeled sites decreased,indicating that flutamide did not completely block the binding of T-BSA-FITC to cell membrane.Pre-incubation with excess free T,followed by T-BSA-FITC,detected almost no green fluorescent label,indicating that pre-incubation of T could completely block the binding of T-BSA-FITC to the cell membrane.(Fig.2)3.Study on the type of hippocampal neurons expressing androgen membrane receptors(excitatory / inhibitory)Two groups of different neuronal types were co-localized with vGLUT1 antibody labeled glutamatergic neurons(excitatory)and GABA antibody-labeled GABAergic neurons(inhibitory)and T-BSA-FITC had common standard.In the study of glutamatergic neurons labeled with vGLUT1 antibody,a total of 224 neurons were observed.Among them,140 neurons expressed vGLUT1,and the proportion of glutamatergic neurons was62.50%;122 neurons were simultaneously labeled by T-BSA-FITC,and the ratio of double-labeled neurons to excitatory neurons was 87.14%(Fig.3).In the study of GABA-labeled GABAergic neurons,207 neurons were observed.Among them,73 neurons expressed GABA,and the proportion of GABAergic neurons was 35.27%(Fig.4);52 neurons were simultaneously labeled byT-BSA-FITC,and the ratio of double-labeled neurons to inhibitory neurons was 71.23.%.(Table 1)4.The effect of androgen on the synaptic protein PSD95 in the primary hippocampal neuronsPSD95 Western blot results showed that compared with the Con group(0.209±0.016),the optical density of the T group(0.374±0.033)and the T-BSA group(0.363±0.026)bands relative to the internal reference GAPDH increased significantly(P<0.05).There was no difference between T group and T-BSA group.(Fig.4 and Table 2)5.The effect of androgen on the CaM/CaMK II signaling pathway in primary hippocampal neuronsCaM Western blot results showed that compared with the Con group(0.146±0.021),the optical density of the T group(0.247±0.037)and the T-BSA group(0.241±0.024)bands relative to the internal reference GAPDH increased significantly(P<0.05).There was no difference between T group and T-BSA group.CaMK? and phosphotyrosine Western blot results showed that in the results of p-CaMK II,compared with the Con group(0.061±0.011),the optical density of the T group(0.145±0.013)and the T-BSA group(0.138±0.011)bands relative to the internal reference GAPDH increased significantly(P<0.05).There was no difference between T group and T-BSA group.In the results of CaMK II,compared with the Con group(0.252±0.015),the optical density of the T group(0.244±0.016)and the T-BSA group(0.257±0.016)bands relative to the internal reference GAPDH had no difference.There was no difference between T group and T-BSA group.(Fig.6,Fig.7 and Table 3)6.The effect of androgen membrane receptor mediated CaM/CaMK II signaling pathway on the synaptic protein PSD 95 of hippocampal neuronsPSD95 Western blot results showed that compared with the Con group(0.261±0.014),the optical density of the T group(0.437±0.050),the T-BSA group(0.420±0.037)and the K+T group(0.350±0.026)bands relative to the internal reference GAPDH increased significantly(P<0.05).The K+T groupwas lower than T and T-BSA groups(P<0.05)? There was no difference between T group and T-BSA group.(Fig.8 and Table 4)Conclusion:1.There are androgen membrane binding sites in hippocampal neurons of SD rats.There are characteristic differences between androgen membrane binding sites and intracellular receptors.Androgen membrane binding sites can be competitively bound by free T.The majority of neurons expressing the androgen membrane binding site are glutamatergic neurons(excitatory)and minority GABAergic neurons(inhibitory).2.Androgen rapidly increased the expression of synaptic protein PSD95 in hippocampal neurons,and activated the CaM/CaMKII signaling pathway in hippocampal neurons in a short period of time.CaM/CaMKII signaling pathway is involved in the membrane receptor mediated effects of androgen on synaptic protein PSD95 in hippocampal neurons.