Rapid Modulation Of Testosterone On Synaptic Plasticity Of Primary Hippocampal Neurons And Involvement Of ERK/MAPK And CREB Signaling Pathway

Androgens are biosynthesized mainly in the interstitial Leydig cells, with the critical factors responsible for the development of the male phenotype during embryogenesis and the achievement of sexual maturation at puberty. In addition, androgens remain essential for the maintenance of male reproductive function and behavior in adulthood. Interestingly, androgens are also synthesized locally and secreted in hippocampal neurons, and for adult male,level of hippocampal androgens is higher than that of plasma androgens. Thus,more and more scholars have turned their attention to the neuroprotective effect of androgens, such as modulating the brain development, sexual dimorphism, learning and memory, cognition and mood, increasing viability of neurons and protecting them from injury induced by oxidative stress, serum deprivation and Aβ. Here, we focused on the modulation on synaptic plasticity induced by androgen.With the increasing of age, epidemiological data suggested that, androgens level in plasm become lower and lower. Correspondingly, the cognitive competence of brain becomes weaker. Therefore, we speculated that the decrease of androgen level related with aging constituted the risk factors of Alzheimer’s disease. Our laboratory have previously reported that, decrease of androgen induced by castration could drop the spine density of SAMP8 mice,injure the ability of learning and memory, form cognition impairment, but supplemented androgen treatment could reverse the drop of spine density.Therefore, for the purpose of understanding the etiology of AD, this study investigated whether physiological dose of androgens are necessary to maintain synaptic plasticity.Just like other steroid hormones, androgens exert their physiologicalfunction through two pathways. One is genomic pathway. This classic genomic model for steroid hormone action presumes that steroid hormones can freely cross the plasma membrane, enter the cytoplasm, and bind to and activate specific intracellular steroid receptor proteins. The bound steroid receptors act as transcription factors and bind as homodimers or heterodimers to specific DNA response elements in target gene promoters, causing activation or repression of transcription and subsequently protein synthesis.While the other is a rapid nonclassical action. The nongenomic mechanism is identified by one of the following characters:(1) Speed: the effects should occur in a time frame(seconds to minutes).(2) Membrane mediated.(3) the action wouldn’t be blocked by Flutamide, a classical inhibitor of i ARs(except that the structure of the inhibitor is partly related with the site of m ARs, and the substructure is simultaneously shared by both m ARs and i ARs);(4)Lacking transcription/translation machinery activation: experiments in the presence of inhibitor of transcription and translation, that certain steroid responses can be elicited.There are evidences showed that androgen modulated the synaptic structural plasticity in a nongenomic pathway. Previously, our laboratory have demonstrated that there exists a novel androgen-binding sites or receptors(m ARs) on the membrane of hippocampal neurons. Now, we designed to observe the rapid action from T-BSA, a large molecule conjugated with bovine serum albumin(BSA), since it is prohibited from entering deep into the cytoplasm or from translocating to the nucleus, the rapid modulation induced by testosterone is mediated by membrane androgen receptors(m ARs). In addition, by using Flutamide, a classical inhibitor of i ARs, we detected weather it can block the rapid modulation driven by testosterone.Previous studies on spinogenesis induced by androgen usually chose either animal model or brain slices as observed objective, few experiments on cellular level are reported. This study used primary cultured hippocampal neurons and assessed the rapid modulation on dendritic spine density driven by testosterone. Compared with animal model, primary cultured hippocampalneurons possess such advantages as: fewer influence factors, simple interference from organism, easily control of experiment conditions. What’s more, the cultures are characterized by defined stages of maturation,interconnection with each other, morphological features and expression pattern and localization of neuronal proteins that seem to be essentially identical to those of neurons developed within the intact brain. So it must be a suitable alternate to neurons in vivo. As far as observed objective is concerned,this experiment, in some degree, completed the study system of rapid modulation on synaptic structural plasticity induced by androgen, contributing to understand the way by which androgens produce physiological effects.Synaptic proteins play an important role in maintaining synaptic functions normal. Active zones located in presynaptic terminals are rich in synaptic vesicles filled with neurotransmitters, during the course of neurotransmitters releasing, the activities of synaptic vesicles are influenced by presynaptic proteins and their complex. Synaptophysin(SYN) is a major integral protein of the synaptic vesicle membrane. It is widely expressed in central and peripheral neurons as well as in neuroendocrine cells and represents a major cytoplasmic Ca2+binding site of synaptic vesicle preparations. With the functions of involvement in not only the fusion of synaptic vesicle membrane and presynaptic membrane, but also the release of neurotransmitters,synaptophysin is regarded as a specific marker of synaptic terminals. The amount of synaptophysin reflects indirectly density of synapse, so it is an important reference index for synaptic plasticity.Under electron microscope, postsynaptic membranes of excitatory synapses present a thickened postsynaptic density, composed of neurotransmitters and receptors, signal-transducing proteins and cytoskeletal proteins, which are associated with a variety of synaptic function, such as ion channels, synaptic activity and intracellular signal transduction pathway. Densities-95(PSD-95)is a main component of the postsynaptic density protein. As a very important scaffold protein at the postsynaptic membrane, PSD-95 is responsible for assembling and organization, the core in the course of postsynaptic signalingtransduction. In addition, PSD95 also interacts with the regulatory subunits of N-methyl-D-aspartate(NMDA) receptors and their associated signaling proteins to participate in the information storage process. At present, PSD95 has been used as a postsynaptic specific markers and an important reference index for synaptic plasticity.Belonging to a family of ionotropic glutamate receptors,N-Methyl-D-Aspartate Receptors(NMDARs) are selectively activated by NMDA or glutamic acid, associated with excitatory neurotransmission in the central nervous system. Therefore, it is essential for higher level brain activity such as learning and memory. NMDARs have several subunits, among which NR1 is a basic unit. Expressed widely in the central nervous system, NR1 is thought to be required for the assembly of a functional Glu N channel.In spite of the effects on synaptic plasticity of androgen and the close association between synaptic plasticity and specific synaptic proteins, there is few reports about the mediation on synaptic proteins expression induced by androgen. Our laboratory have showed that testosterone and dihydrotestosterone both up-regulate expression of synaptic proteins such as SNAP25, Syt1, SYN, PSD95, NR1 and Drebrin, consequently have influence on synaptic plasticity in a nongenomic pathway. A recent experiment from our laboratory suggested that dihydrotestosterone also increases the protein expression of SYN, PSD95 and Drebirn in castrated SAMP8 mice in MCI. To further discuss this issue, we explored the expression changes of synaptic protein SYN, PSD95 and NR1 in cultured hippocampal neurons treated with testosterone.Although a large number of scholars have devoted themselves to the study on rapid action of androgen on synaptic plasticity, the underlying mechanisms remain obscure. It is well known that a lot of signaling pathways take part in the modulation on synaptic plasticity, and some have associated with androgen’s nongenomic pathway. For example, ERK/ MAPK, PKA, PKC,p38/ MAPK, LIMK as well as Ca MKⅡ. This study focused on the involvement of extracellular signal-regulated kinase(ERK) and c AMPresponse element binding protein(CREB) signaling pathway.A compelling body of evidence suggested that ERKs are activated in neurons in response to excitatory glutamatergic signaling, which controls many forms of synaptic plasticity(including functional plasticity-LTP and structural plasticity-spinogenesis) that are thought to underlie higher brain processes such as learning and memory. The development of studies on CREB have undergone a long period of time. Studies from invertebrates to rodents all indicate that CREB-dependent transcription is essential for many forms of learning and memory. Phosphorylation of CREB at Ser133 leads to recruitment of other transcription machinery to CREs to regulate gene transcription. Therefore, it is probable that CREB phosphorylation at Ser133 is an important step in the induction of gene expression that is essential to learning. Our previous study on SAMP8 mice in MCI showed that,dihydrotestosterone up-regulated phosphorylation level of ERK/MAPK and CREB of hippocampal neurons, thus, we speculate that ERK/MAPK and CREB signaling pathway could involve in the modulation on synaptic structural plasticity induced by androgen. This study emphasized the participation of signaling pathway of ERK/MAPK and CREB in modulation spinogenesis of cultures driven by testosterone.All in all, with primary hippocampal neurons as observed objective, this study investigated the rapid modulation on spine density induced by physiological dose of testosterone, discussed the influence on protein and m RNA expression of specific synaptic markers, SYN, PSD95 and NR1,explored the underlying mechanism by which physiological dose of androgen effects on synaptic structural plasticity, verified whether ERK/MAPK and CREB signaling pathway is involved.Part One Testosterone rapid modulates spine density of primaryhippocampal neuronsobjectives: Using primary cultured hippocampal neurons as observed objective, the current study aimed at assess the modulation on spine density driven by appropriate dose of testosterone in a short time;Verify that the rapidaction on synaptic plasticity induced by testosterone was mediated in a nongenomic pathway with the evidence from T-BSA, which was prohibited from entering deep into the cytoplasm or from translocating to the nucleus;Investigate whether these m ARs are identical to classical i ARs by flutamide,an inhibitor of classical i ARs, to determine its binding specificity.Methods:Experiment 1 Culture and identification of primary hippocampal neuronsChose embryonic day 17 SD rats, anesthetized it and took out the embryos,decapitated fetal rat and picked up the brains, dissected hippocampus, broke the tissues and digested it with Accutase, a super alternative of tyrisin, pumped it gently and carefully then cultivated the cell suspension. The hippocampal neurons were not utilized as samples until they were cultured for 18 days in vitro. Hippocampal neuron purity was assessed by exposing cultures to anti-rabbit MAP2 antibody and goat anti-rabbit FITC(1:100) antibody to mark neurons, then Hoechst 33258 to stain nuclei. The proportion of cells that were MAP2-positive with total stained nuclei was quantified.Experiment 2 time-effect experiment of rapid action on spine density induced by testosteroneAccording to time of treatment, DIV18 primary hippocampal neurons were randomly divided into a control group(0 min group), 15 min group(15 min group), 30 min group(30 min group), 1 h group(1 h group) and 2 h group(2 h group). Based on references, the concentration of testosterone administrated was 10 n M, and control group was treated with the same dose of DMSO as testosterone. After disposure, immunofluorescence cytochemical staining of Drebrin was conducted, for the purpose of observing the alternation of spine density induced by testosterone at different point of time. Observed with 600×oil immension lens under light microscope, dendrite segments 20 μm in length,from two to three dendrites per neuron, were sampled 50 μm away from the cell body, followed by counting the density of spine.Experiment 3 Dose-effect experiment of rapid action on spine density induced by testosteroneAccording to different dose of treatment, DIV 18 primary hippocampal neurons were randomly divided into 1 n M group(1 n M group), 10 n M group(10 n M group), 100 n M group(100 n M group) and 1000 n M group(1000 n M group). Based on results of time-effect experiment above, the optimization administration time was 1 h. After disposure, immunofluorescence cytochemical staining of Drebrin was conducted, for the purpose of observing the alternation of spine density induced by different dose of testosterone.Experiment 4 Rapid action on spine density induced by testosteroneDIV 18 primary hippocampal neurons were randomly divided into a control group(group Con), treated with testosterone group(group T), treated with T-BSA group(group T-BSA) and Flutamide pretreated for 1 h and testosterone another hour group(group F+T). Based on the results above, we took it for granted that the optimization administration time and dose were 1 h and 10 n M respectively. After disposure, immunofluorescence cytochemical staining of Drebrin was conducted, for the purpose of observing the alternation of spine density induced by 10 n M testosterone for an hour’s action.Results:1 Identification purity of hippocampal neuronTo mark neurons, we exposed cultures to anti-rabbit MAP2 antibody and goat anti-rabbit FITC(1:100) antibody, then Hoechst 33258 to stain nuclei.MAP2 positive presented red in soma, and nuclei stained with Hoechst 33258 presented blue. Hippocampal neuron purity was defined as proportion of cells that were MAP2-positive with total stained nuclei, the purity of cultures was excellent at no less than 90% at in vitro day 18.2 Time-effect experiment of rapid action on spine density induced bytestosteroneUsing immunofluorescence cytochemical staining of Drebrin, we analyzed the density of spine at different point of time to determine the best administration time. Enhancements of spine density were observed with the administration of testosterone, it was at the time point of 1 h, spine density peaked at 2.08±0.15 thorns/μm, there were significant differences comparedwith the other four groups(P < 0.01). In the subsequent time point(2 h), the density of spine(1.75±0.15 thorns/μm) decreased slightly compared to that at1 h, with statistically significant difference(P < 0.05).3 Dose-effect experiment of rapid action on spine density induced bytestosteroneTreated with T of different dose, spine density of cultures in each group showed: 1 n M group, 1.54±0.16 thorns/μm; 10 n M group, 2.15±0.14thorns/μm; 100 n M group, 1.92±0.18 thorns/μm; 1000 n M group, 1.39±0.18thorns/μm; It was in 10 n M group the spine density presented the highest,with significant difference(P < 0.05). Spine density was higher in group 100 n M than group 1 n M and 1000 n M, the differences were significant(P <0.05).4 Rapid action on spine density induced by testosteroneCompared with group Con(1.12±0.10), group T(1.96±0.19), group T-BSA(1.90±0.18) and group F+T(1.34±0.14) all showed higher density of spine,differences were statistically significant(P < 0.05); The Result of group F+T was lower than group T and T-BSA(P < 0.05), but there was no significant difference between group T and group T-BSA(P > 0.05).Conclusion:1 With the treatment of testosterone, different dose of T all up-regulates spine density of primary hippocampal neurons rapidly, the optimization treatment time and dose are 1 h and 10 n M respectively.2 Since both T and T-BSA increase spine density of cultures, the action is mediated mainly by m ARs in a nongenomic pathway.3 Flutamide, an inhibitor of classical i ARs, couldn’t block the effect on spine density induced by testosterone entirely, which suggested that m ARs and i ARs are not identical completely. Maybe, in the very period, testosterone exerts influence in both classical pathway and nongenomic effect.Part Two Testosterone rapid modulate expression of synaptic proteins ofprimary cultured hippocampal neuronsObjectives: Using primary cultured hippocampal neurons, the current studyaimed to investigate the rapid modulation on synaptic markers: SYN, PSD95 and NR1 induced by physiological dose of testosterone, including protein and m RNA expression. This study explored the relationship between rapid modulation by androgen and synaptic proteins, contributing to helping understand the pattern by which testosterone exerts physiological action.Methods:Experiment one Immunofluorescence cytochemistry staining for synaptic proteins SYN, PSD95 and NR1DIV 18 primary hippocampal neurons were randomly divided into a control group(group Con), treated with testosterone group(group T), treated with T-BSA group(group T-BSA) and Flutamide pretreated for 1 h and testosterone another hour group(group F+T). Based on the results of part one, we administrated T and T-BSA for 1 h, with the concentration of 10 n M and 0.36 n M respectively, followed by immunofluorescence cytochemical staining of SYN, PSD95 and NR1. After images acquisition, mean fluorescence intensity(MFI) were calculated and analyzed for the purpose of observing the changes of protein expression induced by testosterone.Experiment two Western blotting for synaptic proteins SYN, PSD95 and NR1The observed objectives, group division and method of administration were as same as experiment one. After disposure, protein of cultured hippocampal neurons was extracted proceeding for western blotting. Bands of SYN, PSD95 and NR1 were detected using an Odyssey IR fluorescence scanning imaging system and relative Optical density(OD) were analyzed to assess the protein expression of SYN, PSD95 and NR1, GAPDH was used as an internal control.Experiment three q-PCR for m RNA expression of synaptic proteins SYN,PSD95 and NR1The observed objectives, group division and method of administration were as same as experiment one. After disposure, m RNA of cultured hippocampal neurons was extracted followed by q-PCR. After the first strand synthesis, the m RNA level of SYN, PSD95 and NR1 were provided by real-timequantitative PCR detecting system, GAPDH was used as an internal control.Results:1 Immunofluorescence cytochemical staining results for synaptic proteinsSYN, PSD95 and NR1Results of SYN detected by immunofluorescence cytochemical staining showing that, compared with group Con(0.064±0.003), group T(0.089±0.004), group T-BSA(0.085±0.004) and group F+T(0.070±0.004) all presented higher MFI, with significant difference(P < 0.05); Group F+T presented lower MFI than group T and T-BSA(P < 0.05), while there was no difference between group T and T-BSA(P > 0.05).Results of PSD95 detected by immunofluorescence cytochemical staining showing that, compared with group Con(0.047±0.004), group T(0.074±0.004), group T-BSA(0.070±0.004) and group F+T(0.070±0.005) all presented higher MFI, with significant difference(P < 0.05); While there was no difference between any two groups of T, T-BSA and F+T(P > 0.05).Results of NR1 detected by immunofluorescence cytochemical staining showing that, compared with group Con(0.031±0.004), group T(0.074±0.004), group T-BSA(0.074±0.003) and group F+T(0.044±0.003) all presented higher MFI, with significant difference(P < 0.05); Group F+T presented lower MFI than group T and T-BSA(P < 0.05), while there was no difference between group T and T-BSA(P > 0.05).2 Western Blotting results for synaptic proteins SYN, PSD95 and NR1Results of SYN detected by Western Blotting showing that, compared with group Con(1.478±0.074), group T(2.787±0.153), group T-BSA(2.629±0.155)and group F+T(2.164±0.144) all presented higher protein expression, with significant difference(P < 0.05); Group F+T presented lower relative OD value than group T and T-BSA(P < 0.05), while there was no difference between group T and T-BSA(P > 0.05).Results of PSD95 detected by Western Blotting showing that, compared with group Con(0.276±0.017), group T(0.460±0.035), group T-BSA(0.463±0.015) and group F+T(0.427±0.025) all presented higher proteinexpression, with significant difference(P < 0.05); While there was no difference between any two groups of T, T-BSA and F+T(P > 0.05).Results of NR1 detected by Western Blotting showing that, compared with group Con(0.031±0.004), group T(0.069±0.003), group T-BSA(0.070±0.004)and group F+T(0.037±0.003) all presented higher relative OD value, with significant difference(P < 0.05); Group F+T presented lower relative OD value than group T and T-BSA(P < 0.05), while there was no difference between group T and T-BSA(P > 0.05).3 q-PCR results for m RNA expression of synaptic proteins SYN, PSD95 and NR1The results of SYN detected by q-PCR showing that, compared with group Con, group T(28.22±3.57), group T-BSA(32.57±3.62) and group F+T(11.63±0.29) all presented much higher m RNA expression, with significant difference(P < 0.05); Group F+T presented lower than group T and T-BSA(P< 0.05), while there was no difference between group T and T-BSA(P > 0.05).The results of PSD95 detected by q-PCR showing that, compared with group Con, group T(5.94±0.22), group T-BSA(5.58±0.21) and group F+T(5.40±0.08) all presented much higher m RNA expression, with significant difference(P < 0.05); While there was no difference between any two groups of T, T-BSA and F+T(P > 0.05).The results of NR1 detected by q-PCR showing that, compared with group Con, group T(20.64±0.45), group T-BSA(20.04±0.62) and group F+T(3.21±0.10) all presented much higher m RNA expression, with significant difference(P < 0.05); Group F+T presented lower than group T and T-BSA(P< 0.05), while there was no difference between group T and T-BSA(P > 0.05).Conclusion:1 Testosterone rapidly up-regulates the protein and m RNA expression of synaptic markers: SYN, PSD95 and NR1.2 Flutamide partly blocked the modulation on protein and m RNA expression of synaptic markers:SYN and NR1 induced by testosterone, but not blocked the increase of protein and m RNA expression of PSD95.3 Testosterone effects on the protein and m RNA expression of synaptic markers: SYN, PSD95 and NR1 in a nongenomic pathway mainly.Part Three ERK/MAPK and CREB signaling pathway involves in therapid modulate on synaptic structural plasticity induced bytestosteroneObjectives: With primary cultured hippocampal neurons, the current study was designed to test the rapid alternation of phosphorylation level of ERK/MAPK and CREB induced by testosterone; In order to demonstrate the specificity of this signaling pathway, we investigated whether ERK/MAPK and CREB were still activated by testosterone in the presence of U0126, and discussed whether U0126 blocked the up-regulation of spine density by testosterone. The study further explored the underlying mechanism by which testosterone exert action on synaptic structural plasticity in a nongenomic pathway.Methods:Experiment one Effects on phosphorylation level of ERK/MAPK and CREB by testosterone with x MAP technologyDIV 18 primary hippocampal neurons were randomly divided into a control group(group Con), treated with testosterone group(group T), treated with T-BSA group(group T-BSA) and Flutamide pretreated for 1 h and testosterone another hour group(group F+T), with the same method of administration as part one. protein was extracted and proceeded for flexible Multi-Analyte Profiling(x MAP) technology in order to detect the phosphorylation level of ERK/MAPK and CREB induced by testosterone.Experiment two Phosphorylation level of ERK/MAPK and CREB by testosterone in the presence of U0126The observed objectives and group division were as same as experiment one.DIV 18 cultures were treated with U0126 for 1 h before administration of T and T-BSA, followed by western blotting to detect the phosphorylation level of ERK/MAPK and CREB induced by testosterone.Experiment three Enhancement of spine density by testosterone in thepresence of U0126The observed objectives, group division and administration methods were as same as experiment two. Immunofluorescence cytochemistry staining for Drebrin to mark spines, analyzed the alternation of spine density induced by testosterone, in order to investigate the effect of U0126 during the course.Results:1 x MAP results for phosphorylation level of ERK/MAPK and CREBThe results of ERK/MAPK detected by x MAP technology showing that:compared with group Con(0.069±0.007), group T(0.357±0.026), group T-BSA(0.364±0.050) and group F+T(0.178±0.031) all presented much higher phosphorylation level, with significant difference(P < 0.01); group F+T presented lower than group T and T-BSA(P < 0.05), while there was no difference between group T and T-BSA(P > 0.05).The results of CREB detected by x MAP technology showing that:compared with group Con(0.243±0.045), group T(0.862±0.121), group T-BSA(0.890±0.086) and group F+T(0.551±0.017) all presented much higher phosphorylation level, with significant difference(P < 0.01); Group F+T presented lower than group T and T-BSA(P < 0.05), while there was no difference between group T and T-BSA(P > 0.05).As far as the total protein expression of ERK/MAPK and CREB were concerned, x MAP technology detected that there were no significant differences between any two groups(P > 0.05).2 Western blotting Results for the phosphorylation level of ERK/MAPK and CREB in the presence of U0126The results of p ERK1/MAPK and p ERK2/MAPK in the presence of U0126 detected by western blotting showed that: Group Con(0.345±0.054,0.034±0.002); Group T(0.288±0.029, 0.035±0.002); Group T-BSA(0.350±0.014, 0.036±0.003) and Group F+T(0.403±0.038, 0.032±0.002). The phosphorylation level of ERK1 and ERK2 showed no significant differences between any two groups(P > 0.05).The results of p CREB in the presence of U0126 detected by westernblotting showed that: Group Con(0.168±0.007); Group T(0.161±0.007);Group T-BSA(0.178±0.001) and Group F+T(0.164±0.007). The phosphorylation level of CREB showed no significant differences between any two groups(P > 0.05).3 Results for spine density in the presence of U0126Cultures were pretreated with U0126 for an hour, followed by administration of T and T-BSA, then proceeded for immunofluorescence cytochemistry staining of Drebrin to assess the spine density. Results in all the four groups were: Group Con 1.01±0.18, group T 0.98±0.19, group T-BSA1.03±0.21 and group F+T 1.02±0.14. Based on statistical analysis of the density of spine, no significant differences were observed in any two groups(P > 0.05).Conclusion:1 Both T and T-BSA have effects on inducing phosphorylation of ERK/MAPK and CREB.2 U0126 is able to block the phosphorylation of ERK/MAPK and CREB induced by testosterone.3 U0126 is able to reverse the increase of spine density driven by testosterone.4 ERK/MAPK and CREB signaling pathway involves in the rapid modulation on synaptic structural plasticity induced by testosterone.