Detecting The Presence Of Hippocampus Membrane Androgen Receptors In SAMP8 Mice And Their Induced Synaptic Plasticity

Androgens, biosynthesis mainly in the interstitial Leydig cells, are the critical factors responsible for the development of the male phenotype during embryogenesis and for the achievement of sexual maturation at puberty. In adulthood, androgens remain essential for the maintenance of male reproductive function and behaviour. Apart from their effects on reproduction,androgens affect a wide variety of non-reproductive tissues such as skin, bone,muscle, and brain. This classic genomic model for steroid hormone action presumes that steroid hormones can freelycross 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.The biological actions of androgens via transcriptional regulation of target genes by the i ARs are referred to as “genomic action”. In addition to transcriptional or genomic mode of action, accumulating evidence has demonstrated that androgens can exert novel, non-classical effects in various cell types and tissues. This mechanism of androgen action became known as“non-genomic action”.Similar to the non-genomic actions of other steroids, there are certain basic criteria/categories for an androgen induced response to be considered non-genomic in nature.(i) Speed: the effects should occur in a time frame(seconds to minutes) not sufficiently long enough to allow gene transcription/translation. The classical genomic model predicts that the latency between steroid exposure and observed responses can be no shorter than the time it takes for the steroid to trigger gene transcription followed by proteinsynthesis.(ii) Membrane mediated: the response should involve membrane embedded or associated receptors or binding proteins, and with an action that can be induced even when the steroid is conjugated to molecules that prohibit it from entering deep into the cytoplasm or from translocating to the nucleus when bound to a receptor. The most common example is the use of testosterone(T) conjugated to large molecules such as bovine serum albumin(BSA).(iii) Lacking transcription/translation machinery activation:experiments use either that cell lines that lack the necessary machinery for a genomic response or identify androgen effects which are insensitive to inhibitors of transcription and translation, demonstrate that certain steroid responses can be elicited in systems where gene transcription or protein synthesis is unlikely or impossible.The non-genomic effect of steroids could potentially be mediated(i) by direct binding to a specific-binding site of the target molecule, in the absence of a AR,(ii) through the classical intracellular-AR,(iii) by a distinct non-classical transmembrane-AR, i.e., transmembrane G-protein coupled receptor, or(iv) via changes in membrane fluidity.Although the molecular identity of membrane androgen-binding sites or receptors(m ARs) remains elusive, non-genomic effects of androgens have now been convincingly demonstrated in several tissues, in particular in the reproductive, cardiovascular, immune and musculoskeletal systems. The hippocampus is involved in learning and memory processes and is a known target for the modulatory actions of sex hormones. Because the hippocampus is rich in androgen receptors, indicating that it is a target for the action of androgens, a reasonable interpretation of these findings may be that androgens regulate synapse density via an androgen receptor-dependent mechanism. This study sought to determine the existence of m ARs and their functional consequence in the hippocampus.Senescence-accelerated-prone mouse 8(SAMP8) has been proposed as a suitable, naturally derived animal model for investigating the fundamental mechanisms of Alzheimer’s disease(AD). In addition, serum testosterone(T)levels decrease quickly in the natural growth process of this model. Recently,we reported that androgen plays an important role in sustaining and regulating structural synaptic plasticity in the hippocampus of male SAMP8 mice,whereas androgen deficiency may contribute to the etiology of age-related cognitive impairment, corresponding to the decline in endogenous androgen production. The hippocampus is attractive as a center of learning and memory and is one of the major target areas for the neuromodulatory actions of androgens. There is little doubt that the hippocampus retains some degree of androgen-regulated neuroplasticity, and extensive studies have been performed to investigate their role in modulating hippocampal plasticity and function. Classical genomic actions modulate synaptic plasticity via gene transcription and the synthesis of synaptic proteins in neurons. Now, we focus on the problem of whether androgen mediates rapid, non-genomic synaptic plasticity in the hippocampus.In recent years, accumulating evidence has indicated that the mammalian brain, in areas such as the hippocampus, synthesizes estrogen and androgen by cytochrome P450 s, hydroxysteroid dehydrogenases and 5-alpha-reductase. For hippocampus-derived sex hormones, one of the essential functions may be the rapid and repetitive modulation of synaptic plasticity and cognitive functions,in addition to genomic slow modulation. Rapid modulation particularly favors locally synthesized sex hormones rather than circulating hormones which travel a long distance before reaching the brain. Immunoelectron microscopic analysis has provided direct evidence that these sex hormone receptors localize not only in the nuclei but also in extranuclear sites, including dendritic spines and axon terminals. Increasing evidence suggests that membrane ERs are involved in the rapid synaptic plasticity and the non-genomic actions of estrogen, which may play a major role in the modulation of neuroplasticity.Although there is increasing evidence that androgen interferes with synaptic remodeling, the evidence of its rapid, non-genomic actions in the hippocampus is limited compared with estrogen.The current study investigated the presence and specificity of m ARs ofthe hippocampus in SAMP8 mice.A fluorescent membrane impermeable testosterone-bovine serum albumin-fluorescein isothiocyanate(T-BSA-FITC)macromolecular was used to label m ARs.To further verify whether these m ARs are identical to classical i ARs,we used flutamide(F,antagonist of the classical AR)and anti-AR antibody(Ab)to determine its binding specificity.Although biochemical,molecular and physiological studies have added to the understanding of the rapid,non-genomic actions of androgens,few studies have addressed how m ARs induce synaptic plasticity in the hippocampus.Here we analyzed the rapid effects of T and dihydrotestosterone(DHT)on the density of dendritic spines,and T-BSA was also investigated.To explore the protective mechanisms and neurological basis of T and DHT,we determined whether they altered the expression of synaptic proteins,which play crucial roles in cognitive function and synaptic plasticity.Part One Detecting the presence of hippocampus membrane androgenreceptors in SAMP8 miceObjectives: The current study investigated the presence and specificity of m ARs of the hippocampus in SAMP8 mice. A fluorescent membrane impermeable testosterone-bovine serum albumin-fluorescein isothiocyanate(T-BSA-FITC) macromolecular was used to label m ARs. To further verify whether these m ARs are identical to classical i ARs, we used flutamide(F,antagonist of the classical AR) and anti-AR antibody(Ab) to determine its binding specificity.Methods:Experiment 1 Flow cytometry analysis for the presence and quantification of m ARsMale 7-month-old SAMP8 mice were randomly divided into a control group, BSA-FITC group and T-BSA-FITC group. To ensure consistent dosing,only one batch of stock was used for all of the treatment groups. BSA-FITC was used as the control for the nonspecific FITC signal, whereas T-BSA-FITC measured the specific m AR signal. The mice were implanted with cannulas into the lateral ventricle and injected with PBS(0.5 h), BSA-FITC(0.5 h) andT-BSA-FITC(15 min, 0.5 h, 1 h and 2 h). Groups of mice were deeply anesthetized with 6% chloral hydrate and were rapidly decapitated.Hippocampal tissue samples were immediately dissected from the brains, and the cells were harvested, washed and resuspended in PBS. To determine the levels of FITC, the cells were analyzed with excitation at 488 nm and a 525 nm band-pass filter in the emission path. The mean fluorescent intensity(MFI)was derived using Cell Quest Pro software by flow cytometry. The relative fluorescence intensity(RFI) was calculated as the ratio of the MFI of specific staining to that of control staining.Experiment 2 Flow cytometry analysis for the binding specificity of m ARs using i AR antagonistMale 7-month-old SAMP8 mice were randomly divided into a control group, BSA-FITC group, T-BSA-FITC group and F+T-BSA-FITC group.Groups of mice were implanted with cannulas into the lateral ventricle and injected with PBS(0.5 h), BSA-FITC(0.5 h), T-BSA-FITC(0.5 h) and F+T-BSA-FITC(pre-administration of F for 0.5 h). The mice were deeply anesthetized with 6% chloral hydrate and rapidly decapitated. Hippocampal tissue samples were immediately dissected from the brains and the cells were harvested, washed and resuspended in PBS. The MFI was derived using Cell Quest software by flow cytometry. The RFI was calculated as the ratio of the MFI of specific staining to that of control staining.Experiment 3 Flow cytometry analysis for the binding specificity of m ARs using anti-AR AbMale 7-month-old SAMP8 mice were randomly divided into membrane-intact groups(control group, FITC-Ig G group and anti-AR Ab +FITC-Ig G group) and permeabilized groups(control group, FITC-Ig G group and anti-AR Ab + FITC-Ig G group). Groups of mice were deeply anesthetized with 6% chloral hydrate and rapidly decapitated. Hippocampal tissue samples were immediately dissected from the brains and the cells were harvested. The cells of permeabilized groups were added to fixative(contains 2%paraformaldehyde and 0.1% Triton X-100 detergent in PBS). Classical i AR inintact- and permeabilized-cells were detected with anti-i AR antibodies and FITC-labeled Ig G by flow cytometry analysis. The MFI was derived using Cell Quest software by flow cytometry. The RFI was calculated as the ratio of the MFI of specific staining to that of control staining.Results:1 Flow cytometry analysis for the presence and quantification of m ARsWe performed flow cytometry analysis to determine the presence and quantification of m ARs using a fluorescent membrane impermeable T-BSA-FITC macromolecular complex. Strong fluorescence enhancements were observed with the injection of T-BSA-FITC at 15 min(P < 0.01). In the subsequent time period(0.5 h and 1.0 h), the fluorescent intensity did not increase or decrease compared to that at 15 min(P > 0.05). However,approximately 2 h after injection of T-BSA-FITC, the fluorescent intensity was decreased compared with other T-BSA-FITC groups(P < 0.01).BSA-FITC did not bind to neurons under the same injection conditions(P >0.05), showing that the binding was mediated by T in the T-BSA-FITC macromolecule. Plasma membrane labeling in the T-BSA-FITC group indicated that membrane androgen binding sites were present in hippocampal neurons.2 Flow cytometry analysis for the binding specificity of m ARs using i AR antagonist and anti-AR AbTo further verify whether these m ARs are identical to classical i ARs, we used the F and anti-AR Ab to determine its binding specificity. Strong fluorescence enhancements were observed with the injection of T-BSA-FITC at 0.5 h(P < 0.01). Flow cytometry analysis showed that pre-administration of F reduced FITC signal intensity compared to the coupling action of the T-BSA-FITC(P > 0.05), but did not completely block membrane binding(P <0.01, compared with control group and BSA-FITC group). In addition, we performed an immunoreactive assay with anti-AR Ab. In membrane-intact groups, we found that membrane labeling was not detectable, especially in the anti-AR Ab group(P > 0.05). However, we were able to detect that anti-ARAb binds to the intracellular androgen receptors in hippocampal neurons of permeabilized groups.Conclusion: This study identified the presence and specificity of membrane androgen binding sites and the quickly functional consequence in hippocampal neurons of male SAMP8 mice. 1 The hippocampal neurons exhibited membrane androgen binding sites. 2 Pretreatment with i AR antagonist, flutamide(F), failed to completely prevent the coupling action of the T-BSA-FITC membrane binding. 3 Classical i ARs did not localize to the membrane of hippocampal neuronsPart Two Effects of androgen on dendritic thorns of hippocampus in maleSAMP8 mice induced by membrane androgen receptorsObjectives: The current study demonstrated the rapid effects of androgen on dendritic thorns of hippocampus in male SAMP8 mice induced by membrane androgen receptors.Methods: Experiments included dose-effect and time-effect experiments.Male 7-month-old SAMP8 mice were randomly divided into Sham group(sham-operated control group), Cast group(castration group), T group(castration plus T group), T-BSA group(castration plus T-BSA group) and DHT group(castration plus DHT group). Three days after castration, the groups were implanted with cannulas into the lateral ventricle. Dose-effects were examined by injecting different doses of T(25, 50, 100 μg/5μl), T-BSA(50, 100, 200 μg/5μl) or DHT(15, 30, 60 μg/5μl) into lateral ventricles for 2 h.Afterwards, time-effects were examined by injecting T, T-BSA and DHT for0.5 h, 1 h, 2 h and 4 h. The groups of mice were deeply anesthetized with 6%chloral hydrate and rapidly decapitated. The brains were removed, and tissue samples were immediately dissected from the superior colliculus to the optic chiasma. The tissue blocks were processed for Golgi–Cox staining using a Rapid Golgi Stain Kit. Secondary and tertiary apical dendrites were selected by quantitative analysis in which 10 μm segments were magnified 1000× in the digitized images. The dendritic spine density values were expressed as the number of thorns/10μm of dendrite.Results: The secondary and tertiary apical dendrites in the CA1 region of the hippocampus were observed in the digitized images. The dendritic spine density in the hippocampal CA1 region of the Cast group was 0.69 thorns/μm,revealing a significant difference(P < 0.01) compared with the Sham group(0.92 thorns/μm). Following the treatment with androgen(T, T-BSA and DHT),dendrites had significantly more thorns than the Cast group. Dose-effects were examined by injecting different doses of T, T-BSA and DHT into the lateral ventricle for 2 h. In the T group, the enhancing effect on the density of dendritic spines was most significant at 50 μg/5μl T(0.89 thorns/μm)compared with 25 μg/5μl(0.71 thorns/μm) and 100 μg/5μl(0.87 thorns/μm) T.In the T-BSA group, the enhancing effect on the density of dendritic spines was most significant at 100 μg/5μl T-BSA(0.88 thorns/μm) compared with 50μg/5μl(0.70 thorns/μm) and 200 μg/5μl(0.85 thorns/μm) T-BSA. In the DHT group, the enhancing effect on the density of dendritic spines was most significant at 30 μg/5μl DHT(0.92 thorns/μm) compared with 15 μg/5μl(0.73thorns/μm) and 60 μg/5μl(0.90 thorns/μm) DHT. Afterwards, time-effects were examined by injecting 50 μg/5μl T, 100 μg/5μl T-BSA and 30 μg/5μl DHT for 0.5 h, 1 h, 2 h and 4 h. In the T group, the enhancing effects on the density of dendritic spines were 0.73 thorns/μm(0.5 h), 0.85 thorns/μm(1 h),0.90(2 h) thorns/μm, and 0.90 thorns/μm(4 h). In the T-BSA group, the enhancing effects on the density of dendritic spines were 0.72 thorns/μm(0.5h), 0.79 thorns/μm(1 h), 0.89(2 h) thorns/μm, and 0.87 thorns/μm(4 h). In the DHT group, the enhancing effects on the density of dendritic spines were0.74 thorns/μm(0.5 h), 0.89 thorns/μm(1 h), 0.92 thorns/μm(2 h), and 0.95thorns/μm(4 h). Because the treatments of injecting 50 μg/5μl of T, 100μg/5μl T-BSA and 30 μg/5μl DHT into the lateral ventricle for 2 h were effective for the density of dendritic spines, these dosages and time of T and DHT were used in the following investigations.Conclusion: 1 There was a significant decrease in dendritic spines density in the castrated SAMP8 mice. 2 Both T and DHT induced a rapid increase in the density of dendritic spines.Part Three Effects of androgen on synaptic proteins of hippocampus inmale SAMP8 mice induced by membrane androgen receptorsObjectives: The current study demonstrated the rapid effects of androgen on synaptic proteins of hippocampus in male SAMP8 mice induced by membrane androgen receptorsMethods: Male 7-month-old SAMP8 mice were randomly divided into Sham group, Cast group, T group and DHT group. Three days after castration,the groups were implanted with cannulas into the lateral ventricle. The dosages and time of T and DHT were generated in accordance with dose and time effects.(1) Immunohistochemical staining. The computer image analysis system Image-Pro Plus 6.0 was used to determine the average values of the optical density(OD) of SNAP25, Syt1, SYN, PSD95, NR1 and Drebrin in the hippocampal CA1 and CA3 region. The sections were performed by nucleus counter staining with hematoxylin showed blue. Ten sections were analyzed for each mouse and the average OD value was determined for each mouse.(2)Western blotting. SNAP25, Syt1, SYN, PSD95, NR1 and Drebrin were detected using an Odyssey IR fluorescence scanning imaging system and quantified by densitometric analysis using an ALPHA analytical system.GAPDH was used as an internal control.Results:1 About immunohistochemical staining for SNAP25, Syt1, SYN,PSD95, NR1 and Drebrin, the Cast group was lightly stained, and the OD value in the CA1 and CA3 region were significantly lower than the Sham group(P < 0.01). T and DHT administration enhanced the level of SNAP25,Syt1, SYN, PSD95, NR1 and Drebrin in hippocampal neurons, the OD value were significantly higher compared to Cast group(P < 0.01). T and DHT administration enhanced the level of SNAP25, Syt1, SYN, PSD95, NR1 and Drebrin in hippocampal neurons, the OD value were significantly higher compared to Cast group(P < 0.01).2 Western blotting analysis showed that the SNAP25, Syt1, SYN,PSD95, NR1 and Drebrin expression in the Cast group were lower, and theIOD value were statistically significant difference compared to P8-Sham group(P<0.01).The SNAP25,Syt1,SYN,PSD95,NR1 and Drebrin IOD value were increased after T and DHT administration,which showed significant difference compared to the Cast group(P<0.01).Conclusion: 1 Castration resulted in a down-regulation of the protein levels of SNAP25, Syt1, SYN, PSD95, NR1 and Drebrin. 2 T and DHT treatment clearly and significantly improved the level of SNAP25, Syt1, SYN,PSD95, NR1 and Drebrin protein in a relatively short time. 3 Androgens rapidly increased morphological synaptic plasticity, which may partly be achieved through SNAP25, Syt1, SYN, PSD95, NR1 and Drebrin.