Exogenous dehydroisoandrosterone sulfate reverses the dendritic changes of the central neurons in aging male rats

Experimental Gerontology 57 (2014) 191–202

Contents lists available at ScienceDirect

Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

Exogenous dehydroisoandrosterone sulfate reverses the dendritic changes of the central neurons in aging male rats Jeng-Rung Chen a,⁎, Guo-Fang Tseng b, Yueh-Jan Wang b, Tsyr-Jiuan Wang c,⁎⁎ a b c

Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan Department of Anatomy, College of Medicine, Tzu-Chi University, Hualien, Taiwan Department of Nursing, National Taichung University of Science and Technology, Taichung, Taiwan

a r t i c l e

i n f o

Article history: Received 12 September 2013 Received in revised form 6 June 2014 Accepted 11 June 2014 Available online 12 June 2014 Section Editor: Christian Humpel Keywords: Somatosensory cortex Hippocampus Dendritic structures DHEAS Aging male rat

a b s t r a c t Sex hormones are known to help maintaining the cognitive ability in male and female rats. Hypogonadism results in the reduction of the dendritic spines of central neurons which is believed to undermine memory and cognition and cause fatigue and poor concentration. In our previous studies, we have reported age-related regression in dendrite arbors along with loss of dendritic spines in the primary somatosensory cortical neurons in female rats. Furthermore, castration caused a reduction of dendritic spines in adult male rats. In light of this, it was surmised that dendritic structures might change in normal aging male rats with advancing age. Recently, dehydroepiandrosterone sulfate (DHEAS) has been reported to have memory-enhancing properties in aged rodents. In this study, normal aging male rats, with a reduced plasma testosterone level of 75–80%, were used to explore the changes in behavioral performance of neuronal dendritic arbor and spine density. Aging rats performed poorer in spatial learning memory (Morris water maze). Concomitantly, these rats showed regressed dendritic arbors and spine loss on the primary somatosensory cortical and hippocampal CA1 pyramidal neurons. Exogenous DHEAS and testosterone treatment reversed the behavioral deficits and partially restored the spine loss of cortical neurons in aging male rats but had no effects on the dendritic arbor shrinkage of the affected neurons. It is concluded therefore that DHEAS, has the efficacy as testosterone, and that it can exert its effects on the central neuron level to effectively ameliorate aging symptoms. © 2014 Elsevier Inc. All rights reserved.

1. Introduction We reported recently that dendritic spines on the primary somatosensory cortical neurons in normal adult male rats were substantially reduced 2 weeks after removal of the testis. Very interestingly, the dendritic spine reduction was reversed following exogenous testosterone treatment (Chen et al., 2013). The same results were found in female rats, the cortical pyramidal neurons changed their spine density cyclically during estrous cycle and this appeared to be regulated by the accompanying changes of gonadal hormones (Chen et al., 2009b; Wang et al., 2014). It would appear therefore that as with estrogen, testosterone has a similar effect on the integrity of dendrites. In this connection, the dendritic spines rather than the dendritic length or branching profuseness of somatosensory cortical pyramidal neurons were modulated by sex hormones (Chen et al., 2009b, 2013). Hypogonadism, commonly associated with aging or for purpose of disease treatment, has been

⁎ Correspondence to: J.-R. Chen, Department of Veterinary Medicine, National ChungHsing University, No. 250, Kuo Kuang Road, Taichung 402, Taiwan. ⁎⁎ Correspondence to: T.-J. Wang, Department of Nursing, National Taichung University of Science and Technology, No. 193, Section 1, Sanmin Rd., Taichung, Taiwan. E-mail addresses: [email protected] (J.-R. Chen), [email protected] (T.-J. Wang).

http://dx.doi.org/10.1016/j.exger.2014.06.010 0531-5565/© 2014 Elsevier Inc. All rights reserved.

shown to affect memory and impair cognition. It has been reported that androgen positively affects cognitive performance (Almeida et al., 2004; Hirshman et al., 2004; Janowsky, 2006) and mood (Barrett-Connor et al., 1999b; Seidman, 2003b) in human beings and the cognitive functions of laboratory animals (Flood et al., 1995). In addition, many studies have also demonstrated age-related shrinkage in the dendritic arbors and in dendritic spine loss of cortical pyramidal neurons in rodents (Luine et al., 2011; Wang et al., 2009), dogs (Mervis, 1978), primates (Peters et al., 1998) and humans (de Brabander et al., 1998; Dickstein et al., 2007, 2012; Nakamura et al., 1985; Scheibel and Scheibel, 1975). Based on the above reports and along with our previous results concerning the changes of dendritic structure in aging female rats (Wang et al., 2009, 2014), it would be reasonable to speculate that similar changes might also occur in the somatosensory cortical neurons and hippocampal CA1 pyramidal neurons in aging male rats. Androgen deficiency in older men is common, and the potential sequelae are numerous. The main symptoms of hypogonadism are muscle atrophy and weakness, reduced sexual functioning, increased adiposity, low bone mass, depressed mood, and fatigue. Interest on androgen replacement therapy is growing, but the current evidence supporting hormonal replacement as a neuroprotective therapy remains inconclusive. Testosterone and dehydroepiandrosterone, precursors of sex hormone secreted by the adrenal gland, represent two possible treatments for

192

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

androgen therapy. It is well documented that dehydroepiandrosterone (DHEA) declines with aging and is used clinically to treat hypogonadalism and menopause syndromes (Genazzani et al., 2007; Saltzman and Guay, 2006). In the blood, most DHEA is found as DHEAS with levels that are about 300 times higher than that of free DHEA. The conversion of DHEA to DHEAS is reversibly catalyzed by sulfotransferase primarily in the adrenal gland, liver and small intestine (Parker, 1999). Recently, DHEA treatment has been reported to improve cortical functions reversing the dendritic spine loss in hippocampal CA1 pyramidal neurons (MacLusky et al., 2006; Parducz et al., 2006; Vallee et al., 2001). These findings have led us to explore whether exogenous androgen could also reverse the age-related changes of dendritic structure on the primary somatosensory cortical and hippocampal CA1 pyramidal neurons in male rats. 2. Materials and methods 2.1. Animals Twenty-five young male rats (10–12 weeks, CD® IGS, BioLasco, Ilan, Taiwan) and 15 normal aging male rats (18–20 months of age, CD® IGS, BioLasco, CD® IGS, BioLasco, breeders retired at 5–7 months of age from BioLasco) were used in this study. Animals were housed in a temperature (24 ± 1 °C), humidity (60% ± 5%) and light (light on at 06:00 h and off at 18:00 h)-controlled environment. Experimental protocols were approved by the National Chung-Hsing University's Intramural Animal Care and Use Committee. Animals were fed with normal diet and water ad libitum. In the first experiment, five young male rats were given vehicle (sesame oil, Sigma-Aldrich, St Louis, MO) injection as the sham control. Fifteen aged male rats were divided into 3 groups: of which 10 of them received intraperitoneal DHEAS injection (4 mg/kg, Sigma-Aldrich, Aging + D) or testosterone (15 mg/kg, Sigma-Aldrich, Aging + T), the remaining 5 rats received the vehicle (n = 5) injection daily starting five days before being sacrificed. All animals were subjected to Morris water maze task (please see below) starting 3 days before scheduled to be sacrificed. Another experiment was conducted to verify the function of DHEAS on young adult and castrated rats. Twenty rats were divided into 2 groups: the first group (n = 10) was castrated and survived for 2 weeks; the other group (n = 10) was sham-operated control. Half of each group (n = 5) received intraperitoneal DHEAS injection (4 mg/kg, n = 5) and the other half received the vehicle (n = 5) injection daily starting five days before being sacrificed.

pH 7.3, for 30 min. The brain was immediately removed and sectioned with a vibratome into 350-μm-thick coronal slices (5–6 slices for the somatosensory cortex and 2–3 slices for the hippocampus). Most of the thick slices were processed by intracellular dye injection to reveal the dendritic arbor of selective individual cells. The remaining brain slices, which contained anterior commissure in the midline, were postfixed in 4% paraformaldehyde in PB for 2 days. They were then resectioned into 20-μm thick sections (Chen et al., 2003) and stained with crystal violet for studying the cytoarchitecture.

2.4. Intracellular dye injection and subsequent immunoconversion of the injected dye The brain slices were treated with 0.1 M PB containing 10−7 M 4′, 6diamidino-2-phenyl-indole (DAPI; Sigma-Aldrich) for 30 min to make all cell nuclei fluoresced blue under the filter set that visualized the yellow fluorescence of intracellular dye Lucifer yellow (LY, Sigma-Aldrich) adopted (Chen et al., 2010). DAPI-treated slides showed clear six cortical laminae under the fluorescence microscope and allowed a precise selection of neurons for dye injection. The brain slice was placed in a shallow chamber on the stage of a fixed-stage, fluorescence microscope (Olympus BX51) and covered with 0.1 M PB. Intracellular micropipette filled with 4% LY in water was carefully positioned with a three-axial hydraulic micromanipulator (Narishige, Tokyo, Japan) to select neurons for dye injection. The injection current was generated by an intracellular amplifier (Axoclamp-IIB). Four to five neurons each from layer III, layer V or CA1 pyramidal neurons per slide were randomly selected for injection when the dendritic structures were invisible before immunoconversion. At the end of injection, the slice was rinsed in 0.1 M PB and postfixed in 4% paraformaldehyde in 0.1 M PB before cryoprotected and sectioned into 60-μm-thick serial sections for subsequent immunoconversion. The above tissue sections were first incubated with 1% H2O2 in PB for 30 min to remove endogenous peroxidase activity and then incubated in PBS containing 2% Bovine Serum Albumin (Sigma-Aldrich) and 1%

2.2. Morris water maze task The spatial learning and memory performance were evaluated by Morris water maze, and the protocols were modified from our previous study (Chen et al., 2009a). Animal performance was recorded with a video camera for subsequent analysis of the path and swimming speed. The maze apparatus consisted of a circular pool 200 cm in diameter and 60 cm deep. The pool was filled with water at approximately 23–25 °C to a height of 50 cm. A transparent platform (diameter 15 cm) was placed at a constant position 2–3 cm below the surface of the water. The visual cues arrayed around the room were available for the rats to learn the location of the hidden platform. The rats were tested for 3 consecutive days with two trials per day. Rats were allowed to remain on the platform for 20 s if escaped within 180 s, or alternatively placed on the platform and remained there for 20 s if failed to locate the underwater platform within 180 s. A recovery period of 10 min was allowed between the two trials. The escape times and swimming paths of the two trials conducted each day were recorded and averaged. 2.3. Tissue preparation Animals processed for fixed tissue intracellular dye injection were deeply anesthetized as described above and perfused with a fixative containing 2% paraformaldehyde in 0.1 M phosphate buffer (PB),

Fig. 1. Blood testosterone level and soma area of cortical pyramidal neurons. Effects of aging and DHEAS treatment on blood testosterone (A) and soma area of layer III and layer V pyramidal neurons (B). Aging + D, aging rats treated with DHEAS; Aging + T, aging rats treated with testosterone. *, p b 0.05 between the marked and control rats; #, p b 0.05 between the marked and aging rats; &, p b 0.05 between the marked and aging + D groups.

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

Triton X-100. Sections were then treated with biotinylated rabbit antiLY (1:200; Molecular Probes, Eugene, OR) in PBS for 18 h at 4 °C and then with standard avidin–biotin HRP reagent (Vector, Burlingame, CA) for 3 h at room temperature. They were then reacted with 0.05% 3-3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich) and 0.01% H2O2 in 0.05 M Tris buffer. Reacted sections were mounted onto slides for 3-dimensional reconstruction.

2.5. Blood testosterone measurement To measure the blood testosterone level, 1.5 ml blood was withdrawn from the heart before perfusion at 9:00 a.m. The blood was then centrifuged (3000 ×g, 15 min, 4 °C) and the serum stored at −20 °C until measurement. Level of testosterone was assayed with an automated ADVIA Centaur® Immunoassay System (Bayer, Germany) with ADVIA Centaur TSTO ReadyPack (07207660) commissioned by a clinical laboratory (UM Clinical Laboratory, Taichung, Taiwan).

193

2.6. Data analysis To estimate the soma area of pyramidal neurons in the section stained with crystal violet, we used a camera lucida drawing tube and 40 × objective lens at the final magnification of 380 × in a twodimensional plane. A total of 5 brain sections at the level of the anterior commissure across the midline from each rat were scrutinized for the soma area of layer III and V pyramidal neurons. In total, 10 layer III and 10 layer V pyramidal neurons (contained nuclei) of each section derived from each rat were randomly selected, drawn and analyzed with PC-based software (Freeman Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA). Demarcations between soma and dendrites were taken as the points where the convex curvature of the soma became concave (Chen et al., 2003). The mean of the soma area of the selected neurons of each animal is the soma area of the particular type of neuron of the animal. To study the changes of dendritic arbor and length of cortical pyramidal neurons, the complete dendritic arbors of 8 neurons from each rat were reconstructed 3-dimensionally with Neurolucida

Fig. 2. Behavioral tests with spatial learning and memory in rats. Effects of aging and DHEAS on spatial learning and memory evaluated with Morris water maze. The escape time and swimming path of 3 tests of each session are plotted in A and B. The escape time and swimming path on the 3rd day are analyzed in C and D. The swimming speed is plotted in E. *, p b 0.05 between the marked and control rats; #, p b 0.05 between the marked and aging rats.

194

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

(MicroBrightField, Williston, VT). Some injected neurons in the superficial layer of a brain slide were excluded from the analysis because of incomplete apical dendrite or basal dendrites. The mean of the dendritic lengths of the 8 neurons of each animal is the dendritic length of the particular type of neuron of the animal. To estimate the density of dendritic spines, we randomly analyzed 8 hippocampal CA1, layer III and layer V pyramidal neurons each from each animal of each treatment group, representatively. Dendrites of the studied pyramidal neurons

were differentiated into proximal and distal segments of the apical and basal dendrites following the criteria described before (Chen et al., 2009a, 2013). Briefly, for layer III pyramidal neurons, proximal and distal basal dendrites were defined as the location between the first to second branch (about 25–75 μm) and the last one or two branches (about 100–150 μm) from the soma, respectively. For the relatively larger layer V pyramidal neurons, proximal and distal basal dendrites were defined as the location between the first to second branch

Fig. 3. 3D reconstructed layer III somatosensory cortical pyramidal neurons. Representative reconstructed layer III pyramidal neurons of the control, aging, DHEAS and testosterone treated cortices (A–D). Roman numerals and horizontal bars on the left of each drawing mark the cortical layers. The dendrogram of respective neuron is shown below. Bar = 50 μm for A–D.

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

(50–100 μm) and the last one or two branches (150–200 μm) from where they originate from the soma, respectively. In both layer III and V pyramidal neurons, proximal apical dendrite was the first or second branch of the apical trunk, and distal apical dendrite was the terminal dendrite after the last branch point. For hippocampal CA1 pyramidal neurons, basal dendrites were defined as those in the stratum oriens, and apical dendrite was on the other side of the cell body layer. The proximal segment of CA1 pyramidal neurons was in the stratum radiatum, and distal segment was in the stratum lacunosum-moleculare (Chen et al., 2009a). Three random segments of each category of dendrites were sampled in each neuron and the mean represents the spine density of the particular segment of the neuron. The means of dendritic segment of the 8 neurons studied in each animal were then averaged yielding the spine density of the particular dendritic segment of the type of neuron of each animal. Data were expressed as mean ± SE (standard error) unless otherwise indicated. N represents the number of animals. Statistical significance was tested with one-way analysis of variance (ANOVA)

195

followed by the S–N–K test to determine any difference between treatment groups. 3. Results As expected, aging males had a low blood testosterone level; DHEAS or vehicle treatment alone did not affect the blood testosterone level; testosterone injection significantly increased the blood testosterone level (Fig. 1A; F(3, 16) = 22.9, p b 0.05). The primary somatosensory cortex of aged male rats remained six-layered in structure, but the thickness of the gray matter was slightly reduced (data not shown). The neurons in layers III and V remained pyramidal in outline; furthermore, there was no noticeable change in cell size of these neurons (Fig. 1B; F(3, 16) = 0.39, p = 0.76 for layer III pyramidal neurons; F(3, 16) = 0.11, p = 0.95 for layer V pyramidal neurons). For hippocampus-related function, we assessed the rat spatial memory with water maze task (Fig. 2). The aging rats utilized a longer duration

Fig. 4. 3D reconstructed layer V somatosensory cortical pyramidal neurons. Representative reconstructed layer V pyramidal neurons of the control, aging, DHEAS and testosterone treated cortices (A–D). The apical dendritic trunk is in red while the filled blue circle represents cell body. Branches of each basal dendritic trunk are displayed with the same color. Roman numerals and horizontal bars on the left of each drawing mark the cortical layers. Bar = 100 μm for A–D.

196

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

to locate the hidden platform than the control rats. Exogenous DHEAS and testosterone treatments significantly decreased the escape time of aging rats in water maze task (Fig. 2A and C; F(3, 16) = 6.07, p b 0.05 for the first day of training; F(3, 16) = 20.3, p b 0.05 for the second day of training; F(3, 16) = 10.32, p b 0.05 for the third day of training). Although the aging rats had a lower swimming speed, they traveled a longer distance to locate the hidden platform (Fig. 2B and D; F(3, 16) = 1.78, p = 0.16 for the first day of training; F(3, 16) = 5.54, p b 0.05 for the second day of training; F(3, 16) = 5.89, p b 0.05 for the third day of training). Exogenous DHEAS treatment slightly increased the swimming speed, but it was statistically insignificant as compared to the untreated aging rats (Fig. 2E; F(3, 16) = 2.99, p b 0.05). To investigate the morphological changes of aging effect on dendritic structures, we analyzed the layer III and layer V pyramidal neurons of

the primary somatosensory cortex. The full dendritic arbors of pyramidal neurons were reconstructed with Neurolucida®. The 3D reconstructed layer III and layer V pyramidal neurons were shown and their dendrograms were plotted in Figs. 3–5. The dendritic arbors, including dendritic length (Fig. 6A and B; F(3, 16) = 3.94, p b 0.05 for basal dendritic length of layer III pyramidal neurons; F(3, 16) = 4.75, p b 0.05 for apical dendritic length of layer III pyramidal neurons; F(3, 16) = 5.91, p b 0.05 for basal dendritic length of layer V pyramidal neurons; F(3, 16) = 5.28, p b 0.05 for apical dendritic length of layer V pyramidal neurons), number of terminal ends (Fig. 6C and D; F(3, 16) = 5.47, p b 0.05 for basal terminal ends of layer III pyramidal neurons; F(3, 16) = 2.99, p b 0.05 for apical terminal ends of layer III pyramidal neurons; F(3, 16) = 12.34, p b 0.05 for total terminal ends of layer III pyramidal neurons; F(3, 16) = 1.87, p b 0.05 for basal terminal ends of layer V pyramidal neurons; F(3, 16) = 5.31, p b 0.05 for apical terminal ends of

Fig. 5. 3D reconstructed hippocampal CA1 pyramidal neurons. Representative reconstructed hippocampal CA1 pyramidal neurons of the control, aging, DHEAS and testosterone treated cortices (A–D). The apical dendritic trunk is in red while the filled blue circle represents cell body. Branches of each basal dendritic trunk are displayed with the same color. The abbreviation and horizontal bars on the right of each drawing mark the hippocampal layers. SLM, stratum lacunosum-moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Bar = 100 μm for A–D.

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

197

Fig. 6. Dendritic arbors analysis of layer III and V pyramidal neurons. The dendritic length (A and B), number of terminal ends (C and D), and dendritic branching profuseness (E and F, Sholl's analysis) were analyzed. For Sholl's analysis, dendritic crossings on the concentric circles drawn from the center of soma at 50-μm increments in radii were adopted. *, p b 0.05 between the marked and control rat.

layer V pyramidal neurons; F(3, 16) = 13.34, p b 0.05 for total terminal ends of layer V pyramidal neurons) and dendritic branching profuseness (Fig. 6E and F), were found to be withdrawn in aging rats. A similar alteration was observed in the dendritic arbor of hippocampal CA1 pyramidal neurons which appeared to be retracted in aging male rats (Fig. 8A and B; F(3, 16) = 6.7, p b 0.05 for basal terminal ends of CA1 pyramidal neurons; F(3, 16) = 2.02, p b 0.05 for apical terminal ends of CA1 pyramidal neurons; F(3, 16) = 4.64, p b 0.05 for total terminal ends of CA1 pyramidal neurons; F(3, 16) = 16.5, p b 0.05 for basal terminal ends of CA1 pyramidal neurons; F(3, 16) = 2.75, p b 0.05 for apical terminal ends of CA1 pyramidal neurons; F(3, 16) = 8.7, p b 0.05 for total terminal ends of CA1 pyramidal neurons). Exogenous DHEAS or testosterone did not promote the change in shape of the dendritic arbor of central neurons (Figs. 6 and 8). The total dendritic lengths of both layer III and V pyramidal neurons of the aged rats were significantly shorter than their young counterparts, respectively (Fig. 6A and B; F(3, 16) = 5.91, p b 0.001 for total dendritic length of layer III pyramidal neurons; F(3, 16) = 13.11, p b 0.001 for total dendritic length of layer V pyramidal neurons). The reduction appeared to be slightly greater in apical than basal dendrites, 18% and 20% versus 14% and

19% for layer III and layer V pyramidal neurons, respectively. Sholl's analysis of the dendritic branching profuseness between pyramidal neurons of the aged and young adult rats revealed further differences such as fewer branches and shorter boundary (Fig. 6E and F). To find out whether the shortening of dendrites in pyramidal neurons of the aged cortex was accompanied by alteration of dendritic spines, we studied the density of their dendritic spines (Fig. 7). In the aged cortex, the spine density on the proximal and distal apical dendrites was reduced by 47% and 49% in layer III, and 35% and 49% in layer V pyramidal neurons as compared with those of young animals, respectively. The spine density on proximal and distal basal dendrites was reduced by 31% and 44% in layer III, and 36% and 28% in layer V pyramidal neurons of the aged cortex, respectively (Fig. 7C; F(3, 16) = 23.39, p b 0.05 for proximal basal dendrite of layer III pyramidal neurons; F(3, 16) = 25.91, p b 0.05 for distal basal dendrite of layer III pyramidal neurons; F(3, 16) = 64.26, p b 0.05 for proximal apical dendrite of layer III pyramidal neurons; F(3, 16) = 77.43, p b 0.05 for distal basal dendrite of layer III pyramidal neurons; F(3, 16) = 20.83, p b 0.05 for proximal basal dendrite of layer V pyramidal neurons; F(3, 16) = 24.73, p b 0.05 for distal basal dendrite of layer V pyramidal neurons; F(3, 16) = 26.5, p b 0.05 for

198

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

or hippocampal CA1 pyramidal neurons, but the spine density remained lower in density compared with that of young rats (Figs. 7C and 8D). Exogenous testosterone treatment in aging male rats also significantly improved the spine loss on layer III and layer V somatosensory cortical neurons or hippocampal CA1 pyramidal neurons, but in some segments the effect of testosterone was less evident than DHEAS as manifested at the distal segment of basal dendrite and all segments of apical dendrite on layer III and layer V cortical pyramidal neurons and distal segment of apical dendrite on CA1 pyramidal neurons (Figs. 7C and 8D). We next aimed to ascertain whether DHEAS would exert its effect on young male or hypogonadal rats. The results showed that the spine density on all segments of layers III and V pyramidal neurons was significantly decreased 2 weeks after castration, and that DHEAS treatment significantly restored the spine density. Remarkably, the spine density of DHEAS-treated normal rats also exceeded that of the vehicle-treated rats (Fig. 9; layer III pyramidal neurons: F(3, 16) = 39.16, p b 0.05 for spine density of proximal basal dendrite; F(3, 16) = 61.34, p b 0.05 for spine density of distal basal dendrite; F(3, 16) = 62.07, p b 0.05 for spine density of proximal apical dendrite; F(3, 16) = 72.68, p b 0.05 for spine density of distal apical dendrite; layer V pyramidal neurons: F(3, 16) = 68.12, p b 0.05 for spine density of proximal basal dendrite; F(3, 16) = 76.57, p b 0.05 for spine density of distal basal dendrite; F(3, 16) = 70.61, p b 0.05 for spine density of proximal apical dendrite; F(3, 16) = 51.87, p b 0.05 for spine density of distal apical dendrite). 4. Discussion

Fig. 7. Spine density analysis of layer III and V pyramidal neurons. Representative micrographs showing the dendritic spines of layer III (A) and V (B) pyramidal neurons of the somatosensory cortices in control, aging, DHEAS and testosterone treated rats. Changes of spine density are analyzed in C. *, p b 0.05 between the marked and control rat; #, p b 0.05 between the marked and aging rats; &, p b 0.05 between the marked and aging + D groups. Bar = 10 μm for A and B.

proximal apical dendrite of layer V pyramidal neurons; F(3, 16) = 72.28, p b 0.05 for distal basal dendrite of layer V pyramidal neurons). Like the somatosensory cortex, the spine density on the basal dendrite and proximal and distal apical dendrites was reduced respectively by 18%, 19% and 38% in hippocampal CA1 pyramidal neurons compared with that in the young rats (Fig. 8C and D; F(3, 16) = 13.81, p b 0.05 for basal dendrite of CA1 pyramidal neurons; F(3, 16) = 51.3, p b 0.05 for proximal apical dendrite of CA1 pyramidal neurons; F(3, 16) = 54.3, p b 0.05 for distal basal dendrite of CA1 pyramidal neurons). Interestingly, exogenous DHEAS treatment in aging male rats significantly regained the dendritic spines on layer III and layer V somatosensory cortical neurons

The major finding of this study is that the widespread functions of androgen could partially restore the age-related spine loss in layer III, layer V somatosensory cortical neurons and hippocampal CA1 pyramidal neurons. Associated with this, androgen enhances the performance of aging rats in water maze task. As in aging female rats, the layer III, layer V primary somatosensory cortical neurons or hippocampal CA1 pyramidal neurons in normal aging male rats exhibited a significant regression in the dendritic arbors and loss of dendritic spines (Wang et al., 2009, 2014). In general, dendritic spines are the primary sites of excitatory input on most cortical pyramidal neurons and their density may affect the synaptic transmission. Loss of dendritic spines in hippocampal CA1 pyramidal neurons has also been demonstrated in normal aging rat and senescence-accelerated mice (del Valle et al., 2012). The deterioration of spatial learning in adult and aging rats is consistent with the notion that the hippocampus is required for learning and memory. The concomitant reduction of dendritic spines on the hippocampal neurons would be the underlying cause. It would appear therefore that dendritic change is a common feature affecting most central neurons such as pyramidal neurons in the medial prefrontal cortex (Grill and Riddle, 2002), motor cortices (Nakamura et al., 1985) and hippocampus (Luine et al., 2011), cerebellar Purkinje neurons (Zhang et al., 2010) and retinal ganglion cells (Samuel et al., 2011). It is suggested that regression in dendritic arbor and loss of dendritic spines on layer III, layer V primary somatosensory cortical neurons or hippocampal CA1 pyramidal neurons as observed in this study in aging rats may reflect different degrees of degenerative changes that might be induced by regional changes in trophic support (Chen et al., 1997) or in neuronal activity (Grill and Riddle, 2002). In the clinic, such dendritic arbor regression and reduction in dendritic spine density of cortical pyramidal neurons may contribute to behavioral dysfunctions as observed in aging including cognitive loss, anxiety, fatigue, poor concentration and memory, and confusion. Regression of the dendritic arbors as well as loss of dendritic spines both in layer III and layer V somatosensory cortical pyramidal neurons was observed in both normal aging female (Wang et al., 2009, 2014) and male (present results) rats. We reported previously that castration for up to 24 weeks did not alter the dendritic arbor; instead, it reduced the spine density of the above-mentioned neurons. It is therefore suggested that the dendritic arbors and spines of cortical pyramidal

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

199

Fig. 8. Dendritic arbors and spine density analysis of hippocampal CA1 pyramidal neurons. The dendritic length (A) and number of terminal ends (B) were analyzed. Representative micrographs showing the dendritic spines of distal apical dendrite of hippocampal CA1 pyramidal neurons in control, aging and DHEAS treated rats (C). Changes of spine density are analyzed in D. *, p b 0.05 between the marked and control rats; #, p b 0.05 between the marked and aging rats; &, p b 0.05 between the marked and aging + D groups. Bar = 10 μm for C.

neurons are differentially regulated (Chen et al., 2013). In general, dendritic spines may be largely maintained by gonadal hormones (Chen et al., 2009b, 2013) or synaptic activity (Saneyoshi and Hayashi, 2012), whereas the dendritic arbors may be more dependent on trophic factors (Andrews and Cowen, 1994; Ma and Taylor, 2010). As reported in our previous study, androgen modulates the spine density in somatosensory cortical neurons of castrated rats (Chen et al., 2013). Here we show that the testosterone supplement also can recover the spine loss of somatosensory cortical pyramidal neurons in normal aging rats. A similar action of androgen on spine synapses has been identified in the hippocampus of adult male rodents (Leranth et al., 2003) and hippocampus and prefrontal cortical neurons of non-human primates (Hajszan et al., 2008). Another novel finding of this study is that exogenous DHEAS can improve the deteriorated performance of water maze task and restore the spine loss of hippocampal CA1 pyramidal neurons. This is consistent with the suggestion that DHEA promotes synaptogenesis of hippocampal pyramidal neurons (MacLusky et al., 2004). Interestingly, the synaptogenic function of DHEAS was not only confined or restricted to the hippocampal pyramidal neurons, but also appears to regulate the somatosensory cortical neurons. In addition, it is striking that DHEAS promotes the spine density of somatosensory pyramidal neurons two weeks after castration in comparison with the normal aging rats. It is noteworthy that in some dendritic segments of cortical pyramidal neurons, the efficacy of DHEAS on recovery of spine loss is better than testosterone treatment (Figs. 7 and 8). In castrated rats, DHEAS can fully restore the spine loss on layer III and V somatosensory pyramidal neurons or even surpassing numerically that of the control rats (Fig. 9 and Chen et al., 2013). Although direct evidence of the signal pathway of DHEAS

modulating the spine density of cortical pyramidal neurons is lacking, the following possible mechanisms may be considered. Recently, some electrophysiological studies by Chen et al. have shown that DHEAS treatment significantly facilitates the long-term potentiation of hippocampal CA1 pyramidal neurons (Chen et al., 2006a,b). Studies with the antagonist treatment have suggested that DHEAS mainly acts on NMDA receptor via sigma-1 (σ1) receptor in the induction of DHEAS-facilitated LTP (Chen et al., 2006a). Further study shows that DHEAS functions through an activation of NMDAr/Src-mediated signal amplification mechanism followed by a MAPK/ERK signaling cascade (Chen et al., 2006b). Their results indicated that in the DHEAS-treated rats the peak amplitude of intracellular Ca2+ elevation increased up to approximately 2.4-fold of control rats (Chen et al., 2006b). Intracellular calcium is an important secondary message in the spine plasticity modulation of cortical pyramidal neurons (Murakoshi et al., 2011; Nikonenko et al., 2002; Oertner and Matus, 2005). Live cell imaging studies have suggested that changes in Ca2+ concentration in the dendritic spine alter the arrangement and dynamics of the spine actin cytoskeleton leading to changes in spine morphology and stability (Fischer et al., 1998; Majewska et al., 2000). Thus, the efficacy of DHEAS on recovery of spine loss of cortical pyramidal neurons may depend on the intracellular Ca2+ transients. Some studies also indicate that DHEAS can play a neuromodulatory role and alter synaptic transmission in the hippocampus through selective postsynaptic actions on inhibitory synaptic transmission (Ffrench-Mullen and Spence, 1991; Meyer et al., 1999; Steffensen, 1995). It has been reported that DHEA binding to the androgen or estrogen receptor is lower because its affinity is at least an order of magnitude less than that of the endogenous ligand (Waterhouse et al., 2007). In the present study, exogenous DHEAS is

200

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

Fig. 9. The effects of DHEAS treatment in castrated rats. Representative micrographs showing the dendritic spines of proximal apical dendrite of layer III and V pyramidal neurons (A) of the somatosensory cortices in control (Control) and 2 weeks castrated (C2w) rats with vehicle or DHEAS treatment. The spine density of layer III (B) and layer V (C) pyramidal neurons was analyzed. *, p b 0.05 between the marked and vehicle treated control rats; #, p b 0.05 between the marked and 2 week castrated rats with vehicle treated rats. Bar = 10 μm for A.

superior to testosterone in terms of its effect in regaining spine loss of cortical pyramidal neurons. Thus, the effects of DHEA do not appear to be mediated by direct binding to the androgen or estrogen receptor. An alternative explanation for DHEAS action on cellular function is through its metabolism to other steroids that do have a high affinity

for estrogen and androgen receptors. DHEAS may be converted into estrogen or testosterone in tissues that have the appropriate steroidogenic enzymes (Miller, 2008). This suggests that circulating DHEA and DHEAS exist as a precursor pool for steroid hormone synthesis in appropriate tissues and under appropriate conditions. These steroidogenic enzymes do exist in brain areas such as the cerebral cortex, thus raising the possibility that DHEA can be produced de novo in the brain, or can be converted to another active steroid by local cellular metabolism (Cascio et al., 2000; Hajszan et al., 2004; Ishii et al., 2007; MacLusky et al., 2004; Mukai et al., 2006a,b). Recent studies reported that sex hormones, including estrogen and testosterone, could modulate the spine density of hippocampal pyramidal neurons (Leranth et al., 2003; Woolley et al., 1996) and primary somatosensory pyramidal neurons (Chen et al., 2009b, 2013). Further data have suggested that DHEA treatment increases CA1 spine synapse density, an effect that is blocked by a nonsteroidal aromatase inhibitor (Hajszan et al., 2004). They thus propose that the ability of DHEA to increase spine density is mediated by the aromatization of DHEA in the CA1 pyramidal neurons, and could be through activation of either estrogen or androgen receptors (Hajszan et al., 2004; Ishii et al., 2007; Mukai et al., 2006b). In aging male rats, DHEAS may improve the spine loss of cortical pyramidal neurons through either the sigma-1 receptor or steroidogenic enzymes. In the lack of experimental evidence, this remains purely speculative. It has been hypothesized that such a change in androgen levels may contribute to variations in cognitive function, as well as to the incidence of certain types of neurological disorders, with cognitive dysfunction being their prominent symptom. Several studies provide support for this idea, as investigating androgen levels in patients with Alzheimer's disease has demonstrated a reduction in the circulating concentrations of testosterone and DHEA-sulfate, in comparison to normal controls (Hogervorst et al., 2001; Rosario et al., 2010; Sunderland et al., 1989; Yanase et al., 1996) Subsequent research has also found androgens to have clear positive effects on cognitive performance in human beings, as well as in laboratory animals (Almeida et al., 2004; Flood et al., 1995; Hirshman et al., 2004). In addition, androgens appear to elicit a well-characterized positive influence on mood (Almeida et al., 2004; Barrett-Connor et al., 1999a; Seidman, 2003a). Until now, there is a lack of large-scale, long-term studies assessing the benefits and risks of testosterone replacement therapy in hypogonadism. Many reports indicate that testosterone replacement therapy may produce a wide range of benefits including improvement in fatigue, sexual function, bone density, muscle mass, body composition, mood, erythropoiesis, cognition, quality of life, and cardiovascular disease (Bassil, 2011; Pinsky and Hellstrom, 2010). The most controversial area of testosterone replacement therapy is the issue of risk, especially the possible stimulation of prostate cancer by testosterone, even though there is no evidence to support this. Other possible risks include worsening symptoms of benign prostatic hypertrophy, liver toxicity, hyperviscosity, erythrocytosis, worsening untreated sleep apnea, or severe heart failure (Bassil and Morley, 2010; Maas et al., 1997). On the other hand, no serious or adverse effects related to DHEA have been reported. The present results suggest that both the gonadal hormone precursors DHEAS and testosterone are effective in restoring the agerelated or hypogonadism-induced loss of dendritic spines associated with the pyramidal neurons in the somatosensory cortex and hippocampus in male rats. Thus DHEAS, a choice supplement for ameliorating syndromes associated with hypogonadism and menopause in the female, may also be used in male hypogonadism for its supporting activity of dendritic spines. The fact that DHEAS or testosterone was effective in restoring the dendritic spines following 5 consecutive days of intraperitoneal injection demonstrates that effect is immediate or early in onset. This indicates the responsiveness of cortical neurons subjected to extended period of deprivation of gonadal hormones. Together, the present results strongly argue that in addition to treating the spine loss caused by hypogonadism, DHEAS treatment may offer a potential and effective therapeutic strategy restoring the loss of cortical neuronal

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

dendritic spines resulted from senility or diseases such as Alzheimer's disease and dementia. 5. Conclusion Like the female counterpart, aging in males reduced the blood testosterone and at the same time the brain structures notably the output neurons were altered. Aging rats performed poorer in Morris water maze and all pyramidal neurons of their primary cortical and hippocampal CA1 pyramidal neurons had reduced dendritic arbors and spine density. Like testosterone, DHEAS appeared to act on central neurons to maintain their excitatory synaptic connection for effectively alleviating age-related behavioral deficit and might be a better candidate for hormone replacement therapy. Acknowledgments This work was supported by research grants from the National Science Council of Taiwan to Chen, J-R (NSC102-2320-B-005-001-MY3) and Tseng, G-F (NSC100-2320-B-320-001) and grants from the NUTC to Wang, T-J (101T23302) and Tzu-Chi University to Wang, Y-J and Tseng, G-F (TCIRP98006). References Almeida, O.P., Waterreus, A., Spry, N., Flicker, L., Martins, R.N., 2004. One year follow-up study of the association between chemical castration, sex hormones, beta-amyloid, memory and depression in men. Psychoneuroendocrinology 29, 1071–1081. Andrews, T.J., Cowen, T., 1994. Nerve growth factor enhances the dendritic arborization of sympathetic ganglion cells undergoing atrophy in aged rats. J. Neurocytol. 23, 234–241. Barrett-Connor, E., Goodman-Gruen, D., Patay, B., 1999a. Endogenous sex hormones and cognitive function in older men. J. Clin. Endocrinol. Metab. 84, 3681–3685. Barrett-Connor, E., Von Muhlen, D.G., Kritz-Silverstein, D., 1999b. Bioavailable testosterone and depressed mood in older men: the Rancho Bernardo Study. J. Clin. Endocrinol. Metab. 84, 573–577. Bassil, N., 2011. Late-onset hypogonadism. Med. Clin. North. Am. 95, 507–523. Bassil, N., Morley, J.E., 2010. Late-life onset hypogonadism: a review. Clin. Geriatr. Med. 26, 197–222. Cascio, C., Brown, R.C., Liu, Y., Han, Z., Hales, D.B., Papadopoulos, V., 2000. Pathways of dehydroepiandrosterone formation in rat brain glia. J. Steroid Biochem. Mol. Biol. 75, 177–186. Chen, L., Hamaguchi, K., Hamada, S., Okado, N., 1997. Regional differences of serotoninmediated synaptic plasticity in the chicken spinal cord with development and aging. J. Neural Transplant. Plast. 6, 41–48. Chen, J.R., Wang, Y.J., Tseng, G.F., 2003. The effect of epidural compression on cerebral cortex: a rat model. J. Neurotrauma 20, 767–780. Chen, L., Dai, X.N., Sokabe, M., 2006a. Chronic administration of dehydroepiandrosterone sulfate (DHEAS) primes for facilitated induction of long-term potentiation via sigma 1 (sigma1) receptor: optical imaging study in rat hippocampal slices. Neuropharmacology 50, 380–392. Chen, L., Miyamoto, Y., Furuya, K., Dai, X.N., Mori, N., Sokabe, M., 2006b. Chronic DHEAS administration facilitates hippocampal long-term potentiation via an amplification of Src-dependent NMDA receptor signaling. Neuropharmacology 51, 659–670. Chen, J.R., Wang, T.J., Huang, H.Y., Chen, L.J., Huang, Y.S., Wang, Y.J., Tseng, G.F., 2009a. Fatigue reversibly reduced cortical and hippocampal dendritic spines concurrent with compromise of motor endurance and spatial memory. Neuroscience 161, 1104–1113. Chen, J.R., Yan, Y.T., Wang, T.J., Chen, L.J., Wang, Y.J., Tseng, G.F., 2009b. Gonadal hormones modulate the dendritic spine densities of primary cortical pyramidal neurons in adult female rat. Cereb. Cortex 19, 2719–2727. Chen, J.R., Wang, T.J., Wang, Y.J., Tseng, G.F., 2010. The immediate large-scale dendritic plasticity of cortical pyramidal neurons subjected to acute epidural compression. Neuroscience 167, 414–427. Chen, J.R., Wang, T.J., Lim, S.H., Wang, Y.J., Tseng, G.F., 2013. Testosterone modulation of dendritic spines of somatosensory cortical pyramidal neurons. Brain Struct. Funct. 218, 117–1407. de Brabander, J.M., Kramers, R.J., Uylings, H.B., 1998. Layer-specific dendritic regression of pyramidal cells with ageing in the human prefrontal cortex. Eur. J. Neurosci. 10, 1261–1269. del Valle, J., Bayod, S., Camins, A., Beas-Zarate, C., Velazquez-Zamora, D.A., GonzalezBurgos, I., Pallas, M., 2012. Dendritic spine abnormalities in hippocampal CA1 pyramidal neurons underlying memory deficits in the SAMP8 mouse model of Alzheimer's disease. J. Alzheimers Dis. 32, 233–240. Dickstein, D.L., Kabaso, D., Rocher, A.B., Luebke, J.I., Wearne, S.L., Hof, P.R., 2007. Changes in the structural complexity of the aged brain. Aging Cell 6, 275–284.

201

Dickstein, D.L., Weaver, C.M., Luebke, J.I., Hof, P.R., 2012. Dendritic spine changes associated with normal aging. Neuroscience. http://dx.doi.org/10.1016/j.neuroscience.2012. 1009.1077. Ffrench-Mullen, J.M., Spence, K.T., 1991. Neurosteroids block Ca2+ channel current in freshly isolated hippocampal CA1 neurons. Eur. J. Pharmacol. 202, 269–272. Fischer, M., Kaech, S., Knutti, D., Matus, A., 1998. Rapid actin-based plasticity in dendritic spines. Neuron 20, 847–854. Flood, J.F., Farr, S.A., Kaiser, F.E., La Regina, M., Morley, J.E., 1995. Age-related decrease of plasma testosterone in SAMP8 mice: replacement improves age-related impairment of learning and memory. Physiol. Behav. 57, 669–673. Genazzani, A.D., Lanzoni, C., Genazzani, A.R., 2007. Might DHEA be considered a beneficial replacement therapy in the elderly? Drugs Aging 24, 173–185. Grill, J.D., Riddle, D.R., 2002. Age-related and laminar-specific dendritic changes in the medial frontal cortex of the rat. Brain Res. 937, 8–21. Hajszan, T., MacLusky, N.J., Leranth, C., 2004. Dehydroepiandrosterone increases hippocampal spine synapse density in ovariectomized female rats. Endocrinology 145, 1042–1045. Hajszan, T., MacLusky, N.J., Leranth, C., 2008. Role of androgens and the androgen receptor in remodeling of spine synapses in limbic brain areas. Horm. Behav. 53, 638–646. Hirshman, E., Merritt, P., Wang, C.C., Wierman, M., Budescu, D.V., Kohrt, W., Templin, J.L., Bhasin, S., 2004. Evidence that androgenic and estrogenic metabolites contribute to the effects of dehydroepiandrosterone on cognition in postmenopausal women. Horm. Behav. 45, 144–155. Hogervorst, E., Williams, J., Budge, M., Barnetson, L., Combrinck, M., Smith, A.D., 2001. Serum total testosterone is lower in men with Alzheimer's disease. Neuro. Endocrinol. Lett. 22, 163–168. Ishii, H., Tsurugizawa, T., Ogiue-Ikeda, M., Asashima, M., Mukai, H., Murakami, G., Hojo, Y., Kimoto, T., Kawato, S., 2007. Local production of sex hormones and their modulation of hippocampal synaptic plasticity. Neuroscientist 13, 323–334. Janowsky, J.S., 2006. The role of androgens in cognition and brain aging in men. Neuroscience 138, 1015–1020. Leranth, C., Petnehazy, O., MacLusky, N.J., 2003. Gonadal hormones affect spine synaptic density in the CA1 hippocampal subfield of male rats. J. Neurosci. 23, 1588–1592. Luine, V.N., Wallace, M.E., Frankfurt, M., 2011. Age-related deficits in spatial memory and hippocampal spines in virgin, female Fischer 344 rats. Curr. Gerontol. Geriatr. Res. 2011, 316386. Ma, C.H., Taylor, J.S., 2010. Trophic responsiveness of purified postnatal and adult rat retinal ganglion cells. Cell Tissue Res. 339, 297–310. Maas, D., Jochen, A., Lalande, B., 1997. Age-related changes in male gonadal function. Implications for therapy. Drugs Aging 11, 45–60. MacLusky, N.J., Hajszan, T., Leranth, C., 2004. Effects of dehydroepiandrosterone and flutamide on hippocampal CA1 spine synapse density in male and female rats: implications for the role of androgens in maintenance of hippocampal structure. Endocrinology 145, 4154–4161. MacLusky, N.J., Hajszan, T., Prange-Kiel, J., Leranth, C., 2006. Androgen modulation of hippocampal synaptic plasticity. Neuroscience 138, 957–965. Majewska, A., Brown, E., Ross, J., Yuste, R., 2000. Mechanisms of calcium decay kinetics in hippocampal spines: role of spine calcium pumps and calcium diffusion through the spine neck in biochemical compartmentalization. J. Neurosci. 20, 1722–1734. Mervis, R., 1978. Structural alterations in neurons of aged canine neocortex: a Golgi study. Exp. Neurol. 62, 417–432. Meyer, J.H., Lee, S., Wittenberg, G.F., Randall, R.D., Gruol, D.L., 1999. Neurosteroid regulation of inhibitory synaptic transmission in the rat hippocampus in vitro. Neuroscience 90, 1177–1183. Miller, W.L., 2008. Steroidogenic enzymes. Endocr. Dev. 13, 1–18. Mukai, H., Takata, N., Ishii, H.T., Tanabe, N., Hojo, Y., Furukawa, A., Kimoto, T., Kawato, S., 2006a. Hippocampal synthesis of estrogens and androgens which are paracrine modulators of synaptic plasticity: synaptocrinology. Neuroscience 138, 757–764. Mukai, H., Tsurugizawa, T., Ogiue-Ikeda, M., Murakami, G., Hojo, Y., Ishii, H., Kimoto, T., Kawato, S., 2006b. Local neurosteroid production in the hippocampus: influence on synaptic plasticity of memory. Neuroendocrinology 84, 255–263. Murakoshi, H., Wang, H., Yasuda, R., 2011. Local, persistent activation of Rho GTPases during plasticity of single dendritic spines. Nature 472, 100–104. Nakamura, S., Akiguchi, I., Kameyama, M., Mizuno, N., 1985. Age-related changes of pyramidal cell basal dendrites in layers III and V of human motor cortex: a quantitative Golgi study. Acta Neuropathol. 65, 281–284. Nikonenko, I., Jourdain, P., Alberi, S., Toni, N., Muller, D., 2002. Activity-induced changes of spine morphology. Hippocampus 12, 585–591. Oertner, T.G., Matus, A., 2005. Calcium regulation of actin dynamics in dendritic spines. Cell Calcium 37, 477–482. Parducz, A., Hajszan, T., Maclusky, N.J., Hoyk, Z., Csakvari, E., Kurunczi, A., Prange-Kiel, J., Leranth, C., 2006. Synaptic remodeling induced by gonadal hormones: neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids. Neuroscience 138, 977–985. Parker Jr., C.R., 1999. Dehydroepiandrosterone and dehydroepiandrosterone sulfate production in the human adrenal during development and aging. Steroids 64, 640–647. Peters, A., Sethares, C., Moss, M.B., 1998. The effects of aging on layer 1 in area 46 of prefrontal cortex in the rhesus monkey. Cereb. Cortex 8, 671–684. Pinsky, M.R., Hellstrom, W.J., 2010. Hypogonadism, ADAM, and hormone replacement. Ther. Adv. Urol. 2, 99–104. Rosario, E.R., Carroll, J., Pike, C.J., 2010. Testosterone regulation of Alzheimer-like neuropathology in male 3xTg-AD mice involves both estrogen and androgen pathways. Brain Res. 1359, 281–290. Saltzman, E., Guay, A., 2006. Dehydroepiandrosterone therapy as female androgen replacement. Semin. Reprod. Med. 24, 97–105.

202

J.-R. Chen et al. / Experimental Gerontology 57 (2014) 191–202

Samuel, M.A., Zhang, Y., Meister, M., Sanes, J.R., 2011. Age-related alterations in neurons of the mouse retina. J. Neurosci. 31, 16033–16044. Saneyoshi, T., Hayashi, Y., 2012. The Ca2+ and Rho GTPase signaling pathways underlying activity-dependent actin remodeling at dendritic spines. Cytoskeleton 69, 545–554. Scheibel, M.E., Scheibel, A.B., 1975. Dendrite bundles, central programs and the olfactory bulb. Brain Res. 95, 407–421. Seidman, S.N., 2003a. The aging male: androgens, erectile dysfunction, and depression. J. Clin. Psychiatry 64 (S10), 31–37. Seidman, S.N., 2003b. Testosterone deficiency and mood in aging men: pathogenic and therapeutic interactions. World J. Biol. Psychiatry 4, 14–20. Steffensen, S.C., 1995. Dehydroepiandrosterone sulfate suppresses hippocampal recurrent inhibition and synchronizes neuronal activity to theta rhythm. Hippocampus 5, 320–328. Sunderland, T., Merril, C.R., Harrington, M.G., Lawlor, B.A., Molchan, S.E., Martinez, R., Murphy, D.L., 1989. Reduced plasma dehydroepiandrosterone concentrations in Alzheimer's disease. Lancet 2, 570. Vallee, M., Mayo, W., Le Moal, M., 2001. Role of pregnenolone, dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging. Brain Res. Brain Res. Rev. 37, 301–312.

Wang, T.J., Chen, J.R., Wang, Y.J., Tseng, G.F., 2009. The cytoarchitecture and somadendritic arbors of the pyramidal neurons of aged rat sensorimotor cortex: an intracellular dye injection study. Neuroscience 158, 776–785. Wang, T.J., Chen, J.R., Wang, W.J., Wang, Y.J., Tseng, G.F., 2014. Genistein partly eases aging and estropause-induced primary cortical neuronal changes in rats. PLoS One 9 (2), e89819. http://dx.doi.org/10.1371/journal.pone.0089819. Waterhouse, R.N., Chang, R.C., Atuehene, N., Collier, T.L., 2007. In vitro and in vivo binding of neuroactive steroids to the sigma-1 receptor as measured with the positron emission tomography radioligand [18F]FPS. Synapse 61, 540–546. Woolley, C.S., Wenzel, H.J., Schwartzkroin, P.A., 1996. Estradiol increases the frequency of multiple synapse boutons in the hippocampal CA1 region of the adult female rat. J. Comp. Neurol. 373, 108–117. Yanase, T., Fukahori, M., Taniguchi, S., Nishi, Y., Sakai, Y., Takayanagi, R., Haji, M., Nawata, H., 1996. Serum dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) in Alzheimer's disease and in cerebrovascular dementia. Endocr. J. 43, 119–123. Zhang, C., Zhu, Q., Hua, T., 2010. Aging of cerebellar Purkinje cells. Cell Tissue Res. 341, 341–347.