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Androgens Increase Survival of Adult-Born Neurons in the Dentate Gyrus by an Androgen Receptor-Dependent… Hamson, Dwayne K.; Wainwright, Steven R.; Taylor, J. R.; Jones, B. A.; Watson, Neil Verne; Galea, Liisa A.M. Sep 1, 2013

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1 Androgens Increase Survival of Adult Born Neurons in the Dentate Gyrus by an Androgen Receptor 1 Dependent Mechanism in Male Rats. 2 D. K. Hamson1, S. R. Wainwright2, J. R. Taylor, B. A. Jones, N. V. Watson3, L. A. M. Galea*1,2  3 1. Department of Psychology, University of British Columbia, Vancouver, British Columbia, Canada4 2. Program in Neuroscience, University of British Columbia, Vancouver, British Columbia, Canada5 3. Department of Psychology, Simon Fraser University, Burnaby, British Columbia, Canada6 * Corresponding Author: L. A. M. Galea, Department of Psychology, 2136 West Mall, Vancouver,7 British Columbia, Canada, V6T 1Z4.  Tel: 604 822 6536, Fax: 604 822 6923, email: lgalea@psych.ubc.ca 8 9 Key Words: adult neurogenesis, dentate gyrus, hippocampus, androgens, androgen 10 receptor, neuron survival 11 12 D.K.H., S.R.W., J.R.T., B.A.J., N.V.W., L.A.M.G. have nothing to declare 13 Text word count: 5476 14 *Published as: Hamson D.K., Wainwright S.R., Taylor J.R., Jones B.A., Watson N.V. , Galea L.A.M. (2013) Androgens Increase Survival of Adult Born Neurons in the Dentate Gyrus by an Androgen Receptor Dependent but Cell Non-Autonomous Mechanism. Endocrinology, 154: 3294-3304. ) 2 Abstract 15 Gonadal steroids are potent regulators of adult neurogenesis.  We previously reported that androgens, 16 such as testosterone (T) and dihydrotestosterone (DHT), but not estradiol, increased survival of new 17 neurons in the dentate gyrus of the male rat. These results suggest androgens regulate hippocampal 18 neurogenesis via the androgen receptor (AR). To test this supposition, we examined the role of ARs in 19 hippocampal neurogenesis using two different approaches. In Experiment 1, we examined neurogenesis in 20 male rats insensitive to androgens due to a naturally occurring mutation in the gene encoding the AR 21 (termed TFMs) compared to wild type males. In Experiment 2, we injected the AR antagonist, flutamide, 22 into castrated male rats and compared neurogenesis levels in the dentate gyrus of DHT and oil-treated 23 controls. In Experiment 1, chronic T increased hippocampal neurogenesis in wild type males but not in 24 androgen-insensitive TFM males. In Experiment 2, DHT increased hippocampal neurogenesis via cell 25 survival, an effect that was blocked by concurrent treatment with flutamide.  DHT, however, did not 26 affect cell proliferation. Interestingly, cells expressing doublecortin, a marker of newborn immature 27 neurons, did not colabel with ARs in the DG, but ARs were robustly expressed in other regions of the 28 hippocampus.  Together, these studies provide complementary evidence that androgens regulate adult 29 neurogenesis in the hippocampus via the AR but at a site other than the dentate gyrus.  Understanding 30 where in the brain androgens act to increase the survival of new neurons in the adult brain may have 31 implications for neurodegenerative disorders. 32   33  3 Introduction 34 Neurogenesis in the adult is observed in the dentate gyrus (DG) in rodents (1) and humans (2).  35 Despite its evolutionarily conserved nature, the role of adult neurogenesis remains to be fully elucidated, 36 although studies suggest adult hippocampal neurogenesis is an important factor in learning, memory (3) 37 and depression (4).  38 Neurogenesis in the hippocampus is a multistep process that begins with the proliferation of cells, 39 differentiation into neurons, migration to the granule cell layer, survival and integration of these new 40 neurons into the circuitry. Adult hippocampal neurogenesis can be increased via independent modulation 41 of any one of these stages. For example, spatial learning increases neurogenesis in the hippocampus via 42 enhancement in cell survival independent of changes in cell proliferation (5, 6).  Conversely, chronic 43 antidepressant treatment increases neurogenesis in the hippocampus via increasing cell proliferation 44 independent of cell survival (7). 45 Androgens and estrogens are potent modulators of adult hippocampal neurogenesis. Chronic 46 estradiol decreases neurogenesis in adult female rats (8). In males, however, chronic treatment with 47 testosterone or dihydrotestosterone (DHT), but not estradiol, increases hippocampal neurogenesis via cell 48 survival (9). Given that testosterone can be converted to estradiol via aromatase or to the non-49 aromatizable androgen, DHT via 5α reductase, this previous finding suggests that androgens mediate 50 hippocampal neurogenesis via binding to the androgen receptor (AR) in male rats. However, this is 51 somewhat equivocal as DHT can be reduced to another metabolite, 5α-androstane-3α,17β-diol (3α-52 androstanediol; 3α-Diol) (10) that may act via a non AR mediated mechanism (11, 12). Thus, it is unclear 53 whether androgens mediate neurogenesis via ARs or through some other mechanism. Although there are 54 no reported ARs in the DG in the majority of rat strains (13,14, but see 15) it is possible that there is 55 transient AR expression in immature neurons which may suggest a direct mechanism for androgens to 56 promote survival of immature neurons. 57  Thus, the current study examined the hypothesis that androgens increase neurogenesis via cell 58 survival in the adult DG via ARs using genetic (Experiment 1) and pharmacological (Experiment 2) 59  4 methods.    In Experiment 1, we used male rats harboring a mutation in the gene encoding the AR, the 60 testicular feminization mutation (TFM) rats. These chromosomal males are insensitive to androgens due 61 to a missense mutation in the steroid-binding domain (16), producing a non-functional AR at 62 physiological androgen concentrations (17). In Experiment 2, we block the ability of DHT to bind ARs 63 via systemic treatment with the competitive AR antagonist, flutamide, and examine neurogenesis and AR 64 expression in different regions of the hippocampus.  We hypothesize that if androgens are enhancing 65 neurogenesis in the DG by acting on ARs, androgens should not affect neurogenesis in TFM-affected or 66 in flutamide-treated male rats.  67 Methods 68 Animals-  69 In Experiment 1, 7 wild type (wt) Sprague Dawley males and 6 males carrying a mutation in the 70 gene encoding the androgen receptor (termed testicular feminization mutation males, or TFMs) were 71 generated at Simon Fraser University.  Sprague Dawley females previously identified as carrying the 72 TFM allele (XwtXtfm) were mated with males and, after weaning, pups were identified at 30 days of age as 73 having a male (large anogenital distance, scrotal sac, penis, no nipple line) or a female (short anogenital 74 distance, no scrotal sac, nipple line) phenotype.  Because males carrying the testicular feminization 75 mutation (XtfmY) cannot be distinguished phenotypically from females (18, 19, 20), TFMs were positively 76 identified as being male and having a mutated AR via a PCR assay of genomic DNA extracted from ear 77 tissue (see 21 for details).  In Experiment 2, 23 adult male Sprague-Dawley rats were generated from our 78 breeding colony at the University of British Columbia.  Animals were housed in clear polyurethane bins 79 with wood chip bedding, maintained on a 12hr light:12hr dark schedule in a temperature controlled room, 80 and given access to tap water and lab chow ad libitum. All protocols were approved by the institutional 81 animal care committee (UBC and SFU) and conformed to the regulations set by the Canadian Committee 82 for Animal Care.  All efforts were made to reduce animal suffering. 83 Surgeries and Steroid Manipulations-  84 5 Experiment 1: Wild type males (n=7) and TFMs (n=7) were castrated at approximately 60 days 85 old under aseptic conditions using isoflurane. At the time of surgery, animals within each group were 86 given either 2 20mm Silastic (Dow Corning, Midland, MI) implants filled with Testosterone propionate 87 (Steraloids Inc; internal diameter = 1.57mm; external diameter = 3.18mm) or 2 20mm Silastic implants 88 filled with silicone (blanks), placed sc on the back of the neck.  The testosterone propionate and blank 89 Silastic capsules were constructed and prepared following Smith, Damassa, and Davidson (22).  90 Berndtson et al, (23) reported serum testosterone levels in the high physiological range of males 91 implanted with the same sized capsules containing testosterone propionate.  Capsules of this size are also 92 expected to release approximately 1.5mg testosterone propionate every 24 hours (23).  Following 93 recovery, all animals were injected with BrdU (200mg/kg) intraperitonially, prepared the same as above.  94 This experiment was carried out at Simon Fraser University.  95 Experiment 2: Castrations were performed at approximately 60 days old and under aseptic 96 conditions using isoflurane. Animals were monitored closely and weighed daily for one week after 97 surgery. After recovery, all animals received a single intraperitoneal injection (ip) of 5-98 bromodeoxyuridine (BrdU; 200mg/kg; Sigma-Aldrich, St. Louis, MO) to label dividing cells and their 99 progeny. BrdU was prepared fresh by dissolving 20mg/ml of BrdU in warm 0.9% saline buffered with 100 0.7% NaOH.  One day following BrdU injection, animals received subcutaneous (sc) injections for 30 101 days of either: 1) vehicle (oil; n=6), 2) dihydrotestosterone (DHT; 0.25mg/day; n=5), 3) flutamide 102 (2.5mg/day; n=6), or 4) a combination of DHT and flutamide (DHT 0.25mg/day: flutamide 2.5mg/day; 103 n=6) delivered in a 0.6cc bolus.  The dose of DHT chosen previously increased neurogenesis in castrated 104 male rats (9). The dose of flutamide, a competitive androgen receptor antagonist, has been shown to 105 decrease the weight of the bulbocavernosus and levator ani (BC/LA) muscles (24). This experiment was 106 carried out at the University of British Columbia. The experimental timeline is shown in Figure 1. 107 Tissue Preparation- After 30 days of steroid treatments, all animals were given an overdose of 108 sodium pentobarbital or CO2 and transcardially perfused with either 0.1M phosphate buffered saline 109 (PBS; Experiment 1) or normal saline (0.9%; Experiment 2), followed by 4% phosphate buffered 110  6 paraformaldehyde (PFA). Brains were harvested, placed in 4% paraformaldehyde for 24 hours, then 111 placed in 30% sucrose and stored at 4°C until sectioning. Brains were sectioned on a microtome at 40µm 112 through the entire rostro-caudal extent of the DG and divided into 10 series of sequential sections.  Tissue 113 was stored an antigen-sparing solution (0.05M tris-buffered saline, 30% ethylene glycol, and 20% 114 glycerol).  All cell counts and areas were quantified on every 10th section from the entire hippocampus.  115 The average number of sections per series was 14.5 +/- 1.2.  For Experiment 2, the BC/LA muscles, 116 located at the base of the penis, were also harvested at the time of perfusion via bilateral cuts of the 117 ischiocavernosus and careful dissection from the anus (see 25 for a review).  Muscles were then blotted 118 dry and weighed.  119 Histology 120 1. Immunocytochemistry 121  A. BrdU: All rats were perfused 32 days after BrdU injection and thus the majority of BrdU-122 labelled cells would be 31 days old.  BrdU and Ki67 were visualized in the GCL and hilus on every 10th 123 section using the avidin-biotin peroxidase method.  Free-floating sections were rinsed 3 times for 10 124 minutes in tris-buffered saline (TBS; pH 7.4) in between each step listed below (unless otherwise stated).  125 To eliminate endogenous peroxidase activity, sections were incubated for 10 minutes in 0.6% hydrogen 126 peroxide. To denature DNA and facilitate exposure of the BrdU antigen sites, tissue was incubated in 2 N 127 hydrochloride acid (HCl) for 30 mins in a water bath (37°C), immediately followed by 10 minutes in 0.1 128 M borate buffer (pH 8.5) to neutralize the HCl.  Sections were blocked in 3% normal donkey serum 129 (NDS; Chemicon, Temecula, CA, USA) diluted in TBS for 30 minutes at room temperature and then 130 incubated in a mouse anti-BrdU monoclonal primary antibody (1:200 in TBS, 3% NDS, and 0.1% Triton-131 X 100; Roche Diagnoistics, Laval, Quebec, Canada) for 48 hours. Sections were then incubated in a 132 donkey anti-mouse secondary antibody (1:100 in TBS; Vector Laboratories, Burlington, ON, Canada) for 133 4 hours at room temperature followed by incubation in avidin-biotin horseradish peroxidase complex for 134 2 hours at room temperature (ABC Elite Kit; 1:100; Vector Laboratories, Burlington, ON, Canada). The 135 chromogen, diaminobenzidine (DAB; Sigma), was used to visualize antigen sites. Tissue was reacted for 136  7 5 minutes in 0.002% DAB diluted in TBS containing 0.003% hydrogen peroxidase and 0.03% nickel 137 chloride for enhancement. Sections were mounted onto Super-frost/Plus (Fisher Scientific, Napean, ON, 138 Canada) slides and allowed to dry overnight at room temperature. The following day, sections counter 139 stained with cresyl violet and cover slipped with Permount (Fisher Scientific).  140  B. Ki67:  Ki67 is expressed during the active phase of the cell cycle except for G0 and part of G1 141 and is thus an excellent endogenous marker of cell proliferation (see 26 for review).  Free-floating 142 sections were rinsed 3 times for 10 minutes in 0.1M phosphate buffered saline (PBS) in between each step 143 listed below unless otherwise stated. To eliminate endogenous peroxidase activity, sections were 144 incubated for 10 minutes in 0.6% hydrogen peroxide diluted in deionized water.  Sections were incubated 145 overnight at 4°C in a rabbit polyclonal primary directed against Ki67 (1:3000; Vector VPK 451), diluted 146 in PBS + 0.04% Triton-x, and 3% normal goat serum (NGS).  The following day, sections were rinsed 5 147 times for 10 minutes in PBS and then incubated overnight at 4°C in a goat anti-rabbit secondary (1:500; 148 Vector) diluted in PBS.  Sections were then washed 5 times for 10 minutes in PBS and then incubated in 149 an avidin-biotin complex for 1 hour and 15 minutes.  This was followed by washing tissue 2 times for 2 150 minutes in sodium acetate buffer (0.175M) before being reacted in DAB (SK-4100; prepared following 151 manufacturer’s instructions) for 15 minutes.  Sections were mounted onto glass sides, counterstained, and 152 cover slipped as previously stated.  153 2. Immunofluorescence 154  To determine a possible site of androgen action on cell survival, we used double label 155 immunofluorescence for doublecortin (DCX) and androgen receptor (AR). DCX is a microtubule-156 associated phosphoprotein that preferentially labels newborn neurons in the GCL (27).  Additionally, we 157 determined the cellular phenotype of a subset of randomly chosen newborn cells in the GCL of the DG 158 using double label immunofluorescence for BrdU and the neuron specific marker, NeuN, to give an 159 estimate of the total number of neurons produced in the adult hippocampus (28).  160  Every tenth section containing the dentate gyrus was used to visualize DCX/AR or BrdU/NeuN.  161 In between each step listed below, tissue was rinsed 3 times for 10 minutes in TBS unless otherwise 162  8 stated.  Tissue was blocked before each antibody incubation listed below for 30 minutes in 3% NDS 163 diluted in TBS and containing 3% Triton-X 100.  Additionally, all antibodies were diluted in TBS 164 containing 1% normal donkey serum (NDS) and 3% Triton-X 100.  To label DCX and AR, tissue was 165 incubated for 24 hours in both the goat anti-DCX (1:200; C18, sc8066, Santa Cruz Biotechnology, Santa 166 Cruz, CA, USA) and rabbit anti-AR (1:200; Epitomics, Burlingame, CA, USA) primary antibodies.  167 Tissue was subsequently incubated in a donkey anti-goat Alexa488 secondary (1:200; Invitrogen; 168 Burlington, ON, Canada) to label DCX and a donkey anti-rabbit Alexa549 secondary (1:200; Invitrogen) 169 to label AR. Sections were mounted on Superfrost/Plus slides and cover slipped with diazobicyclooctane 170 (TBS, 2.5% DABCO, 10% polyvinyl alcohol, and 20% glycerol) to prevent fading.   171  For BrdU and NeuN, tissue was incubated for 48hours in a mouse anti-NeuN (1:100; Chemicon) 172 at 4°C. Tissue was then incubated overnight at 4°C in donkey ant-rabbit FITC secondary (1:200; 173 Invitrogen).  Sections were fixed in 4% paraformaldehyde diluted in TBS followed by two rinses in 174 normal saline.  Tissue was incubated in 2 N hydrochloride acid (HCl) for 30 mins in a water bath (37°C), 175 and immediately incubated for 10 mins in 0.1 M borate buffer (pH 8.5) and then incubated for 48 hours at 176 4°C in a rat anti-BrdU primary (1:250; Roche Diagnostic), followed by a donkey anti-rat secondary (Cy3; 177 1:200; Invitrogen).  Tissue was mounted and cover slipped with the anti-fade agent, as stated above.  178 No immunoreactivity was observed in any of the tissue processed when the primary antibody was 179 omitted. 180 Microscopy 181  An experimenter blind to treatment conditions counted BrdU-labelled cells in the GCL and the 182 hilus at 1000x magnification on a light microscope (Nikon Eclipse 600).  All BrdU-labelled cells were 183 counted bilaterally through the entire dentate gyrus (13-15 sections in total). BrdU-labelled cells observed 184 within 50µm of the inner edge of the GCL (i.e., in the subgranular zone; SGZ) were combined with 185 counts from the GCL. Cells in the hilus were counted separately for many reasons: 1) to account for 186 potential changes in the blood-brain barrier permeability by treatment; 2) new neurons in the hilus are 187 considered ectopic; and 3) new cells in the hilus give rise to a different population of cells than new cells 188  9 in the GCL. Ki67-expressing cells were counted on all sections containing the subgranular zone.  An 189 estimate of the total number of BrdU-labelled and Ki67-expressing cells per rat was calculated by 190 multiplying the number of cells by 10 (9).  The sampling frame consisted of the entire dentate gyrus and 191 to avoid duplication of counting, cells in the uppermost plane of focus were not counted.  Areas of each 192 section were quantified from digitized images for both the GCL and hilus.  Volume estimate was 193 calculated by multiplying the sum of the area of each section by the section thickness (Cavalieri’s 194 principle).  195  In order to determine whether androgens increase neurogenesis directly in the DG by acting on 196 immature neurons, we examined whether DCX-expressing cells were double labeled for ARs.  Fifty 197 DCX-expressing neurons were randomly selected from the GCL of each rat. The percentage of DCX-198 expressing cells colabelled with AR was determined at a magnification of 600x.  Doublecortin is a 199 cytoplasmic protein expressed from day 1- 21 after proliferation (29) and because of this, DCX-200 expressing cells display a wide range of the maturation and extent of processes.  We thus examined the 201 morphology of the processes of DCX-expressing cells and classified them into 1 of 3 categories based on 202 criteria established by (30) and used in a number of studies (31, 32, 33, 34).  Briefly, DCX-expressing 203 cells were classified as 1) “immature” if they displayed no or very short processes (<10µm):, 2) 204 “intermediate” if they displayed longer processes than the immature cells, but the processes only reached 205 within the GCL or touched the molecular cell layer but did not extend further or 3) “postmitotic” if a 206 single thick dendrite extended and branched into the molecular layer or if the dendrites were fine and 207 displayed multiple branch points within the GCL.    208  In addition to examining AR immunofluorescence in DCX-expressing cells, we also assessed AR 209 immunofluorescence in the CA1 and CA3 regions of the hippocampus, the ventromedial hypothalamus 210 (VMH), and the posterodorsal division of the medial amygdala (MePD) using a 4 point subjective rating 211 scale (robust- +++; intermediate- ++; light- +, absent – 0).  The distribution of AR in these areas served as 212 a positive control and the subjective ratings of the immunofluorescence were compared to previously 213 studies (13, 14). 214  10 To determine if BrdU-labelled cells were of a neuronal phenotype, 50 cells from each animal 215 were randomly selected from the GCL (25 from dorsal, 25 from ventral DG) and the percentage of BrdU-216 labelled cells colabelled with the neuron specific marker, NeuN, was determined at 600x magnification.  217 The total number of BrdU-labeled cells was then multiplied by the percentage of BrdUNeuN labeled 218 neurons, for an estimation of “total neurogenesis” (35, 36).   219 Statistical Analyses 220 For Experiment 1, total BrdU-labelled and Ki67-expressing cells, the volume of the GCL and 221 hilus were each analyzed separately using repeated measures analysis of variance (RM-ANOVA) with 222 brain region (GCL, hilus) as a within-subjects factor and androgen treatment (DHT, Oil) and anti-223 androgen treatment (flutamide, Oil) as between-subjects factors.  The phenotypes of DCX-expressing 224 cells were represented as a percent and were thus arc sine transformed to facilitate statistical analysis.  A 225 RM-ANOVA was run on the transformed values with phenotype (immature, intermediate, mature) as a 226 within-subjects factor and androgen treatment and anti-androgen treatment as between-subjects factors.  227 The mass of the BC/LA muscles and net neurogenesis (percent BrdU/NeuN double labeled neurons x 228 total BrdU labeled cells) were analyzed using a univariate ANOVA with androgen treatment and anti-229 androgen treatment as between-subjects factors. For Experiment 2, RM-ANOVA was used to separately 230 analyze volume and total BrdU-labelled cells with brain region (GCL, hilus) as a within subjects factor 231 and treatment (Testosterone, blank) and genotype (wild type male, TFM) as between-subjects factors. 232 Test statistics were considered significant if their probability value was ≤ 0.05.  Where appropriate, post 233 hoc analyses were carried out using the Neuman-Keul’s procedure. 234 Results 235  Experiment 1: 236 1. Androgen treatment did not affect the volume of the DG in wild type males and TFMs. 237 As expected, the volume of the hilus was larger compared to the volume of the GCL (main effect 238 of region: F (1, 11)=130.3,p<0.0001).  However there were no other significant main or interaction effects 239 of androgens on the volume of the GCL or hilus (all p’s >0.26; see Table 1). 240  11 2. Testosterone implants increased cell survival in wild type males, but not in TFMs. 241  Testosterone-treated wild type males had more BrdU-labelled cells in the GCL+SGZ compared 242 to all other groups (interaction between region, genotype, and androgen treatment: F (1, 11)=6.13, 243 p=0.03; all p’s < p=0.008).  Androgen treatment was completely ineffective at increasing cell survival in 244 the GCL+SGZ of TFM-affected males, as the number of BrdU-labelled cells in the testosterone -treated 245 TFM-affected males did not differ significantly from the blank-treated TFM-affected males (p=0.59).  246 Finally, the blank-treated wild type males did not differ from both the testosterone-implanted and blank-247 implanted TFM-affected males on the number of BrdU-labelled cells (all p’s >0.8).   There were no other 248 significant main or interaction effects (all p’s> 0.07).  See Figure 2). 249 Experiment 2: 250 1. There were no significant group differences in the volume of the DG. 251 As expected, the hilus was larger than the dentate gyrus (main effect of region: p<0.0001), 252 however, there were no other main or interaction effects (all p’s > .36; see Table 2).  253 2. DHT increased BC/LA muscle mass, an effect that was blocked by flutamide. 254  We examined the muscle mass of the highly androgen sensitive perineal muscles (37), the 255 BC/LA, to ensure our dose of flutamide (2.5mg/rat/day) was able to block the effects of DHT. As 256 expected, the mass of the BC/LA muscle in the DHT treated group was significantly higher compared to 257 all other groups (all p’s<0.0001; interaction of androgen and anti-androgen treatment (F (1, 19)=62.95, 258 p<0.0001). Flutamide alone did not have any anabolic effects on muscle mass, as the weights did not 259 differ significantly compared to the oil treated group (p=0.21). See Table 2. 260  3. DHT treatment increased the number of BrdU-labelled cells in the GCL, an effect that was blocked by 261 co-treatment with flutamide. 262 We examined the total number of BrdU-labeled cells in the GCL+SGZ and hilus following 30 263 days of treatment of either oil, DHT, DHT + flutamide, or flutamide in order to determine whether 264 androgens increased BrdU-labeled cells by acting directly through ARs.  Systemic injections of DHT 265 increased the total number of BrdU-labelled cells in the GCL+SGZ compared to the oil-treated castrates 266  12 (p=0.0002) and as we hypothesized, flutamide treatment blocked the ability of DHT to increase cell 267 survival in the GCL+SGZ (p=0.0003: interaction between region, androgen treatment, and anti androgen 268 treatment: F (1, 19) =4.68, p=0.04).  However, when administered alone, flutamide had no significant 269 effect on the total number of BrdU-labeled cells in the GCL+SGZ (vs oil treated group; p=0.86).  270 Androgen or anti androgen treatment did not affect the total number of BrdU-labelled cells in the hilus 271 compared to oil controls (all p’s >0.9) (see Figure 3A and 3D).  We have also analyzed the average 272 number of BrdU-labeled cells per section, and the pattern of results is the same as for the analysis of the 273 total number of cells. That is, DHT increased the average number of BrdU-labeled cells per section and 274 flutamide blocked this androgenic increase (data not shown). 275 4. Flutamide, but not DHT exposure, reduced the percentage of BrdU-labeled cells that were colabelled 276 with NeuN. 277 We examined the colabelling of BrdU and NeuN (BrdU/NeuN) in the GCL to determine how 278 many BrdU-labeled cells were neurons.  Approximately 79-84% were colabelled with the neuron specific 279 marker, NeuN, suggesting that the majority of BrdU-labelled cells were neurons (see Table 3).  Flutamide 280 treatment reduced the percentage of BrdU/NeuN labeled cells compared to oil treated controls (main 281 effect of anti-androgen treatment: F (1,19)=9.1, p=0.007).  However, there were no other significant 282 effects (all p’s>0.15) and DHT treatment did not affect the percentage of BrdU/NeuN labeled cells 283 (p=0.29; see Table 3).  284 5. DHT treatment increased the number of neurons in the GCL, but this effect was blocked by co 285 administration with flutamide. 286 To obtain an estimation of the overall number of neurons produced in the adult GCL (i.e., “net 287 neurogenesis”) we multiplied the total number of BrdU-labeled cells by the proportion of BrdU-labeled 288 cells that also expressed the neuron specific marker, NeuN (35, 36).  There were more new neurons 289 (BrdU/NeuN-labeled) in the DHT treated males compared to the DHT + flutamide, flutamide, and oil 290 treated groups (all p’s <0.01;Table 3; interaction between androgen and anti-androgen treatment: F 291 (1,19)=5.6, p=0.029). 292  13 6. Thirty days of DHT treatment did not significantly affect cell proliferation in the DG. 293 In order to determine whether systemic DHT administration affected cell proliferation, we 294 examined the total number of Ki67-expressing cells in the GCL+SGZ and the hilus.  In contrast to the 295 effects of androgen treatment on cell survival, there were no significant main or interaction effects on the 296 total number of Ki67-expressing cells (all p’s >0.4; Figure 3B, 3D). 297 7. DHT administration did not significantly affect the proportion of doublecortin-expressing immature 298 neurons displaying immature, intermediate or mature processes in the GCL. 299 We examined the morphology of the processes of DCX-expressing neurons in the GCL in order 300 to determine whether DHT treatment affected the dendritic maturation of these neurons. The proportion 301 of DCX-expressing neurons displaying the mature phenotype (~49%) was significantly higher compared 302 to the immature (~31%; p= 0.013) and intermediate (~20%; p= 0.0006) phenotypes (main effect of 303 phenotype: F (2, 36)= 8.66, p= 0.0008, all other main or interaction effects (all p’s> 0.13). Figure 4 (A-F, 304 K-M) depict examples of DCX-expressing immature neurons in the GCL.  Figure 4N contains a 305 histogram of the percentages of DCX phenotypes expressed by each treatment group. 306 8. No ARs were observed in DCX-expressing neurons in the DG. 307 In order to determine whether androgens increase neurogenesis in the DG by acting directly upon 308 immature neurons in the adult hippocampus, we examined the colocalization of DCX and AR in the GCL. 309 None of the DCX-expressing neurons displayed nuclear AR immunofluorescence (see Figure 4G-J). In 310 fact, there were no nuclear AR expressing cells throughout the entire extent of the DG. However, DHT-311 treated males displayed robust AR immunofluorescence in the CA1 (see Figure 5B) of the hippocampus 312 and the ventromedial hypothalamus (VMH).  While AR immunofluorescence was also observed in the 313 CA3 (see Figure 5D that depicts AR immunofluorescence in the CA3 of a representative animal treated 314 with DHT) and in the medial amygdala, it was not as robust compared to the CA1 and VMH.  315 Conversely, androgen receptor expression was completely absent in these regions of oil-, flutamide-, and 316 DHT + flutamide-treated groups (see Figure 5E that depicts a lack of AR immunofluorescence in the CA3 317 of a representative animal treated with DHT+ flutamide).  318  14 Discussion 319 This is the first study to provide direct evidence that androgens affect survival of newborn 320 neurons in the hippocampus of adult male rats by acting directly through the AR using both genetic and 321 pharmacological methods. First, we replicate a previous report showing that systemic DHT treatment 322 increased the survival of new neurons in the DG (9).  Second, we extend these results by showing that the 323 survival promoting effects of DHT on neurogenesis can be blocked via systemic injection of the AR 324 specific antagonist, flutamide. We further demonstrate that a competent AR is necessary for androgen-325 induced neurogenesis in the hippocampus, as chromosomal males carrying a mutation in the gene 326 encoding the androgen receptor (TFM-affected males) do not show an androgen-induced increase in 327 neurogenesis compared to wild type (wt) males.  Finally, given our observation that ARs are not 328 expressed by newborn neurons in the DG, the data support the conclusion that androgens mediate the 329 survival of new neurons by acting on ARs somewhere other than the DG.  330 Androgens increase the survival of new neurons in the DG 331  In the current report, androgen treatment (both DHT and T) in adult wildtype male rats increased 332 the survival of new neurons in the DG. In Experiment 2 BrdU was injected 24 h prior to hormone 333 treatment, thus BrdU-labeled cells would have been daughter cells at the time of the first androgen 334 treatment as the length of the cell cycle is approximately 24 h (28). There was no effect of castration or 335 DHT treatment after 30 days on cell proliferation (Ki67-expressing cells) in the DG.  This is consistent 336 with previous reports that showed short- or long-term castration did not significantly affect cell 337 proliferation in the hippocampus (9, 38, 39).   DHT treatment also did not alter the phenotype of DCX 338 expressing cells or the percentage of cells double-labeled for BrdU and NeuN.  Together these data 339 suggest androgens increase adult neurogenesis in the DG by affecting neuron survival independent of 340 affecting cell proliferation or differentiation of new neurons. 341  The duration of androgen administration is an important factor in promoting the survival of new 342 neurons. We found that 30 days of systemic androgen treatment increased cell survival in the DG of adult 343 male rats (current study, and 9). However, a shorter duration (15 days) of androgen treatment did not 344  15 enhance DG neurogenesis (40), suggesting that the optimal time period for androgens to increase cell 345 survival is between 16-30 days, and given the time frame involved, likely via a genomic mechanism. 346 Androgens Increase Neurogenesis via the Androgen Receptor 347 Flutamide, a competitive AR antagonist, blocked the ability of DHT to increase both the total 348 number of BrdU-labeled cells and the overall number of neurons generated in the GCL (i.e., “net 349 neurogenesis”).  Given that flutamide prevented the DHT induced increase in the mass of the BC/LA 350 muscles, and blocked the expression of ARs in the hippocampus, amygdala, and ventromedial 351 hypothalamus, all which are AR mediated effects (14, 41, 42, 43) suggests our dose of flutamide was 352 effective in antagonizing ARs systemically. Thus, data from Experiment 2 support the conclusion that 353 androgens mediate adult neurogenesis in the GCL via the AR and not through the transformation of DHT 354 to a metabolite that acts via GABAa or progesterone receptors.   355 Repeated estradiol administration in adulthood results in a decrease in cell survival in female, but 356 not in male, rats (8), suggesting a sex difference in the neurogenic response to estradiol.  Given the lack of 357 an effect of chronic estradiol on hippocampal neurogenesis in adult males in adulthood strongly suggests 358 androgens may have an organizational (i.e., developmental) effect in determining the response to 359 estradiol.  Further, given that the androgen receptor plays an important role in the masculinization of 360 sexually dimorphic structures in the nervous system, and that TFM males do not have a functional 361 androgen receptor throughout their entire life, one possibility that could account for the results in 362 Experiment 1 is that TFM-affected males may have responded in a feminine manner to estradiol.  In the 363 current study, we used capsules filled with testosterone propionate, and thus estradiol would have been 364 formed in both males and TFMs despite a non-functional AR protein (see 44).  If TFMs are feminine in 365 their response to estradiol, then the overall lower levels of neurogenesis observed in the TFMs compared 366 to wildtype males may have been due to a decrease in cell survival due to a lack of AR stimulation as we 367 have proposed or the effects of estradiol to decrease neurogenesis in TFMs.  It has previously been 368 reported that certain regions of the TFM nervous system are not fully masculine (45), however, some 369 aspects are fully masculinized despite the mutation in the AR gene (reviewed in 46). What is more, there 370 16 are no published reports suggesting the dentate gyrus in TFMs is feminine and thus we have no reason to 371 suspect a feminine response to estradiol.  Finally, given that the results in Experiment 1 and Experiment 2 372 are similar despite the different androgens used, we conclude androgens mediate new neuron survival in 373 the DG via the androgen receptor.  374 Adult Born Immature Neurons Do Not Express Androgen Receptors. 375 In the current report, we observed nuclear AR immunoreactivity in the medial amygdala, VMH, 376 CA1 and CA3 regions but not within the DG.  This is consistent with a variety of published reports 377 showing that both nuclear AR mRNA and protein are expressed in the CA1/CA3 regions of the 378 hippocampus, the amygdala, and hypothalamus, but not in the DG  (13, 14, 47, 48). To our knowledge, 379 this is the first study to examine whether immature neurons express ARs in the DG. We did not find 380 evidence of DCX and AR colabelling indicating that immature neurons do not express ARs. While it is 381 possible that ARs are transiently expressed within immature neurons, given that DCX is expressed 382 anywhere from 1-21 days after birth of the neuron, we believe that this is not the case. Furthermore we 383 did not observe AR immunofluorescence independently of DCX in the SGZ, thus, we are confident ARs 384 are not expressed within the DG. 385 Given that we did not observe AR expression in immature neurons or elsewhere in the DG leads 386 to the parsimonious conclusion that androgens act in a non cell-autonomous manner, and in another 387 region to affect neuron survival. The CA3 region is of interest as newborn granule neurons extend axons 388 to CA3 pyramidal cells within 4 days after mitosis (49), CA3 pyramidal cells contain ARs, and 389 destruction of this region decreases survival, but not proliferation, of new neurons in the DG (50).  All of 390 these findings suggest that the CA3 is an important region regulating adult hippocampal neurogenesis. 391 Further, terminals from granule neurons synapse on to specialized structures of CA3 pyramidal neurons, 392 termed thorny excrescences (51).  The induction of thorns is dependent upon AR as an AR antagonist 393 (hydroxyflutamide) blocks the ability of androgens to induce production of thorns (51).  Thus, androgens 394 appear to be coordinating the necessary structural changes in CA3 that may lead to increased survival of 395 newborn neurons. 396  17  How do androgens increase new neuron survival in the DG via a non cell autonomous 397 mechanism? We propose that androgens act upon neurons in the CA3 region of the hippocampus, which 398 then send an as yet unidentified retrograde survival factor to newborn neurons in the DG.  A similar 399 proposal of non cell-autonomous action has been put forth for the androgen dependent survival of motor 400 neurons in the spinal nucleus of the bulbocavernosus (SNB), and thus may be a common mechanism of 401 androgen action in the nervous system. Androgens act on the target muscles, BC/LA, to spare SNB motor 402 neurons from programmed cell death during development, possibly via ciliary neurotrophic factor (52).   403 It should be noted that other hormones affect neurogenesis in the adult dentate gyrus via 404 regulation of neuron survival.  Dehydroepiandrosterone (DHEA) can bind to ARs (53) and can increase 405 neurogenesis by affecting survival, similar to androgens, but can also affect proliferation, unlike 406 androgens (54).  In male mice, 3 days of injections of progesterone increases the survival of 7, 28 and 56 407 day old neurons (55), while an escalating dose of progesterone can also stimulate cell proliferation in 408 male rats (56).  Finally, chronic estradiol (15 days) increases new neuron survival in the hippocampus in 409 female, but not male, rats (57), although 5 days of exposure to estradiol can increase neurogenesis in male 410 meadow voles when administered 6-10 days after BrdU (58).  Interestingly, a percentage of newborn 411 neurons can express estrogen receptors suggesting estradiol may regulate survival directly (59). 412 Intriguingly there is a sex difference in the percentage of new neurons that express estrogen receptors (59, 413 60).  For a more detailed review the reader is directed to Galea et al (61). 414 Conclusions 415  We provide evidence that androgens promote neurogenesis in the adult hippocampus by 416 increasing the survival of newborn neurons via an androgen receptor dependent mechanism.  However, 417 androgens did not significantly affect cell proliferation, the morphology of immature neurons or the 418 percentage of new cells differentiating into neurons in the adult hippocampus.  The data support the 419 conclusion that androgens do not promote neurogenesis by acting directly on adult born neurons, but 420 instead act via a cell non-autonomous process that is accomplished outside of the dentate 421 gyrus.   Determining how, why and where androgens are working to promote adult neurogenesis in the 422  18 hippocampus may prove important in developing new therapeutic treatments for neurodegenerative 423 disease.  424  425  426  427  428  429  430  431  432  433  434  435  436  437  438  439  440  441  442  443  444  445  446  447  448  19 Acknowledgements 449 We would like to thank Anne Cheng and Alice Chan for animal care.  Funding for this research was 450 provided by the Canadian Institutes of Health Research (LAMG) and from the Natural Sciences and 451 Engineering Research Council of Canada (NVW). 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Zhang Z, Yang R, Zhou R, Li L, Sokabe M, Chen L (2010) Progesterone promotes the survival of 606 newborn neurons in the dentate gyrus of adult male mice. Hippocampus 20:402-412.  607 56. Barha CK, Ishrat T, Epp JR, Galea LA, Stein DG (2011) Progesterone treatment normalizes the levels 608 of cell proliferation and cell death in the dentate gyrus of the hippocampus after traumatic brain injury.  609 Exp Neurol 231:72-81. 610 57. Barker JM, Galea LAM (2008) Repeated estradiol administration alters different aspects of 611 neurogenesis and cell death in the hippocampus of female, but not male, rats. Neuroscience 152:888–902.  612 58. B.K. Ormerod BK, T.T.-Y. Lee TT-Y, L.A.M. Galea LAM (2004) Estradiol enhances neurogenesis in 613 the dentate gyri of adult male meadow voles by increasing the survival of young granule neurons. 614 Neuroscience 128(3): 645–654 615 59. Mazzucco CA, Lieblich SE, Bingham BI, Williamson MA, Viau V, Galea LAM (2006) Both estrogen 616 receptor α and estrogen receptor β agonists enhance cell proliferation in the dentate gyrus of adult female 617 rats. Neuroscience 141:1793–1800.  618 60. Isgor C, Watson SJ (2005) Estrogen receptor alpha and beta mRNA expressions by proliferating and 619 differentiating cells in the adult rat dentate gyrus and subventricular zone. Neuroscience 134:847-856. 620 61. Galea LA, Uban KA, Epp JR, Brummelte S, Barha CK, Wilson WL, Lieblich SE, Pawluski JL (2008) 621 Endocrine regulation of cognition and neuroplasticity: our pursuit to unveil the complex interaction 622 between hormones, the brain, and behaviour. Can J Exp Psychol 62:247-260. 623  624  625  626  627  628  629  630  631  632  26 Figure Captions 633 Figure 1. Graphical depiction of the protocol used in Experiment 2. 634 Males were castrated at 60 days of age and allowed to recover for 1 week.  Following recovery, animals 635 were injected with bromodeoxyuridine (BrdU), and then treated with either oil, flutamide, 636 dihydrotestosterone, or dihydrotestosterone and flutamide for 30 days.  Animals were perfused and brains 637 and bulbocavernosus/levator ani muscles were harvested at day 100. 638 Figure 2. Testosterone implants increased the total number of BrdU labeling in wild type males only. 639 Mean total BrdU-labelled cells in wild type males (Male) implanted with either testosterone propionate 640 (TP) or blank capsules (Bl) and chromosomal males carrying the testicular feminization mutation (TFMs) 641 implanted with either TP or Bl capsules.  TP was only effective in increasing cell survival in the granule 642 cell layer (GCL) of males; there was no effect of TP on survival in TFMs. *= significantly different 643 compared to Bl males, TP TFM, and Bl TFM. 644 Figure 3. Antagonism of the androgen receptor (AR) by flutamide decreased the survival of cells in the 645 granule cell layer. 646 A) Mean total BrdU-labelling in the dentate gyrus and subgranular zone (DG + SGZ) of animals treated 647 with oil, flutamide (Flut), dihydrotestosterone (DHT), or dihydrotestosterone and flutamide (DHT + Flut).  648 DHT treatment increased the number of BrdU-labelled cells compared to all other groups (compared to 649 oil-treated group (p<0.0002), DHT+Flut (p<0.0003), Flut (p<.0003)). The ability of DHT to increase the 650 number of BrdU-labelled cells was blocked by co-treatment with flutamide. *= significantly different 651 compared to all other groups. B) Mean total number of Ki67 labeled cells in the granule cell layer (GCL) 652 and hilus.  There were no significant effects of DHT or flutamide treatment on cell proliferation in the 653 subgranular zone (SGZ) or hilus. C) The colocalization of the neuron specific marker, NeuN (arrow), with 654 bromodeoxyuridine (BrdU; arrow) in the same cell from a representative animal suggests this is a new 655 adult born neuron.  Scale bar = 20μm. D) Representative examples of BrdU (arrow in first panel) and 656 Ki67 (arrow in second panel) immunoreactivity in the GCL and SGZ, respectively.  Scale bar = 20μm. 657  27 Figure 4.  Dihydrotestosterone treatment did not affect the ratio of immature, intermediate, or mature 658 doublecortin expressing neurons. Panels A and B- Low power (using a 10X objective) photomicrograph 659 of representative DCX immunofluorescence in a male treated with dihydrotestosterone (A) and a male 660 treated with dihydrotestosterone and flutamide (B; scale bars = 200um).  Arrows (A) and arrowheads (B) 661 point to examples of DCX expressing neurons in the GCL.   Panels C, D, E, and F- the same DCX 662 expressing neurons from A and B magnified 400x. Scale bar = 20um.  Panels G, H, I, and J- no AR 663 immunofluorescence was found in any of the exemplar DCX expressing neurons.  Scale bar = 20um. 664 Panels K, L, M- representative examples of neurons used in classifying DCX expressing neurons as (K) 665 “immature”, (L) “intermediate”, and (M) “mature”.  Panel N Doublecortin morphology of immature 666 neurons in the granule cell layer (GCL) of males treated with Oil, flutamide (Flut), Dihydrotestosterone 667 (DHT), and DHT and flutamide (DHT+Flut).  There were no statistically significant differences between 668 the groups in the percentage of cells displaying the immature, intermediate, or mature phenotypes. Note: 669 K and L were taken using a 40X objective and M was taken using a 100X oil immersion objective.   670 Figure 5. Androgen receptor (AR) immunofluorescence in the CA1 and CA3 regions of the 671 hippocampus. Panels A, B, and C- Representative low power photomicrograph of doublecortin (DCX; A) 672 and androgen receptor (AR; B) immunofluorescence in the hippocampus.  The arrow in A points to 673 neurons in the granule cell layer (GCL) expressing DCX from a male treated with dihydrotestosterone 674 (DHT).  The arrowheads in B point to nuclear AR immunofluorescence localized to the pyramidal cell 675 layer of the CA1/CA3 region of the hippocampus in the same DHT treated male. Panel C is the overlay of 676 DCX and AR immunofluorescence. Scale bar = 500um. Panels D and E- representative nuclear AR 677 immunofluorescence in pyramidal cells of CA3 in a male treated with DHT (D) and a lack of AR 678 immunofluorescence in a male treated with both DHT and flutamide (E).  Arrowheads in E point to 679 presumptive CA3 region.  Scale bar = 200um.1 680 Table 1. Volume of the granule cell layer (GCL) and Hilus of animals in Experiment 1. Genotype Treatment GCL (mm3) Hilus (mm3) Male Testosterone (n=4) 2.9±0.27 7.0±0.87 Blank (n=4) 3.3±0.31 9.2±0.90 TFM Testosterone (n=4) 3.1±0.27 8.1±0.93 Blank (n=3) 3.1±0.31 8.5±1.10 There were no statistically significant differences in the volume of both the GCL and hilus among groups.  As expected, the volume of the hilus was larger than the GCL. Table 2. Volume (mean+/-S.E.M.) of the granule cell layer (GCL), Hilus of animals, and weight (mean+/-S.E.M.) of the bulbocavernosus/levator ani muscle (BC/LA) in Experiment 2. There were no statistically significant differences in the volume of both the GCL and hilus among the groups in Experiment 2, however, the volume of the hilus was larger than the GCL.  The mass of the BC/LA muscle (in grams; gm) was higher in the DHT treated group compared to the oil treated group.  Flutamide alone did not affect the mass of the BC/LA weight, however, flutamide blocked the anabolic effects of DHT on BC/LA muscle mass. Statistical analysis revealed the mass of the BC/LA was highest in the DHT treated males compared to all other groups. The mass of the BC/LA did not differ between the oil-, flutamide-, and DHT + flutamide groups.  Note: * = p<0.001 for all post hoc comparisons. Treatment GCL (mm3) Hilus (mm3) BC/LA (gm) Oil (n=6) 2.6±0.17 7.7±0.55 0.36±0.03 Flut (n=6) 2.4±0.17 6.8±0.55 0.44±0.04 DHT (n=5) 2.6±0.19 6.9±0.60 1.05±0.05 * DHT+Flut (n=6) 2.3±0.17 6.6±0.55 0.46±0.04 Table 3.  Percentage (mean+/-S.E.M.) of BrdU-labeled cells colabeled with NeuN and Net Neurogenesis (mean+/-S.E.M.) in the granule cell layer of the dentate gyrus. Treatment BrdU/NeuN (%) Net Neurogenesis Oil (n=6) 84.33+/-0.01 3044.55+/-387.07 Flutamide (n=6) 82.67+/-0.01 2811.90+/-408.10 DHT (n=5) 84.80+/-0.01 4881.21+/-571.24 * DHT + Flutamide (N=6) 79.43+/-0.01 2753.05+/-224.36 The average number of neurons (i.e., BrdU/NeuN labeled) reaching survival was lowest in the groups that received flutamide.  Overall, the number of neurons produced (percent BrdU/NeuN x total BrdU labeled cells = net neurogenesis) was highest in the animals that received DHT compared to the other groups. Note: * = p<0.001 for all post hoc comparisons regarding “Net Neurogenesis”. 


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