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The activational and organizational influences of androgens on stress-induced changes in adult hippocampal… Kambo, Jaspreet Singh 2006

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T H E A C T I V A T I O N A L A N D O R G A N I Z A T I O N A L I N F L U E N C E S OF A N D R O G E N S O N S T R E S S - I N D U C E D C H A N G E S I N A D U L T H I P P O C A M P A L C E L L P R O L I F E R A T I O N A N D D E F E N S I V E B E H A V I O U R by J A S P R E E T S I N G H K A M B O B . Sc., The Univerisity of Toronto, 2003 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E In T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Neuroscience) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A December 2005 ©Jaspreet Singh Kambo, 2005 Role of androgens in stress-induced changes in adult cell proliferation Abstract Adult male laboratory rats demonstrate a suppression in hippocampal cell proliferation in response to an acute predator odour stress. This stress-induced suppression in cell proliferation is not seen in adult females and past studies have shown that activational levels of ovarian hormones do not regulate the stress-induced suppression. The present study examines the effects of activational and organizational levels of androgens on stress-induced changes in hippocampal cell proliferation, corticosterone (CORT) and defensive and non-defensive behaviours in adult male rats. In the first experiment, adult male Sprague-Dawley rats were castrated and exposed to trimethyl thiazoline (TMT) . Androgen status did not significantly affect TMT-induced suppression in cell proliferation or expression of defensive behaviours. However, castrated males did not show an increase in duration of stretch attends, a risk assessment behaviour. In the second experiment, male pups were injected with the androgen receptor antagonist flutamide, while females were injected with testosterone proprionate (TP) postnatally. Early T P treatment in adult females increased dentate gyrus volume, prevented a regular estrous cycle, increased adult body weight and decreased levels of cell proliferation without causing a stress-induced suppression in cell proliferation compared to oil-treated females. Flutamide-treated males did not significantly differ from oil-treated males on any measure. The results of these studies suggest that the sex difference in stress-induced suppression of hippocampal cell proliferation is not directly regulated by organizational or activational levels of androgens. Role of androgens in stress-induced changes in adult cell proliferation i i i T A B L E O F C O N T E N T S Abstract i i Table of Contents i i i List of Tables iv List o f Figures v Acknowledgements v i i Introduction 1 Methods 6 Results 16 Discussion 41 Activational levels of androgens do not mediate the stress-induced suppression of hippocampal cell proliferation in male rats 42 Activational levels of androgens do not alter the expression of defensive or non-defensive behaviours in response to predator odour stress in adult male rats ... 45 Injecting and marking male neonatal pups appears to alter basal and stress levels of cell proliferation in adulthood 48 Neonatal injections of TP to females reduces basal cell proliferation without inducing a stress-induced suppression 50 Organizational levels df androgens do not alter the expression of defensive behaviour to TMT; non-defensive behaviour is altered by neonatal manipulation 52 Considerations of litter effects 54 Future Studies ". 55 8 References 58 Role of androgens in stress-induced changes in adult cell proliferation iv LIST O F T A B L E S Table 1. Granule cell layer (mm 3) volumes as a function of stress and androgen status 16 Table 2. Frequency and duration of non-defensive behaviours as a function of stress and adult androgen levels (Experiment 1) 22 Table 3. Correlations between G C L cell proliferation and behavioural measures (Experiment 1) 25 Table 4. Various measures of maternal behaviour 27 Table 5. Mean body weight in adulthood as (PND 60) a function of organizational hormonal status (Experiment 2) 28 Table 6. The ano-genital distance and body weight of pups at P N D 12 as a function of hormonal treatment 28 Table 7. Raw cell count estimates 32 Table 8. Frequency and duration of stretch attends directed away from the vial as a function of stress and organizational hormonal status (Experiment 2) 33 Table 9. Frequency and duration of rearing as a function of stress and organizational hormone status (Experiment 2) 37 Table 10. Frequency and duration of grooming as a function of stress and organizational hormonal status (Experiment 2) 38 Table 11. Percentage of BrdU-labeled cells that are co-labeled with D C X (Experiment 2) 41 Table 12. Correlations between G C L cell density and behavioural measures (Experiment 2) 41 Role of androgens in stress-induced changes in adult cell proliferation v LIST OF F I G U R E S Figure 1. The relationship between the H P A and H P G axes 4 Figure 2. Timeline for Experiment 2 11 Figure 3 A & B . A ) BrdU-labeled and B) fluorescence-labeled cells 14 Figure 4 A & B . The mean number of BrdU-labeled cells in S G Z and hilus 18 Figure 5 A & B . The mean frequency and duration of defensive burying 19 Figure 6 A & B . The mean frequency and duration of the stretch response 20 Figure 7A & B . The mean frequency and duration of direct contact with vial 23 Figure 8. Plasma testosterone levels of S H A M operated animals were significantly reduced in TMT-exposed animals at time of perfusion (n = 5 per group) 24 Figure 9. Corticosterone levels of castrates and non-castrates in response to T M T stress 24 Figure 10A & B . Mean volume of granule cell layer and hilar volume (Experiment 2) 30 Figure 11A & B . The mean density of BrdU-labeled cells in S G Z and hilus (Experiment 2) 31 Figure 12A & B . The frequency and duration of defensive burying (Experiment 2 ) . . . . 34 Figure 13A & B . The frequency and duration of stretch attends (Experiment 2) 35 Role of androgens in stress-induced changes in adult cell proliferation v i Figure 14A & B . The frequency and duration of direct contact with the vial (Experiment 2) 36 Figure 15. Corticosterone levels of hormone-treated, oil-treated and unmarked rats in response to T M T stress 40 Role of androgens in stress-induced changes in adult cell proliferation v i i A C K N O W L E D G E M E N T S First and foremost, I would like to thank Dr. Li isa Galea for her faith, support and guidance over the years that we have worked together. Under her tutelage I became a stronger student and person. Thank you. I would like to also thank Lucille Hoover, Annie Cheng and Al ice for their incredible technical expertise and helpfulness. I would also like to thank Dr. Victor Viau and his laboratory for their help and advice with the R I A sampling. I would like to thank my lab mates and 4 t h floor companions for making my experience at U B C memorable and fun. Whether it was helping me on my thesis or joining me for a drink at Koerner's, I w i l l never forget the colleagues and friendships I have made here. Thanks to: Katia Sinopoli, Christine Mazzucco, Jodi Pawluski, Jenn Barker, Mark Spritzer, Stephanie Lieblich, Hope Walker, Jon Grosshuesch, Souraya Mansour, Sarah Walker, Sarah N g , Naureen Ismail, Matt H i l l , Marie Tse, Orsolya Magyar, A l i n a Webber, Andrea Olsen, Brennan Eadie, Van Redilia, Carl Ernst and anyone I may have forgotten. Role of androgens in stress-induced changes in adult cell proliferation 1 T H E A C T I V A T I O N A L A N D O R G A N I Z A T I O N A L I N F L U E N C E S OF A N D R O G E N S O N S T R E S S - I N D U C E D C H A N G E S I N A D U L T H I P P O C A M P A L C E L L P R O L I F E R A T I O N A N D D E F E N S I V E B E H A V I O U R The hypothalamic-pituitary-adrenal (HPA) axis is the organism's primary endocrinological stress response system and has been well characterized (reviewed in Handa et al, 1994; Viau , 2002). Briefly, neurosecretory cells in the paraventricular nucleus of the hypothalamus release corticotropin-releasing hormone (CRH) and vasopressin into the portal blood to the anterior pituitary gland, which then act synergistically to stimulate the release of adrenocorticotropic hormone ( A C T H ) into the bloodstream. A C T H in turn stimulates the production and release of the major glucocorticoid for a particular species (corticosterone (CORT) in rats, Cortisol in humans) from the adrenal cortex. The H P A axis also involves a negative feedback system involving both glucocorticoid receptors: Type I receptors (also known as mineralocorticoid receptors) and type II receptors (also known as glucocorticoid receptors). Although not traditionally considered as being a part of the H P A axis, the hippocampus, which has numerous type I and type II receptors (Evans and Arrizza, 1989), is a major site of glucocorticoid action and plays a substantial role in the negative feedback action of C O R T (Herman et al, 1993; McEwen , Weiss and Schwartz, 1968; see Figure 1). Because of its abundance of glucocorticoid receptors, the hippocampus is particularly vulnerable to the effects of acute and chronic stress. Glucocorticoid receptor levels are decreased in the hippocampus in male rats after acute stress (Racca et al, 2005; Role of androgens in stress-induced changes in adult cell proliferation 2 Filipovic, Gavrilovic, Dronjak, Radojcic, 2005). Electrophysiologically, acute stress enables long-term depression (Xiong et al, 2004) while it impairs long-term potentiation in the C A 1 region of the hippocampus (Xiong et al, 2004; Shakesby, A n w y l and Rowan, 2002) in the male rat hippocampus. Furthermore, there are gender differences in the way acute stress alters hippocampal morphology and functioning. Acute foot-shock increases spine density in the C A 1 region of male rats, but decreases spine density in C A 1 region of female rats (Shors, Chua and Falduto, 2001) which is coincident with a facilitation in learning on a hippocampus-dependent task (classical eye-blink conditioning) in males, and an impairment in learning on this same task in females (Bangasser and Shors, 2004; Beyl in and Shors, 1998; Wood and Shors, 1998). The facilitation in learning seen in males may be specific to the particular stressor or the learning task as acute predator odour stress impairs spatial learning the Morris Water Maze (Sandi et al, 2005). Adult mammalian neurogenesis was first reported in the dentate gyrus of the hippocampus by Altman in i962. Neurogenesis can be subdivided into two functionally distinct processes: cell proliferation and cell survival. Cel l proliferation is the production of daughter cells from progenitor cells, which in the hippocampus, are located in the subgranular zone of the dentate gyrus. These daughter cells have the potential to become mature glia or neurons. Cel l survival refers to new cells that survive until maturity; daughter cells could be progenitors, glia, neurons or could die before they mature. New granule neurons express mature neuronal markers, receive synaptic input, extend axons into the C A 3 region of the hippocampus, and show normal electrophysiological responses, indicating that these newly born cells are functional (van Praag et al, 2002; Hastings and Gould, 1999; Markakis and Gage, 1999; Stanfield and Trice, 1988). Role of androgens in stress-induced changes in adult cell proliferation 3 Cel l proliferation can be up-regulated by a number of factors including: serotonin (Banasr et al, 1991), estrogen (after 4 hours; Ormerod et al, 2003), neurosteroids (Karishma and Herbert, 2002), nitric oxide (Zhang et al, 2001) and trophic factors (Jin et al, 2003, 2002; Trejo et al, 2001). Conversely, cell proliferation can be down-regulated by a number of factors including: stress (Holmes and Galea, 2002; Tanapat et al, 2001), high levels of adrenal steroids (Cameron and Gould, 1994), glutamate (Cameron et al, 1995) and estradiol after 48 hours (Ormerod et al, 2003). Acute stress has been shown to suppress cell proliferation in a number of species, including monkeys (Gould et al, 1998), tree shrews (Galea et al, 1996) and rats (Bain et al, 2004; Falconer and Galea, 2003; Holmes and Galea, 2002; Tanapat et al, 2001) an effect that persists up to one week later but is no longer evident after three weeks indicating that acute stress suppresses cell proliferation but not cell survival (Tanapat et al, 2001). This stress-induced suppression is mediated by corticosterone, as adrenalectomizing animals eliminates this suppression; conversely, replacing corticosterone in adrenalectomized animals restores this suppression (Cameron and Gould, 1994). The acute stress-induced suppression in cell proliferation may vary by type of stressor, species and gender. Specifically mice do not show an acute stress-induced suppression of cell proliferation, while there are conflicting reports as to how cell proliferation is affected by acute restraint stress (Bain, Dwyer and Rusak, 2004; Pham, Nacher, H o f and McEwen , 2003). Intriguingly, acute predator odour stress suppresses cell proliferation in male but not female rats, an effect that is not modulated by adult levels of ovarian hormones in the female rats (Falconer and Galea, 2003). Role of androgens in stress-induced changes in adult cell proliferation 4 There is a relationship between the H P A and hypothalamic-pituitary-gonadal axis. In male rats, the relationship is inhibitory (Viau, 2002, Handa et al, 1994), while in female rats, the relationship is excitatory (Handa et al, 1994). For instance, females have higher basal and stress-induced levels of C O R T than do males (Handa et al, 1994; Burgess and Handa, 1992; Kant et al, 1983; Gaskin and Kitay, 1971; Critchlow et al, 1963; Kitay, 1961). Androgens inhibit H P A function at the level of the hypothalamus likely via afferents from the medial preoptic area (Viau and Meaney, 1996; Bingaman et al, 1994; Almeida et al, 1992); in contrast, estrogen enhances the stress response directly Negative Feedback Hippocampus C O R T Hypothalamus C R H GnRH Pituitary Gland Estrogen A C T H L H / F S H Adrenal Glands Estrogen Medial Preoptic Area Androgens Gonads Figure 1: The relationship between the H P A and HPG axes. The paraventricular nucleus of the hypothalamus secretes corticotropic releasing hormone into the pituitary gland, which in turns releases adrenocorticotropic hormone (ACTH). A C T H acts on the adrenal glands to release corticosterone which acts on the hippocampus to regulate negative feedback of the H P A axis. Gonadotropin releasing hormone (GnRH) acts on the pituitary gland to release leutenizing and follicle-stimulating hormone which act on the gonads to release androgens and estrogens in males and females, respectively. Androgens inhibit the H P A axis via the medial preoptic area (MPOA), while estrogen stimulates the H P A axis directly at all levels of the axis. Role of androgens in stress-induced changes in adult cell proliferation 5 at all levels of the H P A axis (Burgess and Handa, 1992; V i a u and Meaney, 1992; Bohler et al, 1999; Haas and George, 1989; Raps et al, 1971; Kitay et al, 1963). This differential regulation of the H P A axis by androgens and estrogen coupled with the findings that adult manipulation of ovarian steroids did not eliminate the sex difference in stress-induced suppression of cell proliferation in rats suggest that stress-induced cell proliferation may be regulated by either to activational (adult) or organizational (developing) levels of androgens. Gonadal hormones have developmental (organizational) effects on the H P A axis induced suppression of cell proliferation in rats suggest that stress-induced cell proliferation may be regulated by either to activational (adult) or organizational (developing) levels of androgens. (Seale et al, 2005; McCormick and Mahoney, 1999; Cratty et al, 1995; Gr imm and Frieder, 1987; et al, 1997; Naftolin et al, 1975; McCormick et al, 1998). For instance, a single injection of testosterone propionate to females within 24 hours of birth has been shown to decrease basal and stress-induced C O R T , corticotropin releasing hormone, arginine vasospressin levels, and increase glucocorticoid receptor m R N A levels in female rats (Seale et al, 2005). Conversely, males treated with flutamide, an androgen receptor antagonist, within 24 h of birth showed elevated basal and stress-induced levels of C O R T relative to oil-injected animals (McCormick and Mahoney, 1999), while neonatal castration causes an increase in stress, but not basal, levels of C O R T in male rats (McCormick et al, 1998). 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) is a compound that has been isolated from fox feces and has been shown to produce behavioural (Falconer and Galea, 2003; Holmes and Galea, 2002; Morrow, Elsworth and Roth, 2002; Wallace and Rosen, 2000), Role of androgens in stress-induced changes in adult cell proliferation 6 and endocrinological (Morrow et al, 2002; Morrow, Redmond, Roth and Elsworth, 2000) stress responses. Behaviourally, the effects of T M T depend on the environment. Studies using small chambers lined with bedding (Falconer and Galea, 2003; Holmes and Galea, 2002) show an increase in defensive burying, risk assessment (stretch attends) and avoidance, while freezing is reported in environments without bedding (Wallace and Rosen, 2000). The purpose of the present study was to study the activational and organizational effects of androgens on the stress-induced suppression in cell proliferation in adult male rats. In Experiment 1, adult male rats were castrated (or sham operated) and exposed to either stress or control odour. If adult levels of androgens mediate the stress-induced suppression, then removing circulating androgens should eliminate the suppression in cell proliferation in response to stress. In Experiment 2, male pups were postnatally injected with flutamide and female pups were postnatally injected with testosterone propionate. If organizational levels of androgens are responsible for the stress-induced suppression of cell proliferation in adult males, then 1) flutamide-injected male rats should not show a stress-induced suppression, and/or 2) testosterone-injected female pups should show a stress-induced suppression in cell proliferation as adults. Defensive and non-defensive behaviours in response to stress were also measured and it was anticipated that stress would cause an increase in defensive and risk assessment behaviours. Methods Experiment 1 Subjects Role of androgens in stress-induced changes in adult cell proliferation 7 Subjects were 2 month old adult male Sprague-Dawley rats (n= 32) weighing between 350 and 450g at the time o f testing. Animals were obtained from the University of British Columbia Animal Care Centre. They were housed singly on a 12 h: 12 h light:dark cycle (lights on at 0700) and they were provided free access to food (Purina Rat Chow) and water. A l l animal research was conducted in accordance with the guidelines of the Canadian Council on Animal Care and the policies of the University of British Columbia. Every effort was made to minimize the number of animals used per group. Apparatus A l l testing was done in 30cm x 30cm x 45cm Plexiglas chambers that were lined with 5 cm of corn cob bedding. A 20 ml scintillation vial with a tissue (Kimwipe) inside was placed in the front right-hand corner of each chamber. The chambers were placed in a fumehood along with two other chambers and the chambers were visually isolated from each other using wax paper. Black construction paper was used as a backdrop such that all animals had the same visual environment. ^ Procedure Prior to any testing, all animals were anesthetized using 2-bromo-2-chloro-1,1,1-trifluoroethane (halothane, flow rate 4%, M T C Pharmaceuticals, Cambridge, Canada) and either bilaterally castrated ( C S T X ) or given a sham surgery ( S H A M ) and given one week to recover before testing began. Briefly, an incision was made into the scrotal sac and underlying muscle wall . The blood vessels were tied off above the testes before they were removed. S H A M animals were anesthetized only. The animals were then handled Role of androgens in stress-induced changes in adult cell proliferation 8 for 5 days (5 minutes per day) and habituated to the test chambers for another 5 days (20 minutes per day) between 10 am and 3 pm. During the habituation phase, animals were brought into the hallway adjacent to the testing room. Three animals were habituated at a time, while the rest remained in the hallway. A l l subjects were tested the day after the final habituation phase. On test day, half o f the animals from each surgical group were exposed to 150 ul T M T (n-8/surgical treatment) while the other half were exposed to 150 ul control odour (distilled water n=8/surgical treatment). The T M T and water were applied on the Kimwipe inside the scintillation vial. T M T is a main component odour-causing component of fox feces and has been used as a predator odour stress in rats (Falconer and Galea, 2003; Holmes and Galea, 2002; Morrow et al, 2002; Tanapat et al, 2001; Funk and Ami r , 2000; Morrow et al, 2000; Wallace and Rosen, 2000; Vernet-Maury, Polak and Demael 1984). The animals were exposed to either T M T or control odour for one hour in their chambers. They were videotaped for the first 15 minutes of the session. After 15 minutes, all rats were injected with bromodeoxyuridine (BrdU) (200mg/kg, i.p.; Sigma, St. Louis, U S A ) , a cell synthesis marker that incorporates itself into D N A during the S-phase of mitosis (Cameron and M c K a y , 1998). Animals were given an overdose of sodium pentobarbital (2 ml/kg somnotol, M T C Pharmaceuticals) 24 hours later, perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, and the brains extracted. Arterial blood was collected at the time of perfusion, stored at 4°C for 24 hours. The samples were then spun at 8000 revolutions per minute for 20 minutes, serum extracted and stored at -20°C and later analyzed for serum testosterone (MP Biomedicals, Orangebury N Y ) and corticosterone levels (Inter Medico, Markham ON). Brains were removed and processed as described below. Role of androgens in stress-induced changes in adult cell proliferation 9 In order to evaluate the phenotype of BrdU-labeled cells, a subset group of the above animals (n=12, n=3/group) were left in their home cages for the next 4 days and the perfused on the day 5th post-testing or four days after B r d U injection. Four days was chosen based on past studies showing that maximum D C X labeling of immature neurons is seen at this time (Brown et al, 2003) On test day, in this subset of animals, tail blood was also taken from the animals at five different time points for the purposes of measuring C O R T levels: 1 hour before testing (basal), 30 minutes after the onset of exposure to T M T , 1 hour after the onset o f exposure and 2 hours after the onset o f testing. For the C O R T sampling procedure, animals were brought into the hallway adjacent to the testing room. Animals were individually brought into a separate room where they were placed in restraint tubes. Tailblood was taken by nicking and "milking" the tail. The entire procedure took under 5 minutes. Brains were double-stained with immunofluorescent probes to assess either B r d U - and D C X - immunoreactivity. Experiment 2 Subjects A l l test animals were raised from birth. Nine dams (obtained from U B C animal breeding centre) were bred with males in wire mesh cages (1-2 females were placed with one male for a week), and were checked daily for vaginal plugs to ensure successful impregnation. The dams were placed in individual cages after 7 days and left undisturbed (aside from weekly cage changes) until parturition. A t parturition, the nine litters were culled to 12 pups / litter (6 male, 6 female). In 2 of the litters, the pups developed ringtail and were discarded from this experiment. Within each litter, 2 animals were assigned to either the drug condition, oi l condition or the unmarked condition. In Role of androgens in stress-induced changes in adult cell proliferation 10 adulthood, animals were randomly assigned (such that pups from the same litter and given the same neonatal manipulation were placed in different treatment groups in adulthood) to be either exposed to T M T or control odour. In each group, most animals were sacrificed 24 hours later; a subgroup of animals were allowed to survive an extra 4 days in order to be assessed for cell fate determination. The animals were weaned at P N D 24 and housed in same sex/same treatment groups of four until adulthood, at which point they were housed individually as per the same conditions described in Experiment 1. A t the time of testing all animals were 2 months old; males weighted between 400 and 500g, while females weighted between 220 and 320 g. Procedure In order to study the organizational effects of androgens on stress-induced changes in cell proliferation, male pups were given daily injections on post-natal day (PND) 0-5 of either 40 mg/kg (s.c.) of the androgen receptor antagonist flutamide ( F L U ; Sigma, St. Louis, M O , in sesame oil) or sesame oi l alone (OIL). This hormone regime has been used successfully in the past to alter H P A activity in adulthood (McCormick and Mahoney, 1999; Husmann and McPhaul , 1991). Female pups were treated with 300 ug of testosterone propionate (TP; Sigma-Aldrich, St. Louis M O , in 0.05 cc sesame oil) or sesame oi l on P N D 3 and 5, a protocol used by Roof and Havens (1992) that masculinized the female dentate gyrus. A separate group of male and female rats were left undisturbed in the cages aside from routine cage changing (unmarked). In order to identify the pups, drug-treated animals were covered in blue food dye Role of androgens in stress-induced changes in adult cell proliferation 11 Perfuse Male: Flu/Oil (0-5) Female: TP/Oil (3, 5) Weaning Handling / Habituation Test/ BrdU I—H 0 5 24 60 70 71 PND Figure 2: Timeline for Experiment 2. while oil-treated animals were covered in green food dye (Food Club, Scott-Bathgate Ltd., Vancouver, B C ) . A t P N D 12, the animals were weighed and measured for anogenital distance to test for potential early developmental effects of the drugs. These animals were then left undisturbed until 2-3 months of age, at which time they were given the same habituation, testing and post-mortem analysis as the rats in Experiment 1. Briefly, 8 animals from each treatment group (males: F L U , OIL , unmarked; females: TP, OIL, unmarked) were handled for 5 days and habituated for the following 5 days. After habituation, half of the animals in each group were exposed to T M T while the other half was exposed to distilled water. A s per experiment 1, behaviours were videotaped for the first 15 minutes of the session, and then B r d U (200mg/kg) was administered. Most of the animals (n=2-5/group) were perfused 24 hours after testing. In order to evaluate the phenotype of the cells, a separate group of animals (n=24; 3/group) were left in their home cages for the next 4 days and then perfused on the 5th day post-testing or four days after B r d U injection. On test day, the tail blood was also taken from the animals at five different time points for the purposes of measuring C O R T levels in response to T M T exposure: 1 hour before testing (basal), 30 minutes after the onset of testing, 1 hour after the onset of testing, 2 hours after the onset of testing and 4 Role o f androgens in stress-induced changes in adult cell proliferation 12 hours after the onset of testing. In other words, rats received the same procedure as per Experiment 1 except that they were perfused 4 days (instead of 24 h) after testing and blood was collected from their tail veins for C O R T levels. Each blood letting session took under 5 minutes. Brains were triple-stained with immunofluorescent probes to assess either B r d U - and N e u N - or B r d U - and G F A P immunoreactivity. BrdU Immunocytochemistry Brains were sliced into 40 um coronal sections with a Leica vibratome (OTS1000). Sections that included the hippocampus were collected and processed for immunocytochemistry. Briefly, sections were rinsed in tris-buffered saline (TBS). They were then incubated in 2 N hydrochloric acid (HC1) for thirty minutes at 37°C in order to denature the D N A . Following this was a 20 min incubation period in normal horse serum (Vector, Elite Ki t ) and then sections were left overnight in anti-BrdU mouse monoclonal antibody (Roche, 1:100 dilution) on a shaker at 4°C. Sections were rinsed in T B S and incubated in biotinylated antibody anti-mouse IgG (Vector, Elite kit; 1:100 dilution) on a shaker at room temperature. Sections were rinsed in T B S , reacted using an A B C reagent (Vector, Elite kit) with 0.2% diaminobenzidine ( D A B ) , counterstained for Niss l substance with cresyl violet, and coverslipped with Permount. Fluorescence Immunocytochemistry Separate brain sections from the rats from experiments 2 and 3 were also double stained with immunofluorescent probes to assess D C X (a marker for immature neurons (Brown, et al., 2003), B r d U . Slides were rinsed in T B S , blocked in 3% normal donkey serum (Sigma-Aldrich, St. Louis, U S A ) for 30 minutes and then incubated for 24 hr in goat monoclonal an t i -DCX (1:200; Jackson Immunoresearch, West Grove, U S A at 4°C. Role of androgens in stress-induced changes in adult cell proliferation 13 Sections were then rinsed, incubated in 3% normal donkey serum rinsed, and incubated in donkey anti-goat Cy3 (diluted to 1:100 to visualize D C X ; Jackson Immunoresearch, West Grove, U S A ) for 24 hrs at 4°C. Sections were then rinsed, fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, U S A ) for 10 minutes, rinsed in 0.9% saline, incubated in 2 N HC1 (Fisher Scientific, Ottawa, C A N ) for 30 minutes, blocked in 3% normal donkey serum and incubated for 48 hours in rat anti-BrdU (Oxford Biotechnology, Kidlington, U K ) at 4°C . Sections were then rinsed, blocked in 3% N D S and incubated overnight i n donkey anti-mouse F I T C (diluted to 1:100 to visualize B r d U ; Jackson Immunoresearch, West Grove, U S A ) . Sections were rinsed and cover-slipped with the anti-fading agent diazobicyclooctane ( D A B C O ; 2.5% D A B C O , 10% polyvinyl alcohol and 20% glycerol in T B S ; Sigma). Behavioural Measures The animals were videotaped and scored for the frequency and duration of following behaviours during the first 15 minutes of the testing period: Defensive burying (pushing bedding towards the vial in an attempt to bury it), stretch attends (planting the rear paws, allowing the rat to elongate its body and sniff the vial), stretch attends away from the vial , physical contact with vial , rearing (standing up on hind legs) and grooming. Maternal Behaviour In order to account for differences in maternal behaviour between litters, dams and their litters were videotaped for pup sniffing, l icking, hovering, retrieval, mouthing and nest building for 15 minutes on P N D 2,4 , 6, 8,10. The data from these individual trials were averaged. Role o f androgens in stress-induced changes in adult cell proliferation 14 BrdU-ir cells Sections were examined under lOOOx magnification on a Nikon E600 microscope. Every 10 t h section was examined for measurement o f BrdU-labeled cells on peroxidase-treated tissue (see Figure 3). Oval or round BrdU-labeled cells were counted in either the granule cell layer, subgranular zone (approximately 50um border as defined by Palmer, Wilhoitte and Gage, 2000) or hilus. A stereological estimate was produced from the raw counts by multiplying the number of counted cells by 10. The cell proliferation data in Experiment 1 is presented as an estimate of the total number of BrdU-positive cells in the dentate, whereas in Experiment 2 it is presented as a density measurement. This is done to account for the differences in dentate gyrus volume reported in Experiment 2. BrdU-ir cell phenotyping In order to determine cell phenotype, tissue processed for combined immunofluorescence was examined using a confocal laser scanning microscope (Nikon Type 120) on a 63x objective. 25 BrdU-labeled cells from the middle portion of the Hilus GCL 10um Figure 3: A) Confocal photomicrograph of BrdU-labeled cells (green) in the granule cell layer . All BrdU-labeled cells are also co-labeled with DCX (red). B) Photomicrograph of BrdU-labeled cells in the granule cell layer. Role of androgens in stress-induced changes in adult cell proliferation 15 dentate gyrus (four-six sections per brain) was examined with Z-sections taken at 1 um intervals. Optical stacks were created and channels were merged in ImageJ and imported into Adobe Photoshop for channel merging. Testosterone and Corticosterone RIA Serum testosterone was measured using an R I A kit from I C N Biomedicals (MP Biomedicals, Orangebury N Y ) . Samples were run in duplicates and extracted into standard tubes and vortexed with sex-binding globulin inhibitor solution, then mixed with 125 I tracer. Samples were then incubated in primary anti-testosterone antibody at 37°C for 2 hours and then incubated at 37°C for 60 minutes in secondary antibody. Next, samples were centrifuged (2500 rpm for 15 minutes), decanted and the precipitate counted in a gamma counter. The intra-assay variation was 13.6%. The detection limit of the assay was 0.1 ng/ml. Protocol adapted from Viau et al, 2003. Serum corticosterone was measured using an R I A Rat Corticosterone Coat-A-Count kit (Diagnostic Products Corporation, Markham, ON) . The intra-assay variation was 10.6%o. The detection limit o f the assay was 20 ng/ml. Estrous cycle Females from Experiment 2 were smeared in order to establish estrous cycle length and regularity for ten days during the handling and habituation phase. Vaginal smears were extracted using an eye dropper, placed on a slide and examined under a microscope (40x magnification). Data Analyses Role of androgens in stress-induced changes in adult cell proliferation 16 Separate analyses of variance ( A N O V A ) were conducted for all dependent variables (the density and total number of BrdU-labeled cells, the frequency and duration of each behavior, the percentage of BrdU/NeuN cells and B r d U / G F A P cells, and the serum testosterone and C O R T levels) with stress treatment ( T M T or control), androgen status ( C S T X or S H A M in Experiment 1; F L U , TP, OIL or unmarked in Experiment 2) and gender (Experiment 2 only) as between-subject factors. Unless otherwise indicated, Newman-Keuls was used for post-hoc testing (a=0.05). Pearson product-moment correlation tests were conducted between total B r d U cells (stereological estimate) and behaviour scores (frequency and duration). In all statistical procedures, significance was set at a=0.05. Whilep<0.05 w i l l be referred to as statistically significant, these p values reflect the probability of the effect occurring solely by chance. Results Experiment 1 TMT-exposure suppressed cell proliferation regardless of androgen status in adulthood Regardless of androgen status, there is a significant region by odour exposure interaction (Figure 4; F(i i i6) = 7.58, p = 0.014) but no main effect of androgen status (p < 0.9) or any other significant interactions (0.41 < p < 0.59). Post-hoc analyses show that there was a TMT-exposure induced suppression in the granule cell layer (p < 0.0002) but not in the hilus. Table 1: Granule cell layer (mm3) volumes as a function of stress and androgen status. There are no significant differences (n = 4-5 per group). Hormonal status Control odour TMT SHAM 3.6 ± 0 . 2 5 3.1 ± 0 . 0 8 CSTX 3.3 ± 0 . 1 8 3.4 ± 0 . 4 7 Role of androgens in stress-induced changes in adult cell proliferation 17 There were no significant effects on volume of the dentate gyrus (Table 1; main effect of stress: p = 0.78; androgen status: p = 0.41; interaction: p = 0.15), suggesting that the treatments did not significantly alter dentate gyrus volume. TMT-exposure increased the frequency and duration of defensive burying regardless of androgen status in adulthood Regardless of androgen status, TMT-exposed males showed a greater frequency (F(i, 28) = 5.23, p = 0.03; see figure 5) and duration (F(i, 28) = 3.89; p = 0.058) of defensive burying than the control-odour exposed group. There was no significant effect of androgen status (frequency: p = 0.49; duration: p = 0.28), nor was there a significant interaction effect (frequency: p = 0.57; p = 0.29) on defensive burying. TMT-exposure increased the frequency of stretch attends towards the vial regardless of androgen status, but increased the duration only in intact males. A s seen in Figure 6, the TMT-exposed males showed a significant increase in the frequency of stretch attends towards the vial relative to the control group (F(i ; 28) = 10.7; p = 0.002). There was no significant main effect of androgen status (p = 0.28) nor was there a significant interaction (p = 0.28) on stretch attends. There was a significant interaction between stress treatment and androgen status in duration of stretch attends (F(i5 28) = 4.81; p - 0.037). Post -hoc analyses show that a TMT-induced increase in the duration of stretch attends was present only in S H A M (gonadally-intact) rats (p = 0.008). There were no significant main effects or interactions for stretch attend postures directed away from vial (all p's > 0.2; see Table 2). Role of androgens in stress-induced changes in adult cell proliferation 18 Figure 4: The mean number of BrdU-labeled cells in A) granule cell layer (GCL) and B) hilus. TMT-exposed rats exhibited a reduction of BrdU-labeled cells regardless of adult androgen status (n=5 per group). Role of androgens in stress-induced changes in adult cell proliferation 19 • S H A M • C S T X • C o n t r o l T M T Figure 5: The mean frequency and duration of defensive burying. There is a stress-induced increase in the A) frequency and B) duration of defensive burying independent of activational levels of androgens. Duration approached but did not reach significance (p = 0.06) (n=8 per group). Role of androgens in stress-induced changes in adult cell proliferation 20 B 2 1 l H S H A M I I CSTX _T_ X C o n t r o l T M T Figure 6: The mean frequency and duration of the stretch response. There is a stress-induced increase in the A) frequency and B) duration of stretch attends towards the vial independent of activational levels of androgens. S H A M operated animals demonstrated a significantly greater duration of stretch attends than C S T X animals in response to stress (n=8 per group). Role of androgens in stress-induced changes in adult cell proliferation 21 TMT-exposure suppressed the frequency and duration of direct vial contacts regardless of androgen status in adulthood Regardless of androgen status, the TMT-exposed group avoided contact with the vial compared to the control-odour group (main effect of stress treatment: frequency: F ( i , 2 8 ) = 17.29; p = 0.0002; duration: F ( i > 2 8 ) = 10.26; p = 0.003; figure 7). There was no significant effect of androgen status (frequency: p = 0.43; duration: p = 0.65), nor a significant interaction effect (frequency: p = 0.24; duration: p = 0.48) on direct contact with the vial. Castration in adulthood increases the duration of grooming regardless of odour exposure; rearing expression unaffected by stress or gonadal status in adulthood A s seen in Table 2, C S T X animals showed a greater duration of grooming (F(i> 28) = 4.46; p = 0.044), but not for frequency (p = 0.71) regardless of stress treatment. There were no significant effects of stress treatment on grooming (frequency: p = 0.13; duration: p = 0.42), nor a significant interaction effect on grooming (frequency: p = 0.49; duration: p = 0.65). There were no significant main or interaction effects on rearing (main effect of stress: frequency: p = 0.08, duration: p = 0.16; Table 2; main effect of androgen status: frequency: p = 0.44, duration: p = 0.63; interaction: frequency: p = 0.3, duration: p = 0.26). Role of androgens in stress-induced changes in adult cell proliferation 22 Table 2: Frequency and duration o f non-defensive behaviours as a function o f stress and adult androgen levels (Experiment 1). Castrated animals demonstrate an increase in grooming relative to shams (n = 5 per group). Stretch away Group Frequency Duration (s) Control/SHAM 0.75 ±0.31 0.8 ±0.46 Control/CSTX 0.5 ± 0.27 0.58 ±0.31 TMT/SHAM 0.875 ±0.3 1.07 ±0.068 TMT/CSTX 0.5 ± 0.27 0.15 ±0.08 Rearing Control/SHAM 16.88 ±4.2 61.62 ±8.25 Control/CSTX 17.88 ±4.1 75 ±25.14 TMT/SHAM 14.38 ±3.1 55.58 ±26.6 TMT/CSTX 8.13 ± 1.5 22.43 ± 5.42 Grooming Control/SHAM 5.38 ±1.08 87.78 ± 24.4 Control/CSTX 6.63 ± 1.53 169.79 ±42.64 TMT/SHAM 4.38 ±0.94 76.38 ±22.88 TMT/CSTX 4± 1 129.28 ±34.01 Acute exposure to TMT suppressed testosterone 24 hours later Figure 8 shows the group means and standard error of the means (SEMs) for serum testosterone level 24 h after exposure to TMT. Testosterone levels in intact males were suppressed for the TMT-exposed group relative to the control group 24 h later (F(i j 16) = 6.32; p = 0.023). Furthermore, there is no correlation between granule cell layer BrdU counts and levels of testosterone in intact animals (p = 0.54). Corticosterone elevates after exposure to either control or TMT Figure 9 shows the mean and SEMs for serum CORT levels. Serum CORT levels were elevated in both control odour groups and TMT groups 30 mins and 1 hour after the onset of exposure, but returned to basal levels 2 hours later (F(3 t 12) = 17.69; p < 0.001). Role of androgens in stress-induced changes in adult cell proliferation A 23 Figure 7: The mean frequency and duration of direct contact with vial. TMT-exposed animals directly contacted the vial significantly less than water-exposed animals in terms of both A) frequency and B) duration. There was no effect of C S T X (n = 8 per group). Role of androgens in stress-induced changes in adult cell proliferation * 6 i Control T M T Figure 8: Plasma testosterone levels of SHAM operated animals were significantly reduced in TMT-exposed animals at time of perfusion (n = 5 per group). 600 500 S 400 300 u 200 GO 100 r*~! I I I SHAM/Control 1 S H A M / T M T I CSTX/Control • C S T X / T M T I I Baseline 30 mins 1 hr 2 hrs Figure 9: Corticosterone levels of castrates and non-castrates in response to TMT stress. There was a significant elevation in at 30 mins and 1 hour after the onset of exposure, regardless of odour condition or androgen status (n = 2-3 per group). Role of androgens in stress-induced changes in adult cell proliferation 25 There were no other main effects (odour: p < 0.45; gonadal status: p < 0.86), nor were there any significant interactions (p < 0.37). Cell proliferation did not significantly correlate with any behaviours As seen in Table 3, there are no significant correlations between cell proliferation and the frequency or duration of any behaviours. There was also no significant correlation between defensive burying and stretch attends (p > 0.1). T a b l e 3: C o r r e l a t i o n s b e t w e e n G C L ce l l p r o l i f e r a t i o n and b e h a v i o u r a l measures ( E x p e r i m e n t 1). The re are no s ta t is t i ca l ly s i g n i f i c a n t co r re la t ions . R P Defensive burying freq 0.09 0.19 Defensive burying dur 0.09 0.2 Stretch towards freq 0.17 0.07 Stretch towards dur 0.02 0.5 Stretch away freq 0.0 0.93 Stretch away dur 0.0 0.8 Contacts freq 0.07 0.25 Contacts dur 0.0 0.88 Rearing freq 0.0 0.71 Rearing dur 0.05 0.35 Grooming freq 0.0 0.88 Grooming dur 0.06 0.3 Experiment 2 Maternal behaviour Table 4 outlines various measures of maternal behaviour observed on the dams and averaged across all 5 test days. A one-way ANOVA shows that mom 7 had a higher latency to retrieve all of her pups (F(9; 38) = 2.54, p = 0.02), but also demonstrated a trend towards sniffing her pups more frequently (F^ 38) = 2.08, p = 0.057). Furthermore, moms 13 and 16 spent more time licking the AG region of their pups than moms 7, 8, 9 and 14 (F(9,38) = 3.66, p = 0.002). Mom 16 also spent more time hovering over her pups than did mom 9 (F(9> 38) = 2.7, p = 0.016). There was also an effect of frequency of mouthing (F^ Role of androgens in stress-induced changes in adult cell proliferation 26 38) = 2.38, p = 0.03); post-hoc tests show that mom 14 demonstrated a trend towards mouthing her pups more often than mom 16 (p = 0.058) and mom 11 (p = 0.069). M o m 4 showed greater frequency ( F ^ g ) = 4.09, p = 0.001) and duration (F(9; ^ = 3.01, p = 0.008) of nest building relative to all o f the other moms. There were no other significant effects for any of the other measures of maternal behaviour (0.18 < p < 0.81). Pearson product-moment correlations revealed that frequency of defensive burying is positively correlated with maternal nest building (frequency: r 2.= 0.46, p = 0.05; duration: r 2 = 0.35, p = 0.095), while duration o f defensive burying is positively correlated with both the frequency (r = 0.48, p = 0.04) with a trend towards duration (r = 0.4, p = 0.07) of maternal pup sniffing. Both rearing frequency (frequency: r 2 = 0.69705, p = 0.01; duration: r 2 = 0.6, p = 0.014) and duration (frequency: r 2 = 0.74, p = 0.003; duration: r 2 = 0.77, p = 0.002) are positively correlated with maternal body licking. Testosterone propionate prevented regular cycling in females in adulthood Females injected with TP spent 84.1% of time in estrus, while oil-injected and unmarked females cycled regularly (i;e. demonstrated normal transitions from proestrus to estrus to metestrus to diestrus), suggesting that the TP manipulation was successful in masculinizing females. Estrous is defined as a predominance of anucleated, cornified cells. Metestrous is identified by the presence of cornified cells, nucleated epithelial cells and leukocytes. Diestrous shows a majority of leukocytes, while proestrous is defined by the dominance of nucleated epithelial cells (Marcondes, Bianchi and Tanno, 2002). Adult body weight There was a significant sex by treatment interaction (Table 5; Fp, 72) = 12.86, p < 0.001). Post-hoc analyses show that male unmarked animals weigh significantly more Table 4: Various measures of maternal behaviour Litter Retrieval Latency (s) Pups in nest (s) Crouching (s) Hovering (s) Pup sniff (#) Pup sniff (s) Pup body lick (#) Pup body lick (s) 6 68.9 ±40.6 341.7 ± 149.6 273.6 ± 100.5 279.9 ± 59.6 13.2 ± 1.6 17.2 ±2.7 12 ± 3 . 9 65.22 ±25.6 7 26.5 ±6.8 472.8 ±211.8 257.1 ± 116.9 340.4 ± 70.9 30.0 ±9.1 43.0 ±20.8 17 ± 5.8 79.8 ± 29.7 8 16.5 ±6.2 101.9 ±28.6 319.6 ±68.6 229.2 ± 36.3 14.2 ±2.5 14.3 ±3.2 26 ± 11.3 86.8 ± 33.3 9 18.6 ± 6 115.3 ±25.4 328.3 ± 109.6 176.9 ±28.6 14.5 ±5.3 15.4 ±6.4 18 ± 8.5 64.8 ±26.1 10 20.6 ± 3.7 77.7 ± 10.9 231.7 ±70.6 442.0 ± 47.8 9.6 ±3.7 11.4 ±3.8 9.4 ± 2.4 45.4 ± 15 11 39.1 ± 12.8 106.6 ±34.9 333.6 ± 109.7 345.1 ± 102.9 10.6 ±2 .6 15.1 ±4.3 9.6 ± 1.8 53.1 ± 11.6 13 27.7 ± 4.2 135.2 ±36.1 303.8 ± 100.8 392.4 ± 78 7.4 ±2 .0 7.3 ±2 .6 10.6 ±2.2 48.8 ± 11.8 14 12.7 ± 1.2 50.5 ±6.2 331.9 ±49.8 400.9 ± 23.2 11.6 + 2.7 12.2 ±4.8 6.6 ± 0.3 30.3 ± 7.4 15 31.4 ±5.9 128.4 ±44.2 234.6 ± 107.4 494.4 ± 97.6 11.8 ± 2.2 19.2 ±6.6 13 ± 1.3 71.9 ±6.5 16 46.1 ±5.9 143.6 ±53.0 113.2 ±67.7 539.7 ±75.1 13.2 ±5.8 19.5 ±9.6 12 ±2.1 97.8 ± 25.3 Pup AG lick (#) AG lick (s) Mouthing (#) Mouthing (s) Nest building (#) Nest building (s) Grooming self (#) Grooming self (s) 6 7.2 ±2.6 96.7 ± 36.2 5.6 ±2.2 4.45 ±2.6 8.8 ± 1.4 14.7 ±4.6 5.2 ± 1.3 104.1 ±34.9 7 8.2 ±3.3 37.9 ±21.5 9.5 ± 2 2.8 ±0.6 5.5 ±1 .9 4.2 ± 1.8 2.5 ±0 .5 61.05 ± 18.1 8 6.8 ±2.5 69.3 ± 24.9 13 ±3.2 10.8 ±2.8 5.2 ± 1.4 4.4 ±2.6 3.4 ± 1 64.76 ± 23.9 9 5 ± 1.7 51.3 ± 18.2 10.5 ±3.4 12.4 ± 7 20 .2±6 .2 31.4 ± 10.7 5 ± 1.5 59.97 ± 15.3 10 7.2 ± 1.9 118.2 ±33.5 10 ±2.9 7.5 ±3.6 4.2 ± 1.7 2.4 ± 0.8 7 ±0.4 221.6 ± 37.1 11 5.8 ± 1.4 167.2 ±52.3 1.2 ± 1.5 0.8 ±0.5 2.4 ± 1.2 3.1 ±2.2 3.4 ± 1.4 121.4 ±49.3 13 11 ± 1.9 206.2 ± 39.7 3 ±0.9 1.1 ±0.3 5 ±4.9 6.7 ±5.1 8.4 ±2.2 180.4 ±25.1 14 5.2 ± 0.7 62.8 ± 14.8 18 ±9.2 18.8 ± 13.2 8.2 ±2.3 13.1 ±9.9 6.6 ±2.1 134.7 ±47.1 15 9.8 ± 1.5 137.8 ± 13.5 3.4 ± 1.8 1.5 ±0.9 1.2 ±0.4 0.6 ± 0.23 6 ± 1.3 171 ±40 16 -11.6 ± 1.4 234.8 + 53.9 1.2 ±0.7 0.7 ±0.4 1.4 ±0.7 2.7 ±2.22 3 ± 0.5 112.3 ±30.5 28 than male oi l (p = 0.02) or flutamide-treated (p < 0.02) animals, while testosterone-treated females weigh more than oil-treated (p < 0.001) or unmarked (p < 0.001) females. Table 5: Mean body weight in adulthood as (PND 60) a function of organizational hormonal status (Experiment 2). Unmarked males are heavier than other males, while TP females are heavier than other females. Overall, males are heavier than females (n = 10-16 per group). Group Control TMT Male/Unmarked 423.6 ± 8 . 6 424 ± 11.2 Male /Oi l 400.8 ± 8 . 1 397.7 ± 8 . 4 Male/Flu 406.6 ± 9 . 1 398.3 ± 9 . 2 Female/Unmarked 243.7 ± 6 . 5 239.1 ± 8 . 3 Female/Oil 259.5 ± 8 249.8 ± 1 1 . 5 Female/TP 284.5 ± 3 . 9 287 ± 9 . 7 Anogenital distance and body weight at PND 12 Males had a larger A G D at P N D 12 than females (Table 6; M a i n effect of sex: F(i, 57) = 161.12, p < 0.001). There was no significant main effect of treatment (p < 0.11), nor an interaction effect (p = 0.69). There were no significant differences in body weight at P N D 12 across groups (main effect of sex: p = 0.51; main effect of treatment: p = 0.11; interaction effect: p = 0.13). Males also had a larger A G D per body weight at P N D 12 (F(i ; 57) = 136.7, p < 0.001). There was no significant main effect of treatment (p = 0.93), nor was there an interaction (p = 0.63). Table 6: The ano-genital distance and body weight of pups at PND 12 as a function of hormonal treatment. Males have a greater ano-genital distance than females, regardless of hormone treatment (n = 9-13 / group). Group AGD (cm) Body Weight (g) AGD/Body weight Male/Unmarked 7.8 ± 0 . 3 8 25.3 ± 1 0.31 ± 0 . 0 1 Male /Oi l 8.1 ± 0 . 2 4 25.7 ± 1,06 0.32 ± 0 . 0 1 Male/Flu 8.16 ± 0 . 3 2 25.3 ± 1.1 0.32 ± 0 . 0 1 Female/Unmarked 4.76 ± 0 . 2 6 22.2 ± 0 . 9 7 0.22 ± 0 . 0 1 Female/Oil 5.27 ± 0.11 25.2 ± 1 . 2 0.21 ± 0 . 0 1 Female/TP 5.58 ± 0.15 27.1 ± 1 . 4 0.21 ± 0 . 0 1 Role of androgens in stress-induced changes in adult cell proliferation 29 Overall unmarked males had a greater volume of granule cell layer and hilus compared to unmarked females; testosterone partially masculinizes granule cell layer volume in females There was a significant sex by treatment interaction effect on granule cell layer (Figure 10a; F(i > 35) = 3.39, p < 0.05) and hilar volume (Figure 10b; F(i, 35) = 4.50, p = 0.02). Post-hoc analyses show that male unmarked rats had significantly larger granule cell layer volumes relative to female unmarked rats (p = 0.002). In the hilus, the male unmarked rats had significantly greater volumes than all of the other groups (p < 0.006 for all unmarked or o i l treated females (p = 0.05). For both the dentate gyrus and hilus, there was also a significant main effect of sex (dentate gyrus: F(i j 35) = 15.37, p < 0.002; hilus F(i j 35) = 6.55, p = 0.015). There are no other significant main effects in the dentate gyrus or hilus G C L (0.09 < p < 0.94). TMT-induced suppression in cell proliferation in the granule cell layer of unmarked males only Because there was a significant difference between groups on granule cell volume, density measure we examined cell density in Experiment 2. In males (Figure 10a) there were no significant main effects of hormone treatment (p < 0.31), or odour condition (p < 0.24) on BrdU-labeled cell density in the granule cell layer. There was a main effect of region (F ( i , i 5 ) = 523.1, p < 0.001) such that there was greater cell density in the granule cell layer compared to the hilus. There was no significant 3-way interaction on cell density (F(2,i5)= 2.06, p < 0.16). Because a priori we were interested in effects of T M T exposure on cell proliferation in the granule cell layer, we conducted comparisons on T M T vs. water-exposed males. In the unmarked males only, control Role of androgens in stress-induced changes in adult cell proliferation 30 A Figure 10: Mean volume of a) granule cell layer and b) hilar volume. Males have a greater volume relative to females. TP-injected females had a larger granule cell layer than unmarked and oil-treated females Cn = 2-5 ner gronnt. Role of androgens in stress-induced changes in adult cell proliferation 31 Figure 11: The mean number of BrdU-labeled cells in a) SGZ and b) hilus. Control odour-exposed unmarked males demonstrate a higher cell density in the G C L relative to all other males, while TP-treated females showed a lower cell density than unmarked and oil-treated females (n = 2-5 per Role of androgens in stress-induced changes in adult cell proliferation 32 odour-exposed males had higher cell density than TMT-exposed males (p < 0.008). There were no other significant main or interaction effects for cell density (0.22<p<0.31). In females (Figure 11) there was a main effect of region, with the granule cell layer having greater density of BrdU-labeled cells compared to the hilus ( F ( i ; 2 0 ) = 441.03, p < 0.001). There were no significant main effects of drug treatment (p < 0.15) or odour exposure (p < 0.58) and no significant region by treatment interaction (p = 0.15) on density of BrdU-labeled cells. A priori we were interested in the effect of post-natal testosterone treatment on cell proliferation in adulthood and comparisons revealed that testosterone-treated females had lower B r d U cell density in the granule cell layer only, regardless of odour treatment, than o i l (p < 0.015) and unmarked females (p < 0.056). There were no other significant main or interaction effects on cell density (0.56 < p < 0.86). Table 7: Raw cell count estimates from Experiment 2 Male Female TMT Control TMT Control Unmarked 8403.33 ± 1556 10790 ±1570 7170 ±669 7600 ± 818 Oil 6560 ±252 7335 ± 857.5 7107.5 ± 1374 7730 ± 624 Flu/TP 8446 ±360 7660 ±1173 6754 ± 560 7348 ±673 TMT-exposure increased defensive burying in OIL-treated male rats, and increased the frequency of burying in females Figure 12 shows a significant treatment by odour interaction for males in the a) frequency (Fp, 31) = 4.9, p = 0.01) and a near significant interaction for the b) duration Role of androgens in stress-induced changes in adult cell proliferation 33 (F(2,31) = 2.9, p = 0.07) of defensive burying. Post-hoc analyses reveal that only the OIL-treated groups showed an increased in the frequency (p < 0.002) and duration of burying in response to TMT stress (p < 0.008). For females, there was a near significant increase in burying frequency in response to TMT (Figure 12a; F 37) = 3.6, p = 0.06), while there was no effect of treatment (p < 0.65) nor was there an interaction (p < 0.8). There were no significant effects of burying duration (p < 0.9). TMT-exposed rats exhibited more stretch attends towards the vial than control rats The TMT-exposed rats showed an increase in the frequency (Figure 13 a; F(i, 68) = 54.13, p = 0.000) and duration (Figure 13b; F ( i ; 6 8 ) = 23.02, p = 0.000) of stretch attends towards the vial. There were no other significant main effects (0.18 < p < 0.83) or interactions (0.13 < p < 0.79). There was a trend towards a TMT-induced increase in stretch attends away from the vial (Table 8; frequency: F(i, 68) = 3.59, p = 0.06; duration: F(i, 68) = 3.85, p = 0.054). There were no other significant main effects (0.1 < p < 0.32) or significant interactions (0.21 < p < 0.96). Table 8: Frequency and duration of stretch attends directed away from the vial as a function of stress and organizational hormonal status (Experiment 2). There was a trend towards increased frequency and duration for TMT-exposed rats (n = 4-8 per group). Control TMT Group Frequency Duration (s) Frequency Duration (s) Male/Unmarked 0.2 ± 0.2 0.13 ±0.13 0.2 ± 0.2 0.31 ±0.31 Male/Oil 0.75 ±0.41 1.5 ±0.96 1.2 ±0.3 2.41 ±0.95 Male/Flu 0.57 ±0.3 0.37 ±0.25 0.67 ±0.5 0.99 ±0.77 Female/Unmarked 0 0 1.14 ± 0.51 1.37 ±1.1 Female/Oil 1.25 ±0.62 1.29 ±0.85 1.25 ±0.5 1.48 ±0.62 Female/TP 0.13 ±0.13 0.21 ±0.21 1.5 ±0.6 3.07 ± 1.51 Role of androgens in stress-induced changes in adult cell proliferation 34 2 16 1 4 12 1 0 l^l^l Contro l I I T M T S 2 - i i U n m a r k e d O i l F l u U n m a r k e d O i l T P B M ale Fem ale 2 1 4 1 2 1 0 S 6 U n m a r k e d O i l I Flu Unmarked O i l i TP ^ • H Control I I T M T M a l e Fem ale Figure 12: T h e f r e q u e n c y a n d d u r a t i o n o f de fens ive b u r y i n g . T M T - e x p o s u r e inc reased the a) f requency and b ) d u r a t i o n o f de fens ive b u r y i n g i n rats. U n m a r k e d females s h o w e d m o r e b u r y i n g than u n m a r k e d males ( n = 4-8 per g r o u p ) . Role of androgens in stress-induced changes in adult cell proliferation A 35 2 w GO I Control • T M T 6 H I B 2 H i U n m a r k e d O i l F l u U n m a r k e d O i l T P M ale Female 2 w 12 n 10 4 1 C o n t r o l n T M T U nm arked O i l F lu U nm arked O i l T P M ale Fem ale F i g u r e 13: T h e frequency a n d d u r a t i o n o f s t re tch at tends. T M T - e x p o s u r e increased the a) f r e q u e n c y and b ) d u r a t i o n o f s t re tch at tends t o w a r d s the v i a l regardless o f t rea tmen t ( n = 4 - 8 per g r o u p ) . Role of androgens in stress-induced changes in adult cell proliferation 36 A 2 w 2 16 14 12 10 • Control • T M T B 4 H M ale A U n m a r k e d O i l Flu Unmarked O i l T P Fem ale •5 80 60 4 40 •2 20 U nm arked O i l • Control • T M T Flu U n m a r k e d O i l T P M ale Fem ale Figure 14: The frequency and duration of direct contact with the vial. TMT-exposure decreased the a) frequency and b) duration of direct contacts with the vial regardless of treatment (n = 4-8 per group). Role of androgens in stress-induced changes in adult cell proliferation 37 TMT-exposure suppressed the number of direct contacts with the vial Figure 14 shows TMT-exposed animals made direct contact with the vial less frequently (F ( i , 6g) = 42.05, p < 0.001) and for less duration ( F ( 1 , 6 8 ) = 13.02, p < 0.001) than control odour-exposed animals. There were no other significant main effects (0.21 < p < 0.91), nor were there any significant interactions (0.44 < p < 0.97). TMT-exposure increased rearing in unmarked animals only Table 9 shows a significant hormone treatment by odour interaction for frequency of rearing (Fp, 68) = 3.67, p = 0.03) and a trend for a hormone treatment by odour Table 9: Frequency and duration of rearing as a function of stress and organizational hormone status (Experiment 2). TMT-exposure increased the duration of rearing in A F R animals only (n = 4-8 per group). Control TMT Group Frequency Duration (s) Frequency Duration (s) Male/Unmarked 37 ± 4 . 2 123.36 ± 2 5 . 7 1 6 ± 4 46.58 ± 16.2 Male /Oi l 26.25 ± 2.8 83.44 ± 15 29.3 ± 7.3 86.7 ± 2 6 . 8 Male/Flu 31.7 ± 2 . 2 83.77 ± 9 . 4 26.2 ± 4.2 82.77 ± 14.6 Female/Unmarked 41 ± 7 . 6 151.83 ± 5 9 . 7 20.9 ± 3 . 7 62.76 ± 10.45 Female/Oil 27.9 ± 4 100.73 ± 18.5 30.9 ± 7 . 9 92.5 ± 30.2 Female/TP 35.4 ± 6 . 7 115.06 ± 2 4 . 1 28.5 ± 8 . 3 84.7 ± 2 3 . 1 interaction for duration of rearing (Fp, 68) = 2.96, p = 0.06). Post-hoc analysis revealed that control odour-exposed unmarked animals showed less rearing than TMT-exposed unmarked animals (frequency: p = 0.02; duration: p = 0.01). There were no other significant effects (0.22 < p < 0.99). Males groom more than females; TMT-exposure increases the duration of grooming There was a significant main effect of sex, as males groomed more than females (Table 10; frequency: F(i, 68) = 4.03, p = 0.05; duration: F(i, 68) = 4.1, p = 0.05). There Role of androgens in stress-induced changes in adult cell proliferation 38 were no significant main effects for hormone treatment (frequency: p = 0.1; duration: p = 0.36).or odour (frequency: p = 0.71) on grooming. However there was a main effect of Table 10: Frequency and duration of grooming as a function of stress and organizational hormonal status (Experiment 2). Males groomed more than females, and TMT-exposed rats groomed more than control-exposed rats (n = 4-8 per group). Control TMT Group Frequency Duration (s) Frequency Duration (s) Male/Unmarked 2.8 ± 0 . 8 24.79 ± 8 . 6 6 5 ± 1.1 108.2 ± 16.2 Male /Oi l 5.88 ± 1.1 74.54 ± 2 0 . 0 5 4.3 ± 1 . 2 49.2 ± 14.63 Male/Flu 3.57 ± 1 50.16 ± 2 1 3.5 ± 0 . 7 2 43.88 ± 16.73 Female/Unmarked 2.5 ± 0 . 2 9 14.61 ± 4 . 1 6 2.14 ± 0 . 5 5 39.54 ± 10.7 Female/Oil 3.88 ± 1.13 48.8 ± 13.47 3.75 ± 0 . 9 68.6 ± 2 8 . 3 Female/TP 2.5 ± 0.53 23.98 ± 5 . 9 8 3.63 ± 0 . 9 4 56.43 ± 19.36 duration on grooming, with TMT-exposed animals grooming for longer periods of time (F(i, 68) = 4.1, p = 0.05). There were no significant interactions (0.11 < p < 0.98). Corticosterone There was a significant four-way interaction effect on serum levels of corticosterone (F( 4 j 22) = 3.09, p < 0.04; see Figure 15). Post-hoc analyses (Fisher's L S D ) reveal that unmarked males show elevation in serum C O R T levels 30 min after onset of odour exposure regardless of the odour type (water (p < 0.006) or T M T (p < 0.004)). Both groups also show a return to baseline levels of C O R T 2 hrs after onset of exposure (p < 0.002 for both groups). Oil-injected males exposed to T M T do not show an elevation in serum C O R T levels 30 mins after onset of odour exposure (p < 0.44), where as O I L males exposed to water do show an elevation 30 mins post onset of exposure (p < 0.007) and a return to baseline levels 2 hrs post onset of exposure (p < 0.004). F L U males showed a trend towards a significant elevation in serum C O R T 30 mins post exposure to T M T (p < 0.07), Role of androgens in stress-induced changes in adult cell proliferation 39 while F L U males given water exposure showed an elevation 30 mins post exposure and then subsequent decline at 2 h(p < 0.001). Unmarked females given water exposure do not show any differences in serum C O R T across time (0.1 < p < 0.6). Unmarked Females given T M T exposure show a significant elevation in serum C O R T 2 hrs after onset of stressor relative to baseline (p = 0.02). OIL females exposed to T M T show an elevation in serum C O R T 30 mins after onset of exposure and a return to basal levels at the 2hr time-point (p < 0.001). Water exposure for O I L females did not significantly alter serum C O R T levels across time (0.64 < p < 0.96). TP females did not show a TMT-related elevation in serum C O R T (0.74 < p 0.98) while TP females given water exposure did show an elevation 30 mins after onset and subsequent decline 2 hrs later (p < 0.001). More BrdU-labeled cells are co-labeled with an immature neuronal marker The majority o f BrdU-labeled cells were co-labeled with an immature neuronal marker, D C X (Table 11). Furthermore more BrdU-labeled cells were co-labeled with D C X in females compared to males (Table 9; F ( i j i 4 ) = 6.1, p = 0.03). There were no other significant main effects (0.43 < p < 0.78), or interaction effects (0.42 < p < 0.92). Cell proliferation is not associated with any behavioural measures A s seen in Table 12, there are no significant correlations between cell proliferation and behaviour. Role of androgens in stress-induced changes in adult cell proliferation A 600 500 400 300 I 200 I 100 ii I I Male/Unmarked/Water ! Male/Unmarked/TMT I Male/Oil/Water 1 Male/Oil/TMT I Male/Flu/Water Male/FIu/TMT I I , I1O1L Baseline 30 min 2 hrs B Fem ale/Unmarked/Water Female/Unmarked/TMT Female/Oil/Water J Female /Oi l /TMT • Female/TP/Water B F e m a l e / T P / T M T I I Baseline 30 min 2 hrs Figure 15: Corticosterone levels of hormone-treated, oil-treated and unmarked rats in response to T M T stress. Females (a) demonstrate higher levels of C O R T than males (b), regardless of time of sampling, while most groups showed an elevation of C O R T 30 mins after the onset of T M T or control exposure (n = 2-3 per group). Role of androgens in stress-induced changes in adult cell proliferation 41 Table 11: Percentage of BrdU-labeled cells that are co-labeled with D C X (Experiment 2). Females show a greater amount of co-labeled cells than males (n = 2-3 per group). % Co-labeling Group Control TMT Male/Unmarked 56 ± 0 54 ± 2 Male /Oi l 64 ± 6 . 1 66 ± 10 Male/Flu 58 ± 10 66 ± 2 Female/Unmarked 68 ± 8 72 ± 8 Female/Oil 77.3 ± 7 . 1 73.3 ± 9 . 6 Female/TP 80 ± 8 64 ± 4 Table 12: Correlations between G C L cell density and behavioural measures (Experiment 2). There are no statistically significant correlations. P Defensive burying freq 0.06 0.11 Defensive burying dur 0.07 0.08 Stretch towards freq 0.02 0.39 Stretch towards dur 0.02 0.39 Stretch away freq 0.02 0.32 Stretch away dur 0.0 0.87 Contacts freq 0.0 0.75 Contacts dur 0.02 0.37 Rearing freq 0.02 0.38 Rearing dur 0.02 0.34 Grooming freq 0.02 0.35 Grooming dur 0.01 0.52 Discussion It was hypothesized that i f adult levels of androgens regulated the stress-induced suppression in cell proliferation in males, then castrating male rats would eliminate this suppression. Experiment 1 showed that this was not the case as adult male rats showed a TMT-induced suppression of cell proliferation 24 hours later regardless of androgen status. Furthermore, there was nd significant effect of castration on cell proliferation in Role of androgens in stress-induced changes in adult cell proliferation 42 the dentate gyrus, indicating that androgens do not appear to alter cell proliferation consistent with previous studies (Ormerod and Galea, 2003; Galea and McEwen , 1999). T M T exposure did result in a suppression in cell proliferation in adult male rats in Experiment 1, consistent with previous studies (Falconer and Galea, 2003; Holmes and Galea, 2002; Tanapat et al, 2001). The suppression in cell proliferation was selective for the granule cell layer and not due to an overall change in blood-brain barrier permeability, as there were no significant effects of stress or androgen status in the hilus, another area of the adult mammalian brain that shows cell proliferation. This study was the first study to show that adult levels of testosterone do not regulate the stress-induced suppression in cell proliferation in the male adult dentate gyrus. Furthermore, activational (or adult) levels of androgens do not appear to influence the expression of defensive and most non-defensive behaviours in the presence of T M T . However, adult androgen status did affect risk assessment behaviour, as castration decreased the duration of stretch attends in response to T M T exposure. T M T exposure suppressed testosterone levels 24 hours after exposure, consistent with previous findings (Orr and Mann, 1991). Activational levels of androgens do not mediate the stress-induced suppression of hippocampal cell proliferation in male rats Previous research has shown that TMT-exposure produces a rapid suppression in cell proliferation in male but not female rats (Falconer and Galea, 2003): This stress-induced suppression in cell proliferation was not dependent on adult levels of ovarian hormones and this suggests that activational or organizational levels of androgens could mediate this sex difference. In the current study, adult males showed a TMT-induced Role of androgens in stress-induced changes in adult cell proliferation 43 suppression of cell proliferation regardless of androgen status. This finding is not completely unexpected. The H P A and H P G axes inhibit each other (Viau and Meaney, 1996) and as such it would be expected that castrated adult male rats would have higher stress levels of C O R T because they are no longer inhibited by the presence of androgens. Because stress-induced increases in C O R T mediate the stress-induced suppression in cell proliferation (Tanapat et al, 2001), castration of adult male rats would actually be expected to further decrease cell proliferation, rather than increase it. In fact, although not statistically significant, the stress-induce difference in cell proliferation between the castrated groups was larger than the sham groups (Figure 4). Castration has clearly been shown to increase the C O R T response to stress (Viau, 2002; V iau and Meaney, 1996; Handa et al, 1994). This, coupled with the fact that T M T has been shown to elevate C O R T levels (Holmes and Galea, 2002; Morrow et al, 2002; Tanapat et al., 2001), suggests C O R T levels should be enhanced in the castration group. However, in the present study, C O R T levels were not significantly affected by androgen status. This may be due to our sampling methods as at the basal time point all groups had elevated levels of C O R T (greater than 200 ng/ml and previous papers have reported basal levels of C O R T in the range o f 50-70 ng/ml, Viau , Lee, Sampson and W u , 1996). The fact that both control and TMT-exposed groups experienced an elevation in C O R T (at both basal levels and 30 minutes after the onset of odour exposure) suggests that the sampling procedure was not successful in obtaining basal levels of C O R T . Curiously, castration did not cause a larger stress-induced suppression in cell proliferation in the present study. This may be a result of serotonergic activity in the hippocampus in relation to cell proliferation. Serotonin has been shown to up-regulate Role of androgens in stress-induced changes in adult cell proliferation 44 cell proliferation (Banasr, Hery, Printemps and Daszuta, 2004; Veenema, Koolhaas and de Kloet, 2004; Banasr, Hery, Brezun and Daszuta, 2001; Brezun and Daszuta, 1991). Recent studies by Huang and Herbert (2000a, b), however, reported that depleting serotonin by itself had no effect on adult hippocampal cell proliferation (2000a). They provide evidence that serotonin mediates the CORT-induced suppression of cell proliferation (2000b). Interestingly, testosterone inhibits serotonin (Zhang et al, 1999; Martinez-Conde et al, 1985; Engel et al, 1979). Therefore, castration may cause two opposing effects on stress-induced cell proliferation. Castration in adulthood increases stress levels of C O R T (Viau, 2002), which would decrease cell proliferation, and it increases serotonin, which would increase cell proliferation. Given the complexities of the serotonin-testosterone-corticosterone relationship, this system requires further study. In the present study, androgen status alone did not affect cell proliferation. This finding is consistent with previous studies showing that reproductive status in male meadow voles did not alter cell proliferation in the dentate gryrus (Ormerod and Galea, 2003). Although androgens do not appear to significantly alter cell proliferation, androgens do appear to enhance neurogenesis through cell survival (Ormerod and Galea, 2003; Spritzer and Galea, 2005) and androgens have been shown to be neuroprotective in the hippocampus of stressed rats (Frye and McCormick, 2000a; Frye and McCormick, 2000b; Pike, 2001; Hammond et al, 2001; Mizoguchi , Kunishita, Chui and Tabira, 1992). A number of studies have shown that castration increases cell death in response to stress (Frye and McCormick , 2000a; Frye and McCormick, 2000b; Mizoguchi et al, 1992), which suggests that the neuroprotection afforded by testosterone may be a decrease in cell death (and not an increase in cell birth) following stress. Although, cell death was Role of androgens in stress-induced changes in adult cell proliferation 45 not examined in the present study, it has been speculated that androgens suppress cell death through the hippocampal G A B A e r g i c system (Frye and McCormick , 2000b). Activational levels of androgens do not alter the expression of defensive or non-defensive behaviours in response to predator odour stress in adult male rats Consistent with previous studies, T M T exposure in the present study elicited an increase in defensive behaviours (Falconer and Galea, 2003; Holmes and Galea, 2002). The effects of activational levels of androgens on stress-induced changes in defensive and non-defensive behaviours were also examined (Figures 5-7; Table 2). Regardless of androgen status, T M T exposure increased defensive burying, while decreasing the frequency and duration of direct contact with the vial. Most non-defensive behaviours (including contacts with vial) were also unaffected by T M T exposure and androgen status. However, risk assessment behaviour (stretch attend) was affected by androgen status, as a TMT-induced increase in the duration of stretch attends was only seen in the intact male rats. This result is somewhat surprising, as testosterone is normally thought to be anxiolytic (Fernandez-Gusati and Martinez-Mota, 2005; Perrot-Sinal, Ossenkopp and Kavaliers, 2000) - as such, it would be expected that i f defensive burying and stretch attends are reflections of anxiety, both would be increased in response C S T X animals. However, this result is not seen. In fact, stretch attends actually show the opposite effect, where duration of stretch attends is actually decreased in C S T X animals. The disparity between that study and the present study may be due to species of animals and/or manipulation of androgen status as in study by Perrot-Sinal et al (2000) meadow voles were used instead of rats in the present study and androgen status was manipulated by Role of androgens in stress-induced changes in adult cell proliferation 46 reproductive status in Perrot-Sinal et al (2000) as opposed to adult castration in the present study. Furthermore, it could also be speculated that stretch attends are not an expression of anxiety, but meagerly of assessing the danger without an emotional "anxiety" component. In fact, stretch attends has been referred to as a risk-assessment behaviour (Blanchard et al., 1993). In order to validate this hypothesis, the effects of common anxiolytic drugs on stretch attends should be examined. The fact that testosterone does not appear to influence the expression of defensive burying in the present study could be due to the type of stressor. Previous studies examining defensive burying and testosterone have used a shock prod as the stressor, which is both a physical and psychological stressor, while T M T or predator odour is a psychological stressor alone. Although distinct in appearance, defensive burying and stretch attends are behaviours that promote survival in the animal. Defensive burying is an active effort to rid the animal's environment of threatening stimulus, while stretch attends evaluates the level of risk. However in the present study there was no significant correlation between the expression of defensive burying and stretch attends in the present study. Furthermore, these behaviours are regulated by distinct neural regions. Defensive burying is regulated by parts of the limbic system such as the posterior parts of the septal region, the dorsal hippocampus, the caudal shell of the accumbens and the dorsal raphe nuclei (de Boer and Koolhaas, 2003), while stretch attends are regulated by the dorsal premammilary nucleus o f the hypothalamus (Blanchard et al, 2003). Defensive burying is in part regulated by G A B A e r g i c activity (de Boer and Koolhaas, 2003), while stretch attends is in part regulated by the serotonergic system (Shepherd et al, 1992). There are Role of androgens in stress-induced changes in adult cell proliferation 47 some commonalities between the two behaviours. Both appear to be under some degree of influence of norepinephrine (Morilak et al, 2005; Blanchard, Shepherd, Rogers, Magee and Blanchard, 1993), and there are extensive connections between the neural regions regulating each behaviour. Thus it may not be surprising in the present study that there was an effect of androgen status on stretch attends but not on defensive burying. Perhaps androgens influence the neural mechanisms of stretch attends more than defensive burying. Interestingly, there is a mi ld correlation between frequency of stretch attend expression and cell proliferation. Previous studies report no such association (Falconer and Galea, 2003; Holmes and Galea, 2002). A s mentioned, the expression of stretch attends are modulated by serotonin and norepinephrine, both which have been shown to up-regulate hippocampal cell proliferation (Kulkarni, Jha and Vaidya, 2002; Brezun and Daszuta, 1999). It is conceivable that cell proliferation and stretch attends are indirectly linked via one of these neurotransmitter systems. It is important, however, to keep in mind that a large number of correlations were calculated at an alpha level set at 0.05. Given that no other studies have reported a relationship between these measures, it is conceivable that this relationship is a statistical artifact. Experiment 2 Experiment 2 examined the effects of organizational (or early) levels of androgens on the stress-induced suppression in adult hippocampal cell proliferation in both males and female rats. It was hypothesized that i f organizational levels of androgens are responsible for sex differences in stress-induced suppression in cell proliferation, then injecting males postnatally with flutamide (an androgen receptor Role of androgens in stress-induced changes in adult cell proliferation 48 blocker) and/or injecting females postnatally with testosterone would eliminate the stress-induced suppression in adulthood. The data is presented as a density measurement in order to account for the differences in granule cell layer volume reported in Figure 10. However, as seen in Table 7, the raw cell counts are comparable to those in Experiment 1 (Figure 3) indicating consistency in cell proliferation measurements across studies. A s seen in Figure 1 l a , only unmarked males (males that were not manipulated in early life but reared in the facility) demonstrated the stress-induced suppression in cell proliferation relative to their control. Furthermore, postnatal hormonal treatment did appear to alter the expression of defensive behaviours in response to T M T , as only O I L injected males showed an increase in defensive burying. Stretch attends were unaffected by postnatal treatment. This is partially consistent with a previous study that reported no effect of neonatal castration on performance in the elevated plus maze in adulthood (Gonzalez, Albonetti, Siddiqui, Farabollini and Wilson, 1996). Unmarked animals did, however, show more rearing in response to T M T than did O I L or drug treated animals. Injecting and marking male neonatal pups appears to alter basal and stress levels of cell proliferation in adulthood A s seen in Figure 1 l a , only unmarked males demonstrated the stress-induced suppression in hippocampal cell proliferation relative to their control. Males that were injected with oi l (or flutamide) did not show a stress-induced suppression, suggesting that early handling altered the system's response to stress. The lack of the stress-induced suppression in cell proliferation in these groups appears to be due to low levels of cell proliferation in the control odour groups. Regardless of early treatment (oil or flutamide) Role of androgens in stress-induced changes in adult cell proliferation 49 all male control odour groups had fewer BrdU-labeled cells in the hippocampus compared to the control odour unmarked group. Thus the postnatal manipulations in both the OIL- and FLU-injected groups may have acted as a form of early life stress and suppressed cell proliferation i n adulthood. Indeed, it has been shown that other forms of early life stress, such as daily maternal separation (for 3 hours per day) results in higher basal and stress levels of corticosterone in adulthood (Plotsky and Meaney, 1993; L i u et al, 2000). In fact, early maternal separation suppresses basal levels of cell proliferation in adulthood (Mirescu et al, 2004). It is possible that early injection stress and/or the application of coloured dye may act similarly to maternal deprivation stress in causing an increase in H P A activity in adult males. The present study reports that adult body weight is lower in F L U and OIL treated rats in adulthood, relative to unmarked males (Table 4). This is consistent with previous reports that show that early life stressors decrease body weight in adulthood (Yamazaki et al, 2005; Panagiotaropoulos et al, 2004). There is reason to believe that the flutamide and o i l manipulation in the present study was contaminated by the early life stress caused by the injecting/marking procedure. Flutamide administered during the neonatal period should decrease body weight and ano-genital distance (McCormick and Mahoney, 2001; Hib and Ponzio, 1995). Table 6 shows that there were no significant differences between O I L and F L U injected animals in either of these measures at any time point. Both body weight and A G D in the OIL and F L U groups however were suppressed relative to unmarked animals, further supporting the hypothesis that the procedure had a greater effect in males than the actual drug or odour treatment in adulthood. Role of androgens in stress-induced changes in adult cell proliferation 50 Neonatal injections of TP to females reduces basal cell proliferation without inducing a stress-induced suppression Experiment 2 also examined the effects of neonatal administration of testosterone propionate to females on stress-induced changes in adult hippocampal cell proliferation (Figure 1 la). Testosterone propionate has been shown to masculinize the female dentate gyrus when administered on P N D 3 and 5 (Roof and Havens, 1992). It was hypothesized in the present study that testosterone propionate could cause a "masculine" response to stress in terms of cell proliferation. In other words, testosterone propionate-treated females should exhibit a TMT-induced suppression in cell proliferation. A s seen in Figure 11 this did not occur, testosterone propionate-treated females, much like OIL-treated and unmarked females did not demonstrate a TMT-induced suppression in cell proliferation. Testosterone propionate, however, induced a number of important changes in females: changes in body weight, estrous cycle, dentate gyrus volume and cell proliferation suggesting that testosterone propionate exposure in the present study effectively "masculinized" females. Testosterone treated females weighed more in adulthood than O I L and unmarked females in adulthood (Table 5). Furthermore, TP females did not experience a normal estrous cycle in adulthood, spending a significantly greater amount of time in estrus compared to OIL and unmarked females, consistent with previous results (Shors and Miesegaes, 2002). TP treatment resulted in larger D G volumes relative to unmarked and O I L females, consistent with previous findings (Roof and Havens, 1992). Lastly, TP females had lower levels of cell proliferation than did unmarked and O I L females, which is consistent with previous studies reporting that females have higher levels of cell proliferation than males (Falconer and Galea, 2003; Role of androgens in stress-induced changes in adult cell proliferation 51 Tanapat et al., 1999). Interestingly, testosterone propionate masculinized dentate gyrus volume and lowered basal levels of cell proliferation without causing a stress-induced suppression in cell proliferation. In males, the stress-induced suppression appears to be mediated by serotonin (Huang and Herbert, 2000b). It could be hypothesized that the sex differences in stress-induced changes in cell proliferation may involve the organization of the serotonergic system. Previous studies have shown that neonatal testosterone depletes serotonin levels in the brain (Sundblad and Eriksson, 1997). Furthermore, neonatal ablation of the serotonergic system decreases basal levels of cell proliferation in adulthood (Ueda, Sakakibara and Yoshimoto, 2005). Moreover, it has also been suggested that adrenal steroids suppress cell proliferation via NMDA-media ted input from the entorhinal cortex (Cameron et al, 1995; Gould et al, 1994). Sex differences in the organization of the serotonergic system and N M D A system may account for the absence of a stress-induced suppression of cell proliferation in androgenized females. Interestingly, the effects of early life stress that appear to have contaminated the data from the male/flutamide study does not seem to be present with the females. Basal levels of cell proliferation in the OIL-treated females were not different from those in the unmarked females. Al so , granule cell layer volume in OIL-treated females were identical to the unmarked female group. The reason for this lack of contamination could lie in the different injection/marking protocols used for males and females. Females were only injected/marked beginning on P N D 3. Previous research has found that maternal separation that begins on P N D 2 does not alter stress reactivity in adulthood (Rhees et al, 2001). The suggestion that neonatal stress is sensitive to when it is administered during the neonatal period is intriguing as most other neonatal stress paradigms such as Role of androgens in stress-induced changes in adult cell proliferation 52 immobilization (Gilad, Rabey, Eliyayev and Gilad, 2001) and predator (Weidenmyer and Barr, 2001) commence after P N D 7, mimimalizing their relevance to the current study in relation to the importance of the effects of maternal separation studies, which often commence earlier (Mirescu et al, 2004; Kalinichev et al, 2002). Organizational levels of androgens do not alter the expression of defensive behaviour to TMT; non-defensive behaviour is altered by neonatal manipulation Defensive and non-defensive behaviours in response to T M T exposure in adulthood were also examined in Experiment 2 (Figures 12-14). Previous research shows that there were no sex differences in the behavioural response to T M T (Falconer and Galea, 2003). The present study reports that in males, TMT-exposure increased the frequency and duration of defensive burying only in OIL-treated males. Unmarked and FLU-treated males did not demonstrate a TMT-induced increased in defensive burying. The finding that unmarked animals do not show a stress-induced increase in defensive burying is rather surprising, as no other studies report this finding in control animals (Falconer and Galea, 2003; Holmes and Galea, 2002). Furthermore, the finding that flutamide-treated animals do not show a stress-induced elevation is also somewhat surprising, as previous studies have shown that the organizational activity of testosterone does not alter exploration or anxiety-like behaviour (Gonzalez et al 1996). The frequency of defensive burying was increased in response to T M T in females, regardless of gender. Furthermore, TMT-exposed animals showed more stretch attends towards the vial and less direct contact with the vial , regardless of gender or neonatal treatment. These results are interesting in light of the fact that forms of early life stress are known to either Role of androgens in stress-induced changes in adult cell proliferation 53 increase anxiety-like behaviours (i.e. maternal separation). The fact that early life stress does not alter the expression of stretch attends indicates that the neural mechanisms underlying stretch attends differ from those that regulate adult neurogenesis (where early life stress does have an effect; Figure 11). The finding that flutamide does not alter the expressions of stretch attends in males is consistent with previous literature showing no effect of neonatal testosterone in exploratory or anxiety behaviours (Gonzalez et al, 1996). The fact that testosterone did not alter the expression of any defensive or risk assessment behaviours in females is somewhat surprising, as previous studies have shown neonatal testosterone administration to increase the expression of anxiety like behaviours (Wilson et al, 1992). Whether or not the behaviours recorded in the present study can be considered anxiety behaviours is debatable, as they could simply reflect normal behavioural coping mechanisms of animals in the face of stressful stimuli. This could help to explain the discrepancy between the present studies and past research. Interestingly, there was a positive correlation between specific maternal behaviours (next building, licking) and the expression of defensive burying in adulthood. This indicates that the maternal response to the brief separation during the injecting/marking period produces an anxiogenic response to stress in adulthood. This is consistent with previous studies that show that the effects of early life manipulations in pups are due to the changes in maternal behaviour (Levine, 1994). This is the first study to demonstrate that organizational levels of gonadal hormones do not appear to mediate the expression of defensive behaviour towards predator odour stress. Unmarked animals demonstrated more rearing than other groups, in T M T -exposed rats only (Table 5). The effect of neonatal handling on stress behaviour is still Role of androgens in stress-induced changes in adult cell proliferation 54 unclear as there has been some controversy in the literature. Some report an increase in activity and exploratory behaviour (Parfitt et al, 2004; Bodnoff, Suranyi-Cadotte, Quirion and Meaney, 1987; Levine, Haltmeyer, Karas and Denenberg, 1967), while others report a decrease in time spent in open arms of an elevated plus maze (Kalinichev, Easterling, Plotsky and Holtzmann, 2002) or an increase in immobility time in a forced swim test (Papaiouannou et al, 2002). The decrease in rearing (which is potentially an exploratory behaviour) in the present study suggests that brief handling decreases exploratory behaviour in the presence of predator odour. Considerations of litter effects A s mentioned earlier, rats i n adulthood show alterations in stress reactivity in accordance with the type of early life stress/manipulation that they undergo. Neonatal handling (daily separations of pups from moms for 3-15 minutes) has been shown to be anxiolytic on numerous endocrinological and behavioural measures of stress (Cameron et al, 2005; Plotsky and Meaney, 1993; L i u et al, 2000; Meaney et al, 1989; Viau etal; 1993; Panagiotaopoulos et al, 2004; Pappaioannou et al, 2002; Kalinichev et al, 2002). Furthermore, it has been suggested that these changes are a result of alterations in maternal responsive to her pups (Levine, 1994). For instance, handling increases licking/grooming behaviour in moms (Liu et al, 1997; Lee and Will iams, 1975), which produces the aforementioned anxiolytic effects. Indeed, it was shown that certain measures of maternal care were correlated with the expression of defensive burying and rearing in adulthood. The increase in care that these animals received as a result of being separated while the drug and oi l groups were being injected/marked could very well have altered stress-reactivity in adulthood, possibly accounting for the lack of a stress-induced Role of androgens in stress-induced changes in adult cell proliferation 55 increase in defensive burying in unmarked male rats in adulthood. However, as seen in Table 4, there are also individual differences in specific aspects of maternal care, consistent with previous literature (Cameron et al, 2005). B y utilizing a within-litter experiment design (i.e. animals from all treatment conditions are raised by each dam), these effects are accounted for. There are drawbacks to such a design as the alteration in maternal care given to the treated pups could also be carried over to the untreated pups, bringing into question the validity of the "unmarked" group as a control. A between-litters study could have accounted for this, such that treatment-induced changes in maternal care would not have influenced animals from another treatment. For instance, dams prefer the odour o f male pup urine to female pup urine (Moore, 1985). It is possible in the present study that the hormonal manipulation altered the urinary cues in both the flutamide-treated males and the testosterone-treated females such that maternal licking behaviour was altered for all pups. The change in l icking (due to this treatment) would certainly affect the unmarked animals, both males and females, which would compromise their status as true controls. In a between-litter experimental design, such a confound is avoided because all animals in a litter are given the same treatment. The disadvantage to such a design is that individual differences in maternal care are not accounted for - thus it is unknown i f any effects are due to the treatment or the quality of maternal care. It is important to consider the advantages of disadvantages of both designs when conducting experiments with offspring at such a young age. Future studies The handling procedure for the male oi l and flutamide groups may have altered the H P A axis reactivity in males due to excessive handling. Interestingly, the handling Role of androgens in stress-induced changes in adult cell proliferation 56 procedure did not affect the females. This may be in part due to the different injection protocols. Males were injected daily from P N D 0-5, while females were only injected on postnatal days 3 and 5. This means that males had dye applied within 4 hours of birth while females were left untouched for 3 days and that males were injected for 6 days while females were injected for only 2 days. It should also be noted that it appears as though handling has different behavioural and biochemical effects in adult males and females (Papaioannou et al, 2002; Smythe, McCormick, Rochford, and Meaney, 1994), showing that males and females respond differently to neonatal stress, which could also account for the fact that neonatal handling in our study in the females was not as disruptive as neonatal handling in the males. In the future, it may be advisable to use a different method of labeling pups, such as toe-clipping. Another solution would be treat all the pups within a litter with the same drug, such that labeling individual pups is unnecessary. This latter solution, however, requires that the experimenter control for individual differences in maternal behaviour and potential litter effects. Another problem in the present study was that the C O R T sampling procedure seemed faulty, as basal levels of stress were elevated and C O R T increased in both treatment and control groups. From the present data, it would appear as i f androgens are not responsible for sex differences in the stress-induced suppression in adult hippocampal cell proliferation. It may be advisable to examine sex differences in neurotransmitter functioning in relation to stress and cell proliferation. Previous studies suggest that C O R T elevations suppress cell proliferation via serotonergic activity (Huang and Herbert, 2000b) or N M D A activity (Cameron et al, 1995; Gould et al, 1994). There are sex differences in serotonergic Role of androgens in stress-induced changes in adult cell proliferation 57 (Zhang et al, 1999; Martinez-Conde et al, 1985; Engel et al, 1979) and glutamatergic (Romeo et al, 2005) activity which may in part explain the sex difference in stress-induced cell proliferation. Finally, studies examining the effects of flutamide should examine the effects of flutamine in utero. There are two times when testosterone is elevated during development, which are thought to organize the male brain: gestational day 18-20 and within 1-3 hours of birth (Corbier, Kerdelhue, Picon and Roffi , 1978). Injecting flutamide before the first testosterone surge would help to eliminate both of these surges. Indeed numerous studies have used this protocol to successfully block the effects of testosterone in adulthood (Goto et al, 2005; Shors and Miesegaes, 2002). Although androgens do not appear to regulate cell proliferation, they have been shown to increase cell survival (Spritzer and Galea, 2005; Ormerod and Galea, 2003). Interestinlgy, the TMT-induced suppression in cell proliferation in males has been shown to last between 1-3 weeks (Tanapat et al, 2001). It is possible that androgens may mediate stress-induced changes in cell proliferation via cell survival. 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