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Organizational effects of gonadal hormones on the hypothalamic-pituitary-adrenal axis and glucocorticoid… Innala, Leyla 2016

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ORGANIZATIONAL EFFECTS OF GONADAL HORMONES ON THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS AND GLUCOCORTICOID RECEPTOR RESPONSES IN MALE AND FEMALE RATS by  Leyla Innala  B.Sc., The University of British Columbia, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2016  © Leyla Innala, 2016  ii Abstract Neonatal gonadal hormones during critical periods of development can irreversibly alter the adult hypothalamic-pituitary-adrenal (HPA) axis. The aims of this thesis were to investigate the role of neonatal gonadal hormones to 1. have direct organizational effects on the HPA axis or via indirect effects on corticosterone binding globulin that lead to compensatory changes in HPA output and 2. if changes in HPA output are met by changes in glucocorticoid receptor (GR) responses. To assesses these questions, the neonatal hormone milieu in Long Evan rats were manipulated in males by blocking the conversion of testosterone to estradiol with an aromatase blocker (ATD) and in females by administering testosterone propionate (TP). As adults, we assessed the influence of neonatal hormone manipulations on plasma corticosterone levels and GR activation in response to acute and repeated restraint exposure. GR responses were assessed using western blots to analyze GR translocation (nuclear/ nuclear and cytoplasm) and phosphorylation of GR at the serine 211 site (Ser211). We then assessed if changes in GR translocation and Ser211 followed restraint induced changes in total and estimated free CORT levels. Our results showed significant differences in corticosterone levels in neonatal ATD treated male and TP treated female rats compared to their same sex control groups under basal-naïve conditions and after restraint exposure, respectively. In both sexes, for GR translocation and Ser211, there was a main effect of restraint stress exposure, and overall significant positive correlations with CORT (total and estimated free) levels. GR translocation was lower in neonatal TP treated females, with no effect in Ser211. Differences between male neonatal ATD and Sham groups were observed compared to untouched controls in Ser211, indicating the effects of the neonatal ATD treatment appear to be due to effects of surgery and gonadal hormone milieu exposure. Adult gonadal hormone levels differed between female neonatal groups, which are  iii likely due to organizational effects of the neonatal treatments. The current study demonstrates neonatal gonadal hormones have long lasting effects on adult corticosterone outputs and sequential GR responses.    iv Preface This thesis was designed and planned by Leyla Innala and Dr. Victor Viau. Leyla Innala carried out experimental work with assistance from Rokzanna Basi, Judy Chang, Carly Moody, Adam Anonuevo, and Yi Yang for the animal work, from Lesley Hill for the corticosterone RIA data, and from Mingqi Zhang for the GR translocation, liver gene expression, and adrenal weight data. Maternal care observations were carried out by Carly Moody with supervision from Leyla Innala. Lesley Hill independently measured all the CBG data.  Under the supervision of Dr. Victor Viau, Leyla Innala carried out the statistical analyses, with assistance from Dr. Tiffany Lee, and the writing, with feedback from Dr. Victor Viau, Dr. Tiffany Lee and Dr. Megan Gray. The work and methodology employed in this thesis was approved by the University of British Columbia Animal Care Committee under number A13-0251.   v Table of Contents Abstract........................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ........................................................................................................................... v List of Tables ................................................................................................................................ viii List of Figures ..................................................................................................................................ix List of Abbreviations .......................................................................................................................x Chapter 1: Introduction.................................................................................................................. 1 1.1 Sex differences in the hypothalamic-pituitary-adrenal (HPA) axis ........................................1 1.1.1 HPA axis habituation.......................................................................................................4 1.1.2 Corticosteroid-binding globulin .......................................................................................5 1.2 The role of the Glucocorticoid receptor to modulate HPA output ........................................5 1.3 Regulation of corticosterone-activation and translocation of the glucocorticoid receptor ....7 1.3.1 The hippocampus as a major site of GR-mediated negative feedback ................................8 1.4 Hormonal origins of sex differences in the brain .................................................................9 1.4.1 Hormonal organization of the HPA axis output ............................................................... 10 1.4.2 Neonatal organization of GR expression in the hippocampus .......................................... 12 1.5 Summary and specific hypotheses .................................................................................... 12 Chapter 2: Methods...................................................................................................................... 14 2.1 Animals ........................................................................................................................... 14 2.2 Surgeries.......................................................................................................................... 14 2.3 Maternal care observations.............................................................................................. 15  vi 2.4 Body weight measurements ............................................................................................. 16 2.5 Restraint paradigm .......................................................................................................... 16 2.6 Tissue collection............................................................................................................... 18 2.7 Gonadal hormone assays ................................................................................................. 18 2.8 Corticosterone ................................................................................................................. 19 2.9 Corticosteroid binding globulin......................................................................................... 20 2.10 Free corticosterone .......................................................................................................... 21 2.11 Adrenal weight measurement .......................................................................................... 21 2.12 Protein extraction ............................................................................................................ 21 2.13 Western blots .................................................................................................................. 22 2.14 Western blots analysis ..................................................................................................... 23 2.15 Liver gene expression ....................................................................................................... 24 2.16 Statistical analysis ............................................................................................................ 25 Chapter 3: Results ........................................................................................................................ 27 3.1 Maternal care .................................................................................................................. 27 3.2 The effect of neonatal gonadal manipulations on basal CORT levels .................................. 27 3.3 The effect of neonatal gonadal hormones on acute and repeat restraint stress induced CORT levels ...................................................................................................................... 29 3.4 The effect of neonatal manipulations and restraint stress exposure on CBG binding .......... 31 3.4.1 Strain differences in CBG binding properties .................................................................. 33 3.5 The effect of neonatal gonadal hormones on adult adrenal size ........................................ 33 3.6  Organization effects of neonatal gonadal manipulation to influence adult gonadal hormone levels ............................................................................................................................... 36 3.7 GR translocation in the hippocampus ............................................................................... 39  vii 3.8 GR phosphorylated at the serine 211 site in the hippocampus........................................... 42 3.9 The effect of neonatal manipulations on body weight....................................................... 45 3.10 Growth hormone regulated genes in the liver ................................................................... 46 Chapter 4: Discussion ................................................................................................................... 48 4.1 Neonatal ATD treatment in males alters basal-naïve total CORT output, while neonatal TP treatment alters restraint stress CORT output................................................................... 49 4.2 Males showed differences in CBG binding levels after restraint exposure .......................... 51 4.3 Similarities between total and estimated free CORT in response to neonatal manipulations is restraint-dependent in both males and females............................................................. 53 4.4 Neonatal manipulations and restraint stress effects on adrenal size .................................. 53 4.5 Markers of corticosterone activated glucocorticoid receptors differentially respond to neonatal hormone milieu in males and females ................................................................ 54 4.6 Neonatal TP decreases adult estradiol levels in females, with no effect of neonatal ATD on adult testosterone levels in males .................................................................................... 56 4.7 Effects of Neonatal treatments on body weights............................................................... 58 4.8 Methodological considerations in experimental design of neonatal manipulations ............ 59 4.9 Effect of cross-contamination between nuclear and cytoplasmic fractions on calculated GR translocation ................................................................................................................... 61 4.10 Future considerations ...................................................................................................... 61 4.11 Summary ......................................................................................................................... 62 References .................................................................................................................................... 64   viii List of Tables  Table 1: Correlation between GR translocation (nuclear/ nuclear + cytoplasmic) in the hippocampus of males and females and plasma total and estimated free CORT ......................... 41 Table 2: Correlations between GR phosphorylated at the serine 211 site in the hippocampus of males and females and plasma total and estimated free CORT .................................................... 45 Table 3: Mean pre-pubertal and adult body weights.  ................................................................... 46 Table 4: Mean relative mRNA expression of cyp2c11, cypt2c12, and hnf-6 in the liver............. 47   ix List of Figures Figure 1: The hypothalamic-pituitary-adrenal axis......................................................................... 3 Figure 2: Timeline of experimental design ................................................................................... 15 Figure 3: Adult treatment groups .................................................................................................. 18 Figure 4: Mean + SEM of basal levels of plasma CORT and CBG binding ................................ 30 Figure 5: Mean + SEM of restraint stress induced plasma levels of  CORT and CBG binding... 32 Figure 6: Mean + SEM of plasma CBG binding levels ................................................................ 34 Figure 7: CBG binding properties in Long Evans and Sprague Dawley rats.  .............................. 35 Figure 8: Mean + SEM of adrenal size and the adrenal-body weight ratio .................................. 37 Figure 9: Mean + SEM of plasma levels testosterone in males and estradiol in females.  ............ 38 Figure 10: Mean + SEM of GR translocation in the hippocampus of males and females............ 40 Figure 11: Mean + SEM of Ser211 in the hippocampus of males and females ............................ 44   x List of Abbreviations ACTH Adrenocorticotropic hormone  ANCOVA Analysis of covariance  ANOVA Analysis of Variance ATD 1, 4, 6-androstatriene-3, 17-dione AVP Arginine vasopressin  Bag1 BCL-2 associated athanogene  BPA Bisphenol A CBG Corticosteroid biding globulin  CORT Corticosterone  CRH Corticotropin releasing hormone  Cyp2c11 Cytochrome P450, subfamily 2, polypeptide 11 Cyp2c12 Cytochrome P450, family 2, subfamily c, polypeptide 12 DCC Dextran-coated charcoal  fEBP fetoneonatal estrogen binding protein  Fkbp51 FK506 binding protein 5  Fkpb52 FK506 binding protein 4  GDX Gonadectomy GR Glucocorticoid receptor  GRE Glucocorticoid receptor element Hnf-6 ONECUT transcription factor HNF6 beta HPA Hypothalamic-pituitary-adrenal  Hsp90 Heat shock protein 90  Kd dissociation-rate constant  LE Long Evan  MR Mineralocorticoid receptors  neo-GDX Neonatal gonadectomy  OVX Ovariectomy PBS phosphate buffered saline  PND Postnatal day  Ppid Peptidyl-prolyl isomerase D  PVN Paraventricular nucleus of the hypothalamus  RIA Radioimmunoassay SC Silastic capsule SD Sprague Dawley  Ser211 GR receptor phosphorylated at the serine 211 site Sham Sham surgical control TP Testosterone propionate   1 Chapter 1: Introduction 1.1 Sex differences in the hypothalamic-pituitary-adrenal (HPA) axis The hypothalamic-pituitary-adrenal (HPA) axis is activated in response to a stressor, which is defined as a real or perceived threat to homeostasis. The end product of the activation of the HPA axis is the release of glucocorticoids, corticosterone (CORT) in the rat and cortisol in the human, to mobilize energy to meet the demands of the stressor and to restore the organism to homeostasis. Even though activation of the HPA axis is imperative for survival, prolonged elevations in glucocorticoids are known to be associated with mood, metabolic, immune, and cardiovascular disorders, with many of these conditions showing a sex bias in prevalence (as reviewed in Goel et al., 2014). The sex of an individual is also known to affect their risk for certain diseases, including mental illness, with women more likely to suffer from depression, anxiety disorders, phobias, and panic attacks, while men are more likely to develop anti-social behaviours and substance abuse (as reviewed in Kudielka and Kirschbaum, 2005). Thus it is important to understand the interplay between the sex of an individual and their HPA responses to stressors.  The direction and presence of a sex difference in glucocorticoid output can be dependent on the species and/or stressor, in rodents exposed to a psychological stressor, like restraint stress, females have higher CORT output. Although this is contradictory to human data, since in humans cortisol output in response to many stressors is equivalent between men and women or higher in young adult males, with sex differences in the rate of depression emerging during puberty when gonadal hormones are on the rise (as reviewed in Kudielka and Kirschbaum, 2005, Goel et al., 2014). Further, in rodents sex differences in CORT output have been largely attributed to circulating gonadal hormones, with testosterone in males having inhibitory effects  2 and estradiol in females having excitatory effects on HPA axis activity (Viau et al., 2003, Handa and Weiser, 2014) HPA output is determined by the sum of activational drive to the axis and negative feedback of the axis, with the influence of gonadal hormones dependent on the type of stressor. Figure 1 describes the hormonal cascade involved in the activation and termination of the HPA axis. There are two main types of stressors that activate the HPA axis: physical and psychological stress. Physical stress involves the activation of the sympathetic nervous system and the HPA axis through ascending pathways from the brain stem, while psychological stress involves the activation of the HPA axis through descending pathways from the limbic system, which is a group of brain regions that are capable of relaying emotional and memory information to the HPA axis (Herman et al., 2005).  Psychological stressors, such as restraint stress which is a widely used stressor in rodents that involves primarily the activation of the limbic system, and is known to demonstrate robust sex differences in HPA output. These sex differences have been attributed to differences in both activation and negative feedback of the HPA axis (as reviewed in Goel et al., 2014). The focus of this thesis will be on exploring the ability for neonatal sex steroids to organize CORT output and glucocorticoid receptor (GR) mediated negative feedback regulation of the HPA axis.     3  Figure 1: In response to acute stress exposure, the hypothalamic-pituitary-adrenal axis  (HPA) is activated, causing the release of corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) from the paraventricular nucleus of the hypothalamus (PVN). CRH then stimulates the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary into circulation and acts on the adrenal cortex to stimulate the release of corticosterone (CORT) in the rat. CORT then acts to limit the magnitude and duration of the stress response through glucocorticoid receptor mediated negative feedback of the HPA axis directly or indirectly through limbic structures such as the hippocampus.    HippocampusHypothalamusPituitaryAdrenalCRHAVPACTHCORTNegative Feedback 4 1.1.1 HPA axis habituation Understanding sex differences in basal and acute stress induced CORT output provides a starting point for understanding how gonadal hormones during development are shaping the HPA axis. However, this does not address one of the main factors leading to mental illness which is exposure to chronic stress, which is often associated with impaired negative feedback of the HPA axis (Cattaneo and Riva, 2016). As mentioned previously activation of the HPA axis is imperative to survival when there is a real or perceived threat to homeostasis, however it is also imperative for an organism to be able to learn and adapt their response when faced with the same stressor repeatedly. This is often referred to as HPA axis habituation, which is when CORT output decreases with repeated exposure to the same homotypic stressor (Herman, 2013). HPA axis habituation is likely regulated by both changes in HPA activation and negative feedback, as Cole et al. (2000) has demonstrated that blocking mineralocorticoid receptors (MR) on the last day of repeated restraint exposure was able to block the expression of habituation in male rats. These results suggest that negative feedback of the HPA axis are involved in the expression of habituation. Interestingly, though there are no sex differences in the degree of HPA axis habituation (Babb et al., 2014, Innala and Viau, 2014), however there are known sex differences in learning and memory after a stressful experience, with males showing enhanced memory following exposure to an acute stressor (Shors, 2016) and females showing enhanced memory following chronic stress exposure (Luine, 2002). Further, CORT is known to promote memory consolidation via GR-mediated responses (Oitzl et al., 2010), and analysis of MR and GR translocation showed no difference in MR translocation in response to acute and repeat restraint, while GR translocation increased after exposure to acute but not repeat restraint (Innala and Viau, 2014). Therefore, even in the absence of a sex difference in the degree of HPA  5 axis habituation, this does not discount the potential for underlying differences in the regulation of negative feedback of the HPA axis. 1.1.2 Corticosteroid-binding globulin  In addition to sex differences in CORT output, there are also sex differences in the same direction in corticosteroid biding globulin (CBG), which is the carrier protein for CORT, with females having roughly double the amount of CBG compared to males (Gala and Westphal, 1965). Since up to 95% of CORT in circulation is bound to CBG, variations in CBG levels can dramatically alter the amount of free CORT available to bind to its receptors (Perogamvros et al., 2012, Delehanty et al., 2015). Previous work in the lab and others have also demonstrated that although females have higher basal and stress levels of CORT, once CBG is accounted for there is no sex difference in the amount of free CORT (Tinnikov, 1999, Innala and Viau, 2014). Further CBG levels can also be altered by the hormone milieu in adult male rats; CBG levels can be negatively regulated as a function of circulating testosterone levels (Viau and Meaney, 2004) and estradiol replacement in gonadectomized male rats has either no effect or increases CBG levels (Kurabekova et al., 1988, McCormick et al., 2002). In humans, there is a lack of sex differences in CBG and rarely one in cortisol output, however sex differences in stress induced mood disorders and negative feedback of the HPA functioning have been reported (as reviewed in Goel et al., 2014). Therefore, even in the absence of a sex differences in free glucocorticoid output, this does not discount the possibility that there is differential regulation occurring between the sexes that is leading to this convergent response in free CORT.  1.2 The role of the Glucocorticoid receptor to modulate HPA output There are two corticosteroid receptors found in the brain that are involved in negative feedback of the axis, GR and MR. Although CORT is the major ligand for both receptors in the brain, MR  6 has a 10-fold higher affinity for CORT compared to GR, which leads to only 10% of GRs in the nucleus under basal CORT levels, while MR occupancy in the nucleus is maintained at >90% under both basal and stress CORT levels (Reul and Kloet, 1985). While GR and MR are both known to regulate the HPA axis, GR is the main receptor responsible for attenuating elevated glucocorticoid levels, and MR has primarily been implicated in regulating basal CORT levels and involved in activating the HPA axis (Joels et al., 2008).  There are two main mechanisms that GR can act to regulate the HPA axis 1. fast acting that occurs on a time line of seconds to minutes and 2. the classic genomic effects that takes 15 minutes to hours. The fast acting mechanisms are through membrane bound GR and cytoplasmic GR, which once bound to ligand can activate second messenger pathways (Tasker and Herman, 2011). The fast acting mechanisms are primarily inhibitory influences via GABAergic and endocannabinoid transmission (Tasker et al., 2006). While genomic actions of GR are slower to modulate the HPA axis since they involve CORT-activated GR to translocate from the cytoplasm to the nucleus, then bind to glucocorticoid receptor elements (GREs) to activate or repress transcription. GRs ability to act as a transcription factor provides the HPA axis the ability to facilitate appropriate long-term responses to stimuli (Kumar and Thompson, 2005, Oakley and Cidlowski, 2013). It has also been demonstrated in the hippocampus there are two populations of GR-DNA binding sites, one which GR binds to at all CORT concentration, while GR binds the other DNA sites only in the presence of high CORT (Polman et al., 2013). Sex differences have also been seen in the pattern of GR-DNA binding sites, in rodents whose CORT output is sexually dimorphic (Duma et al., 2010). This allows the HPA axis the ability to regulate its output to ensure appropriate responses and termination of the CORT output.  7 1.3 Regulation of corticosterone-activation and translocation of the glucocorticoid receptor In addition to the ability for varying concentrations of circulating glucocorticoids to regulate the genomic actions of GR, there are also co-factors and chaperone proteins that are involved in the translocation of GR to the nucleus. In general, under basal conditions GRs are predominantly found in the cytoplasm bound to Heat shock protein 90 (Hsp90) and FK506 binding protein 5 (Fkbp51), then when CORT binds to GR and FKBP51 is exchanged for FK506 binding protein 4 (Fkbp52) to allow translocation to the nucleus (Grad and Picard, 2007). Additionally, since GR belongs to the same steroid receptor superfamily, it shares many of the same co-factors and chaperone proteins as the progesterone receptor, androgen receptor, and estrogen receptors (Tranguch et al., 2005, Jaaskelainen et al., 2011), which is likely why gonadal hormones have the ability to regulate the expression of GR’s co-factors and chaperone proteins. While the majority of the work showing gonadal hormone regulation has been done to date in vitro, it has demonstrated that co-chaperone peptidyl-prolyl isomerase D (Ppid) and FKBP52, which facilitate translocation, are upregulated by estradiol and highly expressed in breast cancer cells, respectively (as reviewed in Bourke et al., 2012). FKBP51 and co-chaperone BCL-2 associated athanogene (Bag1) are involved in preventing translocation in GR, with FKBP51 being upregulated by estradiol (as reviewed in Bourke et al., 2012). Bag1 and Ppid expression has also been shown to increase in adult female, with a positive correlation with estradiol levels after chronic adolescence stress (Bourke et al., 2013). Although limited work has been done exploring the sex differences in the expression of these co-factors after HPA activation, the ability for them to be regulated by gonadal hormones suggest they may contribute to the sex differences observed in negative feedback.     8  1.3.1 The hippocampus as a major site of GR-mediated negative feedback Although GR is found throughout the brain the major sites of GR mediated negative feedback occurs in a direct fashion through the anterior pituitary and hypothalamus, while indirectly through the hippocampus (Reul and Kloet, 1985). The hippocampus is often regarded as the major site of GR-mediated negative feedback of the HPA axis (Jacobson and Sapolsky, 1991). Many studies have also shown conditions that are associated with reduced GR levels in the hippocampus, like chronic stress exposure, are accompanied with a phenotype of impaired negative feedback (Laryea et al., 2015). Further, site-specific reductions in cytoplasmic GR levels have also been implicated in causing a delayed return to basal CORT levels following stress induced HPA activation (Sapolsky et al., 1984). Lastly, even though GR is found through HPA regulating regions of the limbic system, studies looking at GR co-localization in the nucleus, which represents CORT activated GR, found nuclear GR was higher in the hippocampus, when compare to the pre-frontal cortex even though microdialysis studies demonstrated equivalent levels of glucocorticoids in both regions after stress exposure (Kitchener et al., 2004).   In addition to glucocorticoid regulation of GR levels in the hippocampus, there are also sex dependent changes in GR expression that are regulated by sex steroid actions directly in the hippocampus. Overall, studies have shown estradiol and androgens suppress GR expression, however only in animals that were adrenalectomized (ADX) and/or gonadectomized (GDX) with intact animals showing no sex difference in GR mRNA (Turner, 1997). A previous study from the lab has also found similar results when GR protein levels were analyzed by western blot (Innala and Viau, 2014). Lastly, studies looking at GR binding have not observed any sex  9 differences in the binding capacity or affinity for CORT in intact rats (Turner, 1997). These studies suggest that there are effects of sex steroids to regulate GR expression, however since no sex difference is observed in intact animals this may be due to differential organization leading to a convergent response.  1.4 Hormonal origins of sex differences in the brain  It has long been known that gonadal hormone exposure during development has long lasting organizational effects on the adult phenotype of mammals. The original work showing the role of gonadal hormones on brain development occurred in the late 1950’s when Phoenix et al. (1959) showed that androgens during the neonatal period were capable of irreversibly altering the phenotype of female rats as adults to show male sexual behaviour. This founding study has led to the organizational hypothesis that defines organizational effects as those that are irreversible after a critical window is closed, while activational effects can be reversed at any time (Wallen, 2009). Testosterone has both organizational and activational effects on the brain. During in utero and neonatal development there are two well characterized testosterone surges in male rats occurring at embryonic day 18-19, and within the first few hours after birth which is when testosterone has organizational effects on the brain, while as adults, testosterone has reversible effects on the brain (Rhoda et al., 1983, Handa et al., 1994, Viau and Meaney, 1996).  Since the original work showing that neonatal testosterone administration can organize the female brain to have the potential to show male sexual behaviour, further work has demonstrated that estradiol which is a metabolite of testosterone is sufficient to organize the female brain to have the potential to display male sexual behaviour (McCarthy, 2008). Further, the neonatal surge in circulating testosterone in the male rat also corresponds with an increase in hypothalamic estradiol (Rhoda et al., 1984). The corresponding increase in hypothalamic  10 estradiol is due to the local metabolism of testosterone by aromatase to estradiol, which in turn is responsible for organizing the male brain (McEwen et al., 1977, Whalen and Olsen, 1981). Although females also have circulating testosterone during the perinatal period, circulating testosterone levels and brain aromatase activity are significantly lower in females compared to males (Lieberburg et al., 1979, Maclusky et al., 1985). Females are also protected from their own endogenous estradiol during this period by fetoneonatal estrogen binding protein (fEBP), which is also known as alpha fetoprotein and is found in the neonatal brain and blood (McEwen et al., 1976). More recent work has built upon this framework to further elicit the mechanisms by which estradiol acts to organize the brain during the neonatal period, which has now been demonstrated to occur in some brain regions through unleashing the epigenetic suppression seen in females (Nugent et al., 2015).    1.4.1 Hormonal organization of the HPA axis output The organizational effects of gonadal hormones during the neonatal period have also been shown to alter the HPA axis responses during adulthood. Overall, studies have shown that females given neonatal estradiol or testosterone have lower CORT compared to control females and the removal of estradiol or testosterone during the neonatal period in males results in higher CORT during adulthood (Patchev et al., 1995, McCormick et al., 1998, Seale et al., 2005a, Seale et al., 2005b, Bingham and Viau, 2008). Although these studies demonstrate similar overall findings, there are inconsistencies in the results, such as whether neonatal gonadectomy (neo-GDX) results in higher basal CORT in male rats, or if testosterone administration during the neonatal period can completely reverse the effects of neo-GDX (McCormick et al., 1998, Bingham and Viau, 2008). There are also discrepancies in the extent to which testosterone has an organizational or activational role in lowering CORT levels in females since Goel and Bale  11 (2008) found in mice that neonatal testosterone was insufficient to lower stress induced peak CORT as both neonatal testosterone and oil treated mice ovariectomized (OVX) and given testosterone replacement at the beginning of puberty through adulthood when testing occurred had similar effects on adult CORT. Since the testosterone was given prior to puberty, this does not discount an organizational role for testosterone, as puberty is another critical period for sex steroids to organize the brain (Sisk and Zehr, 2005), therefore the exposure of testosterone during puberty may have had a greater organizational effect on adult CORT levels than the neonatal testosterone treatment. There is also the possibility that the discrepancy in the results may be due to species differences and/or the timing and type of gonadal hormone replacement, since OVX female rats replaced with oestradiol as adults, show an organizational role for neonatal testosterone to modulate the adult CORT response (Seale et al., 2005a).  The above studies addressing the effects of neonatal gonadal hormones to modulate CORT output analyzed total CORT output, however CBG levels are also organized by gonadal hormones during development. This was first discovered in the 1960’s by (Gala and Westphal) where they demonstrated that neo-GDX in males resulted in adult CBG levels equivalent to female levels, with neonatal testosterone injection reducing adult levels in both males and females. Further studies looking at the effect of a single injection of neonatal testosterone has failed to reverse the effect of neo-GDX in males (McCormick et al., 1998). In addition, blocking androgens and estrogens actions during both the perinatal and neonatal period has also failed to alter CBG levels in males (Seale et al., 2005b). Although these studies appear to contradict the role of androgens during the neonatal period to organize CBG levels, Jansson et al. (1989) showed that neo-GDX (postnatal day (PND) 1-2) had a greater effect on reducing CBG levels compared to males GDX at PND 25. Another study looked at the effect of GDX with and  12 without testosterone treatment from PND 1-33 and found that only testosterone treatment between PND 29-33 was able to irreversibly masculinize CBG levels (Kurabekova et al., 1991). Therefore, the impact of gonadal hormones on CBG levels in rodents can have substantial influence on the extent of neonatal gonadal manipulations to alter CORT levels.  1.4.2 Neonatal organization of GR expression in the hippocampus Sex differences in GR protein levels in the hippocampus are apparent during the neonatal period in rats and disappear by the third week of life. These sex differences are observed in the protein levels of the major isoform of GR during the first two weeks of life, with females having less GR protein at PND 5 and 15 (Ordyan et al., 2008). This timeline fits with a postnatal increase in estrogen receptor binding in the hippocampus from PND 1-10 in both sexes (Okeefe and Handa, 1990). This leaves open a potential critical window when estrogenic mechanisms from the neonatal testosterone surge in males could organize GR expression and activity.  1.5 Summary and specific hypotheses Neonatal gonadal hormones are known to irreversibly alter adult HPA axis output in both male and female rats (Patchev et al., 1995, McCormick et al., 1998, Seale et al., 2005a, Seale et al., 2005b, Bingham and Viau, 2008) Although, it is unknown if these changes are due to direct organizational effects on the HPA axis or due to effects on the CBG leading to compensatory changes in HPA output. Further, the mechanism by which changes in HPA output are still largely unknown, as is the role of neonatal sex steroids to organize GR responses under acute and repeat stress conditions. The aim of this thesis was to address these question, and based on presented literature we had two main hypotheses:  13 1. Neonatal sex steroid manipulations in male and female Long Evan rats will alter CORT output, with the blockade of estrogenic actions in males resulting in higher CORT output and the administration of TP resulting in lower CORT outputs.    2. Neonatal sex steroids will organize GR responses in response to acute and repeat restraint stress, with the blockade of estrogenic actions in males resulting in higher levels GR responses and the administration of TP resulting in lower GR responses.     To address these hypotheses, the neonatal hormone milieu in male and female Long Evan rats were manipulated by giving males an aromatase blocker to block the ability for testosterone to be converted to estradiol and females were given testosterone. Then as adults we assessed if altering the neonatal hormone milieu would alter CORT output and GR responses under restraint naïve conditions and after exposure to acute and repeat restraint stress. Since previous work in the lab addressing 1. the influence of neonatal gonadal hormones on adult HPA axis function (Bingham and Viau, 2008, Bingham et al., 2011, Bingham et al., 2012) and 2. CBG responses to repeated restraint exposure (Innala and Viau, 2014) was done in Sprague Dawley rats, we also assessed if there were any strain differences in CBG binding properties which have the ability to alter CORT and CBG binding levels.  GR responses were assessed using western blots to analyze GR translocation and phosphorylated GR. For stress time points, brains were collected at 30 min from onset of restraint exposure, since plasma CORT levels and GR translocation in the hippocampus have previously been demonstrated to peak at this time point (Kitchener et al., 2004).      14 Chapter 2: Methods 2.1 Animals 221 Long Evan rats (Charles River) that were bred from 22 multiparous female rats (2 previous litters) and 10 virgin adult males were used in this thesis. Rats were housed under controlled temperature (23 ± 2 C) and lighting conditions (12:12-hour light:dark cycle, lights on at 0700 hours) with food and water available ad libitum.  All protocols were approved by the University of British Columbia Animal Care Committee. 2.2 Surgeries  During pregnancy, timed-pregnant dams were handled daily and pair housed during gestation until 4 days prior to due date, then single housed with nesting material. Pregnant dams were checked through the day for pups and surgeries were performed once all pups in the litter had a milk band present and within 12 hours of birth. All litters used had at least 5 male pups and 5 female pups, and if needed, pups were cross-fostered to normalize litter composition between dams, with all litters been culled to a maximum of 12 pups.  Gonadal manipulations were performed pharmacologically by surgically implanting a silastic capsule (7 mm length; 1.96 mm outer diameter) filled with 5 mg of crystalline drug (1, 4, 6-androstatriene-3, 17-dione; ATD in males or Testosterone propionate; TP in females) under isoflurane gas anesthetic. To control for the effect of the neonatal surgery, a subset of males and females were subject to a sham surgery. Each litter contained up to 2 drug, 2 sham, and 2 untouched male and female rats, thus to identify manipulated pups, the drug, sham, and cross-fostered pups were marked with a tattoo on the paw and ear notched at the time of surgery. To remove surgical pups, the dam was removed from the cage, then pups were counted and sexed, the dam was then returned to the non-surgical pups while the manipulations were  15 occurring.  After the surgeries were complete and the pups were ready to be re-introduced to the dam, the dam was then removed from the non-surgical pups, the surgical pups were mixed with the non-surgical pups by gently rolling them together with the dam’s urine/ and or soiled bedding, the dam was then placed back with the entire litter.    On PND 21, the drug treated pups had their silastic implant surgically removed by a single incision under isoflurane gas anesthetic. Once recovered from the surgery, the manipulated and un-manipulated animals were either returned to their dam and weaned at 28 days or re-grouped with littermates and weaned (PND 21 wean). Animals were then group housed, then prior to testing they were pair housed into treatment groups. See Figure 2 for full timeline of experimental design.    Figure 2: Timeline of experimental design 2.3 Maternal care observations Variations in maternal care are known to alter neuroendocrine responses in rats, with significant effects on CORT and GR levels in the hippocampus (Liu et al., 1997). Although previous work demonstrates that silastic capsules do not alter maternal care (Bingham et al., 2011), that study SC implants:Males - ATDFemales - TESTImplants removedBasal-NaïveAcuteBasal-RepeatAcute-RepeatPND 0* PND 21** PND 70-80*Within 12 hrs of birth**Weaned PND 21-28Age (PND) 16 involved the manipulation of only 3 pups per litter, and this study entailed 8 manipulated pups per litter. Furthermore, in an effort to minimize the amount of litters needed, we were unable to fully empirically control for litter effects (Abbey and Howard, 1973) since not all adult groups were represented in each litter. Therefore, to validate that our results obtained were due to our neonatal manipulation and not a result of variations in maternal care, maternal care observations were made from PND 1 – 8. During this time, each dam was observed three times during the light phase at 9:00, 13:00, and 17:00 for 1 hour with observation made every 3 minutes, leading to 60 observations per day. During these observations dams were scored on maternal behaviours as described previously by Champagne et al. (2003) and the maternal interactions with pups, maternal self-care, and pup vocalization were recorded. A variable for licking and grooming and arched-back nursing was derived by summing all the observation of licking and grooming and arched-back nursing. 2.4 Body weight measurements Rats were weighed weekly through development, starting from surgery (PND21) and/ or weaning (PND21 or 28). Prior to restraint exposure, or termination (naïve groups) rats were weighed for 4 consecutive days. Rats in the repeat restraint groups were weighed at the end of each restraint exposure prior to being returned to their home cage.  2.5 Restraint paradigm The experimental design is outlined in Figure 3, with all procedures occurring between 0900-1200 (0700 lights on). Prior to restraint exposure, or termination in Naïve groups, all rats were handled and weighed for 4 consecutive days, with restraint exposure and termination occurring no earlier than PND66 and 70, respectively. Restraint exposure involved placing the rats into Plexiglas restrainers for either a 2 hours or 30 min exposure. Restrainer size was based on the  17 size of the individual rats to unsure they could move their head and limbs, while preventing them from turning around in the restrainer. There were four adult treatment groups (n’s=7-10 per neonatal treatment group):  1. Basal-Naïve: rats were sacrificed under restraint-naïve conditions. 2. Acute: rats were sacrificed at the termination of a single 30 min restraint exposure. 3. Basal-Repeat: Rats were restrained for 2 hours per day for 4 consecutive days. Each day following restraint exposure, rats were weighed and returned to their home cages, then left undisturbed until the next day. On day 5, restraint exposure rats were sacrificed under basal conditions. 4. Acute-Repeat: Rats were restrained for 2 hours per day for 4 consecutive days. Each day following restraint exposure, rats were weighed and returned to their home cages, then left undisturbed until the next day. On day 5, restraint exposure rats were sacrificed at the termination of a 30 min restraint exposure. Tail blood samples were collected on day 1 and day 5 from a razor nick over the lateral tail vein 30 min from restraint onset. Blood samples of 300 ul were collected into ice-chilled, EDTA-treated tubes, centrifuged at 4 C, then plasma was collected and stored at -20C.  18  Figure 3: Adult treatment groups 2.6 Tissue collection Rats were sacrificed by decapitation and trunk blood was collected into EDTA-treated tubes, centrifuged at 4 C, then plasma was collected and stored at -20C. The liver, kidneys, adrenal glands, pituitary gland, and brains were dissected, and immediately frozen on dry ice and stored at -80C until protein or mRNA extractions. Prior to freezing the fresh brains, the hippocampus was dissected, and individual hippocampi were immediately frozen on dry ice and stored at -80C until protein extractions. 2.7 Gonadal hormone assays Plasma levels of testosterone1, and estradiol2 were measured in duplicate using commercial Radioimmunoassay (RIA) kits from 1MP Biomedicals, Inc. (Solon, OH, USA) and 2Inter Medico (Markham, ON, Canada), with [I125] as tracer, as per manufacturer’s instructions. The intra- and interassay coefficients of variation for all assays typically ranged from 1-7 and 2-13%, respectively. The standard curve ED50 for testosterone was 0.698 ng/ml, and the detection limit 0.1 ng/ml. The standard curve ED50 for estradiol was 122 ng/ml, and the detection limit 1.4 pg/ml.  30 min restrainDecap@ t=30min1 2 3 430 min restrainDecap@ t=30min1 2 3 4Basal-NaïveAcuteDaily weights (4)Decap @ t=01Daily weights (4) 1  Daily weights (4)Restraint 2 hrs x 4 days Decap @ t=05Basal-RepeatAcute-Repeat5Restraint 2 hrs x 4 daysDaily weights (4) 19 2.8 Corticosterone Plasma levels of CORT were measured in duplicate by RIA, with [3H]-corticosterone as the radiolabeled tracer. First, plasma samples were diluted with 0.1 M Citric acid buffer (pH=3.0) and heated to 60°C for 30 min to inactivate CBG. Samples were then neutralized with 0.1M Tris buffer (pH=9.0), leading to a final sample dilution of 1:100 for stress samples and 1:50 for basal samples. The standard curve was diluted in a standard buffer made from a pool of plasma from unstressed male rats that had CBG inactivated and stripped of endogenous CORT. CBG was inactivated as described above, and standard buffer was diluted to a final dilution of 1:100. Endogenous CORT was then removed by incubating diluted plasma in dextran-coated charcoal (DCC) for 30 min at room temperature, followed by centrifugation. The supernatant was then collected and used as the diluent for the standard curve. Known standards were then made by serial dilution of a stock solution of unlabeled CORT (5mg/ml in PBS) in standard buffer to final concentrations of 25ng/ml to 0.195ng/ml. In addition, background radiation was measured using a sample containing excess unlabeled CORT (100ng/ml in standard buffer) and standard buffer alone was used as the no hormone control (Bo). Samples and known standards were then incubated overnight at 4°C with [3H]-corticosterone and EMD Millipore anti-corticosterone (1:2500).  Samples were then incubated with DCC for 10 min at 4°C, followed by centrifugation. The supernatant was then collected and the amount of antibody bound [3H]-corticosterone in the supernatant was determined in a scintillation spectrophotometer. Counts per minute (cpm) for known and unknown samples were then used to calculate the amount of bound antibody using the following equation [(sample-background)/non-specific binding] x 100. The standard curve was then generated by plotting the amount of labelled antibody against the log[known concentration of CORT in pg/ml]. A sigmoid curve was  20 then fitted using Graphpad to interpolate unknown values. The corticosterone antibody cross-reacts slightly with 11-dehydrocorticosterone (0.67%), Deoxycorticosterone (1.5%), 18-OH-DOC (<0.01%), Cortisone (<0.01%), Cortisol (<0.01%) and Aldosterone (0.2%). The minimum detectable concentration for this assay was 0.195ng/ml, with intra- and inter- assay coefficients of variation of less than 7.5%. 2.9 Corticosteroid binding globulin Binding properties of corticosteroid binding globulin were analyzed in order to estimate the biologically active corticosteroid in circulation (free CORT). This was done by analyzing the CBG-corticosterone binding capacity using an established ligand-saturation assay (Hammond and Lahteenmaki, 1983). Briefly, plasma samples were diluted with phosphate buffered saline (PBS) and incubated in dextran-coated charcoal (DCC) for 30 min to remove endogenous CORT. Labeled ligand, [3H]-corticosterone, was then added to samples in the absence or presence of unlabeled corticosteroid in order to monitor for non-specific binding. DCC was then added for 10 min to separate CBG from its ligand, followed by centrifugation at 0°C. The amount of CBG-bound [3H]-corticosterone in the supernatant was determined in a scintillation spectrophotometer.  Since CBG-corticosteroid binding is time dependent, an assay to determine the apparent dissociation rate (off-rate) was performed to determine the percentage of [3H]-corticosterone bound at 10 min. This was performed as described above for the ligand-saturation assay, except the apparent dissociation rate of CBG-bound corticosteroid was assessed after incubating with DCC for different durations of time (0-18 min) at 0°C (Hammond and Lahteenmaki, 1983). The dissociation-rate constant (Kd), was then determined by Scatchard analysis at 4°C. This was performed as previously described (Smith and Hammond, 1991). Briefly this involved  21 incubating plasma samples with increasing amounts of [3H]-corticosterone. The apparent dissociation rate and the dissociation-rate constant were measured in both Sprague Dawley (SD) and Long Evan (LE) rats since previous studies from the lab addressing 1. the influence of neonatal gonadal hormones on adult HPA axis function (Bingham and Viau, 2008, Bingham et al., 2011, Bingham et al., 2012) and 2. CBG responses to repeated restraint exposure (Innala and Viau, 2014) was done in SD rats. The effect of testosterone on CBG-CORT binding was also assessed in male SD and LE rats, using a competition assay where plasma samples were incubated with increasing concentrations of testosterone (20M – 0.3M) in the presence of [3H]-corticosterone. 2.10 Free corticosterone  Total CORT and CBG values were then used to estimate free CORT using a variation of the mass action equation, as previously outlined by Delehanty et al. (2015), with a Kd of 13 nM, as previously reported in LE rats (Viau and Meaney, 2004).  2.11 Adrenal weight measurement Individual frozen adrenals were thawed on ice, and fat peeled off to isolate the adrenal gland. They were then weighed on an analytical balance (Mettler Toledo, Mississauga, ON, Canada). The weight of the two adrenals in milligrams was then averaged. Since the size of the adrenal gland is proportional to body weight (Bailey et al., 2004), the weight of the rat at termination in grams was then divided by the average weight of the adrenals in milligrams to analyze treatment effects on the adrenal gland.  2.12 Protein extraction  Individual hippocampi from each rat were used to extract whole-cell protein or nuclear and cytoplasmic fractions. Whole-cell protein homogenate was prepared by homogenizing individual  22 hippocampi with RIPA buffer (50 mm Tris, 150 mm NaCl, 10% sodium dodecyl sulphate, 1% IGEPAL, 0.5% Sarkosyl, pH 8.0) containing protease and phosphatase inhibitors (complete protease inhibitor cocktail and phosphatase inhibitor cocktail tablets; Roche Diagnostics, Laval, Quebec, Canada) using a dounce all-glass tissue grinder (Kimble Chase, Rockwood, TN, USA), then centrifuged at 16,000 x g for 30 minutes at 4 °C and supernatant collected. Cytoplasmic and nuclear fractions were separated by centrifugation using an established protocol (Bourke et al., 2013). Briefly, individual hippocampi were homogenized using a dounce all-glass tissue grinder (Kimble Chase) in cytoplasmic extract buffer (50 mM Tris, 1 mM EDTA, 6 mM MgCl2, 10% sucrose, pH 7.2) on ice, followed by centrifugation at 2,000 x g for 5 minutes at 4 °C, the supernatant was then collected for cytoplasmic extraction and pellet was kept for nuclear extraction. Cytoplasmic supernatant was then further centrifuged at 30,000 x g for 60 minutes at 4 °C, with the sequential supernatant collected as the cytoplasmic extract. The nuclear pellet was then washed two times by re-suspending in cytoplasmic extraction buffer, followed by centrifugation at 2,000 x g for 5 minutes at 4 °C. The washed nuclear pellet was then re-suspended in a nuclear extract buffer (cytoplasmic extraction buffer with 0.5M NaCl), and incubated at 0 °C for 1 hour, followed by centrifugation at 8,000 x g and the supernatant collected as the nuclear extract. Protein concentrations were determined using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) and adjusted to ~1-2 ug/ul. Samples were denatured with Laemmli sample buffer (Bio-Rad Laboratories, Mississauga, ON, Canada) with β-mercaptoethanol and frozen at -20C. 2.13 Western blots  Protein extracts (20ug) were loaded onto TGX 4-15% gradient gels (Bio-Rad Laboratories), and separated by gel electrophoresis. The separated proteins were then blotted onto a 0.2 µm  23 nitrocellulose membrane (Bio-Rad Laboratories) at using Trans-Blot Turbo rapid western blotting transfer system (Bio-Rad Laboratories), then washed briefly in phosphate-buffered saline (PBS) and blocked for 1 hour at room temperature in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA). Membranes were probed for with the following primary antibodies in Odyssey blocking buffer with 0.1% Tween 20 (Sigma-Aldrich) overnight at 4°C: anti-GR M20, 1:250 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phosphorylated GR (Ser211), 1:2,000 (Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH, 1:10,000 (Abcam Inc, Toronto, ON, Canada) anti-Histone H3, 1:5,000 (Abcam Inc), anti-β-actin, 1:50,000 (Sigma Aldrich, Oakville, ON, Canada). Membranes were then rinsed 3 times for 5 min in PBST, then incubated with the appropriate secondary antibody (LI-COR anti-mouse and anti-rabbit IRDye secondary, 1:10,000) in Odyssey blocking buffer with 0.1% Tween 20 for 1 hour at room temperature. Membranes were then rinsed 3 times for 10 minutes in PBST, followed by 3 rinses for 10 minutes in PBS. The Odyssey Imaging System (LI-COR Biosciences) was then used to visualize and quantify protein levels.  2.14 Western blots analysis  Translocation of the GR to the nuclease is reported as a within gel calculation of the (nuclear signal)/ (nuclear signal + cytoplasmic signal) using the 95kDa band. Cross-contamination was also reported as a within gel calculation of the (nuclear GAPDH signal)/ (cytoplasmic GAPDH signal) for the nuclear fraction and the (cytoplasmic H3 signal)/ (nuclear H3 signal) for the cytoplasmic fraction. For whole cell Ser211 protein levels since all treatment groups (neonatal and adult) where not represented within each gel, the signal of Ser211 using the 95kDa band and β-actin for each sample of interest was normalized to a control sample (sample of interest/ control sample) to reduce the effect of between blot variation in signal intensity and to allow for  24 comparison between all treatment groups. To control for loading errors, the final relative value for Ser211 protein levels was expressed as a ratio (normalized Ser211/ normalized β-actin).   2.15 Liver gene expression A small portion of the frozen livers were homogenized in 1ml of TRIzol (Invitrogen, Carlsbad, CA, USA), then RNA was isolated using RNeasy Mini kit (Qiagen, Hilden, Germany) as per manufacturer’s instructions. RNA quality and quantity was then assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Total RNA was then reversed transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA). as per manufacturer’s instructions. The expression of Cyp2c11, Cyp2c12, and Hnf-6 gene transcripts were assessed using quantitative real-time PCR was carried out using the 7900HT Fast Real-Time PCR System (Applied Biosystems). Quantification was done using 20ng of total cDNA using the standard curve method, and expression of all genes of interests were normalized to Gapdh. The following primer/ probe sets were used to detect: Cyp2c11(IDT Assay-Rn.PT.58.14115485), Probe: -5’-/56FAM/TCTGAGGTA/ZEN/TGGACTCCTGCTGCT/3IABk FQ/3’, Primer 1: 5’-ACGTTCTATCTCTTCCTGGACT-3’, Primer2: 5’-GGTGGCTACTGTAA CTGACAT-3’; Cyp2c12 (IDT Assay -Rn.PT.58.44751238), Probe: -5’-/56-FAM/CTTCCTCCT/ ZEN/GGCCCTACTCCTCT/3IABkFQ/3’, Primer 1: 5’-AATTGGATATTGATTGACGGAT GTC-3’, Primer 2: 5’-GTCCTTTCTGCTTCTCCTGTATC-3’; Hnf-6 (IDT Assay -Rn.PT.58.14190623), Probe: 5’-/56-FAM/TCTGTCCTT/ZEN/CCCGTGTTCTTGCTC/3IAB kFQ/3’, Primer 1: 5’-GCATGTAGAGTCCGACGTTG-3’, Primer 2: 5’-AGTTCCAGCGCATG TCG-3’; Gapdh (IDT Assay - Rn.PT.58.35727291), Probe: 5’ -/56-FAM/CACACCGAC/ZEN /CTTCACCATCTTGTCT/3IABkFQ/3’, Primer 1: 5’ – GTAACCAGGCGTCCGATAC-3’, Primer 2: 5’-TCTCTGCTCCTCCCTGTTC-3’.  25 2.16 Statistical analysis All analyses were performed using IBM SPSS Statistics for Mac, version 24 (Armonk, NY: IBM Corp). Unless otherwise stated all variables were analyzed using a two-way analysis of variance (ANOVA) for males and females, with neonatal manipulation (untouched, sham, and ATD or TP) and adult restraint state (basal-naïve, acute, basal-repeat, and acute-repeat) as between subject variables. The effect of neonatal manipulation on restraint stress induced CORT (total and free) and CBG in males and females were analyzed using mixed model ANOVAs, with neonatal treatment (untouched, sham, and ATD or TP) as the between-subject variable and restraint day (1 and 5) as the within-subject variable. Pearson’s product-moment correlations were performed to examine the relationship between CORT (total and free) and markers of GR activation (percentage of GR in the nucleus and phosphorylated at the serine 211 site) in the hippocampus. To examine the effect of neonatal treatment and repeat restraint exposure on adrenal measures in males and females, a two-way ANOVAs were conducted with neonatal manipulation (untouched, sham, and ATD or TP) and adult previous restraint state (acute and repeat) as between subject variables. The acute group included both the basal-naïve and acute restraint exposed rats and the repeat group included both the basal-repeat and acute-repeat rats. To examine the effect of neonatal treatment on body weight, an analysis of covariance (ANCOVA) was used in males and females, with age (PND) as the covariate, and neonatal manipulation (untouched, sham, and ATD or TP) as the between-subject variable. Post hoc test were performed using the Bonferroni procedure.  There were two situations where animals were excluded from the analysis. 1) In analysis of basal CORT, when an animal’s CORT levels were 2 standard deviations away from the group mean. This was done since non-specific activation of the HPA axis did occur in some rats  26 euthanized under non-restraint (basal) condition. This resulted in 3 male and 4 female rats being excluded leading to group sizes of 8-10 rats per neonatal treatment in each basal condition. 2) In the analysis of GR translocation, any animals whose nuclear or cytoplasmic fraction had cross-contamination higher than the average or incorrect loading as determined by nuclear and cytoplasmic loading controls were excluded from the analysis. This resulted in 18 male and 8 female rats being excluded leading to group sizes of 7-10 for male neonatal untouched, 4-9 for male neonatal sham, 7-10 for male neonatal ATD treated, 7-10 for female untouched, 8-10 for female neonatal sham, and 6-9 for female neonatal TP treated in each adult condition.   27 Chapter 3: Results 3.1 Maternal care Variations in maternal care, including licking and grooming behaviour of the dam towards the pups are known to organize HPA output and GR levels in the hippocampus (Liu et al., 1997), therefore prior to analyzing HPA output and GR responses a one-way analysis was performed in males and females. This revealed there was no significant difference in the licking and grooming and arched-back nursing behaviours of the dams between the neonatal treatment and the adult condition for males (F2,99=0.367, p=0.694 and F3,98=1.063, p=0.369, respectively) and females (F2,99=0.710, p=0.494 and F3,99=0.729, p=0.537, respectively). Therefore, all remaining analyses did not include maternal care as a variable.  3.2 The effect of neonatal gonadal manipulations on basal CORT levels In males, figure 4 shows basal levels of total CORT levels (a), estimated free CORT levels (c), and CBG binding levels used to calculate estimated free CORT (e). A two-way analysis demonstrated an interaction between neonatal manipulation and previous restraint exposure in total CORT (F2,49=5.177, p=0.009) and estimated free CORT (F2,49=4.775, p=0.013). There was a main effect of neonatal manipulations to alter basal total CORT levels (F2,49=5.517, p=0.007) and a trend to alter estimated free CORT levels (F2,49=2.910, p=0.064). There was no main effect of previous restraint exposure in both total CORT (F1,49=0.006, p=0.937) and estimated free CORT (F1,49=1.662, p=0.203). Post hoc analysis revealed that males treated with ATD had significantly higher total CORT (p’s=0.003) and no significant difference in estimated free CORT (p’s>0.121) under basal-naïve conditions compared to both male control groups. There was no significant difference in both total and estimated free CORT between male control groups (p’s=1.000) under basal-naïve conditions.  After repeated restraint exposure, there was a  28 significantly higher estimated free CORT (p=0.012) and trend in the same direction for total CORT (p=0.052) in the neonatal sham males compared to the neonatal untouched males. There was no significant difference in total (p’s>0.682) and estimated free CORT (p’s>0.116) between neonatal control (untouched and sham) and ATD treated males after repeated restraint exposure. Within neonatal treatment groups, repeated restraint exposure significantly increased total (p=0.023) and estimated free (p=0.003) CORT in the neonatal sham males, and in neonatal ATD treated males there was a significantly decreased total CORT (p=0.033), with no effect on estimated free CORT (p=0.287). There was no effect of repeated restraint exposure on both total and estimated free CORT (p’s=0.929) in the neonatal untouched males. A two-way analysis of CBG binding revealed no effect of neonatal manipulations (F2,51=1.880, p=0.163), and a main effect of previous restraint exposure (F1,51=34.712, p<0.0001) with a trend towards an interaction between neonatal manipulations and previous restraint exposure (F2,51=3.006, p=0.058). Post hoc analysis revealed a significant decrease in CBG binding after previous restraint exposure in neonatal sham males (p<0.0001), and neonatal ATD treated males (p=0.002), with a trend towards a decrease in neonatal untouched males (p=0.082).  In females, figure 4 shows basal levels of total CORT levels (b), estimated free CORT levels (d), and CBG binding levels used to calculate estimated free CORT (f). A two-way analysis, demonstrated a main effect of previous restraint exposure to alter basal total (F1,46=6.516, p=0.014) and estimated free CORT (F1,45=7.164, p=0.010) levels however in both total and estimated free CORT there was no significant effect of neonatal manipulations (F2,46=0.828, p=0.443 and F2,45=1.241, p=0.299, respectively) or interaction (F2,46=0.160, p=0.853 and F2,45=0.061, p=0.941, respectively). A two-way analysis of CBG binding revealed a significant interaction between neonatal manipulations and previous restraint exposure  29 (F2,49=3.631, p=0.034), but no main effect of neonatal manipulation (F2,49=0.268, p=0.766) or previous restraint exposure (F1,49=1.644, p=0.206). Post hoc analysis showed no significant effects, however there were trends toward higher CBG binding after repeated restraint exposure in the neonatal untouched (p=0.068) and the neonatal TP treated (p=0.098) females, and no effect in the neonatal sham females (p=0.146).   3.3 The effect of neonatal gonadal hormones on acute and repeat restraint stress induced CORT levels In males, Figure 5 shows total CORT levels (a), estimated free CORT levels (c), and CBG binding levels used to calculate estimated free CORT (e) at 30 minutes from restraint onset on day 1 and day 5 of daily repeated restraint exposure. A mixed model ANOVA revealed a main effect of repeated restraint exposure on both total CORT levels (F1,23=23.618, P<0.0001) and estimated free CORT levels (F1,23=12.799, p=0.002), with no interaction between neonatal treatment and repeated restraint exposure in both total CORT (F2,23=0.122, p=0.886) and estimated free CORT (F2,23=0.066, p=0.936). There was also no significant effect of repeated restraint exposure (F1,23=0.207, p=0.654) and no interaction with neonatal manipulations (F2,23=0.766, p=0.476) on CBG binding levels.  In females, Figure 5 shows total CORT levels (b), estimated free CORT levels (d), and CBG binding levels used to calculate estimated free CORT (f) at 30 minutes from restraint onset on day 1 and day 5 of daily repeated restraint exposure. A mixed model ANOVA revealed a main effect of repeated restraint exposure on both total CORT levels (F1,26=16.103, p<0.0001) and estimated free CORT levels (F1,26=4.237, p=0.05), with a trend towards an interaction  between neonatal treatment and repeated restraint exposure in both total CORT (F2,26=2.864,  30 Figure 4: Mean + SEM of basal levels of total (a & b) and estimated free (c & d) CORT, and CBG binding (e & f) in males and females under restraint-naïve conditions and 24 hours after repeat restraint exposure. Neonatal ATD treated males had higher basal total CORT levels under naïve conditions compared to neonatal control groups (*p’s=0.003), and neonatal sham treated males had higher estimated free CORT after repeated restraint exposure compared to neonatal untouched males (*p=0.012). Neonatal sham males had significantly higher total and estimated free CORT (#p’s<0.033 vs basal-naïve) and neonatal ATD treated males had significantly lower total CORT (#p=0.033 vs basal-naïve) after repeated restraint exposure. Females had higher total and estimated free CORT levels after repeat restraint exposure (#p’s<0.014 vs basal-naïve). Neonatal sham and ATD treated males had lower CBG binding levels after repeat restraint exposure (#p’s<0.002 vs basal-naïve). Basal-Naive Basal-Repeat0204060UntouchedShamATDTotal CORT [ng/ml]Basal-Naive Basal-Repeat050100150UntouchedShamTPTotal CORT [ng/ml]Basal-Naive Basal-Repeat0.000.501.001.502.00Free CORT [ng/ml]Basal-Naive Basal-Repeat0.000.200.400.600.801.00Free CORT [ng/ml]Basal-Naive Basal-Repeat050010001500CBG [nM]Basal-Naive Basal-Repeat0100020003000CBG [nM]Males Females*#*a) b)c) d)e) f)##### 31 p=0.075) and estimated free CORT (F2,26=3.115, p=0.061). Post hoc analysis revealed females treated as neonates with TP had significantly less total CORT, compared to both female control groups (p’s<0.035), with no significant difference between female control groups (p=1.000) on day 1. There was no significant difference between neonatal treatment groups in estimated free CORT on day 1 (p’s>0.240) and in both total and estimated free CORT on day 5 (p’s>0.722, and p’s>0.773, respectively). There was also a significant decrement in total CORT from day 1 to day 5 in female control groups (p’s<0.006) with neonatal TP treatment group showing no significant difference between day 1 and day 5 (p=0.723). Estimated free CORT showed similar findings with a significant decrement in untouched controls (p=0.015) and a trend in the sham group (p=0.073), with no difference between day 1 and day 5 observed in neonatal TP treated females (p=0.425). There was also no significant effect of repeated restraint exposure (F1,26=0.028, p=0.869), or interaction with neonatal manipulations (F2,26=0.305, p=0.740) on CBG binding levels.  3.4 The effect of neonatal manipulations and restraint stress exposure on CBG binding   Figure 6 shows CBG binding levels in response to acute and repeat restraint exposure in males (top) and females (bottom). In Males, there a trend towards an interaction between neonatal manipulation and restraint exposure (F6,96=2.166, p=0.053) and a main effect of restraint exposure (F3,96=30.892, p<0.0001), with no effect of neonatal manipulation (F2,96=0.711, p=0.494).  Post hoc analysis revealed in untouched and sham controls there was significant more CBG binding under acute restraint conditions compared to basal-naïve (p’s<0.042), with no difference in neonatal ATD treated males (p=0.941). After repeated restraint exposure, in the neonatal sham and ATD treated males, there was significant more CBG binding after restraint exposure compared to their basal counterparts (p’s<0.028), with no difference in neonatal   32  Figure 5: Mean + SEM of restraint stress induced plasma levels of total (a & b) and estimated free (c & d) CORT, and CBG binding (e & f) levels in males and females  at 30 minutes from restraint onset on day 1 and day 5 of daily repeated restraint exposure. In males, there was a significant decrease in both total and estimated free CORT levels from day 1 to day 5 (#p<0.0001 & #p=0.002, respectively). In females, neonatal TP treated rats had lower total CORT on day 1 compared to neonatal control groups (*p’s<0.035) and there was a significant decrease in total CORT levels from day 1 to day 5 in the neonatal control groups (#p’s<0.006), with only neonatal untouched controls showing a significant decrease in estimated free CORT (#p=0.015). Day 1 Day 50100200300400500UntouchedShamATD30 min Total CORT [ng/ml]Day 1 Day 502004006008001000UntouchedShamTP30 min Total CORT [ng/ml]Day 1 Day 505010015030 min Free CORT [ng/ml]Day 1 Day 502040608030 min Free CORT [ng/ml]Day 1 Day 5050010001500CBG [nM]Day 1 Day 501000200030004000CBG [nM]Males Females*a) b)c) d)e) f)#### 33 untouched males (p=1.000). Basal CBG binding levels were also significantly lower after repeat restraint exposure in the neonatal sham and ATD treated males compared to their basal-naïve counterparts (p’s<0.035), with no difference in the neonatal untouched males (p=0.792). In Females, there was no interaction between neonatal manipulation and restraint exposure (F6,98=1.406, p=0.220), and no main effect of neonatal treatment (F2,98=0.030, p=0.971) or restraint exposure (F3,98=1.961, p=0.125). 3.4.1 Strain differences in CBG binding properties Previous work in the lab has utilized Sprague Dawley rats, however the current study used Long Evans rats. Therefore, we analyzed whether there were strain differences in CBG binding properties. Figure 7 shows the apparent dissociation rate (off-rate) (a) and Scatchard plot (b) of CBG-CORT binding in adult Long Evans and Sprague Dawley rats. The percentage bound at 10 minutes was higher in the LE compared to the SD rats. The Scatchard plot shows that LE rats have a higher binding affinity (Kd=0.82) than SD rats (Kd=0.53) at 0°C. In addition, there was no difference in half maximal inhibitory concentration (IC50) of testosterone on CBG-CORT binding in SD (IC50=6.27uM & 7.47uM) and LE (7.26uM & 8.09uM) rats Figure 7c.  3.5 The effect of neonatal gonadal hormones on adult adrenal size  Figure 8 shows the effect of neonatal manipulation on adrenal size (mg) in males (a) and females (c). In males, a two-way analysis of adrenal size revealed no interaction (F2,104=1.124, p=0.329) between neonatal manipulations and previous repeated restraint exposures, and no significant effect of either neonatal manipulation (F2,104=0.687, p=0.505) or previous repeated restraint exposure (F2,104=0.933, p=0.336). In females, there was a main effect of neonatal manipulations in females (F2,105=3.921, p=0.023), with no effect of previous repeated restraint exposure (F1,105=0.166, p=0.685) or interaction between neonatal manipulation and previous restraint   34   Figure 6: Mean + SEM of plasma CBG binding levels in males and females under basal conditions and at 30 min of acute and repeat restraint exposure. In males, there was higher CBG after restraint exposure in the neonatal control groups under acute conditions (#p’s<0.042 vs basal-naïve). In the male neonatal sham and ATD treated rats after repeat restraint exposure there was a lower CBG under basal-repeat conditions (#p’s<0.035 vs basal-naïve) and higher CBG in the acute-repeat condition (##p’s<0.028 vs basal-repeat). There were no significant differences in CBG binding in the females. Basal-Naive Acute Basal-Repeat Acute-Repeat050010001500UntouchedShamATDCBG [nM]Basal-Naive Acute Basal-Repeat Acute-Repeat0100020003000UntouchedShamTPCBG [nM]###MaleFemale##### 35  Figure 7: CBG binding properties in Long Evans and Sprague Dawley rats.  The apparent dissociation rate (a) was higher in the SD rats compared to LE rats. The Scatchard plot (b) shows LE have a higher binding affinity (Kd=0.82) compared to SD (Kd=0.53). The testosterone competition assay (c) showed the half maximal inhibitory concentrations (IC50) were equivalent between SD (IC50=6.27uM & 7.47uM) and LE (7.26uM & 8.09uM) rats. 0 5 10 15 20020406080100 Long EvansSprague DawleyTime (min)% Bound0.0 0.1 0.2 0.30.00.10.20.30.4 Long EvansSprague DawleyKd = 0.82Kd = 0.53Bound (nM)Bound/Free1 10 100020406080100 Sprague DawleyLong EvansTestosterone (uM)% Corticosterone Bounda)b)c) 36 exposure (F1,105=0.042, p=0.959). Post hoc analysis in females showed the neonatal TP treated rats had significantly larger adrenals compared to the untouched control group (p=0.034) and trend towards larger adrenals compared to the sham control group (p=0.081). There was no significant difference in adrenal size between the two control groups (p=1.000). Since adrenal size is influenced by body weight (Bailey et al., 2004), the ratio of adrenal size (mg)/ body weight (g) was calculated and presented in Figure 8 (b & d). Once differences in body weight were accounted for, in males, there was a main effect of previous restraint exposure to decrease the adrenal-body weight ratio (F1,103=10.981, p=0.001), with no effect of neonatal manipulation (F2,103=0.853, p=0.429), or interaction (F2,103=2.141, p=0.123). In females, there was no effect of neonatal manipulation on the adrenal-body weight ratio (F2,105=1.710, p=0.189), and a trend towards a main effect of previous restraint exposure (F2,105=2.949, p=0.089). There was no interaction between neonatal manipulation and previous restraint exposure (F2,105=1.870, p=0.721).  3.6 Organization effects of neonatal gonadal manipulation to influence adult gonadal hormone levels  Figure 9 shows plasma levels of testosterone in males (top) and estradiol (bottom) in females under basal and stress conditions. In males, a two-way analysis revealed a main effect of restraint exposure on testosterone levels (F3,98=13.342, p<0.0001), but no effect of neonatal manipulations (F2,98=0.198, p=0.846) or interaction (F6,98=0.578, p=0.747). Post hoc analysis revealed higher testosterone levels in rats exposed to acute restraint compared to basal-naïve rats (p<0.0001). Rats exposed to repeated restraint showed no difference in testosterone levels between the basal-repeat and repeat restraint rats (p=1.000) or compared to basal-naïve rats (p=1.000).   37 In females, a two-way analysis revealed a main effect of neonatal manipulation on estradiol levels (F2,99=4.735, p=0.011), with no effect of restraint exposure (F3,99=0.254, p=0.858) or interaction (F6,99=1.238, p=0.294). Post hoc analysis showed there were significant differences between the neonatal TP and sham treated rats (p=0.010). Although there were no significant differences between the control groups (p=0.987), the neonatal untouched rats were not significantly different than neonatal TP treated rats (p=0.139).   Figure 8: Mean + SEM of adrenal size (mg) (a & c) and the adrenal-body weight ratio (b& d) in males and females. Neonatal TP treated females had larger adrenal size (*p=0.034 vs untouched). In males exposed to repeated restraint stress had a smaller adrenal-body weight ratio (#p=0.001 vs acute). Untouched Sham ATD0510152025Adrenal weight [mg]Acute Repeat0102030UntouchedShamATDAdrenal-body weight ratioAcute Repeat051015UntouchedShamTPAdrenal-body weight ratioUntouched Sham TP010203040Adrenal weight [mg]Malesa)c)b)Femalesd)#* 38   Figure 9: Mean + SEM of plasma levels testosterone in males and estradiol in females. In males, there was higher testosterone levels after acute restraint exposure (#p<0.0001 vs basal-naïve). In females, neonatal TP treated rats had lower estradiol compared to the neonatal sham controls (*p=0.010).  Basal-Naive Acute Basal-Repeat Acute-Repeat050100150200250UntouchedShamTP****Estradiol [pg/ml]Basal-Naive Acute Basal-Repeat Acute-Repeat0.001.002.003.004.005.00UntouchedShamATD#Testosterone [ng/ml]MalesFemales 39 3.7 GR translocation in the hippocampus Figure 10 shows GR translocation in males (top) and females (bottom) under basal and stress states in the hippocampus. In males, a two-way analysis demonstrated no interaction between neonatal treatment and restraint exposure (F6,80=0.300, p=0.935). There was a significant main effect of restraint stress exposure (F3,80=19.091, p<0.0001) and no significant main effect of neonatal manipulation (F3,80=1.875, p=0.160). Post hoc analysis revealed there was significantly higher GR translocation after restraint exposure under both acute and repeat condition compared to their basal counterparts (p’s<0.0001), with no difference between the naïve-basal and repeat-basal conditions (p=1.000). In females, there was no interaction between neonatal treatment and restraint exposure (F6,91=0.726, p=0.629), with a significant main effect of both restraint stress exposure (F3,91=11.879, p<0.0001) and neonatal manipulation (F3,91=3.626, p=0.031). Post hoc analysis revealed there was significantly higher GR translocation after restraint exposure under both acute (p=0.018) and repeat (p<0.0001) condition compared to their basal counterparts. There was no difference between the naïve-basal and repeat-basal conditions (p=1.000). There was also a significantly lower GR translocation in the neonatal TP treated females compared to the untouched controls (p=0.018), with a trend in the same direction with neonatal sham treated females (p=0.087). Overall, there was a significant positive correlation between GR translocation and CORT (total: r=0.248, p<0.001 and free: r=0.247, p=0.001). Within group Pearson correlations are shown in Table 1, there were no significant correlations between total and free CORT in male neonatal ATD treated, male untouched controls, and female control groups, while there were significant correlations in male sham controls (r=0.474, p=0.012 and r=0.425, p=0.027, respectively) and female TP treated rats (r=0.397, p=0.024 and r=0.492, p=0.004).   40  Figure 10: Mean + SEM of GR translocation (nuclear/ nuclear + cytoplasmic) in the hippocampus of males and females under basal conditions and at 30 min of acute and repeat restraint exposure. In males and females, there was higher GR translocation after acute restraint exposure (*p’s<0.018 vs basal-naïve), and after repeat restraint exposure (**p<0.0001 vs basal-repeat). In females, neonatal TP treated rats had significantly lower GR translocation (#p=0.018 vs untouched).  Basal-Naive Acute Basal-Repeat Acute-Repeat0.000.200.400.60ATDUntouchedSham###GR Nucleus [% whole cell]Basal-Naive Acute Basal-Repeat Acute-Repeat0.000.100.200.300.40TPUntouchedSham****###GR Nucleus [% whole cell]MalesFemalesGR (95 kDa) ®GAPDH (37 kDa) ®H3 (18 kDa) ® 41 Table 1: Correlation between GR translocation (nuclear/ nuclear + cytoplasmic) in the hippocampus of males and females and plasma total and estimated free CORT    Total CORT Free CORT Male untouched Pearson Correlation 0.135 0.157 p value 0.476 0.407 N 30 30 sham Pearson Correlation 0.474 0.425 p value 0.012 0.027 N 27 27 ATD Pearson Correlation 0.188 0.182 p value 0.311 0.327 N 31 31 Female untouched Pearson Correlation 0.258 0.224 p value 0.140 0.203 N 34 34 sham Pearson Correlation 0.155 -0.063 p value 0.368 0.717 N 36 36 TP Pearson Correlation 0.397 0.492 p value 0.024 0.004 N 32 32 Significant correlations are bolded.  To examine if changes in GR translocation were due to the amount of protein loaded, or cross-contamination between fractions two-way analysis were performed. These revealed no significant differences between groups in loading controls (GAPDH: male: treatment F2,80=1.185, p=0.311, state F3,80=1.106, p=0.352 and female: treatment F2,91=0.096, p=0.909, state: F3,91=1.969, p=0.124 and H3: male: treatment F2,80=0.614, p=0.543, state F3,80=1.688, p=0.176 and female: treatment F2,91=0.567, p=0.569, state: F3,91=0.806, p=0.494) or nuclear contamination in the cytoplasm fraction (male: treatment F2,80=0.625, p=0.538, state F3,83=2.274, p=0.086 and female: treatment F2,91=0.222, p=0.801, state: F3,91=0.815, p=0.489) and  42 cytoplasmic contamination in nuclear fraction (male: treatment F2,80=1.334, p=0.261, state F3,80=0.503, p=0.681 and female: treatment F2,91=1.209, p=0.303, state: F3,91=2.446, p=0.069). 3.8 GR phosphorylated at the serine 211 site in the hippocampus Figure 11 shows levels of GR phosphorylated at the serine 211 site (ser211) in the hippocampus.  In males, a two-way analysis of ser211 revealed an interaction between restraint exposure and neonatal manipulation (F6,93=9.553, p<0.0001), and significant main effects of both restraint stress exposure (F3,93=4.098, p=0.009) and neonatal manipulation (F2,93=12.254, p<0.0001). Post hoc analysis revealed differential ser211 levels within male neonatal manipulation groups after repeated restraint exposure, but not in response to acute restraint exposure (p’s>0.111). Firstly, both male control groups had significantly different ser211 levels compared to their basal-naïve counterparts, however the untouched control group displayed a significant decrease (p=0.001) and sham controls displayed a significant increase (p=0.003), with no change observed in ATD treated rats (p=1.000). Secondly, after the last day of restraint stress sham controls had lower ser211 compared to their repeat-basal counterparts (p=0.018), with no difference observed in the untouched controls and ATD treated rats (p’s>0.598). After repeated restraint stress exposure, there were also significant differences between the neonatal manipulation groups under basal and stress conditions. Under basal-repeat conditions neonatal untouched rats had significantly lower ser211 compared to both the neonatal sham and ATD treated rats (p<0.0001), with no significant difference between these two groups (p=0.769). Under repeated stress conditions all neonatal manipulations were significantly different with ATD rats having the greatest amount of ser211, followed by sham controls (p=0.043 vs untouched and p=0.001 vs ATD) then untouched controls (p<0.0001).    43 In females, a two-way analysis of ser211 revealed a significant main effects of restraint stress exposure (F3,92=3.481, p=0.019) with no significant main effect of neonatal manipulation (F2,92=0.723, p=0.488) or interaction (F2,92=0.252, p=0.957) between restraint stress exposure and neonatal manipulation. Post hoc analysis revealed there was significantly higher Ser211 under repeat conditions after restraint exposure compared to their basal counterparts (p=0.015), with no differences between basal-repeat rats (p=0.220) or acute restraint exposed rats (p=1.000) compared to the basal-naïve rats.  Overall, there is a significant positive correlation between ser211 and both total and free CORT (r=0.200, p=0.004 and r=0.182, p=0.009, respectively). Within group Pearson correlations are shown in Table 2. In females, there was a significant positive correlation with free CORT in the untouched control groups (r=0.368, p=0.038), with a trend towards a positive correlation in the sham control (r=0.317, p=0.056) and TP treated groups (r=0.310, p=0.090). Female control groups also showed a trend towards a positive correlation with total CORT (untouched r=0.293, p=0.098 and sham r=0.302, p=0.070), while there was no correlation in TP treated groups. In males there were no significant correlations with total or free CORT.     44  Figure 11: Mean + SEM of Ser211 in the hippocampus of males and females under basal conditions and at 30 min of acute and repeat restraint exposure. In males after repeat restraint exposure, under basal conditions the neonatal untouched rats had lower Ser211 (#p=0.001 vs basal-naive) and the neonatal sham treated rats had higher Ser211 (#p=0.003 vs basal-naive), with the neonatal sham and ATD treated rats having higher ser211 (*p’s<0.0001 vs untouched). Then, after restraint exposure neonatal sham treated males have lower Ser211 (##p=0.018 vs basal-repeat), with neonatal ATD treated males having highest Ser211 levels (*p=0.043 vs untouched & **p=0.001 vs sham), followed by neonatal sham treated males (*p<0.0001 vs untouched). In females after repeat restraint exposure they had higher Ser211 levels (##p=0.015 vs basal-repeat),  **Basal-Naive Acute Basal-Repeat Acute-Repeat0.000.501.001.50UntouchedShamATD###**#Ser211/ actin **Basal-Naive Acute Basal-Repeat Acute-Repeat0.000.200.400.600.801.00UntouchedShamTP##Ser211/ actinSer211 (95 kDa) ®b-actin  (42 kDa) ®MalesFemales 45 Table 2: Correlations between GR phosphorylated at the serine 211 site in the hippocampus of males and females and plasma total and estimated free CORT    Total CORT Free CORT Male untouched Pearson Correlation 0.115 -0.176 p value 0.509 0.311 N 35 35 sham Pearson Correlation 0.061 0.024 p value 0.735 0.896 N 33 33 ATD Pearson Correlation 0.229 0.174 p value 0.192 0.325 N 34 34 Female untouched Pearson Correlation 0.293 0.368 p value 0.098 0.038 N 33 32 sham Pearson Correlation 0.302 0.317 p value 0.070 0.056 N 37 37 TP Pearson Correlation 0.254 0.310 p value 0.169 0.090 N 31 31   Significant correlations are bolded. 3.9 The effect of neonatal manipulations on body weight Body weight is a parameter that is known to be sexually dimorphic in rats, therefore to validate neonatal manipulations, one-way analyses with age used as a covariate were performed in males and females to see if neonatal ATD in males and neonatal TP in females altered body weight pre- and post-pubertal. There was no effect of neonatal manipulations on pre-pubertal body weight in both males (F2,107=2.285, p=0.107) and females (F2,107=0.179, p=0.836). As adults, a one-way analysis controlling for age revealed a main effect of neonatal manipulations in both males (F2,106=9.051, p=0.001) and females (F2,106=66.213, p<0.0001). Post hoc analysis indicated that neonatal ATD treated males had lower body weights compared to both their control groups  46 (p=0.049 vs untouched and p<0.0001 vs sham), and neonatal TP treated females had higher body weight compared to both their control groups (p<0.0001).  In both males and females there was no significant difference in body weight between the control groups (p=0.227 and p=1.000, respectively). Results are summarized in Table 3. Table 3: Mean pre-pubertal and adult body weights.  sex treatment Pre-pubertal Adult Male Untouched Mean 102 421 N 37 37 Std. Error of Mean 1.4 7.1 Sham Mean 105 435 N 36 36 Std. Error of Mean 1.4 7.2 ATD Mean 100 *393 N 37 37 Std. Error of Mean 1.4 7.1 Female Untouched Mean 96 274 N 36 36 Std. Error of Mean 1.2 4.5 Sham Mean 97 273 N 40 40 Std. Error of Mean 1.2 4.3 TP Mean 96 *337 N 34 34 Std. Error of Mean 1.3 4.7 *In males and females, the neonatal gonadal hormone manipulation rats had lower and higher body weights (p’s<0.049 vs untouched and sham), respectively as adults.  3.10 Growth hormone regulated genes in the liver Since there were observed differences in adult body weights in both males and females neonatally treated with ATD and TP, respectively, and their control groups we analyzed three sexually dimorphic liver genes that regulate growth hormone pulsivity (Waxman and Holloway,  47 2009). A one-way analysis showed no significant effect of neonatal manipulations on genes analyzed in males (cyp2c11 – F2,23=2.176, p=0.699, cyp2c12 – F2,23=2.176, p=0.136, and hnf-6 – F2,22=1.088, p=0.354) or females (cyp2c11 – F2,21=1.033, p=0.373, cyp2c12 – F2,23=0.194, p=0.825, and hnf-6 – F2,21=0.163, p=0.851). A second one-way analysis of revealed sex differences in all three genes; cyp2c11 was predominantly expressed in males (F1,48=52.217, p<0.0001) while cyp2c12 and hnf-6 were predominantly expressed in females (F1,50=44.406, p<0.0001 and F1,47=13.354, p=0.001, respectively). Results are summarized in table 4. Table 4: Mean relative mRNA expression of cyp2c11, cypt2c12, and hnf-6 in the liver.  sex treatment cyp2c11 cyp2c12 hnf-6 Male Untouched Mean 1.053 0.035 0.066 N 7 8 8 Std. Error of Mean 0.267 0.009 0.040 Sham Mean 0.816 0.069 0.188 N 9 8 8 Std. Error of Mean 0.200 0.017 0.066 ATD Mean 1.038 0.042 0.111 N 10 10 9 Std. Error of Mean 0.208 0.009 0.062 Female Untouched Mean 0.008 0.562 0.642 N 9 9 9 Std. Error of Mean 0.004 0.101 0.185 Sham Mean 0.049 0.493 0.538 N 9 10 9 Std. Error of Mean 0.036 0.155 0.275 TP Mean 0.010 0.611 0.745 N 6 7 6 Std. Error of Mean 0.004 0.126 0.272 There were no significant differences of neonatal treatment groups in males and females in the relative expression of any of the genes analyzed in the liver.   48 Chapter 4: Discussion The goal of this thesis was to examine the effects of neonatal gonadal hormone manipulation on adult HPA regulation through GR signaling. To achieve this goal, we antagonized the normal conversion of testosterone to estrogen in males by administering ATD over the neonatal period, whereas females received TP over this same interval (PND 1 to PND 21). Based on previous findings that neonatal ATD treatment results in increased CORT responses in adult males (Seale et al., 2005b, Bingham et al., 2011, Bingham et al., 2012) and neonatal TP treatment can decrease the magnitude of the CORT response in adult females (Seale et al., 2005a), we hypothesized we would observe similar responses in CORT levels and this would be reflected in the magnitude of the GR responses under acute and repeat restraint stress conditions. In summary, we expected the results to find, overall, higher CORT and GR activation in adult males treated with ATD as neonates, and conversely, lower CORT and GR activation in adult females treated with TP as neonates.  Overall, the work in this thesis has demonstrated significantly different CORT levels in neonatal ATD treated male and TP treated female rats compared to their same sex neonatal control groups. These differences occurred in the predicted direction as stated above, however they were not fully recapitulated in all stress conditions, as adult males treated with ATD during the neonatal period expressed higher total CORT levels compared to the neonatal male control groups only under basal, stress naïve conditions, and expressed equivalent CORT habituation to the male neonatal control groups. Adult females with neonatal TP treatment had lower total CORT responses to acute restraint stress exposure and failed to show CORT habituation after repeated restraint exposure, unlike the female neonatal control groups who expressed equivalent CORT habituation. In summary, we observed that neonatal estrogens modulated adult restraint- 49 naïve basal CORT levels while neonatal androgens were found to alter restraint induced changes in CORT levels. Since increasing levels of CORT are known to activate GR as described in chapter 1, we expected to see markers of GR activation increase with increasing levels of CORT in circulation. Therefore, we hypothesized that GR phosphorylation, as measured by Ser211 levels, and the percentage of GR translocation to the nucleus would follow observed changes in plasma CORT levels. As predicted, there was an overall weak, but significant positive correlation (r’s<0.248) between CORT and markers of activated GR when all groups were combined. However, subgroup investigation of the neonatal treatments in males and females revealed that only male neonatal sham treated and female neonatal TP treated rats had significant positive correlations between total CORT and GR translocation and no significant correlations were observed between Ser211 and total CORT. This indicates, there is likely an interaction between neonatal manipulation and GR activation, however we only observed an interaction between neonatal manipulation and restraint exposure in Ser211 levels in the hippocampus of males. These results suggest, as seen with the effect of neonatal gonadal hormones on adult CORT responses, that there are differential effects of these neonatal treatments on adult GR activation. 4.1 Neonatal ATD treatment in males alters basal-naïve total CORT output, while neonatal TP treatment alters restraint stress CORT output Adult male rats treated with neonatal ATD showed higher basal total CORT levels under restraint-naïve conditions, compared to neonatal control rats. After restraint exposure (acute and repeat), there was no longer a difference between male neonatal ATD treated rats and the male control groups in both basal and stress induced CORT levels. Further, neonatal ATD treatment did not alter the ability for these rats to express CORT habituation, since they showed equivalent  50 decrements in total CORT levels from day 1 to day 5 of repeated restraint exposure as seen in the male neonatal control rats. With respect to basal CORT levels in unstressed animals, this study replicates previous work by Seale et al. (2005b) where they also observed higher mean averages of total CORT across a 24 hour time period in adult male rats treated with ATD in utero and as neonates. However, they also observed higher CORT levels after exposure to noise stress. Higher acute restraint induced CORT levels in male rats treated with ATD compared to those receiving a sham surgery as neonates has also been observed previously in our lab using a similar neonatal ATD treatment and restraint stress protocol (Bingham et al., 2011, Bingham et al., 2012). However, both the Bingham et al. (2011), Bingham et al. (2012) and Seale et al. (2005b) utilized SD rats, while this study utilized LE rats which may have contributed to the discrepancy in findings.  Neonatal administration of TP in females showed differential effects compared to their same sex control groups on total CORT levels as those seen with neonatal ATD treatment in males. As adults, neonatal TP treated rats had similar basal levels of total CORT compared to the female neonatal control rats. Differences in total CORT levels were observed between the female neonatal TP treated rats and their same-sex control groups after exposure to acute restraint stress resulting in neonatal TP treated rats showing lower total CORT levels at 30 minutes from the onset of restraint exposure. Then, on day 5 of consecutive repeated restraint exposure these same rats failed to express CORT habituation at the 30 min time point as seen in the female neonatal control groups. These results follow the same trend that females exposed to estradiol or testosterone during the neonatal period results in lower CORT levels as adults (Patchev et al., 1995, Seale et al., 2005a). In addition to replicating the effects of neonatal gonadal hormone  51 exposure on acute stress induced CORT levels, this study demonstrates the ability for neonatal TP exposure to prevent CORT habituation following repeat restraint exposure.  In summary, we observed effects of neonatal estrogens to act on adult restraint-naïve basal CORT levels and neonatal androgens to act on restraint induced changes in CORT levels, thus these hormones likely alter the gene expression and methylation profiles of GR targets differentially during development in males and females. In light of the current results, it would be worthy of pursuit to analyze whether GR genes within the two distinct population of GR-DNA binding sites identified by Polman et al. (2013), show changes in gene expression and DNA methylation patterns based on the restraint state the neonatal hormone treatment altered, since one population of these GR-DNA binding sites responds to all levels of CORT, while the other only responds under high levels of CORT. Based on the current observed effects of neonatal gonadal hormones on adult CORT levels, we would predict to see changes in the population of genes that respond at low CORT levels in males and with those that respond to high CORT levels altered in females. 4.2 Males showed differences in CBG binding levels after restraint exposure In addition to analyzing total CORT responses, we also examined CBG levels to determine if changes in total CORT output by neonatal manipulation or restraint exposure were reflected in the estimated free CORT levels since this is often considered the biological active fraction (Mendel, 1989). In rodents, CBG is known to be higher in females, which as discussed in chapter 1 is a result of organizational effects of testosterone during development in males (Gala and Westphal, 1965, Jansson et al., 1985, Kurabekova et al., 1991). In this study we did not observe any main effects of neonatal manipulation on CBG levels in males and females, which is in agreement with previous literature employing similar neonatal hormone manipulation (Seale et  52 al., 2005a, Seale et al., 2005b), and our neonatal manipulations were performed before the critical period for organizational effects of testosterone to occur (Jansson et al., 1985, Kurabekova et al., 1991). CBG levels are also known to decrease in response to chronic (Spencer et al., 1996, Deak et al., 1999, Tinnikov, 1999) and acute stress exposure (Fleshner et al., 1995, Tannenbaum et al., 1997), which we did observe in male rats exposed to repeated restraint; they exhibited lower CBG binding capacities compared to restraint-naïve males. Since this result was only seen in males and testosterone levels differed based on restraint exposure, this suggests a possible interaction between the acute and repeat restraint stress induced differences in testosterone and CBG levels. Interestingly, previous work in the lab utilizing SD rats did not observe this decrement in CBG levels following acute or repeated restraint exposure (Innala and Viau, 2014). In addition to lower CBG binding levels 24 hours after 4 consecutive days of restraint exposure, we also see higher levels of CBG at the termination of a 30 min acute restraint exposure in male neonatal control groups, with no differences observed in females. To address the discrepancies in CBG results between the two strains of rats, we measured half maximal inhibitory concentration (IC50) of testosterone on CBG-CORT binding and observed no difference. However, there were differences in binding affinity of CBG between the strains with LE rats having a lower binding affinity for CORT compared to SD rats.  This would mean at equivalent total CORT levels, the LE rats are actually exposed to higher free CORT than SD rats. Given that stress exposure can decrease CBG levels this suggests LE rats are more susceptible to stress induced changes in CBG levels than SD rats.  53 4.3 Similarities between total and estimated free CORT in response to neonatal manipulations is restraint-dependent in both males and females As adults, males showed no significant differences between total and estimated free CORT as a result of neonatal treatment under stress conditions, but not basal conditions. In females, the opposite was true as we saw differences between total and estimated free CORT levels under stress conditions, but not basal conditions. As discussed in chapter 1, since estimated free CORT is calculated using measurement of both total CORT and CBG binding capacity, variations in CBG levels and binding capacities can greatly affect the amount of circulating free CORT even in the absence of statistical differences between groups as seen in the females under stress conditions.  4.4  Neonatal manipulations and restraint stress effects on adrenal size  The size of the adrenal gland is known to be sexually dimorphic with females having larger adrenal glands compared to males (Sencar-Cupovic and Milkovic, 1976, Majchrzak and Malendowicz, 1983). We did observe this expected size difference in adrenal weight between neonatal control males and females. In females treated with neonatal TP, we observed larger adrenal glands compared to their same-sex controls. However, once adrenal weights were adjusted for body weight there was no longer an effect of neonatal TP. In males, there was no difference in adrenal size regardless of adjustment for body weight even though the neonatal ATD treated rats were significantly lighter than their same-sex controls. In males we observed a significantly lower adrenal-body weight ratio in those subject to repeated restraint exposure, which differed from recent work showing no effect of repeated restraint exposure on adrenal-body weight ratio in males and females (Babb et al., 2014), however they used a different restraint paradigm. Further, since there was no observed difference in adrenal size, the effect of  54 restraint stress to decrease the adrenal-body weight ratio was likely due to decreased body weight gains observed during repeated restraint exposure.     4.5 Markers of corticosterone activated glucocorticoid receptors differentially respond to neonatal hormone milieu in males and females In order for GR to bind to glucocorticoid receptor elements (GREs) it must first be ligand-activated, then translocate to the nucleus. As discussed in chapter 1, this process is dependent on co-factors, which can be regulated by gonadal hormones (Bourke et al., 2012), but are also in large part due to the concentration of CORT in circulation under basal conditions, as well as in response to stress. Based on potential organizational influences of the gonadal hormones during the neonatal period to alter the magnitude of the CORT response, we predicted similar changes in GR activation. Therefore, we sought to examine the effect of neonatal hormone manipulations and its interaction with acute and repeated restraint stress on GR activation, through the measurement of GR translocation and the phosphorylation of the GR receptor at the serine 211 site (Ser211). As expected, there were significant effects of restraint exposure on GR translocation and Ser211 in the hippocampus of both sexes. Also, positive overall correlations were observed between CORT (total and estimated free) and both GR translocation and Ser211. Our current results indicate that GR activation does not fully follow CORT levels in the hippocampus, since we observe similar increases in GR translocation in response to both acute and repeats restraint stress regardless whether we observed differences between day 1 and day 5 of repeated restraint exposure. In addition, we only observed differences in Ser211 levels under repeat restraint conditions, with significant increases as a result of restraint exposure occurring in all female groups and only in males treated with neonatal ATD. These results however differ from previous  55 results in the lab showing GR translocation follows CORT habituation as we observed decrements in GR translocation under repeat compared to acute restraint exposure, which followed the decrements in CORT (Innala and Viau, 2014). These differences might be due to differences strain differences since the previous study utilized SD rats. One potential explanation for this discrepancy is potential differences in free CORT following repeated restraint exposure, as mentioned previously, CBG in SD rats has a higher binding affinity for CORT and we did not observe changes in CBG levels in this strain using a similar restraint paradigm. Therefore, the combination of these two factors has the potential to significantly reduce the levels of free CORT in a SD compared to LE rats. Although both males and females had increases in GR translocation in response to restraint exposure, we did observe females treated with TP as neonates had significantly lower GR translocation compared to female untouched controls and a trend in the same direction in female sham controls. This observation followed our prediction under acute restraint conditions, as neonatal TP treated females had lower total CORT and overall lower GR translocation. In contrast though, we observed no effect of neonatal manipulation in females on Ser211 levels. Taken together, since GR translocation is simply a marker of the percentage of total GR in the nucleus it does not describe the amount of activated GR in the nucleus, whereas Ser211 is a better marker to indicate the amount of activated GR in the nucleus, since levels of Ser211 are known to be ligand-dependent and transcriptional activity of GR is positively correlated with levels of Ser211 (Wang et al., 2002). Further, given Seale et al. (2005a) showed neonatal TP treatment in females, in the absence of gonadal hormones, had significantly greater GR mRNA in the PVN compared to neonatal oil treated females, this suggest that our neonatal treatment  56 may have increased the receptor pool and the decrease in GR translocation does not indicate lower levels of GR activation.  In contrast to GR translocation, we observed a main effect of neonatal manipulation in males, but not females, on Ser211 levels in the hippocampus. However, these differences occurred between all three neonatal conditions suggesting effects observed in the neonatal ATD rats were due to both the neonatal surgery and/ or ATD treatment. Given, GR levels are well known to be altered by the neonatal environment, including neonatal handling (Ladd et al., 2005, Champagne, 2013, Meaney et al., 2013) and our surgical procedure increased the amount of neonatal handling in the sham and ATD treated rats, this is one possible explanation for the effects observed in both the surgically treated rats relative to the untouched control rats. In addition, there are also reported sex specific effects of neonatal stress exposure that are known to mediate changes in adult GR levels (McCormick et al., 1995), which is a possible explanation as to why we only saw this effect in males. In summary, the Ser211 findings in males within acute and repeat restraint conditions do suggest a role of both neonatal surgery and gonadal hormone exposure to alter the capacity for CORT to activate GR. 4.6 Neonatal TP decreases adult estradiol levels in females, with no effect of neonatal ATD on adult testosterone levels in males Since the pharmacological manipulation employed in this study did not involve any direct manipulation of the gonads, we measured testosterone in males and estradiol in females, which are the predominant gonadal hormones circulating in each sex as adults, to address if any changes observed were due to potential activational effects of the steroids, as discussed in chapter 1. We did not observe any main effects of neonatal manipulation on testosterones level. However, we did observe a higher testosterone levels under acute restraint conditions. Previous  57 finding in SD rats that demonstrate higher acute CORT levels in neonatal ATD treated rats did not observe any changes in circulating testosterone in response to restraint stress (Bingham et al., 2012). Therefore, it would be worthy of pursuit to determine if adult GDX and replacing with equal testosterone levels would unmask an organizational effect of neonatal ATD treatment on adult restraint stress induced CORT levels in LE rats.   In females, neonatal TP treatment resulted in significantly lower estradiol levels compared to female neonatal sham controls, with no difference observed with female neonatal untouched controls. Since estradiol was only measured at one-time point and we did not control for estrous cycle, we cannot determine if there were any changes in the cyclicity of estradiol between neonatal groups lead to the observed differences in estradiol levels. Although, previous studies do suggest the estradiol differences between the neonatal TP treated rats and the sham controls may be due to the neonatal TP treatment, since they have demonstrated that neonatal administration of testosterone in the female rats arrested the estrous cycle at the estrus phase and renders these rats infertile (Foecking et al., 2008, Ongaro et al., 2015). Since HPA output is known to vary across the estrous cycle (Viau and Meaney, 1991), the effects of our neonatal testosterone treatment on CORT in response to restraint stress may have been influenced by the potential lack of cyclicity. In addition, the neonatal TP treated rats may not have been responding to the estradiol in a similar manner to the control females as it has been demonstrated that neonatal androgens can impair the ability for a female rats to respond to estradiol replacement when OVXed as adults (Gerall et al., 1972). This was observed in their running rate on a Wahmann activity wheel, were they observed an inverse relationship between the dose of neonatal TP and the running rate as adults. Therefore, as in males, it would be worthy of pursuit to determine if adult OVX and replacement with equal estradiol levels would unmask whether  58 neonatal TP treatment on acute restraint induced CORT levels is due to an organizational effect of neonatal TP treatment on adult restraint stress induced CORT levels in LE rats.  4.7 Effects of Neonatal treatments on body weights  We did not see expected stress induced changes in HPA axis output in the male rats treated during the neonatal period with ATD as previously shown (Bingham et al., 2011, Bingham et al., 2012). Since it is well known that testosterone and its metabolites can have numerous organizational effects on the body during the neonatal period (Ongaro et al., 2013), in addition to looking at the effects on glucocorticoid output and receptor responses, body weights were analyzed in the same animals. This was primarily done to verify the effectiveness of neonatal hormone manipulation, which was done by analyzing body weight during pre-puberty and as adults, prior to restraint exposure. Body weight is an appropriate proxy measurement of drug effectiveness since it is well documented in adult female rats and mice treated with testosterone during the neonatal period exhibit increased body weights (Beatty et al., 1970, Jansson and Frohman, 1987, Cecilia Ramirez et al., 2010, Cecilia Ramirez et al., 2014, Ongaro et al., 2015). In this experiment, we saw a marked changed in the body weight gain of both neonatal treatment groups, with TP treated females having greater body weight gain, as expected, and ATD treated males having lower body weight gains compared to their same sex control groups. These results indicated that although we did not affect stress induced CORT output with neonatal ATD treatment in the males, this drug did have organizational effects on the phenotype of these rats since we did not observe differences in testosterone levels as adults between the male groups. To further explore the effects of neonatal hormone manipulations on body weight, we examined the possibility that these changes were due to changes in growth hormones. Growth hormone levels are involved in the regulation of body weight growth, and there are distinct  59 secretion patterns between males and females, which regulate the sexually dimorphic expression of cytochrome P450 genes in the liver (Waxman and O'Connor, 2006). Therefore, we analyzed the expression of cyp2c11, and cyp2c12 in the liver that are known to be sexually dimorphic and regulated by growth hormone secretion patterns (Waxman and O'Connor, 2006, Cecilia Ramirez et al., 2010). Although we observed marked differences in body weights, these changes were likely independent of adult secretion patterns of GH, since neither of the neonatal treatments affected the expression profile of these genes. In addition to analyzing cytochrome P450 genes, hnf-6 was also analyzed as a previous study in mice has demonstrated a single injection of TP on PND2 resulted in defeminized mRNA level of hnf-6 in TP-treated mice, resulting in a complete reversal of the female specific expression of this gene (Cecilia Ramirez et al., 2014); however, our neonatal TP treatment in female rats failed to produce similar results. Our lack of neonatal treatment effects on CBG further supports the notion that our neonatal treatments did not alter GH secretion patterns since they also contribute to regulating the sex specific levels of CBG (Jansson et al., 1989). Although the data in this thesis suggest our neonatal manipulations did not alter sexually dimorphic GH secretion patterns, a full timecourse of GH levels would be required to definitively answer this question. 4.8 Methodological considerations in experimental design of neonatal manipulations Methodological considerations are always important when interpreting results, especially when determining whether neonatal hormones are having organizational versus activational effects during adulthood. Although the goal of this thesis was to assess the organizational effects of gonadal hormones during the neonatal period, we did not control for adult levels of circulating gonadal hormones by performing adult GDX and replacement with equivalent levels of activational gonadal hormones, instead we were interested in the ability for neonatal gonadal  60 hormones to alter CORT and GR responses in the presence of endogenous levels of circulating gonadal hormones during adulthood. In this thesis, neonatal gonadal hormone manipulations were found to affect levels of circulating gonadal hormones. This effect reduced our ability to unmask potential organizational effects of our neonatal treatments; however, one is only able to distinguish between organizational versus activational effects of a neonatal gonadal hormone manipulation if, as adults, there were similar levels of gonadal hormones (McCarthy et al., 2012). One main caveat to the results in this thesis is we did not observe the expected higher restraint stress induced HPA output in males treated with neonatal ATD compared to male neonatal control groups (Seale et al., 2005b, Bingham et al., 2011, Bingham et al., 2012). This lack of effect in restraint stress induced HPA output in males does not discount a role of neonatal gonadal hormones to alter stress induced HPA output in males, however based on previous findings in the literature it does suggest that the action of the testosterone surge has already had organizational effects on the HPA axis since estradiol levels have been shown to peak in the brain within 2 hours of birth (Rhoda et al., 1984). Although previous work in the lab has demonstrated that blocking aromatase activity within 12 hours is capable of altering HPA function (Bingham et al., 2011, Bingham et al., 2012), these studies were done in a different strain of rat which may have been more susceptible to this treatment since it is known that not all strains of rats will respond to early life events in a similar manner and there are strain and species differences in the degree of sexual dimorphism (Yanai, 1979, Anisman et al., 1998). Although the female rats were manipulated at the same time point, timing of the testosterone treatment is less critical since the critical window for organization effects of gonadal hormones lasts longer as they do not have an endogenous surge in gonadal hormones during the neonatal period  61 (McCarthy et al., 2012). This is one main reason many studies looking at the effects of the neonatal testosterone surge are performed in female rodents.  4.9 Effect of cross-contamination between nuclear and cytoplasmic fractions on calculated GR translocation Although there was no significant difference in the level of cross-contamination between samples included in the analysis of GR translocation, as assessed in males and females using a two-way ANOVA with neonatal treatment and adult conditions as between-subject factors, the calculated levels of cytoplasmic contamination in the nuclear fraction were higher than previously seen in the lab and by Bourke et al. (2013) using the same extraction protocol. However, we did observe similar relative levels of GR translocation as previously seen in samples with lower calculated levels of cytoplasmic contamination in the nuclear fraction (Innala and Viau, 2014) and slightly lower GR translocation than those reported using the same extraction method in response to a 5 min forced swim with no observed cross-contamination between fractions (Bourke et al., 2013). However, one would expect increasing levels of cytoplasmic contamination to result in increased calculated GR translocation since the majority of GR resides in the cytoplasm, which was not observed.  4.10 Future considerations This thesis demonstrates the ability for neonatal manipulations to alter HPA output in both gonadal intact males and females. The differences in CORT output in this study do not support the hypothesis that neonatal manipulation are altering GR-mediated negative feedback of the HPA axis as circulating levels of CORT cannot be fully explained in any of the neonatal groups, in both sexes, by markers of CORT-activated GR (GR translocation and Ser211 protein levels). However, this does not discount a potential role for GR to be mediating the changes in CORT  62 levels between neonatal groups since we did not asses the role of activated GR on restraint stress induced changes on upstream markers of HPA activation like PVN CRH and AVP expression, which have been demonstrated to increase as a result of neonatal ATD treatment in males (Seale et al., 2005b) and decrease in females treated with neonatal TP in the absence of circulating adult gonadal hormones (Seale et al., 2005a).  Although previous work did not demonstrate any differences in the translocation of MR to the nuclease at 30 min from stress onset (Innala and Viau, 2014), and it has been proposed that membrane bound MR acts initially to amplify the stress response, while membrane and genomic GR work to contain the stress response and consolidate the memory (Oitzl et al., 2010), this does not discount a role for MR in mediating glucocorticoid-mediated feedback. Recent work has demonstrated MR is likely a key player in regulating CORT levels through membrane bound MR (Ter Heegde et al., 2015). Further, MR, but not GR, has also been shown in male rats to block the expression of CORT habituation following repeated restraint exposure (Cole et al., 2000). Therefore, further studies should look at the effect of neonatal testosterone treatment in female rats on MR activity since this manipulation prevented the expression of HPA axis habituation.     4.11 Summary Overall the work in this thesis has demonstrated significantly different CORT levels in neonatal ATD treated male and TP treated female rats compared to their same sex control groups under basal-naïve conditions and after restraint exposure, respectively. In respect to markers of CORT-activated GR, measured by the percentage of GR in the nucleus and the amount of GR phosphorylated at the serine 211 site, we observed an overall positive correlation between these markers and CORT, and differential responses in these markers between neonatal treatment groups. Effects of the neonatal manipulations observed on Ser211 appeared to be due to effects  63 of the neonatal surgery and the neonatal gonadal hormone milieu in males. Further, even in situations where differences were seen between the control groups and the neonatal treatment groups, we cannot fully claim these changes are solely due to organizational effects of gonadal hormones during the neonatal period as we did not control for adult gonadal hormone levels. This data does demonstrate that neonatal gonadal hormone manipulations can have long lasting effects on the phenotype of male and female rats. Therefore, any perturbation that has the capability of altering the in utero and neonatal testosterone surges in males as seen with prenatal stress and prenatal alcohol exposure (Ward et al., 2002, Ward et al., 2003) or causes females to be exposed to gonadal hormones during this same period has the capability to cause long-term changes in the HPA axis. Further, a recent meta-analysis looking at the impact of Bisphenol A (BPA), which is known to have estrogenic properties and found at high prevalence in the environment, on child neurobehavioral function found the majority of studies analyzed showed altered child neurobehavioral function with BPA exposure and some studies reported sex specific changes based on the timing of the exposure in utero and postnatal (Mustieles et al., 2015). The work in this thesis highlights the ability for alteration in the neonatal gonadal hormone milieu to cause long lasting effects on the phenotype of both males and females.     64 References Abbey H, Howard E (1973) Statistical procedure in developmental studies on species with multiple offspring. Developmental Psychobiology 6:329-335. Anisman H, Zaharia MD, Meaney MJ, Merali Z (1998) Do early-life events permanently alter behavioral and hormonal responses to stressors? International Journal of Developmental Neuroscience 16:149-164. 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