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The genetic basis for adrenal gland structure : a focus on the stress response Di Curzio, Domenico Luciano 2009

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THE GENETIC BASIS FOR ADRENAL GLAND STRUCTURE: A FOCUS ON THE STRESS RESPONSE  by  Domenico Luciano Di Curzio  B.A. Hons., The University of Winnipeg, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies  (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2009  © Domenico Luciano Di Curzio, 2009  ABSTRACT Adrenal gland function is mediated through secreted hormones, and these hormones play a vital role in the autonomic and HPA-axis mediated stress response. Stress is a complex phenomenon, and the genetic underpinnings of it can be approached using a quantitative trait locus (QTL) analysis. This method has been used to investigate genomic regions associated with variation in complex phenotypes in the mouse CNS and anatomical variation in organ weights. However, it has not been previously used to explore the structure of the adrenal gland. In this study, we used QTL analyses to identify candidate genes underlying adrenal weight and adrenal cortical zones and medulla widths. We used 64 BXD recombinant inbred (RI) strains of mice (n = 571) and 2 parental strains (C57BL/6J and DBA/2J; n = 20) to measure adrenal weights. These 64 BXD RI strains and the progenitor strains were then examined to measure adrenal zone widths. We performed QTL linkage analysis (with WebQTL) to identify QTLs associated with adrenal weight. We found significant QTLs on chromosome 3 for females and Chr 4 for males and suggestive QTLs on Chrs 1, 3, 10, and 14 for females and Chrs 2, 4, 10, 17, and X for males. We identified a significant QTL on Chr 10 and a suggestive QTL on Chr 13 for male adrenal total width. For male adrenal medulla width, we found a significant QTL on Chr 5 and a suggestive QTL on Chr 1. In addition, we identified significant QTLs on Chrs 10 and 14 for male X-zone width. Finally, there are 114 genes that mapped within the significant QTL intervals, of which we identified 5 candidate genes associated with adrenal structure and/or function. In summary, this study is an important first step for detecting genetic factors influencing the structure of the adrenal component of the HPA-axis using QTL analysis, which may relate to adrenal function and provide further insights into elucidating genes critical for stress-related phenotypes.  ii  TABLE OF CONTENTS Abstract……………………..……..…………….…………………………………………………………ii Table of Contents …………………………………………………………………………………………iii List of Tables…………………………… ………………………………………………………………....v List of Figures………………………………….………………………………………………………….vi Acknowledgements…………………………………………… …………………………………………vii 1 Introduction…………………………………………………… ……………………………………...1 1.1 Adrenal gland development, structure, and function……… ………………………………………1 1.2 The adrenal gland and the stress response….. ……………………………………………………..3 1.3 Genes and adrenal gland development, structure, and function……………………………… …...5 1.4 The relationship between adrenal gland structure and function….. …………………….................8 1.5 QTL and RI analyses……………………………………………………………………………….9 1.6 References…… …………………………………………………………………………………...12 2 The Genetic Basis For Adrenal Gland Structure………………………………………………….20 2.1 Introduction……… ……………………………………………………………………………….20 2.2 Methods….……………………………………………………………………………………..…23 2.2.1 Animals………………………………………………………… ....………………………....23 2.2.2 Adrenal removal and weight analysis…………………………………… ………..………...23 2.2.3 Tissue preparation and histology………………………………………… .............................23 2.2.4 Microscopy………………………………………………………… …………………..……24 2.2.5 Adrenal cortex and medulla measurements…………………………………… ………..…..24 2.2.6 Statistical analysis and QTL mapping……………………………………….… …………....24 2.3 Results……………………………………………… ………………………………………….…25 2.3.1 Adrenal weight measurements……………………………… ………..……………………...25 2.3.2 QTL influencing adrenal weight……………………………………………. ……………….26 2.3.3 Adrenal width measurements……. …………………………………………………………..27 2.3.4 QTL influencing adrenal width…… ……………………………………………………..….28 2.3.5 X-zone and adipose analysis…. ……………………………………………………………..29 2.3.6 Genes in QTL regions…. …………………………………………………………………….30 2.4 Discussion………….…………………….. ……………..…………………………………….…31 2.4.1 Overview…..………………………………… ..………………………………………….….31 2.4.2 Differences between parental strains.… ……………………………………………………..31 2.4.3 Interaction between adrenal size and weight…. ……………………………………………..34 2.4.4 Adipose and x-zone analysis… ………………………………………………………………34 2.4.5 Adrenal weight QTL…………… ……………………………………………………………36 2.4.6 Adrenal width QTL….. ………………………………………………………………………37 2.4.7 Candidate genes….. ………………………………………………………………………….39 2.4.8 Stress and anxiety QTL…. …………………………………………………………………..41 2.4.9 Conclusion….. ……………………………………………………………………………….43 2.5 References……….. ……………………………………………………………………………….59 3 General Discussion…………………………………………………………………… ……………..66 3.1 Research conclusions and discussion………….. …………………………………………………66 3.2 Strengths and weaknesses of the research………………………………………………………...67 3.3 Differences between C57BL/6J and DBA/2J mice and the adrenal gland… …………………….68 3.4 The genetic examination of adrenal gland structure…………………………… ………………...72 3.5 Future directions and potential applications of research findings………………………………...73 3.6 References………………………………………………… ……………………………………...76 iii  Appendices……………………………………………………………….…… ………….………………80 Appendix A……………………………………………………………………………… …..………….80  iv  LIST OF TABLES  Table 1.1  BXD stress-related and adrenal structural measures correlations………………..…………..11  Table 2.1  Female mice: Age, body weight, and total adrenal weight measurements…………… ……..44  Table 2.2  Male mice: Age, body weight, and total adrenal weight measurements…………….….……45  Table 2.3  Sex, age, body weight, and adrenal weight correlations…………….. …….…………..….....46  Table 2.4  Female mice: Adrenal total width and zone width measurements..……… ………………....47  Table 2.5  Male mice: Adrenal total width and zone width measurements….…. ………….… ……..…48  Table 2.6  Heritability of phenotypes…………. ……………..………………………………….….…..49  Table 2.7  Age, body weight, adrenal weight, and adrenal width correlations for females… …………..50  Table 2.8  Age, body weight, adrenal weight, and adrenal width correlations for males……. …………50  Table 2.9  BXD adrenal phenotype-related loci and genes ……………………………………….…….50  v  LIST OF FIGURES  Figure 2.1  Sample adrenal width measurements.……..……… ………………….…………………….52  Figure 2.2  Genome-wide linkage map of female BXD adrenal weight …………….…………….……53  Figure 2.3  Genome-wide linkage map of male BXD adrenal weight …………………….……………54  Figure 2.4  Parental strain means for the adrenal width measurements…. ……………………………...55  Figure 2.5  Genome-wide linkage map of male BXD adrenal total width…….……………… …..........56  Figure 2.6  Genome-wide linkage map of male BXD adrenal medulla width…….……………….. …..57  Figure 2.7  Genome-wide linkage map of male BXD x-zone width…………..…………………. ….....58  vi  ACKNOWLEDGEMENTS Animals used in this study were provided primarily by Oak Ridge National Laboratory. Generous funding from the National Institutes of Health (NIH) enabled this project to be performed (DA020677 and AA016666). We thank Richard Cushing, Meifen Lu, Dr. Elissa Chesler, Barbara Jackson, Leslie Galloway, Darla Miller and Dr. Robert Williams for their expert assistance, as well as Dr. Nicole GalloPayet, Dr. James P. Herman, and Dr. Yvonne Ulrich-Lai for their expertise and assistance with adrenal gland endocrinology. We also thank those in the Goldowitz Lab who assisted with this project including Suvina To, Gurjit Rai, Mussawar Ahmed, Christopher Yeh, Derek Rains, and Ann Lu.  vii  1 INTRODUCTION 1.1  Adrenal gland development, structure, and function  The adrenal gland is an endocrine organ, and its function is mediated through the secretion of hormones (Shelton and Jones, 1971; Ehrhart-Bornstein et al., 1998; Li et al., 2002). In all adult vertebrates, the adrenal gland is comprised of the adrenal medulla, the adrenal cortex that surrounds the medulla, and the thin layer of the adrenal capsule that surrounds the organ (Nussdorfer, 1986; Deschepper et al., 2004). The adrenal cortex consists of 3 layers: the outer zona glomerulosa (ZG), the middle zona fasciculata (ZF), and the inner zona reticularis (ZR). In mammalian development, the medulla is derived from the ectodermal neural crest, while the cortex derives from the mesoderm, but they fuse to form the adrenal gland during embryogenesis (Rüsse and Sinowatz, 1998; Wurtman, 2002). In humans, the adrenal cortex begins to develop first at about 4 weeks postconception from a condensation of coelomic epithelial cells on the urogenital ridge, which also form the kidney and gonadal structures (Mesiano and Jaffe, 1997; Keegan and Hammer, 2002). Shortly after, the fetal adrenal and bipotential gonadal cells separate, and the neural crest cells that will form the adrenal medulla begin to migrate to the centre of the gland at around 8 weeks (Fujieda and Tajima, 2005). The fetal adrenal consists of the definitive zone and fetal zone, and during the second trimester, the fetal zone expands until it is larger than the kidney. The definitive zone begins to differentiate into the ZG and ZF at approximately embryonic week 28, while the ZR does not emerge until the end of the third year of life (Fujieda and Tajima, 2005). In early perinatal development, the fetal zone shrinks and degenerates, and cortical zonation from the definitive zone occurs and merges with the adrenal medulla to form the adrenal gland (Keegan and Hammer, 2002; Fujieda and Tajima, 2005). Despite these distinct patterns of development, it is still uncertain how these changes are initiated (Vinson, 2003). The adrenal medulla and the three cortical regions have many unique and important functions associated with endocrine hormone synthesis, such that the medulla and each cortical layer is associated with particular steroidogenic enzymes that secrete distinct steroid hormones and/or neurotransmitters (Parker et al., 1993; Keegan and Hammer, 2002; Fujieda and Tajima, 2005; Ehrhart-Bornstein and Bornstein, 2008). The adrenal medulla is associated with the sympathetic adrenomedullary hormonal system (AHS) and secretes the catecholamines, epinephrine and norepinephrine, and several neuropeptides (Li et al., 2002; Ulrich-Lai et al., 2006; Ehrhart-Bornstein and Bornstein, 2008). The outer ZG secretes the mineralocorticoid, aldosterone, which functions to regulate salt homeostasis and fluid balance. In the middle ZF, glucocorticoids are secreted and have many functions associated with glucose metabolism, among other roles. Cortisol is the major glucocorticoid in humans and various mammals, and it is the primary hormone secreted from the ZF during the HPA-axis mediated stress response. 1  However, the inner ZR also produces glucocorticoids, including cortisol, and also is important for the HPA-axis stress response (Ehrhart-Bornstein and Bornstein, 2008). In addition, the ZR secretes adrenal androgens, which include dehydroepiandrosterone (DHEA) and androstenedione (Andro) that are precursors of estrogen and testosterone, respectively. Despite the customary role that the adrenal gland plays in hormone synthesis and secretion, there are some characteristics and functions that are unique to the murine endocrine system and mice in particular. For instance, the work of Deanesly (1958) suggested that mice secrete androgens via the adrenal cortex, like humans, but it was later found that mice and some other rodents cannot secrete adrenal androgens (VanWeerden et al., 1992; Keegan and Hammer, 2002). Further, unlike other mammals, mice are unable to synthesize cortisol; instead, corticosterone is the primary steroid secreted from the ZF in mice (Heikkilä et al., 2002). The inability of mice to secrete both adrenal androgens and cortisol has been linked to the lack of 17α-hydroxylase expression in the adrenal gland (VanWeerden et al., 1992; Heikkilä et al., 2002). In this regard, mice do not have a functionally distinct ZR, and it likely secretes corticosterone as well. Although Frith (1983) indicated that the ZR is indistinguishable from the ZF in mice, research on various strains of mice shows that the ZR is clearly present (Deschepper et al., 2004), and histological examination of the mouse cortex can be used to clearly distinguish the three cortical layers based on varying cell morphology (Zelander, 1959; Shelton and Jones, 1971; Nussdorfer, 1986). In addition to these layers, cortical zonation in mice includes a thin layer referred to as the zona intermedia (Shelton and Jones, 1971; Nussdorfer, 1986), which is present in only some mammalian species, and a unique transient cortical layer referred to as the X-zone. The X-zone was first identified by Masui and Tamura (1924), as well as by Howard-Miller (1927) and Deanesly (1931). In both male and female mice, the X-zone manifests between approximately postnatal days 10-14 (Keegan and Hammer, 2002). However, this zone regresses in males at about 5-6 weeks of age when they reach sexual maturity (Howard-Miller, 1928; Deacon et al., 1986; Keegan and Hammer, 2002). Meanwhile, the X-zone is found to exist in nulliparous females into adulthood until their first pregnancy, at which point it apparently degenerates as well (Masui and Tamura, 1924; Howard-Miller, 1927; Deanesly, 1931; Sato, 1968; Holmes and Dickson, 1971; Deacon et al., 1986; Tanaka and Matsuzawa, 1995). Although it has been viewed potentially as the equivalent to the fetal zone in human adrenal development (Nussdorfer, 1986; Mesiano and Jaffe, 1997; Keegan and Hammer, 2002; Vinson, 2003), its function is debated and is still uncertain (Gersh and Grollmann, 1939; Keegan and Hammer, 2002; Hershkovitz et al., 2006). Further sex-dependent differences have been found in the structure and function of adrenal glands in mice during development and adulthood. First, pubertal and adult female mice have been shown to exhibit significantly higher adrenal gland weights compared to males (Moog et al., 1954; Badr and 2  Spickett, 1971; Tanaka and Matsuzawa, 1995; Bielohuby, 2007), where both adrenal cortex and medulla weights are found to be higher in females, despite females having significantly lower body weights relative to male mice. This is in contrast to findings with humans, where females have significantly lower adrenal gland and total body weights than males (Rössle and Roulet 1932; Sasano et al., 1956; Kreiner and Dhom, 1979), whereas human adrenal medulla weights were not shown to have a significant sex difference (Kreiner, 1982). Furthermore, Bielohuby (2007) has shown that female mice have larger adrenal glands by volume, as well as show significantly higher ZF cell numbers, and elevated serum corticosterone levels in the ZF compared to males from approximately five weeks onward. In terms of the adrenal cortex volume, the ZG volume is not typically found to be significantly different between male and female mice. Although it is not certain if adrenal cortex volumes are larger in parous females with X-zone regression, based on the previous aforementioned studies with adult mice showing females with higher adrenal gland weights, it is believed that female adrenal cortex volumes would be larger regardless of the presence of the X-zone. This is because the largest cortical zone, the ZF, can account for more than two thirds of the volume of the adrenal gland, medulla included, and it is found to be significantly larger in female mice compared to males (Bielohuby, 2007). Lastly, histochemical staining techniques have shown that although there is a developmental increase in pockets of adipose or unstained fatty tissue visible in both male and female adrenal tissue (Li et al., 2004), female adrenal glands are shown to contain more adipose stored lipids than males (Bielohuby, 2007).  1.2  The adrenal gland and the stress response  Stress is a phenomenon associated with physiological, behavioural, and emotional responses that occur as a consequence of an organism responding inappropriately to real or presumed threats to the organism (Selye, 1956). It was popularized as a scientific concept by the physiologist and endocrinologist Hans Selye (1936), and he defined stress as the nonspecific response of the body to any demand imposed upon it (Selye, 1974). The stress response is the set of physiological, behavioural, and emotional changes that the organism makes in response to the threats or stressors (Solberg et al., 2006; Goldstein and Kopin, 2008). Stress has been viewed as a threat to homeostasis (Solberg et al., 2006; DeRijk et al., 2008), and the stress response was first identified by the physiologist Walter Cannon as a coordinated system of the sympathetic nervous system and the adrenal gland hormone epinephrine (adrenaline) to maintain homeostasis during what he referred to as “fight-or-flight” situations (Cannon and Lissak, 1939). In addition to the sympathoadrenal system, which involves activation of the AHS, stress activates the HPA axis (Goldstein and Kopin, 2008). Acute activation of these systems results in a cascade of compensatory and adaptive behavioural and physiological responses, such as heighted arousal, decreased appetite, and 3  increased respiratory rate, which enhance the survival of the organism, whereas chronic stress can trigger pathophysiologies, including cardiovascular disease, hypertension, diabetes, depression, and anxiety disorders (Chrousos, 1998; Solberg et al., 2006; Goldstein and Kopin, 2008). There are numerous common stress symptoms, such as elevated heart rate, muscle tension, headaches, irritability, anxiety, among others, and there is considerable variability in the responses to acute and prolonged stress exposure. In addition, the stress response is influenced by a variety of genetic and environmental factors (Dumas et al., 2000). As indicated, the adrenal gland plays a vital role in both the acute and prolonged mammalian stress response with the cortex and medulla are known to be activated distinctly during the stress response (Parker et al., 1993). More specifically, when there is a threat to homeostasis, the AHS becomes activated and adrenal medullary chromaffin cells release epinephrine, and to a lesser extent norepinephrine, directly into the blood stream. The release of both catecholamines is stimulated primarily by cholinergic innervation through the splanchnic nerve, which is activated during “fight or flight” situations (Ehrhart-Bornstein and Bornstein, 2008). Meanwhile, the stress response of the adrenal cortex is regulated by neuroendocrine hormones through the HPA axis, where cortisol is released in humans and corticosterone secreted by mice (Keegan and Hammer, 2002; Ehrhart-Bornstein and Bornstein, 2008). During the HPA axis-mediated stress response, the paraventricular nucleus (PVN) of the hypothalamus secretes corticotropin-releasing hormone (CRH), which coordinates and activates the neuroendocrine and behavioural responses necessary to deal with the stressor (Heinrichs and Koob, 2004). CRH triggers increased release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland, which then increases corticosteroid (cortisol and corticosterone) secretion from the adrenal cortex (DeRijk et al., 2008). During acute stress, the elevated levels of corticosteroids induce a negative feedback loop to the pituitary gland, where they bind to low affinity glucocorticoid receptors to regulate the responsiveness of the stress system (de Kloet et al., 1998; de Kloet et al., 2005). Chronic stress can lead to hypercortisolism or constant high levels of circulating glucocorticoids and dysregulation of the HPA axis, which can further affect the regulation of CRH, catecholamines, and serotonin that are associated with the precipitation of depression (Chrousos, 1998; DeRijk et al., 2008). The adrenal medulla and cortex tissues derive from different embryological origins and have different biochemical properties, so it was conventionally viewed that these regions of the adrenal gland were clearly separated and that the AHS and HPA-axis acted independently of one another (Ehrhart-Bornstein et al., 1998). However, these two endocrine tissues are not only morphologically interconnected, but a common mechanism also links and regulates their functions (Nussdorfer, 1996; Ehrhart-Bornstein et al., 1998; Wurtman, 2002; Ulrich-Lai and Engeland, 2005; Ehrhart-Bornstein and Bornstein, 2008). In terms 4  of the stress response, it was formally believed that circulating epinephrine activated the pituitary gland to control the release of ACTH, leading to corticosteroids being released from the adrenal cortex (Wurtman, 2002). However, research by Wurtman and Axelrod (1965), and later Pohorecky and Wurtman (1971), showed that the reverse was occurring, whereby glucocorticoids control epinephrine synthesis and secretion. In their studies, they discovered that high concentrations of glucocorticoids are delivered from the adrenal cortex to the adrenal medulla via an intra-adrenal portal vascular system. In adrenomedullary chromaffin cells, glucocorticoids induce and regulate phenylethanolamine N-methyltransferase (PNMT) (Wurtman, 2002; Ehrhart-Bornstein and Bornstein, 2008), which was found to be the rate-limiting enzyme that synthesizes epinephrine from norepinephrine (Kirshner, 1959). Adrenocortical and adrenomedullary interactions involving glucocorticoids and PNMT are vital for the biosynthesis of epinephrine, which is critical for the autonomic stress response. Thus, such intra-adrenal interactions have been shown to be important for the regulation of the stress response (Wong et al., 2002; EhrhartBornstein and Bornstein, 2008). A recent meta-analysis showed further evidence for a close association between adrenomedullary and HPA axis adrenocortical responses across a variety of stressors, where mean plasma levels of epinephrine and ACTH were highly correlated (r = 0.93), and favoured the concept of a unitary adrenal system (Goldstein and Kopin, 2008).  1.3  Genes and adrenal gland development, structure, and function  As indicated, the adrenal gland is composed of the adrenal cortex and medulla that derive from different embryological origin, and these two endocrine tissues are intimately linked. Furthermore, the structure and function of the adrenal gland are determined by a specific development plan, but it is still uncertain what triggers this distinct developmental pattern to occur. From a genetic perspective, numerous genes have been examined in humans and rodents that are associated with adrenal development and function, which provide important clues for elucidating the nature of this developmental plan of the adrenal. A review of numerous gene mutations found to be related with human and/or rodent diseases or dysfunction of the adrenal gland and adrenogonadal development is provided by Keegan and Hammer (2002). From this review, various genes found to be critical for mouse adrenal development include Wt1, Wnt4 and Wnt11, Sf1, Dax1, Smpd1, Acd, Pomc, and Cited2, while genes associated with obvious biochemical pathways that are important for adrenal function via steroid production and secretion are also well known, and include StAR, Pomc, Sgk1, as well as Sf1, Dax1, and Acd. In terms of adrenal development, Wilms’ tumor 1 (Wt1) is found to be crucial for urogenital ridge development in determining adrenal, kidney, and gonadal cell lineages, and Wt1-knockout (KO) in mice is embryonically lethal (Moore et al., 1999; Vidal and Schedl, 2000). Wingless-related mouse mammary tumor virus integration site 4 (Wnt4) (Vainio and Uusitalo, 2000) and Wnt11 (Lako et al., 1998) also 5  appear to be important for urogenital development, and Wnt4-KO mice show an unspecified adrenal defect and XX females are masculinized (Vainio et al., 1999). Steroidogenic factor 1 (Sf1) and dosage sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome 1 (Dax1) are transcription factors vital for the development and segregation of adrenal cortex and gonads from the adrenogonadal primordium (Keegan and Hammer, 2002; Fujieda and Tajima, 2005). Sf1-KO is lethal with null mice exhibiting no development of the adrenal gland and gonads (Luo et al., 1994). Dax1 appears to play a sexually dimorphic role in gonadal but not adrenal development because Dax1-KO mice do not display adrenal insufficiency and X-zone regression does not occur in both males and females, while only females, and not males, are fertile and show normal gonadal development (Yu et al., 1998). Meanwhile, KO mice of Smpd1 (sphingomyelin phosphodiesterase 1, acid lysosomal) (Carsia et al., 2000b) and mice with a spontaneous mutation of Acd (adrenocortical dysplasia) (Beamer et al., 1994) both display an absence of X-zone formation and abnormal development of the adrenal cortex, but Acd mutants also show adrenal dysplasia. Proopiomelanocortin (Pomc) KO mice exhibit smaller adrenal glands with abnormal cellular composition both pre- and postnatally (Hochgeschwender et al., 2001), and they show severe adrenal gland atrophy in adulthood (Yaswen et al., 1999). Lastly, a mouse KO of Cited2 (CBP/p300-interacting transactivator with ED-rich tail 2) leads to complete adrenal aplasia (Bamforth et al., 2001). Studies have shown that some genes associated with adrenal development and growth are also directly related to adrenal function. Sf1 is associated with regulating the expression of various adrenal hydroxylase genes and is involved with steroid production (Lala et al., 1992). Despite Dax1 not showing sexually dimorphic effects on adrenal development, in humans, it seems to affect adrenal function in males and females differently (Merke et al., 1999) because it is found to interact with androgen and estrogen receptors (Zhang et al., 2000; Holter et al., 2002) and is believed to be expressed at higher levels in females because androgens can downregulate DAX1 transcription (Morohashi et al., 2000). In addition to the growth and development effects, the Acd mutation leads to elevated ACTH, and reduced corticosterone (Beamer et al., 1994), and Pomc-KO mice are shown to exhibit and secrete decreased levels of aldosterone. Since ACTH is integral to regulating the growth of the adrenal cortex, and it is a cleavage product of POMC (Keegan and Hammer, 2002), this suggests that ACTH is essential for normal aldosterone secretion (Coll et al., 2004). Other research has also found evidence of genes important for adrenal steroid production and secretion. Steroidogenic acute regulatory protein (StAR) is believed to be vital to steroidogenesis (Lin et al., 1995; Stocco, 2001), and the StAR-KO mouse exhibits lipoid hyperplasia in the adrenal cortex and significantly low levels of steroids, yet heightened levels of ACTH and CRH (Caron et al., 1997). In 6  addition, mutations or deletions of StAR, along with 21-hydroxylase, 3β- hydroxysteroid dehydrogenase, 17α-hydroxylase, and 11β- hydroxylase, also cause congenital adrenal hyperplasia (CAH), which is a group of diseases associated with an enzymatic defect in the steroidogenesis pathway often leading to glucocorticoid and mineralocorticoid deficiency (Speiser and White, 2003; Fujieda and Tajima, 2005). Serum- and glucocorticoid-regulated kinase-1 (Sgk1) has been determined to be a mediator of aldosterone action, and there is evidence in intact mice that aldosterone leads to a significant increase in Sgk1 expression in the distal tubule of the kidney (Connell and Davies, 2005). Moreover, further studies have shown the significance of Sgk1 in aldosterone action using a transgenic mouse model whose Sgk1 lacks a functional kinase and display mild pseudohypoaldosteronism, among other defects (Wulff et al., 2002). There is also evidence showing significant relationships between circadian-based hormone secretion and gene expression in the adrenal gland in rodents (Wilson et al., 1976; Takahashi et al., 1977; Swan et al., 1978) and rhesus macaque monkeys (Lemos et al., 2006). In the study by Lemos et al. (2006), genome-wide expression profiling analyses were used to show temporal gene expression occurring with a 24-hour rhythm for numerous genes involved in 1) catecholamine synthesis, 2) cholesterol cleavage and dehydroepiandrosterone sulfate(DHEAS) synthesis, 3) protein synthesis and turnover, and 4) the circadian clock mechanism. Among the 322 transcripts identified in the adrenal gland in this study, two Period genes (Per1 and Per2) were shown to display circadian rhythm expression. The circadian expression of these Period genes is suggested to be strongly related to the rhythmic profile of various adrenal functions in mice (Bittman et al., 2003; Ishida et al., 2005). Interestingly, Per1 and Per2 showed peak expression in mice (Bittman et al., 2003) that was opposite to the peak expression of these genes in monkeys (Lemos et al., 2006), which coincided with the nocturnal vs. diurnal nature of mice and monkeys, respectively. Moreover, these studies showed that peak expression of these genes corresponded directly with peak levels of corticosterone in mice and cortisol in monkeys. It has also been shown that light can up-regulate Per1 expression in the adrenal gland of Per1-luc transgenic mice (Albrecht et al., 2001). Lastly, it was found that Per1 and Per2 display different levels of expression between the adrenal cortex and medulla in mice, but both regions show synchronous oscillation of both Per transcripts that are suggested to be regulated by normal light-dark environmental conditions (Bittman et al., 2003; Ishida et al., 2005). Despite the abundance of genetic research associated with adrenal gland development and function, there is much less known about genes related to the structural integrity of the adrenal. In this regard, research in this area has often only found genes associated with the mouse-specific cortical X-zone. In particular, Caspase 2 (Casp2) (Carsia et al., 2000a) and Dax-1-KO (Yu et al., 1998) mice show a disruption in X-zone regression, whereas mice with a spontaneous mutation of Ex (earlier X-zone 7  degeneration) exhibit premature X-zone regression (Shire and Spickett, 1968). However, a spontaneous mutation of Ezg (extent of zona glomerulosa) reduced the growth and size of the ZG (Shire, 1969). In addition, the size and cellular composition of the adrenal gland are affected in Pomc-KO mice (Hochgeschwender et al., 2001). Although the structure of the adrenal gland is intimately linked to a developmental plan, which also connects adrenal gland structure and function, it is not certain how genes, such as Pomc, that are associated with adrenal development and function are directly related to adrenal gland structure. Thus, further genetic examination of adrenal structure is necessary to determine if genetic links are associated with adrenal development, structure, and function, or if the genetic bases of adrenal gland structure is primarily independent of its development and function, which is unlikely the case.  1.4  The relationship between adrenal gland structure and function  The development of the adrenal gland as a single organ with regions derived from different embryological origins that are closely interwoven and have coordinated functions suggest that the structure of the adrenal is linked to its function. With respect to the stress response, the close association between the AHS and HPA activation and the regulation of adrenomedullary epinephrine by adrenocortical glucocorticoids seems to show that the adrenal gland is structured in a manner that enables these distinct but unified functions to be synchronized and optimized for effective response to stress. However, the adrenal gland has other important functions besides the stress response, and stress activates multiple regions of the body and brain. Thus, adrenal structure is potentially affected by each of these multiple functions, which would suggest that its structure may have little to do with its ability to respond to stress. Alternately, the structure of the adrenal gland may be associated more closely to the stress response, compared to other adrenal functions, and examination of this possibility could substantiate the developmental pattern of the adrenal gland that intimately links adrenal regions with unified stress response systems. From this perspective, studies have shown that stress plays an important role in affecting adrenal gland anatomy and structure. In particular, adrenal gland size has been shown to be partially regulated by ACTH stimulation (Bransome, 1968), which is known to regulate stress corticosterone levels during HPA axis activation due to an acute physical or psychological stressor (Solberg et al., 2006). In addition, chronic stress not only leads to elevated basal plasma corticosterone levels in rats (Zelena et al., 2003; Ulrich-Lai et al., 2006) but also increases adrenal weight (Herman et al., 1995; Prewitt and Herman, 1997; Ulrich-Lai et al., 2006). Moreover, a study by Akana et al. (1983) with male rats showed that the trophic effect of ACTH on the adrenal ZF was the most robust determinant of the significant interstrain 8  variability in adrenal weight. In addition, the study by Ulrich-Lai et al. (2006) showed that the stressrelated increases in adrenal weight were coupled with region specific hyperplasia in the ZF, hypertrophy in the ZF and adrenal medulla, and decreased cell size in the ZG. In humans, elevated plasma cortisol levels and adrenal enlargement have been found individuals suffering from chronic stress, such as depressed patients (Amsterdam et al., 1987; Nemeroff et al., 1992), and increases in adrenal weight have been found in individuals who have committed suicide (Dorovini-Zis and Zis, 1987; Szigethy et al., 1994; Dumser et al., 1998). In mice, one study not only showed that significant interstrain variability in adrenal weight existed for male mice, but that lower relative adrenal weight to body weight was found for more stress resistant strains compared to more stress reactive strains (Deschepper et al., 2004). In the study by Deschepper et al. (2004), two inbred strains of mice examined that showed this stressrelated link to adrenal weight were C57BL/6J (B6) and DBA/2J (D2). B6 and D2 mice have been found to differ significantly for various phenotypes, including behavioural measures of stress (Kakihana et al., 1968; Roberts et al., 1992; Tarricone et al., 1995) and anxiety (Trullas and Skolnick, 1993; Ponder et al., 2007). In addition, the divergent genotypes of B6 and D2 strains have been fully sequenced, which has enabled genetic examination of these strains, particularly in association with anxiety- and/or stress-related phenotypes (Roberts et al., 1992; Tarricone et al., 1995; Ponder et al., 2007). Genetic linkage studies have also been conducted for these complex phenotypes, where repeated inbreeding of F2 progeny from B6 and D2 mice have produced large panels of BXD recombinant inbred (RI) strains (Roberts et al., 1995; Tarricone et al., 1995; Mogil et al., 1997; Brigman et al., 2009). After examining the structure of the adrenal gland in B6, D2, and BXD RI mice, unpublished research has shown that adrenal weight and region specific adrenal size is highly correlated with a variety of stress-related measures, including restraint stress, open field behaviour (OFB), and elevated-plus maze (EPM) activity (Table 1.1). These correlational measures were obtained using rich databases (WebQTL) associated with behavioural measures on the mouse, including the differences between B6 and D2 for several stress measures, and showed that robust adrenal structural functional correlations exist. Furthermore, this analysis suggests that a potential genetic link might exist between adrenal structure and function. This possible genetic association can be examined with these strains of mice and comparing complex phenotypes of adrenal structure and function using a gene linkage mapping approach.  1.5  QTL and RI analyses  A Quantitative Trait Locus (QTL) is a genomic region that is associated with or modulates the variation in a measurable phenotype (Williams et al., 1998). A QTL analysis is an invaluable tool for examining the genetic bases for quantifiable and complex phenotypic measures. In understanding the 9  genetic variation associated with complex phenotypes, it is believed that a multitude of genetic loci each share a relatively small modulation of such traits (Lande, 1981), with some of these loci expected to have a more substantial effect on observed phenotypes (Lai et al., 1994). In particular, certain QTLs may account for a comparatively large portion of the variance in different complex traits, which could be analyzed by studying the pattern derived when separating alleles from opposing spectrums at a major QTL (Williams et al., 1998). This approach has been used to investigate genomic regions associated with variation in complex phenotypes in the mouse CNS and anatomical variation in organ weights (Neuschl et al., 2007), as well as physiological and/or behavioural responses to stressors (Tarricone et al., 1995; Mogil et al., 1997; Dumas et al., 2000; Desautes et al., 2002; Jaworski et al., 2002; Cui et al., 2003), but it has not been used to examine the adrenal gland of the mouse. Thus, a QTL analysis of adrenal gland weight and adrenal cortex and medulla size may provide important insight for locating genes or genomic regions that influence adrenal gland structure. Moreover, since the adrenal gland is critical for the stress response, and adrenal structure may be closely associated with the stress response, one potential useful strategy for analyzing the genetics underlying the stress response can involve the genetic examination of the adrenal gland using a QTL approach. In QTL analyses, RI strains are often used to map the chromosomal positions of polymorphic loci that control variance in complex phenotypes. These strains are inbred strains whose chromosomes include a unique and permanent set of homozygous loci that represent recombinations of chromosomes with proportional genetic contributions from the parental strains (Gill et al., 1996; Peirce et al., 2004). They are derived by crossing two inbred strains to produce an F1 generation, which is followed by inbreeding different sets of F2 progeny for 20 or more consecutive generations of brother x sister mating (Taylor, 1978; Plomin and McClearn, 1993; Lad et al., 2007). They are typically studied in sets or panels, and the larger the set of RI strains, the greater the statistical power and mapping resolution that phenotypes are associated with particular genomic regions. In this regard, when there is accessible genotypic data compiled and an abundance of animals available for investigation of each RI strain (Lad et al., 2007; Reiner et al., 2008), RI analyses are useful for examining complex traits and performing genetic linkage mapping of QTLs because the genes associated with specific phenotypes in the progenitor strains are located separately across the different RI strains, and certain associations that occurred during inbreeding of the parental lines are likely to be dissociated (McClearn et al., 1991). Lastly, broad-sense heritability estimates can also be conducted using RI analyses, which is beneficial for optimizing trait expression of varying phenotypes that can be genetically mapped and correlated (Chesler et al., 2005; Plomin et al., 2005).  10  In the current study, adrenal structural measures were performed using BXD RI strains because of the observable phenotypic differences found between their progenitor strains for stress and anxiety-related measures. From this perspective, the aim of this study was two-fold. First, a direct examination of the morphology of the adrenal gland in the parental and BXD RI strains was used to determine if there were strain and sex differences in adrenal gland weight and size, which may be associated with specific QTLs. Second, by using inbred strains of mice that differ significantly in terms of anxiety and the stress response, an indirect examination of genotypic and/or phenotypic connections between adrenal structure and function was conducted using bioinformatics and correlational analyses of previously investigated phenotypic measures and QTLs associated with stress. In this regard, although QTL approaches have been used to examine adrenal weight while performing genetic analysis of the stress response in rats (Llamas et al., 2005; Solberg et al., 2006), to our knowledge, this paper reports the first exploration of QTLs involved in two aspects of the adult mouse adrenal: weight and anatomical structure.  Table 1.1: BXD stress-related measures correlated with adrenal structural measures Male Adr Weight  Fem Adr Weight -0.538*  Male Tot Width  Restraint Stress -0.350 -0.262 Saline 10min Restraint Stress -0.409 -0.603* -0.415 Saline 5min OFB Dist per -0.249 -0.472 -0.157 20-25min OFB Dist per -0.357 -0.422 -0.126 15-20min EPM # of Arm -0.112 -0.034 -0.268 Entries EPM % Time in 0.173 0.133 0.389 Open Arms EPM Open Arm 0.107 0.014 0.118 Entries Cort Resp to -0.476 0.250 0.214 Forced Swim Cort. Level 7hr 0.082 0.223 0.130 After EtOH Cort. Level 1hr 0.346 0.172 0.406 After Saline *Correlation is significant at the 0.05 level (2-tailed).  Fem Tot Width -0.613*  Male Fasc Width  Fem Fasc Width  Male Med Width  Fem Med Width  -0.175  -0.308  -0.300  -0.129  -0.665*  -0.151  -0.383  -0.405  -0.188  -0.593*  0.139  -0.313  -0.386  -0.196  -0.536*  0.182  -0.210  -0.447  -0.182  -0.154  -0.371  -0.560*  0.076  0.264  0.110  -0.101  -0.211  0.694*  -0.250  -0.115  -0.471  -0.564*  0.553*  -0.064  0.292  0.167  0.167  0.454  0.690  0.401  -0.199  0.095  0.160  0.530*  -0.096  0.573*  0.164  0.150  0.267  11  1.6  References  Akana, S.F., Shinsako, J, and Dallman, M.F. (1983). Relationships among adrenal weight, corticosterone, and stimulated adrenocorticotropin levels in rats. Endocrinology, 113: 226-231. Albrecht, U., Zheng, B., Larkin, D., Sun, Z.S., and Lee, C.C. (2001). MPer1 and mper2 are essential for normal resetting of the circadian clock. J Biol. Rhythms, 16: 100-104. Amsterdam, J.D., Marinelli, D.L., Arger, P., and Winokur, A. (1987). 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Wulff, P., Vallon, V., Huang, D.Y., Volkl, H., Yu, F., Richter, K., Jansen, M., Schlunz, M., Klingel, K., Loffing, J., Kauselmann, G., Bösl, M.R., Lang, F., and Kuhl, D. (2002). Impaired renal Na(+) retention in the sgk1-knockout mouse. Journal of Clinical Investigation, 110: 1263-1268. Wurtman, R.J. (2002). Stress and the adrenocortical control of epinephrine synthesis. Metabolism, 51(6 Suppl 1): 11-14. Wurtman, R.J., and Axelrod, J. (1965). Adrenaline synthesis: Control by the pituitary gland and adrenal glucocorticoids. Science, 150: 1464-1465. Yaswen, L., Diehl, N., Brennan, M.B., and Hochgeschwender, U. (1999). Obesity in the mouse model of proopiomelanocortin deficiency responds to peripheral melanocortin. Nat. Med., 5: 1066-1070. Yu, R.N., Ito, M., Saunders, T.L., Camper, S.A., and Jameson, J.L. (1998). Role of Ahch in gonadal development and gametogenesis. Nat. Genet., 20: 353-357.  18  Zelander, T. (1959). 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In all adult vertebrates, the adrenal gland is comprised of the adrenal medulla and three layers of the adrenal cortex that surround the medulla (Nussdorfer, 1986; Deschepper et al., 2004). These cortical regions include the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR). In mammalian development, the medulla and cortex are derived from different germ layers that later fuse to form the adrenal gland during embryogenesis (Rüsse and Sinowatz, 1998; Wurtman, 2002). The medulla and each cortical region have many important functions associated with endocrine hormone synthesis, and the cortex and medulla are known to be activated distinctly during the stress response (Parker et al., 1993). Adrenal medullary chromaffin cells contain catecholamines and several neuropeptides (Li et al., 2002; Ulrich-Lai et al., 2006) that are released directly into the blood stream during “fight or flight” situations, while the ZF of the adrenal cortex secretes cortisol during the stress response and is regulated by neuroendocrine hormones through the hypothalamic-pituitary-adrenal (HPA) axis (Keegan and Hammer, 2002; Fujieda and Tajima, 2005; Ehrhart-Bornstein and Bornstein, 2008). However, both stress response systems are intimately linked (Wurtman, 2002; Ehrhart-Bornstein and Bornstein, 2008; Goldstein and Kopin, 2008) and activate the adrenal cortex and medulla despite their divergent germ layer development, which suggests that the structure of the adrenal gland plays a role in its functions. Hence, an examination of adrenal structure may provide important findings to help narrow the search for understanding more about adrenal function and the stress response. Although the adrenal gland has similar structural features and functions in all mammalian species, including the stress response, there are some structural characteristics and functions that are unique to the murine endocrine system and mice in particular. For instance, it was found that mice and some other rodents cannot secrete adrenal androgens from the ZR (VanWeerden et al., 1992; Keegan and Hammer, 2002), and they secrete corticosterone from the ZF because they are unable to synthesize cortisol (Heikkilä et al., 2002). The inability of mice to secrete both adrenal androgens and cortisol has been linked to the lack of 17α-hydroxylase expression in the adrenal gland (VanWeerden et al., 1992; Heikkilä et al., 2002). In terms of structure, histological examination of the mouse cortex shows that in addition 1  A version of this chapter will be submitted for publication. Di Curzio, D.L. The Genetic Basis for Adrenal Gland Structure.  20  to the three primary cortical zones distinguished by varying cell morphology (Zelander, 1959; Shelton and Jones, 1971; Nussdorfer, 1986; Deschepper et al., 2004), mice also display a thin layer referred to as the zona intermedia (Shelton and Jones, 1971; Nussdorfer, 1986) and a transient cortical layer referred to as the X-zone, which was first identified by Masui and Tamura (1924), Howard-Miller (1928), and Deanesly (1931). There are also sex-dependent differences in the structure and function of the adrenal gland in mice. In particular, pubertal and adult female mice exhibit significantly higher adrenal cortex, adrenal medulla, and whole adrenal gland weights compared to males (Moog et al., 1954; Badr and Spickett, 1971; Tanaka and Matsuzawa, 1995; Bielohuby, 2007) despite females having significantly lower body weights relative to male mice. Female mice also have larger adrenal glands by volume, show elevated serum corticosterone levels, and contain more adipose stored lipids compared to males from approximately 5 weeks of age onward (Bielohuby, 2007). In addition, although the function of the X-zone is debated and is still uncertain (Gersh and Grollmann, 1939; Keegan and Hammer, 2002; Hershkovitz et al., 2006), this zone typically disappears in males at about 5-6 weeks of age (Howard-Miller, 1928; Deacon et al., 1986; Keegan and Hammer, 2002), yet it remains present in nulliparous females into adulthood until their first pregnancy when it degenerates as well (Masui and Tamura, 1924; Howard-Miller, 1928; Deanesly, 1931; Deacon et al., 1986; Sato, 1968; Holmes and Dickson, 1971; Tanaka and Matsuzawa, 1995). Adrenal gland development, structure, and function have also been examined from the genetic perspective (Badr and Spickett, 1971; Pawlus, 1983; Janat and Shire, 1987; Tanaka et al., 1994; Lin et al., 1995; Moore et al., 1999; Vidal and Schedl, 2000; Lemos et al., 2006; Solberg, 2006). Many studies have documented genes associated with adrenal structure and function in rodents and humans. A review of numerous gene mutations related to human and/or rodent diseases or dysfunction of the adrenal gland and adrenogonadal development, as well as rodent gene knockout experiments, is provided by Keegan and Hammer (2002). Their review indicates that some mutations in different genes can result in comparatively similar adrenal-related phenotypes. Mutations of these same genes, however, also results in multiple adrenal structural and functional alterations indicating that the genetic underpinnings of adrenal gland structure and function are also complex. When examining the genetic nature of complex phenotypes, such as adrenal structure and function, quantitative trait locus (QTL) analyses are often effective for locating regions in the genome that are associated with variation in such phenotypes (Williams et al., 1998). As the adrenal gland is critical for the stress response, one useful strategy for analyzing the genetics underlying both adrenal structure and the stress response is the use of groups of inbred and recombinant inbred (RI) mice that express genetically mediated differences in stress response and anxiety. RI analyses are valuable for examining complex traits and performing QTL mapping, when 21  there is accessible genotypic data compiled and an abundance of animals available for investigation of each RI strain (Lad et al., 2007; Reiner et al., 2008). In the current study, BXD RI strains were used. These mice are derived from the C57BL/6J (B6) and DBA/2J (D2) strains, which have been found to differ significantly for various phenotypes, including behavioural stress responses (Kakihana et al., 1968; Roberts et al., 1992; Tarricone et al., 1995) stress responses to and consumption of alcohol (Eleftheriou and Elias, 1975; Belknap et al., 1993; Gill et al., 1996) as well as behavioural displays of anxiety (Trullas and Skolnick, 1993). The D2 mice show heightened anxiety-related behavioural responses during dark–light (DL) testing (Võikar et al., 2005), elevated plus maze (EPM) measures (Yilmazer-Hanke et al., 2003; Võikar et al., 2005), fear-sensitized acoustic startle response (ASR) paradigms (Yilmazer-Hanke et al., 2003), open field (Tarricone et al., 1995; Ponder et al., 2007a), and elevated zero maze (EZM) testing and contextual fear conditioning (Ponder et al., 2007a), as well as restraint stress (Tarricone et al., 1995) compared to B6 mice. Due to the observable phenotypic differences found between these inbred strains, a multitude of studies have endeavoured to identify “candidate” genes for QTLs that may cause or influence the phenotypic differences between B6 and D2 mice (Gill et al., 1996). Thus, the aim of this study was two-fold. First, we directly analyzed the structure of the adrenal gland in the parental and BXD RI strains to determine if there were strain differences in adrenal gland weight and size that could be quantified and examined with a QTL approach. Second, by using inbred strains of mice that differ significantly in terms of anxiety and stress response, we wanted to indirectly examine whether there are genotypic and/or phenotypic connections between adrenal structure and function, particularly with respect to the adrenal stress response. In the present study, we measured total left and right adrenal weights and obtained adrenal total, cortical, and medulla regional width measurements in 64 BXD RI and both parental strains of mice. These measurements were performed to collect data that was used to detect QTLs and candidate genes that might be associated with strain- and sex-related anatomical differences in adrenal gland size. We identified two significant QTLs for adrenal gland weight on Chr 3 and 4 and suggestive QTLs on multiple chromosomes. For the adrenal width measurements, we found significant QTLs on Chr 5, 10, and 14 for the male adrenal measures but not for the female adrenal measures. However, we did find multiple suggestive QTLs for both male and female measures on various chromosomes with some of the regions overlapping. We also draw attention to a list of 114 genes located within these loci that are suggested to account for phenotypic differences amongst the parental and RI strains analyzed. From the QTL analyses and a bioinformatics based investigation of the QTLs, we identified five candidate genes potentially linked to adrenal gland structure. Lastly, although QTL approaches have been used to examine adrenal 22  weight while performing genetic analyses of the stress response in rats (Llamas et al., 2005; Solberg et al., 2006), to our knowledge, this paper reports the first exploration of QTLs involved in two aspects of the adult mouse adrenal: weight and anatomical structure.  2.2 2.2.1  Methods Animals  A total of 591 mice were used in this study, which included 64 BXD RI strains (n = 571, mean = 8.92 mice per strain) and 2 parental strains (C57BL/6J and DBA/2J, n = 20). There were approximately an equal number of males and females used (n = 294 and 297, respectively). The mice were examined between 40 and 86 days of age (mean ± standard error of the mean = 57.69 ± 0.35). The animals were produced at the Oak Ridge National Laboratory (ORNL) and then were transferred to the University of Tennessee Animal Facility in a temperature controlled van. At the facility, the mice were housed in a specific pathogen-free environment, which was kept at an approximate temperature of 23.5 ºC on a 12-h light/dark cycle with 45-50% humidity. They were maintained on a diet of 5% fat Agway Prolab 3000 rat and mouse chow. All experimental procedures were conducted according to the Principles of Laboratory Animal Care protocols (NIH publication No. 86-23, revised 1985) and were approved by the Institutional Animal Care and Use Committee at the University of Tennessee Health Science Center.  2.2.2  Adrenal removal and weight analysis  Whole cages of BXD mice were obtained and placed on a cart at one end of the room, separated as far as they can be from the area where the mice were sacrificed. Mice were then removed from each cage and weighed, and then placed in a separate cage on its own. After 10 minutes, each mouse was then quickly cervically dislocated, and their ventra were opened to provide access to the abdominal cavity. Whole kidneys were then extracted from the body one at a time, careful to include the adrenal glands located at the anterior tip of each organ. The adrenal glands were then carefully dissected from the kidneys and surrounding tissue under a Zeiss dissecting microscope. Lastly, the adrenal glands were weighed on a Mettler Toledo scale to a tenth of a milligram and placed in vials overnight in Bouin's Fixative (75ml Picric Acid: 25ml 37% Formalin: 5ml Glacial Acetic Acid).  2.2.3  Tissue preparation and histology  After fixation, the adrenals were embedded in paraffin blocks and positioned in a horizontal plane for sectioning. There were 2-5 adrenals embedded in each block that were placed together according to histological date sacrificed and by sex. The embedded adrenal glands were sectioned using a Leitz 23  rotating microtome (Leitz 1512; Leitz, Wetzlar, Germany) at 8 microns (μm). Two consecutive sections out of every 20 were mounted on Superfrost Plus slides (Fisher Scientific, Hampton, NH, USA) and stained with hematoxylin and eosin (H & E) for histological observation. After identifying the section that contained the most extensive region of each adrenal, additional sections were mounted +/- 10 from this section and stained with H & E. These latter sections were examined to select the section with the largest adrenal area, and structural measures were performed on this adrenal section.  2.2.4  Microscopy  All adrenal gland sections were analyzed using bright-field light microscopy. Morphometric examination was conducted using a Zeiss Axiovert 200 inverted light microscope with a Zeiss AxiocamHR colour camera, which was attached to a computerized Zeiss Axiovision 4.6 imaging system (Zeiss, Jena, Germany). The examination included quantitative measurements of the adrenal cortical zones and medulla widths, as well as qualitative assessment of the presence of the X-zone region and adipose tissue (lipoid zone). For the total width and some medulla measurements, images of the adrenal sections were taken using a 5X objective. To obtain more precise measures of the cortical regions and the medulla, the tissue was typically viewed with a 10X objective and optovar setting at 1.60.  2.2.5  Adrenal cortex and medulla measurements  The adrenal width measurements were taken at the approximate “midline” where the width of the adrenals was the greatest across the horizontal sections analyzed. The total width and width of each region was measured along this midline, with the cortical zone widths being averaged for the measures taken on either side of the medulla (n = 2). The H & E staining provided visible morphological borders between each of the zones and medulla (See Figure 2.1).  2.2.6  Statistical analysis and QTL mapping  The adrenal weight and regional width measurements were acquired from 64 BXD lines and both parental strains in this study. A linear model was used to examine the effects of factors that may affect the adrenal measures being investigated. Sex, age, body weight, and the respective phenotypes of each BXD strain were taken into consideration in the analyses. Statistical analyses were conducted using the SPSS 16.0 software program. Both quantitative and qualitative assessments were analyzed separately for the different BXD strains and for male and female mice with MANOVA and GLM analyses. Two-tailed Student’s t-tests were conducted to compare the male and female data. In addition, separate Pearson’s R correlations were performed to compare the adrenal width data for each region with the total adrenal 24  weight data. For all analyses, p values ≤ 0.05 were deemed statistically significant. In addition, broadsense heritability measures were conducted, which was performed using a method derived from Hegmann and Possidente (1981) that compares between-strain and total differences, with the calculation h2 = VA/(VA + 2VE), where VA = genetic variance and VE = environmental variance. QTL mapping analyses were performed for the adrenal gland weight and size measures. These analyses classified the BXD strains according to their genotypes using distinct chromosomal markers and compared them separately with the adrenal weight and width phenotypic variables. The significant and suggestive genome-wide loci were generated using 1000 permutation tests that randomly reassign trait values across the strains and compare the permuted and original data to examine the significance of the QTL(s). Both the likelihood ratio statistic (LRS) and logarithmic of odds (LOD) values (LOD = LRS/4.61) were used to measure the linkage between quantitative phenotypic variations and genetic differences at a particular genetic locus (QTLs). The QTL maps were all generated using GeneNetwork (WebQTL) (www.genenetwork.org). Lastly, we surveyed the gene list mapped within the significant QTLs using the National Center for Biotechnology Information (NCBI) Entrez Gene website (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) and the Jackson Laboratory’s MGI Mouse Genome Database project (www.informatics.jax.org) to identify potential candidate genes.  2.3 2.3.1  Results Adrenal weight measurements  The overall measures of whole adrenal weights for both parental strains and all 64 BXD lines for the female and male mice show that females (2.43 ± 0.03 mg) have significantly heavier adrenal glands than males (1.72 ± 0.02 mg; F1,589 = 542.25, p < 0.0005). As would be expected, the relative adrenal weight to body weight was also significantly higher for females compared to males (F1,589 = 986.18, p < 0.0005). In addition, there are significant strain differences in actual adrenal weight (F65,525 = 2.72, p < 0.0005) and relative adrenal weight (F65,525 = 1.93, p < 0.0005), and these strain differences are apparent for both measures in females (F65,231 = 7.10, p < 0.0005; F65,231 = 9.40, p < 0.0005) and males (F65,228 = 6.36, p < 0.0005; F65,228 = 6.24, p < 0.0005). The strain means for adrenal weight, age, body weight, and relative adrenal weight to body weight are shown separately for females and males in Tables 2.1 and 2.2, respectively. Age, sex, body weight, and left-right adrenal weight were also analyzed using Pearson product-moment correlations, and the results show that both age and sex are positively correlated with adrenal weight, while body weight is negatively correlated with adrenal weight (Table 2.3). Sex shows a highly significant correlation with adrenal weight (r = 0.69) and accounts for 48% of the variance in adrenal weight. When controlling for sex, age was only significantly correlated with adrenal weight in 25  females (r = 0.39, p < 0.0005) but not males. Meanwhile, the significant negative correlation between body weight and adrenal weight (r = -0.23, p < 0.0005) reflects the fact that males have lighter adrenal glands but weigh more (21.79 ± 0.19 g) than the females (17.95 ± 0.15 g) who have heavier adrenal glands. Lastly, broad-sense heritabilities (h2) confirm the robust link between sex and adrenal weight because the heritability values for adrenal weight for males and females are much higher than when heritability is calculated across both sexes (Table 2.6). For the parental strains, the main effects for both body weight and adrenal weight show that they do not differ significantly (p = 0.281 and p = 0.237, respectively), despite the B6 mice showing lower values for average body weight (19.89 ± 0.80g) and adrenal weight (1.83 ± 0.17mg) compared to D2 mice, which have an average body weight of 21.50 ± 1.13g and average adrenal weight of 2.12 ± 0.17mg. When accounting for sex differences within the parental strains, we find that body weight and adrenal weight still show no significant difference for both males and females; however, the D2 mice still exhibit slightly higher values for both males and females for body weight (23.22 ± 1.90 and 20.07 ± 1.19g) and adrenal weight measures (1.91 ± 0.23 and 2.30 ± 0.23mg) compared to the B6 male (22.30 ± 0.24g and 1.39 ± 0.14mg) and female (17.96 ± 0.41g and 2.18 ± 0.16mg) mice. Despite this, for both body weight and adrenal weight, there are significant differences between males and females across the parental strains (p = 0.007 and p = 0.016, respectively) with males having a higher body weight (22.81 ± 1.02g) and lower adrenal weight (1.68 ± 0.16mg) compared to females that have an average body weight of 19.11 ± 0.72g and average adrenal weight of 2.25 ± 0.14mg.  2.3.2  QTL influencing adrenal weight  The QTL mapping of adrenal weight was performed for each sex using the 64 BXD RI and parental strains. An interval genome-wide QTL map of female adrenal weight shows a significant QTL located on Chr 3 and four suggestive QTLs are found on Chrs 1, 3, 10, and 14 (Figure 2.2A). The significant QTL is termed Fawq1 for “female adrenal weight QTL,” and Figure 2.2B shows a closer view depicting it mapped at a distal region of Chr 3. After performing a marker regression analysis using WebQTL, Fawq1 is found to have a LRS value of 20.605 (LOD score = 4.470) (Table 2.9). This QTL spans a fairly small region of Chr 3, with the 1-LOD confidence interval between 127.0-129.5 Mb. Meanwhile, an interval whole-genome QTL map of male adrenal weight reveals a significant QTL mapped on Chr 4 along with several suggestive peaks on Chr 4, as well as five suggestive QTLs shown on Chrs 2, 10, 17, and X (Figure 2.3A). The significant QTL is termed Mawq1 for “male adrenal weight QTL” and is depicted in Figure 2.3B to be mapped at a distal region of Chr 4. Marker regression analysis using  26  WebQTL calculates a LRS value of 19.226 (LOD score = 4.170) for Mawq1 (Table 2.9). The QTL spans a fairly small region of Chr 4, with the 1-LOD confidence interval between 99.5-102.5 Mb.  2.3.3  Adrenal width measurements  The adrenal width data from the 64 BXD and parental strains for female mice analyzed (Table 2.4) show that the total width, as well as the ZF and X-zone widths are typically larger than those obtained from the 64 BXD and parental strains for male mice tested (Table 2.5), which often exhibit larger adrenal medulla and slightly larger ZR measures than females. Both tables also show that there are substantial strain differences in all regional adrenal measures, except for the female ZG (p = 0.064) and ZI (p = 0.052) width measures. In addition, broad-sense heritabilities (h2) were calculated across both sexes and separately for males and females for adrenal width. The results show that higher heritability values correspond to the adrenal phenotypes shown in Tables 2.4 and 2.5 that vary significantly between the strains and sexes (Table 2.6). Correlational analysis of the adrenal gland regional measures of width for the BXD and parental strains were compared with each other and with adrenal weight. Total adrenal width correlates significantly with all the regional width measures (data not shown) and with adrenal weight (r = 0.62, p < 0.0005) and accounted for 39% of the variance in adrenal weight (F1,438 = 275.917, p < 0.0005). Adrenal medulla width correlates positively with total adrenal width (r = 0.45, p < 0.0005) and negatively with ZF width (r = - 0.17, p < 0.0005), X-zone width (r = -0.20, p < 0.0005), and with adrenal weight (r = -0.10, p = 0.034). The ZG width shows significant correlations with total adrenal width (r = 0.20, p < 0.0005), ZI width (r = 0.30, p < 0.0005), and ZF width (r = 0.17, p < 0.0005). Aside from the ZG, the ZI width also correlates significantly with total adrenal width (r = 0.14, p = 0.003) and ZF width (r = 0.22, p < 0.0005). Meanwhile, ZF width correlates significantly with all the width measures, except for the ZR, as well as with adrenal weight (r = 0.47, p < 0.0005). Besides the total width, the ZR width only correlates significantly with the X-zone width (r = 0.11, p = 0.028), but it also shows a significant correlation to adrenal weight (r = 0.14, p = 0.003). Lastly, the X-zone correlates significantly with most width measures, except for the ZG and ZI, and shows a robust correlation with adrenal weight (r = 0.71, p < 0.0005), accounting for 51% of the variance in adrenal weight (F1,438 = 447.316, p < 0.0005). As sex plays a prominent role in accounting for differences in adrenal weight, the adrenal width measures were controlled by sex and compared separately for males and females. The results show that sex also has a significant main effect on most of the adrenal width measures, including the total, medulla, ZF, ZR, and X-zone widths, and it accounts for a significant amount of the total variance for these measures ranging from 1.1% to 76%. For the female data, several correlations are similar to the total data 27  across sex, with some less and others more significantly robust (Table 2.7). Despite this, various correlations are no longer significant for the female width data, including those between the total width and ZI, the medulla and the ZF and X-zone, respectively, and the ZF with the X-zone and weight, respectively. The male length data also shows disparity with the total data with the correlations displayed in Table 2.8. Among these various differences, the medulla width is no longer significantly correlated with the ZF and X-zone; the ZF is no longer significantly correlated with the ZG, but is now significantly correlated with the ZR (r = 0.14, p = 0.037); and the ZR is no longer significantly correlated with adrenal weight. For the parental strains, the adrenal width measurements show significant differences for the medulla (p = 0.039), the ZF (p = 0.001), and ZI (p = 0.022) measures. Although the average total width of the adrenal for the B6 mice (1127.80 ± 30.33μm) is higher than the mean for the D2 mice (1058.51 ± 53.99μm), they are not shown to differ significantly (p = 0.307). Meanwhile, the ZG, ZR, and X-zone exhibit similar width measures between the parental strains. In terms of the significant differences, the D2 mice (483.10 ± 26.40μm) have a larger medulla width compared to the B6 mice (412.94 ± 12.78μm), whereas the B6 mice have larger ZF (169.85 ± 13.34μm) and ZI (18.66 ± 1.47μm) widths in comparison to the D2 mice (113.06 ± 7.25μm and 14.03 ± 1.16μm, respectively). Again, we accounted for sex differences within the strains and find that only the ZF width is significantly larger for the B6 mice for both males (143.66 ± 7.76μm, p = 0.029) and females (190.80 ± 18.91μm, p = 0.006) compared to the D2 mice (110.98 ± 8.63μm and 114.78 ± 11.91μm, respectively). Meanwhile, the medulla width is only significantly larger for the D2 female mice (523.91 ± 40.02μm, p = 0.032) and not the male mice (p = 0.754) compared to the B6 mice, while the ZI width is no longer significantly different between the strains for the female mice (p = 0.165) and only approaching significance for the male mice (p = 0.074). In addition, the total adrenal width is now significantly larger for the B6 male mice (1065.04 ± 39.83μm) in comparison to the D2 male mice (906.12 ± 38.56μm; p = 0.025). Lastly, the ZR width displays a unique pattern, where the male mice are significantly different (p = 0.013) with the B6 mice (65.39 ± 6.60μm) exhibiting a wider ZR region than the D2 mice (44.88 ± 1.94μm), whereas the female mice are approaching significance (p = 0.059) but the D2 mice show a larger ZR (70.50 ± 4.41μm) than the B6 mice (52.49 ± 7.49μm) (Figure 2.4).  2.3.4  QTL influencing adrenal width  The QTL mapping for all adrenal width measurements was calculated separately for the male and female mice using the 64 BXD RI and parental strains. There are some suggestive QTLs shown for most of the regional width measures for both males and females. For the female data, two suggestive QTLs are 28  located on Chr 2 for total adrenal width; several suggestive QTLs are located on Chr 4 and one on Chr 17 for ZF width; one suggestive QTL is located on Chr 7 for ZG width; two suggestive QTLs on Chr 7 for ZI width; suggestive QTLs are found on Chr 3, 10, and 17 for ZR width; one suggestive QTL on Chr 3 for X-zone width; and three suggestive QTLs on Chr 3, two on Chr 16, and one on Chr 7 for medulla width (data not shown). Meanwhile, for the male data, the ZF width shows several suggestive QTLs on Chr 4, 7, and 10, two on Chr 6, and one each on Chr 13 and 19. In addition, the male ZI width shows a suggestive QTL on Chr 3, while a suggestive QTL is found on Chr 13 for ZR width (data not shown). Significant QTLs were found for total adrenal, ZF, and X-zone width measures but only for the males. An interval genome-wide QTL map of male total width displays a significant QTL located on Chr 10 and a suggestive QTL on Chr 13 (Figure 2.5A). When examining Chr 10 more closely, Figure 2.5B shows the interval map, where the significant QTL is mapped at a distal region and is termed Mawdq1 for “male adrenal width QTL.” A marker regression analysis reveals a LRS value of 18.771 (LOD score = 4.072) for Mawdq1 (Table 2.9), and a 1-LOD confidence interval displays it spanning a small region between 119.0-120.0 Mb on Chr 10. Meanwhile, an interval genome-wide QTL map of male adrenal medulla width depicts a significant QTL mapped on Chr 5 and a suggestive QTL on Chr 1 (Figure 2.6A). By viewing Chr 5 more closely, Figure 2.6B shows the interval map, where the significant QTL is mapped at a distal region and termed Mmwdq1 for “male medulla width QTL.” A marker regression analysis reveals a LRS value of 19.262 (LOD score = 4.178) for Mmwdq1 on Chr 5 (Table 2.9), and a 1-LOD confidence interval displays it spanning a region between 140.7-143.5 Mb. Lastly, an interval genome-wide QTL map of male X-zone width shows significant QTLs located on Chr 10 and 14 (Figure 2.7A). The significant QTLs are termed Mxwdq1 for “male X-zone width QTL 1” on Chr 10 and Mxwdq2 for “male X-zone width QTL 2” on Chr 14. Upon closer inspection of Chr 10, Figure 2.7B depicts the interval map, where Mxwdq1 is mapped at a distal region. Marker regression analysis shows a LRS value of 12.934 (LOD score = 2.806) for the peak of Mxwdq1 on Chr 10 (Table 2.9), and a 1-LOD confidence interval depicts it spanning a region between 106.7-110.5 Mb. When looking closer at Chr 14, Figure 2.7C depicts the interval map, where Mxwdq2 is mapped at a proximal region. Marker regression analysis shows a LRS value of 12.918 (LOD score = 2.802) for Mxwdq2 on Chr 14 (Table 2.9), and a 1-LOD confidence interval displays it spanning a region between 36.5-39.5 Mb.  2.3.5  X-zone and adipose analysis  As expected, the X-zone is present in all female mice analyzed, since they are all nulliparous, but unexpectedly, the X-zone is also present in male mice of some BXD strains. For the females, the width of the X-zone varies significantly amongst the BXD strains from 27.42µm for BXD67 to 86.36µm for 29  BXD69 (F65,155 = 2.59, p < 0.0005). This strain-related disparity seems to be associated with the presence of fatty adipose tissue. In the adrenal glands examined, adipose is primarily found between the ZR and X-zone, and it is only present in female mice. It typically appears as a series of densely-packed globules that develop and permeate the morphological border between these two inner cortical regions, forming a distinct lipoid zone. In 20 of the 64 female BXD lines examined, the X-zone is significantly larger compared to all strains for the width (F1,293 = 22.18, p < 0.0005). In 19 of these 20 strains, adipose tissue is densely dispersed throughout the inner cortical region, while four other strains show some adipose tissue developing along this morphological border. Furthermore, the presence of this lipoid zone is associated with significantly higher adrenal weights in the 23 strains compared to all female strains examined (F1,303 = 9.24, p = 0.003). Meanwhile, eight of the 64 male BXD strains examined (BXD15, 16, 18, 33, 36, 38, 51, and 56) possess a visible X-zone region. Although the X-zone region is significantly smaller in these male strains in terms of width (F1,245 = 72.24, p < 0.0005) compared to female mice tested, five of these eight strains show the X-zone present in all animals tested within the strains. In addition, age is negatively correlated with X-zone width (r = -0.54, p = 0.005) in these eight strains. Moreover, in the three BXD lines where only some of the animals in each strain display a visible X-zone, age is still negatively correlated with Xzone width (r = -0.62, p = 0.032), such that those with a visible X-zone are typically younger than those not displaying an X-zone.  2.3.6  Genes in QTL regions  In total, there are 114 genes mapped within the six significant QTL regions (Table 2.9). There are 39 genes mapped in the significant QTL regions for adrenal weight. For the female mice tested, 19 genes are located within the Chr 3 interval of 127.0-129.5 Mb. For the male mice, there are 20 genes located in Chr 4 in the QTL interval of 99.5-102.5 Mb. Meanwhile, there are 11 genes within the significant QTL region for total adrenal width. These 11 genes are located in the Chr 10 interval of 119.0-120.0 Mb for the male mice data. In addition, there are 29 genes within the significant QTL regions for male adrenal medulla width. The 29 genes are located in the interval of 140.7-143.5 Mb on Chr 5. Lastly, for the male X-zone width data, there are 35 genes located in the significant QTL regions. The first significant QTL region has 26 genes located in the interval of 106.7-110.5 Mb on Chr 10, while the second QTL region has 9 genes within the significant interval of 36.5-39.5 Mb on Chr 14.  30  2.4 2.4.1  Discussion Overview  Stress is a complex physiological and behavioural response, and the genetic underpinnings of this phenomenon have been examined in various studies. The adrenal gland is the efferent limb of the HPAaxis, and it is critical for physiological responses to stress. The structure of the gland is such that different regions have distinct functional roles, including specific responses to stress. The adrenal cortex is involved in the HPA-axis mediated stress response, while the adrenal medulla is associated with the autonomic “fight or flight” stress response. From this perspective, the gross structure of the gland is directly related to its function. In this study, we wanted to look further into the structure and determine if the size the gland and its sub-regions were associated with specific genes or genomic loci. Since we found significant and suggestive QTLs for our adrenal structural measures, we are able to determine if these phenotypes are linked to the stress response and whether there are potential genetic associations between adrenal structure and function. We performed various structural measurements of adrenal width and adrenal gland weight phenotypes for a multitude of BXD recombinant inbred strains derived from two sequenced strains of mice – C57BL/6J and DBA/2J. We measured whole adrenal weights, along with measures of width for the adrenal medulla and cortical zones. The variation in these measures was substantial among the multiple strains assessed, as well as between males and females. Further, statistically significant variation in these traits was highly correlated to gene variants that were mapped to Chr 3 (female adrenal weight), Chr 4 (male adrenal weight), Chr 5 (male adrenal medulla width), Chr 10 (male adrenal total width and X-zone width), and Chr 14 (male X-zone width). Among these significant genomic regions, a total of 114 genes were identified to be potentially associated with the structural phenotypic differences observed between the sexes and strains of mice examined. Of these multiple genes, some have been documented to be related to specific phenotypes and functions involving the adrenal glands, associated structures, and/or the stress response. These particular genomic regions and candidate genes are briefly discussed here, along with the other research highlighting strain differences in adrenal gland structure and function and potential connections to the results obtained in this study. Overall, the results indicate that there are significant genomic regions associated with the adrenal structural phenotypes that vary across strains and between sexes, which may also be involved with adrenal function.  2.4.2  Differences between parental strains  The D2 and B6 mice, when considering both sexes, did not differ significantly for either adrenal weight or total adrenal width. However, the relative adrenal weights to body weight for D2 mice were 31  heavier compared to the B6 mice for both males and females in our study. Despite this, D2 male mice had significantly smaller adrenals than B6 male mice, and adrenal weight still did not differ significantly between the males. Meanwhile, the females of the parental strains did not differ for either measure. Interestingly, the width of the ZF was significantly larger in B6 mice compared to the D2 mice, regardless of sex. In addition, D2 females had a significantly larger medulla than B6 mice. These findings coincide with previous reports indicating that the genetic background of the mouse strain plays a critical role in adrenal gland size and adrenal cortical zone morphology (Pawlus, 1983; Tanaka and Matsuzawa, 1995, Tanaka et al., 1995; Deschepper et al., 2004; Bielohuby, 2007), as well as postnatal adrenal development (Badr et al., 1968; Badr and Spickett, 1971). However, only Deschepper et al. (2004) examined adrenal weight in B6 and D2 mice, and their data demonstrates the same trends that we find relative to B6 and D2 mice for both females and males. In their study, the B6 female mice had slightly higher values for relative adrenal weights but did not differ significantly from the D2 female mice. Meanwhile, they not only showed that significant interstrain variability in adrenal weight existed for male mice, but that lower relative adrenal weight to body weight was found for more stress resistant strains, including B6 mice, compared to more stress reactive strains, such as D2 mice. D2 mice exhibit more stress and anxiogenic behavioural responses in various testing paradigms compared to B6 mice (Trullas and Skolnick, 1993; Tarricone et al., 1995; Yilmazer-Hanke et al., 2003; Võikar et al., 2005; Ponder et al., 2007a), and B6 have been referred to as stress resistant (Shanks et al., 1990; Anisman et al., 1998). Thus, we expected the adrenal glands of D2 mice to be significantly heavier and larger than the B6 mice. This is because research has shown that chronic stress leads to increased basal plasma corticosterone levels in rats (Herman et al., 1995; Prewitt and Herman, 1997; Zelena et al., 2003; Ulrich-Lai et al., 2006), elevated plasma cortisol levels in chronically stressed depressed patients (Amsterdam et al., 1987; Nemeroff et al., 1992), and adrenal enlargement and increased adrenal weight in both groups. Furthermore, interstrain variability in adrenal weight has also been postulated to be associated with responsiveness or level of activation of the HPA axis by Deschepper et al. (2004). In addition, previous research with male rats has shown significant interstrain variability in adrenal weight, in which the trophic effect of ACTH on the adrenal ZF was the most robust determinant of adrenal weight differences (Akana et al., 1983). This would suggest that adrenal gland weight and size are associated with function, where adrenal morphology is related to hormone and steroid secretion for the stress response. More specifically, since the adrenal medulla secretes catecholamines and the ZF secretes corticosterone, which are critical for the autonomic and HPA-axis mediated stress response, respectively, an association between structure and function would suggest that D2 mice exhibit larger ZF and medulla regions, as they are more stress-reactive and likely have a more sensitive stress response than B6 mice. 32  However, the results from this study for the B6 and D2 mice and for female mice in the Deschepper et al. (2004) analysis suggest that the relationship between adrenal gross anatomy and steroid secretion for the stress response is more complex than expected. The negative relationship between the weight and size of the male adrenals for the parental strains and the similarity in adrenal size and weight for the females in our study suggests that cell number, density, and size may play important roles not only in linking adrenal structure to adrenal weight, but also connecting structure to function. In this regard, the work by Prewitt and Herman (1997) showed that acute restraint stress in rats does not significantly affect adrenal weight or lead to adrenal hypertrophy, but chronic variable stress does induce both adrenal weight increases and adrenal hypertrophy. Furthermore, Ulrich-Lai et al. (2006) showed that chronic stress induces adrenal hypertrophy and hyperplasia in the ZF and hypertrophy in the adrenal medulla, i.e., the regions that specifically secrete stress hormones. Since the female D2 mice exhibited a larger medulla region compared to the B6 mice in our study, this result may coincide with a higher cell density in this region, potentially linking structure to the autonomic stress response. Meanwhile, the B6 mice may have a larger ZF region because they may have a more sensitive HPA-axis mediated stress response than D2 mice, and/or they may have more cells releasing corticosterone that bind to more glucocorticoid receptors, activating the negative feedback loop of the HPA axis and leading to a more efficient stress response than the more anxious D2 mice. However, since the autonomic and HPA-axis mediated stress responses are intimately linked (Wurtman, 2002; Ehrhart-Bornstein and Bornstein, 2008; Goldstein and Kopin, 2008), it is unlikely that a direct link between adrenal structure and function would exist only for the ZF in B6 mice and for the adrenal medulla in D2 mice. Instead, the link between adrenal structure and stress responsivity in these divergent strains may be hidden by the conditions for which they were examined in our study. We performed adrenal measures only under basal conditions, without performing acute or chronic stress paradigms. Since D2 mice are stress reactive and show a larger adrenal medulla region compared to B6 mice, the autonomic stress response system may be the driving force that affects significant adrenal structural and weight changes in this strain during stress. Meanwhile, the negative feedback loop of the HPA-axis stress response may play a modulatory role that reverses the structural and weight increases in the D2 strain, and the ZF might be particularly affected by these changes, which may explain why the size of the ZF is significantly smaller in the D2 mice compared to B6 mice during basal conditions. These hypotheses suggest that adrenal structure and function may be causally linked, but such a connection likely involves multiple factors that were not examined fully in this study. In this regard, structural differences were evident between these strains, but it is not certain how adrenal structure corresponds to adrenal function, particularly the adrenal stress response.  33  2.4.3  Interaction between adrenal size and weight  In this study, we found that adrenal weight and total width showed a robust correlation, yet the QTL analyses for these phenotypes revealed only two overlapping suggestive QTL regions between them, suggesting that these factors have little effect on one another. This is interesting considering that the regression analysis showed total width accounting for 39% of the variance in adrenal weight. Moreover, it would logically make sense that the larger the adrenal gland (determined by width), the heavier it would weigh. Since total cell number, cell density, and cell size measures were not performed for this study, these factors may account for why only a few overlapping QTL regions existed between the weight and width phenotypes. Instead, it is likely a combination of adrenal size and cell density that influences adrenal weight, and an analysis of this possibility may identify more suggestive and significant QTLs in similar genomic regions as those found for adrenal weight. Regardless, there were a few overlapping significant and suggestive QTLs between adrenal weight and the cortical and medulla width measures. In particular, the female ZR and X-zone width showed suggestive QTLs on Chr 3 that mapped within the significant QTL for female adrenal weight (Figure 2.2), while the female total width showed a suggestive QTL on Chr 2 and the male ZF width on Chr 4 that mapped in similar regions to suggestive and significant QTLs for male adrenal weight (Figure 2.3). Moreover, both male and female adrenal weight shared suggestive QTL regions on Chr 10, which mapped in a similar region to a suggestive QTL for female ZR width, along with a suggestive QTL for male ZF width and the significant QTLs on Chr 10 for male adrenal total width (Figure 2.4) and X-zone width (Figure 2.6). Interestingly, the adrenal total width and respective regional measures that showed overlapping QTLs with adrenal weight were the same separate regions in males and females that showed the most robust correlations with adrenal weight, which further strengthened the possible genetic link on Chr 3, 4, and 10 for these adrenal phenotypes. However, since various unique significant QTL regions were found for males and females for adrenal weight and the width phenotypes, the QTL data suggests that these adrenal gland phenotypes may have little effect on each other and that distinct genetic factors may be associated with specific adrenal morphology.  2.4.4  Adipose and x-zone analysis  It was found that adipose tissue was only present in female mice, and it was primarily confined to the region between the ZR and X-zone. The presence of adipose tissue solely in female mice concurs with the work of Deschepper et al. (2004), but its location varies slightly. Although both studies show that adipose develops within an inner cortical region, they found that it develops directly adjacent to the medulla, whereas we typically found it forming more externally with the X-zone separating it from the 34  adrenal medulla. They deemed this region as a lipoid zone and showed that there is significant variability in its presence amongst the 13 mice strains they examined. This variability included the B6 and D2 strains, where the lipoid zone was absent in B6 mice and present but poorly developed in D2 mice. Although we did not find a lipoid zone in both the B6 and D2 mice tested, we did observe significant interstrain variability for this region in the female BXD lines examined. Contrary to both findings, previous research has shown that adipose develops in both male and female mice in the adrenal cortex as they age (Bielohuby, 2007). Since the mice tested in our study ranged from late puberty to early adulthood (~7-12 weeks of age) and 10 weeks for the Deschepper study, this suggests that females may develop adipose tissue in the adrenal gland earlier than males. Furthermore, the location of the lipoid zone adjacent to and within the transient X-zone region may indicate this it is essential for reproductive purposes, as the X-zone degenerates after the first pregnancy. Along these lines, several male strains tested exhibited a visible X-zone region, but none of these strains showed any adipose tissue in the Xzone or the adrenal cortex. This phenomena also coincides with the supposition that this early developing adipose tissue, present only in nulliparous female mice tested, may play an important role in reproduction. In Deschepper et al. (2004), they propose that the lipoid zone may derive from the degenerescence of adrenocorticol cells, but this hypothesis has yet to be elucidated. Meanwhile, although all female mice displayed a visible X-zone region, not all the female BXD strains showed adipose tissue in the X-zone or any cortical region. Moreover, for the male BXD strains that displayed a visible X-zone region, the average age of the mice in these eight lines was about 9 weeks, which is significantly older than the expected age of X-zone degeneration in male mice at approximately 5-6 weeks of age (Deacon et al., 1986; Keegan and Hammer, 2002). Related to this, Deschepper et al. (2004) also found significant interstrain differences for both males and females regarding the presence and/or development of the X-zone. In particular, some male strains clearly exhibited an X-zone at 10 weeks, including the B6 mice, while a few female strains did not display an X-zone at 10 weeks. We did not observe a visible X-zone in our B6 males, but this is not surprising as it was poorly developed in the B6 males from their study. Regardless, from these intriguing results, both these studies not only confirm the results from previous research exemplifying sex differences in adrenal gland development and morphology, but they also indicate that strain differences play an important role in X-zone morphology. Furthermore, the strain variation for female X-zone morphology may be associated directly with adipose tissue morphology. This is because we observed the female BXD lines with a visible lipoid zone showed both significantly larger X-zone regions and had heavier adrenal glands compared to all female BXDs examined. However, from a genetic perspective, the QTL analyses revealed significant QTLs only for male X-zone width that overlapped with both male and female adrenal weight QTLs, but the female data 35  did reveal a suggestive QTL overlapping with a female adrenal weight QTL. Thus, although these analyses indicate that there are both sex and strain differences in X-zone size and adrenal weight, which may be linked genetically, our results did not provide striking evidence that such a genetic link extends to adipose morphology. However, research has shown that the X-zone may be controlled by hypophyseal gonadotropic hormones (Deacon et al., 1986), and its development is likely controlled by genes within specific loci (Janat and Shire, 1987; Tanaka et al., 1994; Tanaka et al., 1995), including the genes Sf-1, Dax-1, and Acd (Beuschlein et al., 2002; Keegan and Hammer, 2002). Although it has yet to be elucidated, the hormonal and genetic factors involved in the development and morphology of the X-zone may also be associated with adipose development and the lipoid zone.  2.4.5  Adrenal weight QTL  By performing separate QTL analyses for male and female mice, we showed that there were unique significant QTLs on Chr 3 and 4 for adrenal weight for females and males, respectively. In addition, the female adrenal weight data revealed separate suggestive QTLs compared to the male data on Chr 1, 3, and 14, while the male adrenal weight data showed unique suggestive QTLs from the female data on Chr 2, 4, 17, and X. This suggests that sex differences in adrenal gland weight may be linked to genetic differences between males and females, and that adrenal weight may be influenced by separate genomic loci in males and females. Despite this, there were overlapping suggestive QTLs located on the distal end of Chr 10 for the male and female weight data. These findings suggest that although there are observable sex differences in adrenal gland weight, genotypic strain differences also appears to play an important role in regulating phenotypic differences in adrenal weight, as significant QTLs were detected for both males and females. Moreover, although most of the QTLs detected were unique for males and females, the overlapping suggestive QTL region on Chr 10 indicates that some similar genes may account for strain variation in adrenal weight. In this regard, if adrenal weight is related to adrenal function, such variation could underlie strain specific differences in anxiety and stress responses observed for B6 and D2 mice (Shanks et al., 1990; Trullas and Skolnick, 1993; Tarricone et al., 1995; Yilmazer-Hanke et al., 2003; Võikar et al., 2005; Ponder et al., 2007a). However, the results from the D2 and B6 mice suggest that adrenal weight is only partially related to adrenal function because adrenal weight did not differ between these strains. Thus, further analysis of adrenal weight and adrenal function must be conducted to determine whether a genetic link exists between these phenotypes.  36  2.4.6  Adrenal width QTL  After performing the QTLs analyses for the adrenal cortical and medulla width measurements for the BXD and parental strains, only one measure revealed no significant or suggestive QTLs, i.e., the male ZG width. Meanwhile, other measures showed one or more suggestive but no significant QTLs, including the ZG, ZI, ZF, and ZR for both males and females. Actually, none of the female width measures revealed any significant QTLs. This result was unexpected because of the robust strain differences for the females on most of these structural measures. In terms of the width measures for the ZG and ZI, the size of these regions was fairly consistent across the strains and between males and females, so it was not surprising that these cortical zones did not show unique significant genomic regions associated with their phenotypic data. Conversely, both the ZF and ZR measures showed significant strain and sex differences, so it was expected that unique significant QTLs would be associated with these phenotypes. Since there were no significant QTLs for these measures, it is difficult to identify genomic regions that may have a substantial influence on these structure phenotypes. Furthermore, since this study was examining the genetic underpinnings of adrenal structure as a first step towards searching for genetic links between adrenal structure and the stress response, these results impede such future analyses. More specifically, when the HPA-axis mediated stress response is activated in rodents, corticosterone is secreted from the ZF. In addition, research has shown that the mouse ZR cannot secrete adrenal androgens and is considered functionally indistinguishable from the ZF (VanWeerden et al., 1992; Heikkilä et al., 2002; Keegan and Hammer, 2002), suggesting that corticosterone is also released from the ZR. Thus, these two regions were of particular interest in trying to relate adrenal structure to the stress response. However, since the stress resistant B6 mice showed a larger ZF in both males and females, and males showed a larger ZR than the stress reactive D2 animals, structural measures of width does not necessarily display a direct association to the HPA-axis stress response. Instead, the connection between adrenal structure and function may be more associated with cell density or cell size in the adrenal cortex, which would coincide with the results from previous research showing ZF hypertrophy and hyperplasia occurring with chronic stress (Ulrich-Lai et al., 2006). Despite this, the QTL analyses for the ZF and ZR measures revealed numerous suggestive QTL regions. Additionally, both the male and female data showed that many of these suggestive regions overlapped with those found for the respective adrenal weight QTLs, including the significant region for male adrenal weight on Chr 4 with male ZF width, female adrenal weight on Chr 3 with female ZR width, and male and female adrenal weight on Chr 10 with male ZF and female ZR width, respectively. Thus, these matching sex-specific suggestive overlapping genomic regions indicate that a potential genetic link 37  may exist for the structural phenotypes associated with these cortical regions. Furthermore, it suggests that a possible association might exist between the size of the ZF and ZR and the stress response, but further experimental evidence would be required to substantiate this link. Although no significant QTLs were found for the measures indicated, the male data did show significant QTLs for the adrenal total, adrenal medulla, and X-zone width. For the total width, the significant QTL was located on Chr 10, which mapped to a similar distal region where overlapping suggestive QTLs were found for male and female adrenal weight. This is the same region that overlapped with suggestive QTLs found for male ZF and female ZR width, and it is close to the significant peak on Chr 10 for male X-zone width. This significant QTL for male X-zone width did overlap with the suggestive QTLs for adrenal weight, which further strengthens the potential genetic link on distal Chr 10 with these various morphological phenotypes. In regards to the male X-zone data, another significant QTL was located on Chr 14. Since research has shown that the presence of the X-zone varies between strains of mice examined for both males and females (Deschepper et al., 2004), and because we found strain differences in the presence of the X-zone only in males, it was not surprising that only males showed significant QTLs for the width data. For the female measures, although there were significant strain differences, this disparity was often related to the formation of adipose tissue along the X-zone/ZR border. In this regard, a genetic link for the strain differences in female X-zone morphology may be associated with the presence of this adipose tissue (lipoid zone), which was not specifically analyzed by performing structural measures of width. Despite this, there may be a genetic link for the strain differences in female X-zone because of the suggestive QTL on Chr 3 for the female X-zone data that overlapped with the significant QTL found for female adrenal weight. This said, the significant and suggestive QTLs identified for the X-zone data would coincide with research suggesting that X-zone development is controlled by genes within specific loci (Janat and Shire, 1987; Tanaka et al., 1994; Tanaka et al., 1995). However, the QTL regions identified on Chr 3, 10, and 14 for the X-zone measures do not include genes linked to X-zone development, such as Sf-1, Acd, and Dax-1 (Beuschlein et al., 2002; Keegan and Hammer, 2002), which are located on mouse Chr 2, 8, and X, respectively. The last significant QTL identified was on Chr 5 for male medulla width. This QTL region was unique to the medulla measures, as no other phenotypic measure performed in this study yielded any suggestive or significant QTLs on Chr 5. Since the adrenal medulla is specifically associated with the autonomic “fight or flight” stress response, this QTL may be related to both adrenal medulla size and the stress response, linking adrenal structure to function. Related to this, the stress reactive D2 mice had a larger adrenal medulla region than the more stress resistant B6 mice. In addition, the results from the Ulrich-Lai study (2006) showed that hypertrophy occurred in the adrenal medulla of rats subjected to a 38  chronic stress paradigm, which would coincide with the overall adrenal enlargement they experienced. Although these results point towards a direct relationship between medulla size and function, it does not explain why our results did not show significant QTLs for the female medulla measures. Both males and females exhibited similar strain variability in the width measures, but only the D2 females had a significantly larger adrenal medulla region compared to the B6 mice. However, the overall size of the male medulla was significantly larger than the female medulla, which could suggest that the males have a more highly sensitized autonomic stress response than female mice if a link does exist between adrenal size and the stress response. However, the diverging QTL results for male and female medulla measures, along with the conflicting medulla size difference between B6 and D2 males and females, suggest that there may not be a direct genetic link between medulla size and the stress response. Despite this, like the ZF data, the female medulla width did show various suggestive QTLs, which could be associated with a genetic link for the phenotypic variability in the female strains analyzed. Overall, the adrenal width QTL results suggest that there are similar genomic regions contributing to both adrenal weight and adrenal gland regional morphology, but there are also other regions uniquely influencing these phenotypes. In particular, for the measures that showed significant QTLs, each phenotype was not only associated with a unique genomic region, including the adrenal weight measures, but they were also different between males and females. This suggests that there are genes contributing to adrenal gland regional morphology that are specific to each cortical region and medulla, which are also separate from those influencing adrenal weight. Furthermore, the genetic influence on adrenal structure also seems to be sexually dimorphic based on these results. Thus, strain and sex differences in adrenal weight may be somewhat associated with but cannot account for strain and sex differences in specific adrenal zone structure. Regardless, our analyses indicate that there are sex and strain differences in adrenal size, and specific regional morphology is likely to be influenced by distinctive genetic loci. Lastly, although the QTL analyses revealed that different genetic loci are associated with certain strain and sex differences in adrenal structure, it is not certain whether such morphological variability is related to adrenal function.  2.4.7  Candidate genes  Using WebQTL, we found a total of 114 different genes mapped within the significant QTL intervals for the weight and structural measures. Despite this large number of genes, the majority of these genes have only been identified by location, while their functions are yet to be elucidated. Further, many other genes have been identified in association with multiple biological functions, but they have not been determined to be associated with the adrenal gland or the endocrine stress response. Thus, we were able 39  to reduce the number of candidate genes considerably by focusing only on those genes that have a biological relevance to the adrenal gland structure, development, and/or function. By taking advantage of the search and web-link capabilities of WebQTL, we sorted through all the genes by connecting to the NCBI Entrez Gene and Jackson Laboratory’s MGI websites. For the adrenal width measures, although there were 75 genes mapped within the significant QTL regions for the male data, none of them displayed any specific relevance to adrenal structure or function. Thus, no interesting candidate genes were identified for the male total, adrenal medulla, and X-zone width measures. Meanwhile, for the female adrenal weight, one candidate gene that is of particular interest in the significant QTL region is paired-like homeodomain transcription factor 2 or pituitary homeobox 2 (Pitx2). It has been implicated in regulating gene transcription in the pituitary gonadotrope of adrenal 4 binding protein/steroidogenic factor-1 (Ad4BP/Sf-1), which is essential for animal reproduction and endocrine regulation (Shima et al., 2008). In addition, Pitx2 has been shown to be vital in hypothalamic and subthalamic nucleus development, as migration of the subthalamic nucleus and hypothalamic neurons was significantly arrested in Pitx2 (cre/null) embryos (Skidmore et al., 2008). Considering its role in hypothalamic development and endocrine regulation, Pitx2 may be linked proper functioning of the HPA axis. Meanwhile, the hypothalamic norepinephrine level (Hnl) gene has been found to be near a QTL for novelty/stress-induced locomotor activation (Nsila3) in Chr 3 when using an AXB/BXA RI mouse strategy, which involves B6 progenitors who exhibit more activation of locomotor activity in an open field compared to the A/J progenitor strain (Gill and Boyle, 2005). It was also previously shown that Hnl is located around 131.843 Mb in Chr 3 in a BALB/cBy x C57BL/6By cross study, where B6 mice exhibited lower levels of hypothalamic norepinephrine compared to BALB/c mice (Eleftheriou, 1974). The mapping location of Hnl in Chr 3 is just outside the significant QTL region detected from our study and may also be associated with HPA-axis mediated adrenal function. For the male adrenal weight, two noteworthy candidate genes are phosphodiesterase 4B, cAMP specific (Pde4b) and the leptin receptor (Lepr). Pde4b appears to be associated with adrenal gland function because it has been shown to contribute to the signaling pathways that influence anxiogenic-like effects on behaviour as Pde4b-/- mice exhibited increased anxiogenic-like behavior and heightened plasma corticosterone levels (Zhang et al., 2008). Meanwhile, Lepr influences the effects of leptin action of brown adipose tissue (Oldfield et al., 2002; Cannon and Nedergaard, 2004) sympathetic nervous system tone (Bates et al., 2004), and hypothalamic regulation of feeding (Oldfield et al., 2002). In addition, Lepr has been suggested to provide mechanistic pathways for understanding how stress-related traits may be associated with risk factors for cardiovascular disease (Liu et al., 2007). Lastly, another gene mapped in the significant QTL for male adrenal weight is interferon alpha (Ifna), which was found 40  to be located in a QTL linked to anxiety and referred to conveniently as Anxiety (Anxty) in Chr 4 (Nakamura et al., 2003). This gene is particularly interesting because it has been linked to increased anxiety in both mice and humans (Bonaccorso et al., 2002) and may also be linked to adrenal gland function.  2.4.8  Stress and anxiety QTL  Although this study is the first to use a QTL approach when examining the adrenal gland structure, numerous studies have performed QTL analyses to investigate genomic regions that may be associated with stress and anxiety phenotypes. As stress and anxiety are complex phenomena, researchers have examined various behavioural and physiological measures of stress and anxiety using mouse or rodents models. In the mouse studies, QTL analyses have identified significant genomic regions on all 19 autosomal chromosomes and the X chromosome associated with these stress and anxiety-related phenotypes. However, after more extensive examination of these QTL results with different strains of mice and larger sample sizes, some of the significant QTL results have not been replicated or have been attributed to other behavioural responses rather than stress and anxiety. Thus, only the more consistent QTL results will be briefly discussed here, particularly those that overlap with the significant and suggestive QTLs found in our study. When examining the genetic basis of stress and anxiety using QTL analyses, numerous researchers consistently find significant QTLs on Chr 1, 4, and 15. Studies have shown that loci on Chr 1 influence anxiety-related behaviours (Henderson et al., 2004; Gill and Boyle, 2005), emotionality (Flint et al., 1995; Willis-Owen and Flint, 2006; Thifault et al., 2008), exploratory behaviour (Turri et al., 2001), fearlike behaviours (Gershenfeld and Paul, 1997), restraint stress (Tarricone et al., 1995), and corticosterone response to ethanol (EtOH) (Roberts et al., 1995). The Chr 1 locus identified from many of these studies is the best replicated QTL for any mouse behaviour (Willis-Owen, 2006). Moreover, research by Yalcin et al. (2004) identified the gene Rgs2, which encodes a regulator of G protein signaling, within this Chr 1 locus that modulates emotionality and anxiety-like behaviours in mice, and they also identified Rgs18 and Brinp3 as candidate genes in this QTL that affect other components of anxiety. Meanwhile, significant loci on Chr 15 have also been shown to influence anxiety-related behaviours (Flint et al., 1995; Turri et al., 2001), emotionality (Flint, 2002), and restraint stress (Tarricone et al., 1995), as well as avoidance behaviour (Turri et al., 2001), suppression of activity in anxiogenic or threatening environments (Flint, 2001; Henderson et al., 2004), stress-induced hyperthermia (Thifault et al., 2008), and acoustic startle response (Joober et al., 2002). In regards to significant QTLs for Chr 4, although these loci have been found when examining behavioural measures of anxiety using open field and elevated plus paradigms, 41  among others, further investigation has typically attributed them to locomotor activity, rather than influencing anxiety (Flint, 2001; Turri et al., 2001; Flint, 2002; Henderson et al., 2004). Despite this, other studies have identified significant QTLs on Chr 4 believed to influence stress responsiveness, including corticosterone levels seven hours after EtOH administration (Roberts et al., 1995), stressinduced hypothermia (Thifault et al., 2008), and tail-suspension test induced hyperthermia (Liu et al., 2007). As our study examined morphological adrenal phenotypes, rather than behavioural responses, it was not surprising that we did not show any significant QTLs on Chr 1 or Chr 15. Despite this, the male medulla measures showed a suggestive QTL in a distal region of Chr 1 that mapped within the significant QTL identified by Flint et al. (1995) and close to the genomic regions repeatedly identified by Turri et al. (2001) that were associated with behavioural responses to various tests of anxiety, including open field, EPM, elevated square maze, DL box, and the mirror chamber. Furthermore, the significant QTL and suggestive QTLs on Chr 4 for male adrenal weight, as well as the suggestive QTLs for both male and female ZF width, overlapped with some of the QTLs influencing both the locomotor activity and stressrelated phenotypes. These results suggest that genomic regions influencing adrenal morphology appear to be mostly independent of those commonly associated with stress and anxiety, but there also seems to be some potential genetic links between these complex phenotypes. With this in mind, we also found that the significant and suggestive QTLs we identified on Chr 3, 5, 10, and 17 overlapped with several of the stress and anxiety-related QTLs from the aforementioned and other studies. For Chr 3, a significant QTL influencing EPM behaviour (Turri et al., 2004) and one associated with corticosterone levels six hours following EtOH administration (Roberts et al., 1995) mapped in a similar region to a suggestive QTL for female adrenal medulla width. In addition, a significant QTL related to emotional reactivity in open field (Thifault et al., 2008) mapped in a similar region to the significant QTL for female adrenal weight. On Chr 17, significant QTLs associated with corticosterone levels six hours postsaline administration (Roberts et al., 1995) mapped in a similar region to a suggestive QTL for female ZR width, while significant QTLs on Chr 17 influencing DL box measures and EPM behaviour (Turri et al., 2004) were located in regions close to a suggestive QTL for male adrenal weight. Moreover, a study by Williams 4th et al.(2009) examined a candidate gene implicated in anxiety-like behaviour in mice and various psychiatric disorders in humans, glyoxalase 1 (Glo1), that is mapped near the same suggestive QTL for male adrenal weight. Meanwhile, more compelling evidence for genetic links between our adrenal phenotypes and stress and anxiety measures are found on Chr 5 and 10. We identified a significant and some suggestive QTLs for male adrenal medulla width on Chr 5. Numerous studies have found significant QTLs on Chr 5 associated with stress and anxiety-related phenotypes, including restraint stress open field rearings 42  (Tarricone et al., 1995), corticosterone levels one and seven hours following EtOH administration (Roberts et al., 1995), several behavioural measures of emotionality (Flint, 2002), basal immobility during the tail suspension test (Liu et al., 2007), and novelty/stress-induced activation (Gill and Boyle, 2005). In addition, a recent study by Dai et al. (2009) revealed that the GABAA receptor α2 (Gabra2) gene on Chr 5 plays a critical role in the stress response. Despite some of these QTLs and Gabra2 not mapping in similar QTL regions to the male medulla measures, the results for Chr 5 indicate that there are significant genomic loci that influence adrenal morphology, which may be linked to stress and anxiety. With this in mind, the distal end of Chr 10 showed the most consistent QTL results for our adrenal data. Several studies have also identified significant QTLs in a distal region of Chr 10 associated with behavioural measures of emotionality (Flint, 2002), anxiety-like behaviours related to learned and innate fear (Ponder et al., 2007b), seizure susceptibility (Gershenfeld et al., 1999), as well as exploratory and fear-like behaviours in mice (Gershenfeld and Paul, 1997; Gershenfeld et al., 1999; Zhang et al., 2005). Moreover, the significant QTL for male total adrenal width and the suggestive QTLs for male and female adrenal weight mapped in a region of Chr 10 that encompassed a QTL termed Exq1 for exploratory and excitability that is associated with exploratory and fear-like behaviours in mice involving open field ambulation and DL box paradigms (Gershenfeld et al., 1999; Zhang et al., 2005). These results indicate that the pleiotropic locus, Exq1, which has been found to affect exploration, fear-like behaviours, and seizure susceptibility, may also influence adrenal measures of size and weight.  2.4.9  Conclusion  Since B6 mice are considered stress resistant and D2 mice are stress reactive, we took advantage of the natural variation in stress and anxiety phenotypes and genotypes in these inbred strains while examining adrenal gland weight and structure. We have utilized quantitative measures, bioinformatics, and statistical approaches to effectively identify significant QTLs and intriguing candidate genes implicated in adrenal gland structure using a BXD RI mouse strategy. We have detected a significant QTL on Chr 3 along with suggestive QTLs on Chr 1, 3, 10, and 14 for female adrenal gland weight, as well as a significant QTL on Chr 4 and suggestive QTLs on chromosomes 2, 4, 10, 17, and X associated with male adrenal gland weight. In addition, we found significant QTLs on Chr 5, 10, and 14 for the male adrenal width measures, along with several overlapping and unique suggestive QTLs on multiple chromosomes for both male and female adrenal phenotypes. We identified candidate genes mapped within these significant QTLs, and some of them have been previously linked to adrenal gland structure, development, and/or function. Furthermore, many of the significant and suggestive QTL regions associated with our adrenal measures mapped in similar genomic regions as those shown to influence a variety of stress and anxiety-related 43  behavioural phenotypes. This analysis is the first step towards identifying genes controlling adrenal gland morphology and may be associated with adrenal gland function. However, further studies must be performed to more accurately identify and examine such candidate genes and clarify their role in adrenal gland structure and function.  Table 2.1 Strain  BXD1 BXD2 BXD6 BXD8 BXD9 BXD11 BXD12 BXD13 BXD14 BXD15 BXD16 BXD18 BXD19 BXD20 BXD21 BXD24 BXD27 BXD28 BXD29 BXD31 BXD32 BXD33 BXD34 BXD36 BXD38 BXD39 BXD40 BXD42 BXD43 BXD44 BXD45 BXD48 BXD50 BXD51 BXD55 BXD56 BXD60 BXD61 BXD62 BXD63 BXD65 BXD66 BXD67  Female mice: Age, body weight, and total adrenal weight measurements Total cases  4 4 8 4 4 4 4 4 4 4 4 8 4 4 4 4 8 4 4 4 4 4 4 8 4 4 4 8 4 4 4 8 4 4 4 4 8 4 4 4 4 4 4  Age (days)  Body weight (g)  55.00 ± 2.31 56.25 ± 0.95 51.25 ± 3.41 56.50 ± 6.06 62.00 ± 3.46 52.75 ± 4.40 54.50 ± 0.96 61.00 ± 8.39 58.75 ± 2.25 48.50 ± 0.29 67.75 ± 5.01 58.75 ± 3.59 60.00 ± 4.00 54.75 ± 2.25 52.25 ± 2.32 61.25 ± 1.60 51.50 ± 0.50 50.50 ± 2.50 53.75 ± 2.59 44.25 ± 3.59 82.00 ± 0.00 72.00 ± 7.00 53.25 ± 1.75 57.50 ± 2.12 58.75 ± 3.15 60.50 ± 5.50 48.00 ± 1.22 54.88 ± 1.13 66.00 ± 0.00 49.00 ± 0.00 54.00 ± 1.73 57.38 ± 2.95 71.25 ± 2.10 60.25 ± 2.36 65.25 ± 0.75 58.25 ± 0.25 53.50 ± 2.31 48.50 ± 2.06 73.00 ± 1.00 66.00 ± 5.20 56.75 ± 4.01 56.00 ± 1.00 59.00 ± 0.00  16.73 ± 0.37 20.55 ± 0.64 16.71 ± 0.89 14.50 ± 1.23 18.70 ± 1.18 14.55 ± 0.94 15.38 ± 0.36 19.23 ± 1.66 19.53 ± 0.84 21.40 ± 0.66 23.88 ± 0.84 19.20 ± 0.73 15.33 ± 0.93 17.95 ± 1.85 17.55 ± 1.44 18.48 ± 0.92 14.53 ± 0.60 18.05 ± 0.80 15.15 ± 0.79 12.98 ± 0.64 21.53 ± 0.43 17.70 ± 0.87 18.78 ± 0.46 17.13 ± 0.80 17.38 ± 0.57 18.55 ± 0.78 15.68 ± 0.26 16.39 ± 0.26 19.20 ± 0.40 16.75 ± 0.65 15.60 ± 0.73 18.88 ± 0.56 16.65 ± 0.56 18.10 ± 0.60 19.70 ± 0.36 16.68 ± 0.09 19.09 ± 1.45 19.25 ± 0.79 18.98 ± 0.65 18.55 ± 1.40 17.18 ± 1.29 16.03 ± 0.97 19.88 ± 0.59  Adrenal weight L (mg) 2.13 ± 0.08 2.48 ± 0.11 2.76 ± 0.15 2.45 ± 0.21 2.53 ± 0.26 3.03 ± 0.10 2.38 ± 0.14 2.50 ± 0.16 2.35 ± 0.17 2.58 ± 0.24 3.45 ± 0.10 3.10 ± 0.24 1.93 ± 0.08 2.63 ± 0.14 2.35 ± 0.18 2.25 ± 0.10 2.34 ± 0.08 3.48 ± 0.11 2.05 ± 0.13 2.23 ± 0.13 3.40 ± 0.15 2.75 ± 0.23 2.88 ± 0.18 2.43 ± 0.14 2.90 ± 0.17 2.63 ± 0.28 2.23 ± 0.17 2.60 ± 0.15 2.35 ± 0.12 1.60 ± 0.04 2.48 ± 0.15 2.69 ± 0.15 2.75 ± 0.19 2.95 ± 0.06 2.13 ± 0.11 2.93 ± 0.22 2.36 ± 0.16 2.23 ± 0.09 3.33 ± 0.13 2.75 ± 0.16 2.65 ± 0.17 2.85 ± 0.12 2.45 ± 0.09  Adrenal weight R (mg) 1.90 ± 0.11 2.53 ± 0.11 2.34 ± 0.15 2.00 ± 0.07 2.25 ± 0.12 3.03 ± 0.17 2.15 ± 0.06 2.63 ± 0.33 2.08 ± 0.06 2.30 ± 0.19 2.68 ± 0.13 2.49 ± 0.12 1.80 ± 0.11 2.43 ± 0.10 2.13 ± 0.06 2.15 ± 0.10 2.26 ± 0.09 3.53 ± 0.21 2.13 ± 0.06 1.85 ± 0.13 3.65 ± 0.16 2.55 ± 0.09 2.73 ± 0.10 2.23 ± 0.15 2.90 ± 0.24 2.53 ± 0.12 2.10 ± 0.07 2.31 ± 0.10 2.25 ± 0.06 1.53 ± 0.09 2.30 ± 0.12 2.64 ± 0.15 2.35 ± 0.21 2.55 ± 0.19 1.78 ± 0.14 2.55 ± 0.14 2.11 ± 0.12 2.10 ± 0.12 2.90 ± 0.10 2.33 ± 0.10 2.33 ± 0.10 2.55 ± 0.10 2.25 ± 0.16  Avg. Adrenal weight (mg) 2.01 ± 0.08 2.50 ± 0.06 2.55 ± 0.14 2.23 ± 0.13 2.39 ± 0.19 3.03 ± 0.13 2.26 ± 0.10 2.56 ± 0.11 2.21 ± 0.10 2.44 ± 0.21 3.06 ± 0.10 2.79 ± 0.18 1.86 ± 0.04 2.53 ± 0.12 2.24 ± 0.09 2.20 ± 0.10 2.30 ± 0.08 3.50 ± 0.11 2.09 ± 0.07 2.04 ± 0.09 3.53 ± 0.14 2.65 ± 0.16 2.80 ± 0.13 2.33 ± 0.15 2.90 ± 0.19 2.58 ± 0.20 2.16 ± 0.12 2.46 ± 0.12 2.30 ± 0.09 1.56 ± 0.05 2.39 ± 0.11 2.66 ± 0.15 2.55 ± 0.20 2.75 ± 0.07 1.95 ± 0.12 2.74 ± 0.16 2.24 ± 0.14 2.16 ± 0.07 3.11 ± 0.08 2.54 ± 0.13 2.49 ± 0.13 2.70 ± 0.08 2.35 ± 0.11  Adrenal weight/ Body weight (%) 0.012 0.012 0.015 0.015 0.013 0.021 0.015 0.013 0.011 0.011 0.013 0.015 0.012 0.014 0.013 0.012 0.016 0.019 0.014 0.016 0.016 0.015 0.015 0.014 0.017 0.014 0.014 0.015 0.012 0.009 0.015 0.014 0.015 0.015 0.010 0.016 0.012 0.011 0.016 0.014 0.014 0.017 0.012  44  Strain  Total cases  Age (days)  Body weight (g)  BXD68 4 63.75 ± 0.75 18.10 ± 0.39 BXD69 4 57.00 ± 1.15 21.00 ± 0.64 BXD70 4 58.25 ± 0.25 16.78 ± 0.26 BXD71 4 56.50 ± 0.50 17.43 ± 1.40 BXD73 8 57.25 ± 3.36 16.71 ± 0.78 BXD75 4 56.75 ± 4.75 20.83 ± 0.77 BXD77 2 57.00 ± 0.00 24.05 ± 0.05 BXD80 4 57.50 ± 0.87 16.48 ± 1.17 BXD83 4 67.75 ± 1.31 16.55 ± 0.72 BXD84 4 62.25 ± 0.75 17.13 ± 0.77 BXD85 4 50.00 ± 0.58 21.83 ± 0.71 BXD86 4 53.75 ± 3.75 16.90 ± 1.34 BXD87 4 59.75 ± 0.85 15.60 ± 0.70 BXD89 4 61.50 ± 1.44 19.35 ± 0.75 BXD90 4 64.50 ± 1.44 20.78 ± 0.88 BXD92 4 58.25 ± 6.25 19.23 ± 0.62 BXD96 4 53.50 ± 3.18 18.35 ± 0.52 BXD97 4 56.75 ± 2.25 17.45 ± 0.36 BXD98 4 53.25 ± 3.66 17.00 ± 0.92 BXD99 4 54.50 ± 0.87 17.03 ± 0.45 BXD100 4 67.00 ± 0.00 20.85 ± 0.37 C57BL/6J 5 61.00 ± 0.00 17.96 ± 0.41 DBA/2J 6 59.33 ± 2.74 20.07 ± 1.19 All data are shown as mean ± standard error of the mean.  Table 2.2 Strain  BXD1 BXD2 BXD6 BXD8 BXD9 BXD11 BXD12 BXD13 BXD14 BXD15 BXD16 BXD18 BXD19 BXD20 BXD21 BXD24 BXD27 BXD28 BXD29 BXD31 BXD32 BXD33 BXD34 BXD36  Adrenal weight L (mg) 2.80 ± 0.07 3.28 ± 0.21 2.40 ± 0.08 1.75 ± 0.06 2.10 ± 0.09 2.45 ± 0.06 2.05 ± 0.15 1.95 ± 0.09 2.60 ± 0.07 2.78 ± 0.10 2.60 ± 0.14 1.95 ± 0.16 2.65 ± 0.16 2.33 ± 0.21 2.08 ± 0.19 3.05 ± 0.16 2.43 ± 0.13 2.70 ± 0.12 2.63 ± 0.09 2.43 ± 0.14 2.68 ± 0.06 2.48 ± 0.23 2.32 ± 0.15  Adrenal weight R (mg) 2.73 ± 0.10 2.93 ± 0.11 2.28 ± 0.03 1.63 ± 0.08 1.91 ± 0.06 2.08 ± 0.06 2.00 ± 0.00 1.70 ± 0.04 2.20 ± 0.18 2.50 ± 0.08 2.40 ± 0.12 1.85 ± 0.13 2.73 ± 0.17 2.15 ± 0.20 2.18 ± 0.21 2.68 ± 0.19 2.20 ± 0.14 2.43 ± 0.09 2.18 ± 0.15 2.38 ± 0.11 2.30 ± 0.04 1.88 ± 0.20 2.28 ± 0.35  Avg. Adrenal weight (mg) 2.76 ± 0.09 3.10 ± 0.15 2.34 ± 0.04 1.69 ± 0.06 2.01 ± 0.07 2.26 ± 0.01 2.03 ± 0.08 1.83 ± 0.05 2.40 ± 0.12 2.64 ± 0.07 2.50 ± 0.10 1.90 ± 0.14 2.69 ± 0.16 2.24 ± 0.19 2.13 ± 0.20 2.86 ± 0.16 2.31 ± 0.13 2.56 ± 0.10 2.40 ± 0.12 2.40 ± 0.12 2.49 ± 0.05 2.18 ± 0.16 2.30 ± 0.23  Adrenal weight/ Body weight (%) 0.015 0.015 0.014 0.010 0.012 0.011 0.008 0.011 0.015 0.015 0.011 0.011 0.017 0.012 0.010 0.015 0.013 0.015 0.014 0.014 0.012 0.012 0.011  Male mice: Age, body weight, adrenal weight, and total adrenal weight measurements Total cases  4 4 8 4 3 4 4 4 4 4 4 8 4 4 4 4 8 4 4 4 4 4 4 4  Age (days)  Body weight (g)  62.00 ± 4.62 56.50 ± 0.87 53.63 ± 2.07 67.75 ± 4.94 61.00 ± 1.00 46.75 ± 1.75 54.00 ± 1.15 66.25 ± 7.43 62.50 ± 1.50 48.25 ± 0.75 55.00 ± 3.46 60.63 ± 4.45 65.75 ± 5.66 57.00 ± 0.00 47.25 ± 0.75 56.00 ± 1.78 56.00 ± 2.05 57.50 ± 9.82 54.25 ± 1.25 53.00 ± 1.73 77.75 ± 0.25 74.50 ± 1.85 57.00 ± 0.00 53.25 ± 0.25  24.28 ± 0.96 19.53 ± 2.83 21.18 ± 0.40 21.68 ± 1.38 22.73 ± 0.65 18.90 ± 1.31 21.15 ± 0.47 23.98 ± 1.73 25.43 ± 1.20 23.83 ± 1.60 23.55 ± 1.06 25.06 ± 0.78 20.45 ± 0.90 22.23 ± 0.09 19.00 ± 1.29 16.75 ± 2.07 21.00 ± 0.85 21.13 ± 0.36 20.35 ± 0.86 22.68 ± 0.77 23.48 ± 1.66 23.48 ± 0.74 24.53 ± 0.31 19.63 ± 0.70  Adrenal weight L (mg) 1.75 ± 0.10 1.60 ± 0.12 2.11 ± 0.10 1.78 ± 0.10 1.50 ± 0.06 2.10 ± 0.07 2.03 ± 0.09 1.68 ± 0.06 2.10 ± 0.08 1.90 ± 0.07 2.48 ± 0.14 1.99 ± 0.08 1.53 ± 0.14 1.83 ± 0.05 1.98 ± 0.05 1.80 ± 0.13 1.80 ± 0.13 2.33 ± 0.06 1.70 ± 0.00 2.00 ± 0.09 1.90 ± 0.07 1.70 ± 0.20 2.13 ± 0.08 1.68 ± 0.03  Adrenal weight R (mg) 1.70 ± 0.11 1.35 ± 0.18 2.14 ± 0.11 1.80 ± 0.18 1.50 ± 0.15 1.93 ± 0.13 2.20 ± 0.04 1.55 ± 0.13 1.73 ± 0.05 1.80 ± 0.08 2.25 ± 0.06 1.66 ± 0.19 1.53 ± 0.09 1.93 ± 0.08 2.00 ± 0.15 1.65 ± 0.13 1.86 ± 0.13 2.25 ± 0.13 1.55 ± 0.05 1.90 ± 0.15 1.68 ± 0.05 1.68 ± 0.17 2.20 ± 0.15 1.75 ± 0.09  Avg. Adrenal weight (mg) 1.73 ± 0.05 1.48 ± 0.11 2.13 ± 0.08 1.79 ± 0.10 1.50 ± 0.10 2.01 ± 0.09 2.11 ± 0.02 1.61 ± 0.08 1.91 ± 0.02 1.85 ± 0.04 2.36 ± 0.10 1.83 ± 0.11 1.53 ± 0.11 1.88 ± 0.06 1.99 ± 0.07 1.73 ± 0.13 1.83 ± 0.12 2.29 ± 0.09 1.63 ± 0.03 1.95 ± 0.04 1.79 ± 0.05 1.69 ± 0.08 2.16 ± 0.08 1.71 ± 0.05  Adrenal weight/ Body weight (%) 0.007 0.008 0.010 0.008 0.007 0.011 0.010 0.007 0.008 0.008 0.010 0.007 0.007 0.008 0.010 0.010 0.009 0.011 0.008 0.009 0.008 0.007 0.009 0.009  45  Strain  Total cases  Age (days)  Body weight (g)  BXD38 4 53.50 ± 1.44 22.18 ± 0.06 BXD39 4 57.00 ± 0.00 15.48 ± 0.53 BXD40 4 54.00 ± 0.00 22.13 ± 0.50 BXD42 8 51.75 ± 1.10 21.23 ± 0.39 BXD43 4 50.00 ± 0.00 18.58 ± 0.92 BXD44 4 58.00 ± 0.00 21.98 ± 0.43 BXD45 4 60.50 ± 3.18 22.83 ± 2.44 BXD48 8 54.25 ± 1.66 21.81 ± 0.73 BXD50 4 66.75 ± 2.66 18.33 ± 0.48 BXD51 4 65.00 ± 0.58 21.33 ± 0.20 BXD55 4 68.50 ± 2.50 21.05 ± 1.67 BXD56 4 65.50 ± 0.29 24.00 ± 0.39 BXD60 8 52.25 ± 2.34 20.50 ± 1.59 BXD61 4 55.00 ± 3.00 22.65 ± 0.44 BXD62 4 51.25 ± 1.70 21.35 ± 1.01 BXD63 4 59.00 ± 3.72 22.33 ± 1.70 BXD65 4 62.00 ± 0.00 21.63 ± 0.60 BXD66 4 54.50 ± 0.29 20.38 ± 1.17 BXD67 4 59.00 ± 3.00 22.50 ± 0.45 BXD68 4 62.00 ± 0.00 24.65 ± 0.99 BXD69 4 48.00 ± 2.00 17.68 ± 0.88 BXD70 4 56.25 ± 2.25 19.18 ± 1.91 BXD71 4 57.00 ± 4.49 21.63 ± 0.52 BXD73 8 50.00 ± 2.39 18.00 ± 0.79 BXD75 4 54.25 ± 4.61 21.98 ± 1.40 BXD77 6 58.33 ± 2.84 29.13 ± 1.93 BXD80 4 59.00 ± 0.00 22.85 ± 0.39 BXD83 4 64.00 ± 4.53 22.28 ± 0.70 BXD84 4 65.00 ± 1.00 22.73 ± 0.60 BXD85 4 58.00 ± 5.20 27.20 ± 2.19 BXD86 4 52.00 ± 0.00 19.33 ± 0.25 BXD87 4 48.50 ± 0.29 17.10 ± 1.12 BXD89 4 54.00 ± 6.93 21.70 ± 2.12 BXD90 4 66.25 ± 0.75 24.30 ± 0.52 BXD92 4 58.50 ± 2.02 20.03 ± 1.20 BXD96 4 48.75 ± 0.75 22.28 ± 0.54 BXD97 4 49.00 ± 0.00 19.80 ± 0.41 BXD98 4 68.50 ± 2.50 23.05 ± 0.87 BXD99 4 55.50 ± 0.50 23.33 ± 0.56 BXD100 4 62.00 ± 0.58 25.88 ± 0.63 C57BL/6J 4 61.00 ± 0.00 22.30 ± 0.24 DBA/2J 5 61.40 ± 2.75 23.22 ± 1.90 All data are shown as mean ± standard error of the mean.  Table 2.3  Adrenal weight L (mg) 1.78 ± 0.03 2.03 ± 0.11 1.50 ± 0.08 1.64 ± 0.09 1.65 ± 0.03 1.40 ± 0.04 1.65 ± 0.12 1.88 ± 0.09 1.45 ± 0.09 1.90 ± 0.11 1.28 ± 0.13 1.68 ± 0.03 1.78 ± 0.07 1.65 ± 0.03 2.18 ± 0.03 1.68 ± 0.08 1.73 ± 0.06 1.60 ± 0.09 1.58 ± 0.17 1.88 ± 0.03 1.75 ± 0.10 1.55 ± 0.10 1.50 ± 0.07 1.55 ± 0.09 1.65 ± 0.06 1.58 ± 0.06 1.63 ± 0.08 1.68 ± 0.05 1.70 ± 0.04 1.70 ± 0.04 1.48 ± 0.03 1.75 ± 0.03 1.58 ± 0.05 1.60 ± 0.11 1.73 ± 0.03 2.05 ± 0.09 1.65 ± 0.12 1.80 ± 0.16 1.63 ± 0.03 2.05 ± 0.10 1.55 ± 0.12 1.90 ± 0.28  Adrenal weight R (mg) 1.78 ± 0.05 2.10 ± 0.08 1.65 ± 0.10 1.40 ± 0.05 1.68 ± 0.08 1.50 ± 0.09 1.38 ± 0.11 1.86 ± 0.07 1.55 ± 0.03 1.70 ± 0.07 1.13 ± 0.06 1.50 ± 0.04 1.64 ± 0.07 1.65 ± 0.09 1.75 ± 0.06 1.58 ± 0.11 1.43 ± 0.06 1.55 ± 0.09 1.40 ± 0.04 1.75 ± 0.06 1.73 ± 0.03 1.38 ± 0.09 1.30 ± 0.07 1.49 ± 0.11 1.45 ± 0.05 1.53 ± 0.07 1.45 ± 0.10 1.68 ± 0.17 1.73 ± 0.05 1.63 ± 0.05 1.45 ± 0.09 1.63 ± 0.08 1.58 ± 0.16 1.48 ± 0.15 1.30 ± 0.11 2.03 ± 0.10 1.45 ± 0.10 1.53 ± 0.14 1.85 ± 0.06 1.85 ± 0.09 1.23 ± 0.20 1.92 ± 0.22  Avg. Adrenal weight (mg) 1.78 ± 0.03 2.06 ± 0.09 1.58 ± 0.08 1.52 ± 0.07 1.66 ± 0.04 1.45 ± 0.05 1.51 ± 0.10 1.87 ± 0.07 1.50 ± 0.04 1.80 ± 0.09 1.20 ± 0.06 1.59 ± 0.03 1.71 ± 0.07 1.65 ± 0.05 1.96 ± 0.02 1.63 ± 0.09 1.58 ± 0.05 1.58 ± 0.08 1.49 ± 0.07 1.81 ± 0.03 1.74 ± 0.06 1.46 ± 0.09 1.40 ± 0.07 1.52 ± 0.10 1.55 ± 0.05 1.56 ± 0.05 1.54 ± 0.07 1.68 ± 0.10 1.71 ± 0.04 1.66 ± 0.04 1.46 ± 0.05 1.69 ± 0.05 1.58 ± 0.10 1.54 ± 0.13 1.51 ± 0.05 2.04 ± 0.07 1.55 ± 0.10 1.66 ± 0.05 1.74 ± 0.04 1.95 ± 0.09 1.39 ± 0.14 1.91 ± 0.23  Adrenal weight/ Body weight (%) 0.008 0.013 0.007 0.007 0.009 0.007 0.007 0.009 0.008 0.008 0.006 0.007 0.008 0.007 0.009 0.007 0.007 0.008 0.007 0.007 0.010 0.008 0.006 0.008 0.007 0.005 0.007 0.008 0.008 0.006 0.008 0.010 0.007 0.006 0.008 0.009 0.008 0.007 0.007 0.008 0.006 0.008  Sex, age, body weight, and adrenal weight correlations Sex  Sex  1  Age  0.023  Age  Body Weight  L Adrenal Weight  R Adrenal Weight  Avg. Adrenal Weight  1  46  Body Weight  Sex  Age  Body Weight  0.544**  0.320**  1  L Adrenal 0.697** 0.157** -0.235** Weight R Adrenal 0.629** 0.142** -0.203** Weight Avg. Adrenal 0.692** 0.156** -0.229** Weight **Correlation is significant at the 0.01 level (2-tailed). Table 2.4 Strain BXD1 BXD2 BXD6 BXD8 BXD9 BXD11 BXD12 BXD13 BXD14 BXD15 BXD16 BXD18 BXD19 BXD20 BXD21 BXD24 BXD27 BXD28 BXD29* BXD31 BXD32 BXD33 BXD34 BXD36 BXD38 BXD39 BXD40 BXD42 BXD43 BXD44 BXD45 BXD48 BXD50 BXD51 BXD55 BXD56 BXD60 BXD61 BXD62 BXD63  Total cases 2 2 8 2 4 2 2 2 2 4 2 8 3 3 2 2 4 4 1 4 2 2 3 8 2 2 4 8 4 2 2 4 3 4 2 3 8 4 3 2  L Adrenal Weight  R Adrenal Weight  Avg. Adrenal Weight  1 0.841**  1  0.962**  0.956**  1  Female mice: Adrenal total width and adrenal zone width measurements Total width (μm) 1135.51 ± 9.21 1253.38 ± 30.37 1347.98 ± 55.39 1064.81 ± 113.91 1271.12 ± 22.82 1358.63 ± 51.86 1067.68 ± 47.25 1279.96 ± 22.03 1037.70 ± 5.80 1214.74 ± 52.26 1409.34 ± 251.39 1302.56 ± 33.97 1076.30 ± 23.60 1395.26 ± 72.98 1245.78 ± 94.33 1247.53 ± 54.42 1271.53 ± 33.43 1425.21 ± 79.37 1255.87 1195.88 ± 52.81 1411.23 ± 88.96 1217.19 ± 6.40 1203.64 ± 80.53 1289.41 ± 40.19 1330.00 ± 16.74 1303.38 ± 5.52 1155.41 ± 35.14 1116.28 ± 76.27 1287.82 ± 34.91 1037.93 ± 12.87 1396.88 ± 59.09 1344.69 ± 65.39 1406.93 ± 37.16 1514.22 ± 32.23 1193.36 ± 2.01 1348.00 ± 32.78 1298.57 ± 35.69 1163.37 ± 78.37 1436.28 ± 57.70 1408.99 ± 10.28  Medulla width (μm) 450.79 ± 29.61 490.49 ± 22.78 528.88 ± 42.65 449.65 ± 64.51 581.75 ± 29.97 493.20 ± 78.43 404.61 ± 22.80 445.34 ± 4.20 412.07 ± 42.07 568.50 ± 38.42 428.64 ± 191.71 453.85 ± 26.95 435.34 ± 30.43 622.83 ± 68.02 528.37 ± 103.85 521.17 ± 24.04 541.24 ± 30.41 459.63 ± 47.17 422.34 463.32 ± 44.84 516.87 ± 20.83 458.63 ± 41.58 500.30 ± 46.41 564.64 ± 25.09 534.47 ± 115.48 441.09 ± 16.85 445.71 ± 25.48 348.74 ± 45.20 602.81 ± 37.47 416.12 ± 3.24 633.32 ± 31.23 615.52 ± 67.46 634.25 ± 38.47 582.92 ± 37.60 548.75 ± 0.27 516.26 ± 20.33 525.20 ± 14.51 463.87 ± 59.91 338.86 ± 96.00 646.42 ± 4.59  Glomer width (μm) 44.03 ± 2.66 46.98 ± 0.79 46.96 ± 3.30 38.61 ± 3.91 41.95 ± 4.78 41.90 ± 3.35 31.41 ± 4.51 45.89 ± 1.60 32.01 ± 2.73 45.05 ± 2.09 49.25 ± 0.71 42.40 ± 4.79 41.13 ± 1.01 38.74 ± 3.41 45.48 ± 4.10 41.04 ± 5.69 47.89 ± 5.42 43.19 ± 5.42 47.20 42.73 ± 4.94 33.42 ± 1.09 33.59 ± 2.61 46.02 ± 1.48 40.33 ± 2.13 44.51 ± 2.88 50.29 ± 3.31 39.89 ± 2.32 46.39 ± 4.72 40.29 ± 0.89 46.44 ± 2.76 51.90 ± 3.67 41.49 ± 2.47 46.91 ± 2.18 55.37 ± 4.38 43.65 ± 0.98 50.00 ± 1.74 48.51 ± 2.14 44.61 ± 1.12 54.27 ± 3.47 47.18 ± 7.57  Inter width (μm) 14.27 ± 0.12 15.91 ± 0.41 22.63 ± 4.16 15.66 ± 0.84 14.27 ± 1.42 15.47 ± 0.84 10.52 ± 1.81 14.97 ± 0.11 11.94 ± 0.55 13.82 ± 0.15 17.50 ± 5.45 14.87 ± 1.52 13.68 ± 0.91 15.34 ± 1.55 16.81 ± 0.17 12.51 ± 1.52 15.00 ± 1.78 12.65 ± 0.49 13.63 13.97 ± 0.64 8.78 ± 0.35 11.30 ± 0.61 16.59 ± 1.09 14.34 ± 0.82 14.18 ± 3.04 20.19 ± 1.01 14.04 ± 1.08 16.87 ± 1.17 15.57 ± 0.19 14.32 ± 1.31 11.14 ± 1.50 14.74 ± 2.16 14.22 ± 0.28 14.98 ± 0.67 12.56 ± 0.76 14.37 ± 0.79 18.53 ± 1.20 13.35 ± 0.55 15.70 ± 1.55 14.17 ± 0.42  Fasc width (μm) 136.56 ± 13.50 182.12 ± 14.88 213.74 ± 9.81 142.96 ± 0.75 153.21 ± 6.79 210.45 ± 18.35 150.62 ± 6.05 201.72 ± 14.95 142.49 ± 6.50 164.18 ± 11.97 221.85 ± 16.17 126.44 ± 9.30 154.22 ± 15.60 147.14 ± 8.46 153.65 ± 18.82 184.07 ± 21.53 143.87 ± 8.40 136.28 ± 6.42 175.15 178.25 ± 11.31 102.86 ± 7.39 195.99 ± 34.13 179.93 ± 11.42 127.68 ± 7.41 171.70 ± 23.78 224.44 ± 17.50 199.28 ± 8.24 155.73 ± 13.55 163.20 ± 10.59 155.08 ± 2.29 189.82 ± 20.07 184.47 ± 12.04 156.53 ± 10.13 232.24 ± 13.26 145.49 ± 0.87 214.72 ± 8.40 176.87 ± 10.25 173.87 ± 4.63 259.65 ± 0.81 212.68 ± 12.40  Retic width (μm) 68.45 ± 26.22 76.21 ± 5.40 53.97 ± 2.61 46.85 ± 12.16 54.23 ± 4.42 68.48 ± 0.64 68.40 ± 1.13 66.92 ± 2.73 67.89 ± 2.86 42.96 ± 6.81 86.39 ± 17.68 89.31 ± 12.55 53.86 ± 5.77 73.84 ± 1.08 64.73 ± 5.82 63.84 ± 5.77 63.86 ± 5.67 73.72 ± 12.74 52.85 55.18 ± 2.60 62.71 ± 24.37 71.29 ± 14.59 61.76 ± 7.84 81.45 ± 8.40 80.07 ± 21.85 72.34 ± 4.09 50.38 ± 2.96 71.76 ± 8.38 45.60 ± 6.05 46.25 ± 0.78 58.13 ± 1.24 48.04 ± 2.57 76.02 ± 11.59 71.63 ± 10.85 54.16 ± 1.45 68.63 ± 6.83 58.15 ± 5.95 54.79 ± 6.34 96.25 ± 11.81 47.74 ± 0.32  X-zone width (μm) 37.89 ± 1.45 66.47 ± 16.87 41.87 ± 5.49 50.10 ± 12.57 42.92 ± 7.18 79.29 ± 30.06 58.87 ± 4.63 74.81 ± 1.04 51.45 ± 5.01 40.75 ± 3.05 66.28 ± 3.12 77.17 ± 9.47 43.97 ± 6.37 65.75 ± 3.25 47.29 ± 6.91 54.34 ± 5.92 61.79 ± 4.12 36.99 ± 7.09 33.91 39.21 ± 7.31 40.90 ± 14.70 57.45 ± 9.77 58.91 ± 8.17 63.56 ± 8.38 57.62 ± 1.14 64.72 ± 4.10 46.24 ± 1.51 57.07 ± 10.21 48.40 ± 4.59 36.67 ± 3.64 57.95 ± 10.02 44.26 ± 6.28 60.23 ± 12.86 66.82 ± 6.07 53.93 ± 2.60 51.74 ± 4.59 47.28 ± 5.97 44.93 ± 1.79 74.49 ± 10.12 43.61 ± 1.40  47  Total Total width (μm) Medulla width Glomer width cases (μm) (μm) BXD65 4 1209.61 ± 10.54 453.62 ± 20.17 42.04 ± 2.69 BXD66 3 1305.66 ± 14.97 509.54 ± 24.25 39.36 ± 1.98 BXD67 4 1118. 29 ± 51.13 413.49 ± 38.66 49.17 ± 5.62 BXD68 2 1236.97 ± 108.46 471.13 ± 60.67 49.75 ± 2.20 BXD69 3 1286.81 ± 44.48 475.64 ± 30.68 40.32 ± 2.61 BXD70 3 1230.03 ± 23.78 522.08 ± 20.69 46.15 ± 0.86 BXD71 3 1067.72 ± 58.14 500.74 ± 40.06 43.77 ± 1.15 BXD73 4 1233.93 ± 29.37 527.33 ± 37.50 47.22 ± 4.46 BXD75 4 1185.87 ± 24.32 483.71 ± 16.16 44.70 ± 0.83 BXD77 2 1024.55 ± 83.02 390.20 ± 34.32 48.38 ± 4.18 BXD80 3 1130.10 ± 31.54 530.51 ± 14.04 35.53 ± 1.64 BXD83 3 1199.17 ± 11.42 501.56 ± 18.73 42.10 ± 3.15 BXD84 3 1272.53 ± 56.52 517.29 ± 33.35 45.36 ± 0.96 BXD85 2 1215.21 ± 16.10 517.88 ± 6.03 44.42 ± 0.53 BXD86 3 1078.08 ± 93.66 534.32 ± 74.53 42.50 ± 4.51 BXD87 2 1248.08 ± 27.68 486.16 ± 36.93 39.86 ± 3.16 BXD89 2 1177.55 ± 67.22 457.99 ± 59.43 38.40 ± 8.04 BXD90 4 1285.69 ± 57.49 551.22 ± 16.56 50.19 ± 6.30 BXD92 4 1264.53 ± 53.36 485.91 ± 32.79 42.04 ± 1.35 BXD96 3 1269.19 ± 44.37 556.90 ± 9.15 46.28 ± 1.02 BXD97 3 1189.60 ± 12.90 429.61 ± 14.31 42.87 ± 1.65 BXD98 4 1235.60 ± 38.30 555.21 ± 37.64 43.08 ± 4.37 BXD99 4 1239.37 ± 68.59 533.79 ± 58.41 41.82 ± 1.22 BXD100 3 1350.56 ± 20.40 601.36 ± 18.01 34.32 ± 1.43 C57BL/6 5 1178.02 ± 30.69 402.23 ± 20.25 41.18 ± 2.34 DBA/2 6 1185.50 ± 50.99 523.91 ± 40.02 38.10 ± 2.18 All data are shown as mean ± standard error of the mean *Only 1 female adrenal weighed from BXD29 available for width measures Strain  Table 2.5 Strain BXD1 BXD2 BXD6 BXD8 BXD9 BXD11 BXD12 BXD13 BXD14 BXD15 BXD16 BXD18 BXD19 BXD20 BXD21 BXD24 BXD27 BXD28 BXD29 BXD31 BXD32 BXD33 BXD34 BXD36  Total cases 2 3 6 3 3 3 2 2 2 2 3 7 2 4 3 4 8 3 3 2 2 3 3 3  Inter width (μm) 14.48 ± 0.76 14.14 ± 1.23 13.94 ± 0.76 15.58 ± 0.21 14.21 ± 0.73 13.05 ± 0.99 15.30 ± 0.40 16.04 ± 1.31 15.64 ± .063 13.06 ± 0.24 12.02 ± 0.64 13.58 ± 1.37 16.41 ± 0.42 15.01 ± 0.63 13.10 ± 0.25 14.88 ± 0.12 11.05 ± 0.03 14.77 ± 1.99 14.92 ± 0.49 15.23 ± 0.18 14.62 ± 0.19 15.05 ± 1.56 16.05 ± 0.90 13.91 ± 1.16 17.92 ± 2.64 13.54 ± 1.50  Fasc width (μm) 175.09 ± 13.02 195.73 ± 3.34 185.49 ± 11.99 178.44 ± 29.53 172.48 ± 11.54 138.40 ± 4.28 149.03 ± 10.08 162.34 ± 16.09 148.82 ± 3.15 136.40 ± 12.73 132.49 ± 6.98 164.47 ± 9.03 184.07 ± 12.86 156.94 ± 22.64 130.53 ± 5.85 157.14 ± 22.24 120.34 ± 32.53 141.63 ± 12.05 173.93 ± 1.61 166.88 ± 14.65 165.24 ± 6.82 167.79 ± 12.54 156.44 ± 5.18 203.06 ± 3.21 190.80 ± 18.91 114.78 ± 11.91  Retic width (μm) 71.50 ± 4.74 74.41 ± 5.70 44.24 ± 2.29 51.09 ± 10.78 86.12 ± 10.68 69.70 ± 6.01 38.43 ± 3.31 43.23 ± 3.00 67.22 ± 3.08 54.76 ± 7.21 51.59 ± 6.71 58.17 ± 2.85 59.09 ± 8.42 59.19 ± 4.67 36.69 ± 1.78 72.74 ± 13.99 51.99 ± 1.26 61.53 ± 6.25 75.92 ± 12.56 55.37 ± 5.43 66.60 ± 3.88 50.98 ± 2.92 66.48 ± 4.78 61.52 ± 2.47 52.49 ± 7.49 70.50 ± 4.41  X-zone width (μm) 71.18 ± 6.33 69.27 ± 4.70 27.42 ± 2.01 53.58 ± 8.03 86.36 ± 11.17 74.66 ± 9.31 28.36 ± 2.60 37.74 ± 7.29 65.53 ± 10.00 56.49 ± 16.92 51.84 ± 1.20 57.12 ± 8.30 54.34 ± 6.70 70.81 ± 18.28 36.32 ± 2.48 80.77 ± 4.35 34.32 ± 2.55 53.14 ± 1.98 70.66 ± 8.70 58.25 ± 1.85 78.33 ± 4.39 47.94 ± 6.10 64.74 ± 6.26 51.24 ± 4.17 36.52 ± 5.19 47.14 ± 5.80  Retic width (μm) 70.66 ± 8.50 61.04 ± 5.03 67.73 ± 3.56 59.86 ± 0.68 69.41 ± 4.65 79.20 ± 7.64 70.32 ± 7.78 66.98 ± 8.81 70.29 ± 4.82 44.54 ± 2.20 69.57 ± 5.09 48.61 ± 3.11 49.85 ± 3.07 60.13 ± 15.29 76.57 ± 7.41 60.30 ± 2.30 57.30 ± 3.44 47.26 ± 3.41 64.72 ± 0.89 51.20 ± 1.68 64.68 ± 10.30 59.30 ± 8.50 85.43 ± 2.92 54.65 ± 3.30  X-zone width (μm) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 19.25 ± 1.58 38.52 ± 4.40 12.06 ± 4.35 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 7.30 ± 7.30 0.00 ± 0.00 27.89 ± 1.36  Male mice: Adrenal total width and adrenal zone width measurements Total width (μm) 1131.54 ± 76.02 1029.55 ± 41.70 1210.67 ± 44.36 1064.63 ± 28.29 1131.33 ± 48.23 1031.25 ± 84.29 962.84 ± 39.16 1019.48 ± 10.49 960.72 ± 54.01 1130.34 ± 27.53 1249.22 ± 49.27 1145.36 ± 31.53 996.24 ± 216.93 1204.90 ± 4.15 1038.31 ± 33.06 1170.92 ± 60.80 1159.81 ± 13.81 1173.54 ± 31.51 964.04 ± 18.38 1074.44 ± 41.15 1060.65 ± 125.58 1210.14 ± 52.47 1194.71 ± 21.01 1084.26 ± 38.21  Medulla width (μm) 579.68 ± 63.88 497.75 ± 43.26 511.88 ± 36.99 526.28 ± 45.26 580.49 ± 29.92 451.38 ± 58.55 471.99 ± 20.71 529.29 ± 46.07 457.25 ± 46.47 657.67 ± 24.67 514.58 ± 52.29 599.24 ± 18.13 443.77 ± 113.11 509.84 ± 21.76 504.37 ± 17.65 587.04 ± 65.55 584.81 ± 23.50 629.44 ± 7.91 483.57 ± 22.85 597.37 ± 90.60 565.12 ± 67.51 650.39 ± 35.33 596.10 ± 6.05 552.73 ± 50.79  Glomer width (μm) 39.74 ± 3.47 40.66 ± 2.86 53.42 ± 3.18 42.77 ± 0.81 43.80 ± 0.90 37.00 ± 2.14 32.11 ± 1.02 36.99 ± 2.42 33.00 ± 0.90 43.31 ± 2.93 44.66 ± 4.33 47.85 ± 3.32 39.26 ± 2.63 48.11 ± 6.99 42.02 ± 3.17 50.04 ± 3.66 41.83 ± 2.91 33.85 ± 5.35 41.17 ± 0.48 36.59 ± 3.69 36.11 ± 4.29 49.87 ± 1.36 45.07 ± 2.40 42.64 ± 1.16  Inter width (μm) 14.46 ± 0.16 12.30 ± 0.34 20.75 ± 2.25 13.25 ± 0.91 15.41 ± 1.28 13.26 ± 0.28 10.48 ± 0.91 11.54 ± 0.43 13.17 ± 1.79 12.88 ± 0.62 14.51 ± 0.28 16.44 ± 1.76 10.97 ± 3.13 19.38 ± 2.48 15.49 ± 0.80 15.39 ± 1.38 19.38 ± 1.29 14.91 ± 0.60 13.36 ± 0.79 12.61 ± 0.64 12.58 ± 0.77 16.86 ± 2.61 14.67 ± 0.78 12.04 ± 0.92  Fasc width (μm) 144.29 ± 3.03 137.80 ± 3.46 168.83 ± 12.71 143.04 ± 14.90 113.96 ± 6.38 146.11 ± 2.20 117.24 ± 4.08 116.84 ± 12.45 125.47 ± 13.80 99.86 ± 0.78 189.72 ± 5.62 113.08 ± 9.43 150.65 ± 24.81 178.80 ± 10.97 119.10 ± 6.45 142.65 ± 18.14 143.33 ± 8.35 142.56 ± 13.12 109.18 ± 0.93 113.02 ± 26.61 129.93 ± 12.04 123.07 ± 12.61 143.80 ± 9.10 115.70 ± 4.56  48  Total Total width (μm) Medulla width cases (μm) BXD38 4 1156.26 ± 32.94 542.62 ± 31.73 BXD39 3 1037.65 ± 41.89 485.41 ± 36.61 BXD40 3 958.60 ± 61.95 488.16 ± 50.58 BXD42 4 1101.15 ± 73.34 462.21 ± 72.20 BXD43 4 1059.56 ± 67.29 554.21 ± 38.19 BXD44 3 981.13 ± 16.99 458.59 ± 18.63 BXD45 2 1026.12 ± 81.35 383.76 ± 5.19 BXD48 8 1262.81 ± 37.31 664.31 ± 22.23 BXD50 4 1063.93 ± 23.90 605.43 ± 19.77 BXD51 2 1271.22 ± 39.42 576.78 ± 55.21 BXD55 4 1084.97 ± 62.89 560.38 ± 67.70 BXD56 2 1057.65 ± 128.58 490.60 ± 118.86 BXD60 7 1068.83 ± 47.57 494.70 ± 38.85 BXD61 3 1091.13 ± 32.46 567.20 ± 7.66 BXD62 4 1144.47 ± 98.03 542.94 ± 70.83 BXD63 2 1249.07 ± 72.74 712.67 ± 0.39 BXD65 3 1038.56 ± 19.94 476.24 ± 7.45 BXD66 3 1101.78 ± 33.07 534.08 ± 38.96 BXD67 4 1104.05 ± 51.58 516.67 ± 39.70 BXD68 3 1102.79 ± 37.20 547.20 ± 37.02 BXD69 3 1111.58 ± 60.22 469.35 ± 34.24 BXD70 4 969.45 ± 73.63 518.65 ± 68.50 BXD71 3 1149.26 ± 22.06 592.02 ± 13.35 BXD73 7 1057.40 ± 27.30 559.71 ± 39.81 BXD75 3 1022.65 ± 53.28 482.71 ± 36.98 BXD77 3 1020.94 ± 26.47 510.18 ± 25.38 BXD80 2 1023.00 ± 75.94 538.11 ± 71.45 BXD83 2 1002.44 ± 12.94 565.91 ± 6.59 BXD84 3 1111.89 ± 61.54 533.75 ± 45.78 BXD85 4 1112.88 ± 27.83 539.44 ± 20.80 BXD86 2 1041.82 ± 12.61 548.81 ± 9.79 BXD87 2 1175.94 ± 27.03 599.59 ± 40.73 BXD89 2 1115.33 ± 65.14 667.32 ± 48.63 BXD90 4 1165.52 ± 37.21 632.02 ± 21.81 BXD92 2 1061.99 ± 23.94 528.20 ± 14.41 BXD96 2 1093.57 ± 136.90 518.86 ± 118.23 BXD97 3 1033.61 ± 47.86 490.34 ± 20.61 BXD98 3 1169.91 ± 30.65 634.35 ± 19.50 BXD99 4 1162.54 ± 28.54 640.21 ± 30.27 BXD100 2 1330.42 ± 85.43 580.97 ± 67.68 C57BL/6 4 1065.04 ± 39.83 426.34 ± 13.75 DBA/2 5 906.12 ± 38.56 434.14 ± 18.25 All data are shown as mean ± standard error of the mean Strain  Glomer width (μm) 40.91 ± 3.77 43.30 ± 0.57 39.11 ± 3.09 51.12 ± 1.71 39.25 ± 3.51 43.19 ± 1.24 45.33 ± 3.18 47.33 ± 1.92 41.40 ± 1.78 61.52 ± 6.78 42.57 ± 3.64 50.26 ± 9.32 48.90 ± 4.11 41.38 ± 2.60 40.69 ± 1.68 50.80 ± 2.55 42.54 ± 4.45 38.97 ± 2.35 48.00 ± 3.43 39.66 ± 1.90 42.61 ± 1.15 35.78 ± 1.18 46.09 ± 2.99 47.42 ± 2.86 44.04 ± 3.06 43.32 ± 4.21 41.26 ± 3.33 37.22 ± 1.30 39.39 ± 1.36 43.47 ± 1.58 40.22 ± 1.82 39.91 ± 2.59 38.53 ± 0.46 43.71 ± 3.06 41.84 ± 1.46 38.51 ± 4.73 42.30 ± 2.89 42.42 ± 3.41 38.81 ± 1.41 45.57 ± 0.67 46.56 ± 3.55 45.55 ± 4.97  Inter width (μm) 13.72 ± 0.75 13.10 ± 0.93 11.85 ± 0.88 14.63 ± 0.83 12.01 ± 0.34 14.85 ± 0.81 17.25 ± 1.67 13.99 ± 0.80 12.73 ± 0.90 12.98 ± 0.48 12.51 ± 0.48 11.15 ± 0.49 15.02 ± 1.61 12.88 ± 0.86 13.51 ± 0.45 14.69 ± 4.58 13.18 ± 0.73 14.83 ± 1.03 17.00 ± 0.84 13.69 ± 0.70 13.70 ± 0.65 12.91 ± 1.13 14.21 ± 0.38 18.12 ± 1.43 14.67 ± 0.03 14.30 ± 0.82 15.30 ± 0.76 10.05 ± 0.13 12.69 ± 0.67 14.57 ± 0.72 14.67 ± 0.05 14.41 ± 1.48 15.29 ± 1.63 14.06 ± 0.55 14.21 ± 0.74 13.66 ± 0.42 14.37 ± 1.31 13.73 ± 0.64 14.14 ± 0.91 13.87 ± 0.78 19.58 ± 0.92 14.61 ± 1.96  Table 2.6 Heritability of phenotypes Phenotype Combined (Across Sexes) Adrenal Weight 16% Total Adrenal Width 20% Adrenal Medulla Width 22% Zona Glomerulosa Width 14% Zona Intermedia Width 18% Zona Fasciculata Width 21% Zona Reticularis Width 17% X-Zone Width 0%  Fasc width (μm) 131.99 ± 19.88 121.93 ± 6.18 108.82 ± 10.51 136.71 ± 14.28 122.65 ± 8.43 120.15 ± 6.52 143.78 ± 19.76 135.83 ± 9.72 97.39 ± 6.16 154.48 ± 17.55 121.09 ± 12.70 128.77 ± 9.67 132.88 ± 12.02 117.66 ± 7.98 138.69 ± 7.18 134.86 ± 23.00 125.61 ± 12.88 128.03 ± 4.87 137.16 ± 10.04 132.93 ± 2.03 164.70 ± 8.14 108.38 ± 6.51 109.33 ± 6.28 104.44 ± 5.94 119.45 ± 8.37 107.22 ± 6.44 107.76 ± 1.19 100.42 ± 2.47 124.47 ± 4.79 127.22 ± 13.12 114.48 ± 6.92 135.80 ± 14.16 109.48 ± 3.83 116.35 ± 7.89 115.57 ± 1.30 134.49 ± 1.92 127.74 ± 11.85 121.41 ± 1.91 116.57 ± 4.24 148.21 ± 39.43 143.66 ± 7.76 110.98 ± 8.63  Males 55% 32% 27% 20% 30% 33% 47% 77%  Retic width (μm) 57.87 ± 6.34 75.22 ± 0.89 64.26 ± 2.15 74.42 ± 7.85 60.99 ± 3.13 72.89 ± 2.19 66.94 ± 14.20 76.55 ± 7.89 59.44 ± 2.44 48.08 ± 6.07 57.44 ± 7.63 49.48 ± 1.21 62.29 ± 4.39 78.82 ± 7.16 97.16 ± 16.38 45.67 ± 1.95 82.21 ± 3.85 83.26 ± 6.97 55.98 ± 4.92 81.12 ± 2.02 83.48 ± 2.77 58.02 ± 4.72 72.81 ± 5.86 53.05 ± 5.02 85.39 ± 9.23 78.95 ± 2.77 71.19 ± 7.56 59.85 ± 1.48 98.78 ± 8.05 80.00 ± 7.65 67.24 ± 0.78 90.00 ± 10.29 38.13 ± 0.78 60.43 ± 1.42 81.59 ± 5.09 81.17 ± 4.58 76.52 ± 6.16 72.79 ± 2.51 69.60 ± 3.05 108.24 ± 20.08 65.39 ± 6.60 44.88 ± 1.94  X-zone width (μm) 36.83 ± 9.87 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 14.15 ± 14.15 0.00 ± 0.00 20.99 ± 1.30 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00  Females 59% 38% 28% 10% 14% 56% 26% 31% 49  Table 2.7  Age, body weight, adrenal weight, and adrenal width correlations for female mice  Age Age  Body Weight  Adrenal Weight  Adrenal Width  Table 2.8  Inter Width  Fasc Width  Retic Width  X-Zone Width  1 .612**  1  .155*  -.045  1  .037  -.015  .237**  1  .367**  -.029  .237**  .207**  1  .392**  -.069  -.033  -.040  -.035  1  .275**  -.026  -.053  -.120  -.012  .644**  1  Age, body weight, adrenal weight, and adrenal width correlations for male mice  Age  Body Weight  Adrenal Weight  Adrenal Width  Medulla Width  Glom Width  Inter Width  Fasc Width  Retic Width  X-Zone Width  1  Body .400** 1 Weight Adrenal -.116 .149* 1 Weight Adrenal .009 .167* .292** 1 Width Medulla .133 .177** .141* .719** Width Glom -.035 -.070 -.013 .301** Width Inter -.080 .009 .026 .271** Width Fasc -.129 -.017 .306** .478** Width Retic -.103 .094 .088 .217** Width X-Zone -.048 .082 .178** .180** Width **Correlation is significant at the 0.01 level (2-tailed). *Correlation is significant at the 0.05 level (2-tailed).  Table 2.9 Adrenal Measures Phenotype Female Adrenal Weight (Fawq1)  Glom Width  1  Body .378** 1 Weight Adrenal .390** .327** 1 Weight Adrenal .292** .189** .525** Width Medulla .093 .080 .031 Width Glom -.027 -.040 .035 Width Inter -.208** -.124 -.031 Width Fascic -.005 -.042 .125 Width Retic .261** .213** .422** Width X-Zone .233** .114 .349** Width **Correlation is significant at the 0.01 level (2-tailed). *Correlation is significant at the 0.05 level (2-tailed).  Age  Medulla Width  1 .013  1  .018  .3832**  1  -.121  .114  .270**  1  -.078  -.058  -.089  .141*  1  .025  .021  -.118  .169*  -.148*  1  BXD adrenal phenotype-related loci and genes Chr 3  Mapping Location  LRS  Gene List  127.0 - 129.5Mb  20.605  Larp7  Neurog2  Alpk1  1500005C15Rik  4930422G04Rik  T2bp  BC002199  5730508B09Rik  9830132P13  D030025E07Rik  4933424H11Rik  Pitx2*  Gm132  Enpep  LOC435755  Elovl6  Egf  6330410L21Rik  Hnl *  50  Adrenal Measures Phenotype Male Adrenal Weight  Chr 4  Mapping Location  LRS  Gene List  99.5 - 102.5Mb  19.226  Pgm2  Ror1  Ube2u  Cachd1  Raver2  Jak1  E130102H24Rik  0610043K17Rik  Ak3l1  Dnajc6  Leprot  C130073F10Rik  Lepr*  RP23-149D11.4  B020004J07Rik  RP23-149D11.5  RP23-149D11.7  Pde4b*  Sgip1  Ifna *  4930564G21Rik  Grip1  4930483C13Rik  Helb  Irak3  Tmbim4  1190005P17Rik  Hmga2  1700006J14Rik  9230105E05Rik  EG231836  Ftsj2  Nudt1  Snx8  Eif3b  Chst12  Grifin  Lfng  Ttyh3  Iqce  AA881470  Amz1  Gna12  Card11  Sdk1  3200001G23Rik  Kdelr2  D330005C11Rik  Foxk1  C330006K01Rik  Slc29a4  D930005D10Rik  4921504P13Rik  Papolb  Mmd2  Wipi2  B130019G13Rik  Tnrc18  Zfp469  Lin7a  3110033O22Rik  Myf5  Myf6  Ptprq  LOC628877  Ppp1r12a  C430003N24Rik  Pawr  Syt1  A830054O04Rik  5330428N10Rik  C030018G05Rik  EG368203  C030044C18Rik  2900074G08Rik  A130086K04Rik  6430709C05Rik  3110043J17Rik  Nav3  A830061P03Rik  9230102K24Rik  E2f7  1700020G17Rik  Csrp2  4930596D02Rik  4930474N05Rik  Gcap14  Rgr  Lrit1  Lrit2  Pcdh21  Ghitm  Nrg3  (Mawq1)  Male Total Width  10  119.0 -120.0Mb  18.771  (Mawdq1)  4930471E19Rik Male Medulla Width  5  140.7 - 143.5Mb  19.262  (Mmwdq1)  Male X-Zone Width  10  106.7 – 110.5Mb  12.934  (Mxwdq1)  Zdhhc17 Male X-Zone Width (Mxwdq2)  14  36.5 – 39.5Mb  12.918  *candidate genes  51  A  B  Figure 2.1: Sample width measurements from female (A and C) and male (B and D) adrenal glands sectioned horizontally.  52  A  B  Figure 2.2: Genome-wide linkage map of female BXD adrenal weight. The blue trace shows the LRS for female BXD adrenal weight. (A) Interval genome-wide QTL map showing a significant QTL detected on chromosome 3 and four suggestive QTLs detected with one each on chromosomes 1, 3, 10, and 14. (B) Interval QTL map with bootstrap analysis of chromosome 3 using female adrenal weight data. The lower gray horizontal line represents suggestive LRS genome-wide threshold at p  0.63. The upper pink horizontal line represents significant LRS genome-wide threshold at p  0.05. A positive additive coefficient (green line) indicates that DBA/2J alleles increase trait values. In contrast, a negative additive coefficient (red line) indicates that C57BL/6J alleles increase trait values. Orange seismograph marks indicate SNP density.  53  A  B  Figure 2.3: Genome-wide linkage map of male BXD adrenal weight. The blue trace shows the LRS for male BXD adrenal weight. (A) Interval genome-wide QTL map showing a significant QTL and several suggestive peaks detected on chromosome 4, as well as five suggestive QTLs with one each on chromosomes 2, 17, and X and two on chromosome 10. (B) Interval QTL map with bootstrap analysis of chromosome 4 using male adrenal weight data.  54  Figure 2.4: Bar graph depicting the parental strain means for the adrenal width measurements. The data is separated by sex for the B6 and D2 mice phenotypes. Error bars show the 95% confidence internal for the standard error of the mean. * indicates significance at the 0.05 level (2-tailed).  55  A  B  Figure 2.5: Genome-wide linkage map of male BXD adrenal total width. The blue trace shows the LRS for male BXD adrenal total width. (A) Interval genome-wide QTL map showing a significant QTL detected on chromosome 10, as well as a suggestive QTL detected on chromosome 13. (B) Interval QTL map with bootstrap analysis of chromosome 10 using male adrenal total width data.  56  A  B  Figure 2.6: Genome-wide linkage map of male BXD adrenal medulla width. The blue trace shows the LRS for male BXD adrenal medulla width. (A) Interval genome-wide QTL map showing a significant QTL detected on chromosome 5, as well as a suggestive QTL detected on chromosome 1. (B) Interval QTL map with bootstrap analysis of chromosome 5 using male adrenal medulla width data.  57  A  B  C  Figure 2.7: Genome-wide linkage map of male BXD X-zone width. The blue trace shows the LRS for male BXD Xzone width. (A) Interval genome-wide QTL map showing significant QTLs detected on chromosomes 10 and 14. (B) Interval QTL map with bootstrap analysis of chromosome 10 using male X-zone width data. (C) Interval QTL map with bootstrap analysis of chromosome 14 using male X-zone width data.  58  2.5  References  Akana, S.F., Shinsako, J, and Dallman, M.F. (1983). 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Mammalian Genome, 16(5): 306-318.  65  3 GENERAL DISCUSSION 3.1  Research conclusions and discussion  The adrenal gland is the efferent limb of the HPA axis, and it is critical for physiological responses to stress. Stress is a complex behavioural and physiological response, and the genetic underpinnings of this phenomenon have yet to be elucidated fully. The structure of the gland is such that different regions have distinct functional roles, including specific responses to stress. The adrenal cortex is involved in the HPA-axis mediated stress response, while the adrenal medulla is associated with the autonomic “fight or flight” stress response. However, these stress response systems are intimately linked via an intra-adrenal circuit between the adrenal cortex and medulla (Nussdorfer, 1996; Ehrhart-Bornstein et al., 1998; Wurtman, 2002; Ulrich-Lai and Engeland, 2005; Ehrhart-Bornstein and Bornstein, 2008). From this perspective, the gross structure of the gland is directly related to its function. In this study, we wanted to look further into the structure and determine if the weight of the gland and the width of its sub-regions were associated with specific genes or genomic loci. Further, we wanted to determine if the regional measures were correlated to its function in terms of the stress response and whether there were potential genetic associations between structure and function. We performed adrenal gland weight measures and various structural measurements in BXD RI strains derived from two fully sequenced strains of mice – B6 and D2. We measured whole adrenal weights, along with measures of width for the adrenal medulla and cortical zones. The variation in these structural measures was substantial among the multiple strains assessed, as well as between males and females. Further, statistically significant variation in these traits was highly correlated to genomic variants that were mapped to Chr 3 (female adrenal weight), Chr 4 (male adrenal weight), Chr 5 (male adrenal medulla width), Chr 10 (male adrenal total and X-zone width), and Chr 14 (male X-zone width). Within these significantly associated genomic regions, there are a total of 114 genes. Of these multiple QTLs, some candidate genes have been documented to be related to specific phenotypes and functions involving the adrenal glands, associated structures, and/or the stress response. These particular genomic regions and candidate genes were briefly discussed, along with other research highlighting strain differences in stress and anxiety measures, and their potential connections to the results obtained in this study. Overall, the results indicated that there are significant genotypic regions associated with adrenal structural phenotypes and that adrenal structure not only varies across strains and between sexes, but also may be involved directly with adrenal function. In this regard, we took advantage of the natural variation in stress and anxiety phenotypes and genotypes in the B6 and D2 inbred strains while examining adrenal gland weight and structure. We have utilized quantitative measures, statistical approaches, and bioinformatics to effectively identify significant QTLs and intriguing candidate genes implicated in adrenal gland structure 66  using a BXD RI mouse strategy. This analysis is the first of its kind and a step towards identifying genes that influence adrenal gland morphology that may be associated with adrenal gland function. However, further studies must be performed to more accurately identify and examine such candidate genes and clarify their role in adrenal gland structure and function.  3.2  Strengths and weaknesses of the research  First of all, this is only the third study to examine QTLs for adrenal weight (Llamas et al., 2005; Solberg et al., 2006) and the first to use a QTL approach when investigating this phenotype in mice. Furthermore, it is the first study to examine and identify QTLs for adrenal gland structure. In terms of these phenotypes, our QTL analyses revealed significant QTLs for adrenal weight and various structural measures. The results suggest that specific and unique genomic regions are potentially associated with our phenotypic measures, which offers the opportunity to investigate the genes located in these genomic regions and try to connect some or all of them to the adrenal gland. Related to this, our study involved performing the QTL analyses using a RI panel with both a large set of BXD strains and a large sample size. In any study, the larger the sample size the more statistical power is associated with the results, but the use of numerous RI strains is particularly effective for our significant QTL results. This is because finer resolution mapping of QTLs is associated with increased numbers of RI strains being analyzed for complex phenotypes (Peirce et al., 2004). Third, there were significant differences obtained for several adrenal measures between the B6 and D2 parental strains, as well as between the BXD lines, which indicates that adrenal structure is influenced by strain background. Furthermore, this suggests that there may be a genetic link associated with the structural differences between these strains. The BXD panel is important for examining this genetic link because the unique, homozygous recombination of the chromosomes across these RI strains from the B6 and D2 progenitor strains allows one to determine the allelic distribution of a specific phenotype, like adrenal weight, at a particular genomic location (McClearn et al., 1991; Gill et al., 1996; Peirce et al., 2004). Fourth, there were significant phenotypic differences between males and females that matched previously established sex differences in adrenal size and weight, which provides evidence for the internal validity of our phenotypic measures. Lastly, adrenal weight has been previously linked to stress using a QTL approach (Solberg et al., 2006). Furthermore, some of their significant QTLs associated with rat adrenal weight mapped to some of the orthological genomic locations found for our significant and suggestive QTL regions for male and female mouse adrenal weight. Thus, examining adrenal weight and structure with a QTL approach is a potentially beneficial first step towards not only identifying genomic regions or specific genes associated adrenal gland morphology but also genomic loci that may influence the stress response. 67  With this in mind, unlike previous studies that have investigated QTLs associated with adrenal weight and stress (Llamas et al., 2005; Solberg et al., 2006), our current study did not perform physiological or behavioural measures of stress. Instead, we focused on analyzing the anatomy of the adrenal and performed adrenal weight and regional width measurements. Thus, a direct comparison between adrenal structure and function was not conducted for the BXD strains and parental mice in our study. Despite this, we did find several significant correlations between our adrenal structural measures and behavioural and physiological measures of the stress response in various BXD strains. In addition, some of our significant and suggestive QTLs for the adrenal measures overlapped with suggestive and significant QTLs identified previously for stress and anxiety-related phenotypes (Flint et al., 1995; Roberts et al., 1995; Turri et al., 2001; Turri et al., 2004; Ponder et al., 2007b; Thifault et al., 2008). Thus, such indirect measures showed potential phenotypic and genetic links between adrenal structure and function, but a direct comparison is appropriate to substantiate this possible connection. In regards to the structural measures, our study involved conducting total, cortical zones, and medulla width measurements for the adrenal gland. From our knowledge, width analysis is not a conventional method of examining adrenal structure. Instead, adrenal volume (Bielohuby, 2007) or total regional cell counts (Ulrich-Lai et al., 2006; Bielohuby, 2007) may be more appropriate measures of adrenal structure. However, such conventional analyses would have been exhaustingly difficult for our study, particularly because large numbers of mice were being assessed. Instead, our width measurements were more appropriate while compiling a large quantity of data, and they often showed relatively small within strain variability for the various BXD strains, as well as for the B6 and D2 mice. Therefore, based on the relative consistency of these structural measurements for the adrenals examined, our width analyses are a novel and valid method of analyzing adrenal gland structure.  3.3  Differences between C57BL/6J and DBA/2J mice and the adrenal gland  In our study, we examined B6 and D2 strains of mice because their genomes are fully sequenced, and they are known to vary significantly for several complex phenotypes. We were particularly interested in their divergent responses to stress and anxiety-related measures (Kakihana et al., 1968; Roberts et al., 1992; Tarricone et al., 1995; Yilmazer-Hanke et al., 2003; Võikar et al., 2005; Ponder et al., 2007a). Since the adrenal gland is vital to the stress response and has not been examined in these inbred strains, we performed our analysis as a first step towards investigating genes or genomic regions associated with adrenal gland structure, but also for the stress response. Based on these previous studies showing D2 mice exhibiting heightened physiological and behavioural responses to stress compared to B6 mice, which are referred to as stress resistant (Shanks et al., 1990; Trullas and Skolnick, 1993; Anisman et al., 68  1998), we hypothesized that adrenal gland structure would be linked to its function in terms of the stress response, and D2 mice would have heavier adrenal weight than B6 mice. Furthermore, we expected D2 mice to exhibit a larger adrenal gland than B6 mice, particularly because we expected the D2 mice to have larger ZF and adrenal medulla regions compared to B6 mice. This is because the ZF is associated with the HPA-axis mediated stress response system, and the adrenal medulla is involved in the AHSmediated autonomic stress response. However, only the relative, but not actual, adrenal weight differed significantly between the strains, while B6 mice exhibited larger ZF size than D2 mice for both males and females, the B6 male mice displayed larger adrenal glands than D2 males, and only the female D2 mice had a larger medulla region than B6 mice. Thus, the results from our study suggest that the relationship between adrenal anatomy and the stress response is more complex than expected. The negative relationship between the weight and size of the male adrenals for the parental strains and the similarity in adrenal size and weight for the females in our study suggests that other measures, such as cell size, number, and/or density, may play important roles not only in linking adrenal structure to adrenal weight, but also connecting structure to function. Since the female D2 mice exhibited a larger medulla region compared to the B6 mice in our study, this result may coincide with a higher cell density in this region, potentially showing a sex-specific link between adrenal structure and the autonomic stress response. Meanwhile, the B6 mice may have a larger ZF region because they may have a more sensitive HPA-axis mediated stress response than D2 mice, and/or they may have more cells releasing corticosterone that bind to more glucocorticoid receptors, activating the negative feedback loop of the HPA axis and leading to a more efficient stress response than the more anxious D2 mice. In terms of relating cell size and density to the stress response, the study by Ulrich-Lai et al. (2006) with rats showed that chronic stress induces adrenal hypertrophy and hyperplasia in the ZF and hypertrophy in the adrenal medulla, i.e., the regions that specifically secrete stress hormones. However, work by Prewitt and Herman (1997) showed that acute restraint stress in rats does not significantly affect adrenal weight nor lead to adrenal hypertrophy, whereas chronic variable stress does induce both adrenal weight increases and adrenal hypertrophy. Furthermore, both studies showed significant increases in plasma corticosterone levels in the control rats for acute and chronic stress conditions, regardless of the adrenal structural changes. In addition, the autonomic and HPA-axis mediated stress responses are intimately linked (Wurtman, 2002; Ehrhart-Bornstein and Bornstein, 2008; Goldstein and Kopin, 2008), thus, it is unlikely that a direct link between adrenal structure and function would exist only for the ZF in B6 mice and for the adrenal medulla in D2 mice. Instead, the link between adrenal structure and stress responsivity in these divergent strains may be hidden by the condition for which they were examined in our study. We performed adrenal measures only under basal conditions, 69  without performing acute or chronic stress paradigms. Since D2 mice are stress reactive and overall show a larger adrenal medulla region compared to B6 mice, the autonomic stress response system may be the driving force that affects significant adrenal structural and weight changes in this strain during stress. Meanwhile, the negative feedback loop of the HPA-axis mediated stress response may play a modulatory role that reverses the structural and weight increases in the D2 strain, and the ZF might be particularly affected by these changes, which may explain why they size of the ZF is significantly smaller in the D2 mice compared to B6 mice during basal conditions. If we consider this hypothesis further, there are no studies that have examined differences in stressinduced epinephrine and norepinephrine levels or adrenal medulla catecholamine concentrations under basal or stress-induced conditions in B6, D2, or other strains of mice. Instead, the hormone secretion of the HPA axis is often the primary focus of stress research, where CRH, ACTH, and/or cortisol or corticosterone levels have been investigated under various conditions (Nemeroff et al., 1992; Herman et al., 1995; Roberts et al., 1995; Prewitt and Herman, 1997; Zelena et al., 2003; Llamas et al., 2005; Solberg et al., 2006; Ulrich-Lai et al., 2006). In the study by Roberts et al. (1995), they investigated corticosterone response to EtOH in B6, D2, and BXD strains of mice. They found that B6 and D2 mice did not differ significantly in corticosterone levels one hour after EtOH or saline administration, but D2 mice showed elevated corticosterone response to EtOH and saline following withdrawal from initial EtOH administration compared to B6 mice, which coincides with previous work indicating that D2 show an adverse response to EtOH compared to B6 mice (Eleftheriou and Elias, 1975; Roberts et al., 1992; Belknap et al., 1993; Gill et al., 1996). These results showed that initial corticosterone response was not significant, whereas a previous study by Roberts et al. (1992) found that D2 mice experienced more severe acute EtOH withdrawal seizures and elevated plasma ACTH and corticosterone levels from acute EtOH administration, which would be associated with the HPA-axis mediated stress response and the ZF region. Meanwhile, both studies also involved performing handling-induced convulsion measures, which examines drug withdrawal hyperexcitability (Goldstein, 1973). In the earlier study, both strains showed increased handling-induced convulsions, but no strain differences, when administered corticosterone, while the later study found that D2 mice exhibited significantly more convulsions compared to B6 mice during EtOH withdrawal. Another study by Roberts et al. (1991) found strain differences in handlinginduced convulsions during EtOH withdrawal that was increased by acute and chronic corticosterone treatment in the seizure-prone strain but not in the seizure-resistant strain. These studies suggest the glucocorticoids are required for EtOH withdrawal seizure activity, and the HPA-axis mediated stress response may be associated with the strain differences in handling-induced convulsions between B6 and D2 mice. Despite this, the inconsistent results on the handling-induced convulsion measure for these 70  stress-divergent strains may be associated with the autonomic “fight or flight” stress response, which involves the adrenal medulla. This idea would potentially coincide with our hypothesis about the AHSmediated stress system being the driving force in adrenal structural changes during stress, including handling-induced convulsions. In this regard, some studies have shown that the noradrenergic system actually plays an anticonvulsant role (Tsuda et al., 1990), where norepinephrine and other adrenergic agonists reduced handling-induced convulsions (Chermat et al., 1981). In addition, both norepinephrine and neuropeptide Y synergy decreased handling-induced convulsions (Weinshenker et al., 2001). These results contradict our AHS hypothesis, but neither study examined the adrenal medulla, epinephrine, or the autonomic stress response, and another study found that the noradrenergic system may potentiate sound-induced seizures (Horton et al., 1980). Thus, the AHS-mediated stress response may be associated with increased epinephrine release during handling-induced convulsions, which may account for increased convulsions in the D2 mice compared to B6 mice, but further investigation would be required to test this hypothesis. There was also research that examined the differences between B6 and D2 mice while performing a tail suspension test (TST), which seems to provide further evidence for this medulla-driven hypothesis. The TST is used to examine antidepressant activity in pharmacological studies (Cryan et al., 2005), and immobility during the TST is indicative of despair and an index of an animal’s depressive state in a stressful situation (Lad et al., 2007). In the study by Lad et al. (2007), they found that D2 mice spent markedly less time immobile and exhibited significantly more transitions from immobile to mobile (increased mobile frequencies) compared to B6 mice. Depression is viewed as a chronic stress-related disorder (Carroll et al., 1976; Rubin et al., 1987; Ulrich-Lai et al., 2006), and this would suggest that stress reactive D2 mice should have shown more time immobile than the stress resistant B6 mice. However, the B6 mice appear to be more “depressed” than D2 mice from these results. Thus, immobility on the TST measures despair, which may be separate from anxiety and stress associated with handling during this test. Similarly, like the handling-induced convulsion measures, D2 mice may have struggled more throughout the TST compared to B6 mice because their “fight or flight” stress response was being activated during testing. From this perspective, the hypothesis suggests that adrenal structure and function may be causally linked, or the connection involves multiple factors that were not examined directly in our study. Regardless, adrenal structural differences are evident between these strains, but it is not certain how adrenal structure corresponds to adrenal function, particularly the adrenal stress response.  71  3.4  The genetic examination of adrenal gland structure As indicated, this is the first study to use a QTL approach to examine adrenal gland structure. It is  also the second study, following Solberg et al. (2006), to identify significant QTLs for adrenal gland weight. QTL analyses are efficient for establishing statistical associations between phenotype and genotype, as well as enable one to narrow the search across the genome for candidate genes that may have a strong influence on complex phenotypes, such as adrenal gland weight and structure. We found several significant and suggestive QTLs for both adrenal weight and structural measures of the adrenal cortex and medulla. Many of these QTLs were unique to the specific structural phenotype examined, which indicates that there are multiple and distinct genomic loci that potentially influence these phenotypes. However, there were a few overlapping significant and suggestive QTLs between adrenal weight and the adrenal cortical and medulla measures. These overlapping regions were found primarily on Chr 3, 4, and 10, and they suggest that some genes in these genomic regions may play a pleiotropic effect by influencing adrenal weight and region specific structure. Interestingly, when analyzing total adrenal width and widths of specific adrenal zones, the regions in males and females that exhibited the most robust correlations with adrenal weight were the structural measures that showed overlapping QTLs with adrenal weight, which further strengthened the possible genetic link on Chr 3, 4, and 10 for these adrenal phenotypes. In this regard, our analysis provides an avenue for which the genetic underpinnings of adrenal gland structure can be further investigated. In our study, we also examined the results from previous studies that have identified significant and suggestive QTLs for stress and anxiety-related phenotypes. Since the adrenal gland is critical for the stress response, we wanted to determine if there are genomic regions that overlapped between our adrenal structural measures and those associated with stress and anxiety, which would suggest that there is a genetic link between adrenal structure and function. With this in mind, we found that significant and suggestive QTLs on Chr 3, 10, and 17 for our adrenal measures overlapped with several stress and anxiety-related QTLs from previous studies. For Chr 3, a significant QTL influencing EPM behaviour (Turri et al., 2004) and one associated with corticosterone levels six hours following EtOH administration (Roberts et al., 1995) mapped in a similar region to a suggestive QTL for female adrenal medulla width, while a significant QTL related to emotional reactivity in open field (Thifault et al., 2008) mapped in a similar region to the significant QTL for female adrenal weight. On Chr 17, significant QTLs associated with corticosterone levels six hours postsaline administration (Roberts et al., 1995) mapped in a similar region to suggestive QTLs for female ZR width, while significant QTLs on Chr 17 influencing DL box measures and EPM behaviour (Turri et al., 2004) were located in regions close to a suggestive QTL for male adrenal weight. Moreover, a study by Williams 4th et al.(2009) examined a candidate gene 72  implicated in anxiety-like behaviour in mice and various psychiatric disorders in humans, glyoxalase 1 (Glo1), that is mapped near the same suggestive QTL for male adrenal weight. In addition, the distal end of Chr 10 showed several overlapping QTLs for our adrenal weight and structural measures, and various studies have also identified significant QTLs in a distal region of Chr 10 associated with behavioural measures of emotionality (Flint, 2002), anxiety-like behaviours related to learned and innate fear (Ponder et al., 2007b), seizure susceptibility (Gershenfeld et al., 1999), as well as exploratory and fear-like behaviours in mice (Gershenfeld and Paul, 1997; Gershenfeld et al., 1999; Zhang et al., 2005). Furthermore, the significant QTL for male total adrenal width and the suggestive QTLs for male and female adrenal weight mapped in a region of Chr 10 that encompassed a QTL termed Exq1 for exploratory and excitability that is associated with exploratory and fear-like behaviours in mice involving open field ambulation and DL box paradigms (Gershenfeld et al., 1999; Zhang et al., 2005). These results indicate that the pleiotropic locus, Exq1, which has been found to affect exploration, fear-like behaviours, and seizure susceptibility, may also influence adrenal measures of size and weight. Thus, although we did not measure the stress response or other adrenal function in our current study, we did find potential genetic connections between adrenal gland structure and function based upon previous work in mice. In this regard, our approach embodies the important statement written by Crick and Koch (2005), which states, "In biology, if seeking to understand function, it is usually a good idea to study structure." With respect to this, our study was the first step towards understanding more about the genetic bases for adrenal gland structure, as a means of also investigating the genetic underpinnings of the adrenal gland stress response.  3.5  Future directions and potential applications of research findings  QTL analyses are effective for examining genomic regions associated with complex phenotypes. This study uses a QTL approach to identify genomic loci associated with adrenal gland structure, which enables us to examine specific genes located in these loci that may have a particularly strong influence on adrenal structure. Once these genes are identified, it would be important to determine if the same genes or gene homologues are expressed in the human adrenal gland. In this sense, it would be essential to determine if these genes or genomic loci are also associated with adrenal gland function, which would provide genetic evidence for a close link between adrenal gland structure and function. Furthermore, our results could provide the basis for determining the function of genes identified in the significant QTL regions for which there is no known function or identify novel roles for such genes. Thus, future studies would have to be conducted to investigate such potential applications of our research. In terms of future directions for our specific results, there are a number of studies that could be performed, especially since 73  our QTL analyses revealed significant QTLs for the adrenal weight and structural measures. After investigating the genes located in the significant genomic regions, we found five interesting candidate genes with functions that have been associated with the adrenal gland and/or its function. It would be beneficial to follow-up on these genes and examine their connection to the adrenal gland further. Thus, one future study could involve determining if these candidate genes are expressed in the adrenal gland at levels that co-vary with the structural phenotypes in the BXD lines analyzed in our study. If there is a high covariance between expression levels and the respective structural phenotypes, it would provide substantial evidence that these phenotypes are influenced by the candidate genes. Our study was also the first to use a RI BXD mouse panel to analyze the adrenal gland. Although we found significant strain differences for several structural measures, we have only scratched the surface for examining adrenal gland structure and anatomy. Thus it might be effective to analyze whole adrenal gland and region specific volumes to determine if similar and/or unique QTL regions are associated with another structural measure. Furthermore, no study has examined cell size, number, or density in the adrenal glands of B6, D2, and BXD strains of mice, nor have the adrenal sub-regions been dissected and weighed separately. Thus, it is not certain whether there would be significant differences between these strains for such morphological measures. Hence, it would be beneficial to conduct such analyses because they may account for strain differences in adrenal structure that were not recognized by our adrenal width analyses. During our research, we found that previous studies suggest that there is a direct link between adrenal gland structure and function, including the stress response. Thus, it would be beneficial to examine this relationship between adrenal structure and the stress response more closely from a behavioural, chemical, molecular, and/or genetic perspective. For behavioural measures, it may be beneficial to examine adrenal gland structure coupled with behavioural testing using stress and anxiety paradigms, like open field behaviour and EPM (Turri et al., 2001) or restraint stress (Tarricone et al., 1995) and chronic stress paradigms (Prewitt and Herman, 1997; Ulrich-Lai et al., 2006). By focusing on the adrenal regions associated with the stress response, a direct comparison of adrenal structure and function might be obtained by determining if the BXD lines showing the largest sizes for the ZF and adrenal medulla correspond to more anxiogenic or higher stress-related behavioural responses compared to BXD lines with smaller ZF and adrenal medulla regions. The same behavioural tests could be performed on the BXD lines with the lowest and highest adrenal weights to determine if similar positive correlations exist between adrenal gland weight and stress-related function. Since chronic stress has been found to produce long-lasting increases in glucocorticoid and ACTH secretion and adrenal hypertrophy (Herman et al., 1995), an additional study could involve obtaining 74  plasma ACTH or corticosterone levels in various BXD lines or the D2 and B6 mice to examine whether larger or heavier adrenals are associated with elevated plasma or corticosterone levels. In this regard, behavioural testing could be incorporated into the analysis, such that basal corticosterone levels could be examined prior to stress-related behavioural testing, and then stress corticosterone levels could be assessed in a manner similar to studies performed by Llamas et al.(2005) and Solberg et al. (2006). This study would determine if physiological and behavioural measures of stress are correlated with adrenal gland size and weight. To examine this relationship between adrenal structure and function further, it would also be effective to conduct histological analysis of the adrenal gland by staining the adrenal tissue and examining stress-related effects on the ZF and medulla. Using techniques performed by Engeland et al. (2005) and Ulrich-Lai et al. (2006), one experiment could involve conducting chronic stress behavioural testing with various BXD or B6 and D2 strains, and then performing immunohistochemical and stereological analyses of the adrenal medulla and ZF to determine if region specific hyperplasia and/or hypertrophy occurs and whether there are strain differences in these measures. Alternately, another experiment could utilize the immobilization stress paradigm performed by Sanchez et al. (2003) to examine basal concentrations of epinephrine and norepinephrine or stress-induced changes in the concentrations of these catecholamines in the adrenal medulla of BXD lines with small and large adrenal medulla regions to determine if concentration levels are correlated with medulla size. These experiments could provide direct evidence that adrenal gland structure and function are specifically linked and that stress may have a particular effect on adrenal gland weight and structure, which could be further mediated by genetic differences in the strains that are examined.  75  3.6  References  Anisman, H., Lacosta, S., Kent, P., McIntyre, D.C., and Merali, Z. (1998). Stressor-induced corticotrophinreleasing hormone, bombesin, ACTH and corticosterone variations in strains of mice differentially responsive to stressors. Stress, 2: 209-220. Belknap, J.K., Crabbe, J.C., and Young, E.R. (1993). Voluntary consumption of ethanol in 15 inbred mouse strains. 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Anxiety-related behavior and densities of glutamate, GABAA, acetylcholine and serotonin receptors in the amygdala of seven inbred mouse strains. Behavioural Brain Research, 145(1-2): 145-159. Zelena, D., Mergl, Z., Földes, A., Kovács, K.J., Tóth, Z., and Makara, G.B. (2003). Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint. Am. J. Physiol. Endocrinol. Metab., 285: E1110E1117. Zhang, S., Lou, Y., Amstein, T.M., Anyango, M., Mohibullah, N., Osoti, A., Stancliffe, D., King, R., Iraqi, F., and Gershenfeld, H.K. (2005). Fine mapping of a major locus on chromosome 10 for exploratory and fear-like behavior in mice. Mammalian Genome, 16(5): 306-318.  79  APPENDICES Appendix A  80  

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