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Unmasking the functional anatomy of medial preoptic nucleus-influences on the hypothalamic-pituitary-adrenal… Williamson, Martin Alexander 2009

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UNMASKING THE FUNCTIONAL ANATOMY OF MEDIAL PREOPTIC NUCLEUSINFLUENCES ON THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS  by MARTIN ALEXANDER WILLIAMSON B.Sc, Dalhousie University, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies  (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  January 2009  © Martin Williamson, 2009  ABSTRACT The hypothalamic-pituitary-adrenal (HPA) axis is a critical mediator of the stress response system. However, despite clear evidence for an inhibitory role of testosterone on stress-induced activation of the HPA axis, the routes and mechanisms have not been addressed. To first determine where testosterone acts in the brain to regulate stress-related input to the HPA axis, I used a combined retrograde transport and immunohistochemical procedure to characterize the anatomical nature by which androgen targets in the brain communicate with the paraventricular nucleus (PVN) of the hypothalamus, the initial point of the neuronally mediated stress response. The findings suggest that androgens could act throughout the brain, and on a large assortment of brain regions that innervate the PVN. Among the brain regions identified, neurons of the medial preoptic nucleus (MPN), highly express androgen receptors and project abundantly to the PVN, suggesting that the MPN stands out as a potential site of integration between testosterone and the HPA axis. To test the functional role of these cells, I tested whether lesions of the MPN alter the inhibitory effects of testosterone on the HPA axis. By selectively removing cells in the MPN, testosterone regulation of the PVN and HPA axis was eliminated. Together, these findings demonstrated that the integrity of the MPN is essential in maintaining the regulatory effects of testosterone on the brain's response to stress. Finally, to clarify whether the MPN is the seat of, or an obligatory relay for the central effects of testosterone, I tested the effects of implanting the androgen receptor antagonist hydroxyflutamide into the MPN, on the stress-induced activation of the PVN and HPA output. The differential effects of androgen exposure in the MPN on the biosynthetic capacity and activational responses of the PVN and its extended circuitries suggested that the MPN is capable of bridging converging limbic influences to the HPA axis with changes in gonadal status.  ii  TABLE OF CONTENTS ABSTRACT  ii  LIST OF TABLES  vi  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS  ix  ACKNOWLEDGEMENTS  xi  CO-AUTHORSHIP STATEMENT  xii  CHAPTER 1 : INTRODUCTION  1  1.1 Literature Review  3  1.2 The hypothalamic-pituitary-adrenal (HPA) axis: general 1.2.1 Components of the HPA axis 1.2.2 Regulation of glucocorticoids 1.2.3 Regulation of ACTH release  3 4 4 6  1.3 Stress-related HPA activation and negative feedback 1.3.1 Activation of the HPA axis 1.3.2 Negative feedback regulation of the HPA axis  9 10 10  1.4 Candidate stress neurocircuits 13 1.4.1 Neuroanatomical organization of the PVN 13 1.4.2 Acute stressors and inducible early-genes 15 1.4.3 Categorization of stressors 17 1.4.4 Neural regulation of the PVN 18 1.4.4.1 Pontine-medullary 18 1.4.4.2 Circumventricular organs 20 1.4.4.3 Limbic system 20 1.4.4.4 Subcortical relays: the bed nucleus of the stria terminalis and hypothalamus.... 22 1.5 The dynamic HPA axis 1.5.1 The dynamic HPA axis: impact of testosterone 1.5.2 Gender mediation of abnormal HPA function  23 25 27  1.6 Androgens and androgen receptors: mechanism of action 1.6.1 Regulation of the androgen receptor 1.6.2 Coregulators of the androgen receptor 1.6.3 Functional role of immediate early-genes  28 31 33 34  1.7 Steroid regulation of the HPA axis 1.7.1 The adrenal gland: regulation by gonadal steroids 1.7.2 The anterior pituitary gland: regulation by gonadal steroids 1.7.3 The PVN: regulation by gonadal steroids 1.7.4 Expression of androgen receptors within afferent regulators of the PVN  35 36 38 38 40  1.8 Evidence for testosterone regulation upstream of the PVN  40  1.9 Thesis objectives and hypotheses  42  1.10 References  43 iii  CHAPTER 2 : Androgen receptor expressing neurons that project to the paraventricular nucleus of the hypothalamus in the male rat 63 2.1 Introduction  63  2.2 Methods 2.2.1 Animals 2.2.2 Mapping candidate androgen-responsive afferents to the PVN 2.2.3 Tissue preparation 2.2.4 Localization and connectivity of androgen-sensitive neurons 2.2.5 Imaging and analysis  65 65 66 68 70 72  2.3 Results 2.3.1 FG injection and retrograde labeling 2.3.2 Forebrain retrograde- and androgen receptor-labeled neurons 2.3.2.1 Limbic-related; PFC, septum, amygdala, and hippocampus 2.3.2.2 Bed nuclei of the stria terminalis 2.3.2.3 Circumventricular organs 2.3.2.4 Hypothalamus 2.3.2.5 Periventricular zone 2.3.2.6 Thalamus 2.3.3 Hindbrain retrograde- and androgen receptor-labeled neurons 2.3.3.1 Reticular core 2.3.3.2 Pons-medulla  73 73 75 75 76 81 81 89 89 90 90 96  2.4 Discussion  101  2.5 References  115  CHAPTER 3 : Selective Contributions of the Medial Preoptic Nucleus to TestosteroneDependant Regulation of the Paraventricular Nucleus of the Hypothalamus and the HPA axis 135 3.1 Introduction  135  3.2 Methods 3.2.1 Animals 3.2.2 Treatment 3.2.3 Tissue and blood collection 3.2.4 Immunoassays and hybridization histochemistry 3.2.5 Imaging and statistics  137 137 138 140 141 145  3.3 Results 3.3.1 Medial preoptic nucleus lesions 3.3.2 Body weights 3.3.3 Testosterone replacement and HPA hormones 3.3.4 Parvicellular PVN Fos-ir 3.3.5 Median eminence CRH- and AVP-ir 3.3.6 Amygdala CRH and AVP mRNA  146 146 149 150 152 152 155  3.4 Discussion  160  3.5 References  167  iv  CHAPTER 4 : The medial preoptic nucleus integrates the central influences of testosterone on the paraventricular nucleus of the hypothalamus and its extended circuitries 172 4.1 Introduction  172  4.2 Methods 4.2.1 Animals 4.2.2 Intracerebral microimplants 4.2.3 Tissue and blood collection 4.2.4 Immunoassays and hybridization histochemistry 4.2.5 Imaging and statistics  174 174 174 175 176 178  4.3 Results 4.3.1 Implant placements and control studies 4.3.2 Testosterone secretion 4.3.3 Stress hormone secretion 4.3.4 Implants effects on peptide expression in the PVN 4.3.5 Implants effects on peptide expression in amygdala and BST 4.3.6 Implants effects on PVN activational responses to acute restraint stress  180 180 184 184 187 187 193  4.4 Discussion  199  4.5 References  205  CHAPTER 5 : General Discussion  210  5.1 Contributions to original knowledge  210  5.2 Methodological considerations 5.2.1 Retrograde tract-tracing and cell counting 5.2.2 Medial preoptic nucleus lesions and testosterone replacement 5.2.3 Androgen receptor antagonism 5.2.4 Immediate early genes and cellular activation  211 211 213 214 216  5.3 Future considerations 218 5.3.1 Gonadal regulation of neurochemical systems 218 5.3.2 Implications of androgen receptor containment within the pre-autonomic part of the PVN 220 5.3.3 Role of androgens in repeated stress paradigms 222 5.3.4 Implications of androgens in brain plasticity and disease 223 5.4 References Appendices Appendix A  226 232 232  v  LIST OF TABLES TABLE 1. RELATIVE DENSITY OF AR STAINING WITHIN CANDIDATE AFFERENT MEDIATORS OF GONADAL STATUS ON THE P V N PROPER AND SURROUND  105  LIST OF FIGURES FIGURE 1-1. DIAGRAM OF THE HYPOTHALAMIC-PITUITARY-ADRENAL (HPA) AXIS 5 FIGURE 1-2. SCHEMATIC DEPICTING THE ROSTROCAUDAL COMPARTMENTALIZATION OF THE PVN.  14 FIGURE 1-3. A DIAGRAM DEPICTING THE CONVERSION OF CHOLESTEROL TO SEX STEROID HORMONES FIGURE 1-4. A DIAGRAMMATIC REPRESENTATION OF THE STRUCTURAL AND FUNCTIONAL ORGANIZATION OF THE ANDROGEN RECEPTOR FIGURE 1-5. MECHANISM OF ACTION OF STEROID HORMONE RECEPTORS, INCLUDING THE ANDROGEN RECEPTOR ( A R )  29 30 32  FIGURE 2-1. PHOTOMICROGRAPHS SHOWING THE APPEARANCE OF IONTOPHORETIC AND CRYSTALLINE FLUOROGOLD INJECTION SITES 69 FIGURE 2-2. PHOTOMICROGRAPHS SHOWING THE APPEARANCE OF FLUOROGOLD-LABELED CELLS IN THE MEDIAL AMYGDALA 77 FIGURE 2-3. ESTIMATED CELL COUNTS OF FLUOROGOLD-LABELED NEURONS AND AR CONTAINMENT WITHIN THE BED NUCLEI OF THE STRIA TERMINALIS 79 FIGURE 2-4. PHOTOMICROGRAPHS SHOWING THE ACCUMULATION OF FLUOROGOLD WITHIN THE POSTERIOR DIVISION OF THE BED NUCLEUS OF THE STRIA TERMINALIS AT THE LEVEL OF THE ROSTRAL EXTENT OF THE P V N 80 FIGURE 2-5. ESTIMATED CELL COUNTS OF FLUOROGOLD-LABELED NEURONS AND AR  CONTAINMENT THROUGH THE ROSTROCAUDAL EXTENT OF THE MEDIAL HYPOTHALAMUS. ... 82 FIGURE 2-6. ESTIMATED CELL COUNTS OF FLUOROGOLD-LABELED NEURONS AND AR CONTAINMENT THROUGH THE ROSTROCAUDAL EXTENT OF THE PERIVENTRICULAR AND LATERAL HYPOTHALAMUS 83 FIGURE 2-7. PHOTOMICROGRAPHS SHOWING THE ACCUMULATION OF FLUOROGOLD AT THE LEVEL OF THE MEDIAL PREOPTIC AREA 85 FIGURE 2-8. ESTIMATED CELL COUNTS OF FLUOROGOLD-LABELED NEURONS AND AR CONTAINMENT THROUGH THE HINDBRAIN RETICULAR FORMATION AND SENSORY ASSOCIATED NUCLEI 91 FIGURE 2-9. ESTIMATED CELL COUNTS OF FLUOROGOLD-LABELED NEURONS AND AR CONTAINMENT THROUGH THE HINDBRAIN RETICULAR CORE NUCLEI 92 FIGURE 2-10. PHOTOMICROGRAPHS SHOWING THE ACCUMULATION OF FLUOROGOLD IN THE PERIAQUEDUCTAL GRAY 93 FIGURE 2-11. ANATOMICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF FLUOROGOLD AND A R IMMUNORACNVE CELLS IN THE VACINITY OF THE LOCUS COERULEUS 95 FIGURE 2-12. ANATOMICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF FLUOROGOLDL A B E L E D AND A R IMMUNOREACTIVE CELLS WITHIN THE VENTRAL MEDULLA 99 FIGURE 2-13. ANATOMICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF FLUOROGOLDL A B E L E D AND A R IMMUNOREACTIVE CELLS WITHIN THE DORSOMEDIAL MEDULLA 100 FIGURE 2-14. SCHEMATIC SUMMARIZING THE ORGANIZATION OF CELL GROUPS IDENTIFIED AS PROJECTING TO THE P V N REGION AND DISPLAYING A R IMMUNOREACTIVITY 113 FIGURE 3-1. HISTOLOGICAL IDENTIFICATION OF IBOTENIC LESIONS IN THE MEDIAL PREOPTIC NUCLEUS (MPN) FIGURE 3-2. SCHEMATIC REPRESENTATION TO DESCRIBE CELL DAMAGE THROUGH THE ROSTROCAUDAL EXTENT OF THE M P N  147 148  vii  FIGURE 3-3. MEAN ± SEM PLASMA ACTH AND CORTICOSTERONE CONCENTRATIONS IN RESPONSE TO A SINGLE 30 MIN EPISODE OF RESTRAINT 151 FIGURE 3-4. MPN LESIONS AND TESTOSTERONE INTERACT ON THE INDUCTION OF FOS EXPRESSION IN THE MEDIAL PARVOCELLULAR, DORSAL PART OF THE PVN PROVOKED BY ACUTERESTRAINT 153 FIGURE 3-5. RELATIVE STRENGTH OF FOS INDUCTION IN THE AUTONOMIC-RELATED, DORSAL PARVOCELLULAR AND MEDIOVENTRAL PARVICELLULAR SUBDIVISIONS OF THE P V N 154 FIGURE 3-6. ANATOMICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF CRH AND AVP STAINING IN THE MEDIAN EMINENCE 156 FIGURE 3-7. MPN LESIONS AND TESTOSTERONE INTERACT ON MEDIAL PARVOCELLULAR AVP TERMINAL FIBERS IN THE MEDIAN EMINENCE UNDER BASAL CONDITIONS 157 FIGURE 3-8. HYBRIDIZATION HISTOCHEMICAL LOCALIZATION OF CRH M R N A IN THE CENTRAL NUCLEUS OF THE AMYGDALA ( C E A ) 158 FIGURE 3-9. HYBRIDIZATION HISTOCHEMICAL LOCALIZATION OF AVP M R N A IN THE MEDIAL NUCLEUS OF THE AMYGDALA ( M E A ) 159 FIGURE 4-1. HISTOLOGICAL ASSESSMENT OF THE APPROXIMATE DISTANCE OF DIFFUSION OF T E S T O S T E R O N E F R O M W A X I M P L A N T S D I R E C T E D A T T H E M E D I A L P R E O P T I C NUCLEUS ( M P N ) IN GONADECTOMIZED MALE RATS 181 FIGURE 4-2. HISTOLOGICAL IDENTIFICATION OF IMPLANTS DIRECTED AT THE MEDIAL PREOPTIC NUCLEUS ( M P N ) IN GONADECTOMIZED RATS 183 FIGURE 4-3. REPRESENTATIVE DARKFIELD PHOTOMICROGRAPHS TO AVP M R N A LOCALIZATION AT THE LEVEL OF THE POSTERIOR DIVISION OF THE STRIA TERMINALIS (PBST) 185 FIGURE 4-4. MEAN ± SEM PLASMA ACTH AND CORTICOSTERONE CONCENTRATIONS PRIOR TO AND FOLLOWING AN ACUTE EPISODE OF RESTRAINT 186 FIGURE 4-5. HYBRIDIZATION HISTOCHEMICAL LOCALIZATION OF AVP AND CRH EXPRESSION IN THE MEDIAL DORSAL PARVOCELLULAR PART OF THE P V N FOLLOWING MEDIAL PREOPTIC NUCLEUS IMPLANTS 188 FIGURE 4-6. DIFFERENTIAL ANDROGEN RECEPTOR BINDING IN THE MPN INTERACTS ON AVP AND CRH M R N A EXPRESSION WITHIN THE PARVOCELLULAR PART OF THE PVN 189 FIGURE 4-7. HYBRIDIZATION HISTOCHEMICAL LOCALIZATION OF AVP M R N A IN THE MEDIAL NUCLEUS OF THE AMYGDALA ( M E A ) 191 FIGURE 4-8. DIFFERENTIAL ANDROGEN RECEPTOR BINDING IN THE MPN INTERACTS ON AVP EXPRESSION IN THE MEDIAL NUCLEUS OF THE AMYGDALA 192 FIGURE 4-9. DIFFERENTIAL ANDROGEN RECEPTOR BINDING IN THE MPN INTERACTS ON THE INDUCTION OF C-FOS MRNA EXPRESSION IN THE MEDIAL PARVOCELLULAR P V N PROVOKED BY ACUTE RESTRAINT 194 FIGURE 4-10. DIFFERENTIAL ANDROGEN RECEPTOR BINDING IN THE MPN INTERACTS ON THE INDUCTION OF FOS-IR IN THE MEDIAL PARVOCELLULAR P V N PROVOKED BY ACUTE RESTRAINT 196 FIGURE 4-11. ASSESSMENT OF THE NUMBER OF RESTRAINT-INDUCED FOS-IR NEURONS AS A FUNCTION OF TREATMENT STATUS THROUGH THE ROSTROCAUDAL EXTENT OF THE POSTERODORSAL PART OF THE MEDIAL AMYGDALA 197 FIGURE 4-12. DIFFERENTIAL ANDROGEN RECEPTOR BINDING IN THE MPN INTERACTS ON THE INDUCTION OF FOS-IR IN THE LATERAL SEPTUM PROVOKED BY ACUTE RESTRAINT 198 FIGURE 4-13. SCHEMATIC VIEW OF THE RAT BASAL FOREBRAIN SHOWING CANDIDATE CIRCUITS AND MECHANISMS MEDIATING THE CENTRAL INFLUENCE OF TESTOSTERONE ON THE P V N AND ITS EXTENDED CIRCUITRIES 203  viii  LIST OF ABBREVIATIONS A ac ACTH AHA ap AQ AR ARH AVP AVPV BST C CBG CAl CeA CRH CS CUN DMH DR DTN ER FG fx GABA GAD 65 GAD 67 GFAP GR HPA axis HIP IEG int KF LC LHA LPO LS ME MeA MEPO mlf MPN MPO MR MRN NTS  noradrenergic cell groups (CI, C2, C3) anterior commisure adrenocorticotropin hormone anterior hypothalamic area area postrema cerebral aqueduct of Sylvius androgen receptor arcuate hypothalamic nucleus arginine vasopressin anteroventral periventricular nucleus bed nuclei of the stria terminalis adrenergic cell groups (Al, A2, A6) corticosterone binding globulin field CAl, Ammon's horn, hippocampus central nucleus of the amygdala corticotropin-releasing hormone superior central nucleus raphe cuneiform nucleus dorsomedial hypothalamic nucleus dorsal nucleus raphe dorsal tegmental nucleus estrogen receoptor Fluorogold columns of the fornix gamma-aminobutyric acid glutamic acid decarboxylase 65 glutamic acid decarboxylase 67 glial fibrillary acidic protein glucocorticoid receptor hypothalamic-pituitary-adrenal axis hippocampus immediate early-gene internal capsule Kolliker-Fuse subnucleus of the Parabrachial nucleus locus coeruleus lateral hypothalamic area lateral preoptic area lateral septal nucleus median eminence medial nucleus of the amygdala median preoptic nucleus medial longitudinal fascicle medial preoptic nucleus medial preoptic area mineralocorticoid receptor median nucleus raphe nucleus of the solitary tract  ix  NTSm och ot PAG PB PB1 PBm PCG PFC PH PMv POMC PoT PPN PS PVN PVNam PVNap PVNdp PVNlp PVNm PVNmm PVNmp PVNmpd PVNmpv PVNp PVNpm PVNpv PVT RE RM scp SFO SON SPF SUBv V3 V4 VBS VMH VNAB ZI  nucleus of the solitary tract, medial division optic chiasm optic tract periaqueductal gray parabrachial nucleus parabrachial nucleus, lateral division parabrachial nucleus, medial division pontine central gray prefrontal cortex posterior hypothalamic nucleus ventral premammillary nucleus proopiomelanocortin posterior thalamic complex pedunculopontine nucleus parastrial nucleus paraventricular nucleus of the hypothalamus paraventricular nucleus of the hypothalamus, anterior magnocellular part paraventricular nucleus of the hypothalamus, anterior magnocellular part paraventricular nucleus of the hypothalamus, dorsal parvicellular part paraventricular nucleus of the hypothalamus, lateral parvicellular part paraventricular nucleus of the hypothalamus, magnocellular division paraventricular nucleus of the hypothalamus, medial magnocellular part paraventricular nucleus of the hypothalamus, medial parvicellular part paraventricular nucleus of the hypothalamus, medial parvicellular part, dorsal zone paraventricular nucleus of the hypothalamus, medial parvicellular part, ventral zone paraventricular nucleus of the hypothalamus, parvicellular division paraventricular nucleus of the hypothalamus, posterior magnocellular part, lateral zone paraventricular nucleus of the hypothalamus, periventricular part paraventricular nucleus of the thalamus nucleus reunions nucleus raphe magnus superior cerebellar peduncle subfornical organ supraoptic nucleus subparafascicular nucleus of the thalamus subiculum, ventral part third ventricle fourth ventricle variable burrow system ventromedial nucleus of the hypothalamus ventral noradrenergic bundle zona incerta  x  ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Dr. Victor Viau. Thanks for your incredible mentorship, your philosophy on research and your constant enthusiasm. You have always given me freedom, encouraged me to pursue my interests and have always been available whenever I sought advice. I am thankful for having developed an appreciation for the fine art of neuroanatomy, for having inherited the eye for things aesthetically pleasing, for the many ski days and for that extra half-slice of pizza, whether I wanted it or not. Thanks! My adventures in Vancouver and at the University of British Columbia may not have been dreamt without the helping hand of Dr. Richard Brown. I am very grateful for your support and guidance while we worked tirelessly on the Jax Phenome project. Nothing could have prepared me better for grad school than the work ethic I inherited from you. But above all, without your guidance, I may still be wandering. I am also very thankful to Dr. Rick Thompson for contributing meaningfully to this doctoral work. You provided me with a comprehensive technical training, and were (coincidentally?) always in Vancouver or near a phone to assist me. I would also like to thank my committee members Drs. John O'Kusky, Bill Honer, Kiran Soma and especially, my honorary member, Liisa Galea, for their invaluable input and guidance. This work is stronger and clearer because of your honest criticism and helpful suggestions. I am indebted to all every member of the Viau lab, past and present, Brenda Bingham, Patricia Lee, Megan Gray, Piam Kiarostami, Ian Linfoot, Steve Sra and Jenny Yu for their support and friendship. But especially Brenda, my partner in crime, you truly impacted my academic career and personal endeavors in the most positive of ways. Thank you!! I am also grateful to the faculty, staff and students who took part in the Friedman project, and also to members of the Neuroscience Program and the department of Cellular and Physiological Sciences. It also goes without saying that many friends, particularly those of the Andale family, made things a little more interesting along the way! Finally, I thank my Mom and Dad for their love and unequivocal support they have provided me over the years, without which this work would not have been possible. You have encouraged me to pursue my goals and ambitions without hesitation. I did it!!!  xi  CO-AUTHORSHIP STATEMENT  Williamson M, Viau V. 2007. Androgen receptor expressing neurons that project to the paraventricular nucleus of the hypothalamus in the male rat. J Comp Neurol 503: 717-740.  I performed all the experiments and data analysis presented in this paper. I wrote the manuscript that was subsequently revised and edited by Dr. Victor Viau.  Williamson M, Viau V. 2008. Selective contributions of the medial preoptic nucleus to testosterone-dependant regulation of the paraventricular nucleus of the hypothalamus and HPA axis. Am J Physiol Regul Integr Comp Physiol 295: R1020-R1030.  I performed all the experiments and data analysis presented in this paper. I wrote the manuscript that was subsequently revised and edited by Dr. Victor Viau.  Williamson M, Viau V. 2008. The medial preoptic nucleus integrates the central influences of testosterone on the paraventricular nucleus of the hypothalamus and its extended circuitries. In preparation.  I performed all the experiments and data analysis presented in this manuscript. I wrote the manuscript that was subsequently revised and edited by Dr. Victor Viau.  xii  CHAPTER 1 : INTRODUCTION In mammals, including humans and rodents, the hypothalamic-pituitary-adrenal (HPA) axis is a critical mediator of the stress response system. The HPA axis is initiated by the activation of a specialized collection of neurons in the brain, situated within the paraventricular nucleus (PVN) of the hypothalamus. Activation of these cells induces the synthesis and sequential release of a cascade of hormones, culminating with the release of glucocorticoids from the adrenal gland (Cortisol in humans, corticosterone in the rat). While normal adaptation to stress relies on the effective short-term activation of the PVN and HPA axis, sustained activation produced by chronic stress is detrimental and is implicated in several types of affective and neurodegenerative disorders. Therefore, tight control of the HPA axis and glucocorticoid release is essential to health. Interestingly, the reproductive system in males has evolved a capacity for inhibiting HPA activity that could provide a buffer against excessive or maladaptive adrenal responses. Testosterone, for example, inhibits stress HPA function in male rodents and in humans, consistent with the strong negative relationship between the magnitude of the HPA stress response and circulating testosterone concentrations, under normal as well as abnormal conditions (reviewed in Williamson et al., 2005). Interesting, hyperactivity of the HPA axis in depressed male patients is associated with a decrease in circulating testosterone, and this relationship is reversed following effective antidepressant therapy. These findings reflect an important inhibitory role for testosterone on stress-related HPA function and affect. At stake, is where testosterone acts within the central nervous system. In the very least, we do know that the HPA response to stress is initiated via converging excitatory and inhibitory input to the PVN that arise from several brain regions, including limbic forebrain, hypothalamic and medullary-pontine cell groups (reviewed in Herman et al., 2003). Further, a significant 1  portion of this input arrives from brain regions that contain androgen receptors (Simerly et al., 1990; Bingaman et al., 1994a; Zhou et al., 1994). To date, where and how testosterone acts in the brain to regulate input to the PVN and pituitary-adrenal output has not been systematically examined. We believe that these potential androgen targets in the brain hold a key to understanding the functional relationship between systems regulating testosterone and glucocorticoid release. Therefore the aim of my thesis is to reveal the routes and mechanisms mediating the effects of testosterone on the HPA response to stress. To achieve this goal, the following experiments were carried out in three major sections. To explore where testosterone acts in the brain to regulate stress-related input to the PVN, in Chapter 2,1 will characterize the anatomical nature by which androgen receptor-expressing cells project to the PVN directly, and assess the relative densities of androgen receptor staining within these candidate afferent cell groups. In Chapter 3,1 will test the functional role of the medial preoptic nucleus in mediating the inhibitory effects of testosterone on the PVN and HPA axis. Based on the involvement of the medial preoptic nucleus, in Chapter 4 I will assess whether androgen receptors in the MPN are functionally relevant to the PVN. The findings from the proposed experiments will together reveal where and how testosterone acts in the brain to regulate the brain's response to stress. I believe that individual predisposition to disease rests with the MPN and its ability to register changes in circulating testosterone levels. Affective disorders in human males are not only met by demonstrable increases in HPA activity, but by genuine decreases in testicular function. Thus, our findings are extremely relevant to the human condition, aimed at revealing the central nervous system bases by which testosterone regulates stress and sculpts behaviour.  2  1.1  Literature Review The following review of the literature will provide information as well as rationale for the  experimental designs of this project. The review will first provide some background information on the HPA axis, including its role and components, and the neuropeptides and circuitries underlying its activation. This will be followed by a description of how the HPA axis responds to varying environmental conditions or contexts and how these relationships are impacted by sex steroids. Finally, this chapter will wrap up by shedding some light on where and how sex steroids, including testosterone, can directly affect components of the HPA axis and adaptive responses to stress. 1.2  The hypothalamic-pituitary-adrenal (HPA) axis: general Mammals respond to challenging or potentially life-threatening situations with  characteristic changes in behaviour that are coupled to changes in autonomic, immune and neuroendocrine systems aimed at reinstating homeostasis (Dallman, 2003; McEwen and Wingfield, 2003). These adaptive physiological coping strategies help orchestrate the organism's adaptation to potentially harmful situations, by the redirection of both energy (e.g., gluconeogenesis, inhibition of reproductive systems and containment of inflammatory responses) and behaviour (e.g., increased arousal and vigilance, and suppression of feeding and reproductive behaviour) (Dallman, 2003; McEwen and Wingfield, 2003). Common to most stressors, is the activation of the hypothalamic-pituitary-adrenal (HPA) axis, which results in a rise of blood levels of glucocorticoids. While central to the mobilization of glucose, glucocorticoids also orchestrate a variety of other adaptive responses at the neuroendocrine, autonomic, and behavioral levels (for reviews see Dallman et al., 2002; McEwen and Wingfield, 2003). While normal adaptation to stress relies on the effective short-term activation of the HPA axis, sustained activation produced by prolonged and repeated exposure to 3  an unavoidable stressor can lead to dysregulation of the HPA axis. The consequences are pathological changes, involving thymus weight loss and proliferation of adrenal tissue. In fact, this HPA dysregulation has also been causally linked to human psychiatric disorders, including anxiety disorders and depression (refer to section 1.5.2) (for reviews see Chrousos, 1998; McEwen, 2000; Young, 2004). Therefore, tight control of the HPA axis and glucocorticoid release is essential to health. 1.2.1  Components of the HPA axis In mammals, including humans and rodents, the hypothalamus, pituitary and adrenal  glands form a neuroendocrine axis, which regulates the synthesis and release of glucocorticoids. This well-characterized neuroendocrine circuit (refer to Figure 1-1) is governed first and foremost centrally by a specialized collection of neurons in the brain situated within the paraventricular nucleus (PVN) of the hypothalamus, which is the final common pathway through which the brain can regulate the neuroendocrine HPA axis (Swanson and Sawchenko, 1980; 1983). The synthesis and release of corticotropin-releasing hormone (CRH) and several other neuropeptides into pituitary portal circulation stimulate the de novo synthesis and release adrenocorticotropin (ACTH) and species-specific glucocorticoids (corticosterone; the major glucocorticoid in rodents, Cortisol in primates), which act at multiple levels to redirect bodily energy sources, including feedback inhibition of the HPA axis (refer to section 1.3.2) (Kovacs et al., 1986; Kovacs and Mezey, 1987; Swanson and Simmons, 1989). 1.2.2  Regulation of glucocorticoids Proper maintenance of normal homeostasis and successful adaptation to any challenge  ultimately rests on the regulation of adrenal glucocorticoids. Seminal experiments examining the regulation of glucocorticoids demonstrated that hypophysectomy (the removal of the pituitary gland, thereby removing the source of ACTH) results in atrophy of the adrenal cortex and makes 4  BRAIN  mpd-PVN CRH + AVP  1 Anterior Pituitary ACTH  l Adrenal Cortex Glucocorticoid ~* hormones  Figure 1-1. Diagram of the hypothalamic-pituitary-adrenal (HPA) axis. Threats to homeostasis (eg. stress), activate specialized populations of cells within the hypothalamus that results in the synthesis and release of neuropeptides such as corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). These neuropeptides then stimulate the synthesis and release of andrenocorticotropin (ACTH) from anterior pituitary corticotrophs, which in turn, stimulates the adrenal cortex to synthesize and release glucocorticoid hormonesfromthe adrenal cortex (corticosterone in the rat).  5  the animal unresponsive to most stressors, with respect to the lack of an adrenal glucocorticoid response (Baxter and Tyrell, 1987). Furthermore, the injection or infusion of ACTH alone restored both adrenal weight and corticosteroidogenesis in hypophysectomized animals (Baxter and Tyrell, 1987). ACTH released from anterior pituitary corticotropes causes the synthesis and secretion of corticosterone from cells of the zona fasciculata of the adrenal cortex (Baxter and Tyrell, 1987; Mountjoy et al., 1992). It appears that ACTH may be the only secretogogue that drives the adrenal zona fasciculata cells to synthesize corticosterone (Baxter and Tyrell, 1987). Although, there are factors that can modulate the rates of corticosterone synthesis and release, including down-regulation of ACTH receptors, which has been shown to occur with sustained stimulation of ACTH. It is also important to mention that the glucocorticoid response to ACTH saturates at low circulating ACTH concentrations and that the maximal adrenal response at higher concentrations integrates the ACTH concentrations over time and simply persists longer (KellerWood et al., 1983). 1.2.3  Regulation of ACTH release Central to the regulation of glucocorticoid synthesis and release is the peptide ACTH.  From studies of anterior pituitary cells, it is estimated that corticotrope cells represent about 310% of the entire anterior pituitary cell population (Westlund et al., 1985). From studies on pure anterior lobe corticotrope cell populations, it has been shown that ACTH is synthesized as a part of a large precursor protein, the proopiomelanocortin (POMC) molecule (Eipper and Mains, 1980). The proteolytic cleavage and processing of this molecule results in a variety of peptides that are also found within secretory vesicles: a large N-terminal fragment, which yields gammaalpha- and beta-melanocyte stimulating hormones (MSH-gamma; MSH-alpha; MSH-beta), ACTH, beta-lipotropin, beta-endorphin, and possibly other unidentified cleavage products (Eipper and Mains, 1980). Studies of primary cell cultures of anterior pituitary cells in vitro have 6  revealed that the corticotropes are most sensitive to the ACTH-releasing action of CRH (Vale et al., 1983). CRH stimulates ACTH secretion through an adenylate cyclase, a cyclic AMPmediated mechanism, and also stimulates the increased synthesis of the POMC molecule (Vale et al., 1983). The mechanisms regulating ACTH secretion in vivo are multifactorial, with the stimulation effect of hypothalamic-releasing factors, mainly CRH, secreted into hypophysialportal circulation via the external zone of the median eminence and binding specific receptors located in the plasma membrane of the pituitary corticotrope (Antoni, 1986; 1993). An excellent review examines the factors and mechanism by which corticotropes are stimulated in greater detail (Antoni, 1986). The 41-amino acid peptide, CRH, was first isolated in 1981 by Vale and colleagues (1981) and determined to be the principal ACTH secretogogue on the basis of its ability to markedly increase the secretion of ACTH from anterior pituitary corticotropes. Advances in the study of the CRH function have revealed that its expression (Thompson et al., 1987; Imaki et al., 1991), receptors (Chalmers et al., 1995; Van Pert et al., 2000) and immunoreactivity (Liposits et al., 1983a; Swanson et al., 1983) are widely distributed throughout the brain, including the hypothalamus. However, studies seeking the identity of hypophysiotropic CRH neurons responsible in the regulation of ACTH release from the anterior pituitary have identified the PVN as the major source of CRH that stimulates ACTH synthesis and release from corticotropes (Tilders et al., 1982; Liposits et al., 1983b; Bruhn et al., 1984). Within the PVN, CRH immunoreactive perikarya are selectively contained within the medial parvocellular subdivision of the PVN (Swanson and Kuypers, 1980; Swanson et al., 1983). And, consistent with this, studies combining immunohistochemical staining with retrograde tract tracing have also confirmed that cells in the medial parvocellular division of the PVN comprise the major source of anterior pituitary-directed CRH. Once the neuropeptide is synthesized within the PVN, CRH is transported down to, and stored in axon terminals that reside in the external zone of the median 7  eminence, forming terminal varicosities that abut on pericapillary space surrounding pituitary portal capillaries. Upon stimulation, CRH is released from these nerve terminals into the hypophysial portal stimulation (Antoni, 1986). As described earlier, the control of ACTH release is multifactorial. Importantly, studies examining corticotrope function have demonstrated that other prominent peptides have been shown to regulate corticotrope ACTH release, foremost among these is arginine vasopressin (AVP). AVP is a nine amino-acid peptide and that is primarily synthesized within magnocellular neurons of the PVN and supraoptic nucleus, which project to the neurophysis, or posterior lobe of the pituitary. Interestingly, it was initially believed that the neurohypophysial system controlled ACTH secretion via the release of AVP from magnocellular neurons (McCann and Brobeck, 1954; Martini and Monpurgo, 1955). However, in the 1970's it became evident that AVP was also expressed by medial parvocellular, CRH-expressing neurons of the PVN, which synthesize and release AVP directly into the hypophysial portal blood system from the external zone of the median eminence (Zimmerman et al., 1977; Whitnall et al., 1985). In fact, a study by Whitnall and colleagues (1995) demonstrated that almost 50% of CRH-positive axon terminals in the external zone of the median eminence also stain strongly for AVP. Furthermore, removing glucocorticoids by adrenalectomy, and thus eliminating negative feedback inhibition of the HPA axis (described in section 1.3.8), can cause virtually all the CRH-positive terminals in the median eminence to show staining for AVP (Whitnall et al., 1985). This suggests that, in the very least, a majority of CRH-expressing cells housed within the medial parvocellular PVN, have the capacity to co-express AVP. On its own, AVP is a weak ACTH secretogogue, but when secreted from axons at the external zone of the median eminence into long portal blood vessels, CRH and AVP synergistically stimulate ACTH secretion from corticotrope cells of the anterior pituitary, with CRH being permissive for the actions of AVP (Yates et al., 1971; reviewed in Antoni, 1993). Of note, it has more recently been demonstrated that neurohypophysial magnocellular neurons can 8  also send axon collaterals to the anterior pituitary (hypophysial portal system) in much the same way that the parvocellular AVP neurons do, and thus, may also participate in the regulation of ACTH (Rittmaster et al., 1987; Antoni, 1993; and see Wotjak et al., 2002). However, this mechanism remains unclear. In any event, since CRH and AVP are co-expressed in a subset of parvocellular neurons (Tramu et al., 1983; Kiss et al., 1984; Whitnall and Gainer, 1988) and costored in a subset of neurosecretory granules in the external zone of the median eminence (Whitnall et al., 1985), it is likely that increases in plasma ACTH concentrations involve the combined effects of CRH and AVP. The neurohypophysial peptide oxytocin, localized within magnocellular neurons of the PVN, can also modulate ACTH release. Preliminary studies demonstrating a stimulatory influence of oxytocin on ACTH secretion have been well documented (Plotsky et al., 1985; Dohanics et al., 1991); however, more recently, inhibitory influences of this peptide on ACTH have also been shown (Neumann et al., 2000a; Neumann et al., 2000b). The mechanism responsible for this bidirectional influence on the HPA axis remains unclear, though this directional distinction is believed to be situation or context-specific, possibly relying on other factors. The anatomical specificity of this regulatory influence is also unclear, as unlike AVP, oxytocin is not co-expressed and released with CRH, suggesting that its contribution to the HPA axis occurs via the magnocellular system or upstream from the PVN. However, the possibility remains that pre-terminal release of oxytocin from axons of passage in the interior zone of the median eminance (posterior pituitary-directed) could provide a route of entry into the pituitary portal system (Holmes et al., 1986). 1.3  Stress-related HPA activation and negative feedback. Common to most stressors is the fact that an individuals' adaptive response to threatened  homeostasis includes the activation of the HPA axis, which is an essential part of the stress 9  response system. The activation of this neuroendocrine axis involves a series of neuronal and hormonal events, leading to successful adaptation and return to equilibrium following environmental perturbations. A wealth of studies has collectively provided the basis for the idea that the characteristics of a stressor are recognized and appraised throughout the central nervous system (Sawchenko et al., 2000; Pacak and Palkovits, 2001; Herman et al., 2003). However, given that the PVN is the final common pathway through which the brain can regulate the adrenal glucocorticoid response to stress, the integration of these events ultimately depends on the PVNCRH neurosecretory system (Swanson and Sawchenko, 1980; 1983). 1.3.1  Activation of the HPA axis As discussed, the hypothalamic PVN is the final point of neuronally-mediated stress  response (Swanson and Sawchenko, 1980; 1983; Sawchenko et al., 1996). Components of the HPA axis that are sequentially recruited during stress rely on the activation of hypophysiotropic neurons, represented by the dorsal medial parvocellular cell group in the PVN. Activation of these neurosecretory neurons induces the release of peptide stores from the median eminence into the pituitary portal circulation, and foremost among these are CRH and AVP. At the level of the anterior pituitary corticotrope, these peptides synergize on ACTH release into the general circulation (Antoni, 1986; 1993; Aguilera, 1994). At the level of the adrenal gland, ACTH promotes the synthesis and release of glucocorticoids into general circulation. 1.3.2  Negative feedback regulation of the HPA axis Under basal conditions, ACTH secretion is controlled by hypothalamic releasing factors  secreted under the influence of the central circadian rhythm and tonic inhibition by adrenal glucocorticoids (Antoni, 1986; Dallman et al., 1987; de Kloet, 1991). Not surprisingly, removal of glucocorticoids by adrenalectomy results in a marked activation of the HPA axis with increases in expression and secretion in both CRH and AVP from the parvicellular PVN neurons, 10  subsequently followed by increases in ACTH (Plotsky and Sawchenko, 1987; Sawchenko, 1987). Similarly, the rapid activation of the HPA axis and the magnitude of the ACTH response caused by acute stress also depend on the level of glucocorticoids (Dallman et al., 1987). For example, ACTH responses to acute stress are higher at the trough than at the peak of the diurnal variation (Akana et al., 1992a), and increasing basal corticosterone levels with pellets of steroid causes a dose-dependent decrease of ACTH responses to acute stress (Akana et al., 1992b). Glucocorticoids can inhibit the HPA axis directly at the level of the pituitary corticotrope and at different sites in the brain, including the PVN and hippocampus (Dallman et al, 1987; Aguilera, 1994). In fact, it is important to mention that the pituitary is sensitive to the inhibitory effects of dexamethasone (a synthetic glucocorticoid), Cortisol and corticosterone on ACTH synthesis and secretion in vitro and in vivo. However, because anterior pituitary corticotrope cells also contain elevated levels of corticosteroid-binding globulin (CBG) (Koch et al., 1976; de Kloet et al., 1977), this likely makes them less sensitive to the actions of corticosterone in the brain (Dallman et al., 1987; Dallman, 2005). Recently however, several experiments have also highlighted that CBG is expressed throughout the rat central nervous system, including the hypothalamus, though not the PVN (Mopert et al. 2006; Jirikowski et al. 2007). Despite this, the brain remains very sensitive to the negative feedback actions of glucocorticoids. In an important study by Plotsky and Vale (1984), rats that were treated with dexamethasone or corticosterone did not show an increase in secretion of CRH following stress (hemorrhage), suggesting that this steroid acts in the brain. Consistent with these findings, Mor and colleagues (1986) later examined the inhibitory role of corticosterone on stimulus-evoked activity in the medial parvocellular PVN following photic stimuli. They revealed a dose-dependent inhibition of this stimulus-evoked activity within the PVN, when physiological levels of corticosterone were injected 30 min prior to stimulus presentation (Mor et al., 1986). One limitation however, was that these findings could not distinguish between a direct inhibitory effect of glucocorticoids on 11  PVN neurons, or whether it occurred upstream, within afferent regulators of the PVN. Coincidentally, Kovacs and colleagues (1986) investigated this directly by assessing the effects of crystalline microimplants of dexamethasone in adrenalectomized rats implanted near the PVN on ACTH secretagogues, CRH and AVP. As expected, without circulating plasma corticosterone, the adrenalectomized rats showed enhanced staining for CRH and AVP in the medial parvocellular part of the PVN. Interestingly, unilateral implants of dexamethasone prevented the increased staining of CRH and AVP on the ipsilateral, but not the contralateral, side of the brain in adrenalectomized rats. These results demonstrated that there was a clear effect of dexamethasone in the brain and suggest that the PVN neurons may be a primary site of glucocorticoid feedback. In summary, it is now acknowledged that corticosterone can act throughout the brain, including perhaps at the level of the pituitary corticotrope (Viau and Meaney, 2004). Corticosterone acts on two types of receptors: the type-1 high-affinity (Kd [0°C]: 0.5-1.0 x 10"9 M) mineralocorticoid receptors (MR), and the type-2 low-affinity (Kd [0°C]: 2.5-5.0 x 10"9 M) glucocorticoid receptor (GR) (Reul and de Kloet, 1985). The primary site for feedback inhibition under basal conditions is the hippocampus, which contains type-1 MR steroid receptors sensitive to non-stressed levels of circulating glucocorticoids (de Kloet, 1991). Type-2 GR becomes occupied with stress levels of circulating glucocorticoids and this constitutes the major proportion of receptors in the pituitary and PVN (Reul and de Kloet, 1985). Consistent with this, using the specific antibodies to MR and GR, Ratka and colleagues (1989) demonstrated that MR elevates, while GR occupancy has little, if any effects on the basal plasma corticosterone levels. Mounting evidence suggests that, beyond their well-known genomic actions, glucocorticoids can also affect cell function via non-genomic mechanisms (Revelli et al., 1998; de Kloet et al., 2008; Haller et al., 2008; Joels et al., 2008). Such mechanisms operate in many major systems and organs including the cardiovascular, immune, endocrine, and nervous systems. 12  Recent findings demonstrate that these non-genomic effects are exerted by direct actions on membrane lipids (affecting membrane fluidity), membrane proteins (e.g. ion channels and neurotransmitter receptors), and cytoplasmic proteins (e.g. MAPKs, phospholipases, protein kinases, etc.). However, this will not be discussed in great detail here (for reviews see Dallman, 2005; Watts, 2005). 1.4 1.4.1  Candidate stress neurocircuits Neuroanatomical organization of the PVN The PVN provides the interface between the central nervous and endocrine systems,  translating neural information into a blood-borne code. The PVN contains a heterogeneous population of neurons that are differentiated on the basis of their neuropeptide content and their pattern of efferent connections (see Swanson and Kuypers, 1980; Swanson and Sawchenko, 1980; 1983, for reviews). Two classes of neuroendocrine cells comprise the nucleus: (i) magnocellular cells that release vasopressin or oxytocin into general circulation at the level of the posterior pituitary, and (ii) parvocellular neurons that secrete a variety of hormones into the hypophysial portal system to influence the synthesis and release of anterior pituitary hormones. The PVN also contains descending neurons, which project to the brainstem and spinal cord that are presumably involved in coordinating autonomic (sympathetic and parasympathetic) and somatomotor responses with endocrine activity. In all, eight distinct cell groups are contained in the PVN. Fortunately, the PVN is highly compartmentalized (Figure 1-2), with descending pre-autonomic, anterior pituitary- and posterior pituitary-directed neurons being largely segregated (Swanson and Sawchenko, 1980). Magnocellular neurons are clustered in three nuclear groups and can easily be distinguished in Nissl stained preparations by their density and size; called anterior (am), medial  13  dp  B  D "v-,'.' mpd mp<  \ \ an \  V«*£-N  I /I,:--' pv—-  pv--  p v - -,  IP'  V /  pv-»- y  mpv  Figure 1-2. Schematic depicting the rostrocaudal compartmentalization of the PVN. The PVN is illustrated at four rostrocaudal levels (A, -1.33; B, -1.53; C, -1.78; D, -2.0 mm from Bregma). Structures labeled for reference: am, anterior magnocellular; dp, dorsal parvocellular; lp, lateral parvocellular; mpd, dorsal medial parvocellular; mm, medial magnocellular; mpv, ventral medial parvocellular; pm, posterior magnocellular; pv, periventricular part of the PVN.  14  (mm) and posterior (pm) magnocellular. Descending pre-autonomic cells are also distinctly clustered into three nuclear groups and can be identified based on their efferent connections; called dorsal (dp), ventral medial (mpv) and lateral (lp) parvocellular. Finally, anterior pituitarydirected, parvocellular neurons, are centered in the dorsal medial parvocellular (mpd) and periventricular (pv) part of the PVN. As described earlier, the HPA axis is controlled by a discrete set of hypophysiotropic neurons in the medial parvocellular division of the PVN, the seat of CRH neurons and a cocktail of other factors, including AVP, that modulate ACTH release. Beyond pituitary-adrenal control, efferent projections from the parvocellular division are also directed to the brainstem, midbrain, and spinal cord (pre-autonomic; non-neurosecretory), while neurosecretory neurons of the magnocellular division send their axons to the posterior pituitary gland, releasing their contents into systemic circulation (Swanson and Sawchenko, 1980; 1983; Sagaretal., 1988). 1.4.2  Acute stressors and inducible early-genes There are many central neural circuits that are in a position to regulate CRH-expressing  PVN neurons. These cells become activated when they receive afferent neuronal signals that arise from several brain regions, including limbic forebrain, hypothalamic and medullary-pontine cell groups, either directly or indirectly carrying stress-related information. During the last two decades, the induction of immediate-early gene (IEG) expression has been widely used as a marker for neuronal activation. A common feature of all IEGs is their potential to be rapidly induced in cells in response to activating stimuli. IEGs include proteins belonging to the Fos family (e.g. Fos and FosB), Jun family (e.g. c-Jun and JunB) and several other proteins (comprehensively reviewed by Herdegen and Leah, 1998). Separate genes encode all these proteins, and the spatial and temporal patterns of these genes (and their proteins) are highly variable (Chan et al., 1993; Herdegen and Leah, 1998; Girotti et al., 2006; Weinberg et al., 2007). 15  Among all the IEGs used, the c-fos IEG and its protein counterpart Fos have been the most widely studied. In fact, cases in which the induction of other IEGs is not accompanied by the induction of the c-fos gene and protein are very uncommon (Herdegen and Leah, 1998, p. 407). Consistent with this, the induction of c-fos also generally only occurs in the subset of neurons that are metabolically and electrophysiologically active (Sharp et al., 1993; Chaudhuri, 1997). Importantly, the induction of c-fos also only takes place in neurons responding to transsynaptic activation (neurons separated by at least one synapse), and must therefore depend upon the action of chemical neurotransmitters (Morgan et al., 1987; Sagar et al., 1988). For this reason, it is believed that the expression of c-fos mRNA and its protein Fos, may serve as an effective method for mapping functional pathways in the central nervous system. Importantly, numerous studies have examined the spatial and temporal induction of c-fos mRNA and/or Fos protein in PVN neurons in response to a variety of stress paradigms, including novelty (Handa et al., 1993), swim stress (Cullinan et al., 1995), restraint (Viau and Sawchenko, 2002; Girotti et al., 2007; Weinberg et al., 2007), adrenalectomy (Brown and Sawchenko, 1997), and many more. There is a fairly broad set of neural structures that are activated by acute stressors. Only some of these stress circuits may be required for the HPA axis, depending on the specific stimulus. Nonetheless, there appears to be a common group that expresses fos-like activity after several acute stressors (see references above). These include the frontal cortex, the hippocampus (in general), the lateral septum, the bed nucleus of the stria terminalis, the major amygdalar subnuclei, the nucleus accumbens, the parvocellular PVN, the periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, the dorsal raphe nucleus, the nucleus of the solitary tract, and the ventrolateral medulla. Collectively, it is clear that there is a broad set of neural structures that are activated under acute stress conditions. While not all of these regions may be actually involved in the regulation of the HPA axis, the aminergic, visceral, limbic and cortical cell groups appear to be quite 16  uniformally activated by stressors, and these groups probably act to prepare the organism for further onslaughts through coordinated behavioral, autonomic and neuroendocrine changes. Nonetheless, based on the marked similarities and heterogeneity in central activational responses to a variety of stressors, numerous researchers have made attempts to distinguish between the types of stressors and the pathways they use to activate CRH expressing cells in the PVN. 1.4.3  Categorization of stressors Many researchers involved in mapping the stress 'neurocircuitry' have accumulated data  that support the notion that the brain categorizes stressors and uses at least partially separate, category-specific neural pathways to generate subsequent stress responses. Consequently, a rich collection of studies have provided data suggesting that the brain uses distinct neurocircuits to generate stress responses to stimuli that are 'actual threats' to homeostasis (physical or systemic stressors) compared to stimuli that threaten the organisms 'state', either real or perceived (neurogenic, processive, emotional or predictive stressors) (Sawchenko et al., 2000; Dayas et al., 2001a; Pacak and Palkovits, 2001; Herman et al., 2003). Real threats represent genuine challenges to homeostasis (e.g. somatic or visceral pain, humoral inflammatory signals, stimulation of baroreceptors or osmoreceptors). The brain recognizes these stressors, via somatic, visceral, or circumventricular sensory pathways in the midbrain and brainstem that directly activate stress centers in the hypothalamus (e.g. the PVN) (Sawchenko et al., 2000; Dayas et al., 2001a; Herman et al., 2003). On the other hand, predicted threats (e.g. anticipation/recognition of predators) or danger associated with new environments (e.g. restraint stress), lead to an activation of the HP A axis in absence of physiological challenge. These stressors, termed neurogenic stressors, are primarily processed by limbic brain regions, which include the hippocampus, amygdala, and prefrontal cortex, as well as their respective relays en route to the PVN (discussed in section 1.4.4.4) (Sawchenko et al., 2000; Dayas et al., 2001a; Herman et al., 2003). 17  1.4.4  Neural regulation of the PVN The PVN is innervated by virtually every region of the brain, including most major  forebrain nuclei, and several midbrain, pontine, and medullary nuclei (Sawchenko et al., 2000; Herman et al., 2003). Thus, the PVN is poised to receive a variety of multimodal input, including those conveying, for example, limbic-related (forebrain); homeostatic, blood-borne (hypothalamic); visual, auditory, and nociceptive (midbrain, pontine); and somatovisceral and somatosensory (medullary) information. Gleaned from cytoarchitectonic, connectional, phenotypic, and functional studies, all of this input ultimately impinges onto topographically organized and functionally distinct sub-regions of the PVN, and different classes of effector motor neurons occupy each of these (refer to Figure 1-2) (Swanson and Kuypers, 1980; Swanson and Sawchenko, 1983). In addition to the medial parvocellular neurosecretory population that target the anterior pituitary, the PVN also houses neurosecretory neurons that course through the median eminence to terminate and release oxytocin and vasopressin from the posterior pituitary, and parvicellular (non-neurosecretory) neurons directed at the autonomic- and sensory-related centers of the central gray, medulla, and spinal cord. (Rivest and Rivier, 1991; Cullinan et al., 1995; Larsen and Mikkelsen, 1995; Cullinan et al., 1996; Kovacs and Sawchenko, 1996; Li et al., 1996; Herman and Cullinan, 1997; Li and Sawchenko, 1998; Dayas et al., 1999; Campeau and Watson, 2000; Dayas et al., 2001b; Hoffman and Lyo, 2002; Herman et al., 2003; Palkovits et al., 2004; Serrats and Sawchenko, 2006) 1.4.4.1 Pontine-medullary Direct inputs to the PVN arising from catecholaminergic cell groups in midbrain and brainstem (e.g. dopamine, noradrenaline, adrenaline) constitute the densest aminergic fields in the brain and generally play a role in excitation of the hypophysiotropic zone of the PVN and HPA axis, promoting CRH and ACTH release (Plotsky, 1987; Plotsky et al., 1989). Ascending 18  noradrenergic inputs to the PVN arise from distinct cell groups of the caudal medulla and pons, which comprise the Al cell group of the caudal ventrolateral medulla, and the Al cell group in the caudal nucleus of the solitary tract (McKellar and Loewy, 1981; Sawchenko and Swanson, 1982; Cunningham and Sawchenko, 1988). Fibers from the ventrolateral medulla (Al cell group) region preferentially innervate the mpd neurosecretory cells of the PVN, where CRH expressing cells are amassed, at the expense of magnocellular neurosecretory cells, which are primarily innervated by the nucleus of the solitary tract (A2 cell group). Noradrenergic cells of the dorsolateral pons, comprising the locus coeruleus (A6 cell group) also contribute to HPA axis regulation. However, this region does not directly innervate the mpd cells of the PVN, likely modulating the PVN via transsynaptic relays (Cunningham and Sawchenko, 1988). Conversely, PVN input from adrenergic cells arise from the more rostrally-positioned cells in the ventrolateral medullary (CI cell group), the rostral nucleus of the solitary tract (C2 cell group), and the C3 group in the rostral dorsomedial medulla. The adrenergic cell groups project primarily to the CRH-expressing cells in the PVN, with little to no input to the magnocellular neurons of the PVN (Cunningham and Sawchenko, 1988). Catecholaminergic projections ascend through two separate pathways, (i) the central tegmental tract, and (ii) the ventral noradrenergic bundle (VNAB). These tracts converge at the rostral level of the pons, and terminate on the PVN via the lateral hypothalamus (Sawchenko and Swanson, 1982; Cunningham and Sawchenko, 1988; Cunningham et al., 1990). Stimulation of the Al, A2, A6 cell groups or the VNAB increases the firing rate of PVN neurons (Saphier, 1989; Saphier and Feldman, 1989) and HPA axis, as indicated by increases in CRH and ACTH release (Plotsky, 1987; Plotsky et al., 1989). In addition, the effects of central noradrenaline or adrenaline administration or VNAB stimulation on CRH release or ACTH/corticosterone secretion can be blocked by either a-1 (Plotsky, 1987) or a-2 (Daniels et al., 1993) adrenoreceptor antagonists. However, it is important to note that the relationship between 19  catecholamine release and HPA activation varies with stimulus modality, indicating that their release is not the sole determinant of the stress response (Pacak et al., 1995). The release of noradrenaline or adrenaline in the PVN can be observed following the exposure to a variety of different stressors (e.g. restraint, foot shock, conditioned fear), and this ascending system appears to be preferentially involved in response to systemic stressors (e.g. interleukin-1, hypoglycemia, ether) (reviewed in Herman et al., 2006). 1.4.4.2 Circumventricular organs The PVN is also positioned to receive information on fluid and electrolyte status by way of circumventricular organs, which include the subfornical organ, median preoptic nucleus, organum vasculum and lamina terminalis, which are blood-brain barrier-free regions. These regions are critically involved in the control of osmoregulation and drinking behaviour, and consequently, lesions among these regions cause adipsia and dehydration (Johnson et al., 1996). These specialized sites bordering the third and fourth ventricles possess highly vascularized capillary fenestrations and provide another means by which blood-borne information can be sent to the PVN directly. Salt loading, which increases blood osmolality, decreases CRH mRNA in parvocellular neurons of the PVN and up-regulates AVP mRNA in magnocellular neurons of the PVN to promote water retention (Imaki et al., 1992). 1.4.4.3 Limbic system The HPA is also under substantial control of the limbic system, including the hippocampus, amygdala and prefrontal cortex. The hippocampus and prefrontal cortex are largely (but not exclusively) inhibitory to HPA axis secretion, whereas the amygdala is implicated in activation of the HPA axis (Jacobson and Sapolsky, 1991; Feldman et al., 1995; Herman and Cullinan, 1997). Hippocampal stimulation in the rat and human decreases glucocorticoid secretion (Rubin et al., 1966; Dunn and Orr, 1984), while hippocampal lesions in rats significantly 20  elevate medial parvocellular PVN CRH and AVP mRNA levels (Herman et al., 1992; Herman et al., 1995). Interestingly, a study by Herman and colleagues (1995) demonstrated that the inhibitory effects of the hippocampus on HPA secretion and CRH and AVP mRNA expression in the PVN are primarily regulated by ventral-subicular neurons located in the caudotemporal CA1 region. However, hippocampal regulation of HPA activation and secretion appears to be stressorspecific, as some data suggests that the hippocampus may also play a stimulatory role in HPA axis regulation (refer to Table 1, Herman et al., 2005). The medial prefrontal cortex is also heavily involved in HPA axis regulation. Lesions of the anterior cingulate and prelimbic divisions of the medial prefrontal cortex enhance HPA hormonal output and PVN c-fos mRNA induction following restraint (Diorio et al., 1993; Figueiredo et al., 2003b). However, recent findings suggest that the infralimbic and prelimbic/anterior cingulate components of the prefrontal cortex play different roles in the regulation of the PVN. In a recent study by Radley et al. (2006), lesions to the prelimbic part enhance restraint-induced Fos and CRH mRNA expression in the mpd PVN, while ablation of the infralimbic and anterior cingulate parts decrease stress-induced Fos protein and CRH mRNA expression in this compartment, but also increase Fos induction in PVN regions involved in preautonomic control. These results support a topographical organization of the medial prefrontal cortex, which differentially regulates the neuroendocrine and autonomic regions of the PVN. The influence of the amygdala on the PVN and HPA axis is largely mediated by its central and medial amygdaloid nuclei, representing the principle projection neurons to the forebrain and brainstem (Swanson and Petrovich, 1998). In contrast with the hippocampus and prefrontal cortex, the amygdala appears to have a stimulatory role on the PVN and HPA axis. Large lesions of either the central or medial amygdaloid nuclei decrease corticosteroid secretion following ether inhalation (Knigge, 1961), olfactory stimulation (Feldman and Conforti, 1981) and leg break (Allen and Allen, 1974). Conversely, direct stimulation of the amygdala increase corticosteroid 21  secretion in the rat (Redgate and Fahringer, 1973; Saito et al., 1989). However, like other limbic regions, the influence of the amygdala appears to be region- and stressor specific. For example, many studies have demonstrated that the medial amygdala shows intense induction in c-fos mRNA and its protein counterpart, Fos, following stressors such as restraint, swimming, and predator odor exposure (Cullinan et al., 1995; Figueiredo et al., 2003a; Figueiredo et al., 2003b), though substantially less following interleukin-1 injection, hypoxia or hemorrhage (Sawchenko et al., 1996; Thrivikraman et al., 1997; Figueiredo et al., 2003a; Figueiredo et al., 2003b). In addition, Dayas and colleagues (1999) have also demonstrated that lesions of the medial amygdala, but not the central amygdala, greatly reduce restraint-induced activation of cells in the mpd PVN, suggesting that the medial rather than the central amygdala is critical to the neuroendocrine cell responses during an emotional stressor (e.g. restraint). 1.4.4.4 Subcortical relays: the bed nucleus of the stria terminalis and hypothalamus Despite the prominent involvement of the hippocampus, medial prefrontal cortex and amygdala in HPA axis regulation, there is little evidence of direct innervation of the PVN from these nuclei. Instead, these regions appear to depend on first- or second-order relays, by projecting to a number of forebrain and/or brainstem regions, which in turn, project to the PVN (Herman et al., 2003). The bed nucleus of the stria terminalis and hypothalamus are ideally positioned as relays between the limbic system and the PVN. Both receive and reciprocate information with the limbic system, and send substantial input to mpd neurons of the PVN (Sawchenko and Swanson, 1983; Sagar et al., 1988; Simerly and Swanson, 1988; Dong et al., 2001; Herman et al., 2003; Dong and Swanson, 2004). For example, the bed nucleus of the stria terminalis, preoptic area, and dorsomedial hypothalamic nucleus project heavily to the mpd PVN and express Fos protein following swim stress (Cullinan et al., 1996). In addition, there exist sitespecific effects within the bed nucleus of the stria terminalis on HPA responses to stress, and 22  these are likely due to the fact that many cell groups within the bed nucleus of the stria terminalis receive considerable overlap of afferent inputs, while others are highly topographically organized with respect to their inputs (e.g. amygdalar vs. hippocampal) (refer to Dong et al., 2001; Dong and Swanson, 2004). There are also numerous hypothalamic connections to the mpd PVN, though only a few have been systematically studied with respect to PVN and HPA function. Both the medial preoptic and dorsomedial hypothalamic nuclei are acknowledged to be putative regulators in PVN function. Lesions of both of these nuclei produce an increase of HPA secretions (Bealer, 1986; Viau and Meaney, 1996), while their stimulation reliably inhibits PVN neurons (Saphier and Feldman, 1986; Boudaba et al., 1996). It is important to note, however, that notwithstanding their important role as relays for limbic stress-modulatory circuitry, many of these regions also carry their own specialized role within the nervous system and should not be simply regarded as relays (Viau, 2002; Herman et al., 2006; Williamson and Viau, 2007). 1.5  The dynamic HPA axis There are functional shifts in the HPA axis that allow the animal to differentially adapt to  challenges in its internal and external environment. Such changes have been documented to occur as a function of development, social status, season, and reproductive experience (Blanchard et al., 1995; Dallman et al., 2002; Wingfield and Sapolsky, 2003; Tamashiro et al., 2005; Williamson et al., 2005; Spritzer et al., 2008). As a function of development, much research attention has focused on the programming effects of the HPA axis in early life and on understanding HPA function in adulthood. However, there has been relatively little research on adolescence/puberty, a time when many of the neural regions implicated in the control of the HPA axis during adulthood, such as the medial prefrontal cortex, medial preoptic area, bed nucleus of the stria terminalis, hippocampus, and amygdala (reviewed in Herman et al., 2003) are undergoing significant developmental changes (reviewed in 23  Spear, 2000). Not surprisingly, HPA function in rats differs in adolescence compared to adulthood in responses to both acute and chronic stressors. One consequence of exposure to stressors in prepubertal rats appears to be a more prolonged exposure to ACTH and glucocorticoids, which may be critical for ongoing brain development, and programming of future behavioral and physiological responses. For example, in males rats exposed to either intermittent foot shock (Goldman et al., 1973), ether vapors (Vazquez and Akil, 1993), or restraint (Romeo et al., 2004), corticosterone levels of prepubertal males takes longer (e.g. 45-60 min) to return to baseline compared to adults (post-pubertal rats). Shifts in HPA axis function have also been documented as a function of social status. One of the hallmarks of the various animal models of social stress is altered responsivity of the HPA axis. For example, the visual burrow system (VBS) involves housing mixed gender groups of rats in a semi-naturalistic environment as a model of social hierarchy and chronic psychosocial stress (Blanchard et al., 1995; Tamashiro et al., 2005). Exposure to chronic social stress is well known to result in a spectrum of responses that have been thought to allow the organism to better cope with short-term or acute stressors (e.g. competition for resources: space, access to a reproductive partner, food or water). Most models of chronic social stress, including the VBS, demonstrate that subordinate male rats exhibit severe weight loss and increased basal plasma corticosterone levels as compared with dominant males (Ely and Henry, 1978; Blanchard et al., 1993; Blanchard et al., 1995; Ely et al., 1997; Tamashiro et al., 2004). In addition, dominant animals also appear to have a more efficient HPA negative feedback regulation compared to subordinates, as plasma corticosterone returns to basal levels more rapidly in dominants when exposed to a novel stressor, whereas plasma corticosterone levels return to basal levels much later in subordinates (McKittrick et al., 2000).  24  1.5.1  The dynamic HPA axis: impact of testosterone It is interesting that the majority of studies examining HPA function are performed in  males under the assumption that males represent a relatively stable gonadal system compared to females. However, gonadal activity in males is subject to variations, as seen by marked changes in testosterone releases patterns as a function of reproductive experience, season, across development and even social status (Blanchard et al., 1993; Blanchard et al., 1995; Wingfield and Sapolsky, 2003; Tamashiro et al., 2004; Romeo and McEwen, 2006). Furthermore, circulating testosterone levels also vary dynamically over circadian rhythm (Plymate et al., 1989) and in response to stress (Rivier and Rivest, 1991; Wingfield and Sapolsky, 2003; Bingham et al., 2005). It is also important to note that these dynamic shifts in gonadal activity appear to be coupled to changes in HPA function. For example, pubertal increases in testosterone levels have been suggested to alter central regulation of HPA responses (Gomez et al, 2004). Work by Romeo and colleagues (2004) suggests that the ongoing development of the medial preoptic area, a site of sex hormone regulation of HPA function (Viau and Meaney, 1996), may be responsible for the reduced effectiveness of testosterone to inhibit stress-induced corticosterone release in prepubertal/early adolescent males. In addition, reduced levels of androgen receptors in the medial preoptic area have been found in prepubertal rats compared to adult males (Romeo et al., 2000). Certainly, many of the neural circuits carrying stress-related information, including the limbic and hypothalamic regions, are undergoing developmental changes during puberty (reviewed in Spear, 2000). It is possible that these changes are essential in facilitating the increase in sensitivity of the HPA axis to sex hormones seen after puberty. In support of this, research by Viau et al. (2005) suggests that the changes in function within PVN afferents may be responsible for the prolonged release of glucocorticoids in response to a stressor in prepubertal rats. For example, despite similar levels of basal CRH mRNA in the parvocellular PVN, adolescent rats showed heightened activational responses to stress, illustrated by increased Fos 25  protein expression and AVP heteronuclear mRNA expression in the parvocellular PVN in response to restraint compared to adult males (Viau et al., 2005). These findings suggest that puberty-related shifts in parvocellular PVN neurosecretory function in male rats may be emphasized by shifts in stress-induced neuronal activation. However, while the mechanisms regulating the development of in HPA function during puberty remain unclear, the available evidence suggests that these changes, including the maturation of the brain, may be closely tied to changes in circulating testosterone. In the VBS social stress model, subordinate male rats consistently show decreased testosterone secretion during chronic social stress, while dominant rats show maintained or elevated levels of testosterone (Mallick et al., 1994; Blanchard et al., 1995). In addition, reductions in plasma testosterone can occur as early as three days of VBS housing and occur prior to elevated basal levels of corticosterone, suggesting that changes in glucocorticoid levels may be explained by changes in plasma testosterone or gonadal status. Moreover, primates also exhibit changes in gonadal status associated with chronic social stressors. For example, olive baboons with an established social hierarchy have relative rank-related gonadal changes in response to capture stress. Dominant males show increases in testosterone while subordinates exhibit significant decreases in testosterone from basal (control) levels (Sapolsky, 1982; 1985; 1986). Numerous rodent and primate studies have been conducted to determine the behavioral correlates of the differences in testosterone levels between dominant and subordinate animals, aiming to elucidate the potential role of androgens in mediating and/or maintaining social hierarchy formation. Using the VBS model, Nguyen et al. (2004) demonstrated that gonadectomized male rats do not form a hierarchy, compared to intact and testosterone-replaced rats that establish social rank. These data suggest that testosterone is important for hierarchy formation, which can ultimately predict the HPA axis response to stress. Moreover, shifts in HPA function occurring as a function of repeated and chronic stress and disease, are also paralleled by changes in gonadal  function. Thus, the extent to which the HPA axis adapts may very well depend on gonadal status (reviewed in Williamson et al., 2005). 1.5.2  Gender mediation of abnormal HPA function Dysfunction in both resting and stress-induced elevations of the HPA axis has been well  described in affective disease states, including anxiety and depression (reviewed in Chrousos, 1998; McEwen, 2000; Young, 2004). Numerous studies have shown that HPA dysfunction or hyperactivity is marked by hypersecretion of CRH, increased Cortisol levels in plasma and cerebrospinal fluid, exaggerated Cortisol responses to ACTH, and enlarged pituitary and adrenal glands. Importantly, these pathological changes are also seen in individuals suffering from severe mood disorders. Hypersecretion of CRH causing hypercortisolaemia may be a result of impaired feedback mechanisms resulting from GR abnormalities, such as decreased receptor number or altered function. Indeed this view is supported by the demonstration of GR abnormalities in postmortem studies of patients with affective disease states (Webster et al., 1999). Intriguingly, gender is an important determinant of human health, and there are genuine differences in the prevalence rates of several mental disorders, such as depression and anxiety, which have primarily highlighted a role for estradiol in women (Rubinow and Schmidt, 2002; Shors, 2002). Importantly, additional findings also point to a potential role for testosterone in the predisposition and treatment of mood disorders related to HPA dysfunction by the association of depressive illness with hypogonadism, at least in older men (Schmidt et al., 2004; Shores et al., 2005). And recently, individual differences in stress reactivity have been proposed as a potentially important risk factor for gender-specific health problems in men and women, in addition to genetic, socio-cultural, hormonal and developmental factors (Goldstein, 2002; Hamann and Canli, 2004; Young and Altemus, 2004; Kajantie and Phillips, 2006). The instability of androgens and estrogens in males and females, respectively, and the potency by which sex 27  steroids operate on the HPA axis (discussed in greater detail below), clearly implicates the gonadal axis as providing a compelling link between normal and abnormal HPA function and affective disease states. 1.6  Androgens and androgen receptors: mechanism of action Androgens belong to a family of hormones that are synthesized from cholesterol called  steroid hormones. The most predominant form of androgen, testosterone, is a gonadal steroid hormone since it is produced in the Leydig cells of the interstitial compartment of the testis by an enzymatic sequence of steps from cholesterol (summarized in Figure 1-3). However, two important androgens, testosterone and its metabolite dihydrotestosterone, can affect a diversity of responses in a variety of tissues. Androgens mediate their effects by regulating gene transcription by interacting with a specific DNA sequence in a ligand-dependent manner via the androgen receptor (AR). Similar to other steroid hormone receptors (refer to Figure 1-4), the AR comprises a transactivation, DNA binding, nuclear localization (hinge), and ligand-binding domains (Jenster et al., 1991; Simental et al., 1991). Of these domains, the N-terminus is the most variable, while the other regions are highly conserved. Within the N-terminus, the region of amino acids from 141-338 consists of polyglutamine and polyproline residues, which appear to be critical for transcriptional activation (Chang et al., 1988b). The DNA-binding domain consists of 67 amino acids, which fold into two zinc-fingers capable of binding DNA. The remaining 290 amino acids form the C-terminus and encode the nuclear localization signal and ligand-binding domain (Chang et al., 1988b). The androgen receptor, a member of the steroid hormone receptor family, was initially cloned in 1988, identified as a 110 kD nuclear protein which consists of approximately 918 amino acid residues (Chang et al., 1988b; a; Lubahn et al., 1988). As demonstrated by a variety of methods, the AR is present in most tissues. The use of 3H-androgen binding by injection of 3H28  HO  P450 cl7 Pregnenolone HO  Dehydroepiandrosterone  3p-HSD aromatase 170-HSD  Progesterone  P450 cl7 17p-HSD  ^  17p-Estradiol  *£  *?  5a-reductase Testosterone  Dihydrotestosterone  Figure 1-3. A diagram depticing a illustrating the conversion of cholesterol to sex steroid hormones, including androgens.  NH2-  COOH  • *<?  &  •  ^^  &  V  #  Figure 1-4. A diagrammatic representation of the structural and functional organization of the androgen receptor. The domains responsible for their specific functions are also indicated with the numbers of amino acid residues.  testosterone, and the eventual development of anti-AR antibodies and cRNA probes have complemented earlier autoradiographic methods, and facilitated the detection of AR protein and mRNA in most tissues (Gustafsson and Pousette, 1975; Chang et al., 1988a; Chang et al., 1989; Husmann et al., 1990). These methods were able to demonstrate that all male sexual organs in the rat show strong positive nuclear signal staining for the AR, whereas other tissues including hepatic, renal, neuronal and muscular exhibit weaker staining. 1.6.1  Regulation of the androgen receptor AR expression is modified during fetal and sexual development, aging, and malignant  transformation (Husmann et al., 1991; Takeda and Chang, 1991). Regulation of AR activity occurs as a function of ligand-binding interactions, target gene transcription, and posttranslational modifications (refer to Figure 1-5) (Chang et al., 1995). Although AR function is influenced by various factors, this occurs in a tissue- and cell-dependent manner. A primary factor that determines how androgens can achieve distinct effects on various genes and cells rests with the fate of the ligand, as different cells possess unique or overlapping combinations of enzymes capable of converting testosterone to different and biologically active forms (Figure 13). The ultimate actions of testosterone on various cells populations must be considered in terms of its two principle metabolites, dihydrotestosterone and estradiol, although several other precursors and end products are also possible (Figure 1-3) (Keller et al., 1996; Lund et al., 2006). Interestingly, while the actions of 5a-reductase on testosterone result in the production of dihydrotestosterone, both of these androgens mediate their effects by altering gene expression within target tissues via the AR (refer to Figure 1-5) (Keller et al., 1996). However, despite the binding of these steroids to a single receptor, the effect of these hormones can be quite distinct. Several groups have now shown that testosterone associates with AR three times faster than dihydrotestosterone (Wilson and French, 1976; Grino et al., 1990) (Keller et al., 1996). In 31  Figure 1-5. The ligand or hormone can be generated in three different ways: (i) an active hormone is synthesized in a classical endocrine organ and enters the cell, (ii) the hormone may be generated from a precursor or prohormone within a target cell, and (iii) the hormone may be a metobolite synthesized within the target cell. In a hormone-free state, the receptor may be localized in the nucleus (not shown). However, some receptors are cytoplasmic due to their association with chaperone proteins, such as hsp-90 and hsp-56. Hormone binding induces dissociation of the complex and translocation to the nucleus. Once in the nucleus, the receptors regulate transcription by binding, generally as dimers, to hormone response (HRE) located in the regulatory regions of genes, which alters the transcription and translation of proteins.  32  agreement with these differential kinetic profiles, Zhou and colleagues (1995) have demonstrated that testosterone is less effective at stabilizing AR in its active conformational state than dihydrotestosterone. Further, differences in dissociation rates are directly related to the capacity of androgens to stimulate gene transcription (Deslypere et al., 1992). This suggests that the reduced form of testosterone, dihydrotestosterone, is capable of amplifying or redirecting the biological effects of androgens in cells expressing 5a-reductase. Finally, testosterone can also be converted to estradiol by the aromatase enzyme, thereby producing effects through the estrogen receptor (ER), a member of the steroid hormone receptor family (discussed in section 1.6.1). Taken together, testosterone is capable of altering gene transcription either directly, in concert with dihydrotestosterone, or indirectly via its conversion to estradiol (Figure 1-3). Thus, the nature by which testosterone operates within afferent mediators of PVN visceromotor function is likely mediated by various metabolites and sex steroid hormone receptors. 1.6.2  Coregulators of the androgen receptor After binding to androgens, the AR is also able to recruit general transcription factors to  its target gene promoters. In addition to its direct interaction with several factors of the general transcriptional machinery as described above, it has become very clear that the transcriptional activity of the AR is regulated by coregulators, including both coactivators and corepressors (Heinlein and Chang, 2002; Wang et al., 2005; Heemers and Tindall, 2007). For example, coactivators are factors that can directly interact with AR and enhance its transcriptional activity. On the other hand, corepressors are factors that associate with AR and repress its transcriptional activity. Both types of coregulators are necessary for efficient modulation of AR target gene transcription. AR coregulators differ from general and specific transcription factors in that they do not affect the basal rate of transcription and typically do not bind to DNA (Heemers and Tindall, 2007). Numerous comprehensive reviews provide an overview of AR coregulators that 33  have been reported to date, revealing a daunting level of functional diversity among these proteins (Heemers and Tindall, 2007; Table 2). Taken together, the diversity of coregulator function and their distribution pattern helps control AR transactivation in a sophisticated and complex manner. The increasing characterization of novel AR coregulators leads to the tantalizing suggestion that new pathways that participate in regulation of AR activity remain to be discovered, beyond the classical signaling pathway described in Figure 1-5. 1.6.3  Functional role of immediate early-genes Like coregulatory proteins, IEGs can also modulate the expression of target genes by  interacting with specific DNA sequences. The IEGs Fos and Jun have been shown to participate in the formation of a homo or heterodimeric complex that binds DNA at the AP-1 site and modulate gene expression expression (reviewed in Curran and Morgan, 1995; Herdegen and Leah, 1998; Ziolkowska and Przewlocki, 2002). Although all combinations of Fos-Jun or Jun-Jun dimmers can bind the consensus AP-1 target element, functional assays have revealed some differences in the ability to bind unique AP-1 sites in various promoters, stability of binding, and transcriptional activation, which renders clarification of the functional significance of any one transcription factor complicated (Herdegen and Leah, 1998). In fact, it has been postulated that the Fos-Jun complex may regulate transcription of late response genes due to their interaction with the AP-1 binding site in the promoter region of target genes (Chiu et al., 1988). In this regard, genes encoding for hypothalamic neuropeptides (e.g. AVP, enkephalin, dynorphin, somatostatin and cholecystokinin) (Gall and Isackson, 1989; Mohr and Richter, 1990; Sonnenberg et al., 1989) or neurotransmitter-related biosynthetic enzymes (e.g. tyrosine hydroxylase) (Cambi et al., 1989; Lewis et al., 1987) that display the AP-1 consensus sequence in the promoter region can be putative functional targets.  34  The Fos and Jun subunits can also participate in transcriptional regulation via mechanisms other than AP-1 interactions. Direct protein-protein interactions between GRs and either Fos or Jun can either reciprocally antagonize one another's transcriptional activity (Schule et al., 1990; Yang-Yen et al., 1990). In fact, down-regulation of functional GRs with induced Fos expression has been proposed to be a possible way by which CRH may stimulate POMC gene transcription in pituitary cells (Autelitano, 1994; Boutillier et al., 1991). Furthermore, estrogen can interact with the Fos-Jun complex to increase the transcriptional efficiency of target genes at the AP-1 site (Gaub et al., 1990; Umayahara et al., 1994; Webb et al., 1995). These interactions are believed to increase the repertoire of possible regulatory complexes that can play a critical role in the regulation of transcription of specific genes. In light of this, the stimulation-induced patterns of IEGs should not be necessarily viewed as end-points, by virtue of their many anatomical relationships with neuropeptides and neurotransmitter systems. 1.7  Steroid regulation of the HP A axis Numerous studies have shown that stress and HPA activation inhibits reproductive  function and behaviour in a variety of species, including rodents, humans and non-human primates (Rivier et al., 1986; Rivier and Rivest, 1991). However, this relationship is by no means unidirectional, because reproductive status and gender heavily impact on both basal and stressinduced HPA activity, indicating that the gonadal and adrenal systems are intimately entwined. Importantly, numerous studies support the existence of sex differences in a number of elements of the HPA axis. Not surprisingly, sex differences in both basal and stress-induced HPA activation are also apparent in a variety of species, including rodents, humans and non-human primates (Handa et al., 1994a; Seeman et al., 1995; Young, 1995; Seeman et al., 2001). Compared to males, females secrete higher levels of ACTH and glucocortocoids under basal conditions and in  35  response to stress. The following sections will discuss how steroids, including androgens and estrogens, can directly regulate components of the HPA axis, although focusing on the rodent. In female rats, chronic estrogen treatment enhances the corticosterone response to stress, and delays the recovery from stress in rats compared to overiectomized female rats (Burgess and Handa, 1992). Studies by Viau and Meaney (1991) demonstrated that the enhanced ACTH and corticosterone responses to acute stress observed in female rats are largely pronounced during the proestrous phase of the estrous cycle, with the effects eliminated by ovariectomy (Seale et al., 2004; Young and Altemus, 2004), highlighting the marked stimulatory effects of ovarian hormones on the HPA axis. However, not all studies have demonstrated enhanced HPA responsiveness associated with estrogens, suggesting that the role of ovarian hormones may be more complex than once thought. For example, while studies using supraphysiological doses of estradiol reliably show enhanced corticosterone responses to stress (Burgess and Handa, 1992; Carey et al., 1995; Ochedalski et al., 2007), estradiol concentrations in the lower-physiological range, inhibit the HPA response to stress (Redei et al., 1994; Young, 1995). In male rats, the sex difference on HPA function is attributed to the inhibitory effect of testosterone occurring via an androgen receptor-mediated mechanism, as ACTH and corticosterone responses to acute stress in male rats are increased by gonadectomy, and this effect is reversed with testosterone or dihydrotestosterone replacement, the reduced non-aromatizable form of testosterone (Handa et al., 1994b; Viau and Meaney, 1996). 1.7.1  The adrenal gland: regulation by gonadal steroids Gonadal steroids have the capacity to modulate corticosterone concentrations at multiple  levels of the HPA axis. At the adrenal cortex, estradiol can produce a faster onset of corticosterone secretion, leading to a faster rate of rise of circulating corticosterone concentrations (Jones et al., 1972). However unlike estrogens, the actions of androgens in males do not appear to  modulate corticosterone release directly. In a study by Baxter and Tyrell (1987), the addition of testosterone to rat adrenal slices did not affect corticosterone secretion. However, while it would appear that testosterone does not directly affect the rates of corticosterone secretion, testosterone has been shown to inhibit several key enzymes involved in glucocorticoid biosynthesis in the zona fasciculata of the adrenal gland (Baxter and Tyrell, 1987). The synthetic step in question, involves the conversion of pregnenolone to progesterone (the glucocorticoid precursor) and the final conversion of deoxycorticosterone to corticosterone. In bovine adrenal homogenates, testosterone inhibits 21-hydroxylation of pregnenolone and 17-hydroxypregnenolone as well as 11 P-hydroxylation of deoxycorticosterone (Sharma et al., 1963; Sharma and Dorfman, 1964). Furthermore, gonadectomy in male rats increases, while testosterone replacement decreases adrenal pregnenolone synthesis in vitro (Malendowicz, 1976). Therefore, since the conversion of cholesterol to pregnenolone is the rate-limiting step in adrenal steroid biosynthesis, which depends on ACTH, this does support a potential inhibitory role of testosterone on adrenal sensitivity to ACTH. However, this possibility has not yet been addressed. In addition, numerous studies have shown that sex differences in basal and stress-induced adrenal activity are not attributed to differences in glucocorticoid clearance rate and metabolism. CBG levels are approximately two-fold higher in adult female rats compared to males (Gala and Westphal, 1965; Keller et al., 1966). This sex difference has been attributed to androgenic-effects in males, as treatment with testosterone or dihydrotestosterone, reverses the stimulatory effects of gonadectomy on plasma CBG (Gala and Westphal, 1965; Keller et al., 1966; Viau and Meaney, 2004). In contrast, plasma CBG levels in females are not altered by ovariectomy or estrogen treatment (Gala and Westphal, 1965; Keller et al., 1966). A decrease in glucocorticoid clearance rate has been associated with increased plasma CBG (Ballard, 1979), suggesting that the metabolism of corticosteroids should be faster in males compared to females. Kinetic studies have revealed however, that males show a reduced capacity for corticosteroids to be hepatically  metabolized, the major site of glucocorticoid metabolism (Kitay, 1961; 1963a). Thus, androgenic regulation of glucocorticoid clearance may be more closely tied to hepatic metabolism rather than androgen effects on plasma CBG. Nonetheless, differences in CBG could alter the capacity for corticosterone to enter the brain to effect glucocorticoid-mediated feedback. 1.7.2  The anterior pituitary gland: regulation by gonadal steroids As described earlier, androgens and estrogens exert potent effects on ACTH levels during  stress. The presence of cognate receptors within a variety of anterior pituitary cells types in the anterior pituitary, including corticotropes also highlights a potential direct role for estrogens on ACTH synthesis and release (Thieulant and Duval, 1985). However, the decreased plasma ACTH levels during stress in gonadal intact males, compared to gonadectomized male rats, are not due to direct effects of testosterone on anterior pituitary activity. Studies examining androgen-regulation of corticotrope activity fail to reveal any direct inhibitory effects of testosterone on ACTH secretion. Stimulation of ACTH release in response to pituitary stalk and median eminence extracts in vitro is greater in pituitaries from gonadectomized male rats compared to testosteronereplaced donors (Coyne and Kitay, 1971). However, it is important to mention that this does not necessarily reflect an androgen-dependant effect on corticotrope sensitivity to ACTH releasing factors, as gonadectomy produces an increase in corticotrope ACTH content (Kitay, 1963b). Although ARs are located in the anterior pituitary (Thieulant and Duval, 1985), they are not contained in ACTH-producing corticotropes (Morel et al., 1984). In addition, the anterior pituitary does not contain aromatase activity (Naftolin et al., 1975; McEwen, 1980), thus ruling out an indirect effect mediated through the estrogen receptor (ER). 1.7.3  The PVN: regulation by gonadal steroids At the level of the PVN, many reports have indicated that estradiol stimulates CRH  mRNA expression (Patchev and Almeida, 1996; Li et al., 2003; Ochedalski et al., 2007). 38  Although, not surprisingly, many others have shown that estradiol has no effect on its expression (Redei et al., 1994), or may even decrease PVN CRH mRNA (Paulmyer-Lacroix et al., 1996). It is also important to note that estrogenic effects in the central nervous system depend largely on its binding to its cognate receptors, namely the ER isoforms a and p\ The expression of ERa is lacking in the PVN (Shughrue et al., 1997; Laflamme et al., 1998). On the other hand, while ERP is highly expressed within posterior pituitary-directed magnocellular neurons, this receptor subtype is not expressed by ACTH-regulating, CRH-expressing cells of the PVN (Hrabovszky et al., 1998; Isgor et al., 2003; Bingham et al., 2006). Intruigingly however, the work of Lund and colleagues (2006) illustrated that the DHT metabolite 3|3-diol and the ER|3-subtype-selective agonist diarylpropionitrile suppressed pituitary-adrenal output, and c-fos mRNA responses to restraint stress in a manner similar to DHT. Interestingly, their findings suggested that the metabolism of DHT to 3|3-diol and subsequent binding to ER|3 contained within the PVN, thereby inhibiting HPA reactivity, which may be one of the possible mechanisms mediating the inhibitory effects for the action of DHT. However, these findings remain controversial given the neuroanatomical localization of ER|3 within the PVN (as discussed above). Alternatively, it remains possible that a small subset of ER|3-containing neurons could occupy the pre-autonomic zone of the PVN, mediating their effects indirectly, via reciprocal hindbrain connections (e.g. ventrolateral medulla or spinal cord (refer to Figure 1-2). Nonetheless, while it is clear that estrogens can act on the HPA axis, their effects are likely complex, and may depend on the level of circulating estradiol, the duration of estradiol exposure and the intensity of the stressor. Taken together, estrogen-mediated effects on the HPA axis likely occurs upstream from the PVN, acting . on brain neurotransmitter and neuropeptide systems that regulate the HPA axis (refer to section 1.4.4). In males, numerous reports have demonstrated that androgens prevent increases in AVP and CRH following gonadectomy under basal conditions (Bingaman et al., 1994b; Viau and  Meaney, 1996), and can decrease both the cellular and transcriptional activation of medial parvocellular neurons under stress conditions (Viau et al., 2003; Lund et al., 2004). However, recent mapping studies show that the ARs are not expressed by ACTH-regulating PVN neurons, but are instead distributed within the pre-autonomic division of the PVN directed at the brainstem and spinal cord (Simerly et al., 1990; Zhou et al., 1994; Bingham et al., 2006). Thus, these findings place androgenic influences of the HPA axis upstream from the PVN. 1.7.4  Expression of androgen receptors within afferent regulators of the PVN The pattern and density of AR expressing cells using autoradiographic, in situ  hybridization histochemistry and immunocytochemical techniques have revealed that ARs are distributed throughout the brain (Sar and Stumpf, 1975; Simerly et al., 1990; Bingaman et al., 1994a; Lisciotto and Morrell, 1994; Kerr et al., 1995; Romeo et al., 2000; Murphy and Hoffman, 2001; Hamson et al., 2004). AR-expressing cells are found within the frontal cortex, to and through the most caudal reaches of brainstem nuclei. In the forebrain, medial (e.g. medial preoptic, stria terminalis and ventral medial hypothalamic) and periventricular zone nuclei (e.g. periventricular preoptic, anteroventral periventricular and posterior periventricular) contain the highest densities of AR expressing cells. In the brainstem, moderate to high densities of AR expressing cells are contained by the posterior thalamic complex, periaqueductal gray, lateral parabrachial and central gray nuclei, and relatively lower densities of AR are found within the midline pontine (e.g. Raphe) and medullary (e.g. NTS, VLM) nuclei. Importantly, therefore, ARs are contained within numerous cell groups that are known to regulate and/or project to the PVN region. 1.8  Evidence for testosterone regulation upstream of the PVN There is strong evidence to suggest that testosterone can act at sites upstream from the  PVN to regulate the HPA axis. Testosterone implants into the medial preoptic area, a brain region 40  rich in ARs, decrease plasma ACTH and corticosterone responses to restraint, as well as decrease AVP, but not CRH levels in the median eminence, effects similar to those of peripheral testosterone implants (Viau and Meaney, 1996). Furthermore, large bilateral lesions of the medial preoptic area attenuate the inhibitory effects of testosterone on stress-induced ACTH and corticosterone release under stress conditions (Viau and Meaney, 1996). In addition, systemic testosterone stimulates CRH and AVP mRNA expression within the anterior fusiform and posterior bed nucleus of the stria terminalis, respectively, and enhances the expression of AVP within the medial nucleus of the amygdala (Viau et al., 2001). Emerging findings also indicate that differences in gonadal status can influence the pattern and the magnitude of stress-induced expression of the c-fos gene, and its protein counterpart, Fos, within several putative central regulators of the HPA axis (Da Costa et al., 1996; Kerr et al., 1996; Cheung et al., 1997; Nappi et al, 1997; Greco et al., 1998; Rachman et al., 1998; Figueiredo et al., 2002; Ceccarelli et al., 2003; Viau et al., 2003; Viau et al, 2005; Ceccarelli et al., 2006). For example, a study by Kerr and colleagues (1996) demonstrates that gonadectomy potentiates, whereas dihydrotestosterone replacement inhibits the induction of c-fos mRNA in the CAl region of the hippocampus in adult male rats in response to novel environment exposure. In addition, gonadectomized male rats show a heightened induction of Fos protein in the paraventricular nucleus of the thalamus and in the arcuate nucleus of the hypothalamus compared to gonadalintact or testosterone-replaced rats following noxious stimulation (Ceccarelli et al., 2003). These studies reveal that male gonadal hormones exert inhibitory effects within circuits related to behavioral coping and neuronal responses to novelty and nociceptive stimuli. On the whole, these findings suggest that the central effects of testosterone on the HPA axis involve multiple components of the limbic/hypothalamic circuitry discussed above.  41  1.9  Thesis objectives and hypotheses.  In this thesis, I describe three research objectives: 1)  Androgen receptors are distributed throughout the central nervous system and are  contained by a variety of nuclei that are known to project to or regulate the PVN. Since testosterone implants in the vicinity of the MPN reduce the plasma ACTH and corticosterone responses to restraint, and high testosterone replacement levels in the periphery that normally suppress the magnitude of the HPA stress response fail to do so in rats bearing large electrolytic lesions of the medial preoptic area, I hypothesize that PVN-projecting neurons in the MPN will express the androgen receptor. 2)  To test the requirement of the medial preoptic nucleus (MPN) in mediating the central  effects of testosterone in circulation, I will compare the effects of low- and high-testosterone in the periphery on basal and stress-induced indices of HPA axis in animals bearing lesions of the MPN. I hypothesize that lesions of the MPN will block the inhibitory effects of testosterone on stress-related input to the PVN and HPA axis. 3)  To test the extent to which androgen receptors in the MPN regulate HPA effector neurons  of the PVN directly, I will compare the effects of microimplants of testosterone and the androgen receptor antagonist hydroxyflutamide into the MPN on acute restraint induced activation and/or neuropeptide expression levels within the PVN, and its extended circuitries. I hypothesize that testosterone and hydroxyflutamide MPN implants will enhance and block, respectively, the inhibitory effects of peripheral testosterone on basal and stress-induced indices of HPA function.  1.10 References Aguilera G. 1994. Regulation of pituitary ACTH secretion during chronic stress. Front Neuroendocrinol 15:321-350. Akana SF, Dallman MF, Bradbury MJ, Scribner KA, Strack AM, Walker CD. 1992a. 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Vasopressin and corticotropin-releasing factor: an axonal pathway to portal capillaries in the zona externa of the median eminence containing vasopressin and its interaction with adrenal corticoids. Ann N Y Acad Sci 297:405-419.  62  CHAPTER 2 : Androgen receptor expressing neurons that project to the paraventricular nucleus of the hypothalamus in the male rat 1 2.1  Introduction In the vertebrate central nervous system, adaptive responses to homeostatic threat (i.e.,  stress) rely on the integrative capacity of several major visceromotor systems. Depending on the nature of the stressor (e.g., intensity, duration, physiology), at least four major responses systems are recruited to various degrees and overlap, including those mediating behavior, autonomic, neuroendocrine, and immune output responses (Dallman, 2003; McEwen and Wingfield, 2003). Proper maintenance of normal homeostasis and successful adaptation to any challenge rests not only within the accuracy by which any one of these systems act, but also within the extent to which the end products or outcomes of these systems interact. A well-documented fact is that stress can influence the output of the hypothalamic-pituitary-gonadal axis, including effects on the synthesis and release of the sex steroids estrogen, progesterone, and testosterone (Vreeburg et al., 1984; Rivier et al., 1986; Rivier and Rivest, 1991; Henry, 1992; Van den Berghe, 2002; Wingfield and Sapolsky, 2003). Conversely, the magnitude of the hypothalamic-pituitary-adrenal (HPA) response to stress also varies as a function of gonadal status in males and females (Viau and Meaney, 1991; Handa et al., 1994; Patchev and Almeida, 1998; Young, 1998; Rhodes and Rubin, 1999; Figueiredo et al., 2002; Isgor et al., 2003; Viau et al., 2003; Lund et al., 2004; Young and Altemus, 2004; Williamson et al., 2005). Of interest here, testosterone can act and interact with corticosterone on almost every conceivable aspect of the HPA axis, including inhibiting the recruitment of hypophysiotropic neurons that are activated during stress, the regulation of the principle adrenocorticotropin co-secretagogues, corticotropin-releasing hormone and arginine  ' A version of this chapter has been published. Williamson M, Viau V. 2007. J Comp Neurol 503:717-740. © 2007 Wiley-Liss. Reprinted with permission of Wiley-Liss, Inc. a subsidiary of John Wiley & Sons. Inc. 63  vasopressin, and cooperatively on the process of glucocorticoid-mediated negative feedback (Viau et al., 1999). More detailed descriptions of how testosterone and corticosterone interact on HPA function are reviewed elsewhere (Viau, 2002; Williamson et al., 2005). The shared inhibitory characteristics by which testosterone and corticosterone operate on the HPA axis would place such influences within the central nervous system and the PVN. Studies examining the distribution of sex steroid hormone receptors within the PVN on spatial (Rhodes et al., 1982; Simerly et al., 1990; Zhou et al., 1994) and connectional grounds (Stern and Zhang, 2003; Bingham et al., 2006) indicate that the androgen receptor and the estrogen receptorbeta isoform are not expressed by cells occupying the medial parvocellular part of the PVN directed at the median eminence, but by cells projecting to the spinal cord and medulla. While this signifies a means by which sex steroids can influence autonomic and sensory function within the PVN directly, these findings also likely place androgenic (and estrogenic) influences on HPA function upstream from the PVN (but see Lund et al., 2006). Androgen receptors are distributed throughout the brain, including within several cortical, hypothalamic, limbic, and brainstem cell groups that regulate HPA function and/or project to the PVN region (Sar and Stumpf, 1975; Simerly et al., 1990; Bingaman et al., 1994; Lisciotto and Morrell, 1994; Kerr et al., 1995; Romeo et al., 2000; Murphy and Hoffman, 2001; Hamson et al., 2004). Because circulating levels of testosterone vary as a function of age, sexual experience, social status and in response to stress (Bartke et al., 1973; Sencar-Cupovic and Milkovic, 1976; De Goeij et al., 1992; Blanchard et al., 1993; Romero et al., 1995; Gomez and Dallman, 2001; Seeman et al., 2001; Dallman et al., 2002; Gomez et al., 2004; Romeo et al., 2004; Tamashiro et al., 2004), situation-specific and statedependent changes in gonadal status may, therefore, act to harmonize several independent, yet converging influences on PVN function. This could hold the key for successful adaptation to complex homeostatic challenges demanding multiple types of system responses.  Tract tracing studies used in combination with immediate-early-gene and phenotypic approaches have proven incredibly instructive in determining the specificity by which distinct classes of homeostatic threat (neurogenic, systemic) influence different central pathways and effector motor neurons in the PVN (reviewed in Cullinan et al., 1995, Sawchenko et al., 2000, Dayas et al., 2001a, Herman et al., 2003, Day, 2005). Further, changes in gonadal and reproductive status in males and females can influence the pattern and the magnitude of stressinduced expression of the c-fos gene, and its protein counterpart, Fos, within the PVN and several putative central regulators of the HPA axis (Da Costa et al., 1996; Kerr et al., 1996; Cheung et al., 1997; Nappi et al., 1997; Rachman et al., 1998; Figueiredo et al., 2002; Ceccarelli et al., 2003; Viau et al., 2003; Viau et al, 2005; Ceccarelli et al., 2006). However, the extent to which regional differences in androgen receptors contribute to this influence, and their containment within PVN-projecting nuclei has not been determined. In the current study we sought to build on the latter by employing a combined retrograde transport and immunohistochemical procedure to 1) characterize the distribution of androgen receptors within PVN-projecting cell groups directly, and 2) assess the relative densities of androgen receptor staining within these candidate afferent mediators of gonadal status. Superimposing the results of these two aims onto the results of previous anterograde experiments, allowed us to examine the potential by which testosterone could influence different classes of effector motor neurons in the PVN. Portions of these results have been presented previously in abstract form (Williamson et al., 2004). 2.2  Methods  2.2.1  Animals Sixty adult male Sprague-Dawley rats (Charles River, St. Constant, Canada) were used,  weighing from 250-260 grams on arrival (40 days old) and from 345-365 grams when sampled (-60 days old). Animals were pair housed under controlled temperature and lighting conditions  (12:12 hour light: dark cycle, lights on at 0600 hours), with food and water available ad libitum. All experimental protocols were approved by the University of British Columbia Animal Care Committee (Appendix A). 2.2.2  Mapping candidate androgen-responsive afferents to the PVN To identify AR-ir neurons in brain regions that project to the region of the PVN, a  combined retrograde transport and immunohistochemical procedure was used in all animals bearing appropriate tracer injections into the PVN as previously described (Li and Sawchenko, 1998). Under ketamine-xylazine-acepromazine anesthesia-analgesia (25, 5, and 1 mg/ml respectively, 1 ml/kg sub-cutaneously), animals received stereotaxically-guided iontophoretic injections or crystalline implants of the retrogradely-transported tracer, Fluorogold (FG; Fluorochrome, Denver, CO), directed at the PVN. Stereotaxic coordinates were adapted from Bregma according to Swanson (1998), AP: -1.45 mm; ML: 0.25 mm; DV: -7.4 mm; bite bar set at 3 below interaural zero. Iontophoretic injections of FG (2% w/v in 0.9% saline) were performed using a glass micropipette (20-25 mm outer diameter; Sutter Instruments, Novato, CA) backfilled with a 2% FG solution. The pipette was positioned to the desired coordinates and left in place for 5 min prior to iontophoresis (5 mA alternating current, 7 sec on/off, 5 min), then left in place for an additional 5 min before removal. Crystalline implants were performed using a glass micropipette (100-175 mm outer diameter; Sutter Instruments, Novato, CA) filled with a suspension of FG (100% w/v in MilliQ) that was dried at 37°C for 48 hours to produce a tracer plug. This assembly was aimed 0.5 mm dorsal to the PVN and left in place for a period of 1 min. The FG plug was then ejected with a wire plunger (76 mm diameter; Hamilton Company, Reno, NV) extending 0.5 mm beyond the pipette tip. The pipette and plunger were left in place for an additional 10 min before removal to minimize the potential uptake and transport of tracer along the pipette and plunger track.  Both of these methods proved effective in delivering FG to the region of the PVN in terms of providing a concentrated amount of tracer directed at the caudal 2/3 of the nucleus (Swanson et al., 1981). Although tracer accumulation was noticeably more intense or complete within individual cells using the crystal implant technique, the total number of FG cells encountered within most forebrain cell groups was, nonetheless, relatively stable using both delivery methods. In the brainstem, both methods produced similar patterns of FG distribution. However, relatively greater disparities in cell staining intensity and density were encountered amongst animals bearing iontophoretic injections. In contrast, retrograde labeling within individual brainstem neurons was much more intense and uniform using the crystal implant technique. This discrepancy likely reflects differences in the amount of tracer deposited, detectable only over long transport distances. The brainstem results described, therefore, were taken only from animals bearing the most appropriate PVN crystal deposits. Tracer deposits and resulting patterns of retrograde accumulation were further controlled, by including initial assessment of the extent to which any deposit (either by injection or implant) was centered and concentrated within the caudal 2/3 of the PVN. Most of the major functional groups of the PVN are found at (although not entirely restricted to) this level, including neurosecretory cell groups that project to the median eminence and posterior pituitary, and preautonomic cell groups giving rise to preganglionic brainstem and spinal cord projections. Assisted by redirected sampling of adjacent thionin-stained sections and alternately viewing the sections under darkfield illumination, material showing tracer delivery that was not centered within the posterior region of the PVN, or not fully encompassing its boundaries served as controls. Accumulation beyond the nuclear boundaries of the nucleus was expected (see Sawchenko and Swanson, 1983). Cases in which this labeling was extended to the contralateral PVN were not included. Controlling FG delivery using these criteria rendered stable patterns and densities of retrograde accumulation between animals. Guided by previous anatomical retrograde  and anterograde transport studies, 10 animals were ultimately used to describe forebrain afferents (5 iontophoretic and 5 crystal injections), and 5 animals (crystal only) were used to describe hindbrain afferents to the PVN. Illustrations representing appropriate iontophoretic and crystal FG injections into the PVN are shown in Figure 2-1. We also relied on animals showing FG deposits that missed the PVN (Figure 2-ID), to help describe projections to the PVN surround. A total of 30 cases showed independent and overlapping profiles of FG that were concentrated dorsal, ventral, and lateral to the caudal part of the PVN. 2.2.3  Tissue preparation Two weeks following retrograde tracer injections into the region of the PVN, optimal for  FG transport (Schmued and Fallon, 1986; Moga and Saper, 1994; Tillet et al, 2000), rats were deeply anesthetized with chloral hydrate (200 mg/kg) and perfused via the ascending aorta with ice-cold 0.9% saline (125 ml), followed by 500 ml of ice-cold 4% paraformaldehyde (pH 9.5). The brains were post-fixed for 4 hours in a solution of the same fixative and cryoprotected in 15% sucrose in 0.1 M potassium phosphate-buffered saline (KPBS, pH 7.4), overnight at 4°C. Five adjacent l-in-5 series of 30-um-thick frozen sections were collected and stored in cryoprotectant (30% ethylene glycol and 20% glycerol in 0.05 M KPBS buffer) at -20°C until histochemical processing. One series was used to examine the injection site and the distribution of retrogradely labeled cells. One series was counter-stained with thionin and alternately compared with darkfield illumination to morphologically mark the location of retrograde labeling. Based on the quality and confinement of the tracer deposit (see above), the remaining adjacent series were processed to determine the relative extent to which cell groups innervating the PVN region localize AR-ir.  Figure 2-1. Photomicrographs showing the appearance of an iontophoretic (A) and crystalline (B) fluorogold injection site two weeks after delivery. Precise locations of the injections were determined by locating the core and spread of the deposit under uv illumination and alternately viewing the section under darkfield illumination (A and C, respectively). The schematic representation (D) describes the injection core (dark blue) and local uptake and transport (light blue) in animals showing appropriate iontophoretic (left) and crystalline tracer (right) injections (n = 5 each). Animals showing tracer injections and implants that were not centered within the posterior region of the PVN (green), or not fully delimited to its boundaries (red) served as controls (N = 30). Structures labeled for reference: AHN, anterior hypothalamic nucleus; fx, fornix; mpd, medial parvicellular, dorsal part (PVN); pm, posterior magnocellular part (PVN); RE, nucleus reunions; V3,3rd ventricle; ZI, zona incerta. Scale bar = 250 um (applies to A-C).  69  2.2.4  Localization and connectivity of androgen-sensitive neurons To detect cells displaying nuclear AR-ir and cytoplasmic tracer accumulation, we  employed a sequential nickel- and non-nickel-intensified avidin-biotin-immunoperoxidase procedure, respectively. This was performed by first localizing AR-ir using antiserum directed at the N-terminal fragment of the AR, followed by incubation in rabbit anti-FG serum. AR-ir was initially localized using the PG-21 antiserum, raised against N-terminal amino acids 1-21 (Chemicon 06-680, Lot number 26042, Temecula, CA; 0.67 ug/ml). However, a portion of the material was processed using the N-20 rabbit anti-AR antibody raised against N-terminal amino acids 2-21 (Santa Cruz Biotechnology sc-816, Lot number El004, Santa Cruz, CA; 0.025 ug/ml; 1:8,000) due to a limited supply of PG-21. Both antibodies are specific to the N-terminal of AR (Prins et al., 1991; Kritzer, 1997), and consistent with the findings of Kritzer (2004), our control studies showed that both of these antibodies produce similar distribution and density profiles for AR in adjacent tissue series taken from the same animal. Free-floating sections were first rinsed in KPBS buffer to remove cryoprotectant, and then pre-treated with 0.3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. This was followed by four rinses in KPBS, and then in sodium borohydride (1% w/v in KPBS) for 5 min to reduce free aldehydes. Sections were then incubated for 48 hours at 4°C in a KPBS-Triton (0.3% Triton-X; Sigma-Aldrich, Oakville, Ont.) solution containing 2% normal goat serum and the primary antiserum to detect AR. AR primary antiserum was detected using a conventional nickel-intensified, avidin-biotinimmunoperoxidase (Vectastain Elite ABC kit; Vector laboratories, Burlington, CA) procedure (Li and Sawchenko, 1998). This procedure was then followed by a non-nickel variant of the procedure described above using primary antisera against FG (Chemicon AB153, Lot number 24010565, Temecula, CA; 1:15,000).  Concurrent immunofluorescence detection of AR and FG involved the N-20 anti-AR antibody and tissue pre-treatment as described above, except that the hydrogen peroxide step was eliminated. Primary antiserum against AR, in this case, was detected with a conjugated antirabbit IgG fluorescent secondary antibody (Alexa 594, Invitrogen, Burlington, Ont; 1:500). Cells displaying nuclear AR-ir and cytoplasmic FG were identified under fluorescence wavelength and ultraviolet excitation, respectively. The pattern of AR staining in the brainstem (Sar and Stumpf, 1975; Simerly et al, 1990; Hamson et al., 2004), either alone or in combination with FG labeling, suggested a distribution within several catecholamine-producing, PVN-projecting cell groups (Chan and Sawchenko, 1994; Palkovits et al, 1997; Li and Sawchenko, 1998; Buller et al., 2001; Dayas et al., 2001b). Histochemical characterization of AR-ir neurons in this population was performed by using a retrograde transport-double immunohistochemical labeling technique including a mouse-derived monoclonal antibody against tyrosine hydroxylase purified from rat phenochromocytoma (TH; Pel-Freeze P80101-0, Lot number 15827, Rogers, AK; 1:2,000; see Liao et al., 1996) and sheep polyclonal antibody against phenyl ethanolamine-N-methyltransferase purified from bovine adrenals (PNMT; Chemicon AB146, Lot number 0602021851, Temecula, CA; 1:5,000; see Legradi and Lechan, 1998). Free-floating tissue encompassing the brainstem from animals bearing PVN tracer implants were prepared as described above with slight modifications of these methods to optimize double labeling for AR-ir and TH- or PNMT-ir, including 1) the elimination of hydrogen peroxide pre-treatment, and 2) using bovine serum albumin as a blocking agent. Primary antisera against AR and TH or PNMT were detected using conjugated anti-rabbit (Alexa 594, Invitrogen; 1:500) and anti-mouse IgG (Alexa 488, Invitrogen; 1:500) or anti-sheep IgG (Alexa 488, Invitrogen; 1:500) fluorescent secondary antibodies, respectively. Concurrent immunofluorescence detection of AR-ir, TH- or PNMT-ir and FG-accumulating neurons was achieved under appropriate fluorescence wavelength and ultraviolet excitation. Counts using 71  antisera against TH and PNMT were obtained from adjacent series of sections from the same animal (n = 4). Control experiments, in which the primary antiserum to AR was preadsorbed for 24 hours at 4°C with 6.7 uM (10-fold excess) synthetic peptide immunogen, corresponding to N-terminal amino acids 1-21 (MEVQLGLGRVYPRPPSKTYRG; SynPep, Dublin, CA) or N-terminal amino acids 2-21 (sc-816 P, Santa Cruz Biotechnology, Santa Cruz, CA) of the rat androgen receptor, failed to yield any evidence of specific AR staining (Bingham et al., 2006). Additional control experiments for antisera cross-reactivity, involving the omission of either primary or secondary antibody, yielded no specific labeling. Finally, the staining pattern for either AR or FG was similar whether detected alone or processed in combination. 2.2.5  Imaging and analysis To provide an estimate of the relative number of AR expressing neurons contained within  PVN-projecting cell groups, cell counts taken from immunoperoxidase material in animals bearing appropriate FG deposits were determined under brightfield conditions using a 40X objective (NA 0.8). Doubly labeled (AR+FG) cells within each population of interest were defined as those showing a black and brown reaction product in the nucleus and cytoplasm, respectively. Counts were taken in complete, regularly spaced (150 um intervals) series of sections through the rostrocaudal extent of the cell groups in question and corrected for doublecounting error using Abercrombie's formula (Abercrombie, 1946; and see Guillery, 2002), factoring in regional differences in cell diameter, where appropriate. Data describing the distribution of AR-ir profiles within cells concurrently displaying cytosolic tracer are extrapolated estimates derived from the total corrected number of profiles encountered in each series of sections multiplied by the sectioning interval of five. Parceling of the rat brain followed the mapping of FG accumulation and AR staining as defined by the morphological features provided 72  by thionin staining of adjacent series of tissue, based on the terminology of Swanson (1998), and of Dong and Swanson (2004,2006c) to describe the major bed nuclei of the stria terminalis. Light-, dark- and fluorescence-level images were captured using a Retiga 1300 CCD digital camera (Q-imaging, Burnaby, BC), analyzed using Macintosh OS X-driven, Open Lab Image Improvision software v. 3.0.9 (Quorum Technologies, Guelph, Ontario), and exported to Adobe Photoshop (v. 10.0, San Jose, CA), where standard methods were used to adjust contrast and brightness, and final assembly at a resolution of 300 dpi. 2.3  Results  2.3.1  FG injection and retrograde labeling As detailed above, several criteria were imposed to ensure that FG injections were  centered at the caudal 2/3 of the PVN region, specifically. Discrete and concentrated injections were produced when delivered iontophoretically; and intense, but less restricted deposits when delivered by crystal implantation. The crystalline deposit also produced minimal spread along the pipette tract, which typically occurs with volume or iontophoretic injections (see also Lind, 1986; Li and Sawchenko, 1998). In most experiments, the tracer injections were concentrated in, but not completely restricted to the morphological confines of the PVN (Figure 2-1). The largest deposits spread dorsally to the nucleus reunions of the thalamus and the zona incerta or ventrally to involve aspects of the anterior hypothalamic area. The injections never extended laterally into or beyond the fornix. In several instances, injections made outside the PVN proper were useful in describing potential indirect sources of androgen-sensitive, limbic forebrain-related input to the PVN (Roland and Sawchenko, 1993; Herman et al, 2003; Herman et al., 2005). As described below, and in agreement with Rinaman et al. (1995), the pattern and density of FG labeling in the brainstem (e.g., nucleus of the solitary tract, ventrolateral medulla) in animals bearing the smallest and most concentrated injections in the PVN was comparable to those showing larger tracer 73  deposits encompassing the anterior 1/3 of the PVN, the ventral tip of the nucleus reunions, and/or deposits centered towards the dorsal aspect of the PVN nucleus. As previously described, these observations indicate that neurons adjacent to, or in the immediate vicinity of the PVN, do not substantially contribute to retrograde labeling in the brainstem. These findings are consistent with previous phenotypic studies describing only meager adrenergic input to neighboring cell groups such as the thalamic nucleus reunions and anterior hypothalamic area (Swanson et al., 1981), and in agreement with previous anterograde studies confirming the existence of brainstem afferents to discrete aspects of the parvicellular and magnocellular divisions of the PVN (Cunningham and Sawchenko, 1988; Cunningham et al., 1990). The pattern of retrograde labeling in the hypothalamus was predominately ipsilateral to the side of the injection, although bilateral labeling was most evident in those animals showing tracer accumulation or spread medially to the opposite PVN. This was most obvious when injections were biased towards the dorsal part of the PVN and/or encroached upon the ventromedial tip of the ipsilateral nucleus reuniens. Bilateral retrograde labeling was evident in the lower brainstem, even when the smallest injections were entirely confined within the nuclear boundaries of the PVN. Although the clear majority of brainstem structures labeled ipsilaterally, in some instances bilateral (albeit sparse) retrograde labeling was observed within the lateral tegmental nucleus, the ventrolateral divisions of the periaqueductal gray, and the caudal aspect of the ventrolateral medulla. To minimize potential differences in the amount of tracer delivered to the PVN, so that numerical assessments in FG accumulation between regions of interest could be made with confidence, we limited quantification to those animals showing comparable levels of tracer spread and accumulation within the immediate vicinity of the nucleus. This was initially determined in unreacted material, in which FG transport and accumulation was visualized under UV illumination, using size, shape, and contralateral diffusion as an index of placement and  accumulation. Some discrepancy between the two methods of tracer delivery was expected at the cellular and regional levels, depending on the area of interest and distance required for tracer transport (described in Methods section 2.2.2). Subsequent light-level analysis of immunoperoxidase reacted tissue indicated comparable numbers and distribution of detectable FG-labeled cells in the forebrain using either tracer delivery method, in general. Although in some cases, the circumscribed injections produced by iontophoresis were more effective in identifying the most local of afferent sources to the PVN (e.g., projections from the anterior hypothalamic nucleus). On the other hand, we observed far less variance in the number of detectable neurons in the brainstem of animals bearing discrete crystalline implants (e.g., pontine and medullary neurons). Thus, the forebrain data presented here were derived using both injection methods, while the brainstem data presented is from animals bearing crystal injections only. Controlling FG injections as such provided a reliable means with which to gauge the relative contributions of AR-expressing afferents to the PVN. 2.3.2  Forebrain retrograde- and androgen receptor-labeled neurons  2.3.2.1 Limbic-related; PFC, septum, amygdala, and hippocampus In agreement with previous anatomical studies, no area of the prefrontal cortex (Sesack et al., 1989; Hurley et al., 1991), septum (Risold et al., 1994; Risold and Swanson, 1997; Radley et al., 2006), amygdala (Canteras et al., 1995; Prewitt and Herman, 1998) or hippocampus (Canteras and Swanson, 1992) showed reliable retrograde labeling following the most discrete tracer injections. In animals bearing FG deposits centered or extending beyond the nuclear boundaries of the PVN, retrograde labeling was occasionally observed in the prefrontal cortex (0-25 labeled neurons per section), lateral septum (15-35), medial amygdala (15-35), and hippocampal ventral subiculum (0-25). Injections placed along the dorsal borders of the PVN, impinging upon the ventral aspects of the nucleus reunions and zona incerta, yielded a cluster of FG-labeled cells in 75  the infralimbic and cingulate cortex (areas 25 and 24/29, respectively) without any apparent dorsal-ventral bias. On the other hand, tracer deposits extending ventrally and into the surrounding subparaventricular zone, and impinging upon the anterior hypothalamic area, consistently yielded tracer uptake in the septal complex, almost always exclusively within the ventral lateral part. In these cases, labeling within the intermediate and dorsal parts of the lateral septum and in the medial septum was absent. Within the amygdala, tracer deposits showing a ventral bias in the PVN often labeled the postero-dorsal and -ventral parts of the medial amygdala (Figure 2-2), while the central and basolateral amygdaloid nuclei were consistently devoid of retrograde labeling (Silverman et al, 1981; Ono et al., 1985; Canteras et al., 1992, 1995; Prewitt and Herman, 1998; Campeau and Watson, 2000). Similarly, FG deposits which invaded the subparaventricular zone, resulted in reliable retrograde accumulation within the temporal aspect of the ventral subiculum (Canteras and Swanson, 1992; Cullinan et al., 1993). Androgen receptors are abundantly expressed in the lateral septum, medial amygdala, and subiculum, and to a much lesser extent in the prefrontal cortex (Sar and Stumpf, 1975; Simerly et al., 1990; Clancy et al., 1992). In animals bearing PVN injections that were off-centered, only modest numbers of doubly labeled (AR+FG) cells were detected in these regions, never exceeding approximately 30% of the total number of FG-labeled cells encountered. 2.3.2.2 Bed nuclei of the stria terminalis Parceling and terminology used for describing FG accumulation followed Dong and Swanson (2004,2006c), in which we compared the pattern of FG labeling and AR staining against the morphological features of adjacent thionin stained sections. Within the anterior division of the bed nuclei of the stria terminalis (BST), FG labeling was detected within the anteromedial group, including the anterodorsal and anteroventral nuclei, in agreement with  Figure 2-2. Photomicrographs showing the appearance of fluorogold (FG)-labeled cells in the medial amygdala, typically encountered when retrograde tracer injections diffused beyond the borders of the PVN or were centered in the subparaventricular zone. Darkfield view of retrograde labeling within the posterodorsal and posteroventral parts of the medial amygdala (A). Enlarged brightfield view (B) of the boxed region showing dual immunoperoxidase labeling for nuclear AR-ir and cytoplasmic FG-ir. Solid arrows show doubly labeled neurons and open arrowhead marks a AR-positive, FG-negative cell. Structure labeled for reference: ot, optic tract; MeApd and MeApv, posterodorsal and posteroventral medial amygdala. Scale bar = 50 um in B; 250 urn in A.  77  several previous retrograde and anterograde mapping studies (Sawchenko and Swanson, 1983; Weiss and Hatton, 1990; Cullinan et al, 1993; Moga and Saper, 1994; Cullinan et al., 1996; Prewitt and Herman, 1998; Spencer et al., 2005). The incidence of AR+FG double labeling was very consistent in both the anterodorsal (31%) and anteroventral (24%) nuclei. While very high numbers of AR-ir cells were encountered within the adjacent lateral, juxtacapsular and oval nuclei, doubly labeled cells in these nuclei were rarely encountered. Within the medial part of the rostrocaudal extent of the BST, FG injections appropriately centered in the PVN routinely labeled a dense packing of cells in the dorsomedial, ventral magnocellular, and fusiform bed nuclei (Figure 2-3), consistent with recent anterograde surveys (Dong and Swanson, 2006a, 2006b, 2006c). Moderate numbers of doubly labeled cells were reliably encountered within all of these nuclei, consistently highest within the dorsomedial nucleus. The posterior division of the BST constitutes a major source of input to the PVN (Ju and Swanson, 1989; Cullinan et al., 1996; Gu et al., 2003; Dong and Swanson, 2004), and is concentrated by both estrogen and androgen receptors (Simerly et al., 1990; Simerly, 1993; Shughrue et al., 1997; Laflamme et al., 1998; Greco et al., 2001; Auger and De Vries, 2002; Simerly, 2002). Not surprisingly, this aspect of the BST showed a very high incidence of double labeling (Figure 2-3), predominately within the principle nucleus (Figure 2-4), which preferentially innervates the medial parvicellular part of the PVN (Dong and Swanson, 2004). Relatively moderate numbers of double labeled cells were encountered within the interfascicular and transverse nuclei. Anterograde analysis by Dong and Swanson (2004) indicate that the transverse BST nucleus innervates, at best, the anterior parvicellular part of the PVN. Clearly distinct, the interfascicular BST nucleus innervates regions located outside or immediately adjacent to the PVN proper, including projections to the ventral aspect of the nucleus reuniens, the dorsomedial portion of the anterior hypothalamic area and the subparaventricular zone of the  78  1200-, 1000-1  CZZl Total FG %AR  o 800o o 0)  .a E 400-  AM  DM  FU  PR  IF  TR  Figure 2-3. Estimated cell counts of FG labeled neurons and AR containment within bed nuclei of the stria terminalis. Mean ± SEM total number of FG neurons (stacked open bars) and the number of doubly labeled (AR+FG) neurons (solid bars) detected per BST region. The number above each solid bar indicates the % of double labeling, providing a relative index of AR containment between each region (n = 10). AM, anteromedial area; DM, dorsomedial nucleus;FU, fusiform nucleus; PR, principal nucleus; IF, interfasciculamucleus; TR, transverse nucleus.  Figure 2-4. Photomicrographs showing the accumulation of FG within the posterior division of the bed nucleus of the stria terminalis at the level of the rostral extent of the PVN. The darkfield view illustrates a high density of FG labeling within anterior parvicellular PVN neurons and a scattered profile of retrograde labeling in the immediate (lateral) vicinity of this PVN population, including within the principle nucleus of the BST (boxed region). AR staining was detected throughout the bed nucleus, but not within anterior parvicellular PVN neurons. Enlarged brightfield view (B) of the boxedregion showing dual immunoperoxidase labeling for AR-ir and FG-ir. Solid arrows show doubly labeled neurons and open arrowhead marks AR-positive, FG-negative cell, ap, anteroparvicellular PVN; fx, fornix; V3, third ventricle. Scale bar = 50 urn in B; 500 um in A.  80  hypothalamus (Dong and Swanson, 2004). Perhaps consistent with this unique projection profile, our initial assessment of injection placement in the PVN suggested a passing influence of ventral FG deposits on retrograde labeling in the region occupied by the interfascicular nucleus. 2.3.2.3 Circumventricular organs A moderate number of FG-labeled cells were consistently found within the lateral parts of the subfornical organ, to a lesser extent within the region of the organ of the lamina terminalis, and not at all within the area postrema (Berk and Finkelstein, 1981; Sawchenko and Swanson, 1983; Plotsky et al., 1988; Weiss and Hatton, 1990; Thellier et al., 1994; Pan et al., 1999). Androgen receptor detection was low to moderate in the subfornical organ and lamina terminalis, and most concentrated in the area postrema (Sar and Stumpf, 1975; Simerly et al., 1990; Hamson et al., 2004), and doubly labeled cells were scarcely, if at all, detected in these regions. 2.3.2.4 Hypothalamus Almost all major areas of the hypothalamus showed retrograde accumulation and showed double labeling to various degrees (Figures 2-5 and 2-6). While the uptake of FG in most of the forebrain proved comparable between the most circumscribed crystal deposits and discrete iontophoretic injections into the PVN, we relied more heavily on the iontophoretic method when gauging tracer accumulation in the immediate vicinity of the PVN, including within the anterior hypothalamic area. Otherwise, comparable numbers of FG accumulating cells in the remainder of the hypothalamus were achieved in animals bearing the smallest crystal injections and the most circumscribed iontophoretic injections of tracer in the PVN. Within the diagonal band complex, small numbers of FG-positive cells were occasionally found. The density of FG-labeled cells in this region appeared to vary with tracer injection size, likely delivered by terminals occupying the perimeter of the PVN (see Larsen et al., 1994). Small to moderate numbers of AR-ir neurons  81  1750-,  Total FG %AR  MPA MPN AHNc DMH VMHd VMHv  Figure 2-5. Estimated cell counts of FG labeled neurons and AR containment through the rostrocaudal extent of the medial hypothalamus. Mean ± SEM total number of FG neurons (stacked open bars) and the number of doubly labeled (AR+FG) neurons (solid bars) detected per hypothalamic region (n = 5). MPA, medial preoptic area; MPN, medial preoptic nucleus, AHNc, central part of the anterior hypothalamic nucleus; DMH, dorsomedial hypothalamic nucleus; VMHd and VMHd, ventromedial hypothalamic nucleus, dorsal and ventral part.  800Total FG %AR (ft  600-  o CD  400-  E 13  200-  PS AVPV PVpo PVp  LHA PMv  Figure 2-6. Estimated cell counts of FG labeled neurons and AR containment through the rostrocaudal extent of the periventricular and lateral hypothalamus. Mean ± SEM total number of FG neurons (stacked open bars) and the number of doubly labeled (AR+FG) neurons (solid bars) detected per hypothalamic region (n = 5). PS, parastrial nucleus; AVPV, anteroventral periventricular nucleus; PVpo, preoptic periventricular nucleus; PVp, posterior periventricular nucleus; LHA, lateral hypothalamic area; PMv, premamillary nucleus, ventral part.  83  were scattered amongst, but seldom co-localized to FG-labeled cells in the diagonal band complex. The medial preoptic area represents a major source of input to the PVN (Tribollet and Dreifuss, 1981; Sawchenko and Swanson, 1983; Simerly and Swanson, 1988; Weiss and Hatton, 1990; Larsen et al., 1994; Cullinan et al., 1996; Champagne et al., 1998; Tribollet et al., 1999; Campeau and Watson, 2000). Consistent with this profile, a very high density of FG-labeled cells was reliably found throughout this region (Figure 2-7). FG-accumulating neurons were also encountered in the lateral part of the preoptic area, but to a much lesser extent (approx 350 cells). This number increased when tracer injections were biased towards, or extended beyond the lateral border of the PVN. Most striking was the amount of FG-labeling in the medial preoptic nucleus (Figure 2-7). Notably, the medial preoptic nucleus projects strongly and preferentially to the dorsal medial parvocellular part of PVN (Simerly and Swanson, 1988). Of all the forebrain regions surveyed, the medial preoptic nucleus showed the highest number of FG-accumulating neurons in total (Campeau and Watson, 2000), and almost 70% of these stained for AR (Figure 25). As discerned along morphological grounds, by redirected sampling of adjacent thionin stained sections, a relatively small to moderate population of doubly-labeled cells was found within the medial (27%) and lateral (16%) aspects of the preoptic area, and within the preoptic periventricular nucleus (see Figure 2-7). Retrograde labeling was moderate, but consistently detected through the rostrocaudal extent of median preoptic nucleus, and with an apparent greater density towards the posterior aspect of the nucleus adjoining the medial preoptic area. Low levels of AR staining was detected throughout the median preoptic nucleus, however, the incidence of double labeling was scarce at best (5-10%). The PVN is strongly innervated by the parastrial nucleus (Sawchenko and Swanson, 1983; Swanson, 1987; Simerly and Swanson, 1988; Cullinan et al., 1996; Thompson  PS  Figure 2-7. Photomicrographs showing the accumulation of FG at the level of the medial preoptic area. The darkfield view illustrates a unilateral distribution of FG uptake (A), prominent within the periventricular and medial zones of the hypothalamus, including a dense collection of cells within the medial preoptic nucleus (boxed region). Note also, dense clusters of FG labeled cells within fusiform (Fu) and parastrial (PS) nuclei. Enlarged view (B) of the boxed region showing concurrent fluorescent retrograde tracer uptake (blue cytoplasm) and AR staining (red nucleus) in the medial preoptic nucleus. Solid arrows show doubly labeled neurons and open arrowhead marks a AR-positive, FG-negative cell, ac, anterior commissure; och, optic chiasm; V3, third ventricle. Scale bar = 50 um in B; 1000 um in A.  85  and Swanson, 2003). While moderate levels of AR staining were most readily detected along the dorsal and ventral borders of the parastrial nucleus (Figure 2-7), the number of double labeled cells encountered varied considerably between animals (Figures 2-6 and 2-7). Consistent with previous experiments (Sawchenko and Swanson, 1983; Watts and Swanson, 1987), scattered numbers of FG-labeled cells (10-15 cells per section) were consistently found within the medioventral part of the suprachiasmatic nucleus in animals bearing the most circumscribed FG injections. In contrast, considerably greater numbers were encountered in animals bearing injections that were centered in or invaded the subparaventricular zone of the PVN. This finding agrees with the anterograde experiment of Watts et al. (1987) suggesting that the suprachiasmatic nucleus provides minor input to the PVN proper. AR staining was not detected in the suprachiasmatic nucleus, consistent with the absence of the AR transcript in this structure (Simerly et al., 1990). A very large assortment of FG-labeled cells were scattered throughout the rostrocaudal extent of the anterior hypothalamic area. While comparable numbers of FG cells were found in the anterior and central parts of the anterior hypothalamic area (approximately 500 and 750 cells, respectively), the incidence of double labeling was consistently higher in the central part (34%, see Figure 2-5) compared to the anterior part (14%). In comparison, relatively few FG-labeled cells were found in the posterior part of the anterior hypothalamic area (approximately 50 cells), but with an incidence of double labeling comparable to that of the central part (34%). At the level of the posterior part of the PVN, noticeable clusters of AR-ir cells were reliably detected within the perifornical part of the anterior hypothalamic area (see Roland and Sawchenko, 1993; • Cullinan et al., 1996; Champagne et al., 1998; Campeau and Watson, 2000). Small, but generally consistent proportions of FG accumulating cells in this region stained positively for AR (approximately 15%).  A substantial number of FG-labeled cells were consistently found within the ventromedial and dorsomedial hypothalamic nuclei (Figure 2-5), in agreement with previous findings (Canteras et al., 1994; Cullinan et al., 1996; Thompson and Swanson, 1998; 2003). The distribution and concentration of tracer accumulating neurons was quite uniform across all parts of the dorsomedial nucleus in animals showing the most circumscribed injections of FG. In contrast, fewer, but no less uniform, numbers of retrograde-labeled cells were observed in animals bearing FG injections that occurred either above of below the PVN proper. These results are in agreement with several previous anterograde (Ter Horst et al., 1984; Ter Horst and Luiten, 1986; 1987; Thompson et al., 1996) and retrograde tracing experiments (Sawchenko and Swanson, 1983; Levin et al., 1987; Fodor et al., 1994; Champagne et al., 1998). While the dorsomedial nucleus reliably stained for AR, the incidence of double labeling was scarce, if at all detectable. Within the small circular anterior part of the ventromedial nucleus, at the level of the posterior part of the anterior hypothalamic area, few and variable numbers of scattered retrograde labeled cells were detected. FG labeling was most evident towards the posterior aspect of the nucleus, concentrated within the larger cell-dense dorsomedial and ventrolateral parts of the ventromedial nucleus (Silverman et al., 1981; Tribollet and Dreifuss, 1981; Sawchenko and Swanson, 1983; Campeau and Watson, 2000). FG labeling was relatively sparse within the diagonally oriented central part separating the dorsal and ventral parts of the ventromedial nucleus. Consistent with previous anterograde tracing studies (Saper et al., 1976; Canteras et al., 1994), the number of FG-labeled cells within the ventromedial nucleus increased dramatically in animals bearing FG injections that were biased towards the subparaventricular zone of the PVN. All parts of the ventromedial nucleus, with the exception of the cell-poor central part, stained strongly for AR. While the dorsomedial and ventrolateral parts contained similar numbers of tracer accumulating and AR-expressing cells in total, the incidence of double labeling was consistently higher within the dorsomedial part (Figure 2-5). 87  Tracer accumulating cells were found scattered throughout the extent of the lateral hypothalamic area, and distributed within each of its sub-regions (Saper et al., 1979; Silverman et al., 1981; Larsen et al., 1994; Cullinan et al, 1996). Specifically, FG-labeled cells were concentrated within the ventral zone of the nucleus, located just dorsal to the optic tract, in agreement with the profile described by Larsen et al. (1994). AR staining was scattered throughout the lateral hypothalamic region without any obvious topographical bias. Doublelabeled cells were encountered in all parts of the lateral hypothalamic area (26%), and a clear majority of these were located within the ventral zone of the nucleus. A moderate number of tracer-accumulating neurons were distributed throughout the rostrocaudal extent of the arcuate nucleus. The highest density of FG labeling was reliably encountered along the medioventral aspect of the nucleus, at the level of the dorsal extent of the dorsomedial hypothalamic nucleus (see Silverman et al., 1981; Sawchenko et al., 1982; Baker and Herkenham, 1995). AR staining was scattered through the rostrocaudal extent of the arcuate nucleus, and the incidence of double labeling was likewise represented by a scattered complement of cells (12%). Within the most caudal reaches of the hypothalamus, low to moderate numbers of FGlabeled cells were routinely detected within the dorsal premamillary nucleus. Most conspicuous, was the distribution of retrograde accumulation within the ventral premamillary nucleus, particularly its anterior part (Figure 2-6), in addition to the posterior periventricular nucleus (Silverman et al., 1981; Sawchenko and Swanson, 1983; Campeau and Watson, 2000). Approximately, 44% of FG-labeled cells in the ventral premamillary nucleus stained positively for AR. Higher numbers of cells were detected within the ventral premammillary nucleus in animals bearing injections that were biased towards the anterior part of the PVN. Taken together, the profile of FG accumulation and sensitivity to injection placement are in line with the  88  anterograde study of Canteras et al. (1992b), showing that the premammillary nucleus projects primarily to the anterior and periventricular regions of the PVN. The medial and lateral mammillary nuclei were consistently devoid of FG labeling. On the whole, a relatively dense number of FG-labeled cells (approximately 250 cells) were detected throughout the posterior hypothalamic area (see Simerly et al., 1990; Yokosuka and Hayashi, 1996), and a reliable number of these stained for AR (24%). 2.3.2.5 Periventricular zone Consistent with previous findings (Tribollet and Dreifuss, 1981; Sawchenko and Swanson, 1983), a very high density of tracer accumulating cells was found through the median preoptic, anteroventral, preoptic, and posterior periventricular nuclei, and within the ventrally displaced suprachiasmatic preoptic nucleus. FG labeling was occasionally detected in the vicinity of the posterior part of the vascular organ of the lamina terminalis, but clearly biased towards the caudal extent of the anteroventral periventricular nucleus. AR staining was prevalent throughout the periventricular zone of the hypothalamus, however the incidence of double labeling was evidently highest in the anteroventral and posterior periventricular nuclei (Figure 2-6). 2.3.2.6 Thalamus Small contingents of tracer-accumulating neurons were routinely identified within the paraventricular nucleus of the thalamus. In agreement with previous retrograde experiments (Sawchenko and Swanson, 1983; Campeau and Watson, 2000), FG-labeled cells were scattered through the rostrocaudal extent of the nucleus, and always distributed ipsilateral to the side of the injection. The density of retrograde labeling in this cell group was typically greater when the injections were placed more dorsally, invading the zona incerta. AR-ir neurons were not detected in the paraventricular nucleus of the thalamus. The highest density of FG-labeling was observed within the posterior thalamic nuclei, including the parvicellular part of the subparafascicular 89  nucleus, in addition to the peripeduncular and posterior intralaminar thalamic nuclei (Campeau and Watson, 2000). Within the parvicellular part of the subparafascicular nucleus, specifically, retrograde labeling was reliably detected in two discrete groups of cells, including a small cluster of horizontally oriented cells near the lateral borders of the ventral posteromedial thalamic nucleus and beneath the fasciculus retroflexus (LeDoux et al., 1985; Price, 1995). This pattern of FG labeling extended caudally into the peripeduncular and posterior intralaminar thalamic nuclei. Within the posterior intralaminar nucleus, retrograde labeling was most apparent along the dorsal borders of the substantia nigra, pars compacta, and a significant complement of FG-labeled cells along this continuum stained for AR (Figure 2-8). 2.3.3  Hindbrain retrograde- and androgen receptor-labeled neurons  2.3.3.1 Reticular core The pattern of FG labeling (Figures 2-8 and 2-9) is in agreement with several previous studies revealing sparse to moderate labeling throughout the hindbrain (Berk and Finkelstein, 1981; Tribollet and Dreifuss, 1981; Sawchenko and Swanson, 1982; Campeau and Watson, 2000). Retrograde labeling was substantial throughout the periaqueductal gray and its pontine continuation, including the lateral, ventrolateral and dorsolateral columns (Bandler and Keay, 1996; Bandler et al., 2000). In the dorsolateral column of the periaqueductal gray, FG labeling was most pronounced in animals bearing tracer injections biased towards the dorsal border of the PVN, in which the rostral division of the nucleus reunions was invaded (Risold et al., 1997). FG labeling was highest and most reliable within the ventrolateral column (Figures 2-9 and 2-10), consistent with previous retrograde (Tribollet and Dreifuss, 1981) and anterograde tracing experiments (Cameron et al., 1995; Floyd et al., 1996) showing that this aspect of the periaqueductal gray provides a rich and unique source of input to the PVN. AR staining was prominent throughout the rostrocaudal extent of the PAG (see Sar and Stumpf, 1975; Simerly et  2000-, Total FG %AR .22  1500-  o CD  1000-  .Q  £  z  500  PoT  PBI  PBm NTSm VLM PPN  Figure 2-8. Estimated cell counts of FG labeled neurons and AR containment through the hindbrain reticular formation and sensory associated nuclei. Mean ± SEM total number of FG neurons (stacked open bars) and the number of doubly labeled (AR+FG) neurons (solid bars) detected per region (n = 5). PoT, posterior thalamic complex; PBL, lateral parabrachial nucleus; PBm, medial parabrachial nucleus; NTSm, nucleus of the solitary tract, medial division; VLM, ventrolateral medulla; PPN, pedunculopontine nucleus.  91  Total FG %AR  5 PAGvl PAGdl DT  8 LDT  LC  RMg  Figure 2-9. Estimated cell counts of FG labeled neurons and AR containment through the hindbrain reticular core nuclei. Mean ± SEM total number of FG neurons (stacked open bars) and the number of doubly labeled (AR+FG) neurons (solid bars) detected per region (n = 5). PAGvl and PAGdl, periaqueductal gray, ventrolateral and dorsolateral column; DT, dorsal tegmentum; LDT, laterodorsal tegmental nucleus; LC, locus coeruleus; RMg, raphe magnus nucleus.  Figure 2-10. Photomicrographs showing the accumulation of FG in the periacqueductal gray. The darkfield view (A) illustrates a very high density of FG labeling particularly within the ventrolateral column (PAGvl). Enlarged brightfield view (B) of the boxed region showing dual immunoperoxidase labeling for AR-ir and FG-ir (see Fig. 9). Solid arrows show doubly labeled neurons and open arrowheads mark AR-positive, FG-negative cells. Aq, aqueduct of Sylvius; IC, inferior colliculus; mlf, medial longitudinal fasciculus; scp, superior cerebellar peduncle. Scale bar = 50 um in B; 500 um in A.  93  al., 1990; Hamson et al., 2004), and double labeling was most pronounced within the ventrolateral part and to a lesser extent within the dorsolateral part of the periaqueductal gray (Figures 2-9 and 2-10). Small clusters of FG-labeled cells were reliably detected within the cuneiform, central tegmental and mesencephalic reticular retrorubral fields, and within the superior colliculus and the rostral linear nucleus (Silverman et al., 1981; Sawchenko and Swanson, 1983). Androgen receptors were detected within all of these nuclei, although the incidence of double labeling was scarce, if at all indicated (0-5%). Substantial numbers of FG-labeled neurons were consistently detected within and in the vicinity of the dorsal tegmental nucleus (Berk and Finkelstein, 1981; McKellar and Loewy, 1981; Tribollet and Dreifuss, 1981; Rye et al., 1987; Bittencourt et al., 1991; Champagne et al., 1998). FG labeling was sparse to undetectable within the largest expanse of the dorsal tegmental nucleus, and this number increased significantly towards the region occupying the caudal extent of the nucleus. FG distribution at this level was most noticeable within the dorsolateral part of the pontine central gray, in the vicinity of the rostral part of the locus coeruleus and Barrington's nucleus (Figure 2-11). A small cluster of FG-labeled cells was also detected within the diffuse part of the nucleus incertus, spanning the midline below the floor of the cerebral aqueduct at the level of the caudal part of the locus coeruleus (Berk and Finkelstein, 1981; Sawchenko and Swanson, 1982; 1983). AR staining was most conspicuous within the nucleus incertus, and a small, but reliable number of FG-labeled cells in this nucleus stained for AR (18%). FG injections in the PVN gave rise to retrograde labeling within distinct mesencephalic 5hydroxytryptamine cell groups, most prominent at the level of the midbrain dorsal (B7), median (B8), and pontine raphe magnus (B3 cell group) nuclei (see Silverman et al., 1981; Sawchenko and Swanson, 1983; Sawchenko et al., 1983; Petrov et al., 1992; Larsen et al., 1996; Champagne et al., 1998). Within the nucleus raphe magnus, FG accumulation was highest towards the rostral  Figure 2-11. Anatomical and immunohistochemical characterization of FG-labeled and AR-ir cells in the vicinity of locus coeruleus (LC). A photomicrograph of separate avidinbiotin immuno-peroxidase and -fluorescent preparations combined to show a weak superimposition of FG accumulating cells (brown) over tyrosine hydroxylaseimrnunoreactive (TH-ir) positive cells (green) in the locus coeruleus. Enlarged view (B) of the boxed region to show concurrent fluorescent retrograde tracer uptake (blue cytoplasm) and AR staining (red nucleus). Note that the bulk of doubly labeled cells locate within the central gray and a tendency for this distribution to occur along the medial border of the locus coeruleus. Enlarged view (C) of the boxed region to show concurrent double immunofluorescent detection of TH (green) and AR (red) stained cells within the locus coeruleus. Estimated % (mean ± SEM) of locus coeruleus TH positive neurons displaying AR staining, and of locus coeruleus FG positive neurons displaying TH and/or both TH and AR (D). This was determined in separate preparations, obtained from adjacent series of sections from individual animals (n = 4). Solid arrows show doubly labeled neurons and open arrowheads mark AR-positive, FG-negative cells. CG, central gray; DT, dorsal tegmental nucleus; V4, fourth ventricle. Scale bar =100 um in B-C; 400 um in A.  95  extent of the nucleus, situated just dorsal to the medial lemniscus, and the number of FG cells detected showed little variation between animals bearing appropriate tracer injections or implants. In contrast, retrograde labeling in the dorsal and median raphe varied considerably as a function of injection size and type. Animals bearing the smallest and most circumscribed tracer injections showed considerably fewer numbers of FG cells in the dorsal (350 cells) and median (175 cells) raphe nuclei compared to animals bearing large injections that invaded the PVN surround. These findings are in agreement with the anterograde tracing study by Larsen et al. (1996), indicating that the raphe magnus provides the largest amount of input directed at the PVN proper, in contrast to the dorsal and median raphe nuclei, which preferentially innervate the PVN surround. AR staining was scattered throughout the midline of the midbrain, pons, and medulla, and all three raphe nuclei showed low to moderate levels of AR staining, in agreement with Simerly et al. (1990) and Hamson et al. (2004). A low, but consistent degree of double labeling was observed within all three raphe nuclei, highest within the median raphe nucleus (9%, see Figure 9) and to a lesser extent in the dorsal (5%) and median raphe (5%) nuclei. 2.3.3.2 Pons-medulla A substantial number of FG-labeled cells were detected in the pontine parabrachial nucleus. Consistent with previous retrograde surveys (Larsen and Mikkelsen, 1995; Pan et al., 1999) and in agreement with anterograde tracing studies (Saper and Loewy, 1980; McKellar and Loewy, 1981; Fulwiler and Saper, 1984; Jhamandas et al., 1992; Bester et al., 1997), the vast majority of retrograde labeling was found within the lateral subdivision of the nucleus and only very few FG-labeled cells were detected in the medial subdivision of the parabrachial nucleus. Based on cytoarchitectonic criteria provided by Fulwiler and Saper (1984), FG labeling was most concentrated within the dorsal and central parts of the lateral parabrachial subdivision, and to a much lesser extent within the external lateral part, consistent with previous retrograde  experiments specifically involving the caudal half of the PVN (Larsen and Mikkelsen, 1995; Pan et al., 1999). Anterograde studies indicate that each of the lateral parabrachial nuclei are preferentially directed at the parvicellular division of the PVN (Saper and Loewy, 1980; Jhamandas et al., 1992), including its dorsal parvocellular part (Bester et al., 1997). AR staining was concentrated throughout the lateral parabrachial subdivision, although to a much lesser extent within the medial subdivision of the nucleus. A high degree of double labeling was reliably observed within the dorsal and central parts of the lateral parabrachial division nucleus, and to a minor extent within the medial division (Figure 2-8). FG injections appropriately centered in the PVN consistently labeled significant numbers of cells in the ventral medulla (the CI and Al cell groups) and moderate numbers in the dorsal medulla, primarily within the nucleus of the solitary tract, representing the C2 and A2 cell groups (McKellar and Loewy, 1981; Sawchenko and Swanson, 1982; Sawchenko et al., 1985; Cunningham and Sawchenko, 1988; Cunningham et al., 1990; Rinaman et al., 1995; Pan et al., 1999; Card et al., 2006). Reliable, but far less retrograde labeling (approximately 50 cells total) was observed in the locus coeruleus (A6 cell group). Double labeling (FG+AR) was highest in the ventral medulla (Figure 2-8), observed as a conspicuous cluster of cells situated between the principle and subtrigeminal parts of the lateral reticular nucleus (Sawchenko et al., 1985; Rinaman et al., 1995). In the nucleus of the solitary tract, the majority of FG-labeled cells were detected within the medial part of the nucleus, and relatively less retrograde labeling was encountered within the lateral part of the nucleus (Sawchenko and Swanson, 1982; Sawchenko et al., 1985). AR staining was uniformly scattered throughout the solitary tract nucleus and locus coeruleus (Simerly et al., 1990; Hamson et al., 2004). Double labeling was reliably encountered within the medial part of the solitary tract nucleus (Figure 2-8) and only a very small contingent of FG labeled cells in the locus coeruleus stained for AR (Figure 2-9).  97  The spatial profile of AR staining within FG-accumulating cells in the locus coeruleus, medulla, and dorsal vagal complex suggested a possible distribution within the catecholaminergic cell groups (Sawchenko and Swanson, 1982; Sawchenko et al., 1985; Rinaman et al., 1995). Thus, we used a combined retrograde transport-double immunohistochemical-labeling technique to gauge the extent to which AR expressing, PVN afferents within these cell groups are represented by noradrenergic and adrenergic neurons (Figures 2-11, 2-12 and 2-13). In agreement with previous findings (Sawchenko et al., 1985), a clear majority of FG-labeled cells spanning the rostrocaudal extent of the ventrolateral medulla, the nucleus of the solitary tract, and in the locus coeruleus (70-85%) stained positively for TH (FG+TH, panel D from Figures 2-11,2-12 and 213). As expected, PNMT-ir was not detected in the locus coeruleus (A6 cell group; see Sawchenko and Swanson, 1982; Sawchenko et al., 1985). Independent assessment of AR and PNMT double labeling suggested an almost even distribution of AR staining amongst the noradrenergic (60%) and adrenergic (40%) neurons of the ventrolateral medulla, and a reliable proportion (20-45%) of the retrogradely-labeled, THpositive cells through the extent of the ventrolateral medulla also stained for AR (FG+TH+AR, Figure 2-12D). Similar to the identity of AR cells in the ventral medulla, roughly half the total of AR cells spanning the rostrocaudal extent of the solitary tract nuclei stained for PNMT. A significant proportion (88%) of TH cells in the nucleus of the solitary tract stained positively stained for AR (TH+AR, Figure 2-13D), however, only rarely and unreliably (0-40%) were triplelabeled (FG+TH+AR) cells encountered (Figure 2-13D). In the locus coeruleus, TH and AR double labeling was considerable (66%), however, triple labeling was inconsistent (0-25%; Figure 2-1 ID). In sum, of the PVN projecting catecholaminergic cell groups, AR was most largely contained within CI and Al neurons of the ventral medulla. Based on the inherent limitations and difficulties in concurrently visualizing and superimposing fluorescent material, the results of the triple labeling experiments, at best, provide 98  D  l  !!• TH + AR  FG + TH  FG + TH + AR  Figure 2-12. Anatomical and immunohistochemical characterization of FG-labeled and AR-ir cells within the ventral medulla. Darkfield photomicrograph of an immunofluorescent preparation to show a TH-ir cell group (A). Higher magnification view (B) of the ventrolateral medulla to show concurrent fluorescent retrograde tracer uptake (blue cytoplasm) and AR staining (red nucleus). Enlarged view (C) of the same preparation in panel A to show concurrent double immunofluorescent detection of TH (green) and AR (red) stained cells. Estimated % (mean ± SEM) of TH positive neurons displaying AR staining, and of FG positive neurons displaying TH and/or both TH and AR in the ventrolateral medulla (D). Solid arrow in each panel identifies the same cell in the ventral medulla showing TH (A), FG and AR (B), and TH and AR staining (C). Scale bar = 100 urn in B-C; 200 urn in A.  99  Figure 2-13. Anatomical and immunohistochemical characterization of FG-labeled and AR-ir cells within the dorsomedial medulla. Darkfield photomicrograph of an immunofluorescent preparation to show TH-ir cell groups in the nucleus of the solitary tract (A). Higher magnification view (B) of the boxed region to show concurrent fluorescent retrograde tracer uptake (blue cytoplasm) and AR staining (red nucleus) in the NTS. Enlarged view (C) of the boxed region of the same preparation in panel A (left A2 group) to show concurrent double immunofluorescent detection of TH (green) and AR (red) stained cells. Estimated % (mean ± SEM) of TH positive neurons displaying AR staining, and of FG positive neurons displaying TH and/or both TH and AR in the NTS (D). Solidarrow in each panel identifies the same TH positive cell (boxed in panel A), showing FG and AR (B), and TH and AR staining (C). ap, area postrema; Aq, aqueduct of Sylvius; NTSm, nucleus of the solitary tract, medial division; V4, fourth ventricle. Scale bar = 50 um in B-C; 375 um in A.  100  a qualitative estimate of AR containment between catecholaminergic afferents. However, noninclusive assessment of AR and TH staining within FG cells from separate experiments provided some indication that our triple labeling estimates were valid. Thus, the amount of triple labeling predicted by the arithmetic product of (FG+AR) x (FG+TH) percent values in the ventrolateral medulla, solitary tract nucleus, and in the locus coeruleus, yielded values of 19.8 ± 2.0%, 11.1 ± 1.2%, and 6.1 ± 0.6%, respectively, overlapping considerably with the mean percentage and range of FG positive cells displaying both TH and AR in these regions, 30 ± 6.1%, 11.3 ± 7.9%, and 8.8 ± 5.5%, respectively (FG+TH+AR, panel D from Figures. 2-11, 2-12 and 2-13). 2.4  Discussion In the present study we characterized the anatomical nature by which androgen sensitive  targets in the brain communicate directly with the paraventricular nucleus (PVN) of the hypothalamus. Several criteria were imposed with respect to retrograde tracer placement in the PVN, allowing us to quantify the relative densities of androgen receptor containment within candidate afferent cell populations. Taking previous anterograde surveys into account (see Table 1), our current findings provide an index of the potential influence of testosterone on various visceromotor systems of the PVN. To determine potential sites of androgen influence, we employed a dual immunohistochemical procedure to concurrently label cells displaying nuclear androgen receptor (AR) staining and cytoplasmic fluorogold (FG) accumulation in the PVN. Delivery of FG by iontophoresis produced discrete injection sites, although occasionally this yielded sub-optimal neuronal tracer uptake. Crystalline tracer deposits, on the other hand, produced denser injections and more intense labeling, although tracer spread typically extended beyond the border of the PVN proper. Both delivery methods were controlled by examining patterns of tracer diffusion and accumulation within individual animals (see Methods, section 2.2.2). As such, we restricted  our analysis only to those animals displaying the most circumscribed FG injections, centered towards the caudal portion of the PVN. Analysis was further restricted to animals showing comparable numbers in FG cell counts, and patterns of accumulation similar to previous retrograde transport experiments (see results). Stable numbers in retrograde labeling were reliably observed in the parastrial, medial preoptic, dorsomedial, and in ventromedial hypothalamic nuclei, for example, and FG cells were rarely encountered within the lateral septal and amygdaloid complex (Cullinan et al., 1996). Achieving reliable FG cell counts between animals provided a stable framework for indexing regional differences in AR containment within PVN projecting cell populations. FG injections centered within the PVN proper yielded a pattern of staining that complemented several previous retrograde transport based characterizations (see Results), and will not be reviewed in great detail here. Briefly, PVN-projecting cells were located throughout the rostrocaudal extent of the brain, extending from the anterior division of the stria terminalis to the caudal aspects of the ventral lateral medulla and nucleus of the solitary tract. Within the forebrain, significant uptake was consistently observed within the periventricular and medial zone nuclei, with the strongest projections arising from the preoptic and hypothalamic cell groups. In the hindbrain, extensive FG labeling was detected within several midline structures including the periaqueductal gray, and within several laterally displaced regions, including the posterior thalamus, lateral parabrachial nucleus, and throughout the ventrolateral catecholaminergic cell groups of the medulla. Within the forebrain, the highest densities of androgen receptor expressing, PVN afferents encountered were found within the periventricular and medial zones of the preoptic area and hypothalamus, and within the bed nuclei of the stria terminalis. Doubly labeled cells were generally detected in lower densities throughout the brainstem, although reliable complements of AR+FG cells were found within the posterior thalamic complex, in the lateral part of the 102  parabrachial nucleus, and in particular, the ventral lateral part of the periacqueductal gray. The pattern of AR staining within FG-accumulating neurons in the medulla and brainstem suggested a possible distribution within catecholaminergic cell groups. Using a combined retrograde transport-double immunohistochemical-labeling technique, our findings indicated that the most reliable contingent of AR staining was found within both noradrenergic and adrenergic components of the ventral medulla. In contrast, AR staining within cells identified as projecting to the PVN was rare to inconsistent within the locus coeruleus and solitary tract nuclei. Taken together, the extensive distribution of androgen-receptor expressing, PVN afferents indicate that a vast set of forebrain and hindbrain nuclei are positioned to mediate androgen-sensitive input to the PVN. In drawing conclusions as to the potential targets of candidate populations described, it is important to emphasize that the borders between the classes of major effector neurons in the PVN are neither clear nor distinct, that neurons of one output class can be found in compartments dominated by other cell types, and that dendrites of neurons of any subdivision seldom respect nuclear boundaries (Swanson and Sawchenko, 1983; Swanson et al., 1987; Rho and Swanson, 1989). Nonetheless, the findings of Rho and Swanson (1989) indicate that the bulk of the dendritic mass of any functionally related cell group in the PVN is confined to the same PVN . compartment. Therefore, based on the results of several previous anterograde transport studies, taken together with our current findings, inferences can be made with some degree of confidence as to which functional cell types in the PVN are preferentially targeted by candidate ARexpressing, PVN projecting cell populations, as summarized in Table 1. Importantly, the topographic distribution of double-labeled (AR+FG) cells throughout the forebrain and brainstem suggests a potential modulation of neurosecretory and non-neurosecretory (autonomic) cells in the PVN, and some capacity for affecting certain cell types uniquely.  Another important consideration is that many brain regions regulating the PVN and HPA axis do not directly innervate cells occupying the PVN proper, but instead act via polysynaptic relays. Thus, in contrast to direct PVN projections arising from the fusiform nucleus of the stria terminalis, anterior hypothalamic, dorsal tegmental or pedunculopontine nuclei, for instance, a number of projections described here ramify at least as prominently and quite often preferentially, to regions immediately adjoining the boundaries of the PVN (see Table 1). A collection of cell groups surrounding the caudal PVN, including the medial aspects of the zona incerta, the perifornical region, the dorsal aspects of the anterior part of the anterior hypothalamic area and the rostral (anterior parvicellular) aspects of the PVH, contain androgen receptors and provide substantial input to the hypophysiotropic zone of the PVN (Roland and Sawchenko, 1993; Boudaba et al., 1996; Bowers et al., 1998; Herman et al, 2002a; Bingham et al., 2006). These cell groups closely positioned around the PVN are thought to constitute an organized perinuclear zone which may serve to integrate information provided by afferents that distribute poorly, if at all, within the borders of the PVN proper, or to activate local inhibitory neurons. As reviewed by Herman and colleagues (Herman et al., 2002a, 2002b, 2003, 2005), this gamma-aminobutyric acid (GABA)-rich PVN surround is ideally positioned to integrate input from the limbic forebrain, including the prefrontal cortex, lateral septum, ventral subiculum, and medial amygdaloid nuclei (and see Results). The peri-PVN region also receives significant cholinergic and serotonergic input, which also greatly exceeds that of the PVN proper (Sawchenko et al., 1983; Ruggiero et al., 1990), and it is clear that this peri-PVN complex represents a principle target of both ascending and descending regulatory systems. Of note, several structures comprising the PVN surround, including the dorsal anterior hypothalamic area and the perifornical region, contain significant complements of GABA producing, AR-expressing cells (see Bingham et al., 2006). Several additional forebrain limbic-related and hindbrain cell groups target the PVN only indirectly, communicating with such upstream relay centers as the medial preoptic area, the bed  Table 2-1. Relative density of AR staining within candidate afferent mediators of gonadal status on the PVN proper and surround 1>2-3 P V N Compartment Cell Group  mp  pm  dp/lp/mpv  Forebrain Limbic Prefrontal cortex Lateral septum Medial amygdala Ventral subiculum Ventral premammillary n. Bed n. Stria terminal is Anteromedial area Dorsomedial n. Dorsolateral n. Fusiform n. Principle n. Interfascicular n. Transverse n. Preoptic area Anterovenlrat periventricular n. Preoptic periventricular n. Median preoptic n. Medial preoptic area Medial preoptic n, Lateral preoptic area Hypothalamus Paraslrial n. Anterior hypo. n. Lateral hypo, area Arcuate n. Ventromedial n. Posterior hypo, area  ++ +++  ++ +++  ++ +++  + +++  + +++  -  +++ ++ + ++ +++ +  +++ ++ + ++ +++ +  +++  + ++ ++ + +++  + ++ ++ + +++ ++  + ++  +  + ++ +  + ++  ++  PVN Surround  References  + ++ + + +++  14,28 22,23 5,21  ++ ++ + ++  10,20  6 3,26  8 8 8 9 9 9  -  +++ +++ ++  -  +  -  +  -  -  +  -  +++ ++ + ++ +++ +  29,32 26,30 26,32  26 29 26, 30, 17  29,32 22,24,26 12, 17, 26 33 4,26 23  ++ + +++  Thalamus Posterior complex H indbrain-Sensory Solitary tract n., media] Parabrachial n„ lateral Para brachial n., medial Hindbrain-Reticular Periaqueductal gray, ventral lateral Periaqueductal gray, dorsal lateral Locus coeruleus Dorsal raphe n. Median raphe n. Raphe magnus n. Dorsal tegmentum Pedunculopontine n. Ventrolateral medulla  f+  +  + + + + +  + + + + + ++ ++  ++ ++  -  ++ +  1, 15, 16, 19 19,25  +  ++ ++ + + + +  11 23 7 18,27 18 18 13.23 23 7  -  + + ++  ++  AR, androgen receptor; PVN, paraventricular nucleus of the hypothalamus. 1. Density of AR-iinmunoreactivily (AR-ir) in select cell groups showing the highest and most reliable incidence of AR +FG double labeling and projecting to (he PVN region, asbased on previous anterograde labeling studies. Nomenclature based on the atlas of Swanson [31]. 2. A three point rating scale was used for comparison in which (+ + + ) represents AR staining in a substantial majority ( > 4 0 % ) o f FG-labeled neurons in a given cell group, (+++) represents a moderale density (20-39%), and (+ ) represents a low, but consistent density (5-19%) of AR staining within scattered populations of retrogradely labeled cells. Cell groups showing very low and unreliable (0-5%) detection are not included (see Results). 3. Based on previous anterograde tracing experiments, (-) indicates a lack of identified projections to the PVN region. 4. Projections lo one or more of the autonomic-related compartments of the PVN, described in detail elsewhere (see appropriate reference). 1. Bester el al., 1997. 2, Campeau and Watson, 2000. 3. Cameras et al„ 1992. 4. Cameras et al., 1994. 5. Cameras el al., 1995. 6. Cullinan et al., 1993. 7. Cunningham and Sawchenko, 1988. 8. Donget al., 2001. 9. Dong and Swanson, 2004. 10. Dong and Swanson, 2006a. 11. Floyd etal., 1996.  12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.  Goto et al., 2005. Groenewegen and Van Dijk, 1984. Hurley et al., 1991. Jhainandas etal., 1992. Krukoffelal., 1993. Larsen el al„ 1994. Larsen el al., 19%. McKellar and Loewy, 1981. Mulders el al., 1997. Prewitt and Herman, 1998. Risold'et al., 1994.  23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.  Risold et al., 1997. Roland and Sawchenko, 1993. Saper and Loewy, 1980. Sawchenko and Swanson, 1983. Sawchenko el al., 1983. Sesack et al., 1989. Simerly and Swanson. 1988. Swanson, 1976. Swanson, 1998. Thompson and Swanson, 2003. Watts et al., 1987.  105  nuclei of the stria terminalis, the dorsomedial hypothalamic nucleus, the lateral hypothalamic area, and the solitary tract nucleus (Herman et al., 2003). The medial preoptic area stands out as an important focal point in this regard, as a large contingency of neurons within the medial preoptic area receive and reciprocate input to other androgen receptor rich groups that, in turn, impinge upon the PVN region, including the bed nuclei, the dorsomedial hypothalamus and the solitary tract nucleus (Simerly and Swanson, 1986; 1988). Varying degrees of AR-ir were detected within each of these cell groups, thus imparting several additional levels of gonadal influence on converging homeostatic information to the PVN. The present findings would suggest a very close if not overlapping relationship between systems governing reproduction and HPA control. Indeed, many forebrain and hindbrain structures that contained extensive distributions of androgen receptor-expressing, PVN afferents are also known to subserve reproductive function (Coolen et al., 1998; Greco et al., 1998b). Thus, regions of the medial basal forebrain known to mediate copulatory behavior, such as the medial preoptic and ventromedial hypothalamic nuclei (McGinnis et al., 1996; Hull et al., 1997) contained high densities of FG+AR cells. Similarly, structures thought to control the production of gonadotropic hormones, including the periventricular preoptic and anteroventral periventricular nuclei (Wiegand and Terasawa, 1982) also showed significant complements of double labeled cells. This distribution suggests that androgenic regulation of gonadotropin-releasing hormone release and behavior is not entirely restricted to components of the gonadal axis, but may also rely on concurrent regulation of both the neuroendocrine and autonomic-related branches of the PVN (Selvage and Rivier, 2003; Selvage et al., 2004). This is consistent with the disruptive effects of adrenalectomy and sympathetic blockade on testosterone secretion, androgenic regulation of gonadotropin-releasing hormone and luteinizing hormone release, and sexual behavior (reviewed in Kalra and Kalra, 1983; Levine et al., 1991; Herbison, 2006, and see Poggioli et al., 1984).  Beyond the realm of reproduction, the prevalence of androgen receptors within thalamic and brainstem nuclei, suggest that multiple sensory systems may be under the influence of testosterone (Gandelman, 1983; Simerly et al., 1990; Hamson et al., 2004). Recent work speaks to several possible routes through which this could be imposed. Ceccarelli and colleagues (2003) revealed that gonadectomized male rats show a heightened induction of Fos in the arcuate hypothalamic nucleus compared to their gonadal-intact counterparts in response to noxious formalin challenge. Indirect influences mediated via the forebrain and hindbrain centers that receive collaterals from the principle spinothalamic pathway have also been examined. Thus, substantial projections from the spinal dorsal horn to the medial and lateral preoptic and hypothalamic nuclei, the posterior thalamic complex, the ventrolateral periacqueductal gray and parabrachial nuclei, with minor inputs to the perifornical and anteroventral periventricular nuclei, have been identified (Giesler et al., 1994). As characterized here, all of these contain a significant assortment of androgen receptors. The somato-visceral and -sensory systems could also serve as a viable target for the inhibitory influences of testosterone on HP A output, consistent with the detection of moderate to large densities of AR+TH double labeling throughout the catecholaminergic cell groups of the caudal brainstem and the reliable incidence of AR staining within identified PVN afferents spanning the Al and CI cell groups of the medulla (reviewed in Sawchenko and Swanson, 1982). Previous studies have indicated a profound influence of gonadectomy and hormone replacement in the rat on several brain monoaminergic systems, including TH activity. Although, this has been primarily studied with respect to dopamine regulation (see Engel et al. 1979; Simerly 1989; Kritzer 2003). The results of our double and triple labeling experiments, directed at determining the percentage of FG positive neurons displaying both TH and AR directly (see Results) indicate that the bulk of any potential androgenic influence on catecholaminergic afferents would have to be placed within the ventral medulla.  The remarkable display of AR staining by TH positive neurons in the locus coeruleus remains worthy of discussion, considering that this number exceeded the capacity of FG+AR labeled cells detected within the locus coeruleus and the number of TH positive, AR stained cells in the dorsal and ventral medulla. On one hand, these findings speak to and confirm the small contribution of the A6 cell group relative to the mass of catecholaminergic afferents provided by the dorsal and ventral medulla. On the other hand, the locus coeruleus is known to heavily project to several PVN and HPA regulating structures (Wyss et al., 1979; Swanson 1982; Simerly and Swanson 1986; Carstens et al. 1990; Risold and Swanson 1997; Thompson and Swanson, 1998), including within major homeostatic- (dorsomedial hypothalamic nucleus, medial preoptic nucleus, ventral tegmental area), sensory- (medial thalamus, substantia nigra) and limbic-related relays (hippocampus, lateral septum). Taken together with the impressive distribution of AR within the locus coeruleus, the possibility that testosterone could alter the activity of any of these pathways should not be underestimated. Modest populations (20-25%) of cells in the nucleus of the solitary tract and ventral medulla identified as PVN projecting are not catecholaminergic (see Rinaman et al., 1995), and these may be occupied by P-inhibin and somatostatin neurons (Sawchenko et al., 1988; Sawchenko et al., 1990; Verbalis et al. 1995). At present, the connectivity of these cell phenotypes and identity with respect to AR staining within these areas has yet to be established. While p-inhibin secretion (Roberts et al., 1989; Dalkin et al., 1994; Harvolson et al., 1994) and somatostatin expression in the forebrain (Baldino et al., 1988; Argente et al., 1990) respond to changes in sex steroid hormone levels, the impact of gonadal status on these phenotypes in the medulla, within PVN projecting afferents specifically (see Chibbar et al., 1990), has not been examined. Based on the extensive containment of androgen receptors within pathways conveying somatosensory and nociceptive information to the PVN, it seems reasonable to assume that  changes in gonadal status could play an important role in altering or sustaining adaptive visceromotor responses to several types of homeostatic challenges. As mentioned above, our findings would also place several aspects of the forebrain into this design, including the medial preoptic and bed nuclei of the stria terminalis, which were consistently found to contain neurons identified as both PVN projecting and containing androgen receptors. In addition to receiving substantial input from putative ascending sensory systems, the medial preoptic and bed nuclei also distribute information flow between several limbic regions, including the prefrontal cortex, lateral septum, and amygdala. Beyond implicating the medial preoptic and bed nuclei as androgen sensitive mediators of sensory information, these nuclei would also appear well suited for bridging emotional and neuroendocrine responses to changes in gonadal status (Rubinow and Schmidt, 1996; Toufexis et al., 2006). While several indications of this potential have been shown in separate studies (Gu et al. 2003, Polston and Simerly, 2003), concurrent effects on both behavioral and PVN visceromotor responses have yet to be examined experimentally. The histochemical identification of neurotransmitter and neuropeptide receptors on or surrounding the paraventricular neurons has provided some essential insight into the chemical nature of PVN control (Sawchenko and Swanson, 1982; Cunningham and Sawchenko, 1988; Cole and Sawchenko, 2002; Herman et al., 2002b). The activation of paraventricular motor neurons ultimately depends on the integration of excitatory and inhibitory signals arising from first order forebrain and hindbrain relays (Sawchenko and Swanson, 1982; 1983). Accordingly, the prime candidate neurotransmitters implicated in PVN control include gamma-aminobutyric acid (GABA), norepinepherine, and glutamate (Cunningham and Sawchenko, 1988; Van den Pol et al., 1990; Brann, 1995; Boudaba et al., 1996), and a variety of neuropeptides, including arginine vasopressin, corticotrophin releasing hormone, galanin, and enkephalin (Simerly et al., 1986; Bittencourt et al., 1991; Moga and Saper, 1994; Champagne et al., 1998).  Testosterone has been shown to interact with several of these neurotransmitter and peptidergic systems and within numerous cell groups identified as projecting to the PVN or regulating the HPA axis (De Vries et al., 1986; Diano et al., 1997; Herbison, 1997; Bloch et al., 1998; Ronnekleiv and Kelly, 2005). GABAergic projections emanating from the MPN (and its afferent relays) stand out as a potential mediator of the upstream inhibitory influences of testosterone on PVN visceromotor function. While a wide variety of neurotransmitters are expressed by neurons of the MPN, GAB A is the most prolific and highly sensitive to gonadal status (Simerly et al., 1986; Bowers et al., 1998; Herman et al., 2004). A clear majority of GABAergic neurons in the MPN express androgen receptors, gonadectomy and local androgen receptor blockade decrease GABAergic activity within this structure (Grattan and Selmanoff, 1993; 1994; Grattan et al., 1996), and flutamide and testosterone implants in the vicinity of the MPN increase and decrease, respectively, the plasma adrenocorticotropin hormone (ACTH) response to restraint stress (Viau and Meaney, 1996; Williamson and Viau, 2006). As reflected by the expression of glutamic acid decarboxylase (GAD) mRNA, acute and repeated restraint exposure activate multiple GABAergic circuits in the brain, particularly within numerous cell groups that we have identified as both PVN projecting and containing androgen receptors, including the MPOA, the peri-PVN region, and the perifornical nucleus (Bowers et al., 1998). These GABAergic pathways warrant further examination as afferent mediators of circulating testosterone on the PVN, considering that normal HPA adaptation to repeated restraint exposure is reduced by gonadectomy and androgen receptor blockade (Bingham et al., 2005). We have also previously observed a strong association between the inhibitory effects of testosterone on stress-induced ACTH release and the expression of AVP mRNA within the medial amygdala and the posterior subnuclei of the stria terminalis (Viau et al., 2001; Bingham et al., 2005). Moreover, opposite to the effects of AVP originating from neurosecretory neurons of the PVN, central AVP pathways appear to exert an inhibitory influence on HPA output (Makara et 110  al., 1996; Wotjak et al., 1996). Similar to GAB A regulation, AVP expression within the medial amygdala and posterior bed nuclei depend heavily on gonadal status, as AVP mRNA levels and immunoreactivity within these regions and their afferent connections are practically abolished by gonadectomy, effects readily reversed with testosterone replacement (De Vries et al., 1986; De Vries and Miller, 1998). Although several independent lines of research support a role for AVP in coordinating the central effects of testosterone on behavioral and visceromotor adaptation (reviewed in Williamson et al., 2005), the anatomical basis for this has yet to be resolved. Thus, while the posterior aspect of the bed nucleus of the stria terminalis is known to project strongly and preferentially to the hypophysiotropic zone of the PVN (Dong and Swanson, 2004), for example, the extent to which this projection contains AR- and AVP-expressing neurons and responds to changes in testosterone secretion remain worthy of pursuit (see Koolhaas et al., 1998; Kalsbeek et al., 2002). Given the potency by which variations in circulating testosterone levels alter HPA output, we would like to believe that the nuclei identified in the present study form an important network that registers fluctuations in testosterone release associated with changes in homeostatic demand. However, the structures identified probably underestimate the substrate and the mechanisms of sex steroid hormone regulation involved. Androgen receptors are distributed throughout the limbic system, including within their respective relays to the PVN, as discussed above. Several of these HPA-regulating nuclei, in addition to those we have identified as PVN-projecting, show various degrees of overlap with respect to estrogen receptor (alpha and beta) distribution (Stumpf, 1970; Pfaff and Keiner, 1973; Stumpf et al., 1975; Simerly et al., 1990; Shughrue et al., 1997; Greco et al., 1998a; Laflamme et al., 1998; Greco et al., 2001) and aromatase activity (Roselli and Resko, 1993; Wagner and Morrell, 1997; Zhao et al. 2007). Thus, situation-dependent and region-specific changes in aromatase activity could very well play a critical role in determining how fluctuations in circulating testosterone levels alter the type and flow of information to the 111  PVN, beyond what could be attributed to androgen receptor-mediated events. Androgen receptors can regulate gene expression directly by binding to potential androgen responsive elements located within the promoter region of target genes or indirectly, by interacting with other steroid hormone receptors (Chen et al., 1997), transcription factors, and second messenger systems (Chang et al., 1995; Heinlein and Chang 2001). While we have described several neurotransmitter and peptidergic systems that reliably respond to changes in gonadal status, the cellular mechanisms governing these responses remain unclear (but see Bao et al., 2006). Nevertheless, the impressive distribution of androgen receptors within several extended circuitries of the PVN provides a tenable basis for assuming that testosterone could act to bridge several independent, yet converging influences to the PVN and HPA axis. As discussed above, many advances have been made in clarifying the anatomical and functional organization of stress-related circuits in the brain, which have been critical in identifying putative regulators of PVN and HPA function. It is clear that stimuli that elicit endocrine, autonomic and behavioral responses rely on complex and centrally coordinated patterns of neuronal activation (Sawchenko et al., 2000; Herman et al., 2003). Further, a common consensus is that different stimuli recruit independent and overlapping neural systems, which in turn project to the PVN, either directly or indirectly (Li and Sawchenko, 1998; Dayas et al., 2001a; but see Pacak and Palkovits, 2001; Day, 2005). Based on the present results (Figure 2-14), and on those of previous retrograde and anterograde studies (refer to Table 1), the PVN is innervated by a substantial number of forebrain and hindbrain structures that receive first or second order somatosensory, visceral and humoral information. Because androgen receptors are contained by a clear majority of these, this clearly places the gonadal axis amongst the principle response systems guarding the organism against homeostatic threat. The challenge now is to determine how changes in circulating testosterone levels are registered within the circuits  112  Figure 2-14. Schematic summarizing the organization of cell groups identified as projecting to the PVN region and displaying AR-ir. The number of dots provides an index of AR containment, where each dot represents a 5% unit of colocalization within a cell group of interest (e.g. 65% in the MPN). Brain regions in italics represent AR-rich structures providing indirect input to the PVN (see Table).  113  described, and how this impacts the stress-induced activation of these projections to the PVN. Our findings provide an important starting point for this pursuit.  114  2.5  References  Abercrombie M. 1946. Estimation of nuclear population from microtome sections. 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Zhou L, Blaustein JD, De Vries GJ. 1994. Distribution of androgen receptor immunoreactivity in vasopressin- and oxytocin-immunoreactive neurons in the male rat brain. Endocrinology 134:2622-2627.  CHAPTER 3 : Selective Contributions of the Medial Preoptic Nucleus to TestosteroneDependant Regulation of the Paraventricular Nucleus of the Hypothalamus and the HPA •  2  axis 3.1  Introduction The vast distribution of different types of sex steroid hormone receptors within the central  nervous system place this class of hormones well beyond the realm of reproductive function (Williamson and Viau, 2007a). Indeed, testosterone and estrogen exert reliable inhibitory and stimulatory effects, respectively, on activity of the hypothalamic-pituitary-adrenal (HPA) axis, a neuroendocrine system essential for survival (Williamson et al., 2005). Moreover, individual and gender based differences in normal and abnormal HPA function can be attributed to variations in sex steroid hormone release (Viau and Meaney, 1991; Viau et al., 2005). Threats to homeostasis activate the HPA axis by triggering the sequential release of a chain of hormones. This is initiated by the recruitment of neurosecretory neurons in the paraventricular nucleus (PVN) of the hypothalamus that secrete peptide stores from the median eminence, primarily corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) (Swanson and Sawchenko, 1983). CRH and AVP synergize on the release of adrenocorticotropin hormone (ACTH) from the anterior pituitary that, in turn, stimulates the release of glucocorticoids from the adrenal gland, Cortisol in humans and corticosterone in the rat (Antoni, 1993; Aguilera, 1994). A fine balance between glucocorticoid negative feedback inhibition and stress-induced drive to the HPA axis ultimately determines the magnitude of the ACTH response to stress (Dallman et al., 1987; Watts, 2005). Testosterone can operate on all of these elements, by exerting inhibitory actions on the cellular and transcriptional activation of PVN motor neurons,  2  A version of this chapter has been published. Williamson M, Viau V. 2008. Am J Physiol Regul Integr Comp Physiol 295:R1020-1030. © 2008 the American Physiological Society. Reprinted with permission of the American Physiological Society.  ACTH secretagogue synthesis and release, as well as by enhancing glucocorticoid negative feedback efficacy (Viau et al., 1999; Viau and Meaney, 2004). There are multiple sites and pathways mediating the central actions of androgens on the HPA axis. Various metabolites of testosterone, including 5a-dihydrotestosterone and its 3p-diol metabolite, are capable of acting locally to inhibit stress-induced levels of PVN Fos mRNA and plasma ACTH and corticosterone (Lund et al., 2006). Our connectional studies predict that androgens can also act within a very large assortment of brain regions relaying sensory and limbic information to the PVN region. The medial preoptic nucleus (MPN) stands out in this regard, as we found that it contains the highest concentration of androgen receptor positive efferents to the PVN (Williamson and Viau, 2007a). In line with this connectional data, testosterone implants into the vicinity of the medial preoptic nucleus reduce the plasma ACTH and corticosterone responses to restraint (Viau and Meaney, 1996). Moreover, high testosterone replacement levels in the periphery that normally suppress the magnitude of the HPA stress response fail to do so in rats bearing large electrolytic lesions of the medial preoptic area (Viau and Meaney, 1996). At this point, several uncertainties remain. First, while these electrolytic lesions in the medial preoptic area encompassed the MPN, they were also large enough to have damaged several neighboring subcortical relays to the PVN region. This would include the anterior hypothalamic area; the ventral noradrenergic ascending bundle, which travels through the lateral hypothalamic and lateral preoptic areas; and the posterior medial region of the bed nucleus of the stria terminalis, which markedly inhibits HPA responses to acute stress (Choi et al., 2007; Choi et al., 2008). Second, while systemic changes in testosterone can operate on CRH and AVP expression and stress-induced levels of Fos within PVN motor neurons (Viau et al., 2003; Lund et al., 2004), the extent to which a functioning MPN is necessary for these synthetic and cellular stress responses to occur remains to be seen.  In the present study we superimposed two levels of testosterone replacement in the periphery in animals receiving sham or small volume, bilateral injections of ibotenic acid into the MPN. This allowed us to assess how testosterone acts and interacts with the MPN on ACTH secretagogue synthesis, HPA output, and intervening levels of Fos activation within different compartments of the PVN. Of note, the MPN receives and sends input to the medial nucleus of the amygdala (Simerly and Swanson, 1988; Canteras et al., 1995), one of many limbic regions expressing androgen receptors (Williamson and Viau, 2007a) and regulating the HPA axis (Dayas et al., 1999). Testosterone stimulates AVP mRNA and peptide in the medial amygdala (de Vries and Panzica, 2006), and several lines of evidence continue to relate the inhibitory influence of the gonadal axis on HPA function in males to testosterone dependent increases in extrahypothalamic AVP (Gomez et al., 2004; reviewed in Williamson et al., 2005). Based on the potential for the MPN to influence the HPA axis to and through AVP neurons in the medial amygdala, we gauged whether the AVP response to testosterone within this structure also depends on the integrity of the MPN. Our findings indicate that the MPN is integral for testosterone to act on the PVN, as well as its extended circuitries. 3.2  Methods  3.2.1  Animals Eighty-eight adult male Sprague-Dawley rats (Charles River, St. Constant, Canada) were  used, weighing 250 g on arrival (56 days old) and 375 g when sampled (~75 days old). Animals were pair housed under controlled temperature (23±2°C) and lighting conditions (12:12-hour light: dark cycle, lights on at 0600 hours), with food (Labdiet; Rat diet 5012) and water available ad libitum. All experimental protocols were approved by the University of British Columbia Animal Care Committee (Appendix A).  3.2.2  Treatment To explore whether and at what concentrations testosterone interacts with the medial  preoptic nucleus, body weight matched animals were gonadectomized and randomly assigned to one of the following testosterone replacement-MPN treatment combinations 1) low testosteronesham, 2) low testosterone-lesion, 3) high testosterone-sham, and 4) high testosterone-lesion, and sampled under basal and/or restraint stress conditions.  Gonadectomy and testosterone replacement. Testes were removed via a scrotal incision under ketamine-xylazine-acepromazine anesthesia (25, 5, and 1 mg/ml, respectively; 1 ml/kg subcutaneously). Each testis was delivered separately through the scrotal incision and removed by severing the vas deferens and spermatic artery, which was ligated to maintain hemostasis. Gonadectomy was completed by closing the scrotal incision with 4-0 non-absorbable suture. Testosterone replacement was performed at the same time, using two subcutaneous Silastic implants (each 35 mm in length, 0.062 mm inner diameter, 1.25 mm outer diameter; Dow Corning, Midland, MI) that were packed with crystalline testosterone (Sigma Aldrich, Oakville, Canada) to a length of 10 mm or 60 mm, for low- and high testosterone replacement, respectively (22). As shown in the Results, these implants provide plasma testosterone concentrations in the physiological range observed in adult male rats (Viau and Meaney, 1996; Viau et al., 2003).  Medial preoptic nucleus lesions. Comparing the effects of MPN lesions in gonadal-intact and gonadectomized (GDX) rats with or without testosterone replacement was initially considered. However, this is not the most informative approach for establishing that the MPN is an obligate target for the central actions of testosterone on the HPA axis to occur. As our previous connectional experiments suggest (Williamson and Viau, 2007a), there are likely several candidate structures in the brain supplying androgen sensitive input to the PVN region, in addition 138  to the MPN. Importantly, GnRH neurons are diffusely distributed throughout a continuum of the medial basal forebrain, including within the region of the MPN (Silverman et al., 1987; Herbison, 2006). As lesions in the vicinity of the MPN can decrease circulating testosterone levels in animals with testes (Kalra and Kalra, 1983), this approach would not allow us to discern a role for the MPN independently from changes in testosterone release. GDX rats show increases in HPA activity similar to those bearing MPN lesions (Viau et al., 2003). Thus, data gleaned from these animals would also lack the specificity for attributing the central effects of testosterone to the MPN. To alleviate these concerns, in the present study we superimposed two levels of testosterone replacement in GDX animals bearing MPN lesions, both to control plasma testosterone and to specifically address whether the MPN is required for the HPA axis to register differences in testosterone in circulation. Three days after gonadectomy and testosterone replacement surgeries, rats were anesthetized by injection of sodium pentobarbital (50 mg/kg, ip) and were placed in a Kopf stereotaxic apparatus. To produce discrete, axon sparing lesions (see 23), rats received bilateral volume injections of ibotenic acid (100 nl, 5 ug/ul in 0.1 M PBS, pH 7.2; Sigma Chemicals, Oakville, Canada) or phosphate buffered saline (Sham lesion; PBS, 0.1 M, pH 7.2) into each MPN sequentially (0.15 mm rostral to bregma, 0.4 mm lateral to the midsagittal sinus, 7.75 mm below the pial surface; bite bar set at 3 mm below the ear bars) using a Hamilton microsyringe (32 gauge blunt needle; Hamilton Company, Reno, USA). The syringe was lowered and kept in position for 5 min prior to injection, which was then delivered at a rate of 10 nl per minute over a ten minute period. To reduce diffusion along the pipette track, the syringe was left in place for an additional 15 min prior to removal. One series of sections through the region of the MPN was counterstained with thionin to examine needle track location and neuron loss. Two additional adjacent series were processed to determine patterns of gliosis and androgen receptor staining. As  139  described in greater detail in the Results, tissue and blood samples from animals bearing improper or ineffective MPN lesions were removed from the analysis. 3.2.3  Tissue and blood collection Animals were weighed daily and allowed to recover for 14 days prior to restraint  exposure, which involved placing rats into flat bottom Plexiglas restrainers (8.5 x 21.5 cm; Kent Scientific, Litchfield, CT) for 30 min and then returning them to their home cage for an additional 30 min period. Blood samples obtained from the tail vein were collected into ice-chilled Aprotinin-EDTA-treated tubes (3.75 mg EDTA/100 uL of blood), centrifuged at 3000 g for 20 min, and stored at -20°C until assayed. Blood samples were obtained immediately following removal from the home cage (0 min), at the end of restraint (30 min), and 30 min following the termination of restraint (60 min). All testing was performed during the light phase of the cycle, beginning at 0800 hrs. Rats were anesthetized for perfusion after a tail blood sample was taken, either following home cage removal or 60 min following the onset of restraint. Based on our previous time course studies, the 30 min poststress interval is optimal for detecting, within individual animals, both the relative differences in stress-induced indices of HP A function and intervening levels of Fos in PVN attributable to differences in gonadal status (Viau et al., 2003). As verified by corneal and pedal pinch reflexes, deep anesthesia was reliably achieved within 1-2 min of chloral hydrate administration (40% w/v in dt^O; 1 ml per 100 g body weight; intraperitoneal). Rats were perfused via the ascending aorta with 0.9% saline, followed by 4% paraformaldehyde (pH 9.5), both at 4°C. Saline and fixative were delivered over 5 and 20 min, respectively, at a flow rate of 20 ml/min. Brains were post-fixed for 4 hours and cryoprotected overnight with 15% sucrose in 0.1 M potassium PBS (KPBS; pH 7.3) before slicing. Five l-in-5 series of frozen 30 um-thick coronal sections through the length of the brain were collected and stored in antifreeze (30%  ethylene glycol and 20% glycerol in sterile DEPC-treated water) at -20°C until processing. Adjacent series of tissues from each animal were used for in situ hybridization- and immunohistochemical analyses. In all cases, an additional series was counterstained with thionin and alternately compared with dark- and bright-field illuminations for morphological and anatomical reference. 3.2.4  Immunoassays and hybridization histochemistry  Radioimmunoassays. Plasma testosterone (25 ul), corticosterone (5 ul, diluted 1:200 as per kit instructions), and ACTH (50 ul) concentrations were measured using commercial RIA kits (MP Biomedical; Solon, OH). For corticosterone, the plasma samples were diluted 1:100 and 1:200 for pre-stress and post-stress time intervals, respectively, to render hormone detection within the linear part of the standard curve. The intra and interassay coefficients of variation for all of the assays typically ranged from 3 - 6 and 10 - 12%, respectively, and 125I-labeled ligands were used as tracer in all cases. The standard curve ED-50 for the corticosterone RIA was 17 ug/dl, with a detection limit of 0.625 ug/dl. The ACTH antibody cross-reacts 100% with ACTHi_39 and ACTH] 24, but not with P-endorphin, a- and P-MSH, and a- and P-lipotropin (all <0.8%). The standard curve ED-50 for the ACTH RIA was 82 pg/ml, with a detection limit of 20 pg/ml.  Immunohistochemistry. Lesion placement in the MPN was determined by analyzing patterns of Nissl staining, glial cell infiltration (glial fibrillary acidic protein, GFAP), and androgen receptor staining within adjacent tissue series. Glial cell infiltration was identified using a primary antiserum purified from bovine glial fibrillary acidic protein (AB5804, Lot number 0506002852; Millipore, Billerica, MA; 1:2k). Androgen receptor (AR) immunoreactive neurons were localized using a primary antiserum (0.025ug/ml; 1:8k) raised against the N-terminal amino acids 2-20 of the androgen receptor (sc-816, Lot number El004; Santa Cruz Biotechnology, Santa Cruz, CA).  Restraint-responsive neurons in the PVN were localized using Fos immunoreactivity as a marker of cellular activation using a primary antiserum (1:45k) raised against amino acids 4-17 of the human Fos protein (Ab-5, Lot number 4191-1-1; Oncogene Research Products, Boston, MA). Free-floating sections were first rinsed in KPBS buffer to remove cryoprotectant, and then pre-treated with 0.3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. This was followed by four rinses in KPBS, and then in sodium borohydride (1% w/v in KPBS) for 5 min to reduce free aldehydes. Sections were then incubated for 48 hours at 4°C in a KPBSTriton (0.3% Triton-X; Sigma-Aldrich, Oakville, Ont.) solution containing 2% normal goat serum and the primary antiserum to detect AR or Fos, as described above. AR and Fos primary antisera were detected using a conventional nickel-intensified, avidin-biotin-immunoperoxidase (Vectastain Elite ABC kit; Vector laboratories, Burlington, CA) procedure (24). GFAP was detected by using a non-nickel variant of the procedure, as previously described (25). Control experiments, in which the primary antisera to AR or Fos were preadsorbed for 24 hours at 4°C with excess synthetic peptide immunogen, corresponding to N-terminal amino acids 2-21 of the rat androgen receptor (0.25 uM, sc-816-P, EVQLGLGRVYPRPPSKTYRG, Santa Cruz Biotechnology, Santa Cruz, CA) or to amino acids 4-17 of the human c-fos (50 uM, PP10, SGFNADYEASSSRC, Oncogene Research Products, Boston, MA), failed to yield any evidence of specific AR or Fos staining. Additional control experiments involving the omission of either primary or secondary antibody yielded no specific labeling. Discrete localization of Fos-ir profiles to the medial dorsal parvocellular (mpd; neurosecretory, anterior pituitary-regulating), and to the dorsal and medial ventral parvocellular (dp and mpv, respectively; nonneurosecretory, autonomic regulating) populations of the PVN was assisted by redirected sampling of an adjacent series of thionin-stained sections. Total cell number estimates of Fos positive cells were generated by counting bilaterally the number of Fospositive cells through each region of interest, averaged by dividing the total number of cell counts 142  by slice number, corrected for double counting errors (Guillery, 2002), and multiplying this product by a factor of five to account for slice frequency (one in five sections). Characterization of CRH and AVP staining in the median eminence was performed using a dual immunohistochemical labeling technique, including a rabbit polyclonal antibody against CRH (T-4037, Lot number 970177-1; Bachem, Torrance, CA; 1: 2k) and a guinea pig antibody against AVP (T-5048, Lot number 061305; Bachem, Torrance, CA, 1: 25k). Free-floating tissue were prepared as described above, with slight modifications of these procedures to optimize double labeling for CRH- and AVP-ir, including 1) the elimination of hydrogen peroxide pretreatment, 2) using bovine serum albumin as a blocking agent, and 3) incubating tissue sections in primary antisera for 24 hours at 4°C. Primary antisera against CRH and AVP were detected using conjugated anti-rabbit IgG (Alexa 594, Invitrogen; 1:500) and anti-guinea-pig IgG (Alexa 488, Invitrogen; 1:500) fluorescent secondary antibodies, respectively. Concurrent immunofluorescence detection of CRH- and AVP-ir in the median eminence was achieved under appropriate fluorescence wavelength conditions, using a Texas red filter (Leica TX2 no. 513843) to detect Alexa 594 under 480 ± 40 nm excitation and 527 ± 30 emission, and a fluorescein isothiocyanate filter (Leica L5 no. 513840) to detect Alexa 488 under 560 ± 40 nm excitation and 645 ± 75 emission wavelengths. Control experiments in which the primary antisera to CRH and AVP were preadsorbed with excess levels of their respective immunogens failed to yield any evidence of specific staining. Furthermore, experiments involving the cross adsorption of excess amounts of CRH and AVP, in addition to the omission of either primary or secondary antibody, provided no evidence of cross-reactivity of the CRH primary to detect AVP nor the AVP primary antibody to detect CRH. A Leica 40X HCL PL Fluotar objective was used to quantify CRH- and AVPimmunoreactivity localized to the external lamina of the median eminence, the anterior pituitarydirected part of the structure. Optical images from ten regularly spaced (150 um) sections  through the median eminence were acquired and binarized using constant acquisition and threshold parameters. Binarized images were further skeletonized, and the total average density of pixels was recorded as a measure of staining intensity. Parvocellular PVN neurosecretory neurons are acknowledged as the principal source of CRH-ir terminals in the external zone of the median eminence (Zimmerman et al., 1977; Lennard et al., 1993). Determination of AVP staining within terminals specific to this PVN cell population was achieved by redirected sampling of CRH and AVP staining, and by quantifying only those AVP profiles superimposed by CRH positive nerve terminals.  Hybridization histochemistry. A hybridization approach was used under basal conditions to identify how testosterone acts independently or interacts with the MPN on the relative levels of CRH and AVP mRNA in the central and medial nucleus of the amygdala, respectively. CRH and AVP mRNA hybridization histochemistry were carried out using [33P] UTP-labeled (GE Healthcare Bio-Sciences, Baie d'Urfe, Canada) antisense cRNA probes transcribed from a full length (1.2kb) cDNA encoding the rat CRH gene and a 230-bp fragment from the 3' end of exon C encoding the rat vasopressin gene. Techniques for riboprobe synthesis, hybridization, and the patterns of hybridization for these probes in the amygdala are described in greater detail elsewhere (Simmons et al., 1989; Viau et al., 2001). Based on the strength of the hybridization signal on x-ray film (B-max, Amersham) the hybridized slides were then coated with Kodak NTB2 liquid autoradiographic emulsion and exposed at 4°C in the dark with desiccant. Exposure time to emulsion was optimized to ensure that mRNA levels detected were within the linear range of the assay and could be quantified by making relative comparisons in OD levels (12 d for CRH mRNA in the central amygdala; 24 d for AVP mRNA in the medial amygdala). Using a standard reference frame, average OD values were determined bilaterally on 6 and 4 regularly spaced (150 \xm) intervals through the central and medial amygdala, respectively, and corrected by  background subtraction. Hybridized tissue series were aligned using white matter morphology illuminated under darkfield conditions and the cytoarchitectonic features provided by an adjacent series of Nissl stained material. 3.2.5  Imaging and statistics  Imaging. Parceling of the rat brain followed the mapping of Fos immunoreactivity in the PVN, and CRH and AVP mRNA in the amygdala as defined by the morphological features provided by thionin staining of adjacent series of tissue, based on the terminology of Swanson (Swanson, 2004), and of Swanson and Kuypers (Swanson and Kuypers, 1980), Swanson and Simmons (Swanson and Simmons, 1989), and Viau and Sawchenko (Viau and Sawchenko, 2002) to describe the PVN, of Dong and Swanson (Dong and Swanson, 2004) to describe the BST, and of Swanson and colleagues (Canteras et al., 1995; Dong et al., 2001) to describe the central and medial amygdala. Features and terminology to describe the MPN were based on Simerly and Swanson (Simerly et al., 1986; Simerly and Swanson, 1986; 1988). Light- and dark-level images were captured using a Retiga 1300 CCD digital camera (Q-imaging, Buraaby, BC), analyzed using Macintosh OS X-driven, Open Lab Image Improvision v. 3.0.9 (Quorum Technologies, Guelph, ON) and ImageJ v. 1.38 software (NIH, Bethesda, MD), and exported to Adobe Photoshop (v. 10.0, San Jose, CA), where standard methods were used to adjust contrast and brightness, and final assembly at a resolution of 300 dpi.  Statistics. Data are expressed as the mean ± SEM and were analyzed by using a two- and threeway ANOVA to detect testosterone replacement and MPN lesion effects on Fos under stress conditions, as well as CRH- and AVP-based data under basal conditions. ACTH and corticosterone were analyzed by ANOVA using one repeated measure (time of sample). Significant ANOVAs were followed using Neuman-Keuls post hoc test. Immuno- and in situ  hybridization-histochemical and hormone comparisons were made observer-blind by assigning coded designations to the data sets in advance. 3.3  Results  3.3.1  Medial preoptic nucleus lesions Our pilot studies indicated that bilateral lesions that were centered within, but biased  towards the caudal part of the nucleus, were most reliable in terms of inducing elevated plasma ACTH responses to stress. Pilot studies employing a unilateral lesion approach in gonadal intact animals demonstrated a complete loss of androgen receptor staining in the ipsilateral, but not in the contralateral MPN. Furthermore, beyond sparing the amount of damage to fibers of passage (Dayas et al., 1999), the volume and concentration of ibotenic acid used was effective in producing lesions that were histologically distinct and consistently uniform in the animals chosen for analysis. The extent of the excitotoxin lesions was reliably demarcated by examining local patterns of GFAP induction and loss of androgen receptor staining (Figures 3-1 and 3-2). The caudal part of the MPN is conspicuously composed of magnocellular neurons (Simerly et al., 1986; Simerly and Swanson, 1986; 1988), and shows a high density of androgen receptor staining that almost completely mirrors its contours. Compared to shams, a lesion was deemed effective in animals showing a loss of androgen receptor staining and magnocellular material, as well as glial infiltration that was centered within the caudal part of the MPN. In general, cellular damage was restricted to the MPN; however, slight damage was occasionally observed within cells occupying the neighboring medial preoptic area and ventral portions of the posterior division of the bed nucleus of the stria terminalis (BST), including the interfascicular nucleus (see Figure 3-1). While we cannot rule out completely the possible influence of these regions, the interfascicular nucleus has little direct input to the PVN (Dong and Swanson, 2004). Furthermore, the principal  Figure 3-1. Histological identification of ibotenic lesions in the medial preoptic nucleus (MPN). Representative adjacent bright- and darkfield photomicrographs to show androgen receptor (A, B) and glial fibrillary acidic protein staining (C, D) through a comparable level of the MPN in animals bearing sham (A, C) and MPN lesions (B, D). Compared to shams (A, C), ibotenic lesions (B-D) caused a marked reduction in androgen receptors and glial infiltration in the area occupied by the caudal part of the nucleus. Structures labeled for reference: fx, fornix; GPe, globus pallidus, external segment; pBST, bed nucleus of the stria terminalis, posterior division. Scale bar = 500 um (applies to all)  Figure 3-2. Schematic representations to describe cell damage through the rostrocaudal extent (A-D) of the medial preoptic nucleus, assisted by cell morphology, androgen receptor, and glial infliltration patterns within adjacent tissue series. The red line describes the nuclear boundaries of the MPN. The areas colored in light- and dark blue describe the full extent and common regions of damage, respectively, in animals showing proper ibotenic lesions. AP values describe the relative distance from bregma (mm) for each slice interval (atlas plates based on Swanson, 2004).  nucleus, clearly spared in all of the animals examined, appears to be the major subnucleus of the posterior BST responsible for regulating the HPA axis (Choi et al., 2007; Choi et al., 2008). Rats showing unilateral or non-uniform bilateral lesions were removed from subsequent analysis. Furthermore, animals bearing lesions that were focused beyond the intended caudal part of the MPN or showing damage that extended well into the lateral preoptic and anterior hypothalamic areas were also excluded. Peptide immunoreactivity, mRNA, and stress hormone data were analyzed in only those animals showing proper lesions in the MPN, as independently verified by an observer who was blind to the experimental design. Based on the exclusion criteria described, a final 'n' of 6 was achieved for each of the four testosterone replacement-MPN treatment combinations.  3.3.2  Body weights There was a main effect of lesion [F(l,44) = 4.3; P < 0.05] and a significant lesion x time  interaction [F(2,88) = 15.0; P < 0.01] on body weight gain, but no effect of testosterone replacement. Differences in body weight gain during the first post-surgical week contributed to this interaction. During this interval there was a significant effect of lesion [F(l,44) = 19.6; P<0.01], as shams showed significantly higher body weight gains (P < 0.05) than did MPN-lesion animals, 4.6 ± 0.5 and 0.8 ± 0.6 g/day, respectively. However, there was no main effect of testosterone (P = 0.83) and no lesion x testosterone interaction (P = 0.98) on body weight gain during the first post-surgical week. By the second post-surgical week immediately prior to stress testing, body weight gains were comparable between sham and MPN-lesion animals, 6.1 ± 0.4 and 6.8 ± 0.5 g/day, respectively. Analysis of absolute body weights on the final day of testing indicated a main effect of testosterone replacement [F(l,44) = 5.5; P < 0.05], but no significant effect of lesion or a significant testosterone x lesion interaction. Low testosterone replaced animals showed higher body weights than their high testosterone replaced counterparts: 370.5 ±  4.4 and 355.5 ± 5.2 g, respectively. Taken together, these findings indicate that the destruction of the MPN did not contribute to changes in growth by the time of testing, whereas testosterone contributed to differences in body weight. 3.3.3  Testosterone replacement and HPA hormones In gonadectomized, low- and high-testosterone replaced rats, plasma testosterone  concentrations were 0.48 ± 0.07 and 2.58 ± 0.08 ng/ml, respectively, validating the reliability of our testosterone replacement regimen. Analysis of plasma ACTH revealed significant main effects of lesion [F(l,20) = 9.3; P < 0.01], testosterone [F(l,20) = 27; P < 0.01], and restraint [F(2,40) = 216; P < 0.01]. Lesion x testosterone [F(l,20) = 5.7; P < 0.05] and lesion x testosterone x restraint [F(2,20) = 6.5; P < 0.05] interactions were both significant. Post hoc analysis revealed no differences in pre-stress levels of ACTH. Compared to shams, rats with MPN lesions showed higher levels of ACTH under stress conditions, at both 30 and 60 min of restraint exposure (Figure 3-3 A). Analysis of plasma corticosterone revealed significant main effects of lesion [F(l,20) = 9.3; P < 0.01] and restraint [F(2,40) = 233; P < 0.01]. There was no main effect of testosterone and no significant lesion x testosterone interaction. Thus, in contrast to the ACTH response within shams, there was no apparent inhibitory effect of testosterone on corticosterone levels at 30 and 60 min of restraint. However, both lesion x restraint [F(2,40) = 6.2; P < 0.01] and lesion x testosterone x restraint [F(2,40) = 3.6; P < 0.05] interactions were significant, signifying a capacity for the MPN lesions to influence corticosterone. Indeed, post hoc analysis confirmed a stimulatory effect of MPN lesions on plasma corticosterone levels immediately prior to and during restraint, regardless of testosterone replacement (Figure 3-3B).  150  A  I I Low T, Sham ^m High T, Sham ^ Low T, IBO High T, IBO  0 min  30min  60 min  Figure 3-3. Mean ± SEM plasma ACTH (A) and corticosterone (B) concentrations prior to (0 min), and 30 and 60 min following an acute 30 min episode of restraint in gonadectomized, low- and high testosterone replaced rats bearing sham- and ibotenic (IBO) lesions in the medial preoptic nucleus (n = 6 per group). *p<0.05 versus low-T sham and both low and high testosterone replaced, MPN lesion animals (A). *P<0.05 indicates a main effect of MPN lesions on corticosterone at 30 min of restraint, regardless of testosterone replacement (B). Likewise, a main effect of MPN lesions was detected on corticosterone at 0 min of restraint (0.42 ± 0.06 and 0.95 ±0.18 jxg/dl in sham and lesion animals, respectively, P<0.05).  151  3.3.4  Parvicellular PVN Fos-ir Quantitative assessment of the number of Fos positive cells within regions of the  parvicellular division of the PVN (Figures 3-4 and 3-5) revealed significant main effects of lesion [F(l,40) = 6.2; P < 0.05], testosterone replacement [F(l,40) = 10.7; P < 0.01], and restraint [F(l,40) = 1763; P < 0.01], as well as a significant lesion x testosterone x restraint x region [F(2,80) = 9.4; P < 0.01] interaction. There were no main effects of lesion or testosterone under basal conditions, both between and within the medial parvocellular regions analyzed. As a function of stress, there was no effect of restraint on the numbers of cells recruited to express Fos protein in the dorsal parvocellular (dp) part of the PVN [F(l,40) = 2.4; P = 0.1] (Figure 3-5A). Whereas both the medial dorsal (mpd) [F(l,40) = 1550; P < 0.01] and medial ventral parvocellular (mpv) [F(l,40) = 493; P < 0.01] parts showed elevations in stress-induced Fos-ir (Figures 3-5 A and 3-5C). For the mpv part of the PVN, there was a significant lesion x testosterone x restraint interaction [F(l,40) = 8.9; P < 0.01]. Sham, high-testosterone replaced animals contributed to this interaction, showing the highest levels of Fos cell numbers under stress conditions (Figure 3-5C). Significant effects of lesion [F(l,40) = 9.8; P < 0.01] and testosterone [F(l,40) = 16; P < 0.01] and a significant lesion x testosterone interaction [F(l,40) = 8.0; P < 0.01] were revealed in the mpd part of the PVN. Post hoc analysis confirmed that the inhibitory effect of testosterone on the number of Fos-ir cells in the mpd occurred in sham, but not in MPN lesion animals (Figure 3-5B).  3.3.5  Median eminence CRH- and AVP-ir Our previous findings indicated that the inhibitory effect of testosterone on stress-induced  ACTH release is associated with changes in AVP, but not CRH content in the median eminence (Viau and Meaney, 1996). Content measures of median eminence AVP provide, for the most  Figure 3-4. MPN lesions and testosterone interact on the induction of Fos expression in the medial parvocellular, dorsal part of the PVN provoked by acute restraint. Brightfield photomicrographs through a comparable level of the PVN in sham lesion rats bearing lowand high-testosterone replacement (A and B, respectively) at 30 min following restraint exposure. The spatial pattern of Fos induction schematized (C) was cytoarchitectonically defined by redirected sampling of Nissl staining patterns aligned to adjacent corresponding brightfield images. Note that the other parvicellular autonomic-related subdivisions (dp, mpv) are also responsive to stress, and the relative increment in mpv Fos displayed in high testosterone replaced animals (see Figure 3-5). Mean ± SEM number of Fos-ir neurons in the medial parvocellular, dorsal (mpd) part of the PVN (D) to illustrate the interaction between lesions and testosterone on Fos induction. **P<0.01 vs low testosterone sham and MPN lesion animals (n = 6 per group). Structures labeled for reference: dp, dorsal parvocellular; mpd and mpv, medial parvocellular, dorsal and ventral parts; and pm, posterior magnocellular part of the PVN. Scale bar = 150 um (applies to A-C).  PVN-dp  o  Low T High T Sham  B  1000n  Low T  High T IBO  PVN-mpd  750  o 500250  Low T High T Sham 200  w  "55 O  Low T  High T IBO  PVN-mpv  150100 50-  Low T High T Sham  Low T  High T IBO  Figure 3-5. Relative strength of Fos induction in the autonomic-related, dorsal parvocellular (A) and medioventral (B) parvicellular subdivisions of the PVN. Mean ± SEM number of Fos-ir neurons 30 min following restraint exposure in gonadectomized, low- and high testosterone-replaced rats bearing sham- and ibotenic (IBO) lesions in the medial preoptic nucleus. *P<0.05 vs low testosterone sham and MPN lesion animals (n = 6 per group).  154  part, an index of magnocellular activity, and both magnocellular and parvocellular neurosecretory neurons contribute to AVP in pituitary portal plasma (Antoni, 1993). To provide a more precise index of parvocellular activity, in the current study we used a dual immunohistochemical approach to detect CRH staining in the external zone of the median eminence and to assess the relative levels of AVP contained by these CRH terminals of medial parvocellular origin (Figure 36). For CRH-ir, there were no main effects of lesion and testosterone. The lesion x testosterone interaction approached significance [F(l,20) = 3.8; P = 0.06], as reflected by a tendency for MPN lesion rats to show lower levels of CRH staining under high testosterone replacement (Figure 37A). Analysis of AVP staining revealed significant effects of lesion [F(l,20) = 6.2; P < 0.05], testosterone [F(l,20) = 10.7; P < 0.01], and a significant lesion x testosterone interaction [F(l,20) = 10.4; P < 0.01]. As revealed by post hoc analysis, the lesion x testosterone interaction was based within the high testosterone replaced group. In high testosterone rats, AVP staining was significantly higher in sham compared to MPN lesion animals (Figure 3-7B). In low testosterone rats, AVP staining was comparable between rats bearing sham and MPN lesions (P = 0.15). 3.3.6  Amygdala CRH and AVP mRNA Densitometric analyses of CRH mRNA through the extent of the central nucleus of the  amygdala under basal conditions (Figure 3-8) indicated a significant effect of testosterone [F(l,20) = 4.6; P < 0.05], but revealed no significant effect of lesion and no significant lesion x testosterone interaction. The effect of testosterone was attributed to an overall inhibitory effect of high testosterone replacement on CRH expression in both sham and MPN lesion groups (Figure 3-8D). Analysis of AVP mRNA in the anterodorsal part of the medial amygdala (Figure 3-9) revealed significant effects of lesion [F(l,20) = 15.1; P < 0.01] and testosterone [F(l,20) = 62; P < 0.01], and a significant lesion x testosterone interaction [F(l,20) = 16.8; P < 0.01]. Post hoc  Figure 3-6. Anatomical and immunohistochemical characterization of CRH and AVP staining in the median eminence of a control rat to illustrate a method for quantifying peptide staining intensity within nerve terminals directed at the anterior pituitary. Photomicrographs to show that CRH distribution is unique to the external zone of the median eminence (A), whereas AVP staining occurs within both the internal lamina and the external zone of the structure (B). A concurrent double immunofluorescent detection method shows a strong superimposition of AVP staining within CRH terminals of the external zone (C). Assessment of the relative levels of AVP contained by these terminals of medial parvocellular origin was achieved by redirected sampling of the pattern of AVP juxtaposed to the profile of CRH staining in the external zone. The product of this sampling and subtraction procedure is shown in panel D. Structures labeled for reference: zi, internal zone; ze, external zone. Scale bar = 75 urn (applies to all).  A  5000-,  & 400005  c •o  30002000  x o  1000 Sham  B f  IBO  400-, • LowT ^HighT  300  c  55 200 J2  D.  100-  Sham  IBO  Figure 3-7. MPN lesions and testosterone interact on medial parvocellular AVP terminal fibers in the median eminence under basal conditions. Mean ± SEM terminal fiber staining densities of CRH (A) and AVP (B) within the external zone of the median eminence in gonadectomized, low- and high testosterone replaced rats bearing sham- and ibotenic (IBO) lesions in the medial preoptic nucleus. *P<0.05 versus sham low testosterone replaced counterpart (n = 6 per group).  D  30  c Q  20-  HLowT IHighT  JL  JL  O < Z  a: E x a: o  10-  Sham  IBO  Figure 3-8. Hybridization histochemical localization of CRH mRNA in the central nucleus of the amygdala (CeA). Representative darkfield photomicrographs of coronal sections through a comparable level of the CeA to show the relative strength of hybridization signal of sham animals that were GDX and replaced with low (A) or high (C) testosterone, and of MPN lesion animals with high testosterone replacement (B). Structure labeled for reference: ot, optic tract. Scale bar = 250 um (applies to A-C). Mean ± SEM relative levels of CeA mRNA to show a main effect of testosterone, but no MPN by testosterone interaction (D). *P<0.05 vs low testosterone replaced counterpart (n = 6 per group).  158  D  20 CZlLowT MHighT  •2  c Q  O < E Q.  3 Sham  IBO  Figure 3-9. Hybridization histochemical localization of AVP mRNA in the medial nucleus of the amygdala (MeA). Representative darkfield photomicrographs of coronal sections through a comparable level of the MeAto show the relative strength of hybridization signal of sham animals that were GDX and replaced with low (A) or high (C) testosterone, and of MPN lesion animals with high testosterone replacement (B). Structure labeled for reference: ot, optic tract. Scale bar = 250 um (applies to A-C). Mean ± SEM relative levels of AVP mRNA to show a MPN x testosterone interaction (D). *P<0.05 vs low testosterone replaced; tP<0.05 vs high testosterone replaced counterpart (n = 6 per group).  159  analysis revealed that the AVP response to high testosterone replacement was significantly higher in shams compared to MPN lesion animals (Figure 3-9D). 3.4  Discussion Our previous experiments showed that lesioning a large extent of the medial preoptic area  blocked the inhibitory effects of a single dose of testosterone replacement on HPA function (Viau and Meaney, 1996). In the current study we used four treatment groups, encompassing two background levels of testosterone replacement in GDX adult male rats bearing sham and MPN lesions specifically. Thus, our current design allowed us to make new inroads on how central and peripheral components of the HPA axis responds to differences in circulating testosterone levels and whether the MPN is required for the HPA axis to register these differences in testosterone. The data implicate testosterone sensitive pathways from the MPN in mediating both the activatonal response to stress and biosynthetic capacity of PVN neurosecretory neurons. Testosterone exerted a dose-related inhibitory effect on restraint-induced levels of Fos within the dorsal medial parvocellular (mpd) part of the PVN as well as ACTH levels in plasma. Within the high testosterone replacement group, rats bearing MPN lesions showed higher levels of stress induced Fos in the mpdPVN and plasma ACTH compared to shams. These findings suggest that testosterone inhibition of HPA effector neurons in the PVN is mediated by androgen receptors located outside the nucleus and that the MPN is required for this mechanism to occur. The extent to which the lesions reflect the removal of testosterone actions that normally occur within or distal to the MPN cannot be ascertained at this point. However, microimplants of the androgen receptor antagonist hydroxyflutamide into the MPN can increase the ACTH response to restraint (Williamson and Viau, 2007b). MPN lesioned animals showed significantly higher levels of corticosterone both under basal and stress conditions than did sham-lesioned rats (Figure 3-3B). However, we found no 160  interaction between lesions and testosterone. This was obviously a consequence of the sham group of animals, showing only a slight testosterone dependent decrement in corticosterone levels at 30 min of restraint exposure. This departure between ACTH and corticosterone disagrees with our previous experiments showing negative relations between testosterone and stress-induced ACTH and corticosterone in animals with testes (Viau and Meaney, 1996; Viau et al., 2003). Our previous GDX + testosterone experiments also showed that stress induced levels of ACTH and corticosterone vary strongly and negatively with testosterone (Viau and Meaney, 1996), in animals replaced with testosterone levels that encompassed the entire range of naturally occurring differences in plasma testosterone (~ 0.2 to 7 ng/ml). These findings could explain why we were unable to detect an inhibitory effect on corticosterone using a single, high dose of testosterone replacement. One may still argue that testosterone is of limited significance to the glucocorticoid response, at least in the current study. However, because of our restricted replacement regimen, we can only interpret our data in so far as determining the relative capacity of different central and peripheral components of the HPA axis to respond to a unique level of testosterone. Our current design, nevertheless, exposed a potential autonomic involvement, perhaps at the level of the PVN, on how testosterone contributes to the net glucocorticoid response. In support of this possibility, sham animals bearing high testosterone replacement showed higher numbers of Fos-ir neurons in the medial parvocellular ventral (mpv) part of the PVN than low testosterone replaced animals under stress conditions (Figure 3-5B). As the mpv cells contribute to the long descending influences of the PVN on the preganglionic spinal cord neurons controlling the adrenal response to ACTH (reviewed in Williamson et al., 2005), this increment in restraint induced mpv Fos could account for the dissociation observed between ACTH and corticosterone release in sham animals with high testosterone. The mpv Fos response to high testosterone replacement was muted, however, in animals bearing MPN lesions, despite showing higher restraint-induced levels of corticosterone. This paradoxical finding is rescued, perhaps, when 161  considering that the magnitude of the corticosterone response to stress occurs as a function of autonomic outflow in addition to ACTH release as executed by the recruitment of the mpd PVN motor neurons (Engeland and Arnhold, 2005; Bornstein et al., 2008). The connective properties of the cellular populations in the PVN that are differentially recruited as a function of testosterone and MPN lesions have not yet been defined. Thus, the extent to which testosterone acts and interacts with the MPN on PVN mpv cells identified as projecting to the preganglionic spinal cord neurons requires further clarification. Nevertheless, it should be noted that the androgen receptor and the estrogen receptor-P isoform are uniquely distributed within autonomic-related cells of the PVN, including the dorsal, lateral, and ventral components of the medial parvocellular division (Bingham et al., 2006). Thus, the mpv PVN neurons appear as ideal candidates for mediating the actions of testosterone on autonomic function directly, whereas the influence of testosterone on hypophysiotropic function appears to be indirect. We conclude that testosterone normally acts on both the autonomic and neuroendocrine arms of the PVN to effect the net glucocorticoid response to stress, and that these actions rely on the integrity of the MPN. In response to high testosterone replacement, unstressed sham animals showed no change in CRH, but showed a substantial increase in AVP staining localized to CRH positive terminals in the external lamina of the median eminence (Figures 3-6 and 3-7). AVP is a weak ACTH secretagogue, but potently enhances the stimulatory effects of CRH on ACTH release (Antoni, 1993). Thus, the stimulatory effect of testosterone and the opposing influence of MPN lesions on AVP content in the median eminence under basal conditions would appear contradictory to effects observed on ACTH under stress conditions. It is generally conceived that resting state levels of CRH- and AVP-ir in the external zone of the median eminence reflect the capacity of the mpd neurons of the PVN to synthesize these peptides (Antoni, 1993). As several previous studies have indicated, however, the relative release patterns and contributions of CRH and AVP to the ACTH response are stressor and context specific, and cannot be inferred solely on the basis of basal 162  measures of peptide content in the median eminence alone (Antoni, 1993; Aguilera and RabadanDiehl, 2000). Our current findings indicate that testosterone requires a functioning MPN to inhibit the stress-induced activation of mpd neurons, as well as to redirect the capacity of these neurons to express AVP in favor of CRH. Taken together, this suggests that the inhibitory effect of testosterone on stress induced ACTH does not occur as a consequence of the capacity of medial dorsal parvocellular (mpd) neurons to express AVP, but may be functionally coupled to the number of mpd neurons recruited to release peptide stores. The extent to which AVP release actually contributes to testosterone regulation of the HPA axis remains to be determined, and worthy of pursuit, as AVP is thought to be the key variable imparting situation specific alterations in the magnitude of the ACTH response to stress (Antoni, 1993; Aguilera and Rabadan-Diehl, 2000). Our lesions targeted the caudal half of the MPN, which houses primarily gammaaminobutyric acid (GABA)-ergic neurons in addition to the peptide galanin (Bowers et al., 1998; Polston and Simerly, 2003). Although galanin has been implicated in the neuroendocrine regulation of reproduction and energy balance (Hohmann et al., 2003), its involvement in HPA regulation has not been approached. While a dependence of cellular activation and peptide expression in the PVN on the integrity of MPN GABA inputs have yet to be established directly, several lines of evidence support this possibility. Testosterone induces GABA activity in the MPN (Grattan and Selmanoff, 1994; Yoo et al., 2000); and several GABA-rich projections to the PVN, including the MPN, are recruited to express Fos protein and GAD mRNA during stress exposure (Bowers et al., 1998; Sarkar et al., 2007). The MPN shows strong bidirectional connections with the medial amygdala (Simerly and Swanson, 1988; Canteras et al., 1995), and AVP expression in this region is extremely sensitive to changes in circulating testosterone levels (de Vries and Panzica, 2006). Several lines of evidence suggest an involvement of extrahypothalamic AVP neurons in mediating the central actions of  testosterone on the HPA axis (Gomez et al., 2004; reviewed in Williamson et al., 2005). Further, the medial amygdala is critical for the HPA response to stressful stimuli, particularly emotional stressors, such as restraint (Feldman et al., 1994; Dayas et al., 1999; Herman et al., 2003; Ma and Morilak, 2005). Thus, we wondered whether the MPN lesions could influence the AVP response to testosterone within this region of the amygdala (Figure 3-9). As expected, sham animals displayed an increase in AVP mRNA levels in the medial nucleus of the amygdala in response to high testosterone replacement. This increment in AVP expression, however, was significantly reduced in animals bearing MPN lesions. Assessment of the relative levels of CRH mRNA in the central nucleus of the amygdala revealed no interactions between lesions and testosterone (Figure 3-8), consistent with the fact that the MPN shows no direct projections to this region of the amygdala (Simerly and Swanson, 1988). Unlike the medial amygdala, the central nucleus (CeA) appears to be less important for HPA activation in response to restraint (Feldman et al., 1994; Prewitt and Herman, 1997; Dayas et al., 1999). Thus, our findings argue against a role for the CeA in mediating the central actions of testosterone, at least in response to acute forms of psychological stressors. However, as we observed a main negative effect of testosterone on CRH expression in the CeA, variations in testosterone may differentially prepare the HPA response under more physical or systemic types of challenges (Prewitt and Herman, 1997; Dayas et al., 1999; Xuetal., 1999). Virtually all AVP cells in the medial amygdala of the rat are immunoreactive for androgen receptors (de Vries and Panzica, 2006). While this signifies a local mode of action, our current findings challenge the notion that testosterone regulates AVP neurons in the medial amygdala directly, subject to the influences of the MPN. The medial amygdala, like most limbic regions, has little or no projections to the hypophysiotropic zone of the PVN (Prewitt and Herman, 1998; Herman et al., 2003; Choi et al., 2007). The functional influences of the medial amygdala nuclei on the HPA axis, if at all mediated by AVP neurons, may instead involve potential relays in the  bed nucleus of the stria terminalis and various hypothalamic structures, including the MPN (Herman et al., 2003; Williamson et al., 2005; Choi et al., 2007). The extent to which any of these projections contain AVP, rely on testosterone, and depend on a functioning MPN, remains worthy of pursuit. AVP pathways originating from the medial amygdala have been shown to contribute to a broad, but linked array of behaviors associated with autonomic, emotional, and coping responses to stress (Liebsch et al., 1996). Taken together with our findings, the MPN stands out as an important neural substrate for harmonizing the central actions of testosterone on behavior and neuroendocrine stress responses. It is interesting that the functional effects of MPN lesions were discriminated, for the most part, in high-, but not in low-testosterone replaced rats. Circulating levels of testosterone vary as a function of age, sexual experience, social status, time of day, and in response to stress (Williamson et al., 2005). Insofar as the MPN is recruited to modulate neuroendocrine, autonomic, and behavioral responses to stress, this may very well depend, therefore, on gonadal status and situation-specific changes in testosterone secretion (Gomez et al., 2004). It is important to note, at least in the current study, that despite massive changes in central stress pathways and at the pituitary, a single dose of high testosterone did not have a large impact on the net corticosterone response. Previous studies have implicated a critical role for AVP in sustaining corticotroph responsiveness during chronic stress (Aguilera and Rabadan-Diehl, 2000). Taken together, our findings may be relevant to understanding how testosterone determines normal adaptation under repeated stress conditions, in addition to affective disease states associated with changes in adrenal function and gonadal status. Indeed, genuine gender differences in depression and anxiety (Rubinow and Schmidt, 2002) and the association of depressive illness with hypogonadism in males (Schmidt et al., 2004; Shores et al., 2004) suggest a potential role for testosterone in the predisposition of mood disorders related to HPA dysfunction. Taken together with the instability of testosterone levels in rodents and humans, and the strength to which the 165  MPN and testosterone interact on the HPA axis, the MPN may be integral to individual differences in HPA function attributed to variations in testosterone release. Our present findings underscore how testosterone can bridge several independent, yet converging influences on the PVN. The anatomical specificity by which the MPN influences the inhibitory effect of testosterone on HPA axis function still remains unsettled, given the connectivity of the MPN with several other extended circuitries of the PVN, also rich in androgen receptors (Simerly and Swanson, 1988; Williamson and Viau, 2007a). 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Ann N Y Acad Sci 297:405-419.  171  CHAPTER 4 : The medial preoptic nucleus integrates the central influences of testosterone on the paraventricular nucleus of the hypothalamus and its extended circuitries.3  4.1  Introduction The medial preoptic nucleus (MPN) has long been recognized as an important nodal point  for integrating sensory and sex steroid hormonal influences on gonadotropin secretion, as well as maternal, reproductive, social, and sexual behaviors (Simerly and Swanson, 1988; McGinnis et al., 1996; Greco et al., 1998). Emerging evidence also underscores a role for the MPN in integrating the central influences of testosterone on the hypothalamic-pituitary-adrenal (HPA) axis responses to homeostatic threat (reviewed in Williamson et al., 2006). This may be mediated in large part by a direct influence of androgen sensitive projections from the MPN to the paraventricular nucleus (PVN) of the hypothalamus, as the MPN contains the highest number of androgen receptor positive cells in the forebrain identified as projecting to the PVN region (Williamson and Viau, 2007). Threats to homeostasis initiate the recruitment of neurosecretory neurons in the PVN to secrete peptide stores from the median eminence, primarily corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). CRH and AVP synergize on the release of adrenocorticotropin hormone (ACTH) from the anterior pituitary that, in turn, stimulates the release of glucocorticoids from the adrenal gland (Antoni, 1993; Aguilera, 1994). Testosterone implants in the vicinity of the MPN inhibit the stress-induced release of ACTH and corticosterone in the rat, effectively mirroring the inhibitory response of the HPA axis to elevated testosterone levels in circulation (Viau and Meaney, 1996). Discrete MPN lesions reduce the normal effect of  3  A version of this chapter is being prepared in manuscript form. Williamson M, Viau V. 2008. The medial preoptic nucleus integrates the central influences of testosterone on the paraventricular nucleus of the hypothalamus and its extended circuitries.  high testosterone levels to inhibit the stress-induced activation of PVN neurosecretory neurons, as well as the capacity of these neurons to synthesize AVP to effect ACTH release (Williamson and Viau, 2008). While the weight of evidence suggests a direct influence of androgen sensitive MPN inputs to the PVN (Williamson and Viau, 2007), the MPN extensively projects to regions of the forebrain and brainstem that regulate a variety of behaviors and physiological mechanisms related to homeostasis (Swanson, 1976; Chiba and Murata, 1985; Simerly and Swanson, 1988). The septum and medial amygdala stand out as important relays in this regard, as both are involved with the regulation of the HPA axis and receive substantial input from the MPN (Chiba and Murata, 1985; Simerly and Swanson, 1988; Risold and Swanson, 1997). AVP pathways originating from the medial amygdala, including those projecting to the lateral septum, regulate a variety of behaviors associated with autonomic, emotional, and coping responses to stress (Liebsch et al., 1996). As testosterone and MPN lesions interact on the capacity of medial amygdala nuclei to express AVP (Williamson and Viau, 2008), the MPN might be in a position to modify multiple afferent mediators of PVN motor function. In the present study, we sought to determine the extent to which the MPN represents the seat of the central actions of testosterone on the PVN and its extended circuitries. Our strategy incorporated a unilateral steroid/antagonist implant approach to exploit the unilateral projections of the MPN, and because bilateral approaches in the MPN could influence testosterone secretion, to prevent secondary effects from occurring within additional androgen sensitive projections to the HPA axis.  173  4.2 4.2.1  Methods Animals Fifty-seven adult male Sprague-Dawley rats (Charles River, St. Constant, Canada) were  used, weighing 250 g on arrival (56 days old) and 375 g when sampled (~75 days old). Animals were pair-housed in a colony room under controlled temperature and lighting conditions (12:12hour light: dark cycle, lights on at 0600 hours) with food and water available ad libitum. Testing was performed during the light phase of the cycle, beginning at 0800 hrs. All experimental protocols were approved by the University of British Columbia Animal Care Committee (Appendix A).  4.2.2  Intracerebral microimplants Body weight matched animals were anesthetized and received stereotaxically guided,  unilateral implants of testosterone (Sigma Chemicals, Oakville, ON, Canada) or the androgen receptor (AR) antagonist hydroxyflutamide (Toronto Research Chemicals, North York, ON, Canada) using 22 ga cannula ejectors aimed to terminate 0.5 mm dorsal to the MPN (based on Lund et al., 2006). Coordinates used for MPN microimplants were 0.29 mm rostral to bregma, 0.8 mm lateral to the midline, and 7.0 mm ventral from the pia. Testosterone and hydroxyflutamide were dissolved in a heated suspension of beeswax (VWR International, Buffalo Grove, IL) to a final concentration of 0.3 M (Christensen and Gorski, 1978; Lund et al., 2006), packed to a height of approximately 400 um, and ejected using a 28 ga wire that extended 0.5 mm beyond the tip of the cannula. Control animals received stereotaxically guided implants of beeswax alone. To avoid the possibility of functional differences attributed to laterality, half of the animals received unilateral implants on either the left and right sides of the MPN.  To determine the extent of diffusion after microimplantation and coordinates for proper placement in the MPN, testosterone and hydroxyflutamide were unilaterally implanted into the MPN in a separate group of animals that were gonadectomized. As AR staining is virtually abolished by the removal of endogenous testosterone, we used the pattern and quality of AR staining induced by these compounds to provide an index of transport and delivery. To verify that the effects of the implants were not attributed to any physical disruption of the MPN, we also compared central measures of HPA function and neuropeptide expression in an additional subset of control, gonadal-intact rats that received either sham surgeries (anesthesia only) or unilateral implants of beeswax alone into the MPN. 4.2.3  Tissue and blood collection After 14 days of recovery, during which time animals were handled, blood samples were  obtained via the tail vein immediately following removal from the home cage and/or at the termination of a single exposure to stress, which involved placing rats into flat bottom Plexiglas restrainers (8.5 x 21.5 cm; Kent Scientific, Litchfield, CT) for 30 min. Based on our previous time course studies, the 30 min timepoint is reliable for simultaneously detecting within individual animals, changes in HPA activity and intervening levels of Fos activation in the PVN attributable to differences in gonadal status (Viau et al., 2003). Blood samples were collected into ice-chilled Aprotinin-EDTA-treated tubes (3.75 mg EDT A/100 ul of blood), centrifuged at 3000 g for 20 min, and stored at -20°C until assayed. Plasma testosterone (25 ul), corticosterone (5 ul), and ACTH (50 ul) concentrations were measured using commercial RIA kits (MP Biomedical; Solon, OH). For corticosterone, the plasma samples were diluted 1:100 and 1:200 for pre-stress and post-stress time intervals, respectively, to render hormone detection within the linear part of the standard curve. The intra and interassay coefficients of variation for all of the assays typically ranged from 3 - 6 and 10 -12%, respectively, and 125I-labeled ligands were used as tracer in all 175  cases. The standard curve ED-50 for the testosterone RIA was 1.2 ng/ml, with a detection limit of 0.1 ng/ml. The standard curve ED-50 for the corticosterone RIA was 17 u,g/dl, with a detection limit of 0.625 ug/dl. The standard curve ED-50 for the ACTH RIA was 82 pg/ml, with a detection limit of 20 pg/ml. Immediately following blood sampling, rats were perfused under deep chloral hydrate anesthesia (350 mg/kg, i.p.), which was reliably achieved within 1 to 2 min of drug administration. Rats were sequentially perfused via the ascending aorta with ice-cold 0.9% saline and then 4% paraformaldehyde (pH 9.5), at a flow rate of 20 ml/min and over 5 and 20 min, respectively. Brains were post-fixed for 4 hours and stored overnight with 15% sucrose in 0.1 M potassium PBS (KPBS; pH 7.3) prior to slicing. Five l-in-5 series of frozen 30 ^m-thick coronal sections through the length of the brain were collected and stored in cryoprotectant (30% ethylene glycol and 20% glycerol in sterile DEPC-treated water) at -20°C. Adjacent tissue series from each animal were used for separate immunocytochemical and in situ hybridization analyses. In all cases, an additional series was counterstained with thionin and alternately compared with darkand bright-field illuminations for morphological and anatomical reference.  4.2.4  Immunoassays and hybridization histochemistry AR immunoreactivity was localized using a primary antiserum (0.025 pig/ml; 1:8k) raised  against the N-terminal amino acids 2-20 of the AR (sc-816, Lot number E1004; Santa Cruz Biotechnology, Santa Cruz, CA). Restraint-responsive neurons were localized using Fos immunoreactivity as a marker of cellular activation using a primary antiserum (1:45k) raised against amino acids 4-17 of the human Fos protein (Ab-5, Lot number 4191-1-1; Oncogene Research Products, Boston, MA). Immunohistochemistry was performed using a conventional nickel-intensified, avidin-biotin-immunoperoxidase (Vectastain Elite ABC kit; Vector laboratories, Burlington, CA) procedure (Li and Sawchenko, 1998). Before addition of primary  antisera, free-floating sections were pre-treated with hydrogen peroxide (0.3%) to quench endogenous peroxidase activity and with sodium borohydride (1%) to reduce free aldehydes. Discrete localization of Fos-ir profiles to defined regions of the PVN was assisted by redirected sampling of an adjacent series of thionin-stained sections. Total cell number estimates of Fos-ir cells within the PVN, medial amygdala, and the lateral septum were generated by counting bilaterally the number of Fos-positive cells through each region of interest, averaged by dividing the total number of cell counts by slice number, corrected for double counting errors (Guillery, 2002), and multiplying this product by a factor of five to account for slice frequency (one in five sections).  Hybridization histochemistry. An in situ hybridization approach was employed to determine relative levels of CRH, and AVP in the PVN, amygdala, and BST under basal conditions and cfos induction in the PVN during restraint using [33P]UTP-labeled (GE Healthcare Bio-Sciences, Baie d'Urfe, Canada) antisense cRNA probes. The CRH mRNA probe was transcribed from a full-length (1.2kb) cDNA encoding the rat CRH gene [Dr. K. Mayo, Northwestern University, Evanston USA (Imaki et al., 1991)]. The AVP mRNA probe was transcribed from a 230-bp fragment of the 3' end of exon C encoding the rat vasopressin gene (Dr. D. Richter, University of Hamberg, Germany). The c-fos mRNA probe was transcribed from a full-length (2.1kb) cDNA encoding the rat c-fos gene [Dr. T. Curran, The Roche Institute (Curran and Morgan, 1985)]. Techniques for riboprobe synthesis, hybridization, and the patterns of hybridization for these probes are described in greater detail elsewhere (Simmons et al., 1989; Chan et al., 1993; Viau et al., 2001; Viau et al., 2005). Briefly, free-floating sections were first rinsed in KPBS to remove cryoprotectant and then mounted and vacuum dried on glass slides overnight. After postfixation with 10% formaldehyde for 30 min at room temperature, sections were digested in proteinase K (10 mg/ml, 37°C) for 30 min, acetylated for 10 min (2.5 mM acetic anhydride, 0.1 M  tnethanolamine, pH 8.0), rapidly dehydrated in ascending ethanol concentrations (50-100%), and then vacuum dried. Radionucleotide antisense cRNA probes were used at concentrations approximating 2.5 x 107 cpm/ml in a solution of 50% formamide, 0.3 M NaCl, 10 mM Tris (pH 8.0), ImM EDTA, 0.05% tRNA, and 10 mM dithiothreitol, IX Denhardt's solution, and 10% dextran sulfate and applied to individual slides. Slides were coverslipped and then incubated overnight at 57.5°C, after which the coverslips were removed and the sections washed three times in 4X standard saline citrate (SSC; 0.15 M NaCl, 15 mM citric acid, pH 7.0) at room temperature, treated with ribonuclease A (20 fig/ml) for 30 min at 37°C, desalted in descending SSC concentrations (2-0. IX SSC), washed in 0.1 X SSC for 30 min at 60°C, and dehydrated in ascending ethanol concentrations. Based on the strength of the hybridization signal on x-ray film (6-max, Amersham, Piscataway, USA) the hybridized slides were then coated with Kodak NTB2 liquid autoradiographic emulsion and exposed at 4°C in the dark with desiccant. Exposure time to emulsion was optimized to ensure that mRNA levels detected were within the linear range of the assay and could be quantified by making relative comparisons in OD levels. Slides were developed at 14°C with Kodak D-19 for 3.5 min, briefly rinsed in distilled water for 15 sec, fixed in Kodak fixer for 6.5 min, and then washed in running water for 45 min at room temperature. Using a standard reference frame, average OD values were determined bilaterally on regularly spaced (150 pirn) intervals through each nucleus of interest and corrected by background subtraction. Hybridized tissue series were aligned using white matter morphology illuminated under darkfield conditions and the cytoarchitectonic features provided by an adjacent series of thionin stained material. 4.2.5  Imaging and statistics Parceling of the rat brain, as defined by the morphological features provided by thionin  staining, was based on the terminology of Swanson (2004), and of Swanson and Kuypers (1980)  to describe the PVN, of Dong and Swanson (2004) to describe the anterior and posterior divisions of the bed nucleus of the stria terminalis, and of Swanson and colleagues (Canteras et al., 1995; Dong et al., 2001a) to describe the central and medial amygdala. Features and terminology to describe the MPN were based on those of Simerly and Swanson (Simerly et al., 1986; Simerly and Swanson, 1986,1988). Light- and dark-level images were captured using a Retiga 1300 CCD digital camera (Q-imaging, Burnaby, BC), analyzed using Macintosh OS X-driven, Open Lab Image Improvision v. 3.0.9 (Quorum Technologies, Guelph, ON) and Image J v. 1.38 software (NIH, Bethesda, MD), and then exported to Adobe Photoshop (v. 10.0, San Jose, CA), where standard methods were used to adjust contrast and brightness for final assembly and labeling. Immuno- and in situ hybridization-histochemical and hormone comparisons were made observerblind by assigning coded designations to the data and tissue sets in advance. ACTH and corticosterone were analyzed by two-way ANOVA (treatment, time) with repeated measures (time). Grouped data from the immunoperoxidase and hybridization histochemical analyses were compared using two-way ANOVA (treatment, side). When main effects and interactions between treatment and side were found to be significant (P < 0.05), additional comparisons were made using Neuman Keuls post hoc tests to explore effects between treatments and by Student t tests (two-tailed) a priori to assess statistical significance of side within treatments. Data were analyzed using absolute measures in all cases (Statview v 5.0, SAS Institute Inc., Cary, NC). To underscore effects lateralized to the sides of the nuclei ipsilateral to the MPN implants, Fos- and neuropeptide-based data are illustrated as the mean ± SEM percent of the contralateral (nonimplanted) side.  179  4.3 4.3.1  Results Implant placements and control studies Surgical shams were initially piloted against animals receiving unilateral control implants  (beeswax only) to determine whether physically disrupting the MPN could induce unilateral downstream effects on stress-induced c-fos mRNA in the PVN, as well as CRH and AVP mRNA in the PVN, BST, and amygdala of unstressed gonadal intact animals. Importantly, the relative levels of these indices of cellular activation and neuropeptide expression were comparable between sham and control groups. Comparison of the relative levels of these transcripts between the ipsilateral and contralateral sides of the nuclei of interest within control animals indicated no lateralized effects. Based on these results, the effects of steroid/antagonist implants within experimental animals were compared against control animals bearing beeswax implants only. The caudal part of the MPN is conspicuously composed of magnocellular neurons (Simerly et al., 1986; Simerly and Swanson, 1986, 1988), and shows a high density of AR staining that almost completely mirrors its contours (see Figure 4-1). Along its rostrocaudal axis, the highest number of AR-positive cells in the MPN that project to the PVN are biased towards the caudal part of the nucleus (Williamson and Viau, 2007). Unilateral implants of testosterone and hydroxyflutamide directed to this part of the MPN were first tested using a separate group of gonadectomized rats. Since gonadectomized rats do not display AR-ir in the absence of testosterone in circulation, the radius and pattern of AR-ir induced by these implants could be used to determine proper placement in the MPN, as well as to develop criterion for avoiding other AR-rich regions of the forebrain that project to the PVN and/or regulate the HPA axis, most problematic within the region of the posterior part of the bed nuclei of the stria terminalis neighboring the caudal extent of MPN (discussed below).  Figure 4-1. Histological assessment of androgen receptor staining to approximate the diffusion characteristics of testosteronefromwax implants directed at the medial preoptic nucleus (MPN) in gonadectomized male rats. Following unilateral implants of testosterone in the MPN, AR-ir was confined to an annulus surrounding the wax implant on ipsilateral side (A), but not on the contralateral side (B). Structures labeled for reference: fx, fornix; GPe, globus pallidus, external segment; MPN, medial preoptic nucleus; oc, optic chiasm; pBST, bed nucleus of the stria terminalis, posterior part; sm, stria medullaris. Scale bar = 400 um.  181  In general, the radius of diffusion was spherical in nature and fairly consistent between test animals, never exceeding greater than 400 ytvci from the edge of the wax implant. AR staining was confined to an annulus surrounding the wax implant on the ipsilateral side only, with no apparent induction within distal ventricular spaces (see Figures 4-1 and 4-2). Gonadectomized rats bearing proper implants within the intended caudal level of the MPN (Figure 4-1) showed induction of AR-ir in the MPN proper and surround, including parts of the medial preoptic area. Rostrally, the induction of AR extended throughout the medial parts of the nucleus, but not within the anterior part of the MPN bordering the anterior division of the BST, nor within the anterior division of the BST. At the level of the implant, AR staining in the preoptic periventricular nucleus located immediately adjacent to the MPN, but not within other periventricular structures, nor within the anterior part of the anterior hypothalamic nucleus. At the most caudal extent of the MPN, at the level of the posterior division of the bed nucleus of the stria terminalis (BST), AR-ir was occasionally induced within the medial ventral part of the interfascicular nucleus, but never as far as its dorsal and lateral parts. Importantly, AR induction did not occur within the transverse and principal nuclei of the posterior BST, regions normally concentrating AR and arginine vasopressin (de Vries et al., 1994; de Vries and Miller, 1998), and providing a strong inhibitory influence on HPA effector neurons in the PVN (Choi et al., 2007; Herman et al., 2003). By contrast, gonadectomized animals showing implants that were ^ 300 pim removed from the intended caudal part of the MPN, failed to show substantial induction of AR-ir within the MPN proper. Implants that were biased towards the caudal most part of the MPN and/or located just dorsal to the nucleus induced patterns of AR staining that encroached upon the anterior part of the PVN, as well as within the medial and posterior parts of the BST. Implants invading the ventricular space caused a massive induction of AR-ir within the anteroventral nucleus of the third ventricle, as well as within the anterior periventricular part of the PVN.  182  Figure 4-2. Representative brightfield photomicrographs to show the unilateral induction of androgen receptor (AR) immunoreactivity following implants of hydroxyflutamide (A-C) and testosterone (D-F) in gonadectomized male rats. Low magnification shows that AR is restricted to the area surrounding the hydroxyflutamide (A) and testosterone (D) implants. High magnification views of the ipsilateral side show that AR is diffusely located within the cytoplasm in response to hydroxyflutamide (B), while testosterone concentrates AR in the nucleus (E). High magnification views of the contralateral side show the absence of AR staining in animals bearing hydroxyflutamide (C) and testosterone (F) implants. Structures labeled for reference: ac, anterior commissure; oc, optic chiasm; 3V, third ventricle. Scale bar = 25 um in B, C, D, and E; 500 um in A and D.  183  On the basis of these findings in gonadectomized animals, experimental (gonadal intact) animals showing implants that either missed the MPN completely, or were not sufficiently removed from other putative regulators of the HPA axis, centered greater than 300 pm. from the intended caudal part of the MPN, or showing levels of AR-ir that suggested ventricular contamination were removed from subsequent analysis. Based on these exclusion criteria, a final 'n' of 6 was achieved for each of the treatment groups under basal and stress conditions. Additional comparisons on the ability of MPN implants to induce local effects in the posterior BST were performed by quantifying relative levels of AVP mRNA and numbers of ARir cells between animals bearing MPN and control implants. There were no significant effects for treatment and side, and no significant treatment x side interactions for either of these variables (P > 0.5 in all cases). As AR-ir and AVP mRNA levels within the region of the posterior BST are extremely sensitive to androgens (van Leeuwen et al., 1985; de Vries, 2006), these findings provide some indication that the region of the posterior BST was effectively avoided in the experimental animals chosen for analysis (refer to Figure 4-3). 4.3.2  Testosterone secretion Testosterone is capable of acting within a wide assortment of brain regions to regulate the  HPA axis. Thus, plasma testosterone was measured from blood samples taken at 0 min of restraint to rule out individual differences in gonadal status. There was no overall effect of treatment (P = 0.74) on the concentration of testosterone in plasma: hydroxyflutamide (FLU) = 2.36 ±0.31; testosterone (TEST) = 2.00 ± 0.57, and control (CTL) = 1.83 ± 0.50 ng/ml. 4.3.3  Stress hormone secretion Restraint reliably increased ACTH and corticosterone secretion (P < 0.0001), but there  were no significant effects of treatment (P ^ 0.065), and no significant interactions between treatment and time (P £ 0.08) for both ACTH and corticosterone (Figure 4-4).  Figure 4-3. Representative darkfield photomicrograph to AVP mRNA localization at the level of posterior division of the stria terminalis (pBST) (A). Mean ± SEM optical densities of AVP mRNA expression within the pBST (B) (n = 6 per group). Structures labeled for reference: fx, fornix; ic, internal capsule; oc, optic chiasm. Scale bar = 500 um.  185  Q.  O  0 min  30 min  0 min  30 min  B  I10 Of  8 °-5  Figure 4-4. Mean ± SEM plasma ACTH (A) and corticosterone (B) concentrations prior to (0 min) and 30 min following an acute episode of restraint in rats bearing hydroxyflutamide (HF), testosterone (TEST) or control (CTL) implants in the MPN (n = 6 per group).  186  It is important to mention that testosterone and corticosterone can interact on the process of glucocorticoid-mediated negative feedback regulation of the HPA axis within several brain regions (Viau et al., 1999; Viau et al., 2001), including those investigated in the current study. Therefore, our inability to detect major changes in testosterone secretion and HPA outflow as a function of MPN implants further underscores the value of using a unilateral implant approach in so far as it allowed us to determine effects related to ARs within the MPN directly, independent of other glucocorticoid- and androgen-sensitive afferent regulators of the HPA axis. 4.3.4  Implants effects on peptide expression in the PVN Densitometric analysis of relative levels of CRH mRNA in the hypophysiotropic [dorsal  medial parvicellular part (mpd)] zone of the PVN as a function of treatment status under basal conditions revealed no effect of treatment (P = 0.92) and no significant interaction between treatment and side (P = 0.63) on CRH mRNA, whose relative strength and distribution was similar across treatment and control groups (Figures 4-5 and 4-6). Material from the same experiment revealed a significant effect of treatment (P = 0.0002) and a significant interaction between treatment and side (P = 0.0004) on AVP mRNA in the PVN mpd region (Figure 4-5). The basis for the interaction occurred as a function of lateralized effects within both treatment groups, however, opposite in nature. Thus, relative to the contralateral (non-implanted) side, animals displayed significantly higher and lower levels of AVP mRNA on the side of the PVN ipsilateral to the implants of hydroxyflutamide and testosterone, respectively (Figure 4-6).  4.3.5  Implants effects on peptide expression in amygdala and BST As we have previously observed, CRH mRNA in the central amygdala increases in  response to elevations in testosterone in circulation, but does not rely on a functioning MPN for this effect to occur (Williamson and Viau, 2008). Further, testosterone can act independently of corticosterone to induce CRH expression within the anterior division of the BST (Viau et al., 187  Figure 4-5. Representative darkfield photomicrographs to show the spatial pattern of AVP (A-C) and CRH (D-F) mRNA expression in the medial dorsal parvocellular (mp) and posterior magnocellular (pm) parts of the ipsilateral PVN in animals bearing hydroxyflutamide (HF), testosterone (TEST) and control (CTL) implants in the MPN. Note the selective influences of the implants on AVP (A-C), but not on CRH (D-F), within the neuroendocrine-related subdivision of the ipsilateral PVN. Structures labeled for reference: mpd, medial parvocellular, dorsal part of the PVN; pm, posterior magnocellular part of the PVN. Scale bar = 200 um (applies to all).  188  [ZZ] Contra Ipsi  Figure 4-6. Differential and selective effects of hydroxyflutamide (HF) and testosterone (TEST) implants in the implants on neuropeptide expression within the hypophysiotropic zone of the PVN. Mean ± SEM relative optical density measures (% of contralateral values-within treatment) of AVP (A) and CRH (B) mRNA expression within the medial parvocellular part of the PVN. *P<0.05 versus the contralateral side (n = 6 per group).  189  2001), particularly within the fusiform nucleus. The MPN shows no direct projections to the central amygdala, but projects to and/or through multiple regions of the anterior BST capable of relaying limbic information to the PVN, including within the anteromedial, fusiform, and oval nuclei (Herman et al., 1994; Dong et al., 2001b; Dong and Swanson, 2006). The relative levels of CRH were determined as function of treatment status under basal conditions to confirm the independence of CRH expression in the amygdala and to test the influence of the implants within anterior BST. There was no overall effect of treatment (P = 0.81) and no significant interaction between treatment and side (P = 0.89) on CRH in the central amygdala, consistent with the lack of input from the MPN to this region of the amygdala (Simerly and Swanson, 1988). Despite the prevalence of MPN projections throughout the anterior division of the BST (Simerly and Swanson, 1988), there were no significant effects of treatment (P £ 0.68 in all cases) and interactions (P £ 0.69 in all cases) for CRH within anterior BST. AVP expression in the medial amygdala depends on testosterone in circulation (de Vries et al., 1994). However, the AVP response in the medial amygdala to elevations in plasma testosterone levels is markedly reduced in animals bearing discrete bilateral lesions of the MPN (Williamson and Viau, 2008). Thus, the relative levels of AVP mRNA expression through the rostrocaudal extent of the medial amygdala were determined as function of treatment status under basal conditions to test an indirect influence of ARs in the MPN. There was no significant effect of treatment (P = 0.61), but a significant interaction between treatment and side (P = 0.0074). The basis for the interaction occurred as a function of lateralized effects within both treatment groups, however, opposite in nature (Figure 4-7). Relative to the contralateral (non-implanted) side, animals displayed significantly lower and higher levels of AVP mRNA on the side of medial amygdala ipsilateral to the implants of hydroxyflutamide and testosterone, respectively (Figure 48).  190  Figure 4-7. Hybridization histochemical localization of AVP mRNA in the medial nucleus of the amygdala (MeA). Darkfield photomicrographs of coronal sections through a comparable level of the MeA to show the relative strength of hybridization signal in rats bearing hydroxyflutamide (A), testosterone (B) or control (C) implants in the MPN under basal conditions. Structures labeled for reference: ot, optic tract. Scale bar = 250 um (applies to all).  191  •sr20(h  I  I Contra Ipsi  Figure 4-8. Differential effects of hydroxyflutamide (HF) and testosterone (TEST) implants in the MPN on AVP expression in the medial nucleus of the amygdala (MeA). Mean ± SEM relative optical density measures of AVP mRNA through the rostrocaudal extent of the MeA. *P<0.05 versus the contralateral side (n = 6 per group).  192  4.3.6  Implants effects on PVN activational responses to acute restraint stress We initially surveyed the effects of MPN implants on c-fos mRNA levels as a marker of  neuronal activation in the PVN under basal conditions and at the termination of 30 min restraint using the same animals sampled for stress hormones. The 30 min timepoint is not suitable for detecting c-fos induction within the lateral septum and medial amygdala in response to restraint (Pace et al., 2005), but represents an interval that is optimal for detecting in this paradigm testosterone- and MPN-dependent changes in c-fos mRNA and protein induction within PVN motor neurons (Viau et al., 2003; Williamson and Viau, 2008).  Paraventricular nucleus of the hypothalamus. Rats that did not experience restraint displayed minimal levels of c-fos mRNA in the PVN, however, acute restraint caused a marked increased in c-fos mRNA levels within the mpd region (Figure 4-9). Comparison of c-fos induction as a function of treatment status revealed significant effects of treatment (P = 0.0012) and side (P = 0.0384), and a significant interaction between treatment and side (P = 0.0043). The basis for these effects occurred as a consequence of lower levels of c-fos mRNA within the testosterone treatment group only, focused on the side of the PVN ipsilateral to the side of the testosterone implant (Figure 4-9). Based on the foregoing analyses and our previous findings showing that testosterone and MPN lesions can interact on the number of Fos-ir neurons in PVN (Williamson and Viau, 2008), we then extended our survey to the number of cells recruited to express Fos protein within different sub-regions of the PVN using an adjacent series of tissue from the same animals. Comparisons of Fos cell counts obtained at the termination of 30 min of restraint revealed no significant effects of treatment and side and no significant interactions between treatment and side within the posterior magnocellular (pm) part of the PVN related to posterior pituitary function, nor within the posterior magnocellular (pm) part of the PVN related to  Figure 4-9. Darkfield photomicrographs through the caudal part of the PVN to show c-fos expression under basal conditions (A) and at 30 min of restraint exposure (B). Note that the expression of c-fos is dampened on the ipsilateral side compared to the contralateral side following a unilateral implant of testosterone (refer to panel B) in the MPN. Mean ± SEM relative optical density measures of c-fos mRNA expression (C) within the parvocellular part of the PVN in rats bearing hydroxyflutamide (HF), testosterone (TEST) or control (CTL) implants. *P<0.05 versus the contralateral side (n = 6 per group). Scale bar =150 um (A and B).  posterior pituitary function, nor within the dorsal parvocellular (dp) and medial ventral parvocellular (mpv) autonomic-related parts (P £ 0.34 in all cases). However, significant effects of treatment (P < 0.05) and side (P < 0.01), and a significant interaction between treatment x side (P < 0.05) were revealed within the mpd region. Identical to the effects seen for c-fos mRNA induction, the interaction was once again based within the testosterone treatment group only, in which animals showed a lower number of mpd cells recruited to express Fos protein on the side of the PVN ipsilateral to the testosterone implant (Figure 4-10).  Medial amygdala. On the basis of previous connectional studies (Chiba and Murata, 1985; Simerly and Swanson, 1988) the MPN issues significant and largely unilateral input to the medial amygdala. The dominant site of Fos induction occurred within the posterodorsal part (Figure 10), with fewer cells detected within the posteroventral and anteroventral parts of the nucleus. Quantitative assessment of the number of restraint-induced Fos-ir neurons as a function of treatment status through the rostrocaudal extent of the posterodorsal part of the medial amygdala revealed no significant effects of treatment (P = 0.86) and side (P = 0.78), and no significant interaction (P = 0.60) between treatment and side (Figure 4-11).  Lateral septal nuclei. On the bases of functional and connectivity studies, the lateral septum is in a position to relay the influences of the MPN on visceromotor responses in the PVN (Simerly and Swanson, 1988; Risold and Swanson, 1997). Determined from the same material used to survey the PVN and medial amygdala under basal and stress conditions, the dominant site of Fos induction in the septal region was localized to the intermediate lateral septal nuclei, with fewer cells detected within the ventral lateral septal nuclei (Figure 4-12). Labeling in the medial septal nuclei was quite sparse, and for the most part absent in the dorsal lateral and posterior septal nuclei. Quantitative assessment of the number of restraint-induced Fos-ir neurons as a function of 195  Figure 4-10. Brightfield photomicrographs to show Fos-ir staining within medial parvocellular neurons on the sides of the PVN ipsilateral (B) and contralateral (A) to the side of the testosterone implant at 30 min of restraint exposure. Note the relative decrement in Fos displayed on the ipsilateral (B) compared to the contralateral (A) side of the nucleus. Mean ± SEM relative numbers of Fos-ir neurons in rats bearing hydroxyflutamide (HF), testosterone (TEST) or control (CTL) implants (C). *P<0.05 versus the contralateral side in animals bearing testosterone implants (n = 6 per group). Scale bar =100 urn.  196  Figure 4-11. Brightfield photomicrograph to show Fos-ir in the medial amygdala (MeA) at 30 min of restraint exposure (A). Structures labeled for reference: cpd, cerebral peduncle; ot, optic tract; st, Stria terminalis. Scale bar =150 um. Mean ± SEM relative numbers of Fos-ir neurons (B) in rats bearing hydroxyflutamide (HF), testosterone (TEST) or control (CTL) implants (n = 6 per group).  B .'V'*  '\ > •  .v.,  •»•»  • it  3jJ# • - : , < •. ,36? - • ( , . *•.!?/&•»•;.  " ^  •  .•  •  :  -  Figure 4-12. Brightfield photomicrographs to show Fos-ir staining on the sides of the lateral septal nucleus ipsilateral (B) and contralateral (A) to the side of the testosterone implant at 30 min of restraint exposure. Structures labeled for reference: LSi, lateral septum, intermediate part; LV, lateral ventricle. Scale bar =150 urn. Note the relative decrement in Fos displayed on the ipsilateral (B) compared to the contralateral (A) side of the nucleus. Mean ± SEM relative numbers of Fos-ir neurons in rats bearing hydroxyflutamide (HF), testosterone (TEST) or control (CTL) implants (C). *P<0.05 versus the contralateral side in animals bearing implants of testosterone (n = 6 per group).  treatment status within the lateral septum revealed significant effects of treatment (P = 0.0313) and side (P = 0.003), and a significant interaction between treatment and side (P = 0.0038). Similar to the PVN, the interaction was based within the testosterone treatment group only, in which animals showed a lower number of cells recruited to express Fos protein on the side of the lateral septum ipsilateral to the testosterone implant (Figure 4-12). 4.4  Discussion Our previous studies have shown that the normal inhibitory effects of testosterone in  circulation on stressed-induced PVN activity and HPA outflow are attenuated in animals bearing discrete bilateral lesions of the MPN (Williamson and Viau, 2008). The high containment of ARs within MPN cells identified as projecting to the PVN region directly could form a basis for this functional dependency (Williamson and Viau, 2007). However, the influence of the MPN might also be mediated indirectly through its projections to and/or through various limbic and forebrain structures implicated in the regulation of stress-induced ACTH release. To make inroads on the underlying circuitries mediating the effects of androgens in the MPN and to determine whether the local actions of testosterone depend on ARs directly, we compared the effects of unilateral implants of testosterone and the AR antagonist hydroxyflutamide into the MPN. These results support an AR involvement of the MPN to modulate the PVN while concurrently altering the capacity of neurons to express AVP in the medial amygdala and stressinduced responses in lateral septal nuclei. As both of these putative regulators of the HPA axis are extensively targeted by the MPN, the present findings position the MPN as an important neural substrate for registering changes in gonadal status and distributing this information to multiple afferent mediators of PVN visceromotor function. In line with the unilateral projections of the MPN to the PVN, medial amygdala, and septal nuclei (see Figure 4-12), the basic effects of the implants were lateralized to the sides of the nuclei 199  ipsilateral to the MPN implants. The testosterone and hydroxyflutamide implants exerted inhibitory and stimulatory effects, respectively on AVP, but not on CRH expression within the mpd region of the PVN. This finding remains in keeping with a selective and upstream influence of testosterone on the capacity of PVN neurosecretory neurons to express AVP, executed by ARs in the MPN (Bingham et al., 2006; Williamson and Viau, 2008). Based on these findings, and the capacity of testosterone in circulation to operate on both the stress-induced activation and synthesis of AVP neurosecretory neurons (Viau et al., 2003), we anticipated similar opposing influences of the implants on indices of cellular activation in the PVN in stressed animals. Lower levels of c-fos mRNA and smaller numbers of mpd cells recruited to express Fos protein occurred on the sides of the PVN ipsilateral to the testosterone implants, but there was no such asymmetry for either of these parameters in animals bearing hydroxyflutamide implants. The MPN has been subdivided into multiple compartments, each showing unique and/or overlapping distributions of neurotransmitters, androgen and estrogen receptors, aromatase activity, as well as different input and output projection profiles (Simerly et al., 1986; Simerly and Swanson, 1986,1988; Simerly et al., 1990; Zhao et al., 2007). Cells within the caudal division of the MPN that project most heavily to the PVN concentrate androgen receptors and both estrogen receptor-alpha and -beta subtypes (Simerly et al., 1990; Shughrue et al., 1997; Laflamme et al., 1998; Williamson and Viau, 2007), although the nature by which aromatase is contained by and regulated within this compartment has yet to be resolved (Zhao et al., 2007). Nonetheless, our findings suggest a role for estrogen receptors in the MPN that may be uniquely involved in the regulation of the stress-induced activation, but not the biosynthetic capacity of PVN visceromotor neurons. We previously noted that the functional effects of MPN lesions on the cellular activation and synthesis of AVP neurosecretory neurons in the PVN are discriminated in animals bearing 200  levels of testosterone in circulation in excess of 3-4 ng/ml (Williamson and Viau, 2008). As the animals in the current study showed plasma testosterone concentrations in the order of 2 ng/ml, the sex steroid hormone receptor bases in the MPN by which the dose-related effects of testosterone on the HPA axis occur remains to be seen. This does not indict the utility of our design as it confirmed the MPN as an important substrate for the central actions of testosterone independent of other sources of androgenic input, and unmasked a tonic inhibitory role for ARs of the MPN to regulate AVP expression in the PVN. We also found differential effects of testosterone and hydroxyflutamide implants in the MPN on the capacity of medial amygdala neurons to express AVP. However, the nature of these changes was opposite to the effects observed for AVP within the hypophysiotropic zone of the PVN, in which ARs of the MPN appeared to exert a stimulatory influence on AVP expression in the medial amygdala. Since the vast majority of AVP expressing cells in the medial amygdala of the rat stain for ARs (de Vries and Panzica, 2006), our current findings continue to challenge the notion that testosterone regulates AVP neurons in the medial amygdala directly (and see Williamson and Viau, 2008). AVP neurons in the medial amygdala target a vast number of brain regions, including the lateral septum, to effect a broad array of behaviors associated with emotional and coping responses, as well as stress-induced changes in autonomic and HPA activity (Buijs and Kaisbeek, 1993; Kalsbeek et al., 2002; Landgraf and Neumann, 2004). Several lines of evidence continue to relate the inhibitory influence of the gonadal axis on HPA function in males to testosterone dependent increases in extrahypothalamic AVP, including within the medial amygdala (de Vries et al., 1994). Further, the medial amygdala is critical for the HPA response to stressful stimuli, particularly emotional stressors such as restraint (Dayas et al., 1999). Furthermore, the work of Feldman and colleagues (1990) showed that MPN lesions inhibit the increase in plasma corticosterone levels after electrical stimulation of the medial amygdala. While the extent to 201  which any of these AVP circuits may come to rely upon ARs in the MPN to control the HPA axis remains to be determined, the MPN would appear, nonetheless, to be an ideal candidate for harmonizing the central actions of testosterone on behavior and neuroendocrine stress responses. The lateral septum is a major recipient of multiple limbic efferents and regions of the telencephalon associated with the processing of somatosensory information, and projects to numerous HPA-regulating nuclei of the hypothalamus, as well as regions immediately surrounding the PVN, schematized in Figure 4-13 (Risold and Swanson, 1997; Herman et al., 2003). Based on this connectivity, perhaps predictably, testosterone implants decreased Fos expression within the lateral septum on the ipsilateral side, strikingly similar to the Fos response in the PVN. While an androgenic- (or estrogenic-) dependent involvement of the MPN may be inferred, the nature or extent to which the lateral septal nuclei are actually responsible for driving the PVN visceromotor responses to stress cannot be ascertained at this point. We have yet to map patterns of stress-induced Fos within septal nuclei identified as projecting to the PVN region or in receipt of input from the MPN. However, the effects of testosterone appeared preferentially localized to Fos responding neurons within the ventrolateral part of the lateral septum, which contains a small but reliable number of cells known project to regions immediately surrounding the PVN (Staiger and Wouterlood, 1990; Li and Sawchenko, 1998; Williamson and Viau, 2007), and thus capable of exerting an inhibitory influence on the PVN and HPA output (Roland and Sawchenko, 1993; Boudaba et al., 1996). Our current findings provide every indication of a close, if not overlapping, relationship between systems governing reproduction and HPA control (Williamson and Viau, 2007), and that the MPN provides a formidable link between these systems. Emotional stressors, such as restraint, are stimuli that target one or more exteroceptive sensory modalities and involve distinct cognitive and affective components (Li et al., 1996; Dayas et al., 2001; Herman et al., 2003). It should not be surprising that stressors of this type recruit a set of highly interconnected cell 202  w cc  i . _ BSTpr t j.E-sJOSj E R ot^y  \  y  ACTH Figure 4-13. Possible circuitry mediating testosterone regulation of the HPA axis. The present results suggest that testosterone acts within the MPN to bridge converging limbic influences to the PVN, including the medial amygdala and lateral septal nuclei. While the anatomical connectivity between these nuclei has been formally established, the chemical identity and sensitivity of these PVN afferents to changes in gonadal status have yet to be determined. The implants employed in the present study targeted the caudal half of the MPN, which houses primarily gamma-aminobutyric acid (GABA)-ergic neurons. Thus, the cellular activation and peptide expression in the PVN may come to rely on testosterone dependent changes in GAB A activity within the MPN, as well as within the PVN surround (see Bingham et al., 2006; Williamson and Viau, 2008). Structures labeled for reference: cc, corpus callosum; vhc, ventral hippocampal commissure; ac, anterior commissure; ot, optic tract; pc, posterior commissure.  203  groups in the limbic forebrain, including the lateral septum, amygdala, bed nucleus, hypothalamus, hippocampus, and frontal cortex (Cullinan et al., 1996; Campeau et al., 1997; Li and Sawchenko, 1998; Dayas et al., 2001). And while the MPN has been historically implicated in facets of reproductive regulation (for reviews see Swann et al., 2003; Balthazart and Ball, 2007), our current findings prime the MPN as playing an integral role amongst several central stress responding systems. 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Gender and puberty interact on the stressinduced activation of parvocellular neurosecretory neurons and corticotropin-releasing hormone messenger ribonucleic acid expression in the rat. Endocrinology 146(1):137-146. Viau V, Lee P, Sampson J, Wu J. 2003. A testicular influence on restraint-induced activation of medial parvocellular neurons in the paraventricular nucleus in the male rat. Endocrinology 144:3067-3075. Viau V, Meaney MJ. 1996. The inhibitory effect of testosterone on hypothalamic-pituitaryadrenal responses to stress is mediated by the medial preoptic area. J Neurosci 16:18661876. Viau V, Soriano L, Dallman MF. 2001. Androgens alter corticotropin releasing hormone and arginine vasopressin mRNA within forebrain sites known to regulate activity in the hypothalamic-pituitary-adrenal axis. J Neuroendocrinol 13:442-452. Williamson M, Bingham B, Viau V. 2005. Central organization of androgen-sensitive pathways to the hypothalamic-pituitary-adrenal axis: implications for individual differences in responses to homeostatic threat and predisposition to disease. Prog Neuropsychopharmacol Biol Psychiatry 29:1239-1248. Williamson M, Viau V. 2007. Androgen receptor expressing neurons that project to the paraventricular nucleus of the hypothalamus in the male rat. J Comp Neurol 503:717-740. Williamson M, Viau V. 2008. Selective contributions of the medial preoptic nucleus to testosterone-dependant regulation of the paraventricular nucleus of the hypothalamus and the HPA axis. Am J Physiol Regul Integr Comp Physiol 295:R1020-1030. Yoo MJ, Searles RV, He JR, Shen WB, Grattan DR, Selmanoff M. 2000. Castration rapidly decreases hypothalamic gamma-aminobutyric acidergic neuronal activity in both male and female rats. Brain Res 878:1-10. Zhao C, Fujinaga R, Tanaka M, Yanai A, Nakahama K, Shinoda K. 2007. Region-specific expression and sex-steroidal regulation on aromatase and its mRNA in the male rat brain: immunohistochemical and in situ hybridization analyses. J Comp Neurol 500:557-573.  CHAPTER 5 : General Discussion 5.1  Contributions to original knowledge The studies presented in this thesis contribute to the growing knowledge base of gonadal  regulation of the PVN and HPA axis. In particular, I utilized a functional neuroanatomical approach to systematically determine where and how testosterone can act in the brain to modulate the brain's response to stress, with an emphasis on the medial preoptic nucleus. Below, I've listed my contributions to original knowledge in science. 1.  Many advances have been made in clarifying the anatomical and functional organization  of stress-related circuits in the brain, which have been critical in identifying putative regulators of PVN and HPA function. However, while numerous studies highlight the potent effects of gonadal steroids on HPA function, their sites and mechanism of action remain unresolved. My studies are the first to characterize the anatomical nature by which androgen sensitive targets in the brain may communicate with the PVN directly. 2.  An important finding here is that the connectional data demonstrate that androgens can act  on a large assortment of multimodal inputs to the PVN, including those involved with the processing of various types of somatosensory and limbic information, and these findings also provide an index of the potential influence of testosterone on various effector systems in the PVN. One critical finding of the present study is that of all the forebrain regions studied, the MPN contained the highest number of PVN-projecting cells in total, and almost 70% of these stained positively for ARs. 3.  AR containing projections from the MPN to the PVN have been implicated in mediating  the inhibitory actions of testosterone on the HPA outflow. The findings from my lesion experiments confirm that the inhibitory effects of testosterone depend on the functional integrity of the MPN. 210  4.  My results also indicate that the effects of MPN lesions were discriminated, in high-, but  not low-testosterone replaced rats. Since levels of circulating testosterone vary as a function of age, sexual experience, time of day and social status, the role of the MPN on the brain's response to stress may very well depend on situation-specific changes in gonadal status. 5.  Therefore, I sought to determine the extent to which the MPN represents the seat of the  central actions of testosterone on the PVN and its extended circuitries. My strategy incorporated a unilateral steroid/antagonist implant approach to exploit the unilateral projections of the MPN to the nuclei of interest, and because bilateral approaches in the MPN could influence testosterone in circulation, to prevent secondary effects from occurring within additional androgen sensitive projections to the HPA axis. My findings demonstrate that AVP biosynthesis in the PVN and MeA appear to depend on the AR in the MPN. In contrast, stress-induced Fos expression in the PVN and lateral septal nuclei responded to testosterone, but not to hydroxyflutamide implants in the MPN. Thus, estrogen (receptor) mediation of the effects of testosterone in the MPN remains a real possibility, at least with respect to cellular activation. 6.  The anatomical specificity by which this occurs remains unresolved given the connectivity  of the MPN with several other extended circuitries, also rich in both androgen and estrogen receptors. Nonetheless, our findings provide several lines of evidence to suggest that the MPN forms part of a circuit that involves both the medial amygdala and lateral septum to integrate the central effects of testosterone on PVN function and HPA outflow. 5.2 5.2.1  Methodological considerations Retrograde tract-tracing and cell counting In Chapter 2,1 characterized the distribution of ARs within staining within PVN-  projecting cell groups using a retrograde fiber tract-tracing approach. To ensure that retrograde tracer deposits were effective at identifying cell groups that project to the neuroendocrine part of 211  the PVN spanning the entire rat brain, I used iontophoretic and crystalline implants of the retrograde tracer, Fluorogold (FG). The total number of FG cells encountered within most cell groups was generally stable using both delivery methods, however, there were benefits inherent to each technique. The discrete deposits produced by iontophoresis were more effective in identifying the most local of afferent sources to the PVN. On the other hand, I observed less variance in the number of detectable neurons in the brainstem following crystalline implants, as tracer accumulation was noticeably more complete within individual cells following retrograde accumulation over longer transport distances. While I did not confirm the reciprocal anatomical connectivity of each candidate cell population using an anterograde fiber tract-tracing approach, the fact that there was relatively little disparity within and between both methods of tracer delivery, along with our effective use of controls (i.e. offset tracer placements) guided by previous retrograde and anterograde anatomical transport studies, I remain confident that my approach provided a reliable means with which to gauge the relative contribution of AR-expressing afferents to the PVN. With respect to my methodology for counting cells, I counted profiles of neuronal cell bodies on every fifth section, corrected for cell size using the Abercrombie method, and then multiplied by five to achieve total cell number estimates (Abercrombie, 1946). As the Abercrombie method is designed for use with spherical objects of relatively constant diameter, such as counting nucleoli as target objects, which are in fact almost perfectly spherical, and under the assumption that neurons possess only one nucleolus per neuron (Abercrombie, 1946). A reason for potential concern is that the neuronal cell bodies are not spherical, which introduces some bias with respect to the shape of the intended target objects (i.e. FG-containing neuronal bodies) (Guillery, 2002). This biased method indicates that my estimates of cell number in the various brain nuclei may not be absolutely accurate. However, it is likely that the degree to which the shapes of neuronal cell bodies within various nuclei depart from spherical do not differ much 212  from one nucleus to another. Therefore, proportional assessments made both between and among individual nuclei remains a valid approach. 5.2.2  Medial preoptic nucleus lesions and testosterone replacement In Chapter 3,1 tested the functional role of the MPN on PVN and HPA activity using  neurotoxic lesions of the MPN (Williamson and Viau, 2008). Previous ablation studies in the MPN, in addition to my own, have employed the use of electrolytic lesions (Viau and Meaney, 1996; Williamson and Viau, 2005). However, because many axons pass through the immediate vicinity of the MPN, including within the lateral preoptic and anterior hypothalamic areas (e.g. the VNAB) (Swanson, 1987; Rothwell, 1989; King, 2006), the effects of such electrolytic lesions on neuroendocrine responses could have reflected the interruption of fibers of passage independently or in addition to the MPN. In the present studies (Chapter 3), I employed discrete excitotoxic lesions of the MPN to avoid this problem. However, previous studies have also reported that larger amounts of ibotenic acid may also damage fibers of passage (Coffey et al., 1990; and see Dayas et al., 1999). Therefore, to minimize this risk I used injections of a relatively small amount and volume of ibotenic acid (5 ng/nl, 500 ng per MPN injection site), which effectively blocked high testosterone inhibition of PVN activation and HPA outflow, but without major signs of gliosis in the animals chosen for analysis. As well, analysis of AR immunoreactivity further confirmed that the lesions were confined, in largest part, to the body of the MPN. While in certain cases animals bearing bearing ibotenic acid lesions displayed GFAP immunoreactivity outside of the MPN, encroaching on the BST (refer to Figure 3-1), no decrement of AR immunoreactivity was detected outside the MPN, suggesting that regions surrounding the MPN (i.e. BST) involved in homeostatic and HPA regulation remained unaffected. Importantly, GnRH neurons are diffusely distributed throughout a continuum of the medial basal forebrain (Silverman et al., 1987; Herbison, 2006), including the region of the MPN.  Thus, it is possible that large lesions in the vicinity of this nucleus could interrupt drive to the gonadal axis, and ultimately systemic levels of testosterone (Smith et al., 1977). I avoided this potential confound by supplying fixed levels of testosterone replacement in the periphery of gonadectomized male rats. The replacement levels of testosterone selected have been previously used to characterize the dose-related inhibitory effect of testosterone in circulation on stress HPA activity (Viau and Meaney, 1996; Williamson and Viau, 2006). Just as important, the low- and high- levels of replacement provided circulating concentrations of plasma testosterone within the normal physiological range of the adult male Sprague-Dawley rats (Viau, 2002; Williamson et al., 2005). Thus, while previous experiments showed that large lesions of the medial preoptic area block the inhibitory effects of high physiological levels of testosterone (Viau and Meaney 1996), the current approach allowed us to determine how varying levels of testosterone interact specifically with the MPN on stress HPA function. However, it is important to mention that testosterone is released in a pulsative manner in gonadal intact rats, and constant replacement, even at physiological levels, is a limitation of the approach. The pulsatile secretion of testosterone in gonadal-intact male rats permits rapid and marked increases in mean hormone concentrations, which may allow preferential engagement of rate-sensitive cellular signaling pathways (reviewed in Veldhuis et al. 2008). Therefore, it is possible that our constant testosterone secretion regiment may have masked or blocked the physiological effects associated with testosterone pulsatility (e.g. changes in pulse size or pulse number) distinct from differences in mean hormone concentrations (Veldhuis et al. 2008). 5.2.3  Androgen receptor antagonism In Chapter 4,1 employed intracerebral microimplants of the AR antagonist  hydroxyflutamide or testosterone to characterize the role of MPN ARs. Because the AR agonists and antagonists were implanted directly into the caudal MPN it is likely that the actions were  mediated locally, restricted to ARs within this region. Our experimental design offered several advantages over my previous studies using gonadectomy and peripheral steroid replacement to manipulate gonadal steroid concentrations. First, because efferents of the MPN are limited to the ipsilateral side of the brain (Simerly and Swanson, 1988; Williamson and Viau, 2007), the use of a unilateral pharmacological antagonist enabled us to unambiguously block the effects of androgens in the MPN without interfering with the contralateral side. Second, the use of unilateral implants in gonadal-intact animals allowed us to interfere with local AR binding, but without interfering with other factors that may be involved in the regulation of GnRH, and hence the downstream regulation of testosterone in circulation. Third, the antagonist enabled differentiation of the actions of testosterone mediated by AR from those that might be inferred by ER, given the concentration of this sex steroid hormone and aromatase activity within the MPN. Hydroxyflutamide is the active metabolite of flutamide, a specific nonsteroidal AR antagonist that has no known agonist properties (Neri et al., 1972; Poyet and Labrie, 1985). As flutamide itself has negligible receptor antagonist activity in nerve tissue in vitro (Clark and Nowell, 1980), hydroxyflutamide was the antagonist of choice in this study. Fourth, by administering hydroxyflutamide directly into the caudal aspects of the MPN, I was able to achieve site-specific antagonism of androgen action locally, but without affecting steroid-dependent processes affecting neuronal plasticity, synaptic connectivity, and neural-glial interactions (Garcia-Segura et al., 1994a; Garcia-Segura et al., 1994b) within the remainder of the central nervous system. While hydroxyflutamide concentrations were not measured directly, it is unlikely that this compound diffused a significant distance from the implant site, as verified by assessing local patterns of AR immunoreactivity induced in a subset of gonadectomized rats (refer to Figures 4-1 and 4-2). Furthermore, using a similar approach, Petersen and Barraclough (1989) demonstrated that implants of the nonsteroidal estrogen antagonist, keoxifene, blocked ovulation in female rats  when administered into the rostral MPOA, but not when placed in locations as little as 500 um rostral or 1 mm lateral to their rostral MPOA coordinates. There was an initial concern that the effects of the implants may be attributed to physical disruption of the MPN. To address this, we compared central measures of HPA function and neuropeptide expression in an additional subset of control, gonadal-intact rats that received either sham surgeries (anesthesia only) or unilateral implants of beeswax alone into the MPN. Since these measures did not differ between groups of animals, we determined that hydroxyflutamideand testosterone-dependent effects were not attributed to space-occupying lesions of our wax implants. 5.2.4  Immediate early genes and cellular activation The majority of my experiments relied heavily on the IEG c-fos, and its protein  counterpart Fos, as markers of cellular activation. The utility of c-fos gene as an anatomical marker for synaptic activation has been extensively discussed, though it is important to keep in mind that not all neurons that are activated in some way (electrophysiologically or metabolically), will necessarily respond with induction of c-fos mRNA or protein, and therefore no conclusions can be drawn from a lack of induction. However, despite this limitation, many advantages can be taken from the presence of c-fos, or its protein counterpart. This early-gene is very sensitive and its induction in some neurons indicates that these neurons were affected in some way by the stimulus. Furthermore, its induction can be detected with a cellular resolution, combined with the visualization of other protein or mRNA markers (e.g. enzymes or receptors), and assessed in a quantitative manner (Morgan and Curran, 1991; Chan and Sawchenko, 1994; reviewed in Ziolkowska and Przewlocki, 2002). Stress-induced c-fos mRNA and Fos expression has also been complemented by the use of markers of neuropeptide utilization. Measuring hnRNA levels (rather than mRNA) is a more 216  precise way to monitor changes in the rate of gene transcription (Herman et al., 1991). That is, within a given cell, there exists a pre-existing pool of mRNA, so the detection of stimulus-induced changes in mRNA can be masked by the presence of mRNA species that have been previously transcribed. In addition, changes in mRNA levels are the result of different factors governing gene expression, including transcription and post-transcriptional processing. Furthermore, as described previously, levels of mRNA can also be affected by the rate of degradation. Thus, the measurement of primary RNA transcripts using hnRNA probes allows the detection of more precise and acute changes in the rate of transcription. My experiments relied on the mature transcript as I sought to assess whether my manipulations altered the steady state expression of peptides. However, subsequent experiments should now exploit more sensitive markers such as changes in hnRNA, which would be more closely tied to the stimulus in question (e.g. restraint) and the utilization or state of peptide stores (e.g. CRH or AVP). Several groups have examined whether neuropeptide secretion and gene activation share the same temporal response patterns as HPA function during stress. For example, following either hypovolemia (Tanimura et al., 1998) or restraint (Girotti et al., 2006), stimuli whose physiological onsets are easily identifiable, the rapid and transient increases in CRH hnRNA paralleled the induction of c-fos mRNA in the PVN, which also correlated positively with plasma ACTH levels (Tanimura et al., 1998; Girotti et al., 2006). Tanimura and colleagues (1998) also emphasized that CRH neurons respond to a stress event in a stimulus-specific manner in terms of both the profiles of secretion and gene expression. That is, the initial response takes place when secretion of stored CRH into the hypophyseal circulation occurs, followed by the induction of cfos mRNA and CRH heteronuclear (hn) RNA (intronic mRNA) expression to replenish CRH stores. These results indicated CRH gene transcription plays an initial and central role in regulating activation of the HPA axis, and that monitoring hnRNA responses further supports the  217  temporal and functional validity of employing IEG induction as an index of cellular activation (for reviews see Watts, 2005; Watts et al., 2006). 5.3  Future considerations  5.3.1  Gonadal regulation of neurochemical systems How do we begin to sort out the neurochemical specificity by which sex steroids,  including androgens, exert their effects on the HPA axis? Superimposing neurochemical systems with our anatomical characterization of ARs within PVN-projecting cell groups directly, will provide us with a very manageable anatomical framework for understanding how gonadal status could contribute to individual differences in HPA function. Based on my findings and those of others, the hypophysiotropic and neural AVP systems explored, could represent one of many circuits in the brain orchestrating the animal's ability to act and interact with its environment. In fact, the effects of gonadal steroids on the neural AVP systems in the BST and the MeA, are among the most dramatic reported for a neurotransmitter system. Several other neuropeptide systems also show dramatic fluctuations in their expression under the influence of gonadal steroids (discussed below); however, complete elimination of the expression of a particular neuropeptide by gonadectomy has been reported only for AVP cells (de Vries et al., 1986). And since these vasopressin neurons project to many forebrain areas, such as the hippocampus and lateral septum, it is not entirely surprising that AVP immunoreactivity within these areas also respond to changes in gonadal status. For example, following gonadectomy of adult male rats, AVP fiber density in the lateral septum decreases gradually, until virtually no fibers can be detected after a few weeks (de Vries et al., 1984; de Vries et al., 1994). Furthermore, similar changes are also observed when there is a more ethological or relevant cause for the decreased gonadal activity, such as during aging (Fliers et al., 1985; Goudsmit et al., 1988) or as a function of reproduction (Buijs et al., 1986; Bittman et al., 1996). It is important to note 218  that in contrast with gonadal steroids, adrenal steroids do not appear to be a strong influence on this neural AVP system. While removal of adrenal steroids by adrenalectomy suppresses AVP gene expression in the MeA, this occurs by an initial suppression of testosterone secretion and a subsequent decrease of AVP (Viau et al., 2001). Thus, it is likely that testosterone-coupled shifts in this AVP system may render it malleable to changing environments, particularly those concerned with marked shifts in gonadal activity, including development, puberty, ageing, social and reproductive status, and stress (refer to section 1.5) (reviewed in Williamson et al., 2005). However, considering the distribution of the gonadal sensitive vasopressinergic systems, it is important to emphasize that these projections do not terminate directly on the PVN (Kalsbeek et al., 2002). Therefore, any modulatory role on the PVN and HPA axis must arrive by transsynatic relay (as discussed in section 1.4.4.4). Interestingly, the projection sites of this neural AVP system do show a very close, if not overlapping relationship with many forebrain and hindbrain structures that I identified as ARcontaining and PVN-projecting (either directly or indirectly) (Williamson and Viau, 2007). This clearly places the sexually dimorphic AVP system among the principle candidate regulators of androgen-regulation of the PVN and HPA axis. The challenge now, is to determine how testosterone-dependant shifts in this AVP system impacts either the recruitment or patterns of gene expression within such PVN afferents. However, it is important to note that, in addition to AVP, there is an incredible assortment of substrate dedicated to regulating input to the HPA-regulating AVP/CRH neurosecretory PVN neurons (Herman et al., 2006). Another possible neurotransmitter that may be involved in the suppressive effects of testosterone on HPA activity is GABA, whose innervations account for over half of the synaptic inputs to the PVN (Decavel and Van den Pol, 1990; Roland and Sawchenko, 1993; Herman et al., 2002). GABA transmission is a key regulator of converging input to the hypophysiotropic PVN and HPA activity (Herman et al., 2004), and is reliably 219  sensitive to changes in testosterone (Grattan and Selmanoff, 1993; 1994; Grattan et al., 1996; Yoo et al., 2000). For example, GABA activity in the MPN is strikingly decreased following gonadectomy (Williamson and Viau, unpublished observations), and this effect is reversible with testosterone replacement (Grattan and Selmanoff, 1994). Interesting, GABA synthesizing enzymes (glutamic acid decarboxylase, GAD-65 and -67) are upregulated following restraint stress (Bowers et al., 1998) and are also sensitive to gonadal steroids (Grattan and Selmanoff, 1993; Grattan et al., 1996). In response to the chronic cannulas containing the AR antagonist hydroxyflutamide aimed at the MPN, the steady state expression of both GAD-65 and -67, though preferentially GAD-67, are significantly increased. Thus, it remains quite possible that the GABAergic system represents a key player in androgenic regulation of the PVN and its upstream putative regulators. Thus, future experiments should more systematically assess the role of GABAergic neurons in androgen-regulation of the HP A axis. As mentioned earlier, there is an incredible assortment of substrate dedicated to regulating input to the hypophysiotropic, HPA-regulating zone of the PVN (Herman et al., 2006). Moreover, sex steroids including testosterone, have been shown to interact with several of these neurotransmitter and peptidergic systems, as well as within numerous cell groups identified as projecting to the PVN directly or regulating the HPA axis indirectly (de Vries et al., 1986; Diano et al., 1997; Herbison, 1997; Bloch et al., 1998; Ronnekleiv and Kelly, 2005). Superimposing further these neurochemical systems onto our anatomical characterization of ARs (as well as ERs) localized to the PVN-projecting cell groups should shed considerable light on our current framework. 5.3.2  Implications of androgen receptor containment within the pre-autonomic part of the PVN. Numerous connectional and phenotypic studies continue to expand upon the descending  influences of the PVN on terminal fields such as the central gray, the intermediolateral cell 220  column, and more recently, the dorsal horn marginal layer of the spinal cord (Hallbeck et al., 2001; Crane et al., 2005). Taken together with my findings showing the distribution of ARexpressing PVN-afferents within the hindbrain (Williamson and Viau, 2007), and the previous findings from our group showing the predominance of AR within pre-autonomic PVN cells that give rise to these descending projections (Bingham et al., 2006), clearly incorporates gonadal status into the realm of somatosensory and autonomic function. Moreover, another emerging dimension is the capacity of this autonomic outflow to influence adrenocortical steroidogenic responses directly (Engeland and Arnhold, 2005). In fact, functional and anatomical evidence suggest that departures between ACTH and corticosterone release under basal and stress conditions can be attributed by altered activity within the pre-autonomic PVN (refer to Figure 35) (Williamson and Viau, 2008), and to differences in adrenal sensitivity to ACTH controlled by sympathetic preganglionic spinal cord neurons (Strack et al., 1989; Motawei et al., 1999; UlrichLai et al., 2006; reviewed in Bornstein et al., 2008). Interestingly, estrogen also appears to impose itself on this autonomic regulation of adrenocortical function. Work by Figueiredo and colleagues (2004) demonstrated that estrogen potently enhances adrenal sensitivity to ACTH in females, despite a clear absence of estrogen receptor isoforms in the adrenal cortex. Also, female rats display a reduced corticosterone response to acute restraint following ER-B antagonism in the PVN (Isgor et al., 2003). Finally, injection of estrogenic and androgenic agonists into the PVN attenuate restraint induced ACTH and corticosterone in gonadectomized male rats (Lund et al., 2006). Taken together with the findings of Bingham et al. (2006) showing a distribution of AR as strict as the profile for ER-B among populations of pre-autonomic neurons in the PVN, my findings underscore a role for androgens in modulating the activity of these non-neurosecretory PVN cells and highlight a need for examining whether androgens (or estrogens) are capable of bridging autonomic and adrenocortical function.  5.3.3  Role of androgens in repeated stress paradigms Our findings reflect the ability of testosterone to modulate the brain's response to acute  restraint stress. However, there are marked differences in the regulation of behavioral, neuroendocrine and metabolic responses between acute stress models and repeated stress models. In most cases, acute stress produces a generalized, non-selective response that involves simultaneous targeting of multiple tissue types and organ systems to achieve a major redistribution of the body's resources in response to a challenge (Selye, 1976; Chrousos et al., 1998; Pacak and Palkovits, 2001; McEwen and Wingfield, 2003; Day, 2005). For example, an acute episode of restraint stress includes adrenocortical (glucocorticoids), adrenomedullary (sympathetic catecholamines), behavioral responses and cognitive activation. On the other hand, over time, repeated stress involves the targeting of a selective subset of these systems or tissues so as to achieve a circumscribed adjustment appropriate for handling a specific challenge (Dallman et al., 2002; Day, 2005). Moreover, sustained drive to the HPA axis produced by prolonged or chronic stress can cause long-term changes in HPA function (reviewed in Aguilera, 1994; Herman et al., 2006). Furthermore, the overall impact of any given repeated stress paradigm on HPA axis function is highly dependent on the experimental design. For example, repeated exposure to some stressors (e.g. restraint) can cause habituation of the HPA response (Aguilera and Rabadan-Diehl, 2000; Girotti et al., 2006; Herman et al., 2006). However, the habituation of the response is stressor-specific (Bhatnagar and Dallman, 1998; Cole et al., 2000; for review see Herman et al., 2006). Because testosterone attenuates the HPA stress response, it would be reasonable to consider that this is adaptive, consistent with the idea that it is optimal to attenuate the pathogenic effects of elevated glucocorticoids levels. Notwithstanding the fact that elevations in glucocorticoids serve to meet the metabolic and physiological demands of stress, inhibition of the stress response by testosterone may not always be an obligatory requirement. For example, 222  chronic cold stress exposure is best survived in male rats, provided normal decreases in testosterone levels are allowed to occur (Gomez and Dallman, 2001). On the other hand, our recent work has also made it clear that gonadal status has profound permissive effects on the regulation of HPA habituation (Bingham et al., 2005). For example, habituation of the HPA response to repeated restraint exposure appears to be met by an increase in testosterone secretion; gonadectomy prevents this habituation, although it is restored with testosterone replacement (Bingham et al., 2005). Therefore, it would be more reasonable to believe that the adaptive nature lies within the capacity of the adrenal axis to respond to variations in testosterone as needed. To the extent that illustrating that changes in gonadal status can intervene on the capacity of the HPA axis to adjust or adapt to repeated stress paradigms, the sites and mechanism by which the gonadal system regulates the HPA response under repeated stress conditions remains unclear and worthy of pursuit. As described earlier, while there are a variety of sites downstream from the PVN at which testosterone could act to regulate subsequent declines in glucocorticoid synthesis and release in response to repeated stress (refer to section 1.7), the registry of changes in gonadal status within the central nervous system would seem to be a requirement at this point of our studies. Similarly, given the role of gonadal steroids in depression and anxiety (refer to section 1.5.2), how circulating levels of testosterone contribute to changes in affect during chronic stress also remains worthy of pursuit. 5.3.4  Implications of androgens in brain plasticity and disease It was the seminal work of Raisman and Field (1971) and soon after by Gorski and  colleagues (1978) who identified a clear sexual difference in the organization of neuronal connections and the size of cells in the MPN, respectively, following neonatal exposure to testosterone. These observations had an enormous impact on the field because they demonstrated that gonadal hormones could cause profound changes in anatomy. Today, there is no shortage of 223  studies showing that gonadal steroids participate in the shaping of the developing rat brain, and a growing body of evidence now shows that gonadal steroids can remodel synapses in adults, illustrated by alterations in neuron and synapse numbers (Kurz et al., 1986; MacLusky et al., 2005), as well as in dendritic and synaptic morphology (Langub et al., 1994; Parducz and GarciaSegura, 1993; Parducz et al., 1993). Much of the work on sex steroid-induced neuroplasticity has focused on the effects of estrogens, though it has become clear that androgens are also capable of modulating structural synaptic plasticity in the adult brain. For example, approximately half of the hippocampal CA1 spine synapse pool is lost following gonadectomy in male rats, and this effect is reversed with testosterone and dihydrotestosterone replacement (Leranth et al., 2003). There also is growing evidence that the prefrontal cortex retains considerable potential for structural (synaptic) plasticity in adulthood. For example, administration of the non-competitive NMDA receptor antagonist, phencyclidine, to rats and monkeys impairs performance in prefrontal cortex-dependent cognitive tasks, and their performance is positively correlated with the number of asymmetric spine synapses in the prefrontal cortex (Jentsch et al., 1997a; Jentsch et al., 1997b). Why is structural synaptic plasticity in limbic brain areas so important? Because based on mounting evidence, it appears that remodeling of synapses within limbic areas, including the hippocampus and prefrontal cortex, may play a critical role in the mechanisms of depression and antidepressant therapy (Hajszan and MacLusky, 2006; Henn and Vollmayr, 2004; MacLusky et al., 2006; Parducz et al., 2006). Given the wealth of information on synaptic remodeling induced by gonadal hormones, researchers have just begun to recognize that synaptic remodeling might play a critical role in the neuropathology of a set of diseases. A critical question is whether the synaptogenic potential of gonadal steroids, androgens or estrogens, could be employed to reverse synaptic pathology in the clinical management of the above-mentioned disorders. Based on our own findings and those of others, androgens (and perhaps estrogens) have the capacity to regulate a vast and multimodal array of neural systems, 224  including the limbic system. Furthermore, the incredible assortment of substrate dedicated to mediating the central effects of testosterone underscores, on one level, how individuals can prosper and defend against a tremendous range of environmental and physiological demands. On another level, it is not hard to imagine how disruptions to these pathways could produce widespread and devastating effects such as those associated with anxiety and depression. The instability of testosterone in males and the potency by which testosterone acts throughout the central nervous system, clearly incorporates the gonadal system as compelling link between abnormal HPA function and affective disease states.  5.4  References  Abercrombie M. 1946. Estimation of nuclear population from microtome sections. Anat Rec 94:239-247. Aguilera G. 1994. Regulation of pituitary ACTH secretion during chronic stress. Front Neuroendocrinol 15:321-350. Aguilera G, Rabadan-Diehl C. 2000. Vasopressinergic regulation of the hypothalamic-pituitaryadrenal axis: implications for stress adaptation. Regul Pept 96:23-29. Bhatnagar S, Dallman M. 1998. Neuroanatomical basis for facilitation of hypothalamic-pituitaryadrenal responses to a novel stressor after chronic stress. Neuroscience 84:1025-1039. 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Activation in neural networks controlling ingestive behaviors: what does it mean, and how do we map and measure it? Physiol Behav 89:501-510. Williamson M, Bingham B, Viau V. 2005. Central organization of androgen-sensitive pathways to the hypothalamic-pituitary-adrenal axis: implications for individual differences in responses to homeostatic threat and predisposition to disease. Prog Neuropsychopharmacol Biol Psychiatry 29:1239-1248. Williamson M, Viau V. 2005. Role of the medial preoptic nucleus in regulating the hypothalamicpituitary-adrenal axis. Soc Neurosci Abstr 31:181.111. Williamson M, Viau V. 2006. Lesions of the medial preoptic nucleus alter the hypothalamicpituitary-adrenal (HPA) axis response to plasma testosterone. Soc Neurosci Abstr 32:563.567. Williamson M, Viau V. 2007. Androgen receptor expressing neurons that project to the paraventricular nucleus of the hypothalamus in the male rat. J Comp Neurol 503:717-740. Williamson M, Viau V. 2008. Selective contributions of the medial preoptic nucleus to testosterone-dependant regulation of the paraventricular nucleus of the hypothalamus and the HPA axis. Am J Physiol Regul Integr Comp Physiol 295:R1020-R1030. Yoo MJ, Searles RV, He JR, Shen WB, Grattan DR, Selmanoff M. 2000. Castration rapidly decreases hypothalamic gamma-aminobutyric acidergic neuronal activity in both male and female rats. Brain Res 878:1-10. Ziolkowska B, Przewlocki R. 2002. Methods used in inducible transcription factor studies: focus on mRNA. In: Kaczmarek L, Roberton HA, editors. Immediate Early Genes and Inducible Transcription Factors in Mapping of the Central Nervous System Function and Dysfunction. San Diego: Elsevier, p 1-31.  231  Appendices Appendix A UBC  THE UNIVERSITY OF BRITISH COLUMBIA  w  ANIMAL CARE CERTIFICATE Application Number: A07-0235 Investigator or Course Director: Victor Viau Department: Cellular & Physiological Sc. Animals:  Start Date:  April 1,2004  ^  0 V a l  September 8,2008  Funding Sources: Funding Agency: Funding Title: Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR) Androgen sensitive pathways to the paraventricular nucleus of the hypothalamus Natural Sciences and Engineering Research Council of Canada (NSERC) Critical influence of neonatal testosterone on stress pathways in the adult brain  Unfunded title: N/A  The Animal Care Committee has examined and approved the use of animals for the above experimental project This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102,6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  

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