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Local and distal origins of limbic-related projections to the paraventricular nucleus of the hypothalamus Anonuevo, Adam Manuel Smith 2013

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 LOCAL AND DISTAL ORIGINS OF LIMBIC-RELATED PROJECTIONS TO THE PARAVENTRIUCLAR NUCLEUS OF THE HYPOTHALAMUS    by ADAM MANUEL SMITH ANONUEVO B.Sc., University of British Columbia, 2010     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  The Faculty of Graduate and Postdoctoral Studies (Neuroscience)      THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2013 ? Adam Manuel Smith Anonuevo, 2013  ii  Abstract  Cortico-limbic circuits are often activated in response to emotional stress to provide salient information about individual stimuli. This allows organisms to recruit adequate response mechanisms, one of which is the hypothalamic-pituitary-adrenal axis. Cellular groups directly adjacent to the paraventricular nucleus of the hypothalamus (PVN) are in position to integrate and relay cortico-limbic projections to the PVN directly. Using a neuroanatomical approach we discovered unique patterns and strengths of cortico-limbic projections to separate cell groups in the PVN surround. Further, in response to restraint stress, dual retrograde labeling with the cellular activation marker FOS suggests these PVN surround circuits are involved in adaptive responses to emotional stress. Of the PVN surround subregions, the zona incerta (ZI) showed particularly dense connectivity with the medial prefrontal cortex (mPFC), lateral septum, and paraventricular thalamus. Due to the strength of this connectivity we sought to confirm that it can act as a relay site to the PVN through targeted injections of anterograde tracer. ZI injections of anterograde tracer led to terminally labeled fibers in the PVN confirming the capacity to influence hypophysiotropic neurons of the PVN. Additionally, there is the potential for the ZI to share functional cross-talk with other PVN surround subregions. In our final experiment we directed injections of retrograde tracer into the PVN in concert with anterograde injections in the mPFC resulting in convergence of tracer labeling within the ZI. The abutted signals strongly indicate the PVN surround is a promising site for limbic influence on HPA.   iii  Preface  This thesis contains research that I largely conducted independently with supervision from Dr. Victor Viau during my MSc studies. This includes experimental design, execution, data collection, and analysis. A manuscript of the principal findings is in preparation to be submitted to a peer-reviewed scientific journal. The work and methodology employed in this thesis was approved by the University of British Columbia Animal Care Committee under number: A09-0512.   iv  TABLE OF CONTENTS ABSTRACT .............................................................................................................................. ii PREFACE  ...............................................................................................................................iii TABLE OF CONTENTS ......................................................................................................... iv LIST OF TABLES .................................................................................................................... x LIST OF FIGURES ................................................................................................................. xi LIST OF ABBREVIATIONS ................................................................................................. xii ACKNOWLEDGEMENTS ................................................................................................... xiv CHAPTER 1: INTRODUCTION .......................................................................................... 1 1.1 Literature Review ............................................................................................................. 2 1.2 The hypothalamic-pituitary-adrenal axis ....................................................................... 3  1.2.1 Importance of the paraventricular nucleus on stress response ...................... 5 1.3 Limbic centers have the capacity to influence HPA activity......................................... 7  1.3.1 Lateral septum ............................................................................................... 7  1.3.2 Medial prefontal cortex ................................................................................. 8  1.3.3 Amygdala ...................................................................................................... 9 v   1.3.4 Hippocampus/subiculum ............................................................................... 9  1.3.5 Summary ..................................................................................................... 10 1.4 Connectivity to the PVN ................................................................................................. 10  1.4.1 Limbic connectivity to the PVN ................................................................. 11 1.5 The PVN surround is in position to relay limbic signals to the PVN ......................... 12  1.5.1 Projections to the PVN surround ................................................................ 12 1.6 Summary and specific hypotheses ................................................................................. 14  1.6.1 Chapter 2. Hypothesis 1 .............................................................................. 15  1.6.2 Chapter 3. Hypothesis 2 .............................................................................. 15  1.6.3 Chapter 4. Hypothesis 3 .............................................................................. 15 CHAPTER 2: Characterization of forebrain regions that project to the paraventricular nucleus of the hypothalamus surround .............................................................................. 17 2.1 Introduction ..................................................................................................................... 17 2.2 Methods ............................................................................................................................ 18  2.2.1 Animals ....................................................................................................... 18  2.2.2 Surgery ........................................................................................................ 18 vi   2.2.3 Tissue collection ......................................................................................... 19  2.2.4 Immunohistochemistry................................................................................ 19  2.2.5 Imaging and analysis ................................................................................... 20 2.3 Results  .............................................................................................................................. 20  2.3.1 Limbic-related; PFC, LS, MeA, PVT ......................................................... 21  2.3.2 Hypothalamus and bed nuclei of the stria terminalis  ................................. 27  2.3.3 Summary ..................................................................................................... 28 2.4 Discussion......................................................................................................................... 28  2.4.1 PVN surround shows unique patterns of cortico-limbic connectivity ........ 29  2.4.2 Heterogeneous efferents from the LS to PVN surround ............................. 29  2.4.3 The ZI receives robust projections from the mPFC .................................... 30  2.4.4 GABAergic polarity .................................................................................... 31 2.5 Summary .......................................................................................................................... 32 CHAPTER 3: Characterization of stress-responding limbic-related inputs to the paraventricular nucleus of the hypothalamus surround ................................................... 33 3.1 Introduction ..................................................................................................................... 33 vii  3.2 Methods ............................................................................................................................ 34  3.2.1 Animals ....................................................................................................... 34  3.2.2 Surgery ........................................................................................................ 34  3.2.3 Tissue collection ......................................................................................... 35  3.2.4 Dual Immunohistochemistry ....................................................................... 36  3.2.5 Imaging and analysis ................................................................................... 37 3.3 Results  .............................................................................................................................. 37  3.3.1 Limbic related; mPFC, LS, MeA, PVT ...................................................... 37 3.4 Discussion......................................................................................................................... 40  3.4.1 Regions of the PVN surround are in receipt of unique patterns of stress   activated circuits .................................................................................................. 40  3.4.2 Tonic and phasic regulation of the HPA axis through the PVN surround .. 42  3.4.3 The PVT may drive ZI related HPA input .................................................. 43  CHAPTER 4: Characterization of stress-responding limbic-related inputs to the paraventricular nucleus of the hypothalamus surround ................................................... 45 viii  4.1 Introduction ..................................................................................................................... 45 4.2 Methods ............................................................................................................................ 46  4.2.1 Animals ....................................................................................................... 46  4.2.2 Surgery ........................................................................................................ 47  4.2.4 Tissue collection ......................................................................................... 48  4.2.5 Immunofluorescence ................................................................................... 49  4.2.6 Imaging and analysis ................................................................................... 49 4.3 Results  .............................................................................................................................. 49  4.3.1 The mPFC sends efferents to the ZI ............................................................ 50  4.3.2 The PVN is in receipt of afferents from the ZI ........................................... 52  4.3.3 A circuit relay between the mPFC and PVN exists in the ZI ..................... 52 4.4 Discussion......................................................................................................................... 54  4.4.1 The ZI is uniquely positioned to integrate multiple sensory modalities ..... 55  4.4.2 PVN surround integration ........................................................................... 55 CHAPTER 5: General discussion ....................................................................................... 58 5.1 Overview .......................................................................................................................... 58 ix   5.1.1 Chapter 2 ..................................................................................................... 58  5.1.2 Chapter 3 ..................................................................................................... 59  5.1.3 Chapter 4 ..................................................................................................... 60 5.2 Methodological considerations ...................................................................................... 60  5.2.1 Injection of neural tracers ........................................................................... 61  5.2.2 Injection placement ..................................................................................... 61  5.2.3 Microscopy.................................................................................................. 61 5.3 Future considerations ..................................................................................................... 61  5.3.1 Functional role of the ZI ............................................................................. 61  5.3.2 Effect of chronic stressors on PVN surround circuits ................................. 62 5.4 Conclusions ...................................................................................................................... 62 References .............................................................................................................................. 64   x  List of Tables Table 3-1 RELATIVE NUMBER OF ACUTE STRESS SENSITIVE CORTICO-LIMBIC  NEURONS TO THE PVN SURROUND ............................................................ 39  xi  List of Figures Figure 1-1 A SCHEMATIC OF THE HYPOTHALAMIC-PITUITARY ADRENAL (HPA)  AXIS ...................................................................................................................... 4 Figure 1-2 HETEROGENEITY OF THE PVN ....................................................................... 6 Figure 1-3 THE RELATIVE LOCATIONS OF PVN SURROUND SUBREGIONS WITH  RESPECT TO THE PVN .................................................................................... 13   Figure 2-1 AFFERENT PROJECTIONS TO THE PVN ...................................................... 22 Figure 2-2 AFFERENT PROJECTIONS TO THE PVN SURROUND ............................... 23 Figure 2-3 PHOTOMICROGRAPH SHOWING THE PATTERN OF FLUOROGOLD  ACCUMULATION IN THE mPFC IN ANIMALS BEARING RETROGRADE  INJECTIONS IN THE ZI .................................................................................... 24 Figure 2-4 PHOTOMICROGRAPH SHOWING THE PATTERN OF FG  ACCUMULATION IN THE LS IN ANIMALS BEARING RETROGRADE  INJECTIONS IN THE sPVZ AND AHA............................................................ 26   Figure 3-1 REPRESENTATIVE PHOTOMICROGRAPH SHOWING FOS+FG DUAL  LABELED CELLS IN CORTICO-LIMBIC SITES ........................................... 38 Figure 3-2 SCHEMATIC SUMMARIZING MAJOR STRESS SENSITIVE AFFERENTS   TO THE PVN SURROUND ................................................................................ 41   Figure 4-1 PHOTOMICROGRAPH SHOWING THE PATTERN OF mPFC  PROJECTIONS TO THE ZIr .............................................................................. 51 Figure 4-2 PHOTOMICROGRAPH DEMONSTRATING AXONAL ELEMENTS IN THE  PVN AND SURROUND AFTER INJECTION OF ANTEROGRADE TRACER  IN THE ZIr........................................................................................................... 53 Figure 4-3 PHOTOMICROGRAPH INDICATING mPFC PROJECTIONS FORM  ABUTMENTS WITH PVN PROJECING CELLS IN THE ZIr ......................... 56   xii  List of Abbreviations aBST bed nuclei of the stria terminalis, anterior part ACC anterior cingulate cortex ACTH adrenocorticotropin hormone AHA anterior hypothalamic area AVP arginine vasopressin BDA biotinylated dextran amine BST bed nuclei of the stria terminalis BSTam bed nuclei of the stria terminalis, anteromedial part BSTif bed nuclei of the stria terminalis, intrafascicular part BSTpr bed nuclei of the stria terminalis, principal part CeA central nucleus of the amygdala CNS central nervous system CRH corticotropin-releasing hormone DMH dorsomedial hypothalamic nucleus DV dorsal-ventral coordinate E epinephrine ER estrogen receptor fa forceps anterior of the corpus callosum FG fluorogold IEG immediate early gene IL infralimbic part of the medial prefrontal cortex LS lateral septum LSc lateral septum, caudal part LSr lateral septum, rostral part LSv lateral septum, ventral part MeA medial nucleus of the amygdala MeApd medial nucleus of the amygdala, posterodorsal part ML medial-lateral coordinate mPFC medial prefrontal cortex MPN medial preoptic nucleus NE norepinephrine pBST bed nuclei of the stria terminalis, posterior part PFA perifornical area PL prelimbic part of the medial prefrontal cortex PS parastrial nucleus PVN paraventricular nucleus of the hypothalamus PVNdp paraventricular nucleus of the hypothalamus, dorsal parvicellular part PVNmpd paraventricular nucleus of the hypothalamus, medial parvicellular  part, dorsal zone xiii  PVNmpv paraventricular nucleus of the hypothalamus, medial parvicellular  part, ventral zone PVNpm paraventricular nucleus of the hypothalamus, posterior magnocellular  part, lateral zone PVT paraventricular nucleus of the thalamus RC rostral-caudal coordinate sPVZ subparaventricular zone V3 third ventricle  VMH ventromedial nucleus of the hypothalamus vSUB ventral subiculum of the hippocampus  ZI zona incerta ZIr zona incerta, rostral part   xiv  Acknowledgements  Since I joined the Viau lab as an undergraduate almost five years ago I have had the opportunity to learn from so many amazing people. I want to especially thank Brenda, Megan, and Martin for training me in the arts. You gave me a target to reach for and I think I might have a shot at the tissue mounting speed title. Leyla, this would have been impossible without all your technical assistance and your vision in the darkroom. Also a real big up to Nirupa! From cashew fruits to Ultimate Frisbee strategies I think we both learned a lot from our banter sessions. I can?t forget or underestimate the help I got from the undergraduates either. Jacques, the hot chocolate/coffee concoction you created has gotten me through many arduous days. And I really owe a great deal of thanks to Qiu Di. Working with you was always so easy. You will be a great veterinarian or veteran.   Last but not least of all the lab members, a big thanks to Victor. I am truly appreciative of the freedom you have given me to pursue my interests and your enthusiasm and mentorship allowed me to grow both academically and personally. Not to mention all those lunches that allowed me to grow physically.  My committee members, Dr. Seamans, Dr. Borgland, and Dr. Floresco your knowledge and feedback helped me to create a better product. I am sincerely greatful!   Finally, I need to thank my family and friends for their support over the years. Mom and Grandma, you both support me entirely regardless of circumstance. Terri, who I gain so much strength from, you taught me the merits of hard work dedication. I am lucky to have you in my life.  1  Chapter 1: Introduction  A stressor is any real or perceived threat to the homeostatic status of an organism. As such, without an adequate response mechanism, prolonged periods of stress invariably lead to aberrant physiological functioning. With this in mind, stress can be categorized further into emotional and physical aspects that show distinct but overlapping patterns of neural activation. These overlapping patterns of neural activation, however, ultimately coincide with the activation of the hypothalamic-pituitary-adrenal (HPA) axis. The purpose of this thesis is to explore how information from distal stress responding brain regions can converge on a single nucleus that is responsible for driving HPA activity.  Conservation of the HPA axis across mammalian lineages emphasizes its importance to preserve normal physiological functioning. Activation of this axis involves communication and amplification of signals from the paraventricular nucleus (PVN) of the hypothalamus to the anterior pituitary and finally to the adrenal cortex, where glucocorticoids are synthesized and released de novo. Due to the hierarchal structure of the axis, the PVN ultimately drives the entire system. As such, the PVN can be seen as an integrative center that is sensitive to inputs from multiple brain regions and ultimately, it is the integration of all these signals that directs HPA activity.  While transient activation of the HPA axis is adaptive to meet environmental demands, chronically elevated glucocorticoid levels have been linked to major depressive disorders, post-traumatic stress disorder and a myriad of affective illnesses (Dinan, 2001, Wheatland, 2005). Therefore, tight regulation of HPA activity is essential to quickly respond to homeostatic threat and then inhibit extended periods of glucocorticoid secretion that can become pathogenic. 2   Glucocorticoids released by the adrenal glands inhibit their own release through a negative feedback mechanism both in the brain and at the level of the anterior pituitary. Within the brain, glucocorticoids have the capacity to act at several sites distal to the PVN that have been intimately linked to the HPA response to stress. Included in these sites are cortico-limbic regions that, in anatomical tracing studies, have failed to demonstrate more than trace amounts of first order connectivity to the PVN (reviewed in: Herman et al. (2002b)). The PVN represents the final point where neural signals converge to influence HPA activity. Understanding the nature of circuitry both to and throughout the PVN and the immediate surround is critical in elucidating the roles of both externally and internally driven cues for HPA activation.  We seek to determine how these cortico-limbic sites showing influence over HPA activity are capable of manipulating neurons of the PVN in lieu of any direct connectivity. Cell groups in the PVN surround are in position to act as a relay to the adjacent PVN. Candidate PVN afferents in the PVN surround include the medial zona incerta, anterior hypothalamic area, subparaventricular zone, and perifornical area. Multiple studies have suggested a local influence of these neurons on HPA activity could represent part of a multi-synaptic pathway for limbic regulation of HPA activity (Roland and Sawchenko, 1993, Boudaba et al., 1996). Expanding on these findings, we seek to determine the relative connectivity of these PVN surround regions to distal limbic sites and assess their role in gating phasic stress information to the PVN. 1.1 Literature Review  This literature review will describe previously drawn hypotheses and models of function as they pertain to the HPA and its regulation by cortico-limbic regions.  3  1.2 The hypothalamic-pituitary-adrenal (HPA) axis  The hypothalamic-pituitary-adrenal (HPA) axis is a vital means by which vertebrates respond to homeostatic insults or threats that are real or perceived (Meaney et al., 1993, Viau and Sawchenko, 2002). Activation of the HPA axis by these stimuli induces a hormonal cascade culminating in synthesis and secretion of glucocorticoids from the adrenal cortex into systemic blood circulation. The hypophysiotropic cells of the PVN synthesize and secrete corticotropin releasing hormone (CRH) into the hypophyseal portal system. CRH binds receptors on corticotropic cells in the anterior pituitary, which stimulates the synthesis and secretion of adrenocorticotropic hormone (ACTH) into the system blood circulation. Subsequently, ACTH then travels to zona fasciculata layer of the adrenal cortex where it stimulates the de novo synthesis and secretion of glucocorticoids (Figure 1-1). The primary rodent glucocorticoid is corticosterone, while in humans it is cortisol. Glucocorticoids, in turn, incite broad physiological and behavioral changes to return the body to its original basal state (Munck et al., 1984, McEwen and Stellar, 1993). These activities include mobilization of energy stores via catabolisis and gluconeogenesis, suppression of immune function, and increasing the pool of glucose available to the central nervous system. In clinical settings, application of synthetic glucocorticoid cortisone can emulate these actions to, as an example, reduce chronic swelling. However, extended and chronic exposure of elevated glucocorticoids can cause a myriad of problems. Such activation of the HPA axis can lead to immunodeficiency, depression, anxiety, decreased lean body mass and increased fat deposit  (Dinan, 2001, Wheatland, 2005). In fact, the aforementioned clinical uses of cortisone can lead to iatrogenic disease in cases of prolonged treatment.   4    Figure 1-1. Schematic of the hypothalamic-pituitary-adrenal axis. Central stress circuitry converges in the hypothalamus to stimulate the release of CRH from hypophysiotropic cells. CRH, in turn, stimulates adrenocorticotropes in the anterior pituitary to release ACTH into system circulation. ACTH then drives the release of CORT from the adrenal cortex. 5   Modern living places an undue amount of strain on our stress response system and this has led to increased incidences of HPA associated disease which poses a substantial burden to global health and medical care resources (Wittchen, 2002). To curtail this growing issue there is a great need to increase our understanding of the HPA axis. Through further characterization of the neural circuits involved we can discover new means for treatment of HPA. 1.2.1 Importance of the paraventricular nucleus on stress response  The multifaceted role of the (PVN) is well indicated by the anatomical organization of separate and distinct cell groups. Connectional and phenotypic observations ramify the various PVN outputs and their role on whole body homeostasis (Swanson and Sawchenko, 1983). These cell groups include posterior magnocellular (pm), medial parvocellular dorsolateral (mpd), dorsal parvocellular (dp), and medial parvocellular ventral (mpv) (Figure 1-2) (Swanson and Sawchenko, 1983, Herman et al., 2002a, Viau and Sawchenko, 2002).  The dp and mpv PVN regions are collectively autonomic and send efferents that stimulate release of norepinephrine (NE) and epinephrine (E) from the medulla of the adrenal glands (Motawei et al., 1999). Catecholamine release acts systemically to increase blood pressure and contractility of skeletal muscle in a manner characteristic of the fight/flight response or alternatively, the alarm phase of Hans Selye?s general adaptive syndrome theory of stress (Selye, 1973).   6    Figure 1-2. Heterogeneity of the PVN. Nissl stain of the PVN indicating the medial parvocellular and laterally displaced magnocellular subregions. dp, dorsal parvocellular; mpv, ventral medial parvocellular; mpd, dorsal medial parvocellular; pm, posterior magnocellular; pv, periventricular part of the PVN.  7   Magnocellular neurosecretory cells of the pm regions directly release oxytocin and vasopressin into the posterior pituitary and thus into systemic circulation (Herman et al., 2002a). Phenotypically similar neurons of the supraoptic nucleus coincide in their activity with the pm region of the PVN.  Finally, the mpd cells represent the hypophysiotropic arm of the PVN. Connectional and phenotypic studies clearly identify the mpd as driving the HPA response (Swanson and Sawchenko, 1983, Herman et al., 2002a, Viau and Sawchenko, 2002). The expression of immediate early genes (IEGs) FOS is reliably induced within the PVN upon exposure to an acute stress stimulus. Variability in expression pattern exists among PVN regions but induction of CRH mpd neurons persists in all stress stimulus conditions (Ceccatelli et al., 1989, Chen et al., 1993).  1.3 Limbic centers have the capacity to influence HPA activity  Physiological indicators of HPA activity, in response to basal and stress-induced conditions, demonstrate a substantial role for cortico-limbic regions in the regulation of the  HPA axis (reviewed in:Herman et al. (2005)). The limbic regions include, but are not limited to, the lateral septum (LS), medial prefrontal cortex (mPFC), amygdala, and ventral subiculum (vSUB) of the hippocampus (Herman et al., 2005). These regions will be briefly described here. 1.3.1 Lateral septum    The lateral septum is a forebrain structure that has been implicated as having a role in HPA activity regulation. There is evidence that LS has a role in the suppression of HPA activity. Multiple studies that performed lesions in the LS found increased measures of HPA activity (Usher et al., 1974, Dobrakovov? et al., 1982, Seggie, 1987, Singewald et al., 2011). 8  In addition, it was observed that lesions of the LS result in increased scores of anxiety (Yadin et al., 1993).  The lateral septum is a particularly large structure and can be divided into multiple regions: the ventral (LSv), intermediate (LSi), and dorsal (LSd) (Risold and Swanson, 1997). Of particular note, the LSv sends a large number of efferents to the hypothalamus (Risold and Swanson, 1997) and appears to be preferentially activated in response to stress as evident through assay for IEG indicators (Cullinan et al., 1996, Li and Sawchenko, 1998). 1.3.2 Medial prefrontal cortex  Based on functional activity and efferent connectivity, the medial prefrontal cortex (mPFC) can be broken into subregions which include the prelimbic (PL) and infralimbic (IL) regions (Fuster, 2008). On the basis of efferent connectivity, the IL projects strongly to other limbic structures including the lateral septum and amygdala as well as hypothalamic relays which include the bed nucleus of the stria terminalis (BST) and preoptic nuclei. Alternatively, the PL projects primarily to the thalamus, basolateral amygdala, and raphe nuclei (Vertes, 2004). Note that the anterior cingulate cortex (ACC) extends dorsally from the PL and is often included as an anatomical extension of the PL or vice versa.  Expression of FOS is increased in both the mPFC and PVN in response to restraint, which is an emotional stressor. This suggests that, with regards to emotional stress, the mPFC plays a role in HPA activity (Figueiredo et al., 2003). Further, bilateral mPFC lesion potentiates the HPA axis response to restraint stress without altering the response to ether exposure (Diorio et al., 1993). Finally, supporting evidence demonstrates that lesions to the dorsal mPFC (anterior cingulate and prelimbic arms) leads to increased CRH and Fos 9  expression in the hypophysiotropic zone of the PVN suggesting loss of inhibitory activity (Radley et al., 2006).  1.3.3 Amygdala  The amygdala is comprised of multiple subregions which are the cortical, basolateral, central amygdala (CeA) and medial amygdala (MeA). However, the bulk of projections from the amygdala that influence the HPA originate from the MeA and CeA (Swanson and Petrovich, 1998). Evidence suggests the amygdala exerts a stimulatory influence on the HPA axis. Physiological indicators of HPA activity, such as ACTH and corticosterone, are decreased when the MeA and CeA are lesioned either selectively or in concert (Allen and Allen, 1974, Beaulieu et al., 1986, Van de Kar et al., 1991, Feldman et al., 1994, Dayas and Day, 2002). In agreement with this data, stimulation of these same parts of the amygdala can drive HPA activity as observed through substantial increases in ACTH and corticosterone (Matheson et al., 1971, Redgate and Fahringer, 1973, Dunn and Whitener, 1986).  1.3.4 Hippocampus/subiculum  The ventral subiculum (vSUB) of the hippocampus represents the major HPA regulatory output of the hippocampus. Directed lesion studies, as well as fiber transection experiments that effectively obliterate functional vSUB efferents result in increases of HPA activation (Herman et al., 1992, Bradbury et al., 1993, Herman et al., 1995, Herman et al., 1998).  The hippocampus is an important structure in HPA regulation due to the high expression of glucocorticoid and mineralocorticoid receptors representing a prominent site for negative feedback regulation (Reul and Kloet, 1985, Reul and De Kloet, 1986, Aronsson et al., 1988, Arriza et al., 1988, Herman et al., 1989a, Herman et al., 1989b). 10  1.3.5 Summary  Although there is substantial evidence that the aforementioned cortico-limbic regions play a role in HPA regulation, it still remains unclear what neurocircuitry is involved in conveying this information. Other previously described relays, such as those found in the BST, suggest that this information can take disynaptic routes to the PVN(Radley et al., 2009). The goal of this thesis is to characterize and confirm additional routes cortico-limbic regions take to send information to the PVN.  1.4 Connectivity to the PVN  Direct projections to the PVN originate from nuclei that deal with physiological stimuli and interoceptive fields. Due to the nature of these direct connections, stimuli that represent substantial physiological threats can induce direct activation of the PVN to modify HPA activity. For example, in response to hemorrhage or peripheral inflammation there is cellular activation within the hindbrain regions of the nucleus of the solitary tract, ventrolateral medulla, locus coeruleus, and parabrachial nucleus (Thrivikraman et al., 1997, Hollis et al., 2004). These hindbrain regions then directly stimulate activation of the HPA axis.   In contrast to stressors that result in strong cellular activation in hindbrain regions, cortico-limbic sites are predominantly activated by stressors described as emotional, predictive, or processive. Examples of these are restraint stress, forced swim, open-field, and social stress among others (Cullinan et al., 1995, Herman and Cullinan, 1997, Li and Sawchenko, 1998, Dayas et al., 2001). The defining feature of these stressors is they do not, on their own, represent imminent homeostatic threat. Instead, individual interpretation of such stressors results in recruitment of cortico-limbic centers previously described. 11   There are opportunities for interaction between cortico-limbic and hindbrain circuitries. Footshock stress, a test that is both a processive and physiological (pain) stressor, both hindbrain activation in addition to limbic activation in the prefrontal cortex, lateral septum, and medial amygdala is observed (Li and Sawchenko, 1998). This suggests that while medullary and hindbrain nuclei directly drive the hypophysiotropic cells of the PVN, limbic nuclei can modulate these effects either through connections to hindbrain regions or through integration at relay sites. 1.4.1 Limbic connectivity to the PVN  The consequence of the hierarchical construction of the HPA axis is that all information pertaining to HPA activity must converge at the level of the PVN to form a single concerted response. Though cortico-limbic regions play a substantial role in directing HPA activity, neural tracing studies have failed to demonstrate significant projections from these regions directly to the hypophysiotropic cells of the PVN (Herman et al., 2002b).   Previously, the BST has been described as a relay for limbic signals to converge onto the PVN and influence HPA activity. Described limbic regions converging onto both the anterior BST (aBST) and posterior BST (pBST) include the subiculum, dorsal mPFC including the PL and anterior cingulate cortex (ACC), CeA, and MeA (Cullinan et al., 1993, Prewitt and Herman, 1998, Radley et al., 2006, Choi et al., 2007, Radley et al., 2009).   Terminal fields from the mPFC, vSUB, LS, and medial amygdala (MeA) also aggregate in the hypothalamic regions directly adjacent to the PVN (Oldfield et al., 1985, Sesack et al., 1989, Hurley et al., 1991, Cullinan et al., 1993, Canteras et al., 1995, Risold and Swanson, 1997). However, as the dendrites of PVN neurons do not extended beyond the morphological confines of the PVN (Van Den Pol, 1982), limbic signals in the surround 12  cannot contact hypophysiotropic mpd cells of the  PVN. This suggests cortico-limbic circuits acting through the PVN surround require a relay akin to what is seen in the BST. 1.5 The PVN surround is in position to relay limbic signals to the PVN  As previously mentioned, cortico-limbic regions send terminal fibers to the PVN surround. The population of cells within the PVN surround are predominantly GABAergic (Roland and Sawchenko, 1993, Boudaba et al., 1996, Risold and Swanson, 1997, Swanson and Petrovich, 1998, Herman et al., 2002b). It is estimated that one third of all input to the PVN is GABAergic (Mikl?s and Kov?cs, 2002). Therefore, it is reasonable to hypothesize the immediately adjacent and GABA rich PVN surround is an important origin of this input. These GABAergic synapses are found both in the parvocellular and magnocellular regions but stronger GABAergic perikarya staining is observed in the parvocellular regions which drive the HPA axis (Van Den Pol, 1982, Roland and Sawchenko, 1993).  The use of retrograde tracer deposits directed at the PVN implicates distinct cell clusters in the surround with the capacity to innervate the PVN. These clusters include the anterior hypothalamic area (AHA), perifornical area (PFA), subparaventricular zone (sPVZ) and zona incerta (ZI) (Figure 1-3) (Roland and Sawchenko, 1993). However, the extent to which the regions of the PVN surround are in receipt of distinct or overlapping limbic innervation remains to be determined. Further, the capacity for cortico-limbic terminals to interact specifically with PVN projecting neurons of the PVN surround is unclear.  1.5.1 Projections to the PVN surround  The PVN surround is in receipt of afferents from all of the divisions of the MeA (Canteras et al., 1995) as well as the LS (Risold and Swanson, 1997), interfascicular nucleus   13    Figure 1-3. The relative locations of PVN-surround subregions with respect to the PVN. These specific regions have been implicated as having the capacity to project to the PVN proper. V3, third ventricle; ZI, zona incerta; PFA, perifornical area; AHA, anterior hypothalamic area; sPVZ, subparaventricular zone; PVN, paraventricular nucleus of the hypothalamus. 14  of the BST, and all of the regions of the medial preoptic nucleus (MPN). Clearly the PVN surround is an area that may contribute substantially to limbic influences on HPA activity.  There is also evidence that the brain stem sends input to the PVN surround. These include cholinergic projections from the nucleus of the solitary tract (Cunningham and Sawchenko, 1988, Cunningham et al., 1990) as well as serotonergic fibers from midbrain raphe nuclei (Larsen et al., 1996). Note that medial serotonergic nuclei of the brainstem do not show significant projections to the PVN itself. Therefore, any ascending serotonergic inputs are likely able to exert their effects over HPA axis through the PVN surround. 1.6 Summary and specific hypotheses  Forebrain cortico-limbic regions have a strong influence over HPA activity, but this is in lieu of any substantial direct connectivity to the PVN itself. The PVN surround is in a position to send efferents to the PVN and tracing experiments have demonstrated that the surround is in receipt of cortico-limbic projections. However, the extent to which the regions of the PVN surround are in receipt of distinct or overlapping degrees of limbic innervation remains to be determined. Further, the capacity for cortico-limbic terminals to interact with PVN projecting neurons of the PVN surround is unclear. For these experiments we utilized neuronal tracing and immunohistochemical procedures to 1) determine the relative amounts of cortico-limbic neurons that project to the PVN surround, 2) assess the capacity of PVN surround projecting neurons to respond to emotional stress, and 3) confirm the presence of a cortico-limbic to PVN relay that exists in the PVN surround.   15  1.6.1 Chapter 2. Hypothesis 1 Different regions of the PVN surround are in receipt of unique patterns and strengths of cortico-limbic input.  It has been demonstrated that terminal fibers from cortico-limbic regions converge upon the PVN surround; however, it remains to be determined what the relative strengths of these connections are to the distinct PVN surround sub-regions which include the AHA, sPVZ, ZI, and PFA. Further, it is unclear what subregions of these distal cortico-limbic regions are sending efferents to the PVN surround. To explore this, we made discrete injections of retrograde tracer fluorogold (FG) in each of these sub-regions. This allowed us to make qualitative comparisons about the relative strengths of connectivity as well as observe the pattern of retrograde signal accumulation in distal sites. 1.6.2 Chapter 3. Hypothesis 2 Cortico-limbic inputs to the PVN surround are sensitive to emotional/psychological stressors.  Cortico-limbic regions are involved in modifying HPA activity in response to emotional/psychological stressors as described above (Section 1.3). Therefore, we predicted that associated projections to the PVN surround would be activated in response to an acute episode of restraint stress, a potent emotional stressor. Through the use of combined IEG FOS + FG immunohistochemistry we were able to determine the relative contributions of stress reactive cells to each of the PVN surround sites.  1.6.3 Chapter 4. Hypothesis 3 In the PVN surround, cortico-limbic fiber terminals synapse with neurons that have the capacity to project onward to the PVN.   For this experiment we used dual injections of neural tracer. Retrograde injections of FG were directed at the PVN while anterograde injections of biotinylated dextran amine (BDA; 10000MW) were targeted to the mPFC. We chose to focus on the mPFC as this 16  region possessed the greatest number of cells and stress-reactive cells projecting to the PVN surround. This was determined from the previous experiments detailed in this thesis.   17  CHAPTER 2: Characterization of forebrain regions that project to the paraventricular nucleus of the hypothalamus surround 2.1 Introduction  As summarized in Chapter 1, multiple cortico-limbic regions are intimately involved in the regulation of HPA activity. However, as there are no substantial first order connections to bridge cortico-limbic and hypophysiotropic cells of the PVN, these signals must be relayed through distal intermediaries.   Multiple regions of the PVN surround, including the AHA, sPVZ, PFA, and ZI, are in position to receive cortico-limbic afferents and relay signals onward to the PVN (Roland and Sawchenko, 1993). Further, multiple forebrain cortico-limbic regions, as indicated through anterograde tracing studies, show substantial densities of terminal fibers in the PVN surround. These forebrain groups include the vSUB, mPFC, LS, MeA, and PVT (Hurley et al., 1991, Canteras et al., 1995, Moga et al., 1995, Risold and Swanson, 1997, ?ng?r et al., 1998).   We sought to determine the relative strength and overlapping patterns of cortico-limbic connections to the individual candidate relays of the PVN surround which include the AHA, sPVZ, PFA, and ZI. Understanding the strength and contributions of cortico-limbic afferents to each of the PVN surround regions will allow us to begin addressing how these individual subregions can guide HPA activity.  To accomplish this we targeted injections of retrograde tracer FG at each of the PVN surround regions (AHA, sPVZ, PFA, ZI). Using stringent criteria for tracer deposit size, intensity, and location, we were able to make relative comparisons about the number of retrogradely labeled cells distally. Finally, the cortico-limbic structures measured are not monolithic structures so we wanted to explore the pattern of retrograde accumulation in these 18  distal sites. This allows us to gauge the role afferents from different sections of a single cortico-limbic site may have on the PVN surround.   2.2 Methods 2.2.1 Animals  Adult male Sprague-Dawley rats (Charles River, St. Constant, Canada) were used, weighing 270-290g on arrival and 290-310g on day of surgery. Animals were pair-housed under controlled temperature and lighting conditions (12:12-hour light:dark cycle, lights on at 0700 hours) with food and water available ad libitum. All experimental protocols were approved by the University of British Columbia Animal Care Committee. 2.2.2 Surgery  Animals were induced and kept at a surgical plane of anaesthesia using isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane) gas in conjunction with pure oxygen as a carrier gas. At time of induction animals also received prophylactic analgesic subcutaneous injection of NSAID (meloxicam; 0.3mg/kg body weight) and lactated Ringer?s saline. Local subcutaneous injection of mepivacaine (0.25%) above the skull acted as a local analgesic.   Following the incision and exposure of the skull, a small hole was drilled to expose the brain and allow introduction of the injection unit. Bregma was determined from skull suture marks and used as a landmark to determine rostral-caudal (RC) coordinate. Medial-lateral (ML) coordinates were based off the superior sagittal sinus, exposed through drilling, which runs between hemispheres of the brain.  Dorsal-ventral (DV) coordinates were derived from distance the dura meningeal layer. Using these guidelines we based our stereotactic coordinates according to Swanson (2004). ZI ? RC: +1.30 mm, ML: -1.00 mm, DV: -6.8mm; sPVZ ? AP: +1.20 mm, ML: -0.25 mm, DV: -7.20 mm; PFA ? AP: +1.20 mm, ML: -1.00 19  mm, DV: -7.00 mm; AHA ? AP: +1.20 mm, ML: -0.50 mm, DV: -7.50 mm; PVN ? AP: +1.20 mm, ML: -0.25mm, DV: -7.00mm.   Glass micropipettes (20-25?m outer diameter; Sutter Instruments, Novato, CA) were backfilled using Hamilton syringe with FG 2% (w/v). The parameters for iontophoresis were 2.5?A alternating current, 7s on/off, 2.5mins. Micropipette tips were descended into the brain and left for 5mins before and after application of current to prevent the movement of tracer deposits along the micropipette tract.  2.2.3 Tissue collection  Optimal transport time for FG transport is fourteen days (Schmued and Fallon, 1986, Moga and Saper, 1994, Tillet et al., 2000), therefore, two weeks post-surgery animals were subjected a single episode of restraint stress for 60mins before being terminally anaesthetized with chloral hydrate (200mg/kg) and perfused via ascending aorta with 0.9% saline (125mL; 4?C) preceding 4% paraformaldehyde (pH 9.5; 4?C; 500mL). Brains were then collected and post-fixed for 4hrs in paraformaldehyde before being transferred to 15% sucrose in 0.1M potassium phosphate-buffered saline (KPBS, pH 7.4) and left 12-16hrs at 4?C. Brains were then sectioned into 5 adjacent series of 30um thick sections into cryoprotectant (30% ethylene glycol, 20% glycerol in 0.05M potassium phosphate buffer solution (KPBS) buffer) and stored at -20?C. 2.2.4 Immunohistochemistry  To detect tracer deposits and distal FG accumulating cells, cryoprotectant was removed from the tissue with serial KPBS rinses before being treated with H2O2 (0.3%) to remove endogenous peroxidase activity followed by treatment with reducing agent NaBH4 (1% w/v) to reduce paraformaldehyde linkages and unmask antigens. These treatment steps were 20  interceded by serial rinses of KPBS to remove residual reaction products. Tissue was then incubated in primary FG rabbit anti-sera (1:10,000; Millipore, Billerica, MA) for 48hrs at 4?C in a KPBS-Triton solution (0.3% Triton-X; Sigma, Oakville, Ont.) containing 2% normal goat serum (NGS).  Sections were rinsed with KPBS before incubation in secondary anti-rabbit biotinylated antibody (1:450) for 1 hour at room temperature preceding detection using a non-nickel-intensified avidin-biotin-immunoperoxidase procedure (Vectastain Elite ABC kit; Vector Laboratories, Burlington, CA). 2.2.5 Imaging and analysis  To estimate the relative contributions of cortico-limbic afferents to the various regions of the PVN surround, FG-ir cell counts were taken throughout the forebrain.  Using light microscope (LeicaDMR) under brightfield conditions, individual positive cells were manually counted throughout entire brain nuclei. Labeled cells were characterized by accumulation of brown reaction product within the cytoplasm. Neuronal borders of the soma were sharply lined by reaction product allowing clear differentiation of individual cells. Counts were multiplied by five to account for the sectioning interval. Therefore, cell numbers are an estimate of total brain nucleus labeling versus a single series of tissue. Cell counts are expressed as the mean ? SEM. 2.3 Results  Using retrograde tracer fluorogold we compared the relative afferent contributions of distal brain nuclei to each of the several PVN surround sites that include the zona incerta (ZI), anterior hypothalamic area (AHA), subparaventricular zone (sPVZ), and perifornical area (PFA). We also directed iontophoretic injections at the PVN to use as a comparative control. 21  The use of iontophoresis allowed us to create restricted injections into each site. To make qualitative comparisons between groups, only those injections of comparable intensity, size, and location in three-dimensional space were used. Finally, by following these guidelines for strict injection evaluation and comparison we found the overwhelming majority of retrograde labeling on the side ipsilateral to injection site.  2.3.1 Limbic-related; PFC, LS, MeA, PVT  In agreement with previous studies, the retrograde injections directed at the PVN showed limited uptake in cortico-limbic regions including the PFC (Sesack et al., 1989, Hurley et al., 1991, Radley et al., 2006), LS (Risold et al., 1994, Risold and Swanson, 1997), and amygdala (Canteras et al., 1995, Prewitt and Herman, 1998) (Figure 2-1). This serves to confirm the placement and concentration of our injections within the PVN as any tracer spreading into the PVN surround will result in substantial cortico-limbic labeling as described below. Further, retrograde accumulation in the parastrial nucleus (PS) is unique to the PVN and provides another means of comparative control between injections made in the PVN surround and those infringing upon the PVN.  Very large numbers of retrogradely labeled cells were detected in the mPFC in animals bearing retrograde injections within the ZI (Fig. 2-2B). The majority of the mPFC labeled cells were found within the prelimbic (PL) subregion (1438.57 ? 403.66), though uptake extended ventrally into the infralimbic (IL) (587.14 ? 122.72) arm and dorsally into the anterior cingulate cortex (ACC) (293.57 ? 109.88) (Fig. 2-3). Strong tracer accumulation in the mPFC was observed almost exclusively in animals bearing injections in the ZI. This is in affirmation of past work that described substantial mPFC uptake when injections of   22    Figure 2-1. Photomicrograph of the appearance of iontophoretic injections of fluorogold (FG) into the PVN (A) and the number of FG accumulating cells in distal forebrain regions (B) (n=8). mPFC, medial prefrontal cortex; LS, lateral septum; aBST, anterior bed nuclei of the stria terminalis; pBST, posterior bed nuclei of the stria terminalis; MPN, medial preoptic nucleus; DMH, dorsomedial nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus; MeA, medial amygdala; PVT, paraventricular nucleus of the thalamus. Scale bar = 500?m 23   Figure 2-1. Photomicrograph of the appearance of iontophoretic injections of fluorogold (FG) into the distinct regions of the PVN-surround (A,C,E) and the number of FG accumulating cells in distal forebrain regions (B,D,F) (B, n=8; D, n=6; F, n=8). mPFC, medial prefrontal cortex; LS, lateral septum; aBST, anterior bed nuclei of the stria terminalis; pBST, posterior bed nuclei of the stria terminalis; MPN, medial preoptic nucleus; DMH, dorsomedial nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus; MeA, medial amygdala; PVT, paraventricular nucleus of the thalamus. Scale bar = 500?m 24    Figure 2-3. Pattern of retrograde accumulation in animals bearing injections of FG in the ZI. Darkfield view demonstrates FG labeled cells in deep layers of the mPFC in the PL and IL subregions. Note that labeled cells were also found dorsal to the PL in the anterior cingulate cortex. PL, prelimbic; IL, infralimbic. Scale bar = 500. 25  retrograde tracer, directed at the PVN, extended dorsally beyond the morphological confines of the PVN (Williamson and Viau, 2007).   In comparison, retrograde uptake in the amygdala was observed in animals bearing injections in either the AHA (525.00 ? 155.54) and sPVZ (501.88 ? 79.87) regions of the PVN surround (Figure 2-2 D&F). The bulk of retrograde signal accumulation in the amygdala was overwhelmingly in the posterodorsal portion of the medial amygdala (MeA). The central and basolateral amygdala possessed only limited and scattered labeled cells. Again, this is in agreement with previous work that found retrograde injections made ventral to the PVN resulted in retrograde uptake in the MeA (Williamson and Viau, 2007).  The paraventricular nucleus of the thalamus (PVT) also demonstrated strong accumulation when injections were made within the PVN surround. Animals bearing ZI injections showed the greatest number of labeled cells (987.78 ? 323.50) though injections into the sPVZ (571.88 ? 84.56) also resulted in strong uptake in the PVT (Figure 2-2B, F)  Strong uptake within the lateral septum was observed in animals with injection in the AHA (1762.50 ? 171.50), ZI (1880.00 ? 521.29), PFA (1360.63 ? 222.58), and sPVZ (2900.00 ? 549.79) (Figure 2-2B, D, F). Additionally, there were characteristic differences in the pattern of accumulation in the LS between injection groups. Those animals bearing injections of tracer in the sPVZ showed dense labeled cells in the ventrolateral subdivision of the LS (LSvl). Alternatively, animals with injections made in the AHA, ZI, and PFA showed comparable diffuse scattering of labeled cells in the LSvl which extended into the dorsal subdivision of the LS (LSd) (Figure 2-4).      26    Figure 2-4. Pattern of retrograde accumulation in animals bearing injections of FG in the sPVZ (A) and AHA (B). Darkfield image demonstrates the differences in the pattern of FG accumulation in the LS. Injections made in the sPVZ resulted in dense numbers of cells in the LSv while those injections in the AHA more cells in the LSd. Retrograde injections made in the ZI were similar to those made in the AHA and represented in (B). Scale bar = 500um. 27  In summary, when considering cortico-limbic accumulation of retrograde tracer, those injections directed at the ZI resulted in the greatest number of labeled cells in the mPFC and PVT. Further, there was considerable uptake found within the LS making the ZI a particularly promising region of the PVN surround for cortico-limbic influences that may converge upon the HPA axis. 2.3.2 Hypothalamus and bed nuclei of the stria terminalis  The number of labeled cells in the anterior bed nucleus of the stria terminalis (aBST) was consistent across regions bearing retrograde injections: PVN (340.63 ? 53.15), ZI (218.89 ? 75.77), sPVZ (265.63 ? 31.16), and AHA (259.17 ? 43.21). Of the subregions of the aBST, the majority of cells were located in the anteromedial (am) division (PVN:174.38?30.63; ZI:120.56?47.69; sPVZ:158.13?15.35; AHA:176.67?30.65). This region of the aBST is particularly important in HPA regulation (Choi et al., 2007)   The posterior bed nucleus of the stria terminalis (pBST) projects most heavily to the sPVZ (585.00 ? 55.90) and AHA (743.33 ? 156.41) with moderate projections to the ZI (314.44 ? 86.79) and PVN proper (229.38 ? 30.89). The majority of these pBST projections originate from the principal (BSTpr) (PVN:98.13?24.89; ZI:123.33?38.57; sPVZ:303.75?44.31; AHA:345.83?87.71) and interfascicular (BSTif) (PVN:101.25?14.63; ZI:155.56?43.74; sPVZ:226.88?14.14; AHA:295.00?84.39) subregions.  Injections made into the PVN resulted in direct labeling in the PS (385.00 ? 49.07), medial preoptic nucleus (MPN) (551.25 ? 107.99), and dorsomedial nucleus of the hypothalamus (DMH) (675.00 ? 121.27) and ventromedial nucleus of the hypothalamus (VMH) (554.38 ? 76.15) in agreement with past findings (Cullinan et al., 1996). As previously mentioned, when compared to PVN injection groups, injections made in the PVN 28  surround resulted in minimal labeling of the PS. Further, no regions of the PVN surround were found to be in receipt of any substantial afferents from the DMH.  The MPN projected to all regions of the PVN surround, though injections made in the AHA resulted in the greatest number of cells found (414.17 ? 38.50). The ventromedial nucleus of the hypothalamus (VMH) sends a large number of efferents to the ventral PVN surround, which includes the sPVZ (1346.88 ? 69.46) and AHA (1411.67 ? 128.53). 2.3.3 Summary  Though there is evidence of strong projections from some midline hypothalamic and BST cell groups to the PVN surround, the bulk of afferent connections appear to originate from cortico-limbic sites that include the mPFC, LS, MeA, and PVT.  The opportunity for integration between distal afferents at the PVN surround suggests these regions could hold some measure of responsibility for processing a multitude of sensory information. 2.4 Discussion  The use of immunohistochemical procedures allowed us to visualize the placement and accumulation of retrograde tracer in local and distal sites. Iontophoretic injections ensure discrete tracer placement concentrated within the borders of each targeted brain region. Those injections centered within the morphological confines of the PVN yielded negligible amounts of tracer uptake in distal limbic sites in agreement with previous studies (see Results). Further, injections made into the surround showed little to no uptake in the parastrial nucleus which does not project to any of the explored PVN surround regions (Tsubouchi et al., 2007). Based on this, we are confident in the accuracy of our retrograde tracer injections in the PVN surround as retrograde uptake profiles for each region showed substantial and unique patterns of limbic uptake. 29  2.4.1 PVN surround shows unique patterns of cortico-limbic connectivity  Observing the patterns of retrograde accumulation in cortico-limbic sites, it becomes apparent that PVN surround subregions can be separated by their afferent connectivity. The distinct patterns of cortico-limbic afferent connections to separate cell groups in the PVN surround suggest these regions have unique and separate roles in HPA control.  The ZI acts as the main synapse point for both the mPFC and PVT. Alternatively, the MeA projects strongest to the AHA and sPVZ which lie in the ventral portion of the PVN surround. Interestingly, the LS projects heavily to all regions of the PVN surround and is the main afferent to the sPVZ, PFA, and AHA. However, due to the unique patterns of retrograde accumulation in the LS in different injection groups, it is difficult to speculate on the role these connections may have on HPA. Past research implicates the LSvl and ventral LSi in gating some aspects of emotional stress (Cullinan et al., 1996, Li and Sawchenko, 1998). Given the pattern of retrograde accumulation, this would suggest the sPVZ is the primary PVN surround relay of stress sensitive LS information to the PVN. 2.4.2 Heterogeneous efferents from LS to PVN surround  Our results suggest that the LS sends projections to all the explored regions of the PVN surround. However, as a function of injection site, we observed differences in retrograde accumulation in the LS. Injections of retrograde tracer directed at the sPVZ resulted in very intense cellular labeling in the LSvl. Alternatively, similar injection made in the AHA and ZI produced much more diffuse patterns of labeled cells that encompassed a large dorsal-ventral component of the rostral LS.  As previously mentioned, the bulk of restraint stress activated cells of the LS are located within the LSvl. As the sPVZ had the greatest number of retrograde accumulating 30  cells clustered into this subregion of the LS, it suggests the sPVZ plays the largest role of the PVN surround regions in gating stress related information from the LS to the PVN. However, to further explore, in Chapter 3 we utilized an emotional stressor known to activate cortico-limbic stress circuitry in conjunction with retrograde tracing methods. This allows us to determine individual cells that are activated by emotional stress and project to separate PVN surround subregions. 2.4.3 The ZI receives robust projections from the mPFC  As previously mentioned the rostral ZI is situated at the dorsal aspect of the caudal third of the PVN and extends in a tract laterally and caudally. Previous studies have indicated that the ZI has four main functions related to control of visceral activity (Mok and Mogenson, 1986, Spencer et al., 1988, Sanghera et al., 1991, Tonelli and Chiaraviglio, 1993, 1995), arousal (Shammah-Lagnado et al., 1985, Berry et al., 1986, Power and Mitrofanis, 2001), attention shifting (Ficalora and Mize, 1989, Nicolelis et al., 1992, May et al., 1997, Mitrofanis, 2002), and posture and locomotor behaviour (Mogenson et al., 1985, Milner and Mogenson, 1988, Supko et al., 1991, 1992, Murer and Pazo, 1993, P?rier et al., 2002). Our findings that the ZI is in receipt of afferents from cortico-limbic regions of the brain involved in HPA axis activity suggests the ZI could play a role in directing behavioral adaptations to stress.   Further, in agreement with past findings we found strong uptake of retrograde tracer in the cingulate cortex extending ventrally into the prelimbic (PL) and infralimbic (IL) subregions of the mPFC when injections were centered within the ZI (Roger and Cadusseau, 1985, Mitrofanis and Mikuletic, 1999). The mPFC plays a potent role in regulating the HPA 31  axis (Diorio et al., 1993, Amat et al., 2005) therefore, the ZI may represent a site for integration of cortical signals to modulate HPA activity.   In the mPFC, the majority of labeled cells were in the dorsal aspect in the PL; however, there were still substantial amounts of labeled cells in the IL arm. Interestingly, these two subdivisions of the mPFC have been proposed to have opposing roles on HPA activation.  Lesions of the dorsal mPFC, that encompassed the ACC and PL, resulted in enhanced reactivity to stress stimuli. Alternatively lesions targeting the IL regions attenuated hormonal measures of HPA activity (Radley et al., 2006). That the ZI receives projections from both subregions strengthens the possibility of the ZI acting as a primary mPFC integration center to drive or inhibit HPA activity.   Phenotypically, past studies confirm that the ZI contains both GABAergic (Nicolelis et al., 1995, Kolmac and Mitrofanis, 1999) and glutamatergic cells (Kolmac and Mitrofanis, 1999). Therefore, the capacity for discrete PL and IL circuits to converge upon uniquely GABAergic or glutamatergic cell populations may contribute to the contrasting role in HPA activation and inhibition of the mPFC.  2.4.4 Polarity of the PVN surround  The role of the PVN surround, in regards to activating or inhibiting the HPA axis, is not entirely clear. The main cellular phenotype within the PVN surround is overwhelmingly GABAergic (Roland and Sawchenko, 1993, Boudaba et al., 1996). However, there is evidence of glutamatergic PVN excitation originating from the PVN surround (Boudaba et al., 1997) though the exact origin of these projections in the surround remains unclear. Both cellular phenotypes are responsive to glutamate stimulation via ionotropic glutamate receptors (Boudaba et al., 1997, Ziegler and Herman, 2000) suggesting that glutamatergic 32  projections from cortico-limbic sites could have either inhibitory or stimulatory effects on HPA activity.   GABAA receptor mRNA is also present in the PVN surround, in addition to the hypophysiotropic PVN. This emphasizes the potential for GABA-GABA disinhibition of the PVN which would correlate with stimulatory effects of the MeA on the HPA axis. Alternatively, those cortico-limbic regions that are implicated in inhibitory effects on the HPA axis may exert their effects through glutamatergic projections that act on GABAergic neurons of the surround. 2.5 Summary  This study demonstrates that the PVN surround is a robust recipient of afferents that originate from multiple cortico-limbic sites including the mPFC, LS, MeA, and PVT. Further, distinct PVN surround regions show unique profiles of connectivity to cortico-limbic sites.  Evidently, due to the strength of cortico-limbic connectivity to the PVN surround there exists a very strong likelihood that the PVN surround is an important relay point for limbic to PVN connectivity.   33  CHAPTER 3: Characterization of stress-responding limbic-related inputs to the paraventricular nucleus of the hypothalamus surround 3.1 Introduction  The role of cortico-limbic regions in HPA axis activity is very well described and multiple structures have shown, through IEG indicators, pharmacological manipulation, and surgical lesion, that certain stressors are particularly well suited to activating the limbic HPA circuits. Specifically, cortico-limbic regions are most involved with directing HPA responses to emotional or psychological stressors (Duncan et al., 1993, Li et al., 1996, Li and Sawchenko, 1998). Due to multiple levels of synapses and integration that precede convergence to the PVN, these types of cortico-limbic circuits permit the integration of contextual cues, past experience, and anticipatory reactions in novel circumstances. Comparatively, physiological threats do not require processing and these signals arrive to the PVN via first order afferents.  Alternatively, regions of the brainstem that gate or are involved in relaying information as it pertains to physiological status project directly to the PVN itself. The consequence of this pattern of connectivity is that there are no opportunities for integration with multiple signals. Instead, indicators of physiological status have a direct route to the HPA axis to maintain tight homeostatic control. Further, immediate threats such as hemmorhage will trigger the HPA axis via direct routes, which will supercede other stimuli proceding through secondary, tertiary, and beyond circuits.  We hypothesize cortico-limbic circuits sensitive to stress send efferents to the PVN surround where these signals can then be relayed onward to the hypophysiotropic cells of the PVN. In order to test this, we directed injections of retrograde FG tracer into the PVN 34  surround.  Following exposure to an acute episode of emotional stress, in this case restraint, we are able to characterize cells activated by the stressor, and projecting to the PVN surround.  We accomplished this through the use of FOS expression which is an indicator of cellular activity. Previous studies have shown that exposure to emotional stressors leads to increases in FOS expression in cortico-limbic regions. Therefore, we can conclude that those cells expressing both FOS and retrograde tracer are stress reactive with the capacity to innervate the PVN surround. 3.2 Methods  Animals used for this experiment were the same as detailed in CHAPTER 2. 3.2.1 Animals Adult male Sprague-Dawley rats (Charles River, St. Constant, Canada) were used, weighing 270-290g on arrival and 290-310g on day of surgery. Animals were pair-housed under controlled temperature and lighting conditions (12:12-hour light:dark cycle, lights on at 0700 hours) with food and water available ad libitum. All experimental protocols were approved by the University of British Columbia Animal Care Committee. 3.2.2 Surgery  Animals were induced and kept at a surgical plane of anaesthesia using isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane) gas in conjunction with pure oxygen as a carrier gas. At time of induction animals also received prophylactic analgesic subcutaneous injection of NSAID (meloxicam; 0.3mg/kg body weight) and lactated Ringer?s saline. Local subcutaneous injection of mepivacaine (0.25%) above the skull acted as a local analgesic.   Following the incision and exposure of the skull, a small hole was drilled to expose the brain and allow introduction of the injection unit. Bregma was determined from skull 35  suture marks and used as a landmark to determine rostral-caudal (RC) coordinate. Medial-lateral (ML) coordinates were based off the superior sagittal sinus, exposed through drilling, which runs between hemispheres of the brain.  Dorsal-ventral (DV) coordinates were derived from distance the dura meningeal layer. Using these guidelines we based our stereotactic coordinates according to Swanson (2004). ZI ? RC: +1.50 mm, ML: -1.00 mm, DV: -6.8mm; sPVZ ? AP: +1.20 mm, ML: -0.25 mm, DV: -7.20 mm; PFA ? AP: +1.20 mm, ML: -1.00 mm, DV: -7.00 mm; AHA ? AP: +1.20 mm, ML: -0.50 mm, DV: -7.50 mm.   Glass micropipettes (20-25?m outer diameter; Sutter Instruments, Novato, CA) were backfilled using Hamilton syringe with FG 2% (w/v). The parameters for iontophoresis were 2.5?A alternating current, 7s on/off, 2.5mins. Micropipette tips were descended into the brain and left for 5mins before and after application of current to prevent the movement of tracer deposits along the micropipette tract.  3.2.3 Tissue collection  Optimal transport time for FG transport is fourteen days (Schmued and Fallon, 1986, Moga and Saper, 1994, Tillet et al., 2000), therefore, two weeks post-surgery animals were subjected a single episode of restraint stress for 60mins before being terminally anaesthetized with chloral hydrate (200mg/kg) and perfused via ascending aorta with 0.9% saline (125mL; 4?C) preceding 4% paraformaldehyde (pH 9.5; 4?C; 500mL). Brains were then collected and post-fixed for 4hrs in paraformaldehyde before being transferred to 15% sucrose in 0.1M potassium phosphate-buffered saline (KPBS, pH 7.4) and left 12-16hrs at 4?C. Brains were then sectioned into 5 adjacent series of 30um thick sections into cryoprotectant (30% ethylene glycol, 20% glycerol in 0.05M potassium phosphate buffer solution (KPBS) buffer) and stored at -20?C. 36  3.2.4 Dual Immunohistochemistry  To detect tracer deposits and distal FG accumulating cells in concert with those cell activated by restraint stress we employed a dual immunohistochemical procedure. Initially, cryoprotectant was removed from the tissue with serial KPBS rinses before being treated with H2O2 (0.3%) to remove endogenous peroxidase activity followed by treatment with reducing agent NaBH4 (1% w/v) to reduce paraformaldehyde linkages and unmask antigens. These treatment steps were interceded by serial rinses of KPBS to remove residual reaction products. Tissue was then incubated in primary FOS rabbit anti-sera (1:20,000; Santa Cruz Biotechnology Inc., cat. no. SC-52, Santa Cruz, CA) for 48hrs at 4?C in a KPBS-Triton solution (0.3% Triton-X; Sigma, Oakville, Ont.) containing 2% normal goat serum (NGS).  Sections were rinsed with KPBS before incubation in secondary anti-rabbit biotinylated antibody (1:450) for 1 hour at room temperature preceding detection using a nickel-intensified avidin-biotin-immunoperoxidase procedure (Vectastain Elite ABC kit; Vector Laboratories, Burlington, CA). Before incubation in FG anti-sera, tissue was rinsed with KPBS and treated with additional H2O2 to quench peroxidase activity from the previous avidin-biotin-immunoperoxidase procedure. Tissue was then incubated in primary FG rabbit anti-sera (1:10,000; Millipore, Billerica, MA) for 48hrs at 4?C in a KPBS-Triton solution (0.3% Triton-X; Sigma, Oakville, Ont.) containing 2% normal goat serum (NGS). Tissue then underwent a non-nickel-intensified avidin-biotin-immunoperoxidase procedure.    37  3.2.5 Imaging and analysis  FOS protein is expressed within the cell nucleus while FG accumulates throughout the cytoplasm. We sought to determine the number of cells both projecting to individual regions of the PVN surround that are responsive to stress. The avidin-biotin-immunoperoxidase procedure was employed such that FOS was labeled black (nickel intensified) while FG was labeled brown (non-intensified). This allowed us to easily identify cells where both signals were present (represented in Figure 3-1). Using light microscope (LeicaDMR) under brightfield conditions, individual positive cells were manually counted throughout entire brain nuclei. Counts were multiplied by five to account for the sectioning interval. Therefore, cell numbers are an estimate of total brain nucleus labeling versus a single series of tissue. 3.3 Results  The number of stress activated cells projecting to the PVN surround from the BST and hypothalamus were extremely limited. Therefore, the incidence of dual labeled cells in cortico-limbic nuclei is emphasized. 3.3.1 Limbic-related; PFC, LS, MeA, PVT  As an extension of retrograde findings, in the mPFC, we saw the largest number of dual labeled FOS+FG cells in ZI injection group animals (246.67?32.47) (Table 1). This was predominantly within the PL (135?19.19) but substantial numbers were observed in the IL (59.17?7.35) and ACC (52.50?6.801) as well. Considering the ZI further, we noted a substantial cluster of dual labeled cells within the PVT (67.5?4.233). Finally, we observed some minor labeling in the LS (22.50?4.61).   38    Figure 3-1. Photomicrograph demonstrating incidence of FG + FOS dual. Solid arrows show dual labeled (FG + FOS) cells and the open arrowhead indicates a FOS-positive, FG-negative cell. Scale bar = 100?m 39                   Table 1. Mean ? SEM number of FOS+FG dual labeled cells in animals bearing injections of retrograde tracer in the ZI, sPVZ, and AHA   PVN surround retrograde injection sites     ZI sPVZ AHA Cortico-Limbic sites         mPFC   246.67?32.47 35.83?8.98 21?4.90 LS   22.5?4.61 78.33?13.94 44?8.72 MeA   10.83?6.11 45.83?9.17 15?3.16 PVT   67.5?4.23 26.66?8.82 20?3.54    1. Injections in the AHA (n=5), sPVZ (n=6), ZI (n=6) 40   We detected the largest amount of dual labeled cells in those animals bearing injections of retrograde tracer in the sPVZ (78.33?13.94) followed by injections made in the AHA (44.00?8.72). The pattern of retrograde tracer accumulation in the sPVZ injection group was unique in comparison with the other PVN surround groups (Chapter 2). The area of the greatest retrograde labeling was in the LSvl in the sPVZ injections and this may help emphasize the specific importance of the sPVZ in gating stress information from the LS.  3.4 Discussion  Animals bearing injections of retrograde tracer in the PVN surround were exposed to a single acute episode of restraints stress. Using a dual immunohistochemical approach we were able to describe cells that have both the capacity to project to the PVN surround (FG-ir) and are sensitive to restraint stress (FOS-ir). The presence of these dual labeled cells confirms that the PVN surround has the capacity to relay stress sensitive information the PVN proper.  3.4.1 Regions of the PVN surround are in receipt of unique patterns of stress activated circuits   The nature of dual labeled cells (FOS+FG) describes what regions of the PVN surround are best suited to relaying acute stress-information from specific cortico-limbic brain nuclei (Summarized in Figure 3-2). Although the LS projects heavily to the entire surround, in response to the acute restraint episode, we found the large majority of dual labeled cells were in those animals with injections of FG in the sPVZ. Therefore, when considering the effects of acute stress, it appears the sPVZ may be best suited for relaying LS information onward to the PVN. As described in Chapter 2, the pattern of retrograde   41    Figure 3-2.Schematic summarizing the major stress sensitive afferents to different cell groups in the PVN-surround. These PVN-surround regions (in red), in turn, have the capacity to project onward to hypophysiotropic cells of the PVN. ACTH, adrenocorticotropic hormone; AHA, anterior hypothalamic area; CRH, corticotropin releasing hormone; LS, lateral septum; mPFC, medial prefrontal cortex; MeA, medial amygdala; PVN, paraventricular nucleus of the hypothalamus; PVT, paraventricular nucleus of the thalamus; sPVZ, subparaventricular zone; ZI, zona incerta. 42  accumulation was unique in those animals bearing injections in the sPVZ as accumulating cells were clustered in the LSv and ventral LSi. This aligns with past studies demonstrating heavy IEG expression in the LSv in response to stress (Cullinan et al., 1996, Li and Sawchenko, 1998).  The sPVZ and AHA were both in receipt of stress projections from the MeA. Given the role of the MeA is largely stimulatory to HPA activity (Allen and Allen, 1974, Van de Kar et al., 1991, Feldman et al., 1994) this may be evidence that stimulatory circuits are relayed through the ventral PVN surround regions. However, this is largely speculator and investigations will need to explore the nature of cellular phenotypes and synapses in these regions.   Finally, the ZI receives a significant number of stress sensitive afferents from the mPFC and the PVT. Therefore, the ZI represents a site where higher-level cortical processing circuits can converge to influence HPA activity. Further, the role of the PVT in habituation (Bhatnagar et al., 2002) means the ZI may be unique among the PVN surround circuits when considering habituation effects on the HPA axis. 3.4.2 Tonic and phasic regulation of the HPA axis through the PVN surround  Although there is clear evidence of stress reactive circuits projecting to the PVN surround, the overwhelming majority of retrogradely labeled cells were not responsive to acute restraint stress. Therefore, it appears that bulk of cortico-limbic information that is being projected to the PVN surround is not involved in processing transient stressors. There remains the possibility that the PVN surround acts primarily in tonic regulation of the HPA axis in a manner similar to the aBST which also shares multiple links with cortico-limbic structures (Cullinan et al., 1993, Herman et al., 1994, Radley et al., 2009). 43   Cortico-limbic regions have been shown through lesion studies to alter the basal biosynthetic capacity of the PVN to respond to stressful stimuli. For example, lesions to the PL arm of the mPFC result in an increased expression of CRH in the hypophysiotropic cells of the PVN leading to protracted ACTH and CORT responses to acute stressor (Radley et al., 2006). Therefore, as the PVN surround nuclei are in receipt of multiple limbic projections they may exert a strong role over the basal tone of the HPA axis. This overlaps well with past studies that have observed local tonic GABAergic inhibitory projections to the PVN (Wuarin and Dudek, 1993, Boudaba et al., 1996). Further, to modify basal tone of the HPA-axis, during exposure to chronic stress, neurons of the PVN surround increase GAD65 mRNA expression (Kolmac et al., 1998).    Finally, although acute restraint stress failed to recruit a large number of cortico-limbic projections to the PVN surround we cannot rule out the possibility that other forms of stressors that have been shown to activate cortico-limbic circuits, will lead to activation of these circuits.  3.4.3 The PVT may drive ZI related HPA input   The paraventricular nucleus of the thalamus (PVT) is an important structure in the regulation of habituation to chronic restraint stress (Bhatnagar et al., 2002). Habituation in turn allowing the animal to be responsive to new stimuli without overtaxing the neuroendocrine axis (Bhatnagar and Dallman, 1998). Arginine vasopressin (AVP) acts as a secretogogue with CRH to influence downstream activation of the HPA axis (Aguilera et al., 2008, Gray et al., 2010). The expression of AVP and its receptor V1b is increased in response to chronic stress. Therefore, in addition to influencing hypophysiotropic PVN cells, 44  the ZI may send projections to specifically regulate some components of the vasopressin system in response to chronic stressors.   45  CHAPTER 4: The zona incerta acts as a relay for medial prefrontal cortex efferents to the paraventricular nucleus of the hypothalamus 4.1 Introduction  The ZI showed the greatest cortico-limbic connectivity out of the described PVN surround subregions. The bulk of these connections originated from the mPFC, PVT, and LS. Due to the strength of this cortico-limbic connectivity we directed our focus on further exploring the capacity for the ZI to act as a relay to the hypophysiotropic cells of the PVN. Further, since the overwhelming majority of these limbic afferents to the ZI originated from the mPFC, we sought to explore the nature of mPFC axon projections to the ZI.   The ZI encompasses a large region of the ventral thalamus that extends in a long medial-lateral tract that is also very expansive in the rostral-caudal dimension (Swanson, 2004). Due to the size of this structure, there are multiple levels for afferent and efferent flow to and through this nucleus. Although we can be confident that cortico-limbic regions send projections to the ZI, we cannot be sure where, or if, these projections accumulate in distinct regions of the ZI. In our first experiment, to address this question we directed injections of anterograde tracer at the mPFC. This resulted in a dense accumulation of labeled fibers in the rostral ZI (ZIr) at approximately the same level of the PVN.   Since we observed that projections from the mPFC accumulate predominantly in the ZIr, we sought to determine if this specific subregion of the ZI can project onward to hypophysiotropic cells of the PVN. To accomplish this we targeted injections of anterograde tracer in the ZIr in animals having undergone surgical adrenalectomy. The adrenalectomy procedure allows us to exploit feedback mechanisms and amplify endogenous CRH expression that is unique to hypophysiotropic cells of the PVN. This, in turn, allows us to 46  easily stain CRH expressing cells of the PVN that are responsible for driving the HPA axis. In comparison with anterograde accumulation we can make conclusions about the capacity for ZIr circuitry to be involved in HPA activity regulation.  Finally, to confirm the ZI can act as an intermediary between the mPFC and PVN, we performed a dual injection experiment where animals would receive targeted injections of anterograde tracer into the mPFC in concert with injections of retrograde tracer in the PVN. This allows us to explore the entirety of the ZI for any points where these signals may converge.  Summary  In this chapter, three experiments were performed using separate animal groups. 1) Anterograde tracer targeted at the mPFC. 2) Anterograde tracer targeted to the ZI where mPFC projections accumulate. 3) Dual injections made in the mPFC (anterograde) and PVN (retrograde) to explore capacity for these circuits to directly interact.  4.2 Methods 4.2.1 Animals  Two batches of animals were used. Both were adult male Sprague-Dawley (Charles River, St. Constant, Canada) weighing 270-290g on arrival and 290-310g on day of surgery. One group of animals was ordered having undergone surgical adrenalectomy by Charles River veterinarian staff (Code:ADREX), while the second group was na?ve. Animals were pair-housed under controlled temperature and lighting conditions (12:12-hour light:dark cycle, lights on at 0700 hours) with food and water available ad libitum. All experimental protocols were approved by the University of British Columbia Animal Care Committee. 47   Experiments 1 and 3 used na?ve animals while the second experiment, involving injections of anterograde tracer into the ZIr used adrenalectomized animals.  4.2.2 Surgery  Animals were induced and kept at a surgical plane of anaesthesia using isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane) gas in conjunction with pure oxygen as a carrier gas. At time of induction animals also received prophylactic analgesic subcutaneous injection of NSAID (meloxicam; 0.3mg/kg body weight) and lactated Ringer?s saline. Local subcutaneous injection of mepivacaine (0.25%) above the skull acted as a local analgesic. Adrenalecotmized rats received injections of corticosterone (1mg/kg) pre- and post-surgery to assist with surgical recovery.  Following incision and exposure of the skull, a small hole was drilled to expose the brain and allow introduction of injection unit. Bregma was determined from skull suture marks and used as a landmark to determine rostral-caudal (RC) coordinate. Medial-lateral (ML) coordinates were based off the superior sagittal sinus, exposed through drilling, which runs between hemispheres of the brain.  Dorsal-ventral (DV) coordinates were derived from distance from the dura meningeal layer. Using these guidelines we based our stereotactic coordinates according to Swanson (2004). ZI ? RC: +1.50 mm, ML: -1.00 mm, DV: -6.8mm; PVN ? AP: +1.20 mm, ML: -0.25mm, DV: -7.00mm; mPFC ? RC: -3.50 mm, ML: -0.68 mm, DV: -3.80 mm.   For anterograde injections directed at the mPFC and ZIr we applied biotinylated dextran amine (10 000MW; Invitrogen; D-1956, Burlington, Ont.) via iontophoresis. Glass micropipettes (20-25?m outer diameter; Sutter Instruments, Novato, CA) were backfilled using Hamilton syringe with BDA 5% (w/v). The parameters for iontophoresis were 4.5?A 48  alternating current, 7s on/off, 5mins. Micropipette tips were descended into the brain and left for 5mins before and after application of current to prevent the movement of tracer deposits along the micropipette tract.  To ensure retrograde injections in the PVN labeled the entirety of hypophysiotropic afferents we utilized volume injections of FG 2% (w/v) of 60nl. Volume injections were made using a Hamilton syringe. Syringe was left in brain for 5mins before and after ejection of neural tracer at a rate of 10nl/min. This ensured high volume tracer deposits did not damage tissue or travel along pipette track. 4.2.3 Tissue collection  Animals bearing single injections of BDA anterograde tracer were sacrificed 7-10 days after surgery in alignment with optimal tracer transport time (Schmued et al., 1990, Veenman et al., 1992). Alternatively, animals bearing injection of FG were sacrificed two weeks post-surgery as optimal transport time for FG transport is fourteen days (Schmued and Fallon, 1986, Moga and Saper, 1994, Tillet et al., 2000). All animals were exposed to a single episode of restraint stress for 60mins before being terminally anaesthetized with chloral hydrate (200mg/kg) and perfused via ascending aorta with 0.9% saline (125mL; 4?C) preceding 4% paraformaldehyde (pH 9.5; 4?C; 500mL). Brains were then collected and post-fixed for 4hrs in paraformaldehyde before being transferred to 15% sucrose in 0.1M potassium phosphate-buffered saline (KPBS, pH 7.4) and left 12-16hrs at 4?C. Brains were sectioned into 5 series of 30um thick sections into cryoprotectant (30% ethylene glycol, 20% glycerol in 0.05M KPBS buffer) and stored at -20?C.   49  4.2.4 Immunofluorescence  To detect tracer deposits and distal accumulating cells projections, cryoprotectant was removed from the tissue with serial KPBS rinses before being reacted with reducing agent NaBH4 (1% w/v) to help unmask antigens by reduction of paraformaldehyde linkages. Tissue was then rinsed with KPBS until bubble free at which point two more 10 minute rinses with KPBS were performed to ensure removal of residual NaBH4 . For detection of BDA, tissue was then incubated in streptavidin, Alexa Fluor 488 conjugate (1:500; Invitrogen, Cat. no. S32354, Burlington, Ont.) for two hours followed by 3 x 10 minute rinses with KPBS before mounting onto slides.  Hypophysiotropic cells of the PVN were detected through a similar procedure. Before incubation in fluorescent Alexa Fluor 594 (1:500; Invitrogen, Cat. no. S11227, Burlington, Ont.), tissue was incubated in CRH rabbit anti-sera for 48hrs (1:2000, Bachem, Cat. no. T4037, Torrance, CA).  We utilized the native fluorescent properties of FG to visualize retrograde injections and accumulation. For all fluorescent preparations, ProLong Gold antifade reagent (Invitrogen, Cat. no. P36930, Burlington, Ont.) was used for coverslipping. This serves to preserve signal during storage and from photobleaching. 4.2.5 Imaging and analysis  Alexa fluorophores, labeling cells and associated projections, were detected by fluorescene wavelight excitation. FG native fluorescence was identified via ultraviolet excitation. Images were captured using a LeicaDMR microscope.   50  4.3 Results  Performing dual ipsilateral injections of neural tracer allowed us to assess the capacity for two sites to interact monosynaptically. For this purpose, we directed injection of anterograde tracer BDA in the mPFC to allow us to determine what downstream sites are in receipt of related projections. Alternatively, volume injections of FG into the PVN allow us to identify all upstream sites that can project to the PVN. By observing where these signals converge we can begin to hypothesize where significant candidates for relay points are located.   4.3.1 The mPFC sends efferents to the ZI  The use of anterograde injections into the mPFC allowed us to explore the level at which mPFC cortico-limbic projections accumulate in the ZI. Our injections of anterograde tracer were targeted towards the rostal portion of the mPFC on the border between the PL and IL (Figure 4-1 A). This was to ensure that our tracer did not spread into the rostral LS that is positioned directly caudal to the mPFC. Additionally, our injection of anterograde tracer was made in the ventral mPFC, composed of the ventral PL and IL subregions, that has been implicated as the major mPFC stress circuit origin (Heidbreder and Groenewegen, 2003).  We discovered a heavy projection of anterogradely labeled fibers that course through the ZI and densely accumulate in a restricted band in the medial ZI at approximately the same rostral-caudal level of the hypophysiotropic cells of the PVN (Figure 4-1 B-D). This dense labeling remained in this medial-lateral region of the ZI 600um removed from the PVN at which point the fibers density decreased.    51    Figure 4-1. Photomicrographs showing injection of anterograde tracer BDA in the ventral mPFC (A) and associated projections in the rostral ZI (B,C,D). The ZIr is shown at different rostral-caudal levels (B, 1.33 ;C, -1.78; D, -2.23 mm from Bregma). fa, forceps anterior of the corpus callosum; ZIr, rostral zona incerta. Scale bar = 1000?m in A; 300?m in B, C, D. 52  4.3.2 The PVN is in receipt of afferents from the ZI  To assess the capacity for ZI efferents to contact hypophysiotropic cells of the PVN we performed anterograde injections in the ZI in adrenalectomized animals. We aimed our injections of anterograde tracer into the region of the ZI where we detected mPFC projections (Section 3.3.1). The use of adrenalectomized animals allowed us to easily identify hypophysiotropic cell of the PVN that express CRH through immunofluorescence. In lieu of adrenalectomy, not all hypophysiotropic cell populations capable of altering HPA axis will express CRH at detectable levels. Therefore, our employment of this surgical paradigm ensures that all HPA regulating cells of the PVN are activated.  Our injections in the ZI led to a large projection of fibers that course ventrally into the sPVZ and dorsomedial AHA (Figure 4-2 A). Further, we observed axonal elements in proximity to CRH positive cells within the PVN (Figure 4-2 B). Our findings demonstrate hypophysiotropic cells of the PVN are in receipt of ZI afferents that reaffirm our predictions that this particular PVN surround subregion can influence HPA axis. The pattern of terminal labeling suggests that there is a strong level of cross-talk between the multiple PVN surround sites. 4.3.3 A circuit relay between mPFC and PVN exists in the ZI  In our final experiment we wanted to determine if ZI neurons capable of projecting to the PVN were the same as those in receipt of mPFC projections. Using injections of anterograde tracer made into the ventral mPFC in conjunction with injections of retrograde tracer in the PVN, we were able to explore the capacity for these nuclei to synapse with each other.    53    Figure 4-2. Photomicrographs demonstrating injection of anterograde tracer BDA in the ZIr (A).  Note dense coursing of fibers from site of injection into the sPVZ. Enlarged view of the PVN (B) shows labeled axonal elements (green) juxtaposed to CRH positive cells (red). Scale bar = 500?m in A; 30?m in B. 54   Using light microscopy, we observed labeled axons and axonal elements abutted to a cluster of retrogradely labeled cells in the lateral section of the ZI (Figure 4-3). Of the retrogradely labeled cells in this cluster, 31.6%?4.8 (n=8 observations) were in contact with labeled anterograde elements. This suggests that of those cells in the ZI that project onward to the PVN, one third have the capacity to act as relays for mPFC projections. 4.4 Discussion 4.4.1 ZI is uniquely positioned to integrate multiple sensory modalities  In this Chapter we confirmed the capacity for the ZI to not only relay cortico-limbic projections to the hypophysiotropic zone of the PVN, but to specifically act as an intermediary between the mPFC and PVN.   In addition to cortico-limbic regions that include the mPFC, LS, and PVT, the ZI is also in receipt of afferents originating from various brainstem nuclei such as the reticular nucleus, ventral tegmental area, pedunculopontine nucleus, dorsal raphe, periaqueductal grey, substantia nigra, and superior colliculus (Shammah-Lagnado et al., 1985, Larsen et al., 1996, May et al., 1997, Kolmac et al., 1998). These brainstem regions have been implicated in a multitude of functions including arousal, locomotion, eye movements, drinking, pain, and emotion. These functions align well with implicated role of the ZI as described in Chapter 2, which are in control of visceral activity (Mok and Mogenson, 1986, Spencer et al., 1988, Sanghera et al., 1991, Tonelli and Chiaraviglio, 1993, 1995), arousal (Shammah-Lagnado et al., 1985, Berry et al., 1986, Power and Mitrofanis, 2001), attention shifting (Ficalora and Mize, 1989, Nicolelis et al., 1992, May et al., 1997, Mitrofanis, 2002), posture and locomotor behaviour (Mogenson et al., 1985, Milner and Mogenson, 1988, Supko et al., 1991, 1992, Murer and Pazo, 1993, P?rier et al., 2002).  55    The convergence of cortico-limbic and brainstem projections in the ZI emphasize the unique position of this PVN surround subregion. Descending cortico-limbic signals that describe the processive qualities of a stressor can be integrated in unison with information pertaining to global physiological status conferred by the ascending brainstem projections. This provides an initial level of processing that occurs distal to the hypophysiotropic PVN.   4.4.2 PVN surround integration  In animals bearing injections of retrograde tracer in the ZI, we observed a very dense collection of projections extending ventrally into the sPVZ (Figure 4-2 A). The functional consequence of this connection is currently unknown but presents an interesting opportunity for integration throughout multiple PVN surround sites. For example, information from the PVT and mPFC that has been gated in the ZI could in turn converge upon the sPVZ to influence responses. In turn, the afferents to the sPVZ originating from the LS and MeA could then be gated or integrated with ZI information.  As the separate PVN surround subregions are further characterized, the functional significance of this local circuit could become more apparent. The hypophysiotropic cells of the PVN respond almost exclusively to GABAergic and glutamatergic inputs (Wuarin and Dudek, 1993). Further, while the PVN surround has populations of both GABAergic and glutamatergic neurons (Roland and Sawchenko, 1993, Boudaba et al., 1996, Boudaba et al., 1997), it is unclear where these particular cell types are located. Therefore, projections from the ZI may act to effectively silence competitive regions of the sPVZ. For example, the mPFC has been indicated in inhibiting HPA drive (Diorio et al., 1993). Therefore, if we assume the primary role of the ZI is to relay inhibitory signals to the HPA axis, this could be achieved via GABAergic projections to hypophysiotropic cells of the PVN. Alternatively,   56   Figure 4-3. Photomicrograph showing convergence of terminals from mPFC (green) with PVN projecting cells (blue) in the ZI. Panels B and C are magnified photomicrographs of the outlined areas b and c, respectively. Arrowheads indicate areas where signals are abutted. Scale bar = 300?m in A; 30?m in B and C. 57  inhibition of the PVN could be achieved through glutamatergic projections to GABAergic cells of the sPVZ that project onward to the PVN. Finally, ZI GABAergic projections to glutamatergic neurons of the sPVZ would remove excitatory tone but may also act to silence sPVZ cortico-limbic afferents such as the LS and MeA (Chapter 2). The potential for significant cross-talk between the PVN surround regions is great and requires further exploration of specific cellular phenotype.   58  Chapter 5: GENERAL DISCUSSION 5.1 Overview  This dissertation sought to characterize the strength and pattern of cortico-limbic projections to the PVN surround. While the ability of various cortico-limbic sites to influence HPA activity is very well described (Pezzone et al., 1992, Senba et al., 1993, Silveira et al., 1993, Bonaz and Tach?, 1994, Cullinan et al., 1995, Silveira et al., 1995, Duncan et al., 1996, Matsuda et al., 1996, Campeau and Watson, 1997, Kollack-Walker et al., 1997, Li and Sawchenko, 1998, Sheehan et al., 2000, Dielenberg et al., 2001, Mongeau et al., 2003, Singewald et al., 2003, Timofeeva et al., 2003, Salom? et al., 2004), it remains unclear what circuits are involved in relaying this information. Previous work has shown that various nuclei in the PVN surround are in position to receive input from cortico-limbic sites and in turn have the capacity to project to the PVN (Roland and Sawchenko, 1993). However, the pattern and strength of cortico-limbic connectivity to the PVN surround has never been described. By employing neural tracing methods we first explored cortico-limbic circuits to the PVN surround. We then assessed, if any singular PVN surround circuit could be acting as a relay point that is both in receipt of cortico-limbic connectivity and sending efferents to PVN hypophysiotropic cells. 5.1.1 Chapter 2  The purpose of this initial experiment was to determine the relative strengths of cortico-limbic connectivity to the various subregions of the PVN surround that include the AHA, sPVZ, PFA, and ZI. Using injections of retrograde tracer directed at each PVN surround site we were able to make comparative estimations of cortico-limbic contributions to each region. All regions of the PVN surround were in receipt of substantial afferents from 59  cortico-limbic regions summarized in Table 1. Briefly, injections into all PVN surround regions showed tracer accumulation in the LS. However, the pattern of accumulation within the LS showed substantial variability with injections made in sPVZ most closely mimicking LS IEG expression during emotional stress. The MeA showed the greatest amount of retrograde accumulation when injections were targeted the AHA and sPVZ that lie directly ventral to the PVN. Finally, injections made in the ZI showed very strong retrograde uptake in the LS, PVT, and mPFC. Due to the relative strength of cortico-limbic connectivity to the ZI we focused on characterizing the ZI as a possible relay point for these signals to converge on the PVN.  5.1.2 Chapter 3  Building upon the findings of Chapter 2 we sought to explore the role the PVN surround played in conferring emotional stress information to the PVN. Restraint stress is a prominent emotional stressor and drives cortico-limbic circuits as evidenced by IEG expression that increases following exposure. By looking for cells that were dual labeled with FOS and FG we were able to determine which stress-sensitive cells were projecting to each of the PVN surround sites. The main results are summarized in Table 2. We found that the bulk of stress-sensitive inputs to the PVN surround were actually relayed to the sPVZ. This in large contrast to Chapter 2 results that showed the LS projected strongly to all PVN surround regions. Further, the sPVZ was in receipt of the greatest number of restraint activated cells in the MeA. Patterns of dual labeled cells for injections made in the AHA followed that of the sPVZ but of a reduced magnitude. The PFA appears to play a limited role in response to this particular stressor. Finally, the ZI showed large numbers of dual 60  labeled cells in the PFC, ACC, and PVT leading us to focus our efforts on further characterizing the ZI as a relay site. 5.1.3 Chapter 4  As previously mentioned, of the PVN surround retrograde injections, those directed at the ZI showed the greatest amount of cortico-limbic tracer accumulation largely focused within the mPFC. Due to the strength of this connectivity, we focused on confirming the presence of a circuit relay through the ZI. Using injections of anterograde tracer in the mPFC we discovered the bulk of these projections accumulate densely in the rostral medial ZI.  Additional anterograde experiments then confirmed that this region of the ZI has the capacity to project to the ZI, as well as other PVN surround regions including the AHA and sPVZ. Finally, performing dual injections of retrograde FG at the PVN and BDA in the mPFC in single animals, we explored the capacity for these two signals to overlap in the ZI. The results confirmed that the ZI is in position to relay these signals. Further, the presence of a single synapse relay in the ZI is very likely.  5.2 Methodological considerations 5.2.1 Injection of neural tracers  While iontophoresis is a very useful tool to create discrete injections into the brain, since the tracer deposit were so minute we may have underestimated the total number of cells that project to each PVN surround site. However, as our results in Chapter 2 are a comparison between injection sites it is important to emphasize that only those injections of comparable size, intensity, and placements were used to make relative comparisons. However, in Chapter 3 when we explored the FOS + FG dual labeling in response to restraint stress we may have underestimated the total number of cells involved in each circuit.  61  5.2.2 Injection placement  In Chapter 2 we were initially attempting to characterize the PVN surround based on the relative strength of afferent cortico-limbic connections. Our initial injections were made at the RC level of the PVN. However, this may not necessarily reflect the exact level where specific cortico-limbic structures project into the PVN surround. This was evident in Chapter 4 when injections of anterograde tracer in the mPFC resulted in a band of terminals in the ZI that was most dense ~300?m caudal to the hypophysiotropic region of the PVN. As a consequence we may have underestimated the strength of cortico-limbic projections to particular regions of the PVN surround. Further, as our anterograde injections into the mPFC were focused primarily in the ventral mPFC, due to its importance in HPA activity (Heidbreder and Groenewegen, 2003) we may not have accounted for the entirety of mPFC projections to the ZI. 5.2.3 Microscopy  Light microscopy can give a reasonable approximation of synapse proximity though it remains inferior to electron microscopy for identification of synapses. A reasonable intermediate is the use of confocal laser scanning techniques. Though confocal laser scanning technology cannot definitively define synapses it has been often used, and is generally accepted, as a means of approximating connectivity (Mason et al., 1992, Herman et al., 2008, Hohensee et al., 2008, Geerling et al., 2010). 5.3 Future considerations 5.3.1 Functional role of the ZI  In this thesis we found that the ZI is capable of acting as a relay center between the mPFC and the PVN. Further, the ZI is in receipt of efferents from restraint stress activated 62  cells in these cortico-limbic centers. Determining the specific role of the ZI would be the next natural step to fully elucidating how this region is influencing HPA activity. Although local hypothalamic circuits that converge on the PVN are largely GABAergic, the ZI is composed of a very heterogeneous population of cellular phenotypes that include, but are not limited to, GABA, glutamate, cytokines, amines, CCK, and NPY. It would be difficult to lesion a single cellular phenotype; therefore it would be recommended to first discover which cell phenotypes project to the PVN. This can be accomplished through PVN targeted injections of retrograde tracer and combination immunohistochemistry for different neurotransmitters. 5.3.2 Effect of chronic stressors on PVN surround circuits  In Chapter 2 we exposed animals to an acute episode of restraint stress in order to activate processive circuits that include cells in cortico-limbic regions. Of the cells we observed with the capacity to project to the PVN surround, few were activated by acute restraint stress exposure. However, we cannot discount the potential for PVN surround circuits to be more heavily involved in regulating the tone of the HPA axis through other stress paradigms. This would fall in alignment with past experiments noting increased GAD65 mRNA expression in the PVN surround in response to chronic stress (Kolmac et al., 1998). Alternatively, the PVN surround may be better suited to activation by different types of emotional or processive stressors such as forced swim, social, burying, open field, or chronic variable stress. 5.4 Conclusions  The PVN surround is in position to integrate stress stimulated signals from multiple cortico-limbic sites. Among the various subregions of the PVN surround, we found that the ZI is uniquely positioned to relay afferent signals from the mPFC onward to the 63  hypophysiotropic zone of the PVN. 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