|UFDC Home||myUFDC Home | Help|
This item has the following downloads:
1 A ROLE FOR THE ORGANIC CATION TRANSPORTER3 IN STRESS INDUCED PSYCHOPATHOLOGY By CATHERINE MARCINKIEWCZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010
2 2010 Catherine Marcinkiewcz
3 To my father, for always believing in me
4 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Darragh Devine, for his support and encouragement of all of my endeavors throughout my graduate career. I would also like to acknowledge my committee members: Dr. Thomas Foster, Neil Rowland, Roger Reep, and Robert Yezierski for their thoughtful guidance in bringing my dissertation project to a suc cessful (and timely) conclusion. Special thanks go out to Dr. Reep and his Biological Scientist, Wendi Malphurs, for lending their expertise in histological methods and neuroanatomy to this project.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 GENERAL INTRODUCTION .................................................................................. 15 Clinical Significance ................................................................................................ 15 Pathophysiology of Depression .............................................................................. 16 Genetic Factors ................................................................................................ 16 The serotonin transporter ........................................................................... 17 Brain derived neurotrophic factor ............................................................... 20 Antidepressant response and the 5HT1A receptor .................................... 21 The glucocorticoid receptor ........................................................................ 22 Role of Stress ................................................................................................... 23 Hippocampus ............................................................................................. 25 Prefrontal cortex ......................................................................................... 26 Role of Monoamines ........................................................................................ 27 Nodes of Interaction between Stress and Monoaminergic systems ................. 29 The Organic Cation Transporter 3 .......................................................................... 30 Neuroanatomical Distribution ........................................................................... 30 Pharmacology .................................................................................................. 32 Role in monoamine transport ..................................................................... 32 Regulation by corticosterone ...................................................................... 33 Interactions with the 5HTT and antidepressant response .......................... 34 Role in Psychopathology .................................................................................. 35 Project Rationale .................................................................................................... 36 2 EFFECTS OF ACUTE AND REPEATED STRESS ON EXPRESSION OF THE OCT3 IN AN ANIMAL MODEL OF DEPRESSION ................................................. 39 Introduction ............................................................................................................. 39 Methods .................................................................................................................. 41 Experimental Design ........................................................................................ 41 General Methods .............................................................................................. 42 Experimental animals ................................................................................. 42 Post mortem tissue processing .................................................................. 42
6 Stress Procedures ............................................................................................ 42 Social defeat .............................................................................................. 42 Restraint stress .......................................................................................... 43 Biochemical and Molecular Assays .................................................................. 44 Real time PCR ........................................................................................... 44 Western blot ............................................................................................... 45 Statistical Analyses .......................................................................................... 47 Results .................................................................................................................... 48 Real time PCR Data ......................................................................................... 48 OCT3 gene expression .............................................................................. 48 GR gene expression .................................................................................. 49 5HTT gene expression ............................................................................... 49 Western Blot Results ........................................................................................ 50 OCT3 protein expression ........................................................................... 50 BDNF protein expression ........................................................................... 51 Discussion .............................................................................................................. 52 OCT3 Gene Expression ................................................................................... 52 OCT3 Protein Expression ................................................................................. 56 GR Gene Expression ....................................................................................... 56 5HTT Gene Expression .................................................................................... 57 BDNF Protein Expression in the Hippocampus ................................................ 58 3 ANTIDEPRESSANT EFFECTS OF AN OCT3 ANTAGONIST IN THE WISTAR KYOTO RAT ........................................................................................................... 70 Introduction ............................................................................................................. 70 Methods .................................................................................................................. 71 Experimental Design ........................................................................................ 71 Experimental Animals ....................................................................................... 72 Drugs and Injections ......................................................................................... 72 Forced Swim Test ............................................................................................ 72 Plasma CORT analysis .................................................................................... 73 Statistical Analyses .......................................................................................... 73 Results .................................................................................................................... 73 Behavioral data .......................................................................................... 73 CORT data ................................................................................................. 74 Discussion .............................................................................................................. 74 4 CONCLUSIONS AND FUTURE DIRECTIONS ...................................................... 79 A Working Model of the OCT3 as a Molecular Switch that Regulates Stress Responsiveness and Vulnerability to Psychopathology ....................................... 79 Bimodal Regulation of OCT3 Gene Expression: A Hypothesis ............................... 81 Stress induced Membrane Trafficking of the OCT3 ................................................ 83 OCT3 Antagonists as Putative Antidepressant Agents ........................................... 84 LIST OF REFERENCES ............................................................................................... 87
7 BIOGRAPHICAL SKETCH ............................................................................................ 99
8 LIST OF TABLES Table page 2 1 Real time PCR Primers ...................................................................................... 60 2 2 Antibodies for Western Blot ............................................................................... 60
9 LIST OF FIGURES Figure page 2 1 Schematic of experimental design. ..................................................................... 59 2 2 The social defeat procedure in action.. ............................................................... 60 2 3 Effects of stress on OCT3 gene expression in the hippocampus. ...................... 61 2 4 Effects of stress on OCT3 gene expression in the mPFC. ................................. 62 2 5 Effects of stress on OCT3 gene expression in the striatum.. .............................. 63 2 6 Effects of stress on GR gene expression in the hippocampus. .......................... 64 2 7 Effects of stress on 5HTT gene expression in the hippocampus.. ...................... 65 2 8 Bivariate correlation analysis for OCT3 x GR and OCT3 x 5HTT. ...................... 66 2 9 Effects of stress on plasma membrane expression of the OCT3 in the hippocampus. ..................................................................................................... 67 2 10 Effects of stress on cytosolic expression of the OCT3 in the hippocam pus. ....... 68 2 11 Effects of stress on BDNF protein expression in the hippocampus.. .................. 69 3 1 Schematic of experimental design. ..................................................................... 77 3 2 Pictorial representation of the forced swim test depicting (A) floating and (B) swimming behaviors. .......................................................................................... 77 3 3 Antidepressant effects of decynium 22 in the forced swim test.. ........................ 78 3 4 Effect of forced swim stress on plasma corticosterone levels.. ........................... 78 4 1 Proposed m odel for the effect of differential expression of the OCT3 on 5HT signaling in the hippocampus. ............................................................................ 86
10 LIST OF ABBREVIATION S 5H1AA 5 hydroxy indole acetic acid 5HT 5 hydroxytryptamine or serotonin 5HT1A 5 hydroxytryptamine 1A 5HTT 5 hydroxytryptamine transporterRemember to use a tab between the abbreviations and the definitions 5HTTLPR 5 hydroxytryptamine transporter linked promoter region ACC anterior cingulate cortex ACTH adrenocorticotropic hormone BDNF brainderived neurotrophic factor BNST bed nucleus of stria terminalis CRF corticotropinreleasing factor CSF cerebrospinal fluid CVS chronic variable stress D22 decynium 22 DMH dorsomedial nucleus of the hypothalamus DA dorsal area of the hypothalamus EMT extraneuronal mono amine transporter GR glucocorticoid receptor GRE glucocorticoid response element HPA hypothalamic pituitary adrenocortical HPLC high performance liquid chromatography LE Long Evans MAO monoamine oxidase MAOI monoamine oxidase inhibitor mPFC medial prefront al cortex
11 OCD Obsessive compulsive disorder OCT3 Organic cation transporter 3 PET Positron emission tomography PTSD Post Traumatic Stress Disorder PVN Parvoventricular nucleus of the hypothalamus SSRI Selective serotonin reuptake inhibitor TCA tricyclic an tidepressant WKY Wistar Kyoto
12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF THE ORGANIC CATION TRASNPORTER 3 IN STRESS INDUCED PSYCHOPATHOLOGY By Catherine Marcinkiewcz December 2010 Chair: Darragh P. Devine Major: Medical Sciences Neuroscience The molecular genetic basis of individual susceptibility to stress related psychiatric disorders is a subject of intense interest in the neuroscience community. For example, studies have shown that genetic polymorphisms in the 5hydroxytryptamine transporter (5HTT) gene are associated with an increased likelihood of developing Major Depression, and it is likely t hat multiple other important signaling mechanisms may be disrupted in these knockouts. Genetic expression of organic cation transporter 3 (OCT3), a corticosteronesensitive monoamine transporter, is upregulated in the hippocampus of 5HTT deficient mice, su ggesting that the OCT3 may play an important role in the regulation of cellular function relevant to major depression. In the present study, we characterized basal and stress induced expression of the (OCT3) in the hippocampus, medial prefrontal cortex (mP FC), and striatum of Wistar Kyoto (WKY) and Long Evans (LE) rats by real time PCR and Western blot. The WKY strain exhibits depressivelike behavior, heightened sensitivity to stress and a muted response to conventional antidepressants of the SSRI class, w hereas the LE strain has been described as stress resilient, exhibiting more active coping responses to stress. In our study, 5HTT gene expression was significantly downregulated in the hippocampus of
13 WKY relative to LE rats with a corresponding basal elev ation of the OCT3 in both the hippocampus and mPFC. These results indicate that the OCT3, which serves as a back up transporter when the 5HTT is saturated or in low abundance, may be a critical modulator of 5HT clearance in the WKY rat. Rats were random ly assigned to the following groups: 1) control 2) acute restraint stress and 3) repeated social defeat + acute restraint stress. OCT3 gene expression was upregulated in the WKY rats exposed to acute restraint stress after a history of social defeat, but n ot in WKY rats exposed to acute restraint stress alone. By contrast, acute stress induced a robust increase in OCT3 gene expression in the hippocampus of LE rats, and this effect was not seen in the LE rats that had a history of social defeat. These result s also suggest that the WKY rats exhibit a delayed genomic adaptation to stress. The pattern of stress induced hippocampal gene expression of the glucocorticoid receptor (GR) was similar to the pattern of expression of the OCT3 gene within both strains, bu t the withingroups differences did not reach statistical significance. However, there was a significant correlation between OCT3 and GR gene expression in the hippocampus within each strain, suggesting that glucocorticoid signaling may regulate both GR an d OCT3 gene expression. OCT3 gene expression was not significantly altered by stress in the mPFC or the striatum of WKY rats or LE rats, which is consistent with previous studies in 5HTT knockout mice in which upregulation of the OCT3 was restricted to the hippocampus. Tissues for western blot analysis of OCT3 protein expression were collected 3 hours after the onset of the acute stressor (or equivalent time in the controls).
14 Consistent with our gene expression data, basal expression of OCT3 protein in the hippocampus was significantly elevated overall in WKY compared to LE rats. The WKY rats exhibited an interesting pattern of elevated cytosolic OCT3 but suppressed membrane OCT3 in the acutely stressed rats with a history of social defeat. In the LE rats, there was a trend toward a decrease in plasma membrane OCT3 expression in the hippocampus after repeated stress that did not reach statistical significance. However, there was a significant increase in OCT3 protein expression in the cytosol after repeated stress in LE rats. Taken together, these data suggest that the OCT3 may be internalized when bound by corticosterone, which would explain why the OCT3 is localized to the cytosol 3 hours after stress. Basal elevation of OCT3 mRNA and protein levels in the hippocampus and mPFC of WKY rats compared to LE rats may indicate that the OCT3 has been recruited to maintain 5HT clearance due to the relatively low abundance of 5HTT. These same high levels of OCT3 expression in the hippocampus may allow WKY rats to maintain high rates of serotonin clearance even when corticosterone is present, which may increase their vulnerability to stress induced psychopathology. In order to test this hypothesis, we administered decynium 22 (0, 1 and 10 ug/kg), a specific inhibitor of the OCT3, to WKY rats shortly before behavioral testing on the forced swim test. It was found that both 1 ug/kg and 10 ug/kg decynium 22 produced a profound antidepressant effect, suggesting that the OCT3 may be a useful target for antidepressant pharmacotherapy in patients who, like the WKY rat, are unresponsive to selective serotonin reuptake inhibitors (SSRIs).
15 CHAPTER 1 GENERAL INTRODUCTION Clinical Significance Major depression is one of the leading causes of disability, lost productivity and diminished quality of life across the world. An estimated 121 million people suffer the devastating physical, emotional, and social consequences of depression, which constitutes the fourth leading contributor to the global burden of disease (World Health Organiz ation, n.d.). In fact, it is estimated that by 2020, depression will be the second leading cause of death behind heart disease, making this one of the most pressing public health concerns of our time. Although there are a variety of commercially available drugs that are commonly used to treat depression, it is estimated that between 1030% of depressed patients do not achieve remission after an initial trial with a selective serotonin reuptake inhibitor (SSRI), the most widely prescribed antidepressant drugs. Furthermore, the success rate drops significantly for subsequent trials with antidepressant drugs after the first trial, such that a large proportion of initially unresponsive patients will eventually be classified as refractory to antidepressant tre atment. Even for those that do respond, however, the time to onset of antidepressant action can be rather prolonged (57 weeks) which may not meet the needs of severely depressed patients who cannot otherwise function normally in their daily lives. Yet des pite its demographic prevalence and drastic socioeconomic and interpersonal impact, the etiology of depression remains elusive. From a biomedical standpoint, progress in this field has been limited by the paucity of animal models that recapitulate the neurochemical and physiological sequelae of major depression (ONeil
16 and Moore, 2003). In our studies we have attempted to address this by performing depressogenic (i.e. stress) manipulations in the Wistar Kyoto (WKY) rat, a strain that exhibits innate depres sive like characteristics. We used this model to characterize neurobiological correlates of depression that may lead to the discovery of new potential targets for antidepressant pharmacotherapy. Our focus is on the organic cation transporter 3 (OCT3), a polyspecific monoamine transporter that has been linked with a variety of psychiatric conditions in humans including obsessivecompulsive disorder (OCD) and methamphetamine use disorder (Aoyama et al., 2006;Fujimoto et al., 2007;Nagayama et al., 2007;Lazar et al, 2008). Here we present a series of studies that help to elucidate the role of the OCT3 stress induced psychopathology, particularly depression. We propose that the results of these studies may pave the way toward clinical studies in humans. Pathophy siology of Depression Depression can best be described as a physiological and behavioral syndrome characterized by flattened affect, and loss of motivation, interest and pleasure in daily activities, as well as physical and mental fatigue that is both perv asive and enduring (>2 weeks) (American Psychiatric Association [DSM IV TR], 2000). There is no singular cause of depression but it is instead thought to arise from a complex interplay of genetic, experiential and developmental factors. Genetic Factors Bo th genetic linkage studies and the candidate gene approach have led to useful insights into the genetic basis of depression. Although there is no one gene that causes depression, researchers have identified a number of susceptibility genes that, when expos ed to the toxic milieu of childhood trauma, parental abuse or neglect, or other
17 forms of developmental and social stress, can substantially increase the likelihood of developing depression. Modern estimates of the heritability of depression from genetic linkages studies vary from 30% to 70% depending on the stringency of the criteria used to define depression in clinical studies, suggesting the need for further investigations into the genetic origins of this elusive disease (McGuffin et al., 2007). One recently identified region of interest located on chromosome 15q has been the subject of at 3 independent largescale studies, including the Genetics of Recurrent Early Onset Major Depression (GenRED) study, the EuropeanU.S. Depression Network (DeNt) study, and a Utahbased study (McGuffin et al., 2007). The serotonin transporter Studies have identified a link between specific genetic loci, such as 5 regulatory region of the serotonin transporter (5HTT) gene, and an enhanced susceptibility to depression and post traumatic stress disorder (PTSD) (Lin et al., 2004;Lee et al., 2005;Pezewas et al., 2005). This 5HTT linked promoter region (5HTTLPR), located on 17q11.2, controls transcription of the 5HTT and thus has important implications for 5HT transmission in the central nervous system. The 5HTT is expressed on the postsynaptic membrane of serotonergic neurons and is primarily involved in reuptake of extracellular 5HT from the synapse. This not only terminates 5HT neurotransmission but also facilitates the repack aging of 5HT into synaptic vesicles. One particular variant of the 5HTTLPR polymorphism, the s allele, results in a reduced transcription rate of 5HTT gene and lower 5HTT mRNA expression (Bradley et al., 2005). In contrast, the long (l) allele confers high transcriptional efficacy resulting in greater 5HTT expression. In agreement with these findings, in vitro studies in lymphoblastoid cell lines derived from
18 individuals who are homogyous for the s allele have verified that 5HTT mRNA content is significant ly lower and that 5HT reuptake is reduced by half relative to carriers of the l allele (Lesch et al., 1996). Carriers of the s allele also exhibit anatomical differences in brain regions that mediate mood, attention and sensitivity to threatening or emotionally evocative stimuli. Regions that play a pivotal role in the modulation of emotional responses, such as the anterior cingulate cortex (ACC) and the amygdala, are reduced in volume, consistent with findings from other studies in depressed patients. Inte restingly, the thalamus is enlarged by a similar margin (1112%) in both depressed subjects and those with the ss genotype (Young et al., 2008). This increased capacity of the thalamus may lead to heightened sensitivity to environmental stimuli, which in t urn could overload the limbic system (e.g. the ACC and amygdala) and cause a compensatory downsizing of these brain regions. Further evidence for a direct relationship between thalamic volume and emotional behavior comes from the fact that treatment with antidepressant drugs reverses the trend; that is, people taking antidepressants show a reduction in thalamic volume relative to controls (Young et al., 2008). There is evidence of a strong geneenvironment interaction between the ss genotype and stress, which may be mediated by differential activation of the thalamus and other limbic regions that are modified by 5HTT gene expression. Studies in mice reveal that behavioral differences between 5HTT knockout and wildtype mice are only apparent under stress but not baseline conditions (Murphy et al., 2001). Similarly in macaques, carriers of the s allele only exhibit decreased serotonergic function when reared under elevated stress conditions (Bennett et al., 2002). Thus the effects of ss
19 genotype on emotional behavior appear to be kindled by stress, an observation that has been confirmed in humans. For instance, functional MRI studies have shown that an emotional task which involves identifying fearful facial expressions elicits a greater amygdala response in human carriers of the s allele compared to noncarriers, which again points toward a role of the 5HTT in modulating limbic response to stress and anxiety provoking events (Hariri et al., 2002). This overactivation of the amygdala in response to stress may also produce a corresponding loss of amygdalar volume that has been previously observed in individuals with the ss genotype. The moderating influence of the ss genotype on individual response to stressful life events is aptly demonstrated using epidemiolog ical data from the Dunedin Multidisciplinary Health and Development study (Caspi et al., 2003). The probability of a major depressive episode in adulthood does not vary significantly with genotype in individuals with a relatively normal, stress free upbringing. However, depression rates climb significantly among s carriers as a function of childhood trauma. Even more striking is the fact that individuals of the ll genotype appear to be remarkably resilient to the effects of childhood trauma, with depression rates remaining at a relatively low 30% even among survivors of severe trauma. The same holds true even when the stress occurs later on in adulthood, that is, the incidence of depression among s carriers, but not ll individuals, increases with cumulative life stress between the ages of 1826. Thus the effects of stress on emotional behavior in genetically vulnerable individuals are not limited to the childhood years. These data suggest that genes themselves may not necessarily cause depression but instead interact with complex array of developmental, experiential, and social factors
20 that predispose an individual to depression. Severe or prolonged stress, in conjunction with certain risk genes such as the 5HTT, seems to be a precipitating factor that sets in motion the neurobiological processes that actually cause depression. However, we cannot attribute a multifactorial disease such as depression to a single gene, as Risch et al. remind us in their 2009 metaanalysis of 14 published studies which revealed no significant interaction between stress, depression, and the 5HTT s allele (Risch et al., 2009). Yet the failure to replicate the gene x environment effect for a single gene, the 5HTT gene, does not exclude the possibility of other genetic, epigenetic, or environmental factors that may alter expression or penetrance of a gene of interest and thus neutralize (or augment) its effect on phenotype (e.g. gene x gene x environment interactions). Brain derived neurotrophic factor Brain derived neurotrophic fact or (BDNF) has a wide distribution in parts of the brain known to mediate emotional responses, and studies have found that hippocampal BDNF is not only depleted in depressed subjects (Chen et al., 2001), but plays a key role in the dendritic remodeling observed in the hippocampus following antidepressant treatment. It has even been suggested that failure to upregulate BDNF early on may predict failure to respond to antidepressants (Tadic et al., 2010). Furthermore, there is some evidence of cross talk between BDNF and 5HT pathways which increases the likelihood of a gene x gene interaction effect on depression outcome. The BDNF val(66)met polymorphism has been the subject of new investigations into the genetic basis of depression and has yielded some novel insights into the geneenvironment debate. The BDNF val(66)met polymorphism in the 5 proregion of the BDNF protein results in lower depolarizationinduced BDNF secretion from neurons, which negatively
21 impacts hippocampal excitability and episodic memory (E gan et al., 2003). Studies have shown a direct association between the BDNF val(66)met polymorphism and characteristics that are prevalent in a variety of psychiatric disorders, such as anxiety (Chen et al., 2006) and suicidality (Pregelj et al., 2010), while others have shown that the interaction of BDNF val(66) met polymorphism with childhood adversity (gene x environment) elevates the risk of depression in adulthood (Aguilera et al., 2009). Moreover, there appears to be a higher order interaction between the BDNF val(66)met polymorphism, the 5HTTLPR polymorphism, and childhood events (gene x gene x environment) that exerts a strong influence on the developmental course of depression (Kaufman et al., 2006). Antidepressant response and the 5HT1A receptor So far we have presented evidence that inheritance of multiple susceptibility genes confers sensitivity to the depressogenic effects of chronic life stress. Further evidence suggests that response time to SSRI antidepressants, such as sertraline, is also aff ected by variation in the 5HTT and other genes. Individuals with one or more copies of the s allele of the 5HTT tend to have a slower response time to sertraline (Durham et al, 2004), whereas ll individuals exhibit the most rapid response. This differential response may be explained in terms of the mechanism of SSRI action. SSRIs directly curtail high 5HTT expression generated by the variant of the 5HTTLPR polymorphism, resulting in a better prognosis for pharmacotherapy. On the other hand, the primary targ et of SSRIs is in lower abundance in carriers of the s allele, which may render individual carriers of the s allele less sensitive to the antidepressant effects of SSRIs. Once again, we find that gene x gene interactions affect the outcome of antidepressant
22 therapy, much as they do the etiology of depression itself. Combination of the ss genotype of the 5HTT with the 5HT1A receptor 1018C/G polymorphism results in a phenotype that is refractory to the antidepressant citalopram (Arias et al., 2005). The 5HT1 A receptor plays an important role in the antidepressant response, since ultimately the goal of therapy is to restore 5HT signaling which is mediated through postsynaptic 5HT1A receptors. However, the inhibitory effects of somatodendritic 5HT1A autoreceptors are thought to predominate in the initial phase of treatment when there is an immediate spike in 5HT in the synapse (Artigas et al., 1996). This excess 5HT binds to the autoreceptors, inhibiting further 5HT release which may account for the initial lag phase of 34 weeks in antidepressant treatment (Blier et al., 1987). Continued treatment with SSRIs is thought to desensitize 5HT1A autoreceptors, restoring 5HT release to pretreatment levels so that postsynaptic 5HT1A receptors are activated (Blier and de Montigny, 1998). The contribution of 5HT1A autoreceptors to antidepressant action may help to explain why the 5HT1A 1018 C/G polymorphism, which occurs in a region that controls transcription of the 5HT1A gene, has such a profound effect on antidepressant response time (Arias et al., 2005). The 5HT1A 1018 C/G polymorphism in particular has been associated with higher rates of transcription of the 5HT1A gene, which may translate to more autoreceptors that inhibit firing of 5HT neurons. The glucocorticoid receptor The glucocorticoid receptor (GR) is a key regulator of stress responsive neurohormonal signaling, in which one or more polymorphisms may contribute to vulnerability for depression. In particular, the 22/23EK polymorphism in the GR gene, much like the 5HTTLPR and BDNF val(66)met polymorphism, contributes to depressive psychopathology only in the presence of childhood trauma (Bet et al., 2008). This
23 particular allele affects responsiveness of the GR, rendering it less sensitive to cortisol. The GR i s widely distributed in parts of the brain that send inhibitory inputs to the parvaventricular nucleus (PVN) of the hypothalamus and essentially switch off the stress response, so this desensitized version of the GR would conceivably render the individual more vulnerable to the deleterious effects of stress. Role of Stress Depression has been characterized as a disorder of stress regulation that typically manifests under conditions of prolonged, and/or severe stress against a background of genetic vulnerabi lity. Under normal circumstances, stress is a highly adaptive series of physiological and behavioral responses to perturbations in the environment that have been coopted to restore and maintain homeostasis. This definition of stress as a response of the organism to some somatic or external event is not to be confused with the stimulus that evokes it, which will be referred to as a stressor Activation of brainstem catecholaminergic neurons, the amygdala, and the bed nucleus of stria terminalis (BNST) send converging, excitatory inputs to the paraventricular nucleus (PVN), which serves as the linchpin for activation of the central stress axis. Stimulation of the PVN initiates a neuroendocrine signaling cascade known as the hypothalamic pituitary adrenocortic al (HPA) axis that begins with secretion of corticotropinreleasing factor (CRF) and argininevasopressin into the pituitary portal circulation. This in turn stimulates adrenocorticotropic hormone (ACTH) release from the anterior pituitary, which is releas ed into systemic circulation. The binding of ACTH in the adrenal cortex releases glucocorticoids such as cortisol (corticosterone in rats) into the circulation (for a review see Herman et al., 2005). It is important to note that any gross interruption of t his vital pathway (e.g. adrenalectomy in rats) is fatal without corticosterone
24 replacement, emphasizing that basal function of the HPA axis is essential for survival. When we speak of stress in the negative, we are referring to severe, prolonged or chronic stress. Glucocorticoids exert their effects on the sympathetic nervous system by increasing heart rate, constricting blood vessels, and reducing digestive and other vegetative functions in preparation for the fight or flight response. In the brain they also bind to glucocorticoid receptors (GR), which are enriched in parts of the limbic system that mediate feedback to the PVN during stress. (Sapolsky et al., 1984;Herman et al., 1989;Herman and Cullinan, 1997;Herman et al., 2005). The key players in this feedback pathway will be discussed in the next sections. Prolonged or severe stressors such as childhood abuse, neglect, social isolation, or maternal separation may induce neurobiological adaptations in the stress axis that are conducive to those extreme environments but become maladaptive under normal circumstances. Associations between childhood trauma and perturbations in the HPA response are well documented in the literature, although there are some discrepancies between reports about the directionality of these changes that may be attributed to confounding factors such as genetic variation, the type and timing of abuse, gender, age and comorbid psychiatric or substance abuse disorders. One study reporting elevated ACTH and cortisol in response to th e dexamethasone/CRF suppression test in victims of childhood abuse suggest an exaggerated pituitary response to CRF or insensitivity to the inhibitory effect of glucocorticoids (Heim et al., 2001), which is characteristic of certain types of depression. In fact, childhood abuse sufferers with comorbid depression had the most robust response, which supports the notion that
25 altered stress responsiveness, mediated by childhood experience and genetic vulnerability, can predispose individuals to psychopathology. On the other hand, depressed individuals without childhood adversity exhibited a slightly lower HPA response relative to controls, highlighting that depression is a disease of diverse etiology. Individuals with and without a history of childhood trauma may become depressed, but the developmental trajectories leading to this common endpoint may differ by a considerable margin. Early life stress has also been linked with an increased risk of depression, which is independently associated with a dysregulated HPA response that is marked by an elevated, yet flattened sinusoidal rhythm of cortisol secretion (Stokes et al., 1995). Animal studies concur with these findings, and reports of CRF involvement in depression abound (Binder and Nemeroff, 2010). For instance, those with a genetic predisposition to depression, such as carriers of the ss genotype of the 5HTT gene, are shown to have elevated waking cortisol and ACTH levels compared to other genotypes (Chen et al., 2009;Wst et al., 2009). Hippocampus The hipp ocampus is a limbic structure located inside the medial temporal lobe which derives its name from the Greek hippos meaning horse and kampos meaning seamonster. Although well known for its role in learning and memory, the hippocampus is also a key modul ator of the stress axis as evidenced by the presence of GRs on hippocampal neurons, which provide an important source of negative feedback to the PVN. The hippocampus is also exquisitely sensitive to the deleterious effects of stress on neurogenesis and s ynaptic integrity. Sustained elevations in glucocorticoid levels
26 negatively impact cell proliferation and BDNF expression in the hippocampus, leading to eventual neuronal loss and hippocampal atrophy (Stokes et al., 1995;Gould et al., 1997;Schmidt and Duman, 2007). Thus prolonged periods of stress interrupts negative feedback to the PVN, allowing excessive glucocorticoid release to exert further hippocampal damage. Depression has also been associated with hippocampal volume loss (Chen et al., 2001;Nichols et al., 2001;Marin et al., 2007;Lanfumey et al., 2008) and concomitant learning and memory impairments that tend to worsen with age (Kim and Diamond, 2002;Schmidt and Duman, 2007). In fact, a lifetime history of depression has been identified as a potential risk factor in the cognitive impairment of elderly subjects (Sierksma et al., 2010). Similarly, depressogenic manipulations in animals result in neuronal degeneration in the CA3 region of the hippocampus and profound deficits in neurogenesis. Reversal of these effects is achieved with antidepressant treatment, and in fact may be essential to achieve a therapeutic response to these drugs (Santarelli et al., 2003). Prefrontal cortex The medial prefrontal cortex (mPFC) plays a somewhat more complex role in n egative feedback regulation of the HPA axis, although its overall actions are inhibitory. Stress evokes cfos expression in the anterior cingulate and prelimbic subdivisions of the mPFC, which have been shown to modulate ACTH and corticosterone secretion a nd cfos induction in the PVN, possibly via projections to the ventrolateral preoptic area, dorsomedial hypothalamus, and peri PVN region (Hurley et al., 1991;Sesack et al., 1989). The infralimbic division of the mPFC, on the other hand, opposes the actions of the other two and sends afferent projections to the bed nucleus of stria terminalis
27 (BNST), medial and central amygdala and the nucleus of the solitary tract, all of which activate the HPA axis. However, the inhibitory effects of the mPFC on the HPA ax is predominate, and structural abnormalities in the mPFC have been implicated in the pathophysiology of a variety of psychiatric disorders. Hypofrontality, or reduced activation of the frontal cortex, contributes to the dysregulation of central stress pathways in the brain and inhibition of emotional and behavioral responses to stress, and these effects tend to be lateralized to the left hemisphere. Depression is associated with gray matter loss in the ACC and orbital and ventrolateral PFC based on MRI and postmortem histological analysis with a corresponding decrease in total metabolic activity as reported by positron emission tomography (PET) studies (Drevets et al., 2008). We also know from animal studies that chronic stress, which has depressogenic effe cts, induces dendritic spine loss (Radley et al., 2006), apical dendritic retraction (Radley et al., 2004) and volumetric reductions in the mPFC (Kitayama et al., 2006), paralleling morphological changes seen in the hippocampus under similar experimental conditions. Role of Monoamines The monoaminergic hypothesis of depression emerges from the fact that depression is closely linked with the dysregulation of serotonergic and other monoamine systems in the brain. In the 1950s it was observed that many patien ts taking the antihypertensive drug reserpine, which depletes monoamines, developed symptoms of depression (Musselman et al., 1998), giving rise to animal models of depression that are based on monoamine depletion. Perhaps the most compelling evidence stem s from yet another fortuitous discovery that iproniazid, an experimental drug used to combat tuberculosis infection, has antidepressant effects (Smith et al., 1953;Loomer et al.,
28 1957; Kline et al., 1958;Salzer and Lurie, 1953). Furthermore, iproniazid pretreatment appeared to reverse the depressogenic effects of reserpine (Saunders et al., 1959; Kline, 1959), suggesting a possible mode of action that involved potentiation of monoaminergic activity. Reserpine depletes monoamines by interfering with uptake o f monoamines into synaptic vesicles through the vesicular monoamine transporter (VMAT), leaving them vulnerable to enzymatic digestion by monoamine oxidases (MAOs). Iproniazid, a monoamine oxidase inhibitor (MAOI), prevents this degradation, and as a resul t monoamine concentrations at nerve terminals are vastly increased (Assael et al., 1960). And while this drug and other MAOIs were abandoned in favor of tricyclic antidepressants (TCA) shortly thereafter, they played an indispensible role in the formulation of the so called catecholamine hypothesis of depression (Schildkraut, 1965). It was originally thought that catecholamines, namely norepinephrine and dopamine, were responsible for the antidepressant action of MAOIs and TCAs. This was supported by the fact that imipramine, a TCA, was shown to block reuptake of norepinephrine into presynaptic terminals (Axelrod et al., 1961). A growing body of evidence, however, pointed to serotonin as the culprit. Tryptophan, the precursor to serotonin synthesis, augmented the antidepressant effects of the MAOI tranylcypromine, while tryptophan depletion had the opposite effect on mood. Furthermore, it was found that imipramine blocked serotonin reuptake in platelets. Depressed patients exhibit reduced concentrations of the serotonin metabolite 5hydroxy indole acetic acid (5HIAA) in the cerebrospinal fluid (CSF) and post mortem brain tissue (Owens et al., 1994). Suicide victims, which represent the most severe case of depression, manifest other signs of serotonin defici ency such as increased 5HT2
29 receptor density, decreased 5HTT density and decreased 5HT1A autoreceptor binding (Owens et al., 1994;Drevets, 1998). These findings led to the eventual design of selective serotonin reuptake inhibitors (SSRIs) which averted many of the side effects incurred with previous antidepressants due to their actions on cholinergic systems. These drugs represent the gold standard of antidepressant therapy today, which again lends credence to the monoamine hypothesis of depression, now modified to include serotonin. Nodes of Interaction between Stress and Monoaminergic systems Dysregulation of the stress response, which can lead to certain types of psychopathology including depression, is thought to stem from low serotonergic drive (Stokes, 1995). As noted earlier, depression is associated with impaired negative feedback to the HPA axis which may be mediated by extended glucocorticoid action at the hippocampus and other limbic sites. Serotonin is a key modulator of two important players in t he stress network, hippocampal BDNF and GR, which may be protective against hippocampal damage and diminished feedback drive. Thus, individual differences in serotonergic tone, whether genetically determined or environmentally induced, may confer vulnerabi lity for affective disorders. The hippocampus is an important site of interaction between glucocorticoid and serotonin systems in the brain, given its role in learning and memory processes, stress regulation, and mood disorders. Corticosterone elicits a rapid response from 5HT systems by increasing the activity of tryptophan hydroxylase, the rate limiting enzyme in 5HT synthesis, and increasing the firing rates of 5HT neurons that project to the hippocampus. A variety of acute stressors have also been shown to increase 5HT release in the hippocampus (Keeney et al., 2006;Linthorst and Reul, 2008). This
30 increase in 5HT release in the hippocampus is important for habituation to chronic stress (Keeney et al., 2006), whereas serotonin depletion (Zhou et al., 20 08) or lesions in serotonergic neurons projecting to the hippocampus (Robertson et al., 2005) resulted in loss of glucocorticoid receptors, anxiety like behavior and anhedonia. Studies in rodents have also found that 5HT1A autoreceptors, which inhibit 5HT release from presynaptic terminals, are desensitized in the hippocampus following chronic stress (Buwalda et al., 2005;Lanfumey et al., 2008). These facilitatory actions of glucocorticoids on 5HT neurotransmission are thought to promote behavioral adaptati on through selective modifications in corticolimbic circuitry. Excessive stimulation by glucocorticoids exerts an inhibitory effect on serotonin function through mechanisms which are not yet clearly understood. As mentioned earlier, acute stress stimulates 5HT release in the hippocampus, but this response is inhibited under conditions of prolonged, unmitigated stress. For instance, mice reared in isolation have a blunted 5HT response to novelty stress compared to groupreared mice (Bickerdike et al., 1993; Muchimapura et al., 2002). Furthermore, chronic social defeat stress decreases the number of 5HTT binding sites in the CA3 region of the hippocampus, similar to what has been observed in suicide victims. We propose a role for OCT3 as a molecular interface between glucocorticoid actions at limbic regulatory sites and serotonin signaling in the brain that mediates neurobiological adaptations to stress. The Organic Cation Transporter 3 Neuroanatomical Distribution The OCT3 belongs to a family of polyspecif ic solute carriers that have been shown to play a significant role in the elimination of cationic species in tissues such as
31 the liver and kidney (Prichard et al., 1993;Ullrich, 1994). The OCT1 was the first to be cloned from rat kidney, and both OCT1 and OCT2 subtypes are predominantly expressed in the kidney, liver and to a lesser extent the intestine. More recently OCT3 mRNA was discovered to be abundantly expressed in the placenta, but is also detectable by Northern blot analysis in certain parts of the brain (Kekuda et al, 1998). Further characterization of OCT3 distribution in the brain by in situ hybridization reveals robust expression within the cerebellum, hippocampus, pontine nucleus and cortex (Wu et al., 1998). High levels of expression are also detected along the brainCSF border and choroid plexus (CP), emphasizing the putative role of the OCT3 in regulating the bioavailability of cationic neurotoxins in the brain. However, the OCT3 also has a hand in what is referred to as uptake2, a low affini ty, steroid sensitive, Na+and Cl-independent and phylogenetically older system for removing extracellular monoamines. Given its role in uptake2, OCT3 expression is found to cluster along monoaminergic pathways innervating the dorsal raphe, striatum, c ortex, PVN of the hypothalamus, thalamus, hippocampus and nucleus accumbens (Amphoux et al., 2006;Gasser et al., 2009). The OCT3 also colocalizes with dopamine D1 receptors in parts of the amygdala, but not with tyrosine hydroxylase or dopamine hydroxylase, suggesting that the OCT3 is situated on postsynaptic membranes (Hill et al., 2009). The cellular distribution of the OCT3 is not uniform across the entire brain, although it was originally named the extraneuronal monoamine transporter (EM T) due to its association with glia in some parts of the brain. For instance in the nigrostriatal pathway, the OCT3 is predominantly expressed in astrocytes. In the hippocampus, however, the OCT3 is primarily localized to neurons where it likely participat es in postsynaptic
32 clearance of extracellular monoamines (Cui et al., 2009). In our work we have focused primarily on the GR rich limbic regions of the hippocampus and medial prefrontal cortex where monoaminergic systems and stress pathways converge in the brain. Pharmacology An alternative catecholamine transport system, dubbed uptake2, was proposed by Iversen in 1965 to account for the accumulation of norepinephrine in cardiac muscle after perfusion at very high concentrations (>1 g/ml). (Iversen, 1965) This mode of transport, unlike highaffinity presynpatic reuptake, is characterized as a low affinity, high capacity, Na+and Clindependent system that is driven by membrane potential and is pharmacological sensitive to steroids, cyanine dye derivat ives and O methylated catecholamines such as normetanephrine (Gr ndemann et al., 1997;Wu et al., 1998;Amphoux et al., 2006). The OCT3 has been shown to exhibit all of these properties and was subsequently established as the molecular identity of uptake2 (W u et al., 1998). Role in monoamine transport A variety of monoamines, including norepinephrine, dopamine, serotonin and histamine, can serve as substrates for the OCT3, albeit at concentrations in the micromolar range. ( Gr ndemann et al., 1998;Gr ndemann et al., 1999;Schomig et al., 2006). This low affinity, high capacity transport system is especially salient when the high affinity transporters are saturated or in low abundance. This was aptly demonstrated in mice with a homozygous deletion of the 5HTT, i n which 5HT levels were kept at relatively normal concentrations due to upregulation of the OCT3 in the hippocampus (Schmitt et al., 2003;Baganz et al., 2008). As previously noted, stress
33 provokes the rapid release of monoamines which may provide the necessary conditions for the OCT3 to exert a discernible effect on monoaminergic neurotransmission. Regulation by corticosterone Corticosteroids have been shown to elicit rapid increases in extracellular monoamine concentrations in the dorsomedial hypothalamus (DMH) (Lowry et al., 2001), possibly through their acute inhibitory actions on the OCT3 (Gasser et al., 2006). Previous studies have demonstrated elevated dopamine levels in the DMH of salamanders within 20 minutes of receiving an intracerbroventricular (ICV) injection of CRF. Similar effects were observed following CORT injection except that both dopamine and 5HT concentrations were elevated (Lowry et al., 2001). Acute restraint stress induced simultaneous increases in 5HT, DA and NE concentrations in the dorsal hypothalamic area (DHA) of Lewis rats (Lowry et al., 2003), which is suggestive of extracellular accumulation mediated by blockade of the OCT3. Conversely in Fischer rats, only NE decreases in the DHA after restraint stress, revealing possible str ain dependent differences in stress responsiveness. This blunted monoaminergic response to acute stress in Fischer rats may partially explain their hyperreactivity to stress as indicated by their plasma corticosterone levels. There was also a negative corr elation between 5HT and plasma corticosterone concentrations in unstressed rats, suggesting that 5HT activity in the DHA may suppress excitability of the HPA axis. Other studies have demonstrated that intraDMH injection of the cyanine dye derivative dec ynium 22, which specifically inhibits the OCT3, elicits robust increases in extracellular 5HT as quantified by in vivo microdiaysis (Feng et al., 2005). Contrary to the findings of Lowry and colleagues, this group did not observe a significant increase in 5HT following corticosterone infusion in the DMH. However, it should be noted that
34 Feng et al. used in vivo microdialysis combined with high performance liquid chromatography (HPLC) on live animals to obtain a measure of extracellular monoamine concentrati ons. Lowry, on the other hand, performed HPLC analysis on postmortem tissues, which reflects both intracellular and extracellular levels of the analyte. However, Feng et al. did observe that corticosterone potentiated the effects of fenfluramine on extracellular 5HT in the DMH. Fenfluramine increases serotonin release by disrupting vesicular storage, causing a flood of 5HT in the synapse that overwhelms high affinity transport (Feng et al., 2009). Taken together, these findings illustrate that the contribut ions of the OCT3 to 5HT neurotransmission are unmasked in the presence of unusually high 5HT levels. The same is true when restraint stress is used in place of corticosterone. While acute restraint stress alone did not elevate extracellular corticosteron e or 5HT levels in SpragueDawley rats as measured by in vivo microdialysis, it did augment the effects of decynium 22 (Feng et al., 2010). It should be noted that the effects of OCT3 on 5HT neurotransmission are not limited to the DMH and DHA, as other st udies have noted that decynium 22 reduces 5HT clearance in the CA3 region of the hippocampus using in vivo chronoamperometry. However, these effects were only apparent in mice lacking at least one copy of the 5HTT gene (Baganz et al., 2008), illustrating once again that uptake2 via the OCT3 is important in the homeostatic regulation of 5HT when highaffinity transport is absent or disabled. Interactions with the 5HTT and antidepressant response Recent studies have shown that the OCT3 is upregulated in the hippocampus of mice lacking 5HTT, which may serve to buffer against elevations in synaptic 5HT concentrations (Schmitt et al., 2003;Baganz et al., 2008). This implies that processes that disrupt normal 5HTT function lead to the recruitment of OCT3 for the r emoval of
35 extracellular 5HT, at least in the hippocampus. It stands to reason that the OCT3 may negate the therapeutic benefit derived from antidepressant drugs, which typically exert their effects by increasing synaptic levels of 5HT. Human carriers of the s allele may be especially vulnerable to the buffering effects of OCT3 on extracellular 5HT, since they presumably have lower 5HTT expression which has been associated with OCT3 upregulation in mice. It would be interesting and informative to see if OCT3 is also upregulated in depressed patients with the s variant of the 5HTTLPR polymorphism, which may both explain their unique depressive phenotype and their insensitivity to conventional antidepressants. Furthermore, targeting the OCT3 may offer new therapeutic strategies for treating such patients, as indicated by preliminary studies in which the OCT3 inhibitor decynium 22 effectively reduced depressivelike behavior in 5HTT knockout mice, but not in wildtype controls (Baganz et al., 2008). Role in Psychopathology The OCT3 has been associated with a variety of psychiatric disorders including obsessive compulsive disorder (OCD) and methamphetamine use disorder in humans(Lazar et al., 2008;Aoyama et al., 2006;Fujimoto et al., 2007;Nagayama et al., 2007) and depressiveand anxiety like behavior in rodents (Wieland et al., 2000;Kitaichi et al., 2005;Baganz et al., 2008;Vialou et al., 2008;Wultsch et al., 2009). These results are not surprising given the role of the OCT3 in 5HT transport and clearance, which i s demonstrated in 5HTT knockout mice (Baganz et al., 2008). It has also been shown that OCT3 antisense and OCT3 inhibitors such as decynium 22 and normetanephrine (Kitaichi et al., 2005;Baganz et al., 2008;Schildkraut et al., 2004;Mooney et al., 2008) can exert an antidepressant effect in the Porsolt swim test. Furthermore, genetic deletion of the OCT3 was found to reduce anxiety like behavior on the elevated plus
36 maze and open field tests (Wultsch et al., 2009). Still others have argued that the OCT3 delet ion may disrupt neuronal function (Vialou et al., 2008). This ambiguity with regard to the role of OCT3 in psychiatric morbidity is partially resolved during the course of our study of the comparative expression of OCT3 in the WKY rat, which represents an animal model of depression with both face and construct validity. We provide the first evidence that OCT3 gene expression is differentially regulated in the Wistar Kyoto rat, which may give rise to an aberrant stress response and vulnerability to psychopat hology. Project Rationale The goal of these studies is to characterize the role of the OCT3 in the etiology of stressrelated psychopathology. Chronic stress is associated with the onset and exacerbation of depression, a limbic mediated mood disorder that is marked by anhedonia, despair, and psychomotor retardation. Stress stimulates the hypothalamic pituitary adrenal (HPA) axis and triggers the release of corticosterone (CORT) into circulation, which can directly modulate 5HT signaling through their actions on OCT3. As such, individual differences in susceptibility to Major Depression may be associated with genetic variations in the expression or functionality of the OCT3. In our studies we made use of the Wistar Kyoto (WKY) rat as an animal model of depression. These animals exhibit depressivelike behavior in the Porsolt forced swim test (Par 1994;Armario et al., 1995;Lopez Rubalcava and Lucki, 2000), which is the current standard in screening new compounds for antidepressant efficacy (Porsolt et al., 1 978). Acute stress in these animals also elicits an exaggerated and protracted neuroendocrine response that fails to habituate with repeated stress exposure (Pare and Redei, 1993;Rittenhouse et al., 2002;De la Garza and Mahoney, 2004). Stress
37 provokes inc reased rates of 5HT turnover, increased 5HT1A receptor sites and a decrease in 5HTT sites (De La Garza and Mahoney, 2004), which may be associated with altered expression of the OCT3 in this strain of rats. This idea is supported by the fact that 7 day treatment with corticosterone alters 5HT uptake in the hippocampus of WKY rats in a 5HTT independent fashion (Fernandez et al., 2001). In the experiments reported in Chapter 2 we assessed gene and protein expression of the OCT3 in the WKY rat. We postulated OCT3 expression would be differentially regulated in the WKY rat relative to the more stress resilient Long Evans (LE) strain, which may account for strain specific deficits in serotonergic tone and subsequent expression of depressivelike behavior. In the experiments of Chapter 3 we assessed the effects of an OCT3 antagonist on depressivelike behavior in the forced swim test. Decynium 22, a specific inhibitor of the OCT3, has been shown to induce a rapid increase in extracellular 5HT content in the dors omedial hypothalamus of rats. Elevations in 5HT due to inhibition of OCT3 are associated with a reduction in depressivelike behavior in the Porsolt forced swim test, We hypothesized that decynium 22 would have an antidepressant effect on the forced swim t est in WKY rat. As described earlier, the OCT3 is a highcapacity, low affinity transporter of 5HT that participates in 5HT clearance and regulates serotonin neurotransmission when the high affinity 5HTT is saturated or in low abundance. Such is the case i n human carriers of the s allele of the 5HTT who have both reduced expression of the 5HTT and a higher propensity toward depressive behavior. Individuals with the ss genotype are also refractory to conventional antidepressants of the selective serotonin reuptake inhibitor
38 (SSRI) class. Due to the low abundance of the 5HTT, there is reason to believe that the OCT3 may be upregulated in these individuals and may also serve as a novel target for antidepressant intervention. The WKY rat shares many characterist ics in common with these genetically predisposed individuals including low 5HTT expression and a poor response to SSRI drugs on the forced swim test (Lahmame and Armario, 1996;Griebel et al., 1999;Lopez Rubalcava and Lucki, 2000;Tejani Butt et al., 2003), indicating that the WKY rat may also serve as a model of antidepressant resistance that can be used to test the effectiveness of new pharmacological approaches to treatment, such as OCT3 inhibition.
39 CHAPTER 2 EFFECTS OF ACUTE AND REPEATED STRESS ON EXPRESSION OF THE OCT3 IN AN ANIMAL MODEL OF DEPRESSION Introduction Several lines of evidence indicate that chronic exposure to stress can invoke depressive symptoms in a subset of individuals with certain genetic, developmental and psychosocial profiles. We hav e attempted to identify some of these factors that can predispose an individual to stress induced psychopathology using a model that recapitulates some of the core features of depressive disorders. Although depression is a multi factorial disease with phen otypic heterogeneity that is difficult to reproduce in an animal model, there are at least two symptoms that manifest in almost all cases of depression: anhedonia, or the loss of pleasure in almost all activities and helplessness, and the inability to act in ones own interest to avoid harmful or unpleasant circumstances (Zacharko and Anisman, 1991;Snaith, 1993;ONeil and Moore, 2003; B rgy, 2008). The WKY rat embodies these core elements of depressive illness, exhibiting excessive immobility on the forced swim test (which is used as a proxy for helplessness behavior) and prolonged elevation in plasma CORT levels (which suggests hyperactivity of the HPA axis). In these rats, stress is also accompanied by reduced 5HT levels (De La Garza and Mahoney, 2004), increased 5HT1A receptor binding in the hippocampus and hypothalamus, and a decrease in 5HTT sites in the cortex and hippocampus (P re and Tejani Butt, 1996), whereas in other strains the directionality of these parameters is reversed. This leads us to postulate that stress may differentially affect the 5HT systems of WKY rats compared to other strains, giving rise to neurobiological adaptations in corticolimbic circuitry that manifest as depression.
40 The OCT3 is a stress responsive, monoamine transporter in the brain that has profound influence on 5HT neurotransmission in the limbic system. The OCT3 is acutely inhibited by corticosterone, which produces local increases in 5HT in limbic regions of the brain that may facilitate stress recovery. Previous studies have shown that the OCT3 is upregulated in the hippocampus of mice lacking the 5HTT. Upregulation of the OCT3 can increase the rate of 5HT clearance in the hippocampus which in turn may inhibit behavioral adaptation to stress. In these studies, we examine d strainspecific differences in OCT3 expression between WKY and Long Evans (LE) rats with a view toward elucidating the molecular mechanisms that underlie stress induced psychopathology. We hypothesized that differential expression of OCT3 in the hippocampus of WKY rats may contribute to their stress vulnerable phenotype. Induction of a depressivelike state in animals can be accomplished by exposure to a variety of stressful stimuli that promote physiological and behavioral changes. In the resident intru der paradigm or social defeat model, a large male resident rat comes into brief contact with the test animal, a smaller intruder rat. This interaction, which almost always results in the defeat of the test animal, aims to recapitulate the dominancesubordination hierarchies that impact mood regulation and affect. The social defeat model of depression possesses high face validity, since the most common source of stress in humans is of a psychosocial nature (Bjorkqvist, 2001). Social defeat is positively associated with elevated glucocorticoid and ACTH activity and behavioral sensitization to mild stress (Buwalda et al., 2005); both core symptoms of human depression (Stokes, 1995). Social defeat also invokes dendritic remodeling and inhibition of neurogenesis in tree shrews (Magarinos et al., 1996; Gould
41 et al., 1997), which correlates with the hippocampal atrophy that occurs in human depression (Sala et al., 2004). On a behavioral level, chronic social defeat also induces anhedonia on the sucrose preference test and helplessness behavior on the forced swim test which is attended by histone modifications in the hippocampus (Rygula et al., 2005;Rygula et al., 2006;Hollis et al., 2010). Furthermore, antidepressant s have been shown to reverse these behavioral adaptations induced by chronic social defeat (Rygula et al., 2006). In this study, we applied the social defeat model to the genetically vulnerable WKY rat to simulate the genes environment interactions that give rise to major depression. Our goal was to determine if the OCT3 gene is differentially regulated by stress in WKY rats compared to LE rats. Given that LE rats readily adapt to stress and exhibit none of the behavioral characteristics associated with depression, this strain was used as a control t hroughout all of our experiments. We also analyzed GR gene expression and BDNF protein expression in the hippocampus as biomarkers of stress induced neuroplasticity. In order to investigate the putative correlation between 5HTT and OCT3, we analyzed 5HTT g ene expression in our samples as well. Methods Experimental Design Separate cohorts of WKY and LE rats were used to obtain tissues for real time PCR and Western blot analysis of the OCT3. In each cohort, WKY and LE rats were randomly assigned to control, acute, and repeated stress groups. All rats in the repeated stress group were exposed to daily social defeat for 7 days. On day 8, rats in the acute and repeated stress groups were exposed to 30 minutes of restraint stress, then returned to their home cages for the remainder of 2 hours (real time PCR) or 3
42 hours (Western blot) since the beginning of the restraint session. Control rats were handled daily but were otherwise undisturbed (Figure 21). General Methods Experimental animals Twenty four adult male WKY and and twenty four adult male LE rats weighing 250300 g at the beginning of the experiment were pair housed in standard polycarbonate cages (43 x 21.5 x 25.5 cm) in a climate controlled vivarium with a 12hour light/dark schedule (lights on at 7 am). The rats were allowed ad libitum access to standard laboratory chow (Lab Diet 5001) and tap water. The rats were allowed 7 days to adapt to the housing facility before the experiments began. At the end of the experiment, the animals were terminated by rapid decapitation. Post mortem tissue processing Rat brains were dissected at 4C to remove the mPFC, hippocampus and striatum according to a modified version of the technique first published by Iversen and Glowinski (Iversen and Glowinski, 1966). Dissected tissues were frozen in isopentane maintained at 40C and subsequently stored at 80 C until further processing. Stress Procedures Social defeat Male retired breeder rats of the Long Evans strain weighing at least 600 g at the start of experiments were isolation housed in standard polycarbonate cages in a climate controlled vivarium with a reverse 12hour light/dark schedule (lights on at 7 pm). Rats were allowed 7 days to acclimate to the housing facility before screening for dominance behavior. During the screening test, an intruder rat was placed inside the residents home cage. If the intruder exhibited submissive behavior (supine posture 3
43 times for a minimum of 2 sec each time), the resident was classified as dominant. Once dominance was established, the test session was terminated. This procedure was repeated daily (up to 30 days) until there are 3 consecutive days in which the intruder was defeated 3 times within the 5 min session. Once the residents met the minimum criteria for dominance behavior they were used for subsequent social defeat experiments. The social defeat procedure consisted of two stages. In the first stage, the intruder rat was placed into a 10 x 10 x 15 cm (inner dimensions) doublewalled wire mesh cage protective cage. The rat was then placed, inside the protective cage, into the home cage of the resident rat for 45 minutes. The cage served to separate the rats, avoiding physical contact but maintaining stressful sensory stimuli. During the second stage, the intruder rat was rem oved from the wire mesh cage and placed directly inside the residents home cage for 5 minutes. Dominance/submission was scored as above. This direct interaction between resident and intruder rats continued for 5 min before the test session was terminated. After exposure to the resident rat, the intruder rat was removed and returned to its home cage (Figure 22). Restraint stress Restraint stress was performed according to methods we previously described by our laboratory (Devine et al., 2003;Simpkiss and D evine, 2003;King et al., 2007). Briefly, each rat was individually removed from its home cage and placed into a restraining tube for 30 minutes. The restraint tube was composed of a soft flexible sheet of plastic (11 X 7 ) mounted to a rigid Plexiglas cradle (8 X 3 X 3) by means of two small bolts with convex heads. The securing nuts were on the bottom of the cradle to prevent any
44 discomfort to the animal. The cradle was open on the two long sides to allow the rat to be placed on the plastic shee t while flat. There were ventilation holes at one end to allow unrestricted breathing, and the other end had a vertical slot to allow comfortable placement of the tail during the restraint process. The plastic sheet was then gently rolled around the anim al and held securely in place with two 12 X 1 Velcro strips. Following the 30 min exposure to restraint stress, the rat was returned to its home cage. Biochemical and Molecular Assays Real time PCR RNA isolation was carried out according to the protocol by Molecular Research Center (MRC), Cincinnati OH. Briefly, tissues were homogenized in 1 ml TRI Reagent RT (MRC, Cincinatti, OH) with a sonifier and mixed with 50 ul 4bromoanisole (BAN), then incubated on ice for 5 minutes. Samples were then centrifuged at 12,000 x g for 15 minutes at 4C and 0.5 ml of the aqueous phase was transferred to a new tube for subsequent wash and precipitation steps. The resulting RNA pellet was reconstituted in nucleasefree water (Applied Biosystems/Ambion, Austin, TX) and t he final concentration was quantified using a NanoDrop spectrophotometer. cDNA was generated from total RNA using the High Capacity RNA to cDNA kit according to the manufacturers protocol. (Applied Biosystems, Foster City, CA). Real time PCR was carried out using Taqman Gene Expression reagents with primer/probe sets from Applied Biosystems that are specific for the OCT3 (Assay ID Rn00570264_m1), 5HTT (Assay ID Rn00564737_m1), GR (Assay ID Rn00561369_m1) or eukaryotic 18S (Assay ID Hs99999901_s1) (Table 21). All samples were analyzed in duplicate on the Applied Biosystems 7500 Fast Real time PCR system.
45 Cycle thresholds (Ct) values were used to compute the relative quantification (RQ) for each gene of interest. RQ values were calculated according to the formula below: Ct = Ct (gene of interest) Ct(18S) Ct = mean Ct (sample) mean Ct (reference) RQ = 2Ct relative quantification The mean Ct (reference) used to compute the Ct was the mean control group in cases where both strains were analyzed together or the LE strain was analyzed separately. In cases where the WKY group was analyzed separately, the Western blot Tissues were processed for Western blot analysis using the Plasma Membrane Protein Extraction Kit (Biovision, Mountain View, CA) to obtain separate fractions of cell surface, organelle, and cytosolic proteins. OCT3 and BDNF protein expression was quantified by Western blot using antibodies directed against the rat OCT3 (Abca m, Cambridge, MA). N Ca actin (Cell Signaling Technology, Danvers, MA) were used as endogenous controls in plasma membrane and cytosol extracts, respectively. Protein electrophoresis was performed on a NuPAGE 4 12% Tris glycine gel (Invitrogen, Carlsbad, CA). Each sample was diluted in loading buffer (50mM Tris HCl (pH 6.8), 2% SDS (sodium dodecyl sulfate), 0.1% bromophenol blue, 10% glycerol, 50 parallel. Elec trophoresis was performed at 195 volts for approximately 60 min. The gels were then equilibrated in protein transfer buffer containing 25 mM Tris, 192 mM glycine, and 10% (v/v) methanol. The proteins were electrophoretically transferred onto a
46 polyvinylide ne difluoride (PVDF) membrane using a semi dry transfer apparatus (Biorad, Hercules, CA). After the transfer, the membrane was rinsed with Tris buffered saline (TBS) containing 0.1% Tween 20 (TBS T) with pH of 7.4 and blocked for 1 hour with 5% nonfat dry milk in TBS T. The membrane was then incubated with rabbit anti rat OCT3 or BDNF IgG (Abcam, Cambridge, MA) (Table 22) diluted 1:1000 in 5% nonfat dry milk in TBS T overnight at 4C. After the overnight incubation they were washed three times in TBS T a nd incubated for 1 hr at room temperature in horseradish peroxidase (HRP) conjugated goat anti rabbit (1:2000 goat anti rabbit peroxidase conjugate (Cell Signaling Technology, Beverly, MA) in 5% nonfat dry milk in TBS T. Membranes were washed 3 10 min i n TBST at room temperature with rocking, then developed using Phototope HRP Western Blot Detection System (Cell Signaling Technology) and exposed to Kodak Biomax Light chemiluminescence film. Chemiluminescent bands were quantified by optical densitometry using Image J software. The PVDF membranes were stripped by immersion in SDS stripping buffer (containing 62.5 mM Tris, 2% SDS and 150 mM 2mercaptoethanol, pH 6.8) for 15 min. at 37C. Membranes were then washed 3 x 10 min in TBS T, reblocked in 5% nonf at dry milk in TBS T, and reprobed with an antibody for N cadherin (plasma membrane marker) or actin (cytosolic marker) to verify equivalent expression of this stable marker is present in blots from all the rats in each treatment group. Chemiluminescent bands were quantified by optical densitometry using the MCID camera system and Image J software. The integrated optical density (OD) of bands corresponding to the
47 OCT3, BDNF, Ncad, and actin were measured using this program and used to compute the relative OD for each gene of interest. Adjusted OD = OD (gene of interest)/OD (endogenous control) Relative OD= Adjusted OD (sample)/mean adjusted OD (reference) In cases where both strains were analyzed together and the LE strain was analyzed separately, the reference is the LE control group. In cases where the WKY strain was analyzed separately, the reference is the WKY control group. Statistical Analyses Gene and protein expression data for the OCT3, GR, and 5HTT were analyzed by two way analyses of variance (ANOVAs) using Graphpad Prism software to determine significant main effects of strain and stress and significant strain x stress interactions. Data from all groups are expressed relative to the LE control group. Individual Student t tests were used to de termine significant differences between WKY and LE rats in each stress category. The results of these analyses are presented in the first graph (A) of each figure. In cases where the twoway ANOVAs revealed a significant main effect of stress, we analyzed both strains separately using a oneway ANOVA so that pairwise comparisons could be made between different stress categories with NewmanKeuls post tests. In this case, all data were expressed relative to the LE control group for the comparisons of stress effects across the LE groups and to the WKY control group for comparisons of stress effects across WKY groups. The results of these analysis are depicted in (B) and (C) of each figure.
48 We performed correlation analysis between OCT3 gene expression and 1) GR and 2) 5HTT gene expression within each strain using the Pearsons correlation in Graphpad Prism. Results Real time PCR Data OCT3 gene expression Hippocampus. There were significant main effects of strain (F(1,41)=1630, p<0.0001) and stress (F(2,41)=266, p<0.0001) on hippocampal gene expression of the OCT3, and a significant interaction between strain and stress (F(2,41)=310.3, p<0.0001) (Figure 23A). Student t tests revealed OCT3 gene expression was significantly higher in WKY compared to LE rats in all stress groups. The effects of stress on OCT3 gene expression were also analyzed separately for each strain. A oneway analysis of variance (ANOVA) revealed a significant effect of stress on OCT3 gene expression in the hippocampus of LE rats (F(2,21)=9.804, p=0.001). Post hoc analysis showed that acute, but not repeated stress induces a robust increase in hippocampal OCT3 gene expression compared to all other groups in LE rats (Figure 23B). An identical analysis in WKY rats also revealed a significant ef fect of stress (F(2,20)=23.15, p<0.0001) on hippocampal gene expression of the OCT3 (Figure 23C) In this case, however, it was repeated, but not acute stress that invoked upregulation of the OCT3 compared to all other groups. Medial prefrontal cortex. A main effect of strain on OCT3 gene expression in the mPFC was apparent (F(1,41)=169.6, p<0.0001), but there was no main effect of stress (F(2,41)=1.357), p=0.2688). There was, however a significant stress x strain interaction
49 (F(2,41)=4.863, p=0.0127) (Fi gure 24A). Student t tests revealed a significant elevation in OCT3 in WKY relative to LE rats in all stress categories. Striatum. There were no main effects of strain (F(1,41)=0.03982, p=0.8428) or stress (F(2,41)=0.4063, p=0.6687) on striatal OCT3 gene expression There was no significant interaction between stress and strain (F(2,41)=0.5070, p=0.6061) and (Figure 2 5A). GR gene expression There was no main effects of strain (F(1,41)=0.3087, p=0.5815) on hippocampal GR expression. Although the effect of stress on GR gene expression did not quite reach significance (F(2,41)=2.941, p=0.0641), there was a trend toward an increase after acute stress. There was also no significant interaction stress x strain interaction (F(2,39)=2.236, p=0.1197) (Figure 26A) There was, however, a significant positive correlation between OCT3 and GR expression in both strains (r(20)=0.5056, p=0.0164 for WKY group and r(21)=0.4165, p=0.048 for LE group) (Figure 28A, B). 5HTT gene expression There was a significant main effect of strain (F(1,41)=11.65, p=0.0015) but not of stress (F(2,38)=0.8443, p=0.4372) on 5HTT expression in the hippocampus. There was also no significant stress x strain interaction (F(2,38)=1.196, p=0.3127) (Figure 27A). Student t tests revealed a signif icant decrease in 5HTT expression in WKY rats in the acute and repeated stress groups, but not in the control group. As with the GR, there was a positive correlation between OCT3 and 5HTT expression in the WKY group (r(20)=0.4892), p=0.0209), but not the LE group (r(21)=0.2750,p=0.2154) (Figure 28C, D).
50 Western Blot Results OCT3 protein expression Plasma membrane. OCT3 protein expression was quantified in the plasma membrane fractions of the hippocampus. Significant main effects of both strain (F(1,42)=11. 03, p=0.0019) and stress (F(2,42)=9.417, p=0.0004) were observed (Figure 2 9A). However, there was no significant interaction between stress and strain on OCT3 protein expression in the hippocampus (F(2,42)=0.3682, p=0.6942). Student t tests indicated a si gnificant elevation in OCT3 in WKY relative to LE rats in the control and repeated stress groups. In LE rats there was a trend toward a decrease in hippocampal OCT3 expression at the plasma membrane with stress that did not reach significance (F(2,21)=2.141, p=0.1425) (Figure 29B). In WKY rats there was a significant effect of stress in hippocampal OCT3 protein expression at the plasma membrane (F(2,21)=11.77), p=0.0004), with post tests indicating a significant downregulation of the OCT3 after repeated stress compared to the control and acute stress groups (Figure 29C). Cytosol. Plasma membrane protein expression of the OCT3 did not correspond to our gene expression data, so we decided to perform these assays in cytosolic fractions to account for any O CT3 protein that may be associated with vesicular membranes. There was a significant main effect of stress (F(2,42)=22.8, p<0.0001) but not of strain (F(1,42)=1.876, p=0.1780) on OCT3 protein expression in the cytosol and a significant interaction between stress and strain (F(2,42)=8.516, p=0.0008) (210A). However, strain effects on OCT3 expression may have been missed due to the fact that lower OCT3 expression in WKY rats in the control and acute stress conditions was counterbalanced by higher expression in the repeated stress condition. According to
51 Student t tests, cytosolic OCT3 was lower in WKY relative to LE rats in the control and acute stress conditions and elevated by a similar margin in the repeated stress condition. Stress effects within each st rain were analyzed separately by oneway ANOVA. In LE rats there was an effect of stress (F(2,21)=4.426, p=0.0249) and post tests indicated a significant increase in cytosolic OCT3 in the repeated stress group relative to controls (Figure 2 10B). In WKY ra ts there was a significant effect of stress on OCT3 protein expression in the cytosol (F(2,21)=18.78, p<0.0001) and post hoc analysis revealed that the OCT3 was upregulated after repeated stress relative to all other groups (Figure 210C). BDNF protein expression BDNF protein expression was quantified in cytosolic fractions of the hippocampus. There was a significant main effect of both strain (F(1,42)=4.349, p=0.0431) and stress (F(2,42)=9.001, p=0.0006), but no significant stress x strain interaction on B DNF protein expression in the hippocampus (F(2,42)=1.106, p=0.3403) (Figure 211A). Student t tests did not indicate an effect of strain within individual stress groups. A one way ANOVA detected a significant effect of stress on BDNF protein expression in LE (F(2,21)=5.026, p=0.0165) rats (Figure 211B) and WKY (F(2,21)=5.06, p=0.0161) (Figure 211C) rats. Post hoc analysis indicated that in both strains, BDNF expression was upregulated after repeated stress compared to all other groups.
52 Discussion OCT3 G ene Expression The results we obtained indicate a profound elevation in basal OCT3 gene and protein expression in the hippocampus and mPFC of WKY relative to LE rats which may translate to increased 5HT clearance in these brain regions. Corticosterone, whi ch inhibits the OCT3, elevates extracellular 5HT which in turn facilitates normalization of the HPA axis after stress (Robertson et al., 2005;Keeney et al., 2006;Zhou et al., 2008). This heightened expression of the OCT3 in the WKY rat (roughly 5X that of LE rats) may simply overwhelm the inhibitory actions of corticosterone, such that the change in extracellular OCT3 between stress and basal conditions is low. The passive behavioral response (e.g. floating) of WKY rats in the forced swim test compared to m ost other strains supports this view. Acute injections of drugs that increase extracellular serotonin, including corticosterone, have been shown to increase active swimming responses on the forced swim test. The relative passivity of the WKY rat suggests t hat corticosterone, which is released during swim, fails to produce a sufficient increase in extracellular 5HT. It is notable that OCT3 gene expression was upregulated after acute stress in LE rats, then returned to basal levels after repeated stress exposure. A single exposure to mild stress has a number of beneficial effects on cognition, mood and memory which may be mediated by local increases in monoamine levels, and stress induced 5HT signaling plays a key role in physiological and behavioral adaptati on to stress. It has been suggested that inhibition of the OCT3 by corticosterone may underlie this stress induced increase in extracellular 5HT in the dorsomedial hypothalamus and possibly other brain regions (Lowry et al., 2001; Lowry et al., 2003; Gasser et al., 2006). Thus, the transient upregulation after a single stress exposure may provide an outlet to boost
53 5HT levels in times of stress. The absence of any effect of acute stress on OCT3 gene expression in WKY rats supports this hypothesis, since WKY rats are known to habituate much more slowly to stress. Elevated basal expression of the OCT3 in WKY rats may also inhibit any further upregulation by stress until the cumulative pressure of multiple stress exposures eventually overcomes this initial hurdle. The fact that rats with divergent behavioral and neurochemical profiles could be differentiated based upon their OCT3 gene expression pattern suggests a close association between the OCT3 and emotional responses to stress. WKY rats are slow to adapt to the effects of repeated stress and manifest a helpless, depressive phenotype under stress conditions whereas LE rats adapt more readily to stress and respond in a more aggressive, proactive manner. These behavioral characteristics correlate nicely with gene expression of the OCT3 under acute and repeated stress conditions. Interestingly, the effects of stress observed in the hippocampus were not replicated in the mPFC or the striatum, indicating that the hippocampus may be the locus of interaction between stress and the OCT3. It has been reported in the literature that the OCT3 is mainly localized to astrocytes in the striatum, which is how it acquired its original name of the extraneuronal monoamine transporter (EMT), but in the hippocampus the OCT3 coloc alizes with postsynaptic neurons where it may play a more direct role in monoaminergic signaling and stress adaptation. The apparent dissociation between hippocampal and striatal gene regulation of the OCT3 may also be attributed to their disparate roles i n affect regulation. For instance, 5HT signaling has been shown to modulate dopamine release in the striatal reward circuitry, but in the hippocampus
54 5HT release not only promotes adaptive and behaviorally appropriate responses to stress but also stimulates brainderived neurotrophic factor (BDNF) signaling and neurogenesis. In the mPFC we did observe a basal elevation of the OCT3 in WKY rats, but the effects of stress were not apparent as they were in the hippocampus. This was surprising, given the high density of GRs in the mPFC and its similar role in feedback inhibition of the PVN (Diorio et al., 1993). Although we cannot rule out the possibility that the OCT3 is upregulated in other brain regions that were not included in our analysis, the data we have collected so far suggests that the effects of stress on OCT3 expression are restricted to the hippocampus. This expression pattern is also found in 5HTT knockout mice, suggesting that OCT3 gene expression under stress conditions may be controlled by a sim ilar mechanism, namely excess extracellular 5HT. However, stress also increases extracellular 5HT in the mPFC (Mokler et al., 2007), so we cannot fully explain the differential response of the hippocampus and mPFC at this time. Clearly, stress exerts robus t effects on OCT3 gene expression in both strains, at suggesting that the OCT3 promoter may be responsive to glucocorticoids themselves or some downstream signaling pathway. Any discussion of the precise mechanism of OCT3 gene regulation by stress at this point would be premature since stress invokes a myriad of biochemical pathways in the brain, any one of which may be responsible for the observed effects. However, there are two logical candidates for this role which warrant further exploration due to thei r known interactions with the OCT3; corticosterone and 5HT. Corticosterone interfaces directly with the OCT3 to inhibit monoamine transport, resulting in a transient increase in 5HT levels in limbic regions of
55 the brain. Given the significance of stress in duced 5HT signaling in modulating stress responsiveness, it seems reasonable to postulate that the corticosteroneOCT3 pathway would be tightly controlled. Corticosterone has been shown to regulate transcription of GR receptors, which mediate negative feedback to the HPA axis, and may exert similar effects on expression of the OCT3 gene. In support of this hypothesis, we found that the expression pattern of the OCT3 is highly correlated with that of the GR. Stress has been shown to upregulate GR gene expression via glucocorticoids and may also upregulate OCT3 gene expression by a similar mechanism. However, our data show that the effects of stress on OCT3 gene expression are much more robust, suggesting that the OCT3may not be acting alone to regulate OCT3 gene expression. This idea is further supported by the fact that WKY rats expressed elevated OCT3 mRNA in the hippocampus and mPFC relative to LE rats under basal conditions. Stress is also known to activate 5HT neurotransmission in the hippocampus, and pr evious studies have provided some evidence that upregulation of the OCT3 may be a homeostatic mechanism for regulating extracellular 5HT concentrations. The compensatory upregulation of the OCT3 in the absence of the 5HTT, which creates conditions of exces s extracellular 5HT, lends support to idea that genetic expression of the OCT3 is regulated, directly or indirectly, by 5HT. In fact, since stress induced 5HT release in the hippocampus has been shown to promote habituation to repeated stress by presumably lowering the HPA responsiveness, this mechanism provides built in a feedback loop that would also turn down OCT3 gene expression after repeated expression, which would explain our findings in LE rats. The absence of any change in OCT3 expression after acute stress in WKY rats may reflect weak 5HT signaling which
56 in turn may underlie poor stress coping and a propensity toward depressivelike behavior as previously demonstrated in the forced swim test. OCT3 Protein Expression We obtained cytosolic, plasma m embrane, and organelle membrane fractions from hippocampal lysates and initially used the plasma membrane fractions to quantify OCT3 protein expression. We observe a significant decrease in OCT3 plasma membrane expression in the hippocampus after repeated stress in WKY rats, and although a similar trend was observed in LE rats, this effect did not reach significance. These results stand in contrast to our real time PCR results, which revealed a robust increase in OCT3 expression in both strains of rats afte r stress. To reconcile this, we also quantified OCT3 expression in cytosolic fractions which may contain internalized vesicles with OCT3 embedded in the vesicular membrane. Hippocampal OCT3 protein expression was increased in cytosolic fractions after repeated stress in both WKY and LE rats, which suggests that the OCT3 is being sequestered in vesicles. One possibility is that vesicular OCT3 is internalized shortly after the binding of CORT and relocated to cytosolic vesicles. GR Gene Expression In our stu dy we did not observe any significant effects of stress on GR expression, contrary to previous reports that single exposures to stress upregulate the GR (Marini et al., 2006). There was, however, a trend toward an increase in GR gene expression after acute stress in LE rats and repeated stress in WKY rats. Furthermore, we found a significant correlation between OCT3 and GR expression in both strains, which may indicate that these two genes are regulated by a similar mechanism
57 5HTT Gene Expression Contrary to our hypothesis, basal 5HTT gene expression in the hippocampus did not significantly differ between LE and WKY rats. The basal elevation of OCT3 mRNA in the hippocampus of WKY rats lead us to hypothesize that the OCT3 may have been recruited to compensat e for low 5HTT expression, however this turns out not to be the case. Several lines of evidence led us to believe that the 5HTT gene may be downregulated in WKY rats by stress in our model. One study in WKY rats demonstrated a robust decrease in 5HTT binding after a 21 day chronic variable stress (CVS) regimen that included foot shock (P re and Tejani Butt, 1996), which suggested we may observe a similar downregulation in the WKY rat after repeated social defeat. Furthermore, we observed a robust upregulat ion of the OCT3 in the hippocampus of WKY rats after chronic social defeat. This same effect was reported in 5HTT knockout mice while 5HT levels remained relatively constant compared to wildtype mice, suggesting that the OCT3 is recruited when 5HTT is scarce to keep 5HT levels in check. We considered the possibility that upregulation of the OCT3 may be secondary to stressinduced downregulation of the 5HTT 5HTT expression was significantly lower in the hippocampus of WKY relative to LE rats in the acute an d repeated stress condition, but overall there was no significant change in 5HTT expression within either strain after stress. These data do not support the hypothesis that upregulation of the OCT3 after stress is driven by the downregulation of 5HTT.
58 BDNF Protein Expression in the Hippocampus Previous studies have shown that chronic social defeat stress reduces neurogenesis in the dentate gyrus of the hippocampus (Magarinos et al., 1996; Gould et al., 1997), which may be associated with reduced BDNF prod uction after stress as reported by Smith and colleagues (1995). However, Bergstr m and colleagues report an increase in BDNF expression following a 5week chronic mild stress regimen (Bergstr m et al., 2008). At this time, it is unclear whether this discre pancy is caused by differences in the length or severity of the stress regimen, but in our study we observed an increase in BDNF in both strains of rats consistent with the latter report. This result is also consistent with increased 5HT signaling in the hippocampus subsequent to stress, since BDNF expression is positively regulated by 5HT and vice versa. In summary, we have demonstrated that stress differentially regulates expression of the OCT3 in rats displaying a depressive phenotype and poor stress coping, indicating that the OCT3 may play a significant role in stress adaptation. Further investigations into the mechanism of this regulation and the time course of OCT3 trafficking to and from the plasma membrane are warranted.
59 Social defeat1 7 8 2 hrs/real time PCR 3 hrs/Western blot 1 7 8 1 7 8 Control HandlingAcute Chronic HandlingTerminate Restraint Terminate Terminate RestraintDay Figure 21. Schematic o f experimental design.
60 Figure 22. The social defeat procedure in action. Social defeat consists of a 45 minute indirect interaction phase (A) followed by a 5 minute direct interaction phase (B) in which the intruder is usually defeated by the resident. Table 21. Real time PCR Primers Primer Assay ID OCT3 Rn00570264_m1 SERT Rn00564737_m1 GR Rn00561369_m1 18S Hs99999901_s1 Table 22. Antibodies for Western Blot Label Company Host Dilution Exposure time OCT3 Abcam Rabbit 1:1000 5 min BDNF Abcam Rabbit 1:1000 30 sec N Cadherin Abcam Rabbit 1:2000 10 sec actin Cell Signaling Rabbit 1:1000 10 sec
61 Control Acute Chronic 0 5 10 15LE WKY *** *** ***RQ (relative to LE control) Control Acute Chronic 0 1 2 3**RQ (relative to LE control) Control Acute Chronic 0 1 2 3***RQ (relative to WKY control)A B C Long-Evans Wistar-Kyoto Figure 23. Effects of stress on OCT3 gene expression in the hippocampus. Data are expressed as relative change or relative quantificati on (RQ) of gene expression levels and values are mean SEM. (A) (p<0.05) and *** (p<0.001) denote statistical significance relative to the LE group in each stress category. (B) *** (p<0.001) denotes statistical significance compared to the LE group. (C D) (p<0.05) and ** (p<0.01) denote statistical significance relative to all other groups.
62 Control Acute Chronic 0 1 2 3LE WKY *** *** ***RQ (relative to LE control) Control Acute Chronic 0.0 0.5 1.0 1.5RQ (relative to LE control) Control Acute Chronic 0.0 0.5 1.0 1.5RQ (relative to WKY control)A B C Long-Evans Wistar-Kyoto Figure 24. Effects of stress on OCT3 gene expression in the mPFC. Data are expressed as relative change or relative quantification (RQ) of gene expression level s and values are mean SEM. (A) (p<0.05), ** (p<0.01) and *** (p<0.001) denote statistical significance relative to the LE group in each stress category. (B) *** (p<0.001) denotes statistical significance compared to the LE group.
63 Control Acute Chronic 0.0 0.5 1.0 1.5LE WKY RQ (relative to LE control) Control Acute Chronic 0.0 0.5 1.0 1.5RQ (relative to LE control) Control Acute Chronic 0.0 0.5 1.0 1.5RQ (relative to WKY control)A B C Long-Evans Wistar-Kyoto Figure 25. Effec ts of stress on OCT3 gene expression in the striatum. Data are expressed as relative change or relative quantification (RQ) of gene expression levels and values are mean SEM.
64 Control Acute Chronic 0.0 0.5 1.0 1.5 2.0LE WKY *RQ (relative to LE control Control Acute Chronic 0.0 0.5 1.0 1.5 2.0RQ (relative to LE control) Control Acute Chronic 0.0 0.5 1.0 1.5 2.0RQ (relative to WKY control)A B C Long-Evans Wistar-Kyoto Figure 26. Effects of stress on GR gene expression in the hippocampus. D ata are expressed as relative change or relative quantification (RQ) of gene expression levels and values are mean SEM. (p<0.05) denotes statistical significance relative to the LE group in the repeated stress group.
65 Control Acute Chronic 0.0 0.5 1.0 1.5 2.0LE WKY *RQ (rel to LE control) Control Acute Chronic 0.0 0.5 1.0 1.5 2.0RQ (relative to LE control) Control Acute Chronic 0.0 0.5 1.0 1.5 2.0RQ (Rel to WKY control)A B C Long-Evans Wistar-Kyoto Figure 27. Effects of stress on 5HTT gene expression in the hippocampus. Data are expressed as relative change or relative quantification (RQ) of gene expression levels and values are mean SEM. (A) (p<0.05) denotes statistical significance relative to the LE group in each stress cat egory. (B) ** (p<0.01) denotes statistical significance compared to the LE group.
66 0.0 2.5 5.0 7.5 10.0 0 1 2 3 4 5WKY (r = 0.5056, p = 0.016)GROCT3 0 1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5LE (p=0.4165, p=0.048)GROCT3 0 1 2 3 4 5 0 1 2 3 4 5WKY (r = 0.4892, p=0.02)5HTTOCT3 0 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 LE (r=0.2750, ns)5HTTOCT3A. B. C. D. Figure 28. Bivariate correlation analysis for OCT3 x GR and OCT3 x 5HTT. Data points represent the RQ values for each individual sample.
67 Control Acute Chronic 0.0 0.5 1.0 1.5LE WKY *Rel. OD (OCT3/NCad) Control Acute Chronic 0.00 0.25 0.50 0.75 1.00 1.25Rel. OD (OCT3/NCad) Control Acute Chronic 0.0 0.5 1.0 1.5 *** **Rel. OD (OCT3/NCad)A B C Long-Evans Wistar-Kyoto Figure 29. Effects of stress o n plasma membrane expression of the OCT3 in the hippocampus. Data are expressed as relative optical density (OD) normalized to N cadherin and values are mean SEM. (B) ** (p<0.01) denotes statistical significance relative to the LE group. (D) ** (p<0.01) and *** (p<0.001) denotes statistical significance compared to the indicated group.
68 Control Acute Chronic 0.0 0.5 1.0 1.5LE WKY ** *Rel. OD (OCT3/-actin) Control Acute Chronic 0.0 0.5 1.0 1.5*Rel. OD (OCT3/-actin) Control Acute Chronic 0.0 0.5 1.0 1.5***Rel. OD (OCT3/-actin)Long-Evans Wistar-Kyoto A B C Figure 210. Effects of stress on cytosolic expression of the OCT3 in the hippocampus. Data are expressed as relative optical density (OD) normalized to actin and valu es are mean SEM. (A) (p<0.05) denotes statistical significance relative to the LE group in each stress category. (C) (p<0.05) denotes statistical significance compared to the control group and (D) *** (p<0.001) denote statistical significance relativ e to both control and acute stress groups.
69 Control Acute Chronic 0.0 0.5 1.0 1.5LE WKY Rel. OD (BDNF/-actin) Control Acute Chronic 0.0 0.5 1.0 1.5*Rel. OD (BDNF/-actin) Control Acute Chronic 0.0 0.5 1.0 1.5*Rel. OD (BDNF/-actin)Long-Evans Wistar-Kyoto A B C Figure 211. Effects of stress on BDNF protein expression in the hippocampus. Data are expressed as relative optical density (OD) normalized to actin and values are mean SEM. (C D) (p<0.05) denotes statistical significance compared to all other groups.
70 CHAPTER 3 ANTIDEPRESSANT EFFECTS OF AN OCT3 ANTAGONIST IN THE WISTAR KYOTO RAT Introduction The goal of these studies was to assess whether the OCT3 may be a useful target for antidepressant intervention in an animal model that is resistant to the effects of conventional SSRI antidepressants. Individuals possessing one or more copies of the s allele of the 5HTT typically exhibit a poor response to SSRIs, especially when this polymorphism is combined with the 5HT1A 1018 C/G polymorphism, otherwise known as the g allele of the 5HT1A receptor (Arias et al., 2005). The WKY rat has been extensively characterized as an animal model of depressi ve behavior and heightened stress responsiveness that may shed new light on the neurobiological underpinnings of Major Depression and its pharmacological treatment in humans (Par and Redei, 1993;Redei et al., 1994;De La Garza and Mahoney, 2004; De La Garz a, 2005;Schmidt and Duman, 2007). Compared to most other strains, the WKY rat exhibits low levels of activity in the forced swim test, which reveals a poor adaptive response to stress and a tendency toward depressivelike behavior (Par and Tejani Butt, 19 96;Griebel et al., 1999;Lopez Rubalcava and Lucki, 2000;De La Garza and Mahoney, 2004;Tejani Butt et al., 2003). The forced swim test is commonly used to screen new compounds for their ability to reduce immobility and increase active swimming and climbing behaviors, which is positively correlated with their clinical efficacy as antidepressants (Porsolt et al., 1978). The WKY rat also displays an attenuated response to SSRIs in the forced swim test that is reminiscent of the antidepressant resistance observ ed in a significant
71 number of depressed individuals (Lahmame and Armario, 1996;Griebel et al., 1999;Lopez Rubalcava and Lucki, 2000;Tejani Butt et al., 2003). Furthermore, 5HTT binding in the hippocampus is reduced in WKY rats after stress compared to other strains, but without any concomitant changes in 5HT clearance rates in the hippocampus (Pollier et al., 2000). These data suggest that some other transporter may be playing a compensatory role in 5HT clearance in the WKY rat, much as it does in 5HTT knoc kout mice (Schmitt et al., 2003;Baganz et al., 2008). The OCT3 is upregulated in the hippocampus of WKY rats (see Chapter 2), which may underlie the reduced efficacy of antidepressant drugs in reversing helplessness behavior in the forced swim test. The relatively low expression of 5HTT combined with high expression of the 5HT1A receptor resembles the genetic profile of depressed individuals with the ss/gg genotype who exhibited the poorest outcome during a 12week trial with the SSRI citalopram (Arias et al., 2005). This led us to hypothesize that acute administration of decynium 22, a specific inhibitor of the OCT3, will mitigate depressivelike behavior on the forced swim test in WKY rats. Methods Experimental Design WKY rats were exposed to a 15 minute s wim or pretest 24 hours before the test day. On the test day, WKY rats were randomly assigned to receive 0, 1 or 10 ug/kg decynium 22 1 hour before testing on the forced swim test for 5 minutes during which swimming, climbing and floating behaviors were recorded with an overhead camera. Rats were terminated by rapid decapitation 30 minutes after the beginning of the swim session for measurement of plasma corticosterone concentrations.
72 Experimental Animals Thirty four adult male WKY rats (Charles River Laboratories International, Inc., Wilmington, MA) weighing between 250300 g at the start of experiments were pair housed in standard polycarbonate cages (43 x 21.5 x 25.5 cm) in a climatecontrolled vivarium with a 12hour light/dark schedule (lights on at 7 am daily). The rats were allowed ad libitum access to standard laboratory chow (Lab Diet 5001) and tap water. The rats were allowed 7 days to adapt to the housing facility before the experiments began. At the end of the experiment, the rats were terminat ed by rapid decapitation for blood and tissue collection. Drugs and Injections Decynium 22 (Sigma Aldrich) was dissolved in ethanol to achieve a concentration of 20 ug/ml, and 200 ug/ml, respectively. Each of these solutions was diluted 1:20 in isotonic saline to a final concentration of 5% ethanol and 1 ug/ml and 10 ug/ml of decynium 22. An intraperitoneal injection of 5% ethanol in isotonic saline with the appropriate concentration of decynium 22 (0, 1, or 10 ug/kg) was administered 1 hour prior to behavi oral testing on the forced swim test. Forced Swim Test This method is based on a modified version of that described by Porsolt (Porsolt et al., 1978). Briefly, rats were placed individually into circular containers (45 cm height; 20 cm diameter) containing 30 cm of water at 23 C for 15 minutes during a pretest session. Twenty four hours later, a test session was conducted for 5 min and swimming, climbing, and floating were recorded with a video camera mounted over the test apparatus. At the end of the tes t session, each rat was dried with a clean towel, placed in a heated cage for 15 minutes, and then returned to its home cage for an additional 15
73 minutes before decapitation. Fresh water was used for each rat and the cylinder was cleaned thoroughly with 4% bleach between sessions. A trained observer, blind to the treatment, scored the videotapes for time spent floating (immobile) (Figure 32). A rat was considered immobile when floating and making only the necessary movements to keep its nostrils above the water surface. Plasma CORT analysis Trunk blood ethylenediaminetetraacetic acid (EDTA) and centrifuged at 1000 x g for 5 minutes at 4C to separate the plasma layer from the cellular layer. The supernatant (plasma) was aliquoted and s tored at 80C until further use. Plasma CORT concentrations were measured using a commercially available enzymelinked immunosorbant assay kit (Immunodiagnostic Systems Ltd., UK) according to the manufacturers protocol. Statistical Analyses A one way ANO VA was used to analyze the effects of decynium 22 (0, 1, 10 ug/kg) on immobility behavior during the forced swim test and plasma CORT concentrations 30 minutes after the beginning of the test. Significant effects (p<0.05) were analyzed by NewmanKeuls post tests to make pairwise comparisons between each dose. Results Behavioral data Treatment with decynium 22 (1 and 10 ug/kg) produced a significant decrease in the time spent immobile on the forced swim test. (F(2,31)=9.565, p=0.0006) (Figure 33). Post hoc analysis verified that both 1 ug/kg (t=3.732, p<0.01) and 10 ug/kg (t=3.466,
74 p<0.01) decynium 22 significantly attenuated immobility on the forced swim test relative to vehicle. CORT data Trunk blood was collected from decapitated animals 30 minute after s tress onset on the same day as behavioral testing in rats treated with vehicle and 10 ug/kg decynium 22 only. The remaining rats treated with 1 ug/kg were used as controls (no stress) 1 week later. Swim stress significantly increased plasma corticosterone concentrations in WKY rats (F(2,15)=120.9,p<0.0001) (Figure 34). Post hoc analysis revealed a significant increase in plasma corticosterone levels between stressed rats treated with 10 ug/kg D22 compared to those receiving vehicle. Discussion This is the first study to report that decynium 22, a specific inhibitor of the OCT3, has an antidepressant effect in the WKY rat, a putative animal model of both behavioral depression and antidepressant resistance. Our findings are consistent with studies in mice wh ich also demonstrate a selective antidepressant effect of decynium 22 (1 ug/kg) on the tail suspension test in animals lacking at least one copy of the 5HTT (Baganz et al., 2008), which is sometimes used as a proxy for the low efficacy s allele that has been associated with a higher risk of depression and low responsiveness to antidepressants targeting the 5HTT. The WKY rat also exhibits a poor antidepressant response to SSRI drugs on the forced swim test, but respond well to drugs that block reuptake of norepinephrine and dopamine (Tejani Butt et al., 2003). Decynium 22, which shows pharmacological activity against the OCT3, a polyspecific monoamine transporter, effectively increases synaptic concentrations of 5HT, dopamine and norepinephrine which may be r equired for antidepressant action in the WKY rat.
75 Furthermore, we have shown that the OCT3 is constitutively upregulated in the WKY rat compared to other strains, making the OCT3 an ideal target for antidepressant intervention as is the case with 5HTT knoc kout mice (Baganz et al., 2008). The failure of SSRIs to elicit an antidepressant response, combined with the robust antidepressant effect of decynium 22 in these same animals, suggests that drugs targeting the OCT3 may have utility in the treatment of depressed patients who are refractory to SSRIs. The WKY rat is described as having a depressive phenotype based upon its low level of activity in the forced swim test. Studies have shown that corticosterone, which peaks shortly after presentation of a stressor, has a profound antidepressant effect in the forced swim test in mice (Stone and Lin, 2008). We postulated that in WKY rats, a blunted corticosterone response to swim stress may account for their relative inactivity in the forced swim test. As mentione d earlier, corticosterone elicits a rapid 5HT response, possibly through its inhibitory actions at the OCT3. However, we observed a robust increase in corticosterone levels in the WKY rat after swim stress, in agreement with previous studies which compared the HPA response of the WKY rat with the SpragueDawley rat. In this study, WKY rats actually had an enhanced corticosterone and ACTH response to swim stress relative to the response of the SpagueDawley rats (Rittenhouse et al., 2002). These data suggest that behavioral dysregulation of the stress response in WKY rats occurs at some other level of the glucocorticoid signaling pathway, perhaps involving the interaction between corticosterone and the OCT3. In this case, decynium 22 essentially substitutes f or corticosterone and reduces immobility behavior down to normal levels (~40%).
76 In conclusion, this study provides evidence that inhibition of the OCT3 can act as an antidepressant in our animal model and suggests that further investigation of the use of OCT3 antagonists as therapeutic agents is warranted, either alone or in combination with other antidepressants (Schildkraut and Mooney, 2004;Mooney et al., 2008).
77 Figure 31. Schematic of experimental design. Behavioral testing schedule which consisted of a pretest on day 1 and the forced swim test on day 2. In Experiment 2, rats treated with vehicle or 10 ug/kg D22 were terminated 30 min after the beginning of the forced swim test for assessment of plasma corticosterone levels. Figure 3 2. Pictorial representation of the forced swim test depicting (A) floating and (B) swimming behaviors.
78 0 1 10 0 10 20 30 40 50 60 70 80 90 100** **Dose decynium 22 (ug/kg)% time Figure 33. Antidepressant effects of decynium 22 in the forced swim test. As indicated, rats injected with 1 ug/kg and 10 ug/kg decynium 22 displayed reduced immobility on the forced swim test compared to vehicle. Data are expressed as the % time spent immobile over a 5 min test session. Values are expressed as mean SEM. *** (p<0.001) represents statistical significance over vehicle. Control Vehicle D22 0 100 200 300 400 Stress*** *** *Plasma CORT (ng/ml) Figure 34. Effect of forced swim stress on plasma corticosterone levels. Swim stress causes a significant increase in plasma corticosterone levels that is unmodified by drug treatment with decynium 22 (5 ug/kg). Swim stress induces a significant increase in plasma corticosterone levels that is slightly augmented by pretreatment with decynium 22. Data are expressed as plasma corticosterone concentrations in ng/ml and all values are expressed as mean SEM. (p<0.05) and *** (p<0.001) represents statistical signific ance over stressfree controls or vehicletreated rats as indicated.
79 CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS A Working Model of the OCT3 as a Molecular Switch that Regulates Stress Responsiveness and Vulnerability to Psychopathology The OCT3 is a cor ticosteronesensitive monoamine transporter which has been previously shown to play a role in depressiveand anxiety like behavior in animal models (Wieland et al., 2000;Kitaichi et al., 2005;Baganz et al., 2008;Vialou et al., 2008;Wultsch et al., 2009). Unlike the highaffinity transporters such as 5HTT, NET, and DAT, the OCT3 is located on postsynaptic membranes where it participates in the bidirectional transport of 5HT, NE, DA and histamine (Gr ndemann et al., 1998;Gr ndemann et al., 1999;Schomig et al ., 2006). This low affinity, high capacity transporter is recruited to remove extracellular monoamines when the highaffinity transporters are saturated or in low abundance (Schmitt et al., 2005;Baganz et al., 2008), which typically occurs under stress conditions. Stress triggers 5HT release in the hippocampus which saturates the highaffinity 5HTT, such that the OCT3 becomes a key player in determining the amplitude and duration of 5HT signaling. Corticosterone, which inhibits the transport function of the OCT3, results in local accumulation of extracellular 5HT and the potentiation of 5HT signaling (Lowry et al., 2001;Gasser et al., 2006;Feng et al., 2009). Thus, the temporal and spatial pattern of OCT3 expression and its functional interactions with corti costerone may play significant roles in emotional and behavioral regulation under stress conditions. The importance of hippocampal 5HT, and hence the OCT3, in stress regulation is illustrated by the fact that lesioning of 5HT projections to the hippocampus results in increased anxiety like behavior and poor stress coping ( Robertson et al., 2005)
80 Feedback inhibition of the HPA response is achieved in part by activation of GRs in the hippocampus by corticosterone (Herman et al., 1989;Whitnall, 1993;Herman and Cullinan, 1997), and previous studies have demonstrated that GRs are upregulated in the hippocampus after a single exposure to social defeat stress (Marini et al., 2006). These parallel actions of corticosterone on OCT mediated 5HT signaling and GR acti vation in the hippocampus, and the subsequent upregulation of OCT3 and GR in this same region, may act in synergy to promote more rapid recovery from and long term habituation to stressful stimuli. We demonstrated differential expression of the OCT3 gene i n WKY compared to LE rats under basal, acute and repeated stress conditions. Given the role of the OCT3 in regulating extracellular 5HT concentrations in the presence of corticosterone, these differences in genetic expression of the OCT3 may help to explai n the abnormal neurochemical and behavioral responses of the WKY rat to stress. There is a marked elevation of OCT3 gene expression in the hippocampus and mPFC of WKY rats compared to LE rats, two limbic regions that have been implicated in feedback regulation of the HPA axis. Plasma membrane expression of the OCT3 is also higher in the hippocampus of the WKY relative to LE rats under basal and repeated stress conditions, with a trend toward higher OCT3 in WKY rats in the acute stress condition. Higher leve ls of OCT3 membrane expression may enable the WKY rat to maintain high levels of 5HT clearance under stress conditions, which in turn may hinder appropriate coping responses (Figure 41). This stressindependent upregulation of the OCT3 in WKY rats might be attributed to the relatively low levels of 5HTT gene expression in the
81 WKY rats, as other studies have established a negative correlation between the expression patterns of these two genes (Schmitt et al., 2005;Baganz et al., 2008). Acute stress induces a robust upregulation of OCT3 gene expression in the hippocampus of LE rats, although this elevated mRNA expression in the LE rats was still well below that of the WKY rats (Figure 23). We did not observe a corresponding increase in OCT3 protein express ion at the plasma membrane in the LE rats after acute stress, so it in unknown whether this upregulation in OCT3 gene expression translated to an increase in 5HT clearance. It is possible that we collected tissues for OCT3 protein expression assays too qui ckly (~ 3hrs) to observe the insertion of newly synthesized OCT3 protein into the membrane. In order to resolve this issue, we would have to collect tissues at a later time point (e.g. 24 hrs after the acute stress), in order to determine if the increase i n gene expression eventually translate to an increase in plasma membrane expression. The increase in OCT3 gene expression in LE rats in response to acute stress exposure is blunted in rats that have a history of stress, indicating that whatever was drivin g the increase in OCT3 transcription after acute stress may have also returned to equilibrium. Conversely in WKY rats, the OCT3 is upregulated in rats with a history of repeated stress. This may be a result of impairments in stress recovery which would res ult in excessive bombardment of hippocampal neurons with corticosterone during repeated stress. Bimodal Regulation of OCT3 Gene Expression: A Hypothesis We propose that OCT3 gene expression is differentially regulated under low and high stress conditions. In the low stress state, OCT3 gene expression may be primarily 5HT driven; that is, low levels of 5HTT expression in the WKY rat triggers a
82 compensatory increase in OCT3 expression. This is supported by the observation that OCT3 gene expression is upregul ated in the hippocampus of 5HTT knockout mice, although it is possible that expression of these two genes is driven in parallel by an addition, indirect mechanism. In the high stress state, the OCT3 gene may be regulated by the dual actions of corticosterone and 5HT. The widespread effects of corticosterone on gene transcription are well documented. Glucocorticoids bind to cytosolic receptors and are subsequently translocated to the nucleus where they can interact with glucocorticoid response elements (GREs ) on target genes to activate gene transcription, and one of these genes may be the OCT3 gene. The interaction between corticosterone and the OCT3 also inhibits 5HT uptake and increases extracellular concentrations of 5HT, which may trigger a homeostatic upregulation of the OCT3. This is one potential mechanism by which corticosterone may indirectly regulate OCT3 gene expression. Stress dependent upregulation of the OCT3 in the hippocampus, but not in the mPFC or striatum, of LE rats may be partly explained by the fact that the hippocampus contains the highest density of GR receptors in the brain, since it is these receptors that tranduce the genomic effects of stress (Jacobson and Sapolsky, 1991). This finding also concurs with those of Schmitt and colleagues, in which OCT3 gene expression was upregulated in the hippocampus of 5HTT knockout mice but not in other brain regions including the cortex, striatum, cerebellum or brainstem (Schmitt et al., 2005). We originally hypothesized that WKY rats may have a blunted corticosterone response to stress which would account for their failure to induce OCT3 gene expression after acute stress. However, we report a relatively normal corticosterone
83 response to acute swim stress in WKY rats (see Figure 34), which concur s with previous studies (Rittenhouse et al., 2002). Therefore, we postulate that the abnormal neurochemical and behavioral responses to stress in the WKY rat may occur at the level of the glucocorticoid receptor (GR), the glucocorticoid response element (G RE), or one of the many downstream targets of the glucocorticoid signaling pathway. The failure of acute stress to induce GR gene expression in WKY rats supports this hypothesis, since GR is a key modulator of stress induced gene expression and may play a role in the induction of the OCT3. Also, the fact that OCT3 expression is many times higher in WKY compared to LE rats raises the possibility that the OCT3 competitively inhibits binding of corticosterone to GRs, which may also interfere with corticosteron e driven gene expression. The absence of any effect of acute stress on OCT3 expression in WKY rats may also be the result of an insufficient change in extracellular 5HT levels between basal and stress conditions (Figure 41). Presumably, LE rats adapt fairly normally to repeated stress and both glucocorticoid and 5HT signaling are reduced accordingly. In contrast, glucocorticoid signaling in the WKY is slowly ramped up as a function of repeated stress without the mitigating effect of enhanced 5HT signaling, which may eventually drive increased expression of the OCT3. Stressinduced Membrane Trafficking of the OCT3 We obtained discrepant results with regard to OCT3 gene and protein expression when protein was quantified at the plasma membrane. This led us to question whether the OCT3 was internalized when bound by CORT after stress. We quantified OCT3 protein expression in cytosolic extracts and found that the OCT3 was upregulated after chronic stress in both LE and WKY rats, whereas acute stress had no apparent effect
84 (Figure 2 10). This corresponded well with the observed downregulation of the OCT3 in plasma membrane extracts, leading us to conclude that the OCT3 is translocated to cytosolic vesicles after stress. The absence of any effect of acute stress on plasma membrane or cytosolic OCT3 expression is difficult to explain, but it may have to do with the timing of sample collection in our experiment. We collected brain tissues 3 hours after stress for protein analysis, which means that the internalized O CT3 may have already been metabolized at this point. Newly synthesized OCT3 is in the process of being trafficked to the plasma membrane so that there is no apparent reduction in OCT3 in plasma membrane fractions. After repeated stress, the reduction at the plasma membrane and increase in cytosolic OCT3 becomes more apparent, but is much more pronounced in WKY rats. By this point, OCT3 gene expression has returned to basal levels in LE rats, but in WKY rats there is a robust upregulation. Therefore, the obs erved increase in cytosolic OCT3 in WKY rats may reflect both CORT bound, internalized OCT3 and newly synthesized OCT3. OCT3 Antagonists as Putative Antidepressant Agents Our studies also demonstrate that the OCT3 antagonist decynium 22 is a potent antidepressant agent in WKY rats, which suggests that drugs targeting the OCT3 may hold promise for antidepressant pharmacotherapy in humans, especially in individuals with two copies of the s allele of the 5HTT gene. WKY rats are an excellent model for this genotype since they not only have a depressive phenotype, but they are typically resistant to the effects of SSRI antidepressants and exhibit low levels of 5HTT expression (Griebel et al., 1999;Lopez Rubalcava and Lucki, 2000;Tejani Butt et al., 2003). While currently there are no published studies on OCT3 gene expression in humans with the ss genotype, we hypothesize that the OCT3 will be upregulated in
85 such individuals. Constitutive upregulation of the OCT3, such as that found in the WKY rat, may dampen 5HT activity and increase susceptibility toward depressivelike behavior. We further postulate that heightened expression of the OCT3 may underlie resistance to the antidepressant effects of SSRIs. SSRIs exert their effects by inhibiting the 5HT uptake via the 5HTT, causing the OCT3, if present in sufficient quantities, to usurp this function. For this reason, drugs that antagonize the OCT3 should theoretically have potent antidepressant effects in individuals with high levels of OCT3 gene expression, as they do in the WKY rat. As stated earlier, OCT3 is a low affinity, high capacity transporter of all monoamines including 5HT and is highly expressed in the hippocampus. As such, its contribution to 5HT uptake becomes significant when the highaffinity 5HTT is s aturated or in low abundance. The latter condition is met in the presence of antidepressants such as fluoxetine, which selectively inhibit 5HTT and provoke its subsequent downregulation after chronic treatment (Hrdina et al., 1993). We postulate that chronic treatment with SSRIs will induce expression of the OCT3, which may account for the reduced efficacy of SSRIs over time in certain individuals. As such, OCT3 antagonists could potentiate the antidepressant effects of chronic SSRI treatment by increasing synaptic concentrations of 5HT and other monoamines such as NE and DA. Previous studies have shown that OCT3 inhibition by normetanephrine, the O methylated metabolite of norepinephrine, augments extracellular norepinephrine in patients treated with venlaf axine and may contribute to its antidepressant effects (Rahman et al., 2008).
86 Figure 41. Proposed model for the effect of differential expression of the OCT3 on 5HT signaling in the hippocampus. Blue ovals represent the OCT3 protein and purple dots r epresent 5HT. Corticosterone (CORT) in this model is shown as gray hexagons. WKY rats have elevated basal OCT3 which results in reduced extracellular 5HT levels in the synapse. Corticosterone is released under stress conditions which inhibits 5HT uptake vi a the OCT3, causing local increases in extracellular 5HT in both LE and WKY rats. In WKY rats, however, OCT3mediated 5HT clearance is only partially inhibited by corticosterone, resulting in lower levels of 5HT in the WKY relative to LE rats.
87 LIST OF RE FERENCES Aguilera M, Arias B, Wichers M, Barrantes Vidal N, Moya J, Villa H, et al. (2009). Early adversity and the 5HTT/BDNF genes: new evidence of geneenvironment interactions on depressive symptons in a general population. Psychol Med 39: 14251432. A merican Psychiatric Association (2000). Diagnostic and statistical manual of mental disorders (Revised 4th ed.). American Psychiatric Association: Washington, DC. Amphoux A, Vialou V, Drescher E, Brss M, Mannoury La Cour C, et al. (2006). Differential pharmacological in vitro properties of organic cation transporters and regional distribution in rat brain. Neuropharmacology 50: 941952. Anisman H, Matheson K (2005). Stress, depression, and anhedonia: Caveats concerning animal models. Neurosci Biobehav R 29 : 525546. Aoyama N, Takahashi N, Kitaichi K, Ishihara R, Saito S, Maeno N, et al. (2006). Association between gene polymorphisms of SLC22A3 and methamphetamine use disorder. Alcohol Clin Exp Res 30: 1644 1649. Arias B, Cataln R, Gast C, Gutirrez B, Fa an L (2005). Evidence for a combined genetic effect of the 5HT1A receptor and serotonin transporter genes in the clinical outcome of major depressive patients with citalopram. J Psychopharmacol 10: 166172. Armario A, Gavalda A, Marti J 1995. Comparison of the behavioral and endocrine response to forced swimming stress in five inbred strains of rats. Psychoneuroendocrino 20: 879 890. Artigas F, Romero L, de Montigny C, Blier P (1996). Acceleration of the effect of selected antidepressant drugs in major de pression by 5HT1A antagonists. Trends Neurosci 19: 378 383. Assael M Gabay F, Khazan N, Sulman FG, Winnik, HZ (1960). The mechanism of the combined treatment of depression with iproniazid and reserpine. J Ment Sci 106: 10211026. Axelrod J, Whitby LG, Hertting G (1961). Effect of psychotropic drugs on the uptake of H3Norepinephrine by tissues. Science 133: 383 384. Baganz NL, Horton RE, Calderon AS, Owens A, Munn JL, Watts LT, et al. (2008). Organic cation transporter 3: Keeping the brain on extracellular serotonin in serotonintransporter deficient mice. PNAS 105: 1897618981. Bennett AJ, Lesch KP, Heils A, Long JC, Lorenz JG, Shoaf SE, et al. (2002). Early experience and serotonin transporter gene variation interact to influence primate CNS function. Mo l Psychiatry 7: 118 122.
88 Bet PM, Penninx BWJH, Bochdanovits Z, Uitterlinden AG, Beekman ATF, van Schoor NM, et al. (2009). Glucocorticoid receptor gene polymorphisms and childhood adversity are associated with depression: New evidence for a gene environment interaction. Am J Med Genet Part B 150B: 660 669. Bickerdike MJ, Wright IK, Marsden CA (1993). Social isolation attenuates rat forebrain 5 HT release induced by KCI stimulation and exposure to a novel environment. Behav Pharmacol 4: 231 236. Binder EB, N emeroff CB (2010). The CRF system, stress, depression and anxiety insights from human genetic studies. Mol Psychiatry 15: 57488. Bjorkqvist K (2001). Social defeat as a stressor in humans. Physiol Behav 73: 4435 4442. Blier P, de Montigny C, Chaput Y (198 7). Modifications of the serotonin system by antidepressant treatments: implications for the therapeutic response in major depression. J Clin Psychopharmacol 7: 24S 35S. Blier P, de Montigny C (1998). Possible serotonergic mechanisms underlying the antidepressant and anti obsessive compulsive disorder responses. Biol Psychiatry 44: 313 323 Bradley SL, Dodelzon K, Sandhu HK, Philibert RA (2005). Relationship of serotonin transporter gene polymorphisms and haplotype to mRNA transcription. Am J Med Genet Part B 136B: 5861. Buwalda B, Kole MHP, Veenema AH, Huininga M, de Boer SF, Korte M, et al. (2005). Long term effects of social stress on brain and behavior: a focus on hippocampal functioning. Neurosci Biobehav R 29: 83 97. Brgy M (2008). Phenomenological i nvestigation of despair in depression. Psychopathology 41: 147 156. Caspi A, Sugden D, Moffitt TE, Taylor A, Craig IW, Harrington H, et al. (2003). Influence of life stress on depression: Moderation by a polymorphism in the 5HTT gene. Science 301: 386 389 Chen B, Dowlatshahi D, MacQueen GM, Wang JF, Young LT (2001). Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication. Biol Psychiat 50: 260 265 Chen MC, Joormann J, Hallmayer J, Gotlib IH (2009). Serotonin transpor ter polymorphism predicts waking cortisol in young girls. Psychoneuroendocrino 34: 681686.
89 Chen ZY, Jing D, Bath KG, Ieraci A, Khan T, Siao CJ, et al. (2006). Genetic variant BDNF (Val66Met) polymorphism alters anxiety related behavior. Science 314: 1401 43. Cui M, Aras R, Christian WV, Rappold PM, Hatwar M, Panza J, et al. (2009) The organic cation transporter 3 is a pivotal modulator of neurodegeneration in the nigrostriatal dopaminergic pathway. Proc Natl Acad Sci USA 106: 80438048. De La Garza II R, Mahoney III JJ (2004). A distinct neurochemical profile in WKY rats at baseline and in response to acute stress: implications for animal models of anxiety and depression. Brain Res 1021: 209 18. De La Garza II R (2005). Wistar Kyoto rats exhibit reduced sucrose pellet reinforcement behavior and intravenous nicotine self administration. Pharmacol Biochem Behav 82: 330 7. Devine DP, Hoversten MT, Ueda Y, Akil H (2003). Nociceptin/Orphanin FQ content in decreased in forebrain neurons during acute stress. J Neu roendocrinol 15: 69 74. Diorio D, Viau V, Meaney MJ (1993). The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of the hypothalamic pituitary adrena responses to stress. J Neurosci 13: 38393847. Drevets WC (1998). Functional neuroimaging studies of depression: the anatomy of melancholia. Annu Rev Med 49: 341 361. Drevets WC, Price JL, Furey ML (2008). Brain structural and functional abnormalities in mood disorders: implications for neurocircuitry models of depression. Brain Struct Funct 213: 93 118 Durham LK, Webb SM, Milos PM, Clary CM, Seymour AB (2004). The serotonin transporter polymorphism, 5HTTLPR, is associated with a faster response time to sertraline in an elderly population with major depressive disorder. Psychopharmacology 174: 525 529. Egan MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, Bertolino A, et al. (2003). The BDNF val66met polymorphism affects activity dependent secretion of BDNF and human memory and hippocampal function. Cell 112: 257 269. Feng N, Lowry CA, Lukkes JL, Orchinik M, Forster GL, Renner KJ (2010). Organic cation transporter inhibition increases medial hypothalamic serotonin under basal conditions and during mild restraint. Brain Res 1326: 105 113 Feng N, Telefont M, Kelly KJ, Orchinik M, Forst er GL, Renner KJ, et al. (2009). Local perfusion of corticosterone in the rat medial hypothalamus potentiates D fenfluramine induced elevations of extracellular 5HT concentrations. Horm Behav 56: 149 157.
90 Feng N, Mo B, Johnson PL, Orchinik M, Lowry CA, Renner KJ (2005). Local inhibition of organic cation transporters increases extracellular serotonin in the medial hypothalamus. Brain Res 1063: 69 76 Fernandez F, Durand M, Coomans V, Mormede P, Chaouloff F (2001). Effects of corticosterone ingestion on hipp ocampal [3H]serotonin reuptake in inbred rat strains. Endocr Regul 35: 119 126. Fujimoto Y, Kitaichi K, Nakayama H, Ito Y, Takagi K, Takagi K, et al. (2007). The pharmacokinetic properties of methamphetamine in rats with previous exposure to methamphetamin e: the differences between Long Evans and Wistar rats. Exp Anim 56: 119 29. Gasser PJ, Lowry CA, Orchinik M (2006). Corticosteronesensitive monoamine transport in the rat dorsomedial hypothalamus: Potential role for organic cation transporter 3 in s tress induced modulation of monoaminergic neurotransmission. J Neurosci 26: 87588766. Gasser PJ, Orchinik M, Raju I, Lowry CA (2009). Distribution of organic cation transporter 3, a corticosteronesensitive monoamine transporter, in the rat brain. J Comp Neurol 512: 529 555. Gould E McEwen BS Tanapat P Galea LA Fuchs E ( 1997). Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation. J Neurosci 17: 24922498. Griebel G, Cohen C, Perrault G, Sanger DJ (1999). Behavioral effects of acute and chronic fluoxetine in Wistar Kyoto rats. Physiol Behav 67: 315 20. Grndemann D, Liebich G, Kiefer N, Kster S, Schmig E (1999). Selective substrates for nonneuronal monoamine transporters. Mol Pharmacol 56: 1 10. Grndemann D, Breidert T, Spitzenberger F, Schmig E (1998). Molecular structure of the carrier responsible for hepatic uptake of c atecholamines. Adv Pharmacol 42: 346349. Grndemann D, Babin Ebell J, Martel F, Ording N, Schmidt A, Schmig E (1997). Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC PK1 cells. J Biol Chem 2 72: 10408 13. Hariri AR, Mattay VS, Tessitore A, Kolachana B, Fera F, Goldman D, et al. (2002). Serotonin transporter genetic variation and the response of the human amygdala. Science 297: 400 403. Heim C, Newport DJ, Bonsall R, Miller AH, Nemeroff CB (200 1). Altered pituitary adrenal axis responses to provocative challenges tests in adult survivors of childhood abuse. Am J Psychiatry 158: 575581.
91 Herman JP, Ostrander MM, Mueller NK, Figueiredo H (2005). Limbic system mechanisms of stress regulation: Hypot halamo pituitary adrenocortical axis. Prog NeuroPsychoph 29: 12011213. Herman JP, Cullinan WE (1997). Neurocircuitry of stress: central control of the hypothalamopituitary adrenocortical axis. Trends Neurosci 20: 7884. Herman JP, Sh hompson R, Douglass J, Akil H, et al. (1989). Evidence for hippocampal regulation of neuroendocrine neurons of the hypothalamopituitary adrenocortical axis. J Neurosci 9: 30723082. Hill JE, Connolly CE, Nothem AP, Gasser PJ (2009). Organic cation transporter 3 as a mechanism for stress induced enhancement of catecholaminergic neurotransmission. Society for Neuroscience 39th Annual Meeting, Chicago, IL. Hollis F, Wang H, Dietz D, Gunjan A, Kabbaj M (2010). The effects of repeated social defeat on long term depressive like behavior and short term histone modifications in the hippocampus in male Sprague Dawley rats. Psychopharmacology 211: 69 77. Hrdina PD, Vu TB (1993). Chronic fluoxetine treatment upregulates 5HT uptake sites and 5HT2 receptors in rat brai n: An autoradiographic study. Synapse 14: 324331. Hurley KM, Herbert H, Moga MM, Saper CB (1991). Efferent projections of the infralimbic cortex of the rat. J Comp Neurol 308: 249 276. Iversen LL (1965). The uptake of catecholamines at high perfusion conc entrations in the rat isolated heart: a novel catecholamine uptake process. Br J Pharmac 25: 18 33. Iversen LL, Glowinski J (1966). Regional studies of catecholamines in the rat brain II: Rate of turnover of catecholamines in various brain regions. J Neuro chem 13: 671682. Jacobson L, Sapolsky R (1991). The role of the hippocampus in feedback regulation of the hypothalamic pituitary adrenocortical axis. Endocr Rev 12: 118 134. Kaufman J, Yang BZ, Douglas Palumberi H, Grasso D, Lipschitz D, Houshyar S, et al (2006) BrainDerived Neurotrophic Factor 5 HTTLPR Gene Interactions and Environmental Modifiers of Depression in Children. Biol Psychiat 59: 673 680 Keeney A, Jessop DS, Harbuz MS, Marsden CA, Hogg S, BlackburnMunro RE (2006). Differential effects of ac ute and chronic social defeat stress on hypothalamic pituitary adrenal axis function and hippocampal serotonin release in mice. J Neuroendocrinol 18: 330 338.
92 Kekuda R, Prasad PD, Wu X, Wang H, Fei YJ, Leibach FH, et al. (1998). Cloning and functional char acterization of a potential sensitive, polyspecific organic cation transporter (OCT3) most abundantly expressed in placenta. J Biol Chem 273: 15971 15979. Kim JJ, Diamond DM (2002). The stressed hippocampus, synaptic plasticity and lost memories. Nat Rev N eurosci 3: 453 462. King CD, Devine DP, Vierck CJ, Mauderli A, Yezierski RP (2007). Opioid modulation of reflex versus operant responses following stress in the rat. Neuroscience 147: 174182. Kitaichi K, Fukuda M, Nakayama H, Aoyama N, Ito Y, Fujimoto Y, et al. (2005). Behavioral changes following antisense oligonucleotideinduced reduction of organic cation transporter 3 in mice. Neurosci Lett 382: 195 200. Kitayama N, Quinn S, Bremner JD (2006). Smaller volume of anterior cingulate cortex in abuserelated posttraumatic stress disorder. J Affect Disorders 90: 171174. Kline NS (1958). Clinical experience with iproniazid (Marsilid). J Clin Exp Psychopathol 19: 72 78. Kline NS (1959). Uses of reserpine, the newer phenothiazines, and iproniazid. Res Publ Asso c Res Nerv Ment Dis 37: 218 244. Lahmame A, Armario A (1996). Differential responsiveness of inbred strains of rats to antidepressants in the forced swimming test: are Wistar Kyoto rats an animal model of subsensitivity to antidepressants? Psychopharmacology 123: 191 198. Lanfumey L, Mongeau R, CohenSalmon C, Hamon M (2008). Corticosteroidserotonin interactions in the neurobiological mechanisms of stress related disorders. Neurosci Biobehav R 32: 11741184. Lazar A, Walitza S, Jetter A, Gerlach M, Warnke A, Herpertz Dahlmann B, et al. (2008). Novel mutations of the extraneuronal monoamine transporter gene in children and adolescents with obsessivecompulsive disorder. Int J Neuropsychopharmacol 11: 3548. Lee HJ, Lee MS, Kang RH, Kim H, Kim SD, Kee BS, et al. (2005). Influence of the serotonin transporter promoter gene polymorphism on susceptibility to posttraumatic stress disorder. Depress Anxiety 21: 135 139. Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, Benjamin J, et al. (1996). Associat ion of anxiety related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274: 1527 1530.
93 Lin PY, Tsai G (2004). Association between serotonin transporter gene promoter polymorphism and suicide: results of a metaanalys is. Biol Psychiat 55: 1023 1030. Linthorst AC, Reul JM (2008). Stress and the brain: solving the puzzle using microdialysis. Pharmacol Biochem Behav 90: 163 73. Loomer HP, Saunders JC, Kline NS (1957). A clinical and pharmacodynamic evaluation of iproniazi d as a psychic energizer. Psychiat Res Rep 8: 129 141. Lopez Rubalcava C, Lucki I (2000). Strain differences in the behavioral effects of antidepressant drugs in the rat forced swimming test. Neuropsychopharm 22: 191 199. Lowry CA, Burke KA, Renner KJ, Moore FL, Orchinik M (2001). Rapid changes in monoamine levels following administration of corticotropinreleasing factor or corticosterone are localized in the dorsomedial hypothalamus. Horm Behav 39: 195205. Lowry CA, Plant A, Shanks N, Ingram CD, Lightman SL (2003). Anatomical and functional evidence for a stress responsive, monoamineaccumulating area in the dorsomedial hypothalamus of adult rat brain. Horm Behav 43: 254 262 Magarinos AM, McEwen BS, Flugge G, Fuchs E (1996). Chronic psychosocial stress ca uses apical dendritic atrophy of hippocampal CA3 pyramidal neurons in subordinate tree shrews J Neurosci 16: 3534 3540. Marin MT, Cruz FC, Planeta CS (2007). Chronic restraint or variable stresses differently affect the behavior, corticosterone secretion and body weight in rats. Physiol Behav 90: 2935. Marini F, Pozzato C, Andreetta V, Janssen B, Arban R, Domenici E, et al. (2006). Single exposure to social defeat increases corticotropinreleasing factor and glucocorticoid receptor mRNA expression in rat hippocampus. Brain Res 1067: 2535. McGuffin P, Cohen S, Knight, J (2007). Homing in on depression genes. Am J Psychiat 164: 195 197. Mokler DJ, Torres OI, Galler JR, Morgane PJ (2007). Stress induced changes in extracellular dopamine and serotonin in the medial prefrontal cortex and dorsal hippocampus of prenatally malnourished rats. Brain Res 1148: 226 233. Mooney JJ, Samson JA, Hennen J, Pappalardo K, McHale N, Alpert J, et al. (2008). Enhanced norepinephrine output during long term desipramine treatment : a possible role for the extraneuronal monoamine transporter (SLC22A3). J Psychiatr Res 42: 605 611.
94 Muchimapura S, Fulford AJ, Mason R, Marsden CA (2002). Isolation rearing in the rat disrupts the hippocampal response to stress. Neuroscience 112: 697 705. Murphy DL, Li Q, Engel S, Wichems C, Andrews A, Lesch KP, et al. (2001). Genetic perspectives on the serotonin transporter. Brain Res Bull 56: 48794. Musselman DL, DeBattista C, Nathan KI, KiltsCD, Schatzberg AF, Nemeroff CB (1998). Biology of mood disorders. In: Schatzberg AF, Nemeroff CB (eds). Textbook of Psychopharmacology American Psychiatric Press: Washington, DC. pp 549 588. Nagayama H, Kitaichi K, Ito Y, Hashimoto K, Takagi K, Yokoi T, et al (2007). The role of the organic cation transporter 3 in methamphetamine disposition and its behavioral response in rats. Brain Res 1184: 260269. Nichols NR, Zieba M, Bye N (2001). Do glucocorticoids contribute to brain aging? Brain Res Rev 37: 273 286. ONeil MF, Moore NA (2003). Animal models of depression: Are there any? Hum Psychopharm 18: 239 254. Owens MJ, Nemeroff CB (1994). Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter. Clin Chem 40: 288 295. Par WP, Redei E (1993). Depressive behavior and stress ulcer in Wistar Kyoto rats. J Physiology Paris 87: 229 238. Par WP (1994). Open field, learned helplessness, conditioned defensive burying, and force swim tests in WKY rats. Physiol Behav 55: 433 439. Par WP, Tejani Butt SM (1996). Effect of stress on the behavior and 5HT system in SpragueDawley and Wistar Kyoto rat strains. Integr Phys Beh Sci 31: 112 122. Pezawas L, Meyer Lindenberg A, Drabant EM, Verchinski BA, Munoz KE, Kolachana BS, et al. (2005). 5 HTTLPR polymorphism impacts human cingulateamygdala inter actions: a genetic susceptibility mechanism for depression. Nat Neurosci 8: 828 834. Pollier F, Sarre S, Aguerre S, Ebinger G, Mormede P, Michotte Y, et al. (2000). Serotonin reuptake inhibition by citalopram in rat strains differing for their emotionality Neuropsychopharm 22: 6476 Porsolt RD, Anton G, Blavet N, Jalfre M (1978). Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol 47: 379 91. Pregelj P, Nedic G, Paska AV, Zupanc T, Nikolac M, Balaic J, et al (2010): The association between brainderived neurotrophic factor polymorphism (BDNF Val66Met) and suicide. J Affect Disorders, print copy in press (originally published online July 27, 2 010, at http://dx.doi.org/10.1016/j.jad.2010.07.001)
95 Pritchard JB, Miller DS (1993). Mechanisms mediating renal secretion of organic anions and cations. Physiol Rev 73: 765 796 Radley JJ, Sisti HM, Hao J, Rocher AB, McCall T, Hof PR et al. (2004). Chronic behavioral stress induces apical dendritic reorganization in pyramidal neurons of the medial prefrontal cortex. Neuroscience 125: 1 6. Radley JJ, Rocher AB, Miller M, Janssen WG, Liston C, Hof PR, et al. (2006). Repeated stress induces dendritic spine loss in the rat medial prefrontal cortex. Cereb Cortex 16: 313 320. Rahman Z, Ring RH, Young K, Platt B, Lin Q, Schechter LE, Rosenzweig Lipson S, Beyer CE (2008). Inhibition of uptake 2 (or extraneuronal monoamine transporter) by normetanephrine potentiates t he neurochemical effects of venlafaxine. Brain Res 1203: 68 78. Redei E, Par WP, Aird F, Kluczynski J (1994). Strain differences in hypothalamic pituitary adrenal axis activity and stress ulcer. Am J Physiol 266: R353 R360. Risch N, Herrel R, Lehner T, Li ang KY, Eaves L, Hoh J, et al. (2009). Interaction between the serotonin transporter gene (5HTTLPR), stressful life events, and the risk of depression: A metaanalysis. J Amer Med Assoc 301: 24622471. Rittenhouse PA, L pez Rubalcava C, Stanwood GD, Lucki I (2002). Amplified behavioral and endocrine responses to forced swim stress in the Wistar Kyoto rat. Psychoneuroendocrino 27: 303 318. Robertson DA, Beattie JE, Reid IC, Balfour DJ (2005). Regulation of corticosteroid receptors in the rat brain: the role of serotonin and stress. Eur J Neurosci 21: 15111520. Rygula R, Abumaria N, Flgge G, Fuchs E, Rther E, HavemannReinecke U (2005). Anhedonia and motivational deficits in rats: Impact of chronic social stress Behav Brain Res 162 : 127 134. Rygula R, Abu maria N, Domenici E, Hiemke C, Fuchs E (2006). Effects of fluoxetine on behavioral deficits evoked by chronic social stress in rats. Behav Brain Res 174: 188192. Sala M, Perez J, Soloff P, Ucelli di Nemi S, Caverzasi E, Soares JC, et al. (2004). Stress an d hippocampal abnormalities in psychiatric disorders. European Neuropsychopharmacol 14: 393 405. Salzer HM, Lurie ML (1953). Anxiety and depressive states treated with isonicotinyl hydrazid (isoniazid). Arch Neurol Psychiat 70: 317 324.
96 Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, et al (2003). Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 301: 805 9. Sapolsky RM, Krey LC, McEwen BS (1984). Glucocorticoidsensitive hippocampal neurons are i nvolved in terminating the adrenocortical stress response. Proc Natl Acad Sci USA 81: 6174 6177. Saunders JC, Radinger N, Rochlin D, Kline NS (1959). Treatment of depressed and regressed patients with iproniazid and reserpine. Dis Nerv Syst 20: 3139. Schi ldkraut JJ (1965). The catecholamine hypothesis of affective disorders: A review of supporting evidence. Am J Psychiat 122: 509 522. Schildkraut JJ, Mooney JJ (2004). Toward a rapidly acting antidepressant: the normetanephrine and extraneuronal monoamine t ransporter (uptake 2) hypothesis. Am J Psychiat 161: 909 911. Schmidt HD, Duman RS (2007). The role of neurotrophic factors in adult hippocampal neurogenesis, antidepressant treatments and animal models of depressivelike behavior. Behav Pharmacol 18: 391 418. Schmitt A, M et al. (2003). Organic cation transporter capable of transporting serotonin is upregulated in serotonintransporter deficient mice J Neurosci Res 71: 701 709. Schmig E, Lazar A, Grndemann D (2006). Extraneuronal monoamine transporter and organic cation transporters 1 and 2: a review of transport efficiency. Handb Exp Pharmacol 175: 15180. Sesack SR, Deutch AY, Roth RH, Bunney BS (1989). Topographical organization of the efferent proj ections of the medial prefrontal cortex in the rat: an anterograde tract tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 290: 213 242. Sierksma ASR, van den Hove DLA, Steinbusc HWM, Prickaerts J (2010). Major depression, cognitive dysf unction and Alzheimers disease: Is there a link? Eur J Pharmacol 626: 7282. Simpkiss JL, Devine DP (2003). Responses of the HPA axis after chronic variable stress: effects of novel and familiar stressors. Neuro Endocrinol Lett 24: 97103. Smith JA (1953) The use of the isopropyl derivative of isonicotinylhydrazine (Marsilid) in the treatment of mental disease. Am Pract Dig Treat 4: 519 520. Smith MA, Makino S, Kvetnansky R, Post RM (1995). Stress and glucocorticoids affect the expression of brain derived neurotrophic factor and neurotrophin 3 mRNA's in the hippocampus. J Neurosci 15: 1766 1777
97 Snaith P (1993). Anhedonia: a neglected symptom of psychopathology. Psychol Med 23: 957 966. Stokes PE (1995). The potential role of excessive cortisol induced by H PA hyperfunction in the pathogenesis of depression. Eur Neuropsychopharmacol 5: 7782 Stone EA, Lin Y (2008). An anti immobility effect of exogenous corticosterone in mice. Eur J Pharmacol 580: 135142. Tadic A, Wagner S, Schlict KF, Peetz D, Borysenko L, Dreimuller N, et al. (2010): The early nonincrease of serum BDNF predicts failure of antidepressant treatment in patients with major depression: A pilot study. Prog NeuroPsychoph, print copy in press (originally published online Aug. 20, 2010, at http://dx.doi.org/10.1016/j.pnpbp.2010.08.011) Tejani Butt S, Kluczynski J, Pare WP (2003). Straindependent modification of behavior following antidepressant treatment. Prog Neuropsychph 27 : 7 14. Ullrich KJ (1994). Specificity of transporters for 'organic anions' and 'organic cations' in the kidney. Biochim Biophys Acta 1197: 45 62. Vialou V, Balasse L, Callebert J, Launay JM, Giros B, Gautron S (2008). Altered aminergic neurotransmission in the brain of organic cation transporter 3deficient mice. J Neurochem 1 06: 147182. Wieland A, Hayer Zillgen M, Bnisch H, Brss M (2000). Analysis of the gene structure of the human (SLC22A3) and murine (Slc22a3) extraneuronal monoamine transporter. J Neural Transm 107: 114957. World Health Organization (n.d.). Depression. In: Mental Health Retrieved Oct. 8, 2010, from http://www.who.int/mental_health/management/depression/definition/en/. Wu X, Kekuda R, Huang W, Fei YJ, Leibach FH, Chen J, et al. (1998). Identity of the organic cation transporter OCT3 as the extraneuronal monamine transporter (uptake2) and evidence for the expression of the transporter in the brain. J Biol Chem 273: 3277632786. Wultsch T, Grimberg G, Schmitt A, Painsipp E, Wetzstein H, Breitenkamp AF, et al. (2009). Decreased anxiety in mice lacking the or ganic cation transporter 3. J Neural Transm 116: 689 697. Wst S, Kumsta R, Treutlein J, Frank J, Entringer S, Schulze TG, et al. (2009). Sex specific association between the 5HTT genelinked polymorphic region and basal cortisol secretion. Psychoneuroend ocrino 34: 972982
98 Young, KA, Bonkale WL, Holcomb LA, Hicks PB, German DC (2008). Major depression, 5HTTLPR genotype, suicide and antidepressant influences on thalamic volume. Brit J Psychiat 192: 285 289. Zacharko RM, Anisman H (1991). Stressor induced an hedonia in the mesocorticolimbic system. Neurosci Biobehav R 15: 391 405. Zhou J, Li L, Tang S, Cao X, Li Z, Li W, et al. (2008). Effects of serotonin depletion on the hippocampal GR/MR and BDNF expression during stress adaptation. Behav Brain Res 195: 129138.
99 BIOGRAPHICAL SKETCH Catherine A. Marcinkiewcz was born in Point Pleasant New Jersey. Her family moved to Florida a few years later where she completed high school in St. Petersburg, Florida. She decided to pursue a career in scienc e as an undergraduate at Johns Hopkins University where she completed her Bachelor of Science in biomedical e ngin eering with a concentration in materials s cience. She returned to Florida to pursue a Master of Science in biomedical engineering before joining the Interdisciplinary Program in Biomedical Sciences in the fall of 2005 to resume her lifelong passion for neuroscience. She joined the laboratory of Darragh Devine in the fall of 2007 to study the neurobiology of stress and psychiatric disease and completed her dissertation work in the fall of 2010. She will pursue her postdoctoral work at the University of North Carolina in Chapel Hill starting in January of 2011.