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Glucocorticoid modulation of the mesocortical dopamine system and aspects of executive function Butts, Kelly Ann 2013

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GLUCOCORTICOID MODULATION OF THE MESOCORTICAL DOPAMINE SYSTEM AND ASPECTS OF EXECUTIVE FUNCTION  by Kelly Ann Butts  B.Sc. (Honours), The University of Edinburgh, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  March 2013  © Kelly Ann Butts, 2013  ii Abstract Enhanced dopamine efflux in the medial prefrontal cortex is a well-documented response to acute stress and is associated with deficits in cognitive performance. However, the underlying mechanism(s) for this response is unknown. The mesocortical dopamine system is comprised of dopamine neurons in the ventral tegmental area that receive excitatory input from and send reciprocal projections to the medial prefrontal cortex. We hypothesize that glucocorticoid receptors in the medial prefrontal cortex modulate the activity of this descending glutamatergic input to the ventral tegmental area during stress. Using in vivo microdialysis, we demonstrate that blocking glucocorticoid receptors locally within the rat medial prefrontal cortex, but not in the ventral tegmental area, attenuates mesocortical dopamine efflux to acute tail-pinch stress. Acute stress leads to a significant increase in glutamate release in the ventral tegmental area that is prevented by blockade of glucocorticoid receptors in the medial prefrontal cortex. The functional impact of enhanced mesocortical dopamine efflux evoked by acute stress was demonstrated using cognitive tasks measuring executive function. Exposure to acute tail-pinch stress selectively impaired performance on a working memory and set-shifting task. Conversely, performance on a non- delayed random foraging or reversal learning task that do not require the medial prefrontal cortex were unaffected by stress. Notably, stress-induced impairments in working memory were attenuated by blockade of glucocorticoid receptors in the medial prefrontal cortex. Taken together, these data suggest that glucocorticoids act locally within the medial prefrontal cortex to modulate mesocortical dopamine efflux by potentiation of glutamatergic drive onto dopamine neurons in the ventral tegmental area leading to the executive function impairments observed during acute stress.  iii Preface We published: Butts, K.A. and Phillips, A.G. (2013). Glucocorticoid receptors in the prefrontal cortex regulate dopamine efflux to stress via descending glutamatergic feedback to the ventral tegmental area. The International Journal of Neuropsychopharmacology. This paper is presented in Chapter 3. All the experiments published in this paper were conceived and designed by myself, and I conducted all of the data collection, analysis, and manuscript preparation. Dr. Anthony Phillips provided advice on experimental design and helped with editing the manuscript.  Butts, K.A., Weinberg, J., Young, A.H., and Phillips, A.G. (2011). Glucocorticoid receptors in the prefrontal cortex regulate stress-evoked dopamine efflux and aspects of executive function. Proceedings of the National Academy of Sciences USA 67, 967-983. This paper was comprised of two sections. The first set of experiments examined the role of the glucocorticoid receptor in mediating stress-evoked dopamine efflux. The second set of experiments examined the behavioural consequences of stress exposure using a working memory task. For the purpose of this thesis, Chapter 2 presents the role of prefrontal glucocorticoid receptors in regulating stress-evoked dopamine efflux. The working memory data will be combined with new a set of behavioural experiments and will be presented in Chapter 4. All the experiments published in this paper were conceived and designed by myself, and I conducted all of the data collection, analysis, and manuscript preparation. Dr. Joanne Weinberg contributed an analytical tool and helped with editing the manuscript. Drs. Allan Young and Anthony Phillips provided advice on experimental design and helped with editing the manuscript.  Certificate of approval: The animal studies presented in this thesis were performed with ethics approval from the University of British Columbia Animal Care Committee (certificate # A09-0277 and A09- 0805).   iv Table of Contents  Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iii Table of Contents ................................................................................................................... iv List of Tables .......................................................................................................................... vi List of Figures ........................................................................................................................ vii Acknowledgements .............................................................................................................. viii Dedication ............................................................................................................................... ix Chapter  1: General Introduction .......................................................................................... 1 1.1 Overview ............................................................................................................... 1 1.2 Prefrontal Cortical Function ................................................................................. 2 1.3 The Mesocortical Dopamine System and Executive Function ............................. 4 1.4 The Stress System ................................................................................................. 9 1.5 Factors Influencing the Effect of Stress on the HPA axis, Mesocortical Dopamine System, and Prefrontal Cortical Function ..................................................... 14 1.6 Research Aims and Hypotheses .......................................................................... 20 Chapter  2: Glucocorticoid Receptors in the Prefrontal Cortex Regulate Stress-Evoked Dopamine Efflux ................................................................................................................. 24 2.1 Introduction ......................................................................................................... 24 2.2 Material and Methods ......................................................................................... 26 2.3 Results ................................................................................................................. 30 2.4 Discussion ........................................................................................................... 37  v Chapter  3: Glucocorticoid Receptors in the Prefrontal Cortex Regulate Dopamine Efflux to Stress via Descending Glutamatergic Feedback to the Ventral Tegmental Area ........... 40 3.1 Introduction ......................................................................................................... 40 3.2 Material and Methods ......................................................................................... 42 3.3 Results ................................................................................................................. 46 3.4 Discussion ........................................................................................................... 53 Chapter  4: Glucocorticoid Receptors Regulate Aspects of Executive Functioning During Stress ................................................................................................................................... 57 4.1 Introduction ......................................................................................................... 57 4.2 Material and Methods ......................................................................................... 61 4.3 Results ................................................................................................................. 70 4.4 Discussion ........................................................................................................... 78 Chapter  5: Conclusion........................................................................................................ 88 5.1 Summary of Research Findings .......................................................................... 88 5.2 Limitations and Future Directions ...................................................................... 93 5.3 Clinical Significance ......................................................................................... 101 References ............................................................................................................................ 103   vi List of Tables Table 4.1 Acute stress does not affect motor or motivational processes during set-shifting.. 75 Table 4.2 Acute stress does not affect motor or motivational processes during reversal learning. .................................................................................................................................. 77   vii List of Figures Figure 1.1 Schematic diagram of the Hypothalamic-Pituitary-Adrenal axis .......................... 11 Figure 2.1 Activation of GRs mediates stress-evoked DA efflux in the mPFC. .................... 31 Figure 2.2 GRs in the mPFC mediate stress-evoked DA efflux. ............................................ 33 Figure 2.3 Corticosterone acts locally in the mPFC to enhance DA efflux. ........................... 34 Figure 2.4 GRs in the mPFC do not mediate DA efflux to an incentive stimulus.................. 35 Figure 2.5 Placements of microdialysis probes. ..................................................................... 36 Figure 3.1 Selective GR antagonists attenuates stress-evoked DA efflux in the mPFC......... 47 Figure 3.2 GRs in the mPFC regulate stress-evoked glutamate efflux in the VTA................ 48 Figure 3.3 Ionotrophic glutamate receptors in the VTA mediate stress-evoked DA efflux in the mPFC. ............................................................................................................................... 50 Figure 3.4 Stress-evoked DA efflux in the mPFC is not mediated by CB1 receptors. ........... 51 Figure 3.5 Placements of microdialysis probes. ..................................................................... 51 Figure 3.6 Schematic depiction of the anatomical connections between the mPFC and VTA proposed to mediate stress-evoked DA efflux in the mPFC. .................................................. 52 Figure 4.1 Blockade of GRs in the mPFC reverses stress-induced working memory impairments............................................................................................................................. 71 Figure 4.2 Performance on a non-delayed random foraging task is unaffected by stress. ..... 72 Figure 4.3 Acute stress impairs set-shifting but does not affect initial visual-cue discrimination learning. .......................................................................................................... 74 Figure 4.4 Acute stress does not affect reversal learning or initial response learning. ........... 77    viii Acknowledgements      First and foremost, I thank my supervisor, Dr. Anthony Phillips, for enlarging my vision of science and providing unlimited guidance and financial support to answer my endless questions. Thank you to Dr. Allan Young for providing me with the opportunity to come to Vancouver to do my PhD and for his initial supervision of my thesis research. Thank you to all my committee members Drs. Joanne Weinberg, Stan Floresco and Liisa Galea. In particular, thank you Dr. Weinberg for her supervision and financial support and for introducing me to tail-pinch stress. Thank you to Dr. Floresco for his guidance and allowing me to conduct some of the behavioural experiments for my thesis in his lab. I would like to thank all the members of the Phillips lab. I owe particular thanks to Dr. Soyon Ahn, Kitty So Giada Vacca, Dr. Christopher Lapish, and Dr. Gemma Dalton for teaching me the skills necessary to conduct the microdialysis and behavioural experiments. Thank you to Pretty Verma, Giada Vacca, and Dr. Gemma Dalton for their assistance with the behavioural experiments and to Liya Ma for our many interesting science discussions. Thank you also to all the members of the Weinberg and Floresco labs. In particular, thanks to Linda Ellis, Wayne Yu, and Maric Tse for their support and always making me feel a part of their labs. Finally, thank you to the Leading Edge Endowment Fund (Rix Student Award) and Canadian Institutes of Health Research-funded Integrated Mentor Program in Addictions Research Training (IMPART) fellowship.      Thank you to my parents for providing me with the wonderful opportunity to travel the world through my schooling and for their constant encouragement and support throughout my many years of education. And last but not least, to Lasse Dissing-Olesen for his invaluable support and who taught me the true values of a scientist.  ix Dedication I dedicate this thesis to Mom, Dad, Nanna, Poppa, Lindsey and Lasse to whom I would have not been able to complete this without their love and support.   1 Chapter  1: General Introduction 1.1 Overview      First documented in 1976 by Thierry and colleagues, dopamine (DA) levels are elevated in the prefrontal cortex (PFC) following prolonged exposure to an acute stressor (Thierry et al., 1976). This phenomenon has since been replicated using a variety of stressors (Takahata and Moghaddam, 1998; Feenstra et al., 2000; Del Arco and Mora, 2001) and across species including the rat, monkey and human (Murphy et al., 1996b; Taber and Fibiger, 1997; Lataster et al., 2011). Furthermore, the immediate effect of exposure to acute stress has been demonstrated to impair working memory function in rats, monkeys and humans (Murphy et al., 1996a; Arnsten and Goldman-Rakic, 1998; Morrow et al., 2000; Schoofs et al., 2009). Stress was also shown to be a major factor contributing to the development, recurrence, and treatment outcome in affective disorders, including bipolar disorder (Ellicott et al., 1990; Hammen and Gitlin, 1997). Pronounced neurocognitive dysfunction is commonly reported in patients with bipolar disorder (Young et al., 2004; Watson et al., 2006) and may be attributed to alterations in PFC functioning that occur as a result of interactions between stress and DA function (Schatzberg et al., 1985). Thus, the quest to understand the behavioural, neurochemical, and electrophysiological events that occur during stress is an ongoing area of research. Much has been learned but important questions still remain concerning the basic mechanisms that may explain how exposure to a stressor leads to elevation of DA in the PFC. The aim of my thesis was to investigate the mechanism by which exposure to an acute stressor leads to increased DA efflux in the PFC and to further delineate the changes to PFC- dependent cognitive functions that occur during stress.   2 1.2 Prefrontal Cortical Function 1.2.1 The Prefrontal Cortex      The PFC is the most recently evolved structure in the mammalian brain and is most highly developed in animals known for their diverse and flexible behavioural repertoires including monkeys and humans (Fuster, 2000a; Miller and Cohen, 2001; Wise, 2008). The primate PFC encompasses a large and heterogeneous set of regions that are broadly divided into a medial, dorsolateral, and orbital region (Uylings and van Eden, 1990). These different subdivisions are thought to be involved in different cognitive and emotion functions (Goldman-Rakic, 1996; Robbins, 1996). By comparing connectivity patterns, the rat PFC can also be divided into a medial, lateral, and ventral region (Krettek and Price, 1977; Sesack et al., 1989; Uylings and van Eden, 1990). The rat medial PFC (mPFC) is subdivided into a dorsal component that includes the precentral and anterior cingulate cortices and a ventral component that includes the prelimbic (PL), infralimbic (IL), and medial orbital cortices. The lateral PFC includes the dorsal and ventral agranular insular and lateral orbital cortices, while the ventral PFC encompasses the ventral orbital and ventral lateral orbital cortices.  1.2.2 Executive Function      Studies conducted in humans, monkeys, and rats have provide support for the idea that the PFC is not involved directly in performance of simple, automatic behaviours, but contributes instead to executive functioning. Executive function is a term used to describe a set of cognitive control processes that are necessary for optimal organization of complex sequences of behavior (Baddeley and Della Sala, 1996; Fuster, 2000a; Miller and Cohen, 2001; Brown and Bowman, 2002). Two aspects of executive function include working memory and  3 cognitive flexibility. Working memory involves the temporary on-line storage and manipulation of information essential to guide ongoing behaviour (Fuster, 1973; Goldman- Rakic, 1995; Miller and Cohen, 2001). It can be assessed in rats, monkeys, and humans in the form of a delayed-response task in which subjects are required to remember information acquired prior to a delay in order to complete a choice-task successfully. Cognitive flexibility refers to the flexible updating of those representations in response to novel information and is assessed in rats, monkeys, and humans in the form of attentional set-shifting (Robbins, 1996; Floresco et al., 2008). Miller and Cohen recently proposed a theory of PFC function to refine its role in executive functions (Miller and Cohen, 2001). The fundamental principle of their theory is that information processing in the brain is a competitive procedure in which different sources of information undertaken within specific brain regions, compete for expression in behaviour. The PFC has a unique capacity to actively maintain patterns of activity that represent goals and means to achieve them. Since the PFC is highly connected to nearly all cortical sensory and motor systems as well as subcortical structures of the brain, it is situated in an important region to provide bias signals to resolve any competition and guide neural activity along appropriate pathways that establish the proper mappings between inputs, internal states, and outputs needed to perform a given task. These “top-down control” signals ensure that weak but task-relevant stimulus-response mappings prevail when they are in competition with more habitual signals, especially when cognitive and or behavioural flexibility is required (Miller and Cohen, 2001).     4 1.2.3 Stress and Prefrontal Cortical Function: Relevance to Bipolar Disorder      Stress is a major factor contributing to the development, recurrence, and treatment outcome in affective disorders, including bipolar disorder (Ellicott et al., 1990; Hammen and Gitlin, 1997). Abnormal morphology (Moore et al., 2009) and neural activity (Vizueta et al., 2012) within the PFC have been implicated in the etiology and maintenance of bipolar disorder. Pronounced neurocognitive dysfunction is commonly reported in patients with bipolar disorder (Young et al., 2004; Watson et al., 2006) and may be attributed to alterations in PFC functioning. Therefore, understanding how stress affects PFC function is important for understanding the neurobiological mechanisms that may contribute to bipolar disorder. Accumulating evidence suggests that PFC activity is altered following exposure to stress and both reduced PFC activity (Vijayraghavan et al., 2007; Qin et al., 2009; Qin et al., 2012) and enhanced PFC activity (Grimm et al., 2006; Stark et al., 2006; Wang et al., 2007; Kern et al., 2008) have been observed. Furthermore, reports of enhanced activity within subcortical regions such as the amygdala (Geuze et al., 2012; Henckens et al., 2012) has lead to a prominent theory of PFC function during stress proposed by Arnsten and colleagues that states that high levels of catecholamines released during stress inhibits PFC activity leading in turn to a loss of “top-down control” and rapid switching to more reflexive response strategies mediated by the amygdala and hippocampus (Arnsten, 2009, 2011).  1.3 The Mesocortical Dopamine System and Executive Function 1.3.1      The Mesocortical Dopamine System      The PFC receives a dense projection of dopaminergic fibers from the ventral tegmental area (VTA) (Lindvall et al., 1984). The densest dopaminergic projections are found in the  5 anterior cingulate, prelimbic, and infralimbic cortices (Van Eden et al., 1987; Berger et al., 1991; Sesack et al., 1995). DA terminals form synaptic contacts onto the dendritic shafts of both pyramidal and γ-aminobutyric acid (GABA) interneurons in the PFC (Seguela et al., 1988; Goldman-Rakic et al., 1989; Sesack et al., 1995). The VTA does not contain a functionally homogenous group of DA neurons, but instead, they are heterogeneous in their anatomical location, efferent projections, and electrophysiological properties (Lammel et al., 2008; Bromberg-Martin et al., 2010; Lammel et al., 2011). A subset of these DA neurons located in the medial VTA project directly to the PFC and appears to be activated selectively by aversive stimuli (Lammel et al., 2011; Lammel et al., 2012). DA neurons exhibit different states of activity that are dependent upon regulation by afferent systems (Floresco et al., 2001; Floresco et al., 2003). The VTA receives both excitatory and inhibitory inputs from widely distributed brain areas (Geisler and Zahm, 2005). Importantly, the only major cortical projection to the VTA arises from the PFC, mainly the PL and IL cortices (Oades and Halliday, 1987; Sesack et al., 1989; Sesack and Pickel, 1992; Geisler and Zahm, 2005). These cortical afferents synapse onto DA neurons that project back to the PFC, thereby creating a circuit that allows that PFC to exert feedback regulation of DA function (Carr and Sesack, 2000).      DA is a neuromodulator whose actions are mediated through G-protein-coupled receptors. Five DA receptor subtypes have been characterized and are grouped into two families: D1- type (D1 and D5) and D2-type (D2, D3, and D4). D1-type receptors are coupled to Gαs and stimulate the production of the second messenger cyclic-AMP (cAMP) (Sidhu and Kimura, 1997; Lezcano and Bergson, 2002) whereas D2-type receptors, on the other hand, are coupled to Gαi/o and inhibit the production of cAMP (O'Hara et al., 1996). In the rodent PFC,  6 D1- and D2-type receptors are found on pyramidal and non-pyramidal neurons (Vincent et al., 1993; Jackson and Westlind-Danielsson, 1994; Missale et al., 1998; Benes and Berretta, 2001). However, the distribution of the D1 receptor extends over a greater area as compared with other DA receptor subtypes (Camps et al., 1989; Lidow et al., 1991; Ouimet et al., 1992). D1 receptor activation in the PFC has both excitatory and inhibitory effects, enhancing both NMDA- and GABAA-mediated synaptic currents on pyramidal neurons (Seamans et al., 2001b; Seamans et al., 2001a; Tseng and O'Donnell, 2004). In contrast, D2 receptor activation reduces NMDA-and GABA-mediated currents (Trantham-Davidson et al., 2004; Tseng and O'Donnell, 2004).      Due to low levels of DA transporters in the PFC (Sesack et al., 1998a; Sesack et al., 1998b), the DA synaptic transmission in the PFC is terminated primarily through uptake by the noradrenaline (NA) transporter or via enzymatic degradation by catechol-O- methyltransferase (COMT) (Cass and Gerhardt, 1995; Wayment et al., 2001; Bymaster et al., 2002; Moron et al., 2002). In humans there are two naturally occurring variants of this gene; Met/Met COMT polymorphism results in lower COMT activity and higher basal DA levels whereas Val/Val COMT results in lower basal DA levels due to higher COMT activity (Lachman et al., 1996; Chen et al., 2004; Tunbridge et al., 2004).  1.3.2 Role of Prefrontal Dopamine in Working Memory      A landmark study in 1979 by Brozoski and colleagues revealed that DA depletion in the PFC of monkeys impaired performance on a delayed response task that was reversed after treatment with DA agonists (Brozoski et al., 1979). Many studies have since replicated this work proving the necessity of DA for working memory function (Arnsten et al., 1994;  7 Sawaguchi and Goldman-Rakic, 1994; Goldman-Rakic, 1998; Phillips et al., 2004). Subsequent research has revealed a more complex relationship between prefrontal DA function and cognitive activity characterized as an inverted-‘U’-shape function of D1 receptor stimulation in the PFC in which too much (Murphy et al., 1996b; Zahrt et al., 1997) or too little (Sawaguchi et al., 1990; Seamans et al., 1998) D1 receptor stimulation in the PFC impairs working memory performance. Studies from our laboratory demonstrated that performance on a working memory task depended on the levels of DA during the time of the delay period. Rats that performed poorly on the task had low extracellular levels of DA during the delay period whereas those that performed optimally had higher levels of DA in the PFC (Phillips et al., 2004). Furthermore, local administration of a D1 receptor agonist into the PFC improved the performance of low performing rats but impaired the performance of high performing rats (Floresco and Phillips, 2001). These neurochemical data correlate with electrophysiological studies that demonstrated that prefrontal cortical neurons sustain neural activity during the delay period in a spatially selective manner as monkeys perform a spatial oculomotor delayed-response task (Goldman-Rakic, 1995). Moderate levels of prefrontal DA enhance spatial tuning by suppressing the activity of neurons firing to the non- preferred directions (Vijayraghavan et al., 2007). High levels of DA, as observed during acute stress, significantly decreased delay-related activity for the neurons in both the preferred and non-preferred direction (Vijayraghavan et al., 2007). Indeed, impairments in working memory have been observed when acute stress is applied prior to testing across species (Murphy et al., 1996a; Arnsten and Goldman-Rakic, 1998; Morrow et al., 2000; Schoofs et al., 2009) and can be prevented by pretreatment with a D1 receptor antagonist in rats and monkeys (Murphy et al., 1996b; Arnsten and Goldman-Rakic, 1998). In humans,  8 impaired working memory and PFC deactivation induced by stress was more pronounced in individuals with the Met/Met COMT polymorphism resulting in higher basal DA levels (Qin et al., 2012).  1.3.3 Prefrontal Dopamine and Cognitive Flexibility      Prefrontal DA is also essential for cognitive flexibility; however, the DA receptors involved in a set-shifting task differ from those that mediate working memory performance (Floresco et al., 2006). Unlike working memory that depends on D1 receptor stimulation, blockade of either D1 or D2 receptors in the PFC impairs performance on a set-shifting task (Ragozzino, 2002; Floresco et al., 2006). According to the dual-state theory proposed by Seamans and colleagues, PFC networks can be either in a D1- or D2-dominated state depending on the level of receptor occupancy. Moderate levels of DA preferentially activate D1 receptors that subsequently increase NMDA and GABAA currents allowing for stable persistent activity states that are required to maintain information in working memory until an appropriate response is executed. Conversely, high levels of DA preferentially activate D2 receptors, thereby reducing NMDA and GABAA currents and inhibiting these persistent activity states and reducing inhibition on pyramidal neurons in competing neural networks allowing for fast flexible switching between new incoming representations (Seamans and Yang, 2004). Therefore, cooperation between prefrontal D1 and D2 receptors would be required for optimal performance on a cognitive flexibility task. Activation of D2 receptors would facilitate the ability of PFC networks to disengage from the previous strategy and find alternative response strategies. Once an alternative strategy is developed, D1 receptor activation would act to facilitate the stabilization of this novel strategy (Seamans and Yang,  9 2004). This theory is supported by animal studies demonstrating that D1 receptor agonists selectively increase the number of perseverative errors on a working memory task (Zahrt et al., 1997; Floresco and Phillips, 2001). Conversely, D2 receptor antagonists significantly increase perseverative errors on a set-shifting task (Floresco et al., 2006) possibly as a result of increased D1 receptor activation (Durstewitz and Seamans, 2008).      Relatively few studies have examined the effect of acute stress on cognitive flexibility. A recent study conducted in humans found that exposure to acute psychosocial stress immediately before performing a task switching test impaired performance (Plessow et al., 2012). Conversely, a study using rats found that acute restraint stress given immediately preceding testing had no effect on set-shifting performance (Thai et al., 2012). High levels of PFC DA released during stress would be hypothesized to impair performance on a set- shifting task through excessive stimulation of D1- or D2-type receptors leading to the inability to disengage from the previously relevant strategy or learn and maintain a new previously irrelevant strategy.  1.4 The Stress System 1.4.1 Hans Selye      The biological meaning of the word “stress” was provided in 1936 by Hans Selye as “the non-specific neuroendocrine response of the body” (Selye, 1937). Selye’s work described the complex changes that occur in the body in response to stress known as the “General Adaptation Syndrome” and included the enlargement of the adrenal glands, atrophy of the thymus and lymph nodes, and gastric ulcers (Selye, 1937, 1950; Selye and Fortier, 1950). The definition of stress was later updated and “neuroendocrine” was dropped when Selye  10 realized that in addition to the involvement of the neuroendocrine system, almost every other organ system is affected during the stress response. Indeed, the autonomic side of the stress system described by Walter Cannon’s “Flight or Fight” syndrome included the rapid and massive release of catecholamines (Cannon, 1939, 1940). Since these initial and profound insights were first published, an enormous amount of work has elucidated the cascade of events initiated in response to stressor exposure that involve both the autonomic and neuroendocrine response systems. The autonomic response originates within the brainstem and provides the most immediate, but short-lived, response via stimulation of both sympathetic and parasympathetic outputs leading to rapid alterations in physiological states through neural innervation of target organs (Ulrich-Lai and Herman, 2009). The neuroendocrine response, described by Hans Selye and named for its component parts, is mediated by activation of the hypothalamic-pituitary-adrenal (HPA) axis.  1.4.2 The Hypothalamic-Pituitary-Adrenal Axis      Exposure to a physiological or psychological stressor gives rise to a complex repertoire of behavioural and physiological responses that are mediated by the HPA axis (Fig. 1.1). Stressful stimuli activate neutral inputs to corticotropin releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN). CRH is secreted into the pituitary portal circulation to the anterior pituitary where it acts on corticotropic cells to stimulate the release of adrenocorticotropic hormone (ACTH) into the systemic circulation. Circulating ACTH stimulates the synthesis of glucocorticoid hormones (cortisol in humans; corticosterone in rodents) from the adrenal cortex. Glucocorticoids released into the blood elicit both rapid and prolonged actions in target tissues throughout the body and also travel back to the brain and  11 serve as important controllers for neuronal responses that allow for adaptation and maintenance of homeostasis (de Kloet et al., 2005). Glucocorticoids exert their effects in the brain via two nuclear receptor types: glucocorticoid receptors (GR) and mineralcorticoid receptors (MR). Glucocorticoids have a 10-fold greater affinity for MR (MRs are approximately 70% occupied during basal levels), and therefore, MRs are believed to mediate tonic basal actions of glucocorticoids (McEwen et al., 1986). GRs, occupied at peak levels of glucocorticoid secretion, appear to mediate phasic responses such as those to stress and will be the focus of further discussion.  Figure 1.1 Schematic diagram of the Hypothalamic-Pituitary-Adrenal axis Stressful stimuli activate neutral inputs to parvocellular neurons in the hypothalamic paraventricular nucleus (PVN). The axons of the parvocellular neurosecretory neurons project to the median eminence (ME) where they release corticotropin-releasing hormone (CRH) into blood vessels in the pituitary portal system that carry the peptides to the anterior pituitary gland. CRH acts on corticotropic cells in the anterior pituitary to stimulate the release of adrenocorticotropic hormone (ACTH) into the systemic circulation. Circulating ACTH stimulates the synthesis of glucocorticoid hormones from the adrenal cortex. Glucocorticoids released into the blood travel back to the brain. Figure removed from archived version of thesis due to copyright.  1.4.3 The Glucocorticoid Receptor      GRs are expressed in nuclear and peri-nuclear sites in neuronal cell bodies throughout the entire brain (Reul and de Kloet, 1985) but are particularly dense in brain regions crucial for memory such as the hippocampus, amygdala and PFC (McEwen et al., 1986). Upon binding to these nuclear receptors, the glucocorticoid-receptor complex translocates to the nucleus where it binds to glucocorticoid response elements on the DNA and modulates gene transcription (Beato et al., 1996; Beato and Klug, 2000). This process usually requires hours or days for an effect to become evident due to the time it takes to synthesize sufficient quantities of protein de novo and process/modify/translocate the protein to the plasma  12 membrane. The outcome of this “genomic” pathway is characterized by its sensitivity toward inhibitors of transcription and translation such as actinomycin D and cycloheximide. On the other hand, rapid effects of glucocorticoids have also been observed within seconds or minutes that are independent of transcription and translation and are thus termed “nongenomic” (Falkenstein and Wehling, 2000). This nongenomic mode of glucocorticoid action is mediated by glucocorticoids binding specifically to cellular membrane sites (Suyemitsu and Terayama, 1975; Orchinik et al., 1991) and rapidly influences electrolyte movement across cellular membranes (Avanzino et al., 1987b; Avanzino et al., 1987a; ffrench-Mullen, 1995). There remains an unsettled debate of whether the nongenomic steroid responses are mediated via non-classic receptors (receptors that are not related to the superfamily of nuclear hormone receptors) or classic receptors acting at the membrane (Losel and Wehling, 2008). In 2000, a high-affinity binding site for corticosterone, which appears to meet all of the criteria for a functional membrane-associated corticosteroid receptor, was partially purified and characterized in neuronal membranes from the amphibian brain (Evans et al., 2000). However, the molecular identity for these membrane-associated receptors is still unclear. Recent electron microscopy studies in the rat lateral amygdala have localized GRs to non-nuclear-membrane translocation sites including dendritic spines where they show an affinity for postsynaptic membrane densities (Johnson et al., 2005). Furthermore, Tasker and colleagues, while examining mechanisms that enable the termination of HPA axis activation, discovered functional evidence for a membrane-bound GR. Bath application of dexamethasone (a selective GR agonist) conjugated to bovine serum albumin to PVN slices resulted in a rapid suppression of glutamate-mediated excitatory synaptic currents in CRH neurons (Di et al., 2003). This effect was completely abolished by co-application of a  13 cannabinoid type 1 (CB1) receptor antagonist (Di et al., 2003). Subsequently, it was demonstrated that glucocorticoids bind to a putative membrane bound receptor resulting in the synthesis and release of endocannbinoids, N-arachidonoylethanolamide (AEA) and 2- Arachidonoylglycerol (2-AG), which act as retrograde signaling molecules that bind to CB1 receptors on the presynaptic membrane to dampen excitatory synaptic input to CRH neurons (Di et al., 2003; Tasker et al., 2006).  1.4.4 Glucocorticoid Receptors in the Prefrontal Cortex Regulate Glucocorticoids Via Negative Feedback      Appropriate physiological responses to stressful stimuli initiated by glucocorticoids are crucial to our survival but given the powerful and potentially aversive effects of prolonged exposure to these stress hormones, other mechanisms are required to limit the magnitude and duration of glucocorticoid release (Plotsky et al., 1986; Sapolsky, 2000; Sapolsky et al., 2000). Accordingly, the HPA axis is subject to negative feedback control by endogenous glucocorticoids acting on GRs (Plotsky et al., 1986; Andrews et al., 2012) and the mPFC is a key region in mediating this response (Diorio et al., 1993; Hill et al., 2011; Radley and Sawchenko, 2011). Interestingly, the PL and IL cortices regulate HPA activity differentially. Lesions of the PL cortex enhance corticosterone secretion (Diorio et al., 1993; Radley et al., 2006), whereas, lesions restricted to the IL cortex decrease corticosterone responses to restraint stress (Sullivan and Gratton, 1999; Radley et al., 2006). The anatomy of the mPFC efferents may provide insights into these differences. Glutamatergic outputs from the PL cortex project via inhibitory relays in the bed nucleus of the stria terminalis (BNST) and peri- PVN regions to the PVN and exert an inhibitory influence on CRH neurons (Radley et al.,  14 2006; Radley and Sawchenko, 2011). The rapid inhibitory effect of glucocorticoids on the HPA axis after stress have been shown to be mediated via GR activation of the endocannabinoid system in the PL PFC region (see Section 1.6.2 below and Chapter 3) (Hill et al., 2011). On the other hand, the IL cortex projects extensively to areas implicated in activation of neural responses to stress including the anterior BNST, medial and central amygdala, and the nucleus of the solitary tract (Sesack et al., 1989; Hurley et al., 1991; Radley et al., 2006).  1.5 Factors Influencing the Effect of Stress on the HPA axis, Mesocortical Dopamine System, and Prefrontal Cortical Function 1.5.1  Not All Stressors Are Created Equal      Exposure to stress can have multiple effects on the HPA axis, DA system, and PFC function depending on the nature of the stressor and factors related to the cognitive task. In laboratory settings, stressors can be applied in many ways that vary along several dimensions such as duration, frequency, intensity, and controllability. Additional factors such as the timing and context of stress exposure relative to the cognitive task are also important.  1.5.1.1 Duration and Frequency      Selye’s early work demonstrated that an animal’s stress response decreases over time with continued application of a stressor (Selye, 1946). This process of adaptation can be measured as a decrease in adrenocortical secretion and severity of gastric ulcers when animals are repeatedly subjected to the same stressor (Mikulaj et al., 1974; Keim and Sigg, 1976; Pollard et al., 1976; Armario et al., 1984a, b). Similarly, DA release in the PFC also shows  15 adaptation to repeated (Imperato et al., 1992; Cabib and Puglisi-Allegra, 1996) or chronic (Mizoguchi et al., 2000; Mizoguchi et al., 2008) stress exposure. Chronic stress has been shown to attenuate the number of DA neurons firing in the VTA (Moore et al., 2001; Valenti et al., 2012) as well as impair working memory function (Mizoguchi et al., 2000; Cerqueira et al., 2007; Hains et al., 2009) and behavioural flexibility (Liston et al., 2006; Bondi et al., 2008; Nikiforuk and Popik, 2011; Nikiforuk, 2012, 2013).  1.5.1.2 Intensity      The intensity of a stressor is difficult to equate across different types of stressors. Plasma corticosterone levels can be appropriate to evaluate the intensity of low to moderate stressor intensity but measuring the difference between higher intensities proves difficult as even mild shock procedures are likely to induce maximal or near-maximal glucocorticoid synthesis (Natelson et al., 1988; Koolhaas et al., 2011). The majority of circulating gluococorticoids are bound to a corticosteroid-binding globulin (CBG) that prevents glucocorticoids from entering tissues and binding to their receptors (Hammond et al., 1990; Lewis et al., 2005). It was recently discovered, by combining blood sampling with microdialysis in the hippocampus of rats, that the maximum free corticosterone response in the brain to a forced swim stress was delayed by approximately 20 min compared with the rise in total hormone in the blood (Droste et al., 2008). Free corticosterone levels started to rise at 5-10 min after the completion of the stress procedure and reached maximum levels at 55-60 min. The delay was the result of increased CBG levels that occur after exposure to more intensive stressors such as forced swim and restraint (Qian et al., 2011). In contrast, exposure to milder stressors such as novelty stress increased free corticosterone levels in the  16 hippocampus within 5 min after the start of the stressor with maximal levels reached by 30 min (Droste et al., 2008). Accordingly, there was no increase in plasma CBG levels after novelty stress (Qian et al., 2011). A study examining the effect of stressor intensity on DA efflux demonstrated that low level of foot shock selectively increased the DA levels in the mPFC whereas a high level of foot shock increased DA in most brain regions examined including mPFC, striatum, nucleus accumbens (NAc), amygdala, PVN, and lateral hypothalamus (Inoue et al., 1994). To date the effect of stressor intensity has not been formally examined in a working memory or cognitive flexibility task. However, a variety of stressors including mild noise stress to severe restraint stress have been shown to impair working memory performance (Arnsten and Goldman-Rakic, 1998; Shansky et al., 2006).  1.5.1.3 Controllability      The ability to exert control over a stressor has been shown to alter the impact of the stressor itself (Maier et al., 1986; Maier and Watkins, 2010). To demonstrate this effect, Maier and colleagues employed a paradigm of escapable (controllable) versus non-escapable (yoked) tail shock stress. In this paradigm, a rat exposed to controllable stress is able to terminate the tail shock stimulus by performing wheel-turning behavior. This action also terminates the stress for his yoked partner who has no control over the termination of the shock. Interesting, in rats given a single session, the controllable or uncontrollable shock procedures produced equivalent elevation of ACTH and corticosterone (Maier et al., 1986). However, rats subjected to uncontrollable stress were resistant to the suppressive effects of dexamethasone on HPA axis activity indicating impaired negative feedback (O'Connor et al., 2003). Furthermore, large and sustained increases in DA levels in the mPFC were observed  17 in rats exposed to uncontrollable stress but not to controllable stress (Bland et al., 2003). Rats exposed to escapable stress subsequently exhibited reduced freezing during fear conditioning (Baratta et al., 2007), an effect dependent on the mPFC (Baratta et al., 2008).  1.5.1.4 Timing and Context      The timing and context of stress exposure relative to the behavioural task has been demonstrated to be a crucial factor in understanding the effect of acute stress on working memory performance. Exposure to acute stress within 30 min of the acquisition phase of a working memory task impairs performance (Murphy et al., 1996b; Murphy et al., 1996a; Arnsten and Goldman-Rakic, 1998; Morrow et al., 2000; Schoofs et al., 2009) whereas performance is facilitated when testing takes place 4 hr and 1 d post-stress (Barha et al., 2007; Yuen et al., 2009; Yuen et al., 2011). These effects of timing may be related to the presence of high levels of circulating glucocorticoids and DA immediately after stress exposure. Indeed, infusion of a D1 agonist directly into the PFC prior to the delay period of a working memory task facilitated or disrupted PFC functioning depending on the duration of the delay period. D1 stimulation improved retrieval when the drug was administered after an extended 12-hr delay when extracellular DA levels in the mPFC were low, whereas, it impaired performance when administered after a 30-min delay when extracellular DA levels were high (Floresco and Phillips, 2001). Relatively few studies have examined the effect of acute stress on cognitive flexibility. Acute stress given immediately preceding testing had no effect on set-shifting in rats (Thai et al., 2012) but impaired performance in humans (Plessow et al., 2012).  18      The direction by which acute stress affects cognitive processes may also depend upon the context in which the stressor is applied relative to the task environment. Indeed, both the context where the stressor is applied (Bouton and King, 1983; Joels et al., 2006; Diamond et al., 2007; Schwabe and Wolf, 2009) and whether the stressor is intrinsic (if stress is originated by elements related to the cognitive task) or extrinsic (if stress is originated by elements completely unrelated to the cognitive task) (de Kloet et al., 1999; Sandi and Pinelo- Nava, 2007) are important factors to acknowledge. For example, in the spatial learning water-maze task, rats trained at a cooler temperature (19 C compared to 25 C) had increased corticosterone levels that correlated with the facilitation in memory retention for the platform location (Sandi et al., 1997). Previous experiments examining the immediate effects of acute stress on working memory or behavioural flexibility have exposed rats to an extrinsic stressor in a different context from the task itself (Murphy et al., 1996a; Barsegyan et al., 2010; Thai et al., 2012).      Finally, the memory phase during which stress is applied is also an important factor in determining the effects of stress on cognition. In spatial memory tasks, stress appears to have opposing effects in consolidation and retrieval. Exposure to stress or corticosterone administration immediately after learning enhances memory consolidation whereas stress exposure prior to the retrieval phase impairs this process (Roozendaal, 2002; Barsegyan et al., 2010). Notably, stress-induced impairments in memory retrieval are also dependent on time as deficits are observed at 15 and 105 min, but not 120 min, post-stress (Dorey et al., 2012).    19 1.5.2 Tail-pinch stress      The experiments presented in this thesis examined the consequence of acute tail-pinch stress on changes in DA efflux in the mPFC and the behavioural consequences associated with PFC-dependent cognition. Tail-pinch was performed by applying a clothespin to the base of the rats’ tail for 15 min. This stressor was chosen for our studies as it can be easily applied during both microdialysis (when animals are tethered to a probe) and within the same context in which animals performed a cognitive task. Furthermore, the neurochemical and behavioural effects of this stressor have been examined extensively since its initial use in the mid-1970s. Antelman and Szechtman demonstrated that tail-pinch induces a “consummatory” response in which a sated rat will immediately begin gnawing, licking, or eating if food pellets are present (Antelman and Szechtman, 1975; Antelman et al., 1975). These behaviours are accompanied by a marked increase in DA and NA in the PFC and moderate increases in DA in the striatum and NAc (Thierry et al., 1976; Cenci et al., 1992; Taber and Fibiger, 1997; Di Chiara et al., 1999). The gnawing and licking behaviours are maintained for the duration of the tail-pinch and can be prevented by systemic blockade of DA, but not NA, receptors (Antelman et al., 1975). To our knowledge, tail-pinch stress has never been used to examine the effects of stress on working memory or cognitive flexibility tasks.       20 1.6 Research Aims and Hypotheses 1.6.1 Examine the Role of the Glucocorticoid Receptor in Mediating Stress-Evoked Dopamine Efflux in the Medial Prefrontal Cortex      Glucocorticoids have clear modulating effects on DA function, and therefore, an understanding of how stress may alter mesocortical DA function is important for understanding how stress can cause impairments in executive function. Much of the earlier work has described the role of glucocorticoid regulation of the mesolimbic DA system in promoting reward-seeking behavior (Rouge-Pont et al., 1995; Piazza et al., 1996; Marinelli et al., 1998; Barrot et al., 2001; Marinelli and Piazza, 2002). Relatively few studies have examined the role of glucocorticoids in regulating the mesocortical DA system and very little is known regarding the hormone receptor or anatomical pathways involved in modulation of PFC DA during stress.      Acute stress has been demonstrated to increase population activity, firing rate, and burst firing of DA neurons (Anstrom and Woodward, 2005; Valenti and Grace, 2008). Glucocorticoid hormones may play a role in this process as removal of glucocorticoids by adrenalectomy decreases DA neurotransmission in the mPFC (Mizoguchi et al., 2004). Furthermore, acute stress leads to strengthening of excitatory synapses on DA neurons in the VTA which is prevented by systemic GR blockade (Saal et al., 2003) suggesting a role for GRs in mediating glucocorticoid effects on mesocortical DA release. Previous studies point to the VTA as the primary site for the regulation of DA during stress (Enrico et al., 1998; Marinelli et al., 1998; Cho and Little, 1999; Saal et al., 2003; Minton et al., 2009). However, emerging evidence from the drug addiction field suggests that the PFC may be the primary site of this regulation. Ambroggi et al. used two mouse models in which GR was specifically  21 knocked out in either DA neurons or postsynaptic neurons expressing the DA D1 receptor to determine which location of GR mediated the effects of glucocorticoids on cocaine self- administration (Ambroggi et al., 2009). Deletion of GR in postsynaptic neurons innervated by DA terminals was associated with reduced spontaneous neural activity, firing rate, and frequency of burst events in DA neurons (Ambroggi et al., 2009). The authors attributed this effect to the disruption of feedback control by postsynaptic neurons in the PFC or NAc on VTA DA neurons. Thus, GRs in the mPFC may be in a unique position to potently modulate forebrain DA efflux during stress. Thus, we hypothesize that stress-evoked DA efflux in the mPFC would be reduced by blockade of GRs within the mPFC.  1.6.2 Mechanisms by which the Glucocorticoid Receptor Mediates Stress-Evoked Dopamine Efflux in the Medial Prefrontal Cortex      The activity of DA neurons is dependent upon regulation by afferent projections from both cortico-limbic and midbrain regions (Floresco et al., 2001; Floresco et al., 2003). Several recent observations suggest that the mPFC may directly regulate mesocortical DA efflux to stress by modulating glutamatergic input to the VTA. First, acute stress increase NMDA and AMPA neurotransmission in the PFC and this is prevented by GR blockade (Yuen et al., 2009). Second, blockade of AMPA receptors in the mPFC reduces stress- evoked DA efflux in the mPFC (Takahata and Moghaddam, 1998). Third, local application of ionotropic glutamate receptor antagonists in the VTA decreases stress-evoked DA efflux in the mPFC (Enrico et al., 1998). Finally, as discussed above, deletion of GR in postsynaptic neurons innervated by DA terminals is associated with reduced spontaneous neural activity, firing rate, and frequency of burst events in DA neurons (Ambroggi et al., 2009).  22      Direct evidence for the role of GR modulation of pyramidal cell output from the mPFC comes from studies demonstrating its function in terminating HPA axis activation. Bath application of corticosterone to PFC slices lead to activation of GR-mediated synthesis of the endocannabinoid, 2-AG, in the PL cortex (Hill et al., 2011). 2-AG stimulated presynaptic CB1 receptors on GABAergic interneurons to inhibit GABA release onto mPFC pyramidal projection neurons (Hill et al., 2011). These enhanced glutamatergic outputs from the mPFC were hypothesized to project via inhibitory relays in the BNST and peri-PVN regions resulting in an inhibitory influence on PVN CRH neurons (Hill et al., 2011; Radley and Sawchenko, 2011). Therefore, glucocorticoids released during stress may facilitate mesocortical DA release by directly enhancing glutamatergic input onto DA neurons in the VTA. Enhanced glutamatergic input to the VTA may occur via an endocannabinoid- mediated reduction of GABAergic input onto prefrontal pyramidal cells (Hill et al., 2011). Thus, we hypothesize that blockade of prefrontal GRs can reduce activity of descending glutamatergic input to the VTA, thereby attenuating stress-evoked DA efflux in the mPFC. Blockade of CB1 receptors in the mPFC may also reduce stress-evoked DA efflux.  1.6.3 Examine the Effects of Acute Stress on Aspects of Executive Function      Prefrontal DA is essential for executive functions such as working memory and cognitive flexibility (Brozoski et al., 1979; Phillips et al., 2004; Floresco et al., 2006). This thesis examines the functional impact of stress-evoked DA efflux on tasks that are dependent on mPFC function (working memory and set-shifting) and compare this to similar tasks that do not require the mPFC (random foraging and reversal learning) (Floresco et al., 1997;  23 Floresco et al., 2008). We focused on the immediate consequences of acute stress on cognitive function, as this may be a time when individuals are particularly vulnerable to the effects of recent stress. Furthermore, we will investigate the effect of applying an acute stressor within the context of the working memory or set-shifting task (i.e. on a radial arm maze or in an operant chamber box) to examine the consequences of this exposure on mPFC function. Finally, we will determine if blocking GRs in the mPFC can reverse stress-induced impairments in working memory performance. We hypothesize that performance on both working memory and set-shifting tasks will be impaired after exposure to acute stress. In contrast, we propose that performance on the random foraging or reversal learning task will not be affected by stress. Finally, we hypothesize that blocking prefrontal GRs will attenuate stress-induced impairments in working memory.      The experimental data in this thesis will be presented in three chapters addressing the specific hypotheses outlined above. Each chapter will contain a short introduction, methods, and discussion and will address the specific aspects of the data presented in the chapter. The last chapter in this thesis will be a general discussion that attempts to integrate the findings from each chapter into a broader view of how glucocorticoids modulate the mesocortical DA system and aspects of executive function.   24 Chapter  2: Glucocorticoid Receptors in the Prefrontal Cortex Regulate Stress-Evoked Dopamine Efflux  2.1 Introduction      Exposure to acute stress increases dopamine (DA) efflux in the prefrontal cortex (PFC) and is associated with deficits in working memory performance (Murphy et al., 1996a; Arnsten and Goldman-Rakic, 1998; Morrow et al., 2000). However, the mechanism by which stress increases DA efflux in the PFC remains to be determined. Glucocorticoid hormones, released following activation of the hypothalamic-pituitary-adrenal axis by acute stress, exert their effects on glucocorticoid receptors (GRs) and mineralcorticoid receptors (MRs). Glucocorticoids have a 10-fold greater affinity for MRs than for GRs, and therefore, MRs are substantially occupied at low basal glucocorticoid levels and primarily mediate basal or tonic actions of glucocorticoids (Reul and de Kloet, 1985; McEwen et al., 1986). GRs are only partially occupied at low basal glucocorticoid levels and become progressively saturated when glucocorticoid levels are elevated during the circadian peak or following exposure to stress (Reul and de Kloet, 1985; McEwen et al., 1986). The GR is expressed in both glial cells and neurons throughout the brain (Reul and de Kloet, 1985). Importantly, GRs are present on DA neurons in the ventral tegmental area (VTA) (Harfstrand et al., 1986) and are also highly expressed in the PFC (Reul and de Kloet, 1985; McEwen et al., 1986; Ahima and Harlan, 1990; Diorio et al., 1993).      We used in vivo microdialysis to investigate the role of GRs in mediating the effects of acute stress on DA efflux in the PFC and to determine the anatomical site of action. We show  25 that corticosterone activates GRs located in the medial PFC (mPFC), and not in the VTA, to enhance mesocortical DA efflux during stress.              26 2.2  Material and Methods 2.2.1 Animals      Male Sprague–Dawley rats (Charles River) were pair-housed except for those subjected to a food restriction protocol. The colony was maintained at 21 °C with a 12-h light/dark cycle (lights on at 7:00 PM). Rats had free access to rat chow and water unless otherwise stated. All experimental protocols were approved by the Committee on Animal Care, University of British Columbia, and conducted in compliance with guidelines provided by the Canadian Council of Animal Care. 2.2.2 Drugs      Stock solutions were made and dissolved in artificial cerebrospinal fluid (aCSF): RU- 38486 (Tocris Biosciences) dissolved in ethanol (EtOH). Corticosterone (Tocris Biosciences) dissolved in DMSO. Neither EtOH nor DMSO exceeded 0.1% when dissolved in aCSF. Lidocaine hydrochloride monohydrate (Sigma-Aldrich) was dissolved in aCSF. 2.2.3 In Vivo Microdialysis      Rats (~280–310 g) were implanted with stainless steel guide cannulae (19 gauge × 15 mm) directly above the mPFC (3.0 mm AP and ±0.6 mm ML from bregma, 4.6 mm DV from dura) and/or LV (−0.8 mm AP, ±1.4 mm ML, 4.5 mm DV) and VTA (−5.8 mm AP, ±0.6 mm ML, 8.0 mm DV) (Paxinos, 1997). One week after surgery, microdialysis probes were implanted 14–16 h before experiments. Probes were concentric in design with silica inlet/outlet lines and a dialysis surface consisting of a semipermeable membrane 1 or 2 mm in length (340 μM outer diameter; 65,000 molecular weight cutoff; Filtral 12; Hospal).  27 Typical in vitro probe (2 mm membrane) recovery of an external DA standard is 18 ± 1% at room temperature. Microdialysis probes were connected to an Instech dual-channel liquid swivel and perfused continuously at 1.0 μL/min with aCSF [10 mM sodium phosphate, 1.2 mM CaCl2, 3.0 mM KCl, 1.0 mM MgCl2, 147.0 mM NaCl (pH 7.4)] using a 2.5-mL gas- tight syringe (Hamilton) and a syringe pump (model 22; Harvard Apparatus). Samples were collected at 15-min intervals. Following establishment of a stable baseline of DA (four samples, <10% variability), the perfusion into the LV, VTA, or mPFC was switched to one containing RU-38486, corticosterone, or lidocaine, and DA was continuously sampled from the mPFC. At 45 min after drug administration, animals were subjected to tail-pinch stress (clothespin with Velcro covering on base of tail) for 15 min, and samples were continually collected for another 120 min. 2.2.4 Food Anticipation and Consumption Task      Rats were food restricted to 90% of their free-feeding body weight. Training took place in a chamber divided by a removable perforated Plexiglas screen as previously described (Ahn and Phillips, 1999). Dialysis samples were collected at 10-min intervals from the mPFC. Once a stable baseline was established, the animals were perfused with RU-38486 directly into the mPFC for 40 min. Three grams of Froot Loops (Kellogg Canada Inc.) was then placed behind the screen for a 10-min appetitive period. The screen was removed, and animals had access to the food for 10 min, after which the remaining food, if any, was replaced by another 3 g of the same food for a total of up to 6 g over 20 min.  28 2.2.5 HPLC      DA in microdialysates was separated using HPLC with electrochemical detection. Four systems were used, each consisting of an ESA 582 pump (Bedford), a pulse damper (Scientific Systems Inc.), a Rheodyne Inert Manual Injector (model 7725i, 50-mL injection loop), a Tosoh Bioscience Super ODS TSK column (2-μm particle, 2 mm × 10 mm) an Antec Leyden Intro Electrochemical Detector and VT-03 flow cell with a Ag/AgCl reference electrode (Vapplied = +650 mV). The mobile phase [100 mM sodium acetate buffer, 40 mg/L EDTA and 4 mg/L of SDS (variable between 3.5 and 4.5 mg/L), pH 4.1, 10% methanol] flowed through the system at 0.17 mL/min. EZChrome Elite software (Scientific Software) was used to acquire and analyze chromatographic data. The average concentration of basal DA in dialysates from the mPFC uncorrected for probe recovery was 0.16 ± 0.01 nM. 2.2.6 Corticosterone Radioimmunoassay      Blood samples were taken once from animals used in the microdialysis experiments via tail nick 15, 30, 45, 60, and 75 min after initiation of stress. Basal corticosterone levels were measured immediately after removal of animals from colony room. Samples were taken from the dark phase between 09:00 AM and 1:00 PM. Blood samples were centrifuged at 1,336 × g for 10 min at 4 °C, and plasma was stored at −20 °C until assayed. Total corticosterone (bound + free) levels were measured by radioimmunoassay using a commercially available assay kit (MP Biomedicals) according to the manufacturer's instructions. The antiserum cross-reacts 100% for corticosterone. The minimum detectable corticosterone concentration was 7.7 ng/mL, and the intra- and interassay coefficients of variation were 7.1% and 7.2%, respectively.  29 2.2.7 Histology      Following all experiments, rats were deeply anesthetized with isoflurane. Brains were promptly removed and stored in 20% wt/vol sucrose and 4% vol/vol paraformaldehyde solution for a minimum of 1 wk. Brains were then sliced into 50 μM coronal sections, stained with cresyl violet, and examined for verification of probe placements.  30 2.3 Results 2.3.1 GRs Modulate Stress-Evoked DA Efflux in the mPFC      To investigate the role of the GR in stress-evoked DA efflux, in vivo microdialysis was used in combination with an acute mild tail-pinch stress and a GR antagonist, RU-38486. Tail-pinch stress was accompanied by a fivefold rise in plasma corticosterone (Fig. 2.1A) from 7.96 μg/dL (basal levels) to 41.54 μg/dL after 15 min of stress (n = 6 rats per condition). The increase in corticosterone release differed significantly from basal levels for the initial 45 min following tail-pinch stress [F(6, 35) = 10.35, P < 0.05, Tukey's test]. A separate study again confirmed a rapid and transient increase in DA efflux in the mPFC, reaching maximum values 15 min after the onset of tail-pinch stress and returning to basal values after 90 min (n = 6; Fig. 2.1B). Administration of RU-38486 (11.1 ng/μL) into the lateral ventricle (LV) using reverse microdialysis (n = 4) significantly attenuated stress- evoked DA efflux measured as mean (± SEM) area under the time–response curves (AUC) for vehicle- and RU-38486–treated rats. (Student t test, P = 0.0348; Fig. 1C).   31  Figure 2.1 Activation of GRs mediates stress-evoked DA efflux in the mPFC. (A) Plasma corticosterone (CORT) concentration (μg/dL) in rats subjected to 15 min of tail-pinch stress (n = 6 for each time point). (B) Time course of the change of DA efflux from baseline measured from the mPFC in rats subjected to tail-pinch stress (15 min; gray-shaded box). RU-38486 or vehicle is perfused into the LV using reverse microdialysis (horizontal black line). All values are expressed as percentage change from baseline ± SEM (black squares; n = 6, vehicle, black triangles; n = 4, RU-38486). (C) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 90 min after the initiation of stress (*P < 0.05).   2.3.2 GRs Within the mPFC Modulate Stress-Evoked DA Efflux      To investigate the anatomical site of action for stress-evoked changes in DA efflux, RU- 38486 was administered into the VTA or mPFC using reverse microdialysis, and DA efflux  32 was measured in the mPFC. Application of RU-38486 (11.1 ng/μL) into the VTA did not alter stress-evoked DA efflux (n = 7 per condition; Fig. 2.2A). In contrast, stress-evoked increases in DA efflux were attenuated following reverse dialysis of RU-38486 into mPFC (n = 8 vehicle treatment group, n = 9 RU-38486 treatment group; Fig. 2.2B). Two-tailed Student t tests of mean AUC (± SEM) revealed no differences in DA levels when RU-38486 was perfused into the VTA (P = 0.511; Fig. 2D), whereas a significant attenuation was observed when this GR antagonist was administered into the mPFC (P = 0.00956; Fig. 2.2E).      To examine further the action of glucocorticoids in modulating intrinsic activity within the mesocortical DA pathway, DA cell bodies in the VTA were functionally inactivated by reverse dialysis of the sodium channel blocker lidocaine (20 mg/mL). Confirming previous experiments in our laboratory, basal DA levels in the mPFC were reduced significantly by lidocaine (Taepavarapruk et al., 2008) (average decrease over the initial 45 min of perfusion: ~27 ± 3% below baseline, P < 0.05; Fig. 2.2C). Furthermore, stress-evoked DA efflux in the mPFC was greatly reduced in animals treated with lidocaine compared with vehicle-treated controls (n = 5 vehicle treatment group, n = 8 lidocaine treatment group). Comparison of AUCs generated for the initial 90 min after stress exposure revealed a significant reduction of DA efflux after intra-VTA lidocaine administration (P = 0.0402, two-tailed Student t test; Fig. 2.2F).  33   Figure 2.2 GRs in the mPFC mediate stress-evoked DA efflux. (A–C) Time course of the change of DA efflux from baseline measured from the mPFC while animals are subject to tail-pinch stress (15 min; gray-shaded box). Using reverse microdialysis, RU-38486 (horizontal black line) is perfused into the VTA (A) or mPFC (B) and lidocaine (Lido) is perfused into the VTA (C). All values are expressed as percentage change from baseline ± SEM (black squares; n = 5–7, vehicle, black triangles; n = 7–9, RU-38486 or lidocaine). (D–F) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 90 min after the initiation of stress (*P < 0.05).  34 2.3.3 Corticosterone Enhances DA Efflux in the mPFC      We next perfused corticosterone (50 μM) into the mPFC via reverse microdialysis. An immediate and sustained increase in mPFC DA efflux was observed in the corticosterone group (n = 8 per condition; Fig. 2.3A), as confirmed by a two-tailed Student t test on mean (± SEM) AUC for vehicle- and corticosterone-treated rats over 120 min (P = 0.00516; Fig. 2.3B).  Figure 2.3 Corticosterone acts locally in the mPFC to enhance DA efflux. (A) Time course of the change of DA efflux from baseline measured from the mPFC while corticosterone or vehicle is administered into the mPFC via reverse dialysis. Application of the drug is shown with a horizontal black line. All values are expressed as percentage change from baseline ± SEM (black squares; n = 8, vehicle, black triangles; n = 8, corticosterone). (B) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 120 min after corticosterone perfusion (*P < 0.05).  2.3.4 GRs Within the mPFC Do Not Modulate DA Efflux Evoked by Feeding Behaviour      To determine if the attenuation of DA efflux by RU-38486 was selective to noxious stimuli, we used a food anticipation and consumption task shown previously to induce  35 feeding-evoked DA efflux (Ahn and Phillips, 1999). Presentation and consumption of highly palatable food (Fruit Loops) in the present study caused a rapid and transient increase in DA efflux, with the highest levels observed 20 min after food consumption, returning to basal values after 60 min (n = 7 per condition; Fig. 2.4A). Administration of RU-38486 (11.1 ng/μL) into the mPFC did not attenuate DA efflux triggered by food anticipation or consumption. RU-38486 had no significant effect on DA efflux evoked by food presentation as measured by comparison of AUCs (P = 0.933, two-tailed Student t test; Fig. 2.4B).  Figure 2.4 GRs in the mPFC do not mediate DA efflux to an incentive stimulus. (A) Time course of the change of DA efflux from baseline measured from the mPFC during a food anticipation and consumption task (gray-shaded box). RU-38486 or vehicle is perfused into the mPFC using reverse microdialysis (horizontal black line). All values are expressed as percentage change from baseline ± SEM (black squares; n = 7, vehicle, black triangles; n = 7, RU-38486). (B) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 70 min after presentation of food (*P < 0.05).   36 2.3.5 Histology      Histological analysis confirmed the location of all microdialysis probes from all experiments to be localized in either the lateral ventricle, the prelimbic or infralimbic areas of the PFC, and the VTA (Fig 2.5).   Figure 2.5 Placements of microdialysis probes. Summary of microdialysis membrane placements. The black bar represents the active surface location of the probe in the mPFC, LV, and VTA respectively. Not all probes are shown, because of overlapping placements.  37 2.4 Discussion      Our data demonstrate a unique mechanism by which DA efflux is enhanced in the mPFC during acute stress. Administration of the GR antagonist RU-38486 into the LV or mPFC significantly attenuated DA efflux in the mPFC induced by exposure to acute tail-pinch stress. Previous studies suggest that glucocorticoids may alter DA efflux through activation of GRs on DA neurons in the VTA, leading to changes in DA synthesis and neuronal firing (Marinelli et al., 1998; Cho and Little, 1999; Saal et al., 2003; Minton et al., 2009). However, we find no effect on prefrontal DA efflux following acute stress when RU-38486 is administered locally into the VTA. Local application of corticosterone directly into the mPFC did evoke a significant increase in DA efflux, supporting a role for local activation of GRs in the mPFC in regulating DA efflux.      Previous studies investigating the effects of systemic administration of corticosterone on DA efflux in the mPFC have produced conflicting results. Administration of corticosterone via drinking water or s.c. injections increased, decreased, or had no effect on DA efflux (Imperato et al., 1989; Lindley et al., 1999; Lindley et al., 2002; Ago et al., 2008; Minton et al., 2009). The present study used reverse microdialysis to administer drugs locally into different brain regions. This approach limited any glucocorticoid secretion that may be triggered by s.c. or i.p. injections (Dalm et al., 2008). It is important to note that rat serum lacks the specific binding protein, α-1-acid glycoprotein, for RU-38486, thereby limiting its diffusion across the blood–brain barrier (Heikinheimo and Kekkonen, 1993). Accordingly, the concentration of RU-38486 reaching the rat brain after systemic administration is only 28% of the serum levels (Heikinheimo and Kekkonen, 1993), which may explain why  38 Imperato et al. failed to observe any effect of RU-38486 when examining the role of GRs in stress-evoked DA efflux in the mPFC (Imperato et al., 1991).      Emerging evidence from the drug addiction field supports the suggestion that glucocorticoids act on GRs on DA terminal regions rather than on DA cell bodies in the VTA. Ambroggi et al. used two mouse models in which GR was specifically knocked out in either DA neurons or postsynaptic neurons expressing the DA D1 receptor to determine which location of GR mediated the effects of glucocorticoids on cocaine self-administration (Ambroggi et al., 2009). Deletion of GR in postsynaptic neurons innervated by DA terminals was associated with reduced spontaneous neural activity, firing rate, and frequency of burst events in DA neurons (Ambroggi et al., 2009). The authors attributed this effect to the disruption of feedback control by postsynaptic neurons in the PFC or nucleus accumbens on VTA DA neurons. Our findings support this mechanism, because functional inactivation of DA cell bodies in the VTA with lidocaine led to a significant reduction in stress-evoked DA efflux. Importantly, it has recently been demonstrated that activation of the DA system during stress is inhibited after infusion of TTX into the ventral hippocampus (Valenti et al., 2011). Brief periods of high-frequency stimulation to the ventral hippocampus lead to a robust and long-lasting increase in DA efflux in the mPFC that is partially blocked by reverse microdialysis of AMPA/kainate receptor antagonists into the mPFC (Taepavarapruk et al., 2008). These findings suggest that increased mesocortical DA efflux observed during acute stress may be driven by ventral hippocampus stimulation of mPFC, resulting in increased excitatory drive to the VTA. Glucocorticoids released after acute stress may enhance this glutamatergic transmission in PFC pyramidal neurons through a GR-dependent mechanism (Yuen et al., 2009). Furthermore, the importance of this pathway is further underscored by  39 the recent finding that stress disrupts long-term potentiation in the hippocampal–PFC pathway (Goto and Grace, 2006; Lee and Goto, 2011).      Our data demonstrate a localized role of GRs in the mPFC; and although we favor a direct effect on dopaminergic terminals or alternatively on glutamatergic afferents from the hippocampus or amygdala within the PFC, we cannot exclude the involvement of GRs in other brain regions in the modulation of prefrontal DA efflux by glucocorticoids. Lesions to the central and basolateral nuclei of the amygdala inhibit DA efflux in the PFC during stress (Davis et al., 1994; Goldstein et al., 1996) and also reduce working memory impairments induced by systemic administration or direct injection into the mPFC of a glucocorticoid agonist (Roozendaal et al., 2004). Glucocorticoids are also implicated in modulating DA metabolism (Lindley et al., 1999; Lindley et al., 2002). However, neither the enzymatic activity nor gene expression levels of catechol-O-methyltransferase or monoamine oxidase in the mPFC change after treatment with corticosterone (Lindley et al., 2005). More recent evidence suggests that corticosterone can inhibit a high-capacity monoamine uptake transport system, which plays a key role in monoamine clearance (Hill et al., 2010a). Future studies to examine the intracellular mechanisms governing the release of DA by GRs in the mPFC are warranted.   40 Chapter  3: Glucocorticoid Receptors in the Prefrontal Cortex Regulate Dopamine Efflux to Stress via Descending Glutamatergic Feedback to the Ventral Tegmental Area  3.1 Introduction  Enhanced dopamine (DA) efflux in the medial prefrontal cortex (mPFC) is a well- documented response to acute stress. We have previously shown that administration of the glucocorticoid receptor (GR) antagonist, RU-38486, into the mPFC significantly attenuates DA efflux in the mPFC and working memory impairments induced by exposure to acute tail- pinch stress (Butts et al., 2011). However, the underlying mechanism(s) by which the GR regulates stress-evoked DA efflux in the mPFC remains to be determined.      DA neurons in the ventral tegmental area (VTA) receive excitatory input from and extend reciprocal projections to the mPFC (Carr and Sesack, 2000). Local application of ionotropic glutamate receptor antagonists or functional inactivation of the DA cell bodies with lidocaine in the VTA decreased stress-evoked DA efflux in the mPFC (Enrico et al., 1998; Butts et al., 2011) suggesting that glucocorticoids released after acute stress may enhance descending glutamatergic feedback to the VTA. This is supported by evidence from the drug addiction field showing that deletion of GR in postsynaptic neurons innervated by DA terminals, but not in DA-releasing neurons, was associated with a reduction in spontaneous neural activity, firing rate, and frequency of burst events in DA neurons, along with decreased glucocorticoid-induced cocaine self-administration by mice (Ambroggi et al., 2009).      Enhanced glutamatergic transmission in mPFC pyramidal neurons following exposure to acute stressors occurs through a GR-dependent mechanism (Yuen et al., 2009). Emerging evidence suggests a possible role of the endocannabinoid system in mediating GR activation into changes in glutamatergic transmission in mPFC neurons. Under conditions of acute  41 stress, glucocorticoids trigger the synthesis and release of the endocannabinoid ligands, N- arachidonylethanolamine and 2-arachidonylglycerol (Di et al., 2003; Tasker et al., 2006; Hill et al., 2010). Endocannabinoid release from the postsynaptic cell acts on cannabinoid receptor subtype 1 (CB1) receptors localized on presynaptic glutamatergic, or GABAergic terminals, to inhibit subsequent neurotransmitter release (Di et al., 2003; Tasker et al., 2006; Hill et al., 2010). Activation of GRs in the mPFC can trigger endocannabinoid release which functions presynaptically via cannabinoid CB1 receptors to inhibit GABA release onto prefrontal pyramidal cells. This in turn leads to increased activity in descending glutamatergic input to the hypothalamus, thereby inhibiting the HPA axis (Hill et al., 2011). A similar mechanism within the mPFC may explain how stress-induced elevation of glucocorticoids may mediate indirect activation of glutamatergic afferents to the VTA leading subsequently to increased DA efflux in the mPFC. This hypothesis is tested directly by blockade of CB1 receptors in the mPFC during exposure to acute stress.      We employed in vivo microdialysis to investigate the role of the GR in mediating enhanced glutamatergic drive to the VTA. Using a selective GR antagonist, devoid of any progesterone activity, we demonstrate that local GR blockade in the mPFC attenuates stress- evoked glutamate efflux in the VTA and DA efflux in the mPFC. In contrast, blocking CB1 receptors in the mPFC failed to attenuate stress-evoked DA efflux. Antagonism of ionotropic glutamate receptor in the VTA attenuated DA efflux in the mPFC evoked by tail-pinch stress.      42 3.2 Material and Methods 3.2.1 Animals      Male Sprague–Dawley rats (Charles River) were pair-housed. The colony was maintained at 21 °C with a 12-h light/dark cycle (lights on at 7:00 PM). Rats had free access to rat chow. All experimental protocols were approved by the Committee on Animal Care, University of British Columbia, and conducted in compliance with guidelines provided by the Canadian Council of Animal Care. 3.2.2 Drugs      Stock solutions were made and dissolved in artificial cerebrospinal fluid (aCSF): RU- 38486 (Tocris Biosciences) was dissolved in ethanol (EtOH). CORT 108297 (Corcept Therapeutics) and CNQX, AP5, AM-251, and NIDA41020 (Tocris Bioscience) were dissolved in DMSO. Neither EtOH nor DMSO exceeded 0.1% when dissolved in aCSF. 3.2.3 In Vivo Microdialysis      Rats (~280–310 g) were implanted with stainless steel guide cannulae (19 gauge × 15 mm) directly above the mPFC (3.0 mm AP and ±0.6 mm ML from bregma, 4.6 mm DV from dura) and/or VTA (−5.8 mm AP, ±0.6 mm ML, 8.0 mm DV) (Paxinos, 1997). One week after surgery, microdialysis probes were implanted 14–16 h before experiments. Probes were concentric in design with silica inlet/outlet lines and a dialysis surface consisting of a semipermeable membrane 1 or 2 mm in length (340 μM outer diameter; 65,000 molecular weight cutoff; Filtral 12; Hospal). Typical in vitro probe (2 mm membrane) recovery of an external DA and glutamate standard is 18 ± 1% at room temperature. Microdialysis probes  43 were connected to an Instech dual-channel liquid swivel and perfused continuously at 1.0 μL/min with aCSF [10 mM sodium phosphate, 1.2 mM CaCl2, 3.0 mM KCl, 1.0 mM MgCl2, 147.0 mM NaCl (pH 7.4)] using a 2.5-mL gas-tight syringe (Hamilton) and a syringe pump (model 22; Harvard Apparatus). Samples were collected at 15-min intervals. Following establishment of a stable baseline (four samples, <10% variability), the perfusion into the mPFC or VTA was switched to one containing RU-38486, CORT 108297, CNQX and AP5, AM-251, or NIDA41020 and DA or glutamate was continuously sampled from the mPFC and/or VTA. At 45 min after drug administration, animals were subjected to tail-pinch stress (clothespin with Velcro covering on base of tail) for 15 min, and samples were continually collected for another 60-120 min. 3.2.4 HPLC      DA and glutamate in microdialysates were separated using HPLC with electrochemical detection. 3.2.4.1 DA HPLC      DA in microdialysates was separated using HPLC with electrochemical detection. Four systems were used, each consisting of a LS 110 HPLC pump (Antec Leyden), a pulse damper (Scientific Systems Inc.), a Rheodyne Inert Manual Injector (model 7725i, 50-mL injection loop), a Tosoh Bioscience Super ODS TSK column (2 μm particle, 2 mm × 10 mm) an Antec Leyden Intro Electrochemical Detector and VT-03 flow cell with a Ag/AgCl reference electrode (Vapplied = +650 mV). The mobile phase [100 mM sodium acetate buffer, 40 mg/L EDTA and 4 mg/L of SDS (variable between 3.5 and 4.5 mg/L), pH 4.1, 10% methanol]  44 flowed through the system at 0.17 mL/min. EZChrome Elite software (Scientific Software) was used to acquire and analyze chromatographic data. The average concentration of basal DA in dialysates from the mPFC uncorrected for probe recovery was 0.16 ± 0.01 nM. 3.2.4.2 Glutamate HPLC      Dialysate samples were subjected to precolumn derivatization with o-phthaldialdehyde (OPA) reagent before injection. 1 µL of OPA reagent (37 mM OPA, 50 mM sodium sulphite, 90 mM boric acid, pH 10.4, 5% methanol) was mixed with 9 µL of sample and allowed to react for 2 min before injection into the ALEXYS GABA and Glu Analyzer using an AS 110 autosampler with a UHPLC injector (Antec Leyden). Mobile phase (50 mM Phophoric Acid, 50 mM Citric Acid, 0.1 mM EDTA, 0.5% Acetonitril, pH 3.1), pumped at 200 µL/min by a LS 110S HPLC pump (AntecLeyden), delivered analytes onto an Acquity UPLC HSS T3 column (1.8 particle, 1.0 x 50 mm). After separation on the column, electrochemical detection was performed with Decade II Dual Cell Control (AntecLeyden). Amino acid derivatives were oxidized in a VT-03 flow cell with an Ag/AgCl reference electrode (Vapplied = +800 mV). Clarity software (AntecLeyden) was used to acquire and analyze chromatographic data. The average concentration of basal glutamate in dialysates from the mPFC and VTA uncorrected for probe recovery was 0.62 ± 0.16 and 0.76 ± 0.12 µM, respectively. 3.2.5 Histology      Following all experiments, rats were deeply anesthetized with isoflurane. Brains were promptly removed and stored in 20% wt/vol sucrose and 4% vol/vol paraformaldehyde  45 solution for a minimum of 1 wk. Brains were then sliced into 50 μM coronal sections, stained with cresyl violet, and examined for verification of probe placement.  46 3.3 Results 3.3.1 Blockade of mPFC GRs with a Selective Antagonist Attenuates Stress-Evoked DA Efflux in the mPFC      These data confirmed our previous observation that tail-pinch stress leads to a rapid and transient increase in DA efflux in the mPFC, reaching maximum levels 15 min after the onset of stress and returning to basal values after 90 min (Fig. 3.1A). Importantly, this effect was attenuated by pretreatment with the selective GR antagonist CORT 108297, which unlike RU-38486, is devoid of antiprogesterone action (Asagami et al., 2011; Belanoff et al., 2011). Application of CORT 108297 (10 uM) into the mPFC via reverse microdialysis significantly attenuated stress-evoked DA efflux (n = 9 vehicle treatment group, n = 10 CORT 108297 treatment group; Fig.1a). Comparison of area under the time–response curves (AUCs) over the initial 90 min after stress exposure, in the CORT 108297 group with vehicle controls, revealed that this selective GR antagonist significantly attenuated stress-induced DA efflux in the mPFC(P = 0.0471; Fig. 3.1B).      47  Figure 3.1 Selective GR antagonists attenuates stress-evoked DA efflux in the mPFC.  (A) Time course of the change of DA efflux from baseline measured within the mPFC while animals are subject to tail-pinch stress (15 min; gray-shaded box). CORT 108297 (horizontal black line) was delivered into the mPFC via reverse microdialysis All values are expressed as percent change from baseline ± SEM (black squares; n = 9, vehicle, black triangles; n =10, CORT 108297). (B) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 90 min after the initiation of stress (*P < 0.05).   3.3.2 Prefrontal GR Blockade Attenuates Stress-Evoked Glutamate Efflux in the Ventral Tegmental Area           Tail-pinch stress induced a rapid and transient increase in glutamate efflux in the VTA reaching maximum values 15 min after the onset of stress, returning to basal values at 90 min (Fig. 2a). Infusion of RU-38486 (23 uM) or CORT 108297 (10 uM) into the mPFC via reverse microdialysis significantly attenuated stress-evoked glutamate efflux (Fig. 3.2A). Interestingly, we did not observe an increase in stress-induced efflux of glutamate in the mPFC (Fig. 3.2B). Two-tailed Student t tests of mean AUC (± SEM) revealed a significant reduction of glutamate efflux in the VTA when RU-38486 was perfused into the mPFC (P = 0.0369; Fig. 3.2C) and when CORT 108297 was perfused into the mPFC (P = 0.00876; Fig. 3.2C). There was no significant difference between treatments on glutamate efflux in the  48 mPFC (P = 0.961, two-tailed Student t test; Fig. 3.2D).    Figure 3.2 GRs in the mPFC regulate stress-evoked glutamate efflux in the VTA. (A-B) Time course of the change from baseline of glutamate efflux in the VTA (A) and mPFC (B) of animals subjected to tail-pinch stress (15 min; gray-shaded box). CORT 108297 or RU-38486 (horizontal black line) were delivered via reverse microdialysis, into the mPFC (black squares; n = 10, vehicle, black triangles; n =9, RU-38486) and/or VTA (black squares; n = 10, vehicle, black triangles; n =10, RU-38486, black circles; n = 6, CORT 108297). All values are expressed as percent change from baseline ± SEM. (C-D) Histograms depicting mean AUC (± SEM) for the change in glutamate efflux relative to baseline for 45 min after the initiation of stress (*P < 0.05).    49 3.3.3 Blockade of Ionotropic Glutamate Receptors in the VTA Reduces Stress- Evoked DA Efflux in the mPFC      We next perfused the AMPA receptor antagonist, CNQX (50 μM), and the NMDA receptor antagonist, AP5 (200 μM), into the VTA via reverse microdialysis while measuring DA efflux in the mPFC during tail-pinch stress. Local perfusion of CNQX and AP5 into the VTA has been previously shown to decrease DA efflux in the mPFC during handling stress (Enrico et al., 1998). Blockade of ionotropic glutamate receptors in the VTA significantly decreased stress-evoked DA efflux in the mPFC (n = 8 vehicle treatment group, n = 6 CNQX and AP5 treatment group; Fig 3.3A), measured by AUC (mean ± SEM) over 90 min post stress (P = 0.0256; Fig. 3.3B).      50  Figure 3.3 Ionotrophic glutamate receptors in the VTA mediate stress-evoked DA efflux in the mPFC. (A) Time course of the change from baseline of DA efflux in the mPFC measured from while animals were subject to tail-pinch stress (15 min; gray-shaded box). CNQX or AP5 (horizontal black line) were perfused into the VTA via reverse microdialysis. All values are expressed as percent change from baseline ± SEM (black squares; n = 8, vehicle, black triangles; n =6, CNQX and AP5). (B) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 90 min after the initiation of stress (*P < 0.05).  3.3.4 Cannabinoid CB1 Receptors Do Not Modulate Stress-Evoked DA Efflux in the mPFC          We also examined the possible role of glucocorticoid-induced synthesis and release of endocannabinoids which may act presynaptically via cannabinoid CB1 receptors to inhibit GABA release onto prefrontal pyramidal cells, thus enhancing descending glutamatergic input to the VTA during stress leading to increased DA efflux in the mPFC (Hill et al., 2011). When administered locally into the mPFC, neither the CB1 receptor antagonist AM-251 (10 uM) nor NIDA-41020 (10 uM) had a significant effect on stress-induced DA efflux in the mPFC (n = 8 vehicle, n = 9 AM-251, n = 8 NIDA-41020; Fig. 3.4A) as measured by comparison of AUCs (P = 0.720 and 0.210, respectively, two-tailed Student t test; Fig. 3.4B).  51  Figure 3.4 Stress-evoked DA efflux in the mPFC is not mediated by CB1 receptors. (A) Time course of the change of DA efflux the mPFC from baseline in rats subjected to tail-pinch stress (15 min; gray-shaded box). AM-251 or NIDA41020 (horizontal black line) was perfused into the mPFC via reverse microdialysis. All values are expressed as percent change from baseline ± SEM (black squares; n = 8, vehicle, black triangles; n =7, AM-251, black circles; n = 8, NIDA-41020). (B) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 90 min after the initiation of stress.  3.3.5 Histology      Histological analysis confirmed the location of all microdialysis probes from all experiments to be localized in either the prelimbic or infralimbic areas of the mPFC and the VTA (Fig. 3.5).  Figure 3.5 Placements of microdialysis probes. Each vertical black bar represents the active membrane surface location of a microdialysis probe in the mPFC or VTA. Not all probes are shown, because of overlapping placements.  52   Figure 3.6 Schematic depiction of the anatomical connections between the mPFC and VTA proposed to mediate stress-evoked DA efflux in the mPFC. Specifically, we hypothesize that elevated circulating corticosterone during stress activates putative membrane GRs on prefrontal glutamatergic projection neurons that terminate on DA neurons in the VTA. Increased activation of these projections neurons by corticosterone would subsequently increase VTA DA neuron cell firing thereby augmenting DA release in the mPFC (black line indicates the glutamatergic projection, the gray line depicts the mesocortical dopaminergic projection).         53 3.4 Discussion      The present study provides the novel observation that glutamate efflux in the VTA is increased in a phasic manner after exposure to tail-pinch stress that can be attenuated by reverse microdialysis of the selective GR antagonist CORT 108297 into the mPFC. Furthermore, blockade of ionotropic glutamate receptors in the VTA lead to a significant reduction in stress-evoked DA efflux in the mPFC. These data support the hypothesis that glucocorticoids act locally within the mPFC to modulate mesocortical DA efflux by potentiation of glutamatergic drive onto DA neurons in the VTA. As similar effects were obtained in a recent study with the progesterone and GR antagonist RU-38486 (Butts et al., 2011) , the present data using the selective GR antagonist CORT 108297 (Asagami et al., 2011; Belanoff et al., 2011), suggest that the observed attenuation of stress-induced DA efflux is mediated by a glucocorticoid and not a progesterone receptor. These results also support our contention that the mPFC is the primary site of glucocorticoid regulation of mesocortical DA neurons during stress.      Although we observed a significant increase in stress-induced glutamate efflux in the VTA, a similar increase in glutamate efflux in the mPFC was not seen in the same animals. Previous studies examining the effects of handling stress on glutamate efflux also failed to detect an increase in the mPFC (Timmerman et al., 1999; Del Arco and Mora, 2001). However, these negative findings differ from previous observations demonstrating an increase in glutamate release in the mPFC of rats exposed to acute restraint or tail-pinch stress using brain microdialysis (Moghaddam, 1993; Bagley and Moghaddam, 1997). Furthermore, Hascup and colleagues employed enzyme-based microelectrode arrays to measure an increase in neuronally derived glutamate in the mPFC after exposure to both tail-  54 pinch and restraint stress (Hascup et al., 2011; Hascup et al., 2012). One possible explanation for our failure to observe a significant increase in mPFC glutamate efflux with a tail-pinch stressor may reflect the relatively mild nature of this stimulus. Alternatively, the detection limit of the analytical system used in the present study (0.05 M) may not have had sufficient sensitivity.      Previous studies examining the mechanisms by which acute stress or corticosterone administration regulates glutamatergic transmission have focused on identifying the transcriptional targets of GR activation. Yuen and colleagues demonstrated that the stress- induced delayed and sustained potentiation of NMDAR- and AMPAR-mediated synaptic currents in layer V mPFC pyramidal cells mediated by GR activation occurs through the induction of serum- and glucocorticoid-inducible kinases and the activation of Rab4 that regulates receptor trafficking and function of NMDARs and AMPARs (Yuen et al., 2009; Yuen et al., 2011). Revest and colleagues demonstrated that activation of GRs leads to an increase in the expression of the transcription factor Egr-1 (early gene response-1) in the hippocampus (Revest et al., 2005; Revest et al., 2010). This in turn can increase expression of synapsin-Ia/Ib, the most highly expressed hippocampal pre-synaptic vesicle (SV)- associated phosphoproteins (Revest et al., 2005; Revest et al., 2010), which exert direct control over the release of neurotransmitters, such as glutamate (Nichols et al., 1992; Jovanovic et al., 2000). In contrast, in the present study, the increase in stress-evoked glutamate efflux in the VTA was observed within 15 min. As this short time frame of action is too rapid to be mediated by means of the classical genomic GR pathway as described above, the immediate effects of acute stress may instead be regulated by a nongenomic  55 membrane receptor-mediated mechanism (Prager and Johnson, 2009; Groeneweg et al., 2011).      Emerging evidence suggests that corticosterone may rapidly affect neuronal signaling by binding to membrane-associated corticosterone receptors (Orchinik et al., 1991; Dallman, 2005; Haller et al., 2008; Prager and Johnson, 2009) and anatomical data confirm that GRs are localized to membranes and synapses (Liposits and Bohn, 1993; Johnson et al., 2005). These membrane receptors have been shown to rapidly change neuronal excitability through direct actions on ion channels or intracellular signaling messengers (ffrench-Mullen, 1995; Tasker et al., 2006; Prager and Johnson, 2009; Groeneweg et al., 2012). Relatively few studies have examined the possible role of rapid nongenomic mechanisms in mediating corticosterone signaling in the mPFC (Groeneweg et al., 2011). However, a recent behavioural study conducted by Barsegyan and colleagues demonstrated that administration of corticosterone conjugated to the membrane impermeable protein, bovine serum albumin, into the mPFC impaired performance on a working memory task (Barsegyan et al., 2010). This effect was prevented by co-administration of RU-38486 and discovered to be mediated by a membrane-GR-induced facilitation of a noradrenergic signal transduction pathway within the mPFC (Barsegyan et al., 2010). Accordingly, we propose that corticosterone acts on a putative membrane GR of layer V pyramidal neurons in the mPFC (Fig. 6). Activation of layer V cortical neurons, the principal projection neurons of the mPFC, would in turn enhance glutamatergic drive onto DA neurons in the VTA, leading to an increase in DA efflux in the mPFC (Fig. 6) (Carr and Sesack, 2000). As previously mentioned, the endocannabinoid signaling molecules are a well-studied retrograde messenger system regulated by membrane GRs (Tasker et al., 2006). Therefore, we investigated whether the  56 present findings may be mediated by glucocorticoid-induced synthesis and release of endocannabinoids that would in turn act presynaptically via cannabinoid CB1 receptors to remove GABAergic inhibition of layer V pyramidal neurons in the mPFC (Hill et al., 2011) and thus enhancing DA efflux in the mPFC. However, blockade of CB1 receptors in the mPFC failed to attenuate stress-evoked DA efflux. Although the concentration of AM-251 and NIDA-41020 used in the present study were effective in previous behavioural and electrophysiological experiments (Di et al., 2003; Miller et al., 2007; Sousa et al., 2011), the possibility remains that higher concentration of these drugs are required to produce positive effects. However, given this finding, it is noteworthy that glucocorticoid-induced synthesis and release of endocannabinoids acting presynaptically via cannabinoid CB1 receptors to inhibit GABA release in the mPFC was not observed until 1 hr after stress exposure (Hill et al., 2011). Future studies examining other rapid GR-mediated nongenomic mechanisms regulating corticosterone signaling in mPFC projection neurons are warranted.      In summary, blockade of GRs within the mPFC with a selective antagonist attenuates stress-evoked DA and glutamate efflux in the mPFC and VTA, respectively. Blockade of ionotrophic glutamate receptors in the VTA also reduces stress-evoked DA efflux in the mPFC, thereby confirming an important contribution of mPFC GRs to the modulation of ‘top-down’ feedback control of mesocortical DA neurons (Phillips et al., 2008). Together, these findings may offer a partial explanation of stress-induced disruption and possibly facilitation of cognitive functions mediated by neural activity within the mPFC.     57 Chapter  4: Glucocorticoid Receptors Regulate Aspects of Executive Functioning During Stress  4.1 Introduction      Executive functions describe higher-order cognitive control processes mediated by the PFC that allow for complex, adaptable behavior (Baddeley and Della Sala, 1996; Fuster, 2000a; Miller and Cohen, 2001; Brown and Bowman, 2002). High levels of catecholamines such as DA released in the PFC during stress reduce PFC activity (Vijayraghavan et al., 2007; Qin et al., 2009; Qin et al., 2012) and are hypothesized to impair PFC cognitive control processes (Arnsten, 2009, 2011). Working memory and set-shifting are two types of executive function that can be used to assess the functional consequence of acute stress exposure on PFC-dependent cognitive processing.  4.1.1 Working Memory      Working memory involves the temporary on-line storage and manipulation of information important to guide ongoing behaviour and can be assessed in rats, monkeys, and humans in the form of a delayed-response task in which subjects are required to remember information achieved prior to a delay in order to successfully complete the task (Fuster, 1973; Goldman- Rakic, 1995; Fuster, 2000b). Our laboratory uses the Delayed Spatial Win-Shift (DSWSh) task performed on an eight-arm radial maze to assess working memory function (Olton, 1979; Packard and White, 1989). The radial arm maze can also be used to perform a non- delayed variant of the DSWSh task, known as random foraging, that assesses a rats’ ability to navigate in a spatial environment without knowledge of the location of food. Lesions or inactivation of the mPFC cause deficits in performance on the DSWSh task but not on the  58 random foraging task (Seamans et al., 1995; Floresco et al., 1997; Seamans et al., 1998). Furthermore, an inverted-‘U’-shape function of D1 receptor stimulation in the PFC has been described in which too little or too much DA impair performance on the DSWSh task (Seamans et al., 1998; Floreso and Phillips, 2001).      A number of studies have examined the effects of acute stress on working memory. Exposure to acute stress impairs working memory if testing occurs within 30 min of stress exposure (Murphy et al., 1996b; Murphy et al., 1996a; Arnsten and Goldman-Rakic, 1998; Morrow et al., 2000; Schoofs et al., 2009) but facilitates working memory if testing occurs 4 hr and 1 d post-stress (Barha et al., 2007; Yuen et al., 2009; Yuen et al., 2011). Since DA levels remain elevated in the PFC for the first 30-45 min after acute stress exposure (Imperato et al., 1992; Taber and Fibiger, 1997; Butts et al., 2011), we hypothesize that blockade of prefrontal GRs which attenuates stress-evoked DA efflux should reverse stress- induced working memory impairments.  4.1.2 Cognitive Flexibility      Cognitive flexibility, the ability to update and modify previously learned behavioural responses in a changing environment, is essential for successful adaptation to promising opportunities and for coping with adverse events. Set-shifting and reversal learning represent two main forms of cognitive flexibility that can be assessed reliably in rodents (Birrell and Brown, 2000; Floresco et al., 2008). In set-shifting, an animal switches its focus from an initially appropriate set of stimuli, to those with different characteristics that subsequently convey information about where to respond correctly to obtain reward (i.e., shift from a visual-cue to a response discrimination). In comparison, during reversal learning, an animal  59 shifts responding within the same stimulus dimension (i.e., shift between response locations). Lesions or inactivation studies in rodents have demonstrated that the medial PFC (mPFC) is required for set-shifting but not for reversal learning whereas the orbitofrontal cortex (OFC) mediates reversal learning but not set shifting (Ragozzino et al., 1999; Birrell and Brown, 2000; Chudasama and Robbins, 2006; Boulougouris et al., 2007; Floresco et al., 2008; Ghods-Sharifi et al., 2008). PFC DA is also important for set-shifting; however unlike working memory, D1 and D2 receptor stimulation are both required for optimal performance (Ragozzino, 2002; Floresco et al., 2006). According to the dual-state theory proposed by Seamans and colleagues, activation of D2 receptors would facilitate the ability of PFC networks to disengage from the previous strategy and find alternative response strategies. Once an alternative strategy is developed, D1 receptor activation would act to facilitate the stabilization of this novel strategy (Seamans and Yang, 2004).      Acute stress has been shown to exert deleterious effects on certain forms of cognition mediated by the mPFC. Accumulating evidence using animal models suggests that chronic stress impairs set-shifting (Liston et al., 2006; Bondi et al., 2008; Nikiforuk and Popik, 2011; Nikiforuk, 2012, 2013) and reversal learning (Cerqueira et al., 2005; Cerqueira et al., 2007; Lapiz-Bluhm et al., 2009; Danet et al., 2010). However, relatively few studies have examined the effect of acute stress on cognitive flexibility. A study conducted in mice demonstrated a strain-dependent impairment in reversal learning after exposure to uncontrollable foot shock stress (Francis et al., 1995). In contrast, a recent study found that acute stress given immediately preceding testing facilitated performance on response reversal but had no effect on set-shifting (Thai et al., 2012). In humans, exposure to acute psychosocial stress immediately before performing a task switching test impaired performance (Plessow et al.,  60 2012). We have recently demonstrated that an immediate effect of acute stress is the selective impairment of performance on a working memory task mediated by the mPFC, whereas performance on a random foraging task mediated by the hippocampus is unaffected by stress (Butts et al., 2011). Therefore, we hypothesize that exposure to a similar stressor (15 min of mild tail-pinch stress) given immediately before testing on a set-shifting or reversal learning task would selectively impair performance on a set-shifting task that is dependent on normal functioning of the mPFC.  4.1.3 Aim      The aim of this study was to determine whether exposure to an acute stressor would selectively impair performance on mPFC-dependent tasks (working memory and set-shifting) but not effect performance on mPFC-independent tasks (random foraging or reversal learning). Furthermore, the functional consequence of blocking the interaction between stress and DA efflux was also examined using an mPFC-dependent working memory task. Rats were exposed to mild tail-pinch stress for 15 min immediately preceding the test phase and within the context of the task. We demonstrate that exposure to acute stress significantly disrupted working memory and set-shifting but had no effect on random foraging and reversal learning. Blockade of prefrontal GRs prevented deficits in working memory function observed following exposure to acute stress.      61 4.2 Material and Methods 4.2.1 Animals      Male Sprague–Dawley rats (Charles River) were single-housed. The colony was maintained at 21 °C with a 12-h light/dark cycle. For animals trained on the working memory and random foraging tasks, lights went on at 7:00 PM. For animals trained on the set-shifting and reversal learning tasks, lights went on at 7:00 AM. Rats were food restricted to 85% of their free feeding weight. All experimental protocols were approved by the Committee on Animal Care, University of British Columbia, and conducted in compliance with guidelines provided by the Canadian Council of Animal Care. 4.2.2 Drugs      A stock solution of RU-38486 (Tocris Biosciences) was made in ethanol (EtOH) and dissolved in artificial cerebrospinal fluid (aCSF, pH 5.0). EtOH did not exceed 1.0% when dissolved in aCSF. 4.2.2.1 Delayed Spatial Win-Shift Task      Rats (~280–310 g) were implanted with bilateral microinjection cannulae (Plastics One Inc.) directly above the mPFC (3.0 mm AP, ±0.6 mm ML, −3.6 mm DV). One week after surgery, rats were food restricted to 90% of their free-feeding body weight. Training was conducted on an eight-arm radial arm maze as previously described (Seamans et al., 1995; Floresco et al., 1997; Seamans et al., 1998). Animals received one training trial per day consisting of an acquisition phase, a delay period, and a retrieval phase (Fig. 4.1A). During the acquisition phase, a novel set of four arms were opened, and upon retrieval of all four  62 food pellets (45 mg; Bioserv) from these arms, animals were retained in the last arm visited and lights were turned off, signaling the delay phase. During the retrieval phase, all eight arms were open, and food pellets were located in the arms that were blocked before the delay. Efficient error-free performance is achieved by visiting each of the four previously blocked arms. Errors were scored as reentries into arms that were previously entered either during the acquisition or retrieval phases. Rats were trained with a 15-min delay, until criterion was reached (one error or less for two consecutive training days). On the test day, animals were randomly preassigned into different treatment groups to receive bilateral infusions (3 μL/min for 20 s; Harvard Apparatus pump) of RU-38486 or vehicle (1% EtOH, pH 5.0) into the mPFC 30–40 min (33.50 ± 0.76) before the start of the DSWSh task. During the delay phase, animals were subjected to acute tail-pinch stress for the entire 15 min or given no stress. The latencies to complete the acquisition phase were recorded to ensure RU- 38486 had no effect on motor or motivational responses. Retrieval phase error types were categorized as either across-phase errors (defined as any initial entry into an arm that had been visited during the acquisition phase) or within-phase errors (defined as any reentry into an arm that had been entered in the retrieval phase). 4.2.2.2 Non-delayed Random Foraging Task      Rats were food restricted to 90% of their free-feeding weight. Training was conducted on an eight-arm radial arm maze as previously described (Seamans et al., 1995; Floresco et al., 1997; Seamans et al., 1998). During each daily session, animals were trained to locate food pellets placed at random in a novel set of four of the eight arms (Fig. 4.2A). Animals were trained to a criterion of one reentry error per daily trial for four consecutive days. The following day, animals were exposed to tail-pinch stress in the testing room in their home  63 cage for 15 min. Immediately after stress exposure, animals were placed on the maze for the test trial. Errors were scored as reentries into arms entered previously within a trial.  4.2.3 Cognitive Flexibility Tasks 4.2.3.1 Apparatus      Testing was conducted in twelve operant chambers (30.5 cm × 24 cm × 21 cm; Med- Associates, St. Albans, VT, USA) enclosed in sound-attenuating boxes. Boxes were equipped with a fan to provide ventilation and to mask extraneous noise. Each chamber was fitted with two retractable levers, one located on each side of a central food receptacle, by which food reinforcement (45 mg; BioServ, Frenchtown, NJ) was delivered by a pellet dispenser. A light emitting diode stimulus light was positioned centrally above each lever and served as a stimulus for visual-cue discrimination learning. Each chamber was illuminated by a single 100-mA house light located in the top-center of the wall opposite the levers. All experimental data were recorded by an IBM personal computer connected to the chambers via an interface.  4.2.3.2 Lever Training      Training was conducted as described previously (Floresco et al., 2008; Dalton et al., 2011). Rats were trained for 1 day on a magazine training schedule before being trained under a fixed-ratio 1 schedule to a criterion of 50 presses in 30 min for both levers (counterbalanced left/right between subjects). Rats were then trained on retractable lever pressing for 5 days. These sessions consisted of 90 training trials and began with the levers retracted and the chamber in darkness. Every 20 s, a trial began with illumination of the house light and insertions of one of the two levers into the chamber. If the rat failed to  64 respond on the lever within 10 s, the lever was retracted, the chamber darkened and the trial was scored as an omission. If the rat responded within 10 s, the lever retracted, a single pellet was delivered immediately and the house light remained illuminated for another 4 s. In every pair of trials, the left or right lever was presented once, and the order within the pair of trials was random. Importantly, the stimulus lights above each of the levers were never illuminated during these training sessions. For the last four retractable lever sessions rats were put into operant boxes for 15 min prior to the initiation of the program as this is the time when the stressor would subsequently be applied during the test days. Rats had to achieve a criterion of less than 5 omissions over the 90 trials before proceeding to the next stage of testing. On the last day of retractable lever training, the side bias for the rat was determined. These sessions were similar to the retractable lever sessions, except that both levers were inserted into the chamber. The stimulus lights above the levers were not illuminated during these trials. On the first trial, a food pellet was delivered after responding on either lever. Upon subsequent insertion of the levers, food was delivered only if the rat responded on the lever opposite to the one chosen initially. If the rat chose the same lever as the initial choice, no food was delivered, and the house light was extinguished. This continued until the rat chose the lever opposite to the one chosen initially. After choosing both levers, a new trial commenced. Thus, one trial for the side-bias procedure consisted of responding on both levers. The lever (right or left) that a rat responded on first during the initial choice of a trial was recorded and counted toward its side bias. If the total number of responses on the left and right lever were comparable, the lever that a rat chose initially four or more times over seven total trials was considered its side bias. However, if a rat made a disproportionate number of responses on  65 one lever over the entire session (i.e., greater than a 2:1 ratio), that lever was considered its side bias. Visual-cue discrimination training commenced the following day.  4.2.3.3 Strategy Set-Shift (Shift to Response Discrimination) 4.2.3.3.1 Day 1: Visual-cue discrimination.      The first phase of testing required rats to press the lever underneath the illuminated stimulus light to receive a food reward pellet (Fig. 4.3A). A session began in darkness with the levers retracted.  Each trial was initiated with the illumination of one of the stimulus lights located above a lever. Three seconds later, the house light was turned on and both levers were extended.  Rats had 10 s to make a response.  A press on a lever below the illuminated stimulus light resulted in the delivery of one reward pellet and retraction of both levers.  The house light was extinguished 4 s later.  A press on the inactive lever resulted in the retraction of both levers and the immediate extinction of the house light. Failure to choose either lever within 10 s resulted in the retraction of both levers and the trial was recorded as an omission.  In every pair of trials, the left or right stimulus light was illuminated once, and the order within the pair of trials was randomized.  Trials continued every 20 s until a rat had received a minimum of 30 trials and achieved criterion performance of 10 consecutive correct responses. Omission trials were not included in the trials to criterion measure. Upon completion of a session, the rat was removed from the chamber and placed back in its home cage. For each trial, lever choice and position of the stimulus light was recorded, as were the latencies to respond on a lever after its insertion, and locomotor activity. Strategy set-shift discrimination commenced the day following successful completion of visual-cue discrimination.   66 4.2.3.3.2 Day 2: Shift to response discrimination.      Twenty-four hours after visual-cue discrimination training, rats entered the strategy shift phase of the experiment. During this shift, rats were required to disengage from the previously- relevant visual-cue strategy and shift to using an egocentric response discrimination strategy, requiring them to press the lever opposite to their side bias (left or right lever) to obtain reward, irrespective of the position of the visual-cue (Fig. 4.3A). As with the initial visual-cue discrimination, a stimulus light was illuminated above one of the two levers for 3 s before the insertion of the levers. Trials were given in a manner identical to visual-cue discrimination trials, and for each trial, the lever that the animal chose and the location of the stimulus light was recorded. Trials continued every 20 s until a rat achieved criterion performance of 10 consecutive correct choices, within a maximum of 150 trials. If a rat failed to achieve criterion within this allotted number of trials, its data was included in the analysis and given a score of 150 trials for this measure.  4.2.3.4 Response Reversal Learning 4.2.3.4.1 Day 1: Initial response discrimination.      For this experiment, a separate group of rats were initially trained on the response discrimination task described above. On each trial, rats were required to press the lever opposite their side bias, regardless of the position of the visual-cue stimulus light, which for this experiment, served as a distracter (Fig 4.4A). Trials continued until a rat achieved criterion performance of 10 correct consecutive choices. There was no limit to the number of trials rats received to achieve this criterion. Upon completion of a training session, the rat was removed from the chamber and placed back in its home cage.  67 4.2.3.4.2 Day 2: Response reversal.      Twenty-four hours after response discrimination training, rats were trained on a reversal of this discrimination.  Hence, a correct choice required a press of the lever opposite to that which was reinforced on Day 1 (Fig 4.4A). All other aspects of training were identical to those used on Day 1 of response discrimination training. Trials continued every 20 s until a rat achieved criterion performance of 10 correct consecutive choices, within a maximum of 150 trials.  4.2.3.5 Error analysis      Errors committed during the shift were subdivided into three error subtypes to determine whether stress treatment impaired the ability to either shift away from the previously learned strategy (perseverative errors), or to acquire and maintain the new strategy after perseveration had ceased (regressive or never-reinforced errors). A perseverative error was scored when a rat responded on a lever with the stimulus light illuminated above it on trials that required the rat to press the opposite lever during the initial phase of the shift. For example, following the shift, the rat may now be rewarded for always pressing the left lever. Accordingly, perseverative error was scored when the rat pressed the right lever when the stimulus light was illuminated above it.  Eight out of every sixteen consecutive trials required the rat to respond in this manner (i.e.; press the lever opposite to that indicated by the previously learned light cue rule). As in previous studies (Floresco et al., 2008; Dalton et al., 2011), these types of trials were separated into consecutive blocks of 8 trials each. Perseverative errors were scored when a rat pressed the incorrect lever on 6 or more out of a maximum of 8 trials per block that required the rat to press the lever that did not have the  68 stimulus light illuminated above it. Once a rat made fewer than five perseverative errors in a block for the first time, all subsequent errors of this type were no longer counted as perseverative errors, because at this point the rat was using the original strategy less than 75% of the time.  Instead, these errors were now scored as regressive errors. The third type of error, termed never-reinforced errors, was scored when a rat pressed the incorrect lever on trials where the visual-cue light was illuminated above the same lever that the rat was required to press during the shift. Regressive and never-reinforced errors were used as an index of the animals’ ability to maintain and acquire a new strategy, respectively. Reversal learning errors were also subdivided into perseverative and regressive subtypes and analyzed over blocks of 16 trials (Jones and Mishkin, 1972; Chudasama and Robbins, 2006). Perseverative errors were scored when rats made an incorrect response, and pressed the lever that was reinforced during initial response discrimination training on Day 1. Once a rat made fewer than 10 perseverative errors within a block of 16 trials for the first time, all subsequent errors were scored as regressive.  4.2.3.6 Stress Treatment      Each experiment consisted of 3 groups; 1) control group (no stress), 2) rats that received stress during initial discrimination learning on Day 1, and 3) rats that received stress prior to set shifting or reversal learning on Day 2. Rats in the stress groups were exposed to 15 min of tail-pinch stress (clothespin with Velcro covering on base of tail), within the operant testing chamber. After this 15 min period, the tail-pinch stress was removed and rats were subjected to visual-cue discrimination, response discrimination, set-shift, or response reversal testing. We have previously shown that exposure to this stressor for 15 min significantly increases  69 levels of circulating corticosterone (Butts et al., 2011). Unstressed rats were put into operant chamber for 15 min without stress exposure.  4.2.3.7 Data Analysis      Data are presented as group means ± standard error of the mean. Analysis of the effects of acute stress on set shifting and reversal days was performed using one-way ANOVAs with Stress (no-stress, stress on set-shifting day, and stress on reversal day) as a between subjects factor. The dependent variables for visual-cue discrimination were trials to criterion and the total number of errors committed. The dependent variables for set-shifting were trials to criterion, total errors, perseverative errors, regressive errors, and never-reinforced errors. The dependent variables for response reversal discrimination were trials to criterion, total errors, and perseverative and regressive errors. Tukey’s post-hoc test was used where appropriate.  4.2.4 Histology      Following the working memory experiment on the DSWSh task, rats were deeply anesthetized with isoflurane. Brains were promptly removed and stored in 20% wt/vol sucrose and 4% vol/vol paraformaldehyde solution for a minimum of 1 wk. Brains were then sliced into 50 μM coronal sections, stained with cresyl violet, and examined for verification of microinjection cannulae placement.   70 4.3 Results 4.3.1  Blockade of Prefrontal GRs Reverses Stress-Evoked Cognitive Deficits.      Given that optimal DA levels in the PFC are required for the integration of information held in working memory into a prospective plan to retrieve food in a complex environment (Seamans et al., 1998; Phillips et al., 2004), we hypothesized that an increase in DA efflux arising from acute tail-pinch stress would impair performance on a DSWSh task, and that treatment with RU-38486 would reverse this effect. Rats were given bilateral infusions into the mPFC of either RU-38486 (100 ng/μL) or vehicle before acute tail-pinch stress during the delay period (Fig. 4.1A,E). As shown in Fig, 4.1B, RU-38486 or vehicle treatment alone had no effect on memory for the correct location of food during the retrieval phase of the task (n = 8 per condition). In contrast, retrieval-phase errors were increased after exposure to tail- pinch stress during the delay period, and this was reversed by infusion of RU-38486 into the mPFC. A two-way repeated-measures ANOVA for the factors of treatment and day revealed a significant main effect of day [F (3, 28) = 14.38, P < 0.05]. Tukey's post hoc analysis confirmed that rats made significantly more retrieval-phase errors after stress exposure compared with the day prior (P < 0.05). Planned comparisons revealed that vehicle-treated animals made significantly more errors after exposure to stress compared with RU-38486– treated or control animals (P < 0.05). Furthermore, animals subjected to tail-pinch stress made significantly more across-phase errors (P < 0.05, two-tailed Student t tests; Fig. 4.1C). RU-38486 did not alter motor or motivational processes, as all food pellets were consumed, and performance timing (a measure of total time to complete the acquisition phase) did not differ between treatment groups (P > 0.05, two-tailed Student t tests; Fig. 4.1D).  71  Figure 4.1 Blockade of GRs in the mPFC reverses stress-induced working memory impairments. (A) Illustration of the DSWSh task. (B) Errors made on DSWSh task after exposure to tail-pinch stress (15 min) compared with errors made the day prior and administration RU-38486 or vehicle into the mPFC (n = 8 for all treatment conditions). (C) Type of errors divided into across phase and within phase. (D) Latencies to complete the acquisition phase. (E) Summary of microinjection cannula tip placements in the mPFC (*P < 0.05).   72 4.3.2 Stress does not affect performance on a non-delayed random foraging task      We hypothesized that performance on a non-delayed variant of the DSWSh task, not dependent on the mPFC (Floresco et al., 1997), would be unimpaired by exposure to acute stress. A non-delayed random foraging task was used subsequently to investigate whether acute stress can impair foraging in a spatial environment without prior knowledge of the location of food (Fig. 4.2A). Performance on this task was unaffected by exposure to acute tail-pinch stress before the test phase (n = 6), as confirmed by a two-tailed Student t test comparing mean ± SEM (P > 0.05, Fig. 4.2B).     Figure 4.2 Performance on a non-delayed random foraging task is unaffected by stress. (A) Illustration of the random foraging task. (B) Errors made on the random foraging task after exposure to tail-pinch stress (15 min; n = 6).      73 4.3.3 Acute Stress Impairs Set-Shifting Performance 4.3.3.1 Day 1: visual-cue discrimination learning      To determine whether exposure to acute stress would impair the initial visual-cue discrimination learning, rats were exposed to 15 min of tail-pinch stress immediately prior to the start of visual-cue discrimination learning (Fig. 4.3A). Analysis of these data revealed no significant main effect of treatment on the number of trials to criterion (F (2,44) = 0.552, NS) or errors to criterion (F (2,44) = 0.296, NS).  Rats receiving stress (n = 14) on Day 1 were not impaired in learning a visual-cue discrimination, making a comparable number of trials or errors to control rats (n = 16) and rats designated to receive stress on Day 2 (n = 15) (Fig. 4.3B,C).  There were no differences between groups in average response latency, locomotor activity, or number of omissions (F (2,44) = 1.936, 1.790, 0.207, respectively, NS; Table 4.1). Thus, acute stress does not disrupt the initial learning of a simple visual cue discrimination strategy.  4.3.3.2 Day 2: shift to response strategy To examine the effect of acute stress on altering the ability to shift responding from a visual- cue to a response discrimination strategy, a separate group of rats were exposed to stress immediately prior to the start of the response discrimination task (Fig. 4.3A). Analysis of these data revealed a significant main effect of treatment on both trials (F (2,44) =3.275, p<0.05; Fig. 4.3D) and errors to criterion (F (2,44) =3.266, p<0.05; Fig. 4.3E). Tukey’s post- hoc analysis revealed that rats exposed to stress during the set-shift required significantly more trials and made significantly more errors (p<0.05) than control rats. In contrast, rats that received stress during visual-cue discrimination learning on Day 1 did not differ from  74 controls on these measures. Subsequent analyses on the types of errors made during the set- shift indicated that rats exposed to stress made more perseverative errors than control rats (F (2,44) =3.149, p=0.053; Fig. 4.3F). The number of regressive and never reinforced errors were unaffected by stress (F (2,44) = 1.636 and 0.876, respectively, NS; Fig. 4.3F). There were no differences between groups in average response latency, locomotor activity, or number of omissions (F (2,44) = 0.842, 1.784, 1.109, respectively, NS; Table 4.1). Thus, acute stress impaired shifting from a visual-cue to a response-based discrimination strategy.   Figure 4.3 Acute stress impairs set-shifting but does not affect initial visual-cue discrimination learning. (A) Diagram illustrating the set-shifting task. Rats exposed to 15 min of tail-pinch stress did not differ from controls in the number of (B) trials and (C) errors to achieve criterion during the initial visual-cue discrimination. Rats exposed to stress had a significantly greater number of (D) trials and (E) errors to achieve criterion when they were required to shift from a visual-cue to a response based strategy. (F) Analysis of the type of errors committed during the set-shift revealed that stress caused a selective increase in perseverative errors. Data are expressed as mean ± SEM (white; n = 14, control, grey; n = 16, stress day 1, black; n = 15, stress day 2). *P < 0.05 significant difference versus control.      75  Response Latency (s) Locomotion (beam breaks/min) Omissions Visual-Cue Discrimination (Day 1):  Control 0.83 ± 0.06 33.53 ± 5.47 0.21 ± 0.15 Stress Day 1 0.97 ± 0.05 30.27 ± 2.97 0.25 ± 0.14 Stress Day 2 0.81 ± 0.07 23.29 ± 2.97 0.13 ± 0.09 Response Discrimination (Day 2):  Control 0.70 ± 0.08 32.64 ± 5.15 0.29 ± 0.22 Stress Day 1 0.67 ± 0.05 33.78 ± 3.19 0 Stress Day 2 0.81 ± 0.10 25.04 ± 1.92 0.47 ± 0.34  Table 4.1 Acute stress does not affect motor or motivational processes during set-shifting. Response latency, locomotion, or trial omission data were not different between groups during visual cue discrimination or response discrimination. Data are expressed as mean ± SEM.  4.3.4 Acute Stress Does Not Affect Response Reversal Learning 4.3.4.1 Day 1: response discrimination training In a separate group of rats, exposure to stress had no effect on acquisition of an egocentric response strategy (Fig. 4.4A). Analysis of these data revealed no significant main effect of treatment on the number of trials to criterion (F (2,38) = 0.584, NS) or errors to criterion (F (2,38) = 0.539, NS).  Rats exposed to acute stress (n = 16) on Day 1 were not impaired in learning a simple response discrimination relative to control rats (n = 16) or rats that would receive stress on day 2 (n = 12) (Fig. 4.4B,C). There were no differences between groups in average response latency, locomotor activity, or number of omissions (F (2,38) = 1.071, 1.112, 0.905, respectively, NS; Table 4.2). Thus, exposure to stress does not impair the initial acquisition of a response discrimination strategy.   76 4.3.4.2 Day 2: response reversal To determine whether exposure to acute stress would impair the ability to shift responding between response locations, a separate group of rats were exposed to 15 min of tail-pinch stress immediately prior to the start of the reversal response discrimination task (Fig. 4.4A). Exposure to stress prior to testing on Day 2 had no effect on the number of trials or errors committed during the reversal. Analysis of these data revealed no significant main effect of treatment on both trials (F (2,38) = 0.228, NS; Fig. 4.4D) and errors to criterion (F (2,38) = 0.297, NS; Fig. 4.4E). There was no specific effect of stress on either perseverative or regressive subtypes of errors (F (2,38) = 0.629 and 0.082, respectively, NS; Fig. 4.4F). There was also no difference between groups in average response latency, locomotor activity, or number of omissions (F (2,38) = 0.543, 1.786, 2.159, respectively, NS; Table 4.2). Thus, acute stress does not disrupt the reversal of a response discrimination strategy.      77  Figure 4.4 Acute stress does not affect reversal learning or initial response learning. (A) Diagram illustrating the reversal learning task. Rats exposed to 15 min of tail-pinch stress did not differ from controls in the number of (B) trials and (C) errors to achieve criterion during the initial response discrimination. Rats exposed to stress did not differ from controls in the number of (D) trials and (E) errors during the response reversal. (F) Analysis of the type of errors committed during the reversal did not differ from controls. Data are expressed as mean ± SEM (white; n = 14, control, grey; n = 12, stress day 1, black; n = 13, stress day 2).   Response Latency (s) Locomotion (beam breaks/min) Omissions Response Discrimination (Day 1):  Control 1.03 ± 0.14 35.45 ± 3.71 0.21 ± 0.11 Stress Day 1 1.00 ± 0.10 29.27 ± 2.70 0.58 ± 0.34 Stress Day 2 0.82 ± 0.07 28.61 ± 4.20 0.23 ± 0.17 Response Discrimination (Day 2):  Control 0.77 ± 0.13 31.72 ± 2.81 0.21 ± 0.11 Stress Day 1 0.64 ± 0.07 26.32 ± 3.66 0 Stress Day 2 0.76 ± 0.07 24.12 ± 2.50 1.08 ± 0.65  Table 4.2 Acute stress does not affect motor or motivational processes during reversal learning. Response latency, locomotion, or trial omission data were not different between groups during response discrimination or response reversal. Data are expressed as mean ± SEM.  78 4.4 Discussion 4.4.1 Blockade of Prefrontal GRs Restores Deficits in Working Memory After Acute Stress.      The DSWSh task was used here to examine the effect of acute stress in modulating prefrontal-dependent working memory processes. Previous studies from our laboratory using in vivo microdialysis in conjunction with this task have demonstrated that DA efflux in the mPFC occurs during both the acquisition (acquiring the information before the delay) and retrieval (using the information following the delay) phases of the task (Phillips et al., 2004). Optimal performance was associated with moderate increases in DA efflux during the retrieval phase, whereas inaccurate recall was negatively correlated with the magnitude of DA efflux. This result led us to investigate whether enhanced DA efflux after tail-pinch stress would impair performance during the retrieval phase of this task and whether RU- 38486 could restore performance given its ability to reduce stress-evoked DA efflux in the mPFC. Control studies have confirmed our microdialysis probes have a permeability efficiency in the range of 15% to 20%. Accordingly, the continuous perfusion of RU-38486 at a concentration of 11.1 ng/μL, infused through the probe at a rate of 1 μL/min, over 45- min period, would deliver ~100 ng/μL of drug into the mPFC (Butts et al., 2011). To ensure use of a comparable dose in the DSWSh working memory study, injections of a 100 ng/μL dose of RU-38486 were given 30–40 min before the acquisition phase. We found that exposure to acute tail-pinch stress during the delay period impaired accurate recall and importantly, microinfusion of RU-38486 into the mPFC significantly reduced the numbers of stress-induced errors. The effect of RU-38486 was not due to alterations in motor or motivational processes, because response latencies were unaffected. Furthermore, these  79 effects appear to be related specifically to acute stress, because microdialysis data confirmed that elevated DA efflux during anticipation or consumption of food was not affected by administration of RU-38486 into the mPFC (Butts et al., 2011).      In contrast to the DSWSh task, performance on a non-delayed random foraging task was unaffected by acute stress, thus providing further insight into the neural circuitry mediating cognitive processes that are affected by stress. Projections from the ventral hippocampus to the prelimbic region of the mPFC are important for performance on the DSWSh task in which foraging is guided by knowledge of the probable location of food (Floresco et al., 1997). Conversely, afferent input from the ventral hippocampus to the nucleus accumbens mediates efficient foraging behavior in the absence of information about the location of food (Floresco et al., 1997). In this context it is noteworthy that administration of hydrocorticosterone to healthy human subjects led to deficits in cognitive processes mediated by the PFC (working memory) but not the hippocampus (declarative memory) (Lupien et al., 1999). Furthermore, blocking D1 receptors in mPFC impairs performance on the DSWSh but not the random foraging task (Seamans et al., 1998). D1 receptors also play an important role in modulating hippocampal-evoked activity of PFC neurons (Floresco and Grace, 2003), suggesting that stress-induced increase in DA efflux in the PFC may disrupt D1 receptor modulation of hippocampal inputs to the PFC, thereby impairing performance on spatial working memory tasks.      Our data extend previous findings on the impairment of working memory performance by acute stress (Murphy et al., 1996a; Arnsten and Goldman-Rakic, 1998; Morrow et al., 2000). The DSWSh task we used differs in important ways from delayed alternation tasks used in  80 other rat studies. First, the rat must remember trial-unique spatial information required to form an effective foraging strategy in an eight-arm radial maze (compared with simple alternation between two arms on a T-maze). Second, our task allows a longer delay (15 min) than the 15–30 s in the delayed alternation task. Finally, inactivation of the mPFC before the retrieval phase (but not before the acquisition phase) severely impairs performance, confirming a role for the mPFC in accessing previously acquired information from short-term memory and its integration into a prospective foraging strategy (Seamans et al., 1995). Therefore, acute stress may impair both the ability to actively retain information over a short delay (as assessed using the delayed alternation task) and/or executive functioning related to memory-based prospective planning as observed on the DSWSh task. Rats subjected to an acute stressor committed mainly across-phase errors consistent with a strong preference to revisit previously baited arms. Therefore, stress may disrupt the ability of the mPFC to suppress responses to familiar stimuli previously associated with reward (i.e., the four-baited arms from the acquisition phase), an effect consistent with disruption of behavioral flexibility (Kolb et al., 1974).      It is important to acknowledge that the impact of glucocorticoids on cognitive processes is complex. Indeed, exposure to a more intense acute stressor can facilitate working memory performance. Yuen et al. report that 20 min of forced swim stress enhanced working memory on a delayed alternation task when tested 4 hr and 1 d poststress - an effect attributed to GR- mediated potentiation of NMDAR- and AMPAR-mediated synaptic currents in PFC pyramidal neurons (Yuen et al., 2009). It is worth noting that this study used adolescent, rather than adult, rats. As the impact of acute stress may depend on the developmental stage of the animal (Lupien et al., 2009), further studies examining the effect of acute stress on  81 age-dependent changes in working memory function are warranted. Enhanced glutamatergic transmission allowing for recurrent excitation within PFC pyramidal neuronal networks is essential for working memory (Goldman-Rakic, 1995). Excessive DA levels suppress firing of PFC neurons engaged in a working memory task (Vijayraghavan et al., 2007), and therefore, the differential effects of acute stress exposure on working memory may be related to the timing of the stressor relative to the memory task (Diamond et al., 2007; Sandi and Pinelo-Nava, 2007). The present study focused on the immediate consequences of acute stress on cognitive function, as this may be a time when individuals are particularly vulnerable to the effects of recent stress. Further support for prefrontal GRs in the disruptive effects of acute stress on working memory performance is provided by a report of working memory deficits on a delayed alternation task after prior microinfusion of corticosterone into the mPFC (Barsegyan et al., 2010). This effect is also reversed by coadministration of a GR, but not an MR, antagonist. This impairment of working memory may reflect an increased DA efflux similar to that reported in the present study after reverse dialysis of corticosterone into the mPFC. 4.4.2 Acute Stress Impairs Set-shifting but not Reversal Learning      A strategy set-shifting task was used here to examine the effect of acute stress in modulating flexibility of cognitive processing known to be dependent on the normal functioning of the mPFC. The present data confirmed that exposure to acute stress impaired a rat’s ability to switch between a visual-cue discrimination to a response discrimination to obtain food reward. Animals exposed to stress immediately before the set-shift session required significantly more trials and made significantly more errors to reach criterion than control rats. Analysis of the type of errors indicated that stressed rats made significantly more  82 perseverative errors, suggesting that this impairment is attributable to an inability to disengage from a previously relevant strategy, as opposed to the acquisition or maintenance of a novel strategy. Rats exposed to stress before being required to learn either a visual-cue or response discrimination on Day 1 were unimpaired thereby indicating that the disruptive effect of stress during the set-shift was not due to impaired learning of a novel strategy or stimulus-reward association. Importantly, exposure to acute stress had no effect on response latencies, locomotion or trial omissions thereby ruling out the possible contribution of a general disruption in locomotor or motivational processes.      In contrast to the set-shifting task, performance on a reversal learning task was unaffected by acute stress. In agreement with these results, a similar acute stressor was shown to impair performance on a prospective working memory task known to be dependent on the mPFC, but had no effect on a random foraging task mediated by the hippocampus (Butts et al., 2011). Furthermore, rats subjected to an acute stressor before the working memory task committed mainly perseverative-type of errors as demonstrated by their strong preference to revisit arms baited previously with food reward before the delay period (Butts et al., 2011). Therefore, stress may disrupt the ability of the mPFC to suppress responses to familiar stimuli previously associated with reward, an effect consistent with the increase in perseverative errors observed during the set-shift. It is noteworthy that the selective increase in perseverative responding induced by exposure to acute stress is similar to that observed after inactivation or lesions of the mPFC (Ragozzino et al., 1999; Boulougouris et al., 2007; Floresco et al., 2008). Set-shifting and reversal learning differ in their neural circuitry therefore these data provide further insight into the mechanisms by which stress can alter cognitive processes.  83      Prefrontal dopamine (DA) is essential for set-shifting (Floresco et al., 2006), whereas 5- HT in the OFC is necessary for reversal learning (Clarke et al., 2005; Clarke et al., 2007). Our previous data indicate that activation of prefrontal glucocorticoid receptors (GRs) during acute stress enhances mesocortical DA efflux resulting in mPFC-dependent cognitive deficits (Butts et al., 2011). Blockade of prefrontal GRs attenuated the increased DA efflux during stress and reversed the working memory deficit (Butts et al., 2011). Indeed, excessive DA efflux during stress has been demonstrated to over-stimulate prefrontal D1 receptors and suppress firing of PFC neurons (Zahrt et al., 1997; Arnsten and Goldman-Rakic, 1998) resulting in perseverative-type errors on a working memory task (Zahrt et al., 1997; Floresco and Phillips, 2001). Therefore, this may be a mechanism by which acute stress causes a selective perseverative impairment in set-shifting. Here it is important to take into account that the type of DA receptors required for mediating optimal performance on a set-shifting task differ from those that mediate working memory performance (Floresco et al., 2006).      Previous studies examining the contribution of different mPFC DA receptor subtypes to strategy set-shifting reported that blockade of D1 or D2 receptors impaired performance on a maze-based set-shifting task by increasing perseverative errors (Ragozzino, 2002; Floresco et al., 2006). Surprisingly, performance was unaffected by infusion of either a D1 or D2 receptor agonist suggesting that, unlike working memory, excessive activation of mPFC D1 or D2 receptors does not have detrimental effects on set-shifting (Floresco et al., 2006). According to the dual-state theory proposed by Seamans and colleagues, activation of D2 receptors should facilitate the ability of PFC networks to disengage from the previous strategy and find alternative response strategies. Once a successful alternative strategy is identified, D1 receptor activation would facilitate the stabilization of this novel strategy  84 (Seamans and Yang, 2004). Nevertheless, the fact that set shifting was not impaired by direct infusion of a D1 or D2 agonist into the mPFC, poses difficulties for the hypothesis that stress-induced disruption of set-shifting is attributable to excessive activation of these receptors by increases in mesocortical DA release. On the other hand, higher levels of PFC DA released during stress may impair set-shifting through excessive stimulation of D4 receptors as infusion of a D4 agonist into the mPFC impaired set-shifting in a manner similar to D1 or D2 receptor blockade (Floresco et al., 2006). This conclusion is supported by a recent study in humans showing that homozygous carriers of the COMT Met/Met allele, which results in lower enzyme activity and enhanced PFC DA signaling, were impaired on performance of a behavioural flexibility task (Schulz et al., 2012). Attenuation of the elevation in mesocortical DA by blockade of prefrontal GRs reduces stress-induced working memory impairments (Butts et al., 2011), therefore future studies should assess the role of prefrontal GR in mediating the selective impairing effect of stress on set-shifting.      Although there have been numerous studies demonstrating the deleterious effect of chronic stress on cognitive flexibility (Liston et al., 2006; Cerqueira et al., 2007; Bondi et al., 2008; Lapiz-Bluhm et al., 2009; Nikiforuk and Popik, 2011), relatively few studies have examined the effect of acute stress on these measures. Furthermore, previous studies examining the effect of acute stress on behavioural flexibility have yielded somewhat mixed results. For example, acute elevated platform stress facilitated reversal learning in rats performing a water maze task when the stressor was given immediately before each training trial but not the test trial (Dong et al., 2013). In contrast, mice performing a similar water maze task displayed strain-dependent impairments in reversal learning after exposure to uncontrollable foot shock stress immediately preceding the test trial (Francis et al., 1995). Of  85 particular note, Thai and colleagues demonstrated that exposure to acute restraint stress before testing significantly facilitated reversal learning while having no effect on set-shifting performance in rats performing similar operant tasks to the ones used in the present study (Thai et al., 2012). Finally, performance on a test of task switching was significantly impaired in human subjects by prior exposure to acute psychosocial stress (Plessow et al., 2012). Clearly, these data emphasize that the impact of acute stress on cognitive processes is complex and dependent upon many factors including the timing of the stressor relative to the memory task (Diamond et al., 2007; Sandi and Pinelo-Nava, 2007) and the context in which stress exposure occurs (Bouton and King, 1983; Joels et al., 2006; Diamond et al., 2007; Schwabe et al., 2009; Schwabe and Wolf, 2009). The present study focused on the immediate consequences of acute stress on cognitive function, as mPFC DA levels remain elevated for approximately 45 min following acute tail-pinch stress (Butts et al., 2011), which coincides with vulnerability to the effects of recent stress. Furthermore, unlike other studies that have examined the effects of acute stress on cognitive flexibility, we applied stress within the context of the task itself (i.e. within the operant chamber). In contrast, in the previous study by Thai and colleagues animals were stressed in a different context prior to being placed in the operant chambers (Thai et al., 2012). Therefore, the effects of acute stress on set-shifting functions mediated by the mPFC may be critically dependent on contextual settings, so that when stress is delivered in the same context as testing it may impair set-shifting. Conversely, stress delivered in a different context does not affect set-shifting and may facilitate reversal learning. Notably, in our previous study where we observed a deficit in mPFC-dependent working memory, the stressor was applied within the context of the cognitive task (Butts et  86 al., 2011), adding further support to the notion that stress exposure within a testing environment may have deleterious effects on mPFC functioning.  4.4.3 Summary      Here, we demonstrate that performance on the DSWSh and set-shifting tasks was impaired by acute stress. In contrast, performance on the non-delayed random foraging or reversal learning task was unaffected by acute stress. Thus, our data indicate that the mPFC is sensitive to the immediate and contextual effects of acute stress. Exposure to acute stress appears to impair the ability to suppress a previously learned response thereby preventing the adoption of new forms of behaviour appropriate to changing circumstances. The inability to adopt flexible cognitive and behavioral strategies in the presence of a dominant perseverative cognitive bias may contribute to the development and maintenance of stress-related psychiatric disorders (Beck and Rector, 2005; Beck, 2008). Stress is a major factor contributing to the development, recurrence, and treatment outcome in affective disorders, including bipolar disorder (Ellicott et al., 1990; Hammen and Gitlin, 1997). Deficits in executive function is commonly reported in patients with bipolar disorder (Young et al., 2004; Watson et al., 2006) and may be attributed to alterations in PFC functioning that occurs as a result of interactions between stress and DA function (Schatzberg et al., 1985). Thus, a better understanding of the neural mechanisms by which exposure to stress causes these impairments may aid the development of novel treatment strategies for these disorders.      Prefrontal activation of GRs during acute stress enhances mesocortical DA efflux (Butts et al., 2011). High levels of PFC DA modulate PFC function during stress by inhibiting the activity of neurons firing in the ‘delay’ phase of a working memory task (Arnsten, 2009,  87 2011). Attenuation of this elevation in mesocortical DA by blockade of prefrontal GRs reduces the stress-induced working memory impairments and therefore may represent a unique therapeutic target for treatment of psychiatric disorders. Before chronic treatment with GR antagonists can be contemplated, many factors remain to be clarified given the complex nature of glucocorticoid effects on cognition. Particular attention should be paid to an apparent inverted U-shape function between levels of glucocorticoids and hippocampal- dependent cognitive performance (de Kloet et al., 1999; Lupien et al., 2005). Limited duration of treatment with GR antagonists may be warranted, given our observation of improved neurocognitive function following 7 d of adjunctive treatment with RU-38486 in patients with bipolar disorder (Young et al., 2004; Watson et al., 2012). Similarly, short-term intervention with RU-38486 has also proven to be beneficial in the treatment for psychotic depression (Flores et al., 2006). Thus, acute blockade of GRs may represent a novel strategy for treatment of psychiatric disorders for which stress appears to be a significant mitigating factor.        88 Chapter  5: Conclusion 5.1 Summary of Research Findings 5.1.1 Overview      The stress response refers to a complex set of adaptive behavioural and physiological processes that are crucial for survival. DA is an evolutionarily conserved molecule within the stress response, increasing in both invertebrates (Ottaviani et al., 1992) and vertebrates (Lataster et al., 2011). Since the seminal study performed by Thierry and colleagues in 1976 it has been repeatedly demonstrated by using a variety of stressors that exposure to acute stress leads to a rapid increase in DA efflux in the mPFC in the rat (Thierry et al., 1976), monkey (Arnsten and Goldman-Rakic, 1998), and human (Lataster et al., 2011). The importance of DA release during stress is further highlighted by the fact that extrasynaptic DA levels, as measured during in vivo microdialysis, remain elevated in the PFC for a prolonged period after stress exposure. Experiments in this thesis and work from others have consistently demonstrated that PFC DA levels remain elevated for around 30-45 min following acute stress exposure (Imperato et al., 1992; Taber and Fibiger, 1997; Butts et al., 2011). However, the mechanism(s) by which stress increases PFC DA are still unknown. Here, we demonstrate that blocking GRs locally within the rat mPFC, but not within the VTA, reduces stress-evoked DA efflux in the mPFC. Blocking GRs within the mPFC also significantly attenuated stress-evoked glutamate efflux in the VTA. Finally, blocking ionotropic glutamate receptors in the VTA, thus blocking glutamatergic input to the VTA, reduced stress-evoked DA efflux in the mPFC. Thus, our data suggest that glucocorticoids act locally within the mPFC to modulate mesocortical DA efflux via potentiation of glutamatergic input onto DA neurons in the VTA. The functional impact of acute stress  89 exposure was demonstrated using tasks assessing executive function. Exposure to tail-pinch stress selectively impaired performance on a working memory and set-shifting task mediated by the mPFC but had no effect on a random foraging or reversal learning task that were not dependent on mPFC function (Seamans et al., 1995; Floresco et al., 1997; Seamans et al., 1998). Notably, stress-induced impairments in working memory were attenuated by blockade of prefrontal GRs. Thus, our data suggest that high levels of DA present in the mPFC during stress impair specific types of cognitive processes.  5.1.2 Glucocorticoid Receptors within the Medial Prefrontal Cortex Regulate Mesocortical Dopamine Efflux      Our data demonstrates that the primary site of glucocorticoid regulation of the mesocortical DA system is within the PFC. This is in contrast to previous hypotheses which suggested that the VTA was the primary site of glucocorticoid regulation of DA during stress (Enrico et al., 1998; Marinelli et al., 1998; Cho and Little, 1999; Saal et al., 2003; Minton et al., 2009). Importantly, our results are in agreement with several observations. First, acute stress increases NMDA and AMPA receptor neurotransmission in the mPFC and this is prevented by GR blockade (Yuen et al., 2009). Second, blockade of AMPA receptors in the mPFC reduces stress-evoked DA efflux in the mPFC (Takahata and Moghaddam, 1998). Finally, emerging evidence from the drug addiction field supports the suggestion that glucocorticoids act on GRs on DA terminal regions rather than on DA cell bodies in the VTA. Ambroggi et al. used two mouse models in which GR was specifically knocked out in either DA neurons or postsynaptic neurons expressing the D1 receptor to determine which location of GR mediated the effects of glucocorticoids on cocaine self-administration  90 (Ambroggi et al., 2009). Deletion of GR in postsynaptic neurons innervated by DA terminals was associated with reduced spontaneous neural activity, firing rate, and frequency of burst events in DA neurons (Ambroggi et al., 2009). The authors attributed this effect to the disruption of feedback control by postsynaptic neurons in the PFC or NAc on VTA DA neurons. Glutamate projections to the VTA primarily arise from the PFC (Oades and Halliday, 1987; Sesack et al., 1989) and these cortical afferents synapse onto DA neurons that project back to the PFC (Carr and Sesack, 2000). Thus, prefrontal GRs are an excellent candidate for regulating mesocortical DA efflux during stress.  5.1.3 Prefrontal Glucocorticoid Receptors Regulate Glutamatergic Input to the Ventral Tegmental Area      Increased VTA DA neuron activity is dependent on excitatory inputs from surrounding structures (Floresco et al., 2001; Floresco et al., 2003). Here, we demonstrate that blocking GRs within the mPFC can prevent the stress-evoked increase in VTA glutamate efflux. This suggests that GRs in the mPFC regulate mesocortical DA release by enhancing glutamatergic input to the VTA. This is in agreement with previous observations showing that acute stress leads to strengthening of excitatory synapses on DA neurons in the VTA that is prevented by systemic GR blockade (Cho and Little, 1999; Saal et al., 2003). Furthermore, blockade of ionotropic glutamate receptors in the VTA attenuates stress-evoked DA efflux in the mPFC (Enrico et al., 1998). However, we cannot conclude that the GRs in the mPFC directly modulate DA neurons in the VTA because glutamatergic output from the mPFC may also project to other regions that regulate the VTA. For example, recent data indicate that lateral habenula (LHb) neurons synapse primarily on DA neurons in the medial VTA that project to  91 the mPFC (Lammel et al., 2012). Phasic stimulation of LHb neurons projecting to the VTA cause a strong conditioned place aversion that is blocked by a D1 receptor antagonist in the mPFC (Lammel et al., 2012). The pedunculopontine tegmental nucleus also provides strong excitatory inputs to the VTA (Floresco et al., 2003). Alternatively, the mPFC may indirectly influence the VTA via inputs to the NAc. Activation of the medium spiny GABAergic output neurons in the NAc leads to inhibition of the ventral pallidum, which sends GABAergic projections to the VTA (Floresco et al., 2001) and the LHb (Araki et al., 1988; Ji and Shepard, 2007), thus disinhibiting DA neuron firing.  5.1.4 Acute Stress Impairs Executive Function Processes      The present studies focused on the immediate and contextual consequences of acute stress exposure on executive function, as this may be a time when individuals are particularly vulnerable to the effects of recent stress. Rats were subjected to acute stress immediately preceding the cognitive task and within the same environment of the task (i.e. stress was applied on the radial arm maze or within the operant chamber). We demonstrated that exposure to acute tail-pinch stress impairs performance on both a working memory and set- shifting task. In contrast, performance on a random foraging or reversal learning task was unaffected by stress. These findings are in agreement with Arnsten and colleagues who demonstrated the exposure to loud noise stress impaired performance on the spatial oculomotor delayed-response task which is mediated by the mPFC but had no affect on the mPFC-independent nondelayed variant of this task (Arnsten and Goldman-Rakic, 1998). Furthermore, exposure to a pharmacological stress impaired performance on the delayed alternation working memory task but did not impair performance on a spatial discrimination  92 task (Murphy et al., 1996b). Thus, these results are in line with the proposal that acute stress leads to a loss of complex cognitive functions that require the mPFC (Vijayraghavan et al., 2007; Arnsten, 2009, 2011).  5.1.5 Blockade of Prefrontal Glucocorticoid Receptors Reverses Stress-Induced Working Memory Impairment      We also demonstrate that high levels of DA released during acute tail-pinch stress remain elevated for 30-45 min before returning to basal levels by 90 min post-stress. Therefore, high levels of DA present within 30-45 min post-stress would provide excessive stimulation of D1 receptors in the mPFC leading to inhibition of firing of “delay” neurons (Vijayraghavan et al., 2007; Arnsten, 2009). Attenuation of this elevation in mesocortical DA by blockade of prefrontal GRs may account for the observed reduction of the stress-induced working memory impairments. Further support for prefrontal GRs in the disruptive effects of acute stress on working memory performance is provided by a report of working memory deficits on a delayed alternation task after prior microinfusion of corticosterone into the mPFC (Barsegyan et al., 2010). This effect is also reversed by coadministration of a GR, but not an MR, antagonist (Barsegyan et al., 2010). Unfortunately, the role of GR in modulating stress- induced impairments on the set-shifting task has yet to be examined. We hypothesize that prefrontal GR blockade would attenuate these impairments possibly by preventing excessive stimulation of D1 or D4 receptors in the PFC (Floresco et al., 2006).  93 5.2 Limitations and Future Directions 5.2.1 Other Brain Regions are Activated by Acute Stress      The data presented in this thesis examined the role of GRs within the mPFC in regulating mesocortical DA efflux to acute stress. However, the VTA receives afferent regulation from other regions that are activated during stress. For example, the amygdala and hippocampus contain a high density of GRs and have direct and indirect projections to the VTA, respectively. Since stress-induced DA efflux in the mPFC is not completely blocked by prefrontal GR antagonism, it is possible that that the amygdala and hippocampus also regulate mesocortical DA efflux during stress. Grace and colleagues report that ventral hippocampal inputs regulate VTA DA activity to aversive stimuli (Valenti and Grace, 2008; Valenti et al., 2011; Valenti et al., 2012). Indeed, it was demonstrated that repeated footshock or acute restraint stress can also increase the population activity of VTA DA neurons that is reversed by infusion of the sodium channel blocker, TTX, into the ventral hippocampus (Valenti et al., 2011). Finally, stress-evoked DA efflux in the mPFC was attenuated by lesions to the basolateral amygdala (BLA) (Davis et al., 1994).      Stress-induced modulation of amygdala or hippocampal activity underlies some aspects of glucocorticoid-induced alterations in cognition (Roozendaal et al., 2004; Roozendaal et al., 2009; Cazakoff et al., 2010). Previous experiments performed in our laboratory show that inputs from the hippocampus to the mPFC are also crucial for working memory function (Seamans et al., 1998). Importantly, stress-induced memory retrieval impairments can be restored by blocking MRs or GRs in the dorsal hippocampus at 15 min or 60 min post-stress, respectively (Dorey et al., 2012). Blocking GRs in the BLA or central nucleus of the amygdala (CeA) prevents enhancement of stress-induced fear conditioning responses (Tronel  94 and Alberini, 2007; Kolber et al., 2008). Lesions of the BLA can block working memory impairment induced by a glucocorticoid administered either systemically or directly into the mPFC suggesting a role for the amygdala in stress-induced deficits (Roozendaal et al., 2004). Future experiments could address this issue using in vivo microdialysis, to perfuse a GR antagonist locally within the ventral hippocampus, BLA, or CeA while monitoring stress- evoked DA efflux in the mPFC.  5.2.2 Neuropeptide Release During Acute Stress      Exposure to acute stress not only increases glucocorticoid levels through the cellular actions of CRF in the HPA axis, but also stimulates the release of CRF in extra-hypothalamic brain regions, such as the VTA (Hauger et al., 2006; Wang et al., 2006; Wanat et al., 2008). Interestingly, invertebrates such as the freshwater snail which do not have glucocorticoid hormones increase DA release in response to CRF and ACTH (Ottaviani et al., 1992). In rats, CRF has been shown to act on CRF Type 1 receptors in the VTA to increase DA neuron firing (Tagliaferro and Morales, 2008; Wanat et al., 2008) and is involved in stress-induced drug relapse (Wise and Morales, 2010). CRF acts locally within the NAc to increase DA release through coactivation of the CRF receptors Type-1 and 2 (Lemos et al., 2012). This provides further support to our hypothesis that regulation of stress-induced VTA DA neurons occurs within the DA terminal regions and also highlights the need for an investigation into the role of local CRF within the mPFC in regulating the mesocortical DA system. Blocking CRF receptors locally in the mPFC during stress and examining changes in mPFC DA efflux could determine if CRF plays a role in elevating mesocortical DA efflux. However, infusion of a CRH antagonist into the lateral ventricles failed to attenuate the increase in mPFC DA  95 efflux induced by tail shock stress (Murphy et al., 2003).  5.2.3 Source of Glucocorticoids      In our study we only measured plasma corticosterone levels in response to acute tail-pinch stress and the levels of free corticosterone present in the mPFC at the time when DA levels are elevated remain to be specfied. Using the in vivo microdialysis methods designed by Linthorst and colleagues, it would be valuable to measure the level of free corticosterone in the mPFC during tail-pinch stress (Droste et al., 2008). Similarly, it would also be important to determine if tail-pinch stress is accompanied by an increase in CBG levels thereby delaying transport of corticosterone into the brain as observed with strong stressors such as forced swim and restraint stress (Droste et al., 2008; Qian et al., 2011). Moreover, corticosterone may be also locally synthesized within the brain (Mellon and Deschepper, 1993; Stromstedt and Waterman, 1995; Gomez-Sanchez et al., 1996). Therefore we cannot specify whether the rapid corticosterone-induced DA release in the mPFC is occurring via corticosterone that is synthesized and released from the adrenal gland or locally within the brain. An experiment in which Metyrapone, an inhibitor of 11B- hydroxylase that is required for the synthesis of corticosterone, is locally administered within the mPFC and then examining DA efflux in the mPFC after acute tail-pinch stress should be conducted to determine the possible involvement of brain-derived corticosterone.  5.2.4 Opposing Actions of Prelimbic and Infralimbic Cortices      The microdialysis experiments presented here measured changes in stress-evoked DA efflux in both the PL and IL mPFC regions. However, as previously discussed, the PL and IL  96 have opposing actions on HPA regulation (Diorio et al., 1993; Sullivan and Gratton, 1999; Radley et al., 2006). Furthermore, there are also opposing actions of the role of the PL and IL cortices in modulating cognitive function. PL lesions altered a rat’s capacity for goal-directed action while leaving intact actions driven by habits (Balleine and Dickinson, 1998). Conversely, lesions of the IL cortex delay or prevent the acquisition of habits and leave goal- directed behavior intact (Balleine and Dickinson, 1998; Coutureau and Killcross, 2003). Therefore, further studies to determine if GRs within the PL and IL cortices differentially regulate DA system function and stress-related behaviours are warranted.  5.2.5 Mechanisms Mediating Glucocorticoid Receptor Regulation of Mesocortical Dopamine During Stress      Our results suggest that the mPFC is the primary site of glucocorticoid regulation of mesocortical DA neurons during stress. Indeed, we hypothesize that corticosterone acts on a putative membrane GR on layer V pyramidal neurons in the PFC to enhance glutamatergic drive onto DA neurons in the VTA (Fig. 3.6). In order to determine a mechanism by which GRs regulate descending glutamatergic input it is important to appreciate the effect of timing on stress-induced alterations in PFC functioning. As demonstrated with experiments on working memory, acute stress impairs performance if tested within 30 min of stress exposure (Murphy et al., 1996a; Arnsten and Goldman-Rakic, 1998; Schoofs et al., 2009; Butts et al., 2011) but facilitates working memory performance if tested 4 hr or 1 d post-stress (Barha et al., 2007; Yuen et al., 2009; Yuen et al., 2011). Treatment with RU-38486 before the stress procedure abolished both the impairing and enhancing effects of acute stress on working memory (Yuen et al., 2009; Butts et al., 2011), indicating that GRs regulate functions that are  97 important for the immediate and delayed effects of stress on cognitive function. In examining mechanisms involved in the stress-induced facilitation of working memory, Yuen and colleagues found in vitro that exposure to acute stress induces a delayed (does not occur before 1 hr) and sustained potentiation of the synaptic response and surface expression of NMDA and AMPA receptors in PFC pyramidal neurons (Yuen et al., 2011). This occurs through the induction of serum- and glucocorticoid-inducible kinases and the activation of Rab4 that mediates receptor trafficking and function of NMDA and AMPA receptors (Yuen et al., 2011). Similarly, delayed effects of glucocorticoid signaling were also observed in a study examining the recruitment of prefrontal cortical endocannabinoid signaling by glucocorticoids involved in the termination of stress response (Hill et al., 2011). Application of corticosterone to prefrontal cortical slices suppressed GABA release onto principal neurons in layer V of the prelimbic region only when examined 1 h later (Hill et al., 2011). This effect was prevented by application of a CB1 receptor antagonist (Hill et al., 2011). Thus, glucocorticoids have delayed effects in the brain leading to enhancement of endocannabinoid signaling and NMDA/AMPA receptor trafficking. These delayed effects of glucocorticoid action are important for termination of the stress response and facilitation of working memory performance.      Another possible mechanism to describe the rapid-onset of GR effects that we observe may be through the direct modulation of intracellular signaling cascades such as Egr-1- induced expression of synapsin-Ia/Ib (Revest et al., 2005; Revest et al., 2010). Blocking the expression of synapsin-Ia/Ib in the hippocampus inhibits the glucocorticoid-mediated increase in stress-related memory (Revest et al., 2010). Thus, activation of GRs on layer V PFC projection neurons may lead to increased release of glutamate onto VTA DA neurons  98 through expression of synapsin-Ia/Ib (Nichols et al., 1992; Jovanovic et al., 2000). However, in the present study, the increase in stress-evoked glutamate efflux in the VTA was observed within 15 min. As this short time frame of action is too rapid to be mediated by means of the classical genomic GR pathway as described above, the immediate effects of acute stress may instead be regulated by a nongenomic membrane receptor-mediated mechanism (Prager and Johnson, 2009; Groeneweg et al., 2011).      Emerging evidence suggests that corticosterone may rapidly effect neuronal signaling by binding to membrane-associated corticosterone receptors (Orchinik et al., 1991; Dallman, 2005; Haller et al., 2008; Prager and Johnson, 2009) and anatomical data confirm that GRs are localized to membranes and synapses (Liposits and Bohn, 1993; Johnson et al., 2005). These membrane receptors have been shown to rapidly change synaptic transmission and neuron excitability through direct actions on ion channels or intracellular signaling messengers (ffrench-Mullen, 1995; Tasker et al., 2006; Prager and Johnson, 2009; Groeneweg et al., 2012). Relatively few studies have examined the possible role for rapid nongenomic mechanisms mediating corticosterone signaling in the mPFC (Groeneweg et al., 2011). However, a recent behavioural study has supported a potential role of membrane-GR. Barsegyan and colleagues demonstrated that administration of corticosterone conjugated to the membrane impermeable protein, bovine serum albumin, into the mPFC impaired performance on a working memory task (Barsegyan et al., 2010). This effect was prevented by co-administration of RU-38486 and discovered to be mediated by a membrane-GR- induced facilitation of a noradrenergic signal transduction pathway within the mPFC (Barsegyan et al., 2010). Accordingly, we propose that corticosterone acts on a putative membrane GR of layer V pyramidal neurons in the mPFC (Fig. 3.6). Activation of layer V  99 cortical neurons, the principal projection neurons of the mPFC, would in turn enhance glutamatergic drive onto DA neurons in the VTA, leading to an increase in DA efflux in the mPFC (Fig. 3.6) (Carr and Sesack, 2000). Future studies determining the intracellular mechanisms governing the rapid effects of prefrontal GR activation in modulating glutamatergic drive to the VTA are warranted.  5.2.6 The Effect of Stress of Prefrontal Cortical Function 5.2.6.1 Not All Stress Is Bad      It is important to note that not all aspects of cognition are impaired by acute stress exposure. Indeed, stress has been shown to enhance some forms of cognition that are mediated by the PFC. For example, bilateral microinjection of corticosterone into the mPFC after inhibitory avoidance training enhances memory consolidation (Barsegyan et al., 2010). Although we applied our stressor within the context of the cognitive task, the stressor was not contingent to the particular cognitive processes under study (Sandi and Pinelo-Nava, 2007). This is in agreement with the findings from Diamond and colleagues that showed that exposing rats to an extrinsic stressor during the delay period of a working memory task on a radial arm maze impaired working memory performance but had no effect on long term reference memory (Diamond et al., 1996). Thus, hypotheses can be made about the effect of intrinsic stress on working memory and cognitive flexibility processes employing the DSWSh task or set-shifting task used in the present studies. For example, suppose the only way to escape a particular stressor was to learn which appropriate set of arms would allow the animal to safely return to its home cage after a delay period. In this scenario, it would be hypothesized that acute stress would enhance working memory performance. Similarly, if a  100 rat was exposed to tail shock stress in an operant set-shifting task and had to shift strategies from always pushing the left lever to obtain food reward to always push the lever with a visual-cue illuminated above it to stop the stress, it would be hypothesized that acute stress would facilitate set-shifting.  5.2.6.2 Mesocortical Dopamine Mediates Many Different Behaviours Important During Stress      High levels of PFC DA during stress may be important for facilitating behavioural processes important for stress-related adaptations. A recent paper investigated the dose dependent effects of mPFC DA on a water maze task assessing escape behaviour, the elevated plus maze for anxiolytic/anxiogenic effects, and the tail flick test for nociception. Bilateral infusion of 5 µg of DA into the mPFC increased anxiolytic behaviour and pain sensitivity, whereas, 20 µg of DA enhanced water maze escape and substantially decreased pain sensitivity (Dent and Neill, 2012). In agreement with these findings, a higher tolerance to pain has been linked with excessive DA neurotransmission in humans (Jarcho et al., 2012) and DA depletion within the mPFC has been demonstrated to enhance the susceptibility to stress-induced ulcer formation (Sullivan and Szechtman, 1995). Mesocortical DA may also have a protective function against future stress exposure via its important role in assigning emotional valence to aversive stimuli to determine whether they  should be avoided or not. Activation of the mesocortical DA pathway via phasic stimulation of LHb neurons projecting to the VTA caused a strong conditioned place aversion which was prevented by infusion of a D1 receptor antagonist into the mPFC (Lammel et al., 2012). Furthermore, depletion of DA  101 within the mPFC significantly impaired the extinction of conditioned fear associations (Morrow et al., 1999).      Taken together, these findings suggest that high DA levels released during stress impair complex executive functions but may facilitate stress-related adaptations such as escape from threat, reduced pain sensitivity, and avoidance behaviour. Since the PFC is highly connected to nearly all cortical sensory and motor systems as well as subcortical structures of the brain, it is situated in an important region to provide “top-down control” signals to ensure stress relevant stimulus-response mappings prevail (Miller and Cohen, 2001). More research is required to understand the complex role of prefrontal DA in facilitating other adaptive stress- related behaviours.  5.3 Clinical Significance      Stress is a major factor contributing to the development, recurrence, and treatment outcome in bipolar disorder (Ellicott et al., 1990; Hammen and Gitlin, 1997). Pronounced neurocognitive dysfunction is commonly reported in patients with bipolar disorder (Young et al., 2004; Watson et al., 2006) and may be attributed to alterations in PFC functioning that occur as a result of interactions between stress and DA function (Schatzberg et al., 1985). Our preclinical data indicate that prefrontal activation of GRs during acute stress enhances mesocortical DA efflux by enhancing glutamatergic input to the VTA, resulting in deficits in executive functioning. Attenuation of this elevation in glutamate in the VTA by blockade of prefrontal GRs reduced stress-induced DA efflux in the mPFC and cognitive impairment, and therefore, may represent a unique therapeutic target for treatment of cognitive deficits that accompany bipolar disorder and other forms of psychiatric disorders. Previous studies  102 demonstrate that improved executive function was obtained in patients with bipolar disorder following 7 d of adjunctive treatment with RU-38486 (Young et al., 2004; Watson et al., 2012). Thus, acute blockade of GRs may represent a novel strategy for treatment of psychiatric disorders for which stress appears to be a significant mitigating factor.                     103 References Ago Y, Arikawa S, Yata M, Yano K, Abe M, Takuma K, Matsuda T (2008) Antidepressant- like effects of the glucocorticoid receptor antagonist RU-43044 are associated with changes in prefrontal dopamine in mouse models of depression. Neuropharmacology 55:1355-1363. Ahima RS, Harlan RE (1990) Charting of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system. Neuroscience 39:579-604. Ahn S, Phillips AG (1999) Dopaminergic correlates of sensory-specific satiety in the medial prefrontal cortex and nucleus accumbens of the rat. J Neurosci 19:RC29. Ambroggi F, Turiault M, Milet A, Deroche-Gamonet V, Parnaudeau S, Balado E, Barik J, van der Veen R, Maroteaux G, Lemberger T, Schutz G, Lazar M, Marinelli M, Piazza PV, Tronche F (2009) Stress and addiction: glucocorticoid receptor in dopaminoceptive neurons facilitates cocaine seeking. Nat Neurosci 12:247-249. Andrews MH, Wood SA, Windle RJ, Lightman SL, Ingram CD (2012) Acute glucocorticoid administration rapidly suppresses basal and stress-induced hypothalamo-pituitary- adrenal axis activity. Endocrinology 153:200-211. Anstrom KK, Woodward DJ (2005) Restraint increases dopaminergic burst firing in awake rats. Neuropsychopharmacology 30:1832-1840. Antelman SM, Szechtman H (1975) Tail pinch induces eating in sated rats which appears to depend on nigrostriatal dopamine. Science 189:731-733. Antelman SM, Szechtman H, Chin P, Fisher AE (1975) Tail pinch-induced eating, gnawing and licking behavior in rats: dependence on the nigrostriatal dopamine system. Brain Res 99:319-337. Araki M, McGeer PL, Kimura H (1988) The efferent projections of the rat lateral habenular nucleus revealed by the PHA-L anterograde tracing method. Brain Res 441:319-330. Armario A, Castellanos JM, Balasch J (1984a) Adaptation of anterior pituitary hormones to chronic noise stress in male rats. Behav Neural Biol 41:71-76. Armario A, Castellanos JM, Balasch J (1984b) Effect of acute and chronic psychogenic stress on corticoadrenal and pituitary-thyroid hormones in male rats. Horm Res 20:241-245. Arnsten AF (2009) Stress signalling pathways that impair prefrontal cortex structure and function. Nat Rev Neurosci 10:410-422. Arnsten AF (2011) Prefrontal cortical network connections: key site of vulnerability in stress and schizophrenia. Int J Dev Neurosci 29:215-223. Arnsten AF, Goldman-Rakic PS (1998) Noise stress impairs prefrontal cortical cognitive function in monkeys: evidence for a hyperdopaminergic mechanism. Arch Gen Psychiatry 55:362-368. Arnsten AF, Cai JX, Murphy BL, Goldman-Rakic PS (1994) Dopamine D1 receptor mechanisms in the cognitive performance of young adult and aged monkeys. Psychopharmacology (Berl) 116:143-151. Asagami T, Belanoff JK, Azuma J, Blasey CM, Clark RD, Tsao PS (2011) Selective Glucocorticoid Receptor (GR-II) Antagonist Reduces Body Weight Gain in Mice. J Nutr Metab 2011:235389. Avanzino GL, Ermirio R, Ruggeri P, Cogo CE (1987a) Effects of corticosterone on neurons of reticular formation in rats. Am J Physiol 253:R25-30.  104 Avanzino GL, Ermirio R, Cogo CE, Ruggeri P, Molinari C (1987b) Effects of corticosterone on neurones of the locus coeruleus, in the rat. Neurosci Lett 80:85-88. Baddeley A, Della Sala S (1996) Working memory and executive control. Philos Trans R Soc Lond B Biol Sci 351:1397-1403; discussion 1403-1394. Bagley J, Moghaddam B (1997) Temporal dynamics of glutamate efflux in the prefrontal cortex and in the hippocampus following repeated stress: effects of pretreatment with saline or diazepam. Neuroscience 77:65-73. Balleine BW, Dickinson A (1998) Goal-directed instrumental action: contingency and incentive learning and their cortical substrates. Neuropharmacology 37:407-419. Baratta MV, Lucero TR, Amat J, Watkins LR, Maier SF (2008) Role of the ventral medial prefrontal cortex in mediating behavioral control-induced reduction of later conditioned fear. Learn Mem 15:84-87. Baratta MV, Christianson JP, Gomez DM, Zarza CM, Amat J, Masini CV, Watkins LR, Maier SF (2007) Controllable versus uncontrollable stressors bi-directionally modulate conditioned but not innate fear. Neuroscience 146:1495-1503. Barha CK, Pawluski JL, Galea LA (2007) Maternal care affects male and female offspring working memory and stress reactivity. Physiol Behav 92:939-950. Barrot M, Abrous DN, Marinelli M, Rouge-Pont F, Le Moal M, Piazza PV (2001) Influence of glucocorticoids on dopaminergic transmission in the rat dorsolateral striatum. Eur J Neurosci 13:812-818. Barsegyan A, Mackenzie SM, Kurose BD, McGaugh JL, Roozendaal B (2010) Glucocorticoids in the prefrontal cortex enhance memory consolidation and impair working memory by a common neural mechanism. Proc Natl Acad Sci U S A 107:16655-16660. Beato M, Klug J (2000) Steroid hormone receptors: an update. Hum Reprod Update 6:225- 236. Beato M, Truss M, Chavez S (1996) Control of transcription by steroid hormones. Ann N Y Acad Sci 784:93-123. Beck AT (2008) The evolution of the cognitive model of depression and its neurobiological correlates. Am J Psychiatry 165:969-977. Beck AT, Rector NA (2005) Cognitive approaches to schizophrenia: theory and therapy. Annu Rev Clin Psychol 1:577-606. Belanoff JK, Blasey CM, Clark RD, Roe RL (2011) Selective glucocorticoid receptor (type II) antagonists prevent weight gain caused by olanzapine in rats. Eur J Pharmacol 655:117-120. Benes FM, Berretta S (2001) GABAergic interneurons: implications for understanding schizophrenia and bipolar disorder. Neuropsychopharmacology 25:1-27. Berger B, Gaspar P, Verney C (1991) Dopaminergic innervation of the cerebral cortex: unexpected differences between rodents and primates. Trends Neurosci 14:21-27. Birrell JM, Brown VJ (2000) Medial frontal cortex mediates perceptual attentional set shifting in the rat. J Neurosci 20:4320-4324. Bland ST, Hargrave D, Pepin JL, Amat J, Watkins LR, Maier SF (2003) Stressor controllability modulates stress-induced dopamine and serotonin efflux and morphine-induced serotonin efflux in the medial prefrontal cortex. Neuropsychopharmacology 28:1589-1596.  105 Bondi CO, Rodriguez G, Gould GG, Frazer A, Morilak DA (2008) Chronic unpredictable stress induces a cognitive deficit and anxiety-like behavior in rats that is prevented by chronic antidepressant drug treatment. Neuropsychopharmacology 33:320-331. Boulougouris V, Dalley JW, Robbins TW (2007) Effects of orbitofrontal, infralimbic and prelimbic cortical lesions on serial spatial reversal learning in the rat. Behav Brain Res 179:219-228. Bouton ME, King DA (1983) Contextual control of the extinction of conditioned fear: tests for the associative value of the context. J Exp Psychol Anim Behav Process 9:248- 265. Bromberg-Martin ES, Matsumoto M, Hikosaka O (2010) Dopamine in motivational control: rewarding, aversive, and alerting. Neuron 68:815-834. Brown VJ, Bowman EM (2002) Rodent models of prefrontal cortical function. Trends Neurosci 25:340-343. Brozoski TJ, Brown RM, Rosvold HE, Goldman PS (1979) Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Science 205:929-932. Butts KA, Weinberg J, Young AH, Phillips AG (2011) Glucocorticoid receptors in the prefrontal cortex regulate stress-evoked dopamine efflux and aspects of executive function. Proc Natl Acad Sci U S A 108:18459-18464. Bymaster FP, Katner JS, Nelson DL, Hemrick-Luecke SK, Threlkeld PG, Heiligenstein JH, Morin SM, Gehlert DR, Perry KW (2002) Atomoxetine increases extracellular levels of norepinephrine and dopamine in prefrontal cortex of rat: a potential mechanism for efficacy in attention deficit/hyperactivity disorder. Neuropsychopharmacology 27:699-711. Cabib S, Puglisi-Allegra S (1996) Different effects of repeated stressful experiences on mesocortical and mesolimbic dopamine metabolism. Neuroscience 73:375-380. Camps M, Cortes R, Gueye B, Probst A, Palacios JM (1989) Dopamine receptors in human brain: autoradiographic distribution of D2 sites. Neuroscience 28:275-290. Cannon WB (1939) The Argument for Chemical Mediation of Nerve Impulses. Science 90:521-527. Cannon WB (1940) The Adrenal Medulla. Bull N Y Acad Med 16:3-13. Carr DB, Sesack SR (2000) Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci 20:3864-3873. Cass WA, Gerhardt GA (1995) In vivo assessment of dopamine uptake in rat medial prefrontal cortex: comparison with dorsal striatum and nucleus accumbens. J Neurochem 65:201-207. Cazakoff BN, Johnson KJ, Howland JG (2010) Converging effects of acute stress on spatial and recognition memory in rodents: a review of recent behavioural and pharmacological findings. Prog Neuropsychopharmacol Biol Psychiatry 34:733-741. Cenci MA, Kalen P, Mandel RJ, Bjorklund A (1992) Regional differences in the regulation of dopamine and noradrenaline release in medial frontal cortex, nucleus accumbens and caudate-putamen: a microdialysis study in the rat. Brain Res 581:217-228. Cerqueira JJ, Mailliet F, Almeida OF, Jay TM, Sousa N (2007) The prefrontal cortex as a key target of the maladaptive response to stress. J Neurosci 27:2781-2787.  106 Cerqueira JJ, Pego JM, Taipa R, Bessa JM, Almeida OF, Sousa N (2005) Morphological correlates of corticosteroid-induced changes in prefrontal cortex-dependent behaviors. J Neurosci 25:7792-7800. Chen J, Lipska BK, Halim N, Ma QD, Matsumoto M, Melhem S, Kolachana BS, Hyde TM, Herman MM, Apud J, Egan MF, Kleinman JE, Weinberger DR (2004) Functional analysis of genetic variation in catechol-O-methyltransferase (COMT): effects on mRNA, protein, and enzyme activity in postmortem human brain. Am J Hum Genet 75:807-821. Cho K, Little HJ (1999) Effects of corticosterone on excitatory amino acid responses in dopamine-sensitive neurons in the ventral tegmental area. Neuroscience 88:837-845. Chudasama Y, Robbins TW (2006) Functions of frontostriatal systems in cognition: comparative neuropsychopharmacological studies in rats, monkeys and humans. Biol Psychol 73:19-38. Clarke HF, Walker SC, Dalley JW, Robbins TW, Roberts AC (2007) Cognitive inflexibility after prefrontal serotonin depletion is behaviorally and neurochemically specific. Cereb Cortex 17:18-27. Clarke HF, Walker SC, Crofts HS, Dalley JW, Robbins TW, Roberts AC (2005) Prefrontal serotonin depletion affects reversal learning but not attentional set shifting. J Neurosci 25:532-538. Coutureau E, Killcross S (2003) Inactivation of the infralimbic prefrontal cortex reinstates goal-directed responding in overtrained rats. Behav Brain Res 146:167-174. Dallman MF (2005) Fast glucocorticoid actions on brain: back to the future. Front Neuroendocrinol 26:103-108. Dalm S, Brinks V, van der Mark MH, de Kloet ER, Oitzl MS (2008) Non-invasive stress-free application of glucocorticoid ligands in mice. J Neurosci Methods 170:77-84. Dalton GL, Ma LM, Phillips AG, Floresco SB (2011) Blockade of NMDA GluN2B receptors selectively impairs behavioral flexibility but not initial discrimination learning. Psychopharmacology (Berl) 216:525-535. Danet M, Lapiz-Bluhm S, Morilak DA (2010) A cognitive deficit induced in rats by chronic intermittent cold stress is reversed by chronic antidepressant treatment. Int J Neuropsychopharmacol 13:997-1009. Davis M, Hitchcock JM, Bowers MB, Berridge CW, Melia KR, Roth RH (1994) Stress- induced activation of prefrontal cortex dopamine turnover: blockade by lesions of the amygdala. Brain Res 664:207-210. de Kloet ER, Oitzl MS, Joels M (1999) Stress and cognition: are corticosteroids good or bad guys? Trends Neurosci 22:422-426. de Kloet ER, Joels M, Holsboer F (2005) Stress and the brain: from adaptation to disease. Nat Rev Neurosci 6:463-475. Del Arco A, Mora F (2001) Dopamine release in the prefrontal cortex during stress is reduced by the local activation of glutamate receptors. Brain Res Bull 56:125-130. Dent MF, Neill DB (2012) Dose-dependent effects of prefrontal dopamine on behavioral state in rats. Behav Neurosci 126:620-639. Di Chiara G, Loddo P, Tanda G (1999) Reciprocal changes in prefrontal and limbic dopamine responsiveness to aversive and rewarding stimuli after chronic mild stress: implications for the psychobiology of depression. Biol Psychiatry 46:1624-1633.  107 Di S, Malcher-Lopes R, Halmos KC, Tasker JG (2003) Nongenomic glucocorticoid inhibition via endocannabinoid release in the hypothalamus: a fast feedback mechanism. J Neurosci 23:4850-4857. Diamond DM, Fleshner M, Ingersoll N, Rose GM (1996) Psychological stress impairs spatial working memory: relevance to electrophysiological studies of hippocampal function. Behav Neurosci 110:661-672. Diamond DM, Campbell AM, Park CR, Halonen J, Zoladz PR (2007) The temporal dynamics model of emotional memory processing: a synthesis on the neurobiological basis of stress-induced amnesia, flashbulb and traumatic memories, and the Yerkes- Dodson law. Neural Plast 2007:60803. Diorio D, Viau V, Meaney MJ (1993) The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of hypothalamic-pituitary-adrenal responses to stress. J Neurosci 13:3839-3847. Dong Z, Bai Y, Wu X, Li H, Gong B, Howland JG, Huang Y, He W, Li T, Wang YT (2013) Hippocampal long-term depression mediates spatial reversal learning in the Morris water maze. Neuropharmacology 64:65-73. Dorey R, Pierard C, Chauveau F, David V, Beracochea D (2012) Stress-induced memory retrieval impairments: different time-course involvement of corticosterone and glucocorticoid receptors in dorsal and ventral hippocampus. Neuropsychopharmacology 37:2870-2880. Droste SK, de Groote L, Atkinson HC, Lightman SL, Reul JM, Linthorst AC (2008) Corticosterone levels in the brain show a distinct ultradian rhythm but a delayed response to forced swim stress. Endocrinology 149:3244-3253. Durstewitz D, Seamans JK (2008) The dual-state theory of prefrontal cortex dopamine function with relevance to catechol-o-methyltransferase genotypes and schizophrenia. Biol Psychiatry 64:739-749. Ellicott A, Hammen C, Gitlin M, Brown G, Jamison K (1990) Life events and the course of bipolar disorder. Am J Psychiatry 147:1194-1198. Enrico P, Bouma M, de Vries JB, Westerink BH (1998) The role of afferents to the ventral tegmental area in the handling stress-induced increase in the release of dopamine in the medial prefrontal cortex: a dual-probe microdialysis study in the rat brain. Brain Res 779:205-213. Evans SJ, Murray TF, Moore FL (2000) Partial purification and biochemical characterization of a membrane glucocorticoid receptor from an amphibian brain. J Steroid Biochem Mol Biol 72:209-221. Falkenstein E, Wehling M (2000) Nongenomically initiated steroid actions. Eur J Clin Invest 30 Suppl 3:51-54. Feenstra MG, Botterblom MH, Mastenbroek S (2000) Dopamine and noradrenaline efflux in the prefrontal cortex in the light and dark period: effects of novelty and handling and comparison to the nucleus accumbens. Neuroscience 100:741-748. ffrench-Mullen JM (1995) Cortisol inhibition of calcium currents in guinea pig hippocampal CA1 neurons via G-protein-coupled activation of protein kinase C. J Neurosci 15:903-911. Flores BH, Kenna H, Keller J, Solvason HB, Schatzberg AF (2006) Clinical and biological effects of mifepristone treatment for psychotic depression. Neuropsychopharmacology 31:628-636.  108 Floresco SB, Phillips AG (2001) Delay-dependent modulation of memory retrieval by infusion of a dopamine D1 agonist into the rat medial prefrontal cortex. Behav Neurosci 115:934-939. Floresco SB, Grace AA (2003) Gating of hippocampal-evoked activity in prefrontal cortical neurons by inputs from the mediodorsal thalamus and ventral tegmental area. J Neurosci 23:3930-3943. Floresco SB, Seamans JK, Phillips AG (1997) Selective roles for hippocampal, prefrontal cortical, and ventral striatal circuits in radial-arm maze tasks with or without a delay. J Neurosci 17:1880-1890. Floresco SB, Todd CL, Grace AA (2001) Glutamatergic afferents from the hippocampus to the nucleus accumbens regulate activity of ventral tegmental area dopamine neurons. J Neurosci 21:4915-4922. Floresco SB, Block AE, Tse MT (2008) Inactivation of the medial prefrontal cortex of the rat impairs strategy set-shifting, but not reversal learning, using a novel, automated procedure. Behav Brain Res 190:85-96. Floresco SB, West AR, Ash B, Moore H, Grace AA (2003) Afferent modulation of dopamine neuron firing differentially regulates tonic and phasic dopamine transmission. Nat Neurosci 6:968-973. Floresco SB, Magyar O, Ghods-Sharifi S, Vexelman C, Tse MT (2006) Multiple dopamine receptor subtypes in the medial prefrontal cortex of the rat regulate set-shifting. Neuropsychopharmacology 31:297-309. Francis DD, Zaharia MD, Shanks N, Anisman H (1995) Stress-induced disturbances in Morris water-maze performance: interstrain variability. Physiol Behav 58:57-65. Fuster JM (1973) Unit activity in prefrontal cortex during delayed-response performance: neuronal correlates of transient memory. J Neurophysiol 36:61-78. Fuster JM (2000a) Executive frontal functions. Exp Brain Res 133:66-70. Fuster JM (2000b) Prefrontal neurons in networks of executive memory. Brain Res Bull 52:331-336. Geisler S, Zahm DS (2005) Afferents of the ventral tegmental area in the rat-anatomical substratum for integrative functions. J Comp Neurol 490:270-294. Geuze E, van Wingen GA, van Zuiden M, Rademaker AR, Vermetten E, Kavelaars A, Fernandez G, Heijnen CJ (2012) Glucocorticoid receptor number predicts increase in amygdala activity after severe stress. Psychoneuroendocrinology 37:1837-1844. Ghods-Sharifi S, Haluk DM, Floresco SB (2008) Differential effects of inactivation of the orbitofrontal cortex on strategy set-shifting and reversal learning. Neurobiol Learn Mem 89:567-573. Goldman-Rakic PS (1995) Cellular basis of working memory. Neuron 14:477-485. Goldman-Rakic PS (1996) Regional and cellular fractionation of working memory. Proc Natl Acad Sci U S A 93:13473-13480. Goldman-Rakic PS (1998) The cortical dopamine system: role in memory and cognition. Adv Pharmacol 42:707-711. Goldman-Rakic PS, Leranth C, Williams SM, Mons N, Geffard M (1989) Dopamine synaptic complex with pyramidal neurons in primate cerebral cortex. Proc Natl Acad Sci U S A 86:9015-9019.  109 Goldstein LE, Rasmusson AM, Bunney BS, Roth RH (1996) Role of the amygdala in the coordination of behavioral, neuroendocrine, and prefrontal cortical monoamine responses to psychological stress in the rat. J Neurosci 16:4787-4798. Gomez-Sanchez CE, Zhou MY, Cozza EN, Morita H, Eddleman FC, Gomez-Sanchez EP (1996) Corticosteroid synthesis in the central nervous system. Endocr Res 22:463- 470. Goto Y, Grace AA (2006) Alterations in medial prefrontal cortical activity and plasticity in rats with disruption of cortical development. Biol Psychiatry 60:1259-1267. Grimm S, Schmidt CF, Bermpohl F, Heinzel A, Dahlem Y, Wyss M, Hell D, Boesiger P, Boeker H, Northoff G (2006) Segregated neural representation of distinct emotion dimensions in the prefrontal cortex-an fMRI study. Neuroimage 30:325-340. Groeneweg FL, Karst H, de Kloet ER, Joels M (2011) Rapid non-genomic effects of corticosteroids and their role in the central stress response. J Endocrinol 209:153-167. Groeneweg FL, Karst H, de Kloet ER, Joels M (2012) Mineralocorticoid and glucocorticoid receptors at the neuronal membrane, regulators of nongenomic corticosteroid signalling. Mol Cell Endocrinol 350:299-309. Hains AB, Vu MA, Maciejewski PK, van Dyck CH, Gottron M, Arnsten AF (2009) Inhibition of protein kinase C signaling protects prefrontal cortex dendritic spines and cognition from the effects of chronic stress. Proc Natl Acad Sci U S A 106:17957- 17962. Haller J, Mikics E, Makara GB (2008) The effects of non-genomic glucocorticoid mechanisms on bodily functions and the central neural system. A critical evaluation of findings. Front Neuroendocrinol 29:273-291. Hammen C, Gitlin M (1997) Stress reactivity in bipolar patients and its relation to prior history of disorder. Am J Psychiatry 154:856-857. Hammond GL, Smith CL, Paterson NA, Sibbald WJ (1990) A role for corticosteroid-binding globulin in delivery of cortisol to activated neutrophils. J Clin Endocrinol Metab 71:34-39. Harfstrand A, Fuxe K, Cintra A, Agnati LF, Zini I, Wikstrom AC, Okret S, Yu ZY, Goldstein M, Steinbusch H, et al. (1986) Glucocorticoid receptor immunoreactivity in monoaminergic neurons of rat brain. Proc Natl Acad Sci U S A 83:9779-9783. Hascup ER, Hascup KN, Pomerleau F, Huettl P, Hajos-Korcsok E, Kehr J, Gerhardt GA (2012) An allosteric modulator of metabotropic glutamate receptors (mGluR(2)), (+)- TFMPIP, inhibits restraint stress-induced phasic glutamate release in rat prefrontal cortex. J Neurochem 122:619-627. Hascup KN, Hascup ER, Stephens ML, Glaser PE, Yoshitake T, Mathe AA, Gerhardt GA, Kehr J (2011) Resting glutamate levels and rapid glutamate transients in the prefrontal cortex of the Flinders Sensitive Line rat: a genetic rodent model of depression. Neuropsychopharmacology 36:1769-1777. Hauger RL, Risbrough V, Brauns O, Dautzenberg FM (2006) Corticotropin releasing factor (CRF) receptor signaling in the central nervous system: new molecular targets. CNS Neurol Disord Drug Targets 5:453-479. Heikinheimo O, Kekkonen R (1993) Dose-response relationships of RU 486. Ann Med 25:71-76. Henckens MJ, van Wingen GA, Joels M, Fernandez G (2012) Corticosteroid induced decoupling of the amygdala in men. Cereb Cortex 22:2336-2345.  110 Hill JE, Makky K, Shrestha L, Hillard CJ, Gasser PJ (2010a) Natural and synthetic corticosteroids inhibit uptake(2)-mediated transport in CNS neurons. Physiol Behav. Hill MN, Patel S, Campolongo P, Tasker JG, Wotjak CT, Bains JS (2010b) Functional interactions between stress and the endocannabinoid system: from synaptic signaling to behavioral output. J Neurosci 30:14980-14986. Hill MN, McLaughlin RJ, Pan B, Fitzgerald ML, Roberts CJ, Lee TT, Karatsoreos IN, Mackie K, Viau V, Pickel VM, McEwen BS, Liu QS, Gorzalka BB, Hillard CJ (2011) Recruitment of prefrontal cortical endocannabinoid signaling by glucocorticoids contributes to termination of the stress response. J Neurosci 31:10506-10515. Hurley KM, Herbert H, Moga MM, Saper CB (1991) Efferent projections of the infralimbic cortex of the rat. J Comp Neurol 308:249-276. Imperato A, Puglisi-Allegra S, Casolini P, Angelucci L (1991) Changes in brain dopamine and acetylcholine release during and following stress are independent of the pituitary- adrenocortical axis. Brain Res 538:111-117. Imperato A, Puglisi-Allegra S, Casolini P, Zocchi A, Angelucci L (1989) Stress-induced enhancement of dopamine and acetylcholine release in limbic structures: role of corticosterone. Eur J Pharmacol 165:337-338. Imperato A, Angelucci L, Casolini P, Zocchi A, Puglisi-Allegra S (1992) Repeated stressful experiences differently affect limbic dopamine release during and following stress. Brain Res 577:194-199. Inoue T, Tsuchiya K, Koyama T (1994) Regional changes in dopamine and serotonin activation with various intensity of physical and psychological stress in the rat brain. Pharmacol Biochem Behav 49:911-920. Jackson DM, Westlind-Danielsson A (1994) Dopamine receptors: molecular biology, biochemistry and behavioural aspects. Pharmacol Ther 64:291-370. Jarcho JM, Mayer EA, Jiang ZK, Feier NA, London ED (2012) Pain, affective symptoms, and cognitive deficits in patients with cerebral dopamine dysfunction. Pain 153:744- 754. Jedema HP, Moghaddam B (1994) Glutamatergic control of dopamine release during stress in the rat prefrontal cortex. J Neurochem 63:785-788. Ji H, Shepard PD (2007) Lateral habenula stimulation inhibits rat midbrain dopamine neurons through a GABA(A) receptor-mediated mechanism. J Neurosci 27:6923- 6930. Joels M, Pu Z, Wiegert O, Oitzl MS, Krugers HJ (2006) Learning under stress: how does it work? Trends Cogn Sci 10:152-158. Johnson LR, Farb C, Morrison JH, McEwen BS, LeDoux JE (2005) Localization of glucocorticoid receptors at postsynaptic membranes in the lateral amygdala. Neuroscience 136:289-299. Jones B, Mishkin M (1972) Limbic lesions and the problem of stimulus--reinforcement associations. Exp Neurol 36:362-377. Jovanovic JN, Czernik AJ, Fienberg AA, Greengard P, Sihra TS (2000) Synapsins as mediators of BDNF-enhanced neurotransmitter release. Nat Neurosci 3:323-329. Keim KL, Sigg EB (1976) Physiological and biochemical concomitants of restraint stress in rats. Pharmacol Biochem Behav 4:289-297.  111 Kern S, Oakes TR, Stone CK, McAuliff EM, Kirschbaum C, Davidson RJ (2008) Glucose metabolic changes in the prefrontal cortex are associated with HPA axis response to a psychosocial stressor. Psychoneuroendocrinology 33:517-529. Kino T, Tiulpakov A, Ichijo T, Chheng L, Kozasa T, Chrousos GP (2005) G protein beta interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus. J Cell Biol 169:885-896. Kolb B, Nonneman AJ, Singh RK (1974) Double dissociation of spatial impairments and perseveration following selective prefrontal lesions in rats. J Comp Physiol Psychol 87:772-780. Kolber BJ, Roberts MS, Howell MP, Wozniak DF, Sands MS, Muglia LJ (2008) Central amygdala glucocorticoid receptor action promotes fear-associated CRH activation and conditioning. Proc Natl Acad Sci U S A 105:12004-12009. Koolhaas JM, Bartolomucci A, Buwalda B, de Boer SF, Flugge G, Korte SM, Meerlo P, Murison R, Olivier B, Palanza P, Richter-Levin G, Sgoifo A, Steimer T, Stiedl O, van Dijk G, Wohr M, Fuchs E (2011) Stress revisited: a critical evaluation of the stress concept. Neurosci Biobehav Rev 35:1291-1301. Krettek JE, Price JL (1977) The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat. J Comp Neurol 171:157-191. Lachman HM, Papolos DF, Saito T, Yu YM, Szumlanski CL, Weinshilboum RM (1996) Human catechol-O-methyltransferase pharmacogenetics: description of a functional polymorphism and its potential application to neuropsychiatric disorders. Pharmacogenetics 6:243-250. Lammel S, Ion DI, Roeper J, Malenka RC (2011) Projection-specific modulation of dopamine neuron synapses by aversive and rewarding stimuli. Neuron 70:855-862. Lammel S, Hetzel A, Hackel O, Jones I, Liss B, Roeper J (2008) Unique properties of mesoprefrontal neurons within a dual mesocorticolimbic dopamine system. Neuron 57:760-773. Lammel S, Lim BK, Ran C, Huang KW, Betley MJ, Tye KM, Deisseroth K, Malenka RC (2012) Input-specific control of reward and aversion in the ventral tegmental area. Nature. Lapiz-Bluhm MD, Soto-Pina AE, Hensler JG, Morilak DA (2009) Chronic intermittent cold stress and serotonin depletion induce deficits of reversal learning in an attentional set- shifting test in rats. Psychopharmacology (Berl) 202:329-341. Lataster J, Collip D, Ceccarini J, Haas D, Booij L, van Os J, Pruessner J, Van Laere K, Myin- Germeys I (2011) Psychosocial stress is associated with in vivo dopamine release in human ventromedial prefrontal cortex: a positron emission tomography study using [(1)(8)F]fallypride. Neuroimage 58:1081-1089. Lee YA, Goto Y (2011) Chronic stress modulation of prefrontal cortical NMDA receptor expression disrupts limbic structure-prefrontal cortex interaction. Eur J Neurosci 34:426-436. Lemos JC, Wanat MJ, Smith JS, Reyes BA, Hollon NG, Van Bockstaele EJ, Chavkin C, Phillips PE (2012) Severe stress switches CRF action in the nucleus accumbens from appetitive to aversive. Nature 490:402-406. Lewis JG, Bagley CJ, Elder PA, Bachmann AW, Torpy DJ (2005) Plasma free cortisol fraction reflects levels of functioning corticosteroid-binding globulin. Clin Chim Acta 359:189-194.  112 Lezcano N, Bergson C (2002) D1/D5 dopamine receptors stimulate intracellular calcium release in primary cultures of neocortical and hippocampal neurons. J Neurophysiol 87:2167-2175. Lidow MS, Goldman-Rakic PS, Gallager DW, Rakic P (1991) Distribution of dopaminergic receptors in the primate cerebral cortex: quantitative autoradiographic analysis using [3H]raclopride, [3H]spiperone and [3H]SCH23390. Neuroscience 40:657-671. Lindley SE, She X, Schatzberg AF (2005) Monoamine oxidase and catechol-o- methyltransferase enzyme activity and gene expression in response to sustained glucocorticoids. Psychoneuroendocrinology 30:785-790. Lindley SE, Bengoechea TG, Schatzberg AF, Wong DL (1999) Glucocorticoid effects on mesotelencephalic dopamine neurotransmission. Neuropsychopharmacology 21:399- 407. Lindley SE, Bengoechea TG, Wong DL, Schatzberg AF (2002) Mesotelencephalic dopamine neurochemical responses to glucocorticoid administration and adrenalectomy in Fischer 344 and Lewis rats. Brain Res 958:414-422. Lindvall O, Bjorklund A, Skagerberg G (1984) Selective histochemical demonstration of dopamine terminal systems in rat di- and telencephalon: new evidence for dopaminergic innervation of hypothalamic neurosecretory nuclei. Brain Res 306:19- 30. Liposits Z, Bohn MC (1993) Association of glucocorticoid receptor immunoreactivity with cell membrane and transport vesicles in hippocampal and hypothalamic neurons of the rat. J Neurosci Res 35:14-19. Liston C, Miller MM, Goldwater DS, Radley JJ, Rocher AB, Hof PR, Morrison JH, McEwen BS (2006) Stress-induced alterations in prefrontal cortical dendritic morphology predict selective impairments in perceptual attentional set-shifting. J Neurosci 26:7870-7874. Losel RM, Wehling M (2008) Classic versus non-classic receptors for nongenomic mineralocorticoid responses: emerging evidence. Front Neuroendocrinol 29:258-267. Lupien SJ, Gillin CJ, Hauger RL (1999) Working memory is more sensitive than declarative memory to the acute effects of corticosteroids: a dose-response study in humans. Behav Neurosci 113:420-430. Lupien SJ, McEwen BS, Gunnar MR, Heim C (2009) Effects of stress throughout the lifespan on the brain, behaviour and cognition. Nat Rev Neurosci 10:434-445. Lupien SJ, Fiocco A, Wan N, Maheu F, Lord C, Schramek T, Tu MT (2005) Stress hormones and human memory function across the lifespan. Psychoneuroendocrinology 30:225- 242. Maier SF, Watkins LR (2010) Role of the medial prefrontal cortex in coping and resilience. Brain Res 1355:52-60. Maier SF, Ryan SM, Barksdale CM, Kalin NH (1986) Stressor controllability and the pituitary-adrenal system. Behav Neurosci 100:669-674. Marinelli M, Piazza PV (2002) Interaction between glucocorticoid hormones, stress and psychostimulant drugs. Eur J Neurosci 16:387-394. Marinelli M, Aouizerate B, Barrot M, Le Moal M, Piazza PV (1998) Dopamine-dependent responses to morphine depend on glucocorticoid receptors. Proc Natl Acad Sci U S A 95:7742-7747.  113 McEwen BS, De Kloet ER, Rostene W (1986) Adrenal steroid receptors and actions in the nervous system. Physiol Rev 66:1121-1188. Mellon SH, Deschepper CF (1993) Neurosteroid biosynthesis: genes for adrenal steroidogenic enzymes are expressed in the brain. Brain Res 629:283-292. Mikulaj L, Kvetnansky R, Murgas K (1974) Changes in adrenal response during intermittent and repeated stress. Rev Czech Med 20:162-169. Miller DK, Rodvelt KR, Constales C, Putnam WC (2007) Analogs of SR-141716A (Rimonabant) alter d-amphetamine-evoked [3H] dopamine overflow from preloaded striatal slices and amphetamine-induced hyperactivity. Life Sci 81:63-71. Miller EK, Cohen JD (2001) An integrative theory of prefrontal cortex function. Annu Rev Neurosci 24:167-202. Minton GO, Young AH, McQuade R, Fairchild G, Ingram CD, Gartside SE (2009) Profound changes in dopaminergic neurotransmission in the prefrontal cortex in response to flattening of the diurnal glucocorticoid rhythm: implications for bipolar disorder. Neuropsychopharmacology 34:2265-2274. Missale C, Nash SR, Robinson SW, Jaber M, Caron MG (1998) Dopamine receptors: from structure to function. Physiol Rev 78:189-225. Mizoguchi K, Ishige A, Takeda S, Aburada M, Tabira T (2004) Endogenous glucocorticoids are essential for maintaining prefrontal cortical cognitive function. J Neurosci 24:5492-5499. Mizoguchi K, Shoji H, Ikeda R, Tanaka Y, Tabira T (2008) Persistent depressive state after chronic stress in rats is accompanied by HPA axis dysregulation and reduced prefrontal dopaminergic neurotransmission. Pharmacol Biochem Behav 91:170-175. Mizoguchi K, Yuzurihara M, Ishige A, Sasaki H, Chui DH, Tabira T (2000) Chronic stress induces impairment of spatial working memory because of prefrontal dopaminergic dysfunction. J Neurosci 20:1568-1574. Moghaddam B (1993) Stress preferentially increases extraneuronal levels of excitatory amino acids in the prefrontal cortex: comparison to hippocampus and basal ganglia. J Neurochem 60:1650-1657. Moore GJ, Cortese BM, Glitz DA, Zajac-Benitez C, Quiroz JA, Uhde TW, Drevets WC, Manji HK (2009) A longitudinal study of the effects of lithium treatment on prefrontal and subgenual prefrontal gray matter volume in treatment-responsive bipolar disorder patients. J Clin Psychiatry 70:699-705. Moore H, Rose HJ, Grace AA (2001) Chronic cold stress reduces the spontaneous activity of ventral tegmental dopamine neurons. Neuropsychopharmacology 24:410-419. Moron JA, Brockington A, Wise RA, Rocha BA, Hope BT (2002) Dopamine uptake through the norepinephrine transporter in brain regions with low levels of the dopamine transporter: evidence from knock-out mouse lines. J Neurosci 22:389-395. Morrow BA, Roth RH, Elsworth JD (2000) TMT, a predator odor, elevates mesoprefrontal dopamine metabolic activity and disrupts short-term working memory in the rat. Brain Res Bull 52:519-523. Morrow BA, Elsworth JD, Rasmusson AM, Roth RH (1999) The role of mesoprefrontal dopamine neurons in the acquisition and expression of conditioned fear in the rat. Neuroscience 92:553-564.  114 Murphy BL, Arnsten AF, Jentsch JD, Roth RH (1996a) Dopamine and spatial working memory in rats and monkeys: pharmacological reversal of stress-induced impairment. J Neurosci 16:7768-7775. Murphy BL, Arnsten AF, Goldman-Rakic PS, Roth RH (1996b) Increased dopamine turnover in the prefrontal cortex impairs spatial working memory performance in rats and monkeys. Proc Natl Acad Sci U S A 93:1325-1329. Murphy EK, Sved AF, Finlay JM (2003) Corticotropin-releasing hormone receptor blockade fails to alter stress-evoked catecholamine release in prefrontal cortex of control or chronically stressed rats. Neuroscience 116:1081-1087. Natelson BH, Ottenweller JE, Cook JA, Pitman D, McCarty R, Tapp WN (1988) Effect of stressor intensity on habituation of the adrenocortical stress response. Physiol Behav 43:41-46. Nichols RA, Chilcote TJ, Czernik AJ, Greengard P (1992) Synapsin I regulates glutamate release from rat brain synaptosomes. J Neurochem 58:783-785. Nikiforuk A (2012) Selective blockade of 5-HT7 receptors facilitates attentional set-shifting in stressed and control rats. Behav Brain Res 226:118-123. Nikiforuk A (2013) Quetiapine ameliorates stress-induced cognitive inflexibility in rats. Neuropharmacology 64:357-364. Nikiforuk A, Popik P (2011) Long-lasting cognitive deficit induced by stress is alleviated by acute administration of antidepressants. Psychoneuroendocrinology 36:28-39. O'Connor KA, Johnson JD, Hammack SE, Brooks LM, Spencer RL, Watkins LR, Maier SF (2003) Inescapable shock induces resistance to the effects of dexamethasone. Psychoneuroendocrinology 28:481-500. O'Hara CM, Tang L, Taussig R, Todd RD, O'Malley KL (1996) Dopamine D2L receptor couples to G alpha i2 and G alpha i3 but not G alpha i1, leading to the inhibition of adenylate cyclase in transfected cell lines. J Pharmacol Exp Ther 278:354-360. Oades RD, Halliday GM (1987) Ventral tegmental (A10) system: neurobiology. 1. Anatomy and connectivity. Brain Res 434:117-165. Olton DS (1979) Mazes, maps, and memory. Am Psychol 34:583-596. Orchinik M, Murray TF, Moore FL (1991) A corticosteroid receptor in neuronal membranes. Science 252:1848-1851. Ottaviani E, Caselgrandi E, Petraglia F, Franceschi C (1992) Stress response in the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata): interaction between CRF, ACTH, and biogenic amines. Gen Comp Endocrinol 87:354-360. Ouimet CC, LaMantia AS, Goldman-Rakic P, Rakic P, Greengard P (1992) Immunocytochemical localization of DARPP-32, a dopamine and cyclic-AMP- regulated phosphoprotein, in the primate brain. J Comp Neurol 323:209-218. Packard MG, White NM (1989) Memory facilitation produced by dopamine agonists: role of receptor subtype and mnemonic requirements. Pharmacol Biochem Behav 33:511- 518. Paxinos GW, C. (1997) The rat brain in stereotaxic coordinates, 3rd Edition. San Diego: Academic. Phillips AG, Ahn S, Floresco SB (2004) Magnitude of dopamine release in medial prefrontal cortex predicts accuracy of memory on a delayed response task. J Neurosci 24:547- 553.  115 Phillips AG, Vacca G, Ahn S (2008) A top-down perspective on dopamine, motivation and memory. Pharmacol Biochem Behav 90:236-249. Piazza PV, Marinelli M, Rouge-Pont F, Deroche V, Maccari S, Simon H, Le Moal M (1996) Stress, glucocorticoids, and mesencephalic dopaminergic neurons: a pathophysiological chain determining vulnerability to psychostimulant abuse. NIDA Res Monogr 163:277-299. Plessow F, Kiesel A, Kirschbaum C (2012) The stressed prefrontal cortex and goal-directed behaviour: acute psychosocial stress impairs the flexible implementation of task goals. Exp Brain Res 216:397-408. Plotsky PM, Otto S, Sapolsky RM (1986) Inhibition of immunoreactive corticotropin- releasing factor secretion into the hypophysial-portal circulation by delayed glucocorticoid feedback. Endocrinology 119:1126-1130. Pollard I, Bassett JR, Cairncross KD (1976) Plasma glucocorticoid elevation and ultrastructural changes in the adenohypophysis of the male rat following prolonged exposure to stress. Neuroendocrinology 21:312-330. Prager EM, Johnson LR (2009) Stress at the synapse: signal transduction mechanisms of adrenal steroids at neuronal membranes. Sci Signal 2:re5. Qian X, Droste SK, Gutierrez-Mecinas M, Collins A, Kersante F, Reul JM, Linthorst AC (2011) A rapid release of corticosteroid-binding globulin from the liver restrains the glucocorticoid hormone response to acute stress. Endocrinology 152:3738-3748. Qin S, Hermans EJ, van Marle HJ, Luo J, Fernandez G (2009) Acute psychological stress reduces working memory-related activity in the dorsolateral prefrontal cortex. Biol Psychiatry 66:25-32. Qin S, Cousijn H, Rijpkema M, Luo J, Franke B, Hermans EJ, Fernandez G (2012) The effect of moderate acute psychological stress on working memory-related neural activity is modulated by a genetic variation in catecholaminergic function in humans. Front Integr Neurosci 6:16. Radley JJ, Sawchenko PE (2011) A common substrate for prefrontal and hippocampal inhibition of the neuroendocrine stress response. J Neurosci 31:9683-9695. Radley JJ, Arias CM, Sawchenko PE (2006) Regional differentiation of the medial prefrontal cortex in regulating adaptive responses to acute emotional stress. J Neurosci 26:12967-12976. Ragozzino ME (2002) The effects of dopamine D(1) receptor blockade in the prelimbic- infralimbic areas on behavioral flexibility. Learn Mem 9:18-28. Ragozzino ME, Detrick S, Kesner RP (1999) Involvement of the prelimbic-infralimbic areas of the rodent prefrontal cortex in behavioral flexibility for place and response learning. J Neurosci 19:4585-4594. Reul JM, de Kloet ER (1985) Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117:2505-2511. Revest JM, Di Blasi F, Kitchener P, Rouge-Pont F, Desmedt A, Turiault M, Tronche F, Piazza PV (2005) The MAPK pathway and Egr-1 mediate stress-related behavioral effects of glucocorticoids. Nat Neurosci 8:664-672. Revest JM, Kaouane N, Mondin M, Le Roux A, Rouge-Pont F, Vallee M, Barik J, Tronche F, Desmedt A, Piazza PV (2010) The enhancement of stress-related memory by glucocorticoids depends on synapsin-Ia/Ib. Mol Psychiatry 15:1125, 1140-1151.  116 Robbins TW (1996) Dissociating executive functions of the prefrontal cortex. Philos Trans R Soc Lond B Biol Sci 351:1463-1470; discussion 1470-1461. Roozendaal B (2002) Stress and memory: opposing effects of glucocorticoids on memory consolidation and memory retrieval. Neurobiol Learn Mem 78:578-595. Roozendaal B, McReynolds JR, McGaugh JL (2004) The basolateral amygdala interacts with the medial prefrontal cortex in regulating glucocorticoid effects on working memory impairment. J Neurosci 24:1385-1392. Roozendaal B, McReynolds JR, Van der Zee EA, Lee S, McGaugh JL, McIntyre CK (2009) Glucocorticoid effects on memory consolidation depend on functional interactions between the medial prefrontal cortex and basolateral amygdala. J Neurosci 29:14299- 14308. Rouge-Pont F, Marinelli M, Le Moal M, Simon H, Piazza PV (1995) Stress-induced sensitization and glucocorticoids. II. Sensitization of the increase in extracellular dopamine induced by cocaine depends on stress-induced corticosterone secretion. J Neurosci 15:7189-7195. Saal D, Dong Y, Bonci A, Malenka RC (2003) Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons. Neuron 37:577-582. Sandi C, Pinelo-Nava MT (2007) Stress and memory: behavioral effects and neurobiological mechanisms. Neural Plast 2007:78970. Sandi C, Loscertales M, Guaza C (1997) Experience-dependent facilitating effect of corticosterone on spatial memory formation in the water maze. Eur J Neurosci 9:637- 642. Sapolsky RM (2000) Stress hormones: good and bad. Neurobiol Dis 7:540-542. Sapolsky RM, Romero LM, Munck AU (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55-89. Sawaguchi T, Goldman-Rakic PS (1994) The role of D1-dopamine receptor in working memory: local injections of dopamine antagonists into the prefrontal cortex of rhesus monkeys performing an oculomotor delayed-response task. J Neurophysiol 71:515- 528. Sawaguchi T, Matsumura M, Kubota K (1990) Effects of dopamine antagonists on neuronal activity related to a delayed response task in monkey prefrontal cortex. J Neurophysiol 63:1401-1412. Schatzberg AF, Rothschild AJ, Langlais PJ, Bird ED, Cole JO (1985) A corticosteroid/dopamine hypothesis for psychotic depression and related states. J Psychiatr Res 19:57-64. Schoofs D, Wolf OT, Smeets T (2009) Cold pressor stress impairs performance on working memory tasks requiring executive functions in healthy young men. Behav Neurosci 123:1066-1075. Schulz S, Arning L, Pinnow M, Wascher E, Epplen JT, Beste C (2012) When control fails: influence of the prefrontal but not striatal dopaminergic system on behavioural flexibility in a change detection task. Neuropharmacology 62:1028-1033. Schwabe L, Wolf OT (2009) The context counts: congruent learning and testing environments prevent memory retrieval impairment following stress. Cogn Affect Behav Neurosci 9:229-236.  117 Schwabe L, Bohringer A, Wolf OT (2009) Stress disrupts context-dependent memory. Learn Mem 16:110-113. Seamans JK, Yang CR (2004) The principal features and mechanisms of dopamine modulation in the prefrontal cortex. Prog Neurobiol 74:1-58. Seamans JK, Floresco SB, Phillips AG (1995) Functional differences between the prelimbic and anterior cingulate regions of the rat prefrontal cortex. Behav Neurosci 109:1063- 1073. Seamans JK, Floresco SB, Phillips AG (1998) D1 receptor modulation of hippocampal- prefrontal cortical circuits integrating spatial memory with executive functions in the rat. J Neurosci 18:1613-1621. Seamans JK, Gorelova N, Durstewitz D, Yang CR (2001a) Bidirectional dopamine modulation of GABAergic inhibition in prefrontal cortical pyramidal neurons. J Neurosci 21:3628-3638. Seamans JK, Durstewitz D, Christie BR, Stevens CF, Sejnowski TJ (2001b) Dopamine D1/D5 receptor modulation of excitatory synaptic inputs to layer V prefrontal cortex neurons. Proc Natl Acad Sci U S A 98:301-306. Seguela P, Watkins KC, Descarries L (1988) Ultrastructural features of dopamine axon terminals in the anteromedial and the suprarhinal cortex of adult rat. Brain Res 442:11-22. Selye H (1937) The Significance of the Adrenals for Adaptation. Science 85:247-248. Selye H (1946) The general adaptation syndrome and the diseases of adaptation. J Clin Endocrinol Metab 6:117-230. Selye H (1950) Stress and the general adaptation syndrome. Br Med J 1:1383-1392. Selye H, Fortier C (1950) Adaptive reaction to stress. Psychosom Med 12:149-157. Sesack SR, Pickel VM (1992) Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area. J Comp Neurol 320:145-160. Sesack SR, Snyder CL, Lewis DA (1995) Axon terminals immunolabeled for dopamine or tyrosine hydroxylase synapse on GABA-immunoreactive dendrites in rat and monkey cortex. J Comp Neurol 363:264-280. Sesack SR, Deutch AY, Roth RH, Bunney BS (1989) Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: an anterograde tract- tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 290:213-242. Sesack SR, Hawrylak VA, Guido MA, Levey AI (1998a) Cellular and subcellular localization of the dopamine transporter in rat cortex. Adv Pharmacol 42:171-174. Sesack SR, Hawrylak VA, Matus C, Guido MA, Levey AI (1998b) Dopamine axon varicosities in the prelimbic division of the rat prefrontal cortex exhibit sparse immunoreactivity for the dopamine transporter. J Neurosci 18:2697-2708. Shansky RM, Rubinow K, Brennan A, Arnsten AF (2006) The effects of sex and hormonal status on restraint-stress-induced working memory impairment. Behav Brain Funct 2:8. Sidhu A, Kimura K (1997) A novel regulation of expression of the alpha-subunit of the G stimulatory protein by dopamine via D1 dopamine receptors. J Neurochem 68:187- 194. Sousa VC, Assaife-Lopes N, Ribeiro JA, Pratt JA, Brett RR, Sebastiao AM (2011) Regulation of hippocampal cannabinoid CB1 receptor actions by adenosine A1  118 receptors and chronic caffeine administration: implications for the effects of Delta9- tetrahydrocannabinol on spatial memory. Neuropsychopharmacology 36:472-487. Stark R, Wolf OT, Tabbert K, Kagerer S, Zimmermann M, Kirsch P, Schienle A, Vaitl D (2006) Influence of the stress hormone cortisol on fear conditioning in humans: evidence for sex differences in the response of the prefrontal cortex. Neuroimage 32:1290-1298. Stromstedt M, Waterman MR (1995) Messenger RNAs encoding steroidogenic enzymes are expressed in rodent brain. Brain Res Mol Brain Res 34:75-88. Sullivan RM, Szechtman H (1995) Asymmetrical influence of mesocortical dopamine depletion on stress ulcer development and subcortical dopamine systems in rats: implications for psychopathology. Neuroscience 65:757-766. Sullivan RM, Gratton A (1999) Lateralized effects of medial prefrontal cortex lesions on neuroendocrine and autonomic stress responses in rats. J Neurosci 19:2834-2840. Suyemitsu T, Terayama H (1975) Specific binding sites for natural glucocorticoids in plasma membranes of rat liver. Endocrinology 96:1499-1508. Taber MT, Fibiger HC (1997) Activation of the mesocortical dopamine system by feeding: lack of a selective response to stress. Neuroscience 77:295-298. Taepavarapruk P, Howland JG, Ahn S, Phillips AG (2008) Neural circuits engaged in ventral hippocampal modulation of dopamine function in medial prefrontal cortex and ventral striatum. Brain Struct Funct 213:183-195. Tagliaferro P, Morales M (2008) Synapses between corticotropin-releasing factor-containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic. J Comp Neurol 506:616-626. Takahata R, Moghaddam B (1998) Glutamatergic regulation of basal and stimulus-activated dopamine release in the prefrontal cortex. J Neurochem 71:1443-1449. Tasker JG, Di S, Malcher-Lopes R (2006) Minireview: rapid glucocorticoid signaling via membrane-associated receptors. Endocrinology 147:5549-5556. Thai CA, Zhang Y, Howland JG (2012) Effects of acute restraint stress on set-shifting and reversal learning in male rats. Cogn Affect Behav Neurosci. Thierry AM, Tassin JP, Blanc G, Glowinski J (1976) Selective activation of mesocortical DA system by stress. Nature 263:242-244. Trantham-Davidson H, Neely LC, Lavin A, Seamans JK (2004) Mechanisms underlying differential D1 versus D2 dopamine receptor regulation of inhibition in prefrontal cortex. J Neurosci 24:10652-10659. Tronel S, Alberini CM (2007) Persistent disruption of a traumatic memory by postretrieval inactivation of glucocorticoid receptors in the amygdala. Biol Psychiatry 62:33-39. Tseng KY, O'Donnell P (2004) Dopamine-glutamate interactions controlling prefrontal cortical pyramidal cell excitability involve multiple signaling mechanisms. J Neurosci 24:5131-5139. Tunbridge EM, Bannerman DM, Sharp T, Harrison PJ (2004) Catechol-o-methyltransferase inhibition improves set-shifting performance and elevates stimulated dopamine release in the rat prefrontal cortex. J Neurosci 24:5331-5335. Ulrich-Lai YM, Herman JP (2009) Neural regulation of endocrine and autonomic stress responses. Nat Rev Neurosci 10:397-409. Uylings HB, van Eden CG (1990) Qualitative and quantitative comparison of the prefrontal cortex in rat and in primates, including humans. Prog Brain Res 85:31-62.  119 Valenti O, Grace AA (2008) Acute and repeated stress induce a pronounced and sustained activation of VTA DA neuron population activity. Society for Neuroscience Abstract 47911. Valenti O, Lodge DJ, Grace AA (2011) Aversive stimuli alter ventral tegmental area dopamine neuron activity via a common action in the ventral hippocampus. J Neurosci 31:4280-4289. Valenti O, Gill KM, Grace AA (2012) Different stressors produce excitation or inhibition of mesolimbic dopamine neuron activity: response alteration by stress pre-exposure. Eur J Neurosci 35:1312-1321. Van Eden CG, Hoorneman EM, Buijs RM, Matthijssen MA, Geffard M, Uylings HB (1987) Immunocytochemical localization of dopamine in the prefrontal cortex of the rat at the light and electron microscopical level. Neuroscience 22:849-862. Vijayraghavan S, Wang M, Birnbaum SG, Williams GV, Arnsten AF (2007) Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory. Nat Neurosci 10:376-384. Vincent SL, Khan Y, Benes FM (1993) Cellular distribution of dopamine D1 and D2 receptors in rat medial prefrontal cortex. J Neurosci 13:2551-2564. Vizueta N, Rudie JD, Townsend JD, Torrisi S, Moody TD, Bookheimer SY, Altshuler LL (2012) Regional fMRI hypoactivation and altered functional connectivity during emotion processing in nonmedicated depressed patients with bipolar II disorder. Am J Psychiatry 169:831-840. Wanat MJ, Hopf FW, Stuber GD, Phillips PE, Bonci A (2008) Corticotropin-releasing factor increases mouse ventral tegmental area dopamine neuron firing through a protein kinase C-dependent enhancement of Ih. J Physiol 586:2157-2170. Wang J, Fang Q, Liu Z, Lu L (2006) Region-specific effects of brain corticotropin-releasing factor receptor type 1 blockade on footshock-stress- or drug-priming-induced reinstatement of morphine conditioned place preference in rats. Psychopharmacology (Berl) 185:19-28. Wang J, Korczykowski M, Rao H, Fan Y, Pluta J, Gur RC, McEwen BS, Detre JA (2007) Gender difference in neural response to psychological stress. Soc Cogn Affect Neurosci 2:227-239. Watson S, Thompson JM, Ritchie JC, Nicol Ferrier I, Young AH (2006) Neuropsychological impairment in bipolar disorder: the relationship with glucocorticoid receptor function. Bipolar Disord 8:85-90. Watson S, Gallagher P, Porter RJ, Smith MS, Herron LJ, Bulmer S, Young AH, Ferrier IN (2012) A randomized trial to examine the effect of mifepristone on neuropsychological performance and mood in patients with bipolar depression. Biol Psychiatry 72:943-949. Wayment HK, Schenk JO, Sorg BA (2001) Characterization of extracellular dopamine clearance in the medial prefrontal cortex: role of monoamine uptake and monoamine oxidase inhibition. J Neurosci 21:35-44. Wise RA, Morales M (2010) A ventral tegmental CRF-glutamate-dopamine interaction in addiction. Brain Res 1314:38-43. Wise SP (2008) Forward frontal fields: phylogeny and fundamental function. Trends Neurosci 31:599-608.  120 Young AH, Gallagher P, Watson S, Del-Estal D, Owen BM, Ferrier IN (2004) Improvements in neurocognitive function and mood following adjunctive treatment with mifepristone (RU-486) in bipolar disorder. Neuropsychopharmacology 29:1538-1545. Yuen EY, Liu W, Karatsoreos IN, Feng J, McEwen BS, Yan Z (2009) Acute stress enhances glutamatergic transmission in prefrontal cortex and facilitates working memory. Proc Natl Acad Sci U S A 106:14075-14079. Yuen EY, Liu W, Karatsoreos IN, Ren Y, Feng J, McEwen BS, Yan Z (2011) Mechanisms for acute stress-induced enhancement of glutamatergic transmission and working memory. Mol Psychiatry 16:156-170. Zahrt J, Taylor JR, Mathew RG, Arnsten AF (1997) Supranormal stimulation of D1 dopamine receptors in the rodent prefrontal cortex impairs spatial working memory performance. J Neurosci 17:8528-8535.  

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