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The role of corticotropin-releasing factor in mediating the effect of acute stress on effort-based decision-making Bryce, Courtney 2015

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      THE ROLE OF CORTICOTROPIN-RELEASING FACTOR IN MEDIATING THE EFFECT OF ACUTE STRESS ON EFFORT-BASED DECISION-MAKING  by  Courtney Bryce    B.A., University of Saskatchewan, 2013    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    MASTER OF ARTS  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Psychology)    THE UNIVERISTY OF BRITISH COLUMBIA  (Vancouver)    August 2015    © Courtney Bryce, 2015 	  	   ii Abstract The acute stress response is an adaptive response to threats in the environment, activating numerous coordinating systems to return the organism to homeostasis. Episodes of acute stress can have differential impacts on learning and memory functioning depending on myriad factors including the context, duration or timing of the stress. The manner in which acute stress influences higher-level cognitive function, including decision-making, however, is relatively less known. Decision-making involves weighing the alternative costs and benefits in order to optimize choice behavior. Increasing the amount of effort required in order to obtain a reward is one type of cost that can alter the subjective value of objectively larger rewards. Using an operant chamber assay, rats were required to choose between a low effort/low reward lever (LR; 2 pellets), and a high effort/high reward lever (HR; 4 pellets), with the effort requirement increasing over trial blocks (2, 5, 10, and 20 presses). Normally rats will choose the HR lever more often when the effort cost is low, reducing their preference for this option as the amount of effort increases. Previous research in our lab revealed that one hour of restraint stress reduces choice of the HR option in this task, which was not mimicked by systemic corticosterone (CORT) injection and not blocked by the dopamine (DA) antagonist, flupenthixol (Shafei et al. 2012). The goal of the current study is to elucidate the neurochemical mechanisms underlying the ability of acute stress to reorganize effort-related decision-making preferences and to clarify the regional specificity of this action. Initial experiments found that corticotropin-releasing factor (CRF), which initiates the hypothalamic-pituitary-adrenal (HPA) axis, is primarily involved in mediating the effect of acute stress, as prior CRF antagonism (alpha-helical CRF; 30 µg) ameliorated the effect of one hour of acute restraint stress and central CRF infusion (3 µg) mimicked the effect of acute restraint stress on HR preference. The effect of CRF was not due to 	  	   iii altering the subjective value of objectively larger rewards, as prior CRF administration (3 µg) had no effect on choice behavior when there were no costs associated with reward, however, this manipulation did reduce the motivation to work for reward, indicating that CRF acts in the effort-based decision-making task by reducing the drive to work for reward. Subsequent experiments aimed to investigate the regional specificity of CRF action in reorganizing effort-related preference behavior. With this in mind, we targeted the ventral tegmental area (VTA), as previous experiments revealed that CRF is released in the VTA in response to stress (Wang et al., 2005), and intra-VTA CRF reduces motivation to work for reward (Wanat et al., 2013). Intra-VTA, but not intra-nucleus accumbens (NAc) core, CRF infusion (0.5 µg) mimicked the effect of central CRF and acute restraint stress on HR preference, signifying the importance of this region in mediating the behavioral effect of acute stress on effort choice. Taken together, these experiments highlight the importance of CRF in mediating the effect of effort-based decision-making and indicate that CRF transmission may influence the motivational impairments and abnormal decision-making associated with human depression. 	  	   iv Preface  All experiments were conducted at the University of British Columbia (Vancouver Campus) and carried out by Courtney Bryce. In addition, Courtney Bryce completed all surgeries, performed the statistical analyses, and wrote the current document, with the concept formation and the majority of the editing done by Dr. Stan Floresco, who was the supervisory author on this project. Research for this thesis was approved by the UBC Animal Care Committee, application number A14-0120.	   	  	  	   v Table of Contents Abstract ------------------------------------------------------------------------------------------------------- ii Preface -------------------------------------------------------------------------------------------------------- iv Table of Contents -------------------------------------------------------------------------------------------- v List of Tables ----------------------------------------------------------------------------------------------- vii List of Figures --------------------------------------------------------------------------------------------- viii List of Abbreviations --------------------------------------------------------------------------------------- ix Acknowledgements ------------------------------------------------------------------------------------------ x Dedication ---------------------------------------------------------------------------------------------------- xi Introduction --------------------------------------------------------------------------------------------------- 1 Acute stress-induced alterations in cognitive functioning -------------------------------------------- 2 Behavioral influence of corticotropin-releasing factor (CRF) --------------------------------------- 5 Depression and effort-based decision-making --------------------------------------------------------- 8 Methods ------------------------------------------------------------------------------------------------------ 13 Animals ---------------------------------------------------------------------------------------------------- 13 Apparatus -------------------------------------------------------------------------------------------------- 13 Initial lever training -------------------------------------------------------------------------------------- 13 Effort-based decision-making -------------------------------------------------------------------------- 14 Reward magnitude discrimination --------------------------------------------------------------------- 16 Progressive ratio schedule of reinforcement ---------------------------------------------------------- 16 Surgery ---------------------------------------------------------------------------------------------------- 17 Drugs and Microinfusion Protocols -------------------------------------------------------------------- 18 Experimental procedure --------------------------------------------------------------------------------- 19 Histology -------------------------------------------------------------------------------------------------- 21 Data analysis ---------------------------------------------------------------------------------------------- 25 Results -------------------------------------------------------------------------------------------------------- 27 Experiment 1: Alpha-helical CRF ameliorates the effect of one hour of acute restraint stress on effort-based decision making --------------------------------------------------------------------------- 27 	  	   vi Experiment 2: Central CRF infusion mimics acute stress on effort-based decision making --- 29 Experiment 3: Central CRF infusion does not alter HR preference when costs are equated in a reward magnitude discrimination ---------------------------------------------------------------------- 32 Experiment 4: Central CRF infusion alters motivation to work for reward in the progressive ratio task --------------------------------------------------------------------------------------------------- 34 Experiment 5: Intra-VTA, but not intra NAc, CRF infusion mimics acute stress and central CRF infusion on HR preference ------------------------------------------------------------------------ 36 Discussion --------------------------------------------------------------------------------------------------- 41 The influence of acute restraint stress on effort-based decision-making preference and the mechanisms underlying this effect --------------------------------------------------------------------- 41 Cognitive/motivational alterations induced by central CRF infusion that influence effort-based decision-making ------------------------------------------------------------------------------------------ 44 Effort discounting following central CRF infusion ----------------------------------------------- 44 Reward magnitude discrimination following central CRF infusion ---------------------------- 46 Progressive ratio schedule of reinforcement following central CRF infusion ---------------- 47 The regional specificity of CRF action on effort-based decision-making preference----------- 49 Conclusions ----------------------------------------------------------------------------------------------- 55 References --------------------------------------------------------------------------------------------------- 59     	  	   vii List of Tables 	  Table 1. Acute stress and CRF antagonist administration on effort-based decision-making rate, choice latency, omissions and locomotion --------------------------------------------------------------- 29 Table 2. Central CRF administration on effort-based decision-making rate, choice latency, omissions and locomotion --------------------------------------------------------------------------------- 31 Table 3. Central CRF administration on reward magnitude discrimination choice latency, omissions and locomotion --------------------------------------------------------------------------------- 33 Table 4. Intra-VTA CRF administration on effort-based decision-making rate, choice latency, omissions and locomotion --------------------------------------------------------------------------------- 38 Table 5. Intra-NAc core administration on effort-based decision-making rate, choice latency, omissions and locomotion --------------------------------------------------------------------------------- 40   	  	   viii List of Figures 	  Figure 1. Accurate (and inaccurate) lateral ventricle cannula placement photographs (Experiments 1-4) ------------------------------------------------------------------------------------------- 22	  Figure 2. Representation of accurate ventral tegmental area (VTA) cannula placements (Experiment 5) ----------------------------------------------------------------------------------------------- 23	  Figure 3. Representation of accurate nucleus accumbens (NAc) core cannula placements (Experiment 5) ----------------------------------------------------------------------------------------------- 24	  Figure 4. Effort discounting following CRF antagonist and restraint stress administration (Experiment 1)  ---------------------------------------------------------------------------------------------- 28	  Figure 5. Effort discounting following intracerebroventricular (ICV) CRF administration (Experiment 2) ----------------------------------------------------------------------------------------------- 31	  Figure 6. Reward magnitude discrimination following ICV CRF administration (Experiment 3) ----------------------------------------------------------------------------------------------------------------- 33	  Figure 7. Progressive ratio schedule of reinforcement following ICV CRF administration (Experiment 4)  ---------------------------------------------------------------------------------------------- 35	  Figure 8. Effort discounting following intra-VTA CRF administration (Experiment 5)                ----------------------------------------------------------------------------------------------------------------- 38	  Figure 9. Effort discounting following intra-NAc core CRF administration (Experiment 5)        ----------------------------------------------------------------------------------------------------------------- 40	  	    	  	   ix List of Abbreviations  Hypothalamic-pituitary-adrenal (HPA) axis Corticotropin-releasing factor (CRF) Corticosterone (CORT) Prefrontal cortex (PFC) Glucocorticoid receptor (GR) Dopamine (DA) Corticotropin-releasing factor receptor 1 (CRFR1) Corticotropin-releasing factor receptor 2 (CRFR2) Ventral tegmental area (VTA) Nucleus accumbens (NAc) High effort/high reward (HR) Low effort/low reward (LR) Intracerebroventricular (ICV) Artificial cerebral spinal fluid (aCSF) Bed nucleus of the stria terminalis (BNST) Pedunculopontine nucleus (PPT)  	  	   x Acknowledgements First, I would like to thank my supervisor, Dr. Stan Floresco, for the encouragement and the direction. Second, thank you to Maric, Patrick, Gemma, Meagan, Josh, Colin, Nicole, Dave, Debra and Ryan for the academic and emotional support. I look forward to our continued friendship. I would also like to acknowledge my committee members, Dr. Kiran Soma and Dr. Luke Clark for taking the time to participate in my education and provide valuable feedback. And finally, I want to thank my family and friends. I could not have completed this document, much less the past two years, without your belief and reassurance.   	  	   xi   This is for my grandparents: William and Catherine Bryce, Peter and Margaret Thiessen.  	  	   1 Introduction The acute stress response is an adaptive response that involves activation of numerous systems in a coordinated effort to return the organism to homeostasis and activate energy availability in order to ensure survival. One system that is activated in response to a stressor is the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is initiated when the paraventricular nucleus of the hypothalamus releases corticotropin-releasing factor (CRF) to act on the pituitary to release adrenocorticotropic hormone (ACTH), which stimulates glucocorticoid release from the adrenal glands (Habib, Gold, & Chrousos, 2001). Glucocorticoids influence both body and brain function, orchestrating the behavioral response to acute stress (Habib, Gold, & Chrousos, 2001). The behavioral phenotype elicited by acute stress on learning and memory has been the subject of considerable research and has been fairly well characterized. For instance, acute stress can have varying effects on learning and memory, with both impairments and improvements in these domains reported, depending on a variety of factors including the context and severity of the stressor (Joëls & Baram, 2009; Kim et al., 2007; de Quervain, Roozendaal & McGaugh, 1998; Diamond & Rose, 1994; Yuen et al., 2009). How acute stress mediates higher-level cognitive functioning, including decision-making ability, however, is relatively less known. Decision-making involves choosing between several alternative possibilities after evaluation of the relative costs and benefits in order to optimize choice behavior. Increasing the amount of effort required to obtain a reward is one type of cost that can diminish the subjective value of objectively larger rewards. Additionally, repeated episodes of stress may result in depressive symptoms including anergia, which may reduce the tendency to exert effort in order to obtain reward. With this in mind, the main goal of the current study was to elucidate the underlying mechanisms of how acute stress may influence effort-based decision-making. 	  	   2 Activation of the acute stress response involves multiple coordinating systems acting to return the organism to homeostasis following a physical or psychological threat (Ulrich-Lai & Herman, 2009). The autonomic nervous system (ANS) initially and rapidly responds to stressors through the activation of the sympathetic nervous system, which results in the adrenal glands releasing adrenaline (and noradrenaline), acting to increase heart rate and blood pressure. The hypothalamic-pituitary-adrenal (HPA) axis is another, interacting system that is rapidly activated in response to a stressor. Activation begins with the secretion of corticotropin-releasing factor (CRF) from the paraventricular nucleus (PVN) of the hypothalamus. CRF acts on receptors in the pituitary to release adrenocorticotrophic hormone (ACTH) into the bloodstream, the main target of which is the adrenal glands. ACTH triggers glucocorticoid secretion from the adrenal glands into the bloodstream, with these hormones acting throughout the body and the brain (Habib, Gold, & Chrousos, 2001). Acute stress-induced alterations in cognitive functioning Acute stress can have differential effects on cognitive functioning depending on a variety of factors including the severity, duration and/or context of the stressor (Joëls & Baram, 2009). However, relatively simple learning and memory functioning appears to be consistently impaired following acute stress exposure. For instance, hippocampal-dependent memory is impaired following episodes of acute stress, whereby exposure to a 2-hour variable “audiogenic” stressor 30 minutes prior to acquisition or a foot shock stressor 30 minutes prior to testing, both disrupt spatial memory on the Morris Water Maze task (Kim et al., 2007; de Quervain, Roozendaal & McGaugh, 1998). Object recognition and object in place are similarly impaired following acute stress, as rats subjected to 30 minutes of elevated platform stress prior to testing show impairments on object recognition and object in place performance (Howland & Cazakoff, 	  	   3 2010). Moreover, recognition memory retrieval is significantly disrupted by one hour of acute restraint stress exposure between acquisition and retrieval at a 4- and 24-hour delay for both the object recognition task and the object location task (Li, Fan, Wang, & Tang, 2012).  As opposed to relatively simple learning and memory tasks, which typically do not recruit frontal lobe involvement, the effects of acute stress on more complex forms of cognition mediated by the PFC can be more variable. For example, studies of cognitive flexibility have reported impairments, facilitation, or null effects following acute stress. Cognitive flexibility is traditionally measured with one of two types of tasks. Reversal learning is a comparatively simpler form of flexibility that is partially reliant on the orbitofrontal cortex (OFC), requiring a switch on one dimension of the task (Ghods-Sharifi, Haluk, & Floresco, 2008; McAlonan & Brown, 2003). Set-shifting, on the other hand, is a more complex, medial PFC (mPFC)- mediated task that involves a shift between different rules, strategies or attentional sets (Birrell & Brown, 2000; Floresco, Block, & Tse, 2008; Ragozzino, Detrick, & Kesner, 1999). Acute stress impaired set-shifting but not reversal learning, when the stressor is administered in the context of the task (15 minute tail pinch in the operant chamber; Butts et al., 2013). However, when a 30 minute restraint stressor was administered prior to the same type of reversal learning task used by Butts et al (2013), reversal learning is facilitated, however this stressor had no effect on set-shifting (Thai, Zhang & Howland, 2013). Similarly, on a touchscreen task of reversal learning in mice, three days of swim stress significantly facilitated reversal learning, with stressed mice requiring fewer trials to reach criterion and making fewer errors than nonstressed mice (Graybeal et al., 2011). Taken together, these studies suggest that the effect of acute stress on cognitive flexibility is complex and partially dependent on the context of the stressor. 	  	   4 The influence of acute stress on more complex forms of cognition, including working memory, is similarly controversial, as some studies report impairments while others show evidence of facilitation. For instance, exposure to a novel environment stressor prior to being placed in a radial arm maze caused rats to make more working memory errors, suggesting spatial working memory is impaired following acute stress exposure (Diamond & Rose, 1994). Similarly, exposure to a 15-minute tail-pinch stressor during the delay phase of the delayed win-shift (DSWSh) radial arm maze task caused rats to make more errors, impairing performance on this task (Butts et al., 2011). Conversely, young rats (25-28 days of age) that were exposed to a 20 minute forced swim stressor facilitated performance on the delayed alternation T-maze task of working memory at both a four-hour delay and 24-hour delay (Yuen et al., 2009). Collectively, these studies convey the complexity with which acute stress influences working memory functioning depending on numerous factors, potentially including the duration and timing of the stressor, as well as the age of the animal. Glucocorticoids mediate many of the effects of acute stress on PFC-dependent behavior. Indeed, the working memory impairment in the DSWSh task was blocked by locally infusing a glucocorticoid receptor (GR) antagonist into the PFC, which attenuated the stress-induced potentiation of PFC DA release, suggesting that glucocorticoids in the PFC aid in potentiating PFC DA release in response to acute stressors (Butts et al., 2011). Moreover, a GR antagonist blocked the acute stress-induced facilitation of working memory in the delayed alternation T-maze task (Yuen et al., 2009). Previous evidence suggests that glucocorticoids exert their effects rapidly in some regions, however the primary effect of this hormone is to influence gene transcription on a longer time scale (Joëls & Baram, 2009). Furthermore, glucocorticoids initiate the negative feedback loop of the HPA axis, stopping further glucocorticoid secretion (Joëls & 	  	   5 Baram, 2009). Due to these factors, it is possible that glucocorticoids may not be the primary hormone mediating the rapidly acting central effects of stress-induced disruptions in effort choice behavior. Behavioral influence of corticotropin-releasing factor (CRF) Mounting evidence suggests that corticotropin-releasing factor (CRF), which initiates the HPA axis, acts centrally in the brain to mediate many of the behavioral effects of acute stress. In support of this notion, anxiety-related behavior is mediated, in part, by CRF acting in limbic regions via CRFR1, independent of the role of CRF in HPA axis function (Müller et al., 2003). Acute stress or centrally administered CRF both elicited conditioned place aversion, decreasing preference for the previously stress- or CRF-paired chamber (Cador et al., 1992). Yet, the ability of CRF to induce conditioned place aversion is likely independent of CRF’s ability to activate the HPA axis, as low doses of systemically administered CRF that reliably activate the HPA axis and increase plasma CORT levels (0.005-0.5 µg), did not induce condition place aversion (Cador et al., 1992). Moreover, CRF is involved in the stress-induced reinstatement of cocaine seeking in addiction models, which also occurs independently of stress-induced activation of the HPA axis (Koob, 2010).  Corticotropin-releasing factor (CRF) is a neuropeptide that is primarily expressed in the paraventricular nucleus (PVN) of the hypothalamus, bed nucleus of the stria terminalis (BNST) and the central amygdala (CeA; Joëls & Baram, 2009). CRF binds with high affinity to the CRF1 receptor and with lesser affinity to the CRF2 receptor. Both CRF receptors are primarily Gs-protein coupled receptors that activate a second messenger cascade. When CRF binds to its receptor, the G protein activates cyclic AMP (cAMP), which phosphorylates protein kinase A (PKA), activating cAMP response element binding protein (CREB; Aguilera & Liu, 2012).  	  	   6 CRF acts on two primary receptor types, CRF1 and CRF2. CRFR1, and to a lesser extent, CRFR2, is widely expressed in cortical and subcortical regions, including in the frontal cortex, nucleus accumbens and amygdala and in mesencephalic regions, such as the dopamine-expressing ventral tegmental area (VTA; Van Pett et al., 2000; Bittencourt & Sawchenko, 2000). Therefore, there are numerous brain regions in which CRF may be acting. The VTA is one region of interest as acute stress can stimulate the release of CRF into the VTA and potentiate DA levels in regions receiving VTA input, suggesting that CRF may act in the VTA to mediate stress-induced mesolimbic DA release (Wang et al., 2005). Previous microdiaylsis studies have shown that foot shock stress induces CRF release in the VTA in both rats that had previously undergone cocaine-self administration and in cocaine-naïve rats (Wang et al., 2005).  Additionally, CRF in the VTA influences DA release in a pathway specific manner in order to selectively reprioritize motivated behavior (Wanat et al., 2013). Specifically, CRF acts in the VTA to reduce motivation to work for natural rewards in a progressive ratio task by influencing DA release in the NAc (Wanat et al., 2013). In order to measure motivation to work for reward, this study used an operant chamber assay requiring the rat to press the active lever for one sugar pellet. The ratio of presses required for one sugar pellet was exponentially increased over the duration of the session. Using this assay, investigators found that acute tail-pinch stress decreased the breakpoint (or the last completed number of presses required for the trial), which was blocked by prior administration of alpha-helical CRF, a nonselective CRF antagonist (Wanat et al., 2013). Furthermore, CRF infusion into the VTA dose-dependently decreased the breakpoint on the progressive ratio task, mimicking the effect of acute stress on motivation to work for natural rewards. Using fast-scan cyclic voltammetry, investigators found that CRF infusion in the VTA acts to reduce phasic DA signaling in the NAc during the progressive ratio 	  	   7 task in response to natural rewards but had no effect on DA release to reward-predictive cues. This suggests that CRF can selectively attenuate phasic DA signals in a stimulus-specific manner, requiring CRF to act on specific inputs into the VTA. In order to investigate this, either the bed nucleus of the stria terminalis (BNST) or the pedunculopontine tegmental nucleus (PPT) were stimulated, both of which evoke phasic DA release. Intra-VTA CRF infusion decreased NAc DA release evoked by PPT stimulation, whereas similar treatments actually enhanced DA release evoked by BNST stimulation. In addition, inactivating the PPT with GABA agonists reversed the CRF-induced decrease in breakpoints during the progressive ratio task. Taken together, these results suggest that CRF has diverse effects on behavior and can induce differential effects on DA release in a pathway and stimulus-specific manner. Furthermore, CRF acting in the VTA is a mechanism by which acute stress can reprioritize motivated behavior.  CRF in the NAc acts in a somewhat opposing manner to CRF in the VTA, as intra-NAc CRF infusion potentiates DA release in the NAc and promotes appetitive behavior (Lemos et al., 2012). Additionally, CRF infusion into the NAc or phasic stimulation of VTA DA neurons both induce approach behavior in a conditioned place preference (CPP) assay (Lemos et al., 2012; Tsai et al., 2009). The ability of NAc CRF to induce CPP was dependent on the ability of CRF to potentiate dopamine release, as prior NAc DA depletion via 6-OHDA infusions failed to elicit conditioned place preference (Lemos et al., 2012). Furthermore, in brain slices of mice that were previously exposed to a forced-swim stressor, CRF in the NAc was no longer able to potentiate DA release, indicating that severe stress dysregulates the ability of CRF to control DA release in the NAc. Indeed, the authors of this report also showed that severe stress could switch the action of CRF in the NAc from appetitive to aversive during conditioned place preference, with previously-stressed rats spending less time in the chamber paired with NAc CRF infusion than in 	  	   8 the vehicle-infused paired chamber (Lemos et al., 2012). Alternatively, CRF infusion into the NAc shell induces a depression-like behavioral profile, including increasing immobility in a forced swim test (FST), decreasing sucrose preference, and reducing time spent in the open arm in the elevated plus maze (Chen et al., 2012). These seemingly discrepant findings may be partially due to the protocol of the experiment. By subjecting rats to a stressor (10 minutes of forced swim) prior to testing immobility behavior, experimenters may have caused CRF to switch from appetitive to aversive and thus increased immobility in the FST. Alternatively, this study targeted the NAc shell whereas the previous study concentrated on the NAc core and previous studies have shown dissociable roles for the NAc core and shell (Floresco, 2015), therefore, CRF may have divergent effects in these regions, although this, to the best of our knowledge, has not directly been studied.  Depression and effort-based decision-making Human depression is a stress-related disorder that is characterized by dysregulation of the HPA axis leading to hypercortisolism (Chrousos, 2009; Board, Wadeson, & Persky, 1957) and increased CRF levels in cerebral spinal fluid (Nemeroff et al., 1984). Indeed, there is a significant correlation between elevated cortisol levels and workload, with higher workload (which can be viewed as a chronic stressor) related to higher cortisol levels (Rose et al., 1982; Melamed & Bruhis, 1996). Furthermore, exposure to prolonged or intense periods of stress substantially increases the risk of developing major depression (Ehlert, Gaab, & Heinrichs, 2001). Anhedonia, which is the inability to derive or experience pleasure, has traditionally been seen as a core symptom of major depressive disorder, however, more recent evidence suggests that depression may reduce the motivation to seek reward, rather than reducing the pleasurability 	  	   9 of reward itself (Bylsma, Morris, & Rottenberg, 2008; Treadway et al., 2012). Anergia, or the lack of motivation to seek or exert effort in order to obtain reward, may be measured with effort-based decision-making assays. These tasks were originally developed for animals but have since been backtranslated in order to assess humans. The human effort-based decision-making task requires subjects to choose between two options: a low effort/low reward option (manual button pressing for 7s / $1 monetary reward) or a high effort/high reward (manual button pressing for 21s / $1.24 - $4.30; Treadway et al., 2012). Depressed patients in this task show reduced preference to work harder for larger rewards, choosing the more effortful option significantly less than control subjects (Treadway et al., 2012). This indicates that the core symptom in depression may not necessarily be an affective impairment, but rather, an impairment in the drive to acquire pleasurable experiences. This symptom domain has only recently been examined in animal models related to depression. In one variant of the effort-based decision-making task, conducted in an operant chamber, rats choose between a low effort/low reward option (1 lever press/ 2 sugar pellets), or a high effort/high reward option (2-20 lever presses/ 4 sugar pellets). Using this model, previous research has found that one episode of acute restraint stress decreases the choice of the high reward/high effort option, with rats choosing the low reward/low effort option more frequently (Shafei et al., 2012). The effect of restraint stress on HR choice does not seem to be mediated by alterations in preferences for objectively larger rewards, as rats subjected to one hour of restraint stress choose the HR option almost exclusively when the costs of each option is equated (i.e. one lever press for two sugar pellets or one lever press for four sugar pellets).  It has been well established that intact dopamine functioning plays a key role in enabling animals to overcome effort-related costs during cost/benefit decision-making. Initial studies of 	  	   10 effort-based decision-making utilized an instrumental responding assay that required rats to choose between pressing a lever for sugar pellet reward (FR5 schedule) or eating the normal rat chow placed in the chamber with the lever. Using this task, researchers found that systemic injection of a dopamine (DA) antagonist reduced lever pressing and increased chow consumption (Salamone et al., 1991). Similarly, systemic injection of a DA antagonist decreased choice of the high effort arm in the T-maze task of effort-based decision-making (Salamone, Cousins, & Bucher, 1994). In this task, one of the arms of the T-maze is baited with 2 sugar pellets (LR arm) and the other arm is baited with 4 sugar pellets (HR), however, the rat is required to jump over a metal barrier to obtain the HR option. More recent studies of effort-based decision-making have employed an operant chamber assay, where rats are required to choose between a low effort/low reward lever (LR; 2 pellets), and a high effort/high reward lever (HR; 4 pellets), with the effort requirement for HR choice increasing over trial blocks (2, 5, 10 and 20 presses). Utilizing this method, findings again confirmed that a systemic dose of a DA antagonist decreased choice of the high effort high reward lever, whereas a systemic dose of amphetamine (a DA releaser) had a biphasic effect on choice behavior, with a low dose increasing high effort choice behavior and a high dose decreasing high effort choice behavior (Floresco, Tse, & Ghods-Sharifi, 2008).  DA depletions in the nucleus accumbens (NAc) and infusion of a DA antagonist into the NAc also reduced lever pressing and increased chow consumption in the instrumental responding task and decreased choice of the high effort arm in the T-maze task, whereas medial striatal DA depletions did not alter behavior (Salamone et al., 1991; Cousins, Sokolowski, & Salamone, 1993; Cousins & Salamone, 1994; however, see Walton et al., 2009). Furthermore, the NAc is a heterogeneous structure that can be functionally dissociated into two separate structures: the NAc core and the NAc shell (Floresco, 2015). Inactivation of the NAc core (with either excitotoxic 	  	   11 lesions or GABA A/B agonists) but not the shell reduced preference for the HR option in an operant chamber assay (Ghods-Sharifi & Floresco, 2010; Walton et al., 2009). These studies suggest that the NAc, specifically the NAc core, plays an essential role in the circuitry underlying normal decision-making processing. Additionally, normal DA transmission is important for optimal decision-making. The mesolimbic DA system promotes association between environmental cues and natural rewards, which facilitate behaviors that were previously associated with the acquisition of reward (Palmiter, 2007). This is consistent with the idea that mesolimbic DA is involved in the evaluation of the relative costs and benefits used to inform decision-making behavior.  The brain regions involved in the neural circuitry mediating effort-based decision-making also contribute to acute stress-induced alterations in behavior. Not surprisingly, previous evidence from our lab indicates that one episode of acute stress reprioritizes effort choice behavior in this task (Shafei et al., 2012), however the mechanisms underlying this effect have yet to be substantiated. CRF is a prime candidate as numerous studies have shown that central CRF infusion parallels the behavioral profile elicited by acute stress. The goal of this study is to understand the neurochemical mechanisms underlying the ability of acute stress to reorganize effort-related decision-making preferences and clarify the regional specificity of this action. Our first aim was to substantiate the role of centrally acting CRF by administering a nonspecific CRF antagonist prior to acute stress as well as infusing CRF centrally in order to ascertain if CRF is both necessary and sufficient to alter effort-related preference. To ensure that CRF is not acting to reduce reward valuation of the objectively larger reward, we utilized a reward magnitude discrimination, which equates the costs of the two options. Furthermore, the progressive ratio schedule of reinforcement was used in order to assess the motivation to work for reward. To 	  	   12 elucidate the brain region involved in the action of CRF we infused CRF into two key dopaminergic regions important in the effort-based decision-making circuitry that also express CRF receptors, the VTA and the NAc core. The results of these experiments point to the importance of CRF in altering choice behavior in the face of divergent effort costs and may provide valuable insights into the neurochemical basis of anergia that characterizes human depression.    	  	   13 Methods Animals Separate cohorts of male Long Evans rats weighing 250-275g at the beginning of training were utilized for all experiments. Following one week of colony acclimatization, rats were individually housed and food restricted to 85% of their free-feeding weight prior to the commencement of operant chamber training. Water was provided ad libitum for the duration of the experiment. Body weight was monitored daily and rat chow was provided immediately following operant chamber training each day. Care was taken to limit the amount of animals used and all testing was done in accordance with the Canadian Council of Animal Care and the Animal Care Committee of the University of British Columbia.  Apparatus Behavioral testing was conducted in operant chambers (30.5 x 24 x 21 cm; Med-Associates, St Alban, VT, USA) enclosed in a sound-attenuating box. Each box was equipped with a fan with the purpose of providing ventilation and limiting extraneous sounds. The chamber was fitted with a central food receptacle where sugar pellets (45 mg; Bioserv, Frenchtown, NJ) were dispensed. Two retractable levers were located on either side of the food receptacle. The operant chamber was illuminated by a 100-mA house light located on the top center of the box opposite the food receptacle. Sensors located along the length of the box measured the number of photo beam breaks that occurred, which was used as an indicator of locomotor activity. Experimental data was recorded by a personal computer connected to the operant chambers via an interface. Initial lever training Lever training commenced following at least 5 days of food restriction. Approximately 20 sugar pellets were placed into the rat’s cage on the day prior to the first exposure to the operant 	  	   14 chamber. On the first day of lever training one of two levers was extended and 2-3 crushed sugar pellets were positioned on this lever prior to placing the rat into the operant chamber. Rats were first trained under a fixed-ratio 1 (FR1) to a criterion of 60 presses in 30 minutes on one lever. When the criterion was met FR1 training was conducted on the other lever, ensuring that both levers were experienced. The order of scheduling was counterbalanced so that half of the rats received the left side FR1 procedure first followed by the right side and the other half received the opposite schedule. Rats were subsequently trained on a simplified version of the full task, requiring rats to press the retractable lever within 10 seconds of extension or the trial was counted as an omission. Sessions consisted of 90 training trials with a 40 second inter-trial interval (ITI). Trials were initiated with the illumination of the houselight and the extension of one of the two levers. Trials were scored as omissions if the rat failed to respond to the lever within 10 seconds following lever extension. If this occurred, the lever retracted, the houselight turned off, and the 40 second ITI began. On the other hand, if the rat responded to the lever within 10 seconds of extension, the rat received one sugar pellet. Rats were trained for approximately 5 days to a criterion of 80 successful trials (i.e. < 10 omissions) for 2 consecutive days, after which, rats were trained on the effort-based decision-making task. Effort-based decision-making Separate cohorts of rats in Experiments 1, 2, and 5 were trained 5-7 days a week on the effort-based decision-making task. Each 32 minute daily training session consisted of 48 trials separated into 4 blocks. Trials were initiated by the illumination of the houselight and the extension of both levers every 40 seconds. In order for the animal to be updated on the change in the effort contingencies in the subsequent block of trials, each of the four blocks began with two forced choice trials in which only one of the two levers was randomly extended. The remainder 	  	   15 of the trials within the block were free choice trials, where both levers were presented and the rat had to choose between the two options. Once the rat chose one of two levers by pressing on it, one or both levers (depending on whether the rat chose the high effort or low effort option) would retract. For all sessions and blocks, one lever was designated as the low reward lever (LR) and the other lever was designated as the high reward lever (HR). Levers were counterbalanced (left/right) between rats, however levers remained constant for each rat during the duration of the experiment. Following presentation of the levers, rats were required to make a response by pressing one of two levers within 25 seconds of insertion. The failure to respond to either lever was scored as an omission with the operant chamber returned to an inter-trial state. If the rat chose the LR lever, both levers would retract and the rat would receive two sugar pellets. Conversely, if the rat chose the HR lever, this lever would remain extended and the LR lever would retract. The rat was then required to press the HR lever until the number of presses required for the current block was completed. The number of presses required increased over subsequent blocks of trials during the session. For the initial block, 2 presses were required to complete HR choice, with subsequent blocks requiring 5, 10 and 20 presses for HR choice. When the required number of presses were completed the lever retracted and 4 pellets were delivered 0.5 seconds apart. The houselight remained illuminated for an additional 4 seconds after the delivery of the final pellet. Once the pellet was dispensed the ITI would begin. If the rat failed to complete the required number of presses on the HR lever within 25 seconds of extension, the lever retracted without delivery of any sugar pellets dispensed followed by the ITI. This, however, was not scored as an omission and the rat’s choice was still incorporated into the analysis. Other measures incorporated into the analysis included the time between lever extension and lever choice, the presses per second on the HR lever, and locomotor activity. 	  	   16 Rats were trained on the effort-discounting task until as a group, they met two criterion: 1) chose the HR lever during the first trial block on at least 75% of the free-choice trials, and 2) demonstrated stable baseline levels of discounting for 3 consecutive days. Stability was analyzed using a 3 x 4 repeated measures analysis of variance (ANOVA) with training day (3) and trial block (4) as within-subjects factors. The animals were judged to have achieved stability of choice behavior when the effect of block was significant at the p<.05 level but there was no main effect of day (p>.05) and no day x trial block interaction (p >.10). Reward magnitude discrimination A separate cohort of nine rats in Experiment 3 were initially trained as above, undergoing FR1 training on both levers followed by retractable lever training. However, instead of the effort-based decision-making task, rats were trained on the reward magnitude discrimination. This task equates the costs of the two levers so that the LR lever requires one press for 2 pellets of reward and the HR lever requires one press to receive 4 pellets. Each session consisted of 48 trials, with 12 trials per block (2 trials at the beginning of each block were forced choice trials and 10 trials were free choice trials). This task did not contain an ascending effort requirement on the HR lever as in the effort-based decision-making task (all blocks required only one press for the HR lever). Rats were trained for 9 days on this task and displayed a strong preference for the HR lever (~90%). Progressive ratio schedule of reinforcement A separate cohort of eight animals was trained on the progressive ratio schedule of reinforcement task in order to assess the motivation to work for reward. Throughout this experiment, rats were only exposed to the left lever and this lever remained in place for the duration of each training session. During the initial phase of training, rats were trained on the FR1 schedule for two days 	  	   17 followed by one day of FR2 schedule training and two days of FR5 schedule training. Completing one ratio resulted in one sugar pellet delivery. Next, rats were trained on the progressive ratio schedule, in which the ratio of presses required to obtain a single reward pellet increased after each pellet delivery. The ratio was adapted from the one used by Brown and colleagues (1998) and increased in the following manner: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 693, 737, and 901 presses. Rats had a maximum of 20 min to complete each ratio and obtain reward. Failure to complete a ratio in the allotted time ended the session and the animals were returned to their homecage until the next training day. The primary variables of interest were: 1) the total number of lever presses over the course of a session 2) the number of sugar pellets received and 3) the last ratio obtained before a session terminated (breakpoint). Training continued for 10 days on this task, when rats displayed stable levels of lever pressing and breakpoints for three consecutive days as a group (i.e. less than 15% variation within the group).  Surgery Approximately two days prior to undergoing cannulation surgery, rats were placed back on free-feeding to minimize surgical complications. Rats were anaesthetized using ketamine (100 mg/kg, IP)/xylazine (10 mg/kg, IP) and given an analgesic (anafen, 10 mg/kg, SC) prior to surgery. When rats attained a surgical plane of anesthesia, they were placed into a stereotaxic frame and secured with earbars to maintain a flat skull. Three separate surgeries were performed in this series of experiments. Rats in Experiments 1-4 underwent unilateral cannulation surgery with the cannula lowered to 1 mm dorsal to the right lateral ventricle (coordinates: a/p -1.0, m/l -1.8, d/v -2.5 from bregma). Rats in Experiment 5 underwent bilateral cannulation surgery with cannulae lowered to 1 mm dorsal to the ventral tegmental area (coordinates: -5.5 a/p, 0.7 m/l, -7.0 d/v 	  	   18 from bregma), or aimed 1 mm dorsal to the nucleus accumbens core (coordinates: a/p  +1.6, m/l  +1.8, d/v -6.5 from bregma). Post-operative procedures included daily weighing and subcutaneous analgesic administration (anafen, 10 mg/kg) for at least 2 days following surgery. Drugs and Microinfusion Protocols Experiment 1 tested if a CRF antagonist could block the effect of acute stress on choice behavior in the effort-based decision-making task. Alpha-helical CRF was chosen as it binds to both CRF1 and CRF2 receptors. The alpha-helical CRF dose (30 µg) was based on prior experiments finding that alpha-helical CRF blocked the effect of acute stress on various behavioral measures using doses ranging from 5 µg to 50 µg (Kalin et al., 1988; Cole et al., 1990; Berridge & Dunn, 1989; Nawata et al., 2012; Krahn et al., 1986).  Experiment 2 assessed if centrally infusing CRF could mimic the effect of acute restraint stress. If CRF does indeed influence preference in the effort-based decision-making task we wanted to identify the dose response curve. With this in mind, we infused three different doses of CRF (0.25, 1 µg, 3 µg) in a counterbalanced manner. Although previous experiments have seen alterations in behavior following doses as low as 0.1 µg CRF, doses as high as 10 µg have been infused into the ventricular system with significant effects on behavior (Dunn & Berridge, 1990). Because the maximum solubility of CRF in aCSF is 1µg/1µl, we had to increase the volume of the infusion for the higher 3 µg dose. Although the vehicle infusion volumes differed, there were no significant differences on performance following 1 µl versus 3 µl vehicle infusions (p>.05), therefore all data obtained after treatments with each of the three vehicle doses were averaged for the analyses.  The regional specificity with which CRF mimics the effect of centrally acting CRF and acute restraint stress was investigated in Experiment 5. The dose of CRF for the VTA and NAc 	  	   19 core infusions (0.5 µg; 1 µg/1 µl) was based on previous studies that infused CRF into the NAc, effectively altering behavior in a conditioned place preference task (Lemos et al., 2012) and Pavlovian instrumental transfer (Peciña et al., 2006) and effective in the VTA, altering behavior on a progressive ratio task in a dose-dependent manner (Wanat et al., 2013).  Experimental procedure Following recovery from surgery and retraining to stability, all cohorts of rats received a mock infusion, where the injectors were placed into the cannula for the same amount of time as the subsequent infusion; however, the infusion pump was not turned on. If rats responded normally following the mock infusion (no significant difference between mock infusion performance and baseline performance) then stress and/or infusions test days commenced.  For Experiment 1, half of the rats received intracerebroventricular (ICV) infusion of the CRF antagonists alpha-helical CRF, and the other half received vehicle infusion, 10 minutes prior to being placed in a novel neutral room for 1 hour (no stress condition). On the second day of the testing protocol, rats received the same infusion as the first day, however, 10 minutes after infusion, rats were subjected to a one-hour acute stressor. Specifically, rats were placed into a Plexiglas cylindric tube (83 x 133 x 197 mm; Harvard Apparatus, Massachusetts, USA) that was located in the same, quiet and lit neutral room as the no stress condition. A desktop fan was aimed at the restrainers for the duration of the restraint stress in order to reduce the risk of hyperthermia. The restrainer length was adjusted to keep the rat immobilized without causing pain. Following one hour of restraint stress, rats were taken out of the restrainer tube and placed back into their home cage where they remained undisturbed for 10 minutes, after which they were placed into the operant chamber for testing. Following this two-day test sequence, rats were retrained to stability (approximately 5 days) and then experienced the counterbalanced infusion 	  	   20 following the same protocol. Therefore, rats that previously received alpha-helical CRF, now received vehicle infusions and vice versa.  For Experiment 2, each microinfusion test consisted of a 2 day sequence. On the first day rats received vehicle infusion (artificial cerebral spinal fluid, aCSF; 1 µl or 3 µl volume) 10 minutes prior to being placed in the operant chamber for the effort-based decision-making task. The following day, rats received a counterbalanced dose of CRF (0.25 µg or 1 µg in 1 µl or 3 µg in 3 µl) 10 minutes prior to the commencement of the session. Following the first test sequence, rats were retrained until stable baseline behavior was attained (approximately 2 to 5 days). Rats were subjected to two more rounds of the two-day experimental protocol (separated by drug-free retraining for 2 to 5 days) where they again received vehicle infusion (1 µl or 3 µl) into the lateral ventricle 10 minutes before the operant chamber session on the first day and a counterbalanced dose of CRF (either 0.25 µg or 1 µg in 1µl or 3 µg in 3 µl) on the second day.   Separate cohorts of rats in Experiments 3 and 4 underwent a similar experimental procedure. Mock infusion was carried out as detailed above followed by lateral ventricle vehicle infusion (aCSF; 3 µl) administered the following day, 10 mins prior to being placed in the operant chamber for testing on the reward magnitude discrimination task (Experiment 3) or the progressive ratio schedule of reinforcement task (Experiment 4). On the following day, rats were infused with CRF (3 µg) in a similar manner, 10 mins prior to being placed in the operant chamber for testing.  Animals in Experiments 5 were subjected to a comparable experimental protocol, whereby, following training and mock infusion, rats were split into two groups based on their performance so that rats in each group were matched in performance on HR preference over the previous three days of training. One group of rats was given vehicle infusion (aCSF, 0.5 µl per side), and the 	  	   21 other group was given CRF infusion (0.5µg/0.5 µl) into either the VTA or the NAc core 10 minutes prior to being placed in the operant chamber for testing. Rats were given 1-7 days of retraining to reestablish baseline responding, after which they received a second, counterbalanced infusion.  Histology Following experimental testing, rats were killed with CO2 and brains were removed and fixed in a 4% formalin solution. Brains were sectioned at 50  µm, mounted on gel-coated slides and Nissl stained using Cresyl Violet. Photographs of representative examples of an accurate lateral ventricle cannula placement for Experiments 1-4 are shown in Figure 1. Animals were taken out of the experiment if the cannula placement was outside of the lateral ventricle, which occurred when the placement was either too dorsal or too lateral (2 animals were removed from Experiment 1, and 4 animals were removed from Experiment 2). Figure 2 represents the ventral point of cannula placements in the VTA for all animals in Experiment 5. Eleven rats were tested, however two were removed from the final analysis, as both placements had one cannula that was placed dorsal to the VTA. The ventral extent of the cannula placements in the NAc core is shown in Figure 3. Fifteen rats that were trained and tested were utilized in the final analysis, with all cannula placements within the NAc core.   	  	   22     Figure	  1	                  B) C) D) A) Figure 1. Lateral ventricle (ICV) cannula placements. A) Accurate cannula placement from an animal in Experiment 2 (CRF16). B) Accurate cannula placement from an animal in Experiment 1 (CRF49). C) Missed cannula placement from an animal in Experiment 2 (CRF10). D) Missed cannula placement from an animal in Experiment 1 (CRF43).  	  	   23 Figure	  2	    	     Figure 2. Accurate ventral tegmental area (VTA) cannula placements (n = 9). 	  	   24    Figure 3. Accurate nucleus accumbens (NAc) core cannula placements (n =15). 	  	   25 Data analysis The main dependent variable for all effort-based decision-making tasks (Experiments 1, 2, and 5) was high effort/high reward (HR) choice, which is the ratio of the number of HR choices divided by the total number of trials (multiplied by 100 for percentage choice). Additional measures included the pressing rate, which is the number of presses on the HR lever per second, choice latency, or the time between lever insertion and lever choice (s), number of omissions, where rats fail to choose either lever, and locomotion, which is the number of beam breaks during the course of the session. The CRF antagonist experiment (Experiment 1) was analyzed using a 4 (treatment) X 4 (block) within-subjects ANOVA for all dependent measures. For all dependent measures in the CRF experiment (Experiment 2), we averaged all three vehicle infusion data together, as performance following these infusions did not significantly differ. Choice data for Experiment 2 were analyzed using a 4 (drug) X 4 (block) within-subjects ANOVA. Choice data for Experiment 5 were analyzed using a 2 (drug) X 4 (block) within-subjects ANOVA. Simple main effects and post hocs were used where applicable. A one-way within-subjects ANOVA with two levels (drug vs vehicle) was utilized for all locomotion data analyses. The main dependent variable for the reward magnitude task (Experiment 3) is choice of the high reward option, which is the ratio of the number of high reward choices divided by total number of trials (multiplied by 100 for percentage choice). Further measures for this task were choice latency, omissions and locomotion (rate cannot be tested as this task only requires one lever press). All dependent measures in this experiment were analyzed with a 2 (drug) X 4 (block) within-subjects ANOVA (except locomotion).  The progressive ratio task (Experiment 4) is unlike the other tasks in that it does not measure choice, as the rats were presented with only one lever. The main variables here are the 	  	   26 breakpoint, or the last ratio obtained, number of lever presses completed during the course of the session and the number of sugar pellets obtained during the session. Other measures include the pressing rate on the lever and locomotion, which is the number of beam breaks per minute in order to normalize the data, as the time spent in the chamber varied. All variables were analyzed using a one-way within-subjects ANOVA with 2 levels (drug vs vehicle). Simple main effects and post hocs were used where appropriate. Figure	    	  	   27 Results Experiment 1: Alpha-helical CRF ameliorates the effect of one hour of acute restraint stress on effort-based decision making The primary aim of the first experiment was to replicate the reduction in HR preference following one hour of acute restraint stress and to clarify the underlying mechanisms of this reorganization of preference behavior. Nineteen rats were tested on the effort-based decision-making task, with two removed due to missed placements, for a total of seventeen rats analyzed (see Figure 1 for cannula placement). The results of this experiment show that one hour of acute restraint reduces HR preference and prior administration of a nonspecific CRF antagonist ameliorated the effect of acute restraint stress of choice behavior. Choice data were analyzed with a two-way repeated measures ANOVA with treatment (four levels) and block (4 levels) as two within-subjects factors. As depicted in Figure 4, the ANOVA revealed a significant main effect of treatment (F(3,48)=2.81, p=.05), although there was no significant treatment x block interaction (F(9,144)=1.23, n.s.). Post hoc comparisons using Tukey’s test further confirmed that vehicle infusions (H20; 4 µl) + acute stress induced a significant reduction in choice of the HR compared to the vehicle + no-stress condition, as well as the CRF antagonist (30 µg alpha-helical CRF; 4 µl) + no-stress condition (p<.05). However, of particular interest, these comparisons revealed that pretreatment with CRF antagonist markedly attenuated the ability of acute stress to reduce preference for the HR option in these rats, in as much as their preference for the HR option did not differ significantly from either of the no stress conditions (p>.05).     	  	   28 Figure	  3	      Figure 4. Effort discounting following pre-stress alpha-helical administration (n = 17). A) Percent choice of the HR (high effort/high reward) lever following ICV vehicle (H20) administration and the no stress baseline compared to ICV vehicle (H20) administration prior to one hour of restraint stress. One hour of restraint stress significantly reduced HR preference (p<.05). B) Percent choice of the HR lever following ICV administration of a CRF antagonist (alpha-helical CRF) and the no stress baseline compared to ICV administration of a CRF antagonist (alpha-helical CRF) prior to one hour of restraint stress. No significant effect of stress when subjected to prior CRF antagonist infusion (p>.05). C) Percent choice of the HR lever following either vehicle or CRF antagonist administration prior to no stress or one hour of restraint stress. Figure 1A) and 1B) are combined in this figure to show all experimental conditions for comparative purposes.  	  	   29 Analysis of the choice latency did not reveal any significant differences across the different conditions on this measure (F (3, 48) = 0.53, n.s.; see Table 1). Likewise, none of the treatments induced a significant change in rates of lever pressing on the HR lever relative to the vehicle + no-stress control condition (F(3,48)=0.67, n.s.). Finally, there was no main effect of treatment on locomotion (F(3,48)=1.58, n.s.). Collectively, these results indicate that acute stress reduces preference for the high effort/high reward, switching reward preference toward the low effort/low reward option. However, this effect was attenuated by prior administration of a CRF antagonist, demonstrating that the reduced tendency to work harder to obtain a larger reward induced by acute stress may be driven by an increase in central CRF transmission. 	  	  Experiment 2: Central CRF infusion mimics acute stress on effort-based decision making The previous experiment uncovered a major role of CRF in the acute stress-induced reduction in preference for larger but more costly rewards. Therefore, our next goal was to investigate if central CRF administration could mimic the effect of acute stress in the effort-based decision-making task. We tested the effects of 3 doses of CRF (0.25, 1 and 3 µg) dissolved in either 1 µl (0.25 and 1 µg doses) or 3 µl (3 µg  dose) of aCSF (vehicle) with seventeen rats, however four were taken out due to missed cannula placements, for a total of thirteen rats analyzed. As performance following the low volume vehicle (1 µl) and high volume vehicle infusion (3 µl) were not significantly different (p>.05), we averaged all three vehicle infusion days together in Table 1                  Rate (presses/sec)        Choice latency (s)                                 uuuuuuuuUOmissions uuuuuuuuuULocomotion Experiment 1 Acute stress & CRF antagonism Vehicle/no stress 3.9 (0.3) 2.0 (0.3) 1.1 (1.1) 957 (97) Vehicle/stress 3.4 (0.3) 2.3 (0.3) 5.3 (2.6) 818 (90) CRF ant/ no stress 3.6 (0.4) 2.6 (0.4) 1.6 (1.4) 942 (112) CRF ant/ stress 3.5 (0.3) 2.2 (0.3) 0.4 (0.2) 873 (101) 	  	   30 the final analysis. The 4 (drug) x 4 (block) within-subjects ANOVA, depicted in Figure 5, revealed a significant main effect of CRF (F(3,36)=8.36,p<.001; treatment x block interaction (F(9,108)=1.37,n.s.). Post hoc comparisons using Dunnett’s tests confirmed that treatment with lower dose (0.25 µg) and medium dose (1 µg) of CRF did not alter choice relative to vehicle infusion (p>.05). However the higher CRF dose (3 µg) significantly reduced HR choice compared to average vehicle infusion (p<.05). With respect to other performance measures, there was a significant main effect of CRF on choice latency (F(3,36)=3.37, p=.03), with post hocs revealing a significant increase in choice latency following the high dose of CRF (p<.01), but no effect on latency at the lower doses (p>.05). Additionally, there was a significant main effect of CRF on rate (F(3,36)=30.95,p<.001), with post hocs revealing a significant reduction in pressing rate following the high dose of CRF (3 µg ; p<.05), but not the low (0.25 µg; p>.05) or medium dose (1 µg; p>.05). Along with an increase in latency and rate, CRF infusion significantly increased the omission rate (F(3,36)=10.15,p<.001), with post hocs confirming that the high dose of CRF increased the omissions compared to vehicle infusion (p<.05). Finally, there was no effect of CRF on locomotion (F(3,36)=1.75,n.s.; see Table 2). Together, these data suggest that 3 µg of central CRF infusion reduces preference for the more effortful option, increases hesitation to make a decision, decreases the vigor with which the lever is pressed once it is chosen and reduces the amount of trials completed. There was no evidence of nonspecific motor impairments, however, as locomotion was not altered by CRF infusion.    	  	   31       	      Figure	  4	       Table 2                  Rate (presses/sec)         Choice latency (s)    uuuuuuuUuOmissions   uuuuUuuuuuLocomotion Experiment 2 Central CRF infusion Average vehicle 3.5 (0.2) 2.4 (0.3) 1.0 (0.6) 998 (74) 0.25 µg CRF 3.3 (0.2) 2.5 (0.3) 0.1 (0.1) 966 (81) 1 µg CRF 3.4 (0.2) 2.6 (0.3) 0.4 (0.2) 989 (78)  3 µg CRF 2.3(0.1)* 3.3(0.5)* 9.7(2.8)* 846 (59) Figure 5. Effort discounting following central (ICV) infusion of CRF (n = 13). Percent choice of the HR lever following vehicle administration (averaged across three vehicle days) compared to low (0.25 µg), medium, (1 µg) and high (3 µg) doses of CRF. Neither low nor medium doses of CRF significantly altered HR preference compared to average vehicle infusion (p >.05), however, high CRF infusion significantly reduced preference for the HR option compared to average vehicle infusion (p<.05). 	  	   32 Experiment 3: Central CRF infusion does not alter HR preference when costs are equated in a reward magnitude discrimination In order to further clarify the manner in which increased CRF activity may have affected effort-based choice behavior, we conducted a control experiment using a reward magnitude discrimination task. A separate group of nine rats were trained on a simpler decision making task which equated the costs of the two levers, so that both levers required one press for either 2 sugar pellets (LR) or 4 sugar pellets (HR). This was to determine if the effects of CRF observed in the previous experiment was attributable to a reduced preference for larger vs smaller rewards. In this experiment, depicted in Figure 6a, CRF infusion (3 µg) prior to testing did not significantly alter HR choice behavior (main effect of CRF: F(1,8)=0.86, n.s.), indicating that CRF influences effort-based decision-making, not by altering reward valuation, but rather via adjusting effort cost preference.  Interestingly, central CRF infusion did significantly increase choice latencies in this task, (F(1,8) =8.76, p=.02), as seen in Figure 6b. This was a separate cohort of rats that had never been exposed to the effort-based decision-making task and so had never experienced effort-related costs, yet they hesitated when making a fairly simple choice between less reward or more reward. This effect mimics the influence of one hour of restraint stress in the reward magnitude task, which also increased choice latencies (Shafei et al., 2012). CRF also decreased locomotion (F(1,8)=29.03, p=.001), but CRF had no significant effect on omissions (mean: 0 vs 1.4; see Table 3).  Collectively, these results suggest that central CRF infusion has no effect on choice behavior in the relatively simple reward magnitude discrimination. Although choice was not affected, central CRF infusion increased deliberation time between choosing 2 pellets or 4  	  	   33    Table 3                      Rate (presses/sec)        Choice latency (s)   uuuuuuuUuOmissions   uuuuUuuuuuLocomotion Experiment 3      Reward magnitude task Vehicle N/A 0.4 (0.1) 0.0 (0.0) 1306 (166) 3 µg CRF N/A 1.0 (0.2)* 1.4 (0.8) 830 (96)* Figure 6. Reward magnitude discrimination following central ICV infusion of CRF (n = 9). A) Percent choice on HR lever following vehicle (aCSF) or CRF (3 µg) infusion. Prior CRF administration had no effect on HR lever when costs were equated (p>.05). B) Choice latencies averaged across all four trial blocks. Prior CRF administration significantly increased the latency to make a choice (p<.05). 	  	   34 pellets, mirroring the effect of acute restraint stress on choice latencies in the previous study (Shafei et al., 2012). In opposition to the other tasks, locomotion was significantly decreased following CRF infusion on the reward magnitude task. However, because this was not seen in the prior effort-based decision-making task and because there was no effect of CRF on omissions, this suggests that the effect is most likely not due to any general nonspecific effects of central CRF infusion. Experiment 4: Central CRF infusion alters motivation to work for reward in the progressive ratio task Although central CRF infusion did not alter the subjective value of objectively larger rewards, the possibility remains that CRF may interfere with the motivation to work for reward. This possibility was investigated using a progressive ratio schedule of reinforcement, used in many studies to assess motivation in rodents. If central CRF reduces the motivation to work for reward, then animals subjected to central CRF infusion would reduce instrumental responding on this task. Eight rats were trained and tested on this task, with all animals included in the within-subjects one-way (drug vs vehicle) ANOVA. The ANOVA revealed that central CRF administration significantly reduced instrumental responding, as shown in Figure 7. Specifically, central CRF infusion significantly reduced the breakpoint (or last ratio achieved; F(1,7)=19.60,p=.003; see Figure 7a), the total number of lever presses (F(1,7)=17.86,p=.004; see Figure 7b), and the number of pellets received during the session (F(1,7)=22.39,p=.002; see Figure 7c). In order to analyze the rates of lever pressing, we had to accommodate for the fact that most rats achieved a lower breakpoint after CRF infusions relative to control treatments. As    	  	   35     Figure 7. Progressive ratio schedule of reinforcement following ICV CRF administration (n = 8). A) Number of pellets received during the test session following vehicle (aCSF) or CRF (3 µg) infusion. Prior CRF infusion significantly reduced the number of pellets obtained in the progressive ratio task compared to vehicle infusion (p<.05). B) Number of lever presses made in the progressive ratio task following vehicle (aCSF) or CRF (3 µg) infusion. Prior CRF infusion significantly reduced the number of lever presses made in this task compared to vehicle infusion (p<.05). C) The breakpoint (or last ratio achieved) during the test session following vehicle (aCSF) or CRF (3 µg) infusion. Prior CRF infusion significantly reduced the breakpoint (or last ratio completed) compared to vehicle infusion (p<.05). D) Number of presses/sec for the first four ratios and the last ratio obtained. Prior CRF infusion had no effect during the lower ratios or the last ratio obtained (p>.05), however CRF significantly reduced the pressing rate during the higher ratios (p<.05).  	  	   36 such, we analyzed rate according to the ratio completed for all rats under both conditions (vehicle and CRF infusion), which in this instance, was the fourth ratio (6 presses required), as  well as the pressing rate for the last ratio obtained. Using a 2 (vehicle vs CRF infusion) X 5 (ratio requirement) within-subjects ANOVA, we found a significant main effect of treatment (F(1,7)=13.38,p=.008), a significant main effect of ratio (F(4,28)=12.68,p<.001), and a significant interaction of treatment and ratio (F(4,28)=3.02,p=.03). Simple main effects analysis revealed that rats displayed comparable rates of lever pressing for the first and second ratios of the session after both treatments (p>.05). However, during the later ratios, rates of responding were slower following CRF treatments relative to vehicle (p<.05; Figure 7d). Furthermore, there was a nonsignificant trend toward central CRF infusion reducing the number of beam breaks per minute (mean:16.14, SEM:1.20) compared to vehicle infusion (mean:19.91, SEM:1.65; F(1,7)=4.91,p =.06). Collectively, these data indicate that CRF infusions reduce the motivation of rats to respond at high ratios to obtain food reward.  Experiment 5: Intra-VTA, but not intra NAc, CRF infusion mimics acute stress and central CRF infusion on HR preference The previous studies indicate that CRF plays an important role in mediating the effect of acute stress on HR preference. The next two experiments were conducted to identify potential brain nuclei where CRF may be acting to mediate this effect. We initially targeted the dopamine cell body region in the VTA, as 1) the VTA expresses CRF receptors (Van Pett et al., 2000) 2) CRF is released into the VTA during episodes of acute stress (Wang et al., 2005) and 3) dopamine plays a critical role in promoting choice of larger, high cost rewards (Salamone et al., 1991; Sokolowski & Salamone, 1998; Aberman & Salamone, 1999; Salamone et al., 1994a&b; Cousins & Salamone, 1994; Floresco et al., 2008). Eleven rats were trained and tested on the 	  	   37 effort-based decision-making task, with two rats removed due to cannula placements outside the VTA (refer to Figure 2 for cannulae placement), leaving nine rats in the final analysis. As Figure 8 depicts, intra-VTA administration of CRF (0.5 µg) significantly reduced HR choice compared to vehicle infusion (F(1,8)=7.56, p=.03), suggesting that the VTA is at least one of the sites where CRF acts to reprioritize effort cost valuation. Intra-VTA CRF infusion did not affect other measures in this task, including choice latency (F(1,8)=.48, n.s.), number of omissions (F(1,8)= 2.17, n.s.), or locomotion (F(1,8)=.04, n.s.), as depicted in Table 4. However, there was a nonsignificant trend of intra-VTA CRF on rate (F(1,8)=4.57,p=.07), with intra-VTA CRF administration decreasing the pressing rate on HR lever compared to vehicle administration, in a similar manner to central CRF infusion on rate in the effort-based decision-making task.  The VTA may not be the only brain region in which CRF acts to mediate effort-based decision-making behavior. For example, CRF also acts in the NAc to potentiate DA release and elicit conditioned place preference (Lemos et al., 2012). Therefore, our next aim was to investigate if CRF may be acting in the NAc, in addition to the VTA, to reduce preference for the larger, costlier reward. To this end, we trained and tested fifteen rats on the effort-based decision-making task, infusing 0.5 µg of CRF into the NAc core prior to testing (see Figure 3 for cannula placement). As opposed to the effects of intra-VTA infusions of CRF, intra-NAc infusions did not induce an overall reduction in preference for the HR option relative to control treatments (main effect of treatment; F(1,14)= 0.0, n.s.). However, this analysis did reveal a treatment x block interaction (F(3,42)=4.82,p=.006). As can be observed in Figure 9, intra-NAc core infusions of CRF appeared to flatten the discounting curve, so that CRF infusion reduced HR preference compared to vehicle infusion when the effort requirement was low (2 presses;    	  	   38    Table 4                   Rate (presses/sec)        Choice latency (s)   uuuuuuuuUOmissions   uuuuUuuuuuLocomotion Experiment 5      Intra-VTA CRF infusion Vehicle 2.6 (0.1) 3.5 (0.4) 0.2 (0.2) 1049 (102) 0.5 µg CRF 2.2 (0.2) 3.7 (0.4) 0.8 (0.3) 1082 (137)       Figure 8. Effort discounting following intra-VTA infusion of CRF (n = 9). A) Percent choice of the HR lever following prior vehicle (aCSF) or CRF (0.5 µg) infusion into the VTA. Prior CRF infusion into the VTA significantly reduced HR preference compared to vehicle infusion (p <.05).  	  	  	   39 p<.05), however CRF infusion increased HR preference compared to vehicle infusion when the effort requirement was high (20 presses; p<.05).  There were no nonspecific effects of intra-NAc CRF infusion on behavior, as we did not find any significant main effect of CRF on choice latency (F(1,14)=1.82, n.s.), rate (F(1,14)=2.47, n.s.), number of omissions (F(1,14)=1.08, n.s.), or locomotion (F(1,14)=.54, n.s.; refer to Table 5). Together these results suggest that CRF is not primarily acting in the NAc core to mediate the behavioral effects of acute restraint stress, but that excessive activation of these receptors in the NAc can alter the manner in which animals discount larger rewards associated with different effort costs.   	  	   40   Figure	  5	      Table 5                   Rate (presses/sec)        Choice latency (s) u uuuuuuuUUUOmissions   uuuuuuuuULocomotion Experiment 6      Intra-NAc core CRF infusion  Vehicle 3.7 (0.4) 2.9 (0.3) 0.1 (0.1)  1156 (96) 0.5 µg CRF 3.5 (0.3) 3.2 (0.2) 1.8 (1.7)  1224 (155) Figure 9. Effort discounting following intra-NAc core infusion of CRF (n = 15). A) Percent choice of the HR lever following prior vehicle (aCSF) or CRF (0.5 µg) infusion into the NAc core. Prior CRF infusion into the NAc core resulted in a significant interaction between drug and block, where prior intra-NAc core CRF infusion reduced HR preference in the first block but increased HR preference in the last block (p<.05). 	  	   41 Discussion The goal of the present set of experiments was to clarify the underlying mechanisms through which acute stress rapidly alters preference during effort-based decision-making. First, we replicated the previous finding that one hour of acute restraint stress significantly reduces preference for the high effort/ high reward option. The alteration of effort-based decision-making driven by a reduction in HR preference was ameliorated by a nonspecific CRF antagonist (alpha-helical CRF), which indicates that central CRF mediates the relationship between acute restraint stress and effort-related choice behavior. The acute restraint stress-induced alteration of effort choice behavior was mimicked by centrally administering CRF. This does not appear to be mediated by influencing the general preference for objectively larger rewards, as there was no reduction in HR choice when costs were equated. Central CRF infusion, however, did reduce the motivation to work for rewards in the progressive ratio task, suggesting that CRF acts in the effort-based decision-making task to reduce the motivation to work for reward. We next investigated where CRF may be acting to influence effort choice behavior. Intra-VTA CRF infusion reduced preference for the costlier, more rewarding option in a similar manner as both central CRF infusion and acute restraint stress. CRF infusion in the NAc core, however, did not cause an overall reduction in HR choice, indicating that the effects of CRF in reducing preference for the large, costly reward are regionally specific.  The influence of acute restraint stress on effort-based decision-making preference and the mechanisms underlying this effect A prior study in our laboratory revealed that one-hour, but not twenty minutes, of restraint stress significantly reduced preference for the HR choice and increased choice latency in the effort-based decision-making task (Shafei et al., 2012). The first objective was to replicate this previous 	  	   42 study. Indeed, we found that one hour of acute restraint stress significantly reduced preference for the more effortful option, with rats switching preference to the low effort/low reward option, and in turn reducing the amount of reward obtained in a single session.  Although acute stress reduced HR preference in the effort-based decision-making task, it may have done so by altering some of the motivational processes underlying this task. Although this remains a possibility, several prior experiments suggest that this is not likely. For instance, acute stress may decrease the subjective value of objectively larger rewards, or decrease tolerance for delay that is inherent when effort costs increase, or alternatively, acute stress may decrease motivation to work for reward. The first possibility was previously disputed as restraint stress had no effect on HR preference when effort costs were equated in the reward magnitude discrimination task, suggesting that acute stress does not disrupt the normal preference for larger versus smaller rewards (Shafei et al., 2012). The second alternative explanation is not likely as restraint stress does not alter behavior in a delay discounting task, where delays between choice and reward are increased but effort costs are the same (Shafei et al., 2012). Moreover, restraint stress still reduced preference in an effort-based decision-making task with equivalent delays (Shafei et al., 2012), where the LR lever required the animal to wait for reward for the equivalent duration as the HR lever. Together these prior experiments indicate that restraint stress does not alter choice in the effort discounting task by altering tolerance for delays. Finally, the third possibility was assessed using the progressive ratio schedule of reinforcement task that measures the motivation to work for reward. Utilizing this task, the previous study found that one hour of restraint stress had no effect on breakpoint or the total number of levers pressed during the session (Shafei et al., 2012), suggesting that restraint stress did not disrupt motivational processes that enable the animal to repeatedly respond to high ratios. Together these studies 	  	   43 indicate that acute stress selectively alters the subjective preference of the HR option in the effort-based decision-making task, biasing the animals toward the less effortful, less costly reward. Although one hour of restraint stress reduced effortful choice behavior, it did not always cause an increase in choice latencies. This suggests that the stress-induced alteration of choice behavior is a robust finding, however, the effect of acute stress on choice latency may be weaker and more inconsistent. Other measures of task performance were not altered by one hour of restraint stress, including the pressing rate or the number of omissions, which mimics the previous finding of restraint stress on this task (Shafei et al., 2012), suggesting that acute stress did not disrupt the ability of rats to robustly respond to the lever. One hour of restraint stress, however, did significantly decrease locomotion. The effect of restraint stress on locomotion is not surprising as previous experiments found a nonsignificant reduction in locomotor activity following one hour of restraint stress in the effort discounting task and significantly reduced locomotor activity in the effort discounting with equivalent delays task (Shafei et al., 2012). Prior experiments found that CORT administration (1 mg/kg or 3 mg/kg) did not alter effort choice behavior and a nonspecific dopamine antagonist (flupenthixol) failed to block the effect of acute stress on HR preference, suggesting that neither CORT nor DA primarily mediate the influence of acute stress on choice behavior (Shafei et al., 2012). As such, our second aim was to understand the mechanisms underlying the acute stress-induced reprioritization of effort-related choice behavior. A primary candidate is CRF, which plays a major role in initiating the HPA axis, but also has numerous effects outside of the hypothalamus.  To investigate the role of CRF in the acute stress-induced alterations in effort choice behavior, we infused a nonspecific CRF antagonist, alpha-helical CRF, prior to one hour of 	  	   44 restraint stress and found that prior CRF antagonism ameliorated the effect of restraint stress on HR preference. Alpha-helical CRF had no effect on any other measure of decision-making, including number of omissions, locomotion, choice latency or rate, suggesting that the effect of the CRF antagonist was not due to the drug influencing any nonspecific behaviors in this task. Together these results suggest that CRF, in the context of acute stress, specifically influences the preference for animals to work harder for a larger reward in the effort-based decision-making task.  Cognitive/motivational alterations induced by central CRF infusion that influence effort-based decision-making Effort discounting following central CRF infusion To further investigate how CRF may modulate cost/benefit decision-making, we assessed whether direct CRF infusion could mimic the behavioral profile of acute restraint stress. To this end, we infused CRF into the lateral ventricle prior to effort-based decision-making. CRF infused into the ventricular system activates many different brain regions that are widely distributed, as previous studies have shown c-Fos-induced activation in brain regions that are relatively distant from the lateral ventricle, overlapping with brain regions that contain CRF receptors (Bittencourt & Sawchenko, 2000). Our results showed that high (3 µg), but not low doses (0.25 µg or 1 µg), of CRF into the lateral ventricle significantly reduced HR preference in the effort-based decision-making task. Similarly, 3 µg of CRF administered ICV decreased the pressing rate on the HR lever, increased the hesitation to make a choice, and increased the number of trials omitted. The high dose of CRF did not influence every aspect of effort-based decision-making, however, as locomotion was not significantly altered by this manipulation.   	  	   45 Although these results provide substantial evidence for the role of centrally acting CRF in mediating the acute stress-induced alterations in effortful choice behavior, only relatively high doses of CRF (3 µg) altered HR preference, with relatively low CRF doses (0.25 or 1 µg) having no effect. This was somewhat surprising as previous studies found that CRF displays a dose-response curve on many anxiety-related measures (Campbell et al., 2004; Dunn & Berridge, 1990). For instance, 0.1, 1 and 10 µg of ICV CRF reduce the amount of time spent in the center in a dose dependent manner during an open field test of anxiety and centrally administered CRF had a dose dependent effect on locomotion on doses between 0.1 and 10 µg (Campbell et al., 2004; Dunn & Berridge, 1990). Furthermore, alterations in behavior on the elevated plus maze, social interaction task, and forced swim task were evident following as little as 0.1 to 0.5 µg of ICV CRF infusion (Dunn & Berridge, 1990; Campbell et al., 2004). These findings suggest that more complex tasks, such as effort-based decision-making, require higher concentrations of circulating CRF to alter behavior when compared to relatively simpler or spontaneous behaviors related to anxiety, despair or social investigation. Collectively, these findings reveal a primary role of CRF in mediating the acute stress-induced alterations in effort discounting. Nonetheless, we cannot definitively rule out the possibility that CORT was somehow involved in the alteration of HR preference. This alternative explanation, however, seems unlikely, for numerous reasons. First, the CRF antagonist, alpha-helical CRF, attenuated the restraint stress-induced reduction in HR choice, indicating a primary role of CRF in mediating this effect. Secondly, previous experiments in our lab have administered exogenous CORT, which had no effect on choice behavior in the effort discounting task (Shafei et al., 2012). Although no experiment has directly studied whether the effects of stress on decision making can be blocked by administration of a GR antagonist, the finding that a 	  	   46 CRF antagonist ameliorated the effect of acute stress on choice behavior and central CRF mimics this effect, argues against the idea that CORT has a primary effect on behavior.  The possibility remains, however, that the CRF antagonist may have acted to block further CORT secretion by blocking the initiation of the HPA axis and that centrally infused CRF initiated the HPA axis leading to increases in CORT, and these alternative possibilities may be the underlying reason for alterations in effort choice behavior. These explanations are improbable for two reasons: 1) central CRF acts independently of the HPA axis in other behavioral tasks that are altered by episodes of acute stress and 2) varying doses of centrally infused CRF activate the HPA axis to a similar degree. Specifically, previous studies have demonstrated that the CRF antagonist, alpha-helical CRF (25 µg), blocked acute stress-induced conditioned place aversion (CPA) and ICV CRF mimicked acute stress by inducing CPA, however low doses of systemically administered CRF activated the HPA axis without influencing CPA (Cador et al., 1992). This indicates that CRF acts centrally, independent of the ability of CRF to stimulate the HPA axis, in order to induce CPA. Furthermore, wide-ranging doses of centrally infused CRF (0.1 µg, 1 µg and 10 µg CRF) activate the HPA axis to a similar degree, with all doses increasing plasma CORT levels significantly over baseline (Campbell et al., 2004; Cador et al., 1992). In our hands, only the 3 µg dose of centrally infused CRF reduced HR preference, with the 0.25 µg and 1 µg doses having no effect, suggesting that this effect was independent of HPA axis activation, as presumably all three doses activated the HPA axis and increased plasma CORT levels. Reward magnitude discrimination following central CRF infusion  One potential explanation for the effects of central CRF infusion on the alteration in effort-related choice behavior is that it may have caused a reduction in preference for objectively larger 	  	   47 rewards. In order to investigate this possibility, we trained a separate cohort of animals on the reward magnitude discrimination task, which equates the cost of the two levers so that one lever press will elicit either two sugar pellets or four sugar pellets. The effect of CRF was not due to the ability of CRF to alter preference for objectively larger rewards, as prior central CRF infusion did not alter preference for the larger reward when it was not associated with any costs. This parallels the results of the previous study, finding that one hour of acute restraint stress decreases HR preference in the effort task but had no effect when the costs were equated in the reward magnitude task (Shafei et al., 2012). Interestingly, central CRF infusion significantly increased choice latency times in the reward magnitude task. The increase in deliberation time following CRF administration, is surprising as rats have been well trained on this task and the choice is a relatively simple one, choosing between more or less reward. Although this was surprising, it mirrors the effect of acute stress, as one hour of acute restraint stress significantly increased deliberation time in the reward magnitude task (Shafei et al., 2012). Together, both acute restraint stress and central CRF infusion display a similar behavioral profile in the reward magnitude discrimination task, with neither disrupting the general preference for larger versus smaller rewards. These results provide further evidence of the major role that CRF plays in mediating the behavioral effect acute stress as both of these manipulations cause an identical behavioral profile in divergent behavioral tasks.  Progressive ratio schedule of reinforcement following central CRF infusion The previous experiment revealed that CRF did not alter reward valuation, however the possibility remains that CRF may be acting in the effort-based decision-making task to reduce the motivation to work for reward. To address this alternative explanation, we infused CRF into the lateral ventricle prior to testing on the progressive ratio task, which is a well-validated task 	  	   48 used in numerous studies to assess motivation to work for reward. Results of this experiment reveal that central CRF infusion significantly reduced the breakpoint, total number of lever presses and number of pellets received, all measures of the motivation to work for reward.  The progressive ratio task requires rats to press one lever the designated amount of times in order to receive one sugar pellet reward. In contrast to the effort-based decision-making, the progressive ratio task utilizes only one lever, and as such there are no alternative options, so rats can only choose whether they prefer to continue responding at higher ratios or not. Furthermore, the lever is continuously available throughout the course of the session and so the rat does not have to wait through an inter-trial interval in order to gain lever access. Central CRF infusion acts to reduce work in both of these tasks, in one by pressing the lever less throughout the session and in the other by choosing the other, less effortful, option.  These results are somewhat at odds with previous research that found one hour of acute restraint stress had no effect on the progressive ratio task (Shafei et al., 2012), however other studies have shown reduced breakpoint following only 20 minutes of stress (Wanat et al. 2013). These seemingly discrepant findings may suggest that the high dose of CRF exerts stronger effects on behavior than does one hour of acute restraint stress, with both manipulations significantly reducing HR behavior, and neither altering reward valuation, however CRF infusion reduces the motivation to work for reward whereas restraint stress has an inconsistent effect on this task. This indicates that high doses of CRF reduce the motivation to work for reward and in doing so, causes a reduction in HR choice in the effort-based decision-making task. Indeed, these findings suggest that high doses of central CRF may be more analogous to chronic stress manipulations in a depression model than to acute stress conditions. Recent evidence reveals that chronic stress (14 days of CORT in the drinking water) significantly 	  	   49 reduced the breakpoint and number of lever presses in a progressive ratio task in a similar manner to central CRF infusion in the current study (Olausson et al., 2013). The regional specificity of CRF action on effort-based decision-making preference Subsequent experiments probed the neural locus where CRF may be acting to alter motivation within the context of effort related decision-making. To this end, the VTA was a primary candidate, as this area houses DA nuclei and DA is crucial for normal cost/benefit decision-making, with systemic DA antagonists reducing preference of the larger, costlier reward (Floresco et al., 2008). Furthermore, CRF is released in the VTA during episodes of acute stress and acts to increase the firing rate of DA neurons (Wang et al., 2005; Wanat et al., 2008). Of particular relevance, previous studies have suggested that stress-induced reduction in motivation to work for reward is mediated by activation of CRF receptors within the VTA (Wanat et al., 2013). Specifically, 20 minutes of restraint stress reduced the breakpoint in the progressive ratio task, which was blocked by previous infusion of the nonspecific CRF antagonist, alpha-helical CRF, directly into the VTA and intra-VTA CRF administration mimicked the effect of 20 minutes of restraint stress, reducing breakpoint on the progressive ratio task (Wanat et al., 2013), similar to the effects of ICV infusions of CRF in the present study. In keeping with the above-mentioned findings, we found that CRF infusion into the VTA significantly reduced preference for the larger, costlier reward in the effort-based decision-making task.  The question remains as to how CRF may modulate VTA activity to alter effort-related decision-making. CRF in the VTA acts in complex, often opposing ways, increasing (or decreasing) excitatory and/or inhibitory transmission through numerous mechanisms involving both receptor subtypes. Retrograde tracing studies suggest that CRF is released into the VTA primarily via the bed nucleus of the stria terminalis (BNST), but the VTA also receives CRF 	  	   50 from the central amygdala (CeA) and the paraventricular nucleus (PVN) of the hypothalamus (Rodaros et al., 2007). More recent evidence suggests that the direct CRF projections from the ventral BNST to the VTA provide a mechanism by which stress releases CRF into the VTA (Vrankjkovic et al., 2014), which in turn, may potentiate stress-induced DA release to VTA targets. In slice preparations, the CRF1 receptor acts pre-synaptically on glutamate terminals to potentiate glutamate release onto AMPA receptors (Williams, Buchta, & Riegel, 2014). In addition to its role pre-synaptically, CRFR1 also potentiates DA neuron firing via a PKC enhancement of the inwardly rectifying hyperpolarization-activated current, Ih (Wanat et al., 2008). Additionally, CRFR2 acts post-synaptically to enhance NMDAR-mediated synaptic transmission in DA neurons via CRFR2-PLC/PKC activation, which also requires CRF binding protein (CRF-BP; Ungless et al., 2003). Conversely, there is evidence to suggest that CRFR2 acts pre-synaptically, as CRF2 receptor activation increases GABA release onto GABA-A receptors and spillover GABA activates GABA-B receptors on glutamate terminals in the VTA to attenuate the release of glutamate (Williams et al., 2014). This latter effect would be expected to reduce DA neuron firing. Therefore, it is clear from these studies that the role of CRF in the VTA is mediated through different mechanisms, both pre- and post-synaptically involving both receptor subtypes. Additionally, these somewhat conflicting roles for CRF in the VTA indicate that CRF1 and 2 receptors act in concert to shape VTA neuronal excitability with the CRF1 receptor primarily exciting these neurons via pre- and post-synaptic mechanisms and the CRF2 receptor acting to both inhibit postsynaptic neurons and attenuate glutamate release onto these neurons. The contradictory role of CRFR1 and CRFR2 has also been elucidated in prior behavioral studies, with the CRF1 receptor linked to anxiogenic responses whereas and the CRF2 receptor 	  	   51 associated with anxiolytic behavior (Hillhouse & Grammatopoulos, 2006). Furthermore, since the CRF1 receptor binds CRF with higher affinity than the CRF2 receptor (Aguilera & Liu, 2012), it has been postulated that the CRF1 receptor mediates the stress-induced behavioral effect of CRF, however, as CRF levels increase, CRF binds with the CRF2 receptor and dampens the stress response. Previous studies have shown that systemic administration of amphetamine caused a dose-dependent increase or decrease in HR preference, with lower doses increasing preference and higher doses decreasing preference in the effort-based decision-making task (Floresco et al., 2008). Thus, it is possible that the alteration in effort-related choice behavior by intra-VTA CRF administration may have been mediated by increased VTA activity and corresponding terminal DA release. However, this explanation is unlikely, as a prior study revealed that systemic administration of a nonspecific DA antagonist did not block the effect of acute stress in the effort discounting task (Shafei et al., 2012). The lack of effect in that study suggests that overall increases in DA are not the primary cause of the acute stress- or CRF-induced alterations in decision making. The above research points to the complexity with which VTA CRF acts in vitro, however, this method may not be ideal for analyzing how CRF acts in the VTA during complex behavioral tasks. Indeed, even in vitro, CRF can affect DA neurons in a differential dose-dependent manner. For instance, lower doses of CRF (3-100 nM) in the VTA potentiate EPSCs via CRF1 receptors, however at higher doses (300 nM) VTA CRF attenuates EPSCs via CRF2 receptors (Williams et al., 2014). Although both receptor subtypes are located pre-synaptically, CRF1 receptors are located on glutamate terminals, altering the probability of glutamate release onto DA neurons, whereas CRF2 receptors are found on GABA terminals, altering the probability of GABA 	  	   52 release onto DA neurons (Williams et al., 2014). When CRF2 receptors are activated, the probability of GABA release is increased and this GABA spillover activates presynaptic GABA-B receptors on glutamatergic terminals to attenuate the release of glutamate onto DA neurons.  When integrating the observations of how CRF may modulate DA transmission, it is interesting to note that both ventricular or VTA infusions of CRF (Wanat et al., 2013) alter progressive ratio responding and effort-related decision making in a manner similar to dopaminergic antagonists (Floresco et al., 2008; Salamone et al., 1991; Salamone, Cousins, & Bucher, 1994). This resemblance would suggest that despite the complexity of how CRF may modulate VTA dopamine neuron activity, the net effect of enhanced CRF transmission in these situations may be to reduce dopamine activity. In this regard, intra-VTA CRF infusion in rats performing a progressive ratio task attenuated DA release in the NAc in response to reward delivery but not to reward-predictive cues, highlighting how activation of VTA CRF receptors in the behaving animal can alter DA release into the NAc in a stimulus-specific manner (Wanat et al., 2013). Together this suggests that CRF in the VTA may act in a pathway specific manner by reducing the probability of glutamate release onto DA neurons in the VTA via presynaptic CRF receptors on glutamatergic terminals. With respect to the present study, CRF infusions may have dampened phasic dopamine release triggered by receipts of rewards. Notably, suppression of reward-associated phasic dopamine signalling by stimulation of the lateral habenula also reduced choice of larger, uncertain rewards (Stopper et al., 2014). A similar mechanism may explain the results of the present study, wherein a reduction in reward-associated dopamine signalling by CRF infusions may have reduced the tendency for animals to select the large reward option on a subsequent trial.   	  	   53 CRF in the VTA acts both pre- and post-synaptically to increase glutamatergic transmission onto DA neurons, however there is a relative dearth of information regarding how other neuron subpopulations, such as glutamatergic or GABAergic neurons, in the VTA are influenced by CRF. CRF receptors in the VTA may act pre- and/or post-synaptically to increase glutamatergic drive onto GABAergic VTA neurons, which would increase inhibitory transmission to DAergic neurons and may attenuate DA release. Indeed, a relative minority of CRF-containing terminals contact tyrosine hydroxylase immunoreactive (TH-ir) neurons (42%; Tagliaferro & Morales, 2008), suggesting that CRF release in the VTA acts on DA neurons, however this may be secondary to the influence of CRF on other neuron subpopulations (58% of CRF-ir terminals contacting TH- neurons), such as GABAergic or glutamatergic neurons in the VTA. Further studies are needed to ascertain the relative influence of CRF on VTA neuron subpopulations, ideally in a behaving animal.  Although intra-VTA CRF infusions significantly altered effort choice behavior by reducing preference for the more effortful option, this manipulation did not have any nonspecific effects on behavior. For instance, there was no effect of intra-VTA CRF infusion on choice latency, the number of omissions made, or locomotor activity, indicating that intra-VTA does not influence effort preference behavior by decreasing locomotion, increasing latency or increasing omission rates. Although these measures were not affected, there was a significant trend toward a decrease in pressing rate following intra-VTA CRF infusion, indicating that CRF in the VTA may reduce the motivation and vigor with which animals respond to this lever once it has been chosen. This is in line with previous studies that show that CRF in the VTA reduces the breakpoint and number of presses made on the progressive ratio task (Wanat et al., 2013). 	  	   54 The NAc core is a key dopamine terminal region that regulates effort-related decision-making, as lesions, inactivation or DA depletion in this region leads to a reduction in preference for larger, more costly rewards (Salamone et al., 1991; Sokolowski & Salamone, 1998; Aberman & Salamone, 1999; Cousins et al., 1993; Salamone et al., 1994a; Cousins & Salamone, 1994; Cousins, Sokolowski, & Salamone, 1993; Cousins & Salamone, 1994; Ghods-Sharifi & Floresco, 2010; Walton et al., 2009). Moreover, this nucleus expresses CRF receptors (Van Pett et al., 2000), indicating that CRF may have actions in this region. However, in contrast to the effects of intra-VTA CRF infusions, similar treatments within the NAc core, did not cause an overall reduction in preference for the larger, costlier reward but rather flattened the effort-discounting curve, with rats choosing the HR option less initially when the effort cost was low and preferring the HR option more when the effort cost was high. The flattening effect of intra-NAc CRF may be caused by the ability of CRF to potentiate DA release in the NAc (Lemos et al., 2012). Indeed, CRF in the NAc acts to potentiate presynaptic DA release via coactivation of CRFR1 and CRFR2 (Lemos et al., 2012). Both CRF receptors are co-localized with tyrosine-hydroxylase immunoreactive fibre segments and are expressed on NAc cell bodies, indicating that CRF acts both pre- and post-synaptically in the NAc (Lemos et al., 2012). CRF application to NAc slices potentiates DA release, which was blocked by application of either a CRFR1 or CRFR2 antagonist, demonstrating that coactivation of both receptors is required to increase DA release. Due to the ability of CRF to potentiate DA release, CRF infusion in the NAc is appetitive in a conditioned place preference (CPP) task with rats spending more time in the chamber paired with NAc CRF infusion (Lemos et al., 2012). As DA is involved in signaling the salience of an event (Redgrave et al., 1999), artificially increasing DA via CRF infusion in a region that integrates information regarding cost/benefit evaluations may result in a disruption in 	  	   55 reward saliency. Interestingly, CRF microinjections into the NAc (shell) magnifies the incentive salience of Pavlovian cues previously associated with reward (Peciña et al., 2006), suggesting that CRF acts in the NAc to increase the salience of a positive incentive cue. Thus, we may speculate that the CRF-induced potentiation of DA release into the NAc may potentially cause rats to value both HR and LR levers with relative equivalence in spite of increasing effort costs. Although these studies provide evidence that CRF in the NAc increases DA release in this region, how this increase in DA effects effort discounting is currently unknown. Systemic injections of DA agonist-like compounds, such as amphetamine, exert a biphasic effect on HR preference, increasing preference at the low dose (0.125 mg/kg) and decreasing preference at the high dose (0.50 mg/kg; Floresco et al., 2008). Microdialysis studies show that DA is increased in the NAc during this task (Salamone et al., 1994b; Day et al., 2010), and antagonizing or depleting DA in the NAc results in a reduction of the effortful choice (Salamone et al., 1991; Sokolowski & Salamone, 1998; Aberman & Salamone, 1999; Cousins et al., 1993; Salamone et al., 1994a; Cousins & Salamone, 1994), suggesting that DA in the NAc core is required for normal effort-related decision-making. That being said, no study, to our knowledge, has directly assessed how DA agonists would influence effort choice behavior. It would be interesting to investigate if a DA agonist infused into the NAc core would exert a biphasic dose effect on effort preference, similar to systemic administration (Floresco et al., 2008), or if the effect would be more similar to the flattening of the effort discounting curve seen following the likely DA potentiation due to intra-NAc core CRF infusion (Lemos et al., 2012). Conclusions These studies reveal that both centrally acting and intra-VTA CRF reorganize effort choice preference, biasing animals away from the high effort/high reward option, in a similar manner to 	  	   56 depressed patients in a back-translated human task of effort-based decision-making (Treadway et al., 2012). This indicates that CRF may play a significant role in depression and potentiation of CRF may induce symptoms such as anergia, or the unwillingness to expend effort in order to receive reward. Indeed, central administration of CRF produces a behavioral phenotype remarkably similar to the symptomology encountered in depressed patients; namely, decreased sexual behavior, disturbed sleep, alterations in locomotion, diminished food consumption and sympathetic nervous system activation (Nemeroff, 1988). Additionally, centrally acting CRF is well placed to modulate both serotonin and noradrenaline as there are CRF receptors in serotonergic and noradrenergic regions and it is widely accepted that these two monoaminergic systems are centrally involved in the etiology of depression (Arborelius et al., 1999). In favor of the CRF theory of depression, CRF levels in the cerebral spinal fluid (CSF) of depressed patients were significantly elevated, sometimes as much as twofold (Banki et al., 1987). This elevation is not seen in all psychological disorders though, as there was no difference in CSF CRF levels in schizophrenia or dementia patients compared to normal controls (Nemeroff et al., 1984), however, other stress-related disorders display similarly potentiated CSF CRF levels including patients with post-traumatic stress-disorder (Bremner et al., 1997).  Though depression is a stress-related disorder, not everyone that experiences stress, even multiple severe episodes, goes on to develop this disorder. This suggests that there are individual differences in susceptibility to acquiring depression and CRF levels may contribute to this development. Animal studies support this theory, as animals that were categorized as resilient, with a long latency to defeat (> 300 ms) in a resident intruder model, displayed a decrease in CRF efficacy, suggesting that dysregulation of the CRF system was protective against the potentiation of circulating CRF (Wood et al., 2010). Furthermore, the CRF antagonist antalarmin 	  	   57 (CRFR1 specific), given for 4 weeks following 5 weeks of chronic mild stress (CMS), counteracted some of the behavioral effects of the CMS (Ducottet, Griebel, & Belzung, 2003), suggesting that CRF antagonists may be useful for treating depressed patients. Indeed, a small human trial for treating depressed patients with a specific CRFR1 antagonist was conducted and the results of that trial were promising. Patients given the drug reduced both patient and physician depression and anxiety scores and experienced a subsequent worsening of depression symptomology once drug administration had ceased (Zobel et al., 2000). Although initially CRFR1 antagonists were shown to be effective, further clinical trials were abandoned due to lack of efficacy or liver toxicity of these drugs (Koob & Zorrilla, 2012). The role of CRF transmission, specifically in the VTA, may be an important target of research in human major depressive disorder, as centrally infused CRF, acute restraint stress and intra-VTA CRF each exert behavioral effects on effort preference in a remarkably similar manner to depressed patients in a back-translated human task of effort-based decision-making (Treadway et al., 2012). Taken together, these experiments suggest that the effort-based decision-making task in combination with manipulations of CRF transmission may be utilized as an animal model of depression that may be more behaviorally relevant and translatable. To summarize, acute stress rapidly and robustly reprioritizes cost/benefit valuations, biasing choices toward the less effortful option. This reorganization appears to be mediated by centrally acting CRF, as a nonspecific CRF antagonist ameliorates the effect of acute stress and centrally infused CRF mimics this effect. Neither acute stress nor centrally acting CRF act to reduce the subjective value of objectively larger rewards, suggesting the influence of both is on the effort cost valuation. Centrally acting CRF, however, appears to reduce effortful choice options by reducing the motivation to work for reward. The VTA is one region principally 	  	   58 involved in the motivation to work for reward, as we found a reduction in effortful choice in the effort discounting task and previous studies have shown a reduction in the motivation to work for reward following intra-VTA CRF infusion (Wanat et al., 2013). This influence seems to be regionally specific as CRF infusion into the NAc core did not elicit an overall reduction in effortful choice preference. Decision-making, which involves weighing the relative costs and benefits of each alternative, is a complex cognitive process that is required for normal functioning. The amount of effort required is one type of cost that can influence the subjective value of these alternative possibilities. Episodes of stress can lead to re-evaluation of these alternatives, rapidly and robustly altering decision-making behavior. In humans, two main symptom domains primarily characterize depressive disorders: 1) feelings of sadness (which are difficult to model in animals) and 2) a substantial lack of motivation, or anergia, with depressed patients reluctant to expend effort in exchange for pleasurable experiences. Indeed, it is this lack of motivation, and not an inability to experience pleasure per se, that is the most debilitating symptom of depression, rendering sufferers unable to participate in everyday activities. The influence of CRF on DA transmission and the fact that both CRF and DA are dysregulated in human depression suggest that this interaction may be the mechanism underlying the considerable decline in motivation seen in human depression.   	  	   59 References Aberman, J. E. & Salamone, J. D. (1999). Nucleus accumbens dopamine depletions make rats more sensitive to high ratio requirements but do not impair primary food reinforcement. Neuroscience, 92(2), 545-552. Aguilera, G. & Liu, Y. (2012). 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