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Contributions of nucleus accumbens circuitry to aspects of aversively-motivated behaviors Piantadosi, Patrick T. 2017

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   CONTRIBUTIONS OF NUCLEUS ACCUMBENS CIRCUITRY TO ASPECTS OF AVERSIVELY-MOTIVATED BEHAVIORS by  Patrick T. Piantadosi  B.A., St. Mary’s College of Maryland, 2010 M.A., The University of British Columbia, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Psychology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2017   © Patrick T. Piantadosi, 2017    ii  Abstract The nucleus accumbens is a heterogeneous brain structure involved in the integration of limbic and cortical input and the coordination of motor output during behavior. Made up primarily of two major subregions, the nucleus accumbens core (NAcC) and shell (NAcS), this region has been suggested to contribute to dissociable aspects of appetitive behavior on the basis of differential functions localized within these subregions. Briefly, the NAcC may promote states of behavioral action during reward-seeking, while the NAcS may refine such behavior by actively inhibiting inappropriate or irrelevant actions. In Chapter 1, we discuss relevant research related to the dissociability of the NAcC and NAcS at the circuit and behavioral levels. In Chapters 2, 3, and 4, we examine the contribution of these two NAc subregions, as well as associated cortical and limbic structures, to Pavlovian and instrumental suppression. Results suggested that the NAcC acted to promote behavioral indices of reward-seeking vigor, while the NAcS was necessary for the appropriate instantiation and expression of conditioned suppression. In Chapter 5, we probed the relevance of these NAc subregions to the performance of a novel active/passive avoidance behavior. On this task, rats had to dynamically promote or inhibit their responding, guided by discrete cues, to avoid a painful stimulus. While both NAc subregions were necessary for promoting behavior during active avoidance trials, only the NAcS was required for inhibiting responding during presentations of the passive avoidance stimulus. A control study suggested that neither NAc subregion was necessary for unconditioned responding to foot-shock, indicating that the previous results could not be explained by changes in pain sensitivity. We also probed the role of monoaminergic transmission to motivational conflict and active/passive avoidance by systemically administering d-amphetamine (AMPH) to a subset of animals in Chapter 3 and 4. These results suggested that AMPH promoted punishment induced inhibition of behavior during motivational conflict, but had the opposite effect during passive avoidance trials, inducing iii  pressing despite punishment. Chapter 5 discusses these results in the framework of a dichotomy between response-promotion and response-inhibition, relating these findings to extant literature in the appetitive and aversive domains.                           iv  Lay Summary The ability to inhibit actions that are potentially harmful is an integral part of an organism’s behavioral repertoire. Dysfunction of this behavior has been suggested to contribute to the compulsive actions that characterize disorders such as addiction and obsessive-compulsive disorder. A region within the ventral striatum, the nucleus accumbens, is composed of two subnuclei, the nucleus accumbens core and shell, that may differentially contribute to aspects of response-inhibition. Specifically, the accumbens core promotes reward-seeking, while the accumbens shell acts to inhibit irrelevant information or actions. Whether these two regions contribute to response-inhibition enforced by an aversive stimulus is unknown. Here, we examined the contribution of these subregions to such behavior by using small infusions of pharmacological agents to inhibit neuronal activity. Results suggested that the accumbens shell contributes to aversively-motivated response-suppression, while the accumbens core promotes action in the appetitive and aversive domains.          v  Preface Experimental chapters (2-4) were conducted in the laboratory of Dr. Stan B. Floresco at the University of British Columbia, within the Department of Psychology. Experiments were designed by Patrick T. Piantadosi (P.T. Piantadosi) and Dr. Stan B. Floresco (S.B. Floresco). All data collection was conducted by P.T. Piantadosi and undergraduate students under his direction. Data were analyzed and written by P.T. Piantadosi, with assistance from S.B. Floresco.  - A version of Chapter 4 has been published in the following form:  Piantadosi, P. T., Yeates, D. C. M. M., Wilkins, M., & Floresco, S. B. (2017). Contributions of basolateral amygdala and nucleus accumbens subregions to mediating motivational conflict during punished reward-seeking. Neurobiology of Learning and Memory, 140, 92–105. https://doi.org/10.1016/j.nlm.2017.02.017  P.T. Piantadosi performed all surgeries, and conducted behavioral training and testing with assistance from D.C.M Yeates, M. Wikins, and K. Pezarro (undergraduate volunteers). P.T. Piantadosi wrote the dissertation, with input from S.B. Floresco.  All experimental protocols were approved by the Animal Care Committee (ACC), University of British Columbia, and conducted in compliance with guidelines provided by the Canadian Council on Animal Care (CCAC). ACC certificate numbers: A10-0197 or A14-021    vi  Table of Contents Abstract ......................................................................................................................................................... ii Lay summary ............................................................................................................................................... iv Preface .......................................................................................................................................................... v Table of Contents ......................................................................................................................................... vi List of Tables ............................................................................................................................................. viii List of Figures .............................................................................................................................................. ix List of Abbreviations .................................................................................................................................... x Acknowledgements ...................................................................................................................................... xi Chapter 1: Introduction ................................................................................................................................. 1 1.1 The NAc: A heterogenous interface between affect and action .................................................... 3 1.2 NAc subregion-specific control of action and inhibition .............................................................. 6 1.3 Models of aversive learning and related circuitry ....................................................................... 12 Chapter 2: Cortico-striatal contributions to the acquisition and expression of discriminative conditioned suppression .................................................................................................................................................. 28 2.1 Introduction ................................................................................................................................. 28 2.2 Methods....................................................................................................................................... 33 2.3 Results ......................................................................................................................................... 40 2.4 Discussion ................................................................................................................................... 48 Chapter 3: Investigating functional cortico-striatal or limbic-striatal circuits contributing to the acquisition and expression of discriminative conditioned suppression ......................................................................... 72 3.1 Introduction ................................................................................................................................. 72 3.2 Methods....................................................................................................................................... 75 3.3 Results ......................................................................................................................................... 80 3.4 Discussion ................................................................................................................................... 82 3.5 Conclusion .................................................................................................................................. 86 Chapter 4: The role of NAc core and shell in motivational conflict during reward and punishment ......... 91 4.1 Introduction ................................................................................................................................. 91 4.2 Methods....................................................................................................................................... 94 4.3 Results ....................................................................................................................................... 100 4.4 Discussion ................................................................................................................................. 108 4.5 Conclusion ................................................................................................................................ 120 Chapter 5: Dissociable contributions of NAc core and shell during active/passive avoidance ................ 128 5.1 Introduction ............................................................................................................................... 128 5.2 Methods..................................................................................................................................... 133 vii  5.3 Results ....................................................................................................................................... 143 5.4 Discussion ................................................................................................................................. 148 5.5 Conclusion ................................................................................................................................ 164 Chapter 6: General discussion .................................................................................................................. 171 6.1 Dissociable contributions of NAc subregions to the inhibition and promotion of behavior ..... 172 6.2 AMPH induces task-dependent bidirectional changes in instrumental punishment ................. 178 6.3 Experimental merits and future directions ................................................................................ 181 6.4 Relevance to neuropsychiatric disease ...................................................................................... 186 6.5 Conclusion ................................................................................................................................ 188 References ................................................................................................................................................. 190                      viii   List of Tables Table 1. Mean (±SEM) values for overall locomotion, and the change in locomotor activity during CS+ versus CS- presentations within the conditioning session, for animals manipulated prior to conditioning…………………………………………………………………………………………….64  Table 2. Mean (±SEM) values for total locomotion, rate of lever-pressing, and total lever-presses during the discriminative fear expression test session………………………………………………………....65  Table 3. Mean (±SEM) values for ancillary measures during the conditioning session, induced by BLA-NAcS manipulation prior to conditioning……………………………………………………………...87  Table 4. Mean (±SEM) values for ancillary measures induced by BLA-NAcS or PL-NAcS manipulation.…………………………………………………………………………………………...87  Table 5. Mean (±SEM) values for ancillary measures on the Conflict or No-Conflict task……………………………………………………………………………………………………..122  Table 6. Mean (± SEM) values for ancillary measures during the active/passive avoidance task……………………………………………………………………………………………………..166              ix   List of Figures Figure 1. Discriminative fear task diagram and histology. ......................................................................... 66 Figure 2. Inactivation of mPFC does not impact the acquisition of conditioned suppression .................... 67 Figure 3. Pre-conditioning NAcS, but not NAcC, inactivation diminishes conditioned suppression. ....... 68 Figure 4. Both mPFC subregions control the expression of conditioned suppression. ............................... 69 Figure 5. IL inactivation has no impact on conditioned suppression expression conducted using a standard, single-stimulus design. ................................................................................................................ 70 Figure 6. NAcS, but not NAcC, mediates the expression of conditioned suppression. .............................. 71 Figure 7. Disconnection methodology diagram. ......................................................................................... 88 Figure 8. A BLA-NAcS disconnection does not mediate the acquisition of conditioned fear. .................. 89 Figure 9. A PL-NAcS projection contributes to the expression of conditioned suppression. ..................... 90 Figure 10. Histology schematic for Conflict and No-Conflict task animals ............................................. 123 Figure 11. Task diagram and data from pharmacological manipulation on the Conflict task .................. 124 Figure 12. Task diagram and data from inactivations on the No-Conflict task ........................................ 125 Figure 13. Baseline analysis suggests NAcS and BLA promote reward seeking as a function of task history. ...................................................................................................................................................... 126 Figure 14. Trial structure and survival plot of training for the active/passive avoidance task. ................ 167 Figure 15. NAcC activity is necessary for active, but not passive, avoidance performance. ................... 168 Figure 16. NAcS activity is necessary for active and passive avoidance performance. ........................... 168 Figure 17. AMPH administration selectively provokes passive avoidance failure. .................................. 169 Figure 18. Neither NAc subregion is necessary for foot-shock sensitivity. .............................................. 170    x  List of Abbreviations  ACC  Anterior cingulate cortex ANOVA  Analysis of variance AP  Anteriorposterior BA#  Broca’s area  B/M  Baclofen/Muscimol BLA  Basolateral amygdala CaMKII Calcium calmodulin-dependent kinase II CO2  Carbon dioxide CREB  cAMP response element binding protein CS (or CS+) Conditioned stimulus CS-  Neutral stimulus dACC  Dorsal anterior cingulate cortex DV  Dorsoventral FR  Fixed ratio IL  Infralimbic cortex ML  Mediolateral mPFC  Medial prefrontal cortex NAc  Nucleus accumbens NAcC  Nucleus accumbens core NAcS  Nucleus accumbens shell  OCD  Obsessive compulsive disorder OFC  Orbitofrontal cortex PFC  Prefrontal cortex PIT  Pavlovian-to-instrumental transfer PL  Prelimbic cortex PTSD  Post-traumatic stress disorder  SAL  Saline US  Unconditioned stimulus VI  Variable interval VTA  Ventral tegmental area            xi  Acknowledgements This thesis would not have been possible but for the outstanding mentorship of my advisor, Dr. Stan B. Floresco. Throughout my time in the lab, he has provided excellent guidance and mentorship, providing me with the opportunity to probe questions that have not traditionally been the central focus of the laboratory. His curiosity regarding the brain is contagious, and many of the questions answered in this thesis are a direct testament to that. During my time in the lab, I believe I have grown tremendously, both as a person and an academic. I credit Dr. Floresco with enabling this growth, and cannot thank him enough.   I am additionally grateful to the other members of my supervisory committee, Dr. Todd Handy and Dr. Catharine Winstanley, who have provided valuable insights into the construction of this thesis. In particular, Dr. Handy and Dr. Winstanley encouraged me to connect this series of experiments to a broad literature, which I believe strengthens the conclusions drawn within.   Many members of the Floresco lab have helped this thesis come to fruition over the past four years. Special thanks to Dr. Colin Stopper and Maric Tse for their input on these projects during their formative stages, provided during long hours of surgery and other animal work. Other members of the laboratory, including Meagan Auger, Debra Bercovici, Courtney Bryce, Gemma Floresco, Nicole Jenni, Josh Larkin, Ryan Tomm, and Mieke van Holstein, have provided advice and camaraderie without which I would be at a loss. Other members of the behavioral neuroscience department, including Lucille Hoover, Alice Chan, and Anne Cheng, provided invaluable structural support for the animal work conducted in this thesis.   I am forever grateful to my family, in particular my parents and brother. My parents raised me to be inquisitive and persevering, both qualities that I believe are apparent in the work conducted throughout my time at UBC. I am grateful to my brother, a fellow neuroscientist, for xii  his valuable input on these projects, as well as his friendship. Finally, I cannot express enough gratitude towards my girlfriend, Joyce Miranda, for everything over the past six years. She has sacrificed more for me than I’d care to admit, and without her patience and love, I’m not sure how my Canadian experience might have turned out.                 1  Chapter 1: Introduction Aversive events and the cues that predict them have a tremendous ability to alter animal behavior (Estes & Skinner, 1941; N. E. Miller, 1948). Depending on the particular contextual or situational variables encountered, fearful events may inhibit or invigorate activity. In many cases, such aversively-motivated behaviors are adaptive; for a foraging rodent, hearing a sound within the frequency range of a predator vocalization will elicit a defensive response that may protect it from harm. Survival is predicated on the ability of an animal to both attend and react to predictive cues in the environment that signal when one action (e.g. foraging or approach behavior) is favored over another (e.g. seeking shelter, or suppressing foraging).  This type of ethological situation has been suggested to have real-world implications for modern-day humans (Hagenaars, Oitzl, & Roelofs, 2014; McNaughton, 1982; M. A. Miller, Thomé, & Cowen, 2013; Pearson, Watson, & Platt, 2014; Pellman & Kim, 2016). Although considerations regarding survival during the pursuit of such needs no longer applies to many individuals, other costs of which we are afraid, such as losing wealth, status, employment, etc., weigh against potential benefits in a similar way as primary punishment. This parallel is exemplified by the aberrant approach/avoidance behavior observed in neuropsychiatric conditions. For example, negative consequences such as punishment are less effective at inhibiting behavior in individuals with substance abuse or obsessive compulsive disorder (Everitt, 2014; Feil et al., 2010; Wood & Ahmari, 2015), suggesting a potential deficit in processing or utilizing negative consequences resulting from behavior. In other disorders, aversive events have an inappropriately extreme impact on behavior, such as the elevated and persistent levels of fear and anxiety expressed towards ambiguous or non-threatening stimuli in 2  individuals suffering from anxiety or post-traumatic stress disorders (Duits, Cath, Lissek, Hox, Hamm, Engelhard, Van Den Hout, et al., 2015; Grillon & Morgan, 1999; Lissek et al., 2014).  Given the notable burden placed on individuals, families, and economies by these and other neuropsychiatric conditions (Hjärthag, Helldin, Karilampi, & Norlander, 2010; Ohaeri, 2003; Whiteford et al., 2013; Whiteford, Ferrari, Degenhardt, Feigin, & Vos, 2015), developing a better understanding of the neurobiological bases of aversively-mediated behavior is necessary. As such, the brain mechanisms by which these events are learned about, maintained, and come to alter behavior are a major focus of modern neuroscience. This interest has led researchers to probe the brains of relatively simple model organisms, such as rodents, using increasingly nuanced techniques during situations that provoke fear, or a competition between bivalent motivations.  The aim of this thesis was to examine a potential role for the rodent nucleus accumbens (NAc), as well as associated cortico-limbic afferents, in aversively-motivated behavior. Here, we use the term aversive motivation to refer to any situation during which behavior is altered by the potential delivery of an aversive stimulus. Although the NAc is commonly considered a “reward” nucleus, given its established role in reinforcement learning and appetitive behavior, a bivalent role for this region has been proposed and demonstrated (Aberman & Salamone, 1999; Kim et al., 2017; Levita et al., 2009; Roitman, Wheeler, & Carelli, 2005; Schoenbaum & Setlow, 2003; Setlow, Schoenbaum, & Gallagher, 2003; Soares-Cunha, Coimbra, Sousa, & Rodrigues, 2016). In the following experiments, we examined the contributions of the NAc, specifically its subregions, the shell and core, to situations where motivational drives conflict. Parallel findings from the appetitive conditioning literature implicate these two subregions in partially dissociable aspects of behavior. Such data suggest that, although both subnuclei may mediate some degree of 3  behavioral approach, the NAcS is uniquely responsible for the refinement of behavior by inhibiting inappropriate actions. To date, few studies have examined whether such a functional dichotomy of NAc function exists when response-inhibition or promotion are enforced by an aversive stimulus, rather than by factors relating to reinforcer availability. We evaluate this question using established Pavlovian and instrumental aversive conditioning methods, as well as a novel avoidance paradigm, combined with local pharmacological inactivation of cortico-limbic-striatal regions of interest.  1.1 The NAc: A heterogenous interface between affect and action Prior to delving into the specific functions of the NAc related to aversively-motivated behavior, a discussion of the region’s hodological complexity is necessary. The NAc is a neuroanatomically and functionally heterogeneous structure, made up primarily of two main subregions, a lateral core (NAcC) which surrounds the rostral portions of the anterior commisure, and a shell which borders the core medially and ventrally (NAcS). These two subnuclei are anatomically dissociable based on their expression of various proteins and neuroactive peptides. For example, the calcium binding protein calbindin is enriched in the NAcC (similar to the dorsal striatum), but relatively absent from the medial aspect of the NAcS (Jongen-Rêlo, Voorn, Groenewegen, Voom, & Groenewegen, 1994; Meredith, Pattiselanno, Groenewegen, & Haber, 1996). In comparison, expression of the peptide substance P is higher in the medial NAcS than in the NAcC (Brog, Salyapongse, Deutch, & Zahm, 1993; Jongen-Rêlo et al., 1994). Primarily useful for distinguishing between these two subregions in situ, such neurochemical distinctions hint at potential differences in the functions controlled by the two subnuclei. Although both the NAcS and NAcC receive afferent input from many of the same limbic and cortical regions, the topographic nature of these projections are largely distinct. Generally, 4  the medial NAcS receives afferent input from ventral regions of the medial prefrontal cortex (mPFC), as well as caudal or ventral sections of the basolateral amygdala (BLA) and hippocampus/subiculum, respectively (Berendse, Galis-de Graaf, & Groenewegen, 1992; Brog et al., 1993; French & Totterdell, 2002; Groenewegen, Wright, Beijer, & Voorn, 1999; Sesack, Deutch, Roth, & Bunney, 1989; Vertes, 2004). In comparison, the NAcC receives input from more dorsal regions of the mPFC, as well as a diffuse projection from basolateral amygdala (BLA) and ventral hippocampus/subiculum. Midbrain dopamine neurons make a substantial projection to the NAc, although the particular cell groups that project to each structure are different. The medial A10 neurons in the ventral tegmental area (VTA) project prominently to the medial NAcS, while the more lateral A10 neurons project predominantly to the NAcC (Ikemoto, 2007). Thus, afferent projections to NAc subregions are often oriented topographically, which suggests that behavioral dissociations may be mediated in part by these partially segregated circuits. In addition to the heterogeneity of afferent input received by these regions, the NAcS and NAcC make efferent projections to divergent regions. The NAcC is typically considered to be more tightly linked to motor output, projecting primarily to lateral ventral pallidum, substantia nigra pars compacta (as well as the reticulata), and other motor affector sites (Berendse, Groenewegen, & Lohman, 1992; Heimer, Zahm, Churchill, Kalivas, & Wohltmann, 1991; Pennartz, Groenewegen, & Lopes Da Silva, 1994). In contrast, the NAcS projects to dopaminergic cells in the ventral tegmental area, hypothalamic sites, and medial ventral pallidum to control a diverse array of behavioral functions (Heimer et al., 1991; Pennartz et al., 1994). Although many projections from these subnuclei are segregated, both regions share overlapping inputs to the bed nucleus of the stria terminalis, lateral septum, and lateral habenula. NAc 5  subregions also project throughout the basal ganglia, including intrinsic reciprocal connections between the NAcC and NAcS, which are more extensive from NAcC to NAcS than vice versa (Van Dongen et al., 2005).  Within each structure, the inputs from limbic and cortical regions converge on inhibitory GABAergic, medium spiny projection neurons (French & Totterdell, 2002, 2003), which make up approximately 90% of cells in this nucleus (Meredith, 1999). Physiologically, these medium spiny neurons have a bistable membrane potential, resting at a relatively hyperpolarized membrane potential (“down-state”), and oscillating between this resting potential and a more depolarized potential “up-state” (O’Donnell & Grace, 1995; O’Donnell, Greene, Pabello, Lewis, & Grace, 1999). These up-states can be driven by strong afferent input from limbic (primarily ventral subiculum) or prefrontal regions (Calhoon & O’Donnell, 2013a; Goto & O’Donnell, 2002; Gruber & O’Donnell, 2009; O’Donnell & Grace, 1995; O’Donnell et al., 1999). Once in an upstate, action potential firing can be elicited by activity in critical limbic or cortical afferents, suggesting that the NAc may effectively act as a gate, allowing task-relevant inputs to control NAc output (Gruber, Hussain, & O’Donnell, 2009; Mogenson, Jones, & Yim, 1980). When foraging in an operant environment, for example, coherence between structures mediating spatial navigation, such as the hippocampus, and the NAcC increases, while exploiting an instrumental operant response to receive reward increases coherence with a mPFC to NAcC circuit (Gruber et al., 2009). Results such as these provide support for the hypothesis that the NAc integrates competing input from limbic and cortical afferents, with the specific circuit most relevant for task performance being recruited on demand. Given that differences exist in the specific efferent and afferent projections of each NAc subregion, it is possible that the integration of these inputs may lead to differences in function.  6  1.2 NAc subregion-specific control of action and inhibition In fact, the dissociability of these subregions has been demonstrated across a variety of experimental paradigms, primarily within the appetitive domain (for review, see Floresco, 2015). Although a comprehensive review of these functions will not be undertaken here, findings from the appetitive conditioning literature that may be of direct relevance to action selection following aversive conditioning will be discussed. These functions include the ability of cues to act as incentive stimuli, the refinement of cue-directed action selection, and the regulation of impulsivity.  Incentive salience is a construct that describes the process by which discrete environmental stimuli become imbued with the motivational properties of antecedent primary reinforcers. This process seeks to explain how, in some animals, discrete cues predictive of reward can come to control approach behavior (Berridge, 2012; Dickinson & Balleine, 1994). One way to assess the incentive properties of a cue is by examining the Pavlovian-instrumental transfer (PIT) effect. Following Pavlovian pairing of a stimulus (CS+; e.g., light, lever) with reinforcement (e.g., sucrose), some rats learn to approach and engage the CS+, but not an equivalently presented CS- (similar modality cue, never paired with reinforcement), reflecting a shift in the incentive value of that cue. Although the CS+ is occasionally a manipulanda such as an operant lever, this procedure is purely Pavlovian, with no instrumental response required for reward delivery. During the transfer phase of the PIT procedure, presentation of the CS+ can invigorate instrumental responding if the instrumental response is reinforced with the same outcome as the CS+ (outcome-specific) or a novel substance (outcome-general). Lesions or inactivations of NAc subregions differentially impacts these two types of PIT (Corbit & Balleine, 2011; Corbit, Muir, & Balleine, 2001). Generally, inhibiting activity within the NAcS impairs 7  the outcome-specific form of PIT, while the same manipulation of the NAcC impairs the outcome-general form (Corbit & Balleine, 2011; Corbit et al., 2001). Consistent with an integrative role of the NAc as a limbic-motor interface, this dissociation between the regional specificity of outcome-specific versus general PIT is mediated by BLA-NAcS and BLA-NAcC projections, respectively (Corbit & Balleine, 2005; Shiflett & Balleine, 2010). Recent reports suggest a parallel functional circuit between the ventromedial PFC (vmPFC) and the NAcS that may also mediate outcome-specific PIT (Keistler, Barker, & Taylor, 2015). Thus, NAcS may be particularly sensitive to specific cue-outcome relationships, while NAcC may act more generally to increase motivated output, as a function of differential cortico-limbic input.  Further support for such an incentive-motivational account of NAc function comes from a series of elegant studies examining the meso-cortico-limbic-striatal regulation of response selection, using a discriminative stimulus (DS) appetitive task (Ambroggi, Ghazizadeh, Nicola, & Fields, 2011; Ghazizadeh, Ambroggi, Odean, & Fields, 2012; Ishikawa, Ambroggi, Nicola, & Fields, 2008, 2010; Nicola, Yun, Wakabayashi, & Fields, 2004). This task requires rats to discriminate between a DS that signals reward availability, which can be obtained by pressing an active lever, and another stimulus that is never reinforced (NS). In addition, lever-presses on another, inactive lever are never reinforced. Over the course of training, rats come to both discriminate well between the DS and NS, as well as allocate their instrumental activity towards the active lever exclusively during DS presentations. Thus, appropriate action selection results from the promotion of reinforcement-seeking behavior during the DS, and an inhibition of this same response during all other task phases. The neural correlates of this behavior are observed both in the NAc, as well as the mPFC and BLA (Ambroggi et al., 2011; Ghazizadeh et al., 2012; Ishikawa et al., 2008, 2010). When 8  first acquiring the task, animals learn to refine their behavior by encoding both the relevance of the DS, and the irrelevance of the NS and the inactive lever, as well as other non-rewarded task epochs, such as during the inter-stimulus interval. This acquisition is related to phasic activity within the NAcS that correlates with the inhibition of irrelevant task actions, such as neural responses to the NS (Ghazizadeh et al., 2012). In addition, a separate mechanism promotes the tonic activity of neurons that act to oppose reward-seeking, further supporting a response-inhibitory account of NAcS function. These two inhibitory processes during learning appear to be mediated by a projection from the vmPFC (Ghazizadeh et al., 2012). In parallel, another circuit mediated by the NAcC acts to promote approach behavior during DS presentations (Ambroggi, Ishikawa, Fields, & Nicola, 2008; Ishikawa et al., 2008, 2010). BLA neurons respond to a DS with short latencies, occurring earlier than do responses in the NAcC (Ambroggi et al., 2008). Such results suggest that the BLA drives neuronal responses in the NAcC, contributing to DS-evoked approach activity. The promotion of DS-evoked activity is also driven by a possible circuit involving the dorsomedial PFC (dmPFC) and NAcC (Ishikawa et al., 2008, 2010). Single unit activity related to cue presentation or operant behavior is often larger in magnitude when preceded by a DS, as compared to NS, suggesting that the DS-evoked behavior and neural activity reflect an incentive motivational process.  These electrophysiological studies provide correlative evidence that NAc subregions, in concert with cortico-limbic afferents, dissociably contribute to action selection. To causally identify a role for these regions in response promotion and inhibition, pharmacological compounds can be infused directly into discrete brain regions to affect neuronal activity. When key regions of the PFC, BLA, or NAc are pharmacologically inhibited during performance, behavioral impairments suggestive of deficient response-promotion and response-inhibition are 9  observed (Ambroggi et al., 2011, 2008; Ghazizadeh et al., 2012; Ishikawa et al., 2008, 2010; Nicola et al., 2004; Yun, Wakabayashi, Fields, & Nicola, 2004). For example, the infusion of GABAB and GABAA receptor agonists, baclofen/muscimol (B/M) into the vmPFC unmasks activity within the NAcS that encodes previously inhibited task events, including reward-seeking activity following NS presentation and during lever-presses on the never-reinforced lever (Ambroggi et al., 2011; Ghazizadeh et al., 2012). The same manipulation of the dmPFC or BLA preferentially impacts neuronal activity and behavior in response to the DS (Ishikawa et al., 2008, 2010). When considering the NAc, the inhibition of activity within each subregion produces differential results on DS-evoked reward-seeking, and NS-evoked behavioral inhibition. Infusing B/M into the NAcC selectively decreases motivated reward-seeking behavior driven by presentations of the DS (Ambroggi et al., 2011). Inactivation of the NAcS, in comparison, makes a relatively specific contribution to the suppression of inappropriate or non-rewarded behavior. Taking this subregion offline temporarily disinhibits lever-pressing during the NS, as well as pressing of the inactive (never-reinforced) lever (Ambroggi et al., 2011). Thus, these regions of the ventral striatum integrate afferent input to refine behavior, consistent with its hypothesized role as a limbic-motor interface. However, the manner in which action selection is refined differs by each subregion, with the NAcC allowing for response-promotion in response to an incentive cue, and the NAcS inhibiting task-irrelevant or inappropriate reward-seeking. These finding are paralleled by studies examining the reinstatement of reward-seeking following extinction, which is often exaggerated in animals seeking food, cocaine, or alcohol following NAcS inactivation (Di Ciano, Robbins, & Everitt, 2008; Floresco, McLaughlin, & Haluk, 2008; Millan, Furlong, & McNally, 2010; Peters, LaLumiere, & Kalivas, 2008). Eliminating neural activity within the NAcC, in contrast, typically produces the opposite effect, 10  inhibiting reinstatement (Di Ciano et al., 2008; Floresco et al., 2008). Extinction is a form of behavioral flexibility that is thought to involve the formation of a new, inhibitory association between a stimulus or action that previously produced an outcome, and the diminished incentive value following omission of the outcome (Bouton & Moody, 2004). Inactivation of NAcS during reinstatement may hamper the usage of this inhibitory memory, subsequently reinstating behavior to a level comparable to animals that never underwent extinction. On the other hand, NAcC-inactivation could eliminate phasic activity related to incentive cue presentation, diminishing reward-seeking. Taken together, these results suggest that, while the NAcC is relevant for general motivational drive in response to discrete stimuli, the NAcS may be particularly important for suppressing inappropriate or inefficient response-strategies. The NAc has also been implicated in impulsivity, which is a multifaceted construct that reflects an inability to withhold a response when required (for review, see Basar et al., 2010). Of particular relevance to response-inhibition as conceptualized here are impulsive actions, often operationalized as premature motor responses that occur without foresight. This sort of suppression can be indexed by Go/No-Go or five-choice serial reaction time tasks (5-CSRTT). In a typical Go/No-Go task, discrete cues require either the production (a “Go” response) or inhibition (a “No-Go” response) of a particular instrumental behavior in order to trigger reward delivery. Thus, animals must flexibly and bi-directionally alter their behavior depending on the particular cue presented. Unit recordings within the NAc illustrate that individual neurons encode Go or No-Go stimuli, increasing or decreasing their activity during cue presentation (Roitman & Loriaux, 2014; Setlow et al., 2003). Interestingly, response-suppression during successful No-Go or unsuccessful Go trials has been shown to produce increases in NAc activity that were greater in magnitude than were decreases, implying that elevations in accumbens 11  activity may allow for response-inhibition (Roitman & Loriaux, 2014). Although no studies have examined whether the neural correlates of Go or No-Go performance differ across accumbens subregions, data from other assays of impulsive action, such as the 5-CSRTT, provide insight into the relative contributions of the NAcS and NAcC. The 5-CSRTT requires rats to wait a set period of time prior to the brief illumination of a stimulus light, during which a nosepoke in the illuminated port delivers reward. Responses prior to illumination of the stimulus light provide a measure of impulsive action, known as premature responses, which delay the possibility of reward receipt by restarting the waiting period. Inactivation of NAcS has been shown to increase premature responses, while inactivation of NAcC simply diminishes attentional accuracy on this task (Feja, Hayn, & Koch, 2014). Consistent with the aforementioned vmPFC-NAcS circuit mediating response-suppression, vmPFC inactivation produces the same sort of impulsive actions (Feja & Koch, 2014), which is recapitulated following pharmacological disconnection of this circuit, but not a vmPFC-NAcC projection (Feja & Koch, 2015). While impulsive actions may be particularly within the purview of the NAcS, the NAcC has been shown to contribute to aspects of inhibitory control including impulsive choice (Cardinal, Pennicott, Sugathapala, Robbins, & Everitt, 2001a; Pothuizen, Jongen-Rêlo, Feldon, & Yee, 2005). Impulsive choice represents a more cognitive aspect of impulsivity, where animals shift their choice away from a large reward as the delay associated with reward delivery increases. Such results suggest that the NAcC may incorporate the costs associated with intertemporal choices, while being less responsible for the relatively more rapid impulse control deficits associated with impulsive actions. Therefore, the contribution of the NAcS to impulsive actions seems relatively consistent, however NAcC may also contribute to aspects of response inhibition depending on the type of response required.  12  Taken together, these results implicate accumbens subregions in dissociable aspects of appetitive behavior. In particular, the NAcS mediates the impact that cues have on behavior reinforced by a specific incentive, while actively inhibiting task-irrelevant information and actions to refine action-selection. In contrast, the NAcC drives motivated behavior both generally and in the presence of discrete motivational cues, without a prominent role in behavioral suppression. Similarly, the NAcS may control the inhibition of impulsive actions, while the NAcC promotes response accuracy, as well as the arguably more cognitive facets of waiting impulsivity. That these same psychological principles of NAc function may apply not only to appetitive behavior, but also to aversively-motivated response-inhibition and promotion has received less empirical scrutiny.  1.3 Models of aversive learning and related circuitry The emphasis on action selection evident across studies of NAc function suggests that aversively-motivated behaviors which require response-promotion or inhibition may similarly depend upon this region. In the appetitive domain, these two poles of behavior can be provoked by reward availability versus reward unavailability or the risk of reward omission. In the aversive domain, response-inhibition results from the presentation of an aversive stimulus, such as a minor foot-shock, ocular air-puff, or loud acoustic startle stimulus. Depending on the experimental conditions, response-promotion can also be observed during aversive conditioning, particularly if an animal is given the ability to escape or avoid potential danger. These two poles of aversively-motivated behavior, termed defensive reactions and defensive actions (Moscarello & Ledoux, 2014), make up an essential part of an animals defensive repertoire, and may be differentially regulated by the NAc. To better understand these two functions, and how NAc 13  subregions may contribute to them, a brief review of their psychological and neurobiological underpinnings is necessary.  A variety of methods have been devised to evaluate defensive behaviors, built upon two primary associative learning theories. The first borrows from the tenets of classical conditioning put forth by Pavlov (1926) and others. Commonly termed Pavlovian fear conditioning, this procedure involves the pairing of an initially neutral stimulus (e.g., light, auditory tone, context, etc.) with an aversive unconditioned stimulus (US; e.g., minor foot-shock, ocular air-puff, loud acoustic startle stimulus, etc.) Following repeated pairings of these stimuli, the neutral stimulus becomes a conditioned stimulus (CS+), capable of eliciting a conditioned fear response when presented in the absence of the US. In some designs, presentations of the CS+ can be intermingled with the presentation of an explicitly neutral stimulus (CS-). Such discriminative fear paradigms serve to control for baseline levels of fear and examine potential generalization of the fear response (Likhtik & Paz, 2015; Piantadosi & Floresco, 2014). Importantly, during Pavlovian fear learning, the behavior of an animal has no consequence on the probability of the delivery of the aversive US.  In contrast to Pavlovian methods, the second model, based upon the Skinnerian principle of instrumental conditioning (Skinner, 1938), results when an action is reinforced or punished, depending on the affective valence of outcome itself. Using this methodology, an animal controls the probability of US delivery via the production or inhibition of a particular instrumental response. In the case of punishment, an instrumental action, such as pressing a lever for reinforcement, can be paired with a contingent aversive unconditioned stimulus, such as foot-shock. This pairing results in the expression of fear or anxiety during future situations in which the punished instrumental action is available (Estes & Skinner, 1941; Vogel, Beer, & Clody, 14  1971). In most cases, this procedure is conducted in animals that are in a deprived state, typically from a primary reinforcer such as food or water. Deprivation ensures that motivational conflict is produced during punishment, as animals are highly motivated to seek reinforcement due to deprivation, but also to avoid the aversive punishment that is concurrently delivered.  Whether conducted in a purely Pavlovian or instrumental manner, one can immediately see that the fear produced by either procedure will have a qualitatively similar impact on behavior: ongoing activity is inhibited due to the potential delivery of an aversive stimulus. Despite the inherent difficulty in inferring emotional states in non-verbal species (Ledoux, 2014; Panksepp, 2011), reliable measures of fear during aversive conditioning have been developed based upon the innate defensive reactions expressed by mammals (Bolles, 1970; Moscarello & Ledoux, 2014). The most commonly measured of these defensive reaction is freezing, defined as the cessation of all movement (except respiration) (Blanchard & Blanchard, 1969; Campbell & Teghtsoonian, 1958). Freezing reflects an attempt to evade predator detection (Bouton & Bolles, 1980), and provides researchers with a relatively unambiguous index of fear that can be scored with ease. A secondary measure, which can be used in Pavlovian or instrumental scenarios, is the conditioned suppression of reinforcement-seeking. Animals innately suppress their foraging behavior in the presence of threat (Fanselow & Lester, 1988; Whishaw & Dringenberg, 1991). Similar to conditioned freezing, this behavior is likely caused by a desire to minimize exposure to danger that may occur during foraging. By utilizing these (and other) behavioral indices of fear, one can begin to examine the neural correlates of such affective conditioning.  While these defensive reactions predominate in standard, Pavlovian situations where the behavioral repertoire of an animal is severely curtailed, other, active responses prevail when animals are provided with control over their environment (Berger & Brush, 1975; Mowrer & 15  Lamoreaux, 1946; Whishaw & Dringenberg, 1991). So called avoidance conditioning incorporates Pavlovian and instrumental mechanisms, consisting of an early stage where CS presentations evoke fear following pairing with an aversive US, and a later stage where the performance of an instrumental response (e.g., lever-press, shuttling response) terminates the CS and eliminates the potential delivery of the aversive US (Maia, 2010). Thus, animals can learn to elicit an active approach response, overcoming the initial defensive reactions evoked by CS presentation, to control the probability of receiving a foot-shock or other aversive stimulus.  Investigation of the neural circuitry underlying aversive learning has leaned heavily on basic, Pavlovian fear conditioning. Predominantly using freezing as a readout of fear, a central fear circuit encompassing nodes within the amygdala and prefrontal cortex, as well as midbrain nuclei, has been identified. Briefly, the sensory properties of the CS+ and foot-shock US converge on the lateral segment of the basolateral amygdala (BLA), allowing for the acquisition and expression of conditioned fear (Iwata, LeDoux, Meeley, Arneric, & Reis, 1986; LeDoux, Cicchetti, Xagoraris, & Romanski, 1990; Wilensky, Schafe, & LeDoux, 1999). Projections from basal amygdala to the central nucleus of the amygdala (CeA) trigger freezing (as well as neuroendocrine and autonomic) responses upon re-exposure to the CS+ alone (no foot-shock), via projections to midbrain nuclei (e.g. periaqueductal gray) (Amorapanth, 1999; Fanselow, 1994).  While the initial acquisition of Pavlovian fear is predicated on amygdala integrity, fear expression and extinction appear to require mPFC circuitry (Courtin, Bienvenu, Einarsson, & Herry, 2013; Maren & Quirk, 2004). Generally, the two main subregions of the rodent mPFC, the more dorsal prelimbic (PL) and the more ventral infralimbic (IL), are suggested to play dissociable roles in the expression and extinction of fear conditioning. Specifically, PL mPFC 16  activity promotes the expression of conditioned fear, whereas IL activity inhibits fear, as occurs during extinction (Burgos-Robles, Vidal-Gonzalez, & Quirk, 2009; Corcoran & Quirk, 2007; Milad, Vidal-Gonzalez, & Quirk, 2004; Quirk, Russo, Barron, & Lebron, 2000). Stimulation of the PL enhances, whereas pharmacological inactivation or lesion decreases, freezing behavior in response to an aversively conditioned cue (Quirk et al., 2000; Sierra-Mercado, Padilla-Coreano, & Quirk, 2011; Vidal-Gonzalez, Vidal-Gonzalez, Rauch, & Quirk, 2006). In contrast, IL stimulation diminishes conditioned freezing, enhancing extinction, while the opposite occurs following pharmacological or optogenetic silencing of this subregion (Bukalo et al., 2015; Sierra-Mercado et al., 2011; Vidal-Gonzalez et al., 2006). Single unit activity in these regions faithfully tracks their apparent opposite roles in fear expression. PL activity occurs during tone presentations, and aberrantly elevated PL activity during extinction is correlated with extinction failure (Burgos-Robles et al., 2009). In contrast, IL excitability decreases during conditioning, and increases during extinction learning (Santini, Quirk, & Porter, 2008). It is important to emphasize that, although understood in greater anatomical detail in the rodent, evidence suggesting that the human amygdala and PFC (subdivisions homologous to PL/IL mPFC in rodents) perform similar functions to their rodent counterparts has been reported (Adolphs, Tranel, Damasio, & Damasio, 1995; Bechara et al., 1995; Büchel, Dolan, Armony, & Friston, 1999; Delgado, Nearing, LeDoux, & Phelps, 2008; Hariri et al., 2009; LaBar, Gatenby, Gore, LeDoux, & Phelps, 1998; Milad et al., 2005; Motzkin, Philippi, Wolf, Baskaya, & Koenigs, 2014).  Reliance on the relatively simple, Pavlovian assessment of fear has left a comparative imbalance in the understanding of the circuitry relevant to instrumental punishment. However, recent work has identified structures involved in punishment, including some classically related 17  to fear such as the BLA and mPFC (Bressel & McNally, 2014; Jean-Richard-Dit-Bressel & McNally, 2015; Pascoli, Terrier, Hiver, & Lüscher, 2015; Vento, Burnham, Rowley, & Jhou, 2017). Inactivation of the BLA disinhibits reward-seeking during punishment, consistent with a native role for this region in response-inhibition (Jean-Richard-Dit-Bressel & McNally, 2015). Interestingly, this effect is specific to the manipulation of the caudal aspect of the BLA, as rostral inactivations had no effect on behavior (Jean-Richard-Dit-Bressel & McNally, 2015). Caudal BLA projects more strongly to the NAcS than the NAcC  (Berendse, Galis-de Graaf, et al., 1992; Brog et al., 1993; Groenewegen et al., 1999; Kita & Kitai, 1990; Wright, Beijer, & Groenewegen, 1996), implying that a BLA to NAcS projection may be relevant to punishment-induced response-inhibition. The BLA likely encodes the value associated with a particular event, whether positive or negative, and allows for the appropriate modification of behavior in response. In the case of an aversively conditioned Pavlovian stimulus, the adaptive response would be to freeze, while in an instrumental punishment setting, the conditioned suppression of reward-seeking would be expected. Unlike the general assessment of freezing, conditioned suppression requires the integration of multiple affective signals (e.g., fear, hunger, etc.), for which an interface between the limbic and motor systems, perhaps the nucleus accumbens, is likely required. Unlike the consistency between Pavlovian and instrumental fear responses requiring the BLA, the dissociation between the function of PL and IL cortex is less reliable. For example, pharmacological inactivation of PL or IL has been shown to dramatically disinhibit shocked water-spout licking in thirsty rats (Resstel, Souza, & Guimarães, 2008). Animals become less sensitive to punishment following inactivation of either prefrontal subregion, persevering in reward-seeking despite negative consequences. In contrast, other studies have suggested that 18  lateral segments of the PFC, including the orbitofrontal cortex (OFC) and insula, contribute more to instrumental punishment than do either subregion of the mPFC (Jean-Richard-Dit-Bressel & McNally, 2016). Importantly, both the insular cortex and OFC project to the ventral striatum, including the NAc (Brog et al., 1993; Heilbronner, Rodriguez-Romaguera, Quirk, Groenewegen, & Haber, 2016). The lack of coherence regarding PL/IL cortex function from studies investigating instrumental punishment and conditioned freezing suggests that separable mechanisms may underlie each behavior. Specifically, when inhibiting reward-seeking, subregions of the mPFC may play qualitatively similar roles in the top-down regulation of such behavior.  This suggestion has also been illustrated in studies examining the neural correlates of addiction-like compulsive reward seeking, defined as drug-seeking despite foot-shock punishment (Deroche-Gamonet, Belin, & Piazza, 2004; Everitt et al., 2008). For example, prolonged access to cocaine produces punishment-resistant drug seeking in some animals (Vanderschuren & Everitt, 2004), concomitant with hypofunction of medial prefrontal cortex (mPFC) (Chen et al., 2013). Optogenetic inhibition or activation of mPFC decreases or increases, respectively, the impact of punishment on cocaine seeking (Chen et al., 2013, but see Pelloux, Murray, Everitt, 2013), suggesting that mPFC activity may be causally related to the punishment-mediated inhibition of seeking. Similarly, pharmacological inactivations of the mPFC produce operant responding for both cocaine and sucrose that is insensitive to potential punishment (Limpens, Damsteegt, Broekhoven, Voorn, & Vanderschuren, 2015; Resstel et al., 2008). Thus, prefrontal regions seem to perform a top-down inhibitory function, acting as a break when responding is directly punished, or in the presence of a fear-inducing stimulus. Like the BLA, mPFC projects strongly to regions of the ventral striatum, with dorsal regions of the 19  mPFC projecting to the NAcC, and more ventral regions projecting to the NAcS (Berendse, Galis-de Graaf, et al., 1992; Brog et al., 1993; Sesack et al., 1989; Vertes, 2004).  On the periphery of this fear circuitry is the NAc, a ventral-striatal structure at the nexus of affective, cognitive, and spatial information arriving from numerous cortico-limbic afferents. Long considered a “reward” nucleus based in large part upon the necessity of this region for the production of appetitive motivation (Cardinal, Parkinson, Hall, & Everitt, 2002; Parkinson, Cardinal, & Everitt, 2000; Stopper & Floresco, 2011), numerous re-conceptualizations have attempted to reconcile data suggesting that aversive events are also processed and influenced by NAc activity (Berridge & Kringelbach, 2013; Carlezon & Thomas, 2009; Levita et al., 2009; Reynolds & Berridge, 2002; Roitman et al., 2005; Salamone, 1994; Schoenbaum & Setlow, 2003; Setlow et al., 2003; Soares-Cunha et al., 2016). These later studies illustrate that single neurons in the NAc respond to primary aversive stimuli (e.g. quinine taste), as well as the cues that predict them (Roitman et al., 2005), and are necessary for the ability of such cues to alter behavior (Schoenbaum & Setlow, 2003; Setlow et al., 2003). Although defensive reactions such as freezing are equivocally-related to NAc activity, this nucleus may be more relevant for the modification of reward-seeking behavior by fear (Kim et al., 2017). Finally, the NAc is implicated directly in the avoidance of harm, a function critical to appropriate navigation of approach/avoidance scenarios (Ramirez, Moscarello, LeDoux, & Sears, 2015; Salamone, 1994).  1.4 The NAc and aversively-motivated behavior To postulate that the NAc is responsible for aspects of aversion, an expectation that neurons within this region process aversive stimuli must be met. In fact, unconditioned aversive stimuli have been shown to modulate NAc activity and neuromodulator release (Badrinarayan et al., 2012; Baliki et al., 2013; Budygin et al., 2012; Roitman et al., 2005). For example, neurons 20  within the NAc increase their firing rate to infusion of an aversive quinine taste (Roitman et al., 2005). This is coupled with a decrease in dopamine signaling during the quinine infusion, which may be directly related to encoding of the motivational properties of the substance itself (Roitman, Wheeler, Wightman, & Carelli, 2008). Interestingly, dopamine release may be differentially affected as a function of subregional differences between the NAcC and NAcS in response to primary aversive stimulus delivery (Budygin et al., 2012). Voltammetric recordings from anesthetized rats subject to tail pinch suggests that, while release in the NAcC is time-locked to the delivery of the tail pinch, dopamine release in the NAcS occurs immediately following the cessation of the pinch. This result implies that NAcS may be relatively more important for safety or relief learning, consistent with a variety of findings from animals and humans (Baliki et al., 2013; Fernando, Urcelay, Mar, Dickinson, & Robbins, 2013; Mohammadi, Bergado-Acosta, & Fendt, 2014).  Research has also demonstrated that the NAcS in particular can generate bivalent motivational states via input from cortical subregions (Reynolds & Berridge, 2002; Richard & Berridge, 2013). Infusions of  the GABAA receptor agonist muscimol instigates ingenstive behavior when infused into the rostral NAcS, but biases behavior towards defensive reactions when infused into the caudal NAcS (Reynolds & Berridge, 2002). Interestingly, IL cortex acts to put a break on either of these processes instigated by the NAcS, as activation of this structure decreases feeding or defensive behaviors induced by rostro-caudal disruption of excitatory activity within the NAcS (Richard & Berridge, 2013). By potentiating inhibitory signaling in the NAcS, behaviors that are normally curtailed (e.g., voracious eating, anti-predator behavior when there is no immediate threat) become unmasked. These findings are in general agreement with a response-inhibitory circuit that is mediated by cortico-striatal activity and can bias motivational 21  states. Importantly, these effects on feeding only occur in the rostral portion of the NAcS, and do not generally impact reward-seeking in an operant environment (Hanlon, Baldo, Sadeghian, & Kelley, 2004; Stratford & Kelley, 1997; Zhang, Balmadrid, & Kelley, 2003, but see Wirtshafter & Stratford, 2010). In addition to unconditioned responses, other studies have demonstrated that physiological and neurochemical indices of aversive learning occur in the NAc. For example, when a CS is paired with an aversive event, dissociations have been observed between the release of dopamine within each subnuclei of the accumbens in response to CS delivery (Badrinarayan et al., 2012; Oleson, Gentry, Chioma, & Cheer, 2012). Badrinarayan and colleagues (2012) reported that the presentation of an aversive CS decreases dopamine release probability in the NAcC, while increasing the magnitude of release in the NAcS. Findings regarding the NAcC have be corroborated by Oleson and colleagues (2012), showing that NAcC dopamine decreases during CS presentations following fear conditioning (Oleson et al., 2012). These neurochemical results suggest that NAc subregions differentially encode conditioned stimuli predicting an aversive consequence. Specifically, decreases in NAcC dopamine release during CS presentations may induce a state of hypoactivity during fear (Kelley, Baldo, Pratt, & Will, 2005), while increases observed within the NAcS may signal salience or relief. Direct electrophysiological recordings in the NAc illustrate that fear conditioning potentiates mPFC to NAc afferents to CS+, but not CS-, deliveries, in a manner that is dependent on BLA input (McGinty & Grace, 2008). Interestingly, the majority of recordings conducted by McGinty and colleagues (2008) were localized in the NAcS, coherent with the suggestion that this region may be particularly sensitive to aversive conditioning. Although no studies in humans have assessed the differential contributions of accumbens subregions, activity within the whole NAc does 22  appear to track the valence of aversive cues (Delgado, Jou, Ledoux, & Phelps, 2009; Delgado, Li, Schiller, & Phelps, 2008; Delgado, Nearing, et al., 2008; Jensen et al., 2003; Klucken et al., 2009; Pohlack, Nees, Ruttorf, Schad, & Flor, 2012). Presentations of an aversive CS+ increases activity within the NAc, while CS- presentations result in a smaller change in activity (Jensen et al., 2008; Levita et al., 2009; Romaniuk et al., 2010). Taken together, these findings imply that the NAc may play an integral role in the learning and expression of aversive conditioning. Despite evidence that the NAc is involved in unconditioned and conditioned responses to aversive stimuli, studies investigating the functional contribution of this nucleus to defensive behaviors are essentially equivocal. Some studies implicate the NAc in the acquisition (but not expression) of contextual fear conditioning, while sparing freezing induced by presentation of an aversive cue (Haralambous & Westbrook, 1999; Riedel, Harrington, Hall, & Macphail, 1997). This specific effect on contextual fear conditioning has been suggested to be mediated by the prominent role of the ventral hippocampus/subiculum, which projects strongly to the NAc (Britt et al., 2012; Brog et al., 1993; French & Totterdell, 2002, 2003), in contextual declarative memory. However, still others report that inactivation of the NAcC impairs both the acquisition and expression of fear-potentiated startle towards discrete cues (Schwienbacher, Fendt, Richardson, & Schnitzler, 2004). Finally, recent studies suggest that the ventral striatum, including the NAc, is critical for the extinction of fear (Correia, McGrath, Lee, Graybiel, & Goosens, 2016; Rodriguez-Romaguera, Monte, & Quirk, 2012). Given the diversity of input reaching the NAc, such discrepant results may not be particularly surprising. As outlined previously, the NAc receives dense projections from the BLA, mPFC, and ventral hippocampus, as well as neuromodulatory signals from the midbrain, all of which have been hypothesized to regulate different aspects of aversive and appetitive conditioning (Cardinal et al., 2002; 23  Carlezon  Jr. & Thomas, 2009). Adding to this complexity is that few studies have evaluated the contribution of individual NAc subregions to aversive conditioning. Of the studies separately considering the NAcC and NAcS, the majority have utilized permanent lesions which likely affect multiple aspects of behavior, including learning, consolidation, and expression (Parkinson, Robbins, & Everitt, 1999; Riedel et al., 1997; Wendler et al., 2013).  In addition, none of these previous studies have evaluated the contribution of these regions to the aversion-induced suppression of reward-seeking. This is particularly relevant given that appetitive conditioning studies show that the learned inhibition of behavior may be uniquely under the control of the NAcS, via input from critical cortico-limbic afferents (Ambroggi et al., 2011; Floresco et al., 2008; Ghazizadeh et al., 2012; Peters et al., 2008). The mPFC and BLA have separately been linked to the conditioned inhibition of reward-seeking (Chen et al., 2013; Jean-Richard-Dit-Bressel & McNally, 2015, 2016; Limpens et al., 2015; Resstel et al., 2008), which they may enforce by direction projections to the NAc. Until recently, this hypothesis had not been empirically tested. Kim and colleagues (2017) utilized molecular and optogenetic techniques to interrogate a mPFC to lateral NAcS circuit during conditioned suppression. They found a subset of mPFC neurons projecting to the lateral NAcS that were activated by foot-shock, and whose activity was inversely related to reward-seeking. These neurons were active during suppression, consistent with a role for the mPFC in top-down inhibitory control, while hypoactivity within this projection was related to reward-seeking despite potential punishment. Thus, the NAcS may be a striatal subregion particularly sensitive to the influence of aversive stimuli on reward-seeking. Still, this prior study examined the lateral NAcS, which receives less input from regions previously suggested to be relevant for response-24  inhibition such as the mPFC and caudal BLA. Thus, investigation of the medial NAcS during the conditioned inhibition of reward-seeking is warranted.  While the contribution of the NAc to Pavlovian and instrumental forms of response suppression is uncertain, active behaviors designed to escape predation have been shown to rely upon this nucleus. The learning and expression of active avoidance depends upon intact function and dopaminergic innervation of the NAc. Dopamine release in the NAc increases during active avoidance learning, and depleting dopamine in this region subsequently impairs the learning and expression of this behavior (Boschen, Wietzikoski, Winn, & Cunha, 2011; Gentry, Lee, & Roesch, 2016; McCullough, Sokolowski, & Salamone, 1993; Oleson et al., 2012; Wadenberg, Ericson, Magnusson, & Ahlenius, 1990; Wietzikoski et al., 2012). During a successful avoidance, phasic dopamine release occurs in the NAcC upon avoidance-cue presentation (Oleson et al., 2012). Consistent with a bivalent role for this nucleus, dopamine release is provoked by both reward cues and avoidance cues during performance of a well-trained approach/avoidance task (Gentry et al., 2016). Performance on this task is correlated with cue-selective dopamine release, as poor performing animals show a pattern of dopamine release that is non-specifically higher and less selective for relevant cues (Gentry et al., 2016). Neural activity within the NAcS has also been shown to be necessary for active avoidance performance. Specifically, temporary inactivation of NAcS, or reversible disconnection of the NAcS from its efferent BLA projection, impairs the ability of rats to produce an active avoidance (Fernando et al., 2013; Ramirez et al., 2015). The NAcS may facilitate avoidance by encoding the salience of signaled periods of safety during avoidance, as inactivation of this structure has been shown to impair avoidance in situations where safety signals are not presented (Fernando et al., 2013).  25  In humans, active avoidance is also associated with neural activity in the NAc, suggesting that a conserved avoidance circuit may exist across mammalian species (Delgado et al., 2009; Levita, Hoskin, & Champi, 2012). Activity within the NAc increases during the learning of an active avoidance response, in a manner that is correlated with amygdala activity (Delgado et al., 2009). Thus, similar limbic-striatal interactions may underlie human active avoidance. Human research has also provided insight into the accumbal regulation the opposite pole of avoidance, passive avoidance (Levita et al., 2012). During this behavior, animals must withhold an instrumental response to avoid an aversive stimulus. Levita and colleagues (2012) required participants to make a button press to avoid an aversive consequence during the presentation of one stimulus (active avoidance), and to withhold a button press to avoid an aversive consequence during the presentation of another stimulus (passive avoidance). Participants completed this task within an fMRI, revealing that BOLD activity within the NAc was differentially modulated by active versus passive avoidance cues. Active avoidance provoked an increase in BOLD activity within the NAc, while passive avoidance produced a deactivation in the same region. Methodological limitations prevented this study from evaluating potential subregional-specificity of this effect. Still, it is possible that the NAc and NAcS are differentially required on such a task, in keeping with a potential role for the NAcS in response-inhibition (passive avoidance) and the NAcC in response-promotion (active avoidance).  1.5 Objectives Due to the present ambiguity regarding the necessity of NAc subregions to aversively-motivated behavior, we examined the contribution of these nuclei to three distinct, yet related, behaviors. These experiments were predicated on the general hypothesis that the NAcS may control aspects of aversion-mediated response-inhibition, while the NAcC primarily contributes to approach 26  behavior. One behavioral ramification of Pavlovian fear cue presentation is the rapid reorganization of ongoing behavior, such as during performance of an appetitive task (Estes & Skinner, 1941; Kamin, Brimer, & Black, 1963). Such conditioned suppression of reward-seeking has been proposed to reflect a type of aversive PIT, for which the NAc is necessary (as outline above) in the appetitive domain (Cardinal et al., 2002; Everitt, Cardinal, Parkinson, & Robbins, 2003). A second manifestation of fear on behavior can be examined during instrumentally delivered punishment, such that rats are fearful of approaching a desired stimulus or reinforcer. Assessment of such motivational conflict has revealed roles for major NAc afferents, including regions of the prefrontal cortex (Broersen et al., 1995; Jean-Richard-Dit-Bressel & McNally, 2016; Resstel et al., 2008) and BLA (Jean-Richard-Dit-Bressel & McNally, 2015), suggesting that NAc itself may be integral. Finally, fear can, in certain situations, invigorate behavior, as occurs during avoidance. Such active-avoidance is known to be dependent on NAc circuitry (Delgado et al., 2009; Levita et al., 2012; Ramirez et al., 2015; Wendler et al., 2013). However, another pole of avoidance behavior is passive-avoidance, whereby animals must inhibit responding to avoid punishment. In humans, this behavior has been shown to involve activations or deactivations of the NAc during active and passive avoidance, respectively (Levita et al., 2012). Thus, we aimed to more specifically examine the circuitry involved in these related, but distinct, avoidance behaviors, at the level of the NAcS and NAcC.  Chapter 2: Examined the role of NAc and prefrontal subregions to the acquisition and expression of discriminative Pavlovian conditioned suppression. During these experiments, animals were subjected to discriminative fear conditioning, where one conditioned stimulus terminated with a mild foot-shock (CS+), while another had no consequence (CS-). Fear was assessed by examining the conditioned suppression of reinforcement-seeking during presentation 27  of each CS type. Subregions of the medial PFC and NAc were pharmacologically inactivated prior to acquiring fear, or prior to the expression of fear. This experiment was designed to provide evidence that Pavlovian mechanisms of fear are regulated differentially by the NAcC and NAcS, as well as the PL and IL cortices. Chapter 3: Examined the role of two potential circuits mediating the acquisition and expression of discriminative Pavlovian conditioned suppression. Based on the results of Chapter 2, we utilized a pharmacological disconnection procedure to probe whether a BLA-NAcS circuit mediates the acquisition of conditioned fear, and whether a PL-NAcS circuit mediates its expression. Chapter 4: Examined the role of the NAcS and NAcC in the expression of instrumental punishment during conflict. During this task, rats were enticed to seek reward by a shift in reinforcement from a lean to a rich schedule, however, lever-press responses were concurrently punished by a mild foot-shock. After acquiring this behavior, these two accumbens subregions were pharmacologically inactivated. This experiment was designed to provide evidence that response-suppression mediated by instrumental punishment is sensitive to manipulation of the NAcS, but not NAcC.  Chapter 5: Examined the role of the NAcS and NAcC in active versus passive avoidance. After extensive training, each subregion was pharmacologically inactivated to examine potentially dissociable contributions of the NAcS to response-inhibition (passive avoidance trials) and response-promotion (active avoidance trials). This experiment allowed for the neurobiological dissection of cue-driven instrumental actions, at the level of the NAc. 28  Chapter 2: Cortico-striatal contributions to the acquisition and expression of discriminative conditioned suppression 2.1 Introduction Fear is a powerfully motivating emotion with the ability to have an enduring effect on behavior. For example, fear-inducing stimuli are capable of suppressing reward-seeking, which, in an ethological setting, allows animals to go unnoticed by predators during foraging (Estes & Skinner, 1941; Kamin et al., 1963; Whishaw & Dringenberg, 1991). In modern humans, the maladaptive expression of such suppression has been suggested to underlie psychiatric disorders characterized by compulsions or impulse control deficits, including substance abuse and obsessive compulsive disorder (OCD) (American Psychiatric Association, 2013; Belin-Rauscent, Fouyssac, Bonci, & Belin, 2016; Everitt, 2014; Feil et al., 2010; Figee et al., 2016; Jentsch & Taylor, 1999; Limpens, Schut, Voorn, & Vanderschuren, 2014; Lubman, Yücel, & Pantelis, 2004; Perry & Carroll, 2008). A hallmark of substance abuse, for example, is the seeking of the addictive substance despite adverse consequences, which often include negative effects on physical and mental health, or the loss of occupational or social relationships. These ramifications typically induce fear or anxiety in healthy individuals, curtailing such maladaptive behaviors, but are less effective in these psychiatric populations. Thus, the neural basis of fear-induced response-inhibition may have important implications for our understanding of behavior from both an ethological and translational perspective. Fear conditioning, based upon the associative learning principles outlined by Pavlov (1926), is the most common method used in the interrogation of these circuits. During a typical Pavlovian fear conditioning procedure, a brief, unexpected foot-shock (US) is rapidly associated with co-occurring discrete (elemental) conditioned stimuli (CS). Subsequent re-exposure to these Pavlovian cues will cause a rat to elicit a variety of defensive behaviors, including defensive 29  reactions (Bouton & Bolles, 1980; Fanselow, 1994; Moscarello & Ledoux, 2014). The most commonly measured index of defensive behavior during Pavlovian fear is freezing, typically defined as the cessation of all movement not related to respiration. A second, often complementary measure is the conditioned suppression of reinforcement-seeking, which indexes the withholding of an instrumental, reinforcement-seeking response during the presentation of an aversive CS (Estes & Skinner, 1941; Kamin et al., 1963). This suppression enables animals to minimize potential exposure to danger while foraging during an event that has been unambiguously associated with an aversive consequence (Whishaw & Dringenberg, 1991). Defensive reactions such as conditioned suppression amount to a response-inhibitory mechanism acting to suppress behavior during a potentially dangerous event.  Investigations of these and related behaviors have helped to delineate a central fear circuit encompassing distinct subnuclei of the amygdala (for review, see Fanselow & LeDoux, 1999) and prefrontal cortex (for review, see Courtin, Bienvenu, et al., 2013 and Sotres-Bayon & Quirk, 2010), amongst other regions. Briefly, the sensory components of the aversive US and CS converge in the lateral and basal amygdala, rendering this basolateral (BLA) complex critical for fear acquisition, consolidation, and expression  (for review, see Fendt & Fanselow, 1999). Emerging evidence suggests that the two major subregions of the mPFC, the prelimbic (PL) and infralimbic (IL) cortices, may perform opposing functions during fear expression and extinction (for review, see Maren & Quirk, 2004). PL activity appears to promote, while IL activity inhibits, the expression of conditioned freezing, with IL activity mediating the extinction of this behavior (Corcoran & Quirk, 2007; Sierra-Mercado et al., 2011; Vidal-Gonzalez et al., 2006), although it is relatively unclear if this distinction applies to other defensive reactions, such as conditioned suppression. In fact, studies of conditioned suppression following instrumental 30  punishment suggest that PL and IL similarly promote the inhibition of seeking under threat of danger (Resstel et al., 2008; but see Jean-Richard-Dit-Bressel & McNally, 2016), suggesting that the suppression of reinforcement-seeking may be regulated differently than freezing at the level of the prefrontal cortex.  Notably absent from this canonical fear circuitry is the NAc, a region of the ventral striatum that receives convergent input from prefrontal and amygdala subregions necessary for both appetitive and aversive affective conditioning (Berendse, Galis-de Graaf, et al., 1992; Brog et al., 1993; Groenewegen et al., 1999; Vertes, 2004; Wright et al., 1996). This nucleus is positioned to act as a limbic-motor interface, gating the impact of cortico-limbic input on action selection via its downstream inputs to motor effector sites (Mogenson et al., 1980). Despite the prime anatomical and physiological arrangement of this nucleus relevant to fear conditioning, experimental data is essentially equivocal regarding its involvement in conditioned fear (McDannald & Galarce, 2011; Parkinson et al., 1999; Riedel et al., 1997; Rodriguez-Romaguera et al., 2012; Schwienbacher et al., 2004). For example, previous work suggests that lesions or inactivations of the entire NAc leave instrumental or Pavlovian conditioned suppression intact (McDannald & Galarce, 2011; Rodriguez-Romaguera et al., 2012). Some of this ambiguity may relate to a lack of appreciation for the heterogeneous nature of the NAc itself. Like the aforementioned mPFC, the NAc is composed of at least two distinct subregions, the nucleus accumbens shell (NAcS) and nucleus accumbens core (NAcC), that are often anatomically as well as functionally dissociable (Brog et al., 1993; Floresco, 2015; Zahm & Brog, 1992). The NAcS, which is located on the medial and ventral aspect of the anterior commissure, receives input from ventromedial mPFC, including PL and IL cortex, as well as the caudal aspect of the basolateral amygdala. In contrast, the NAcC, a more lateral nucleus encircling the anterior 31  commissure, receives input from the dorsal mPFC, particularly the anterior cingulate and PL cortex, as well as the full extent of the basolateral amygdala (Berendse, Galis-de Graaf, et al., 1992; Brog et al., 1993; Vertes, 2004; Wright et al., 1996).  Recent descriptions of the dichotomous nature of these subnuclei suggests that, although both NAcC and NAcS may be critical for approach behavior, the NAcS makes a unique contribution to response-suppression (Ambroggi et al., 2011; Floresco, 2015; Ishikawa et al., 2008; Peters et al., 2008; Piantadosi, Yeates, Wilkins, & Floresco, 2017). For example, neurons within the NAcC encode the motivational relevance of an appetitive CS, while those within the NAcS more often encode the appetitive CS as well as unrewarded task events, such as the presentation of a neutral stimulus (Ambroggi et al., 2011). Consistent with this preferential encoding of task-irrelevant events, inactivation of the NAcS disinhibits seeking behavior during portions of the task that are explicitly unrewarded (Ambroggi et al., 2011; Ghazizadeh et al., 2012; Ishikawa et al., 2008). In comparison, the same manipulation of the NAcC decreases responding during presentation of the appetitive CS. Similarly, the reinstatement of reinforcement-seeking following the formation of an inhibitory extinction memory is often exaggerated in animals following inactivation of the NAcS, but not the NAcC (Floresco et al., 2008; Millan et al., 2010; Peters et al., 2008). In many cases, the control of action selection has been shown to involve interactions between the NAc and its key prefrontal (Ghazizadeh et al., 2012; Ishikawa et al., 2008, 2010; Peters et al., 2008) and BLA (Ambroggi et al., 2008; Millan & McNally, 2011) afferents. These regions of the NAc have also been differentially associated with impulsive actions, which occur due to a failure of response-inhibition (Feja et al., 2014; Feja & Koch, 2015; Murphy, Robinson, Theobald, Dalley, & Robbins, 2008). In the context of impulsive action, these studies illustrate that the NAcS, via interactions with the vmPFC, 32  promotes response-inhibition, while NAcC is necessary for task performance (Feja et al., 2014; Feja & Koch, 2015). Such results support a hypothesis that NAcS and NAcC contribute relatively specifically to the promotion or inhibition of actions, respectively, when examining reward-seeking.  Until recently, the possibility that the suppression of reward-seeking induced by aversive consequences relies upon the NAcS has not been experimentally examined. One recent study by Kim and colleagues (2017) used precise genetic targeting and calcium imaging to illustrate that a subset of neurons within the mPFC project to the lateral NAcS to promote suppression following punishment. Activity within this projection was suppressed when an animal sought reward previously associated with foot-shock, but increased when such reward-seeking was inhibited (Kim et al., 2017). Optogenetic activation of this pathway inhibited seeking when under risk of punishment, suggesting a causal role for this projection in response-inhibition mediated by an aversive event. However, the task utilized by Kim and colleagues (2017) delivered the aversive stimulus in an instrumental fashion, leaving open the question of whether the NAcS mediates response-inhibition in response to aversive Pavlovian cues. Moreover, whether medial NAcS and the NAcC perform dissociable roles during the conditioned suppression of reward-seeking is unknown. This chapter aimed to examine whether individual subnuclei of the mPFC and NAc differentially contribute to conditioned suppression, using a discriminative conditioning protocol. Temporary pharmacological inactivations of the PL, IL, NAcC, or NAcS were conducted to probe the involvement of these regions in the acquisition or expression of the discriminative conditioned suppression of sucrose-seeking. All rats were trained to lever-press for sucrose reward, and then were subjected to two critical fear conditioning days. During acquisition, two 33  conditioned stimuli were delivered, one that co-terminated with a mild foot-shock (CS+), and one that was never associated with any consequence (CS-). Following fear learning, rats were given an expression test day where the influence of each type of CS on lever-press suppression was evaluated. We hypothesized that, although none of the subnuclei tested would be necessary for the acquisition of discriminative conditioned suppression, inactivation of either the PL cortex or NAcS prior to the fear expression test would disinhibit sucrose-seeking during the presentation of an aversive CS+, consistent with a role for these regions in generating suppression in response to aversive stimuli. In contrast, we anticipated that inactivation of the IL cortex, which has previously been linked to the extinction of Pavlovian fear, would enhance fear expression, while the same manipulation of the NAcC would simply promote response vigor.  2.2 Methods 2.2.1 Animals All procedures were approved by the Animal Care Committee at the University of British Columbia, in accordance with the Canadian Council on Animal Care guidelines. Separate groups of Long Evans rats (Charles River) arrived weighing 250-300g. Rats were initially housed in groups (4-5 rats/cage) with ad libitum access to food and water. After 5-10 d of acclimatization to the colony, rats were stereotaxically implanted with bilateral stainless-steel guide cannula, described in detail below. During the remainder of the experiment (approximately 4 wks), rats were singly-housed and food-restricted to approximately 90% of their free-feeding weight. Rats were allowed to gain weight following this initial period of restriction, such that they were maintained on a delayed growth curve. Each experimental cohort was composed of 16 rats. To avoid potential cohort effects, care was taken to assign a comparable number of rats to each 34  experimental Treatment condition (B/M vs. SAL), based primarily on matching for the average number of lever-presses made during baseline sessions. 2.2.2 Apparatus Behavior was assessed using eight standard Med Associates operant chambers, enclosed in a sound attenuating chamber (30.5 X 24 X 21 cm; Med Associates, St. Albans, VT, USA). Each operant chamber was assembled in an identical fashion. Two levers, separated by a food receptacle where sucrose reinforcement was delivered (45 mg pellet; BioServ, Frenchtown, NJ, USA), were situated on the right wall of the chamber (as viewed from the open chamber door). Above each lever was a 100 mA cue light, used as part of the compound CS+. On the opposite wall of the chamber (left wall), a single 100 mA house light illuminated the chamber and served as part of a compound CS-. An auditory speaker, which allowed for the delivery of discriminative auditory stimuli via a programmable generator (ANL-926, Med Associates), was located next to the house light. Locomotor activity was measured by four infrared photobeams located just above the grid floor, which was comprised of 19 stainless steel rods spaced 1.5 cm apart. These rods were wired to a shock source and solid-state grid scrambler to allow for foot-shock delivery.  2.2.3 Surgery  Due to changes in institutional policies regarding anesthesia, rats were anesthetized either with a combination of ketamine/xylazine (100/10 mg/ml at 100/10 mg/kg, i.p.) or a half dose of ketamine/xylazine (same mg/ml, i.p) followed by maintenance using Isoflurane anesthetic (2-3% Isoflurane concentration) throughout surgery. Twenty-three gauge bilateral stainless-steel guide 35  cannula were implanted aimed at the PL, IL, NAcS or NAcC according to the following stereotaxic coordinates (in mm): PL – from bregma: AP +3.2; ML: ±0.7; from dura: DV: -2.8 IL – from bregma: AP: +2.8; ML: ±0.7; from dura: DV: -4.1 NAcS – from bregma, AP: +1.3, ML: ±1.0, from dura, DV: -6.3 NAcC – from bregma, AP: +1.6, ML: ±1.8, from dura, DV: -6.3  Four stainless-steel skull screws were inundated with dental acrylic to secure cannula in place. Stainless-steel obturators flush with the end of the guide cannula were inserted after surgery. Rats were given 5-10 d to recover from surgery before beginning behavioral training.  2.2.4 Lever training The day before their initial operant training session, all rats were provided with ~30 sucrose pellets in their home cage, to reduce neophobia to the reinforcer. All training was conducted at a consistent time each day. Rats were initially trained to press the left lever (only lever available during any portion of training/testing) on a fixed ratio 1 (FR1) schedule of reinforcement to a criterion of 40 total presses during the 30 min session. After reaching criterion, rats were trained over three consecutive days on increasing variable interval (VI) schedules, whereby reward was provided after approximately 15 (VI15), 30 (VI30), or 60 (VI60) seconds of pressing (one session at a particular schedule, per day). Rats were then trained on the VI60 schedule for 10-13 d, after which aversive conditioning was conducted. A VI60 schedule engenders a high rate of lever-pressing in rats, while allowing reward rate to remain relatively consistent, allowing for the 36  accurate assessment of conditioned suppression as a proxy for fear (Kamin et al., 1963; McAllister, 1997; Piantadosi & Floresco, 2014; Quirk et al., 2000).  2.2.5 Discriminative fear conditioning 2.2.5.1 Conditioning session Following VI60 training, rats underwent discriminative fear conditioning in an identical fashion as we have reported previously (Piantadosi & Floresco, 2014), based off of discriminative assays used in rodents and humans (Antunes & Moita, 2010; Balog, Somlai, & Kéri, 2013; Jensen et al., 2008). During this protocol, rats received 8 presentations each of a neutral conditioned stimulus (CS-) and an aversive conditioned stimulus (CS+), with an average inter-stimulus interval of 180 s (min: 100 s, max: 240 s). Rats were placed into a chamber and initially received two presentations of a 30 s CS- (1 kHz, 80 dB tone and flashing house-light). Following these two presentations, rats received six more CS- presentations, and seven presentations of the 30 s CS+ (9 kHz, 80 dB tone and flashing house-light co-terminating with a 0.5 mA foot-shock delivered over 0.5 s) in a pseudorandom order. The session ended following one additional CS+ delivery. Previous work in our laboratory suggests that this combination of visual stimuli and order of presentation produces robust and reliable discriminative conditioned suppression in control animals (Piantadosi & Floresco, 2014). The day after this conditioning session, animals were given a baseline VI60 session (no shocks or conditioned stimuli). 2.2.5.2 Expression test session The day after the baseline VI60 session (48 hrs post-conditioning), rats were given a fear expression test session. Rats initially experienced a 5 min period identical to their normal VI60 session, during which they lever-pressed for sucrose reward. Immediately following this period, 37  presentations of the CSs began, initially with four 30 s CS- presentations (five min inter-stimulus interval), followed by four 30 s presentations of the CS+ (no foot-shock; five min inter-stimulus interval). The suppression of lever-pressing during each CS presentation served as an index of fear, as rats suppress seeking behavior in the presence of an aversive CS+ (Kamin et al., 1963; Piantadosi & Floresco, 2014; Quirk et al., 2000; Sierra-Mercado et al., 2011). Suppression was calculated using the formula [(A-B)/(A+B)], where A was the number of lever-presses made in the 30 s epoch prior to CS presentation, and B was the number of lever-presses made during the 30 s CS presentation. Calculated this way, complete suppression is indicated by a value of 1, while a values at 0 or below indicate no suppression or facilitation, respectively. Rarely, rats did not press during a pre-tone and tone period; a suppression value of 1 was applied to all such instances, as in previous reports (Quirk et al., 2000). To ensure that suppression ratios were accurate, an a priori inclusion criteria of greater than 200 presses made during the test session was established. Across all experimental cohorts, data from n = 3 rats were eliminated as a result of this criterion.  2.2.6 Single-stimulus fear conditioning: Pre-test IL inactivation As the impact of IL cortex inactivation during the expression test was unexpected, we conducted an additional experiment to ascertain whether conditioned suppression expression differentially requires the IL as a function of the discriminative versus single-stimulus nature of the design. Thus, animals were implanted with cannula into the IL cortex, and given an identical lever training protocol as described above.  However, during the conditioning session, animals received eight presentations of a single, 30 s CS+ (identical to the CS+ used in the discriminative protocol) only, similar to conditioning procedures used in prior studies examining IL function during fear (Akirav, Raizel, 38  & Maroun, 2006; Sierra-Mercado et al., 2011). Forty-eight hrs later, rats were given a test session that was initially identical to a normal VI60 day. Beginning five min into the session, they received 12 presentations of the 30 s CS+ (no foot-shock), each separated by a three min interstimulus interval.  2.2.7 Microinfusion To examine the acquisition or expression of discriminative fear, separate cohorts of rats were given microinfusion before either the conditioning or expression test sessions. Initially, all rats were given a mock infusion 10 min prior to their final VI60 session before discriminative conditioning. During this session, obturators were removed, mock injectors flush with the indwelling guide cannula were inserted, and animals were allowed to freely move in the infusion enclosure for approximately two min. On the infusion day, obturators were removed and stainless-steel injectors extending 0.8 mm beyond the guide cannula were lowered into the region of interest. Through this injector, rats received bilateral infusion of 0.9% saline (SAL; 0.3 μl/side) or a solution of the GABAB-receptor agonist baclofen and the GABAA-receptor agonist muscimol (B/M; 75 ng/μl of each drug at a volume of 0.3 μl/side). Infusions were conducted over 45, with injectors left in place for an additional 60 s to allow for diffusion of solution from cannula tips. The dose and volume of B/M selected has been used previously to dissociate between the NAcS and NAcC on a wide variety of behavioral measures (Dalton, Phillips, & Floresco, 2014; Floresco et al., 2008; Ghods-Sharifi & Floresco, 2010; Stopper & Floresco, 2011). We have also used the same or larger volumes of a B/M solution to dissociate PFC subregions (Dalton, Wang, Phillips, & Floresco, 2016; St. Onge & Floresco, 2010). We chose to use the same, smaller volume as infused into NAc subregions to limit the potential for diffusion across the dorsoventral axis of the PFC.  39  2.2.8 Histology All rats were euthanized with CO2 and brains were removed and fixed in a 4% phosphate buffered formalin solution. Brains were sectioned at 50 μm, following which tissue was mounted and Nissl stained using Cresyl Violet. Placements were examined under a light microscope, and the ventral extent of each infusion is indicated in Fig. 1B and C.  2.2.9 Data analysis Because there was no lever available during the conditioning session itself, the only behavioral measure available for the assessment of conditioning was locomotion, as assessed by photobeam-breaks/epoch. For each CS delivery, the change in locomotor activity during the CS presentation was calculated, as compared to the overall locomotor baseline (average of all 16 pre-tone periods). These change in locomotion values were then averaged for each CS type, and analyzed using two-way between/within ANOVAs with Treatment group (SAL vs. B/M) as the between-subjects factor, and CS Type (CS+ vs. CS-) as the within-subjects factor. This analysis attempted to clarify the efficacy of conditioning, and its potential alteration by drug-treatment during the conditioning session itself.  During the expression test session, the suppression ratio during each CS presentation was analyzed using between/within-subjects three-way ANOVAs with Treatment group (SAL vs. B/M) as the between-subjects variable, and CS Type (CS+ vs. CS-) and CS Number (1-4) as the within-subjects variables. Separate ANOVAs were conducted on data from animals infused pre-conditioning or pre-test for each brain region (PL, IL, NAcC, and NAcS). Follow-up simple main effects analyses were conducted using one-way ANOVAs or t-tests, where appropriate. Locomotion (photobeam breaks/session) during the conditioning session or expression test were 40  analyzed using separate independent samples t-tests. The rate of lever-pressing (presses/min) in the first 5 min of the expression test session and the total number of lever-presses made during the session were analyzed in an identical fashion. 2.3 Results 2.3.1 PL cortex inactivation pre-conditioning  To assess the contribution of PL activity during the acquisition of discriminative fear conditioning, this region was inactivated immediately prior to the conditioning phase of the task. During this phase, overall locomotor activity was unchanged by Treatment (t(16)=0.26,p>0.79) (Table 1). Rats increased their locomotion significantly more during CS+ presentations, as compared to CS- (F(1,16)=32.67,p<0.001), suggesting that animals behaviorally differentiated between the two stimuli. Treatment had no effect on the change in locomotion induced by either stimulus, as shown by a non-significant main effect of Treatment (F(1,16)=0.39,p>0.54), and a non-significant CS Type x Treatment interaction (F(1,16)=0.03,p>0.86) (Table 1). Thus, rats appeared to respond comparably during conditioning, regardless of treatment condition.  During the expression test, the level of conditioned suppression expressed by rats that had their PL inactivated (n = 7) prior to the conditioning phase rats did not differ from those infused with SAL (n = 11), as illustrated by a non-significant main effect of Treatment (F(1,16)=0.08,p>0.77), as well as a non-significant CS Type x Treatment interaction (F(1,16)=0.13,p>0.72) (Fig. 2A). A significant main effect of CS Type (F(1,16)=57.21,p<0.001), indicated that both groups discriminated between the CS+ and CS- accurately during the expression test, however there was no CS Type x Treatment interaction (F(1,16)=0.13,p>0.72). Additionally, there was no CS Number x Treatment interaction (F(3,48)=2.36,p>0.08). Neither the overall number of lever-presses made throughout the test session (t(16)=0.94,p>0.36), nor the 41  rate of lever-pressing during the first 5 min of the expression test were affected by Treatment (t(16)=0.48,p>0.63) (Table 2). Similarly, overall locomotor activity during the test day did not differ as a result of Treatment (t(16)=-0.46,p>0.65) (Table 2). Thus, PL cortex activity was not necessary for the appropriate acquisition of discriminative fear conditioning, and did not impact general indices of motivated behavior.  2.3.2 IL cortex inactivation pre-conditioning Temporary inactivation of IL cortex immediately prior to conditioning had no effect on the change in locomotion in response to CS- or CS+ presentations during the conditioning phase. The change in locomotor activity was nearly identical across Treatment conditions (F(1,23)=0.001,p>0.99), and both groups expressed a greater change in locomotion during CS+ presentations, as compared to CS- presentations, indicated by a main effect of CS Type (F(1,21)=11.57,p<0.005), and no CS Type x Treatment interaction (F(1,21)=0.77,p>0.39) (Table 1). The overall level of locomotion during the conditioning session did not differ between saline and IL-inactivated groups (t(21)=-0.46,p>0.65) (Table 1). This pattern of results suggests that IL inactivation does not affect within-session changes gross locomotor output, or the CS-specific modulation of behavior.  When tested drug-free during the fear expression test session, control rats (n = 10) expressed similar levels of discriminative conditioned suppression as did rats that underwent IL inactivation (n = 13) prior to the conditioning session (Fig. 2B). Although there was a significant effect of CS Type (F(1,21)=75.37,p<0.001), there was no main effect of Treatment, (F(1,21)=0.17,p>0.68), and no CS Type x Treatment interaction, (F(1,21)=0.66,p>0.42), indicative of intact conditioned suppression. There was no significant CS Type x CS Number interaction, and no three-way interaction, (all F-values < 1.2, all p-values > 0.33). No change 42  was observed in locomotor activity throughout the session, (t(21)=-0.15,p>0.88), or the rate of pressing during the first 5 min of the test session, (t(21)=-0.10,p>0.92), as a function of treatment (Table 2). Finally, the number of presses made during the entirety of the expression test was not different in IL-inactivated animals, as compared to controls (t(21)=0.15,p>0.88) (Table 2). Like the PL cortex, IL activity during fear conditioning acquisition was not necessary for the appropriate expression of discriminative conditioned suppression during the test session.  2.3.3 NAcS inactivation pre-conditioning During conditioning, control and NAcS-inactivated rats made similar CS-induced changes in locomotor activity. There was no main effect of Treatment (F(1,20)=0.004,p>0.94), suggesting that locomotor activity was comparable across drug conditions, and there was no CS Type x Treatment interaction (F(1,20)=2.24,p>0.15) (Table 1). On average, rats locomoted more during CS+ presentations than during CS- presentations, regardless of inactivation status, as evidenced by a significant main effect of CS Type (F(1,20)=6.91,p<0.02). Consistent with this, there was no change in the amount of locomotion across the entire session (t(20)=-0.89,p>0.38) (Table 1). Thus, gross locomotor and cue-induced locomotor activity during the conditioning session were comparable across treatment conditions, with both groups appearing to acquire the CS associations without issue.  Interestingly, inactivation of NAcS (n = 11) during the conditioning session diminished lever-press suppression during the subsequent drug-free expression test, as compared to SAL animals (n = 11) (Fig. 3A). A main effect of Treatment (F(1,20)=7.55,p<0.02), suggested that response inhibition produced by CS presentation was decreased as a result of NAcS inactivation during conditioning. Despite the overall decrease in suppression during CS presentations, rats in the inactivation group still discriminated accurately between the CS- and CS+, as illustrated by a 43  significant main effect of CS Type (F(1,20)=109.99,p<0.001), but no significant CS Type x Treatment interaction (F(1,20)=2.60,p>0.12). Although there was no three-way interaction (F(3,60)=0.98,p>0.40), inspection of the data suggested that suppression allocated towards the CS+ was particularly diminished by inactivation, indicative of a decrease in strength of the fear memory. Neither the rate of lever pressing prior to the first CS presentation (t(20)=0.15,p>0.88), nor the total number of lever-presses made during the expression test session (t(20)=0.22,p>0.83) were altered by treatment (Table 2). Finally, there was no evidence that inactivation of the NAcS prior to conditioning impacted locomotor activity during the expression test (t(20)=0.62,p>0.55) (Table 2). Taken altogether, these results imply that the amount of suppression produced during the expression test session is reduced by inactivation prior to conditioning, suggesting that the fear memory established during learning is less enduring, and more labile. The subsequent impact of this memory on behavior is thus less pronounced, resulting in less conditioned suppression.  2.3.4 NAcC inactivation pre-conditioning Inactivation of NAcC immediately prior to the conditioning session slightly altered locomotor activity during CS presentations. CS presentations (collapsed across CS+ and CS-) tended to cause less of a change in locomotion in NAcC-inactivated animals than it did in control animals, as shown by a trend level Treatment effect (F(1,20)=4.22,p=0.053). Consistent with this, overall locomotor activity throughout the session was decreased in NAcC-inactivated rats (t(20)=3.06,p<0.007) (Table 1). Still, both treatment groups had a greater increase in locomotion during CS+ presentations, as compared to CS- presentations, as shown by a significant main effect of CS Type (F(1,20)=14.36,p<0.002), but no CS Type x Treatment interaction (F(1,20)=0.02,p>0.88) (Table 1). These findings suggest that, despite NAcC animals being less 44  active, they behaviorally distinguished between each CS type, as measured by their change in locomotor activity.  On the drug-free expression test day, rats that received NAcC inactivation (n = 10) prior to conditioning performed similarly to those that received saline (n = 12) (Fig. 3B). The overall level of conditioned suppression was comparable across treatment conditions (F(1,20)=0.14,p>0.71), and both groups distinguished between the CS+ and CS- in a similar manner, as shown by a non-significant CS Type x Treatment interaction (F(1,20)=0.003,p>0.95), and a significant effect of CS Type (F(1,20)=79.72,p<0.001). Additionally, there was no three-way interaction (F(3,60)=0.33,p>0.80). The total number of presses made throughout the session was unchanged by NAcC inactivation (t(20)=-1.62,p>0.12), as was the total amount of locomotor activity (t(20)=-1.38,p>0.18) (Table 2). Finally, the rate of lever-pressing during the first 5 min of the test session was unchanged by previous inactivation of the NAcC (t(20)=-1.21,p>0.24) (Table 2). Like the prefrontal cortex, NAcC activity during conditioning was not necessary for the subsequent expression of discriminative fear conditioning. 2.3.5 PL cortex inactivation pre-expression test In contrast to the null effect of pre-conditioning inactivation, PL cortex activity proved necessary for the appropriate expression of conditioned suppression during the test session (Fig. 4A). These animals were given discriminative conditioning in a drug-free state, and then subjected to inactivation of the PL (n = 13) or saline (n = 12) infusion immediately prior to the expression test session. Here, a significant main effect of Treatment (F(1,23)=13.09,p<0.001), was observed, suggesting that PL inactivation altered conditioned suppression. This was accompanied by a CS Type x Treatment interaction (F(1,23)=11.68,p<0.005), with simple main effects analysis showing that that control rats expressed more fear during the CS+ than the CS- 45  (F(1,11)=26.43,p<0.001), while PL inactivated rats did not (F(1,12)=0.42,p>0.53). There was no CS Number x Treatment interaction (F(3,69)=1.37,p>0.25). Additionally, there was no change in the rate of lever-pressing prior to the first CS presentation (t(23)=0.32,p>0.75), suggesting that the disinhibition of pressing during the CS+ in PL-inactivated animals was not a result of general behavioral activation (Table 2). Further supporting this, locomotor activity throughout the session was not altered by PL inactivation (t(23)=-1.76,p>0.09), nor was the total number of lever-presses made during the session (t(23)=1.28,p>0.20) (Table 2). Thus, PL activity was necessary for the appropriate expression of fear towards a discriminative CS+, with inactivation markedly reducing the suppression of activity typically observed during its presentation.  2.3.6 IL cortex inactivation pre-expression test Temporary inactivation of IL prior to the expression test session had a qualitatively similar effect on discriminative conditioned suppression than did inactivation of the more dorsal PL cortex (Fig. 4B). A main effect of Treatment was observed (F(1,20)=5.60,p<0.03), suggesting that the overall level of suppression across both tone types was lower in IL inactivated rats (n = 12), as compared to controls (n = 10). However, unlike PL cortex, there was no significant CS Type x Treatment interaction (F(1,20)=0.16,p>0.69). There was a significant main effect of CS Type (F(1,20)=4.92,p<0.04), suggesting that, collapsed across treatment conditions, presentation of the CS+ caused more suppression than did presentation of the CS-. As with PL inactivation, there was no significant there-way interaction (F(3,60)=0.95,p>0.42). Locomotion was unchanged following IL inactivation (t(20)=0.75,p>0.46), as was the rate of lever-pressing during the first 5 min of the session (t(20)=-0.09,p>0.92), and the total number of lever-presses made (t(20)=-0.74,p>0.46), suggesting that the impact of IL inactivation was specific to 46  behavioral suppression induced by the conditioned stimuli, and not a general effect of behavioral disinhibition (Table 2).   As the decrease in conditioned suppression following IL manipulation was unexpected, we chose to perform a control experiment aimed at determining whether the suppression-reducing impact of IL inactivation was specific to a discriminative context. When a separate group of rats underwent fear conditioning using a single CS, IL inactivation (n = 8) did not have a significant effect on conditioned suppression, as compared to control animals (n = 8) (Fig. 5B). There was no main effect of Treatment (F(1,14)=1.65,p>0.22), with both groups extinguishing at a comparable rate as indicated by a significant effect of CS Block (F(5,70)=10.02,p<0.001), but no significant Treatment x CS Block interaction (F(5,70)=1.57,p>0.18). Although the rate of pressing at the beginning of the session was the same regardless of Treatment (t(14)=0.14,p>0.89), inactivated animals made more lever presses throughout the session (t(14)=2.84,p<0.013) (Table 2). However, treatment had no impact on overall locomotor activity (t(14)=0.17,p>0.87) (Table 2). This pattern of results suggests that conditioned suppression was not significantly altered by IL inactivation when assessed using a single stimulus, which contrasts with the significant reduction of conditioned suppression observed in the discriminative context. This effect may be mediated in part by a general disinhibition of lever-pressing that appears to have occurred throughout the session, as evidenced by the elevated number of lever presses made by the IL inactivated animals.  2.3.7 NAcS inactivation pre-expression test Like the PL cortex, inactivation of NAcS (n = 13) eliminated the appropriate expression of discriminative conditioned suppression, as compared to control rats (n = 14) (Fig. 6A). There was a significant CS Type x Treatment interaction (F(1,25)=5.02,p<0.035), indicative of a 47  differential pattern of fear expression induced by NAcS inactivation, as compared to control rats. This was driven by less suppression during presentation of the CS+ for animals in the NAcS inactivation group (F(1,25)=4.24,p=0.05). In contrast, lever-pressing during the CS- did not change as a function of treatment, (F(1,25)=0.20,p>0.66). There were no other significant two-way interactions, and no significant three-way interaction (all F-values < 1.3, all p-values > 0.25). NAcS inactivation did not alter the total number of lever-presses made during the session (t(25)=-1.18,p>0.24), nor the rate of pressing during the initial portion of the session (t(25)=0.15,p>0.88) (Table 2). Similarly, there was no change in overall locomotion during the expression test session (t(25)=-1.21,p>0.23) (Table 2). Thus, the NAcS can be shown to play a relatively specific role in producing response-suppression during the presentation of a potentially aversive CS+, without affecting non-specific indices of motivation such as total lever-press rate or locomotion.  2.3.8 NAcC inactivation pre-expression test In contrast to the disinhibitory impact of NAcS inactivation, the same manipulation of the NAcC had no impact on fear expression (Fig. 6B). Following inactivation of NAcC (n = 9) prior to the expression test, no main effect of Treatment was observed (F(1,19)=0.05,p>0.84), indicating that these animals expressed levels of fear comparable to control rats (n = 12). A main effect of CS Type (F(1,19)=102.36,p<0.001), combined with no CS Type x Treatment interaction (F(1,19)=0.54,p>0.47), suggested that animals discriminated between the CS- and CS+ regardless of treatment condition. Additionally, there was no significant three-way interaction (F(3,57)=0.80,p>0.50). Despite the lack of overt effect on suppression during each CS presentations, the overall number of lever-presses was decreased in NAcC-inactivated animals (t(19)=2.23,p<0.04), although the rate of lever-pressing during the first five min of the session 48  was not significantly different from control animals (t(19)=1.74,p>0.09) (Table 2). Similarly, NAcC inactivation decreased locomotion (t(19)=2.80, p<0.02) (Table 2). These results suggest that NAcC promotes behavioral activation, without a particular role in modulating actions based on cues predicting safety or an aversive consequence.  2.4 Discussion Using pharmacological inactivations, we showed that separate subregions of the PFC and NAc uniquely contribute to the acquisition and expression of discriminative Pavlovian fear, as measured by conditioned suppression. Under control conditions, presentation of an aversive CS+ in the absence of foot-shock caused a marked suppression of ongoing reward-seeking, while presentation of a neutral CS- did not alter behavior. Although neither subregion of the mPFC was necessary for the acquisition of discriminative fear, both subregions regulated the expression of acquired suppression, in keeping with a top-down, inhibitory function of the mPFC. The involvement of the NAc, a striatal structure known to integrate cortico-limbic input during response-selection, was dependent on the particular subregion targeted. Inactivation of the NAcC left the acquisition and expression of conditioned suppression intact, but tended to diminish indices of behavioral activation, including locomotion and total lever-presses. In contrast, inactivation of the NAcS diminished conditioned suppression regardless of whether the manipulation was conducted prior to the acquisition or expression phase of the task, implicating this structure in the plasticity associated with fear acquisition, as well as the activity necessary for response-inhibition during subsequent expression.  2.4.1 Discriminative fear acquisition: Prefrontal and accumbal contributions  Of the prefrontal and accumbal subregions tested, only the NAcS was necessary to acquire normal levels of suppression towards a discriminative conditioned stimulus, when 49  assessed during a later expression test. Importantly, none of the regions tested affected the change in locomotion induced by CS- and CS+ presentations during conditioning, indicating that all animals maintained the ability to discriminate between the two conditioned stimuli. Furthermore, locomotion in response to the CS+ in part reflects the burst of activity induced by US delivery, suggesting that unconditioned responses to the foot-shock were not altered by regional inactivations. The finding that the prefrontal cortex is not required for the acquisition of conditioned fear to a CS+ is in keeping with much previous research using single stimulus, Pavlovian designs, and assessing freezing. For example, inactivations or lesions of PL cortex leaves the acquisition of conditioned fear intact (Corcoran & Quirk, 2007; Morgan, Romanski, & LeDoux, 1993; Quirk et al., 2000). The complementary finding reported here that IL cortex is also not involved in the acquisition of conditioned fear is more novel. Still, this result is consistent with the study of a similar defensive behavior, the acquisition of conditioned place aversion following intraplantar formalin injection, which is not affected by IL manipulation (Jiang et al., 2015). These results point to the involvement of other regions in the plasticity associated with fear learning. They also suggest that, when examining fear acquisition, the irrelevance of mPFC activity is comparable to that when examining other defensive reactions, such as freezing.  Despite this apparent lack of necessity during fear acquisition, it is important to recognize that electrophysiological signatures related to fear discrimination learning have been observed in mPFC (Laviolette, Lipski, & Grace, 2005; Orona & Gabriel, 1983). During conditioning, the frequency and burst firing of mPFC neurons increases in response to CS+, but not CS-, presentations (Laviolette et al., 2005). In addition, dopaminergic modulation of the PL cortex during the acquisition of discriminative fear conditioning is capable of altering subsequent neural 50  and defensive responses during presentations of an olfactory CS+ and CS- (Lauzon, Ahmad, & Laviolette, 2012; Lauzon, Bishop, & Laviolette, 2009; Laviolette et al., 2005). Laviolette and colleagues have suggested that dopamine-receptor mediated activity of calcium calmodulin-dependent kinase II (CaMKII), a protein critical for memory formation, can bias the salience of the CS+/CS- depending on the strength of the conditioning procedure (Lauzon et al., 2012, 2009; Laviolette et al., 2005). Here, we utilized temporary inactivations that served to hyperpolarize affected neurons, an effect that fundamentally differs from the targeting of specific dopamine-receptor subtypes, and generally does not impact fear acquisition (Corcoran & Quirk, 2007). Manipulation of specific neuromodulatory targets within the mPFC may alter salience encoding in a fashion that does not directly depend upon changes in neuronal excitability, although the parameters under which this is the case remain to be identified.  Like the two prefrontal subregions tested, NAcC inactivation had no impact on discriminative fear acquisition. This nucleus may be particularly relevant for contextual, but not cued, fear conditioning (Levita, Dalley, & Robbins, 2002; Wendler et al., 2013). Yet, one previous study assessing the conditioned suppression of licking has implicated the NAcC in the formation of a fear memory in response to a discrete cue (Parkinson et al., 1999). Important methodological differences may explain this apparent discrepancy. First, Parkinson and colleagues (1999) utilized permanent lesions, which may impact other processes related to the acquisition of fear or the instrumental licking behavior. In addition, these researchers employed a trace conditioning protocol, which involves a short delay between the delivery of the CS and US. Trace conditioning contrasts with the delay conditioning (CS co-terminates with the US) method employed here, and has been suggested to rely on partially segregated circuitry (Raybuck & Lattal, 2014), for example requiring activity within the PL cortex during acquisition (Gilmartin 51  & McEchron, 2005; Gilmartin, Miyawaki, Helmstetter, & Diba, 2013). As neither PL nor NAcC are generally necessary for the acquisition of delay fear conditioning, the results of Parkinson and colleagues (1999) may relate to the presence of the trace interval between CS presentation and US delivery. Still, the present results continue to support an account of the NAcC in promoting behavioral activation, as inactivation of this nucleus decreased locomotor activity within the fear conditioning session.  Surprisingly, NAcS inactivation during fear acquisition diminished the subsequent expression of conditioned suppression. Although these rats maintained the ability to discriminate between the CS+ and CS-, overall suppression was lower as a result of NAcS inactivation during learning. At first glance, this result appears to contradict previous findings suggesting that the NAcS is not a critical structure for the acquisition of cued fear in rodents (Jongen-Rêlo, Kaufmann, & Feldon, 2003; Parkinson et al., 1999; Riedel et al., 1997). Despite these previous null findings, the NAcS has been shown to control fear learning in some situations, such as when learning a new fear association in the presence of an already established fear-predictive cue (Bradfield & McNally, 2010). In the present design, animals must form two divergent associations during the conditioning phase, one between CS+ and foot-shock, and one between the CS- and nothing. If the NAcS is necessary for updating fear based upon the status of individual cues as fear-predictors, eliminating activity in this subnuclei could subsequently alter the fear expressed towards the CS+ versus CS-. In addition, electrophysiological signatures of discriminative fear learning have been reported to occur in the NAcS. Neurons projecting from mPFC to the NAc (mostly NAcS) encode the aversive nature of an olfactory CS+ (but not a CS-), in a BLA-dependent manner (McGinty & Grace, 2008). 52  In addition, most of the previous studies examining the contribution of the NAcS to fear learning have used a single, discrete stimulus and measured freezing as their dependent measure of fear. Comparison of the present study with these archival reports suggests that the circuitry relevant for freezing may diverge from those necessary for conditioned suppression when considering the involvement of the NAc. Although speculative, as we did not measure freezing in the present study, this dissociation would be in keeping with the role of the NAc in controlling motivated behavior as a function of affective input (Mogenson et al., 1980). Unlike freezing, conditioned suppression requires the integration of affective information with a competing drive (instrumental action leading to reinforcement), which may induce a state of motivational conflict that could require activity within prefrontal and striatal structures (Friedman et al., 2015; Kim et al., 2017; Resstel et al., 2008).  Given that the effect of NAcS inactivation prior to conditioning was observed during a later fear expression test, fear conditioning may induce plasticity within the NAcS as a result of input from efferent regions that encode fear conditioning. One candidate afferent region is the BLA, which projects monosynaptically to the NAcS (Kita & Kitai, 1990; Phillipson & Griffiths, 1985; Wright et al., 1996), and is critical for the encoding of fear conditioning (Fanselow & LeDoux, 1999). The projection from BLA-NAcS has been shown to mediate related aspects of aversive-motivation, including the consolidation of inhibitory avoidance, an assay of passive defensive behavior similar to conditioned suppression (LaLumiere, Nawar, & McGaugh, 2005), as well as the performance of active avoidance, a defensive action employed to remove a potentially aversive stimulus (Ramirez et al., 2015). At the molecular level, foot-shock induces cAMP response element binding protein (CREB) expression in the NAcS, which has been shown to subsequently decreases motivation and impair the extinction of conditioned fear (Muschamp 53  et al., 2011). A similar induction of CREB occurs during fear conditioning within the lateral segment of the amygdala (Yiu et al., 2014), suggesting a potentially common mechanism for fear-ensemble formation during aversive learning. Here, inactivation may prevent the plasticity associated with CREB expression in this nucleus during discriminative fear acquisition, altering the expression of conditioned suppression, and accelerating within-session extinction during the test session.  2.4.2 Discriminative fear expression: Prefrontal and accumbal contributions Separate animals were tested to examine subregional contributions to the expression of discriminative conditioned suppression. In these experiments, we observed that inactivation of either prefrontal subregion disinhibited lever-pressing during CS+ presentations, indicative of a loss of conditioned fear. The observation that the PL mPFC acts to promote Pavlovian fear during an expression test, as illustrated here, is concordant with previous literature (Corcoran & Quirk, 2007; Limpens et al., 2015; Piantadosi & Floresco, 2014; Sangha, Robinson, Greba, Davies, & Howland, 2014; Sierra-Mercado et al., 2011; Vidal-Gonzalez et al., 2006). Inactivation of PL prior to an expression test session resulted in rats engaging in lever-pressing, despite the impending threat posed by the CS+. This effect was apparent from the first CS+ presentation, implying that this alteration was not a product of accelerated extinction. Although the result of this behavioral change was a loss of discriminative conditioned suppression, this effect was driven entirely by a loss of fear towards the aversive cue, suggesting that the irrelevance associated with the neutral CS- remained intact. A model of PL cortex function during the early stages of fear expression and extinction posits that activity within this subregion promotes the expression of defensive reactions such as freezing and conditioned suppression (Pendyam et al., 2013; Sierra-Mercado et al., 2011). Given that the expression of freezing is 54  incompatible with lever-pressing, it is possible that a decrease in freezing explains in part the loss of conditioned suppression. Some evidence against this suggestion comes from our assessment of locomotion, which was not altered by PL inactivation. If PL-inactivated animals froze significantly less than their control counterparts, locomotion may be expected to be higher, which was not the case. In keeping with a particular role in conditioned suppression, PL cortex has been shown to regulate aversion-induced response-inhibition when seeking cocaine (Chen et al., 2013; Limpens et al., 2015) or alcohol (Seif et al., 2013). Similarly, PL (and potentially IL) cortex appear to mediate the response-inhibition enforced during periods of learned cocaine unavailability (Gutman, Ewald, Cosme, Worth, & Lalumiere, 2014; Mihindou, Guillem, Navailles, Vouillac, & Ahmed, 2013). The present study supports these findings, and illustrates that the fear promoting aspect of the PL cortex is specific to a CS+ in a discriminative context. Unlike the unambiguous parallel between the findings of this study and previous studies regarding the PL cortex and fear expression, our observation that IL cortex inactivation decreased conditioned suppression is somewhat surprising. One critical consideration regarding this result is the overall lower level of suppression observed following pre-test IL manipulation (Fig. 4B), when compared to the same manipulation of the more dorsal PL cortex (Fig. 4A). One possible explanation for this difference is that, because our IL cannula were not lowered at an angle during surgery, damage caused to the overlying PL cortex diminished fear expression (Sierra-Mercado et al., 2011). However, this explanation is unlikely, as surgery was conducted in an identical fashion for animals in the pre-conditioning infusion experiments (Fig. 2A and B), where control levels of fear were comparable across prefrontal subregions. Moreover, animals used in the pre-test PL versus IL experiments did not differ in other measures that could have potentially contributed to the difference in baseline conditioned suppression, such as locomotion, 55  overall lever pressing, or the rate of lever pressing (Table 2). Thus, the diminished overall conditioned suppression observed in the IL-manipulated (as compared to PL-manipulated) animals must relate to the infusion being conducted immediately (10 min) prior to the test session. A review of previous studies manipulating IL cortex function during fear expression or extinction shows that animals are typically tested upwards of 30-45 min from the time of infusion, which may abrogate such technical confounds (Akirav et al., 2006; Bravo-Rivera, Roman-Ortiz, Brignoni-Perez, Sotres-Bayon, & Quirk, 2014; Sierra-Mercado et al., 2011). Here, animals were tested 10 min post-infusion to maintain both internal and external consistency, as we have utilized this approach without observing such baseline differences (e.g., Dalton, Wang, Phillips, & Floresco, 2016; Piantadosi & Floresco, 2014; Stopper & Floresco, 2011). However, this may have artificially reduced the amount of conditioned suppression, even under control conditions.  Despite this caveat, we observed a further significant reduction in conditioned suppression induced by inactivation of the IL, as compared to control rats. Using a single-stimulus approach, Sierra-Mercado, Quirk and colleagues (2011) have shown that pharmacological inactivation of IL prolongs conditioned freezing, an effect opposite to that of PL cortex inactivation. Conversely, stimulation of this region has been shown to decrease fear, enhancing extinction either within-session or across sessions (Bukalo et al., 2015; Milad et al., 2004; Vidal-Gonzalez et al., 2006). Here we were interested in the acute impact of each region on fear expression, and did not formally examine the possibility that IL manipulation may affect between-session extinction, which has been shown to depend on IL projections to the amygdala (Bukalo et al., 2015; Do-Monte, Manzano-Nieves, Quinones-Laracuente, Ramos-Medina, & Quirk, 2015). In these previous experiments, conditioned freezing served as the primary 56  dependent measure of fear. Limited experimental evidence suggests that the expression of conditioned suppression, unlike conditioned freezing, is either decreased or not affected by IL inactivation (Jean-Richard-Dit-Bressel & McNally, 2016; Resstel et al., 2008; Sierra-Mercado et al., 2011). For example, inactivation of either PL or IL cortex reduces lever-press suppression induced by instrumental punishment (Resstel et al., 2008), a qualitatively similar effect to that observed here. In addition, a pronounced strain difference in the expression of defensive reactions following IL manipulation has been reported. Lesions of the IL cortex in Long Evans rats (as used here) did not affect freezing in response to a CS+, while the same manipulation in Sprague Dawley rats (used in most previous studies of IL function) abnormally elevated the level of conditioned freezing, delaying extinction (Chang & Maren, 2010). Thus, differences in fear expression circuitry across inbred and outbred rat strains, as well as fundamental differences between the regions necessary for particular defensive reactions, may explain the lack of consistency between the function of the IL observed here and in previous studies.  Although these explanations may shed light on why PL and IL function are not always dissociable, they beg the question as to why we observed an effect, a decrease in conditioned suppression expression following discriminative fear conditioning, of IL inactivation at all. In fact, when we conducted a single-stimulus assessment of conditioned suppression, IL inactivation did not significantly impact fear expression (Fig. 5). These data suggest that the comparable function of PL and IL observed here may additionally relate to the discriminative nature of our task. In support of this, Sangha, Howland and colleagues (2014) have shown that these subregions are not functionally dissociable during performance of a similar Pavlovian discriminative task. In their study, inactivation of PL or IL altered discriminative fear expression in the same manner, decreasing conditioned freezing during the presentation of an aversive cue, 57  while leaving intact the ability of a neutral, safe cue to ameliorate fear (Sangha et al., 2014). Thus, IL cortex may also promote fear during situations that produce a conflict between representations evoked by stimuli encoding safety and fear.   Within the NAc, only the NAcS was relevant for fear expression, with inactivation decreasing conditioned suppression in a manner similar to the PL cortex. NAcC-inactivated rats had no difficulty discriminating between the CS+ and CS-, expressing levels of fear and indifference comparable to control rats. NAcC manipulation was not entirely without effect, as inactivation resulted in rats locomoting less and performing fewer lever-presses than control rats, although their rates of lever-pressing were comparable (Table 2). Such an effect is consistent with previous reports from our and other laboratories suggesting that this nucleus is involved in the invigoration of behavior (Ghods-Sharifi & Floresco, 2010; Nicola, 2010; Stopper & Floresco, 2011). Outside of the aversive domain, the NAcC is known to be involved in the ability of an appetitive Pavlovian conditioned stimuli to invigorate behavior (Ambroggi et al., 2011; Parkinson, Willoughby, Robbins, & Everitt, 2000; Yun et al., 2004). For example, activity and dopamine release within this nucleus is necessary for a cue predicting reward availability to efficiently promote instrumental reinforcement-seeking (Ambroggi et al., 2011; McGinty, Lardeux, Taha, Kim, & Nicola, 2013; Nicola, 2010). Here, NAcC activity was not required for essentially the opposite pattern of behavior, the inhibition of reinforcement-seeking by an aversive Pavlovian conditioned stimulus. Thus, the mechanisms through which the NAc modulate behavior may be biased towards response-promotion, instead of response-inhibition.  On the other hand, NAcS activity proved necessary for rats to appropriately suppress reinforcement-seeking during the presentation of an aversive Pavlovian CS+. This was not the result of general behavioral disinhibition, as indices of general behavioral activation, including 58  the rate of pressing early in the session, the total number of lever-presses made, and locomotor activity were not different than in controls (Table 2). This dissociation points to a more nuanced role for the NAcS, whereby instrumental reward-seeking is impacted specifically by a Pavlovian stimulus previously associated with a negative event. Outside of the negative valence, the NAcS has been suggested to fulfil an inhibitory function during extinction learning for reinforcers including food (Floresco et al., 2008), alcohol (Millan et al., 2010), and cocaine (Peters et al., 2008). Similarly, refining behavior through the learned cessation of instrumental responding during periods of reward unavailability or non-reinforcement is believed to be mediated by an inhibitory NAcS function (Ambroggi et al., 2011; Blaiss & Janak, 2009; Floresco et al., 2008). Populations of neurons that encode task-irrelevant stimuli and behaviors during reward-seeking are more numerous in the NAcS, as compared to the NAcC (Ambroggi et al., 2011), which may provide a neuronal mechanism for the NAcS-specific impact on fear-induced response-inhibition. Moreover, the NAcS is necessary for Pavlovian cues to invigorate instrumental behavior, as assessed by the Pavlovian-to-instrumental transfer (PIT) effect (Corbit & Balleine, 2011; Corbit et al., 2001). Conditioned suppression, which has been described as an aversively-motivated form of PIT (Cardinal et al., 2002), may also depend on this subregions of the NAc.   Given the dense projection from ventromedial PFC, including the ventral PL and IL cortex, to the NAcS (Brog et al., 1993; Sesack et al., 1989; Vertes, 2004), it is important to comment on the qualitative similarity between each region’s effect on conditioned suppression. Based on the present results, the native role for PL cortex during conditioned suppression appears to promote the top-down inhibition of seeking behavior under threat. NAcS may function in a similar manner, although the time-course of inhibition may be somewhat distinct, given that behavioral disinhibition was apparent from the first stimulus presentation following 59  PL inactivation, while the effect of NAcS inactivation did not appear until after the first CS+ delivery (all trials conducted in extinction). It is possible that this function of the mPFC is mediated by its projection to downstream targets, including the NAcS. This hypothesis is supported by previous work suggesting that some neurons projecting from mPFC to NAc (mostly terminating within the NAcS) encode the behavioral relevance of an aversive CS+ and a neutral CS- in a BLA-dependent manner (McGinty & Grace, 2008). Similarly, a recent study identified a microcircuit originating in the mPFC and terminating in the lateral NAcS that promotes suppression following foot-shock (Kim et al., 2017). Activity within this circuit decreased when animals made a seeking response during risk of foot-shock, and activation of this projection inhibited such behavior. Although we targeted the medial NAcS, it is possible that homologous functions are controlled by these topographically adjacent areas. Pharmacological disconnection of these two structures would allow for this hypothesis to be tested. 2.4.3 Relevance to fear circuitry in humans, and psychiatric populations Here, we utilized a discriminative fear conditioning design that is similar to those employed in a translational setting, where CS- presentations serve as a baseline index of fear, and CS+ presentations induce fear. Using such designs, a relatively conserved fear circuit encompassing the amygdala, prefrontal cortex, and ventral striatum has been identified in the human brain (for review, see Adolphs, 2013; Delgado, Nearing, et al., 2008; Milad & Quirk, 2012; Peters et al., 2009). Within the PFC, the dorsal anterior cingulate cortex (dACC; BA32) and ventromedial PFC (vmPFC; BA25) have been suggested to be functionally and anatomically homologous to the rodent PL and IL cortex, respectively (Heilbronner et al., 2016; Milad & Quirk, 2012). Activity in the dACC occurs in response to CS+ presentations, and this activity (as well as the overall thickness of the region) correlates positively with physiological measures of fear in 60  humans (Milad, Quirk, et al., 2007). On the other hand, vmPFC activity appears to track extinction learning in humans, as this region displays patterns of activity consistent with deactivation during conditioning, but activation during extinction (Milad, Wright, et al., 2007; Phelps, Delgado, Nearing, & Ledoux, 2004). Here, we provide tentative support for the dACC-PL homology suggested by these previous studies, as they apply to the expression of conditioned fear. However, our results seem to suggest that IL cortex performs a similar function, promoting conditioned suppression, in a manner inconsistent with human vmPFC activity. This may again stem from the nature of the defensive reaction measured, as freezing (in rats) and skin conductance or verbal scoring (in humans) do not produce a state of motivational conflict similar to that induced by the conditioned suppression of reinforcement-seeking. Although conditioned suppression paradigms exist in humans (Allcoat, Greville, Newton, & Dymond, 2015; Greville, Newton, Roche, & Dymond, 2013), to date, the relevant functional imaging studies have not been performed to evaluate this hypothesis. In addition to prefrontal homology, discriminative aversive conditioning produces activity in the ventral striatum of humans (Delgado et al., 2009; Delgado, Li, et al., 2008; Delgado, Nearing, et al., 2008; Jensen et al., 2003; Klucken et al., 2009; Pohlack et al., 2012). This activity is generally differential, with activity increasing in response to a CS+ to a greater degree than a CS-, a pattern which develops over the course of the conditioning session (Klucken et al., 2009). In addition, activity in this nucleus has been shown to translate fear into motivated action, as learning to avoid an aversive CS+ also recruits the NAc (Delgado et al., 2009). In the present study, NAcS activity was necessary for the appropriate acquisition and expression of discriminative conditioned suppression. Thus, it is possible that the NAc activity observed in human imaging studies of fear learning may reflect preferential activation of the NAcS. 61  Interestingly, only one study has examined subnuclei of the NAc in humans. In this study, diffusion tractography was used to differentiate the NAcS and NAcC in the human brain, with results indicating that the putative NAcS responds in anticipation of thermal pain, while NAcC responds particularly to the offset of a painful stimulus (Baliki et al., 2013). Whether this anticipatory activity relates to behavior is currently unknown, but may partially explain the anticipatory activity observed in NAc prior to presentation of a conditioned aversive stimulus (Jensen et al., 2003).  A number of neuropsychiatric disorders are characterized by the maladaptive influence of affect on decision-making processes. Meta-analytic studies have consistently shown that patients with anxiety disorders express more fear to a CS- than do control individuals (Duits, Cath, Lissek, Hox, Hamm, Engelhard, Van Den Hout, et al., 2015; Lissek et al., 2005). This deficit may be related to aberrant function of prefrontal circuitry, as trait anxiety is associated with diminished coupling between the amygdala and the vmPFC and a heightened coupling between the amygdala and the dorsomedial PFC, patterns that were opposite that observed in healthy comparison subjects (Kim, Gee, Loucks, Davis, & Whalen, 2011). Specifically, vmPFC activity is negatively modulated by similarity to a CS+, while dorsomedial PFC activity is positively modulated by the CS+ similarity. This effect has recently been reported to be disturbed in individuals with PTSD, suggesting that imbalanced prefrontal discrimination mechanisms may contribute to anxiety (Kaczkurkin et al., 2017). In the present study, the fear expressed towards a CS- was normal regardless of treatment. Thus, other regions, such as the BLA, which has been shown to encode the valence of discriminative stimuli in rats, non-human primates, and humans (Genud-Gabai, Klavir, & Paz, 2013; McHugh et al., 2013; Sangha, Chadick, & Janak, 2013; Schiller, Levy, Niv, LeDoux, & Phelps, 2008), may be causally-related to fear generalization.  62  In addition, prefrontal hypofunction appears to be related to inhibitory control deficits in substance abuse (for review, see Goldstein & Volkow, 2011). In cocaine users, deficits in inhibitory control are known to correlate with reduced dACC activity, the same region suggested to promote fear expression previously (Goldstein et al., 2009; Hester & Garavan, 2004; Kaufman, Ross, Stein, & Garavan, 2003; Li et al., 2008). In rats, hypofunction of the functionally homologous PL cortex recapitulates key aspects of addictive behavior, including seeking drug under threat of punishment (Chen et al., 2013; Limpens et al., 2015). Such a deficit may be related to the loss of response-inhibitory function within the PL or dACC, as a function of addiction progression. Moreover, obsessive-compulsive disorder is characterized by aberrant cortico-striatal connectivity, which centers around projections from the orbitofrontal cortex to the ventral striatum (Figee et al., 2016; Wood & Ahmari, 2015). While involving a partially overlapping circuit, hyperactivity (not hypoactivity) of this orbitofrontal-ventral striatal projection appears to mediate compulsive aspects of obsessive-compulsive disorder (Ahmari et al., 2013). Deep brain stimulation of the ventral striatum can improve OCD symptoms (Greenberg et al., 2010; Sturm et al., 2003), possibly due to a normalization of oscillatory activity between ventral striatum and cortex (Figee et al., 2013). The results of the present study suggest that PL cortex or NAcS activation natively promotes aversively-mediated response inhibition, which may not be consistent with an OCD-like phenotype. Further investigation of the cortico-striatal regulation of compulsive-like behaviors relevant to addiction and OCD are necessary to clarify this distinction.  2.5 Conclusion  Investigation of the cortico-striatal basis of conditioned suppression revealed distinct roles for particular subnuclei of these regions. NAcC activity was not necessary for the 63  acquisition or expression of discriminative conditioned suppression, yet this subregion promoted behavioral activation. In contrast, NAcS activity was required for the appropriate acquisition and expression of conditioned suppression, suggesting that this region is critical for aversively-motivated response suppression. Although neither region of the mPFC was involved in fear acquisition, both PL and IL similarly disinhibited reward-seeking during CS+ presentations. These results provide evidence that particular subregions of the NAc dissociably affect conditioned suppression, and implicate a possible mPFC (particularly PL) to NAcS circuit in this effect. In addition, NAcS was shown to play a novel role in fear acquisition, suggesting that an efferent projection known to be involved in aversive learning, possibly BLA, to this region may prime plasticity related to fear learning. Thus, these findings logically lead to circuit-based hypotheses of fear acquisition and expression, involving the NAcS and another structure during acquisition, and a separate PL to NAcS circuit during expression.         64  Table 1. Mean (±SEM) values for overall locomotion, and the change in locomotor activity during CS+ versus CS- presentations within the conditioning session, for animals manipulated prior to conditioning. *: main effect of CS Type during conditioning, p < 0.05. †: p < 0.05 vs SAL. Cannula placement Treatment Locomotion (photobeam breaks) ∆ in activity during CS+ presentations ∆ in activity during CS- presentations PL SAL 1434.82 (±145.92) 0.20 (±0.13) -0.002 (±0.11)*   B/M 1522.29 (±361.36) 0.25 (±0.12) 0.03 (±0.12)*  IL SAL 1581.10 (±250.46) 0.19 (±0.12) 0.06 (±0.12)*  B/M 1444.15 (±180.19) 0.23 (±0.13) 0.02 (±0.10)* NAcS SAL 1981.73 (±222.79) 0.09 (±0.13) 0.03 (±0.11)*  B/M 2353.82 (±353.55) 0.17 (±0.12) -0.04 (±0.09)* NAcC SAL 1523.08 (±126.96) 0.20 (±0.15) 0.06 (±0.15)*  B/M 998.40 (±109.63)† 0.08 (±0.09) -0.07 (±0.07)*                 65  Table 2. Mean (±SEM) values for total locomotion, rate of lever-pressing, and total lever-presses during the discriminative fear expression test session. *: p < 0.05 vs SAL. #: p =  0.09. Infusion timeline Cannula placement Treatment Locomotion (photobeam breaks) Lever-press rate (presses/min) Total lever-presses Pre-conditioning PL SAL 1554 (±208) 17.4 (±2.5) 709.6 (±67.7)  B/M 1699 (±212) 19.5 (±4.0) 836.9 (±134.8)  IL SAL 1465 (±274) 18.7 (±2.5) 794.4 (±98.7)   B/M 1603 (±198) 18.3 (±3.4) 773.2 (±111.5)  NAcS SAL 1764 (±251) 18.9 (±2.1) 807.8 (±81.5)   B/M 1554 (±232) 20.2 (±2.5) 777.9 (±105.9)  NAcC SAL 1975 (±163) 16.4 (±1.3) 688.3 (±49.2)   B/M 2335 (±208) 19.3 (±2.1) 846.3 (±89.8)       Pre-test PL SAL 1557 (±188) 22.2 (±3.8) 1003.5 (±140.9)  B/M 1176 (±115) 23.8 (±3.0) 1245.9 (±126.7)  IL SAL 1560 (±165) 21.5 (±2.8) 1029.6 (±147.0)   B/M 1365 (±195) 21.9 (±3.0) 1173.2 (±127.9)  NAcS SAL 1762 (±218) 21.0 (±3.6) 760.6 (±135.8)   B/M 2285 (±388) 20.3 (±2.9) 1028.7 (±189.2)  NAcC SAL 1709 (±199) 21.9 (±2.9) 751.1 (±93.3)   B/M 989 (±131)* 15.6 (±1.8)# 483.0 (±60.9)*       Single-stimulus (Pre-test) IL SAL 2304 (±333) 24.4 (±5.4) 1450.9 (±242.2)  B/M 2412 (±518) 25.4 (±4.6) 2587.9 (±318.2)*   66   Figure 1. Discriminative fear task diagram and histology. (A) Discriminative fear task diagram. Note that separate groups of animals were infused prior to conditioning and the expression test. (B) Representative histology figure for the pre-conditioning infused, or (C) pre-test infused animals. Blue filled circles represent PL placements, yellow filled circles represent IL placements, red triangles represent NAcS placements, while orange pentagons indicate NAcC placements. Each dot represents the most ventral extent of the infusion, as observed in Nissl stained sections.   67   Figure 2. Inactivation of mPFC does not impact the acquisition of conditioned suppression (A) PL cortex inactivation (B/M) prior to the conditioning session has no impact on the subsequent expression of conditioned suppression during the expression test. Both SAL and B/M treated animals expressed higher levels of conditioned suppression towards the CS+ than the CS-. (B) The same manipulation of IL cortex had no impact on conditioned suppression. Open star represents a main effect of CS Type, p < 0.05.     68   Figure 3. Pre-conditioning NAcS, but not NAcC, inactivation diminishes conditioned suppression. (A) Inactivation (B/M) of the NAcS prior to the conditioning session reduced the amount of conditioned suppression expressed during the expression test, as compared to SAL-infused animals. (B) Pre-conditioning inactivation of NAcC was without effect on the level of discriminative conditioned suppression. Open star represents a main effect of CS Type, p < 0.05, or a simple-main effect analysis of the difference in CS+ expression between B/M and SAL groups.     69   Figure 4. Both mPFC subregions control the expression of conditioned suppression. (A) PL cortex was necessary for the appropriate expression of discriminative suppression, as B/M treatment diminished the degree of suppression to the CS+, as compared to SAL treatment. (B) Inactivation of IL produced a qualitatively similar effect, diminishing overall suppression. Open star represents a main effect of CS Type or Treatment, p < 0.05. n.s.: non-significant.    70   Figure 5. IL inactivation has no impact on conditioned suppression expression conducted using a standard, single-stimulus design. (A) Histology schematic for animals in the single-stimulus fear conditioning experiment. Yellow circles represent the ventral extent of infusion into the IL cortex. (B) Infusion of B/M into the IL had no impact on the expression of conditioned suppression when evaluated using a single-stimulus approach. Open star: Simple main effect breakdown of the CS Block effect, p < 0.05 as compared to the first block of CS presentations.   71   Figure 6. NAcS, but not NAcC, mediates the expression of conditioned suppression.  (A) NAcS inactivation (B/M) selectively diminished the expression of conditioned suppression towards the CS+, as compared to SAL-infused controls. (B) Inactivation of the NAcC, in contrast, had no impact on conditioned suppression expression. Open star represents a main effect of CS Type or Treatment, p < 0.05. Closed star represents a significant difference between the Treatment conditions on suppression towards the CS+, p < 0.05.          72  Chapter 3: Investigating functional cortico-striatal or limbic-striatal circuits contributing to the acquisition and expression of discriminative conditioned suppression 3.1 Introduction In the previous chapter, we established that activity within the NAcS during discriminative Pavlovian fear conditioning was necessary for the appropriate conditioned suppression of reinforcement-seeking during a subsequent expression test. In addition, NAcS and PL partially mediated the expression of conditioned suppression, suggesting that these regions may act in concert to inhibit action during fear. Investigation of the neural circuits that mediate the acquisition and expression of conditioned suppression may provide clinically-relevant insight into the etiology of disorders characterized by affective disturbances such as punishment insensitivity (Deroche-Gamonet et al., 2004; Figee et al., 2016; Limpens et al., 2014). To better understand the circuit mechanisms contributing to these effects, pharmacological disconnections can be utilized to prevent communication between multiple brain regions during particular task events. By inserting cannula into each region of interest in an asymmetric fashion (one cannula in each region, in contralateral hemispheres), infusions can be made to completely abolish communication between the two structures. In contrast, inserting cannula into each region symmetrically (one cannula in each region, in the ipsilateral hemisphere) partially disrupts communication, leaving both structures in one hemisphere intact and capable of maintaining normal behavior. Disruption of behavior following a contralateral disconnection, combined with a null effect of ipsilateral manipulation, implies that the targeted regions form a functional circuit. Our research group has consistently utilized this method to examine the cortico-limbic-striatal regulation of decision-making and executive function (Block, Dhanji, Thompson-Tardif, & Floresco, 2007; Floresco & Ghods-Sharifi, 2007; Jenni, Larkin, & Floresco, 2017; St Onge, Stopper, Zahm, & Floresco, 2012),  73  A number of candidate regions relevant to the acquisition of fear conditioning may exert their effects in part through a projection to the NAcS. Foremost among these is the BLA, a structure vital to fear acquisition (Fanselow & LeDoux, 1999). Lesions or inactivations of this region dramatically diminish the acquisition of defensive reactions and physiological indices of fear (Goosens & Maren, 2001; Helmstetter & Bellgowan, 1994; Koo, Han, & Kim, 2004; LeDoux et al., 1990; Wilensky et al., 1999), consistent with a role for this region in integrating the sensory properties of the CS and US for fear memory formation. Synaptic plasticity within this BLA complex, mediated by glutamate NMDA-receptors, is necessary for fear memory formation (for review, see Johansen, Cain, Ostroff, & Ledoux, 2011; Orsini & Maren, 2012). Blocking these receptors within the BLA using the specific NMDA-receptor antagonist AP-V, for example, has been shown to prevent the normal acquisition of Pavlovian conditioned fear (Maren, Aharonov, Stote, & Fanselow, 1996). The BLA mediates many of the behavioral and autonomic manifestations of fear through projections to the central amygdala, an output nucleus that gates fear expression through downstream interactions with structures including the periaqueductal gray (for review, see Fendt & Fanselow, 1999). However, the BLA also regulates other aspects of aversively-motivated behavior via projections to striatal nuclei, including the NAcS. The BLA projects monosynaptically and primarily ipsilaterally to the NAcS (Brog et al., 1993; Groenewegen et al., 1999; Kita & Kitai, 1990; Shinonaga, Takada, & Mizuno, 1994), forming a functional circuit that is known to mediate active avoidance (Ramirez et al., 2015) as well as the consolidation of inhibitory avoidance (LaLumiere et al., 2005). These types of avoidance conditioning rely in part upon Pavlovian mechanisms, suggesting that the associations made during Pavlovian fear acquisition, and their subsequent effect on conditioned suppression, may be similarly mediated. 74  Additionally, olfactory fear learning modulates mPFC to NAcS activity, an effect which has been shown to depend upon BLA input to the NAcS (McGinty & Grace, 2008).  Because the impact of NAcS inactivation during conditioning was apparent 48 hrs later during a fear expression test (Chapter 2), it is possible that BLA neurons projecting to the NAcS produce glutamate-mediated plasticity (similar to that which occurs in the BLA during conditioning) within this ventral striatal subnucleus. BLA stimulation has previously been shown to increases dopamine and glutamate release in the NAc (Floresco, Yang, Phillips, & Blaha, 1998; Jackson & Moghaddam, 2001), as well as induce plasticity within NAc neurons (Floresco, Blaha, Yang, & Phillips, 2001). Thus, contralateral disconnection of these structures utilizing traditional pharmacological inactivation of the BLA, and a NMDA-R antagonist to eliminate plasticity in the NAcS, may recapitulate the effect of bilateral NAcS inactivation prior to the conditioning session. The results of the previous chapter also implicated a possible circuit between the PL and NAcS that may mediate the expression of conditioned suppression. The mPFC, including the PL and IL cortex (particularly its ventral aspect), projects strongly to the medial NAcS (Brog et al., 1993; Sesack et al., 1989; Vertes, 2004), providing a candidate circuit for this effect. Behaviorally, both PL and NAcS make substantial contributions to other types of response inhibition, including forming a functional circuit mediating impulsive action (Feja et al., 2014; Feja & Koch, 2014, 2015; Resstel et al., 2008). Additionally, interactions between glutamatergic activity in the PL and dopaminergic activity in the NAcS mediates the expression of anxiety (Ahmadi, Nasehi, Rostami, & Zarrindast, 2013). Of direct relevance to fear, a recent study has illustrated that mPFC neurons projecting to the lateral segment of the NAcS promote the conditioned suppression of reward-seeking in an instrumental punishment paradigm (Kim et al., 75  2017). It remains possible that a PL to medial NAcS circuit similarly regulates response inhibition, which can be investigated by performing contralateral disconnections using pharmacological inactivation to eliminate neural activity in each structure, asymmetrically.  Two hypotheses were tested, based on our initial regional inactivation experiments (Chapter 2). The first was whether BLA input to the NAcS during acquisition is necessary for the appropriate expression of discriminative conditioned suppression. The second was whether the PL and NAcS form a functional circuit mediating the expression of conditioned suppression. 3.2 Methods Most experimental procedures were identical to those from Chapter 2. Thus, only notable deviations from the previous procedure will be described here.  3.2.1 Pharmacological disconnection rationale and surgery To establish whether a functional circuit between two regions mediates a particular behavior, one can employ an asymmetric disconnection procedure (Fig. 7A & B). Generally, this technique relies upon the disruption of neural activity within a brain region of interest in one hemisphere, and the disruption of activity in a different region in the contralateral hemisphere. When neural activity in one region (for example, the BLA) is perturbed within one hemisphere, the transmission of task-relevant information to another region of interest (for example, the NAcS) is prevented. In the contralateral hemisphere, neural activity can be disrupted in the efferent target (the NAcS), but not the upstream region (the BLA), such that neither hemisphere has a complete functional circuit with which to regulate the behavior if interest.  Interpretation of this procedure relies upon the assumption that the disruption of activity between these structures in the ipsilateral hemisphere should be without effect, as the intact 76  circuit in the contralateral hemisphere should be able to maintain normal function. To control for this, symmetric disconnections can be performed, where activity is disrupted within each region in the ipsilateral hemisphere. In the event that an ipsilateral disconnection produces an effect, unilateral manipulation of each region individually can be performed to see whether the effect of the symmetric disconnection was due to the partial loss of a functional circuit, or whether the effect is mediated by a single node within this putative circuit.  The first disconnection experiment was designed to probe the circuit basis of the role of the NAcS during the conditioning session. In the previous chapter, we illustrated that inactivation of the NAcS during conditioning resulted in a more labile fear memory during the expression test (Figure 7A). Here, we examined whether this effect may be mediated by a projection from BLA to the NAcS. Because the effect of pre-conditioning NAsS inactivation illustrated in the previous chapter was observed days later (during the expression test), we suspected that BLA may trigger glutamate-mediated plasticity within the NAcS. To eliminate this plasticity, we infused a dose of the glutamate NMDA-receptor antagonist AP-5 into the NAcS, combined with B/M into the BLA to inactivate this region. AP-V has been shown to block long-term potentiation (Davis, Butcher, & Morris, 1992; Morris, 1989), and has previously been used to impair the acquisition of a spatial working memory task when infused into the NAc at this dose (Smith-Roe, Sadeghian, & Kelley, 1999). The second experiment examined the possibility that PL cortex may drive fear expression in part through a projection to NAcS, in keeping with their qualitatively similar effects on the expression of conditioned suppression (Figure 7B). To disconnect the PL and NAcS, rats received infusion of B/M into the contralateral PL and NAcS (asymmetric disconnection), or ipsilateral PL and NAcS (symmetric disconnection). In addition, we conducted unilateral 77  infusions into the PL or NAcS to control for the possibility that the disconnection effects were due to the impact of a single infusion into one hemisphere. A separate group of rats received infusion of saline into the contralateral PL and NAcS. Twenty-three gauge stainless steel guide cannula were implanted aimed at the BLA and NAcS, PL and NAcS, or unilaterally in the PL or NAcS, according to the following stereotaxic coordinates (in mm): BLA – from bregma: AP: -3.2 ML: ±5.3; from dura: DV: -6.3 PL – from bregma: AP +3.2; ML: ±0.7; from dura: DV: -2.8 NAcS – from bregma, AP: +1.3, ML: ±1.0, from dura, DV: -6.3 The particular hemisphere selected for each placement was counterbalanced across experimental conditions, such that roughly equivalent numbers of rats received cannula in each combination of hemispheres. Four stainless-steel skull screws were inundated with dental acrylic to secure cannula in place. Stainless-steel obturators flush with the end of the guide cannula were inserted after surgery. Rats were given 5-10 d to recover from surgery before beginning behavioral training.  3.2.2 Microinfusion For the pre-conditioning disconnection experiment, the glutamate NMDA-receptor antagonist AP-5 (1 μg /0.3 μl saline) was infused into the NAcS, combined with a standard dose (75 ng/μl of each drug at a volume of 0.3 μl/side) of B/M into the BLA to inactivate this region. Some rats received these infusions into the contralateral BLA and NAcS (asymmetric disconnection), while others received infusion into the ipsilateral BLA and NAcS (symmetric 78  disconnection). A separate group of rats received infusion of 0.9% saline (0.3 μl/side) into the contralateral BLA and NAcS. Each infusion was conducted over 45 s, with the microinjector left in place for an additional 1 min to allow for diffusion. Separate groups of animals received contralateral BLANAcS disconnection (n = 13), ipsilateral BLANAcS disconnection (n = 12), or saline infusion (n = 10).  For the pre-test disconnection experiment, rats received infusion of B/M (75 ng/μl of each drug at a volume of 0.3 μl/side) into the contralateral PL and NAcS (asymmetric disconnection), or ipsilateral PL and NAcS (symmetric disconnection). A separate group was given unilateral inactivations of the PL or NAcS (same infusion parameters), to control for the possibility that the disconnection effects were due to the impact of a single infusion into one hemisphere. Control rats received infusion of 0.9% saline (0.3 μl/side) into the contralateral PL and NAcS. Each infusion was conducted over 45 s, with the microinjector left in place for an additional 1 min to allow for diffusion. Separate groups of animals received contralateral PLNAcS disconnection (n = 9), ipsilateral PLNAcS disconnection (n = 9), saline infusion (n = 10), or Uni-PFC (n = 5) and Uni-NAcS (n = 5) infusion. 3.2.3 Histology All rats were euthanized with CO2 and brains were removed and fixed in a 4% phosphate buffered formalin solution. Brains were sectioned at 50 μm, following which tissue was mounted and Nissl stained using Cresyl Violet. Placements were examined under a light microscope, and the ventral extent of each infusion is indicated in Figure 8A (BLANAcS disconnection) and Figure 9A (PLNAcS disconnection).   79  3.2.4 Data analysis Data analysis was conducted in a nearly identical fashion to the previous chapter. For the pre-conditioning BLANAcS disconnection experiment, analyses were identical with the exception that the between-subjects Treatment factor had three levels: saline, contralateral BLANAcS disconnection, and ipsilateral BLANAcS disconnection. Overall locomotor activity during the session was analyzed with a one-way ANOVA, with Treatment as the between-subjects factor.  Analysis of conditioned suppression during the expression test was conducted in an identical manner to the previous chapter, with the exception that the between-subjects Treatment factor for each experiment included more levels. For the pre-conditioning experiment, the Treatment factor include three levels: saline, contralateral BLANAcS disconnection, and ipsilateral BLANAcS disconnection. For the expression test experiment, the Treatment factor was made up of four levels: saline, contralateral PLNAcS disconnection, ipsilateral PLNAcS disconnection, unilateral inactivation (combined across PL and NAcS placements, see Results). Follow-up simple main effects analyses were conducted using one-way ANOVAs, where appropriate. Locomotion (photobeam-breaks/session) during the conditioning session or expression test were analyzed using separate independent samples t-tests. The rate of lever-pressing in the first 5 min of the session and the total number of lever-presses made during the session were analyzed in an identical fashion.   80  3.3 Results 3.3.1 BLA-NAcS disconnection during the acquisition of discriminative conditioned suppression Disconnection of BLANAcS had no effect on CS-induced changes in locomotor activity during the conditioning session, as compared to animals that underwent ipsilateral disconnection or saline treatment (Table 3). There was no main effect of Treatment (F(2,32)=0.59,p>0.56), and there was no CS Type x Treatment interaction (F(2,32)=0.23,p>0.79). There was a main effect of CS Type (F(1,32)=15.29,p<0.001), which indicated that the locomotor increase from baseline was greater during CS+ presentations than CS- presentation, regardless of treatment. Disconnection tended to increase overall locomotor activity, although this effect only approached significance (F(2,32)=3.13,p<0.06). Regardless, disconnection did not affect the differential change in locomotion from baseline caused by CS+ and CS- presentation. Surprisingly, disconnection of the BLA from the NAcS during conditioning had no impact on the subsequent expression of discriminative conditioned suppression (Figure 8B). There was no effect of Treatment (F(2,32)=0.74,p>0.48), no CS Type x Treatment interaction (F(2,32)=1.06,p>0.35), and no three-way interaction (F(6,96)=0.52,p>0.79). Thus, all rats suppressed their reinforcement-seeking more during presentations of the CS+ than the CS-, as indicated by a main effect of CS Type (F(1,32)=101.68,p<0.001). The total number of lever presses made during the expression test session did not differ as a function of Treatment (F(2,32)=2.41,p>0.10), nor did the rate of lever-pressing during the first 5 min of the test session (F(2,32)=2.19,p>0.12) (Table 4). Similarly, total locomotion was not altered by BLANAcS disconnection (F(2,32)=0.91,p>0.41) (Table 4). Thus, it is unlikely that a BLANAcS pathway mediates the role of the NAcS in the acquisition of discriminative conditioned fear.  81  3.3.2 PL-NAcS disconnection during the expression of discriminative conditioned suppression As there was no significant difference between the mean suppression ratio during the CS- and CS+ for animals in the Uni-PFC group (CS-: 0.07±0.06 SEM, CS+: 0.82±0.10 SEM) versus the Uni-NAcS group (CS-: 0.14±0.06 SEM, CS+: 0.85± 0.08 SEM) (F(1,8)=0.39,p>0.55), these groups were combined into a singular unilateral inactivation group for all subsequent analyses. Disconnection of the PL cortex from the NAcS diminished the expression of conditioned suppression, indicated by a significant main effect of Treatment (F(3,34)=3.66,p<0.022), as well as a CS Type x Treatment interaction (F(3,34)=6.46,p<0.001) (Figure 9B). There was no three-way interaction (F(9,102)=1.31,p>0.24). Follow up simple-main effects analyses on the two-way interaction revealed that this effect was due to a difference between the treatment conditions on CS+ trials (F(4,33)=8.42,p<0.001), but not CS- trials (F(4,33)=0.66,p>0.58). Further analysis indicated that suppression during the CS+ was similar between saline animals and the unilaterally inactivated group (F(1,18)=0.88,p>0.36). In contrast, animals in the contralateral PLNAcS disconnection group (F(1,17)=24.48,p<0.001), or ipsilateral PLNAcS disconnection group (F(1,17)=10.07,p<0.006), expressed less conditioned suppression during CS+ presentations, when compared to saline-infused control animals. Contralateral PLNAcS disconnection animals also expressed less conditioned suppression during the CS+ than did unilaterally inactivated animals (F(1,17)=12.16,p<0.003), but the comparison between ipsilateral PLNAcS disconnection and unilateral infusion only approached significance (F(1,17)=3.94,p>0.06).  None of the treatments had an effect the number of lever presses made during the expression test (F(3,34)=0.12,p>0.94), or the rate of lever-pressing made during the initial 82  portion of the test session (F(3,34)=0.30,p>0.82) (Table 4). However, locomotor activity did differ as a function of treatment (F(3,34)=7.02,p<0.001). This was driven by a significant increase in locomotor activity in the ipsilateral disconnection group, as compared to all other groups (all p-values < 0.025). 3.4 Discussion Here, we attempted to identify two functional circuits involving the NAcS that mediate the acquisition or expression of Pavlovian conditioned suppression. Contralateral or ipsilateral disconnection of the NAcS and BLA performed prior to the acquisition of fear conditioning had no impact on subsequent expression, suggesting that the NAcS may interact with another structure during this critical task epoch to mediate fear acquisition. In contrast, disconnection of the PL cortex from the NAcS, whether conducted in a contralateral or ipsilateral manner, decreased the expression of conditioned suppression. Importantly, unilateral inactivation of either structure had no impact on performance. The effect of PLNAcS disconnection was qualitatively similar to the effect of bilateral inactivation of either structure alone, providing evidence that a functional circuit between these structures controls fear-mediated response-inhibition.  3.4.1 A BLA-NAcS circuit does not mediate fear acquisition First, we chose to investigate a possible BLANAcS circuit mediating discriminative fear acquisition, based in large part on the accepted role for this amygdalar region in fear learning (Fanselow & LeDoux, 1999). This projection also mediates other aspects of aversively-mediated behavior, including active and passive avoidance (LaLumiere et al., 2005; Ramirez et al., 2015). By infusing the NMDA-receptor antagonist AP-V into the NAcS in one hemisphere, and 83  reversibly inactivating the BLA in the opposite hemisphere, we aimed to eliminate the relevant communication and plasticity that may occur during fear learning. However, animals that received this contralateral disconnection expressed similar levels of conditioned suppression as did ipsilateral disconnection and saline-infused controls, suggesting that this pathway is not involved in fear acquisition.    Although the BLA is consistently involved in the acquisition of conditioned freezing and other physiological changes, recent data indicate that this effect may be mediated by an intra-amygdala, rather than accumbens, projections. Targeting specific subsets of neurons that project from the BLA to the central nucleus of the amygdala (CeA) or the NAc, Namburi, Tye and colleagues (2015) demonstrated that optogenetic inhibition of the BLA to CeA projecting cells inhibited fear learning, while the same manipulation of the BLA to NAc projectors was without effect. Thus, the BLA may mediate the acquisition of Pavlovian fear exclusively through intrinsic connections with the amygdala. Additionally, there has been some suggestion that conditioned suppression can persist despite damage to the BLA that eliminates conditioned freezing reactions (Lee, Dickinson, & Everitt, 2005; McDannald & Galarce, 2011). For example, after multiple days of conditioning, BLA-lesioned rats never express normal levels of discriminative freezing towards a CS+, but not a CS-, while conditioned suppression develops normally (McDannald & Galarce, 2011). Thus, it is possible that the BLA does not consistently contribute to the acquisition of conditioned suppression, providing a potential theoretical explanation for the null effect of our disconnection procedure.   This lack of effect leads to the question of what region may mediate the observed effect of bilateral NAcS inactivation on fear acquisition. One possible region is the ventral hippocampus (vHPC), which projects strongly to the medial NAcS (Brog et al., 1993; French & 84  Totterdell, 2002, 2003). In fact, the density of the vHPC projection to the medial NAcS has been shown to be larger than that of the BLA or mPFC (Britt et al., 2012). This region has also been shown to be involved in fear acquisition (Bast, Zhang, & Feldon, 2001; Esclassan, Coutureau, Di Scala, & Marchand, 2009; Maren & Holt, 2004). For example, inactivation of vHPC prior to discriminative fear acquisition disrupts the expression of freezing towards a CS+, without altering fear expressed towards a CS- (Chen, Foilb, & Christianson, 2016). This effect is qualitatively similar to that observed in the previous chapter following NAcS inactivation, implicating a potential serial circuit between these two structures. Previous research suggests that a projection from the vHPC to NAcS is critical for other form of learning, including spatially-guided conditioned place preference and foraging (Floresco, Seamans, & Phillips, 1997; Ito, Robbins, Pennartz, & Everitt, 2008). Thus, future studies investigating the relevant efferent projection to NAcS should target this vHPC projection.  3.4.2 Interactions between the PL and NAcS mediate discriminative conditioned suppression Disconnection of a PLNAcS circuit diminished conditioned suppression (Chapter 2). This finding supports the contention that information regarding the aversive nature of the CS+ is serially transmitted from the PL cortex to the NAcS, promoting the top-down regulation of conditioned suppression. Decreased PL activity has previously been associated with reward-seeking despite the threat of punishment in a Pavlovian or instrumental context (Chapter 2; Chen et al., 2013; Limpens et al., 2015; Resstel et al., 2008). This pattern of findings reinforces the notion that the mPFC inhibits behavior during threat, in a top-down manner. Consistent with this, a similar circuit has recently been demonstrated between the mPFC and lateral NAcS which causally enforces response inhibition during periods of reward-seeking under threat of shock 85  (Kim et al., 2017). In that study, a subset of mPFC projection neurons to the NAcS are active during the decision to inhibit reward-seeking behavior, while hypoactivity within this projection is typical of seeking during punishment. Exciting this aversion-sensitive projection decreases the probability of seeking during threat of punishment, consistent with a causal role in response-selection mediated by this mPFClateral NAcS projection (Kim et al., 2017). Here, we illustrate that such a PLmedial NAcS projection is similarly involved in the influence of an aversively-conditioned Pavlovian cue on reward-seeking.   One key point of contention arising from these data is that both contralateral and ipsilateral disconnections of PL and NAcS resulted in a similar behavioral phenotype, decreasing conditioned suppression. A parsimonious interpretation of data arising from disconnection procedures relies upon on the assumption that projections are primarily ipsilateral, such that removal of a single node within each circuit in different hemispheres effectively prevents all communication within the circuit. However, if contralateral projections between these two structures exist, they may remain partially intact and able to impact behavior. In fact, PL cortex projects both ipsilaterally and contralaterally to the NAcS (Brog et al., 1993; Vertes, 2004). A common way to eliminate these ipsilateral connections is to perform a partial callosotomy, severing the contralateral communication between the two structures. Here, our NAcS cannula transected the corpus callosum, which should limit the contralateral connection between these two regions, making this explanation unlikely. Importantly, when we inactivated either structure in isolation, there was no effect on conditioned suppression. Taken together, these results imply that parallel PLNAcS projections within each hemisphere are necessary for normal conditioned suppression. Unilateral nodes within this circuit are neither necessary nor sufficient 86  to produce behavior, providing evidence that interactions between these subnuclei are of critical importance. 3.5 Conclusion Here, we attempted to answer the question of whether particular circuits involving the NAcS were involved in the acquisition or expression of discriminative fear. In the first experiment, we attempted to recapitulate the impact of bilateral NAcS inactivation during the acquisition of discriminative fear by blocking plasticity within the NAcS while the BLA was inactivated in contralateral or ipsilateral hemispheres. Contrary to our expectation, disconnection of this BLANAcS circuit had no impact on the ability to acquire discriminative fear, as measured by conditioned suppression during an expression test. This result suggests that another afferent region, possibly the vHPC, may prime NAcS during acquisition, allowing for subsequent discriminative fear expression. In a separate experiment, we examined whether a direct PLNAcS circuit may mediate the impact of bilateral inactivation of either structure on the expression of conditioned suppression. In fact, we observed that disconnection (contralateral or ipsilateral) of this cortico-striatal circuit recapitulated a qualitatively similar effect, diminishing the expression of conditioned suppression. This result suggests a top-down role for the PL cortex in the regulation of Pavlovian conditioned suppression, mediated via the NAcS. Overall, these studies provide further clarification of how defensive behavior is mediated by discrete cortico-limbic-striatal circuits, implicating regions of the mPFC and NAcS in the inhibition of responding during Pavlovian threat, and the NAcC in the promotion of appetitive vigor.     87  Table 3. Mean (±SEM) values for ancillary measures during the conditioning session, induced by BLA-NAcS manipulation prior to conditioning. Measures included the change in locomotor activity during CS+ versus CS- presentations and total locomotion within the conditioning session. *: main effect of CS Type during conditioning, p < 0.05. Cannula placement Treatment Locomotion (photobeam breaks) ∆ in activity during CS+ presentation ∆ in activity during CS- presentation BLA-NAcS SAL 1314.70 (±183.70) 0.19 (±0.19) -0.003 (±0.12)*   Contra-Disc 1359.23 (±115.75) 0.19 (±0.12) 0.04 (±0.13)*   Ipsi-Disc 2287.17 (±485.26) 0.11 (±0.16) -0.02 (±0.14)*   Table 4. Mean (±SEM) values for ancillary measures induced by BLA-NAcS or PL-NAcS manipulation. Total locomotion, the rate of pressing (first 5 min of the test session), and total lever-presses are reported during the expression test session. *: p < 0.025 versus all other PL-NAcS treatment conditions.  Cannula placement Treatment Locomotion (photobeam breaks) Lever-press rate (presses/min) Total lever-presses BLA-NAcS SAL 2099 (±187) 26.2 (±2.9)  1069.9 (±87.6)  Contra-Disc 2046 (±192) 27.6 (±3.4) 1079.4 (±123.4)  Ipsi-Disc 2464 (±314) 39.2 (±6.8) 1486.3 (±209.3)      PL-NAcS SAL 1651 (±205) 24.3 (±3.3) 968.2 (±114.7)  Contra-Disc 2155 (±272) 23.6 (±4.0) 1056.2 (±217.1)  Ipsi-Disc 3384 (±357)* 23.5 (±3.9) 938.6 (±104.1)  Uni-Inact 2455 (±371) 19.8 (±2.9) 947.5 (±144.5)   88   Figure 7. Disconnection methodology diagram. (A) Cartoon depicting the functional disconnection employed to examine a potential BLA-NAcS circuit mediating the acquisition of conditioned suppression, or a (B) PL-NAcS circuit relevant for the expression of conditioned suppression. The red cartoon structure represents the NAcS, while the afferents are indicated in green (BLA) or blue (PL). The white X in a filled circle represents the pharmacological manipulation of a particular structure. Solid black lines with an arrow indicate intact projections, while broken lines with an interrupted end represent the effect of pharmacological intervention. Note that these diagrams are overly simplified, and do not depict potentially relevant projections to 3rd brain regions.     89   Figure 8. A BLA-NAcS disconnection does not mediate the acquisition of conditioned fear. (A) Histology schematic illustrating the ventral extent of each infusion in the NAcS (left) or BLA (right) Closed circles represent contralateral infusions (SAL or Contra-Disc), and closed triangles represent ipsilateral disconnections (Ipsi-Disc). (B) Contralateral disconnection and ipsilateral disconnection of a BLA-NAcS pathway had no impact on fear expression, as these animals did not differ from saline-infused controls. Open star: Main effect of CS Type, p < 0.05.   90   Figure 9. A PL-NAcS projection contributes to the expression of conditioned suppression. (A) Histology schematic illustrating the ventral extent of each infusion in the NAcS (left) or PL cortex (right). Closed circles represent contralateral infusions (SAL or Contra-Disc), closed triangles represent ipsilateral disconnections (Ipsi-Disc), and grey pentagons represent unilateral inactivation (Uni-Inact). (B) Animals that underwent contralateral disconnection or ipsilateral disconnection expressed less fear towards the CS+, as compared to control animals. Unilateral inactivation was significantly different from contralateral disconnection animals, but comparison between this group and ipsilateral disconnection animals only approached significance. Open star: main effect of CS-type, p < 0.05. Closed star: comparison of suppression to the CS+, p < 0.05 between the SAL group and the Contra-Disc or Ipsi-Disc group, or the Contra-Disc and the Uni-Inact group. #: p = 0.06 between the Ipsi-Disc and Uni-Inact group.     91  Chapter 4: The role of NAc core and shell in motivational conflict during reward and punishment 4.1 Introduction In the previous chapters, we identified that the NAc is functionally heterogeneous when considering Pavlovian fear expression. Instrumental punishment may similarly rely upon discrete NAc subregions, however this hypothesis has not been empirically tested. Considerable research has been dedicated to clarifying the influence of positive reinforcement on decision-making, implicating the NAc and associated cortico-limbic afferents in such reinforcement learning (Cardinal et al., 2002; Floresco, 2015; Parkinson, Cardinal, et al., 2000). In contrast, less is known about how this system guides behavior in response to punishment, a process by which an instrumental action co-occurs with a negative consequence, such as a lever-press-contingent foot-shock in rodents. In a majority of individuals, punishment serves to suppress the instrumental action with which it occurs. However, neuropsychiatric conditions such as substance abuse and obsessive compulsive disorder are characterized by compulsivity, whereby punishment is less effective in curtailing detrimental behavioral patterns (Everitt, 2014; Feil et al., 2010; Figee et al., 2016; Jentsch & Taylor, 1999; Lubman et al., 2004; Perry & Carroll, 2008). As such, investigation of the circuitry underlying punishment-induced inhibitory control may provide insight into the pathophysiological underpinnings of these symptoms in various disease states.  Compulsivity in the face of punishment is recognized by the DSM-5 as a core symptom of substance abuse and other disorders, and pre-clinical findings suggest that these symptoms may be driven by alterations within cortico-limbic circuitry (Chen et al., 2013; Limpens et al., 2014; Pelloux, Murray, & Everitt, 2013; Radke, Jury, et al., 2015; Radke, Nakazawa, & Holmes, 2015). Prolonged access to cocaine produces punishment-resistant drug seeking, concomitant 92  with hypofunction of medial prefrontal cortex (mPFC) (Chen et al., 2013). Optogenetic inhibition or activation of mPFC decreases or increases, respectively, the impact of punishment on cocaine seeking (but see Pelloux, Murray, Everitt, 2013), suggesting that mPFC activity may be causally related to the punishment-mediated inhibition of seeking. Similarly, pharmacological inactivation or lesion of the mPFC produces operant responding for both cocaine and sucrose that is insensitive to potential punishment, whether presented in a Pavlovian or instrumental fashion (Limpens et al., 2015; Resstel et al., 2008). Prefrontal regions seem to perform a top-down inhibitory function, acting as a break when responding is directly punished, or in the presence of a fear-inducing stimulus. Likewise, the basolateral amygdala (BLA) promotes behavioral suppression during punishment. Jean-Richard-Dit-Bressel and McNally (2015) recently showed that inactivation of caudal (but not rostral) BLA eliminated the inhibition of lever-pressing produced by contingent foot-shock. Inactivated rats made more lever-presses during punishment, and did not display the typical increase in latency to press caused by punishment. Thus, both mPFC and BLA may contribute in a similar manner to punishment avoidance during appetitively-motivated behavior. Although the BLA and PFC appear to subserve complementary roles in punishment avoidance, the downstream structure mediating this effect is currently unknown. The nucleus accumbens (NAc) receives dense glutamatergic input from both mPFC and BLA, and is known to regulate various forms of appetitive conditioning via its meso-cortico-limbic efferents (Cardinal et al., 2002; Floresco, 2015; Sesack & Grace, 2010). The NAc is primarily composed of two functionally and anatomically distinct subregions, the more lateral core (NAcC) and more medial shell (NAcS) (Heimer et al., 1997; Zahm & Brog, 1992). These two subregions have been suggested to serve dissociable yet complementary functions during reward-seeking, with the 93  NAcC driving approach towards motivationally-relevant stimuli, and the NAcS facilitating inhibition of inappropriate behaviors (Ambroggi et al., 2011; Floresco, 2015). In this regard, the ventral regions of the mPFC and caudal BLA project strongly to the medial NAcS (Berendse, Galis-de Graaf, et al., 1992; Brog et al., 1993; Groenewegen et al., 1999; Heilbronner et al., 2016; Kita & Kitai, 1990; Wright et al., 1996), suggesting that this nucleus may facilitate inhibition of punished behavior regulated by these upstream cortical and limbic regions. A recent experimental report supports this contention, suggesting that a projection from the mPFC to lateral NAcS is active when suppressing punished reward-seeking, and inhibited when promoting seeking regardless of punishment (Kim et al., 2017). However, whether the medial NAcS or more lateral NAcC contributes to such behavior is unknown. It is therefore possible that NAc subregions may differentially contribute to adjusting behavior in response to punishment, with NAcS suppressing reward-seeking in the face of punishment in a manner similar to the BLA or PFC, and NAcC generally promoting action.  The present series of experiments were designed to both confirm a role for BLA in mediating reward/punishment conflict, and explore the potential differential contribution of NAcS versus NAcC to the same behavior. To this end, separate groups of well-trained rats received reversible inactivation of BLA, NAcS, or NAcC while performing an operant-based “Conflict” task. We also examined the potential monoaminergic contribution to punished reward-seeking by administering the monoamine releaser d-amphetamine (AMPH) in a subset of animals on the Conflict task, as previous studies have suggested that elevations in monoamine activity promotes the punishment-induced suppression of behavior (Killcross, Everitt, & Robbins, 1997; Lazareno, 1979; Leone, de Aguiar, & Graeff, 1983). During the Conflict task, sucrose reward was available on a lean reinforcement schedule, without punishment, during two 94  “Safe/Reward” periods. Interspersed between these periods was a separate “Conflict” period, wherein sucrose was available on a richer schedule, but 50% of lever-presses triggered a foot-shock punishment. Results using this Conflict task, and a “No-Conflict” (identical schedules of reinforcement, but no punishment) control variant, suggested that BLA and NAcS promote punishment-induced behavioral suppression, while NAcC plays a more general role in driving reward-seeking.  4.2 Methods 4.2.1 Animals  All experimental protocols were approved by the Animal Care Committee, University of British Columbia, and conducted in compliance with guidelines provided by the Canadian Council on Animal Care. All reasonable efforts were made to minimize the number and suffering of animals used. Male Long-Evans rats arrived weighing 225-350g (Charles River) and were group housed (4-5 per cage) and allowed 6-7 d of acclimation to the colony. Colony temperature (21° C) and light cycle (12-h light/dark) were kept constant. Prior to operant training, all rats were individually housed and food-restricted to approximately 90% of their free-feeding weight, and allowed to gain weight throughout the course of the experiment on a delayed-growth curve.  4.2.2 Apparatus  Behavioral testing was conducted in eight Med Associates (St Albans, VT, USA) operant conditioning chambers. Each chamber (30.5 cm x 24 cm x 21 cm) was contained in a sound-attenuating enclosure, ventilated by a fan that also served to mask external noise. Within each chamber were two retractable levers along one wall, separated by a food receptacle from which sucrose pellet reinforcement was delivered (45 mg pellet, BioServ, Frenchtown, NJ). For all experiments, only the left lever was extended into the chamber. Each box was outfitted with 95  three 100 mA cue lights, one over each retractable lever, and one over the food receptacle. A single 100 mA house light was situated on the wall opposite the food receptacle. Four infrared photobeans located just above the grid floors were used to index locomotor activity. The chamber floor consisted of 19 stainless steel rods spaced 1.5 cm apart. The rods were wired to a shock source and solid-state grid scrambler for the delivery of foot-shock.  4.2.3 Surgery Rodent anesthesia was conducted slightly differently for BLA and NAc placements, due to changes in institutional policies regarding anesthetic techniques. Animals receiving BLA cannula were anesthetized with a combination of ketamine/xylazine (100 and 20 mg/ml at 100 and 10 mg/kg, i.p.), exclusively. Animals receiving NAc cannula were first anesthetized with a half-dose of ketamine/xylazine (same mg/ml, i.p.), and then maintained on Isoflurane anesthetic (2-3% Isoflurane concentration) throughout surgery. Twenty-three gauge bilateral stainless-steel guide cannula were aimed at the BLA, NAcS, or NAcC according to the following stereotaxic coordinates (in mm): BLA – from bregma, AP: -2.7, ML: ±5.3, from dura, DV: -7.0 NAcS – from bregma, AP: +1.3, ML: ±1.0, from dura, DV: -6.3 NAcC – from bregma, AP: +1.6, ML: ±1.8, from dura, DV: -6.3  Dental acrylic adhered to four stainless-steel skull screws held cannula in place. Stainless-steel obturators flush with the end of the guide cannula were inserted immediately following surgery, and remained in place throughout the experiment. Rats were given approximately 1 wk to recover from surgery before beginning behavioral training.  96  4.2.4 Training  Twenty-four hours before their first operant training session, rats were provided with ~30 sucrose pellets in their home cage, to reduce potential food neophobia. Subsequently, 15 min training sessions were conducted at a consistent time each day, 5-7 days per week. Rats were initially trained for 3 d on a fixed-response 1 (FR1) schedule, such that each press of the lever was rewarded with a sucrose pellet. Animals were then trained for 4-5 d on a variable-interval 15 s/FR1 (VI15/FR1) reinforcement schedule, whereby a lever-press after a 15 s interval was rewarded with a single sucrose pellet. The final portion of training was conducted over 3 days, on a VI15/FR5 schedule, identical to VI15/FR1 except that the 5th press after the variable-interval was rewarded with a single pellet.  The Conflict task was based on procedures used by Broersen et al. (1995), and consisted of three discrete 5 min phases (15 min total session length). During these sessions, the lever remained inserted into the chamber for the entire session. During the first and the third phases, the house-light was illuminated and rats were reinforced for lever-presses on a VI15/FR5 schedule. There was no danger of punishment during these two phases, and thus they were called Safe/Reward phases. In contrast, during the second (middle) block, the house light was turned off, and the left cue-light was illuminated, signaling the 5 min Conflict period. Here, reward was delivered on a FR1 schedule, but, in addition, lever-presses resulted in foot-shock punishment delivered on a random ratio-2 schedule (i.e., 50% of responses were shocked), with no time-out restricting the number of presses or foot-shocks received. This produces a state of anxiety, akin to that in the Vogel conflict task (Vogel et al., 1971).  Shock intensity was individually titrated over the course of training, such that each rat eventually achieved criterion performance of receiving less than 20 shocks per session for two 97  consecutive days. The range of shock intensity across all experimental cohorts was: 0.35 – 0.75 mA. To achieve criterion during the Conflict period, rats in the BLA group required a mean shock intensity of 0.48 mA (±0.02 SEM), those in the NAcS required a mean shock intensity of 0.53 mA (±0.03 SEM), and those in the NAcC of 0.60 mA (±0.02 SEM). Across the different experimental groups, rats required 13.9 (±0.4 SEM; range 10-19) training sessions on the Conflict task to achieve criterion performance. Separate groups of rats were trained on a No-Conflict control version of the task, for which the initial training was the same as the Conflict version. This task was identical to the Conflict task, with the notable exception that no foot-shocks were delivered at any point. This No-Conflict task was designed to assess whether any potential alterations in responding induced by regional inactivation could be attributed to changes in the reinforcement schedules (VI15/FR5 vs. FR1) that occurred during the Conflict task. On the No-Conflict task alone, rats with BLA cannula required 12 days of training, while those with NAc cannula required 15, until they displayed asymptotic levels of responding as a group during the three phases, defined as two consecutive days with < 20% variation in lever-pressing across phases.  4.2.5 Microinfusion and systemic pharmacology Once an individual rat displayed stable behavioral performance, it received a mock infusion 10 min prior to the daily training session. This procedure consisted of removal of the obturators, insertion of a mock injector flush with the end of the guide cannula, and placement in the infusion enclosure for approximately 2 min. All microinfusions were conducted 10 min before animals were placed in their operant chamber. On the infusion day, microinjectors extending 0.8 mm beyond the guide cannula were lowered into the brain and animals received bilateral infusion of 0.9% saline (0.3 μl/side for NAc infusion and 0.5 μl/side for BLA infusion) or a 75 98  ng (NAc) or 125 ng (BLA) dose of the GABA agonists muscimol and baclofen (B/M; same volume/side as saline). Each infusion was conducted over 45 s (NAcS) or 75 s (BLA), with injectors left in place for an additional 60 s to allow for diffusion of solution from cannula tips. This dose and volume of B/M in the NAc has been used previously to dissociate between the NAcS and NAcC on a wide variety of behavioral measures (Dalton et al., 2014; Floresco et al., 2008; Ghods-Sharifi & Floresco, 2010; Stopper & Floresco, 2011). In addition, this dose and volume of B/M infused into the BLA is similar or identical to other studies examining the role of the BLA individually (Ghods-Sharifi, St Onge, & Floresco, 2009), or dissociating between adjacent amygdala subregions (Jean-Richard-Dit-Bressel & McNally, 2015; Millan, Reese, Grossman, Chaudhri, & Janak, 2015). Order of infusion was counterbalanced across animals, such that some rats received SAL prior to B/M, while others received infusion in the opposite order. All animals were re-trained for a minimum of two days prior to receiving their second infusion.  After receiving counterbalanced infusion test days, rats with NAc placements were given two additional test days (at least two days after their final microinfusion test). On the first test day, rats received an injection of saline (SAL; 1 ml/kg, i.p.) 10 min prior to the operant session. The next day, rats were given an injection of d-amphetamine (AMPH; 1 mg/kg at a concentration of 1 mg/ml, i.p.). This experiment did not include animals with BLA placements, as it was conceived of following the completion of that experimental cohort.  4.2.6 Histology  Following the completion of behavioral testing, all rats were euthanized with CO2 and brains were removed and fixed in a 4% formalin buffered saline solution. Once fixed, brains were sliced at 50 μm and mounted on glass slides for placement analysis. The ventral extent of the 99  infusion in each region is displayed in Fig. 10. On the Conflict task, 11 animals completed the experiment with accurate BLA placements (Fig. 10A), 13 with NAcS placements, and another 13 with NAcC placements (Fig. 10B). For the No-Conflict control task, 7 animals were included for each of the NAcS and NAcC (Fig. 10C). Six animals with BLA cannula were tested on the No-Conflict control task. The brains from these animals were sectioned, mounted, stained and initially confirmed to be within the BLA. However, these sections were unfortunately lost prior to plotting them in a figure. Twenty-nine animals were included in the systemic AMPH study, including n = 13 from the NAcS and n = 16 from the NAcC (some animals in the NAcC group that were excluded from microinfusion analysis due to inaccurate cannula placement were included in this analysis as drug was administered systemically).  4.2.7 Data analysis Choice behavior during the Conflict and No-Conflict tasks was analyzed using two-way within-subjects ANOVAs with Treatment (B/M or SAL; AMPH or SAL) and Phase (Safe/Reward 1, Conflict, and Safe/Reward 2) as the two within-subjects factors. For the inactivation experiments, we also conducted a supplementary analysis to compare baseline performance (lever-pressing and pellets received) across the two tasks using a two-way, between/within subjects ANOVA, with the between-subjects factor of Task (Conflict vs. No-Conflict control) and the within-subjects factor Phase (Safe/Reward 1, Conflict, and Safe/Reward 2). In these analyses, baseline data were computed by averaging data obtained on the day prior to each infusion test day (two days total). In addition, due to similarities in the level of responding during the Safe/Reward (VI15/FR5) phases between saline-infused rats trained on the control task, and rats that received BLA or NAcS inactivations on the Conflict task, we also conducted a series of two-way ANOVAs within these two brain regions, with Condition (SAL No-Conflict vs. B/M 100  Conflict) as the between-subjects factor, and Phase (Safe/Reward 1 and 2) as the within-subjects factor. Follow-up or exploratory comparisons were conducted using one-way ANOVAs or paired-samples t-tests, where appropriate. Analysis of supplementary measures (locomotion, pellets received, etc.) were also conducted using one-way ANOVAs or paired-samples t-tests. To examine any potential relationship between foot-shock intensity and Conflict period responding, the Pearson correlation between the mA shock intensity value and lever-presses during the Conflict period was also analyzed. 4.3 Results 4.3.1 Experiment 1: Conflict task 4.3.1.1 BLA inactivation   Under control conditions, rats that were well-trained on the Conflict task (n = 11) apportioned their lever-pressing in an adaptive manner across the three, 5-min phases (Fig. 11A), as they had done during the later phases of training. These animals displayed robust levels of responding during the un-punished, but less frequently reinforced Safe/Reward phases, whereas during the punished Conflict phase, rats showed a dramatic reduction in lever-pressing. BLA inactivation markedly altered this profile (Fig. 11B). Analysis of the lever-pressing data did not reveal a main effect of Treatment (F(1,10)=2.38, not significant (n.s.)), indicating that the total number of responses made during a session did not differ after B/M relative to saline infusions. However, this analysis did produce a significant Treatment x Phase interaction (F(2, 20)=7.59,p<0.05). Subsequent simple-main effects analyses revealed that BLA inactivation produced a dramatic disinhibition of responding during the Conflict phase (F(1,10)=16.42, p<0.05). The degree of disinhibition during Conflict was not significantly correlated with shock intensity (r=-0.07, p>0.8). Yet, this effect was accompanied by a reduction in responding during the first 101  (F(1,10)=6.28,p<0.05), but not second (F(1,10)=2.70, n.s.), Safe/Reward phases. In keeping with this effect on responding, rats obtained more pellets following BLA inactivation (main effect of Treatment: (F(1,10)=16.53, p<0.01), Treatment by Phase interaction: (F(2,20)=15.80, p<0.01) (Table 5). This was driven by an increase in food received during the Conflict phase (F(1,10)=16.36, p<0.005), whereas there was no difference across treatment in terms of pellets obtained during the Safe/Reward phases (both F-values < 1.0, both p-values > 0.40). Locomotor activity did not differ across treatment conditions (t(10)=0.84,p>0.05) (Table 5). Thus, BLA inactivation induced a substantial increase in punished reward-seeking, but simultaneously attenuated responding during unpunished periods during which the effort requirement to obtain these rewards was greater.  4.3.1.2 NAcS inactivation Inactivation of the NAcS (n = 13) produced a profile that was qualitatively similar to that induced by BLA inactivation in some respects (Fig. 11C). Here, data analysis again produced a significant Treatment x Task Phase interaction (F(2,24)=14.01,p<0.001), with this effect driven by an increase in responding during the punished Conflict phase (F(1,12)=7.56, p<0.05), and a reduction in lever-pressing during the first (F(1,12)=14.81,p<0.005), and second (F(1,12)=11.20,p<0.05) Safe/Reward phases. Notably, the disinhibition of responding during the Conflict phase following NAcS inactivation was smaller in magnitude relative to that induced by BLA inactivation. Like BLA inactivation, there was no statistically significant relationship between shock intensity and the degree of disinhibition during Conflict (r=0.16, p>0.6). NAcS inactivation reduced overall lever-pressing measured across the entire session, as revealed by a significant main effect of Treatment (F(1,12)=11.82, p<0.001). Despite this effect, there were no differences in the number of pellets obtained over the session (main effect of Treatment: 102  (F(1,12)=1.62, n.s.), although there was a significant Treatment x Phase interaction (F(2,24)=11.63, p<0.001), meaning that rats received fewer pellets during Safe/Reward phase 1 (F(1,12)=9.46,p<0.01) and 2 (F(1,12)=6.05,p<0.05), and more pellets during the Conflict phase following B/M infusions vs saline (F(1,12)=7.90,p<0.05) (Table 5). NAcS inactivation had no significant impact on locomotion (t(12)=1.44, n.s.) (Table 5). Thus, suppression of neural activity within the NAcS, like BLA, disinhibited Conflict responding and attenuated lever-pressing during the Safe/Reward phases.  4.3.1.3 NAcC inactivation In contrast to the effects of BLA or NAcS inactivation, infusions of B/M into the NAcC (n = 13) produced a substantial decrease in responding across all task phases (main effect of Treatment: (F(2,24)=35.55,p<0.001); Treatment x Task Phase interaction, (F(2,24)=20.45,p<0.001) (Fig. 11D). Simple main-effects analyses confirmed that inactivation reduced responding during Safe/Reward phase 1 (F(1,12)=29.25,p<0.001) and 2 (F(1,12)=29.77,p<0.001), as well as the Conflict phase (F(1,12)=7.23, p<0.05). The reduction in lever-pressing during Conflict in particular was not correlated with shock intensity (r=-0.45,p=.12). Accordingly, animals received fewer rewards over the entire session after NAcC inactivation (main effect of Treatment: (F(1,12)=15.14 ,p<0.001) (Table 5). Inactivation also reduced locomotor activity during the session (t(12)=4.04, p<0.05) (Table 5).  Thus, inactivation of the NAcC resulted in a general suppression of reward-seeking, irrespective of whether responding was punished or not.   4.3.1.4 AMPH administration Previous research suggests that AMPH administration will suppress punished instrumental behavior (Killcross et al., 1997; Lazareno, 1979; Leone et al., 1983). In the present study, AMPH 103  (1mg/kg) was administered systemically to animals following the completion of microinfusion test days (n = 29 total; 13 with NAcS cannula, 16 with NAcC cannula). Here, we did not observe a main effect of Treatment (F(1,28)=0.31,p>0.58), or a significant Treatment x Task Phase interaction (F(2,56)=0.22,p>0.80), although there was a significant effect of Task Phase (F(2,56)=207.61,p<0.001) (Fig. 11E). Due to our a priori prediction that AMPH would decrease punished seeking, we ran a series of exploratory t-tests to analyze whether AMPH produced the hypothesized effect. In fact, although AMPH had no impact on responding during the Safe/Reward phases (both t-values > 0.5, both p-values > 0.65), AMPH significantly decreased punished responding during the Conflict phase (t(28)=2.62, Bonferroni corrected p<0.014). This exploratory analysis provided validation of the experimental protocol, and supported previous findings regarding the role of monoamines in punished reward-seeking behavior. AMPH administration caused rats to receive fewer sugar pellets, as indicated by a significant main effect of Treatment (F(1,28)=6.22,p<0.02) and a significant Treatment x Task Phase interaction (F(2,56)=6.23,p<0.005). Direct comparison suggests that the number of pellets received during the Safe/Reward phases were identical (both F-values < 1, and p-values > 0.5), the number of pellets received during the Conflict phase was decreased by AMPH treatment (F(1,28)=6.88,p<0.02) (Table 5). AMPH treatment did not change the total number of lever-presses made throughout the session (F(1,28)=0.31,p>0.58), but did significantly increase locomotion (t(28)=2.34,p<0.03) (Table 5). These results point to a dissociation between the behaviorally activating impact of AMPH on motoric behavior, and its behaviorally suppressing impact on reward-seeking during punishment.    104  4.3.2 Experiment 2: No-Conflict control task In Experiment 1, inactivation of either the BLA or NAcS (but not NAcC) increased responding during the Conflict period, where lever-presses delivered food on a FR1 schedule but also delivered foot-shocks after 50% of responses. Inactivation of each of these three target nuclei reduced responding during the unpunished Safe/Reward phases, where food was delivered on a leaner, VI15/FR5 schedule. Notably, alterations in responding induced by either BLA or NAcS inactivation did not cause an overall reduction in the amount of reward obtained over the session, with BLA inactivation actually increasing the number of pellets received. This latter observation prompted us to explore whether the reduction in responding during the Safe/Reward periods observed in Experiment 1 was driven by changes in the manner in which animals allocated the relative vigor of responding during the different task phases, or merely by reduced motivation to lever-press for rewards. Thus, separate groups of rats were trained on a No-Conflict control task that used identical schedules of reinforcement as the Conflict task, but no foot-shock punishment was delivered during training (Fig. 12A). 4.3.2.1 Experiment 2: BLA, NAcS and NAcC inactivation In contrast to the profound alterations in response profiles on the Conflict task induced by BLA inactivation, similar treatments did not significantly alter behavior in animals trained on the No-Conflict control task (n = 6) (Fig. 12B). BLA inactivation was without effect on either lever-pressing or pellets received (all F-values<1.73, n.s) or locomotion (t(5)=1.60, n.s.) (Table 5). Similarly, NAcS inactivation (n = 7) did not significantly impact lever-pressing or rewards received (all F-values<2.36, n.s.) (Fig. 12C and Table 5), and also did not affect locomotor activity (t(6)=0.19, n.s.) (Table 5).  105  On the other hand, inactivation of NAcC (n = 7) diminished motivated output during performance of the No-Conflict task (Fig. 12D). Under control conditions, animals in this group displayed noticeably lower rates of responding during the Safe/Reward phases of the task, compared to rats receiving saline infusions into either the BLA or NAcS (Fig. 12B, C, and D). Nevertheless, infusions of B/M into this nucleus reduced responding across all phases of the task (main effect of Treatment: (F(1,6)=9.46,p<0.05); Treatment x Phase interaction (F(1,6)=0.94, n.s.). Accordingly, rats received fewer reward pellets after NAcC inactivation (F(1,6)=24.24,p<0.01) (Table 5). Although NAcC inactivation decreased locomotor activity on the Conflict task (Table 5), the same treatment administered prior to performance on the control task did not significantly affect locomotion (t(6)=1.71, n.s.) (Table 5). Collectively, these results lend support to the idea that NAcC promotes appetitively-motivated responding. In comparison, the lack of effect of BLA or NAcS inactivation on this task implies that alterations in behavior on the Conflict task induced by these treatments are unlikely to be attributed to changes in arousal or motivation for food reward.   4.3.2.2 Experiment 2: Baseline analysis and cross-task comparison during Safe/Reward responding Inactivation of BLA and NAcS differentially affected responding during the V115/FR5 reward phases for rats trained on the Conflict versus No-Conflict tasks. Inactivation of either structure during the Conflict task reduced responding during the Safe/Reward periods, whereas, for rats trained on the No-Conflict task, these same manipulations did not affect performance during these phases. A closer inspection of the data obtained from the two experiments revealed that, under control conditions, rats trained on the two tasks appeared to show different rates of responding during these reward phases (e.g., compare the Safe/Reward panels in Figure 11B & C 106  with those of Figure 12B & C). We also noticed that, by the end of training on the No-Conflict task, baseline rates of responding (collapsed across all three regions of interest) displayed by animals trained on the control task (n = 21) differed considerably from those trained on the Conflict task (n = 37) across all three task phases. To investigate this further, we compared the number of lever-presses made on the days prior to each infusion treatment (baseline) by all rats trained on the two tasks (Fig. 13A), using a two-way between/within subjects ANOVA with Task and Phase as between and within-subjects factors, respectively. Comparison of the lever-pressing data yielded a significant Task x Phase interaction (F(2,112)=59.69, p<0.001). Predictably, rats trained on the control task made many more responses during the middle, FR1 phase (F(1,56)=713.50, p<0.001) compared to those trained on the Conflict task, where lever-presses delivered both food and foot-shocks (Fig. 13B, middle panel). However, rats trained on the No-Conflict task made fewer responses during the first (F(1,56)=22.53,p<0.001) and third (F(1,56)=35.44,p<0.001) Safe/Reward phases relative to those trained on the Conflict version (Fig. 13B, left and right panels). As a consequence, rats trained on the No-Conflict task obtained fewer pellets during the first (F(1,56)=25.37,p<0.001) and second (F(1,56)=26.89, p<0.001) Safe/Reward phases compared to those in the Conflict condition (full Task x Phase interaction (F(2,112)=891.17, p<0.001) (Fig. 13C). Notably, this pattern of results was still observed when only data from a subset of rats trained on the Conflict task for a comparable number of days (< 13) to those trained on the control task (n = 14) were included, thereby equating the relative amount of training received by rats in both groups. Here, we again observed significant Task x Phase interactions for the number of lever-presses (F(2,66)=47.71,p<0.001) and pellets received per phase (F(2,66)=418.712, p<0.001; data not shown). This further suggests that any alteration in baseline behavior displayed by rats trained on the two tasks was driven by their experience 107  with punishment, and not a difference in the amount of instrumental training. Thus, rats trained on the Conflict task appeared to maximize their rates of responding during the unpunished Safe/Reward phases of the task, presumably to accommodate for their suppression of responding during the Conflict period. In comparison, during the No-Conflict control task, where reward was available on identical schedules but in the absence of punishment, rats varied their relative rates of responding over the session in a different manner. Here, they were more lackadaisical during the Safe/Reward phases where the effort requirements were higher, and instead responded more vigorously and obtained more food during the middle, FR1 phase.   We also observed that the lower levels of responding during the Safe/Reward phases by rats trained on the No-Conflict task were comparable to the rates of responding during the same two phases of the Conflict task following inactivation of either the BLA or NAcS. In light of this, we formally compared response rates during the two Safe/Reward phases for saline-infused rats on the No-Conflict task, and animals that received inactivation of BLA or NAcS on the Conflict task, using two separate two-way, between/within subjects ANOVA, with Condition (SAL No-Conflict vs. B/M Conflict) as the between-subjects factor, and Safe/Reward Phase as the within-subjects factor. Within the BLA, there was no main effect of Condition (F(1,15)=0.14, n.s.) or Condition x Phase interaction (F(1,15)=2.99, n.s.) (Fig. 13D, left panel). Similarly, within the NAcS, there was no impact of Condition (F(1,18)=0.16, n.s.), nor a Condition x Phase interaction (F(1,18)=1.38, n.s.) (Fig. 13D, right panel). Thus, for rats trained on the Conflict task, inactivation of either the BLA or NAcS attenuated responding during the unpunished period such that rats behaved in a manner similar to those that received saline infusions into the same brain region, and had never experienced punishment during training. 108  4.4 Discussion 4.4.1 Summary The present findings reveal complementary roles for the BLA and NAcS in mediating responding in situations involving motivational conflict where actions may yield both reward and potential punishment. Neural activity in these two nuclei facilitated response-suppression when lever-presses yielded both food and shock, while in the same context, these regions invigorated responding when the effort requirements to obtain unpunished rewards were greater. These effects on the Conflict task did not appear to be related to shifts in the reinforcement schedule from Safe/Reward (VI15/FR5) to Conflict (FR1) periods, as performance on a control task using the same schedules of reinforcement in the absence of punishment was unaffected by inactivation of these regions. In contrast, the NAcC appears to more generally promote appetitive motivation, irrespective of motivational conflict, as inactivation of this region diminished seeking behavior, including locomotion, across both task conditions. These data suggest that the NAcS, but not NAcC, mediates aversion-mediated response-inhibition in an instrumental setting, similar to their respective roles in Pavlovian conditioned suppression and behavioral activation, as revealed in the previous chapters.  4.4.2 Cooperative roles for the BLA and NAcS in modulating punished reward-seeking The finding that inactivation of the BLA disinhibited responding during the Conflict period is in keeping with a vast literature implicating this nucleus in influencing behavioral responses to aversive or threatening situations (Adolphs, 2013; Fanselow & LeDoux, 1999). Classically, BLA lesions eliminate conditioned fear responses in both humans and rodents (Adolphs et al., 1995; Erlich, Bush, & Ledoux, 2012; LaBar et al., 1998; LeDoux et al., 1990). Recent work suggests that these lesions also shift preference away from smaller, unpunished rewards and towards 109  larger rewards that may also be punished, a finding that is complemented by the results of the present study (Orsini, Trotta, Bizon, & Setlow, 2015). These studies suggest that BLA activity is necessary to appropriately recall and utilize the memory of an aversive event, and subsequently modify behavior. Jean-Richard-Dit-Bressel and McNally (2015) illustrated that the BLA processes aversive consequences in the context of instrumental punishment, independent of its role in Pavlovian fear learning. In their study, inactivation of BLA disrupted the suppression of punished responding, causing rats to approach and engage the lever more often and with faster latencies compared to control animals, similar to what was observed in the present study. Interestingly, this effect on punished responding was subject to a pronounced rostral-caudal dissociation, whereby the caudal (posterior to AP: -2.6), but not rostral (anterior to AP: -2.6), BLA appeared to play a more prominent role in suppressing behavior under these conditions. Although our sample size precluded the examination of whether punishment-resistance was differentially affected by inactivation across the rostral-caudal extent of the BLA, it is notable that our targeted BLA coordinate fell within the caudal range (AP: -2.7) used by Jean-Richard-Dit-Bressel and McNally (2015). Taken together, these findings suggest that the BLA, particularly its caudal aspect, contributes to the modification of responding in situations where actions are either directly punished or no longer rewarded. One neuroanatomical feature that distinguishes the caudal BLA from the more rostral portion is that it sends a relatively dense projection to the medial NAcS (Brog et al., 1993; Groenewegen et al., 1999; Kita & Kitai, 1990; Shinonaga et al., 1994). As such, BLA may confer the appropriate inhibition of reward-seeking in the face of potential punishment in part through its projection to the NAcS. This supposition is supported by the present findings, whereby NAcS inactivation produced an increase in punished responding similar to that 110  produced by BLA inactivation. These findings complement a burgeoning literature implicating the NAcS in avoiding potential aversive consequences, with these functions mediated through direct interactions with the BLA (Fernando et al., 2013; Ramirez et al., 2015). The disinhibition of punished responding following BLA or NAcS inactivation is also coherent with data suggesting that the NAcS, via interactions with BLA, promotes the appropriate attenuation of responding in appetitively-motivated situations. One such construct is the extinction of a conditioned association, which produces a new inhibitory memory that acts to suppress the now irrelevant response. Previous work suggests that inactivation of NAcS can disinhibit responding during the reinstatement of extinguished food, alcohol, or cocaine seeking (Floresco et al., 2008; Millan et al., 2010; Peters et al., 2008). Inactivation of NAcS in these and similar situations also typically releases non-rewarded behaviors from inhibition, with rats producing more operant responses in situations that are never reinforced following inactivation (Ambroggi et al., 2011; Blaiss & Janak, 2009; Floresco et al., 2008). Interestingly, caudal (but not rostral) BLA inactivation produces the same type of exaggerated reinstatement response following extinction of food-seeking (McLaughlin & Floresco, 2007). A similarity in function of the NAcS and caudal BLA has also been demonstrated by Millan and colleagues (2015), who showed that inactivation of either structure disinhibits reward-seeking during periods of reward unavailability. A BLA to NAcS pathway has also been shown to directly mediate the inhibition of alcohol seeking through extinction, as contralateral disconnection of these two structures disinhibits extinguished seeking behavior (Millan & McNally, 2011). Here we extend these observations, illustrating that neural activity within both the BLA and NAcS are crucial for inhibiting reward-seeking that may also yield aversive consequences.  111  It is notable that the effect of NAcS inactivation on responding during the Conflict period was comparatively smaller to that induced by BLA inactivation, suggesting that other, parallel output pathways from the BLA (e.g., the central amygdala or mPFC) may also contribute to behavioral suppression in situations involving punishment. On the other hand, the NAc has been suggested to act as a “limbic-motor interface” (Mogenson et al., 1980), integrating input from cortico-limbic afferents to allow for appropriate action selection (Calhoon & O’Donnell, 2013b; Gruber et al., 2009; O’Donnell & Grace, 1995), suggesting that other inputs to this nucleus may also refine behavior in these situations. For example, the NAcS receives efferent input from mPFC (Berendse, Galis-de Graaf, et al., 1992; Brog et al., 1993; Heilbronner et al., 2016), a region that additionally contributes to punishment-induced suppression of behavior. Previous studies have utilized lesions, dopamine antagonism, or optogenetic silencing of the mPFC to cause persistent instrumental responding for reward despite the potential for punishment (Broersen et al., 1995; Chen et al., 2013; Resstel et al., 2008). Similarly, in situations where an aversively conditioned Pavlovian stimulus is presented during reward-seeking, inactivation of this same region disrupts the typically observed conditioned suppression of lever-pressing, even when punishment is omitted (Limpens et al., 2015). In Chapter 3, we illustrated that a PL mPFC to NAcS projection mediates the expression of Pavlovian conditioned suppression, a process that is likely related to instrumental suppression. A recent study provided additional support for this hypothesis, illustrating that a mPFC neurons projecting to the lateral NAcS are active during, and partially responsible for, the suppression of reward-seeking during potentially punished task phases (Kim et al., 2017). Given this framework, it is plausible that the BLA and mPFC may provide affective and contextual information to the NAcS, which activates neuronal ensembles to execute motor programs that inhibit punished responding. Alternatively, the mPFC may provide 112  top-down control over the NAcS and/or BLA (St Onge et al., 2012), aiding in the refinement of action selection when reward-seeking may result in aversive consequences.  Still, there exists debate on the precise role of mPFC in punishment, as excitotoxic lesions of mPFC prior to cocaine seeking have been shown to not affect punished seeking (Pelloux et al., 2013). In that study, permanent lesions of the mPFC were induced prior to self-administration training, which may have allowed for some degree of long-term compensation from other brain regions. However, a more recent study by Jean-Richard-Dit-Bressel & McNally (2016) used reversible inactivations to illustrate that lateral, but not medial PFC contributes to punishment-induced suppression. Specifically, inactivation of the lateral orbitofrontal cortex (OFC) disinhibited punished lever-pressing, while inactivation of rostral agranular insular cortex (RAIC) spared punished seeking, but increased the choice of the previously punished lever in a shock-free choice test. These lateral frontal regions have been implicated in numerous functions that may be relevant to punishment, including value encoding, interoception, and response-inhibition in animals and humans (Bari & Robbins, 2013; Bechara, Damasio, & Damasio, 2000; Bryden & Roesch, 2015; Clark et al., 2008; Craig, 2009; Morein-Zamir & Robbins, 2015), and may contribute to punishment through their direct, sometimes reciprocal projections with the extended amygdala and NAc (Heilbronner et al., 2016; Reynolds & Zahm, 2005; Shinonaga et al., 1994). Future studies employing pharmacological or pharmacogenetic disconnection of these structures may help determine the directionality of communication between these proposed circuits underlying the punishment-induced inhibition of behavior.  There are a number of potential alternative explanations for the observed disinhibition of punished responding following BLA and NAcS inactivation. For example, disinhibition during Conflict may reflect a simple decrease in the expression of Pavlovian fear. Upon commencement 113  of the Conflict period, internal (e.g., timing) and external (e.g., cue light illumination) cues may act to inhibit responding via the production of conditioned fear behaviors such as freezing. However, we find this explanation unlikely for several reasons. Although we did not measure conditioned fear in the present study, performance on a similar task was found to be uncontaminated by conditioned fear, as freezing levels steadily declined across 5 days of training, and fear-related freezing was unaffected by BLA inactivation (Jean-Richard-Dit-Bressel & McNally, 2015). Given that training on our task was substantially longer (10-20 days), it is likely that any conditioned fear produced by the Conflict period was eliminated over the course of training. We also did not observe any change in locomotor activity following inactivation of the BLA or NAcS, which may have been expected had we affected the expression of long epochs of behavioral arrest. Additionally, freezing in response to shock-associated cues is not dependent on the integrity of the NAcS (Haralambous & Westbrook, 1999; Thomas, Hall, & Everitt, 2002).  It is possible that indices of conditioned fear which incorporate a reward-seeking component, such as conditioned suppression, may differentially depend on accumbens subregions, as illustrated in Chapter 3. It is also unlikely that inactivations affected the unconditioned response to foot-shock, as lesions of the NAc or BLA do not generally alter foot-shock-induced changes in locomotion or lever-pressing (Levita et al., 2002; McDannald & Galarce, 2011; Schwienbacher et al., 2004). Finally, the disinhibition of pressing observed following BLA or NAcS inactivation may have resulted from rats simply being hungrier, or otherwise more motivated to seek food. Muscimol infusion into the NAcS has been shown to produce orexigenic behavior in rats, but only when food is freely available (Hanlon et al., 2004; Stratford & Kelley, 1997). However, infusion of muscimol into the NAcS (at doses similar to those used here) does not impact instrumental responding for food delivered on a progressive ratio schedule (Zhang et al., 2003). 114  Additional evidence against a simple, hunger-based explanation comes from the finding that BLA and NAcS inactivation actually decreased responding during the Safe/Reward periods of the Conflict task, and did not alter responding during any phase of the No-Conflict control task. Given these considerations, we find it unlikely that the increase in punished responding for food induced by BLA or NAcS inactivation was attributable to alterations in Pavlovian fear mechanisms, foot-shock sensitivity, or enhanced motivation to obtain food. Rather, the present findings suggest that these nuclei work in a cooperative manner to reorganize behavior and suppress ongoing reward-seeking when these actions may also yield aversive outcomes.  4.4.3 Differential effects of BLA and NAcS inactivation on unpunished reward-seeking. In addition to increasing punished reward-seeking, inactivation of the BLA and NAcS reduced lever-pressing during the Safe/Reward phases of the Conflict task, when food was delivered on a leaner, VI15/FR5 schedule. Yet, in a separate No-Conflict control experiment, inactivation of these nuclei did not affect responding during the Safe/Reward or FR1 epochs, where rats pressed for food on identical, shifting schedules of reinforcement, but did not experience foot-shocks at any point during training. This lack of effect suggests that the reduction in responding during the Safe/Reward phases of the Conflict task induced by BLA and NAcS inactivation may stem in part from the history of punishment in this context differentially recruiting these regions during appetitive behavior.   In an attempt to clarify the seemingly discrepant effects of BLA/NAcS inactivation on reward-seeking, baseline responses of rats trained on the two tasks were analyzed, revealing noticeable differences in how these groups allocated their relative response rates across the task epochs. Those trained on the Conflict task displayed higher rates of responding during the Safe/Reward phases compared to animals trained on the control task, presumably as a 115  compensatory measure for their reduced responding during the punished Conflict period. Conversely, rats performing the No-Conflict task obtained considerably more food during the middle, FR1 phase than those on the Conflict task, which may explain why their response rates were lower during the Safe/Reward phases. This pattern of results suggests that a history of punished reward-seeking alters the manner in which animals adjust their response rates to changes in schedules of positive and negative reinforcement. Inactivation of the BLA or NAcS prior to the Conflict task altered response profiles, so that behavior over the session resembled that of animals performing the control task that never experienced punishment. Therefore, the impact of BLA or NAcS inactivation on responding during the Conflict task may not simply reflect the involvement of these regions in the suppression of punished responding and/or invigoration of responding when the effort requirements are high. Rather, neural activity within these nuclei may mediate a broader perception of the task context that enables appropriate adjustments in behavioral output to reduce the occurrence of aversive consequences, while at the same time attempting to maximize the amount of reward that may be obtained. 4.4.4 NAcC inactivation and motivated responding - comparisons with NAcS. In contrast to the differential effects on responding induced by BLA or NAcS inactivation, similar infusion of GABA agonists into the NAcC diminished reward-seeking across all phases of both tasks, concomitant with a decrease in locomotion and other indices of motivated output. These observations are perhaps unsurprising, as the NAcC has been shown to be necessary for motivated behavior and flexible approach during appetitive reward-seeking across a variety of experimental paradigms (Ambroggi et al., 2011; Di Ciano et al., 2008; Ghods-Sharifi & Floresco, 2010; Ishikawa et al., 2008, 2010; Nicola, 2010; Parkinson, Willoughby, et al., 2000; Stopper & Floresco, 2011). For example, behavioral responding to a discriminative incentive cue 116  which predicts reward availability is dependent on the NAcC and its cortico-limbic afferents (Ambroggi et al., 2008; Ishikawa et al., 2008, 2010). Relatedly, flexible approach behavior is governed by dopaminergic activity in the NAcC (McGinty et al., 2013; Nicola, 2010). Blockade of dopamine receptors in the NAcC decreases the likelihood that rats trained on a cued FR8 schedule would respond for reward, as a function of spending more time off task. Dopamine activity in the NAcC also appears critical for the ability of a cue to act as an incentive stimulus, becoming imbued with the motivational properties of the reinforcer itself (Saunders & Robinson, 2012). Diminished locomotor activity may also contribute to the decrease in lever-pressing observed following NAcC inactivation. However, locomotor activity was only significantly decreased on the Conflict task, and not the No-Conflict control task, yet lever-pressing was substantially decreased by NAcC inactivation on both tasks. This suggests that the impact of NAcC inactivation on locomotion and lever-press behavior are partially dissociable based on task history, and thus may be mediated by potentially separable mechanisms. Finally, an intact NAcC has been shown to be necessary for appropriate effort expenditure during appetitive behavior, as dopamine blockade (Nunes, Randall, Podurgiel, Correa, & Salamone, 2013; Salamone, Correa, Farrar, & Mingote, 2007) or inactivation (Ghods-Sharifi & Floresco, 2010) of this region decreases the amount of physical effort animals are willing to expend to receive a larger reward. Therefore, the lower degree of task engagement observed following NAcC inactivation in the present study likely reflects a decrease in willingness to exert effort to obtain rewards and/or the impact of incentive stimuli on behavior.    Of particular interest is the dramatic contrast between the effects of NAcC versus NAcS inactivation on these tasks. As described previously, the NAcS appears to be critical for inhibiting punished responding, consistent with a broad literature implicating this region of the 117  NAc in facilitating optimal foraging behavior by inhibiting task-irrelevant behaviors (Ambroggi et al., 2011; Floresco et al., 2008; Ishikawa et al., 2010). In contrast, the NAcC plays a key role in promoting approach behavior towards motivationally relevant stimuli. Inactivation of NAcC profoundly decreased reward-seeking during both tasks, while NAcS inactivation disinhibited punished seeking behavior, and only affected safe reward-seeking in rats that had a history of punishment during training. Furthermore, inactivation of the NAcC tended to cause hypolocomotion and reduced the number of rewards received by rats on both tasks, while neither effect was observed following NAcS inactivation. These findings complement a growing literature that suggests that the NAcC and NAcS may play somewhat opposing, yet complementary, roles in enabling an organism to obtain its goals (Floresco, 2015). Both nuclei, via input from their upstream corticolimbic afferents, act in concert to optimize goal directed behavior, although they appear to do so in distinct manners.   4.4.5 AMPH tends to promote punishment-sensitivity during conflict Previous research has suggested that pharmacological enhancement of monoamine release increases punishment susceptibility, biasing behavior away from an instrumental response that is concurrently rewarded and punished (Broersen et al., 1995; Killcross et al., 1997; Lazareno, 1979; Leone et al., 1983). Here, results of an exploratory analysis provided support for this account, with AMPH decreasing reward-seeking specifically during the Conflict phase. AMPH is a potent releaser of the monoamines, including dopamine and serotonin, both of which have been implicated in response-inhibition (Crockett, Clark, & Robbins, 2009; Killcross et al., 1997; Pascoli et al., 2015; Simon et al., 2011). For example, AMPH alters performance on a task where foot-shock is probabilistically associated with one instrumental response that delivers are large amount of reward, but not another that delivers a small reward (Mitchell, Vokes, Blankenship, 118  Simon, & Setlow, 2011; Orsini, Trotta, et al., 2015; Simon et al., 2011; Simon, Gilbert, Mayse, Bizon, & Setlow, 2009). On this task, AMPH induces a risk-averse phenotype, biasing choice away from the instrumental response that probabilistically delivers large reward and punishment, and towards the instrumental option that is safe, but objectively less rewarding. This potentiation of risk aversion induced by AMPH has been shown to be mediated in part by dopamine, as blockade of the D2 receptor reduces the impact of AMPH on choice (Simon et al., 2011). Although the present task did not allow animals to choose between multiple options during the Conflict period, animals were more likely to withhold responding during this phase, as a function of AMPH treatment.   The risk-aversion induced by AMPH in studies using foot-shock punishment contrasts with other studies that operationalize risk and punishment as reward omission, during which AMPH promotes risk-seeking (St. Onge & Floresco, 2009). This discrepancy has been suggested to relate to the ability of AMPH to enhance the salience of relevant task events (Orsini, Moorman, Young, Setlow, & Floresco, 2015). On tasks employing foot-shock punishment, the delivery of this aversive stimulus is more salient than is the difference in reward magnitude, and thus AMPH induces risk-aversion. On tasks where reward omission serves as punishment, the receipt of a large reward may be the more salient factor, such that AMPH biases choice towards instrumental actions that may result in that reward, which will manifest as risk aversion. In support of this, other tasks with arguably more salient omission periods, such as the rodent gambling task, show the inverse effect of AMPH, with animals becoming more sensitive to reward omission punishment following treatment (Zeeb, Robbins, & Winstanley, 2009). The results of the present study are broadly consistent with this dissociation, as punishment is clearly more salient than the relatively richer schedule of reinforcement during the Conflict period, as 119  evidenced by the large disparity in lever-presses made during the Safe/Reward phases versus the Conflict phase. Under these conditions, AMPH would be expected to produce risk aversion, which was broadly confirmed here.  4.4.6 Relevance for psychiatric disorders The findings that the BLA and NAcS both contribute to suppressing punished reward-seeking may provide insight into how dysfunction of these circuits contributes to the compulsive behaviors observed in a variety of psychiatric disorders. Compulsivity in the face of punishment is a hallmark of drug addiction and obsessive-compulsive disorder (OCD) (Figee et al., 2016; Morein-Zamir & Robbins, 2015). Structures that promote punishment-induced behavioral suppression, such as mPFC and BLA, project directly to NAc and are central to the pathology of both disorders (Wood & Ahmari, 2015). mPFC hypoactivity contributes to deficient top-down inhibition of drug seeking in rodents (Chen et al., 2013; Limpens et al., 2015), and is correlated with inhibitory control deficits in cocaine users (Morein-Zamir, Simon Jones, Bullmore, Robbins, & Ersche, 2013). Furthermore, abstinence from cocaine use is related to improvements in prefrontal cortical function, suggesting that the successful cessation of drug use is either predicated on or causally related to normalized cortical activity (Connolly, Foxe, Nierenberg, Shpaner, & Garavan, 2012). Self-administration of most addictive substances induces dysregulation of the dopaminergic projections to the NAc, combined with altered NAc plasticity (Britt & Bonci, 2013; Grueter, Rothwell, & Malenka, 2012; Russo et al., 2010). Prolonged drug exposure can downregulate dopamine D2 receptor levels in the ventral striatum, of which the nucleus accumbens is a large part, which is thought to produce impulsive behavior (Lee et al., 2009; Volkow, Fowler, Wang, Baler, & Telang, 2009). In OCD, the ventral striatum receives abnormally-elevated afferent input from the orbitofrontal cortex (Abe et al., 2015). Evidence 120  from preclinical models suggests that activity in this pathway may underlie the compulsions observed in individuals with OCD (Figee et al., 2016; Wood & Ahmari, 2015). Although the meso-cortico-limbic-striatal circuit overlap between the two disorders is apparent, more work is required to determine the direction of change and relation to punishment-induced inhibition of responding. Additional exploration of different nodes within meso-cortico-limbic-striatal circuitry that contribute to these aspects of behavior may allow for a better understanding of underlying neuropathophysiology of these disease states. To this end, the present results suggest that abnormal functioning of the BLA and NAcS may be a contributing factor to compulsive behaviors associated with these conditions.  4.5 Conclusion These findings point to complementary roles for the BLA and NAcS in suppressing appetitively-motivated behaviors in the face of punishment. This form of response-suppression mechanism is adaptive, with survival often predicated on weighing potential benefits against punishments when seeking food, or other primary rewards. In addition, all of the regions investigated here played an important role during safe reward-seeking, although NAcS and BLA were selectively recruited following a history of punishment, as performance was spared on a punishment-free control task. We also observed that promoting monoamine release, via systemic treatment with AMPH, resulted in less reward-seeking during punishment, consistent with previous findings. Overall, these results may be relevant for neuropsychiatric disorders where compulsive behavior is resistant to punishment, including substance abuse and obsessive-compulsive disorder (Everitt, 2014; Figee et al., 2016; Morein-Zamir & Robbins, 2015; Wood & Ahmari, 2015). They also provide novel insights into a subregion-specific bivalent function of the rodent NAc, and suggest a possible circuit basis for this divergent effect. In summary, our work suggests that BLA and the 121  NAcS are recruited during punishment-induced inhibition of behavior, while the NAcC is recruited to actively promote seeking behavior, irrespective of punishment.                    122  Table 5. Mean (±SEM) values for ancillary measures on the Conflict or No-Conflict task. The number pellets received in total, partitioned across the three phases of the Conflict and No-Conflict control tasks, and locomotor counts following SAL or B/M infusion into the BLA, NAcS, and NAcC, and systemic AMPH or saline treatment *: p < 0.05 vs. SAL  Total Pellets Safe/Reward 1 Conflict/FR1 Safe/Reward 2 Locomotion (photobeam breaks) Conflict       BLA            SAL 44.0 (±3.7) 16.0 (±1.0) 16.4 (±2.8) 11.6 (±1.2) 478 (±56)    B/M 87.1* (±10.5) 15.1 (±0.8) 60.9 (±10.4)* 11.1 (±0.9) 384 (±44)  NAcS          SAL 42.3 (±3.2) 15.9 (±0.6) 10.5 (±3.1) 15.9 (±1.2) 791 (±79)     B/M 51.2 (±9.0) 12.2 (±0.9) 27.6 (±8.4) 11.3 (±1.3) 698 (±80)  NAcC          SAL 54.9 (±5.9) 15.5 (±1.0) 25.5 (±4.5) 13.9 (±1.3) 885 (±72)     B/M 28.8 (±4.7)* 10.4 (±1.5) 11.6 (±2.3) 6.8 (±1.5) 568 (±80)* AMPH         SAL 51.4 (±7.0) 15.4 (±0.9) 21.2 (±7.1) 14.8 (±1.0) 929 (±61)    AMPH 40.7 (±8.8)* 15.5 (±1.1) 11.0 (±9.0)* 14.2 (±1.2) 1026 (±114)*       No Conflict       BLA            SAL 124.2 (±5.3) 13.2 (±1.1) 101.7 (±4.7) 9.3 (±1.8) 478 (±56)    B/M 121.3 (±6.2) 15.5 (±2.4) 95.8 (±4.0) 10.0 (±0.8) 384 (±44)  NAcS          SAL 127.9 (±4.3) 14.7 (±1.3) 102.7 (±2.4) 10.4 (±1.7) 688 (±34)     B/M 115.1 (±8.5) 10.4 (±0.9) 93.7 (±7.8) 11.0 (±1.4) 686 (±84)  NAcC          SAL 106.4 (±9.9) 9.9 (±0.8) 88.4 (±10.2) 8.1 (±1.8) 605 (±114)     B/M 57.6 (±12.5)* 6.0 (±1.4)* 48.4 (±10.8)* 3.1 (±1.0)* 431 (±94)   123   Figure 10. Histology schematic for Conflict and No-Conflict task animals Histology schematic for Conflict task animals with cannula located in the BLA (A), or NAc subregions (B), as well as on the No-Conflict control task (C). All symbols indicate the most ventral point of infusion in the BLA (A; black squares) or NAc (B, C; black triangles = NAcS placement, grey circles = NAcC placement). Numbers to the left of each representative atlas section indicate distance (mm) from bregma.   124   Figure 11. Task diagram and data from pharmacological manipulation on the Conflict task (A) Flow-chart of a daily Conflict task session indicating the schedules of food reinforcement and punishment. (B, C, D, E) The left, center, and right graphs represent the first Safe/Reward period, the Conflict period, and the second Safe/Reward period, respectively. Data are presented as mean ± SEM. (B) BLA inactivation with baclofen/muscimol (B/M) decreased output during Safe periods (left, right), but dramatically disinhibited punished responding during Conflict (center), relative to saline (SAL) control treatments. (C) Similarly, NAcS inactivation reduced lever-pressing during both Safe/Reward periods (left, right), and disinhibited pressing during the Conflict period (center). (D) NAcC inactivation diminished motivated output, regardless of task phase. (E) Exploratory analysis revealed that AMPH tended to promote response-inhibition during the Conflict phase, without affecting performance during the Safe/Reward phases. Closed star denotes p < 0.05 between SAL and B/M or SAL and AMPH treatment during a particular task phase.  125   Figure 12. Task diagram and data from inactivations on the No-Conflict task  (A) Flow-chart of a daily No-Conflict control task session. Note that this task differs from the Conflict task only in the fact that no punishment was ever delivered during the middle epoch where food was delivered on an FR1 schedule of reinforcement. (B, C, D) The left, center, and right graphs represent the first Safe/Reward period, the “Conflict” period, and the second Safe/Reward period, respectively. Neither BLA (B) nor NAcS (C) inactivation (B/M) had any significant effect on performance. (D) NAcC inactivation decreased reward-seeking across all phases of the No-Conflict control task, similar to the effect of inactivation during the Conflict task. Note the difference in scaling during the FR1 period (middle panels) compared to that used for the conflict period data displayed in Figure 2. Closed star denotes p<0.05 between SAL and B/M infusion during a particular task phase. 126   Figure 13. Baseline analysis suggests NAcS and BLA promote reward seeking as a function of task history. (A) Combined task diagram for the Conflict and No-Conflict control task. Both tasks had identical Safe/Reward periods (left and right columns), but different during the “Conflict” period (middle column). (B, C) The left, center, and right graphs represent the first Safe/Reward period, the “Conflict” period, and the second Safe/Reward period, respectively. (B) Rats trained on the Conflict task pressed maximally during the Safe/Reward periods, and markedly suppressed their pressing during the Conflict period, whereas rats trained on the No-Conflict control task displayed the inverse. (C) The same pattern of results was found when examining pellets received. (D) Direct comparison of lever-press behavior during the Safe/Reward phases. The left graph displays the performance of rats that received intra-BLA B/M during the Conflict task to those that received intra-BLA SAL during the No-Conflict Control task. The right graph displays the performance of rats that received intra-NAcS B/M during the Conflict task to those that 127  received intra-NAcS SAL during the No-Conflict Control task. There was no difference in the number of presses made during the Safe/Reward phases between rats that received SAL during the No-Conflict task and inactivation during the Conflict task for either BLA (left graph) or NAcS (right graph), suggesting that inactivation of BLA or NAcS may induce a behavioral state similar to saline-infused rats trained on the No-Conflict task that never encountered foot-shock during training. Closed star denotes p < 0.05 between task conditions during a particular task phase. n.s. = not significant.                      128  Chapter 5: Dissociable contributions of NAc core and shell during active/passive avoidance 5.1 Introduction When faced with a potential threat, animals may employ one of two main types of defensive behaviors: defensive reactions and defensive actions (LeDoux, 2012; Moscarello & Ledoux, 2014). Defensive reactions are designed to evade predator detection and, in rodents, include forms of behavioral suppression such as freezing. These reactions can facilitate the passive avoidance of dangerous or threatening stimuli. Conversely, defensive actions are typically instrumental behaviors which enable the organism to actively avoid or escape threat. Both active and passive avoidance responses serve adaptive functions, with their flexible application, conducted in accordance with environmental contingencies, being critical to survival.  This dichotomy of active versus passive defensive strategies may be viewed analogously to processes that govern appetitive behavior. For example, Go/No-Go conditioning generally requires an active approach response to receive reward in the presence of one cue (a “Go” response), while another cue signals that suppressing approach (a “No-Go” response) results in reward delivery. It is well-established that different aspects of appetitively-motivated behavior are predicated on activity in meso-cortico-limbic-striatal circuitry. A particularly crucial node in this network is the nucleus accumbens (NAc), which integrates diverse limbic, cognitive, and neuromodulatory input to promote flexible action selection (Calhoon & O’Donnell, 2013b; Floresco, 2015; Gruber et al., 2009; Gruber & O’Donnell, 2009; Mogenson et al., 1980). The NAc has been further partitioned into lateral core (NAcC) and medial shell (NAcS) regions, based on neuroanatomical and functional differences (for review, see Heimer et al, 1997; Zahm and Brog, 1992), with these regions often playing dissociable, yet complementary, roles in guiding motivated behavior.  129  The NAcC has been proposed to promote active approach behaviors, while the NAcS may fulfill a dual role, inhibiting inappropriate responses while also aiding in the production of active behaviors (Ambroggi et al., 2011; Blaiss & Janak, 2009; Floresco, 2015; Floresco et al., 2008; Ghazizadeh et al., 2012; Ghods-Sharifi & Floresco, 2010; Piantadosi et al., 2017). For example, neurophysiological studies have shown that neurons in both the NAcC and NAcS encode a discriminative stimulus that signals reward availability, yet a higher proportion of neurons in the NAcS (as compared to NAcC) also encode a neutral stimulus that signals reward unavailability (Ambroggi et al., 2011). Inactivation of the NAcC preferentially affects behavior elicited by reward-predictive stimuli, while inactivation of NAcS unmasks irrelevant behaviors such as lever-pressing and Pavlovian approach during presentation of non-rewarded stimuli and intertrial intervals (Ambroggi et al., 2011; Blaiss & Janak, 2009). The NAcS (but not NAcC) has also been suggested to actively inhibit extinguished and non-reinforced instrumental behavior during the reinstatement of food (Floresco et al., 2008), alcohol (Millan et al., 2010), or cocaine seeking (Peters et al., 2008). Consideration of these data implies that these two nuclei facilitate reward-seeking in partially distinct ways, with the NAcS enforcing response-inhibition to focus and constrain behavioral output, and the NAcC promoting approach towards relevant stimuli.  Although the NAc is typically viewed as a “reward” nucleus, it is important to note that neurons within this region are also responsive to aversive stimuli and the cues that predict them (Delgado, Li, et al., 2008; Jensen et al., 2003; Roitman, Wheeler, & Carelli, 2005; Schoenbaum & Setlow, 2003; Setlow, Schoenbaum, & Gallagher, 2003). For example, on a mixed valence Go/No-Go task, largely separate populations of NAc neurons develop phasic responses to cues that predict appetitive or aversive outcomes (Setlow et al., 2003). These responses may facilitate behavioral flexibility in both appetitive and aversive contexts, allowing for active responses to be 130  elicited to obtain rewards, while also enabling the response-suppression necessary to avoid punishment. Interestingly, data from a similar Go/No-Go task suggests that NAc neurons track the behavioral response necessitated by a Go or No-Go cue, in keeping with a role for this nucleus in action selection (Roitman & Loriaux, 2014). Consistent with this idea, we have recently shown that subregions of the NAc are differentially responsible for the promotion and inhibition of reward-seeking during punishment (Chapter 4). Specifically, inactivation of the NAcS disinhibited punished reward-seeking, whereas similar inactivation of the NAcC induced a general suppression of instrumental responding for reward (Chapter 4; Piantadosi et al., 2017). Similarly, the NAcS disinhibited reward-seeking despite the presentation of an aversive Pavlovian conditioned stimulus, while NAcC simply promoted behavioral activation (Chapter 2 & 3). However, it remains unclear whether these two NAc subregions perform dissociable functions during action-selection motivated exclusively by cues that predict aversive outcomes. Of particular interest would be whether the NAcS and NAcC are differentially responsible for the performance of defensive reactions versus actions in response to discrete cues. Previous work has separately examined the contribution of the NAc to these two types of defensive behaviors. With respect to defensive actions, the NAc and its dopaminergic input are integral for the learning and expression of “Go”-like actions such as active avoidance (Fernando et al., 2013; Gentry et al., 2016; Ilango, Shumake, Wetzel, & Ohl, 2014; Lichtenberg, Kashtelyan, Burton, Bissonette, & Roesch, 2014; Oleson et al., 2012; Ramirez et al., 2015; Salamone, 1994). In particular, inactivation of NAcS, or disconnection of amygdalar inputs to this nucleus impairs the expression of active avoidance (Fernando et al., 2013; Ramirez et al., 2015). In comparison, avoidance expression does not appear to be affected by NAcC inactivation (Ramirez et al., 2015). Yet, DA release in the NAcC increases during the presentation of an 131  active avoidance cue, suggesting that transmission in this region may be relevant for the execution of this behavior (Gentry et al., 2016; Oleson et al., 2012). Thus, both subnuclei of the accumbens may contribute to aversively-motivated active behaviors that avoid negative consequences.  In comparison to its role in active avoidance, neurotransmission in the NAc has been shown to be necessary for the acquisition, but not expression, of defensive reactions such as passive avoidance, as measured by latency on one-trial step-through tasks (Bracs, Gregory, & Jackson, 1984; De Leonibus et al., 2003; Lorenzini, Baldi, Bucherelli, & Tassoni, 1995; Martínez et al., 2002; Shirayama et al., 2015). When conducted prior to learning, manipulations that perturb NAc functioning cause rats to approach a context previously associated with foot-shock more rapidly than control rats, although these effects are typically absent when conducted prior to expression. Unlike active avoidance, this mnemonic test is acute and not amenable to repeated testing. In addition, the difficulty posed by a No-Go trial during Go/No-Go performance is enhanced by the necessity to accurately discriminate between discrete Go vs. No-Go stimuli, and then withhold a prepotent response. These crucial aspects of passive avoidance behavior are not captured by such one-trial step-through tasks. Thus, development of a task that can adequately measure the flexibility and repetition associated with fully aversively-motivated “Go” vs. “No-Go” performance is necessary. In this regard, gerbils have been trained to perform a two-way active avoidance procedure, whereby two different conditioned stimuli necessitate either a passive or active avoidance response in order to avoid foot-shock (Schulz, Woldeit, Gonçalves, Saldeitis, & Ohl, 2015; Stark, Rothe, Wagner, & Scheich, 2004; Wetzel, Ohl, & Scheich, 2008). During one auditory stimulus, animals were required to make an instrumental shuttling response to avoid a foot-shock, while presentation of the other auditory stimulus 132  required the inhibition of a shuttling response. Acquisition of this task has been shown to increase dopamine release in the prefrontal cortex (Stark et al., 2004), similar to other forms of behavioral flexibility (for review, see Floresco, 2013). Interestingly, coherence between the auditory cortex and ventral striatum, of which the NAc is a primary component, increases following presentation of the active avoidance stimulus over the course of training on this task (Schulz et al., 2015). These later results suggest that the NAc may integrate afferent input to accurately promote or inhibit responding during complex avoidance performance. Additional insight into accumbal contributions to active versus passive avoidance comes from functional imaging studies conducted with human subjects (Levita et al., 2009, 2012). In one study, participants were trained to discriminate between two visual stimuli that instructed them to either press a button to make an active avoidance response or passively withhold a response to avoid an aversive outcome. Performance of an active avoidance response induced an increase in BOLD signal within the NAc, yet successful passive avoidance trials were associated a deactivation in this region (Levita et al., 2012). This pattern of activation/deactivation suggests that the NAc may function to promote active avoidance, while suppression is necessary for appropriate inhibition during passive avoidance. Unfortunately, the constraints on spatial resolution imposed by fMRI in that study did not permit a more detailed characterization of how changes in activation within different subregions of the NAc may be associated with different types of avoidance responses. Developing a preclinical analog of this task would aid in clarifying the role of different brain nuclei in the appropriate promotion versus suppression of aversively-motivated behavior, as well as generally improving our understanding of complex avoidance behaviors. 133  Here we report on the development of a novel operant task that required rats to use discriminative cues that informed them of whether an impending foot-shock could be avoided by either pressing a lever or withholding a lever press, permitting the examination of the neural basis of the active versus passive poles of avoidance behavior. Using reversible inactivation, we explored the contribution of the NAcC or NAcS to these different aspects of behavior. We hypothesized that inactivation of NAcC, which is involved in Pavlovian and instrumental approach, would impair active avoidance selectively. On the other hand, we expected that inactivation of the NAcS would not only impair approach-mediated active avoidance, but also perturb the suppression of behavior during passive avoidance trials. In addition, we probed potential monoaminergic contributions to active/passive avoidance behavior by investigating the effect of systemically administered d-amphetamine (AMPH).  5.2 Methods Active/Passive Avoidance training was adapted from previous reports conducting active avoidance in an operant setting (Fernando, Mar, Urcelay, Dickinson, & Robbins, 2015; Fernando et al., 2013; McCullough et al., 1993; Sokolowski, McCullough, & Salamone, 1994), and based on a paradigm used in humans, as described by Levita et al (2012).  5.2.1 Animals All experimental protocols were approved by the Animal Care Committee, University of British Columbia, and conducted in compliance with guidelines provided by the Canadian Council on Animal Care. All reasonable efforts were made to minimize the number and suffering of animals used. Male Long-Evans rats arrived weighing 250-275 g for active/passive avoidance training and 325-350 g for foot-shock sensitivity (Charles River) and were initially group housed (4-5 per 134  cage) and allowed to acclimatize to the vivarium for 6-7 days. The temperature (21° C) and light cycle (12-h light/dark) were kept constant.  Forty-eight total rats were utilized in the Active/Passive experiments, with three separate cohorts of n = 16 rats tested experimentally over the course of approximately 18-24 mo. For the foot-shock sensitivity experiments, one cohort of animals (n = 12) was given surgery and tested over a 3 wk period. All animals (except those used to test foot-shock sensitivity) were initially food-restricted to approximately 90% of their free-feeding weight, to promote exploration and exploitation of the operant environment, even though the task did not use food as a reinforcer. Throughout the course of the experiment, rats were allowed to gain weight at a rate of approximately 5 g/wk, maintaining a slightly delayed growth curve.  5.2.2 Apparatus Eight standard Med Associates (St. Albans VT, USA) operant conditioning chambers were used for all training and testing, as previously described (Piantadosi et al., 2017). A sound attenuating enclosure (30.5 cm x 24 cm x 21 cm) surrounded the operant chamber, providing ventilation and masking external noise via a fan. Each operant chamber contained two retractable levers on one wall, with a food receptacle in the middle (although no food was delivered in these experiments). Only the left operant lever was inserted into the chamber during these experiments. Each box was outfitted with three 100 mA cue lights, situated above the operant levers and the food receptacle. The opposite wall of the operant chamber contained a centrally located 100 mA house light, and an audio speaker that allowed for delivery of auditory stimuli via a programmable generator (ANL-926, Med Associates). Locomotor activity was monitored by four infrared photobeam sensors located slightly above the stainless-steel grid floor. The grid floor was wired to a shock source and solid-state grid scrambler for the delivery of foot-shock.  135  5.2.3 Initial lever shaping After reaching approximately 90% of their free-feeding weight, rats began to receive daily (conducted 5-7 d per week at a consistent hour) operant sessions. During the first session, rats were placed in the operant box, which was illuminated by the house light. No lever was extended for the duration of this session, and rats were simply allowed to locomote inside the chamber for 1 hr. This session served to reduce the neophobia associated with the novel environment, and allow rats to familiarize themselves with the environment. In turn, the insertion of the lever on the subsequent training day would be a novel stimulus that would elicit approach.  The day after this habituation session, rats underwent a lever-retraction training session, which consisted of the left operant lever being extended into the box under constant illumination of the house-light. During the 60 min lever-retraction training session, a press on the lever caused it to retract for 1 s, followed by its reinsertion. This procedure allowed the rat to learn the mechanics of the operant lever. If rats did not respond on this operant lever during the initial session, a small amount of sucrose powder was placed on the lever to entice the rat to produce an operant response. Note that this was the only point of the entire training where rats may have experienced some food in the chamber. All rats completed lever-retract training in 1-3 days. Rats performed a mean of 155 ± 24 SEM lever-presses during their final lever-retraction training session (range: 15-968 presses). 5.2.4 Active avoidance training After progressing from lever-retraction training, rats began the initial phase of active avoidance training. This task consisted of 20 discrete active avoidance trials, each of which occurred after a 105 (± 30) s ITI. A trial began with the left operant lever being inserted into the chamber and an 136  auditory cue played simultaneously. Across separate rats, the auditory cue varied between three distinct tones: a white noise cue (0 Hz, 80 dB), a high pitch pure tone (9 kHz, 80 dB), and a low pitch pure tone (1 kHz, 80 dB). The tone assigned to signal an active avoidance trial remained consistent throughout the experiment. In the initial portion of training, the signaled active avoidance period was 20 s. A lever-press during this period terminated the tone and resulted in the retraction of the lever. The house light was then extinguished, and a 30 s visual safety signal (illumination of the central cue light, located in between the two retractable levers) was presented. Presentation of a safety signal reinforces avoidance learning by explicitly signaling successful avoidance, and thus, safety (Berger & Brush, 1975; Dinsmoor, 2001; Dinsmoor & Sears, 1973; Fernando et al., 2013; Fernando, Urcelay, Mar, Dickinson, & Robbins, 2014; Morris, 1975). If rats took longer than 20 s to make a lever-press, the active avoidance auditory cue terminated and the escape period began, during which rats received a foot-shock at the end of the 20 s active avoidance period, and then again 5 and 10 s later (i.e. 25 and 30 s post-cue/lever presentation). As escape behavior typically precedes the development of active avoidance performance, successful escapes were also reinforced with the delivery of the same 30 s safety signal (Solomon & Wynne, 1953). Responses within the first 20 s were termed successful active avoidances, while responses made during the subsequent 10 s escape period (following at least 1 foot-shock) were classified as escapes. A lack of a response during either the 20 s avoidance or 10 s escape period caused the lever to retract, termination of the tone and house light, and the trial was scored as an active avoidance failure. On the first day of training, foot-shock intensity was set to 0.2 mA, and then individually titrated in 0.05 mA increments throughout training, such that rats ideally remained motivated via negative reinforcement to perform an active avoidance response (Fernando et al., 2015). Once 137  rats made approximately > 60% active avoidance or escape responses on the initial training task (i.e. < 40% failures), the avoidance period was decreased to 15 s. Rats were then trained on this 15 s active avoidance task to the same criterion (< 40% failure). A small percentage of rats that progressed beyond this portion of the task and began performing poorly were given remedial sessions on active avoidance, in order to rescue performance.  5.2.5 Active/passive avoidance training Following the initial active avoidance training, rats were trained on a blocked version of the active/passive avoidance task. During this task, 12 active avoidance trials (identical to those described previously, 15 s active avoidance period) and 12 passive avoidance trials were presented (Fig. 14A). During passive avoidance trials, one of the three tones not used for active avoidance (counterbalanced across rats) was presented at the same time as insertion of the left operant lever. On these trials, after insertion of the lever, rats were required to withhold a lever press for 15 s to avoid a food-shock. After a successful passive avoidance trial, the lever was retracted, and a 30 s safety cue (same as active avoidance training) was presented. In contrast, a lever press during a passive avoidance trial resulted in the immediate delivery of a foot-shock of the individually titrated intensity, and was scored as a passive avoidance failure. If a rat made a press during a passive avoidance trial, the lever remained extended until the 15 s passive avoidance period elapsed. Thus, rats could make multiple presses during these trials, with each press resulting in foot-shock. The number of lever presses made during passive avoidance trials were recorded.  This initial active/passive avoidance training was conducted in a blocked design. Typically, each session began with 12 active avoidance trials and ended with 12 passive avoidance trials. In order to familiarize rats with the eventual randomized presentation of active 138  avoidance and passive avoidance trials, rats also received days where trials were presented in the opposite order, 12 passive avoidance trials followed by 12 active avoidance trials. Typically, performance on the variant where active avoidance trials preceded passive avoidance trials was better than performance on the opposite (passive followed by active) variant. Thus, the criteria for successful acquisition of the active/passive avoidance contingency was < 50% failure on active avoidance and passive avoidance trials, combined, during the passive followed by active version of the task.  After acquiring the active/passive avoidance contingency in a blocked design, rats began daily sessions of a fully randomized final version of the task (Fig. 14A). Each session again consisted of 12 active avoidance and 12 passive avoidance trials, pseudorandomly presented according to a programmed sequence. All task parameters were otherwise identical to the previous training stage. Rats were trained on this intermixed version of the active/passive avoidance task until reaching a final task criterion of > 50 % success on both active and passive avoidance trials. As with the previous portions of training, a small percentage of rats that progressed beyond this phase of the task and began performing poorly were given remedial sessions on the blocked active avoidance and passive avoidance design, in order to rescue performance. Upon reaching the final active/passive avoidance performance criterion, rats underwent stereotaxic surgery for the implantation of guide cannula into the NAcC or NAcS. Following post-surgical recovery, rats were retrained to criterion before pharmacological testing.    139  5.2.6 Surgery Rats were initially anesthetized with ketamine (50 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.). Following this induction protocol, rats were prepped for surgery, placed in a stereotaxic frame, and maintained on isoflurane anesthesia (2-3% isoflurane concentration) for the duration of the procedure. Twenty-three-gauge bilateral stainless-steel guide cannula were aimed at the NAcS or NAcC, according to the following stereotaxic coordinates (in mm): NAcS – from bregma, AP: +1.3, ML: ±1.0, from dura, DV: -6.3 NAcC – from bregma, AP: +1.6, ML: ±1.8, from dura, DV: -6.3 Four stainless-steel skull screws were inundated with dental acrylic, holding the cannula in place. Stainless-steel obturators flush with the end of the guide cannula were inserted into the guide cannula at the conclusion of surgery. Rats were allowed 7-10 days to recover from surgery prior to either being re-trained on the active/passive avoidance task, or tested for foot-shock sensitivity.  5.2.7 Microinfusion and Systemic AMPH Administration Prior to any mock or microinfusion, all rats (except those used to test foot-shock sensitivity) were required to perform stably across three straight days, with < 25% variation in the percentage of active avoidance and passive avoidance successes. These rats initially received a mock infusion 10 min prior to their regular training session, during which obturators were removed, and a stainless-steel mock injector flush with the end of the guide cannula was inserted for approximately 2 min. During this duration, rats were placed into a small enclosure and allowed to freely move. Following this mock infusion day, rats were subjected to the first of two microinfusion test days. These test days were counterbalanced, such that roughly half of all 140  animals received bilateral infusion of a solution containing the GABA agonists baclofen and muscimol (B/M; 75 ng each in 0.3 ul/side), while the others received infusion of 0.9% saline alone (SAL; 0.3 µl/side). Each infusion took place over 45 s, with the microinfusion injectors left in place for an additional 60 s to allow for the infusate to diffuse from the injector tip. Following the initial microinfusion test day, rats were retrained over the course of at least two days until they again displayed criterion performance, after which they received their second, counterbalanced microinfusion test. We have previously used this dose/volume to behaviorally dissociate between the NAcC and NAcS during an approach/avoidance Conflict task (Piantadosi et al., 2017), as well as a number of other behavioral assays of cognition and motivation (Dalton et al., 2014; Floresco et al., 2008; Ghods-Sharifi & Floresco, 2010; Stopper & Floresco, 2011). To ensure that only data from animals that understood the task contingencies were included in the regional inactivation analyses, a criterion of > 50% successful active avoidance and < 50% failure on passive avoidance during their saline infusion day was set. Details regarding the ramification of this exclusion criteria are described below (see Task Acquisition and Baseline Performance).  Following successful completion of two microinfusion test days, a subset of rats (n = 20; 10 each from NAcC and NAcS) were given an additional two test days, during which they received an injection (i.p.) of 0.9% saline (1 ml/kg) or a 1 mg/kg (delivered in 1ml/kg of 0.9% saline) dose of AMPH. These test days were counterbalanced and separated by at least two re-training sessions. Each injection was given 10 minutes prior to placing the rat in the operant box. This subset of animals included some rats that were excluded from regional inactivation analysis due to missed cannula placements (n = 3). Systemic AMPH manipulation was not conducted on 141  the first cohort of rats (n = 16), as this experiment was conceived of following the completion of this experimental group.  5.2.8 Foot-Shock Sensitivity  Separate groups of animals underwent a foot-shock sensitivity experiment, to examine whether changes in avoidance behavior following NAc subregion inactivation could be explained by alterations in pain sensitivity. Procedures were based off of established protocols (Pang et al., 2010; Quirk et al., 2000; Tian et al., 2011). Animals were initially implanted with guide cannula aimed at the NAcC or NAcS, and allowed to recover (see Surgery). On the first day, all rats were placed into the operant chamber for 1 hr under constant illumination of the house light. The door to the sound attenuating enclosure surrounding the chamber was left open, as animals needed to be visible during foot-shock delivery on the following test days. The day after this locomotion session, rats underwent the first of two foot-shock sensitivity test days. Half of the rats within each region were infused with saline or B/M, in an identical manner as described above (see Microinfusion and systemic AMPH administration). The rat was then placed into the operant chamber, under illumination of the house-light. After 15-20 s, a 0.5 s foot-shock was administered at an intensity of 0.05 mA. An experimenter blind to the experimental treatment scored the following behaviors during each shock delivery: noticing (any noticeable reaction), flinching (hind-paws briefly raised off the grid floor), vocalizing. The current was increased by 0.05 mA and delivered every 10 s, until the rat vocalized. Rats were then given 2-3 d without being placed into the operant box. Following this break, rats were given 2 more locomotion sessions, conducted in a similar manner to their first day. These sessions were aimed at eliminating any contextual fear that may have occurred during the first foot-shock session. The 142  next day, rats were infused with the counterbalanced treatment, and foot-shock delivery and scoring was conducted in an identical manner as above.  5.2.9 Histology After the completion of all test days, rats were euthanized with CO2 and brains were removed and fixed in a 4% formalin buffered saline solution. Following adequate fixation, brains were sliced at 50 µm and mounted on glass slides and Nissl stained using cresyl violet acetate. For animals in the active/passive avoidance experiment, the ventral extent of the infusion bolus is displayed in Fig. 15A for the NAcC, and Fig. 16A for the NAcS. For animals in the foot-shock sensitivity experiment, the location of infusion is displayed in Fig. 18A. 5.2.10 Data analysis For both active and passive avoidance trials, the number of successful avoidances, escapes, and failures were converted into a percentage of total trials of each type. For each brain region, the percentage of successful active avoidances, escapes, and failures were analyzed using one-way ANOVAs, with Treatment (SAL and B/M, or SAL and AMPH) as the within-subjects factor. The percentage of successful passive avoidances and the number of presses made during a passive failure were analyzed using separate one-way ANOVAs, again with Treatment as the within-subjects factor. We also compared the relative importance of the NAcC and NAcS to active avoidance success by first calculating the change between the saline and B/M conditions, and then conducting a one-way ANOVA with Treatment (SAL vs. B/M) as the within-subjects factor.   Locomotor data were converted into a beam-break/min value, and subsequently analyzed using separate one-way ANOVAs. For manipulations that caused a change in locomotion, 143  Pearson correlations were conducted between the locomotor values and the % passive avoidance failure, to determine whether locomotion varied systematically with behavior during passive avoidance trials. The latency to make a response following cue presentation and lever insertion were collected for all trial types (active avoidance, escape, and passive avoidance). Due to the nested nature of this data (multiple and variable numbers of responses for different rats), we choose to analyze this data using a multilevel modelling approach, using the lme4 package in the statistical program R (Bates, Mächler, Bolker, & Walker, 2014; R Core Team, 2017).  For the foot-shock sensitivity experiment, potential order effects of Treatment were first examined using a two-way ANOVA on data from animals infused with SAL on the first or second day. Thus, this between/within-subjects ANOVA had the between subjects factor of Test Day (Day 1 versus Day 2), and the within-subjects factor of Response Type (Noticing, Flinching, and Vocalizing). Then, with separate two-way ANOVAs were conducted across cannula placement. The within-subjects factors were Treatment (SAL and B/M) and Response Type (Noticing, Flinching, and Vocalizing).  5.3 Results 5.3.1 Task Acquisition and Baseline Performance During the initial portion of avoidance training, all rats (n = 48) acquired the active avoidance contingency. All of the following descriptive statistics regarding training are presented as a mean ±SEM. Rats generally completed active avoidance training rapidly, in a mean of 12.96 ±0.91 days, with a range of 4-28 days. On the final day of their active avoidance training, rats performed 77.4% ±3.3 active avoidance responses, 11.1% ±2.2 escapes, and 11.5% ±2.5 active avoidance failures. All rats then progressed to the blocked design, during which they received active avoidance followed by passive avoidance trials, or vice versa. Of the 48 total rats, n = 43 144  successfully reached criterion on this blocked design after an additional 14.4 ±1.2 sessions, over a range of 4-36 days. On the final day of training on the blocked design, performance remained good, with rats making 67.4% ±5.6 active avoidance responses, 8.8% ±2.9 escapes, 23.8% ±5.8 active avoidance failures, and 17.4% ±3.5 passive avoidance failures.  Of the 43 total rats that completed the blocked design, 35 rats reached the final task criterion on the full active/passive avoidance task. Data from 8 rats that did not reach criterion were not included in the final analysis. These 35 successful rats achieved criterion performance after an additional 9.9 ±1.0 training sessions (range: 2-24). Upon reaching criterion, these rats again demonstrated good levels of performance on both active and passive avoidance trials, making 70.5% ±5.4 active avoidances, 10.6% ±4.1 escapes, and 18.9% ±4.9 active avoidance failures, and 23.7% ±5.5 passive avoidance failures. A survival plot of training (Fig. 14B) displays the total number of pre-surgery avoidance training sessions (all training except the initial locomotion and lever-retract training sessions) required by all rats (Fig. 14B, black line) and the high performing rats (Fig. 14B, broken gray line) that reached the final task criterion.  5.3.2 Active/passive avoidance inactivation studies Of the 35 rats that achieved the final task criteria and were implanted with guide cannula, 14 were excluded from the final analysis due to either cannula placements outside of the region of interest (n = 7), unexpected mortality following surgery (n = 4), or poor performance following surgery (n = 3). This resulted in final ns of 10 and 11 for the NAcC (Fig. 15A) and NAcS (Fig. 16A) groups, respectively. The mean shock intensity for NAcC group was 0.30 mA (range: 0.25-0.35 mA), while for the NAcS it was 0.29 mA (rage: 0.25-0.35 mA). These mean intensities were not significantly different (F(1,19)=0.06,p>0.80). 145  5.3.3 Active/passive avoidance: NAcC inactivation Inactivation of NAcC markedly impaired performance on active avoidance trials (F(1,9)=39.51, p<0.001) (Fig. 15B). Subsequent analysis probing this impairment revealed that, although escape behavior was left intact (F(1,9)=1.55,p<0.24) (Fig. 15B),  the incidence of active avoidance failure was dramatically increased (F(1,9)=47.09,p<0.001) (Fig. 15C). As a result of poorer performance on active avoidance trials, rats received more foot-shocks following NAcC inactivation (F(1,9)=44.73,p<0.001) (Table 6). Thus, animals maintained the ability to escape foot-shock at a comparable level as under control conditions, but their ability to proactively utilize the active avoidance cue was potently disrupted. In contrast to the effect on active avoidance trials, passive avoidance behavior was unaffected by NAcC inactivation, as there was no difference in the percentage of passive avoidance failures (F(1,9)=0.19,p=0.67) or the number of lever-presses made during passive avoidance trials  (F(1,9)=0.22,p<0.65) (Fig. 15D and E). Inactivation of NAcC had no impact on the latency to produce an active avoidance (B=0.66, t = 0.95, p>0.34), escape behavior (B = 1.89, t = 1.03, p > 0.32), or passive avoidance (B=-0.52, t = -0.36, p > 0.72) (Table 6). Furthermore, locomotor activity was normal following inactivation of the NAcC (F(1,9)=1.00, p>0.34) (Table 6). These data indicate that activity within the NAcC appears to play a selective role in initiating active defensive responses instigated by cues signaling an impending aversive outcome. Similarly, successful passive avoidance does not require the NAcC, as the presentation of a discriminative stimulus associated with lever-press contingent punishment remained effective at producing response-inhibition. This latter finding also confirms that inactivation of the NAcC did not impair the ability to discriminate between the two auditory cues.    146  5.3.4 Active/passive avoidance: NAcS inactivation Inactivation of the NAcS also resulted in poor performance during active avoidance trials (F(1,10)=24.38,p<0.001) (Fig. 16B). Again, escape behavior was left intact (F(1,10)=1.70,p>0.22) (Fig. 16B). Thus, impaired avoidance behavior resulted from a selective increase in active avoidance failures (F(1,10)=24.40,p<0.001) (Fig. 16C). The increase in active avoidance failure following NAcS inactivation caused animals to receive more foot-shocks (F(1,10)=26.12,p<0.001) (Table 6). Visual inspection of these data suggested that the decrease in active avoidance performance following NAcC inactivation may have been quantitatively more dramatic than the decrement induced by NAcS inactivation. To investigate this, we calculated the percent change in the number of active avoidance successes during the inactivation test day, compared to performance following saline infusion. This analysis revealed a numerically greater decrement in active avoidance performance induced by inactivation of the NAcC (-55.0% ±7.5) versus the same manipulation of the NAcS (-35.3 ±7.7). However, this difference only approached statistical significance (F(1,19)=3.31,p=0.085).  In marked contrast to the effects of NAcC inactivation, similar treatments within the NAcS disinhibited behavior during passive avoidance trials (F(1,10)=12.86,p<0.005), causing rats to erroneously respond more on the lever during presentation of the passive avoidance cue (F(1,10)=8.38,p<0.02) (Fig. 16D and E). This manipulation also significantly increased locomotion during the session (F(1,10)=8.80,p<0.05) (Table 6). However, this increase in locomotion was not correlated with the disinhibition of passive responding observed following inactivation of NAcS (r = -0.23, p>0.49) (Fig. 16F), nor with the decrease in active avoidance (r = -0.29,p>0.39)(data not shown). NAcS inactivation did not affect the latency to respond during active avoidance trials (B = -0.01, t = -0.03, p > 0.9), escapes (B = -0.24, t = -0.14, p > 0.89), or 147  passive avoidance trials (B = 0.54, t = 0.39, p > 0.70) (Table 6). Thus, this pattern of results suggests that NAcS promotes active avoidance while also suppressing inappropriate behavioral activation during passive avoidance.  5.3.5 Active/passive avoidance: Systemic AMPH administration Unlike inactivation of either accumbens subregion, systemic treatment with AMPH (1 mg/kg) had no overt impact on active avoidance performance (F(1,19)=0.79,p>0.38) (Fig. 17A), although animals were significantly quicker to make an active avoidance response (B = -0.83, t = -2.38, p < 0.02), but not other responses (both p-values > 0.01) (Table 6). This manipulation also spared escape behavior (F(1,19)=1.00,p>0.32) (Fig. 17A), and thus did not result in a change in active avoidance failure (F(1,19)=1.51,p>0.23) (Fig. 17B). On the other hand, AMPH administration produced a selective increase in the percentage of passive failures (F(1,19)=10.60,p<0.005) (Fig 17C), without altering the overall number of passive presses made during these failures (F(1,19)=1.10,p>0.30) (Fig. 17D). Thus, although AMPH disinhibited behavior during passive avoidance trials, rats remained susceptible to instrumental punishment, making a comparable number of passive presses during failure as under control conditions. As expected, locomotion was increased following AMPH administration (F(1,19)=33.79,p<0.001) (Table 6), and interestingly, this locomotor increase tended to be positively correlated with passive avoidance failure (r = 0.42, p = 0.06) (Fig. 17E) 5.3.6 Foot-shock sensitivity: NAc inactivations Of the 12 animals allocated to the foot-shock sensitivity experiment, one animal died during surgery, and one animal had a cannula placement outside of the region of interest, resulting in final ns of 6 for the NAcC group, and 4 for the NAcS group (Fig. 18A). First, we examined any 148  potential order effect of infusion, to insure that previous experience with a foot-shock test session did not impact subsequent performance on the second test session. Analysis of animals infused with SAL on the first test day versus the second suggested that there was no order effect, as illustrated by no effect of Test Day (F(1,8)=2.15,p>0.18), and no Test Day x Response Type interaction (F(2,16)=0.87,p>0.43). Thus, data from both days were combined for further within-subjects analysis.   Inactivation of the NAcC did not affect foot-shock sensitivity (Fig. 18B). There was no significant effect of Treatment (F(1,5)=0.46,p>0.52), nor a Treatment x Response Type interaction (F(2,10)=0.46,p>0.64). There was a pronounced main effect of Response Type, (F(2,10)=32.60,p<0.001), which suggested that the current intensity required to elicit each response increased across the three behaviors scored, regardless of treatment (all p-values < 0.025) (Fig. 18B). NAcS inactivation also had no effect on foot-shock sensitivity, as there was no main effect of Treatment (F(1,3)=0.21,p>0.68), and no Treatment x Response Type interaction (F(2,6)=1.17,p>0.37) (Fig. 18C). Again, a significant main effect of Response Type (F(2,6)=22.21,p<0.005), was the result of the current intensity requirement increasing across the three behaviors scored, independent of treatment (all p-values < 0.05) (Fig. 18C). These results imply that the sensitivity to the aversive stimulus used here was not affected by manipulation of either NAc subregion.  5.4 Discussion Although the NAc has long been known to be a key output nucleus in the production of appetitive behaviors, a bivalent role for this nucleus is relatively understudied. In the present experiments, a novel behavioral assay was designed to probe the contribution of NAc subregions and monoamine function to active and passive avoidance. During this task, discriminative stimuli 149  signaled whether the avoidance of an aversive foot-shock could be achieved by either performing or withholding an instrumental action. Our findings revealed that both the NAcC and NAcS contribute to successful active avoidance behavior. However, the NAcS also played a role in suppressing behavior in response to a cue signaling that a passive strategy will avoid punishment, as inactivation of this nucleus alone disinhbited behavioral responding during passive avoidance trials. Furthermore, treatment with the monoamine releaser AMPH selectively enhanced behavioral activation, increasing locomotion as well as passive avoidance failures, suggesting that the excessive release of dopamine and other monoamines may impede the suppression of behaviors that lead to aversive outcomes. 5.4.1 Behavioral considerations Initial training on the active avoidance task produced relatively rapid learning of a lever-press avoidance response in nearly all animals, as compared to previous reports using similar training methodology (Berger & Brush, 1975; Fernando et al., 2015; McCullough et al., 1993; Oleson et al., 2012). Lever-press active avoidance is notoriously difficult to train in rats, particularly when compared to more simple, naturalistic behaviors such as shuttling (D’Amato & Schiff, 1964; Meyer, Cho, & Wesemann, 1960). The enhanced rate of learning observed here may be related to the lower foot-shock current intensity used, as well as the individual titration of current intensity during learning, factors that differed from most previous reports (D’Amato & Schiff, 1964; McCullough et al., 1993; Meyer et al., 1960; Oleson et al., 2012). As illustrated by the foot-shock sensitivity experiment, the shock intensities used to motivate avoidance were able to induce unconditioned responses indicative of discomfort in all rats tested, suggesting that these intensities were sufficient to act as aversive-motivators. In addition, the novel lever-retraction session conducted prior to avoidance training served to establish the instrumental response 150  required for an active avoidance, without any explicit reinforcement contingency associated with a response. This also prevented the need for experimenter-based shaping of behavior oriented towards the lever (McCullough et al., 1993; Oleson et al., 2012). These procedural variations may aid in optimizing lever-press active avoidance procedures for use with rodents in the future.  Following acquisition of active avoidance, a passive avoidance component was added, initially in separate trial blocks, and eventually as randomly presented trials. A protocol consisting of active avoidance training preceding passive avoidance (and not vice versa) was chosen because success on passive trials is indicated by response suppression, for which there needs to be an established active behavior to inhibit. Although a majority of rats were able to acquire the blocked design, a fair amount of experimental attrition was observed during training on the full active/passive avoidance task, where active and passive avoidance trials were intermixed randomly over a session. This attrition stemmed partially from an a priori inclusion criterion for rats to perform well on both trial types, to allow for conclusions to be drawn about the effect of pharmacological manipulation. A small number of rats minimized active avoidance failure by predominantly performing escape responses, a mediating strategy that allowed rats to potentially discern between trial types (if a shock is received following a tone, but prior to any instrumental response, then that trial is an active avoidance). Escape behavior can also represent an intermediate step in active avoidance learning, which may suggest inadequate acquisition of the active avoidance response (Solomon & Wynne, 1953). Other rats simply were unable to maintain active avoidance performance once the discrimination component was introduced, likely developing a learned helplessness-like phenotype due to the receipt of foot-shock without the production of an avoidance response for an extended period of training (Seligman & Beagley, 1975). Although we were not able to include these animals due to our performance 151  criteria, future studies may utilize these poor performing animals in order to provide insight into mechanisms that oppose avoidance, including the pervasive expression of conditioned fear (Martinez et al., 2013).  The full version of the active/passive avoidance task was designed to provide insight into aversively-motivated flexible behavior. In the appetitive (or mixed valence) domain, assays such as the Go/No-Go task probe the ability of animals to utilize cues that necessitate opposing behaviors on a flexible basis. Previous research suggests that NAc activity is modulated by the presentation of Go and No-Go cues (Roitman & Loriaux, 2014; Setlow et al., 2003). This activity is strongly related to the action necessitated by cue presentation, consistent with a role for this nucleus in action selection (Roitman & Loriaux, 2014). The results of the present study are broadly consistent with a parallel role for this nucleus in aversively-motivated flexible behavior. Perhaps more intriguingly, we observed a dissociation between the impact of NAcC and NAcS inactivation that may be related to similar mechanisms underlying appetitive reinforcement-seeking, including a particular role for the NAcS in response-inhibition, and a dual role for these structures in active approach (Ambroggi et al., 2011, 2008; Floresco, 2015; Ghazizadeh et al., 2012; Peters et al., 2008).  5.4.2 Regulation of active behaviors by NAcC Inactivation of the NAcC profoundly impaired the expression of active avoidance, without affecting passive avoidance performance. The involvement of the NAcC in the production of active avoidance is in keeping with a number of previous neurochemical studies. During active avoidance learning, dopamine release within the NAc is positively correlated with successful performance (Dombrowski et al., 2013; McCullough et al., 1993). Similarly, NAc dopamine release occurs during the presentation of the active avoidance cue on successful avoidance trials 152  during established active avoidance expression (Gentry et al., 2016; Oleson et al., 2012). Importantly, these later studies targeted their voltammetric assessment of dopamine release to the NAcC, providing confirmatory evidence that transmitter release in this subregion mediates avoidance.  Although neuromodulatory activity within the NAcC is relevant to active avoidance learning and performance, few studies have investigated how altering neural activity in this subnucleus may affect such behavior. To our knowledge, only one previous study has separately examined NAcS and NAcC function during the expression of well-trained active avoidance (Ramirez et al., 2015). In that study, rats learned a simple two-way active avoidance response over the course of four days, which was not affected by subsequent NAcC inactivation on day five (Ramirez et al., 2015). Two main factors distinguish the present study from the one conducted by Ramirez and colleagues (2015). First, it may be that the auditory stimulus disambiguation required here recruits brain regions that are not necessary for the simple, single-stimulus active avoidance behavior. Consistent with this, a previous study has shown that coherence between auditory cortex and the lateral ventral striatum, which may include the NAcC, increases when learning about a stimulus that necessitates an active avoidance response, but not when associating a different stimulus with a passive avoidance response (Schulz et al., 2015). This finding was interpreted to suggest that plasticity within the auditory cortex and ventral striatum allows for stimulus discrimination and appropriate behavioral output. Thus, it is possible that such a mechanism continues to be necessary for the normal expression of active avoidance, particularly in situations requiring stimulus discrimination. Secondly, the instrumental response required here (lever-press) is a relatively more complex action to produce than is shuttling (Bolles, 1970; D’Amato & Schiff, 1964). Given that neurotransmission in the 153  NAcC is critical for re-engagement during bouts of lever-pressing for reward (McGinty et al., 2013; Nicola, 2010), inactivation of this nucleus could render rats unable to efficiently locate and engage the lever, although gross locomotor activity may remain intact. Such an effect may diminish lever-press avoidance, while sparing the more naturalistic (and less localized) shuttling response required by Ramirez et al. (2015). However, Bravo-Rivera, Quirk and colleagues (2014) examined NAc function on a platform-based avoidance task where animals had the concurrent opportunity to lever-press for sucrose reward. Using infusions that primarily targeted the NAcC, these researchers demonstrated that inactivation impaired avoidance and concomitantly increased freezing during the avoidance stimulus (Bravo-Rivera et al., 2014). Thus, the NAcC may also promote avoidance by suppressing Pavlovian defensive reactions such as freezing. Taken together, these findings raise the possibility that active behaviors instigated by Pavlovian or instrumental mechanisms may require the NAcC, regardless of whether the behavior is aversively or appetitively motivated. Neurotransmission in the NAcC has previously been shown to control flexible approach towards Pavlovian or instrumental stimuli conditioned via appetitive reinforcement (McGinty & Grace, 2008; Nicola, 2010; Saunders & Robinson, 2012). For example, NAcC activity is necessary for the acquisition of discriminative Pavlovian conditioned approach, where rats learn to approach a CS+ that signals reward delivery, but not a CS- that signals no reward (Di Ciano et al., 2008; Parkinson et al., 1999; Parkinson, Willoughby, et al., 2000; Saunders & Robinson, 2012). NAcC activity is also required for the ability of a Pavlovian stimulus to drive appetitively-motivated instrumental behavior (Ambroggi et al., 2011, 2008; Hall, Parkinson, Connor, Dickinson, & Everitt, 2001). Lesions of the NAcC disrupt the general form of Pavlovian-to-instrumental transfer, where a previously learned appetitive CS+ 154  potentiates the vigor with which a novel instrumental response is acquired (Hall et al., 2001). Similarly, instrumental responding during a discriminative stimulus that signals reward availability is diminished by NAcC inactivation (Ambroggi et al., 2011, 2008). These previous findings serve to illustrate that actions motivated by appetitive reinforcement require NAcC activity. In the present study, the ability of a negatively-reinforced auditory stimulus to elicit approach and engagement with a lever to actively avoid foot-shock was impaired following NAcC inactivation. Importantly, this effect was specific to anticipatory behavior, as the number of escapes, which are motivated by the US directly, remained unchanged following NAcC inactivation. This result suggests that the NAcC promotes approach behavior mediated by aversive motivation in a similar manner to this regions role in appetitive motivation.  It is important to note that the effect of NAcC inactivation on active avoidance was not the result of psychomotor slowing, which could manifest as poor active avoidance performance. This consideration is particularly relevant given that inactivation of the NAcC often slows response latencies and decreases locomotor activity during cognitive performance (Ambroggi et al., 2011; Dalton et al., 2014; Feja et al., 2014; Floresco et al., 2008; Ghods-Sharifi & Floresco, 2010; Stopper & Floresco, 2011). Following NAcC inactivation during this active/passive avoidance task, response latencies and locomotion were comparable to control data. This discrepancy illustrates a potential divergence between the mechanisms underlying appetitively and aversively-motivated behaviors. Such a suggestion has implications for our understanding of the aforementioned flexible approach hypothesis (McGinty et al., 2013; Nicola, 2010), which emphasizes that neurons within in this nucleus facilitate locomotor approach and engagement during reinforcement-seeking, in a dopamine-dependent manner. Thus, although there are notable similarities between the behavioral ramifications of NAcC inactivation on approach 155  behavior mediated by reinforcement and punishment, underlying processes such as motor activation and reaction time may be differentially mediated. That NAcC inactivation spared passive avoidance behavior fits with hypotheses suggesting that NAcS, but not NAcC, is uniquely responsible for inhibiting inappropriate behavioral responses (Ambroggi et al., 2011; Blaiss & Janak, 2009; Floresco, 2015). Previous studies have shown that parameters of response inhibition that are affected by NAcS inactivation, such as instrumental responding on an inactive lever or during an explicitly non-rewarded period, are unchanged by NAcC inactivation (Ambroggi et al., 2011, 2008; Blaiss & Janak, 2009; Floresco et al., 2008). Here, we operationalize response-inhibition as the ability to withhold a lever press during the presentation of a cue predicting instrumentally-delivered punishment. We have previously shown that the withholding of a sucrose-seeking response by the presentation of a Pavlovian aversive cue or by instrumental punishment is intact following NAcC inactivation (Chapter 2, 3, and 4; Piantadosi et al., 2017). Other tasks assessing impulsivity, a multifaceted construct that reflects the inability to withhold an action due to motor or cognitive dysfunction, have produced inconsistent results regarding the requirement of NAc subregions. Some studies have suggested that the NAcC promotes response-inhibition (Cardinal, Pennicott, Sugathapala, Robbins, & Everitt, 2001b; Christakou, Robbins, & Everitt, 2004; Pothuizen et al., 2005), while others implicate the NAcS (Feja et al., 2014), or neither structure (Eagle & Robbins, 2003; Murphy et al., 2008). Of the studies supporting a role for the NAcC in impulsive responding, the majority utilized permanent lesions that may severely impact underlying processes, such as the appropriate timing of responding (Singh et al., 2011). In addition, assays of impulsivity commonly operationalize punishment as a loss of opportunity for (more) reward, while the present study enforces response-inhibition via the delivery of foot-shock. Thus, the involvement 156  of the NAcC in response-inhibition may be situationally dependent on the method used to provoke response-inhibition. Future studies are necessary to determine the extent to which passive avoidance taxes NAcC-mediated mechanisms similar to those regulating the multifaceted processes underlying impulsivity. 5.4.3 Dual functions of the NAcS in active and passive behavior Here, we hypothesized that NAcS would contribute to the performance of both active and passive avoidance. Consistent with this, inactivation of NAcS produced a qualitatively similar impairment during active avoidance trials as did NAcC inactivation. One potential explanation for the comparable impact of inactivation of either subnuclei was the spread of the infusate from within the NAcS to the NAcC. We have previously demonstrated dissociable roles for these subregions using similar infusion procedures to great effect (Dalton et al., 2014; Floresco, Ghods-Sharifi, Vexelman, & Magyar, 2006; Floresco et al., 2008; Piantadosi et al., 2017; Stopper & Floresco, 2011), and, in the present study, dissociations between each subregion were observed during passive avoidance trials, as well as locomotor activity. This suggests that spillover of the infusate from NAcS to NAcC cannot fully account for the similar effect on active avoidance. A more likely explanation comes from previous work suggesting that NAcS independently promotes active avoidance, via interactions with the BLA (Ramirez et al., 2015). Our data extend this observation beyond the realm of two-way active avoidance, illustrating that NAcS promotes active responding in an operant environment. As a subnucleus within the limbic-motor interface (Mogenson et al., 1980), the NAcS is positioned to integrate affective information regarding the avoidance stimulus arriving from BLA, and translate this information into defensive action, in this case active avoidance (Martinez et al., 2013; Ramirez et al., 2015). NAcS projects to downstream targets within the ventral pallidum and midbrain dopamine 157  system, pathways which may act to promote the appropriate expression of active avoidance (Ilango et al., 2014; Ilango, Shumake, Wetzel, Scheich, & Ohl, 2012; Saga et al., 2017). Another related possible mechanism contributing to the promotion of avoidance by the NAcS is the invigoration of responding by a safety signal during successful avoidances. Safety signals reinforce avoidance behavior by explicitly indicating that the instrumental response has been successful, potentially coming to act as a conditioned reinforcer (Dinsmoor & Sears, 1973; Fernando et al., 2013, 2014; Morris, 1975). Inactivation or infusion of AMPH into the NAcS (but not NAcC) has been shown to decrease operant active avoidance only during sessions where a safety signal was present (Fernando et al., 2013). Suppressing NAcS neural activity via inactivation may have reduced the motivational impact that the safety signal has on behavior, causing a decrement in active avoidance responding.  The key dissociation observed in the present study was that activity within the NAcS, but not the NAcC, is necessary for the appropriate inhibition of punished responding during passive avoidance trials. Although motor activity was also disinhibited following NAcS inactivation, hyperlocomotion alone cannot explain the resulting deficit in passive avoidance, as there was no correlation between these two measures. This effect is also unlikely to be explained by a general mnemonic impairment, as performance on tasks assessing passive avoidance expression independently are not affected by NAc manipulation (De Leonibus et al., 2003; Lorenzini et al., 1995). Thus, this result is likely related to the demands of the task, requiring rats to balance active versus passive behaviors on a dynamic basis, akin to classic Go/No-Go paradigms. Relatedly, we have recently shown that this particular accumbens subregion (as well as the basolateral amygdala) is required when animals inhibit reward-seeking under threat of punishment on a “conflict” task (Chapter 4; Piantadosi et al., 2017). When seeking sucrose 158  during the conflict period, where the reinforcement schedule is rich but concurrently punished, inactivation of NAcS and BLA, disinhibited lever-pressing despite punishment. The passive avoidance trials employed here are similar to the punished period on the conflict task, during which control animals typically make few inhibitory control errors. Removing the influence of the NAcS eliminates the break on punished responding, causing rats to make passive avoidance errors. Inactivated animals also became less susceptible to instrumental punishment delivery, as they made more passive presses during these errors. This later finding indicates that the impairment in passive avoidance extends beyond an inability to properly respond to predictive conditioned stimuli, and includes a loss of instrumentally administered aversive-motivation following the punishing foot-shock itself. The results of our foot-shock control experiment suggest that this disinhibition of pressing is unlikely to be caused by changes in foot-shock sensitivity per se, as unconditioned responses were normal following NAcS inactivation. Instead, the link between foot-shock receipt and the implementation of suppression may be diminished in the absence of NAcS activity.  The mechanism through which the NAcS regulates punishment-induced response-inhibition may also relate to the ability of this subregion to refine behavior by encoding the disadvantageous nature or irrelevance of stimuli and actions (Ambroggi et al., 2011; Blaiss & Janak, 2009; Floresco et al., 2008; Gal, Schiller, & Weiner, 2005; Millan et al., 2010; Peters et al., 2008; Pothuizen et al., 2005). In the appetitive domain, inactivation or blockade of dopamine function within the NAcS releases inappropriate behavior from inhibition, such as operant responding during task periods that are explicitly not reinforced (Ambroggi et al., 2011; Blaiss & Janak, 2009; Ghazizadeh et al., 2012). Activity within the NAcS is often necessary for the inhibition of behavior following extinction learning, as disrupting transmission within this region 159  disinhibits extinguished behavior (Blaiss & Janak, 2009; Floresco et al., 2008; Peters et al., 2008). Lesions of this subnucleus also prevent the acquisition of aversively-motivated learned irrelevance, which occurs when numerous non-reinforced presentations of a stimulus retard the subsequent association of that stimulus with punishment (Gal et al., 2005; Pothuizen et al., 2005). These results support the contention that the NAcS promotes response-inhibition under some circumstances, independent of the valence of the motivator.  As discussed previously, the literature implicating the NAc in impulsivity is mixed, perhaps due in part to a lack of attention to subregional distinctions and an overreliance on permanent lesions (Basar et al., 2010). One recent study has utilized reversible inactivations to demonstrate that NAcS activity opposes impulsive actions and choices, while NAcC is more necessary for general aspects of motivated behavior (Feja et al., 2014). The NAcS may facilitate response inhibition in concert with dopaminergic input from the midbrain, as blocking dopamine D2 receptors within the NAcS (but not NAcC) exacerbates the performance of impulsive actions in highly impulsive rats (Besson et al., 2009). The present data suggest that the ability to inhibit lever pressing during the presentation of a passive avoidance stimulus dependent in part on NAcS function, which may relate to a generalizable role for this nucleus in response-inhibition.  5.4.4 Monoaminergic correlates of response promotion and inhibition We also probed the ability of the monoamine releaser AMPH to alter the expression of well-learned active/passive avoidance, illustrating that AMPH selectively affected performance on passive avoidance trials. It is well-established that AMPH administration can potentiate the acquisition of active avoidance performance (Barrett, Leith, & Ray, 1972; Kulkarni, 1968; Niemegeers, Verbruggen, & Janssen, 1970). This effect has been suggested to be the result of an increase in general motoric output, allowing animals to overcome the behavioral suppression 160  induced by fear early in active avoidance training (Kulkarni, 1968). Thus, it is perhaps unsurprising that AMPH had no impact on performance in the well-trained animals tested here, as they have already overcome this obstacle to successful active avoidance. Consistent with this, AMPH administration has been shown to be ineffective at altering active avoidance performance in animals performing at asymptote (Rosen & La Flore, 1973). Still, dopaminergic signals in the NAc persist during performance of a similar approach/avoidance task requiring animals to attend to discrete cues that necessitate one of three instrumental responses, an active avoidance response, a sucrose-seeking response, or no action at all (Gentry et al., 2016). Taken together, these results suggest that, although a baseline level of dopamine activity within the NAc is likely necessary for active avoidance, potentiating this signal via AMPH does not impact performance.  In contrast to the null effect on active avoidance, AMPH administration impaired performance on passive avoidance trials. The trend-level correlation observed here between hyperlocomotion and passive avoidance failure suggests that motor disinhibition may contribute to this effect. Broadly, this finding is consistent with previous studies suggesting that AMPH administration can provoke impulsive actions (for review, see Robbins, 2002). For example, at doses similar to those used here, AMPH has been shown to produce premature responses on the five-choice serial reaction time task, which may reflect a loss of inhibitory control over prepotent actions (Baarendse & Vanderschuren, 2012; Cole & Robbins, 1989, 1987; Harrison, Everitt, & Robbins, 1997; Murphy et al., 2008; Pattij, Janssen, Vanderschuren, Schoffelmeer, & Van Gaalen, 2007; Wiskerke et al., 2011). In addition, AMPH administration selectively impairs performance on No-Go trials, without affecting Go responses, in an appetitively-motivated Go/No-Go paradigm (Blackburn & Hevenor, 1996). Interestingly, the loss of inhibitory control induced by AMPH on assays of impulsive action has been shown to be related to dopamine and 161  µ-opiod receptor activation within the NAcS (Pattij et al., 2007; Wiskerke et al., 2011). These findings suggest that AMPH may act directly or indirectly within the NAcS to alter response inhibition, in keeping with the similarity between the two treatments shown here. However, unlike NAcS inactivation, rats treated with AMPH were able to inhibit their passive responding upon receipt of the foot-shock, making the same amount of passive presses as under control conditions. Notably, this finding is unlikely to be due to alterations in foot-shock sensitivity, as previous studies suggest that unconditioned responses to aversive stimuli are unchanged by AMPH administration at the doses given here (Conti, Maeiver, Ferkany, & Abreu, 1990; Mitchell et al., 2011; Orsini, Trotta, et al., 2015; Simon et al., 2009). This discrepancy between the impact of AMPH and NAcS inactivation suggests some degree of dissociation between the mechanisms mediating cue-induced suppression, and the suppression induced by receipt of a punishing unconditioned stimulus.   The suggestion that AMPH produces deficits in response-inhibition during punished trials may appear to conflict with data demonstrating that AMPH decreases the willingness of animals to accept punishment during reinforcement-seeking (Geller & Seifter, 1960; Lazareno, 1979; Mitchell et al., 2011; Orsini, Trotta, et al., 2015; Simon et al., 2009). During these tasks, animals are more reticent to produce an instrumental reward-seeking response during punishment, an effect that is potentiated by AMPH administration. One major difference between these studies and the present work is the presence or absence of response-competition related to the goal of the instrumental response. In the case of the experiments described here, there is no response-competition induced by the instrumental response itself. In comparison, during these other punishment tasks, there is a prominent response-conflict component, as rats are highly motivated to seek reward, yet simultaneously want to limit exposure to potential harm. This distinction 162  suggests that AMPH may bias behavior towards or away from punishment, as a function of the presence or absence of response-competition, respectively.  Although the behavioral effects of AMPH are most commonly attributed to its modulation of dopamine function via blockade of the dopamine transporter, this stimulant also elevates other monoamines, including serotonin (Kuczenski & Segal, 1989; Seiden, Sabol, & Ricaurte, 1993; Sitte & Freissmuth, 2015). Evidence supporting a dopamine-mediated account of the effects of AMPH comes from previous studies on impulsive action, where the effect of AMPH is dramatically reduced by intra-NAc dopamine lesions (Cole & Robbins, 1989). Similarly, blockade of dopamine D2/3 receptors in the NAc blocks the impulsigenic impact of systemic AMPH administration (Pattij et al., 2007). In contrast, depletion of brain serotonin induces impulsive action and prevents the ability of AMPH to potentiate this aberrant behavior (Harrison et al., 1997). Taken together, these results suggest that the AMPH-induced passive avoidance impairment seen here may be mediated by dopamine release, possibly within the NAc. Still, the relation between impulsivity and passive avoidance as operationalized here remains to be established, and more work is necessary to probe the particular brain region and transmitters mediating this effect.    5.4.5 NAc circuitry regulating active/passive avoidance: Relevance for humans Aberrant avoidance behavior is present across a number of neuropsychiatric conditions (Dymond & Roche, 2009; Figee et al., 2016; Maner & Schmidt, 2006; Ottenbreit & Dobson, 2004; Trew, 2011). Therefore, identifying the neural substrates underlying the basic behavior may improve our understanding of disorders characterized by such disturbances. To this end, activity within the human ventral striatum, which contains the NAc, has been associated with active avoidance performance (Delgado et al., 2009; Jensen et al., 2003; Levita et al., 2012). Bilateral ventral 163  striatal activation has been reported during the presentation of an avoidance cue, as compared to a neutral cue. These results indicate that neurons within the ventral striatum are relevant while learning the association between a particular instrumental action and its avoidance outcome (Delgado et al., 2009), as well as while performing an active avoidance response (Jensen et al., 2003; Levita et al., 2012). Although neither study had the spatial resolution necessary to probe specific contributions of ventral striatal subregions to this behavior, they are broadly consistent with the present finding suggesting that this region is necessary for instrumental active avoidance.   Comparatively less attention has been paid to the circuitry underlying passive avoidance in humans. Here we based our task design on one of the few studies to have used functional imaging techniques to probe both poles of avoidance behavior using a button-press active/passive avoidance task (Levita et al., 2012). These researchers illustrated that deactivation within the NAc is observed during passive avoidance trials, while the aforementioned active avoidance trials produced activations within this nucleus. In keeping with the clinical relevance of such data, this pattern of activation/deactivation was correlated with a measure of state anxiety. Given that our manipulation involved temporarily decreasing neuronal activity within the NAc, one might have expected an improvement in passive avoidance performance based on this limited human literature. In fact, we observed the opposite pattern of results, when manipulating the NAcS specifically. One potential explanation for this discrepancy arises from research illustrating that deactivations as measured by fMRI may not necessarily correspond to decreases in neuronal activity, particularly when measured within striatal regions (Hayes & Huxtable, 2012; Mishra et al., 2011). Specifically, comparisons between functional imaging and electrophysiological indicators of neuronal activity suggest that concordance between these 164  measures is low when measured in the striatum, but high when measured in cortical and thalamic regions. These researchers hypothesized that deactivations may instead reflect alterations in neurovascular coupling, rather than changes in neuronal activity. If this is the case, the deactivation observed in the NAc by Levita et al. (2012) may not necessarily imply that diminished activity in this nucleus precedes successful passive avoidance. Additionally, Levita and colleagues (2012) assessed BOLD activity within the entire window around active/passive avoidance cue presentation and behavior, which likely includes the outcome phase. Thus, some of the activity observed in that study may reflect relief or safety processing, functions which may require the NAc (Baliki et al., 2013; Mohammadi et al., 2014). Another level of ambiguity is added by the fact that most fMRI research is not capable of dissociating between subregions of the NAc, which we have shown here to be differentially responsible for aspects of avoidance behavior. To date, the only functional imaging study to have dissociated NAcS and NAcC in the human brain has suggested that activity within the NAcS occurs in anticipation of thermal pain, while NAcC activity occurs following the cessation of a painful stimulus, supporting a role for these subnuclei in aversion (Baliki et al., 2013). Thus, future studies examining active/passive avoidance performance should account for possible functional differences within accumbens subregions.   5.5 Conclusion Using a novel active/passive avoidance task, we illustrate that the two main subregions of the rodent NAc, the NAcS and NAcC, differentially regulate aspects of this behavior. Specifically, both subregions promoted the expression of active avoidance, while only the NAcS contributed to response-suppression during passive avoidance. Administration of the monoamine releaser AMPH also selectively impaired passive avoidance responses. These results are in keeping with 165  previous research differentially implicating these subregions in avoidance, as well as processes that contribute to active approach and impulsive action.                  166  Table 6. Mean (± SEM) values for ancillary measures during the active/passive avoidance task. Overall locomotion, the number of shocks received (during active avoidance failure), and the response latency for active avoidances, escapes, and passive avoidances following regional inactivation or AMPH treatment. * = p < 0.05 vs SAL  Locomotion (photobeam breaks/min) Shocks received (active avoidance) Active avoidance response latency (s) Escape response latency (s) Passive avoidance response latency (s)  NAcC          SAL 24.4 (±3.1) 7.2 (±1.2) 5.5 (±0.7) 2.5 (±0.6) 6.7 (±1.4)     B/M 21.3 (±2.9) 23.8 (±1.7)* 6.3 (±1.0) 4.4 (±1.2) 6.2 (±0.9)        NAcS          SAL 24.3 (±3.2) 4.3 (±0.9) 6.1 (±0.8) 3.5 (±1.3) 5.3 (±1.2)     B/M 51.6 (±8.1)* 14.4 (±2.2)* 5.7 (±0.8) 4.4 (±1.1) 6.2 (±1.1)       AMPH         SAL 23.9 (±1.7) 3.7 (±0.7) 5.5 (±0.4) 2.9 (±0.5) 6.4 (±0.9)    1 mg/kg 58.0 (±6.2)* 5.4 (±1.8) 4.7 (±0.3)* 1.9 (±0.2) 5.7 (±0.6)         167   Figure 14. Trial structure and survival plot of training for the active/passive avoidance task. (A) Layout of a single trial on the active/passive avoidance task. Each trial type and potential outcome are outlined from the branches following Trial Start. LP = lever press, gray outlined lightning bolt = foot-shock delivery. (B) A survival plot showing all rats that reached the full active/passive avoidance task (black line), and a subset of rats that reached the criterion on the final version of the full active/passive avoidance task (broken gray line). 168   Figure 15. NAcC activity is necessary for active, but not passive, avoidance performance. (A) Histology diagram, the ventral extent of each microinfusion within the NAcC is labeled by a filled triangle. (B) NAcC inactivation decreased the percentage of successful active avoidance trials, without affecting escape responses. (C) Inactivation induced more failures during active avoidance trials. (D, E) The percentage of passive avoidance failures and the number of passive presses did not change following NAcC inactivation. Star denotes p<0.05 between the SAL and B/M conditions.  Figure 16. NAcS activity is necessary for active and passive avoidance performance. (A) Histology diagram, the ventral extent of each microinfusion within the NAcS is indicated by a filled circle. (B) Inactivation of the NAcS decreased active avoidance success, but left escape behavior intact. (C) The percentage of active avoidance trials ending in failure was increased by NAcS inactivation. (D) NAcS inactivation induced passive avoidance failures, and (E) increased the total number of presses made during passive failure. (F) Scatterplot comparing locomotion (beam breaks/min) against the number of passive avoidance failures in the NAcS inactivation condition. There was no significant relationship between these measures. Star denotes p<0.05 between the SAL and B/M conditions. 169   Figure 17. AMPH administration selectively provokes passive avoidance failure. (A) Performance of active avoidance and escape behavior was normal following AMPH (1 mg/kg) administration. (B) AMPH treatment had no effect on active avoidance failure. (C) AMPH increased the percentage of passive avoidance failures, without altering (D) the total number of presses made during passive avoidance failure. (E) Scatterplot comparing locomotion (beam breaks/min) against the number of passive avoidance failures in the AMPH condition. There was a trend towards a positive correlation between these two measures. Star denotes p < 0.05 between the SAL and AMPH conditions.   170   Figure 18. Neither NAc subregion is necessary for foot-shock sensitivity. (A) Histology diagram, the ventral extent of each microinfusion within the NAcC (closed triangles) and NAcS (gray circles) are indicated. (B, C) NAcS inactivation (B) or NAcC inactivation (C) had no effect on the current threshold required for animals to notice, flinch, or vocalize following foot-shock delivery. Star denotes simple main effects comparisons at a p < 0.05 level, between each measure of foot-shock sensitivity (regardless of treatment).            171  Chapter 6: General discussion The present experiments examined the function of two major NAc subregions, the NAcC and NAcS, in a variety of related aversively-motivated behaviors. Consistent across these studies was the necessity to inhibit responding during discrete task epochs, while promoting active behavior during others. Response-inhibition was motivated either by purely Pavlovian mechanisms, as in Chapters 2 and 3, or by the potential for instrumental punishment, as in Chapters 4 and 5. Regardless of the conditioning mechanism, the NAcS was necessary for animals to suppress responding, as inactivation of this structure disinhibited lever-pressing during threat. In the case of Pavlovian fear, this effect appeared to be mediated in part by a projection from the PL cortex to the NAcS. In the next two experiments, NAcS inactivation diminished the impact of instrumental punishment on reward-seeking and passive avoidance. Critically, neither accumbens subregion was necessary for normal unconditioned responding to foot-shock alone, suggesting that these results cannot be explained by alterations in pain sensitivity.  Unlike the NAcS, the NAcC was not responsible for aversive motivation, as inactivation of this structure instead affected indices of behavioral activation, such as locomotor activity and response vigor. During the Pavlovian fear task, this region played no role in fear acquisition or expression. Instead, the NAcC appeared to promote locomotor activity, as well as the vigor with which animals pressed the operant lever. A similar effect was observed in Chapter 4, with NAcC inactivation reducing operant reward-seeking, concomitant with a decrease in locomotion, regardless of whether rats were trained on a task delivering instrumental punishment or not. We then illustrated that the promotion of responding mediated by the NAcC was not exclusive to the appetitive context, as active avoidance performance, which required an instrumental response to 172  avoid foot-shock, was powerfully impaired by inactivation. Thus, these results suggest a fundamental role for the NAcC in the invigoration of behavior.   In addition to the interrogation of these ventral striatal subregions, we probed the necessity of relevant cortico-limbic regions to aspects of these aversively-motivated behaviors. In the case of the prefrontal cortex, we observed that the top-down control of Pavlovian conditioned suppression expression was mediated by the PL cortex, and to a lesser extent the IL cortex. Pharmacological disconnection illustrated that a direct projection from the PL cortex to the NAcS was responsible for the former effect. We also evaluated the possibility that a BLANAcS circuit was necessary for fear acquisition, although a disconnection experiment demonstrated that this projection was not involved. Although glutamatergic projections from the BLA to the NAcS did not mediate Pavlovian fear acquisition, intact activity in the BLA was necessary for the inhibition of reward-seeking during punishment. Finally, the promotion of catecholaminergic activity was examined for its effect on Conflict performance, as well as active/passive avoidance, given that both tasks necessitated response-inhibition. Interestingly, AMPH selectively and bidirectionally affected indices of response-inhibition on both tasks, promoting suppression during instrumental punishment, but disinhibiting instrumental actions during punished passive avoidance trials.  6.1 Dissociable contributions of NAc subregions to the inhibition and promotion of behavior Here, we designed a series of experiments to probe the hypothesized involvement of the NAcC and NAcS in aversively-motivated behavior. The dissociability of these regions has primarily been examined in the appetitive domain, using behavioral tasks that assess Pavlovian and instrumental mechanisms contributing to action selection. A particularly instructive illustration of the functional differences (and similarities) between these two regions comes from 173  electrophysiological and pharmacological experiments during the performance of a simple behavioral assay. During this task, animals learn that the presentation of a discriminative stimulus indicates that reward is available for a press on an operant lever, while presses during other epochs, including the presentation of a neutral stimulus, are never reinforced (Ambroggi et al., 2011, 2008; Ghazizadeh et al., 2012; Ishikawa et al., 2008, 2010; Nicola et al., 2004; Yun et al., 2004). Neurons within the NAcS encode task events that acquire irrelevance over the course of training, such as the presentation of a neutral stimulus or pressing a never-reinforced inactive lever (Ghazizadeh et al., 2012). Learning to inhibit these irrelevant responses recruits the NAcS, via a projection from the vmPFC, which promotes the activity of tonically active NAcS neurons that inhibit behavior during non-reinforced task phases (Ghazizadeh et al., 2012).   During performance of this task, the same pattern of results holds true. While NAcS activity preferentially tracks task-irrelevant events, the NAcC is more likely to be activated by the rewarded discriminative stimulus (Ambroggi et al., 2011). Inactivation of the NAcS has a disinhibitory effect on reward-seeking actions during irrelevant task phases (Ambroggi et al., 2011). In contrast, NAcC inactivation decreases behavior during presentation of the discriminative stimulus indicating reward availability (Ambroggi et al., 2011, 2008; Ishikawa et al., 2008). Further evidence for such a dissociation comes from studies illustrating that the NAcS inhibits the reinstatement of reward-seeking for a variety of substances, while the NAcC typically promotes such behavior (Di Ciano et al., 2008; Floresco et al., 2008; Millan et al., 2010; Peters et al., 2008).   Using three separate, but related aversive conditioning paradigms, we observed that the NAcS subserved a response-inhibitory function, while the NAcC simply promoted actions. The aversive Pavlovian or instrumental inhibition of reward-seeking was less pronounced when 174  NAcS was taken silenced using reversible inactivations. This was true regardless of whether the behavior being suppressed was motivated by appetitive reinforcement or by negative reinforcement. These results suggest a consistent role for the NAcS across these distinct conditioning paradigms, in keeping with the conceptualization of this region as a limbic-motor integrator (Mogenson et al., 1980). Such a hypothesis is not incompatible with evidence suggesting that the NAcS is also able to promote actions that enable avoidance or escape of danger, as also illustrated here (Fernando et al., 2014; Ramirez et al., 2015). The NAcS may be recruited to suppress activity in situations where escape or avoidance are not possible, while also facilitating actions to ensure safety when such opportunities are available. The BLA, which integrates valence signals to allow for appropriate behavior, likely contributes to such action (or inaction) selection, as this region is critical for passive defensive responses, as well as active defensive actions (for which a direct projection to NAcS has been demonstrated) (Correia et al., 2016; Jean-Richard-Dit-Bressel & McNally, 2015; Ramirez et al., 2015; Sierra-Mercado et al., 2011).  This later facet of active behavior motivated by aversive consequences also required the NAcC. This is in keeping with a variety of research from the appetitive conditioning literature suggesting that the NAcC motivates active behaviors. As discussed, neurons within the NAcC encode stimuli that signal reward availability, and inactivation of this structure decreases instrumental reward-seeking behaviors (Ambroggi et al., 2011). In addition, this subnucleus plays an important role in enacting the behaviorally activating effects of conditioned stimuli. For example, blocking dopamine activity within the NAcC diminishes the expression of a conditioned approach response mediated by a cue that predicts reward delivery, without altering behavior in animals that do not attribute incentive salience to the cue (Saunders & Robinson, 175  2012). A formal conceptualization of this function, known as the flexible approach hypothesis, suggests that activity (particularly dopamine release) within the NAcC allows animals to appropriately engage and re-engage with instrumental manipulanda in the environment (McGinty et al., 2013; Nicola, 2010). Although we did not assess dopaminergic activity within the NAcC, inactivation of this subregion typically impaired the vigor with which animals engaged in a particular behavior, regardless of task context. By decreasing neural activity in this region, we may have provoked a similar state to that induced by hypo-dopaminergia in previous studies. Relatedly, blockade of neuronal activity or activity at dopamine receptors decreases the amount of effort rats are willing to expend to receive reward (Ghods-Sharifi & Floresco, 2010; Nunes et al., 2013; Salamone et al., 2007), which may contribute to the lower rate of pressing during reward-seeking observed across the two reward-seeking tasks examined.  An open question stemming from these results relates to why the NAcS, in comparison to the NAcC, preferentially regulates response-inhibition. One likely explanation relates to the partially segregated pattern of afferent input made to each region. The NAcS receives projections from regions of the vmPFC and caudal BLA that regulate response-inhibition, while neurons in the dorsal mPFC and rostral BLA that promote behavioral activation project to the NAcC (Berendse, Galis-de Graaf, et al., 1992; Kita & Kitai, 1990; Sesack et al., 1989). Thus, when an animal encounters a cue that predicts punishment, for example, glutamatergic activity from vmPFC or caudal BLA may enhance activity in a subpopulation of neurons within the NAcS that regulate response-inhibition. On the other hand, when the promotion of an active behavior is necessitated, dorsal mPFC and rostral BLA may preferentially be activated to carry out this function. In fact, these afferent projections have in many cases been borne out experimentally (Ambroggi et al., 2008; Ghazizadeh et al., 2012; Ishikawa et al., 2008; McGinty & Grace, 2008; 176  Setlow, Roozendaal, & McGaugh, 2000). Still, this circuit description is clearly oversimplified, as a fully segregated circuit is not supported by physiological or pharmacological analyses, as illustrated in the present data and previous work (Ambroggi et al., 2011, 2008; Ishikawa et al., 2008). For example, both NAcC and NAcS promote actions under some circumstances, such as when performing an active avoidance. It is conceivable that this similarity in effect is mediated by the extant, but potentially more sparse, overlapping projections from these afferent regions. Still, the generation of dissociable functions within these two regions can likely be attributed in part to differential afferent input.   Once these subregions have been activated, they must enact changes in response promotion or inhibition via downstream projections. In comparison to study of the NAc afferents that mediate complex forms of action selection, less is known about the downstream mediators of such effects. Regardless, NAcS and NAcC project to largely distinct target areas, with NAcC maintaining mostly inter-basal ganglia projections to structures like the substantia nigra and lateral ventral pallidum, while NAcS projects to limbic associated structures, including the ventral tegmental area (VTA), lateral hypothalamus, and medial ventral pallidum (Berendse, Groenewegen, et al., 1992; Groenewegen et al., 1999; Ikemoto, 2007; Pennartz et al., 1994; Zahm & Brog, 1992; Zahm & Heimer, 1993). Projections from NAcC to the basal ganglia leave it poised to directly affect motor actions, consistent with the integral role of this nucleus in the promotion of active behaviors reported here and elsewhere (Ambroggi et al., 2008; Ghods-Sharifi & Floresco, 2010; Ishikawa et al., 2008; Salamone et al., 2007; Saunders & Robinson, 2012). For example, the ventral pallidum regulates the interaction between cortico-basal ganglia loops that are necessary for reward-related behavior (for review, see Smith, Tindell, Aldridge, & Berridge, 2010). These researchers propose that the ventral pallidum acts as a “final common 177  pathway” for limbic input to influence approach behavior, in this case mediating reward-seeking. The dense projection from NAcC to the ventral pallidum may also allow this region to promote both appetitive and aversively-motivated actions, as a function of limbic-striatal-pallidal interactions. Compared to the intra-basal ganglia projections made by the NAcC, projections from the NAcS are relatively more diverse, consistent with the notion that this nucleus is a transition zone between the extended amygdala (Alheid, 2003; Heimer et al., 1997). The projection from the NAcS to the VTA may have direct relevance to aversively-mediated response-inhibition. Optogenetic self-stimulation of VTA dopamine induces plasticity in NAcS neurons, increases the excitability of OFC neurons, and produces punishment-resistant seeking of cocaine (Pascoli et al., 2015). As NAcS neurons are primarily GABAergic, activity of these neurons would be expected to inhibit VTA dopamine cells. Such a projection could phasically inhibit VTA dopamine cells, preventing the activity necessary to produce reward-seeking during danger. Supporting this dopamine-disinhibition account of punishment resistance is evidence that silencing a key inhibitory afferent to the VTA, the rostromedial tegmentum, produces reward-seeking during punishment similar to that which was produced by NAcS (or BLA) inactivation here (Vento et al., 2017).  In addition, a pathway from the NAcS to the lateral hypothalamus has been directly linked to the inhibition of drug-seeking (Millan et al., 2010). Following the extinction of alcohol seeking, inactivation of the NAcS enhances reinstatement, while increasing activity in neuropeptidergic cells within the lateral hypothalamus. Silencing the lateral hypothalamus eliminates the effect of NAcS inactivation on reinstatement, suggesting that tonic inhibition of the lateral hypothalamus by the NAcS enforces the learned inhibition of alcohol seeking (Millan 178  et al., 2010). Evidence implicating the lateral hypothalamus in aversion-mediated response-inhibition comes from another study examining input from this region to the VTA during punished sucrose-seeking (Nieh et al., 2015). Stimulation of this lateral hypothalamus to VTA pathway disinhibits punished seeking, while silencing this circuit inhibits the same behavior, an opposite pattern that would be expected if this effect was further mediated by the NAcS. Thus, despite NAcS projecting to the lateral hypothalamus, this target region appears to function in an opposite manner. Such a paradoxical finding implies that these regions may not function in parallel during punishment, instead operating in concert with other relevant afferents. Overall, further work is necessary to identify downstream targets of NAcC and NAcS through which they can accomplish their respective roles in response-promotion and inhibition.  6.2 AMPH induces task-dependent bidirectional changes in instrumental punishment In addition to probing NAc function during motivational conflict and active/passive avoidance, we examined potential monoaminergic contributions to both behaviors by administering a systemic dose of AMPH. AMPH administration provokes the release dopamine and serotonin (Kuczenski & Segal, 1989; Seiden et al., 1993; Sitte & Freissmuth, 2015; Sulzer, Sonders, Poulsen, & Galli, 2005), and has been used extensively to probe constructs such as incentive salience and impulse control. Here, this manipulation provided valuable insight into neurochemical targets related to aversively-motivated response inhibition. Previous research has suggested that AMPH-induced monoamine release may enhance punishment sensitivity, particularly in situations where punishment is associated with reward-seeking behavior (Broersen et al., 1995; Geller & Seifter, 1960; Killcross et al., 1997; Lazareno, 1979; Leone et al., 1983). Thus, when an instrumental action associated with reward-seeking is punished, AMPH or other monoamine-releasers diminish seeking. Results of an exploratory analysis conducted here 179  suggested that AMPH similarly facilitated response-inhibition during punishment, supporting the external validity of our Conflict task.  A similar pattern of behavior has been observed during performance on a more complex decision-making assay, where rats have the option to choose a lever that delivers a small amount of reward, with no chance of punishment, or another lever that delivers a larger reward with a probability of punishment that increases across discrete trial blocks. AMPH administration biases rats away from the lever that delivers a large reward and a probabilistic shock, indicative of enhanced punishment sensitivity (Mitchell et al., 2011; Orsini, Trotta, et al., 2015; Simon et al., 2011, 2009). This effect appears to be mediated by the dopamine D2 receptor, as antagonism of this receptor blocks the impact of AMPH on risky choice (Simon et al., 2011).   These results suggest the intriguing possibility that dopamine D2 receptors within the NAcS may promote response-inhibition during punished reward-seeking. Activity at these receptors in the NAcS has previously been shown to oppose impulsive actions (Besson et al., 2009). Similarly, highly impulsive animals have lower levels of D2 receptor expression within the ventral striatum, an effect which is predictive of enhanced escalation of seeking of the psychostimulant cocaine (Dalley et al., 2007). Impulsivity has also been directly related to the taking of cocaine in a compulsive manner, operationalized as perseverance through foot-shock punishment (Belin, Mar, Dalley, Robbins, & Everitt, 2008). Thus, dopaminergic activity and receptor-expression within the NAcS may similarly relate to putative compulsive reward-seeking, such as perseveration through instrumental punishment.   In contrast to the apparent promotion of suppression mediated by AMPH during conflict, this same manipulation caused rats trained on an active/passive avoidance task to produce more passive avoidance failures, indicative of a loss of response-inhibition. Despite this change in 180  passive failure rate, rats maintained the ability to inhibit responding upon receipt of a painful stimulus, as the total number of passive presses did not differ following AMPH treatment. This later finding implies that pain sensitivity is intact following AMPH administration, suggesting that alterations in punishment-induced response-inhibition were not due to changes in pain threshold.  The effect of AMPH on passive avoidance trials is in keeping with data suggesting that AMPH administration can cause response-inhibitory deficits on No-Go trials of a Go/No-Go task (Blackburn & Hevenor, 1996), and induce impulsive actions, a subtype of impulse control deficit that reflects motor behavior produced without forethought (Pattij et al., 2007). Lesions of the NAcS block the impact of AMPH on impulsive actions (Murphy et al., 2008), as does intra-NAcS blockade of D2./3 receptors (Pattij et al., 2007). Given that both the Conflict and active/passive avoidance tasks assess the withholding of a punished response, it is surprising that AMPH would produce an opposite pattern of results on each task. As outlined in Chapter 4, AMPH has been proposed to affect task performance based on the salience of options or outcomes (Orsini, Moorman, et al., 2015). For example, on the Conflict task, a behavior that provokes a shock is further inhibited by AMPH because the shock is more salient than the relatively richer schedule of reinforcement. Given that rats were trained on the active avoidance portion of the task first, animals apply more salience to the active avoidance cue. This would lead to a bias towards active avoidance, which may enhance the prepotency of this response. Support for such an account is provided by the relatively higher levels of passive avoidance failure, as compared to active avoidance failure observed in rats at baseline. AMPH administration may further enhance this bias, promoting approach behavior to a pathological degree, and causing passive avoidance failures.  181  6.3 Experimental merits and future directions While the present results provide meaningful insight into brain regions that are relevant to aspects of aversive-motivation, a number of methodological issues bear considering. First, these studies were conducted primarily using a single methodology, reversible pharmacological inactivations. This consistency was necessary to facilitate the generalization of findings related to aversively-motivated response-inhibition across tasks. Additionally, inactivations are a preferable first pass technique to traditional permanent lesion studies for examining novel functions of brain nuclei, as they are likely less susceptible to compensatory mechanisms that may obscure the role of the targeted region (Poulos, Ponnusamy, Dong, & Fanselow, 2010; Zelikowsky, Bissiere, Hast, Bennett, & Abdipranoto, 2013). Still, the limitations of this technique warrant discussion. First, we targeted small brain subnuclei, which are often separated by less than 1 mm. This proximity raises the possibility that our effects may be mediated in part by diffusion from the targeted region into neighboring regions. Most studies examining the functional spread of microinfusions conducted in the manner described here have found that functional spread ranges from between 0.5-3 mm in situ (Allen et al., 2008; Edeline, Hars, Hennevin, & Cotillon, 2002; Lorenzini et al., 1995). Thus, there is some possibility that contamination in surrounding regions may explain some of the present observations. Although we cannot exclude this possibility, the key behavioral dissociations observed in the majority of studies described here were in opposite directions, which would be difficult to reconcile based simply on drug diffusion outside of the region of interest. In many of the cases presented here, results fit into a theoretical framework outlined in directional hypotheses, a fact that would be inconsistent with a non-specific drug effect. Similarly, we have used these same infusion parameters to dissociate these two regions on a variety of behavioral tasks, previously (Dalton et al., 2014; Floresco et al., 2008; Ghods-Sharifi & Floresco, 2010; Stopper & Floresco, 2011). 182  Still, new techniques have been developed that will aid in testing the hypotheses generated by the present thesis with a higher degree of specificity. The most relevant of these techniques are optogenetics and chemogenetics, both of which utilize viral or genetic methods to target cells, enabling the expression of engineered channels that can be manipulated precisely without relying on drug diffusion (Britt & Bonci, 2013; Johansen, Wolff, Lüthi, & Ledoux, 2012; Roth, 2016; Stuber, Britt, & Bonci, 2012). Broadly, these techniques are extremely well-suited for the investigation of small brain nuclei. For example, optogenetic manipulations allow particular brain subnuclei to be infected and then targeted with light, minimizing concerns regarding drug diffusion. Similarly, chemogenetic techniques allow for the expression of receptors engineers to respond to a specific, non-bioactive ligand. Once receptors have been infused into a particular subregion, this ligand can be administered systemically at doses that cross the blood-brain barrier (but see Gomez et al., 2017), eliminating the need to directly infuse drug into a brain region, which can potentially impact baseline behavior (see Chapter 2, infralimbic effect prior to test). Similarly, circuit-based investigations can be conducted with more confidence regarding the anatomical specificity of the targeted projection. Using the PLNAcS disconnection experiment from Chapter 3 as an example, virus coding for an excitatory or inhibitory channel could be infused into the PL, which is eventually trafficked in an anterograde fashion and expressed in axon terminals in projection regions. Optic fibers can then be implanted in the terminal region of interest, in this case, the NAcS, allowing for light-based manipulation of PL axon terminals located in the NAcS. Such an experiment eliminates the necessity of ipsilateral control groups, for example, as stimulation of terminals eliminates the possibility that an effect is mediated by projections to the contralateral hemisphere or a third brain region.  183  In addition to the refinement in anatomical targeting, the temporal specificity afforded by these modern manipulations is dramatically better than that provided by pharmacological inactivations. Optogenetic stimulation or inhibition allows for precise, millisecond control over neuronal activity. On tasks such as those conducted here, the bolus infusion of receptor agonists eliminates activity for upwards of two hours (Duuren et al., 2007; Edeline et al., 2002). While pharmacological methods allow for the gross assessment of a region’s contribution to behavior, they preclude the assessment of which specific task epochs the region is involved in. Given that neural activity is often time-locked to particular task events (Ambroggi et al., 2011; Burgos-Robles, Vidal-Gonzalez, & Quirk, 2009; Kim et al., 2017; Nieh et al., 2015), modulating neuronal activity during such periods could refine our understanding of the contributions that each brain region makes to specific components of a given behavior. This may help tease apart effects like those observed on the Conflict task (Chapter 4), where the impact on reward-seeking during safety may be mediated by functions unrelated to the direct inhibition of reward-seeking during punishment, for example.  Such precision is necessary as it is becoming increasingly apparent that even within brain subnuclei, heterogeneous populations of neurons exist that may not have the same impact on behavior. For example, Kim and colleagues (2017) demonstrated that neural activity within a projection from mPFC to the NAcS, but not from mPFC to VTA, was correlated with response-inhibition during potential threat. This circuit was then broken down even further, with only a subpopulation of shock-activated cells within the mPFC to NAcS projection being of crucial relevance to the suppression of reward-seeking during danger. Thus, while pharmacological disconnections may be able to identify the necessity of one projection versus another, the ability to delve deeper into circuit-based mechanisms requires the use of optogenetic or chemogenetic 184  techniques paired with molecular and activity-based tagging of neurons. Some combination of these techniques could be used to probe the circuit-basis of the effects observed in Chapters 4 and 5. For example, the downstream region through which BLA enforces the inhibition of reward-seeking behavior during punishment is currently unknown. Given the results outlined in the present thesis, the NAcS may be one such output region, which could be confirmed by infusing virus coding for an excitatory or inhibitory channel into the BLA, and placing optic fibers into the NAcS to stimulate or inhibit activity within this projection. Similarly, the active/passive avoidance task is likely mediated by cortico-limbic-striatal circuitry, and contains numerous time-locked events that would be amenable to interrogation through a combination of in vivo electrophysiology and optogenetic or chemogenetic manipulation. These experiments could be accompanied by receptor-specific pharmacological manipulations to examine the contribution of various neuromodulators, such as dopamine acting at the D2 receptor within the NAcS, to the behaviors identified in this thesis, as there is no methodological substitute at present for the investigation of these targets (Jenni et al., 2017). A final important limitation of the present data set is that our main outcome measure for Chapters 2-4 was conditioned suppression, which is the absence of a response. This measure was chosen as our stated interest was in the impact of fear on motivated behavior, emphasizing aversively-motivated response-inhibition. While this measure often correlates strongly with other measures of conditioned fear, such as freezing (McDannald & Galarce, 2011; Sierra-Mercado et al., 2011), we can only speculate on what the animal is doing during these task epochs, as our operant chambers are not equipped with cameras for the assessment of other defensive reactions. Given that the expression of conditioned freezing is generally incompatible with ongoing operant behavior, it is possible that some of our results may be explained by changes in conditioned 185  freezing. With regards to motivational conflict, previous studies using similar methodology have demonstrated that freezing is essentially eliminated during punishment training, and is not affected by BLA inactivations that disinhibit instrumental behavior during punishment (Jean-Richard-Dit-Bressel & McNally, 2015). Thus, it is unlikely that these results could be explained by changes in the expression of freezing.  Similarly, previous work has shown that freezing diminishes over the course of training on an active avoidance task where rats concomitantly can lever-press for reward (Bravo-Rivera et al., 2014; Oleson et al., 2012). While not identical to the present active/passive avoidance task, one study illustrates that the refinement of behavior when behavioral responses compete (in this case, active avoidance involves standing on a platform that entirely prevents reward-seeking) involves a decrease in freezing and an increase in reward-seeking and avoidance. However, Bravo-Rivera and colleagues (2014) have shown that inactivation of the NAc (mostly targeting the NAcC) dramatically impairs active avoidance, in part by potentiating freezing during presentations of the active avoidance stimulus. Thus, a possible explanation for the decrease in avoidance or motivational conflict performance in NAcC-inactivated animals is an increase in conditioned freezing. One piece of evidence suggesting that freezing alone may not explain the diminished active avoidance observed following NAcC inactivation is that locomotor activity was normal following NAcC inactivation. If NAcC-inactivation caused freezing to predominate during active/passive avoidance, one might expect that the level of locomotor activity would be lower, which was not the case. Still, concurrent measurement of freezing during these tasks is the only way to truly eliminate this possibility in the future.  186  6.4 Relevance to neuropsychiatric disease  While we view this work through the lens of basic science, it is important to consider what implications the present results have for brain dysfunction, as occurs in numerous neuropsychiatric conditions. Of the most relevance to the present experiments are disorders characterized by compulsive or impulsive patterns of behavior. For example, in substance abuse, reward-seeking often occurs despite negative punishment (American Psychiatric Association, 2013). This phenotype has been suggested to be due to a deficit in the response-inhibition typically induced by an aversive consequence, and can be assessed pre-clinically by using conditioned suppression paradigms (Belin-Rauscent et al., 2016; Chen et al., 2013; Limpens et al., 2014; Nieh et al., 2015; Pascoli et al., 2015). In humans, homologous regions of the prefrontal cortex to those which we showed are involved in suppressing reward-seeking during instrumental punishment and Pavlovian fear in rats, have been shown to be hypoactive during impulse control in cocaine addicts (Goldstein & Volkow, 2011; Morein-Zamir et al., 2013). Interestingly, these prefrontal deficits are related to decreased dopamine D2 receptor expression in the NAc of addicted individuals, even following protracted drug-abstinence (Volkow et al., 2009; Volkow, Wang, Fowler, Tomasi, & Telang, 2011). These D2 receptors are thought to be inhibitory, suggesting that a loss of signaling at this dopaminergic substrate may contribute to inhibitory control deficits (Everitt et al., 2008; Volkow & Morales, 2015). Unfortunately, technological limitations in human imaging have not permitted the subregional assessment of such effects. Taken together, these findings strongly implicate the prefrontal cortex and NAc in aspects of response-inhibition of direct relevance to substance use disorders.  Our results are also potentially relevant to disorders of fear or anxiety, which are characterized by deficits in fear discrimination, extinction, and aberrant avoidance (Duits, Cath, 187  Lissek, Hox, Hamm, Engelhard, van den Hout, et al., 2015; Graham & Milad, 2011; Jovanovic & Norrholm, 2011; Lissek et al., 2014; Maner & Schmidt, 2006). For example, activity within human ventromedial PFC promotes extinction (Milad, Wright, et al., 2007), while activity of the dorsal ACC promotes fear expression (Delgado, Nearing, et al., 2008; Milad, Quirk, et al., 2007). In the present study, we recapitulated a dorsal ACC-like effect by examining the function of PL cortex during early fear extinction, inactivation of which potently inhibited fear expression. IL cortex, which has been described as being functionally homologous to the ventromedial PFC in humans (Heilbronner et al., 2016; Milad & Quirk, 2012), had a similar effect, in contrast to its established role in fear extinction. Although methodological concerns clouded the interpretation of this result, we suggest that ventral regions of the PFC like the IL cortex may promote fear expression under certain conditions, such as when conflict exists between opposing motivational drives. Such a function is consistent with the deficits observed in individuals with damage to the vmPFC on tasks assessing emotion-guided decision-making, such as under conditions of risk (Bechara, Damasio, Damasio, & Anderson, 1994; Bechara et al., 2000; Bechara, Damasio, Damasio, & Lee, 1999; Clark et al., 2008).  Our results further suggest that mPFC and NAc are not critical for fear discrimination, which is characteristically disturbed in individuals suffering from anxiety or post-traumatic stress (Duits, Cath, Lissek, Hox, Hamm, Engelhard, van den Hout, et al., 2015), as none of our manipulations impacted the level of fear expressed towards the CS-. This is particularly interesting given that we have previously shown that mPFC disinhibition can elevate fear expressed towards a CS-, while simultaneously decreasing fear towards a CS+, indicating a loss of discrimination (Piantadosi & Floresco, 2014). Such results imply that regions downstream to the mPFC, such as the basal amygdala, which has been shown to encode the presentation of a 188  neutral CS- in non-human primates (Genud-Gabai et al., 2013), or ventral hippocampus, which is hyperactive during fear generalization in patients with post-traumatic stress disorder (Kaczkurkin et al., 2017), may mediate fear discrimination. Finally, we evaluated response-inhibition and promotion during a fully aversively-mediated active/passive avoidance task. In humans, active avoidance has been associated with ventral striatal activity, which includes the NAc (Delgado et al., 2009; Levita et al., 2012). The degree of NAc activation during active avoidance has been positively correlated with state anxiety, suggesting that high anxiety may co-occur with high levels of avoidance, consistent with clinical findings (Dymond & Roche, 2009). To date, only one study has examined the response-inhibitory pole of passive avoidance, with results suggesting that NAc deactivations may be of critical importance to this behavior (Levita et al., 2012). In this thesis, we observed that NAcS inactivation provoked inhibitory control failures, an effect opposite to what would be predicted from this previous imaging study. Although these results may relate to the difficulty (and possibly inaccuracy) of interpreting BOLD deactivations , they do provide evidence that neurons within the NAc are sensitive to passive avoidance performance (Hayes & Huxtable, 2012; Mishra et al., 2011). Thus, further basic and translational research on this task, ideally utilizing imaging techniques that can dissociate the major subdivisions of the NAc in humans, and employing manipulations with improved anatomical and temporal specificity in rats, is necessary.  6.5 Conclusion Overall, the present results add to a growing body of literature suggesting that the heterogeneity within brain regions may have important functional implications. Here, we have dissociated the two major subregions of the NAc, the shell and core, during aspects of 189  aversively-motivated behavior. Whether assessed in a Pavlovian or instrumental fashion, response-suppression motivated by a potential aversive consequence was mediated by the NAcS, while the NAcC simply promoted motivational vigor. In the case of Pavlovian fear expression, a functional circuit between the PL cortex and NAcS appeared to mediate this effect, while qualitative similarities existed between the functions of the BLA and NAcS during motivational conflict. Similarly, performance of a complex avoidance behavior that required response-promotion and response-inhibition necessitated the function of these subnuclei, with both regions being necessary for normal response-promotion, and the NAcS being necessary for response-inhibition.  This thesis represents some of the first evidence for a dissociable function of these regions in aversively-motivated behavior. These results are generally coherent hypotheses suggesting a role for the NAcC in approach behavior, and the NAcS in response-suppression. They also provide more evidence against a reward-specific interpretation of NAc function (Levita et al., 2009; Salamone, 1994). 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