SOHINI DUTTA, Ph.D., May 2021 BIOMEDICAL SCIENCES

DISSOCIABLE ROLES OF THE NUCLEUS ACCUMBENS CORE AND SHELL

SUBREGIONS IN THE EXPRESSION AND EXTINCTION OF CONDITIONED FEAR

DISSERATION ADVISOR: Dr. AARON M. JASNOW

The nucleus accumbens (NAc), consisting of core (NAcC) and shell (NAcS) sub-regions, has primarily been studied as a locus mediating the effects of drug reward and addiction. However, there is ample evidence that this region is also involved in regulating aversive responses, but the exact role of the NAc and its subregions in regulating associative fear processing remains unclear.

Here, we investigated the specific contribution of the NAcC and NAcS in regulating both fear expression and fear extinction. Using Activity-Regulated Cytoskeleton-Associated protein (ARC) expression as an indicator of neuronal activity, we first show that the NAcC is specifically active only in response to an associative fear cue during an expression test. In contrast, the NAcS is specifically active during fear extinction. We next inactivated each subregion using lidocaine and demonstrated that the NAcC is necessary for fear expression, but not for extinction learning or consolidation/retention of extinction memory. In contrast, we demonstrate that the NAcS is necessary for the consolidation of extinction memory, but not fear expression or extinction learning. Further, inactivation of mGluR1 or ERK signaling specifically in the NAcS disrupted extinction memory but had no effect on fear expression or extinction learning itself. Our data provide the first evidence for the importance of the ERK/MAPK pathway as an underlying neural mechanism facilitating extinction consolidation within the NAcS. These findings suggest that the

NAc subregions play dissociable roles in regulating fear recall and the consolidation of fear

extinction memory, and potentially implicate them as critical regions within the canonical fear circuit.

DISSOCIABLE ROLES OF THE NUCLEUS ACCUMBENS CORE AND SHELL

SUBREGIONS IN THE EXPRESSION AND EXTINCTION OF CONDITIONED FEAR

A dissertation submitted

to Kent State University in partial fulfillment

of the requirements for the

degree of Doctor of Philosophy

Sohini Dutta

May 2021

Except for previously published materials

Dissertation written by

Sohini Dutta

B.Sc., University of Delhi, India, 2012

M.Sc., Bangalore University, India, 2014

Ph.D., Kent State University, Ohio, 2021

Approved by

Aaron M. Jasnow, Ph.D. , Chair, Doctoral Dissertation Committee

Colleen M. Novak, Ph.D. , Members, Doctoral Dissertation Committee

Gail C. Fraizer, Ph.D.

John Johnson, Ph.D.

John Gunstad, Ph.D.

Accepted by

Ernest J. Freeman, Ph.D. , Director, School of Biomedical Sciences Mandy Munro-Stasiuk, Ph.D. , Interim Dean, College of Arts and Sciences

TABLE OF CONTENTS

TABLE OF CONTENTS………………………………………………………………………….v

LIST OF FIGURES……………………………………………………………………………...vii

LIST OF TABLES………………………………………………………………………………..ix

LIST OF ABBREVIATIONS……………………………………………………………………..x

ACKNOWLEDGEMENTS……………………………………………………………………...xii

CHAPTER 1: GENERAL INTRODUCTION………...... ………………………………...... 1

1.1 ANXIETY DISORDERS AND TRAUMA-RELATED DISORDERS………………………2

1.2 GENERAL INTRODUCTION OF NUCLEUS ACCUMBENS………...... …………12

1.3 GLUTAMATERGIC TRANSMISSION IN FEAR EXPRESSION AND FEAR

EXTINCTION...... 16

1.4 ERK/MAPK PATHWAY………………...... …………………………………19

1.5 SPECIFIC AIMS……………………………...... ……………………………………23

1.6 REFERENCES……………………...... ………………………………………………24

CHAPTER 2: THE NUCLEUS ACCUMBENS CORE IS NECESSARY FOR FEAR

EXPRESSION WHEREAS THE SHELL REGULATES FEAR EXTINCTION

CONSOLIDATION………………………………...... ……………36

2.1 INTRODUCTION……………………………………...... ………………………………36

2.2 METHODS.……………………………………………...... …………………………...... 41

2.3 RESULTS...... 48

2.4 DISCUSSION...... 63

2.5 REFERENCES...... 66

CHAPTER 3: MGLUR1 ACTIVATION AND THE ERK/MAPK SIGNALING PATHWAY

WITHIN THE NUCLEUS ACCUMBENS SHELL IS NECESSARY FOR FEAR

EXTINCTION CONSOLIDATION...... 72

3.1 INTRODUCTION...... 72

3.2 METHODS...... 75

3.3 RESULTS...... 80

3.4 DISCUSSION...... 93

3.5 REFERENCES...... 95

CHAPTER 4: GENERAL INTRODUCTION...... 99

4.1 LIMITATIONS AND FUTURE DIRECTIONS...... 108

4.2 REFERENCES...... 110

LIST OF FIGURES

Figure 1 Pavlovian fear conditioning and extinction of conditioned fear in rodents...... 6

Figure 2 Schematic illustrating the signaling pathway from the group 1 metabotropic glutamatergic receptors (mGluR1) to the MAPK/ERK cascade in the neurons...... 22

Figure 3 A simplified schematic of major efferents to the Nucleus Accumbens Core (NAcC) and shell (NAcS) from the Basolateral Amygdala (BLA), Infralimbic Cortex (IL), Prelimbic Cortex

(PL) and the Ventral Tegmental Area (VTA)...... 52

Figure 4 Schematic of ICC analysis experiment...... 62

Figure 5 The nucleus accumbens core is selectively active during fear expression whereas the shell is selectively active following fear extinction...... 64

Figure 6 Confocal images of Calbindin and ARC expression in the NAc core and shell...... 66

Figure 7 Inactivation of the NAc core disrupts fear expression but not extinction learning or the consolidation of extinction...... 69

Figure 8 Inactivation of the NAc shell disrupts the consolidation of fear extinction but has no effect on fear expression or extinction learning...... 72

Figure 9 Group I metabotropic glutamate receptors within the NAc shell mediate the consolidation of fear extinction...... 98

Figure 10 Group I metabotropic glutamate receptors within the NAc core do not mediate fear expression or extinction learning...... 101

Figure 11 Pre-extinction infusions of 1ug/side U0216, within the NAc shell, disrupts fear extinction consolidation whereas 0.5ug/side U0216 has no effect on fear extinction...... 105

vii

Figure 12 Post-extinction infusions of 1ug/side U0216, within the NAc shell disrupts fear extinction consolidation...... 107

viii

LIST OF TABLES

Table 1 Behavioral Statistical Summary of experiments 2.1,2.2, and 2.3...... 74

Table 2 Behavioral Statistical Summary of experiments 3.1,3.2, and 3.3...... 109

LIST OF ABBREVIATIONS

AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

ANOVA Analysis of variance

ARC Activity-regulated cytoskeleton-associated protein

BA Basal amygdala

BDNF Brain-derived neurotrophic factor

BLA Basolateral amygdala

CB-1 Cannabinoid receptor-1

CeA Central amygdala

CREB cAMP-response element binding protein

CS Conditional stimulus

CT Computerized tomography

D1 Dopamine receptor 1

D2 Dopamine receptor 2

DA Dopamine

DSM Diagnostic and Statistical Manual

ERK Extracellular signal-regulated kinase fMRI Functional magnetic resonance imaging

GABA Gamma aminobutyric acid

GDP Guanosine diphosphate

GTP Guanosine triphosphate

IEG Immediate early gene

IL Infralimbic cortex

ITI Inter-tone interval

LA Lateral amygdala

LTD Long-term depression

LTP Long-term potentiation

MAPK Mitogen-activated protein kinase

MEK MAPK/ERK kinase mGLUR Metabotropic glutamatergic receptor mPFC Medial prefrontal cortex

MSN Medium spiny neurons

NAc Nucleus accumbens

NAcC Nucleus accumbens core

NAcS Nucleus accumbens shell

NMDA N-methyl-D-aspartate

OCD Obsessive compulsive disorder

PB Phosphate buffer

PBS Phosphate-buffered saline

PL Prelimbic cortex

PLC Phospholipase C

PTSD Post-traumatic stress disorder

US Unconditional stimulus vmPFC Ventromedial prefrontal cortex

ACKNOWLEDGEMENTS

“Arise, awake, stop not till the goal is reached.” – Swami Vivekananda

The journey of a PhD is a long and arduous one with its fair share of ups and downs. It made me realize that while the joy of running experiments and discovering novelties is an excitement that’s unparalleled, majority of the doctoral training is about failures and patience. The hurdles become easier to handle and overcome when surrounded by people who keep the motivation alive with their love, support, guidance, and mentorship. This is a dedication to the village of wonderful people I was fortunate to be surrounded by and who made it all worth it at the end.

To begin, I would like to thank Dr. Aaron Jasnow for his guidance these last 5 years. He has always pushed me to perform to my highest standard and over the last few years, has managed to bring out the best in me. He has allowed me to make mistakes, learn from them, expand my boundaries to learn new techniques outside of my dissertation projects while guiding me throughout my doctoral dissertation. To Dr. David Riccio, whose years of wisdom and knowledge have been instrumental in learning how to design my experiments better and his kindness has always instilled a sense of calm even in moments of uncertainty. To my committee members, Dr. John Johnson, Dr. John Gunstad, and Dr. Coleen Novak I would like to thank them for their insight and help during my graduate studies.

To Jordan and Samantha, who are women of inspiration in their own right. They have been an unwavering support system throughout my time in the Jasnow lab and have been a constant cheerleader through good times and the not-so-great ones. The special moments we’ve spent together are countless, and I appreciate every single one of them. To Jordan, whose silent support and pranks have been a source of strength and laughter without which life would have seemed a bit dull. To Samantha, whose clarity, organization and experimental designing expertise is only

xii

second to her kindness and empathy. To Maeson, who welcomed me with a wide smile and warmth into the Jasnow lab and I cherish the times spent together, within and outside of lab. To

Dr. T. Lee Gilman, for taking me under her wing when I was just beginning, wide eyed with excitement but lacking majority of the skills required. To all the research assistants, especially

Jasmin and Carly, who’ve now entered the next phase of their education as PhD candidates; their help in running experiments and thus collecting data was a huge contribution to my dissertation.

To Manasi and Monica, who became family in a country thousands of miles away from my own.

They were the work partners in coffee shops, the shoulders to lean on when the days of homesickness hit a little too hard and most importantly, a formidable force that kept me going even during days that never seemed to end. Manasi has also been the person to exchanging scientific ideas with, learning in and outside of lab, to venting out and co-organizing events with.

To Megan, who was my first friend in this country and her passion for science can only be rivalled by her genuine warmth and friendship. To Isha, I’ll always cherish our late-night assignment completion and navigating graduate life together. To Nikita, who lights up the room just by being present.

I wouldn’t be here without my family and friends (in India and around the world) who are my pillars in life. My aunts and uncles, my cousins, my nephews and nieces, their love and support- which have always been present and in abundance. Apart from the weekend video calls, I have had the privilege to take for granted that anytime I call, the person on the other side will always pick up. To my family in Houston- Aaju, Buria didi, Sandipto, Karna and Bheem and Columbus-

Rinku mashi, Raj mesho, Nishant and Prateek, apart from their love, it wouldn’t have been possible to live this far away from home without them being so nearby. From helping me settling in in my first year to fixing my auto and technical issues from miles away and everything in

xiii

between, I knew I could always fall back on them for anything and everything. To my oldest friends, Sreetama, Swet, Shruti, Shefali, Shria, and Shweta, who are more of a family than friends. I can’t remember a time they haven’t been in my life. Even after 2 decades, they continue to fill my life with laughter, and I am privileged that I get to grow up with them and have had their constant council and wisdom throughout this journey and beyond. They are my haven, and words are not enough to express my gratitude for their friendship. To Greeshma for teaching me to never hold a grudge against anyone and for making the biggest troubles seem tiny with her carefree attitude. To Rahul, Naman, and Arya for the fun and laughter and making new memories. To the altruistic community of STEMPeers and all the selfless people I’ve had the fortune to interact with and become friends with in the last 1 year, thank you for the positivity and celebrating every small win. To the furry friends (dogs)- Karna, Hoppy, Bheem, Twinkle and Oscar for their unconditional love and innate ability to cheer up my mood every single time I met any one of them.

Lastly, to the two most important people in my life; my parents. They have pushed me to chase my dreams while ensuring my feet stay firmly rooted to the ground beneath and guided me back whenever I drifted away. They have always believed in me and have made countless sacrifices for my happiness, to provide me with the best of education and cultural experiences. My mother has taught me resilience and my father has been my guiding force and source of wisdom for everything in life. I am what I am because of them, because of their love and support. This one, is for the two of them.

xiv

CHAPTER 1

General Introduction

1.1 Anxiety disorders and trauma- and stressor-related disorders

Anxiety disorders and trauma- and- stressor- related disorders (subsequently referred to as trauma-related disorder for the remainder of this document) such as post-traumatic stress disorder (PTSD), are some of the most predominant categories of neuropsychiatric disorders which rank among the top three global disease burden with an increasing trend in the last few decades. A study across 44 countries found that at any given time 1 out of 14 people are diagnosed with a clinically significant anxiety disorder (Baxter et al., 2013). In addition, a study across 32 countries found that lifetime prevalence of PTSD ranges from 1.3-11.7% (Michael et al., 2007a). Further, adults in the age group of 35-54 years are 20% more likely to have anxiety disorders as compared to older adults with women having a higher likelihood than men to be diagnosed with anxiety disorders (McLean et al., 2011; Baxter et al., 2013). Increased prevalence of anxiety disorders has been associated with substance abuse, decreased workplace productivity, and increased mortality, thus presenting a huge socio-economic burden (Greenberg et al., 1999;

Wittchen, 2002; Andlin-Sobocki and Wittchen, 2005; Kessler et al., 2012). Further, most anxiety disorders are comorbid with other neuropsychiatric disorders such as major depressive disorder and substance abuse disorder and these comorbidities exist across all developmental stages

(Essau et al., 2018). In fact, the rate of co-morbidity for any anxiety disorder is approximately

74.1% whereas that of PTSD is at 81%. Even though a high comorbidity between substance

abuse and anxiety and trauma-related related disorders has been established, the underlying neural neurobiology remain poorly understood. The present study aims to dissect the contribution of the nucleus accumbens subregions, a common node in both the appetitive and aversive domain to shed light on theories suggesting aversive learning engages systems that are associated with appetitive behavior (Abraham et al., 2014). This fundamental knowledge will bridge the gap in our understanding of how disruption of one functioning capacity (fear) affects other regulatory functions (reward, drug- seeking behavior) and in the identification of new and common biomarkers and treatment strategies.

1.1.1 How are Fear and Anxiety Defined?

Anxiety and trauma-related disorders are neuropsychiatric disorders which are characterized by excessive fear and anxiety or persistence of negative emotional states such as fear along with anhedonic, dysphoric or aggressive symptoms, respectively (American Psychiatric, 2013).

Evolutionarily, fear and anxiety have induced appropriate adaptive responses to ensure survival of an organism to a real threat, danger, or conflict, or in the anticipation of threat. These responses include behavioral activation, such as fleeing/avoiding, fighting, or freezing, and autonomic responses such as increased blood pressure and heart rate or increased release of several stress-associated hormones (Katkin, 1965; Ziegler, 2012; Hu et al., 2016; Latsko et al.,

2016). Over the years there have been extensive debate on whether the terms fear and anxiety can be used interchangeably or not. While a growing literature provides evidence for distinguishing fear from anxiety, there remains a lack of clear conceptualization and distinction.

On a theoretical level, if fear and anxiety are different constructs, they must be initiated by different causal events and elicit different consequences. As argued by some theorists and clinical researchers, fear produces a defensive reaction, produced in response to a specific

dangerous or threatening stimulus whereas anxiety does not have a specific inducing stimulus, instead results from prolonged hypervigilance due to anticipation of a threat or in response to a diffuse one (Bolles, 1970; Izard, 1993; Barlow, 2004; Perusini and Fanselow, 2015). In accordance, the American Psychiatric Association associates fear as “post stimulus” whereas anxiety is “pre-stimulus.” Extending the view to pre-clinical research, studies by Davis and his colleagues suggest that fear and anxiety can be distinguished from each other by acute (fear) versus sustained (anxiety) threat using ‘startle’ as a defensive measure of fear. Further, they provide evidence for differences in neuro-anatomical structures involved in fear (central nucleus of amygdala) and anxiety (bed nucleus of stria terminalis). However, for others the two constructs are considered isomorphic and are “psycho-physiologically indistinguishable” wherein anxiety represents the emotional facets of fearful perceptions (Izard, 1993; Beck et al.,

2005). Additionally, the definitions of different anxiety disorders within the Diagnostic and

Statistical Manual of Mental Disorders (DSM) are unclear and often use the two terms - fear and anxiety - to explain each other (American Psychiatric, 2013; Perusini and Fanselow, 2015).

Irrespective of ambiguity in the clinical use of the two terms, one of the key characteristics of fear, anxiety and trauma and stressor related disorder is excessive and persistent fear (American

Psychiatric, 2013). For the purpose of my work, fear refers to the behavioral, autonomic and the physiological response to a real or a perceived threat rather than the subjective ‘feeling’ of fear such as terror.

1.1.2 Aversive associative learning in anxiety and trauma- and-stressor related disorders

Dysfunctional aversive associative learning has been a critical contributing factor to the development of anxiety disorders and trauma-related disorders such as PTSD. Associative

learning refers to the learning of predictive relationships between environmental stimuli, and the associated outcome. Associative learning enables animals to discriminate between an aversive stimulus and a rewarding stimulus and thus guides behavior into approach or avoid responses.

Aversive associative learning – learning of the predictive relationship between an environmental stimulus and an aversive outcome - has evolutionarily helped animals to avoid harmful or dangerous environments ensuring safety and survival of an individual. Further, aversive associative learning processes have been suggested to be one of the fundamental underlying learning processes in the development of anxiety and trauma-related related disorders. Because almost all anxiety and trauma-related related disorders involve some sort of aversive learning, preclinical and clinical models have used the Pavlovian fear conditioning paradigm to understand and model the associative processes contributing to the pathophysiology of anxiety, and trauma- related disorders (Watson and Rayner, 1920; Rescorla, 1988; Pavlov, 2010). The principle underlying Pavlovian fear conditioning is the contingency created between different stimuli and the associated conditional response. Auditory fear conditioning, a commonly used Pavlovian fear conditioning paradigm, is the most established animal model to study the neural basis of anxiety disorders that involve aversive associative conditioning. In this paradigm, a neutral conditional stimulus (CS, e.g., a tone), is paired with an aversive unconditional stimulus (US, e.g., a foot shock). In rodent studies, after successive pairings, animals learn to associate the tone with an aversive shock which elicits a conditional fear response to the CS alone. In rodents, this fear is commonly quantified by measuring the amount of freezing behavior defined by the lack of any movement except breathing (although currently most often automated through video tracking software) displayed during the presentation of the CS alone. Subsequent repeated exposure of the

CS in the absence of the US results in a decline in freezing behavior resulting in extinction of the

fear response. To date, Pavlovian fear conditioning has proved to be an excellent translational model from rodent studies to the human population to understand different fear learning processes such as fear acquisition, fear expression, fear generalization and fear extinction

(Boddez et al., 2013; Lonsdorf et al., 2017). Patients suffering from PTSD have shown deficits in their ability to distinguish between a CS predicting an aversive outcome versus a CS that was not paired with an aversive outcome. PTSD patients also display impaired fear inhibition and delayed fear extinction (Orr et al., 2000; Blechert et al., 2007; Jovanovic et al., 2010).

Additionally, within the human population studies using neuro-imaging and functional MRIs have shown that patients with anxiety disorders display elevated fear response during fear acquisition and fear extinction as compared to healthy controls (Grillon, 2002; Lissek et al.,

2005; Etkin and Wager, 2007; Duits et al., 2015; Fullana et al., 2016; Greco and Liberzon,

2016). Taken together, fear conditioning paradigms provide an appropriate, consistent, and highly controllable method to investigate the neural mechanisms underlying fear memory processing and how they relate to the development, maintenance, and expression of underlying fear- and threat-related processes seen in anxiety disorders.

Figure 1 Pavlovian fear conditioning and extinction of conditioned fear in rodents. (A)

Schematic illustrating the steps of auditory fear conditioning wherein a neutral conditional stimulus (CS, e.g., a tone), is paired with an aversive unconditional stimulus (US, e.g., a foot shock). After successive pairings, animals learn to associate the tone with an aversive shock which elicits a conditional fear response (freezing) to the CS alone. Subsequent repeated exposure of the CS in the absence of the US results in a decline in freezing behavior resulting in extinction of fear. (B) Schematic representation of behavioral protocol and (C) corresponding

freezing behavior of cued fear training, extinction training and extinction retention test in rodents. On day 1, animals are exposed to repeated presentation of tone-shock pairings with gradual increase in freezing behavior (% freezing) as the association is learned. On day 2, mice are exposed to multiple tones, in the absence of the shock, resulting in extinguishing of fear over time with tone-alone presentations. On day 3, extinction retention is tested to the presentation of tones. Successful extinction results in reduced fear. The image was created with biorender.com.

1.1.3 Fear extinction

Anxiety and trauma-related disorders are generally associated with sustained fear expression which can partly be explained due to deficits in fear inhibition or extinction to cues and contexts that are safe (Milad et al., 2006; Graham and Milad, 2011; Maren and Holmes, 2016; Kerry J.

Ressler, 2020). Fear extinction is not the forgetting of the original associative memory, rather it involves the formation of a new memory trace which reduces the original association of the CS with the US (Rescorla, 1996; Bouton, 2002; Myers and Davis, 2002; Bouton et al., 2006; Myers and Davis, 2007). Thus, fear extinction involves new inhibitory learning which results in reduced expression of fear. In line with this thought, there has been tremendous effort in understanding the neural mechanisms associated with fear extinction. Orr and colleagues found that individuals with PTSD displayed deficits in extinction learning as compared to healthy controls (Orr et al.,

2000). This finding was extended to other studies involving extinction in PTSD patients as compared to healthy controls (Peri et al., 2000; Blechert et al., 2007). Differences in extinction learning were also observed between children with anxiety disorders and healthy children

(Waters and Pine, 2016) and in patients suffering from depression and generalized anxiety disorder (Michael et al., 2007b; Otto et al., 2014). From a clinical perspective, fear extinction is significant because it forms the basis of most exposure therapy treatment strategies. Exposure therapy, a prominent component of cognitive behavior therapy, is based on the principle of extinction learning and has been extensively used in the clinical setting for the treatment of a range of anxiety disorders to reduce fear responses to stimuli that provoke anxiety and panic

(Milad and Quirk, 2012; Maren and Holmes, 2016; Craske et al., 2018). Additionally, the

National Institute of Mental Health (NIMH) hopes to improve behavioral and cognitive therapies

for patients suffering from anxiety and trauma-related disorders by focusing on learning and memory mechanisms associated with extinction. Therefore, it is the hope that by identifying the neuronal correlates, such as the different brain structures and the molecular mechanisms, that mediate not only the retrieval of fear memories but also fear inhibition, a number of novel, powerful, and targeted therapeutic interventions can be developed for better treatment of anxiety disorders and trauma-related disorders.

1.1.4 Neural circuits of fear expression and extinction

Studies using pre-clinical models have been powerful in understanding the neural circuits associated with fear learning and its extinction. One of the key brain regions associated with fear learning is the amygdala, which is important not only for the expression of fear memory but also for the acquisition and consolidation of fear and its extinction. Within the amygdala, the basolateral amygdala (BLA) and the central amygdala (CeA) have strongly been implicated in the regulation of fear memories. Anatomical studies have indicated that the nuclei are extensively interconnected. The BLA, further consisting of the lateral (LA) and basal (BA) nuclei is the critical site for convergence of conditional and unconditional stimuli. Further, the

BLA sends out unidirectional axonal projections to the CeA and this flow of information has been shown to be important for fear expression (McDonald, 1982). There are several studies using traditional lesion techniques and pharmacological manipulations implicating a critical role for the BLA in regulating the expression of conditioned fear. For example, Maren and colleagues found that excitotoxic lesions of the amygdala block both cued and context fear expression

(Maren et al., 1996a). Similarly, several studies have reported that inactivation of the BLA using sodium channel blockers, GABA receptor agonists, or NMDA receptor antagonists prior to

context fear conditioning or fear expression tests resulted in reduced fear expression

(Helmstetter, 1992; Helmstetter and Bellgowan, 1994; Maren et al., 1996b; Lee and Kim, 1998;

Goosens and Maren, 2001; Sierra-Mercado et al., 2010). In addition to its role in regulating fear expression, synaptic transmission and intracellular signaling pathways within the amygdala have been implicated in the acquisition and expression of extinction memory using electrophysiological and signaling pathway studies (Quirk et al., 1995; Davis, 2002; Herry et al.,

2006; Sierra-Mercado et al., 2010; Jasnow et al., 2013; Gilman et al., 2018). In line with pre- clinical literature, studies in the human population have shown that fear expression and extinction learning involve the amygdala. Increased activation of the amygdala as observed using fMRI was correlated to extinction learning (LaBar et al., 1998; Phelps et al., 2004). Further, fMRI and CT scan studies not only reported increased amygdala activity during fear expression

(Cheng et al., 2006; Adolphs, 2008) but also showed that bilateral amygdala damage inhibited the expression of fear (Adolphs et al., 1995).

The amygdala has extensive connections with cortical and subcortical brain regions including the hippocampus and the medial prefrontal cortex (mPFC) (Quirk et al., 2003;

Rosenkranz et al., 2003), and it is through these connections that the amygdala coordinates the expression of fear and its extinction. The mPFC by itself has been implicated in both fear conditioning and extinction with the pre-limbic (PL) and the infra-limbic (IL) cortical regions exhibiting differential roles. Specifically, the PL plays a key role in the consolidation and recall of fear memory (Corcoran and Quirk, 2007; Sierra-Mercado et al., 2010), whereas, the IL regulates the acquisition of extinction and extinction memory (Quirk et al., 2000; Laurent and

Westbrook, 2009). In addition, micro-stimulation of the IL enhances fear extinction in rats further supporting its critical role in fear extinction (Milad and Quirk, 2002; Vidal-Gonzalez et

al., 2006). In agreement with the rodent literature, the functional analogue of IL in humans, the ventral-medial prefrontal cortex (vmPFC), is activated during expression of extinction memory

(Gottfried and Dolan, 2004; Phelps et al., 2004; Milad et al., 2007). Thus, the subdivisions of the mPFC, while being equally important in fear learning regulate opposing behavioral outcomes with the IL biased towards fear reduction and PL towards fear expression. Another brain region critical for fear expression and extinction is the ventral hippocampus, which has bidirectional communication with the BLA and the vmPFC (Pitkanen et al., 2000; Hoover and Vertes, 2007).

Several studies have reported the importance of an active hippocampus and underlying signaling pathways for both expression and extinction of cued and context fear (Maren and Holt, 2004;

Bouton et al., 2006; Fischer et al., 2007; Sierra-Mercado et al., 2010).

Regulation of emotional memory is important for both the aversive domain and the appetitive domain. A large portion of the literature has focused on the effect of fear-evoking cues in engaging the amygdala and the related circuitry to regulate defensive behavior, however their impact in motivational neural circuitry is less known. There have been an increasing number of studies which indicate common brain regions and neuronal connections engaged in reward and emotional circuitry. The mPFC which has been extensively implicated in regulating fear expression and extinction as discussed previously, is also involved in facilitating extinction to cues associated with drugs of abuse (Taylor et al., 2009). While inactivation of PL reduced fear expression, it also reduced relapse to drugs of abuse in rats (LaLumiere and Kalivas, 2008).

Additionally, BLA has also been shown to be involved in regulating the extinction of appetitive behavior (Weiskrantz, 1956; Burns et al., 1999). Therefore, it is imperative to study the common neuronal substrates associated with these circuits since malfunction of either circuit is likely to impact both. In addition, associative learning is a multi-faceted phenomenon engaging both

competitive and complimentary learning and memory systems. Over the last several years, there has been emerging evidence of common neural substrates facilitating extinction of both fear and drug memories (Peters et al., 2009; Abraham et al., 2014; Leung et al., 2016). While failure to extinguish fear is an underlying mechanism contributing to PTSD, failure to extinguish appetitive behavior can lead to drug relapse, and patients suffering from anxiety and trauma disorders have an increased likelihood to relapse to drugs of abuse (Shaham et al., 2000; Sinha et al., 2006). Therefore, it is critical to identify common neural circuitry implicated in the extinction of conditioned aversive responses and drug reinforcement. One putative component of such a circuit is the nucleus accumbens (NAc), which acts as a hub guiding both aversive and appetitive behaviors.

1.2 The nucleus accumbens

The nucleus accumbens was first identified as a part of the ventral striatum by the works of

Heimer and Mogenson (Heimer; Mogenson, 1985). Classically, the NAc has been viewed as a critical node for limbic-motor integration. The NAc receives strong projections from the limbic structures such as amygdala, hippocampus and prefrontal cortices, and in turn projects to subcortical motor sites, the substantia nigra and ventromedial globus pallidus to help coordinate behavioral responses. The NAc also projects to the lateral hypothalamus which regulates autonomic and physiological response to the same stimuli (Alexander et al., 1991). There is extensive literature focusing on the role of the NAc in regulating rewarding and appetitive stimuli (Di Chiara, 2002; Ambroggi et al., 2008; Carlezon Jr and Thomas, 2009; Day et al.,

2011; Russo and Nestler, 2013). However, an increasing number of clinical and pre-clinical studies have suggested a broader role of the NAc in processing fear or aversive stimuli. The NAc has been implicated in several neurological disorders, including depression, anxiety disorders,

Parkinson’s and Alzheimer’s diseases and thus has been a target region for several pharmacotherapies (Russo and Nestler, 2013; Salgado and Kaplitt, 2015; Francis and Lobo,

2017; Fu et al., 2017; Nie et al., 2017; Li et al., 2018). While much is known about the role of the

NAc in appetitive and reward behavior, its contribution to aversive emotional learning remains understudied.

1.2.1 Nucleus accumbens composition and connectivity with the canonical fear circuit

Anatomically and histochemically, the NAc is a heterogenous structure, further divided into two distinct sub-regions, identified as the core and shell in rodents, primates and humans

(Záborszky et al., 1985; Zahm and Brog, 1992; Voorn et al., 1994; Meredith et al., 1996; Baliki et al., 2013). The central and the dorsal subunit of the NAc is the core which has been implicated in regulating motor function and closely resembles the dorsal striatum (Pierce et al., 1996;

Groenewegen et al., 1999a; Zahm, 1999). The core is surrounded medially, ventrally, and ventrolaterally by the shell. The NAc shell has been considered to be a part of the extended amygdala due to the histochemical and biochemical similarities between the two regions although other studies have provided evidence of considerable differences for the two to be treated as separate, functionally interconnected regions (Zahm, 1998; De Olmos and Heimer,

1999; Sturm et al., 2003). Structurally, the NAc occupies a large portion of the basal forebrain and is composed of 95% medium spiny GABAergic neurons, and 1% -2% each of interneurons and cholinergic neurons. The medium spiny neurons (MSN) are further classified depending on their dopamine receptor subtype, D1 and D2, classification in addition to the transcription profiles of other genes such as substance P and enkephalin (Gerfen et al., 1990; Lobo et al.,

2006; Francis and Lobo, 2017).

At the circuit level, the NAc is tightly linked to the canonical fear circuit. It is a critical hub for the convergence of inputs from regions heavily implicated in the acquisition, consolidation and retrieval of fear learning and memory processes such as the amygdala, hippocampus and the prefrontal cortex (Beckstead, 1979; Kelley and Domesick, 1982; da Silva

Lopes et al., 1984; Phillipson and Griffiths, 1985; Berendse and Groenewegen, 1990; Wright et al., 1996). The NAc is innervated by excitatory glutamatergic inputs from the amygdala, hippocampus, prefrontal cortex (PFC) and the ventral tegmental area as well as dopaminergic afferents from the ventral tegmental area and substantial nigra (Phillipson and Griffiths, 1985;

Brog et al., 1993; Friedman et al., 2002; Qi et al., 2016). Dopaminergic neurons from the ventral tegmental area synapse onto the medium spiny neurons within the NAc and are regulated by glutamatergic inputs to the NAc (Floresco et al., 2001). The NAc also receives afferents from regions involved in motor control such as the dorsal caudate and globus pallidus (Groenewegen et al., 1999b; Zahm, 1999). This connectional framework makes the nucleus accumbens a critical node where fear and reward circuitry overlap, and it has been suggested that these brain regions with afferents to NAc can influence distinct behavioral outputs through their interactions with the NAc. The role of NAc in regulating reward related behavior and emotion to drugs of abuse and other appetitive behavior has already been well established. However, the involvement of this region in mediating the aversive states of emotion is less understood. I will discuss the state of the current literature below.

1.2.2 Nucleus accumbens in aversive emotional conditioning

Due to dopamine (DA) turnover within the NAc being higher than the rest of the striatum and the extensive evidence in the reward system, most of the work on the NAc in aversive learning has focused on dopaminergic modulation. As a result, dopaminergic signaling within the NAc is now

suggested to be involved in facilitating aversive emotional learning. In the preclinical literature, elevation in levels of DA have been reported in the accumbens in response to aversive and stressful stimuli. For example, in an early study it was found that a stressful stimulus elevated

DA release within the NAc (Abercrombie et al., 1989). This finding was replicated in other studies that found a similar increase in DA transmission within the NAc following a foot-shock, tail pinch or an aggressive interaction (Louilot et al., 1986; Kalivas and Duffy, 1995). While the previous studies involved unsignalled presentations of aversive cues, similar observations were made when studying the role of DA transmission within the NAc in response to associative aversive conditioning. In one study the extracellular DA within the core was elevated during contextual fear expression test as compared to the shell whereas similar increased levels were observed within the shell during cued fear expression test (Pezze et al., 2001). In contrast, another study showed elevated DA levels in both the NAc core and shell following contextual fear conditioning(Martinez et al., 2008). Similar increases in DA transmission were found in the

NAc shell in response to a fear-evoking cue (Badrinarayan et al., 2012). Further, infusions of a dopamine receptor-2 (D2) receptor antagonist within the NAc in rodents impaired fear extinction consolidation and retention (Holtzman-Assif et al., 2010) suggesting that dopaminergic signaling within the nucleus accumbens is involved in regulating fear expression as well as extinction.

Beyond the dopaminergic signaling, several other studies have added to the evidence suggesting the involvement of NAc in regulating associative fear learning. For example, pharmacological inactivation or lesioning of the NAc has been reported to block the acquisition and expression of fear to cues and context, although reports are not always consistent (Schwienbacher et al., 2004).

(Haralambous and Westbrook, 1999). (Riedel et al., 1997). In addition to aversive emotional processing, the NAc has been implicated in mood disorders. The MSNs within the NAc and

changes in their molecular expression and synaptic plasticity have previously been implicated in depression. Evidence from rodent studies for the involvement of NAc in regulating aversive emotional stimuli are further supported by non-human primate and clinical studies. Increased activity within the NAc was observed during freezing in rhesus monkeys (Kalin et al., 2005). A clinical trial conducted by Jensen and colleagues has shown that the ventral striatum gets activated by the anticipation of an aversive event (Jensen et al., 2003). Multiple studies have shown that deep brain stimulation of the ventral striatum reduced anhedonia and produced functional improvements in patients with treatment-resistant depression (Sturm et al., 2003;

Schlaepfer et al., 2008; Malone et al., 2009). Postmortem brain analysis in patients with major depression or bipolar disorder showed reduced volume of NAc (Baumann et al., 1999). One major drawback to these rodent and human studies is that they did not examine the nucleus accumbens subregions individually. Given that the core and shell have distinct connectivity with regions implicated in anxiety and other neuropsychiatric disorders, this suggests a differential functional role in mediating the acquisition, expression or retrieval of aversive learning and memory processes.

1.3 Glutamatergic transmission in fear expression and extinction

Activity dependent synaptic plasticity, long-term potentiation (LTP) or long-term depression

(LTD) is one of the underlying neural mechanisms thought to be responsible for learning (for review see Nakanishi, 1994; Martin et al., 2000; Takeuchi et al., 2014). The changes in the individual synapses that mediate such synaptic efficacy involve several steps including presynaptic transmitter release, action of the neurotransmitters, changes in the post synaptic receptors, activation of post-synaptic signaling cascades and gene activation and protein synthesis. There are several neurotransmitters, receptors and subsequent signaling cascades

which are integral for neuronal information processing and contribute to synaptic plasticity.

Glutamate, being one of the major excitatory neurotransmitters in the brain, has been predominantly implicated in several forms of memory processing. Glutamate acts by binding and activating two general types of receptors- ionotropic glutamate receptors such as N-methyl-d- aspartate (NMDA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) that mediate fast excitatory neurotransmission and G-protein coupled metabotropic glutamate receptors (mGluRs) which are modulatory in nature and mediate slow neurotransmission.

Depending on the G protein the mGluR is coupled to, Gq (stimulatory) or Gi (inhibitory), they can inhibit or potentiate calcium and potassium channels, mediate presynaptic inhibition of transmitter release and also potentiate AMPA and NMDA receptor-mediated synaptic responses

(Swartz and Bean, 1992; Rothe et al., 1994; Davies et al., 1995; Choi and Lovinger, 1996;

Ribeiro et al., 2010). Thus, along with NMDA/AMPA receptor activation, mGluRs activity have been widely implicated in LTP and LTD formation (Bear and Abraham, 1996; Riedel et al.,

1996; Wang et al., 2016b). Further, mGluR1s have been implicated in several neurological disorders, such as epilepsy, Alzheimer’s disease, Parkinson’s disease, Schizophrenia, anxiety disorders, and have been used as therapeutic targets (Meldrum, 2000).

Structurally, the mGluRs contain seven membrane-spanning domains and can be classified into three groups on the basis of their amino acid sequence homology, signal transduction and pharmacology; group I, group II and group III (Pin and Duvoisin, 1995; Anwyl, 1999). Group 1 mGluRs, consisting of mGluR1 and mGluR5, are found post-synaptically in different brain regions implicated in fear learning and memory including the amygdala, hippocampus, cortex and the striatum (Moroni et al.; Kim et al., 2007; Wang et al., 2016b). Inhibition of group 1 mGluRs has produced anxiolytic-like effects in several animal studies (Pilc et al., 2002). Further,

group 1 mGluRs within the hippocampus have been implicated in fear learning and extinction consolidation and appear to be essential for synaptic plasticity (Bajkowska et al., 1999;

Zahorodna and Bijak, 1999). Additionally, mGluR5 modulate synaptic plasticity via interactions with NMDA receptors through scaffolding proteins such as Homer and Shank (Naisbitt et al.,

1999; Tu et al., 1999). Extensive literature has shown that group 1 mGluRs are important for synaptic plasticity and hippocampal- based learning. For example, mGluR1 gene knockout mice showed a reduction in LTP within the hippocampus and a deficit in context specific associative learning (Aiba et al., 1994). Similarly, mice lacking mGluR5 showed reduced LTP in NMDA- receptor dependent pathways along with impaired spatial learning and context fear acquisition

(Lu et al., 1997). Several other studies have implicated mGluR5 in contextual fear conditioning

(Gravius et al., 2006). For instance, intra amygdala infusions of mGluR5 antagonists impaired the acquisition of fear potentiation of startle (Nielsen et al., 1997; Fendt and Schmid, 2002), while more general blockade of group 1 mGluRs specifically within the lateral amygdala inhibited LTP and impaired acquisition of both contextual and cued fear conditioning (Rodrigues et al., 2002). Further, blocking mGluR1 in the amygdala impaired fear extinction acquisition and retention (Kim et al., 2007). Within the infralimbic cortex, mGluR5 inactivation has shown to prevent fear extinction retention but did not affect extinction learning (Fontanez-Nuin et al.,

2010).

Evidence of glutamate and glutamate receptors involvement in anxiety disorders extend into the clinical literature as well; glutamate was found to be elevated in the ACC among generalized social phobic patients as compared to healthy controls and some studies have found that mGluRs are effective targets for anxiety disorders; specifically oral administration the of mGluR2/3 agonist, LY354740, in humans has shown to reduce anxiety as compared to patients

who received placebo (Swanson et al., 2005). Additionally, a mGluR5 antagonist, fenobam, has anxiolytic properties without the side effects accompanying benzodiazepines administration in humans (Pecknold et al., 1982; Goldberg et al., 1983; Porter et al., 2005). Further, both glutamate hypofunction and hyperfunction and thus, both mGluR agonists and antagonists as described above, have been implicated in neuropsychological disorders (for detailed review see

Linden and Schoepp, 2006). Overall, these data provide evidence that metabotropic glutamate receptor activity is highly significant in associative learning and critical for the maintenance of persistence of synaptic plasticity through LTP or LTD. Still, the potential role of modulating mGluR1 activity, specifically, and its implication in anxiety and trauma-related disorder is less known. Through our studies we aim to delve deeper into the contribution of the mGluR1 in fear learning within the NAc subregions, which has not been examined previously.

1.4 ERK/MAPK Pathway

As previously mentioned, mGluRs modulate neurotransmission through intracellular signaling pathways and one of the signaling pathway coupled to mGluR1s is the extracellular signal regulated kinase /mitogen activate protein kinase (ERK/MAPK) (Wang et al., 2007b; Mao and

Wang, 2016; Yang et al., 2017). MAPK are a family of serine/threonine kinases which are densely expressed in postmitotic neurons. ERK, a subclass of MAPK, are highly conserved protein kinases which directly interact with the intracellular domains of group I mGluRs and has been extensively studied for its role in regulating synaptic transmission (for review seeThomas and Huganir, 2004; Cestari et al., 2014; Mao and Wang, 2016; Medina and Viola, 2018). One of the potential pathways in which a group 1 mGluR can initiate the ERK pathway is through the

Gq subunit. The Gq subunit of the activated mGluR1/5 induces the hydrolysis of phospholipase C, resulting in diacylglycerol and inositol-1,4,5, -triphosphate (IP3) production which finally

triggers the Ca2+ and the protein kinase C pathways, respectively. Ca2+ phosphorylates Ras-GDP into Ras-GTP which in turn phosphorylates the MAPK/ERK enzyme (MEK). MEK then phosphorylates ERK1 and ERK2 (Nakielny et al., 1992; Thomas and Huganir, 2004; Wang et al., 2007a; Mao and Wang, 2016). The phosphorylation of ERK and MAPK proteins activates several downstream transcriptional factors such as CREB and Elk-1. Further, these signaling pathways regulate and induce de novo synthesis of proteins implicated in synaptic plasticity such as ARC/ARG 3.1 and BDNF (Sweatt, 2004; Thomas and Huganir, 2004; Bekinschtein et al.,

2008; Antoine et al., 2014). Additionally, the MAPK/ERK pathways have been extensively implicated in fear learning and memory processes and underlying synaptic plasticity. The first evidence of involvement of ERK in synaptic plasticity was demonstrated through MEK inhibition blocking LTP within the hippocampus (English and Sweatt, 1997). The requirement of

ERK in LTP formation was further reported in cortical neurons (Di Cristo et al., 2001). Over the years, the role of ERK in LTP has been extended to different brain regions involved in fear learning and memory such as the amygdala, anterior cingulate cortex, and the prefrontal cortex

(Huang et al., 2000; Hugues et al., 2006; Toyoda et al., 2007). Hyperphosphorylation of ERK1/2 was observed within the CA3 region of the hippocampus during consolidation of contextual fear memory and in the amygdala following cued fear conditioning (Schafe et al., 2000; Sananbenesi et al., 2002). Inhibiting the ERK/MAPK pathway within the CA1 of the hippocampus resulted in reduced fear expression in both cued and context fear conditioning (Trifilieff et al., 2006).

Blocking the ERK/MAPK pathway within the amygdala blocked both the consolidation and reconsolidation of the auditory fear conditioning in one study (Duvarci et al., 2005) whereas it impaired LTP and showed reduced fear expression to the tone in another one (Schafe et al.,

2000). Inhibition of the ERK/MAPK pathway and the subsequent protein synthesis also inhibited

fear extinction consolidation within the prefrontal cortex (Herry et al., 2006). All the evidence above suggests a common cellular and molecular processes, involving ERK/MAPK pathway, underlying fear memory acquisition, consolidation, expression or extinction. Within the nucleus accumbens, while the pathway has not been studied in the capacity of regulating fear memory, there is evidence for its role in reward- related learning (Gerdjikov et al., 2004; Wang et al.,

2016a). Thus, expanding our knowledge of the ERK/MAPK signaling pathways in brain regions where the reward and fear circuit overlap, such as the NAc, will be critical in developing targeted and combined treatment strategies for several comorbid disorders such as anxiety, trauma-related disorders and drug addiction.

Figure 2 Schematic illustrating the signaling pathway from the group 1 metabotropic glutamatergic receptors (mGluR1) to the MAPK/ERK cascade in the neurons. On activation, the Gq subunit of the mGluR1 hydrolyze the Phospholipase C (PLC) to induce the

Ca2+ or the PKC pathways. The Ca2+ phosphorylates Ras-GDP into Ras-GTP which in turn phosphorylates the MAPK/ERK enzyme (MEK). The MEK then phosphorylates ERK1 and

ERK2 which then activate downstream transcriptional factors such as Elk-1 and CREB. This image was made using biorender.com

1.5 Specific aims of dissertation

Specific Aim 1 tests the hypothesis that the NAc core and shell subregions differentially regulate fear expression and fear extinction consolidation. Using Pavlovian fear conditioning, pharmacology, and immunohistochemistry, we’ll investigate the following questions:

a) Using cellular activity mapping, we will define the involvement of the NAc core and shell

in fear expression and fear extinction consolidation

b) Test the hypothesis that inactivating the NAc core is necessary for fear expression

c) Test the hypothesis that inactivating the NAc shell disrupts the consolidation of fear

extinction.

Specific Aim 2 tests the hypothesis that glutamatergic signaling within the NAc is necessary to mediate extinction consolidation

a) Tests the hypothesis that mGluR1 receptors within the NAc shell, but not NAc core, are

necessary for consolidating fear extinction.

b) Tests the hypothesis that the ERK signaling pathway within the NAc shell is essential for

consolidating fear extinction.

1.6 Reference

Abercrombie ED, Keefe KA, DiFrischia DS, Zigmond MJ (1989) Differential Effect of Stress on

In Vivo Dopamine Release in Striatum, Nucleus Accumbens, and Medial Frontal Cortex.

Journal of Neurochemistry 52:1655-1658.

Abraham AD, Neve KA, Lattal KM (2014) Dopamine and extinction: a convergence of theory

with fear and reward circuitry. Neurobiology of learning and memory 108:65-77.

Adolphs R (2008) Fear, faces, and the human amygdala. Current Opinion in Neurobiology 18:166-

172.

Adolphs R, Tranel D, Damasio H, Damasio AR (1995) Fear and the human amygdala. The Journal

of Neuroscience 15:5879.

Aiba A, Chen C, Herrup K, Rosenmund C, Stevens CF, Tonegawa S (1994) Reduced hippocampal

long-term potentiation and context-specific deficit in associative learning in mGluR1

mutant mice. Cell 79:365-375.

Alexander GE, Crutcher MD, DeLong MR (1991) Basal ganglia-thalamocortical circuits: parallel

substrates for motor, oculomotor,“prefrontal” and “limbic” functions. Progress in brain

research 85:119-146.

Ambroggi F, Ishikawa A, Fields HL, Nicola SM (2008) Basolateral amygdala neurons facilitate

reward-seeking behavior by exciting nucleus accumbens neurons. Neuron 59:648-661.

American Psychiatric A (2013) Diagnostic and statistical manual of mental disorders (DSM-5®):

American Psychiatric Pub.

Andlin-Sobocki P, Wittchen HU (2005) Cost of anxiety disorders in Europe. Eur J Neurol 12 Suppl

1:39-44.

Antoine B, Serge L, Jocelyne C (2014) Comparative dynamics of MAPK/ERK signalling

components and immediate early genes in the hippocampus and amygdala following

contextual fear conditioning and retrieval. Brain Structure and Function 219:415-430.

Anwyl R (1999) Metabotropic glutamate receptors: electrophysiological properties and role in

plasticity. Brain Research Reviews 29:83-120.

Badrinarayan A, Wescott SA, Vander Weele CM, Saunders BT, Couturier BE, Maren S, Aragona

BJ (2012) Aversive Stimuli Differentially Modulate Real-Time Dopamine Transmission

Dynamics within the Nucleus Accumbens Core and Shell. The Journal of Neuroscience

32:15779-15790.

Bajkowska M, Brański P, Smiałowska M, Pilc A (1999) Effect of chronic antidepressant or

electroconvulsive shock treatment on mGLuR1a immunoreactivity expression in the rat

hippocampus. Pol J Pharmacol 51:539-541.

Baliki MN, Mansour A, Baria AT, Huang L, Berger SE, Fields HL, Apkarian AV (2013) Parceling

Human Accumbens into Putative Core and Shell Dissociates Encoding of Values for

Reward and Pain. The Journal of Neuroscience 33:16383-16393.

Barlow DH (2004) Anxiety and its disorders: The nature and treatment of anxiety and panic:

Guilford press.

Baumann B, Danos P, Krell D, Diekmann S, Leschinger A, Stauch R, Wurthmann C, Bernstein

H-G, Bogerts B (1999) Reduced Volume of Limbic System–Affiliated Basal Ganglia in

Mood Disorders. The Journal of Neuropsychiatry and Clinical Neurosciences 11:71-78.

Baxter AJ, Scott K, Vos T, Whiteford H (2013) Global prevalence of anxiety disorders: a

systematic review and meta-regression. Psychological medicine 43:897.

Bear MF, Abraham WC (1996) Long-term depression in hippocampus. Annual review of

neuroscience 19:437-462.

Beck AT, Emery G, Greenberg RL (2005) Anxiety disorders and phobias: A cognitive perspective:

Basic Books.

Beckstead RM (1979) An autoradiographic examination of corticocortical and subcortical

projections of the mediodorsal‐projection (prefrontal) cortex in the rat. Journal of

Comparative Neurology 184:43-62.

Bekinschtein P, Cammarota M, Katche C, Slipczuk L, Rossato JI, Goldin A, Izquierdo I, Medina

JH (2008) BDNF is essential to promote persistence of long-term memory storage.

Proceedings of the National Academy of Sciences 105:2711-2716.

Berendse HW, Groenewegen HJ (1990) Organization of the thalamostriatal projections in the rat,

with special emphasis on the ventral striatum. Journal of Comparative Neurology 299:187-

228.

Blechert J, Michael T, Vriends N, Margraf J, Wilhelm FH (2007) Fear conditioning in

posttraumatic stress disorder: Evidence for delayed extinction of autonomic, experiential,

and behavioural responses. Behaviour Research and Therapy 45:2019-2033.

Boddez Y, Baeyens F, Hermans D, Beckers T (2013) A learning theory approach to anxiety

disorders: Human fear conditioning and the added value of complex acquisition

procedures.

Bolles RC (1970) Species-specific defense reactions and avoidance learning. Psychological

Review 77:32-48.

Bouton ME (2002) Context, ambiguity, and unlearning: sources of relapse after behavioral

extinction. Biological psychiatry 52:976-986.

Bouton ME, Westbrook RF, Corcoran KA, Maren S (2006) Contextual and temporal modulation

of extinction: behavioral and biological mechanisms. Biological psychiatry 60:352-360.

Brog JS, Salyapongse A, Deutch AY, Zahm DS (1993) The patterns of afferent innervation of the

core and shell in the "accumbens" part of the rat ventral striatum: immunohistochemical

detection of retrogradely transported fluoro-gold. The Journal of comparative neurology

338:255-278.

Burns LH, Everitt BJ, Robbins TW (1999) Effects of excitotoxic lesions of the basolateral

amygdala on conditional discrimination learning with primary and conditioned

reinforcement. Behavioural brain research 100:123-133.

Carlezon Jr WA, Thomas MJ (2009) Biological substrates of reward and aversion: a nucleus

accumbens activity hypothesis. Neuropharmacology 56:122-132.

Cestari V, Rossi-Arnaud C, Saraulli D, Costanzi M (2014) The MAP(K) of fear: from memory

consolidation to memory extinction. Brain research bulletin 105:8-16.

Cheng DT, Knight DC, Smith CN, Helmstetter FJ (2006) Human amygdala activity during the

expression of fear responses. Behavioral neuroscience 120:1187.

Choi S, Lovinger DM (1996) Metabotropic glutamate receptor modulation of voltage-gated Ca2+

channels involves multiple receptor subtypes in cortical neurons. The Journal of

Neuroscience 16:36.

Corcoran KA, Quirk GJ (2007) Activity in Prelimbic Cortex Is Necessary for the Expression of

Learned, But Not Innate, Fears. The Journal of Neuroscience 27:840-844.

Craske MG, Hermans D, Vervliet B (2018) State-of-the-art and future directions for extinction as

a translational model for fear and anxiety. Philosophical Transactions of the Royal Society

B: Biological Sciences 373:20170025.

da Silva Lopes FH, Arnolds D, Neijt HC (1984) A functional link between the limbic cortex and

ventral striatum: physiology of the subiculum accumbens pathway. Experimental brain

research 55:205-214.

Davies CH, Clarke VRJ, Jane DE, Collingridge GL (1995) Pharmacology of postsynaptic

metabotropic glutamate receptors in rat hippocampal CA1 pyramidal neurones. British

journal of pharmacology 116:1859-1869.

Davis M (2002) Role of NMDA receptors and MAP kinase in the amygdala in extinction of fear:

clinical implications for exposure therapy. European Journal of Neuroscience 16:395-398.

Day JJ, Jones JL, Carelli RM (2011) Nucleus accumbens neurons encode predicted and ongoing

reward costs in rats. European journal of neuroscience 33:308-321.

De Olmos JS, Heimer L (1999) The concepts of the ventral striatopallidal system and extended

amygdala. Annals of the New York Academy of Sciences 877:1-32.

Di Chiara G (2002) Nucleus accumbens shell and core dopamine: differential role in behavior and

addiction. Behavioural brain research 137:75-114.

Di Cristo G, Berardi N, Cancedda L, Pizzorusso T, Putignano E, Ratto GM, Maffei L (2001)

Requirement of ERK activation for visual cortical plasticity. Science 292:2337-2340.

Duits P, Cath DC, Lissek S, Hox JJ, Hamm AO, Engelhard IM, Van Den Hout MA, Baas JMP

(2015) Updated meta‐analysis of classical fear conditioning in the anxiety disorders.

Depression and anxiety 32:239-253.

Duvarci S, Nader K, LeDoux JE (2005) Activation of extracellular signal-regulated kinase–

mitogen-activated protein kinase cascade in the amygdala is required for memory

reconsolidation of auditory fear conditioning. European Journal of Neuroscience 21:283-

289.

English JD, Sweatt JD (1997) A requirement for the mitogen-activated protein kinase cascade in

hippocampal long term potentiation. Journal of Biological Chemistry 272:19103-19106.

Essau CA, Lewinsohn PM, Lim JX, Ho M-hR, Rohde P (2018) Incidence, recurrence and

comorbidity of anxiety disorders in four major developmental stages. Journal of Affective

Disorders 228:248-253.

Etkin A, Wager TD (2007) Functional neuroimaging of anxiety: a meta-analysis of emotional

processing in PTSD, social anxiety disorder, and specific phobia. American journal of

Psychiatry 164:1476-1488.

Fendt M, Schmid S (2002) Metabotropic glutamate receptors are involved in amygdaloid

plasticity. European Journal of Neuroscience 15:1535-1541.

Fischer A, Radulovic M, Schrick C, Sananbenesi F, Godovac-Zimmermann J, Radulovic J (2007)

Hippocampal Mek/Erk signaling mediates extinction of contextual freezing behavior.

Neurobiology of learning and memory 87:149-158.

Floresco SB, Todd CL, Grace AA (2001) Glutamatergic Afferents from the Hippocampus to the

Nucleus Accumbens Regulate Activity of Ventral Tegmental Area Dopamine Neurons.

The Journal of Neuroscience 21:4915.

Fontanez-Nuin DE, Santini E, Quirk GJ, Porter JT (2010) Memory for Fear Extinction Requires

mGluR5-Mediated Activation of Infralimbic Neurons. Cerebral Cortex 21:727-735.

Francis TC, Lobo MK (2017) Emerging role for nucleus accumbens medium spiny neuron

subtypes in depression. Biological psychiatry 81:645-653.

Friedman DP, Aggleton JP, Saunders RC (2002) Comparison of hippocampal, amygdala, and

perirhinal projections to the nucleus accumbens: Combined anterograde and retrograde

tracing study in the Macaque brain. Journal of Comparative Neurology 450:345-365.

Fu K, Miyamoto Y, Sumi K, Saika E, Muramatsu S-i, Uno K, Nitta A (2017) Overexpression of

transmembrane protein 168 in the mouse nucleus accumbens induces anxiety and

sensorimotor gating deficit. PloS one 12:e0189006.

Fullana MA, Harrison BJ, Soriano-Mas C, Vervliet B, Cardoner N, Àvila-Parcet A, Radua J (2016)

Neural signatures of human fear conditioning: an updated and extended meta-analysis of

fMRI studies. Molecular Psychiatry 21:500-508.

Gerdjikov TV, Ross GM, Beninger RJ (2004) Place preference induced by nucleus accumbens

amphetamine is impaired by antagonists of ERK or p38 MAP kinases in rats. Behavioral

neuroscience 118:740.

Gerfen CR, Engber TM, Mahan LC, Susel ZVI, Chase TN, Monsma FJ, Sibley DR (1990) D1 and

D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal

neurons. Science 250:1429-1432.

Gilman TL, Dutta S, Adkins JM, Cecil CA, Jasnow AM (2018) Basolateral amygdala Thy1-

expressing neurons facilitate the inhibition of contextual fear during consolidation,

reconsolidation, and extinction. Neurobiology of learning and memory 155:498-507.

Goldberg ME, Salama AI, Patel JB, Malick JB (1983) Novel non-benzodiazepine anxiolytics.

Neuropharmacology 22:1499-1504.

Goosens KA, Maren S (2001) Contextual and Auditory Fear Conditioning are Mediated by the

Lateral, Basal, and Central Amygdaloid Nuclei in Rats. Learning & Memory 8:148-155.

Gottfried JA, Dolan RJ (2004) Human orbitofrontal cortex mediates extinction learning while

accessing conditioned representations of value. Nature neuroscience 7:1144-1152.

Graham BM, Milad MR (2011) The study of fear extinction: implications for anxiety disorders.

Am J Psychiatry 168:1255-1265.

Gravius A, Barberi C, Schäfer D, Schmidt WJ, Danysz W (2006) The role of group I metabotropic

glutamate receptors in acquisition and expression of contextual and auditory fear

conditioning in rats – a comparison. Neuropharmacology 51:1146-1155.

Greco JA, Liberzon I (2016) Neuroimaging of fear-associated learning.

Neuropsychopharmacology 41:320-334.

Greenberg PE, Sisitsky T, Kessler RC, Finkelstein SN, Berndt ER, Davidson JR, Ballenger JC,

Fyer AJ (1999) The economic burden of anxiety disorders in the 1990s. J Clin Psychiatry

60:427-435.

Grillon C (2002) Associative learning deficits increase symptoms of anxiety in humans. Biological

psychiatry 51:851-858.

Groenewegen HJ, Wright CI, Beijer AVJ, Voorn P (1999a) Convergence and segregation of

ventral striatal inputs and outputs. Annals of the New York Academy of Sciences 877:49-

63.

Groenewegen HJ, Wright CI, Beijer AV, Voorn P (1999b) Convergence and segregation of ventral

striatal inputs and outputs. Annals of the New York Academy of Sciences 877:49-63.

Haralambous T, Westbrook RF (1999) An infusion of bupivacaine into the nucleus accumbens

disrupts the acquisition but not the expression of contextual fear conditioning. Behavioral

Neuroscience 113:925-940.

Heimer L The subcortical projections of allocortex: similarities in the neuronal associations of the

hippocampus, the piriform cortex and the neocortex. In, pp 173-193: Raven.

Helmstetter FJ (1992) Contribution of the amygdala to learning and performance of conditional

fear. Physiology & Behavior 51:1271-1276.

Helmstetter FJ, Bellgowan PS (1994) Effects of muscimol applied to the basolateral amygdala on

acquisition and expression of contextual fear conditioning in rats. Behavioral neuroscience

108:1005.

Herry C, Trifilieff P, Micheau J, Lüthi A, Mons N (2006) Extinction of auditory fear conditioning

requires MAPK/ERK activation in the basolateral amygdala. European Journal of

Neuroscience 24:261-269.

Holtzman-Assif O, Laurent V, Westbrook RF (2010) Blockade of dopamine activity in the nucleus

accumbens impairs learning extinction of conditioned fear. Learning & memory (Cold

Spring Harbor, NY) 17:71-75.

Hoover WB, Vertes RP (2007) Anatomical analysis of afferent projections to the medial prefrontal

cortex in the rat. Brain Structure and Function 212:149-179.

Hu MX, Lamers F, de Geus EJC, Penninx BWJH (2016) Differential Autonomic Nervous System

Reactivity in Depression and Anxiety During Stress Depending on Type of Stressor.

Psychosomatic Medicine 78.

Huang Y-Y, Martin KC, Kandel ER (2000) Both protein kinase A and mitogen-activated protein

kinase are required in the amygdala for the macromolecular synthesis-dependent late phase

of long-term potentiation. Journal of Neuroscience 20:6317-6325.

Hugues S, Chessel A, Lena I, Marsault R, Garcia R (2006) Prefrontal infusion of PD098059

immediately after fear extinction training blocks extinction‐associated prefrontal synaptic

plasticity and decreases prefrontal ERK2 phosphorylation. Synapse 60:280-287.

Izard CE (1993) Organizational and motivational functions of discrete emotions.

Jasnow AM, Ehrlich DE, Choi DC, Dabrowska J, Bowers ME, McCullough KM, Rainnie DG,

Ressler KJ (2013) Thy1-Expressing Neurons in the Basolateral Amygdala May Mediate

Fear Inhibition. The Journal of Neuroscience 33:10396-10404.

Jensen J, McIntosh AR, Crawley AP, Mikulis DJ, Remington G, Kapur S (2003) Direct Activation

of the Ventral Striatum in Anticipation of Aversive Stimuli. Neuron 40:1251-1257.

Jovanovic T, Norrholm SD, Blanding NQ, Davis M, Duncan E, Bradley B, Ressler KJ (2010)

Impaired fear inhibition is a biomarker of PTSD but not depression. Depression and anxiety

27:244-251.

Kalin NH, Shelton SE, Fox AS, Oakes TR, Davidson RJ (2005) Brain Regions Associated with

the Expression and Contextual Regulation of Anxiety in Primates. Biological psychiatry

58:796-804.

Kalivas PW, Duffy P (1995) Selective activation of dopamine transmission in the shell of the

nucleus accumbens by stress. Brain Research 675:325-328.

Katkin ES (1965) Relationship between manifest anxiety and two indices of autonomic response

to stress. Journal of Personality and Social Psychology 2:324.

Kelley AE, Domesick VB (1982) The distribution of the projection from the hippocampal

formation to the nucleus accumbens in the rat: an anterograde and retrograde-horseradish

peroxidase study. Neuroscience 7:2321-2335.

Kerry J. Ressler, M.D. , Ph.D. (2020) Translating Across Circuits and Genetics Toward Progress

in Fear- and Anxiety-Related Disorders. American Journal of Psychiatry 177:214-222.

Kessler RC, Petukhova M, Sampson NA, Zaslavsky AM, Wittchen H-U (2012) Twelve-month

and lifetime prevalence and lifetime morbid risk of anxiety and mood disorders in the

United States. International journal of methods in psychiatric research 21:169-184.

Kim J, Lee S, Park H, Song B, Hong I, Geum D, Shin K, Choi S (2007) Blockade of amygdala

metabotropic glutamate receptor subtype 1 impairs fear extinction. Biochemical and

Biophysical Research Communications 355:188-193.

LaBar KS, Gatenby JC, Gore JC, LeDoux JE, Phelps EA (1998) Human Amygdala Activation

during Conditioned Fear Acquisition and Extinction: a Mixed-Trial fMRI Study. Neuron

20:937-945.

LaLumiere RT, Kalivas PW (2008) Glutamate Release in the Nucleus Accumbens Core Is

Necessary for Heroin Seeking. The Journal of Neuroscience 28:3170-3177.

Latsko MS, Farnbauch LA, Gilman TL, Lynch Iii JF, Jasnow AM (2016) Corticosterone may

interact with peripubertal development to shape adult resistance to social defeat. Hormones

and behavior 82:38-45.

Laurent V, Westbrook RF (2009) Inactivation of the infralimbic but not the prelimbic cortex

impairs consolidation and retrieval of fear extinction. Learning & Memory 16:520-529.

Lee H, Kim JJ (1998) Amygdalar NMDA receptors are critical for new fear learning in previously

fear-conditioned rats. Journal of Neuroscience 18:8444-8454.

Leung HT, Holmes NM, Westbrook RF (2016) An appetitive conditioned stimulus enhances fear

acquisition and impairs fear extinction. Learning & Memory 23:113-120.

Li Z, Chen Z, Fan G, Li A, Yuan J, Xu T (2018) Cell-Type-Specific Afferent Innervation of the

Nucleus Accumbens Core and Shell. Frontiers in Neuroanatomy 12.

Linden A-M, Schoepp DD (2006) Metabotropic glutamate receptor targets for neuropsychiatric

disorders. Drug Discovery Today: Therapeutic Strategies 3:507-517.

Lissek S, Powers AS, McClure EB, Phelps EA, Woldehawariat G, Grillon C, Pine DS (2005)

Classical fear conditioning in the anxiety disorders: a meta-analysis. Behaviour research

and therapy 43:1391-1424.

Lobo MK, Karsten SL, Gray M, Geschwind DH, Yang XW (2006) FACS-array profiling of striatal

projection neuron subtypes in juvenile and adult mouse brains. Nature neuroscience 9:443-

452.

Lonsdorf TB et al. (2017) Don’t fear ‘fear conditioning’: Methodological considerations for the

design and analysis of studies on human fear acquisition, extinction, and return of fear.

Neuroscience & Biobehavioral Reviews 77:247-285.

Louilot A, Le Moal M, Simon H (1986) Differential reactivity of dopaminergic neurons in the

nucleus accumbens in response to different behavioral situations. An in vivo voltammetric

study in free moving rats. Brain Research 397:395-400.

Lu Y-M, Jia Z, Janus C, Henderson JT, Gerlai R, Wojtowicz JM, Roder JC (1997) Mice Lacking

Metabotropic Glutamate Receptor 5 Show Impaired Learning and Reduced CA1 Long-

Term Potentiation (LTP) But Normal CA3 LTP. The Journal of Neuroscience 17:5196.

Malone DA, Dougherty DD, Rezai AR, Carpenter LL, Friehs GM, Eskandar EN, Rauch SL,

Rasmussen SA, Machado AG, Kubu CS, Tyrka AR, Price LH, Stypulkowski PH, Giftakis

JE, Rise MT, Malloy PF, Salloway SP, Greenberg BD (2009) Deep Brain Stimulation of

the Ventral Capsule/Ventral Striatum for Treatment-Resistant Depression. Biological

psychiatry 65:267-275.

Mao L-M, Wang JQ (2016) Regulation of Group I Metabotropic Glutamate Receptors by

MAPK/ERK in Neurons. J Nat Sci 2:e268.

Maren S, Holt WG (2004) Hippocampus and Pavlovian fear conditioning in rats: muscimol

infusions into the ventral, but not dorsal, hippocampus impair the acquisition of conditional

freezing to an auditory conditional stimulus. Behavioral neuroscience 118:97.

Maren S, Holmes A (2016) Stress and Fear Extinction. Neuropsychopharmacology 41:58-79.

Maren S, Aharonov G, Fanselow MS (1996a) Retrograde abolition of conditional fear after

excitotoxic lesions in the basolateral amygdala of rats: absence of a temporal gradient.

Behavioral neuroscience 110:718.

Maren S, Aharonov G, Stote DL, Fanselow MS (1996b) N-methyl-D-aspartate receptors in the

basolateral amygdala are required for both acquisition and expression of conditional fear

in rats. Behavioral neuroscience 110:1365.

Martin SJ, Grimwood PD, Morris RGM (2000) Synaptic plasticity and memory: an evaluation of

the hypothesis. Annual review of neuroscience 23:649-711.

Martinez RCR, Oliveira AR, Macedo CE, Molina VA, Brandão ML (2008) Involvement of

dopaminergic mechanisms in the nucleus accumbens core and shell subregions in the

expression of fear conditioning. Neuroscience Letters 446:112-116.

McDonald AJ (1982) Cytoarchitecture of the central amygdaloid nucleus of the rat. Journal of

Comparative Neurology 208:401-418.

McLean CP, Asnaani A, Litz BT, Hofmann SG (2011) Gender differences in anxiety disorders:

prevalence, course of illness, comorbidity and burden of illness. Journal of psychiatric

research 45:1027-1035.

Medina JH, Viola H (2018) ERK1/2: A Key Cellular Component for the Formation, Retrieval,

Reconsolidation and Persistence of Memory. Frontiers in Molecular Neuroscience 11.

Meldrum BS (2000) Glutamate as a Neurotransmitter in the Brain: Review of Physiology and

Pathology. The Journal of Nutrition 130:1007S-1015S.

Meredith GE, Pattiselanno A, Groenewegen HJ, Haber SN (1996) Shell and core in monkey and

human nucleus accumbens identified with antibodies to calbindin-D28k. Journal of

Comparative Neurology 365:628-639.

Michael T, Zetsche U, Margraf J (2007a) Epidemiology of anxiety disorders. Psychiatry 6:136-

142.

Michael T, Blechert J, Vriends N, Margraf J, Wilhelm FH (2007b) Fear conditioning in panic

disorder: Enhanced resistance to extinction. Journal of abnormal psychology 116:612.

Milad MR, Quirk GJ (2002) Neurons in medial prefrontal cortex signal memory for fear extinction.

Nature 420:70-74.

Milad MR, Quirk GJ (2012) Fear extinction as a model for translational neuroscience: ten years

of progress. Annual review of psychology 63:129-151.

Milad MR, Rauch SL, Pitman RK, Quirk GJ (2006) Fear extinction in rats: Implications for human

brain imaging and anxiety disorders. Biological Psychology 73:61-71.

Milad MR, Wright CI, Orr SP, Pitman RK, Quirk GJ, Rauch SL (2007) Recall of fear extinction

in humans activates the ventromedial prefrontal cortex and hippocampus in concert.

Biological psychiatry 62:446-454.

Mogenson GJ (1985) From motivation to action: a review of dopaminergic regulation of limbic->

nucleus accumbens-> ventral pallidum-> pedunculopontine nucleus circuitries involved in

limbic-motor integration. Limbic motor circuits and neuropsychiatry:193-236.

Moroni F, Nicoletti F, Pellegrini-Giampietro DE Metabotropic glutamate receptors and brain

function. In: Portland.

Myers KM, Davis M (2002) Behavioral and Neural Analysis of Extinction. Neuron 36:567-584.

Myers KM, Davis M (2007) Mechanisms of fear extinction. Molecular Psychiatry 12:120-150.

Naisbitt S, Kim E, Tu JC, Xiao B, Sala C, Valtschanoff J, Weinberg RJ, Worley PF, Sheng M

(1999) Shank, a novel family of postsynaptic density proteins that binds to the NMDA

receptor/PSD-95/GKAP complex and cortactin. Neuron 23:569-582.

Nakanishi S (1994) Metabotropic glutamate receptors: Synaptic transmission, modulation, and

plasticity. Neuron 13:1031-1037.

Nakielny S, Cohen P, Wu J, Sturgill T (1992) MAP kinase activator from insulin‐stimulated

skeletal muscle is a protein threonine/tyrosine kinase. The EMBO Journal 11:2123-2129.

Nie X, Sun Y, Wan S, Zhao H, Liu R, Li X, Wu S, Nedelska Z, Hort J, Qing Z (2017) Subregional

structural alterations in hippocampus and nucleus accumbens correlate with the clinical

impairment in patients with Alzheimer’s disease clinical spectrum: parallel combining

volume and vertex-based approach. Frontiers in neurology 8:399.

Nielsen KS, Macphail EM, Riedel G (1997) Class I mGlu receptor antagonist 1-aminoindan-1,5-

dicarboxylic acid blocks contextual but not cue conditioning in rats. European Journal of

Pharmacology 326:105-108.

Orr SP, Metzger LJ, Lasko NB, Macklin ML, Peri T, Pitman RK (2000) De novo conditioning in

trauma-exposed individuals with and without posttraumatic stress disorder. Journal of

abnormal psychology 109:290.

Otto MW, Moshier SJ, Kinner DG, Simon NM, Pollack MH, Orr SP (2014) De Novo Fear

Conditioning Across Diagnostic Groups in the Affective Disorders: Evidence for Learning

Impairments. Behavior Therapy 45:619-629.

Pavlov PI (2010) Conditioned reflexes: an investigation of the physiological activity of the cerebral

cortex. Annals of neurosciences 17:136.

Pecknold JC, McClure DJ, Appeltauer L, Wrzesinski L, Allan T (1982) Treatment of anxiety using

fenobam (a nonbenzodiazepine) in a double-blind standard (diazepam) placebo-controlled

study. Journal of clinical psychopharmacology 2:129-132.

Peri T, Ben-Shakhar G, Orr SP, Shalev AY (2000) Psychophysiologic assessment of aversive

conditioning in posttraumatic stress disorder. Biological psychiatry 47:512-519.

Perusini JN, Fanselow MS (2015) Neurobehavioral perspectives on the distinction between fear

and anxiety. Learning & Memory 22:417-425.

Peters J, Kalivas PW, Quirk GJ (2009) Extinction circuits for fear and addiction overlap in

prefrontal cortex. Learning & memory (Cold Spring Harbor, NY) 16:279-288.

Pezze MA, Heidbreder CA, Feldon J, Murphy CA (2001) Selective responding of nucleus

accumbens core and shell dopamine to aversively conditioned contextual and discrete

stimuli. Neuroscience 108:91-102.

Phelps EA, Delgado MR, Nearing KI, LeDoux JE (2004) Extinction Learning in Humans: Role of

the Amygdala and vmPFC. Neuron 43:897-905.

Phillipson OT, Griffiths AC (1985) The topographic order of inputs to nucleus accumbens in the

rat. Neuroscience 16:275-296.

Pierce RC, Bell K, Duffy P, Kalivas PW (1996) Repeated cocaine augments excitatory amino acid

transmission in the nucleus accumbens only in rats having developed behavioral

sensitization. Journal of Neuroscience 16:1550-1560.

Pilc A, Kłodzińska A, Brański P, Nowak G, Pałucha A, Szewczyk B, Tatarczyńska E, Chojnacka-

Wójcik E, Wierońska JM (2002) Multiple MPEP administrations evoke anxiolytic- and

antidepressant-like effects in rats. Neuropharmacology 43:181-187.

Pin JP, Duvoisin R (1995) The metabotropic glutamate receptors: Structure and functions.

Neuropharmacology 34:1-26.

Pitkanen A, Pikkarainen M, Nurminen N, Ylinen A (2000) Part VII. The Parahippocampus in

Context: Interaction with Other Brain Regions-Reciprocal Connections between the

Amygdala and the Hippocampal Formation, Perirhinal Cortex, and Postrhinal Cortex in.

Annals of the New York Academy of Sciences-Paper Edition 911:369-391.

Porter RHP, Jaeschke G, Spooren W, Ballard TM, Büttelmann B, Kolczewski S, Peters J-U,

Prinssen E, Wichmann J, Vieira E (2005) Fenobam: a clinically validated

nonbenzodiazepine anxiolytic is a potent, selective, and noncompetitive mGlu5 receptor

antagonist with inverse agonist activity. Journal of Pharmacology and Experimental

Therapeutics 315:711-721.

Qi J, Zhang S, Wang H-L, Barker DJ, Miranda-Barrientos J, Morales M (2016) VTA glutamatergic

inputs to nucleus accumbens drive aversion by acting on GABAergic interneurons. Nature

neuroscience 19:725-733.

Quirk GJ, Repa JC, LeDoux JE (1995) Fear conditioning enhances short-latency auditory

responses of lateral amygdala neurons: parallel recordings in the freely behaving rat.

Neuron 15:1029-1039.

Quirk GJ, Russo GK, Barron JL, Lebron K (2000) The role of ventromedial prefrontal cortex in

the recovery of extinguished fear. The Journal of neuroscience : the official journal of the

Society for Neuroscience 20:6225-6231.

Quirk GJ, Likhtik E, Pelletier JG, Paré D (2003) Stimulation of medial prefrontal cortex decreases

the responsiveness of central amygdala output neurons. Journal of Neuroscience 23:8800-

8807.

Rescorla RA (1988) Pavlovian conditioning: It's not what you think it is. American psychologist

43:151.

Rescorla RA (1996) Preservation of Pavlovian Associations through Extinction. The Quarterly

Journal of Experimental Psychology Section B 49:245-258.

Ribeiro FM, Paquet M, Cregan SP, Ferguson SS (2010) Group I metabotropic glutamate receptor

signalling and its implication in neurological disease. CNS Neurol Disord Drug Targets

9:574-595.

Riedel G, Wetzel W, Reymann KG (1996) Comparing the role of metabotropic glutamate receptors

in long-term potentiation and in learning and memory. Progress in Neuro-

Psychopharmacology and Biological Psychiatry 20:761-789.

Riedel G, Harrington NR, Hall G, Macphail EM (1997) Nucleus accumbens lesions impair context,

but not cue, conditioning in rats. NeuroReport 8:2477-2481.

Rodrigues SM, Bauer EP, Farb CR, Schafe GE, LeDoux JE (2002) The Group I Metabotropic

Glutamate Receptor mGluR5 Is Required for Fear Memory Formation and Long-Term

Potentiation in the Lateral Amygdala. The Journal of Neuroscience 22:5219.

Rosenkranz JA, Moore H, Grace AA (2003) The prefrontal cortex regulates lateral amygdala

neuronal plasticity and responses to previously conditioned stimuli. Journal of

Neuroscience 23:11054-11064.

Rothe T, Bigl V, Grantyn R (1994) Potentiating and depressant effects of metabotropic glutamate

receptor agonists on high-voltage-activated calcium currents in cultured retinal ganglion

neurons from postnatal mice. Pflügers Archiv 426:161-170.

Russo SJ, Nestler EJ (2013) The brain reward circuitry in mood disorders. Nature Reviews

Neuroscience 14:609-625.

Salgado S, Kaplitt MG (2015) The Nucleus Accumbens: A Comprehensive Review. Stereotactic

and Functional Neurosurgery 93:75-93.

Sananbenesi F, Fischer A, Schrick C, Spiess J, Radulovic J (2002) Phosphorylation of

Hippocampal Erk-1/2, Elk-1, and p90-Rsk-1 during Contextual Fear Conditioning:

Interactions between Erk-1/2 and Elk-1. Molecular and Cellular Neuroscience 21:463-476.

Schafe GE, Atkins CM, Swank MW, Bauer EP, Sweatt JD, LeDoux JE (2000) Activation of

ERK/MAP Kinase in the Amygdala Is Required for Memory Consolidation of Pavlovian

Fear Conditioning. The Journal of Neuroscience 20:8177.

Schlaepfer TE, Cohen MX, Frick C, Kosel M, Brodesser D, Axmacher N, Joe AY, Kreft M,

Lenartz D, Sturm V (2008) Deep Brain Stimulation to Reward Circuitry Alleviates

Anhedonia in Refractory Major Depression. Neuropsychopharmacology 33:368-377.

Schwienbacher I, Fendt M, Richardson R, Schnitzler H-U (2004) Temporary inactivation of the

nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats.

Brain Research 1027:87-93.

Shaham Y, Erb S, Stewart J (2000) Stress-induced relapse to heroin and cocaine seeking in rats: a

review. Brain Research Reviews 33:13-33.

Sierra-Mercado D, Padilla-Coreano N, Quirk GJ (2010) Dissociable Roles of Prelimbic and

Infralimbic Cortices, Ventral Hippocampus, and Basolateral Amygdala in the Expression

and Extinction of Conditioned Fear. Neuropsychopharmacology 36:529.

Sinha R, Garcia M, Paliwal P, Kreek MJ, Rounsaville BJ (2006) Stress-induced cocaine craving

and hypothalamic-pituitary-adrenal responses are predictive of cocaine relapse outcomes.

Archives of general psychiatry 63:324-331.

Sturm V, Lenartz D, Koulousakis A, Treuer H, Herholz K, Klein JC, Klosterkötter J (2003) The

nucleus accumbens: a target for deep brain stimulation in obsessive–compulsive- and

anxiety-disorders. Journal of Chemical Neuroanatomy 26:293-299.

Swanson CJ, Bures M, Johnson MP, Linden A-M, Monn JA, Schoepp DD (2005) Metabotropic

glutamate receptors as novel targets for anxiety and stress disorders. Nature Reviews Drug

Discovery 4:131-144.

Swartz KJ, Bean BP (1992) Inhibition of calcium channels in rat CA3 pyramidal neurons by a

metabotropic glutamate receptor. Journal of Neuroscience 12:4358-4371.

Sweatt JD (2004) Mitogen-activated protein kinases in synaptic plasticity and memory. Curr Opin

Neurobiol 14:311-317.

Takeuchi T, Duszkiewicz AJ, Morris RGM (2014) The synaptic plasticity and memory hypothesis:

encoding, storage and persistence. Philosophical Transactions of the Royal Society B:

Biological Sciences 369:20130288.

Taylor JR, Olausson P, Quinn JJ, Torregrossa MM (2009) Targeting extinction and reconsolidation

mechanisms to combat the impact of drug cues on addiction. Neuropharmacology 56:186-

195.

Thomas GM, Huganir RL (2004) MAPK cascade signalling and synaptic plasticity. Nature

Reviews Neuroscience 5:173-183.

Toyoda H, Zhao M-G, Xu H, Wu L-J, Ren M, Zhuo M (2007) Requirement of extracellular signal-

regulated kinase/mitogen-activated protein kinase for long-term potentiation in adult

mouse anterior cingulate cortex. Molecular Pain 3:1744-8069.

Trifilieff P, Herry C, Vanhoutte P, Caboche J, Desmedt A, Riedel G, Mons N, Micheau J (2006)

Foreground contextual fear memory consolidation requires two independent phases of

hippocampal ERK/CREB activation. Learning & Memory 13:349-358.

Tu JC, Xiao B, Naisbitt S, Yuan JP, Petralia RS, Brakeman P, Doan A, Aakalu VK, Lanahan AA,

Sheng M (1999) Coupling of mGluR/Homer and PSD-95 complexes by the Shank family

of postsynaptic density proteins. Neuron 23:583-592.

Vidal-Gonzalez I, Vidal-Gonzalez B, Rauch SL, Quirk GJ (2006) Microstimulation reveals

opposing influences of prelimbic and infralimbic cortex on the expression of conditioned

fear. Learning & Memory 13:728-733.

Voorn P, Brady LS, Schotte A, Berendse HW, Richfield EK (1994) Evidence For Two

Neurochemical Divisions in the Human Nucleus Accumbens. European Journal of

Neuroscience 6:1913-1916.

Wang B, Yang X, Sun A, Xu L, Wang S, Lin W, Lai M, Zhu H, Zhou W, Lian Q (2016a)

Extracellular Signal-Regulated Kinase in Nucleus Accumbens Mediates Propofol Self-

Administration in Rats. Neuroscience Bulletin 32:531-537.

Wang H, Ardiles AO, Yang S, Tran T, Posada-Duque R, Valdivia G, Baek M, Chuang Y-A,

Palacios AG, Gallagher M, Worley P, Kirkwood A (2016b) Metabotropic Glutamate

Receptors Induce a Form of LTP Controlled by Translation and Arc Signaling in the

Hippocampus. The Journal of Neuroscience 36:1723.

Wang JQ, Fibuch EE, Mao L (2007a) Regulation of mitogen-activated protein kinases by

glutamate receptors. Journal of Neurochemistry 100:1-11.

Wang JQ, Fibuch EE, Mao L (2007b) Regulation of mitogen‐activated protein kinases by

glutamate receptors. Journal of neurochemistry 100:1-11.

Waters AM, Pine DS (2016) Evaluating differences in Pavlovian fear acquisition and extinction

as predictors of outcome from cognitive behavioural therapy for anxious children. Journal

of Child Psychology and Psychiatry 57:869-876.

Watson JB, Rayner R (1920) Conditioned emotional reactions. Journal of experimental

psychology 3:1.

Weiskrantz L (1956) Behavioral changes associated with ablation of the amygdaloid complex in

monkeys. Journal of comparative and physiological psychology 49:381.

Wittchen HU (2002) Generalized anxiety disorder: prevalence, burden, and cost to society.

Depress Anxiety 16:162-171.

Wright CI, Beijer AV, Groenewegen HJ (1996) Basal amygdaloid complex afferents to the rat

nucleus accumbens are compartmentally organized. Journal of Neuroscience 16:1877-

1893.

Yang JH, Mao L-M, Choe ES, Wang JQ (2017) Synaptic ERK2 Phosphorylates and Regulates

Metabotropic Glutamate Receptor 1 In Vitro and in Neurons. Molecular Neurobiology

54:7156-7170.

Záborszky L, Alheid GF, Beinfeld MC, Eiden LE, Heimer L, Palkovits M (1985) Cholecystokinin

innervation of the ventral striatum: A morphological and radioimmunological study.

Neuroscience 14:427-453.

Zahm DS (1998) Is the Caudomedial Shell of the Nucleus Accumbens Part of the Extended

Amygdala? A Consideration of Connections. 12:245-265.

Zahm DS (1999) Functional-anatomical implications of the nucleus accumbens core and shell

subterritories. Annals of the New York Academy of Sciences 877:113-128.

Zahm DS, Brog JS (1992) On the significance of subterritories in the “accumbens” part of the rat

ventral striatum. Neuroscience 50:751-767.

Zahorodna A, Bijak M (1999) An antidepressant-induced decrease in the responsiveness of

hippocampal neurons to group I metabotropic glutamate receptor activation. European

Journal of Pharmacology 386:173-179.

Ziegler MG (2012) Chapter 61 - Psychological Stress and the Autonomic Nervous System. In:

Primer on the Autonomic Nervous System (Third Edition) (Robertson D, Biaggioni I,

Burnstock G, Low PA, Paton JFR, eds), pp 291-293. San Diego: Academic Press.

CHAPTER 2

The nucleus accumbens core is necessary for fear expression whereas the shell regulates

fear extinction consolidation

2.1 Introduction

The NAc is a heterogenous structure divided into two distinct sub-regions, the central and the dorsal region is designated as the core, which is surrounded medially, ventrally, and ventrolaterally by the shell. These two subregions differ in their connectional and cellular framework (Zahm and Brog, 1992b; Voorn et al., 1994; Meredith et al., 1996; Baliki et al.,

2013). The NAc is a part of the cortico-striatal-thalamo-cortical loop and the two subregions show distinctive innervation patterns by brain regions, heavily implicated in emotional learning, such as the amygdala, hippocampus, prefrontal cortex and the ventral tegmental area as illustrated in figure 2.1 (Brog et al., 1993; Brauer et al., 2000). The medial shell receives heavy inputs specifically from the infralimbic cortex whereas the prelimbic cortex projects heavily to the core. The hippocampus projects heavily to both core and shell, however the shell receives considerably more dense inputs from the CA1 region as compared to the core (Li et al., 2018).

The basal amygdala sends glutamatergic efferent to both the core and shell but does not preferentially innervate either of the two subregions (Phillipson and Griffiths, 1985; Brog et al.,

1993; Wright and Groenewegen, 1995; Floresco, 2015; Correia et al., 2016). Dopaminergic inputs from the ventral tegmental area preferentially innervate the shell as compared to the core

(Gerfen et al., 1987; Salgado and Kaplitt, 2015). In addition to the differences in the afferents, the two subregions also show differences in their efferent projections. The core predominantly

connects with the premotor areas of the cortex whereas the shell connects with the prefrontal and subcortical motor areas (Alexander et al., 1991; Zahm and Brog, 1992a; Zahm, 1999). While the shell projects to the lateral hypothalamus and the extended amygdala and is considered a part of the transition zone between the two, the core has no connections with either region. Both the core and shell project to the ventral pallidum but the shell innervates the neurotensin-rich area in the ventromedial ventral pallidum whereas the core projects to the dorsolateral ventral pallidum which further communicates with the subthalamic nucleus and substantial nigra (Heimer et al.,

1991; Groenewegen et al., 1993). Further, the shell is an important link in the loop and has been suggested to indirectly influence NAc core connections through a feed-forward loop either through a ventral pallido-thalamic pathway including the prefrontal cortex (Groenewegen et al.,

1993) or through the ventral tegmental area and parts of substantial nigra (Nauta et al., 1978;

Haber et al., 2000). In addition, the core and the shell also send out reciprocal GABAergic projections to each other (Taverna et al., 2004; van Dongen et al., 2005).

At the molecular level, the core subregion consists of densely packed cells with a higher density of calbindin, enkephalin, GABA-A receptors and limbic associated protein whereas the shell, composed of large, loosely packed cells, is more populated by substance P, DA and serotonin receptors (Voorn et al., 1989; Caboche et al., 1993; Prensa et al., 2003). In addition, the shell also contains high levels of neurotensin, cholecystokinin and opioid peptides which makes it analogous to the extended amygdala (Deutch and Cameron, 1992; Prensa et al., 2003; Salgado and Kaplitt, 2015)

The differential structural and functional connectivity along with the cellular composition of the two sub-regions suggest that they may regulate distinct behavioral functions, however, to date, the specific dissociable roles of the two sub-regions haven’t been established. Studies

investigating the dopamine transmission within the motivational circuit to regulate aversive cues have shown the core and the shell respond differently to fear-evoking cues. Aversive cues resulted in decreased DA release within the NAc core whereas the absence of expected aversive outcome, as seen during extinction, increases DA release within the NAc shell (Badrinarayan et al., 2012; Luo et al., 2018). In addition, the small amount of literature that currently exists on the contribution of these two subregions in fear learning result in every possible outcome with very contradictory results. Differential regulation of fear memory processes by shell and core using

NMDA lesions where the shell showed reduced freezing to the context and the cue whereas animals with core lesions did not show altered freezing to either cue or context (Jongen-Rêlo et al., 2003). Rats with selective lesions of the NAc shell did not show impairment to either discrete or contextual cues. In contrast, animals with NAc core lesions showed impaired conditioning to discrete cues and enhanced conditioning to contextual cues (Schwienbacher et al., 2004b). In another study lesions in the NAc core disrupted contextual fear expression (Levita et al., 2002b). Thus, although it seems that the core and the shell play a distinct role in fear conditioning, their specific contribution remain unclear because of the inconsistent findings in the literature. Some of these inconsistencies could be due to procedural differences (e.g., number of training trials, intensity of shock, etc.) between the various studies, or the use of different lesioning techniques (i.e., temporary vs. permanent; excitotoxic vs. electrolytic). Data from a number of labs using a wide variety of behavioral, methods suggest that the NAc is critically involved in aversive behavior and fear learning (McCullough et al., 1993; Salamone, 1994; Beck and Fibiger, 1995; Haralambous and Westbrook, 1999; Levita et al., 2002b; Reynolds and

Berridge, 2002; Thomas et al., 2002; Jongen-Relo et al., 2003; Reynolds and Berridge, 2003;

Schwienbacher et al., 2004a; Pecina et al., 2006; Muschamp et al., 2011; Richard and Berridge,

2011; Lichtenberg et al., 2014; Wendler et al., 2014a; Mika et al., 2015; Ramirez et al., 2015;

McCullough et al., 2016), but the underlying neurobiology is poorly understood and only a few studies have made functional distinctions in NAc anatomy in the same study (Reynolds and

Berridge, 2002; Richard and Berridge, 2011; Richard and Berridge, 2013). In fact, to date the specific dissociable role of the two sub-regions in Pavlovian fear learning remain unclear. The present studies aim to evaluate the specific involvement of the two subregions in fear expression and fear extinction by immunohistochemistry using cell activity markers and systemic inactivation of the sub-regions. Given the strong connections of the shell and the core with the IL and PL respectively, along with non-preferential connections to the basolateral amygdala, we hypothesize that the core subregion is involved in regulation of fear expression and the shell in mediating fear extinction consolidation.

Figure 3 A simplified schematic of major efferents to the nucleus accumbens core (NAcC) and shell (NAcS) from the basolateral amygdala (BLA), infralimbic cortex (IL), prelimbic cortex (PL) and the ventral tegmental area (VTA). The BLA sends glutamatergic projections to both the core and the shell. The PL sends glutamatergic projections specifically to the core whereas the IL projects specifically to the shell. Dopaminergic inputs from the VTA preferentially innervate the shell as compared to the core. The hippocampus sends glutamatergic connections to both core and shell though the latter receives more dense projections from the

CA1 subregion. The image was created with biorender.com.

2.2 Methods

2.2.1 Animals

All experiments used 7-9 weeks old adult male C57BL/6J mice which were generated from a breeding colony in the Department of Psychological Sciences at Kent State University, from breeders originally purchased from Jackson Laboratories (Stock# 000664). All experimental mice were of F4 generation or less to avoid genetic drift. All mice were group housed with 2-5 animals per cage and were on a 12:12 light/ dark cycle with free access to food and water. Mice used for experiments were seven weeks or older. All experiments were conducted with approval from the Kent State Institutional Animal Care and Use Committee

(IACUC) and in a facility accredited by the Association for Assessment and Accreditation and

Laboratory Animal Care (AALAC), in accordance with the NIH guidelines for the Care and Use of Laboratory Animals, 8th edition.

2.2.2 Stereotaxic Surgery

Mice at 7-9 weeks of age underwent stereotaxic surgery for cannula implantation. Mice were anesthetized with a subcutaneous (sc.) injection of ketamine (75mg/kg) + xylazine (10mg/kg) and acepromazine (2mg/kg) cocktail prior to the surgery. Following anesthesia, the mice received a single subcutaneous administration of ketoprofen (5mg/kg) for analgesia. The mice were then mounted on a stereotaxic apparatus (David Kopf instruments, Tujunga, CA) and the scalp of each mouse was retracted. The skull was adjusted so that lambda and bregma were within 0.5mm of each other. Two guide cannulae (26 gauge, 4 mm pedestal height; Plastics One,

Ranaoke, VA) were bilaterally implanted aimed at the NAc core (+1.18mm AP; +2.20mm ML; -

4.52mm DV; angled at 14◦) or NAc shell (+1.30mm AP; +1.60mm ML; -4.40mm DV; angled at

10◦), secured with screws and cranial cement. Dummy cannulae were inserted in the guide

cannulae after the completion of the surgeries. Upon completion of surgeries, the anesthesia was reversed with a subcutaneous injection of atipamezole (0.5 mg/kg). All mice were given one week to recover prior to the start of behavioral procedures.

2.2.3 Apparatus

Fear conditioning was performed in four identical conditioning chambers (7” W x 155 7” D x

12”H) containing two Plexiglas walls, two aluminum sidewalls, and a stainless-steel grid-shock floor, and positioned inside an isolation chamber (Coulbourn Instruments, Allentown, PA). A speaker was positioned along the sidewall to generate a tone to provide the conditional stimulus

(CS). The cued fear training context, Context A, consisted of the conditioning chamber with a polka-dot insert attached to the rear Plexiglas wall, dim illumination, and the stainless-steel grid floors were cleaned with 70% ethanol. Extinction training and retention tests occurred in a distinct context (Context B) which contained no visible illumination (illuminated only with an infrared light) and flat brown Plexiglas floors which were cleaned with 2% Quatricide

(Pharmacal Research Laboratories).

2.2.4 Cued Fear Conditioning and Extinction

All animals were handled for 5 minutes a day, for two consecutive days prior to context exposure. For all experiments, mice were pre-exposed to the Context A for five minutes.

Twenty-four hours later, mice were returned to the conditioning context (Context A) and after a

180s acclimation period, mice received three pairings of the tone CS (75 dB, 6kHz) and unconditional stimulus (US), foot-shock (2s, 0.8 mA). For each of the pairings the US began as soon as the CS ended, and each pairing was separated by an inter-tone interval of 182 seconds.

Mice were removed from the apparatus 30s after the last shock and returned to their home cage.

The following day, animals were tested for fear expression in Context B which also served as

extinction training. For expression testing and extinction training, mice received 21 unreinforced

CS presentations with an ITI of 60 seconds. Twenty-four hours after extinction training, mice were tested for extinction memory in Context B and were presented with 15 unreinforced CS presentations. Following the extinction memory test, infusion sites were verified, and brains collected for immunofluorescence (see below for procedural details). For experiment 1 examining immediate early gene activation in response to fear expression or fear extinction, there were three additional control groups: home cage, tone only, and explicitly unpaired. For the tone only and explicitly unpaired groups, mice were pre-exposed to Context A for five minutes on Day 1. On Day 2, mice in the tone only group were exposed to 3 tones (CS) with an ITI of

182 seconds and then tested in context B, 24 hours later for freezing to the same CS as the day before. For animals in the explicitly unpaired group, training involved exposure to 3 tones (CS) and 3 shock (US) which were unassociated and had an ITI of 60 seconds in between. Expression testing and extinction training paradigm remained the same as before.

2.2.5 Pharmacology and Procedure

All localized infusions were performed through an acute infusion cannula (Plastics One; 33 gauge) inserted into the guide cannula. Infusion cannulae were connected to PE 50 polyethylene tubing ( 0.015″ ID × 0.043″ OD; BD Medical, Franklin Lakes, NJ) that were connected to

Hamilton syringes (Model 85; Hamilton, Reno, NV), and mounted on an infusion pump (PHD

2000, Harvard Apparatus). Pharmacological inhibitors were bilaterally infused at a rate of 0.1

µl/min for a total infusion volume of 0.2 µl. The infusion needle was left in place for an additional minute. To temporarily inactivate the NAc core or shell, mice received localized infusions of either vehicle (Phosphate buffered saline; PBS) or 4% (w/v) lidocaine HCL (pH of ~

7.0; Sigma) dissolved in PBS) dissolved in PBS. Lidocaine is a voltage-dependent sodium

channel blocker and was used to temporarily inactivate the sub-region of interest (Butterworth and Strichartz, 1990) as we have done previously. Mice were trained on the cued fear conditioning task as described above. 24 hours later, mice received an infusion of Lidocaine or vehicle into the NAc core or shell. Infusions lasted two minutes and expression testing/extinction training began 5 minutes after the end of infusions. The mice were then tested for extinction memory 24 hours after extinction training.

2.2.6 Locomotor behavior

Locomotor behavior was assessed using an open field apparatus to confirm that freezing behavior was not altered by lidocaine infusions into the NAc core. We did not verify if NAc shell inactivation had an effect on locomotion because no immediate difference in freezing behavior was observed after lidocaine infusions. Square opaque acrylic open field boxes measuring 42 cm

W × 42 cm D × 39 cm H (Coulbourn Instruments, Allentown PA) served as testing arenas. The boxes were cleaned with 70% ethanol after the testing for each animal was completed. Mice underwent stereotaxic surgeries for guide cannula implantation targeted at the core subregion.

Following one week of recovery, mice received local infusions of either vehicle or lidocaine

(4%) as previously described and 5 minutes later were gently placed in the center of the open field boxes. Locomotor behavior by the mouse was measured for 5 minutes using AnyMaze

(version 5; Stoelting, Wood Dale, IL) and total distance traveled in meters was analyzed. All mice were returned to their home cage following the completion of the test.

2.2.7 Histological verification of cannula placements and immunofluorescence

Calbindin immunohistochemistry is commonly used to delineate the two NAc sub-regions because the core is more densely populated by calbindin positive neurons compared to the shell.

Thus, we performed calbindin immunofluorescence to delineate the shell and core subregions. In

addition, to precisely verify canula placements in the respective subregions and estimate spread of infused pharmacological substances, mice received localized infusions of quisqualic acid

(0.012M), an excitotoxic amino acid which has been used to destroy cholinergic neurons when used in higher concentrations (0.12M). However, when infused at a lower concentration of

0.012M as above, it results in the induction of immediate early genes (IEG) such as cFOS and

ARC (Page et al., 1993). As with all other pharmacological substances, quisqualic acid was bilaterally infused at a rate of 0.1 µl/min for a total infusion volume of 0.2 µl. Thus, we combined local infusions of quisqualic acid (0.012 M) with co-immunofluorescence for calbindin and ARC to verify that our infusions were restricted to the shell or core only.

One hour following completion of infusions, mice were deeply anesthetized with sodium pentobarbitol (Fatal plus; Vortech Pharmaceutics Ltd.). The mice were then transcardially perfused with ice-cold 0.9% saline for 3 minutes followed by 4% paraformaldehyde (PFA) in

0.1M sodium phosphate buffer, pH 7.4 for 7 minutes and the brains were immediately extracted.

Following extraction, the brains were post-fixed in PFA overnight, followed by 24-72 hours cryoprotection in 30% sucrose in 0.1M phosphate buffer solution with 0.02-0.05% sodium azide to avoid bacterial growth. Coronal sections of 40 µm thickness were sectioned on a cryostat and immunofluorescence was performed for ARC and calbindin expression. Coronal brain sections were co-stained with antibodies against ARC and calbindin. Free floating sections were rinsed in 1M phosphate buffer (PB), followed by blocking in 0.1M PBS containing 0.9% NaCl, 0.5%

Triton X-100 and 5% normal alpaca serum for two hours. After blocking, the sections were incubated at 4◦ C for 24 hours with primary antibodies- 1:2000 rabbit anti-ARC (Synaptic systems; 156 003) and 1:500 mouse anti-calbindin (Abcam; ab108404), diluted in 1M PB containing 0.5% Triton X-100. The following day, sections were thoroughly rinsed in 1M PB

and incubated in secondary antibodies (1:1000 anti-rabbit alexafluor 488 and 1:500 anti-mouse

Cy3; Jackson ImmunoResearch) for 2 hours at room temperature followed by 1:2000 DAPI for 1 hour and room temperature. Sections were washed in 1M PB, mounted onto slides, and coverslipped with Prolong Gold Antifade Mountant (Invitrogen).

2.2.8 Immunofluorescence for ARC analysis

For ARC analysis, mice were deeply anesthetized and transcardially perfused as described above either 60 minutes after the end of a fear expression test consisting of 5 tones or 75 minutes after extinction training as described above. Because ARC expression can be elevated for 90 minutes after fear conditioning (Lonergan et al., 2010; Young and Williams, 2013), we chose a slightly longer extinction-to-perfusion interval for the extinction experiment. This was done to ensure that any ARC expression observed was not the result of fear expression to the first several presentations of the CS during the extinction session, which might have obscured ARC expression due solely to extinction (Fig 1). Three additional control groups were included in this experiment: home cage controls, tone only controls, which were only exposed to the CS in the absence of shock, and explicit unpaired training in which the tone and shock presentations were separated by 60 seconds. Coronal sections of 40 µm thickness were cut on a cryostat and immunofluorescence was performed for ARC and calbindin protein. ARC is an IEG which has been implicated in LTP and memory consolidation. It has been found to be localized in dendrites with neuronal stimulation and commonly used as a cell activity marker. Immunofluorescence was conducted as described above. Imaging was conducted on an Olympus FluoView 3000 confocal microscope. The software program FIJI (NIH) was used to quantify the signal intensities of the nucleus accumbens subregions of interest. Each image was despeckled to filter image noise and the background was subtracted using a rolling bar radius of 5.0 . Each image

had a designated upper and lower signal threshold using the auto threshold feature and were then converted to a binary image for each of the different channels. Binary images were then further adjusted using watershed segmentation to automatically separate particles that were touching

(Cullen et al., 2015). Analysis involved counting of the ARC positive cells and DAPI+ cells within each region and expressing the ARC+ cells as a percentage of DAPI positive cells

(ARC+/DAPI+ x 100) within the NAc core and shell for all the different groups keeping the parameters for circularity and cell size constant across all analysis (Lacagnina et al., 2019).

Three bilateral measurements were taken for each region of each mouse from every other consecutive section. The ARC+ cell counts as a percentage of DAPI+ cells for the 3 sections were then averaged together.

2.2.9 Statistical analysis

Freezing during extinction training and extinction testing were analyzed using a two-way repeated measures factorial analysis of variance (ANOVA; treatment X tones) on Graphpad

Prism statistical software. ARC cell count was analyzed using a two-way ANOVA (subregion X behavioral test). Statistically significant ANOVAs were followed up with Sidak’s multiple comparison. Effect sizes were calculated for completed experiments along with post-hoc power analyses using G*Power 3. Please refer to Table 1 for detailed statistical result for each experiment.

2.3 Results

2.3.1 The NAc core is preferentially active during fear expression but not during extinction learning

To identify if the NAc shell and core are preferentially involved in fear expression versus extinction, we first ran mice on cued fear conditioning, which were sacrificed after fear

expression testing (Fig 4 A) or after extinction training (Fig 4 B) and brains processed for ARC expression (Fig 4 C- F). First, calbindin was used as a marker to delineate the two NAc subregions. Calbindin is highly expressed in the NAc core compared to the shell. To confirm our analyses were restricted to the core or the shell, we first verified colocalization of calbindin positive and ARC positive cells in each subregion for each slice. On average across all sections,

27% of the ARC positive cells were colocalized with calbindin in the shell, whereas 64% of the

ARC positive cells were colocalized with calbindin within the core verifying our neuroanatomical specificity of our analyses (Fig 6 B) In addition to a home cage (HC) control group, additional control groups were run using tone only training or explicitly unpaired training protocols. Freezing behavior was as expected in each of the control and experimental groups, with explicitly unpaired and tone only groups displaying minimal freezing during the fear expression test extinction training, whereas the normally trained mice displayed elevated freezing (Fig 5 A). A total of 48 mice were used in the fear expression experiment and a two- way ANOVA revealed a significant main effect of behavioral test (F(3,40) = 111.1 p <0.001), a main effect of subregion (F(1,40) = 57.94, p<.001) and a significant behavioral test X subregion

+ + interaction (F(3,40) = 65.22, p <0.001). There were significantly more Arc /DAPI cells in the

NAc core as compared to the shell during fear expression testing (Fig 5 B, Fig 6 A) (p < 0.05).

Further, there were significantly more Arc+/DAPI+ cells in the NAc core after mice underwent a cued fear expression test as compared to mice that underwent the explicitly unpaired training (p

< 0.05). There was no significant difference in ARC expression in the NAc core or shell between tone only and the explicitly unpaired groups (p > 0.999). Finally, there was no significant difference in Arc+/DAPI+ cells in the NAc shell as compared to the core or shell of mice in the explicitly untrained group suggesting little activation of the NAc shell subregion during the fear

expression test for the two control groups (Fig 5B). These data suggest that the increase in the

ARC activity in the NAc core was due to fear recall and not due to exposure to shock or tones alone and was specific to the NAc core. In contrast, there was significantly more ARC+/DAPI+ cells in the NAc shell as compared to the core in mice that underwent fear extinction (Fig 5 C,

Fig 6 A) (P < 0.05). A total of 48 animals were used in the fear extinction experiment and a two- way ANOVA revealed a significant main effect of behavioral test (F(3,40) = 267.4, p <0.001), a main effect of subregion (F(1,40) = 176.1, p<.001) and a significant behavioral test X subregion interaction (F(3,40) = 163.9, p <0.001). Again, ARC expression in the shell was significantly more in the fear extinction group as compared to the core and shell in the explicitly unpaired group

(Fig 5 C) (p < 0.05). Finally, the number of ARC+/DAPI+ cells in the NAc core subregion of mice that underwent fear extinction was no different than the core or shell subregions in mice of the explicitly unpaired control group suggesting that the NAc core subregion is not active during fear extinction (Fig 5 C).

Taken together, these data suggest that a dissociation between activity of the NAc core and shell subregions during fear expression versus fear extinction. Whereas the NAc core subregion is recruited during fear expression, activity of the NAc shell is recruited during fear extinction and the activity in both regions was dependent upon associate learning.

Figure 4 Schematic of ICC analysis experiment (A) Schematic of behavioral procedure for analysis of ARC+ neurons in the NAc during fear expression. (B) Schematic of behavioral

procedure analysis of ARC+ neurons in the NAc following fear extinction. (C) Animals were transcardially perfused either 60 mins post expression testing or 75 mins post extinction training.

(D) Coronal sections of 40 µm thickness were cut on a cryostat and (E) immunofluorescence was performed for ARC and calbindin expression. (F) Imaging was conducted on an Olympus

FluoView 3000 confocal microscope and signal intensities of the ARC and calbindin fluorescence within the core and the shell subregions were quantified using the software program

FIJI (NIH).

Figure 5 The NAc core is selectively active during fear expression whereas the shell is selectively active following fear extinction.

(A) Behavior during training, expression and extinction. During training (Left), mice receiving traditional tone-shock pairings increased freezing with each successive presentation of the tone.

Tone only mice displayed minimal freezing in response to the tone presentations. Explicitly unpaired mice froze in response to shocks but did not exhibit any association between the shock and tones the next day during the expression or extinction test. Twenty-four hours after training, mice underwent an expression test in context B and were presented with 5 tones (Middle).

Traditionally trained mice (Exp Testing) displayed significantly more freezing compared to the

Explicitly Unpaired and Tone Only groups. During extinction (Right), mice that were trained with tone-shock pairings (Extinction Training) displayed high freezing during the initial tones but then exhibited extinction in response to repeated tone presentations. Mice that were in the

Tone Only or Explicitly Unpaired groups displayed minimal freezing to tones throughout the procedure. Values are displayed as average percent freezing (± SEM). (B) ARC+/DAPI+ cell analysis for fear expression. The NAc core subregion is selectively active during fear expression.

There were significantly more ARC+ cells in the NAc core as compared to the shell after a fear expression test. ARC expression in the NAc shell was near baseline and no different than tone only and explicitly unpaired groups. Values are displayed as mean (Arc+/DAPI+) x 100 (±SEM).

Significance values were set at p < 0.05 (*** = p < 0.01). (C) Arc+/DAPI+ cell analysis for fear extinction. The NAc shell subregion is selectively active during fear extinction. There were significantly more ARC+ cells in the NAc shell as compared to the core after a fear extinction.

ARC expression in the NAc core was near baseline and no different than tone only and explicitly unpaired groups. Values are displayed as mean (Arc+/DAPI+) x 100 (±SEM). Significance values were set at p < 0.05 (*** = p < 0.01).

Arc Core

Calbindin = Red Calbindin = Red Arc = Green

Figure 6 Confocal images of calbindin and ARC expression in the NAc core and shell

(A) Representative image of calbindin expression showing intense staining in the NAc core, but little staining in the shell (Left). Representative image showing absence of colocalization between calbindin and ARC in a the NAc shell subregion (Center), and colocalization between

ARC and calbindin expression in the core (Right). (B) Representative images of ARC expression in the NAc core and shell in mice that underwent normal cued fear conditioning and then tested for fear expression (Top) and after fear extinction (Bottom). ARC expression was significantly greater in the NAc core during a fear expression test versus after fear extinction. In contrast,

ARC expression was significantly greater in the NAc shell after fear extinction versus during fear expression.

2.3.2 Inactivation of the NAc Core disrupts fear expression but has no effect on extinction learning or extinction retention.

To evaluate the influence of the core subregion in regulating fear expression and extinction, all mice were trained and tested for cued fear conditioning and received local infusions of either lidocaine or vehicle into the core subregion five minutes prior to expressing testing/extinction training (Fig 7 A). Twenty-four hours later the animals were tested for extinction retention. A total of 25 mice were used for these experiments, 6 animals were removed from the analysis either due to unilateral infusions or complete missed targets. Mice that had their core subregion inactivated showed attenuated freezing to the tones during the expression testing/extinction training session (Fig 7 B). A two-way ANOVA revealed a significant main effect of treatment

(F(1, 23) = 11.340, p =0.003) and a significant tone X treatment interaction (F(14, 322) = 2.02, p=

0.016). When tested for extinction retention 24 h later, there was no main effect of treatment (F(1,

23) = 0.004, p=0.948), a main effect of tone (F(14, 322)=2.956, p <0.001), and no significant interaction (F(14, 23) = 0.56, p=0.894). Because the NAc core strongly influences motor behavior we wanted to verify that the effects of lidocaine on freezing were not due to its effects on locomotor activity. Thus, mice were infused with lidocaine into the core subregion and tested in an open field. Distance travelled during a 10 min test revealed no significant differences in the total distance traveled between lidocaine-treated and vehicle-treated animals (t(18) = 0.263, p =

0.7953), suggesting that the reduced freezing as a result of NAc core inactivation was not due to alterations in NAc control of motor behavior (Fig 7 C).

Figure 7 Inactivation of the NAc core disrupts fear expression but not extinction learning or the consolidation of extinction. (A) Schematic representation of the experimental paradigm.

All mice underwent cued fear conditioning (FC) in Context A. Twenty-four hours later, mice underwent a fear expression test that also served as extinction training in Context B (Exp

Test/Ext). Mice received infusions of lidocaine (Lid) or vehicle (Veh) 5 minutes before the expression test/extinction training. Twenty-four hours after extinction training, mice were tested

for extinction memory in Context B (Ext Ret). (B) Inactivation of the nucleus accumbens core attenuated fear expression during the expression test, but did not disrupt extinction learning or the consolidation of extinction. Inactivation of the NAc core prior to the fear expression test had no effect on consolidation of extinction. The percent freezing of mice that were infused with lidocaine (filled circles) were compared with those that were infused with vehicle (open circles).

There were no significant differences between lidocaine-treated and vehicle-treated mice during the extinction retention test. Values are represented as % freezing (±SEM). Statistical significance was set at p < 0.05) (C) To verify that temporary inactivation of the nucleus accumbens core did not alter freezing by modulating motor activity, mice were tested in an open field apparatus after an infusion of lidocaine or vehicle in the nucleus accumbens core. Distance travelled during a 10 min test revealed no significant differences in the total distance traveled between lidocaine-treated (filled bars) and vehicle-treated animals (white bars) (t(16) = 0.264, p =

0.7953). (D) Site verification of lidocaine infusions. (Left) Region of interest in the representative image of ARC spread used to verify canula placements in the nucleus accumbens core. Verification of infusions were accomplished through colocalization of calbindin (Red) and

ARC(Green) expression. The core subregion displays elevated calbindin compared to the shell subregion. (Right) Detailed mapping representing the maximum spread (light red), and the average spread (dark red) of ARC expression in mice used in this experiment. ARC expression was largely restricted to the nucleus accumbens core, with minimal spread into the shell subregion.

2.3.3 Inactivation of the NAc shell disrupts the consolidation of fear extinction memory but has no effect on fear expression or extinction learning

Given the ARC expression in the NAc shell associated with fear extinction, we next wanted to evaluate how inactivation of the NAc shell would affect fear expression and extinction. A total of

32 mice were used for these experiments, 9 mice were removed from the analysis either due to unilateral infusions or due to complete missed targets. During the expression test and extinction training session, there was no main effect of treatment (F(1, 30) = 0.09, p = 0.77), a main effect of tone (F(14, 420)=8.650, p <0.001) suggesting successful within session extinction, and no significant treatment X tone interaction ( F(14, 420) = 0.87, p=0.593). However, when the animals were tested for extinction retention 24 h later, a two-way ANOVA revealed that there was a main effect of treatment (F (1, 30) = 6.86, p=0.01) and a significant treatment X tone interaction (F (14,

420) = 1.854, p=0.03). Lidocaine-treated animals froze significantly more to the tone compared to the vehicle-treated animals (Fig 8 B). These data suggest that the shell subregion is necessary for the consolidation and retention of extinction memory. Taken together, these data suggest that the

NAc shell is necessary for extinction memory, but not for fear expression or extinction learning itself.

Figure 8 Inactivation of the NAc shell disrupts the consolidation of fear extinction but has no effect on fear expression or extinction learning. (A) Schematic representation of the experimental paradigm. All mice underwent cued fear conditioning in Context A (FC). Twenty-

four hours later, mice underwent a fear expression test that also served as extinction training in

Context B (Exp Test/Ext). Mice received infusions of lidocaine (Lid) or vehicle (Veh) 5 minutes before the expression test/extinction training. Twenty-four hours after extinction training, mice were tested for extinction memory in Context B (Ext Ret). (B) Inactivation of the nucleus accumbens shell had no effect on fear expression or extinction learning. The percent freezing of mice that were infused with lidocaine (filled circles) were compared with those that were infused with vehicle (open circles). Values are represented as % freezing (±SEM). Significance was set at p < 0.05. However, when mice were tested for extinction retention 24 h later, lidocaine-treated mice froze significantly more to the tone compared to the vehicle-treated mice (p < 0.05) suggesting a disrupted consolidation of extinction. (C) (Left) Region of interest in the representative image of ARC spread used to verify canula placements in the nucleus accumbens shell. (Right) Representative image of ARC spread in the NAc shell. The nucleus accumbens core shows extensive calbindin staining (Red) which is absent in the shell subregion, where ARC expression is restricted (Green). (D) Site verification of lidocaine infusions. Detailed mapping representing the maximum spread (light red), and the average spread (dark red) of ARC expression in mice used in this experiment. ARC expression was largely restricted to the nucleus accumbens shell, with minimal spread into the core subregion.

Statistical F/t %Total Effect Subregion Inactivation test Infusion Test day Comparison Statistic DF variance P * ηp2 Size Power Tone X Treatment 2.021 14, 322 3.744 0.016 * 0.105 0.34 0.99 Exp test/ Ext tr Tone 2.972 14, 322 5.507 <.001 *** 0.147 0.41 1.00 Treatment 11.340 1, 23 15.720 0.003 ** 0.330 0.70 1.00 Core Tone X Treatment 0.561 14, 322 1.204 0.894 ns 0.027 0.17 0.85 Ext Ret Repeated Tone 2.956 14, 322 6.339 <.001 *** 0.128 0.38 1.00 Measures Pre-ext Treatment 0.004 1, 23 0.008 0.948 ns 0.000 0.01 0.05 Two-Way tr Tone X Treatment 0.869 14, 420 1.097 0.593 ns 0.020 0.15 0.85 Systemic ANOVA Exp test/ Ext tr Tone 8.650 14, 420 10.910 <.001 *** 0.179 0.47 1.00 Treatment 0.087 1, 30 0.145 0.77 ns 0.002 0.05 0.13 Shell Tone X Treatment 1.854 14, 420 2.951 0.03 * 0.08 0.29 0.99 Ext Ret Tone 3.261 14, 420 5.191 <.001 *** 0.13 0.38 1.00 Treatment 6.868 1, 30 8.061 0.01 * 0.19 0.48 1.00

Core t Test Open Field Distance travelled 0.264 16 0.80 ns 0.00 0.66 0.37 Core vs Behavior 111.100 3,40 53.170 <0.001 *** 0.89 2.89 1.00 Two- way Shell- NA NA NA ANOVA Interaction 65.220 3,40 31.210 <0.001 *** 0.83 2.21 1.00 Expression Subregion 57.940 1,40 9.242 <0.001 *** 0.01 1.20 1.00 Core vs Behavior 267.40 3,40 53.120 <0.001 *** 0.95 4.47 1.00 Two- way Shell- NA NA NA ANOVA Interaction 163.900 3,40 32.570 <0.001 *** 0.92 3.51 1.00 Extinction Subregion 176.100 1,40 11.660 <0.001 *** 0.81 2.10 1.00

Table 1: Behavioral Statistical Summary of experiments 2.1,2.2 and 2.3

2.4 Discussion

Several studies using lesions, as well as investigations into dopamine signaling within the NAc subregions provide evidence that the core and shell have distinct roles regulating memory, approach, fear conditioning and instrumental conditioning (Parkinson et al., 1999; Corbit et al.,

2001; Bassareo et al., 2002; Jongen-Relo et al., 2003; Blaiss and Janak, 2009). Whereas it is clear that this region plays an important role in regulating aversive motivation (Levita et al.,

2002a; Reynolds and Berridge, 2003; Levita et al., 2009; Chen et al., 2012; Wenzel et al., 2015;

Soares-Cunha et al., 2019), the precise functions of the core and shell in specific fear processes have not been fully delineated. In the current set of experiments, we show that the NAc core subregion is distinctly activated during fear retrieval but not following extinction. In contrast, the

NAc shell is active only after extinction, but not during fear retrieval. Pharmacological inactivation of each subregion separately demonstrated that the NAc core promotes fear expression to an explicit cue but is not necessary for fear extinction learning or extinction memory. In contrast, we found that the NAc shell is necessary for the consolidation of extinction memory, but not for fear expression or extinction learning itself. Together, these data suggest dissociable roles of the NAc core and shell subregions in specific control of fear expression and extinction. In particular, the NAc core is more heavily involved in fear recall and its expression, whereas the NAc shell is critical for the consolidation of extinction memory. Although there is support for “mode” switching based on rostral-caudal gradients within the nucleus accumbens

(Berridge, 2019), here we show that separate “modules” exist between the NAc core and shell regulating fear memory and extinction. We did not, however, investigate these responses based on manipulations along a rostral-caudal axis, nor did we investigate appetitive behaviors in our study. Alternatively, extinction may occur partially through counterconditioning (Newall et al.,

2017; Kang et al., 2018) which would lend support the idea of “mode” switching across the two subregions.

We show that the NAc core is specifically active during fear retrieval versus fear extinction, whereas the NAc shell is specifically active following a fear extinction procedure (Fig 5). We note three prior studies in which results suggest that the NAc shell is involved in the expression of fear or fear memory based on IEG analyses (Campeau et al., 1997; Thomas et al., 2002;

Knapska and Maren, 2009). One of the above studies also found the NAc core to be active during fear expression (Thomas et al., 2002), which is in agreement with our findings here, and a final study found that the NAc was not responsive at all for any fear behavior (Huang et al.,

2013). Several specific methodological differences can explain the discrepancy in the findings, including differences in behavioral tasks (fear-potentiated startle, fear renewal), IEG analysis (zif

268, c-fos), and that we rigorously defined the NAc core and shell based on the known differences in colocalization of calbindin (Meredith et al., 1996; Tan et al., 1999). Overall, our

ARC data suggest that the NAc shell is specifically involved in the extinction of fear to a previously learned CS, which is supported by our additional pharmacological and behavioral experiments (Fig 5,7,8).

We next demonstrated that the core promotes freezing, but is not necessary for extinction learning, whereas the NAc shell promotes the consolidation of extinction memory (Fig 7,8).

Several studies have implicated dopamine actions in the nucleus accumbens as a region in conditioned fear. For example, dopamine activity in the nucleus accumbens is implicated in the blocking of learned conditioned fear responses (Iordanova et al., 2006). Furthermore, infusions of haloperidol, a dopamine receptor-2 antagonist increases fear expression and delays within session extinction as we ass disrupting extinction retention (Holtzman-Assif et al., 2010).

However, a limited number of these studies have examined the different contributions of the core and shell and their signaling mechanisms in fear learning and its extinction. One study examined dopamine release in the nucleus accumbens core and shell subregions during context fear expression in the training context or a novel context. Elevated dopamine was observed in both the shell and core subregions only when rats were tested in the original training context, suggesting that dopamine release in both regions is associated with contextual fear expression

(Martinez et al., 2008) However, a limited number of these studies have examined the different contributions of the core and shell and their signaling mechanisms in fear learning and its extinction. In addition, two separate studies found that lesioning of the core subregion disrupted fear expression to a cue CS (Parkinson et al., 1999; Wendler et al., 2014b). However, in one of the studies the animals were tested in the training context, making it difficult to distinguish between context and pure CS elicited freezing (Wendler et al., 2014b). Here we provide direct evidence that the core subregion is necessary for the expression of cued fear but not for its extinction. Temporary inactivation of the NAc core attenuated freezing to the CS when mice were tested in a different context but had no effect on extinction memory the follow day (Fig 7).

Additionally, the significant difference in ARC expression between the fear expression group when compared to the tone only or the explicitly unpaired group suggests that the NAc core is required for recall of the association between the CS and the US (Fig 5). These data support earlier findings showing increased expression of Zif268 within the core during retrieval of cued fear memories (Thomas et al., 2002). Taken together, our data suggest that the neuronal activity within the NAc core subregion is necessary for expression of fear but is not involved in extinction learning. We confirmed that the reduction of freezing observed here during fear testing could not be interpreted as an effect on locomotor activity using an open field; both

lidocaine-treated, and vehicle-treated groups showed similar locomotor activity during the open field test (Fig 7). Thus, our histological and behavioral pharmacology data suggest that the nucleus accumbens core contributes to the expression of fear during memory recall but is not necessary for extinction learning or its consolidation. Pharmacological inactivation of the NAc shell had a specific disruption of fear extinction memory during the extinction retention test but did not disrupt expression of fear or extinction learning itself (Fig 8). Our evidence for behavioral dissociation here is supported by anatomical evidence in that the core and shell display differential innervation patterns from the prefrontal cortices. The core receives projections specifically from the prelimbic cortex which is involved in fear expression whereas the infralimbic cortex, necessary for fear extinction, projects specifically to the shell subregion

(Brog et al., 1993; Sierra-Mercado et al., 2010). Combined, these data suggest converging input to the nucleus accumbens may support different learned fear responses.

Thus, our findings suggest a dissociation between the NAc core and shell in regulating fear expression/recall and extinction memory, with the core promoting fear expression and the shell necessary for the consolidation of fear extinction memory.

2.5 Reference

Alexander GE, Crutcher MD, DeLong MR (1991) Basal ganglia-thalamocortical circuits: parallel

substrates for motor, oculomotor,“prefrontal” and “limbic” functions. Progress in brain

research 85:119-146.

Badrinarayan A, Wescott SA, Vander Weele CM, Saunders BT, Couturier BE, Maren S, Aragona

BJ (2012) Aversive Stimuli Differentially Modulate Real-Time Dopamine Transmission

Dynamics within the Nucleus Accumbens Core and Shell. The Journal of Neuroscience

32:15779-15790.

Baliki MN, Mansour A, Baria AT, Huang L, Berger SE, Fields HL, Apkarian AV (2013) Parceling

Human Accumbens into Putative Core and Shell Dissociates Encoding of Values for

Reward and Pain. The Journal of Neuroscience 33:16383-16393.

Bassareo V, De Luca MA, Di Chiara G (2002) Differential Expression of Motivational Stimulus

Properties by Dopamine in Nucleus Accumbens Shell versus Core and Prefrontal Cortex.

The Journal of Neuroscience 22:4709.

Beck CH, Fibiger HC (1995) Conditioned fear-induced changes in behavior and in the expression

of the immediate early gene c-fos: with and without diazepam pretreatment. J Neurosci

15:709-720.

Berridge KC (2019) Affective valence in the brain: modules or modes? Nature Reviews

Neuroscience 20:225-234.

Blaiss CA, Janak PH (2009) The nucleus accumbens core and shell are critical for the expression,

but not the consolidation, of Pavlovian conditioned approach. Behavioural brain research

200:22-32.

Brauer K, Hausser M, Hartig W, Arendt T (2000) The core-shell dichotomy of nucleus accumbens

in the rhesus monkey as revealed by double-immunofluorescence and morphology of

cholinergic interneurons. Brain Res 858:151-162.

Brog JS, Salyapongse A, Deutch AY, Zahm DS (1993) The patterns of afferent innervation of the

core and shell in the "accumbens" part of the rat ventral striatum: immunohistochemical

detection of retrogradely transported fluoro-gold. The Journal of comparative neurology

338:255-278.

Butterworth JFt, Strichartz GR (1990) Molecular mechanisms of local anesthesia: a review.

Anesthesiology 72:711-734.

Caboche J, Vernier P, Rogard M, Besson M-J (1993) Haloperidol increases PPE mRNA levels in

the caudal part of the nucleus accumbens in the rat. Neuroreport 4:551-554.

Campeau S, Falls WA, Cullinan WE, Helmreich DL, Davis M, Watson SJ (1997) Elicitation and

reduction of fear: behavioural and neuroendocrine indices and brain induction of the

immediate-early gene c-fos. Neuroscience 78:1087-1104.

Chen YW, Rada PV, Bützler BP, Leibowitz SF, Hoebel BG (2012) Corticotropin-releasing factor

in the nucleus accumbens shell induces swim depression, anxiety, and anhedonia along

with changes in local dopamine/acetylcholine balance. Neuroscience 206:155-166.

Corbit LH, Muir JL, Balleine BW (2001) The Role of the Nucleus Accumbens in Instrumental

Conditioning: Evidence of a Functional Dissociation between Accumbens Core and Shell.

The Journal of Neuroscience 21:3251-3260.

Correia SS, McGrath AG, Lee A, Graybiel AM, Goosens KA (2016) Amygdala-ventral striatum

circuit activation decreases long-term fear. Elife 5:e12669.

Cullen PK, Gilman TL, Winiecki P, Riccio DC, Jasnow AM (2015) Activity of the anterior

cingulate cortex and ventral hippocampus underlie increases in contextual fear

generalization. Neurobiology of learning and memory 124:19-27.

Deutch AY, Cameron DS (1992) Pharmacological characterization of dopamine systems in the

nucleus accumbens core and shell. Neuroscience 46:49-56.

Floresco SB (2015) The Nucleus Accumbens: An Interface Between Cognition, Emotion, and

Action. Annual Review of Psychology 66:25-52.

Gerfen CR, Herkenham M, Thibault J (1987) The neostriatal mosaic: II. Patch-and matrix-directed

mesostriatal dopaminergic and non-dopaminergic systems. Journal of Neuroscience

7:3915-3934.

Groenewegen HJ, Berendse HW, Haber SN (1993) Organization of the output of the ventral

striatopallidal system in the rat: ventral pallidal efferents. Neuroscience 57:113-142.

Haber SN, Fudge JL, McFarland NR (2000) Striatonigrostriatal pathways in primates form an

ascending spiral from the shell to the dorsolateral striatum. Journal of Neuroscience

20:2369-2382.

Haralambous T, Westbrook RF (1999) An infusion of bupivacaine into the nucleus accumbens

disrupts the acquisition but not the expression of contextual fear conditioning. Behavioral

Neuroscience 113:925-940.

Heimer L, Zahm DS, Churchill L, Kalivas PW, Wohltmann C (1991) Specificity in the projection

patterns of accumbal core and shell in the rat. Neuroscience 41:89-125.

Holtzman-Assif O, Laurent V, Westbrook RF (2010) Blockade of dopamine activity in the nucleus

accumbens impairs learning extinction of conditioned fear. Learn Mem 17:71-75.

Huang ACW, Shyu B-C, Hsiao S, Chen T-C, He AB-H (2013) Neural substrates of fear

conditioning, extinction, and spontaneous recovery in passive avoidance learning: A c-fos

study in rats. Behavioural brain research 237:23-31.

Iordanova MD, Westbrook RF, Killcross AS (2006) Dopamine activity in the nucleus accumbens

modulates blocking in fear conditioning. Eur J Neurosci 24:3265-3270.

Jongen-Relo AL, Kaufmann S, Feldon J (2003) A differential involvement of the shell and core

subterritories of the nucleus accumbens of rats in memory processes. Behav Neurosci

117:150-168.

Jongen-Rêlo AL, Kaufmann S, Feldon J (2003) A differential involvement of the shell and core

subterritories of the nucleus accumbens of the rats in memory processes. Behavioral

Neuroscience 117:150-168.

Kang S, Vervliet B, Engelhard IM, van Dis EAM, Hagenaars MA (2018) Reduced return of threat

expectancy after counterconditioning versus extinction. Behaviour Research and Therapy

108:78-84.

Knapska E, Maren S (2009) Reciprocal patterns of c-Fos expression in the medial prefrontal cortex

and amygdala after extinction and renewal of conditioned fear. Learn Mem 16:486-493.

Lacagnina AF, Brockway ET, Crovetti CR, Shue F, McCarty MJ, Sattler KP, Lim SC, Santos SL,

Denny CA, Drew MR (2019) Distinct hippocampal engrams control extinction and relapse

of fear memory. Nature neuroscience 22:753-761.

Levita L, Dalley JW, Robbins TW (2002a) Nucleus accumbens dopamine and learned fear

revisited: a review and some new findings. Behavioural brain research 137:115-127.

Levita L, Dalley JW, Robbins TW (2002b) Disruption of Pavlovian contextual conditioning by

excitotoxic lesions of the nucleus accumbens core. Behav Neurosci 116:539-552.

Levita L, Hare TA, Voss HU, Glover G, Ballon DJ, Casey BJ (2009) The bivalent side of the

nucleus accumbens. NeuroImage 44:1178-1187.

Li Z, Chen Z, Fan G, Li A, Yuan J, Xu T (2018) Cell-Type-Specific Afferent Innervation of the

Nucleus Accumbens Core and Shell. Frontiers in Neuroanatomy 12.

Lichtenberg NT, Kashtelyan V, Burton AC, Bissonette GB, Roesch MR (2014) Nucleus

accumbens core lesions enhance two-way active avoidance. Neuroscience 258:340-346.

Lonergan ME, Gafford GM, Jarome TJ, Helmstetter FJ (2010) Time-dependent expression of Arc

and zif268 after acquisition of fear conditioning. Neural Plast 2010:139891.

Luo R, Uematsu A, Weitemier A, Aquili L, Koivumaa J, McHugh TJ, Johansen JP (2018) A

dopaminergic switch for fear to safety transitions. Nature Communications 9:2483.

Martinez RC, Oliveira AR, Macedo CE, Molina VA, Brandao ML (2008) Involvement of

dopaminergic mechanisms in the nucleus accumbens core and shell subregions in the

expression of fear conditioning. Neurosci Lett 446:112-116.

McCullough KM, Choi D, Guo J, Zimmerman K, Walton J, Rainnie DG, Ressler KJ (2016)

Molecular characterization of Thy1 expressing fear-inhibiting neurons within the

basolateral amygdala. Nat Commun 7:13149.

McCullough LD, Sokolowski JD, Salamone JD (1993) A neurochemical and behavioral

investigation of the involvement of nucleus accumbens dopamine in instrumental

avoidance. Neuroscience 52:919-925.

Meredith GE, Pattiselanno A, Groenewegen HJ, Haber SN (1996) Shell and core in monkey and

human nucleus accumbens identified with antibodies to calbindin-D28k. Journal of

Comparative Neurology 365:628-639.

Mika A, Bouchet CA, Bunker P, Hellwinkel JE, Spence KG, Day HE, Campeau S, Fleshner M,

Greenwood BN (2015) Voluntary exercise during extinction of auditory fear conditioning

reduces the relapse of fear associated with potentiated activity of striatal direct pathway

neurons. Neurobiol Learn Mem 125:224-235.

Muschamp JW, Van't Veer A, Parsegian A, Gallo MS, Chen M, Neve RL, Meloni EG,

Carlezon WA (2011) Activation of CREB in the Nucleus Accumbens Shell Produces

Anhedonia and Resistance to Extinction of Fear in Rats. The Journal of neuroscience : the

official journal of the Society for Neuroscience 31:3095-3103.

Nauta WJH, Smith GP, Faull RLM, Domesick VB (1978) Efferent connections and nigral afferents

of the nucleus accumbens septi in the rat. Neuroscience 3:385-401.

Newall C, Watson T, Grant K-A, Richardson R (2017) The relative effectiveness of extinction and

counter-conditioning in diminishing children's fear. Behaviour Research and Therapy

95:42-49.

Page KJ, Saha A, Everitt BJ (1993) Differential activation and survival of basal forebrain neurons

following infusions of excitatory amino acids: studies with the immediate early gene c-fos.

Experimental Brain Research 93:412-422.

Parkinson JA, Robbins TW, Everitt BJ (1999) Selective excitotoxic lesions of the nucleus

accumbens core and shell differentially affect aversive Pavlovian conditioning to discrete

and contextual cues. Psychobiology 27:256-266.

Pecina S, Schulkin J, Berridge KC (2006) Nucleus accumbens corticotropin-releasing factor

increases cue-triggered motivation for sucrose reward: paradoxical positive incentive

effects in stress? BMC Biol 4:8.

Phillipson OT, Griffiths AC (1985) The topographic order of inputs to nucleus accumbens in the

rat. Neuroscience 16:275-296.

Prensa L, Richard S, Parent A (2003) Chemical anatomy of the human ventral striatum and

adjacent basal forebrain structures. Journal of Comparative Neurology 460:345-367.

Ramirez F, Moscarello JM, LeDoux JE, Sears RM (2015) Active avoidance requires a serial basal

amygdala to nucleus accumbens shell circuit. J Neurosci 35:3470-3477.

Reynolds SM, Berridge KC (2002) Positive and negative motivation in nucleus accumbens shell:

bivalent rostrocaudal gradients for GABA-elicited eating, taste "liking"/"disliking"

reactions, place preference/avoidance, and fear. The Journal of neuroscience : the official

journal of the Society for Neuroscience 22:7308-7320.

Reynolds SM, Berridge KC (2003) Glutamate motivational ensembles in nucleus accumbens:

rostrocaudal shell gradients of fear and feeding. European Journal of Neuroscience

17:2187-2200.

Richard JM, Berridge KC (2011) Nucleus Accumbens Dopamine/Glutamate Interaction Switches

Modes to Generate Desire versus Dread: D1 Alone for Appetitive Eating But D1 and D2

Together for Fear. The Journal of neuroscience : the official journal of the Society for

Neuroscience 31:12866-12879.

Richard JM, Berridge KC (2013) Prefrontal cortex modulates desire and dread generated by

nucleus accumbens glutamate disruption. Biol Psychiatry 73:360-370.

Salamone JD (1994) The involvement of nucleus accumbens dopamine in appetitive and aversive

motivation. Behav Brain Res 61:117-133.

Salgado S, Kaplitt MG (2015) The Nucleus Accumbens: A Comprehensive Review. Stereotactic

and Functional Neurosurgery 93:75-93.

Schwienbacher I, Fendt M, Richardson R, Schnitzler HU (2004a) Temporary inactivation of the

nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats.

Brain Res 1027:87-93.

Schwienbacher I, Fendt M, Richardson R, Schnitzler H-U (2004b) Temporary inactivation of the

nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats.

Brain Research 1027:87-93.

Sierra-Mercado D, Padilla-Coreano N, Quirk GJ (2010) Dissociable Roles of Prelimbic and

Infralimbic Cortices, Ventral Hippocampus, and Basolateral Amygdala in the Expression

and Extinction of Conditioned Fear. Neuropsychopharmacology 36:529.

Soares-Cunha C, de Vasconcelos NAP, Coimbra B, Domingues AV, Silva JM, Loureiro-Campos

E, Gaspar R, Sotiropoulos I, Sousa N, Rodrigues AJ (2019) Nucleus accumbens medium

spiny neurons subtypes signal both reward and aversion. Molecular Psychiatry.

Tan Y, Williams ES, Zahm DS (1999) Calbindin-D 28kD immunofluorescence in ventral

mesencephalic neurons labeled following injections of Fluoro-Gold in nucleus accumbens

subterritories: inverse relationship relative to known neurotoxin vulnerabilities. Brain

Research 844:67-77.

Taverna S, van Dongen YC, Groenewegen HJ, Pennartz CMA (2004) Direct Physiological

Evidence for Synaptic Connectivity Between Medium-Sized Spiny Neurons in Rat

Nucleus Accumbens In Situ. Journal of Neurophysiology 91:1111-1121.

Thomas KL, Hall J, Everitt BJ (2002) Cellular imaging with zif268 expression in the rat nucleus

accumbens and frontal cortex further dissociates the neural pathways activated following

the retrieval of contextual and cued fear memory. The European journal of neuroscience

16:1789-1796. van Dongen YC, Deniau JM, Pennartz CM, Galis-de Graaf Y, Voorn P, Thierry AM,

Groenewegen HJ (2005) Anatomical evidence for direct connections between the shell and

core subregions of the rat nucleus accumbens. Neuroscience 136:1049-1071.

Voorn P, Gerfen CR, Groenewegen HJ (1989) Compartmental organization of the ventral striatum

of the rat: immunohistochemical distribution of enkephalin, substance P, dopamine, and

calcium‐binding protein. Journal of Comparative Neurology 289:189-201.

Voorn P, Brady LS, Schotte A, Berendse HW, Richfield EK (1994) Evidence For Two

Neurochemical Divisions in the Human Nucleus Accumbens. European Journal of

Neuroscience 6:1913-1916.

Wendler E, Gaspar JC, Ferreira TL, Barbiero JK, Andreatini R, Vital MA, Blaha CD, Winn P, Da

Cunha C (2014a) The roles of the nucleus accumbens core, dorsomedial striatum, and

dorsolateral striatum in learning: performance and extinction of Pavlovian fear-conditioned

responses and instrumental avoidance responses. Neurobiology of learning and memory

109:27-36.

Wendler E, Gaspar JCC, Ferreira TL, Barbiero JK, Andreatini R, Vital MABF, Blaha CD, Winn

P, Da Cunha C (2014b) The roles of the nucleus accumbens core, dorsomedial striatum,

and dorsolateral striatum in learning: Performance and extinction of Pavlovian fear-

conditioned responses and instrumental avoidance responses. Neurobiology of learning

and memory 109:27-36.

Wenzel JM, Rauscher NA, Cheer JF, Oleson EB (2015) A Role for Phasic Dopamine Release

within the Nucleus Accumbens in Encoding Aversion: A Review of the Neurochemical

Literature. ACS Chemical Neuroscience 6:16-26.

Wright CI, Groenewegen HJ (1995) Patterns of convergence and segregation in the medial nucleus

accumbens of the rat: Relationships of prefrontal cortical, midline thalamic, and basal

amygdaloid afferents. Journal of Comparative Neurology 361:383-403.

Young E, Williams C (2013) Differential activation of amygdala Arc expression by positive and

negatively valenced emotional learning conditions. Frontiers in Behavioral Neuroscience

7:191.

Zahm DS (1999) Functional-anatomical implications of the nucleus accumbens core and shell

subterritories. Annals of the New York Academy of Sciences 877:113-128.

Zahm DS, Brog JS (1992a) On the significance of subterritories in the “accumbens” part of the rat

ventral striatum. Neuroscience 50:751-767.

Zahm DS, Brog JS (1992b) On the significance of subterritories in the "accumbens" part of the rat

ventral striatum. Neuroscience 50:751-767.

CHAPTER 3 mGluR1 activation and the ERK/MAPK signaling pathway within the nucleus accumbens

shell is necessary for fear extinction consolidation

3.1 Introduction

In addition to identifying brain regions involved in fear expression and extinction, it is also imperative to study the underlying mechanisms within the NAc regulating fear processing.

Glutamatergic modulation is critical for processing of fear memories and as well as in drug addiction. Glutamate modulates neurotransmission either by stimulation of ionotropic glutamate receptors such as N-methyl-d-aspartate (NMDA), and alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) or G protein-coupled metabotropic glutamate receptors

(mGluRs). mGluRs are widely distributed across brain regions critical for aversive emotional regulation, have been implicated in fear learning and extinction consolidation, and also for synaptic plasticity (Bajkowska et al., 1999; Zahorodna and Bijak, 1999; Kim et al., 2007a). In addition, substantial evidence points to a key role of mGluRs in memory consolidation (Riedel et al., 2003; Xu et al., 2009; Menard and Quirion, 2012). Blocking mGluR1 within the amygdala impaired both extinction consolidation as well as retention of extinction memory(Kim et al.,

2007a). Additionally, administration of mGluR5 agonist increased of the IL neurons and when rats were administered with a mGluR5 antagonist, they displayed impaired extinction memory retention (Fontanez-Nuin et al., 2011). As with most areas of the brain, glutamate receptors are expressed on the medium spiny neurons of the nucleus accumbens. In addition to AMPA and

NMDA receptors, the NAc expresses large numbers of postsynaptic group I metabotropic

glutamate receptors (mGluRs; mGluR1 and mGluR5) that are substantially contacted by terminals from the basolateral amygdala and infralimbic cortex (Mitrano and Smith, 2007b;

Mitrano et al., 2008; Mitrano et al., 2010a). Within the NAc, glutamate is one of the key neurotransmitters that influence reward and addictive behaviors. Glutamatergic signaling in the

NAc has been shown to depolarize neurons and interact with dopamine to regulate various behaviors such as motor movement, reward-related behaviors and learning mechanisms. Studies investigating the distribution of the mGluRs in the NAc core and shell didn’t find any gross difference in the distributions of the group I mGluRs (Mitrano and Smith, 2007a), however, mGluR1s-immunoreactive axons were found to be more abundant that mGlur5-labelled axons in both core and shell. Moreover, mGluR1s within the NAc are contacted by inputs from the BLA and the IL (Mitrano et al., 2010b). mGluR1 within the NAc is required for LTP and are dependent on an increased Ca2+ release via the mGluR/PLC pathway induced by the glutamate released during high frequency stimulation of the glutamatergic inputs into the NAc (Schotanus and Chergui, 2008). Thus far, studies examining glutamatergic signaling within the NAc have focused largely on its influences on reward-related behaviors and drugs of abuse. Given the importance of NAc as an intermediate locus for modulating both reward and aversive stimuli, it is imperative to investigate the contribution of mGluRs and the subsequent signaling pathway in regulating fear processes within the NAc subregions.

At the molecular level, signaling via mGluR1 is coupled to the extracellular signal- regulated kinases/ mitogen-activated protein kinases (ERK/MAPK) pathway which has also been strongly implicated in the acquisition, consolidation and retention of extinction memory (Schafe et al., 2000; Santini et al., 2004; Herry et al., 2006b; Fischer et al., 2007). In the prefrontal cortex, inhibition of the ERK/MAPK pathway immediately after extinction training not only

blocked training related LTP but also impaired retention of extinction memory (Hugues et al.,

2004; Hugues et al., 2006).Additionally, Kim and colleagues found elevated phosphorylation of the ERK proteins specifically within the IL, and the PL, following cued fear extinction training

(Kim et al., 2011). Similarly, increased ERK activity was observed in the IL, during extinction training as well as following extinction memory retrieval (Ishikawa et al., 2012). Within the hippocampus, ERK phosphorylation was elevated following extinction training and localized infusions of U021, an inhibitor of MEK, immediately after extinction training reduced retention of the extinction memory (Fischer et al., 2007). These findings were further extended to the basolateral amygdala where Herry and colleagues found that ERK/MAPK phosphorylation was increased during acquisition of fear extinction memory and inhibiting the signaling pathway using the MEK inhibitor, U0216, blocked the extinction acquisition of auditory fear conditioning

(Herry et al., 2006a). Further, intra-BLA infusions of MAPK inhibitor also impaired consolidation of extinction of fear potentiated startle (Lu et al., 2001). Increased ERK phosphorylation has been observed within the nucleus accumbens in response to drugs of abuse

(Valjent et al., 2004) along with evidence of interaction between group 1 mGluRs and the ERK pathway within the nucleus accumbens with respect to alcohol and nicotine addiction (Stevenson et al., 2019; Yang et al., 2020). However, no study has investigated how mGluR1s and the

ERK/MAPK signaling pathway within the NAc regulate fear learning processes. In the next set of studies, we were interested in identifying the role of the mGluR1s and the ERK/MAPK pathway within the core and shell subregions in fear expression and fear extinction.

3.2 Methods

3.2.1 Animals

(IACUC) and in a facility accredited by the Association for Assessment and Accreditation and

Laboratory Animal Care (AALAC), in accordance with the NIH guidelines for the Care and Use of Laboratory Animals, 8th edition.

3.2.2 Stereotaxic Surgery

Ranaoke, VA) were bilaterally implanted aimed at the nucleus accumbens core (+1.18mm AP;

+2.20mm ML; -4.52mm DV; angled at 14◦) or nucleus accumbens shell (+1.30mm AP;

+1.60mm ML; -4.40mm DV; angled at 10◦), secured with screws and cranial cement. Dummy

cannulae were inserted in the guide cannulae after the completion of the surgeries. Upon completion of surgeries, the anesthesia was reversed with a subcutaneous injection of atipamezole (0.5 mg/kg). All mice were given one week to recover prior to the start of behavioral procedures.

3.2.3 Apparatus

Fear conditioning was performed in four identical conditioning chambers (7” W x 155 7” D x

(Pharmacal Research Laboratories).

3.2.4 Cued Fear conditioning and extinction

All animals were handled for 5 minutes a day, for two consecutive days prior to context exposure. For all experiments, mice were pre-exposed to the Context A for five minutes.

Twenty-four hours later, mice were returned to the conditioning context (Context A) and after a

180s acclimation period, mice received three pairings of the tone CS (75 dB, 6kHz) and unconditional stimulus (US), foot-shock (2s, 0.8 mA). For each of the pairings the US co- terminated with the last two seconds of the CS and each pairing was separated by an inter-tone interval of 182 seconds. Mice were removed from the apparatus 30s after the last shock and

returned to their home cage. The following day, animals were tested for fear expression in

Context B which also served as extinction training. For expression testing and extinction training, mice received 21 unreinforced CS presentations with an ITI of 60 seconds. Twenty-four hours after extinction training, mice were tested for extinction memory in Context B and were presented with 15 unreinforced CS presentations. Following the extinction memory test, infusion sites were verified, and brains collected for immunofluorescence (see below for procedural details).

3.2.5 Pharmacology and Procedure

All localized infusions were performed through an acute infusion cannula (Plastics One; 33 gauge) inserted into the guide cannula. Infusion cannulae were connected to PE 50 polyethylene tubing (0.015″ ID × 0.043″ OD; BD Medical, Franklin Lakes, NJ) that were connected to

Hamilton syringes (Model 85; Hamilton, Reno, NV), and mounted on an infusion pump (PHD

2000, Harvard Apparatus). Pharmacological inhibitors were bilaterally infused at a rate of 0.1

µl/min for a total infusion volume of 0.2 µl. The infusion needle was left in place for an additional minute. Mice were trained on the cued fear conditioning task as described above.

Infusions lasted two minutes and expression testing/extinction training began 5 minutes after the end of infusions. The mice were then tested for extinction memory 24 hours after extinction training. To investigate the role of mGluR1s in the core and shell, the mice received bilateral infusions of either 0.55nmol/ side of the mGluR1 antagonist, 1-aminoindan-1,5, dicarboxylic acid (AIDA), or the vehicle (0.9% sodium chloride) 5 minutes prior to extinction training. To test the involvement of ERK/MAPK pathway in facilitating extinction consolidation within the shell, the mice received bilateral infusions of vehicle (50% DMSO) or 1,4-diamino-2,3-dicyano-1,4-bis

(o-aminophenylmercapto) butadiene (U0126), a MEK inhibitor, either 5 minutes prior to or

immediately after extinction training. One of two doses of U0126 were administered - 0.5 µg or l

µg per hemisphere based on prior literature. The mice were then tested for extinction memory 24 hours after extinction training in the absence of drug.

3.2.6 Histological verification of cannula placements and immunofluorescence

Calbindin immunohistochemistry is commonly used to delineate the two NAc sub-regions because the core is more densely populated by calbindin positive neurons compared to the shell.

Thus, we performed calbindin immunofluorescence to delineate the shell and core subregions. In addition, to precisely verify canula placements in the respective subregions and estimate spread of infused pharmacological substances, mice received localized infusions of quisqualic acid

(0.012M), an excitotoxic amino acid which has been used to destroy cholinergic neurons when used in higher concentrations (0.12M). However, when infused at a lower concentration of

0.012M as above, it results in the induction of immediate early genes (IEG) such as cFos and

Arc. As with all other pharmacological substances, quisqualic acid was bilaterally infused at a rate of 0.1 µl/min for a total infusion volume of 0.2 µl. Thus, we combined local infusions of quisqualic acid (0.012 M) with co-immunofluorescence for calbindin and ARC to verify that our infusions were restricted to the shell or core only.

0.1M sodium phosphate buffer, Ph 7.4 for 7 minutes and the brains were immediately extracted.

immunofluorescence was performed for ARC and calbindin expression. Coronal brain sections were co-stained with antibodies against ARC and calbindin. Free floating sections were rinsed in 1M phosphate buffer (PB), followed by blocking in 0.1M PBS containing 0.9% NaCl, 0.5%

Olympus FluoView 3000 confocal microscope. The software program FIJI (NIH) was used to visualize the fluorescence of the nucleus accumbens subregions of interest.

3.2.7 Statistical analysis

Freezing during extinction training and extinction testing were analyzed using a two-way repeated measures factorial analysis of variance (ANOVA) (treatment X tones) on Graphpad

Prism statistical software. Statistically significant ANOVAs were followed up with Sidak’s multiple comparison. Effect sizes were calculated for completed experiments along with post- hoc power analyses using G*Power 3. Please refer to Table 2 for detailed statistical result for each experiment.

3.3 Results:

3.3.1 Group I metabotropic glutamate receptors within the NAc shell regulate the consolidation of extinction memory

Given the effects of inactivating the NAc shell on extinction memory, we next investigated if glutamatergic modulation through mGluR1 in the NAc shell is necessary for regulating extinction. A total of 15 mice were used in these experiments. As in the experiments above, all mice underwent cued fear conditioning and 24 h later were infused with either AIDA or vehicle five minutes prior to fear expression testing/extinction training. There was no main effect of treatment (F(1, 13)=0.750, p=0.40), a main effect of tone (F (14, 182) =4.437, p <0.001) and no significant treatment X tone interaction (F(14, 182)=1.554, p=0.10) on fear expression and extinction training. Thus, there was no effect on within session extinction (Fig 9 B). However, when the mice were tested 24 h later for extinction retention there was a main effect of treatment

(F(1, 13)=20.01, p<0.001), a main effect of tone (F(14,182)=3.0, p<0.001) but no tone X treatment interaction (F(14,182)=1.7, p=0.057). Specifically, AIDA-treated mice displayed more freezing compared to the vehicle-treated animals (Fig 9 B). These data suggest that signaling through mGluR1 activation within the shell is not necessary for fear expression or within-session extinction but is necessary for the consolidation or retention of extinction memory.

Figure 9 Group I metabotropic glutamate receptors within the NAc shell mediate the consolidation of fear extinction. (A) Schematic representation of the experimental paradigm.

All mice underwent cued fear conditioning in Context A (FC). Twenty-four hours later, mice underwent a fear expression test that also served as extinction training in Context B (Exp

Test/Ext). Twenty-four hours after extinction training, mice were tested for extinction memory in

Context B (Ext Ret). Five minutes prior to the expression testing/extinction training, mice received local infusions of AIDA or vehicle as indicated by the arrow. Twenty-four hours later, mice were tested for extinction retention (Ext Ret). (B) mGluR1 within the shell is necessary for the consolidation of extinction. Analysis of freezing between mice receiving AIDA (filled circle) and vehicle (open circle), showed no effect of treatment on fear expression and extinction training. However, when the mice were tested 24 h later for extinction retention there was a main effect of treatment with AIDA-treated mice displayed higher levels of freezing compared to the vehicle-treated animals, suggesting disruption of fear extinction consolidation (p < 0.05). (C)

Site verification of lidocaine infusions within the shell subregion. Detailed mapping representing the maximum spread (light red), and the average spread (dark red) of ARC expression in mice used in these experiments.

3.3.2 Group I metabotropic glutamate receptors within the NAc core do not mediate fear expression or extinction learning.

In the above experiments, we demonstrated that the NAc core is necessary for the expression of fear as measured by freezing, whereas the shell regulates the consolidation of extinction memory through mGluR1. We next investigated if glutamatergic modulation through mGluR1 is necessary for regulating these fear processes within the core. We hypothesized that inactivation of mGluR1 in the core would have no effect on fear expression or extinction. A total of 17 animals were used in this experiment. All mice underwent cued fear conditioning as above and

24 h later were infused with the mGluR1 antagonist, AIDA, or vehicle five minutes prior to expression testing/extinction training. A two- way repeated measures ANOVA revealed no main effect of treatment (F(1, 15)=2.349, p=0.15) and no significant interaction (F(14, 210)=1.131, p=0.33) on fear expression and extinction training. Analysis revealed a main effect of tone (F(14, 210) =

7.65, p<0.001) showing that both groups extinguished their fear to the tones by the end of the extinction session. Additionally, when the mice were tested for extinction retention 24 h later, there was no main effect of treatment (F(1, 15) = 0.010, p=0.92), a main effect of tone ( F(14,210)=

8.4, p<0.001) and no treatment X tone interaction (F(14, 210) = 0.632, p=0.84). These data suggest that mGluR1 activation in the NAc Core is not involved in the expression of fear or its extinction and extinction retention (Fig 10 B).

100

Figure 10 Group I metabotropic glutamate receptors within the NAc core do not mediate fear expression or extinction learning. (A) Schematic representation of the experimental paradigm. All mice underwent cued fear conditioning in Context A (FC). Twenty-four hours later, mice underwent a fear expression test that also served as extinction training in Context B

(Exp Test/Ext). Twenty-four hours after extinction training, mice were tested for extinction memory in Context B (Ext Ret). Five minutes prior to the expression testing/extinction training,

101

mice received local infusions of AIDA or vehicle as indicated by the arrow. Twenty-four hours later, mice were tested for extinction retention (Ext Ret). (B) mGluR1 within the core is not involved in fear expression, extinction learning or the consolidation of extinction. There was no effect of treatment on fear expression and extinction training between mice that received AIDA

(filled circle) and those that received vehicle (open circle). Additionally, when the mice were tested for extinction retention 24 h later, there was no significant effect of treatment, suggesting no effect on the consolidation of extinction. Values are represented as % freezing (±SEM).

Significance was set at p < 0.05. (C) Site verification of lidocaine infusions. Detailed mapping representing the maximum spread (light red), and the average spread (dark red) of ARC expression in mice used in these experiments. ARC expression was largely restricted to the nucleus accumbens core (Left) or shell (Right) for each experiment.

102

3.3.3 The consolidation of rear extinction requires ERK/MAPK in the NAc shell

Given our previous data suggesting that mGluR1 are important for fear extinction memory we hypothesized that their downstream signaling through the ERK/MAPK pathway within the NAc shell would be necessary for fear extinction retention. A total of 66 mice were used for these experiments. Nine animals were removed because they received unilateral infusions. All mice underwent cued fear conditioning as before and initially received bilateral, local infusions of either 0.5ug/side or 1ug/side of the MEK inhibitor, U0216, into the NAc shell five minutes prior to fear expression testing/extinction training session (Fig 11A). These doses have been used previously in the amygdala to investigate the involvement of the ERK/MAPK pathway in auditory fear conditioning (Schafe et al., 2000; Duvarci et al., 2005; Herry et al., 2006b). During the extinction training session, a two-way ANOVA revealed no main effect of treatment (F(2, 24)

= 0.901, p=0.42) and no significant treatment X tone interaction (F(28, 336) = 0.828, p=0.72).

However, when mice were tested 24 hours later for extinction memory there was a main effect of treatment (F(2, 24)=19.290, p<0.001), a main effect of tone ( F(14,366)= 2.398, p=0.003) but no treatment X tone interaction (F(28,336)=1.447, p=0.07). Specifically, mice that received 1ug/side of

U0216 displayed enhanced fear as compared to mice that received 0.5ug/side U0216 and vehicle-treated mice (Fig 11 B). These data indicate that ERK/MAPK signaling within the NAc shell is necessary for extinction memory, but not for fear expression or extinction. However, we did observe a small non-significant within-session extinction training effect of 1ug/side dose with pre-extinction infusions only during the final 2 tones of extinction training. Thus, we ran an additional group that received local infusions of 1ug/side of U0216 immediately after the extinction training session (Fig 12 A). During the extinction training there was no main effect of treatment (F(1,15)= 0.424, p=0.525), there was a main effect of tone, (F(14,210)= 2.278, p=0.006),

103

and no treatment X tone interaction (F(14,210)= 1.734, p=0.051). During the extinction retention test, there was a significant main effect of treatment (F (1,15) = 10.60, p=0.005), a main effect of tone (F(14,210)= 5.394, p<0.001) but no tone X interaction (F(14,210)= 1.333, p=0.190). Specifically. mice that received local infusions of U0126 into the NAc shell immediately after extinction exhibited more freezing to the tones compared to the vehicle-treated group during the extinction retention test, suggesting a disruption of extinction memory (Fig 12 B). These data are the first to show that the ERK/MAPK signaling within the NAc shell is critical for the appropriate consolidation of extinction memory.

104

Figure 11 Pre-extinction infusions of 1ug/side U0216, within the NAc shell, disrupts fear extinction consolidation whereas 0.5ug/side U0216 has no effect on fear extinction (A)

Schematic representation of the experimental paradigm. All mice underwent cued fear conditioning in Context A. Five minutes prior to the expression testing/extinction training, animals received local infusions of either 0.5ug/1ug U0216, or vehicle as indicated by the arrow.

Twenty-four hours after extinction training, mice were tested for extinction memory in Context

105

B. (B) Pre-extinction infusions of 1ug/side of U0216 (black circles) into the NAc shell showed disrupted extinction consolidation, but no effect was observed with a 0.5ug/side (grey circles) infusion as compared to vehicle (white circles). During the extinction training session, a two- way ANOVA revealed no main effect of treatment (F(2, 24) = 0.901, p=0.42) and no significant treatment X tone interaction (F(28, 336) = 0.828, p=0.72). However, when mice were tested 24 hours later for extinction memory there was a main effect of treatment (F(2, 24)=19.290, p<0.001), a main effect of tone ( F(14,366)= 2.398, p=0.003) and no treatment X tone interaction

(F(28,336)=1.447, p=0.70). Values are represented as average % freezing (±SEM). Significance was set at p < 0.05. These data suggest that the ERK/MAPK signaling pathway in the nucleus accumbens shell is necessary for appropriate consolidation of fear extinction memory. (C)

Verification of cannula placements. Detailed mapping representing the maximum spread (light red), and the average spread (dark red) of ARC expression of animals within the nucleus accumbens shell with pre-extinction infusions.

106

Figure 12 Post-extinction infusions of 1ug/side U0216, within the NAc shell, disrupts fear extinction consolidation (A) Schematic representation of the experimental paradigm. All mice underwent cued fear conditioning in Context A. Twenty-four hours later, mice underwent extinction training and immediately after received infusions of U0126 or vehicle as indicated by the arrow. Twenty-four hours later, animals were tested for extinction retention. (B) Post- extinction infusions of 1ug/side of U0216 (black circles) into the NAc shell showed disrupted extinction consolidation as compared to vehicle (white circles). During the extinction training

107

there was no main effect of treatment (F(1,15)= 0.424, p=0.525), there was a main effect of tone,

(F(14,210)= 2.278, p=0.006), and no treatment X tone interaction (F(14,210)= 1.734, p=0.051).

During the extinction retention test, there was a significant main effect of treatment (F (1,15) =

10.60, p=0.005), a main effect of tone (F(14,210)= 5.394, p<0.001) but no tone X interaction

(F(14,210)= 1.333, p=0.190). Mice infused with U0126 post-extinction displayed significantly more freezing to tone when they were tested for extinction memory 24 hours later. Values are represented as average % freezing (±SEM). Significance was set at p < 0.05. (C) Verification of cannula placements. Detailed mapping representing the maximum spread (light red), and the average spread (dark red) of ARC expression of animals within the nucleus accumbens shell with post-extinction infusions.

108

Statistical F/t %Total Effect Subregion Inactivation test Infusion Test day Comparison Statistic DF variance P * ηp2 Size Power Tone X Exp test/ Treatment 1.131 14, 210 3.129 0.33 ns 0.09 0.32 0.99 Ext tr Tone 7.652 14, 210 21.160 <.001 *** 0.41 0.84 1.00 Treatment 2.349 1, 15 4.681 0.15 ns 0.14 0.40 0.99 Core Tone X Treatment 0.632 14, 210 1.958 0.84 ns 0.07 0.28 0.99 Ext Ret Tone 8.449 14, 210 26.160 <.001 *** 0.51 1.02 1.00 Treatment 0.010 1, 15 0.017 0.92 ns 0.00 0.03 0.05 mGluR1 Tone X Exp test/ Treatment 1.554 14, 182 6.078 0.10 ns 0.20 0.50 1.00 Ext tr Tone 4.437 14, 182 17.350 <.001 *** 0.41 0.84 1.00 Treatment 0.750 1, 13 1.390 0.40 ns 0.05 0.24 0.91 Pre-Ext tr Tone X Treatment 1.708 14, 182 5.843 0.057 ns 0.28 0.63 1.00 Ext Ret Repeated Tone 3.006 14, 182 10.280 <.001 *** 0.42 0.84 1.00 Measures Treatment 20.010 1, 13 22.140 <.001 *** 0.61 1.24 1.00 Two-Way Tone X ANOVA Exp test/ Treatment 0.828 28, 336 2.500 0.72 ns 0.05 0.24 0.99 Ext tr Tone 9.146 14, 336 13.800 <.001 *** 0.24 0.56 1.00 Treatment 0.901 2, 24 3.349 0.42 ns 0.07 0.27 0.99 Shell Tone X Treatment 1.447 28, 336 2.966 0.07 ns 0.10 0.33 1.00 Ext Ret Tone 2.398 14, 336 2.458 0.003 ** 0.08 0.30 0.99 Treatment 19.290 2, 24 43.140 <.001 *** 0.61 1.27 1.00 ERK/MAPK Tone X Exp test/ Treatment 1.734 14, 210 4.868 0.051 ns 0.09 0.32 0.99 Ext tr Tone 2.278 14, 210 6.395 0.006 * 0.12 0.37 0.99 Post Treatment 0.424 1, 15 1.285 0.525 ns 0.03 0.17 0.64 extinction Tone X training Treatment 1.333 14, 210 3.627 0.19 ns 0.14 0.40 0.99 Ext Ret Tone 5.394 14, 210 14.670 <.001 *** 0.39 0.80 1.00 Treatment 10.600 1, 15 16.060 0.005 ** 0.41 0.84 1.00

Table 2: Behavioral Statistical Summary of experiments 3.1,3.2 and 3.3

109

3.4 Discussion

We demonstrated that blocking mGluR1 activity within the NAc shell subregion prior to extinction training disrupts consolidation of extinction memory but has no effect on fear expression or extinction learning (Fig 9). In contrast, inactivation of mGluR1 within the NAc core had no effect on fear expression, extinction learning, or extinction memory (Fig 10). The current findings, to the best of our knowledge, are the first to show that mGluR1 activity within the NAc shell subregion is required for consolidation of extinction memories. Further, a major signaling pathway downstream of mGluR1 activation is ERK/MAPK (Ferraguti et al., 1999;

Thandi et al., 2002; Wang et al., 2007; Mao et al., 2008; Willard and Koochekpour, 2013; Mao and Wang, 2016; Yang et al., 2017). The ERK/MAPK pathway within the amygdala, vmPFC and hippocampus has been implicated in extinction consolidation of both auditory and contextual fear conditioning (Hugues et al., 2004; Herry et al., 2006b; Fischer et al., 2007; Peng et al.,

2010), but has not previously been examined in the NAc. We therefore determined if

ERK/MAPK activity is necessary for extinction consolidation within the NAc shell. Pre- extinction training infusions of U0126 disrupted extinction retention, but also had a small non- significant difference in freezing only during the last few tone presentations during extinction training. To eliminate any possible effect of extinction learning on differences we used a post extinction training infusion procedure in the next experiment. Here, we observed that U0126 infusion into the NAc shell disrupted extinction memory (Fig 11,12). These data, to the best of our knowledge, are the first to indicate that the ERK/MAPK signaling within the NAc shell is necessary for the consolidation of fear extinction memory.

Our finding is in line with previous studies which have shown that mGluR1s are necessary for extinction learning. Blocking mGluR1 within the amygdala prior to extinction learning reduced

110

extinction retention. Further, within the amygdala, mGluR1s modulate fear extinction partly through cannabinoid receptor-1 (CB1) -dependent endocannabinoid signaling resulting in long- term depression (LTD) of GABAergic interneurons (Herry et al., 2008; Gunduz-Cinar et al.,

2016). Additionally, mGluR1 activity has been suggested to be involved in fear extinction through the depotentiation of thalamic synapses in the amygdala and therefore implicated in regulating fear extinction through both excitatory and inhibitory pathways (Kim et al., 2007b).

Thus, mGluR1s are thought to influence fear extinction through both excitatory and inhibitory pathways. Within the amygdala, mGluR1s modulate fear extinction partly through cannabinoid receptor-1 (CB1) -dependent endocannabinoid signaling resulting in long-term depression (LTD) of GABAergic interneurons (Herry et al., 2008; Gunduz-Cinar et al., 2016). mGluRs also produce LTD directly through ERK/MAPK signaling (Sanderson et al., 2016), and are necessary within the amygdala for fear extinction learning (Kim et al., 2007a). Our data suggest that this signaling pathway is recruited in the NAc shell to promote the consolidation of fear extinction memory. Further CB1 receptors are predominantly located in the glutamatergic afferents in the

NAc (Mailleux and Vanderhaeghen, 1992; Pickel et al., 2004). Thus, a possible mechanism by which mGluR1 within the NAc shell promotes the consolidation of extinction memory is through

ERK and CB1-mediated LTD of GABAergic neurons.

The ERK/MAPK pathway has been implicated in mechanisms underlying synaptic plasticity and

LTP and LTD necessary for fear extinction learning (Sweatt, 2004; Thomas and Huganir, 2004).

One of the underlying mechanisms of synaptic plasticity requires new mRNA and protein synthesis (for review seeDavis and Squire, 1984; McGaugh, 2000). Local translation of mRNA within the amygdala has been suggested to be dependent on the ERK/MAPK pathway (Herry et al., 2006a). Along the same line, genes, particularly BDNF, involved in synaptic plasticity have

111

been implicated in fear extinction in both animals and humans; mice and humans with a BDNF polymorphism showed deficits in extinction learning and reduced efficacy of exposure therapy respectively (Soliman et al., 2010; Felmingham et al., 2013). Pharmacologically, a study showed that BDNF infusion into the IL induced fear extinction even in the absence of behavioral extinction training (Chhatwal et al., 2006; Peters et al., 2010). These results extend to other regions such as amygdala and hippocampus where high levels of BDNF were correlated with increased fear extinction. The transcription of BDNF in regulated by cAMP response element- binding protein (CREB) which is phosphorylated by the ERK/MAPK pathway. Our studies, in addition to previous results, implicate the BDNF-ERK pathway as a potential biomarker for anxiety and trauma-related disorder. Additionally, since ERK/MAPK pathway has also been implicated inducing LTD (Zhong et al., 2008), therefore it is possible that fear extinction within the nucleus accumbens shell is through synaptic depression promoted by the ERK/MAPK pathway. The contribution of the nucleus accumbens in fear extinction related plasticity remains less clear. However, our studies suggest that one of the neurobiological mechanisms regulating fear extinction in the subregion may involve the ERK/MAPK signaling pathways dependent protein synthesis and plasticity within the NAc shell.

3.5 References

Bajkowska M, Brański P, Smiałowska M, Pilc A (1999) Effect of chronic antidepressant or

electroconvulsive shock treatment on mGLuR1a immunoreactivity expression in the rat

hippocampus. Pol J Pharmacol 51:539-541.

Chhatwal JP, Stanek-Rattiner L, Davis M, Ressler KJ (2006) Amygdala BDNF signaling is

required for consolidation but not encoding of extinction. Nature neuroscience 9:870-872.

112

Davis HP, Squire LR (1984) Protein synthesis and memory: a review. Psychological bulletin

96:518.

Duvarci S, Nader K, LeDoux JE (2005) Activation of extracellular signal-regulated kinase–

mitogen-activated protein kinase cascade in the amygdala is required for memory

reconsolidation of auditory fear conditioning. European Journal of Neuroscience 21:283-

289.

Felmingham KL, Dobson-Stone C, Schofield PR, Quirk GJ, Bryant RA (2013) The brain-derived

neurotrophic factor Val66Met polymorphism predicts response to exposure therapy in

posttraumatic stress disorder. Biological psychiatry 73:1059-1063.

Ferraguti F, Baldani-Guerra B, Corsi M, Nakanishi S, Corti C (1999) Activation of the

extracellular signal-regulated kinase 2 by metabotropic glutamate receptors. European

Journal of Neuroscience 11:2073-2082.

Fischer A, Radulovic M, Schrick C, Sananbenesi F, Godovac-Zimmermann J, Radulovic J (2007)

Hippocampal Mek/Erk signaling mediates extinction of contextual freezing behavior.

Neurobiology of learning and memory 87:149-158.

Fontanez-Nuin DE, Santini E, Quirk GJ, Porter JT (2011) Memory for fear extinction requires

mGluR5-mediated activation of infralimbic neurons. Cerebral cortex (New York, NY :

1991) 21:727-735.

Gunduz-Cinar O, Flynn S, Brockway E, Kaugars K, Baldi R, Ramikie TS, Cinar R, Kunos G, Patel

S, Holmes A (2016) Fluoxetine Facilitates Fear Extinction Through Amygdala

Endocannabinoids. Neuropsychopharmacology 41:1598-1609.

113

Herry C, Trifilieff P, Micheau J, Lüthi A, Mons N (2006a) Extinction of auditory fear conditioning

requires MAPK/ERK activation in the basolateral amygdala. European Journal of

Neuroscience 24:261-269.

Herry C, Trifilieff P, Micheau J, Luthi A, Mons N (2006b) Extinction of auditory fear conditioning

requires MAPK/ERK activation in the basolateral amygdala. The European journal of

neuroscience 24:261-269.

Herry C, Ciocchi S, Senn V, Demmou L, Müller C, Lüthi A (2008) Switching on and off fear by

distinct neuronal circuits. Nature 454:600-606.

Hugues S, Deschaux O, Garcia R (2004) Postextinction Infusion of a Mitogen-Activated Protein

Kinase Inhibitor Into the Medial Prefrontal Cortex Impairs Memory of the Extinction of

Conditioned Fear. Learning & Memory 11:540-543.

Hugues S, Chessel A, Lena I, Marsault R, Garcia R (2006) Prefrontal infusion of PD098059

immediately after fear extinction training blocks extinction‐associated prefrontal synaptic

plasticity and decreases prefrontal ERK2 phosphorylation. Synapse 60:280-287.

Ishikawa S, Saito Y, Yanagawa Y, Otani S, Hiraide S, Shimamura Ki, Matsumoto M, Togashi H

(2012) Early postnatal stress alters extracellular signal‐regulated kinase signaling in the

corticolimbic system modulating emotional circuitry in adult rats. European Journal of

Neuroscience 35:135-145.

Kim J, Lee S, Park H, Song B, Hong I, Geum D, Shin K, Choi S (2007a) Blockade of amygdala

metabotropic glutamate receptor subtype 1 impairs fear extinction. Biochemical and

Biophysical Research Communications 355:188-193.

114

Kim J, Lee S, Park K, Hong I, Song B, Son G, Park H, Kim WR, Park E, Choe HK (2007b)

Amygdala depotentiation and fear extinction. Proceedings of the National Academy of

Sciences 104:20955-20960.

Kim JH, Li S, Richardson R (2011) Immunohistochemical analyses of long-term extinction of

conditioned fear in adolescent rats. Cerebral cortex 21:530-538.

Lu K-T, Walker DL, Davis M (2001) Mitogen-Activated Protein Kinase Cascade in the Basolateral

Nucleus of Amygdala Is Involved in Extinction of Fear-Potentiated Startle. The Journal of

Neuroscience 21:RC162.

Mailleux P, Vanderhaeghen JJ (1992) Distribution of neuronal cannabinoid receptor in the adult

rat brain: a comparative receptor binding radioautography and in situ hybridization

histochemistry. Neuroscience 48:655-668.

Mao L-M, Wang JQ (2016) Regulation of Group I Metabotropic Glutamate Receptors by

MAPK/ERK in Neurons. J Nat Sci 2:e268.

Mao L-M, Zhang G-C, Liu X-Y, Fibuch EE, Wang JQ (2008) Group I Metabotropic Glutamate

Receptor-mediated Gene Expression in Striatal Neurons. Neurochemical Research

33:1920-1924.

McGaugh JL (2000) Memory--a century of consolidation. Science 287:248-251.

Menard C, Quirion R (2012) Group 1 metabotropic glutamate receptor function and its regulation

of learning and memory in the aging brain. Front Pharmacol 3:182.

Mitrano DA, Smith Y (2007a) Comparative analysis of the subcellular and subsynaptic

localization of mGluR1a and mGluR5 metabotropic glutamate receptors in the shell and

core of the nucleus accumbens in rat and monkey. Journal of Comparative Neurology

500:788-806.

115

Mitrano DA, Smith Y (2007b) Comparative analysis of the subcellular and subsynaptic

localization of mGluR1a and mGluR5 metabotropic glutamate receptors in the shell and

core of the nucleus accumbens in rat and monkey. J Comp Neurol 500:788-806.

Mitrano DA, Arnold C, Smith Y (2008) Subcellular and subsynaptic localization of group I

metabotropic glutamate receptors in the nucleus accumbens of cocaine-treated rats.

Neuroscience 154:653-666.

Mitrano DA, Pare JF, Smith Y (2010a) Ultrastructural relationships between cortical, thalamic,

and amygdala glutamatergic inputs and group I metabotropic glutamate receptors in the rat

accumbens. J Comp Neurol 518:1315-1329.

Mitrano DA, Pare JF, Smith Y (2010b) Ultrastructural relationships between cortical, thalamic,

and amygdala glutamatergic inputs and group I metabotropic glutamate receptors in the rat

accumbens. Journal of Comparative Neurology 518:1315-1329.

Peng S, Zhang Y, Zhang J, Wang H, Ren B (2010) ERK in learning and memory: a review of

recent research. International journal of molecular sciences 11:222-232.

Peters J, Dieppa-Perea LM, Melendez LM, Quirk GJ (2010) Induction of Fear Extinction with

Hippocampal-Infralimbic BDNF. Science 328:1288.

Pickel VM, Chan J, Kash TL, Rodriguez JJ, MacKie K (2004) Compartment-specific localization

of cannabinoid 1 (CB1) and μ-opioid receptors in rat nucleus accumbens. Neuroscience

127:101-112.

Riedel G, Platt B, Micheau J (2003) Glutamate receptor function in learning and memory. Behav

Brain Res 140:1-47.

116

Sanderson TM, Hogg EL, Collingridge GL, Corrêa SAL (2016) Hippocampal metabotropic

glutamate receptor long-term depression in health and disease: focus on mitogen-activated

protein kinase pathways. Journal of Neurochemistry 139:200-214.

Santini E, Ge H, Ren K, Pena de Ortiz S, Quirk GJ (2004) Consolidation of fear extinction requires

protein synthesis in the medial prefrontal cortex. The Journal of neuroscience : the official

journal of the Society for Neuroscience 24:5704-5710.

Schafe GE, Atkins CM, Swank MW, Bauer EP, Sweatt JD, LeDoux JE (2000) Activation of

ERK/MAP Kinase in the Amygdala Is Required for Memory Consolidation of Pavlovian

Fear Conditioning. The Journal of Neuroscience 20:8177.

Schotanus SM, Chergui K (2008) Dopamine D1 receptors and group I metabotropic glutamate

receptors contribute to the induction of long-term potentiation in the nucleus accumbens.

Neuropharmacology 54:837-844.

Soliman F, Glatt CE, Bath KG, Levita L, Jones RM, Pattwell SS, Jing D, Tottenham N, Amso D,

Somerville LH, Voss HU, Glover G, Ballon DJ, Liston C, Teslovich T, Van Kempen T,

Lee FS, Casey BJ (2010) A Genetic Variant BDNF Polymorphism Alters Extinction

Learning in Both Mouse and Human. Science 327:863.

Stevenson RA, Hoffman JL, Maldonado-Devincci AM, Faccidomo S, Hodge CW (2019) MGluR5

activity is required for the induction of ethanol behavioral sensitization and associated

changes in ERK MAP kinase phosphorylation in the nucleus accumbens shell and lateral

habenula. Behavioural brain research 367:19-27.

Sweatt JD (2004) Mitogen-activated protein kinases in synaptic plasticity and memory. Curr Opin

Neurobiol 14:311-317.

117

Thandi S, Blank JL, Challiss RAJ (2002) Group-I metabotropic glutamate receptors, mGlu1a and

mGlu5a, couple to extracellular signal-regulated kinase (ERK) activation via distinct, but

overlapping, signalling pathways. Journal of Neurochemistry 83:1139-1153.

Thomas GM, Huganir RL (2004) MAPK cascade signalling and synaptic plasticity. Nature

Reviews Neuroscience 5:173-183.

Valjent E, Pagès C, Hervé D, Girault J-A, Caboche J (2004) Addictive and non-addictive drugs

induce distinct and specific patterns of ERK activation in mouse brain. European Journal

of Neuroscience 19:1826-1836.

Wang JQ, Fibuch EE, Mao L (2007) Regulation of mitogen-activated protein kinases by glutamate

receptors. Journal of Neurochemistry 100:1-11.

Willard SS, Koochekpour S (2013) Glutamate, glutamate receptors, and downstream signaling

pathways. Int J Biol Sci 9:948-959.

Xu J, Zhu Y, Contractor A, Heinemann SF (2009) mGluR5 has a critical role in inhibitory learning.

J Neurosci 29:3676-3684.

Yang JH, Mao L-M, Choe ES, Wang JQ (2017) Synaptic ERK2 Phosphorylates and Regulates

Metabotropic Glutamate Receptor 1 In Vitro and in Neurons. Molecular Neurobiology

54:7156-7170.

Yang JH, Sohn S, Kim S, Kim J, Oh JH, Ryu IS, Go BS, Choe ES (2020) Repeated nicotine

exposure increases the intracellular interaction between ERK-mGluR5 in the nucleus

accumbens more in adult than adolescent rats. Addiction Biology n/a:e12913.

Zahorodna A, Bijak M (1999) An antidepressant-induced decrease in the responsiveness of

hippocampal neurons to group I metabotropic glutamate receptor activation. European

Journal of Pharmacology 386:173-179.

118

Zhong P, Liu W, Gu Z, Yan Z (2008) Serotonin facilitates long-term depression induction in

prefrontal cortex via p38 MAPK/Rab5-mediated enhancement of AMPA receptor

internalization. The Journal of Physiology 586:4465-4479.

119

CHAPTER 4

General Discussion

Anxiety and trauma-related disorders are some of the most common neuropsychological disorders with higher prevalence as compared to other mental disorders globally as evidenced by an exhaustive survey conducted by the World Health Organization in 14 countries spanning two years, reporting that 12-month prevalence of anxiety disorders, including trauma-related disorders

(Kessler et al., 1994; Kessler et al., 1995; Demyttenaere et al., 2004). Additionally, anxiety and trauma-related disorders are generally chronic with prolonged feelings of fear and stress. In the last several years, advancements have been made in elucidating the underlying neurobiological mechanisms of anxiety and trauma-related disorders. Because excessive associative fear is one of the contributing factors to the development and persistence of anxiety and trauma-related disorders, it has been proposed that enhancing existing treatment requires a deeper understanding of the neurobiological mechanisms of fear learning and fear reduction. Further, the NIMH has proposed a framework to better understand the pathophysiology which includes the underlying neuronal circuitry and the dysfunction that leads to anxiety and trauma-related disorders. Thus, anxiety and trauma-related disorders have been viewed as disorders of circuitry involved in fear conditioning and extinction (For review see Insel et al., 2010). It is the hope that data from laboratory-based studies of the brain circuitry associated with anxiety and trauma-related disorders will enhance the clinical management and treatment of the disorders. To this end, it is imperative to identify and study the underlying neuronal mechanisms in brain regions which are not only involved modulating fear in response to a threat but also those which are involved in the reduction

120

of the fear once the threat has passed (e.g., fear extinction). Indeed, fear extinction has been argued as a model for identifying potential biomarkers for several reasons; 1) an inability to appropriately inhibit or extinguish fear is one of the key features of anxiety and trauma-related disorders; 2) exposure therapy, a prominent CBT treatment in clinical settings is based on the animal models of fear extinction and; 3) there are commonalities in the brain regions involved in fear extinction between non-human animals and humans and evident from neuroimaging studies (for review see

Graham and Milad, 2011).

The nucleus accumbens has been considered to be a critical structure for motivational and emotional regulation. While several neurobiological, behavioral, and anatomical studies have implicated the nucleus accumbens in regulating fear, the exact contribution of the nucleus accumbens core and shell subregions in fear processing remain contradictory and unclear. Brain imaging studies also implicate the nucleus accumbens in anxiety disorders by showing NAc activation in response to aversive stimuli and anxiety-dependent active avoidance behavior (Jensen et al., 2003; Levita et al., 2012). Further, no study to date has examined the dissociable roles of the nucleus accumbens subregions in fear expression and fear extinction in the same study. In our studies, we sought to explore whether the two subregions, in lieu of their difference in neural connectivity and structural composition, would play different roles in regulating fear learning processes. We show for the first time, through experiments 2.3.1, 2.3.2 and 2.3.3 that the NAc core is necessary for fear expression specifically whereas the NAc shell is critical for fear extinction consolidation. We first demonstrate the differential contribution of the core and shell in fear expression and fear extinction through IEG expression analysis. We observed enhanced ARC expression in the core following fear expression whereas the shell specifically showed enhanced

ARC activity following fear extinction. Previous studies, based on IEG analysis, have reported the

121

involvement of NAc shell and core subregions in different fear learning processes, however the findings remain inconsistent. While one study showed that the NAc shell is involved in the expression of fear or fear memory based on IEG analyses, another study found that the NAc was not responsive at all for any fear behavior (Campeau et al., 1997; Thomas et al., 2002; Knapska and Maren, 2009; Huang et al., 2013). One of the above studies, however, found the NAc core to be active during fear expression (Thomas et al., 2002), which is in agreement with our findings here. The studies above had several specific methodological differences in behavioral tasks (fear- potentiated startle, fear renewal) and IEG analysis (zif 268, c-fos) that may explain the inconsistent results. In the present work we rigorously defined the NAc core and shell based on the known differences in colocalization of calbindin (Meredith et al., 1996; Tan et al., 1999) and then used very localized infusions of pharmacology into each subregion followed by mapping spread of activity. Combined, these results suggest dissociable roles of the nucleus accumbens core and shell subregions in fear processing. As mentioned, the IEG analysis is further supported with pharmacological manipulations wherein lidocaine was infused into the core and the shell separately to temporarily inactivate each subregion prior to extinction learning. Our data exhibit that inactivating the core subregion reduces expression of fear but does not interfere with extinction learning or its retention. Whereas inactivation of the shell subregion disrupts fear extinction consolidation, it does not disrupt fear expression or extinction learning. The current results strongly support the dissociable roles of the two subregions, which is supported by current neuroanatomical evidence, but has not been demonstrated previously functionally for fear behavior.

The nucleus accumbens has been classically viewed as the reward center, however the idea that this subregion only plays a role in regulating the pleasurable property of a reward has been

122

challenged by several reports (Salamone et al., 1994; Berridge, 2007; Nicola, 2007; Floresco,

2015). It has been argued that the ‘reward’ can have several meanings including ‘a reinforcer, pleasure, appetite motivation’ which can either be obtained by approaching a positive stimulus or also by successfully avoiding a threatening stimulus. The affective valence of the stimulus is processed by the limbic structures and translated into behavioral expressions of approach towards positive stimuli and avoidance of negative stimuli. A ‘module’ hypothesis has been suggested to understand how the brain systems differentiate between a positive and negative stimuli (Berridge, 2019). In this hypothesis a ‘module’ is defined as a neural system, and each such module is responsible for regulating the processing of a single affective valence and the responses associated with it. Our studies suggest that two different modules exist within the nucleus accumbens, the shell which regulates fear extinction learning and memory and the core which is necessary for fear expression. This is line with other studies which implicate the presence of different modules within the nucleus accumbens. For example, studies have shown that GABAergic projections from the nucleus accumbens to the ventral tegmentum and ventral pallidum produced reward-related and defensive motivation (for review see Carlezon Jr and

Thomas, 2009). However, there is also evidence of bivalent ‘modes’ across the rostral-caudal gradient within the shell. The ‘mode’ hypothesis assumes that a particular neuronal module does not have a one-to-one relationship with a single valence but regulates several different valence- related functions (Berridge, 2019). For example, muscimol injections in the caudal part of the

NAc shell caused negative reactions to sucrose and elicited conditioned place avoidance, whereas rostral injections caused positive appetitive responses (Reynolds and Berridge, 2002).

While we did not investigate our manipulation across the rostro-caudal gradient within either of the subregions, it is possible that the rostral part of the NAc shell, one of the regions targeted in

123

the present experiment, drives appetitive responses during extinction consolidation. Thus, by promoting a more ‘rewarding’ outcome the extinction may occur partially through some form of counterconditioning (Newall et al., 2017; Kang et al., 2018) which would also lend support the idea of “mode” switching across the two subregions.

One of the possible interpretations behind the existence of the bivalent modules within the nucleus accumbens is that convergence of potentially competing polysynaptic pathways from different brain regions involved in fear expression and fear extinction such as the amygdala, hippocampus, infralimbic and prelimbic cortices, as well as those between the core and the shell.

A similar competitive interaction has been suggested between the amygdala and the medial prefrontal cortex in fear extinction wherein the projections from the infralimbic cortex to the amygdala compete with other excitatory inputs to influence output of the central amygdala

(Quirk et al., 2003; Paré et al., 2004). Further, the BLA and the IL, both of which are necessary for fear extinction consolidation project to the nucleus accumbens shell, which we show to also be important for fear extinction consolidation (Brog et al., 1993; Li et al., 2018). It is possible that glutamatergic inputs to the shell excite GABAergic interneurons which then inhibit output of the core, thus reducing fear expression and facilitating the extinction of fear. Additionally, the amygdala and the PL, both of which are critical brain regions involved in regulating fear expression, project to the core, which we show is also necessary for fear expression. However, precisely how these inputs drive fear expression through the core or fear extinction through the shell is still not well understood. Overall, these connectivity with other brain regions critical for regulating fear processes implicate that the NAc is critical hub for integration of cortical and subcortical nuclei involved in regulating fear expression and extinction and can be considered a potential target for the treatment of anxiety and trauma-related disorders.

124

While identifying brain regions involved in regulating fear expression and fear inhibition is crucial, it is equally imperative to understand the underlying neurobiological pathways to further understand fear learning related plasticity. Therefore, through experiments 3.1, 3.2 and

3.3 we investigated the role of glutamatergic signaling and subsequent pathways that are necessary for mediating fear expression and fear extinction within the core and the shell subregion. Our data demonstrate that mGluR1 and ERK/MAPK signaling specifically within the nucleus accumbens shell promote the consolidation of extinction but are not necessary for fear expression or extinction learning. Together, these are novel findings and provide evidence of not only the dissociable roles of the NAc core and shell subregions in specific control of fear, but also identify the importance of glutamatergic activity within the NAc, presumably from afferent connections, and ERK/MAPK signaling pathway in facilitating extinction consolidation within the shell.

Because fear extinction forms the basis for exposure therapy in the clinical setting, the identification of the NAc shell as another substrate in regulating fear extinction holds critical importance. Indeed, the involvement of the NAc shell in anxiety disorders has been implicated in clinical studies wherein deep brain stimulation of the shell resulted in significant improvement of anxiety symptoms (Sturm et al., 2003). Further, there may be some overlap in appetitive and aversive circuits within the NAc. For instance, the NAc core is involved in the self- administration of rewarding stimuli (LaLumiere and Kalivas, 2008; Bull et al., 2014), but the

NAc shell is reported to be involved in the context-dependent control of both reinstatement and extinction of drug seeking (Peters et al., 2009; Millan et al., 2010; Gibson et al., 2019). Thus, the regional associations have some overlap for expression and extinction of appetitive and aversive responses but are not strictly segregated. Given the high comorbidity between anxiety disorders

125

and substance abuse disorder, understanding the common domains that regulate both aversive and appetitive memories is crucial. In fact, the mPFC has already been identified as a locus where circuits for fear and reward extinction overlap and one of the prefrontal outputs that mediates extinction of drug seeking is from the IL to the NAc shell (Peters et al., 2009). Thus, it is possible that glutamatergic inputs from the IL to the NAc shell not only mediate extinction of drugs of abuse but also of fear. In addition to commonalities in the neural networks, there is overlap in the cellular and molecular mechanisms governing fear and drug seeking such as dopamine, glutamatergic and endocannabinoid signaling. There is exhaustive evidence of the aforementioned pathways in drug seeking in rodents and humans which are not discussed here

(for detailed review see Di Chiara, 2002; Di Chiara et al., 2004; Xi et al., 2006; Willuhn et al.,

2010; Quintero, 2013; Abraham et al., 2014; Scofield et al., 2016). These pathways have also been identified to play a predominant role in regulating fear memories; there is evidence of increased mGluR1 expression within the shell following fear extinction learning (Sutton et al.,

2003), supporting the findings of experiment 3.3.1, 3.3.2. and 3.3.3 here. One possibility is that mGluR1 activation mediates fear extinction consolidation through CB1-dependent activity of

GABAergic neurons (Kim et al., 2007; Gunduz-Cinar et al., 2016). Further, dopamine D2 receptors, which have been suggested to be important for fear extinction consolidation in the nucleus accumbens (Holtzman-Assif et al., 2010), may modulate the synthesis and release of endocannabinoids for CB1 receptors (Giuffrida et al., 1999; Pickel et al., 2006). While the combined activity of the mGluR1/CB1/D2 pathway has not yet been studied in the nucleus accumbens subregions, the available evidence suggests important interactions between mGluR1, the endocannabinoid, and dopaminergic systems may regulate fear extinction.

126

Excessive associative fear or the inability to suppress fear responses is associated with anxiety disorders and trauma-related disorders such as PTSD. Further, evidence from both clinical and animal studies, combined with our data, suggest that a fundamental pathology in the nucleus accumbens could potentially predispose an individual to both anxiety and trauma- related disorders and potential co-morbid substance abuse. Taken together, the findings suggest that the NAc plays a prominent role in regulating fear memory and the consolidation of fear extinction and could perhaps be considered part of the canonical fear memory circuitry.

Furthermore, this region has the potential to be a target for the development of combined therapeutic intervention strategies of both addiction and fear-related disorders.

4.1 Limitations and future directions

Our studies provide compelling evidence that the nucleus accumbens subregions are involved in fear expression and consolidation of fear extinction. These data suggest that the nucleus accumbens subregions play a similar role as other traditional fear nodes (e.g, BLA, IL,PL) which posits the nucleus accumbens as a critical component of the canonical fear circuitry. Therefore, further exploring how the neuronal connections between NAc core and shell and other critical regions regulating fear learning processes, such as the amygdala, prefrontal cortex, and hippocampus will shed more light on the how the nucleus accumbens subregions integrate within the fear circuit.

While exposure therapy remains one the predominant treatments for individuals suffering from anxiety and trauma-related disorders, one of the major limitations of fear extinction is that the extinction memory is fragile and susceptible to reemergence of fear, thus hindering the efficacy of therapy (Bouton, 2000; Hermans et al., 2006; Vervliet et al., 2013). Thus, one of the major challenges with exposure therapy is to maintain fear reduction over a longer duration.

127

Over the years different forms of fear relapse have been identified such as spontaneous recovery, renewal, and reinstatement. Briefly, renewal of fear occurs to the extinguished CS (tone/context) is presented in a context distinct from where the extinction training took place thus resulting in return of the conditioned response. Spontaneous recovery refers to the loss of an extinguished response to a CS over time, whereas reinstatement refers to the return of fear when an individual is exposed to the US in the absence of the CS following extinction (for detailed review see

Mystkowski et al., 2002; Bouton, 2004; Hermans et al., 2005; Vervliet et al., 2013; Goode and

Maren, 2014). Along similar lines, our data indicate that the nucleus accumbens is a critical node involved in facilitation of fear extinction consolidation. Further study to investigate its role, both at the circuit and molecular level, in various fear relapse phenomenon will add a critical gap in our knowledge on the mechanisms of fear relapse and might identify the NAc as a target region for therapy. Additionally, our study did not investigate the role of nucleus accumbens subregions in fear acquisition. Fear acquisition refers to fear learning through repeated pairings of the CS with the US. Patients with anxiety disorders are more likely to acquire associative fear strongly as compared to healthy controls (Lissek et al., 2005), and it is possible the nucleus accumbens plays an important role in this process. There has been only one other study which reported the role of the nucleus accumbens in the acquisition of fear (Schwienbacher et al., 2004)and no study has investigated the individual involvement of the core and shell subregions in this process.

Another limitation is that we did not examine female mice in our current study. There is clear evidence that the likelihood of a woman experiencing anxiety and trauma- related disorder at any point in their life span is significantly higher as compared to men. Data shows that lifetime prevalence rate of anxiety disorder is women is 30.5% as compared to men at 19.2% and that of

PTSD is at 10.4% in women compared to 5.0% in men (Kessler et al., 1994; Kessler et al.,

128

1995). This trend of increased prevalence of anxiety disorders and trauma- related disorders extend specifically to fear extinction as well, however the underlying neurobiology remain poorly understood (for review see Velasco et al., 2019). However, there is one study which showed that females required a greater number of extinction training sessions as compared to men for ERK phosphorylation (Matsuda et al., 2015). Further, in spite of controversial conclusions, morphometric studies indicate potential dimorphisms in the nucleus accumbens on the basis of sex. (Brabec et al., 2003; Neto et al., 2008; Mavridis et al., 2011; Salgado and

Kaplitt, 2015). Therefore, future studies comparing the role of nucleus accumbens subregions in fear processing between males and females would prove to be step forward in the development of precision medicine for the treatment of anxiety and trauma-related disorders.

4.2 References

Abraham AD, Neve KA, Lattal KM (2014) Dopamine and extinction: a convergence of theory

with fear and reward circuitry. Neurobiology of learning and memory 108:65-77.

Berridge KC (2007) The debate over dopamine’s role in reward: the case for incentive salience.

Psychopharmacology (Berl) 191:391-431.

Berridge KC (2019) Affective valence in the brain: modules or modes? Nature Reviews

Neuroscience 20:225-234.

Bouton ME (2000) A learning theory perspective on lapse, relapse, and the maintenance of

behavior change. Health psychology 19:57.

Bouton ME (2004) Context and behavioral processes in extinction. Learning & memory (Cold

Spring Harbor, NY) 11:485-494.

Brabec J, Krásený J, Petrovický P (2003) Volumetry of striatum and pallidum in man--anatomy,

cytoarchitecture, connections, MRI and aging. Sbornik lekarsky 104:13-65.

129

Brog JS, Salyapongse A, Deutch AY, Zahm DS (1993) The patterns of afferent innervation of the

core and shell in the "accumbens" part of the rat ventral striatum: immunohistochemical

detection of retrogradely transported fluoro-gold. The Journal of comparative neurology

338:255-278.

Bull C, Freitas KCC, Zou S, Poland RS, Syed WA, Urban DJ, Minter SC, Shelton KL, Hauser KF,

Negus SS, Knapp PE, Bowers MS (2014) Rat Nucleus Accumbens Core Astrocytes

Modulate Reward and the Motivation to Self-Administer Ethanol after Abstinence.

Neuropsychopharmacology 39:2835-2845.

Campeau S, Falls WA, Cullinan WE, Helmreich DL, Davis M, Watson SJ (1997) Elicitation and

reduction of fear: behavioural and neuroendocrine indices and brain induction of the

immediate-early gene c-fos. Neuroscience 78:1087-1104.

Carlezon Jr WA, Thomas MJ (2009) Biological substrates of reward and aversion: a nucleus

accumbens activity hypothesis. Neuropharmacology 56:122-132.

Demyttenaere K et al. (2004) Prevalence, severity, and unmet need for treatment of mental

disorders in the World Health Organization World Mental Health Surveys. Jama 291:2581-

2590.

Di Chiara G (2002) Nucleus accumbens shell and core dopamine: differential role in behavior and

addiction. Behavioural brain research 137:75-114.

Di Chiara G, Bassareo V, Fenu S, De Luca MA, Spina L, Cadoni C, Acquas E, Carboni E,

Valentini V, Lecca D (2004) Dopamine and drug addiction: the nucleus accumbens shell

connection. Neuropharmacology 47 Suppl 1:227-241.

Floresco SB (2015) The Nucleus Accumbens: An Interface Between Cognition, Emotion, and

Action. 66:25-52.

130

Gibson GD, Millan EZ, McNally GP (2019) The nucleus accumbens shell in reinstatement and

extinction of drug seeking. European Journal of Neuroscience 50:2014-2022.

Giuffrida A, Parsons LH, Kerr TM, de Fonseca FR, Navarro M, Piomelli D (1999) Dopamine

activation of endogenous cannabinoid signaling in dorsal striatum. Nature neuroscience

2:358-363.

Goode TD, Maren S (2014) Animal Models of Fear Relapse. ILAR Journal 55:246-258.

Graham BM, Milad MR (2011) The study of fear extinction: implications for anxiety disorders.

Am J Psychiatry 168:1255-1265.

Gunduz-Cinar O, Flynn S, Brockway E, Kaugars K, Baldi R, Ramikie TS, Cinar R, Kunos G, Patel

S, Holmes A (2016) Fluoxetine Facilitates Fear Extinction Through Amygdala

Endocannabinoids. Neuropsychopharmacology 41:1598-1609.

Hermans D, Craske MG, Mineka S, Lovibond PF (2006) Extinction in Human Fear Conditioning.

Biological psychiatry 60:361-368.

Hermans D, Dirikx T, Vansteenwegenin D, Baeyens F, Van den Bergh O, Eelen P (2005)

Reinstatement of fear responses in human aversive conditioning. Behaviour Research and

Therapy 43:533-551.

Holtzman-Assif O, Laurent V, Westbrook RF (2010) Blockade of dopamine activity in the nucleus

accumbens impairs learning extinction of conditioned fear. Learning & memory (Cold

Spring Harbor, NY) 17:71-75.

Huang ACW, Shyu B-C, Hsiao S, Chen T-C, He AB-H (2013) Neural substrates of fear

conditioning, extinction, and spontaneous recovery in passive avoidance learning: A c-fos

study in rats. Behavioural brain research 237:23-31.

131

Insel T, Cuthbert B, Garvey M, Heinssen R, Pine DS, Quinn K, Sanislow C, Wang P (2010)

Research Domain Criteria (RDoC): Toward a New Classification Framework for Research

on Mental Disorders. American Journal of Psychiatry 167:748-751.

Jensen J, McIntosh AR, Crawley AP, Mikulis DJ, Remington G, Kapur S (2003) Direct Activation

of the Ventral Striatum in Anticipation of Aversive Stimuli. Neuron 40:1251-1257.

Kang S, Vervliet B, Engelhard IM, van Dis EAM, Hagenaars MA (2018) Reduced return of threat

expectancy after counterconditioning versus extinction. Behaviour Research and Therapy

108:78-84.

Kessler RC, Sonnega A, Bromet E, Hughes M, Nelson CB (1995) Posttraumatic stress disorder in

the National Comorbidity Survey. Archives of general psychiatry 52:1048-1060.

Kessler RC, McGonagle KA, Zhao S, Nelson CB, Hughes M, Eshleman S, Wittchen H-U, Kendler

KS (1994) Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the

United States: results from the National Comorbidity Survey. Archives of general

psychiatry 51:8-19.

Kim J, Lee S, Park H, Song B, Hong I, Geum D, Shin K, Choi S (2007) Blockade of amygdala

metabotropic glutamate receptor subtype 1 impairs fear extinction. Biochemical and

Biophysical Research Communications 355:188-193.

Knapska E, Maren S (2009) Reciprocal patterns of c-Fos expression in the medial prefrontal cortex

and amygdala after extinction and renewal of conditioned fear. Learn Mem 16:486-493.

LaLumiere RT, Kalivas PW (2008) Glutamate Release in the Nucleus Accumbens Core Is

Necessary for Heroin Seeking. The Journal of Neuroscience 28:3170-3177.

Levita L, Hoskin R, Champi S (2012) Avoidance of harm and anxiety: A role for the nucleus

accumbens. NeuroImage 62:189-198.

132

Li Z, Chen Z, Fan G, Li A, Yuan J, Xu T (2018) Cell-Type-Specific Afferent Innervation of the

Nucleus Accumbens Core and Shell. Frontiers in Neuroanatomy 12.

Lissek S, Powers AS, McClure EB, Phelps EA, Woldehawariat G, Grillon C, Pine DS (2005)

Classical fear conditioning in the anxiety disorders: a meta-analysis. Behaviour research

and therapy 43:1391-1424.

Matsuda S, Matsuzawa D, Ishii D, Tomizawa H, Sutoh C, Shimizu E (2015) Sex differences in

fear extinction and involvements of extracellular signal-regulated kinase (ERK).

Neurobiology of learning and memory 123:117-124.

Mavridis I, Boviatsis E, Anagnostopoulou S (2011) Anatomy of the human nucleus accumbens: a

combined morphometric study. Surgical and radiologic anatomy 33:405-414.

Meredith GE, Pattiselanno A, Groenewegen HJ, Haber SN (1996) Shell and core in monkey and

human nucleus accumbens identified with antibodies to calbindin-D28k. Journal of

Comparative Neurology 365:628-639.

Millan EZ, Furlong TM, McNally GP (2010) Accumbens Shell–Hypothalamus Interactions

Mediate Extinction of Alcohol Seeking. The Journal of Neuroscience 30:4626-4635.

Mystkowski JL, Craske MG, Echiverri AM (2002) Treatment context and return of fear in spider

phobia. Behavior Therapy 33:399-416.

Neto LL, Oliveira E, Correia F, Ferreira AG (2008) The human nucleus accumbens: where is it?

A stereotactic, anatomical and magnetic resonance imaging study. Neuromodulation:

Technology at the neural interface 11:13-22.

Newall C, Watson T, Grant K-A, Richardson R (2017) The relative effectiveness of extinction and

counter-conditioning in diminishing children's fear. Behaviour Research and Therapy

95:42-49.

133

Nicola SM (2007) The nucleus accumbens as part of a basal ganglia action selection circuit.

Psychopharmacology (Berl) 191:521-550.

Paré D, Quirk GJ, Ledoux JE (2004) New vistas on amygdala networks in conditioned fear. Journal

of neurophysiology 92:1-9.

Peters J, Kalivas PW, Quirk GJ (2009) Extinction circuits for fear and addiction overlap in

prefrontal cortex. Learning & memory (Cold Spring Harbor, NY) 16:279-288.

Pickel VM, Chan J, Kearn CS, Mackie K (2006) Targeting dopamine D2 and cannabinoid-1 (CB1)

receptors in rat nucleus accumbens. Journal of Comparative Neurology 495:299-313.

Quintero GC (2013) Role of nucleus accumbens glutamatergic plasticity in drug addiction.

Neuropsychiatric disease and treatment 9:1499.

Quirk GJ, Likhtik E, Pelletier JG, Paré D (2003) Stimulation of medial prefrontal cortex decreases

the responsiveness of central amygdala output neurons. Journal of Neuroscience 23:8800-

8807.

Reynolds SM, Berridge KC (2002) Positive and Negative Motivation in Nucleus Accumbens

Shell: Bivalent Rostrocaudal Gradients for GABA-Elicited Eating, Taste

“Liking”/“Disliking” Reactions, Place Preference/Avoidance, and Fear. The Journal of

Neuroscience 22:7308.

Salamone JD, Cousins MS, Bucher S (1994) Anhedonia or anergia? Effects of haloperidol and

nucleus accumbens dopamine depletion on instrumental response selection in a T-maze

cost/benefit procedure. Behavioural brain research 65:221-229.

Salgado S, Kaplitt MG (2015) The Nucleus Accumbens: A Comprehensive Review. Stereotactic

and Functional Neurosurgery 93:75-93.

134

Schwienbacher I, Fendt M, Richardson R, Schnitzler H-U (2004) Temporary inactivation of the

nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats.

Brain Research 1027:87-93.

Scofield MD, Heinsbroek JA, Gipson CD, Kupchik YM, Spencer S, Smith ACW, Roberts-Wolfe

D, Kalivas PW (2016) The Nucleus Accumbens: Mechanisms of Addiction across Drug

Classes Reflect the Importance of Glutamate Homeostasis. Pharmacological Reviews

68:816.

Sturm V, Lenartz D, Koulousakis A, Treuer H, Herholz K, Klein JC, Klosterkötter J (2003) The

nucleus accumbens: a target for deep brain stimulation in obsessive–compulsive- and

anxiety-disorders. Journal of Chemical Neuroanatomy 26:293-299.

Sutton MA, Schmidt EF, Choi K-H, Schad CA, Whisler K, Simmons D, Karanian DA, Monteggia

LM, Neve RL, Self DW (2003) Extinction-induced upregulation in AMPA receptors

reduces cocaine-seeking behaviour. Nature 421:70-75.

Tan Y, Williams ES, Zahm DS (1999) Calbindin-D 28kD immunofluorescence in ventral

mesencephalic neurons labeled following injections of Fluoro-Gold in nucleus accumbens

subterritories: inverse relationship relative to known neurotoxin vulnerabilities. Brain

Research 844:67-77.

Thomas KL, Hall J, Everitt BJ (2002) Cellular imaging with zif268 expression in the rat nucleus

accumbens and frontal cortex further dissociates the neural pathways activated following

the retrieval of contextual and cued fear memory. The European journal of neuroscience

16:1789-1796.

Velasco ER, Florido A, Milad MR, Andero R (2019) Sex differences in fear extinction.

Neuroscience & Biobehavioral Reviews 103:81-108.

135

Vervliet B, Craske MG, Hermans D (2013) Fear Extinction and Relapse: State of the Art. Annual

Review of Clinical Psychology 9:215-248.

Willuhn I, Wanat MJ, Clark JJ, Phillips PEM (2010) Dopamine Signaling in the Nucleus

Accumbens of Animals Self-Administering Drugs of Abuse. In: Behavioral Neuroscience

of Drug Addiction (Self DW, Staley Gottschalk JK, eds), pp 29-71. Berlin, Heidelberg:

Springer Berlin Heidelberg.

Xi Z-X, Gilbert JG, Peng X-Q, Pak AC, Li X, Gardner EL (2006) Cannabinoid CB1 receptor

antagonist AM251 inhibits cocaine-primed relapse in rats: role of glutamate in the nucleus

accumbens. Journal of Neuroscience 26:8531-8536.

136