Contributions of the Renin Angiotensin System to Fear Memory and Fear Conditioned Cardiovascular Responses

by Adam Swiercz

B.S. in Biology, May 2006, The George Washington University M.P.S. in Molecular Biotechnology, May 2009, The George Washington University M.S. in Physiology, May 2011, Georgetown University

A Dissertation submitted to

The Faculty of The Columbian College of Arts & Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

January 10, 2020

Dissertation co-directed by

Paul J. Marvar Associate Professor of Pharmacology and Physiology and David Mendelowitz Professor of Pharmacology & Physiology

The Columbian College of Arts and Sciences of The George Washington University certifies that Adam Swiercz has passed the Final Examination for the degree of Doctor of

Philosophy as of October 2nd, 2019. This is the final and approved form of the dissertation.

Contributions of the Renin Angiotensin System to Fear Memory and Fear Conditioned Cardiovascular Responses

Adam Swiercz

Dissertation Research Committee:

Paul J. Marvar, Associate Professor of Pharmacology & Physiology, Dissertation Co-Director

David Mendelowitz, Professor of Pharmacology & Physiology, Dissertation Co-Director

Abigail Polter, Assistant Professor of Pharmacology & Physiology, Committee Member

Colin Young, Assistant Professor of Pharmacology & Physiology, Committee Member

iii

Acknowledgements

I would like to thank and acknowledge Dr. Paul Marvar, whose mentorship has made this dissertation possible. It has been a pleasure working in your lab, and I am truly grateful for your support and encouragement throughout the years. Thanks to the current and former members of the Marvar lab who have made my time at GW a rewarding and enjoyable experience.

I would also like to thank my co-mentor, Dr. David Mendelowitz, as well as my committee members: Dr. Narine Sarvazyan, Dr. Colin Young, and Dr. Abigail Polter.

You have all been very generous with your time, and your guidance has been incredibly helpful. To Dr. Leo Chalupa and Dr. Peter Nemes, thank you for your help during the defense process. Dr. Vincent Chiappinelli, thank you for your support during my time in the department of pharmacology and physiology. I would also like to thank Dr. Linda

Werling and Marc Wittlif, who I had the pleasure of working with during my first few years in the IBS program.

I am especially grateful for my parents, who instilled in me the determination required to accomplish my personal and academic goals. To my wife, Kelly, thank you for your endless patience, optimism, and support.

Abstract of Dissertation

Contributions of the Renin Angiotensin System to Fear Memory and Fear Conditioned Cardiovascular Responses

Anxiety disorders, such as Posttraumatic stress disorder (PTSD), are associated with an increased risk of developing cardiovascular disease. While the exact mechanisms underlying this relationship are not entirely clear, recent evidence suggests that a hormonal system involved in the maintenance of blood pressure and fluid balance may play an integral role. The renin angiotensin system

(RAS), which modulates autonomic nervous system activity and cardiovascular function, also influences learning processes such as fear memory formation and maintenance. Pharmacologically targeting the RAS may be effective in reducing symptoms of fear and anxiety disorders. This dissertation seeks to improve our understanding of the relationship between fear memory and cardiovascular reactivity, as well as the involvement of the RAS in fear learning processes.

The first study examines the effects of extinction learning on cardiovascular responses to conditioned auditory stimuli. Through simultaneous recording of freezing behavior, blood pressure, and heart rate, it is shown that blood pressure responses are attenuated by repeated conditioned stimulus exposure. In the second study, the role of the angiotensin II type 1 receptor (AT1R) in the reconsolidation of auditory fear memory is investigated. Our findings suggest that blockade of AT1R during reconsolidation leads to long-term reductions in freezing behavior. Results from this study also indicate that treatment with the AT1R antagonist losartan following memory retrieval leads to differential gene expression patterns in the

amygdala. The final study identifies the functional properties of central angiotensin

II type 2 receptors (AT2R) in fear expression and extinction. The regional distribution and characteristics of AT2R+ cells within the amygdala are examined in detail. Furthermore, pharmacological activation of AT2R in the central amygdala was used to determine how these receptors might contribute to fear learning and expression. Finally, AT2R-expressing neurons in the central amygdala are shown to project to the periaqueductal grey, a brain region responsible for mediating freezing behavior.

The studies in this dissertation are the first to incorporate acute cardiovascular responses such as blood pressure into the assessment of extinction and reconsolidation, which are important clinical targets for the treatment of anxiety and fear. Furthermore, evidence is provided that AT1R contributes to the reconsolidation of auditory fear memories, and that AT2R-expressing neurons in the central amygdala modulate fear expression and extinction. These findings advance our understanding of the physiological and neurobiological systems that regulate fear learning, and suggest that compounds targeting the RAS may be useful in the treatment of fear-related psychiatric conditions.

Table of Contents

Acknowledgements ...... iv

Abstract of Dissertation ...... v

Table of Contents ...... vii

List of Figures ...... ix

List of Tables ...... xi

List of Abbreviations ...... xii

Chapter 1:

General Introduction ...... 1 Posttraumatic Stress Disorder ...... 2 Posttraumatic Stress Disorder and Cardiovascular Disease Risk ...... 3 The Renin Angiotensin System ...... 5 AT1R ...... 7 AT2R ...... 8 The Renin Angiotensin System and the Sympathetic Nervous System .....9 The Brain Renin Angiotensin System...... 11 Anxiety and the Brain Renin Angiotensin System ...... 14 Angiotensin II in Learning and Memory ...... 15 Emotion and Memory ...... 16 Fear Memory ...... 18 Pavlovian Fear Conditioning ...... 19 Circuitry of Fear Conditioning...... 21 Fear-Conditioned Physiological Responses ...... 24 Extinction of Fear Memory ...... 26 A Role for the Renin Angiotensin System in PTSD ...... 29 Memory Reconsolidation ...... 30 Genetic Markers of Reconsolidation ...... 32 Summary and Specific Aims ...... 34 References ...... 40

Chapter 2:

Extinction of Fear Memory Attenuates Conditioned Cardiovascular Fear Reactivity Abstract ...... 60 Introduction ...... 61 Materials and Methods ...... 63

vii

Results ...... 67 Discussion ...... 73 References ...... 88

Chapter 3: Evaluation of an Angiotensin Type 1 Receptor Blocker on the Reconsolidation of Fear Memory Abstract ...... 93 Introduction ...... 94 Materials and Methods ...... 95 Results ...... 100 Discussion ...... 105 References ...... 121

Chapter 4: Angiotensin Type 2 Receptor Expressing Neurons in the Central Amygdala Influence Fear-Related Behavior Abstract ...... 127 Introduction ...... 128 Materials and Methods ...... 130 Results ...... 133 Discussion ...... 138 References ...... 156

Chapter 5:

General Discussion ...... 163 Study 1 ...... 164 Study 2 ...... 172 Study 3 ...... 178 General Summary and Conclusions ...... 185 References ...... 187

viii

List of Figures

Figure 1-1. Blood pressure regulation through the renin-angiotensin system cascade .....37

Figure 1-2. Systems involved in fear memory formation ...... 38

Figure 1-3. The neural circuits underlying fear conditioning ...... 39

Figure 2-1. Behavioral and cardiovascular changes during extinction training ...... 80

Figure 2-2. Schematic of fear conditioning and testing protocol for home cage extinction studies ...... 81

Figure 2-3. Pre-extinction training CS-dependent cardiovascular response test 1 (home cage) ...... 82

Figure 2-4. Post-extinction training CS-dependent cardiovascular response tests 2 and 3 (home cage) ...... 83

Figure 2-S1. Baseline cardiovascular measures in training and home cage contexts studies ...... 84

Figure 2-S2. Freezing behavior during fear conditioning ...... 85

Figure 2-S3. Group data of baseline heart rate variability prior to fear conditioning ...... 86

Figure 3-1. Post-retrieval losartan reduces freezing behavior ...... 111

Figure 3-2. Post-retrieval losartan does not alter recall of conditioned cardiovascular responses ...... 112

Figure 3-3. Losartan attenuates stress-induced increases in blood pressure following handling and injection ...... 113

Figure 3-4. Differential BLA gene expression analysis following post-retrieval losartan ...... 114

Figure 3-5. Transcriptional profiles and pathways ...... 115

Figure 4-1. Intra-central amygdala (CeA) angiotensin II type 2 receptor (AT2R) activation reduces fear expression ...... 145

Figure 4-2. The effects of intra-central amygdala (CeA) angiotensin II type 2 receptor (AT2R) activation on basal anxiety measures and plasma corticosterone levels ...... 146

Figure 4-3. AT2R-eGFP expressing GABAergic neurons in CeM but not CeL ...... 147

Figure 4-4. AT2R-eGFP neurons in CeM are predominately projection neurons ...... 148

Figure 4-5. Angiotensin II type 2 receptor (AT2R)-enhanced green fluorescent protein (eGFP) neurons in medial division of the central amygdala (CeM) project to periaqueductal gray (PAG) ...... 149

Figure 4-S1. AT2R-eGFP expressing cells are neuronal...... 151

Figure 4-S2. The effects of intra CeA AT2R activation on an open field test ...... 152

List of Tables

Table 2-S1 Baseline day/night (12 h) mean arterial pressure, heart rate, and activity levels in mice prior to fear conditioning ...... 87

Table 3-S1 Genes differentially expressed in Saline compared to NR group in BLA...... 116

Table 3-S2 Genes differentially expressed in Losartan compared to NR group in BLA...... 119

Table 4-S1 Key Resources ...... 150

List of Abbreviations

1. ACE: Angiotensin converting enzyme

2. ACE2: Angiotensin converting enzyme 2

3. ACTH: Adrenocorticotropic hormone

4. ADH: Antidiuretic hormone

5. AGT: Angiotensinogen

6. Ang II: Angiotensin II

7. Ang III: Angiotensin III

8. Ang 1-7: Angiotensin (1-7)

9. APV: DL-2-amino-5-phosphonovaleric acid

10. AT1R: Angiotensin II receptor type-1

11. AT2R: Angiotensin II receptor type-2

12. BLA: Basolateral amygdala

13. CeA: Central amygdala

14. CGP42112A: N-α-nicotinoyl-Tyr-Lys-(N-αCBZ-Arg)-His-Pro-Ile-OH

15. CVD: Cardiovascular disease

16. CVO: Circumventricular organ

17. C21: Compound 21

18. DVC: Nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve

19. GAD: Glutamate decarboxylase

20. HPA: Hypothalamic pituitary adrenal axis

21. ICV: Intracerebroventricular

22. ICM: Intercalated cell mass

xii

23. IEG: Immediate early gene

24. i.p.: Intraperitoneal

25. LA: Lateral amygdala

26. LH: Lateral hypothalamus

27. LTP: Long-term potentiation

28. NMDA: N-Methyl-D-aspartic acid

29. PAG: Periaqueductal Grey

30. PD 123319: (S)-1-[4-(dimethylamino)-3-methylphenyl]methyl-5-(diphenylacetyl)-

4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acid

31. PLA2: Phospholipase A2

32. PP2A: serine/threonine phosphatase 2A

33. PR: Prorenin

34. PRR: Prorenin receptor

35. PTSD: Posttraumatic Stress Disorder

36. PVN: Paraventricular nucleus of hypothalamus

37. RAS: Renin-angiotensin System

38. RSNA: Renal Sympathetic Nerve Activity

39. RVLM: Rostral Ventrolateral Medulla

40. SHR: Spontaneously Hypertensive Rats

xiii

CHAPTER 1

Introduction

Posttraumatic Stress Disorder

While many individuals experience significant traumatic events at some point in their lives, approximately 7-8% of civilians (Kessler et al., 2005), and up to 20% of veterans (Seal et al., 2009) will develop symptoms of a debilitating psychiatric condition called posttraumatic stress disorder (PTSD). The Diagnostic and Statistical Manual for

Mental Disorders (DSM-5) (American Psychiatric Association, 2013) describes four primary PTSD symptom clusters that occur following exposure to a traumatic event: (1) intrusive symptoms associated with the traumatic event; (2) avoidance behavior and efforts to evade distressing memories and external reminders of the traumatic event; (3) negative mood alterations including detachment, diminished interest, and the inability to experience positive emotions; and (4) hyperarousal characterized by exaggerated startle responses, hypervigilance, sleep disturbances, and reckless or self-destructive behavior.

The heterogeneous nature of this disorder suggests a complex interaction of underlying neurobiological mechanisms contributing to the development and persistence of symptoms (Norrholm and Jovanovic, 2018). In general, PTSD symptoms reflect pathological fear expression and can occur when acquisition or generalization of fear is excessive, and extinction is impaired (Bowers and Ressler, 2015).

Current PTSD treatments result in low and variable rates of success. While approximately 60% of PTSD patients show some improvement on select serotonin reuptake inhibitors (SSRIs) (Steckler and Risbrough, 2012), these drugs can take months to reach full effectiveness and only 20-30% of patients reach complete clinical remission

(Difede et al., 2014). Prolonged exposure therapy has also shown promise as an effective cognitive therapy for PTSD. Most psychotherapies for PTSD include at least some

component of prolonged exposure, which is based on the emotional processing theory and the idea that fear is activated by associative networks containing information about fear stimuli, defensive responses to fear stimuli, and aspects of the meaning of the stimulus to the individual (Foa and Kozak, 1986). Successful treatment begins with activation of the fear network, followed by encoding of new information that is incompatible with existing information in the fear network. Over time, fear associations are weakened as new information about them is incorporated. In this view, the physiological activation and habituation that occurs within and between exposure sessions is indicative of emotional processing. The argument that exposure leads to fear reduction has made this type of therapy one of the most useful for the treatment of PTSD, and follow-up studies have demonstrated long-lasting reduction in PTSD symptoms in responsive patients (Resick et al., 2012). A meta-analysis examining the efficacy of psychotherapies in the military personnel and veterans found that 49-70% attained some level of symptom improvement. However, approximately two-thirds of patients retained their PTSD diagnoses following treatment (Steenkamp et al., 2015). A need remains for the development of more effective evidenced-based approaches for the treatment of

PTSD.

PTSD and Cardiovascular Disease Risk

The effects of PTSD are not limited to psychological symptoms. Early observations of abnormal cardiovascular responses associated with trauma describe a condition called “irritable heart syndrome”, and veterans with this condition exhibited greater heart rate (HR) and respiratory responses to presentations of bright flames and

pistol shots (Meakins and Wilson, 1918). These findings were among the first to establish a link between trauma-related disorders and increased sensitivity of the autonomic nervous system (Orr et al., 2002), and represent an age-old interest in the relationship between the autonomic nervous system and psychiatric disorders related to trauma. To this day, the heightened physiologic responses of patients with PTSD is one of the most robust findings in this area of research (Orr et al., 2002).

PTSD diagnoses are associated with a significantly greater risk of developing heart disease (Boscarino, 2008; Boscarino and Chang, 1999; Kibler et al., 2009; Xue et al., 2012). Recent reports show that veterans diagnosed with PTSD are at approximately

45% greater risk of myocardial infarction and roughly 30% greater risk of developing congestive heart failure and peripheral vascular disease later in life than veterans without

PTSD (Beristianos et al., 2016). The link between PTSD and cardiovascular disease

(CVD) is supported by the long-standing theory that psychological stress contributes to the development and progression of human disease in general (Cohen et al., 2007; Selye,

1956). This relationship is mediated in part by changes in the hypothalamic-pituitary- adrenocortical (HPA) axis, and the sympathetic-adrenal-medullary (SAM) system, both of which can affect autonomic nervous system activity and inflammatory responses. The exact mechanisms underlying the link between PTSD and cardiovascular disease, however, are not completely understood.

Autonomic nervous system activity during times of stress may also contribute to the PTSD-CVD association. The “reactivity hypothesis” proposes that individuals with exaggerated cardiovascular reactivity to psychological stress have a greater risk of developing cardiovascular disease (Manuck et al., 1990). This idea is supported by

multiple studies showing that stressor-evoked cardiovascular activity is associated with adverse future cardiovascular health outcomes (Chida and Steptoe, 2010; Trotman et al.,

2019). In the case of PTSD, imbalances in autonomic responses may be more common.

For instance, direct measurement of muscle sympathetic nerve activity has revealed that veterans with PTSD show exaggerated sympathetic nervous system (SNS) and hemodynamic reactivity to mental stressors, as well as impaired baroreflex sensitivity relative to veterans without PTSD (Park et al., 2017). While the exact mechanisms underlying the link between PTSD and cardiovascular disease are not completely understood, the autonomic nervous system and the hormonal systems that influence autonomic activity are of interest. In recent years, one hormonal system in particular called the renin angiotensin system (RAS), has been shown to play an important role in both the cardiovascular and psychological pathologies associated with fear and anxiety disorders (Khoury et al., 2012a; Phillips and de Oliveira, 2008; Shekhar et al., 2006).

The Renin Angiotensin System

The RAS is a hormonal system best known for its role in the conservation of salt and blood volume and the maintenance of blood pressure (BP). The RAS was discovered over 100 years ago by Robert Tigertedt and Per Gustav Bergman who found that the injection of rabbit kidney extracts into recipient rabbits consistently increased the recipient’s blood pressure. From these experiments, it was concluded that a pressor substance named “renin” existed in the kidney. Later studies would clarify that renin itself was not the potent vasoconstrictive substance responsible for the pressor response;

it was instead a proteolytic enzyme that acts upon the precursor peptide angiotensinogen

(AGT) (Braun-Menendez and Page, 1958).

In response to internal signals indicating low blood pressure such as renal sympathetic activity, reductions in afferent arteriole stretch, decreased salt delivery to the macula densa, and adrenergic stimulation, renin is released from the kidney into the circulation. Renin cleaves the liver-derived AGT protein into a smaller (10 AA) peptide fragment called angiotensin I or “proangiotensin”. The biologically inert angiotensin I is further degraded by another enzyme called angiotensin-converting enzyme (ACE), which is located on the surface of vascular endothelial cells. ACE cleaves angiotensin I into the biologically active octapeptide angiotensin II (Ang II), which is the primary effector peptide of the RAS (Fig. 1-1).

Further processing of Ang II can generate additional peptides that exert their own biological effects. One of the degradation products of Ang II is called Angiotensin III

(Ang III). Ang III regulates volume homeostasis by stimulating vasopressin release during periods of hypovolemia (Reaux et al., 2001; Ruiz-Ortega et al., 2001).

Additionally, the ACE homologue angiotensin converting enzyme II (ACE2) can convert

Ang II to angiotensin 1-7 (Ang 1-7). Ang 1-7 is the endogenous ligand for the receptor

Mas (Gironacci et al., 2011), and can prevent some of the deleterious effects of Ang II such as inflammation, fibrosis, and apoptosis (Cao et al., 2019). Angiotensin IV (Ang

IV) is also derived from Ang II, and while its mechanism of action is still unclear it appears to act within the nervous system to enhance learning and memory (Gard, 2008;

Lew et al., 2003). Currently, an effort is underway to develop Ang IV-based compounds

that might improve compromised memory and motor systems in neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease (Wright et al., 2015).

Prorenin (PR) and its receptor (PRR) are also important contributors to central

RAS function (Pitra et al., 2019). PRR is distributed broadly within neurons located in brain regions that control cardiovascular function (Shan et al., 2010). PR can activate the

PRR, which then causes a conformational change in the PR molecule, allowing it to exert full renin activity even without proteolytic cleavage (Nguyen et al., 2002). This pathway leads to increased conversion of angiotensinogen to Ang I and Ang II, and has been implicated in neurogenic hypertension (Huber et al., 2015). However, while smaller angiotensin peptides and recently discovered receptors appear to mediate effects on both physiological and cognitive function, two Ang II receptors in particular are critical for

RAS function and have been examined in detail.

Angiotensin II Type 1 Receptor

The primary receptor of Ang II is the angiotensin II type 1 receptor (AT1R).

Classical functions of the AT1R include vasoconstriction, increased noradrenaline (NA) release, stimulation of sodium reabsorption in the proximal tubule, cell growth in the heart, and secretion of aldosterone from the adrenal cortex (Unger, 2000). Ang II binding to AT1R can initiate several intracellular signaling cascades. The most common pathway is triggered by interaction with the Gq/11 protein subunit, which activates phospholipase-

C and the second messengers inositol triphosphate (IP) and diacylglycerol (DAG)

(Conchon and Clauser, 2004). AT1R activation can also stimulate phospholipase A2

(PLA2) and arachidonic acid production (Szczepanska-Sadowska et al., 2018). The

metabolism of arachidonic acid produces eicosanoids that regulate contractility of vascular smooth muscle cells (Touyz and Schiffrin, 2000). This receptor is responsible for most of the physiological actions of Ang II in the periphery and is therefore an effective therapeutic target for the treatment of hypertension. Imidazolic compounds like

Dup753 (losartan) bind with high affinity to the AT1R (Rhaleb N E et al., 1991).

Losartan is a competitive antagonist that reduces the interaction of Ang II with its AT1R

(Sica et al., 2005). In humans, approximately 14% of losartan is converted into the pharmacologically active metabolite E 3174, which is up to 40-fold more potent than its parent compound (Sica et al., 2005).

Angiotensin II Type 2 Receptor

The more recently discovered angiotensin II type 2 receptor (AT2R) is bound with high affinity by L-spinacine derivatives like PD123319 (Chiu et al., 1989). Much less is known about the functional roles of this receptor, however it appears to exert anti- inflammatory, antiproliferative, and antihypertensive effects that counteract those of the

AT1R and is therefore considered a protective arm of the RAS (Sumners et al., 2013).

AT2R is a G-protein coupled receptor and signals through the Gi alpha subunit (Gao and

Zucker, 2011). Stimulation of this receptor leads to the activation of phosphotyrosine phosphatases and is associated with the inactivation of mitogen activate protein kinases

(MAPKs) and extracellular signal-regulated kinase (ERKs) (Horiuchi et al., 1999). Ang

II binds with similar affinity to the AT2R as it does the AT1R, and AT2R activity can be inhibited by the selective antagonist PD123319, or activated with the agonist CGP42112

(Bosnyak et al., 2010). Recently, a new highly selective non-peptide agonist was

developed called Compound 21 (C21)(Wan et al., 2004a). Pharmacological activation of the AT2R with C21 has been shown to have beneficial effects in pre-clinical models of stroke and hypertension (Bennion et al., 2018; Foulquier et al., 2012).

The Renin Angiotensin System and the Sympathetic Nervous System

Not long after the discovery of angiotensin, it was demonstrated that Ang II can increase blood pressure by directly acting within the central nervous system to evoke sympathetic discharges. One of the earliest studies identifying the ability of Ang II to activate the central SNS was a cross-circulation study in dogs where infusion of Ang II into the cranial circulation led to an increased blood pressure in both the donor and recipient animals (Bickerton and Buckley, 1961). In order to maintain homeostasis, Ang

II must be able to act rapidly with the sympathetic nervous system to prevent life- threatening losses of intravascular volume, and to act slowly by sensitizing blood vessels to vasoconstrictive agents and promoting vascular growth (Abdel-Rahman et al., 2004).

Ang II increases SNS activity by enhancing ganglionic transmission, facilitating NA release at sympathetic nerve terminals, and strengthening the post synaptic effects of NA.

These effects are mediated in part by the AT1R, as blockade of this receptor can reduce the ability of Ang II to increase sympathetic neurotransmission.

One of the central sites through which circulating Ang II alters sympathetic activity is the area postrema, a circumventricular organ (CVO) in the medulla oblongata

(Reid, 1992). The area postrema has a high density of AT1R and is directly connected to medullary centers that regulate cardiovascular activity. The paraventricular nucleus of the hypothalamus (PVN) is another key brain region responsible for the generation of

sympathetic vasomotor tone. Plasma Ang II levels are thought to affect PVN activity, however this region is located within the BBB and therefore not in direct contact with circulating Ang II. Ang II-mediated PVN activity is most likely explained by the actions of Ang II on CVOs such as the subfornical organ (SFO), which contains abundant AT1R and sends axonal projections to the PVN (Bains and Ferguson, 1995). Cells within the

PVN project to the thoraco-lumbar spinal cord and other premotor regions important for the maintenance of sympathetic outflow and arterial pressure, such as the rostral ventrolateral medulla (RVLM) (Badoer, 2001). Other CVOs known to mediate the actions of Ang II on blood pressure regulation include the organum vasculosum of the lamina terminalis (OVLT), and the anteroventral third ventricle (AV3V) (Vieira et al.,

2010). The role of AT1R in Ang II-mediated changes in SNS activity has made it an important target for the treatment of SNS-related pathologies such as hypertension and congestive heart failure. In addition to the AT1R, the RAS may also act within the CNS to increase sympathetic activity via mineralocorticoid receptors (Zhang et al., 2008), oxidative stress (Hirooka, 2011), or even the PRR (Li et al., 2012). Strong RAS–SNS interactions suggest that the RAS may also be tied to other systems controlled by sympathetic activity, including defensive behaviors.

The primary brain regions controlling sympathetic nerve activity (SNA) and arterial baroreflex regulation are the rostral ventrolateral medulla (RVLM) and the nucleus of the solitary tract (NTS), both of which are located in the medulla oblongata

(Dampney, 1994; Dampney et al., 1996). Baroreflex function is controlled in part by an exchange of information between the RVLM and the NTS which determines peripheral sympathetic nerve activity through projections to sympathetic preganglionic neurons in

the spinal cord (DiBona and Jones, 2001). In the RVLM, Ang II can change the static and dynamic characteristics of neuronal baroreflex responses (Saigusa and Arita, 2014).

AT1R is highly expressed in both the RVLM and the NTS (Ferguson and Washburn,

1998). These nonfenestrated regions lie behind the blood brain barrier (BBB) and are therefore out of reach of blood-borne Ang II, supporting a potential neuromodulatory role of brain-derived Ang II (Allen et al., 1999) in autonomic and cardiovascular function.

The Brain RAS

Ang II is a hydrophilic peptide, and while there is some evidence of receptor- mediated transcytosis allowing transport of such proteins into the brain (Rose and Audus,

1998) hydrophilic peptides do not readily cross the BBB (Laterra et al., 1999). Instead, circulating Ang II is believed to exert its central effects primarily through CVOs which lack a blood brain barrier. For example, Ang II potentiates the pressor response by binding to the area postrema, while its dipsogenic effects are controlled mainly by the subfornical organ (SFO) and the vascular organ of lamina terminalis (OVLT). While

Ang II acts on CVOs to drive the drinking response, increased sympathetic output, and increased secretion of vasopressin, CVO neurons can also relay information to BBB- protected brain regions by using Ang II as neurotransmitter (Lind et al., 1985). How exactly Ang II acts as chemical messenger within the brain is not fully understood, but the distribution of RAS components and receptors throughout the brain suggests that it plays an important role in central functions beyond maintaining water balance and cardiovascular homeostasis (Mogi and Horiuchi, 2013; Wright and Harding, 2013).

Several decades of research support the existence of a functional, independent brain RAS in multiple species (e.g. sheep, dogs, humans, rats and mice) (Paul et al.,

2006). However, the exact source of Ang II and other angiotensin peptides within the brain remains a topic of debate. While the substrate for renin (AGT) is expressed in glial cells (Deschepper et al., 1986; Stornetta et al., 1988) and neurons (Yang et al., 1999), renin itself is present at extremely low levels within the brain and is difficult to detect.

Still, transgenic mice expressing green fluorescent protein (GFP) under control of the renin promoter suggest that renin is expressed in close proximity to angiotensin (Lavoie

Julie L. et al., 2004), and primarily in neurons (Lavoie et al., 2004). Recent studies using a novel knockout mouse model in which the brain-specific renin isoform (renin-b) is deleted bolster the argument that angiotensin is synthesized de novo in the brain

(Shinohara Keisuke et al., 2016; Sigmund et al., 2017). In further support of this theory, it has also been shown that ACE is expressed in astrocytes, alongside Ang II and Ang II receptors (Nakagawa Pablo and Sigmund Curt D., 2017). Interestingly, renin and angiotensinogen are co-expressed in the CEA and the parabrachial nucleus, and expressed in adjacent cells in the RVLM, BST, SFO, and Ca1-3 (Lavoie Julie L. et al.,

2004). In addition to identifying the region-specific expression of RAS components, the functional properties of the brain RAS can also be examined by targeting centrally- located Ang II receptors.

AT1R are expressed in many regions of the rodent brain, particularly those that regulate the cardiovascular and dipsogenic actions of Ang II (e.g. SFO, OVLT, MNPO,

NTS, and the dorsal motor nucleus of the vagus (DVN)) (Rowe et al., 1992; Song et al.,

1992). AT1R expression is also observed in limbic regions such as the amygdala and the

bed nucleus of the stria terminalis (BNST) (McKinley et al., 2003), as well as the hippocampus and piriform cortex (Allen et al., 1999; Lenkei et al., 1997).

Autoradiography and tissue homogenate preparations suggest that Ang II receptor distribution is consistent across many species, including rats, mice, dogs, monkeys, and humans (Wright and Harding, 1997). In humans, autoradiographic and homogenate binding studies show that the AT1R is expressed throughout the forebrain, particularly in the paraventricular hypothalamus (PVN), the median eminence, the substantia nigra, and the putamen and caudate nucleus. Importantly, radioligand binding is also observed in other brain regions like the amygdala, frontal cortex, hippocampus, and periaqueductal grey (Barnes et al., 1993). To aid in the mapping of gene expression in the central nervous system, the GENSAT project developed a mouse called NZ44, which carries a bacterial artificial chromosome (BAC) transgene expressing enhanced green fluorescent protein (eGFP) in the AT1R locus (Gonzalez et al., 2012). The NZ44 mouse shows high

AT1R expression in neurons within the SFO, OVLT, PVN, CEA, and RVLM.

AT2R are also expressed throughout the brain, and have previously been reported in regions like the thalamus, superior colliculus, inferior olive, locus coerelious, and medial amygdala (Gehlert et al., 1991; Rowe et al., 1992). In fact, the expression of

AT2R in sensory processing regions like the thalamus provided some of the first evidence that Ang II receptors have unidentified neuromodulatory functions (Gehlert et al., 1991).

The recent development of an AT2R-eGFP reporter mouse has allowed for better localization and characterization within the rodent brain, and confirmed that these receptors are positioned to modulate blood pressure and stress responses (de Kloet et al.,

2016a). In the brain, AT2R can suppress neuronal activity by facilitating K+ channel

current and decreasing firing rate (Gao et al., 2011). Activation of AT2R can also increase nitric oxide (NO) production and intracellular cyclic 3,5-guanosine monophosphate (cGMP).

Anxiety and the Brain Renin Angiotensin System

A potential link between the RAS and anxiety disorders emerged following early observations that treatment with the ACE inhibitor captopril could decrease depressive symptoms and improve mood status (Zubenko and Nixon, 1984). Notably, these effects were independent of blood pressure because they were not caused by other antihypertensive medications such as a-methyldopa and prazosin. Similar results have been obtained in pre-clinical studies of RAS blockade (Costall et al., 1990).

Administration of the angiotensin receptor blocker (ARB) candesartan has also been shown to decrease anxiety of non-stressed rats in the elevated plus maze (Pavel et al.,

2008). Efforts to localize the brain regions responsible for these effects have found that genetic knockout of the AT1R from the PVN reduces anxiety-like behavior and increases the time spent in the open arms of an elevated plus maze (Wang et al., 2016).

Furthermore, the intraamygdaloid injection of losartan has been shown to have anxiolytic effects in both stressed and non-stressed animals (Lopez et al., 2012). Collectively, it appears that Ang II acts to increase anxiety via centrally located AT1R (Saavedra et al.,

2005). Ang II may exacerbate stress and anxiety directly by increasing SNS activity, however emerging evidence reveals a more complex relationship between RAS and anxiety; one in which RAS activity directly influences cognitive function in brain regions that control fear and anxiety.

Angiotensin II in Learning and Memory

Ang II has been repeatedly shown to affect learning processes, however efforts to determine its exact role have generated conflicting results. Most studies support the proposal that Ang II acts within the brain to reduce cognitive function and impair learning and memory, however there is some evidence that Ang II may in fact enhance learning and memory under certain conditions. Early studies using a conditioned avoidance model in rats found that intracerebroventricular (ICV) injection of Ang II before or after training can facilitate learning and memory and improve memory retention

(Braszko, 2005; Georgiev and Yonkov, 1985). Importantly, ICV Ang II was shown to be anxiogenic and may therefore non-specifically affect outcomes in memory tasks by altering anxiety-like behavior. Peripheral injection of Ang II was also shown to increase re-entry latencies in passive avoidance tests (Braszko and Wiśniewski, 1988). Others have found that ICV Ang II can improve object recognition, an effect that can be blocked by pre-treatment with the AT1R antagonist losartan (Denny et al., 1991; Kułakowska et al., 1996).

The majority of studies, however, suggest that Ang II can impair cognition (Gard,

2002). This theory is supported by findings that Ang II inhibits long-term potentiation in the hippocampus (Denny et al., 1991) and the amygdala (von Bohlen und Halbach and

Albrecht, 1998) of rats. Direct infusion of Ang II into the CA1 region of the hippocampus blocks memory consolidation leading to dose-dependent amnesia in an inhibitory avoidance memory retention test (Kerr et al., 2005). Furthermore, intra- hippocampal Ang II in the avoidance model appears to affect memory specifically, as it

does not alter locomotor activity, exploratory behavior, or anxiety state (Bonini et al.,

2006).

An inhibitory effect of endogenous Ang II on cognitive processes is also supported by studies showing that blockade of Ang II signaling pathways can enhance learning and memory. For example, intraperitoneal (i.p.) injection of losartan facilitates learning in elevated-maze and passive avoidance tasks in mice (Raghavendra et al.,

1998). More recent studies have found that ICV administration of ACE inhibitors, in addition to AT1R antagonists, results in significant enhancements of both short and long- term memory while ICV administration of Ang II leads to memory impairment (Bild et al., 2013). The demonstrated roles of Ang II in regions like the amygdala and hippocampus, and the fact that the effects of Ang II antagonism have for the most part been observed using memory tasks with aversive properties (e.g. passive avoidance, conditioned avoidance, fear conditioning), are consistent with the theory that the brain

RAS plays a functional role in fear learning and memory.

Emotion and Memory

As impressive as our brains may be, they are not capable of remembering every single moment of every single day. Memories are selectively retained, with more weight given to some than others. Evolution has ingrained in many species the ability to quickly associate sensory stimuli with environmental threats, resulting in persistent memories that aid in survival. One process through which this occurs is by the formation of emotional responses.

Memories associated with strong emotions are more readily preserved and longer lasting, owing to neurobiological processes common to both emotion and memory.

Emotional arousal leads to the release of stress hormones, such as cortisol (corticosterone in mice) and epinephrine, that can modulate the consolidation and extinction of long-term memories (McGaugh, 2013). Under conditions of stress, the SNS enters an active state, leading to a brain-wide increase in norepinephrine mediated by the locus coeruleus

(Henckens et al., 2009; Valentino and Van Bockstaele, 2008) which projects to numerous areas responsible for memory processing, such as the amygdala, hippocampus, and prefrontal cortex (Devoto et al., 2005; Petrov et al., 1993).

The effect of catecholamines on memory is demonstrated by an enhancement of the consolidation of novel information following administration of epinephrine or norepinephrine (Gold and Van Buskirk, 1975). Circulating epinephrine acts on vagal afferents that terminate in the NTS (Roozendaal et al., 2006). Neurons project directly from the NTS to the amygdala; which modulates the acquisition and consolidation of emotional memories (Goldstein, 1992; Sah et al., 2003). Glucocorticoid hormones also enhance memory consolidation by acting presynaptically on noradrenergic brainstem cells that project to the basolateral amygdala, and postsynaptically by increasing the efficacy of the B-adrenoceptor-cyclic AMP/protein kinase A system (Roozendaal et al.,

2006). When given either before (Buchanan and Lovallo, 2001) or after (Flood et al.,

1978; Sandi and Rose, 1997) training, glucocorticoids can enhance memory consolidation. Blocking the production of glucocorticoids has the opposite effect, impairing memory consolidation and preventing the memory enhancement caused by epinephrine (Liu et al., 1999; Roozendaall et al., 1996). The ability of stress hormones to

modulate memory strength suggests that certain types of memories, especially those formed following stressful events, are more important for us to remember than others.

Fear Memory

Among the most powerful of emotions, fear is generally perceived as negative despite its crucial role in our survival. The formation and maintenance of fear memories is a complex process requiring the interaction of environmental, neurobiological, and physiological systems (Fig. 1-2). Our ability to rapidly associate potential threats with predictive cues is critical for us to learn from previous experiences and avoid danger in the future.

It should be noted that the term “fear” used in this dissertation and in most of the pre-clinical literature generally refers to the mental state responsible for the behavioral or physiological alterations in animals after aversive conditioning. This nonconscious process is not identical to the conscious state of fear that we experience as humans.

While it is unlikely that rats and mice experience fear in the same way as us, the study of fear in other species has helped inform our understanding of more complex emotional states in humans. Due to the common use of the word fear to describe observations in both rodents and humans, this distinction is important. For a detailed discussion on the semantics of fear terminology, see (LeDoux, 2017).

Fear memory, which is one of the most widely studied forms of memory, has many attributes that make it a useful model for investigation. First, the acquisition of fear memory is an adaptive process that can directly influence the probability of an organism’s survival. Memories formed under conditions of fear are formed rapidly and

last a very long time (Bouton and Bolles, 1979). Second, fear elicits strong responses.

The detection of a potential threat leads to rapid physiological and behavioral changes that prepare the animal for a potentially dangerous encounter. These characteristics make fear memories amenable to the investigation of specific brain regions and signaling pathways required for memory formation and expression.

Another advantage of fear memory models is that the underlying neural circuitry is highly conserved across species, making it possible to translate findings from other species to humans. The key brain regions required for fear memory processing (e.g. amygdala, hippocampus, BNST) perform similar functions in mice and humans (Flores et al., 2018). Identification of fear circuits is made possible by the fact that fear responses are accompanied by predictable physiological and behavioral responses that can be observed and quantified. Such responses include increased release of stress hormones, elevated cardiovascular activity, autonomic nervous system activation, freezing or escape behavior, and attenuation of motivated behavior. Animal modeling of fear phenotypes using Pavlovian conditioning has led to the identification of the circuitry and neurotransmitter systems required for both normal and aberrant fear (Fanselow and

Wassum, 2016).

Pavlovian Fear Conditioning

While it is of great interest to understand how an animal learns, thinks, and remembers, these internal processes are not readily observable. In the early 1900’s, Ivan

Pavlov found that dogs would exhibit a salivary response to previously neutral stimuli

(e.g. metronome or bell) if they were conditioned to perceive the stimulus as a predictor

of food (Todes, 1997). This form of classical conditioning has become one of the most widely studied phenomena in psychology because it demonstrated that associative memories can be paired with predictable physiological or behavioral responses. In other words, the relationship between the mental process of anticipation and the physiological process of preparation allows for the training of an animal to elicit certain measurable outcomes (e.g. freezing behavior, salivation, changes in HR, changes in BP, corticosterone release, etc). The conditioned response can therefore be visually or mechanically quantified and used as an index of the strength of the associative memory.

These early observations laid the groundwork for over a century of research into the discrete neurological and biological processes underlying memory and behavior.

Fear conditioning is a type of associative learning in which a neutral conditioned stimulus (CS) is paired with an aversive unconditioned stimulus (US). Prior to conditioning, the CS would elicit no change in behavior or stress, where the US would cause an unconditioned response such as recoiling or escaping. After a few brief pairings, a “fear” memory is formed, which leads the animal to exhibit a defensive response when it is exposed to the conditioned stimulus alone. Through aversive association, the previously harmless stimulus can come to activate neural defensive circuits and the reflexive autonomic and somatic outputs required for defensive behavior

(Lang et al., 2000). Compared to other behavioral designs such as inhibitory avoidance,

Pavlovian conditioning allows for more accurate parsing of memory phases (Giustino et al., 2016).

Auditory fear conditioning is the process of training an animal to react to a discrete auditory cue. In order to minimalize generalization of fear, the animal is first

acclimated to the fear conditioning environment during a habituation phase. After habituation, the animal is fear conditioned, and an auditory cue is presented in combination with an aversive cue such as a footshock. This pairing can be repeated any number of times, depending on the requirements of the experiment. After fear conditioning, when the animal is presented with the auditory cue, it will respond as though it expects the US to follow. Testing of the conditioned response to an auditory cue is typically done in a novel context that the animal does not associate with the footshock. The natural tendency of the animal to explore the new environment results in a very noticeable difference between levels of freezing, which is the arrest of somatomotor activity that occurs when an animal expresses fear, before or during CS presentation.

Contextual fear conditioning occurs when an animal is exposed to a novel environment or context in which it is exposed to an aversive stimulus. In this model, the context itself becomes the CS. After conditioning, returning the animal to the conditioning context evokes defensive responses such as freezing behavior. Some level of contextual conditioning always occurs during an aversive conditioning experiment, depending on the salience and predictive value of the contextual cues and whether or not more consistent and identifiable cues are present.

Circuitry of Fear Conditioning

One of the key structures responsible for fear learning is the amygdala. The amygdala is an almond-shaped cluster of heterogeneous nuclei located within the temporal lobe that is critical for both auditory and contextual fear conditioning (Gale,

2004). Anatomical divisions of the amygdala that directly control fear expression and learning include the basolateral amygdala (BLA) which consists of the lateral (LA) basal

(BA) and basomedial (BMA) nuclei (Xu et al., 2016), and the central amygdala (CeA) which is described in detail below. Lesioning of the lateral and central nuclei of the amygdala prior to fear conditioning attenuate freezing to both auditory and contextual stimuli, suggesting that the pathways that regulate auditory and contextual fear learning overlap within the amygdala, and are therefore not anatomically dissociable at the level of amygdala nuclei (Goosens and Maren, 2001). During fear conditioning, information about the CS converges with information about the US to alter synaptic plasticity in the lateral amygdala. Subsequent exposure to the CS activates these potentiated synapses to initiate conditioned fear responses.

If the CS is an auditory cue, information is transmitted from the medial geniculate nucleus of the auditory thalamus to the lateral nucleus (LA), which is the main input site of sensory information in the amygdala (Doron and Ledoux, 2000). These pathways arrive at the LA either directly from the auditory thalamus (LeDoux et al., 1985), or indirectly via the auditory cortex (Romanski et al., 1993) (Fig. 1-3). Contextual fear is similarly mediated by the amygdala, however it also requires the dorsal hippocampus in order to consolidate and store representations of the environment (Fanselow, 2000).

The LA communicates with another region of the amygdala called the central nucleus (CeA), which is a specialized autonomic-projecting motor region (Petrovich and

Swanson, 1997). This striatum-like nucleus, consisting mainly of GABAergic medium spiny neurons, is considered the major output station of the amygdala (Duvarci et al.,

2011). There are two major divisions of the CeA consisting of distinct cell populations:

the lateral central nucleus of the amygdala (CeL), and the medial central nucleus of the amygdala (CeM). The CeL receives input from the BLA, and contains GABAergic neurons that project to the CeM (Cassell et al., 1999; Penzo et al., 2014). Of the many neuronal subpopulations in the CeL, two in particular play important, opposing roles in the expression of fear. Cells that express protein kinase C delta (PKC-훿+) are called

CeLoff neurons because they are inhibited in response to a CS following conditioning

(Janak and Tye, 2015). PKC-훿- neurons, on the other hand, are referred to as CeLon neurons and exhibit increased spiking upon CS presentation. Conditioned fear responses occur through a disinhibitory circuit where CeLon neurons inhibit CeLoff neurons projecting to CeM output neurons (Ciocchi et al., 2010; Haubensak et al., 2010). These

CeL neurons are thought to contribute to the network that stores aversive memory traces

(Li, 2019).

The CeM mediates many of the autonomic and behavioral responses to stress by projecting to regions in the lateral hypothalamus (Krettek and Price, 1978), the midbrain

(LeDoux et al., 1988), and the brainstem (Jongen-Rêlo and Amaral, 1998). Sympathetic responses to conditioned fear are mediated by amygdala projections to the lateral hypothalamus, while parasympathetic responses are mediated by projections to medullary structures critical to cardiovascular regulation, such as the NTS and DVN (Schwaber et al., 1982). Evidence from pharmacological (Bandler et al., 1985; Tomaz et al., 1988;

Zhang et al., 1990) and lesion studies (Hopkins and Holstege, 1978; Kim et al., 1993;

LeDoux et al., 1988) suggests that freezing behavior is controlled by projections from the

CeA to the periaqueductal grey region (PAG). In addition to freezing behavior, the periaqueductal grey also mediates flight and conditioned analgesic responses

(Anagnostaras et al., 2014), which are thought to offset some of the potentially painful effects of the threat (Fanselow, 1986). The exact mechanisms responsible for PAG- mediated defensive behaviors remain poorly understood, however recent optogenetic and viral tracing studies suggest that projections from the CeA mediate freezing by targeting

GABAergic interneurons in the ventrolateral periaqueductal grey (vlPAG). The CeA to vlPAG projection disinhibits glutamatergic neurons in the vlPAG that project to premotor targets in the medulla (Tovote et al., 2016). Although inactivation of the CeM prevents the expression of freezing behavior, it does not affect fear memory formation, suggesting that this region of the CeA plays a limited role in aversive memory (Ciocchi et al., 2010).

Fear-Conditioned Physiological Responses

Even before Walter Bradford Cannon coined the term “fight or flight” in the early twentieth century it was apparent that many organisms required the ability to rapidly adjust cardiovascular function to accommodate the physical demands of an ever-changing environment (Cannon, 1916). This requirement is conserved across a broad spectrum of species, indicating strong evolutionary pressure and allowing for the investigation of these responses in a wide variety of test subjects. The neural circuitry of fear processing areas in the brain is also highly conserved, particularly between rodents and humans

(Anderson and Adolphs, 2014).

Adaptations to fear are most commonly assessed by changes in behavior, which are rapid, robust, and relatively easy to observe and quantify (Mahan and Ressler, 2012).

Freezing behavior, for example, is the cessation of all movement aside from that caused

by respiration. This behavior is used to operationally define fear in Pavlovian models of fear conditioning. Freezing is a centrally controlled, coordinated, and complex defensive response that is accompanied by significant physiological adjustments. Although they are easily observed and well-characterized, purely behavioral readouts of fear responses provide little information about the internal changes taking place within an organism.

There is a compelling need for additional measures that reliably indicate the emotional state of animals (Lee et al., 2001; Stiedl et al., 2004). One way to address this is by either directly or indirectly monitoring alterations in autonomic activity, which reflect physiological adjustments to changes in emotional state.

Activation of the autonomic nervous system occurs during CS presentation, resulting in increases of both parasympathetic and sympathetic activity that modify heart rate (Nijsen et al., 1998) and lead to a net increase in blood pressure. Increased heart rate and blood pressure are common responses to fear conditioned stimuli in rabbits, rats, mice, and humans (Burhans et al., 2015; Stiedl and Hager, 2017). In freely moving rats, the pairing of an auditory stimulus with a footshock leads to CS-induced changes in blood pressure and heart rate (LeDoux et al., 1984). Robust conditioned cardiovascular responses are also observed in mice, where even a single CS-US pairing leads to increases in heart rate (Stiedl, 1999; Tovote et al., 2004) and blood pressure (Tovote et al., 2005) when animals are exposed to the CS 24hr later. CeA projections to the dorsal motor nucleus of the vagus contribute to conditioned heart rate responses (Davis, 1992;

Schwaber et al., 1982; Young and Leaton, 1994), while conditioned blood pressure responses are controlled in part by projections to the hypothalamus coming from the

BNST and CeA (Crestani et al., 2013; Keifer et al., 2015a; Resstel et al., 2008).

Although conditioned cardiovascular responses have been successfully used to examine the acquisition of fear, relatively few studies have attempted use them as markers of fear inhibition or extinction learning.

Extinction of Fear Memory

Fear extinction is the reduction of conditioned responses that occurs follows exposure to repeated CS presentations in the absence of negative reinforcement.

Extinction mechanisms are one of the main targets of current psychological treatments for PTSD, which aim to help patients stop experiencing fear in response to traumatic memories by re-exposing them to traumatic stimuli in a safe environment. Importantly, after extinction training the threat expectancy does not disappear, but is instead inhibited

(Bouton, 1993; Pavlov (1927), 2010). Fear learning-induced plasticity in the amygdala remains intact even when the behavioral responses are completely extinguished. In other words, extinction training does not alter the original associative fear memory, but instead consists of a new and distinct learning process (Quirk and Mueller, 2008).

Under normal conditions, extinction learning does not directly modify the original fear memory. Instead, it leads to the formation of a new inhibitory “safety” memory that competes with the initial memory. There are many experimental studies that demonstrate this important distinction, and which identify at least three circumstances in which the original memory may re-emerge after extinction: 1) spontaneous recovery, 2) renewal, and 3) reinstatement. Spontaneous recovery describes the return of conditioned responses after an extended period of time (Robbins, 1990). Renewal occurs when an animal is exposed to the conditioned stimulus in a context different from the one that it

was extinguished in (Bouton and King, 1983). Reinstatement is observed when an animal is re-exposed to the unconditioned stimulus after extinction training (Bouton and

Bolles, 1979; Rescorla and Heth, 1975). Each of these situations can lead to a re- emergence of conditioned fear after extinction training and taken together demonstrate that extinction training leaves the initial fear association intact. Furthermore, these findings reveal the temporary and relatively weak nature of extinction training relative to easily obtained and long-lasting fear memories. These properties of extinction learning might be overcome by pharmaceutical interventions that augment the extinction process and prevent the re-emergence of fear. Such interventions are highly desirable in the clinical setting for the treatment of PTSD.

Extinction learning and expression is supported by intrinsic networks within the amygdala. Inactivation of the BA impairs extinction (Herry et al., 2008), and unit recording studies have revealed different types of neurons within the BA that control the maintenance and extinction of fear (Amano et al., 2011). Rapid switching between these fear and extinction neurons is controlled in part by inhibitory circuits within the BA, and associated with an overall increase of GABAergic inhibition in this region (Duvarci and

Pare, 2014). Extinction is also regulated by intercalated cells (ICM), which prevent the activation of CeM neurons by the BA (Likhtik et al., 2008).

The ventral-medial pre-frontal cortex, which contains the infralimbic and prelimibic cortical regions, is required for extinction (Milad and Quirk, 2002; Morgan et al., 1993; Quirk et al., 2000). A role for the infralimbic cortex in extinction is supported by the findings that low freezing responses to CS exposure are associated with increased firing of infralimbic neurons, and that electrical stimulation of infralimbic neurons during

CS exposure in non-extinguished animals reduces freezing behavior. These findings demonstrate that activity in the infralimbic cortex reduces fear during re-exposure to conditioned stimuli, implicating this region in extinction recall. Importantly, infralimbic neurons are responsive to the CS only after extinction training, and are not activated by the CS during extinction training, suggesting that they are responsible specifically for long-term extinction memory (Milad and Quirk, 2002). The infralimbic cortex likely influences freezing behavior via direct projections to the capsular region of the central amygdala (McDonald et al., 1996) where the activation of ICM can in turn inhibit CeA output.

The hippocampus also plays a critical role in extinction learning and memory.

The BLA is able to process contextual information due to reciprocal connections with the hippocampus (Sotres-Bayon et al., 2006), and inactivation of the dorsal hippocampus prior to the retrieval of extinguished fear memories can disrupt the context-dependent expression of those memories (Corcoran and Maren, 2001). Furthermore, infusion of the

GABA agonist muscimol into the dorsal hippocampus before extinction training can disrupt the contextual encoding of extinction memory, leading to renewal of fear even when tested in the extinction-training context (Corcoran, 2005). Taken together, these findings point to significant involvement of the hippocampus in fear acquisition and extinction processes.

A Role for the Renin Angiotensin System in PTSD

Impaired extinction has been implicated in human anxiety disorders including

PTSD (Krabbe et al., 2018; Milad and Quirk, 2012). Patients with PTSD exhibit heightened skin conductance responses and amygdala activity following extinction training, suggesting a deficiency in the maintenance of extinction memory (Wicking et al., 2016). Extinction deficits in PTSD patients could be due in part to an inability to properly use contextual cues to modulate fear response (Garfinkel et al., 2014).

Dysregulated hippocampal function may prevent the proper generalization of extinction learning, allowing novel contexts to more easily elicit the return of fear (Wicking et al.,

2016).

Given that extinction impairments are observed in PTSD patients, and that cognitive behavioral therapies used to treat PTSD rely heavily upon extinction mechanisms, the pharmacological enhancement of this process could potentially improve current treatment strategies. A number of pre-clinical studies suggest that certain compounds may facilitate extinction learning and retention. Approaches have targeted both GABAergic (Castellano and McGaugh, 1989; McGaugh et al., 1990) and glutamatergic (Ledgerwood et al., 2003; Walker et al., 2002) neurotransmission with some success, however few of these compounds have been tested for clinical utility.

Recently, drugs that target the RAS have been identified as potential modulators of extinction. In an analysis of data from a large cross-sectional study on the genetic and environmental factors that contribute to PTSD, it was found that hypertensive medications targeting the RAS pathway were associated with reduced PTSD symptom severity (Khoury et al., 2012a). Pre-clinical studies in mice have shown that shown that

both systemic losartan (Marvar et al., 2014) and central deletion of AT1R of corticotropin-releasing factor neurons (Hurt et al., 2015) lead to a reduction in conditioned fear. Importantly, the extinction-enhancing effects of AT1R blockade are observed in both males and females (Parrish et al., 2019); an important fact given that females are much more likely than males to meet diagnostic criteria for PTSD (Ramikie and Ressler, 2018). Two recent clinical studies suggest that losartan may enhance the effects of exposure therapy by regulating emotional processing (Pulcu et al., 2019a;

Reinecke et al., 2018). While most of this work suggests that the AT1R modulates extinction learning, the neurobiological mechanisms are not clear.

Memory Reconsolidation

Enduring memories are not formed instantaneously. Instead, newly acquired information persists in a fragile state and consolidates over a period of time following the initial learning or training session (McGaugh, 2000). The perseveration-consolidation hypothesis of memory, developed by Muller and Pilzecker over 100 years ago (Dudai,

2004; Müller and Pilzecker, 1900) proposed that memory takes time to fixate. This theory was based on observations that new stimuli presented shortly after training could impair recall, and would help explain clinical observations of retrograde amnesia

(Burnham, 1903). Since the theory of consolidation became the dominant view of memory formation, the general consensus has been that consolidated memories are unchangeable. Recent findings, however, reveal that the life of a memory is more dynamic than previously believed.

Memory retrieval can initiate two distinct mnemonic processes: extinction and reconsolidation. While the concept of extinction has been around since Pavlov’s studies in the early 1900’s, the stability of consolidated memories was not challenged until the

1960’s, when it was shown that electroconvulsive shocks given after memory retrieval could lead to specific memory impairments at a later time (Misanin et al., 1968). The amnesic effect caused by post-retrieval shock was similar to the amnesia observed when the shock was given immediately after fear conditioning, suggesting that a process similar to consolidation was occurring again after the memory was recalled. A controversial idea eventually emerged, suggesting that the reactivation of a memory renders it temporarily unstable, at which point it can be manipulated (Lewis, 1979). This idea was met with some skepticism, but experienced a resurgence around the turn of the century when Nader and colleagues showed that they could disrupt memories by injecting the protein synthesis inhibitor anisomycin into the BLA shortly after retrieval of a conditioned auditory fear memory (Nader et al., 2000). These studies not only revitalized interest in the reconsolidation theory, but they demonstrated that this process was dependent on de novo protein synthesis – the first neurobiological evidence of the cellular processes shared between consolidation and reconsolidation.

Perhaps one of the most exciting aspects of the reconsolidation theory is its potential to aid in the treatment of fear disorders. The effects of disrupting reconsolidation are long lasting, suggesting that it may be possible to permanently weaken specific memories associated with traumatic events. To date, the beta-blocker propranolol has shown the most promise and has yielded positive results in both rats

(Dębiec and Ledoux, 2004; Dębiec et al., 2011; Villain et al., 2016) and humans (Kindt et

al., 2009). Other reconsolidation inhibitors have also been identified in animal studies, including oxytocin (Hou et al., 2015a), and xenon (Meloni et al., 2014).

The amygdala and the hippocampus are the two primary neural structures known to contribute to the reconsolidation phase. Post-retrieval inactivation of the BLA with tetrodotoxin or lidocaine, which inhibit the propagation of action potentials by blocking voltage-dependent sodium channels, can impair freezing responses to both auditory and contextual fear conditioned stimuli (Baldi et al., 2008; Sacchetti et al., 2007). Glutamate acts in the BLA to influence reconsolidation through NMDA and AMPA ionotropic receptors. When NMDA receptors are blocked before retrieval, reconsolidation is inhibited (Mamou et al., 2006). On the other hand, activation of these receptors with D- cycloserine (DCS) has the opposite effect, strengthening the fear memory (Lee et al.,

2006). The role of AMPA receptors in reconsolidation is less clear. While blockade of this receptor does not directly affect memory reconsolidation (Milton et al., 2013) there is some evidence that a shift from calcium impermeable AMPA receptors to calcium permeable AMPA receptors occurs, which may contribute to reconsolidation in the BLA

(Hong et al., 2013).

Genetic Markers of Reconsolidation

Much like newly formed memories, the reconsolidation of retrieved memories is a protein synthesis-dependent process that initiates similar molecular mechanisms. For example, retrieval of auditory conditioned fear has been shown to increase expression of the activity-dependent inducible immediate early gene (IEG) zif268 in the BLA. zif268 encodes a zinc finger transcription factor of the Egr family; a group of proteins that

modulate synaptic plasticity, memory consolidation, and the induction of long-term potentiation (LTP) (Cole et al., 1989). Mice carrying a zif268 null mutation exhibit long- term deficits to recalled recognition memories (Bozon et al., 2003). Expression of the

CREB-regulated IEG c-Fos is also increased in the BLA when fear memories are retrieved 6hr and 24hr after conditioning (Do-Monte et al., 2015; Hall et al., 2001). IEGs like zif268 and c-Fos are routinely used as indirect measures of neuronal activity because they encode transcription factors that control the expression of downstream target genes and regulate neuronal physiology (Gallo et al., 2018). In addition to these well-known genes, other transcriptional pathways invoked by reconsolidation are beginning to gain attention. One example of this would be activity-regulated cytoskeletal-associated protein (Arc/Arg3.1 ), which is observed in the amygdala following auditory fear memory retrieval (Maddox and Schafe, 2011).

Additionally, the neuronal activity-dependent Neuronal PAS domain protein 4

(Npas4) is an IEG that has recently been implicated in the transcriptional regulation of genes controlling synaptic plasticity and inhibitory synapse development. Both the mRNA and protein product of Npas4 are regulated within the amygdala in a learning- dependent manner, and required for newly acquired and reactivated memories (Ploski et al., 2011). While the expression of these IEGs has been demonstrated in various models of memory retrieval, much less is known about the specific downstream transcriptional targets required for reconsolidation. However, our current state of understanding suggests that the reconsolidation of memory brings about a consolidation-like process

(Alberini and LeDoux, 2013), albeit one with distinct temporal and regional characteristics.

Summary and Specific Aims

Over the last century, substantial progress has been made in describing the mechanisms underlying emotionally charged memories and how dysregulation of these memories can lead to debilitating psychiatric conditions and potentially dangerous alterations in cardiovascular function. However, little is known about how behavioral and pharmacological treatments might alter cardiovascular responses to fear memory recall. Furthermore, the role of Ang II and its receptors in fear learning processes is not clear. In this dissertation, I will describe the effects of pharmacologically targeting RAS receptors on both behavioral and cardiovascular components of the defensive response associated with learned fear. I approached this goal through two specific aims:

Aim 1:

A small number of early studies have laid the groundwork in determining how conditioned fear leads to cardiovascular adjustments in humans and rodents. However, little is known about how these responses change following interventions that modify the behavioral expression of those previously conditioned fear memories. In the first aim, I sought to determine whether Ang II receptors are required for the maintenance of fear memory and the conditioned cardiovascular response to a fear state. Given the fact that pharmacologically targeting Ang II receptors can directly affect cardiovascular function, my first objective was to develop a memory-testing paradigm in which I could separate behavioral responses from the physiological alterations that occur during fear recall. To this end, I used radiotelemetry to monitor cardiovascular parameters in combination with

a modified protocol for extinction learning, which is thought to model the fear reduction processes underlying exposure therapy in humans.

Previous work has identified the AT1R as a potential target for the enhancement of fear extinction (Hurt et al., 2015; Marvar et al., 2014; Parrish et al., 2019; Zhou et al.,

2019). However, the function of the Ang II receptor subtype 2 (AT2R) in similar fear learning tasks is unknown. The second objective was therefore to determine whether brain AT2R plays a similar role in fear memory and expression to its counterpart the

AT1R. This was accomplished by pharmacologically activating AT2R within the CeA in a mouse model of Pavlovian conditioning. The distribution and connectivity of CeA cells expressing AT2R were then determined through a combination of immunohistochemistry and retrograde labeling in an AT2R-eGFP reporter mouse strain.

Aim 2:

In addition to extinction learning, another approach to treating aberrant memory processes has interested researchers and clinicians. It has been known for more than a century that new memories must be consolidated in order to become long-term memories

(McGaugh, 2000). It was also believed that after the period of consolidation, memories become persistent and cannot be disrupted. This dogma has been challenged by recent findings showing that the retrieval or activation of a memory can render it temporarily labile, so that it may be updated with new information (Sara, 2000). Water deprivation models in multiple species (Frenkel et al., 2005; Sierra et al., 2013) suggest that endogenous Ang II contributes to memory reconsolidation via the Ang II type 1 receptor

(AT1R). The second aim of this dissertation was to determine whether blockade of AT1R

during the window of reconsolidation would reduce fear expression to conditioned auditory stimuli. This was accomplished using the ARB losartan, which modulates fear learning in mice (Marvar et al., 2014) and humans (Pulcu et al., 2019a; Reinecke et al.,

2018), in combination with behavioral and cardiovascular measures of fear.

Figure 1-1: Blood pressure regulation through the renin-angiotensin system cascade. Decreases in blood pressure lead to increased secretion of renin by the kidney. Renin cleaves angiotensinogen into Ang I. Pulmonary ACE converts Ang I to Ang II. Ang II stimulates vasoconstriction, salt and water retention, aldosterone release, and increases sympathetic nervous system activity. Adapted from (Bernardi et al., 2016).

Figure 1-2: Systems involved in fear memory formation. Fear memories are formed and maintained through the interaction of various environmental, physiological, and neurobiological systems. Adapted from (Maddox et al., 2019).

Figure 1-3: The neural circuits underlying fear conditioning. Information about the conditioned stimulus (CS) is conveyed from the auditory thalamus and auditory cortex to the lateral nucleus of the amygdala (LA), where it converges with information about the unconditioned stimulus (US) coming from the somatosensory thalamus and cortex. The LA is directly and indirectly connected to the central amygdala (CE), which controls the expression of freezing behavior, blood pressure and heart rate, and hormonal responses, via projections to the central gray (CG), the lateral hypothalamus (LH), and the paraventricular hypothalamus (PVN), respectively. Adapted from (Phelps and LeDoux, 2005).

References:

Abdel-Rahman, E.M., Unger, T., and Schölkens, B.A. (2004). Angiotensin (Berlin ; Springer).

Alberini, C.M., and LeDoux, J.E. (2013). Memory reconsolidation. Current Biology 23, R746–R750.

Allen, A.M., Zhuo, J., and Mendelsohn, F.A. (1999). Localization of angiotensin AT1 and AT2 receptors. J. Am. Soc. Nephrol. 10 Suppl 11, S23-29.

Amano, T., Duvarci, S., Popa, D., and Paré, D. (2011). The fear circuit revisited: contributions of the basal amygdala nuclei to conditioned fear. J. Neurosci. 31, 15481– 15489.

American Psychiatric Association (2013). Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association).

Anagnostaras, S.G., Sage, J.R., and Carmack, S.A. (2014). Pavlovian Fear Conditioning. In Encyclopedia of Psychopharmacology, I.P. Stolerman, and L.H. Price, eds. (Berlin, Heidelberg: Springer Berlin Heidelberg), pp. 1–4.

Anderson, D.J., and Adolphs, R. (2014). A Framework for Studying Emotions across Species. Cell 157, 187–200.

Badoer, E. (2001). Hypothalamic paraventricular nucleus and cardiovascular regulation. Clin Exp Pharmacol Physiol 28, 95–99.

Bains, J.S., and Ferguson, A.V. (1995). Paraventricular nucleus neurons projecting to the spinal cord receive excitatory input from the subfornical organ. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 268, R625–R633.

Baldi, E., Mariottini, C., and Bucherelli, C. (2008). Differential roles of the basolateral amygdala and nucleus basalis magnocellularis during post-reactivation contextual fear conditioning reconsolidation in rats. Neurobiology of Learning and Memory 90, 604– 609.

Bandler, R., Depaulis, A., and Vergnes, M. (1985). Indentification of midbrain neurones mediating defensive behaviour in the rat by microinjections of excitatory amino acids. Behavioural Brain Research 15, 107–119.

Barnes, J.M., Steward, L.J., Barber, P.C., and Barnes, N.M. (1993). Identification and characterisation of angiotensin II receptor subtypes in human brain. Eur. J. Pharmacol. 230, 251–258.

Bennion, D.M., Jones, C.H., Dang, A.N., Isenberg, J., Graham, J.T., Lindblad, L., Domenig, O., Waters, M.F., Poglitsch, M., Sumners, C., et al. (2018). Protective effects

of the angiotensin II AT2 receptor agonist compound 21 in ischemic stroke: a nose-to- brain delivery approach. Clinical Science 132, 581–593.

Beristianos, M.H., Yaffe, K., Cohen, B., and Byers, A.L. (2016). PTSD and Risk of Incident Cardiovascular Disease in Aging Veterans. The American Journal of Geriatric Psychiatry 24, 192–200.

Bernardi, S., Michelli, A., Zuolo, G., Candido, R., and Fabris, B. (2016). Update on RAAS Modulation for the Treatment of Diabetic Cardiovascular Disease. J Diabetes Res 2016.

Bickerton, R.K., and Buckley, J.P. (1961). Evidence for a Central Mechanism in Angiotensin Induced Hypertension. Proceedings of the Society for Experimental Biology and Medicine 106, 834–836.

Bild, W., Hritcu, L., Stefanescu, C., and Ciobica, A. (2013). Inhibition of central angiotensin II enhances memory function and reduces oxidative stress status in rat hippocampus. Progress in Neuro-Psychopharmacology and Biological Psychiatry 43, 79– 88. von Bohlen und Halbach, O., and Albrecht, D. (1998). Mapping of angiotensin AT1 receptors in the rat limbic system. Regulatory Peptides 78, 51–56.

Bonini, J.S., Bevilaqua, L.R., Zinn, C.G., Kerr, D.S., Medina, J.H., Izquierdo, I., and Cammarota, M. (2006). Angiotensin II disrupts inhibitory avoidance memory retrieval. Hormones and Behavior 50, 308–313.

Boscarino, J.A. (2008). A Prospective Study of PTSD and Early-Age Heart Disease Mortality Among Vietnam Veterans: Implications for Surveillance and Prevention. Psychosom Med 70, 668–676.

Boscarino, J.A., and Chang, J. (1999). Electrocardiogram abnormalities among men with stress-related psychiatric disorders: Implications for coronary heart disease and clinical research. Ann. Behav. Med. 21, 227–234.

Bosnyak, S., Welungoda, I., Hallberg, A., Alterman, M., Widdop, R., and Jones, E. (2010). Stimulation of angiotensin AT2 receptors by the non-peptide agonist, Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats. British Journal of Pharmacology 159, 709–716.

Bouton, M.E. (1993). Context, time, and memory retrieval in the interference paradigms of Pavlovian learning. Psychological Bulletin 114, 80–99.

Bouton, M.E., and Bolles, R.C. (1979). Role of conditioned contextual stimuli in reinstatement of extinguished fear. Journal of Experimental Psychology: Animal Behavior Processes 5, 368–378.

Bouton, M.E., and King, D.A. (1983). Contextual control of the extinction of conditioned fear: Tests for the associative value of the context. Journal of Experimental Psychology: Animal Behavior Processes 9, 248–265.

Bowers, M.E., and Ressler, K.J. (2015). An overview of translationally informed treatments for PTSD: animal models of Pavlovian fear conditioning to human clinical trials. Biol Psychiatry 78, E15–E27.

Bozon, B., Davis, S., and Laroche, S. (2003). A Requirement for the Immediate Early Gene zif268 in Reconsolidation of Recognition Memory after Retrieval. Neuron 40, 695– 701.

Braszko, J.J. (2005). Valsartan abolishes most of the memory-improving effects of intracerebroventricular angiotensin II in rats. Clin. Exp. Hypertens. 27, 635–649.

Braszko, J.J., and Wiśniewski, K. (1988). Effect of angiotensin II and saralasin on motor activity and the passive avoidance behavior of rats. Peptides 9, 475–479.

Braun-Menendez, E., and Page, I.H. (1958). A Suggested Revision of Nomenclature— Angiotensin. Nature 181, 1061.

Buchanan, T.W., and Lovallo, W.R. (2001). Enhanced memory for emotional material following stress-level cortisol treatment in humans. Psychoneuroendocrinology 26, 307– 317.

Burhans, L.B., Smith-Bell, C.A., and Schreurs, B.G. (2015). Effects of extinction treatments on the reduction of conditioned responding and conditioned hyperarousal in a rabbit model of posttraumatic stress disorder (PTSD). Behavioral Neuroscience 129, 611–620.

Burnham, Wm.H. (1903). Retroactive amnesia: Illustrative cases and a tentative explanation. The American Journal of Psychology 14, 382–396.

Cannon, W.B. (1916). Bodily Changes in Pain, Hunger, Fear, and Rage: An Account of Recent Researches Into the Function of Emotional Excitement (D. Appleton).

Cao, Y., Liu, Y., Shang, J., Yuan, Z., Ping, F., Yao, S., Guo, Y., and Li, Y. (2019). Ang- (1-7) treatment attenuates lipopolysaccharide-induced early pulmonary fibrosis. Lab Invest.

Cassell, M.D., Freedman, L.J., and Shi, C. (1999). The Intrinsic Organization of the Central Extended Amygdala.

Castellano, C., and McGaugh, J.L. (1989). Retention enhancement with post-training picrotoxin: Lack of state dependency. Behavioral and Neural Biology 51, 165–170.

Chida, Y., and Steptoe, A. (2010). Greater cardiovascular responses to laboratory mental stress are associated with poor subsequent cardiovascular risk status: a meta-analysis of prospective evidence. Hypertension 55, 1026–1032.

Chiu, A.T., Herblin, W.F., McCall, D.E., Ardecky, R.J., Carini, D.J., Duncia, J.V., Pease, L.J., Wong, P.C., Wexler, R.R., Johnson, A.L., et al. (1989). Identification of angiotensin II receptor subtypes. Biochemical and Biophysical Research Communications 165, 196– 203.

Ciocchi, S., Herry, C., Grenier, F., Wolff, S.B.E., Letzkus, J.J., Vlachos, I., Ehrlich, I., Sprengel, R., Deisseroth, K., Stadler, M.B., et al. (2010). Encoding of conditioned fear in central amygdala inhibitory circuits. Nature 468, 277–282.

Cohen, S., Janicki-Deverts, D., and Miller, G.E. (2007). Psychological Stress and Disease. JAMA 298, 1685–1687.

Cole, A.J., Saffen, D.W., Baraban, J.M., and Worley, P.F. (1989). Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340, 474–476.

Conchon, S., and Clauser, E. (2004). AT1 Receptor Molecular Aspects. In Angiotensin Vol. I, T. Unger, and B.A. Schölkens, eds. (Berlin, Heidelberg: Springer Berlin Heidelberg), pp. 269–295.

Corcoran, K.A. (2005). Hippocampal Inactivation Disrupts the Acquisition and Contextual Encoding of Fear Extinction. Journal of Neuroscience 25, 8978–8987.

Corcoran, K.A., and Maren, S. (2001). Hippocampal Inactivation Disrupts Contextual Retrieval of Fear Memory after Extinction. J. Neurosci. 21, 1720–1726.

Costall, B., Domeney, A.M., Gerrard, P.A., Horovitz, Z.P., Kelly, M.E., Naylor, R.J., and Tomkins, D.M. (1990). Effects of captopril and SQ29,852 on anxiety-related behaviours in rodent and marmoset. Pharmacol. Biochem. Behav. 36, 13–20.

Crestani, C., Alves, F., Gomes, F., Resstel, L., Correa, F., and Herman, J. (2013). Mechanisms in the Bed Nucleus of the Stria Terminalis Involved in Control of Autonomic and Neuroendocrine Functions: A Review. Current Neuropharmacology 11, 141–159.

Dampney, R.A. (1994). Functional organization of central pathways regulating the cardiovascular system. Physiological Reviews 74, 323–364.

Dampney, R.A., Hirooka, Y., Potts, P.D., and Head, G.A. (1996). Functions of angiotensin peptides in the rostral ventrolateral medulla. Clin Exp Pharmacol Physiol 23 Suppl 3, S105-11.

Davis, M. (1992). The role of the amygdala in fear and anxiety. Annu. Rev. Neurosci. 15, 353–375.

Dębiec, J., and Ledoux, J.E. (2004). Disruption of reconsolidation but not consolidation of auditory fear conditioning by noradrenergic blockade in the amygdala. Neuroscience 129, 267–272.

Dębiec, J., Bush, D.E.A., and LeDoux, J.E. (2011). Noradrenergic enhancement of reconsolidation in the amygdala impairs extinction of conditioned fear in rats--a possible mechanism for the persistence of traumatic memories in PTSD. Depress Anxiety 28, 186–193.

Denny, J.B., Polan-Curtain, J., Wayner, M.J., and Armstrong, D.L. (1991). Angiotensin II blocks hippocampal long-term potentiation. Brain Research 567, 321–324.

Deschepper, C.F., Bouhnik, J., and Ganong, W.F. (1986). Colocalization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain. Brain Research 374, 195–198.

Devoto, P., Flore, G., Saba, P., Fà, M., and Gessa, G.L. (2005). Stimulation of the locus coeruleus elicits noradrenaline and dopamine release in the medial prefrontal and parietal cortex. J. Neurochem. 92, 368–374.

DiBona, G.F., and Jones, S.Y. (2001). Sodium Intake Influences Hemodynamic and Neural Responses to Angiotensin Receptor Blockade in Rostral Ventrolateral Medulla. Hypertension 37, 1114–1123.

Difede, J., Olden, M., and Cukor, J. (2014). Evidence-based treatment of post-traumatic stress disorder. Annu. Rev. Med. 65, 319–332.

Do-Monte, F.H., Quiñones-Laracuente, K., and Quirk, G.J. (2015). A temporal shift in the circuits mediating retrieval of fear memory. Nature 519, 460–463.

Doron, N.N., and Ledoux, J.E. (2000). Cells in the posterior thalamus project to both amygdala and temporal cortex: A quantitative retrograde double-labeling study in the rat. Journal of Comparative Neurology 425, 257–274.

Dudai, Y. (2004). The Neurobiology of Consolidations, Or, How Stable is the Engram? Annual Review of Psychology 55, 51–86.

Duvarci, S., and Pare, D. (2014). Amygdala Microcircuits Controlling Learned Fear. Neuron 82, 966–980.

Duvarci, S., Popa, D., and Paré, D. (2011). Central Amygdala Activity during Fear Conditioning. J Neurosci 31, 289–294.

Fanselow, M.S. (1986). Conditioned Fear-Induced Opiate Analgesia: A Competing Motivational State Theory of Stress Analgesiaa. Annals of the New York Academy of Sciences 467, 40–54.

Fanselow, M.S. (2000). Contextual fear, gestalt memories, and the hippocampus. Behavioural Brain Research 110, 73–81.

Fanselow, M.S., and Wassum, K.M. (2016). The Origins and Organization of Vertebrate Pavlovian Conditioning. Cold Spring Harbor Perspectives in Biology 8, a021717.

Ferguson, A.V., and Washburn, D.L.S. (1998). Angiotensin II:A peptidergic neurotransmitter in central autonomic pathways. Progress in Neurobiology 54, 169–192.

Flood, J.F., Vidal, D., Bennett, E.L., Orme, A.E., Vasquez, S., and Jarvik, M.E. (1978). Memory facilitating and anti-amnesic effects of corticosteroids. Pharmacology Biochemistry and Behavior 8, 81–87.

Flores, Á., Fullana, M.À., Soriano-Mas, C., and Andero, R. (2018). Lost in translation: how to upgrade fear memory research. Molecular Psychiatry 23, 2122.

Foa, E.B., and Kozak, M.J. (1986). Emotional processing of fear: Exposure to corrective information. Psychological Bulletin 99, 20–35.

Foulquier, S., Steckelings, U.M., and Unger, T. (2012). Impact of the AT2 Receptor Agonist C21 on Blood Pressure and Beyond. Curr Hypertens Rep 14, 403–409.

Frenkel, L., Maldonado, H., and Delorenzi, A. (2005). Memory strengthening by a real- life episode during reconsolidation: an outcome of water deprivation via brain angiotensin II. European Journal of Neuroscience 22, 1757–1766.

Gale, G.D. (2004). Role of the Basolateral Amygdala in the Storage of Fear Memories across the Adult Lifetime of Rats. Journal of Neuroscience 24, 3810–3815.

Gallo, F.T., Katche, C., Morici, J.F., Medina, J.H., and Weisstaub, N.V. (2018). Immediate Early Genes, Memory and Psychiatric Disorders: Focus on c-Fos, Egr1 and Arc. Front. Behav. Neurosci. 12.

Gao, L., and Zucker, I.H. (2011). AT2 receptor signaling and sympathetic regulation. Current Opinion in Pharmacology 11, 124–130.

Gao, J., Zhang, H., Le, K.D., Chao, J., and Gao, L. (2011). Activation of Central Angiotensin Type 2 Receptors Suppresses Norepinephrine Excretion and Blood Pressure in Conscious Rats. Am J Hypertens 24, 724–730.

Gard, P.R. (2002). The role of angiotensin II in cognition and behaviour. European Journal of Pharmacology 438, 1–14.

Gard, P.R. (2008). Cognitive-enhancing effects of angiotensin IV. BMC Neurosci 9, S15.

Garfinkel, S.N., Abelson, J.L., King, A.P., Sripada, R.K., Wang, X., Gaines, L.M., and Liberzon, I. (2014). Impaired Contextual Modulation of Memories in PTSD: An fMRI

and Psychophysiological Study of Extinction Retention and Fear Renewal. J. Neurosci. 34, 13435–13443.

Gehlert, D.R., Gackenheimer, S.L., and Schober, D.A. (1991). Autoradiographic localization of subtypes of angiotensin II antagonist binding in the rat brain. Neuroscience 44, 501–514.

Georgiev, V., and Yonkov, D. (1985). Participation of angiotensin II in learning and memory. I. Interaction of angiotensin II with saralasin. Methods Find Exp Clin Pharmacol 7, 415–418.

Gironacci, M.M., Adamo, H.P., Corradi, G., Santos, R.A., Ortiz, P., and Carretero, O.A. (2011). Angiotensin-(1-7) Induces Mas Receptor Internalization. Hypertension 58, 176– 181.

Giustino, T.F., Fitzgerald, P.J., and Maren, S. (2016). Revisiting propranolol and PTSD: Memory erasure or extinction enhancement? Neurobiology of Learning and Memory 130, 26–33.

Gold, P.E., and Van Buskirk, R.B. (1975). Facilitation of time-dependent memory processes with posttrial epinephrine injections. Behavioral Biology 13, 145–153.

Goldstein, L. (1992). The Amygdala: Neurobiological Aspects of Emotion, Memory, and Mental Dysfunction. Yale J Biol Med 65, 540–542.

Gonzalez, A.D., Wang, G., Waters, E.M., Gonzales, K.L., Speth, R.C., Van Kempen, T.A., Marques-Lopes, J., Young, C.N., Butler, S.D., Davisson, R.L., et al. (2012). Distribution of angiotensin type 1a receptor-containing cells in the brains of bacterial artificial chromosome transgenic mice. Neuroscience 226, 489–509.

Goosens, K.A., and Maren, S. (2001). Contextual and Auditory Fear Conditioning are Mediated by the Lateral, Basal, and Central Amygdaloid Nuclei in Rats. Learn. Mem. 8, 148–155.

Hall, J., Thomas, K.L., and Everitt, B.J. (2001). Fear memory retrieval induces CREB phosphorylation and Fos expression within the amygdala. European Journal of Neuroscience 13, 1453–1458.

Haubensak, W., Kunwar, P.S., Cai, H., Ciocchi, S., Wall, N.R., Ponnusamy, R., Biag, J., Dong, H.-W., Deisseroth, K., Callaway, E.M., et al. (2010). Genetic dissection of an amygdala microcircuit that gates conditioned fear. Nature 468, 270–276.

Henckens, M.J.A.G., Hermans, E.J., Pu, Z., Joëls, M., and Fernández, G. (2009). Stressed Memories: How Acute Stress Affects Memory Formation in Humans. J. Neurosci. 29, 10111–10119.

Herry, C., Ciocchi, S., Senn, V., Demmou, L., Müller, C., and Lüthi, A. (2008). Switching on and off fear by distinct neuronal circuits. Nature 454, 600–606.

Hirooka, Y. (2011). Oxidative stress in the cardiovascular center has a pivotal role in the sympathetic activation in hypertension. Hypertens. Res. 34, 407–412.

Hong, I., Kim, J., Kim, J., Lee, S., Ko, H.-G., Nader, K., Kaang, B.-K., Tsien, R.W., and Choi, S. (2013). AMPA receptor exchange underlies transient memory destabilization on retrieval. PNAS 110, 8218–8223.

Hopkins, D.A., and Holstege, G. (1978). Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat. Exp Brain Res 32, 529–547.

Horiuchi, M., Akishita, M., and Dzau, V.J. (1999). Recent progress in angiotensin II type 2 receptor research in the cardiovascular system. Hypertension 33, 613–621.

Hou, Y., Zhao, L., Zhang, G., and Ding, L. (2015). Effects of oxytocin on the fear memory reconsolidation. Neuroscience Letters 594, 1–5.

Huber, M.J., Basu, R., Cecchettini, C., Cuadra, A.E., Chen, Q.-H., and Shan, Z. (2015). Activation of the (pro)renin receptor in the paraventricular nucleus increases sympathetic outflow in anesthetized rats. Am J Physiol Heart Circ Physiol 309, H880–H887.

Hurt, R.C., Garrett, J.C., Keifer, O.P., Linares, A., Couling, L., Speth, R.C., Ressler, K.J., and Marvar, P.J. (2015). Angiotensin type 1a receptors on corticotropin-releasing factor neurons contribute to the expression of conditioned fear. Genes Brain Behav. 14, 526– 533.

Janak, P.H., and Tye, K.M. (2015). From circuits to behaviour in the amygdala. Nature 517, 284–292.

Jongen-Rêlo, A.L., and Amaral, D.G. (1998). Evidence for a GABAergic projection from the central nucleus of the amygdala to the brainstem of the macaque monkey: a combined retrograde tracing and in situ hybridization study. Eur. J. Neurosci. 10, 2924–2933.

Keifer, O.P., Hurt, R.C., Ressler, K.J., and Marvar, P.J. (2015). The Physiology of Fear: Reconceptualizing the Role of the Central Amygdala in Fear Learning. Physiology 30.

Kessler, R.C., Berglund, P., Demler, O., Jin, R., Merikangas, K.R., and Walters, E.E. (2005). Lifetime Prevalence and Age-of-Onset Distributions of DSM-IV Disorders in the National Comorbidity Survey Replication. Arch Gen Psychiatry 62, 593–602.

Khoury, N.M., Marvar, P.J., Gillespie, C.F., Wingo, A., Schwartz, A., Bradley, B., Kramer, M., and Ressler, K.J. (2012). The Renin-Angiotensin Pathway in Posttraumatic Stress Disorder: Angiotensin-Converting Enzyme Inhibitors and Angiotensin Receptor Blockers Are Associated With Fewer Traumatic Stress Symptoms. J. Clin. Psychiatry 73, 849–855.

Kibler, J.L., Joshi, K., and Ma, M. (2009). Hypertension in relation to posttraumatic stress disorder and depression in the US National Comorbidity Survey. Behavioral Medicine 34, 125–132.

Kim, J.J., Rison, R.A., and Fanselow, M.S. (1993). Effects of amygdala, hippocampus, and periaqueductal gray lesions on short- and long-term contextual fear. Behavioral Neuroscience 107, 1093–1098.

Kindt, M., Soeter, M., and Vervliet, B. (2009). Beyond extinction: erasing human fear responses and preventing the return of fear. Nature Neuroscience 12, 256–258. de Kloet, A.D., Pitra, S., Wang, L., Hiller, H., Pioquinto, D.J., Smith, J.A., Sumners, C., Stern, J.E., and Krause, E.G. (2016). Angiotensin Type-2 Receptors Influence the Activity of Vasopressin Neurons in the Paraventricular Nucleus of the Hypothalamus in Male Mice. Endocrinology 157, 3167–3180.

Krabbe, S., Gründemann, J., and Lüthi, A. (2018). Amygdala Inhibitory Circuits Regulate Associative Fear Conditioning. Biological Psychiatry 83, 800–809.

Krettek, J.E., and Price, J.L. (1978). Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat. Journal of Comparative Neurology 178, 225–253.

Kułakowska, A., Karwowska, W., Wiśniewski, K., and Braszko, J.J. (1996). Losartan influences behavioural effects of angiotensin II in rats. Pharmacol. Res. 34, 109–115.

Lang, P.J., Davis, M., and Öhman, A. (2000). Fear and anxiety: animal models and human cognitive psychophysiology. Journal of Affective Disorders 61, 137–159.

Laterra, J., Keep, R., Betz, L.A., and Goldstein, G.W. (1999). Blood—Brain Barrier. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th Edition.

Lavoie, J.L., Cassell, M.D., Gross, K.W., and Sigmund, C.D. (2004). Localization of renin expressing cells in the brain, by use of a REN-eGFP transgenic model. Physiological Genomics 16, 240–246.

Lavoie Julie L., Cassell Martin D., Gross Kenneth W., and Sigmund Curt D. (2004). Adjacent Expression of Renin and Angiotensinogen in the Rostral Ventrolateral Medulla Using a Dual-Reporter Transgenic Model. Hypertension 43, 1116–1119.

Ledgerwood, L., Richardson, R., and Cranney, J. (2003). Effects of D-cycloserine on extinction of conditioned freezing. Behavioral Neuroscience 117, 341–349.

LeDoux, J.E. (2017). Semantics, Surplus Meaning, and the Science of Fear. Trends in Cognitive Sciences 21, 303–306.

LeDoux, J.E., Sakaguchi, A., and Reis, D.J. (1984). Subcortical efferent projections of the medial geniculate nucleus mediate emotional responses conditioned to acoustic stimuli. J. Neurosci. 4, 683–698.

LeDoux, J.E., Ruggiero, D.A., and Reis, Donald.J. (1985). Projections to the subcortical forebrain from anatomically defined regions of the medial geniculate body in the rat. Journal of Comparative Neurology 242, 182–213.

LeDoux, J.E., Iwata, J., Cicchetti, P., and Reis, D.J. (1988). Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear. Journal of Neuroscience 8, 2517–2529.

Lee, H.J., Choi, J.-S., Brown, T.H., and Kim, J.J. (2001). Amygdalar NMDA Receptors Are Critical for the Expression of Multiple Conditioned Fear Responses. J. Neurosci. 21, 4116–4124.

Lee, J.L.C., Milton, A.L., and Everitt, B.J. (2006). Reconsolidation and Extinction of Conditioned Fear: Inhibition and Potentiation. Journal of Neuroscience 26, 10051–10056.

Lenkei, Z., Palkovits, M., Corvol, P., and Llorens-Cortès, C. (1997). Expression of Angiotensin Type-1 (AT1) and Type-2 (AT2) Receptor mRNAs in the Adult Rat Brain: A Functional Neuroanatomical Review. Frontiers in Neuroendocrinology 18, 383–439.

Lew, R.A., Mustafa, T., Ye, S., McDowall, S.G., Chai, S.Y., and Albiston, A.L. (2003). Angiotensin AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP). J. Neurochem. 86, 344–350.

Lewis, D.J. (1979). Psychobiology of active and inactive memory. Psychological Bulletin 86, 1054–1083.

Li, B. (2019). Central amygdala cells for learning and expressing aversive emotional memories. Curr Opin Behav Sci 26, 40–45.

Li, W., Peng, H., Cao, T., Sato, R., McDaniels, S.J., Kobori, H., Navar, L.G., and Feng, Y. (2012). Brain-targeted (pro)renin receptor knockdown attenuates angiotensin II- dependent hypertension. Hypertension 59, 1188–1194.

Likhtik, E., Popa, D., Apergis-Schoute, J., Fidacaro, G.A., and Paré, D. (2008). Amygdala intercalated neurons are required for expression of fear extinction. Nature 454, 642–645.

Lind, W., Swanson, L.W., and Ganten, D. (1985). Organization of Angiotensin II Immunoreactive Cells and Fibers in the Rat Central Nervous System. NEN 40, 2–24.

Liu, L., Tsuji, M., Takeda, H., Takada, K., and Matsumiya, T. (1999). Adrenocortical suppression blocks the enhancement of memory storage produced by exposure to psychological stress in rats. Brain Research 821, 134–140.

Lopez, L.H.L., Caif, F., García, S., Fraile, M., Landa, A.I., Baiardi, G., Lafuente, J.V., Braszko, J.J., Bregonzio, C., and Gargiulo, P.A. (2012). Anxiolytic-like effect of losartan injected into amygdala of the acutely stressed rats. Pharmacological Reports 64, 54–63.

Maddox, S.A., and Schafe, G.E. (2011). The Activity-Regulated Cytoskeletal-Associated Protein (Arc/Arg3.1) Is Required for Reconsolidation of a Pavlovian Fear Memory. Journal of Neuroscience 31, 7073–7082.

Maddox, S.A., Hartmann, J., Ross, R.A., and Ressler, K.J. (2019). Deconstructing the Gestalt: Mechanisms of Fear, Threat, and Trauma Memory Encoding. Neuron 102, 60– 74.

Mahan, A.L., and Ressler, K.J. (2012). Fear conditioning, synaptic plasticity and the amygdala: implications for posttraumatic stress disorder. Trends in Neurosciences 35, 24–35.

Mamou, C.B., Gamache, K., and Nader, K. (2006). NMDA receptors are critical for unleashing consolidated auditory fear memories. Nature Neuroscience 9, 1237.

Manuck, S.B., Kasprowicz, A.L., and Muldoon, M.F. (1990). Behaviorally-Evoked Cardiovascular Reactivity and Hypertension: Conceptual Issues and Potential Associations. Ann Behav Med 12, 17–29.

Marvar, P.J., Goodman, J., Fuchs, S., Choi, D.C., Banerjee, S., and Ressler, K.J. (2014). Angiotensin Type 1 Receptor Inhibition Enhances the Extinction of Fear Memory. Biological Psychiatry 75, 864–872.

McDonald, A.J., Mascagni, F., and Guo, L. (1996). Projections of the medial and lateral prefrontal cortices to the amygdala: a Phaseolus vulgaris leucoagglutinin study in the rat. Neuroscience 71, 55–75.

McGaugh, J.L. (2000). Memory--a century of consolidation. Science 287, 248–251.

McGaugh, J.L. (2013). Making lasting memories: Remembering the significant. Proc Natl Acad Sci U S A 110, 10402–10407.

McGaugh, J.L., Introini-Collison, I.B., Nagahara, A.H., Cahill, L., Brioni, J.D., and Castellano, C. (1990). Involvement of the amygdaloid complex in neuromodulatory influences on memory storage. Neurosci Biobehav Rev 14, 425–431.

McKinley, M.J., Albiston, A.L., Allen, A.M., Mathai, M.L., May, C.N., McAllen, R.M., Oldfield, B.J., Mendelsohn, F.A.O., and Chai, S.Y. (2003). The brain renin–angiotensin system: location and physiological roles. The International Journal of Biochemistry & Cell Biology 35, 901–918.

Meakins, J., and Wilson, R. (1918). The effect of certain sensory stimulation on respiratory and heart rate in cases of so-called “irritable heart.” Heart, A Journal for the Study of Circulation 17–22.

Meloni, E.G., Gillis, T.E., Manoukian, J., and Kaufman, M.J. (2014). Xenon Impairs Reconsolidation of Fear Memories in a Rat Model of Post-Traumatic Stress Disorder (PTSD). PLoS One 9.

Milad, M.R., and Quirk, G.J. (2002). Neurons in medial prefrontal cortex signal memory for fear extinction. Nature 420, 70–74.

Milad, M.R., and Quirk, G.J. (2012). Fear extinction as a model for translational neuroscience: ten years of progress. Annu Rev Psychol 63, 129–151.

Milton, A.L., Merlo, E., Ratano, P., Gregory, B.L., Dumbreck, J.K., and Everitt, B.J. (2013). Double Dissociation of the Requirement for GluN2B- and GluN2A-Containing NMDA Receptors in the Destabilization and Restabilization of a Reconsolidating Memory. J. Neurosci. 33, 1109–1115.

Misanin, J.R., Miller, R.R., and Lewis, D.J. (1968). Retrograde Amnesia Produced by Electroconvulsive Shock after Reactivation of a Consolidated Memory Trace. Science 160, 554–555.

Mogi, M., and Horiuchi, M. (2013). Effect of angiotensin II type 2 receptor on stroke, cognitive impairment and neurodegenerative diseases: Effect of AT2 in stroke and dementia. Geriatrics & Gerontology International 13, 13–18.

Morgan, M.A., Romanski, L.M., and LeDoux, J.E. (1993). Extinction of emotional learning: Contribution of medial prefrontal cortex. Neuroscience Letters 163, 109–113.

Müller, G.E., and Pilzecker, A. (1900). Experimentelle beiträge zur lehre vom gedächtniss (J.A. Barth).

Nader, K., Schafe, G.E., and Le Doux, J.E. (2000). Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature 406, 722–726.

Nakagawa Pablo, and Sigmund Curt D. (2017). How Is the Brain Renin–Angiotensin System Regulated? Hypertension 70, 10–18.

Nguyen, G., Delarue, F., Burcklé, C., Bouzhir, L., Giller, T., and Sraer, J.-D. (2002). Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J. Clin. Invest. 109, 1417–1427.

Nijsen, M.J., Croiset, G., Diamant, M., Stam, R., Delsing, D., de Wied, D., and Wiegant, V.M. (1998). Conditioned fear-induced tachycardia in the rat; vagal involvement. European Journal of Pharmacology 350, 211–222.

Norrholm, S.D., and Jovanovic, T. (2018). Fear Processing, Psychophysiology, and PTSD: Harvard Review of Psychiatry 26, 129–141.

Orr, S.P., Metzger, L.J., and Pitman, R.K. (2002). Psychophysiology of post-traumatic stress disorder. 23.

Park, J., Marvar, P.J., Liao, P., Kankam, M.L., Norrholm, S.D., Downey, R.M., McCullough, S.A., Le, N.-A., and Rothbaum, B.O. (2017). Baroreflex dysfunction and augmented sympathetic nerve responses during mental stress in veterans with posttraumatic stress disorder: Sympathetic nerve responses in PTSD. The Journal of Physiology 595, 4893–4908.

Parrish, J.N., Bertholomey, M.L., Pang, H.W., Speth, R.C., and Torregrossa, M.M. (2019). Estradiol modulation of the renin–angiotensin system and the regulation of fear extinction. Translational Psychiatry 9, 36.

Paul, M., Poyan Mehr, A., and Kreutz, R. (2006). Physiology of Local Renin- Angiotensin Systems. Physiological Reviews 86, 747–803.

Pavel, J., Benicky, J., Larrayoz-Roldan, I., Murakami, Y., Sanchez-Lemus, E., Zhou, J., And Saavedra, J.M. (2008). Peripherally administered Angiotensin II AT1 receptor antagonists are anti-stress compounds in vivo. Ann N Y Acad Sci 1148, 360–366.

Pavlov (1927), P.I. (2010). Conditioned reflexes: An investigation of the physiological activity of the cerebral cortex. Ann Neurosci 17, 136–141.

Penzo, M.A., Robert, V., and Li, B. (2014). Fear conditioning potentiates synaptic transmission onto long-range projection neurons in the lateral subdivision of central amygdala. J. Neurosci. 34, 2432–2437.

Petrov, T., Krukoff, T.L., and Jhamandas, J.H. (1993). Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat. Brain Res. 609, 81–92.

Petrovich, G.D., and Swanson, L.W. (1997). Projections from the lateral part of the central amygdalar nucleus to the postulated fear conditioning circuit. Brain Res. 763, 247–254.

Phelps, E.A., and LeDoux, J.E. (2005). Contributions of the Amygdala to Emotion Processing: From Animal Models to Human Behavior. Neuron 48, 175–187.

Phillips, M.I., and de Oliveira, E.M. (2008). Brain renin angiotensin in disease. J Mol Med 86, 715–722.

Pitra, S., Worker, C.J., Feng, Y., and Stern, J.E. (2019). Exacerbated effects of prorenin on hypothalamic magnocellular neuronal activity and vasopressin plasma levels during salt-sensitive hypertension. American Journal of Physiology-Heart and Circulatory Physiology 317, H496–H504.

Ploski, J.E., Monsey, M.S., Nguyen, T., DiLeone, R.J., and Schafe, G.E. (2011). The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories. PLoS ONE 6, e23760.

Pulcu, E., Shkreli, L., Holst, C.G., Woud, M.L., Craske, M.G., Browning, M., and Reinecke, A. (2019). The effects of the angiotensin II receptor antagonist losartan on appetitive versus aversive learning – a randomized controlled trial. Biological Psychiatry.

Quirk, G.J., and Mueller, D. (2008). Neural mechanisms of extinction learning and retrieval. Neuropsychopharmacology 33, 56–72.

Quirk, G.J., Russo, G.K., Barron, J.L., and Lebron, K. (2000). The role of ventromedial prefrontal cortex in the recovery of extinguished fear. J. Neurosci. 20, 6225–6231.

Raghavendra, V., Chopra, K., and Kulkarni, S.K. (1998). Involvement of cholinergic system in losartan-induced facilitation of spatial and short-term working memory. Neuropeptides 32, 417–421.

Ramikie, T.S., and Ressler, K.J. (2018). Mechanisms of Sex Differences in Fear and Posttraumatic Stress Disorder. Biological Psychiatry 83, 876–885.

Reaux, A., Fournie-Zaluski, M.C., and Llorens-Cortes, C. (2001). Angiotensin III: a central regulator of vasopressin release and blood pressure. Trends Endocrinol. Metab. 12, 157–162.

Reid, I.A. (1992). Interactions between ANG II, sympathetic nervous system, and baroreceptor reflexes in regulation of blood pressure. American Journal of Physiology- Endocrinology and Metabolism 262, E763–E778.

Reinecke, A., Browning, M., Klein Breteler, J., Kappelmann, N., Ressler, K.J., Harmer, C.J., and Craske, M.G. (2018). Angiotensin Regulation of Amygdala Response to Threat in High-Trait-Anxiety Individuals. Biological Psychiatry: Cognitive Neuroscience and Neuroimaging.

Rescorla, R.A., and Heth, C.D. (1975). Reinstatement of fear to an extinguished conditioned stimulus. Journal of Experimental Psychology: Animal Behavior Processes 1, 88–96.

Resick, P.A., Williams, L.F., Suvak, M.K., Monson, C.M., and Gradus, J.L. (2012). Long-term outcomes of cognitive–behavioral treatments for posttraumatic stress disorder among female rape survivors. Journal of Consulting and Clinical Psychology 80, 201– 210.

Resstel, L.B.M., Alves, F.H.F., Reis, D.G., Crestani, C.C., Corrêa, F.M.A., and Guimarães, F.S. (2008). Anxiolytic-like effects induced by acute reversible inactivation of the bed nucleus of stria terminalis. Neuroscience 154, 869–876.

Rhaleb N E, Rouissi N, Nantel F, D’Orléans-Juste P, and Regoli D (1991). DuP 753 is a specific antagonist for the angiotensin receptor. Hypertension 17, 480–484.

Robbins, S.J. (1990). Mechanisms underlying spontaneous recovery in autoshaping. Journal of Experimental Psychology: Animal Behavior Processes 16, 235–249.

Romanski, L.M., Clugnet, M.-C., Bordi, F., and LeDoux, J.E. (1993). Somatosensory and auditory convergence in the lateral nucleus of the amygdala. Behavioral Neuroscience 107, 444–450.

Roozendaal, B., Okuda, S., de Quervain, D.J.-F., and McGaugh, J.L. (2006). Glucocorticoids interact with emotion-induced noradrenergic activation in influencing different memory functions. Neuroscience 138, 901–910.

Roozendaall, B., Bohus, B., and McGaugh, J.L. (1996). Dose-dependent suppression of adrenocortical activity with metyrapone: Effects on emotion and memory. Psychoneuroendocrinology 21, 681–693.

Rose, J.M., and Audus, K.L. (1998). Receptor-mediated angiotensin II transcytosis by brain microvessel endothelial cells. Peptides 19, 1023–1030.

Rowe, B.P., Saylor, D.L., and Speth, R.C. (1992). Analysis of angiotensin II receptor subtypes in individual rat brain nuclei. Neuroendocrinology 55, 563–573.

Ruiz-Ortega, M., Lorenzo, O., Rupérez, M., Esteban, V., Suzuki, Y., Mezzano, S., Plaza, J.J., and Egido, J. (2001). Role of the renin-angiotensin system in vascular diseases: expanding the field. Hypertension 38, 1382–1387.

Saavedra, J.M., Ando, H., Armando, I., Baiardi, G., Bregonzio, C., Juorio, A., and Macova, M. (2005). Anti-stress and anti-anxiety effects of centrally acting angiotensin II AT1 receptor antagonists. Regulatory Peptides 128, 227–238.

Sacchetti, B., Sacco, T., and Strata, P. (2007). Reversible inactivation of amygdala and cerebellum but not perirhinal cortex impairs reactivated fear memories. European Journal of Neuroscience 25, 2875–2884.

Sah, P., Faber, E.S.L., Lopez De Armentia, M., and Power, J. (2003). The Amygdaloid Complex: Anatomy and Physiology. Physiological Reviews 83, 803–834.

Saigusa, T., and Arita, J. (2014). ANG II modulates both slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rabbit rostral ventrolateral medulla. American Journal of Physiology - Regulatory Integrative and Comparative Physiology 306, R538–R551.

Sandi, C., and Rose, S.P.R. (1997). Training-dependent biphasic effects of corticosterone in memory formation for a passive avoidance task in chicks. Psychopharmacology 133, 152–160.

Sara, S.J. (2000). Retrieval and Reconsolidation: Toward a Neurobiology of Remembering. Learn. Mem. 7, 73–84.

Schwaber, J., Kapp, B., Higgins, G., and Rapp, P. (1982). Amygdaloid and basal forebrain direct connections with the nucleus of the solitary tract and the dorsal motor nucleus. J. Neurosci. 2, 1424–1438.

Seal, K.H., Metzler, T.J., Gima, K.S., Bertenthal, D., Maguen, S., and Marmar, C.R. (2009). Trends and risk factors for mental health diagnoses among Iraq and Afghanistan veterans using Department of Veterans Affairs health care, 2002-2008. Am J Public Health 99, 1651–1658.

Selye, H. (1956). The stress of life (New York, NY, US: McGraw-Hill).

Shan, Z., Shi, P., Cuadra, A.E., Dong, Y., Lamont, G.J., Li, Q., Seth, D.M., Navar, L.G., Katovich, M.J., Sumners, C., et al. (2010). Involvement of the brain (pro)renin receptor in cardiovascular homeostasis. Circ. Res. 107, 934–938.

Shekhar, A., Johnson, P.L., Sajdyk, T.J., Fitz, S.D., Keim, S.R., Kelley, P.E., Gehlert, D.R., and DiMicco, J.A. (2006). Angiotensin-II Is a Putative Neurotransmitter in Lactate- Induced Panic-Like Responses in Rats with Disruption of GABAergic Inhibition in the Dorsomedial Hypothalamus. J. Neurosci. 26, 9205–9215.

Shinohara Keisuke, Liu Xuebo, Morgan Donald A., Davis Deborah R., Sequeira-Lopez Maria Luisa S., Cassell Martin D., Grobe Justin L., Rahmouni Kamal, and Sigmund Curt D. (2016). Selective Deletion of the Brain-Specific Isoform of Renin Causes Neurogenic Hypertension. Hypertension 68, 1385–1392.

Sica, D.A., Gehr, T.W.B., and Ghosh, S. (2005). Clinical pharmacokinetics of losartan. Clin Pharmacokinet 44, 797–814.

Sierra, R.O., Cassini, L.F., Santana, F., Crestani, A.P., Duran, J.M., Haubrich, J., de Oliveira Alvares, L., and Quillfeldt, J.A. (2013). Reconsolidation may incorporate state- dependency into previously consolidated memories. Learning & Memory 20, 379–387.

Sigmund, C.D., Diz, D.I., and Chappell, M.C. (2017). No Brain Renin–Angiotensin System: Déjà vu All Over Again? Hypertension 69, 1007–1010.

Song, K., Allen, A.M., Paxinos, G., and Mendelsohn, F.A. (1992). Mapping of angiotensin II receptor subtype heterogeneity in rat brain. J. Comp. Neurol. 316, 467– 484.

Sotres-Bayon, F., Cain, C.K., and LeDoux, J.E. (2006). Brain Mechanisms of Fear Extinction: Historical Perspectives on the Contribution of Prefrontal Cortex. Biological Psychiatry 60, 329–336.

Steckler, T., and Risbrough, V. (2012). Pharmacological Treatment of PTSD – Established and New Approaches. Neuropharmacology 62, 617–627.

Steenkamp, M.M., Litz, B.T., Hoge, C.W., and Marmar, C.R. (2015). Psychotherapy for Military-Related PTSD: A Review of Randomized Clinical Trials. JAMA 314, 489–500.

Stiedl, O. (1999). Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice. Behavioural Brain Research 104, 1–12.

Stiedl, O., and Hager, T. (2017). Cardiovascular Conditioning: Neural Substrates☆. In Reference Module in Neuroscience and Biobehavioral Psychology, (Elsevier), p.

Stiedl, O., Tovote, P., Ögren, S.O., and Meyer, M. (2004). Behavioral and autonomic dynamics during contextual fear conditioning in mice. Autonomic Neuroscience 115, 15– 27.

Stornetta, R.L., Hawelu-Johnson, C.L., Guyenet, P.G., and Lynch, K.R. (1988). Astrocytes synthesize angiotensinogen in brain. Science 242, 1444–1446.

Sumners, C., Horiuchi, M., Widdop, R.E., McCarthy, C., Unger, T., and Steckelings, U.M. (2013). Protective arms of the renin-angiotensin-system in neurological disease. Clinical and Experimental Pharmacology and Physiology 40, 580–588.

Szczepanska-Sadowska, E., Czarzasta, K., and Cudnoch-Jedrzejewska, A. (2018). Dysregulation of the Renin-Angiotensin System and the Vasopressinergic System Interactions in Cardiovascular Disorders. Curr Hypertens Rep 20, 19.

Todes, D.P. (1997). Pavlov’s physiology factory. Isis 88, 204–246.

Tomaz, C., Brandão, M., Bagri, A., Carrive, P., and Schmitt, P. (1988). Flight behavior induced by microinjection of GABA antagonists into periventricular structures in detelencephalated rats. Pharmacology Biochemistry and Behavior 30, 337–342.

Touyz, R.M., and Schiffrin, E.L. (2000). Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells. Pharmacol. Rev. 52, 639–672.

Tovote, P., Meyer, M., Beck-Sickinger, A.G., von Hörsten, S., Ove Ogren, S., Spiess, J., and Stiedl, O. (2004). Central NPY receptor-mediated alteration of heart rate dynamics in mice during expression of fear conditioned to an auditory cue. Regul. Pept. 120, 205– 214.

Tovote, P., Meyer, M., Pilz, P.K.D., Ronnenberg, A., Ögren, S.O., Spiess, J., and Stiedl, O. (2005). Dissociation of Temporal Dynamics of Heart Rate and Blood Pressure Responses Elicited by Conditioned Fear but Not Acoustic Startle. Behavioral Neuroscience 119, 55–65.

Tovote, P., Esposito, M.S., Botta, P., Chaudun, F., Fadok, J.P., Markovic, M., Wolff, S.B.E., Ramakrishnan, C., Fenno, L., Deisseroth, K., et al. (2016). Midbrain circuits for defensive behaviour. Nature 534, 206–212.

Trotman, G.P., Gianaros, P.J., Zanten, J.J.C.S.V. van, Williams, S.E., and Ginty, A.T. (2019). Increased stressor-evoked cardiovascular reactivity is associated with reduced amygdala and hippocampus volume. Psychophysiology 56, e13277.

Unger, T. (2000). Neurohormonal modulation in cardiovascular disease. American Heart Journal 139, s2–s8.

Valentino, R.J., and Van Bockstaele, E. (2008). Convergent regulation of locus coeruleus activity as an adaptive response to stress. European Journal of Pharmacology 583, 194– 203.

Vieira, A.A., Nahey, D.B., and Collister, J.P. (2010). Role of the organum vasculosum of the lamina terminalis for the chronic cardiovascular effects produced by endogenous and exogenous ANG II in conscious rats. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 299, R1564–R1571.

Villain, H., Benkahoul, A., Drougard, A., Lafragette, M., Muzotte, E., Pech, S., Bui, E., Brunet, A., Birmes, P., and Roullet, P. (2016). Effects of Propranolol, a β-noradrenergic Antagonist, on Memory Consolidation and Reconsolidation in Mice. Front. Behav. Neurosci. 10.

Walker, D.L., Ressler, K.J., Lu, K.-T., and Davis, M. (2002). Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusions of D-cycloserine as assessed with fear-potentiated startle in rats. Journal of Neuroscience 22, 2343–2351.

Wan, Y., Wallinder, C., Plouffe, B., Beaudry, H., Mahalingam, A.K., Wu, X., Johansson, B., Holm, M., Botoros, M., Karlén, A., et al. (2004). Design, Synthesis, and Biological Evaluation of the First Selective Nonpeptide AT 2 Receptor Agonist. J. Med. Chem. 47, 5995–6008.

Wang, L., Hiller, H., Smith, J.A., Kloet, A.D. de, and Krause, E.G. (2016). Angiotensin type 1a receptors in the paraventricular nucleus of the hypothalamus control cardiovascular reactivity and anxiety-like behavior in male mice. Physiological Genomics 48, 667–676.

Wicking, M., Steiger, F., Nees, F., Diener, S.J., Grimm, O., Ruttorf, M., Schad, L.R., Winkelmann, T., Wirtz, G., and Flor, H. (2016). Deficient fear extinction memory in posttraumatic stress disorder. Neurobiology of Learning and Memory 136, 116–126.

Wright, J.W., and Harding, J.W. (1997). Important roles for angiotensin III and IV in the brain renin-angiotensin system. Brain Research Reviews 25, 96–124.

Wright, J.W., and Harding, J.W. (2013). The brain renin-angiotensin system: a diversity of functions and implications for CNS diseases. Pflugers Arch. 465, 133–151.

Wright, J.W., Kawas, L.H., and Harding, J.W. (2015). The development of small molecule angiotensin IV analogs to treat Alzheimer’s and Parkinson’s diseases. Progress in Neurobiology 125, 26–46.

Xu, C., Krabbe, S., Gründemann, J., Botta, P., Fadok, J.P., Osakada, F., Saur, D., Grewe, B.F., Schnitzer, M.J., Callaway, E.M., et al. (2016). Distinct Hippocampal Pathways Mediate Dissociable Roles of Context in Memory Retrieval. Cell 167, 961-972.e16.

Xue, Y., Taub, P.R., Iqbal, N., Fard, A., Wentworth, B., Redwine, L., Clopton, P., Stein, M., and Maisel, A. (2012). Cardiac Biomarkers, Mortality, and Post-Traumatic Stress Disorder in Military Veterans. The American Journal of Cardiology 109, 1215–1218.

Yang, G., Gray, T.S., Sigmund, C.D., and Cassell, M.D. (1999). The angiotensinogen gene is expressed in both astrocytes and neurons in murine central nervous system. Brain Research 817, 123–131.

Young, B.J., and Leaton, R.N. (1994). Fear potentiation of acoustic startle stimulus- evoked heart rate changes in rats. Behav. Neurosci. 108, 1065–1079.

Zhang, S.P., Bandler, R., and Carrive, P. (1990). Flight and immobility evoked by excitatory amino acid microinjection within distinct parts of the subtentorial midbrain periaqueductal gray of the cat. Brain Research 520, 73–82.

Zhang, Z.-H., Yu, Y., Kang, Y.-M., Wei, S.-G., and Felder, R.B. (2008). Aldosterone acts centrally to increase brain renin-angiotensin system activity and oxidative stress in normal rats. Am. J. Physiol. Heart Circ. Physiol. 294, H1067-1074.

Zhou, F., Geng, Y., Xin, F., Li, J., Feng, P., Liu, C., Zhao, W., Feng, T., Guastella, A.J., Ebstein, R.P., et al. (2019). Human extinction learning is accelerated by an angiotensin antagonist via ventromedial prefrontal cortex and its connections with basolateral amygdala. Biological Psychiatry.

Zubenko, G.S., and Nixon, R.A. (1984). Mood-elevating effect of captopril in depressed patients. Am J Psychiatry 141, 110–111.

CHAPTER 2

Extinction of Fear Memory Attenuates Conditioned Cardiovascular Fear Reactivity

Swiercz, A.P., Seligowski, A.V., Park, J., and Marvar, P.J. (2018). Frontiers in Behavioral Neuroscience 12.

(Used with permission)

ABSTRACT

Post-traumatic stress disorder (PTSD) is characterized by a heightened emotional and physiological state and an impaired ability to suppress or extinguish traumatic fear memories. Exaggerated physiological responses may contribute to increased cardiovascular disease (CVD) risk in this population, but whether treatment for

PTSD can offset CVD risk remains unknown. To further evaluate physiological correlates of fear learning, we used a novel pre-clinical conditioned cardiovascular testing paradigm and examined the effects of Pavlovian fear conditioning and extinction training on mean arterial pressure (MAP) and heart rate (HR) responses. We hypothesized that a fear conditioned cardiovascular response could be detected in a novel context and attenuated by extinction training. In a novel context, fear conditioned mice exhibited marginal increases in MAP (∼3 mmHg) and decreases in HR (∼20 bpm) during CS presentation.

In a home cage context, the CS elicited significant increases in both HR (100 bpm) and

MAP (20 mmHg). Following extinction training, the MAP response was suppressed while

CS-dependent HR responses were variable. These pre-clinical data suggest that extinction learning attenuates the acute MAP responses to conditioned stimuli over time, and that

MAP and HR responses may extinguish at different rates. These results suggest that in mouse models of fear learning, conditioned cardiovascular responses are modified by extinction training. Understanding these processes in pre- clinical disease models and in humans with PTSD may be important for identifying interventions that facilitate fear extinction and attenuate hyper-physiological responses, potentially leading to improvements in the efficacy of exposure therapy and PTSD–CVD comorbidity outcomes.

INTRODUCTION

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric disorder in which over- generalization of fear and impaired fear extinction recall can lead to a permanent state of hyperarousal and emotional numbing that can negatively impact daily life (Desmedt et al., 2015). PTSD is in part characterized by an inability to adequately suppress fear responses under safe conditions (Jovanovic et al., 2010; Sijbrandij et al.,

2013) and is often accompanied by exaggerated physiological symptoms [e.g., increased heart rate (HR), blood pressure, and sympathetic drive] (American Psychiatric

Association, 2013; Edmondson et al., 2013; Vaccarino et al., 2013; Park et al., 2017).

Enhanced acute stress responses may contribute to increased rates of acute cardiac events and cardiovascular disease (CVD) risk in PTSD patients (Edmondson et al.,

2013; Edmondson and von Känel, 2017; Myers, 2017), however, the underlying physiological mechanisms remain unclear. Further examination of altered cardiovascular reactivity that occurs in states of fear is therefore required to better understand the link between PTSD and CVD.

Classical rodent and human models of fear conditioning are commonly used to study both the expression and extinction of learned fear, providing practical methods to identify PTSD biomarkers and prevention strategies. For example, extinction training is believed to be analogous to exposure therapy, which is one of the most effective therapeutic treatments for PTSD, phobias, and anxiety disorders (Morrison and

Ressler, 2014). Extinction training results in the formation of a new extinction memory, and a gradual reduction of conditioned response (Quirk, 2002). Extinction recall occurs when the extinction memory is retrieved and expressed later (Quirk et al.,

2000; Milad et al., 2009). Due to its potential as a therapeutic target, many efforts have recently been made to discover treatments that may strengthen or facilitate extinction recall (Bukalo et al., 2014; Bowers and Ressler, 2015). Critical to these efforts is the ability to accurately measure extinction- specific changes to conditioned responses.

While most assessments of extinction in animal models rely entirely on changes in freezing behavior, conditioned cardiovascular responses have previously been shown to serve as important physiological correlates of fear teaching (Gaburro et al., 2011). For example, rodent studies using radiotelemetry demonstrate that HR

(Tovote et al., 2005b), HR variability (Stiedl et al., 2009), and blood pressure (Hsu et al., 2012) are reliable indicators of fear memory acquisition that can be used to distinguish between non-specific and associative threat responses. However, few studies have examined the effects of fear extinction on cue-dependent conditioned cardiovascular responses. Within these studies, the focus has primarily been on reductions in HR reactivity (Stiedl, 1999; Stiedl et al., 2009; Camp et al., 2012; Hager et al.,

2014).

Under certain conditions, re-exposure to a conditioned stimulus causes co- activation of the sympathetic and parasympathetic branches of the autonomic nervous system. Blockade of sympathetic outflow with propranolol decreases fear-associated tachycardia following auditory fear conditioning, while atropine enhances it (Iwata and

LeDoux, 1988). Similar results have also been reported in contextual models of fear conditioning (Carrive, 2006). These findings suggest that the conditioned cardiovascular response consists of activation of the sympathetic nervous system (SNS), which is partially buffered by simultaneous activation of the parasympathetic nervous

system (PSNS). Through cardiac nerves and circulating adrenal catecholamines, sympathetic activation results in an increased HR. Mean arterial pressure (MAP) also increases in response to sympathetically mediated blood vessel constriction (Baudrie et al., 2001). Parasympathetic activation, on the other hand, simultaneously acts to lower

HR via cholinergic modulation of sinoatrial node activity. Given that blood pressure and HR are under autonomic regulation, but with distinct temporal and network control

(Tovote et al., 2005a), both parameters should be considered during assessment of fear expression and extinction recall. To date, the effects of extinction training on conditioned blood pressure responses have not been directly tested.

Here we developed a novel conditioned cardiovascular response behavioral paradigm to examine the effects of extinction training on cue-dependent blood pressure and HR responses. We hypothesized that a fear conditioned cardiovascular response could be detected in a novel context and attenuated by extinction training. Closely evaluating cardiovascular reactivity to conditioned fear may contribute to a better understanding of the hyper-physiological responses in PTSD and associated CVD risk.

Physiological measures of inhibitory learning could also lead to more accurate assessments of extinction efficiency in animal models. The objectives of this study were to examine the real-time behavioral and cardiovascular responses to fear conditioning and extinction using a mouse model, and to examine the effects of extinction training on

MAP and HR responses during extinction recall.

MATERIALS AND METHODS

Animals

Adult male (3–4 months old) C57BL/6J mice from Jackson Laboratory (Bar

Harbor, ME, United States) were used for all experiments. The C57BL/6 strain is a commonly used inbred strain that has been shown to extinguish fear responses well in comparison to other strains (Hefner et al., 2008; Camp et al., 2012). Mice were housed individually in temperature and humidity-controlled polyethylene cages on a 12 h light/dark cycle. Animals were supplied with water and food ad libitum for the duration of the experiments. All procedures were approved by the Institutional Animal Care and

Use Committee at The George Washington University and were in compliance with

National Institutes of Health guidelines.

Radiotelemetry

Telemeter Implantation, Data Collection and Analysis Animals were anesthetized with an IP injection of ketamine/xylazine and maintenance of anesthesia was assessed with toe pinch. HDX-11 transmitters [Data Sciences International (DSI), St.

Paul, MN, United States] were implanted subcutaneously, with a blood pressure transducer inserted into the carotid artery. Animals were allowed to recover for 14 days before beginning behavioral experiments. Blood pressure signals were sampled at a rate of 500 Hz. Blood pressure and activity data were continuously collected during 24 h baseline measurements, fear conditioning, extinction training, and cardiovascular response tests. Blood pressure data were analyzed using Ponemah software version 6.3 (DSI). Baseline day, night, and 24 h averages were calculated from

12 h epochs corresponding with the light/dark cycle. HR was derived from the blood pressure channel.

Fear Conditioning

For 2 days prior to fear conditioning, animals were exposed to the chamber to habituate them to handling and context. Auditory f e a r conditioning was performed in conditioning test cages (7” x 7” x 12”; model H10-11M-TC) equipped with overhead cameras and grid shock floors (H10-11M-TC-SF). Test cages were enclosed in sound attenuating isolation cubicles (Model H10-24T; Coulbourn

Instruments, Holliston, MA, United States). Fear conditioned animals received both the conditioned stimulus and unconditioned stimulus (CS-US group), and were presented with CS-US pairings of a 30 s auditory cue (6 kHz, 75 db) co-terminating with a mild footshock (0.5 s, 0.5 mA). There was a 3 min 30 s inter-trial interval between each pairing. Control (CS group) animals were exposed to the CS under fear conditioning conditions but never received a footshock. Fear conditioning test cages were cleaned thoroughly with 70% ethanol before each session. After conditioning, animals were returned to the home cage for 24 h before extinction training

Extinction Training

Two rounds of extinction training were performed in modified test cages to distinguish them from the fear conditioning context. The shock grid was replaced with a clear plexiglass floor and the clear chamber walls were covered with paper. The chambers were wiped down with water and peppermint soap before each extinction session. Extinction training occurred 24 and 48-h following fear conditioning (Figure

2 - 1A). A 5 min pre-CS period preceded the first tone presentation in each test.

Extinction sessions consisted of either 30 conditioned stimulus tone trials in CS and

CS-US groups, or 35 trials in the Extinction (Ext) group. Each trial lasted 30 s and was followed by a 30 s inter-trial interval. The No Extinction (No Ext) control group was placed into the modified context for the same duration, but was not exposed to the conditioned stimulus during extinction training. A non-conditioned (No US) control group was not included in the home cage extinction experiments based on previous studies showing that the auditory stimulus would evoke only mild, transient cardiovascular effects that do not differ significantly from baseline values (Tovote et al., 2005a). The percentage of time spent freezing was calculated using Freezeframe

3.32 (Coulbourn Instruments). All behavioral experiments occurred during the light phase (7am–7pm).

Cardiovascular Response Tests

The conditioned cardiovascular response was measured in the home cage 24-h after fear conditioning (Cardiovascular Response Test 1), 24-h after the first extinction session (Cardiovascular Response Test 2), and 1-h after the second extinction session

(Cardiovascular Response Test 3) (Figure 2-2). The home cage was placed in a sound attenuating chamber, and a speaker was positioned on top of the cage. Mice were left undisturbed for 1 h before remotely initiating a 4 CS memory test to determine the effects of extinction training on the conditioned cardiovascular response during extinction recall.

Statistical Analysis

Prism 6.0 (Graphpad Software Inc., La Jolla, CA, United States) was used for statistical evaluation of mouse data. Data are presented as the mean ± SEM, with p- values <0.05 considered statistically significant. Analysis of variance (ANOVA) for repeated measures was used for statistical analysis followed by Bonferroni tests for post hoc comparison.

RESULTS

Behavioral and Cardiovascular Responses During Extinction Training in a

Novel Context

To examine the cardiovascular responses to conditioned fear during extinction training, two groups of animals were equipped with radiotelemeters and were either exposed to a fear conditioning protocol (CS-US group) or exposed to five auditory cues without footshocks (CS group). Behavioral and cardiovascular responses

(freezing behavior, MAP, and HR) were simultaneously monitored in the extinction context during two consecutive days of extinction training in the CS-US and CS groups (Figure 2-1A).

Freezing Behavior

As expected, a progressive increase in freezing response to CS exposure was observed only in the CS-US animals (Figure 2-1B). Simultaneous behavioral and cardiovascular responses to extinction trials across 2 days are shown in Figures 2-1C–

K. The average of the first 4 CS (CS1–4) presentations on Day 1 and the last 4 CS

presentations on Day 2 (CS27–30) were taken as measures of acquisition and extinction, respectively (Yang et al., 2016). On Day 1 of extinction training, the CS-US group exhibited increased freezing throughout the 30 CS presentations (Figure 2-1C).

Fear acquisition was demonstrated in the CS-US animals compared to the CS by group differences in freezing during the first 4 CS presentations (71% ± 4 vs. 10% ±

3, p < 0.05) (Figure 2-1E). CS-US animals showed a significant reduction in freezing from Day 1–2, with a significant group x time interaction [F(1,17) = 20.68, p =

0.0003] (Figure 2-1E).

Mean Arterial Pressure and HR in Novel Context

At baseline on Day 1 of extinction training, MAP and HR were similar between groups (Figures 2 -1F,G). In response to the first CS, a trend for a small increase in

MAP (3 mmHg), which remained elevated throughout the extinction session and a corresponding bradycardic HR response were observed in the CS-US group compared to control. Overall, both groups showed a slow reduction in HR throughout the extinction session. Following the last CS presentation, the CS-US group displayed a rapid increase in HR corresponding with the cessation of freezing.

There was a significant group by time interaction [F(48,864) = 5.032, p < 0.0001], with group differences in HR throughout the No CS period (Figure 2-1G).

On Day 2 of extinction training, in response to CS, there was a similar trend for a small transient increase in MAP and bradycardic response within the CS-US animals only during CS 1–4. For the remainder of the session, MAP was similar between groups while HR slowly declined (−100 bpm). Unlike Day 1 of extinction, the CS-US group did not exhibit the sharp increase in HR during the No CS period. MAP responses were then evaluated across days between extinction sessions (Figure 2-1J).

When comparing the first 4 CSs (Day 1 of extinction) and the last 4 CSs (Day 2 of extinction), repeated measures-ANOVA revealed a main effect of time, [F(1,18) = 14.28, p = 0.0014], and a trend for group by time interaction [F(1,18) = 3.736, p = 0.0691]. There was also a reduction in HR in response to CS from Day 1 of extinction to Day 2 of extinction in CS-US animals (Figure 2-1K). However, because a comparison of HR between groups revealed a significant group by time interaction [F(1,18) = 5.936, p =

0.0254] with a main effect of time [F(1,18) = 52.52, p < 0.0001] but not group [F(1,18)

= 0.3778, p = 0.5465], this reduction cannot be attributed solely to extinction learning and is more likely a result of within session HR recovery.

In addition, both groups displayed a slow, gradual recovery of HR throughout each session, but neither MAP nor HR returned to resting baseline levels by the end of the test on either day. This suggests that handling and novel context exposure contribute to the elevations of MAP and HR regardless of whether or not the animals were fear conditioned. Furthermore, because these HR elevations are similar at the beginning of both days of extinction training, the effects of habituation appear to be

minimal. These findings are consistent with previous studies showing that novel environments can induce HR elevations in mice despite previous habituation (Liu et al., 2013). Because elevated baseline cardiovascular measures could potentially mask cardiovascular adjustments caused by the conditioned stimulus, we next examined the effects of extinction training on fear conditioned (CS-US) mouse cardiovascular reactivity in a home cage environment.

CS-Dependent Conditioned Cardiovascular Responses in the Home Cage

Mean Arterial Pressure and HR Response (Cardiovascular Response Test 1)

Conditioned physiological responses are highly dependent on the resting physiological state, which in part influences the cardiovascular response to conditioned stimuli. Therefore, in order to examine both the conditioned cardiovascular responses and the effects of extinction, cardiovascular response tests were conducted in the home cage environment (Stiedl et al., 2004). Two groups of mice (No Ext and Ext) were equipped with radiotelemeters and all animals were fear conditioned as previously described (Figure 2 - 2). 24 h after fear conditioning (prior to extinction training), mice were exposed to 4 CS trials in the home cage (Cardiovascular

Response Test 1). Baseline activity levels, blood pressure and HR during the pre-CS period were significantly lower than in the extinction context in both groups (Figure 2-

S1), while Pre-CS cardiovascular baselines were similar to mean 12-h baselines during the light cycle (Table 2-S1). As shown in Figure 2-3A, a two-phase pressor response was observed during the first CS presentation of the 4 CS test in both groups. This consisted of a rapid rise (general arousal) of approximately 10 mmHg within the first 10 s

of the CS, followed by a slower, steady increase which has previously been attributed to associative learning (Tovote et al., 2005a; Figure 2-3B). Subsequent CS presentations also coincided with a rapid rise in MAP. Peak MAP (No Ext 122 ± 5; Ext 124 ± 3 mmHg) were reached within the first 3 s of the second CS. MAP averaged over the 4

CSs was significantly increased from Pre-CS baselines in both No Ext and Ext groups confirming a strong conditioned CS-dependent MAP pressor response in these animals

(Figure 2-3C). An ANOVA comparing the 4 CS MAP revealed no significant main effect of group [F(1,13) = 1.212, p = 0.2908] or group by time interaction [F(1,13) =

0.7181, p = 0.4121], yet did reveal a significant main effect of time [F(1,13) = 33.38, p < 0.0001]. As shown in Figures 2-3D,E,F), there was an overall significant increase

(∼100 bpm) in HR over the 4 CS trials in both groups relative to pre-CS baseline. An

ANOVA revealed a main effect of time [F(1,11) = 10.59, p = 0.0077], with no group by time interaction [F(1,11) = 0.01081, p = 0.9190] and no main effect of group

[F(1,11) = 0.6541, p = 0.4358]. In summary, these data demonstrate a consistent CS- dependent home cage pressor response that was accompanied by an overall increase in

HR 24 h following fear conditioning.

Extinction of CS-Dependent Cardiovascular Responses

Mean Arterial Pressure and HR Response (Cardiovascular Response Test 2)

The two groups of animals subsequently went through either an Ext or No

Ext training protocol as shown in Figure 2-2. Following extinction training, animals were tested at two time points in order to examine (A) long-term retention of the CS- dependent cardiovascular response and (B) within-session cumulative effects of

additional extinction trials. To examine the long-term retention of extinction learning during the CS- dependent cardiovascular response, a 4-tone cardiovascular response test (#2) was conducted 24 h following extinction training. As shown in Figures 2-4A,C,

5 min pre-CS baseline MAP and HR were similar between Ext and No Ext groups

(MAP: 103 ± 6 vs. 108 ± 4 mmHg; HR: 522 ± 49 vs. 515 ± 28 bpm) and within the range of normal daytime averages (Table 2-S1). Similar to the initial CS response in Figure

2-3A, the No Ext group displayed a biphasic pressure increase that was characterized by a rapid increase in MAP (∼10 mmHg) within the first 10 s, followed by a slower increase that persisted until the end of the first CS. In these animals, peak MAP (132 mmHg) was again reached during the first 3 s of the second CS presentation (Figure 2-

4A). An ANOVA comparing the 4 CS MAP revealed a significant group by time interaction [F(1,13) = 5.164, p = 0.0407] (Figure 2-4B). Post hoc tests revealed that

MAP in the No-Ext group significantly increased from baseline, while MAP in the Ext group did not. In animals that underwent extinction training (Ext group), the initial rise in blood pressure of ∼10 mmHg was observed; however the second phase was distinctively absent (Figure 2-4A). Despite a trend for a change in HR increase to CS in the No Ext group (Figure 2-4C), HR changes were highly variable (Figure 2-4C). An

ANOVA comparing the 4 CS HR revealed no significant group by time interaction

[F(1,13) = 2.463, p = 0.1406] (Figure 2-4D). These results demonstrate that 24 h after training, the conditioned MAP response is significantly blunted in the Ext group while the HR response to CS is not significantly different between groups, and thus may track closely to the extinguished freezing behavior.

Mean Arterial Pressure and HR Response (Cardiovascular Response Test 3)

To further evaluate the effects of extinction on the conditioned cardiovascular response, the Ext group was exposed to a second day of extinction training, which resulted in an extinction effect as determined by freezing responses to CS (Figure 2-

S2). All animals were tested 1 h later in their home cage for CS-dependent cardiovascular responses. Consistent with the two previous cardiovascular response tests, a two-phase blood pressure response was observed in the No Ext animals and the peak MAP (122 ± 7) was reached within the first 3 s of the second CS (Figure 2-4E).

In the Ext group a small, slow increase in MAP was observed throughout the test (∼5 mmHg) but this increase did not appear to be associated with CS onset. Peak MAP in the Ext group was significantly lower than that of the No Ext group (106 mmHg ±

3 vs. 122 ± 7, p < 0.05) (Figure 2-4E). Moreover, both phases of the MAP response were absent in the Ext animals. An ANOVA comparing the 4 CS MAP revealed a significant group by time interaction [F(2,16) = 4.409, p = 0.0298] (Figure 2-4F).

Similar comparisons of HR indicate no group by time interaction [F(1,12) = 1.504, p =

0.2436] (Figure 2-4H). Peak HR were not significantly different between No Ext (555 ±

61) and Ext (523 ± 28) groups (Figure 2-4G) and remained within the range of normal resting HRs (Table 2-S1).

DISCUSSION

The current findings demonstrate that recall of consolidated extinction memories can modulate the conditioned cardiovascular response, which is influenced by context- dependent differences in blood pressure and HR sensitivity. Alterations of the

conditioned MAP response may serve as a novel index in the evaluation of extinction efficiency and may aid in identifying the hyper-physiological underpinnings of PTSD and co-morbid CVD-PTSD.

Conditioned HR (Fitzgerald and Martin, 1971) and BP (Iwata and LeDoux, 1988) during CS presentation have previously been reported in the conditioning context, which elicits contextual and non-specific stress related alterations in cardiovascular activity.

Furthermore, freezing behavior and locomotor activity are closely linked to changes in blood pressure and HR (Vliet et al., 2003) and can potentially confound conditioned cardiovascular responses. To minimize these disruptions, conditioned cardiovascular responses are often evaluated in the resting or home cage environment (Iwata and

LeDoux, 1988; Stiedl et al., 2004, 2009; Camp et al., 2012). In the present study, we predicted that testing in a novel context would reduce contextual fear and minimize non-specific stress enough to observe HR and MAP responses to CS between conditioned (CS-US) and control (CS) groups. Additionally, we hypothesized that blood pressure and HR responses in the extinction context would decrease across days as a result of extinction training.

During the first extinction session in a novel context (Day 1), freezing in response to CS confirmed fear acquisition in conditioned animals, while the magnitude of the cardiovascular response was not significantly different from controls.

Throughout the CS period on Days 1 and 2 of extinction, MAP remained high while

HR gradually fell in both groups over the CS period, indicating a within-session habituation effect on HR but not MAP. While the relatively small conditioned cardiovascular response limits the interpretation of these findings, it is possible that the

small elevation of MAP in the CS- US group on Day 1 was sustained by repeated CS presentation and extinguished by repeated CS exposure, while HR increases were offset by reductions in physical activity during times of freezing.

During the second extinction session in a novel context (Day 2), there was trend for a reduction in MAP in the CS- US group only, while HR was significantly reduced in both groups. These data may suggest an extinction-specific reduction of blood pressure response that was distinct from a generalized habituation-like effect on

HR. Interestingly, following cessation of CS presentation during extinction training on

Day 1, CS- US animals displayed a rapid increase in HR. While this may have been a consequence of increased locomotion due to an immediate reduction in freezing, there were no overall differences in activity levels between groups during this time.

Because conditioned HR responses are controlled by activation of both the sympathetic and parasympathetic components of the autonomic nervous system (Iwata and LeDoux,

1988; Carrive, 2006), and freezing behavior is accompanied by parasympathetically driven HR deceleration (Roelofs, 2017), this increase in HR may be mediated in part by parasympathetic withdrawal. Similarly, on both day 1 and 2 in the novel context we observed an initial brief decelerative HR response upon CS onset (Figure 2-1G,I), which is likely parasympathetically mediated (Iwata and LeDoux, 1988). This deceleration in HR is consistent with prior human fear research using HR (typically associated with fear responding) (Lang et al., 2011; Sege et al., 2017). Taken together, these findings suggest that CS exposure in a novel context may elicit MAP and HR responses at different rates, with changes in autonomic regulation of HR first emerging

upon CS onset (deceleration) and again when the conditioned stressor is removed

(acceleration).

To further investigate extinction-dependent responses in our mouse model, while minimizing context-enhanced basal cardiovascular effects (see Figure 2-S1), we next sought to determine whether repeated CS exposure could extinguish conditioned cardiovascular responses when measured in the home cage. As opposed to previous studies using an extended single CS (Stiedl, 1999; Stiedl et al., 2004, 2007, 2009; Tovote et al., 2005a), we evaluated the cardiovascular response using 4 CS presentations. A 4

CS memory test was used based on the following considerations: (1) the cumulative effect of multiple CS presentations could be determined by using shorter CS presentations spaced with inter-trial intervals; (2) the test duration would be long enough to encompass both the fast, sympathetic-mediated vasoconstriction response

(Baudrie et al., 1997), and the slower humoral-mediated responses (Tovote et al., 2005a);

(3) the low number of CS presentations would minimize extinction in the No Ext group caused by multiple testing sessions.

In the home cage context, both groups of mice showed stable resting MAP and

HR during the pre-CS period. Prior to extinction training and 24-h post-fear conditioning, MAP increased across the 4 CS trials in a CS-dependent manner. This increase reached ∼20 mmHg and was characterized by two phases of rise. The initial, rapid increase is thought to result from general arousal and lasts for approximately 5s. The second phase of the conditioned blood pressure response, which has been shown to result from associative fear learning in mice, allows for distinction between general arousal and associative memory (Tovote et al., 2005a).

While HR was increased in both groups across CS trials, the magnitude of these responses was markedly less than previously described (Stiedl and Spiess, 1997).

Variations in conditioning protocols and testing procedures likely account for these differences (Stiedl and Spiess, 1997; Stiedl, 1999; Stiedl et al., 2004, 2007; Tovote et al., 2005a). Based on the clear presence of an associative blood pressure response in this experiment, and the ability of a 30 CS extinction protocol to reduce long-term fear expression (Marvar et al., 2014), we reasoned that the MAP response would be suppressed during extinction recall.

To evaluate the cardiovascular response during long-term extinction memory recall, animals were re-tested 24 h after extinction training in the home cage. The No Ext group exhibited a CS-dependent MAP increase across 4 CS home cage trials, while extinguished animals did not. Furthermore, the general arousal phase of the MAP response was present, while the associative phase was absent in the Ext group. These data suggest that extinguished animals respond with an acute, generalized arousal similar to non-extinguished animals, while the associative component of the blood pressure response can be modified by non-reinforced CS exposure.

Despite the extinction-dependent reduction in MAP response, there was no significant difference in HR response between groups. Although we observed a trend for increased HR in the No Ext across the 4 CS presentations, it was not significantly different from the pre-CS period at this time point. We attribute the lack of a conditioned HR response to the increased variability and small conditioned HR response observed in this model. Either the fear conditioning protocol did not result in a strong enough conditioned HR response to detect the effects of extinction

between Ext and No Ext groups, or the HR response was so sensitive to CS presentation in the home cage that it was quickly extinguished in both groups following the first home cage test. In either case, the results of this study point to MAP as a reliable index of extinction in mouse models of fear conditioning. Taken together, the

MAP and HR data from these experiments show that that the conditioned blood pressure response is significantly blunted by extinction training while the HR response did not allow for distinction between extinguished and non-extinguished control animals.

Short-term effects of an additional extinction training session were also evaluated during the final home cage test. Consistent with the two previous cardiovascular response tests, a two- phase blood pressure response was observed in the No Ext animals with peak MAP values occurring within the first 3 s of the second CS (Figure 2-4E).

There were no significant HR responses in either group at this time point. The results of this extinction test confirm a reduction in the MAP response in the Ext group and show minimal change of HR in response to CS in either group. Interestingly, conditioned HR responses in our mouse model in the home cage were not as robust as those previously reported by other groups (Stiedl and Spiess, 1997; Tovote et al.,

2005a). As a result, HR increases in resting animals were seemingly extinguished during home cage testing. Future studies will need to address the effects of conditioned stimulus intensity and duration on HR responses.

In summary, the present study demonstrates for the first time that extinction training attenuates the acute blood pressure responses evoked by conditioned stimuli and that in this behavioral paradigm, MAP was a more reliable measure of conditioning

and extinction than HR. Moreover, this reduction of the fear-induced cardiovascular response appears to be independent of activity-related behavioral changes. While there is some evidence to suggest that extinction training can attenuate cardiac responses in humans (Panitz et al., 2015), future studies are required to determine the impact of extinction-based therapeutic interventions on cardiovascular reactivity in PTSD patients. Such studies may potentially lead to improvements in extinction-based therapies and PTSD–CVD comorbidity outcomes.

Figure 2-1: Behavioral and cardiovascular changes during extinction training. Schematic of fear conditioning and 2-day extinction protocol (A). Freezing behavior during fear conditioning (B). Freezing behavior (C,D), mean arterial pressure (F,H), and heart rate (G,I) during 2-day extinction training protocol. Average freezing behavior (E), mean arterial pressure (J), and heart rate (K) during CS 1–4 (Day 1), and CS 27–30 (Day 2) in the extinction context. Error bars indicate standard error of the mean (n = 9–11 per group, *p < 0.05 CS vs. CS-US; #p < 0.05 Day 1 vs. Day 2).

Figure 2-2: Schematic of fear conditioning and testing protocol for home cage extinction studies. All animals were fear conditioned on Day 0. Conditioned cardiovascular responses were measured in the home cage 24 h later (Cardiovascular Response Test 1). Animals were then placed into a novel context and exposed to either 35 CS (Ext group) or no CS (No Ext group) for the first extinction session. On Day 2, a home cage test (Cardiovascular Response Test 2) was conducted prior to the second extinction session. The Ext group was then exposed to another 35CS extinction session. One hour later, all animals were tested in the home cage (Cardiovascular Response Test 3).

Figure 2-3: Pre-extinction training CS-dependent cardiovascular response test 1 (home cage). Mean arterial pressure (A), and heart rate (D) collected during 4 CS presentations and averaged every 3 s. (B,E) Depict second-by-second fluctuations in MAP and HR during the first CS presentation. Average MAP (C) and HR (F) during the 5 min pre-CS period, and over 4 CS presentations (n = 6–9 per group, *p < 0.05 pre-CS vs. 4 CS avg).

Figure 2-4: Post-extinction training CS-dependent cardiovascular response tests 2 and 3 (home cage). Mean arterial pressure (A) and heart rate (C) collected during cardiovascular response test 2. Average MAP (B) and HR (D) during 4 CS presentations. Mean arterial pressure (E), and heart rate (G) collected during cardiovascular response test 3. Average MAP (F) and HR (H) during 4 CS presentations (n = 6–9 per group, *p < 0.05 pre-CS vs. 4 CS avg).

Figure 2-S1: Baseline cardiovascular measures in training and home cage contexts. Activity level (A), mean arterial pressure (B), diastolic pressure (C), systolic pressure (D), and heart rate (E) over the 5 min pre-CS period in each context (n = 9–11 per group. ∗ p < 0.05 Training vs. Home Cage).

Figure 2-S2: Freezing behavior during fear conditioning (A), extinction training Day 1 (B), and extinction training Day 2 (C). All animals received a footshock and CS during fear conditioning (n = 6–9 per group).

Figure 2-S3: Group data of baseline heart rate variability prior to fear conditioning. Light and dark cycle values were calculated from 12hr segments after exclusion of R-R intervals not found within ±2 standard deviations of the mean. The standard deviation of all normal- to-normal R-R intervals (SDNN) (A) reflects the overall heart rate variability. The root mean square of successive differences (RMSSD) (B) is an estimate of the short-term components of the heart rate. The proportion of RR intervals having a difference of > 6 msec (pNN6) (C) reflects cardiac parasympathetic activity (n=12 per group, *p < 0.05 Light vs. Dark, paired t-test).

Table 2-S1: Baseline day/night (12 h) mean arterial pressure (MAP), heart rate (HR), and activity levels in mice prior to fear conditioning.

REFERENCES

American Psychiatric Association (2013). Diagnostic and Statistical Manual of Mental Disorders, 5th Edn. Virginia, NV: American Psychiatric Association.

Baudrie, V., Laude, D., Chaouloff, F., and Elghozi, J.-L. (2001). Genetic influences on cardiovascular responses to an acoustic startle stimulus in rats. Clin. Exp. Pharmacol. Physiol. 28, 1096–1099.

Baudrie, V., Tulen, J. H., Blanc, J., and Elghozi, J. L. (1997). Autonomic components of the cardiovascular responses to an acoustic startle stimulus in rats. J. Auton. Pharmacol. 17, 303–309.

Bowers, M. E., and Ressler, K. J. (2015). An overview of translationally informed treatments for PTSD: animal models of pavlovian fear conditioning to human clinical trials. Biol. Psychiatry 78, E15–E27.

Bukalo, O., Pinard, C. R., and Holmes, A. (2014). Mechanisms to medicines: elucidating neural and molecular substrates of fear extinction to identify novel treatments for anxiety disorders. Br. J. Pharmacol. 171, 4690–4718.

Camp, M. C., Macpherson, K. P., Lederle, L., Graybeal, C., Gaburro, S., Debrouse, L. M., et al. (2012). Genetic strain differences in learned fear inhibition associated with variation in neuroendocrine, autonomic, and amygdala dendritic phenotypes. Neuropsychopharmacology 37, 1534–1547.

Carrive, P. (2006). Dual activation of cardiac sympathetic and parasympathetic components during conditioned fear to context in the rat. Clin. Exp. Pharmacol. Physiol. 33, 1251–1254.

Desmedt, A., Marighetto, A., and Piazza, P. V. (2015). Abnormal fear memory as a model for posttraumatic stress disorder. Biol. Psychiatry 78, 290–297.

Edmondson, D., Kronish, I. M., Shaffer, J. A., Falzon, L., and Burg, M. M. (2013). Posttraumatic stress disorder and risk for coronary heart disease: a meta-analytic review. Am. Heart J. 166, 806–814.

Edmondson, D., and von Känel, R. (2017). Post-traumatic stress disorder and cardiovascular disease. Lancet Psychiatry 4, 320–329.

Fitzgerald, R. D., and Martin, G. K. (1971). Heart-rate conditioning in rats as a function of interstimulus interval. Psychol. Rep. 29, 1103–1110.

Gaburro, S., Stiedl, O., Giusti, P., Sartori, S. B., Landgraf, R., and Singewald, N. (2011). A mouse model of high trait anxiety shows reduced heart rate variability that can be reversed by anxiolytic drug treatment. Int. J. Neuropsychopharmacol. 14, 1341–1355.

Hager, T., Maroteaux, G., du Pont, P., Julsing, J., van Vliet, R., and Stiedl, O. (2014). Munc18-1 haploinsufficiency results in enhanced anxiety-like behavior as determined by heart rate responses in mice. Behav. Brain Res. 260, 44–52.

Hefner, K., Whittle, N., Juhasz, J., Norcross, M., Karlsson, R. M., Saksida, L. M., et al. (2008). Impaired fear extinction learning and cortico-amygdala circuit abnormalities in a common genetic mouse strain. J. Neurosci. 28, 8074–8085.

Hsu, Y. C., Yu, L., Chen, H., Lee, H. L., Kuo, Y. M., and Jen, C. J. (2012). Blood pressure variations real-time reflect the conditioned fear learning and memory. PLoS One 7:e32855.

Iwata, J., and LeDoux, J. E. (1988). Dissociation of associative and nonassociative concomitants of classical fear conditioning in the freely behaving rat. Behav. Neurosci. 102, 66–76.

Jovanovic, T., Norrholm, S. D., Blanding, N. Q., Davis, M., Duncan, E., Bradley, B., et al. (2010). Impaired fear inhibition is a biomarker of PTSD but not depression. Depress. Anxiety 27, 244–251.

Lang, P. J., Wangelin, B. C., Bradley, M. M., Versace, F., Davenport, P. W., and Costa, V. D. (2011). Threat of suffocation and defensive reflex activation: threat of suffocation. Psychophysiology 48, 393–396.

Liu, J., Wei, W., Kuang, H., Zhao, F., and Tsien, J. Z. (2013). Changes in heart rate variability are associated with expression of short-term and long-term contextual and cued fear memories. PLoS One 8:e63590.

Marvar, P. J., Goodman, J., Fuchs, S., Choi, D. C., Banerjee, S., and Ressler, K. J. (2014). Angiotensin type 1 receptor inhibition enhances the extinction of fear memory. Biol. Psychiatry 75, 864–872.

Milad, M. R., Pitman, R. K., Ellis, C. B., Gold, A. L., Shin, L. M., Lasko, N. B., et al. (2009). Neurobiological basis of failure to recall extinction memory in posttraumatic stress disorder. Biol. Psychiatry 66, 1075–1082.

Morrison, F. G., and Ressler, K. J. (2014). From the neurobiology of extinction to improved clinical treatments. Depress. Anxiety 31, 279–290.

Myers, B. (2017). Corticolimbic regulation of cardiovascular responses to stress. Physiol. Behav. 172, 49–59.

Panitz, C., Hermann, C., and Mueller, E. M. (2015). Conditioned and extinguished fear modulate functional corticocardiac coupling in humans. Psychophysiology 52, 1351– 1360.

Park, J., Marvar, P. J., Liao, P., Kankam, M. L., Norrholm, S. D., Downey, R. M., et al. (2017). Baroreflex dysfunction and augmented sympathetic nerve responses during mental stress in veterans with post-traumatic stress disorder. J. Physiol. 595, 4893–4908.

Quirk, G. J. (2002). Memory for extinction of conditioned fear is long-lasting and persists following spontaneous recovery. Learn. Mem. 9, 402–407.

Quirk, G. J., Russo, G. K., Barron, J. L., and Lebron, K. (2000). The role of ventromedial prefrontal cortex in the recovery of extinguished fear. J. Neurosci. 20, 6225– 6231.

Roelofs, K. (2017). Freeze for action: neurobiological mechanisms in animal and human freezing. Philos. Trans. R. Soc. Lond. B Biol. Sci. 372:20160206. doi: 10.1098/rstb.2016.0206

Sege, C. T., Bradley, M. M., and Lang, P. J. (2017). Escaping aversive exposure. Psychophysiology 54, 857–863.

Sijbrandij, M., Engelhard, I. M., Lommen, M. J. J., Leer, A., and Baas, J. M. P. (2013). Impaired fear inhibition learning predicts the persistence of symptoms of posttraumatic stress disorder (PTSD). J. Psychiatr. Res. 47, 1991–1997.

Stiedl, O. (1999). Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice. Behav. Brain Res. 104, 1–12.

Stiedl, O., Jansen, R. F., Pieneman, A. W., Ögren, S. O., and Meyer, M. (2009). Assessing aversive emotional states through the heart in mice: implications for cardiovascular dysregulation in affective disorders. Neurosci. Biobehav. Rev. 33, 181–190.

Stiedl, O., Misane, I., Koch, M., Pattij, T., Meyer, M., and Ögren, S. O. (2007). Activation of the brain 5-HT2C receptors causes hypolocomotion without anxiogenic- like cardiovascular adjustments in mice. Neuropharmacology 52, 949–957.

Stiedl, O., and Spiess, J. (1997). Effect of tone-dependent fear conditioning on heart rate and behavior of C57BL/6N mice. Behav. Neurosci. 111, 703–711.

Stiedl, O., Tovote, P., Ögren, S. O., and Meyer, M. (2004). Behavioral and autonomic dynamics during contextual fear conditioning in mice. Auton. Neurosci. 115, 15–27.

Tovote, P., Meyer, M., Pilz, P. K. D., Ronnenberg, A., Ögren, S. O., Spiess, J., et al. (2005a). Dissociation of temporal dynamics of heart rate and blood pressure responses elicited by conditioned fear but not acoustic startle. Behav. Neurosci. 119, 55–65.

Tovote, P., Meyer, M., Ronnenberg, A., Ögren, S. O., Spiess, J., and Stiedl, O. (2005b). Heart rate dynamics and behavioral responses during acute emotional challenge in corticotropin-releasing factor receptor 1-deficient and corticotropin- releasing factor-overexpressing mice. Neuroscience 134, 1113– 1122.

Vaccarino, V., Goldberg, J., Rooks, C., Shah, A. J., Veledar, E., Faber, T. L., et al. (2013). Post-traumatic stress disorder and incidence of coronary heart disease: a twin study. J. Am. Coll. Cardiol. 62, 970–978.

Vliet, B. N. V., Chafe, L. L., and Montani, J. P. (2003). Characteristics of 24 h telemetered blood pressure in eNOS-knockout and C57Bl/6J control mice. J. Physiol. 549, 313–325.

Yang, W. Z., Liu, T. T., Cao, J. W., Chen, X. F., Liu, X., Wang, M., et al. (2016). Fear erasure facilitated by immature inhibitory neuron transplantation. Neuron 92, 1352–1367.

CHAPTER 3

Evaluation of an Angiotensin Type 1 Receptor Blocker on the Reconsolidation of Fear Memory

Adam P. Swiercz, Laxmi Iyer, Zhe Yu, Allison Edwards, Prashant N.M., Brian Nguyen, Anelia Horvath and Paul J. Marvar

(In preparation)

ABSTRACT

Inhibition of the angiotensin type 1 receptor (AT1R) has been shown to decrease fear responses in both humans and rodents. These effects are attributed to modulation of extinction learning, however the contribution of AT1R to alternative memory processes remains unclear. Using classic Pavlovian conditioning combined with radiotelemetry and whole-genome RNA sequencing, we evaluated the effects of the AT1R antagonist losartan on fear memory reconsolidation. Following the retrieval of conditioned auditory fear memory, animals were given a single injection of losartan (i.p. 10mg/kg) or saline.

Animals that received post-retrieval losartan exhibited significantly less freezing at 24hrs

(LTM-1d) and 1 week (LTM-7d); an effect that was dependent upon memory reactivation. Cardiovascular responses were unaltered by losartan during conditioned stimulus (CS) presentation, however the stress-dependent increases in blood pressure

(associated with handling) during reconsolidation were blunted. Whole-genome RNA sequencing data suggest that peripherally-administered losartan alters gene expression during reconsolidation in the basolateral amygdala. These findings demonstrate that post-retrieval AT1R blockade modifies behavioral markers of conditioned fear memory, supporting a role for this receptor in reconsolidation and as a potential pharmacotherapeutic target for maladaptive fear disorders such as PTSD.

INTRODUCTION

Life-threatening traumatic events (e.g., military combat, assault, or natural disaster) can contribute to maladaptive fear memories. These trauma-associated fear memories and their improper consolidation, reconsolidation, and extinction contribute to mental health disorders such as posttraumatic stress disorder (PTSD). The renin- angiotensin system (RAS) has been identified as a potential pharmacotherapeutic target for PTSD as retrospective clinical studies have shown that treatment with angiotensin- converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARBs) are associated with fewer PTSD symptoms (Khoury et al., 2012a; Nylocks et al., 2015).

Further pre-clinical research demonstrates that peripheral AT1R inhibition with losartan

(Lazaroni et al., 2012; Marvar et al., 2014; Parrish et al., 2019) or deletion of AT1R from select neuronal populations facilitates fear memory extinction (Hurt et al., 2015). More recent evidence in humans indicates that losartan improves early threat discrimination and facilitates threat processing (Reinecke et al., 2018), in addition to accelerating fear extinction (Zhou et al., 2019) and modifying aversive learning (Pulcu et al., 2019a).

Despite these observations in both humans and rodents, the underlying neurobiological mechanisms remain unclear.

Extinction is not the only process that can occur following the retrieval of a previously consolidated memory; memory reactivation can also initiate the process of reconsolidation. Reconsolidation provides a time-limited window of vulnerability to selectively weaken or enhance a previously consolidated memory (Sara, 2000).

Interference with reconsolidation can have amnesic effects on the reactivated memory, and this process which could potentially be manipulated to benefit a range of psychiatric

conditions, including PTSD, obsessive compulsive disorder, delusions, and hallucinations

(Nader et al., 2013). To date, little is known about RAS involvement in reconsolidation, but it is important to determine whether recent findings from extinction studies in rodents and humans might also be explained by interference with reconsolidation.

Interestingly, water deprivation in the arthropod strengthens memory by facilitating reconsolidation via increases in brain angiotensin II (Frenkel et al., 2005). A similar strengthening of contextual fear memory reconsolidation by Ang II-mediated

AT1R activation in the dorsal hippocampus was demonstrated in rats (Sierra et al., 2013).

If endogenous Ang II is responsible for reconsolidation improvements, it may be possible to weaken long-term memories by blocking AT1R activation during the window of reconsolidation. To further probe the neurobiological mechanisms of AT1R inhibition of fear memory, we evaluated the effects of AT1R antagonism following retrieval of a fear memory using classic Pavlovian conditioning, combined with radiotelemetry and whole- genome RNA sequencing. We hypothesized that post-retrieval blockade of AT1R would disrupt reconsolidation and alter key cardiovascular and central transcriptomic pathways.

Our findings indicate that losartan, administered shortly after memory retrieval, reduces long-term freezing responses to conditioned auditory stimuli while altering the differential expression of specific genes in the amygdala. These studies build upon growing evidence linking brain angiotensin receptors to fear-related memory (Hurt et al.,

2015; Kerr et al., 2005; Lazaroni et al., 2012; Marinzalda et al., 2014; Marvar et al.,

2014).

MATERIALS AND METHODS

All procedures were approved by the Institutional Animal Care and Use

Committee at The George Washington University and were in compliance with National

Institutes of Health guidelines. Adult male (3–4 months old) C57BL/6J mice from

Jackson Laboratory (Bar Harbor, ME, United States) were used for all experiments.

Animals were individually housed in temperature and humidity-controlled polyethylene cages on a 12 h light/dark cycle and supplied with water and food ad libitum.

Radiotelemetry: As previously described (Swiercz et al., 2018) HDX-11 radio transmitters (Data Sciences International (DSI); St. Paul, MN) were subcutaneously implanted. A blood pressure catheter was placed into the left carotid artery and advanced to the aortic arch. Animals recovered for 14 days after surgery before beginning behavioral experiments. During telemetry recording, blood pressure signals were sampled at a rate of 500Hz. Blood pressure and activity data were continuously collected during memory retrieval, and memory testing. Blood pressure data were analyzed using

Ponemah software version 6.3 (DSI).

Drugs: The AT1R antagonist losartan (Sigma-Aldrich; St. Louis, MO) was dissolved in sterile saline for intraperitoneal injection (10mg/kg). The total injection volume was 0.2mL per animal. This dose was selected based on previous studies demonstrating an effect on memory extinction (Marvar et al., 2014).

Pavlovian Fear Conditioning: Auditory fear conditioning was performed as previously described (Swiercz et al., 2018; Yu et al., 2019). Mice were individually habituated with the experimenter and conditioning chamber for 20 and 40-min sessions on days 1 and 2. Fear conditioning consisted of five trials of a conditioned stimulus (CS)

tone (30s, 6 kHz, 75 dB) co-terminating with an unconditioned stimulus (US) foot shock

(0.5mA, 0.5s,) spaced by 3min 30s inter-trial intervals.

Retrieval Protocol: Twenty-four hours after fear conditioning, animals were re- exposed to the CS to reactivate the memory and initiate reconsolidation (Clem and

Huganir, 2010). The fear conditioning chamber was modified by replacing the shock grid with a clear Plexiglas floor clear walls of the chamber with patterned construction paper and the chamber was scented with peppermint oil. The first group of animals was given a peripheral injection of either saline (0.2ml ip) or losartan (10mg/kg ip) 10 minutes after retrieval. A baseline period of 120 s, during which the Pre-CS percent freezing average was calculated, preceded the presentation of a single 30s CS. The second group was fear conditioned as described above, and 24hr later placed into the retrieval context, however this time no auditory cue was played. 10 minutes after this sham retrieval session, the second group was also given a peripheral injection of saline

(0.2ml ip) or losartan (10mg/kg ip). These animals were treated identically to the retrieval groups except for the omission of the CS. The behavioral protocol is depicted in

Fig 3-1A.

Retrieval Protocol (plus radiotelemeters): Animals equipped with radiotelemeters were fear conditioned as described above. Memory retrieval and subsequent testing for these animals was performed in the homecage to minimize cardiovascular alterations due to handling. 24hr after fear conditioning, the homecage was placed inside a sound attenuating chamber, with a speaker positioned above the bedding. Animals were left undisturbed for approximately 45min to allow blood pressure and heart rate to return to resting values. The same retrieval protocol used in non-

telemetry animals was remotely initiated and telemetry data was recorded throughout the session. The behavioral protocol for these animals is depicted in Fig. 3-2A.

Memory Testing: Freezing behavior was assessed 24hr (LTM-1d) and 1 week

(LTM-7d) after memory retrieval. Animals were returned to the retrieval context and exposed to 4 CS presentations, with a pre-CS period of 5 min and an inter-trial interval of

30s. After testing, animals were returned to the homecage with no further behavioral intervention until the 1wk test. The same 4CS testing protocol was used at both time points.

We also tested LTM in telemeter-equipped animals. CS-induced changes in blood pressure and heart rate were assessed relative to the pre-CS period. During testing, the homecage was placed into a sound attenuating chamber and the animals were left undisturbed for 45min before the testing protocol was initiated. After each test, the telemetry recording was stopped and the animals were returned to the housing facility.

Tissue Collection, RNA Extraction and whole-genome RNA Sequencing: Mice that received either saline or losartan (10mg/kg) post-retrieval were sacrificed 40 minutes after the tone with brief exposure to CO2 gas. The non-retrieval (NR) control mice were sacrificed directly from the home cage without receiving the retrieval cue (Merlo et al.,

2014). Brains were collected, snap frozen and stored in -80°C. The brains were mounted on Cryostat (CyroStar NX50) and 0.5 millimeter bilateral tissue punches were taken from the basolateral amygdala (BLA) (Paxinos and Watson, 1998).

For whole genome RNA sequencing, the bilateral BLA punches from two mice of the same group were pooled together. Total RNA was extracted from the pooled BLA samples using Trizol reagent (Sigma) according to the manufacturer’s instructions. RNA

quality control (QC), library construction and sequencing were performed by the George

Washington University Genomics Core. RNA quality and quantity were assessed using an Agilent RNA 6000 Nano kit on a 2100 Bioanalyzer. Samples were constructed into libraries using the Illumina TruSeq Stranded Total RNA Human/Mouse/Rat Library Prep kit (Illumina Inc. San Diego, CA), and were sequenced using an Illumina NextSeq 500 with a High Output Kit v2.5-150 cycles.

Sequencing reads were trimmed for adapters and bases with a Q-score under 20 were removed using Flexbar v. 3.0.3 (Roehr et al., 2017). Reads were then pseudo- aligned to Ensembl release 94 GRCm38.p6 Mus musculus transcripts using kallisto v.

0.43.2 (Bray et al., 2016). The counts of reads pseudo-aligned to each transcript by kallisto was imported into R v. 3.6.0 and analyzed for differentially abundance genes between treatments with DESeq2 v. 1.24.0 (Love et al., 2014). Transcript IDs were mapped to gene IDs and genes with false discovery rate-adjusted Q-score under 0.05 were considered to be statistically significant. Mitochondrial genes were filtered out from the analysis. Gene ontology annotation was performed using Panther (Thomas et al.,

2003), and pathway analysis was carried out using Metacore from Clarivate Analytics.

Quantitative PCR (qPCR): As described above RNA was extracted from bilateral

BLA punches and total mRNA was reversely transcribed with qScript cDNA supermix

(Quant Bio) and qPCR was performed using TaqMan® Universal PCR-Master Mix

(Applied Biosystems, Waltham, MA) on a CFX384 Real-time PCR Detection System

(Bio-Rad;, Hercules, CA). The following TaqMan® primers were used: Fos, (Assay ID-

Mm00487425_m1) Arc (Mm01204954_g1) and EGR-1 (Mm00656724_m1). The

comparative Ct method (Livak and Schmittgen, 2001) was used to analyze differences in mRNA levels. The samples were normalized to the housekeeping gene 18S.

Data Analysis: Prism 6.0 (Graphpad Software Inc., La Jolla, CA, United States) was used for statistical evaluation of behavioral and cardiovascular data.

Data are presented as the mean ± SEM. For comparisons between treatment groups over time, one- or two-way analysis of variance (ANOVA) for repeated measures (RM) was employed, followed by Bonferroni tests for post hoc comparisons. When appropriate,

One way ANOVA or t-tests were utilized for group comparisons. P-values <0.05 were considered statistically significant.

RESULTS

Post-retrieval Losartan Disrupts Reconsolidation of Conditioned Auditory Fear:

To determine whether blockade of AT1R with losartan during the window of reconsolidation alters the maintenance of conditioned auditory fear memory, mice were fear conditioned and then 24 hours later received a single-CS retrieval cue (Fig. 3-1A).

All groups exhibited similar acquisition of fear as measured by percent freezing during the conditioning protocol (Fig. 3-1B). Prior to drug treatment, animals from both the saline and losartan groups showed a significant increase in freezing during the single CS retrieval cue (F1, 19= 39.09, p< 0.0001) (Fig. 3-1C). Twenty four hours after retrieval and under drug-free conditions, the LTM-1d test was conducted. During the first 4 CS cued memory test, a significant reduction in freezing behavior was observed in mice that received losartan after memory reactivation (Fig. 3-1D). Two-way RM ANOVA with the between-subjects factor of Drug (Saline and losartan), and the within-subjects factor

100

of Tone (pre-CS and CS) revealed a significant main effect of Tone (F1, 17 = 87.04, p <

0.0001) and a significant interaction of Drug x Tone (F1, 17 = 5.739, p = 0.0284) with no main effect of Drug (F1, 17 = 3.013, p = 0.1007). Bonferroni post hoc analysis revealed a significant reduction in freezing in the losartan group (p<0.05) during CS presentation.

Memory was assessed one-week later using a 4 CS cued memory test. Two-way RM

ANOVA revealed no significant Drug x Tone interaction (F1, 19 = 2.282, p =0.1474); however there were significant main effects of Tone (F1, 19 = 59.33, p < 0.0001) and of

Drug (F1, 19 = 13.33, p =0.0017), indicating a persistent reduction in the freezing behavior of losartan-treated animals relative to controls (Fig. 3-1E).

To determine whether the effects of losartan were specific to the process of reconsolidation, we assessed freezing behavior in a separate control cohort of animals that did not undergo retrieval, thus the auditory fear memory was not reactivated (non- retrieval group). Using a similar injection time-course in the absence of a retrieval cue, animals received either saline or losartan and freezing responses were measured 24hr later. In response to the CS, increased freezing was observed in both groups as indicated by a significant main effect of Tone (F1, 21 = 227.9, p < 0.0001) (Fig. 3-1F). Freezing was not significantly different in losartan-treated animals relative to saline controls (F1, 21

= 3.823, p < 0.0640), and there was no Drug x Tone interaction (F1, 21 =0.4378, p =

0.5154). Similar results were obtained 1 week later during the LTM-7d test, where there was a main effect of Tone (F1, 18 =145.0, p < 0.0001), but no significant effect of Drug

(F1, 18 =1.543, p = 0.2301) or Drug x Tone interaction (F1, 18 =0.0194, p = 0.8908) (Fig. 3-

1G). These results indicate that post-retrieval losartan reduces freezing during both

LTM-1d and LTM-7d tests, and that memory reactivation is required for these effects.

101

Post-retrieval losartan does not affect recall of conditioned cardiovascular responses:

To determine whether reductions in cue-dependent freezing following post-retrieval losartan alters the recall of a conditioned cardiovascular response, we assessed the cardiovascular state of the animals during memory recall. These animals were surgically equipped with radiotelemeters, fear conditioned, and exposed to a single retrieval cue 24 hr later as outlined in the schematic (Fig. 3-2A).

Using a conditioned cardiovascular testing paradigm, the animals were exposed to

4 CS presentations while resting in their homecage at 24 hours (LTM-1d) and 1 week

(LTM-7d) following retrieval (Fig. 3-2A). The auditory cue elicited an increase in MAP

(Main effect of Tone F1, 9 = 20.21, p= 0.0015), with no main effect of Drug (F1, 9 =

0.3128, p = 0.5896) and no Drug x Tone interaction (F1, 9 =0.8311, p = 0.3857)(Fig. 3-

2B, C). A conditioned HR response was also observed during this test, with a significant main effect of Tone (F1, 9 = 17.19, p = 0.0025) but no Drug (F1, 9 = 1.285, p = 0.2862) or

Drug x Tone interaction (F1, 9 = 0.0013, p = 0.9724) (Fig. 3-2D, E).

One week after memory retrieval, the LTM-7d test was performed. MAP increased in both groups during CS presentation, as indicated by a main effect of Tone

(F1, 8= 18.50, p =0.0026) with no main effect of Drug (F1, 8= 0.9117, p =0.3676) and no

Drug x Tone interaction (F1,8= 0.3162, p =0.5893) (Fig. 3-2F, G). HR also increased during CS presentation, with a main effect of Tone (F1, 9= 13.66, p=0.0050), but no effect of Drug (F1, 9= 0.3644, p=0.5610) and no Drug x Tone interaction (F1, 9= 0.2175, p=0.6521) (Fig. 3-2H, I). These findings suggest that unlike freezing behavior, conditioned cardiovascular responses were not affected by post-retrieval losartan.

102

Post-retrieval stress-dependent increases in blood pressure (associated with handling) are attenuated by losartan. One factor that may influence or modulate the strength of reconsolidation is animal handling during an injection procedure, which elicits a stress response including a robust increase in MAP and HR and which may modulate the strength of reconsolidation (Gazarini et al., 2013). Having demonstrated that post- retrieval losartan reduces freezing behavior at two different time points, we next assessed whether these effects might be explained by altered blood pressure modulation via AT1R antagonism immediately following retrieval. To address this, we performed additional injections and monitored the effects on MAP and HR in telemeter-equipped animals to determine how losartan might alter the cardiovascular state during the reconsolidation phase. Each mouse was injected with either saline or losartan and immediately placed back in its homecage while telemetry data was collected (Fig. 3-3A) .

Throughout the 2 hr post-injection period, there was we found a main effect of

Drug (F1, 20 = 10.41, p = 0.0042) and of Time (F39, 780 = 10.09, p < 0.0001), with no significant Drug x Time interaction (F39, 780 = 0.7456, p = 0.8726) (Fig. 3-3B). Two-RM

ANOVA on the mean MAP during the 2 hrs following injection showed a significant main effect of Drug (F1, 20 = 5.703, p = 0.0269), and a significant Drug x Time interaction

(F1, 20 = 7.359, p = 0.0134), with no main effect of Time (F1, 20 = 0.08710, p = 0.7709)

(Fig. 3-3C). There was a main effect of Time (F39, 780= 5.584, p < 0.0001) on HR throughout the 2-hr post-injection period, with no Drug (F1, 20 = 3.557, p = 0.0739) or

Drug x Time interaction (F39, 780 = 0.2498, p > 0.999) (Fig. 3-3D). Similarly, mean HR following the injection showed a significant main effect of Time (F1, 20 = 22.46, p =

103

0.0001), but no Drug (F1, 20 = 0.6690, p = 0.4231) or Drug x Time interaction (F1, 20 =

2.088, p = 0.1639) (Fig. 3-3E). These results suggest that acute losartan administration blunts the stress-induced MAP, but not HR, increases associated with handling and injecting previously conditioned animals.

Transcriptomic analysis of the basolateral amygdala (BLA) following post-retrieval losartan:

Next, we performed a transcriptomic analysis of the basolateral amygdala (BLA) following post-retrieval losartan to determine if losartan modulates genes in the BLA, a key nucleus for the acquisition, persistence, and loss of fear memory. We first performed

RT-qPCR on the BLA for the immediate early-activation genes (IEGs), Fos, Arc and

Egr-1 (Wang et al., 2018). Irrespective of drug treatment, both retrieval groups showed increased expression (2-3 fold-change) compared to non-retrieval (NR or “control”) group (Fig. 3-4A). These data confirm the transcription of IEGs at the 40 minute time point which are independent of losartan.

Next, we expanded this analysis using an unbiased whole transcriptome RNA- sequencing approach. With a p<0.05 cut-off point threshold, we identified 90 differentially expressed genes (DEGs) for saline vs NR group and 38 DEGs for losartan vs NR group (Supplementary Table 3-S1). Volcano plot analysis (Fig. 3-4B) indicated that the number of data points that reached statistical significance (p<0.5; in red) in the

Saline vs NR group were greater than the losartan vs NR comparison. Independent of behavior, direct comparison between saline and losartan groups identified 13 unique

DEGs at a statistical threshold of p<0.1 and one unique gene at p<0.05.

104

As compared to the non-retrieval control group, the differential expression analysis revealed that most of the DEGs were significantly upregulated in the saline- and losartan-treated mice (Fig. 3-4B). Among the DEGs that were significantly upregulated,

23 common genes were identified (Fig. 3-4C). As shown in Fig. 3-4D, the majority of the 23 common genes (white background) upregulated following retrieval are IEGs.

Significantly higher levels of specific gene expression (black background 67 genes, p<0.05) were identified in the saline vs NR group as compared to losartan vs NR (grey background, 15 genes, p<0.05). To further highlight this distinction, the top DEGs of highest statistical (p<0.01) are displayed in the corresponding boxes of Fig. 3-4D.

Gene ontology analysis on the DEGs showed redistribution of the biological processes between the saline vs NR and Losartan vs NR groups. Particular biological processes undergoing changes included biological adhesion (7.8% vs 2.3%), biological regulation (28.4% vs 38.6%) and immune system processes (2.6% vs 0) (Fig. 3-5A). The top three deregulated pathways estimated by Metacore, Clarivate Analytics were

Gonadotropin-Releasing hormone signaling, Neurogensis, and AP-1 in the cellular metabolism, and these pathways were shared between Saline-NR and Losartan-NR groups (Fig. 3-5B). Due to the limited number DEGs in the saline vs losartan groups (13 genes), direct comparison for the pathway analysis was not performed.

DISCUSSION

Our findings demonstrate that losartan administered following memory retrieval reduces long-term freezing behavior and modifies differentially-expressed genes in the amygdala, while leaving conditioned blood pressure and heart rate responses intact.

105

These results suggest that post-retrieval administration of losartan can modify reconsolidation processes, advancing our current understanding for the role of the brain

RAS in fear-related memory.

In addition to losartan, other cardiovascular drug targets (Brunet et al., 2008) and behavioral interventions (Monfils et al., 2009; Zuccolo and Hunziker, 2019) are currently being investigated for their potential to disrupt reconsolidation and reduce the strength of fear memories. For example, the FDA-approved β-adrenergic receptor antagonist propranolol has gained significant attention as a potential pharmacotherapeutic for disrupting reconsolidation in humans with PTSD is (Pitman et al., 2013). However, conflicting findings have been reported in both animal models and human clinical trials.

In some clinical studies, propranolol has been shown to effectively reduce physiological and behavioral fear responses and PTSD symptoms (Brunet et al., 2008; Kindt et al.,

2009), without measurable effects on memory reconsolidation in other studies (McGhee et al., 2009). A discrepancy is also seen between several pre-clinical studies, with some showing that propranolol effectively reduces fear responses and interferes with memory reconsolidation (Dębiec et al., 2011; Taherian et al., 2014; Vetere et al., 2013), while others do not (Muravieva and Alberini, 2010; Villain et al., 2016). While the clinical efficacy for propranolol as a treatment for PTSD continues to be debated, renewed interest in memory reconsolidation has led to the study of many alternative compounds that may be of translational value (Hou et al., 2015b; Meloni et al., 2014).

Similar to propranolol, losartan is an FDA-approved drug used to treat high blood pressure that can also modify memory processes in normotensive subjects (Marvar et al.,

2014; Pulcu et al., 2019a; Zhou et al., 2019). The ability of the RAS to modulate central

106

memory processes may explain early retrospective clinical observations between RAS inhibition and reduced PTSD symptom severity (Khoury et al., 2012a).

To further interrogate the memory effects of RAS blockade, we used Pavlovian auditory fear conditioning in which a single CS retrieval was used to initiate memory destabilization in mice. The necessity of AT1R for this process was determined by pharmacological inhibition during the window of reconsolidation, which in rodents lasts approximately 4-6hr (Nader et al., 2000; Sara, 2000). Given the evidence for its role in the formation (Bonini et al., 2006; Delorenzi et al., 1995; Kerr et al., 2005), expression

(Hurt et al., 2015), and reconsolidation (Frenkel et al., 2005) of fear memories, we hypothesized that blockade of AT1R signaling during the window of reconsolidation would decrease fear responses. In support of our hypothesis, peripheral injection of losartan shortly after memory retrieval reduced freezing behavior both 1 day and 7 days later. Importantly, this reduction was only observed when the losartan was paired with the CS retrieval as losartan had no effect on non-retrieval control animals. These data suggest a reconsolidation-specific drug effect, consistent with previous reports identifying a role for Ang II in memory reconsolidation across species (Frenkel et al.,

2005; Sierra et al., 2013).

We recently showed that the acute conditioned blood pressure response to an auditory stimulus can be attenuated by extinction training, consisting of repeated re- exposure to the stimulus (Swiercz et al., 2018). Here, we sought to determine whether post-retrieval losartan could affect conditioned cardiovascular responses independent of extinction learning. In contrast to the effects we observed on freezing behavior, post- retrieval losartan did not modify the conditioned cardiovascular response 24 hours or 1

107

week post-retrieval. The lack of change in the conditioned cardiovascular response may be due to procedural differences between the telemetry and non-telemetry studies. To our knowledge, this is the first study to examine reconsolidation disruption in rodents by assessing the conditioned cardiovascular response. In keeping with previous reports

(Dębiec and Ledoux, 2004; Dębiec et al., 2011), memory reactivation and testing occurred in the same context, which in this case was the homecage. It is possible that memory reactivation in the homecage does not initiate reconsolidation in the same way as reactivation in a novel context. Future studies are needed to determine how reactivation in the homecage may affect reconsolidation processes.

Recent studies raise the possibility that the physiological or interoceptive state of an animal during reconsolidation may contribute to memory updating. For example in rats, an aversive cue can become a desirable one if the animals are placed in a different physiological state (Robinson and Berridge, 2013) when they first re-encounter the cue.

In our experimental design, mice received an intraperitoneal injection of either losartan or saline 10 minutes following retrieval. It is possible that the increased stress associated with scruffing and handling during the injection could impact the strength of the memory and the expression of associated fear responses. Indeed, we found that the acute post- retrieval administration of losartan significantly blunts the blood pressure response evoked by the scruffing and injection procedure. Given the importance of the physiological state of the animal and the role of emotional valence in memory updating

(Villain et al., 2016), we speculate that this acute and transient blood pressure lowering effect of post-retrieval losartan may contribute to the freezing reductions observed during memory testing.

108

In addition to the cardiovascular and behavioral measures of fear following post- retrieval losartan, we also evaluated whole genome transcriptional differences in the basolateral amygdala (BLA), a key structure required for acquisition, extinction, and reconsolidation of learned fear (Gale, 2004)(Nader et al., 2000). Compared to the NR group, several DEGs were identified 40 minutes after losartan administration and during the window of reconsolidation (48 vs 133 DEGs), with a large overlap of genes (32) between the two treatment conditions. This overlap may be attributed to the behavioral intervention or the reconsolidation process. Most of the common IEGs between the saline and losartan groups are involved in memory and learning processes and controlled by CREB-mediated phosphorylation. These data support previous studies demonstrating the role of Egr-1 (Maddox and Schafe, 2011), Fos (Hall et al., 2001), and Arc (Mamiya et al., 2009) as essential for markers for memory consolidation in the lateral amygdala and hippocampus.

Our RNA sequencing results identified 90 DEGs following retrieval in the saline group, but only 38 DEGs following retrieval in the losartan-treated group in the amygdala. This reduction in DEGs may suggest that post-retrieval losartan down- regulates a majority of genes which are activated during retrieval. For example, Egr2 and

Egr3, involved in neuronal plasticity and immune system activation (O’Donovan et al.,

1999; Taefehshokr et al., 2017), were differentially expressed only in the saline vs NR group. Other major biological processes implicated from the enrichments of DEGs in the losartan vs NR group include the negative regulation of the ERK cascade and MAPK activity, which are both involved in downstream AT1R signaling (Yoshida et al., 2013).

Only the losartan vs NR group comparison showed differential expression for junction

109

proteins Claudin 5 and Gjb6. These results suggest that post-retrieval losartan is associated with a disparity in gene expression patterns and biological responses, but this effect is overshadowed by the dynamic transcriptional and epigenetic processes which are predominant at this early time point of reconsolidation.

In conclusion, we found that post-retrieval losartan contributes to long-term reductions in freezing behavior and alterations in central gene expression that occur independent of conditioned cardiovascular reactivity. These effects may be mediated in part by differences in the physiological state of the animals, as a single post-retrieval losartan injection lowered blood pressure during the window of reconsolidation.

Collectively, these studies add to the growing body of evidence describing RAS contributions to fear learning and memory, and suggest that targeting the AT1R may be a valid therapeutic approach during reconsolidation. The ability of systemic AT1R inhibition to both facilitate extinction (Marvar et al., 2014; Zhou et al., 2019), and to modify reconsolidation as shown here, suggest that AT1R blockade may be unique in its directional effects on fear memory, and important for the development of novel PTSD treatments.

110

Figure 3-1: Post-retrieval Losartan Reduces Freezing Behavior. (A) Schematic of the conditioning, retrieval, and testing protocol. (B) Average freezing during the 5th CS presentation of fear conditioning. (C) Freezing behavior before and during the 1CS retrieval cue. Non-retrieval groups (-) did not receive cue exposure. Freezing response to 4 CS presentations during the 24hr (LTM-1d) (D) and 1wk (LTM-7d) (E) memory tests in retrieval groups. Freezing response to 4 CS presentations during the LTM-1d test (F) and LTM-7d test (G) in non-retrieval groups. (n = 11-13 per group, #saline vs. losartan p<0.05, *Bonferroni post hoc p<0.05)

111

Figure 3-2: Post-retrieval losartan does not alter recall of conditioned cardiovascular responses. (A) Schematic of the conditioning, retrieval, and testing protocol used in the radio telemetry experiments. (B) Average MAP during 5 minute baseline (Pre-CS) and throughout 4 CS presentations during the 24hr (LTM-1d) test. (C) Change in MAP during each 30s CS relative to the Pre-CS period during the LTM-1d test. (D) Average HR during 5 minute baseline (Pre-CS) and throughout 4 CS presentations during the LTM-1d test. (E) Change in HR during each 30s CS relative to the Pre-CS period during the LTM-1d test. (F) Average MAP during 5 minute baseline (Pre-CS) and throughout 4 CS presentations during the 1wk (LTM-7d) test. (G) Change in MAP during each 30s CS relative to the Pre-CS period during the LTM-7d test. (H) Average HR during 5 minute baseline (Pre-CS) and throughout 4 CS presentations during the LTM-7d test. (I) Change in HR during each 30s CS relative to the Pre-CS period during the LTM-7d test. (n = 4-6/group).

112

Figure 3-3: Losartan attenuates stress-induced increases in blood pressure following handling and injection. (A) Schematic of the conditioning, retrieval, and injection protocol used in the radio telemetry experiments. (B) MAP throughout the 2 hr post- injection period (data expressed in 3 min bins) (C) Average mean arterial pressure (MAP) during 30 minute pre-injection period and throughout the 2 hr post-injection period. (D) HR throughout the 2 hr post-injection period (data expressed in 3 min bins). (E) Average heart rate (HR) during 30 minute pre-injection period and throughout the 2 hr post-injection period (n = 4-6/group, *Bonferroni post hoc p<0.05)

113

Figure 3-4: Differential BLA gene expression analysis following post-retrieval losartan. (A) Quantitative RT-PCR showing the increased level of Immediate early genes at 40 minutes in both saline and losartan groups as compared to NR. (n=8 to 10; Error bars are ± SEM. *p % 0.05, ***p <0.001 by One way ANOVA – Tukey’s test). (B) Volcano plots depicting the changes in the expression of 90 genes in saline vs NR and 38 genes in Losartan vs NR. Each dot represents a gene with red dots representing genes with corrected p-value < 0.05 and blue dots with p-value >0.05. (C) Heat map representing the 23 common genes which are differentially expressed in both saline and losartan treated groups after memory retrieval compared to NR. Each line represents a DEG and blue and green indicate the low and high levels of expression respectively. (D) Venn Diagram illustrates the number and names of the common and uncommon genes between the two comparisons. The genes with significantly altered expression in both saline vs NR and losartan vs NR are in white background, genes with differential expression in only saline vs NR but not in losartan vs NR are in black background and genes with differential expression in only losartan vs NR but not in saline vs NR gray background (p<0.05). The names of genes with significant change (p<0.01) in expression are listed for each group with blue and red showing increased and decreased fold change value.

114

Figure 3-5: Transcriptional profiles and pathways (A) Chart showing the biological processes significantly enriched by the differentially-expressed genes (DEGs) in each group. (B) Pathway analysis by PANTHER software of the DEGs in saline vs NR and losartan vs NR groups.

115

Supplementary Tables:

Supplementary Table 3-S1: Genes differentially expressed in Saline compared to NR group in BLA

Saline vs. NR Gene log FoldChange p-value q-value Arc 1.670122738 4.17E-26 7.47E-22 Fos 2.088462087 6.52E-24 5.84E-20 Sgk1 1.192721736 8.56E-23 5.11E-19 Ddit4 1.010506867 7.09E-19 3.18E-15 Junb 1.031365846 5.30E-18 1.90E-14 Nr4a1 1.33370792 8.12E-17 2.42E-13 Sik1 1.049751381 2.37E-16 6.06E-13 Btg2 1.287781238 4.89E-13 1.09E-09 Egr1 1.0766593 6.94E-11 1.38E-07 Dusp1 1.057409908 1.98E-09 3.55E-06 Zfp189 0.727676798 1.00E-08 1.64E-05 Tiparp 0.693838432 2.33E-08 3.47E-05 Rasd2 0.611288341 2.97E-08 4.09E-05 Ccn1 1.119138869 1.23E-07 0.000157343 Npas4 1.020217028 1.98E-07 0.000236099 Tle3 0.732197815 2.22E-07 0.000247913 Arl4d 1.05901029 4.30E-07 0.000453073 Plin4 0.901785364 9.13E-07 0.000860326 Dlgap3 0.450589575 8.87E-07 0.000860326 Irf2bpl 0.508504203 1.30E-06 0.001161593 Fosl2 0.845775374 1.60E-06 0.001361857 Hsph1 0.382680553 2.40E-06 0.001871962 Ergic2 -0.302646481 2.32E-06 0.001871962 Bahd1 0.477595173 2.77E-06 0.0020283 Tob2 0.507015832 2.83E-06 0.0020283 Fosb 1.001849683 3.22E-06 0.002085223 Pim3 0.444052487 3.26E-06 0.002085223 Plekhf1 0.934439218 3.07E-06 0.002085223 Adgrb1 0.421183159 3.89E-06 0.00239905 Egr4 0.949334157 4.29E-06 0.002559197 Chrm4 0.394892239 4.45E-06 0.002571946 Tent5a 0.541699072 4.78E-06 0.00267644 Mycbp 0.694743677 4.97E-06 0.002698782 Gadd45g 0.6376187 5.74E-06 0.003024049 Egr2 1.110948374 6.53E-06 0.003246105

116

Gm49373 1.013010119 7.93E-06 0.003840286 Midn 0.404770846 1.05E-05 0.004810648 Rps13-ps2 -0.032966606 1.07E-05 0.004810648 Gm49496 0.062245404 1.07E-05 0.004810648 Trib1 0.479309763 1.14E-05 0.004893827 Epop 0.555772505 1.21E-05 0.005019025 Nhlrc3 -0.427230841 1.35E-05 0.005487693 Gadd45b 0.488012138 1.68E-05 0.006702126 Zcchc14 0.366885553 1.76E-05 0.00684768 Mycn 0.644459595 2.04E-05 0.007772988 Pitpnc1 0.389941018 2.38E-05 0.008864576 Egr3 0.372435639 2.57E-05 0.009377384 Slc41a1 0.305685844 2.84E-05 0.00979061 Mdm1 -0.383750536 2.79E-05 0.00979061 Irs2 0.462683023 2.83E-05 0.00979061 Gfap 0.333282511 4.16E-05 0.014045403 Nckap5l 0.498871809 4.37E-05 0.014431959 Ier5l 0.48905082 4.43E-05 0.014431959 Actn1 0.347040308 4.77E-05 0.015237411 Rnf165 0.533418872 5.45E-05 0.017124475 Thbs4 0.20545421 6.04E-05 0.01832471 Csrnp1 0.406324712 5.94E-05 0.01832471 Jun 0.429523081 6.30E-05 0.018810349 Tob1 0.33395819 6.44E-05 0.018893197 Rbfox3 0.337711136 6.63E-05 0.019145393 Agap2 0.298345496 7.19E-05 0.020448231 Capn11 0.340976499 7.62E-05 0.02131227 Dact1 0.41583226 8.45E-05 0.02328141 Hlf 0.312229177 9.91E-05 0.024919213 Corin 0.527720519 0.000101455 0.024919213 Sulf2 0.27473799 0.000100525 0.024919213 Dlg4 0.317281306 9.75E-05 0.024919213 Epas1 0.311586363 9.57E-05 0.024919213 Jph3 0.235178646 0.000100324 0.024919213 Nab2 0.294127438 9.93E-05 0.024919213 Rbm39 -0.262112984 0.000102978 0.024919213 Zfp945 -0.292121346 0.000102951 0.024919213 Utp14b 0.307994558 0.000120139 0.028684494 Kctd18 -0.267962572 0.000130691 0.030793288 Mn1 0.318293874 0.000139127 0.03235519 Hipk2 0.310935285 0.000141831 0.032561013 Cacnb3 0.264576268 0.000159668 0.036192068 Samd14 0.291569186 0.000161739 0.036203209

117

Mknk2 0.273985677 0.000164713 0.03641382 Trp53i11 0.307710436 0.000173889 0.037973475 Col5a2 -0.355181554 0.000178038 0.038411221 Cnih2 0.250778087 0.000181393 0.038669161 Ccdc117 0.291344704 0.000186361 0.039260756 Gper1 -0.412769972 0.000198099 0.041248291 Tnk2 0.2567614 0.000206699 0.042209271 Map7d1 0.289178665 0.000207428 0.042209271 Zfp867 -0.313468947 0.000229864 0.046249132 Dusp5 0.314495991 0.000232922 0.046343796 Flrt1 0.274560902 0.000235608 0.046362922 Zbtb7a 0.261714675 0.00027559 0.053077023 Spsb1 0.299425335 0.000275656 0.053077023 Jam2 0.231205878 0.000282436 0.05380399 Jph4 0.260265745 0.000290118 0.054685712 Itga10 0.341668488 0.000324141 0.060462362 Rhobtb2 0.237114466 0.000335996 0.062027676 Gm12895 0.268874973 0.000368495 0.065986448 Gm12896 0.268874973 0.000368495 0.065986448 Inf2 0.261003805 0.000388556 0.068437162 Ptk2b 0.23498572 0.000389825 0.068437162 Cd4 0.214672601 0.000395439 0.068748864 Adra2a 0.283991275 0.000407637 0.069519521 Gm14760 0.024094238 0.000405172 0.069519521 Col16a1 -0.263050954 0.000417527 0.070534423 Pcdhgc3 0.245202896 0.000431667 0.072241654 Armcx5 -0.200870818 0.000468103 0.077614077 Rexo1 0.242841343 0.000497292 0.0816974 Epb41l1 0.185959691 0.000519444 0.083798994 Fmnl1 0.256935585 0.000516161 0.083798994 5031439G07Rik 0.21958427 0.000571919 0.091440703 Tns3 0.229690474 0.000634166 0.091593219 Sox8 0.232179784 0.000626226 0.091593219 Cplx2 0.233528716 0.000639367 0.091593219 Plekhm2 0.218458506 0.000603643 0.091593219 Egln1 0.22551396 0.000597147 0.091593219 Cip2a -0.237648519 0.000579071 0.091593219 Chst2 0.19830471 0.000611035 0.091593219 Rims4 0.199304864 0.000639194 0.091593219 Srcin1 0.235056413 0.000586667 0.091593219 Stau1 0.223289054 0.00061592 0.091593219 Begain 0.221348375 0.000610867 0.091593219 Hepacam 0.228937418 0.000637989 0.091593219

118

Upf1 0.230713817 0.000630057 0.091593219 Tia1 -0.208481949 0.000646531 0.091884372 Ttyh3 0.228269287 0.000670971 0.093388398 Zfp319 0.22730828 0.000672759 0.093388398 Mest 0.204280279 0.000667943 0.093388398 Heatr6 -0.217610011 0.000717196 0.098717538 Mysm1 -0.212321493 0.000722176 0.098717538 Adipor2 0.207384878 0.000729162 0.098917439 Sox11 0.220369216 0.000735288 0.098998503

Supplementary Table 3-S2: Genes differentially expressed in Losartan compared to NR group in BLA

Losartan vs. NR Gene log Fold Change p-value q-value Arc 1.634631797 2.84E-25 4.98E-21 Sgk1 1.169663206 2.18E-22 1.90E-18 Fos 1.961258567 2.96E-21 1.72E-17 Junb 0.912550607 9.03E-15 3.95E-11 Zfp189 1.009161066 2.14E-14 7.48E-11 Ddit4 0.851950774 4.33E-14 1.26E-10 Btg2 1.230584924 2.00E-12 5.01E-09 Nr4a1 1.077036039 6.75E-12 1.48E-08 Sik1 0.794917465 3.54E-10 6.89E-07 Gadd45b 0.921404805 1.34E-09 2.34E-06 Tiparp 0.790532802 1.67E-09 2.66E-06 Dusp1 1.017786685 3.57E-09 5.20E-06 Plin4 1.072486982 1.35E-08 1.81E-05 Arl4d 1.006654771 8.28E-07 0.001035338 Gjb6 0.658234827 1.58E-06 0.001848986 Plekhf1 0.919431881 3.94E-06 0.004309373 Nab2 0.449047825 7.70E-06 0.007926214 Dio2 0.41719727 8.22E-06 0.007990189 Cldn5 -0.647048669 9.53E-06 0.008778521 Tctn1 -0.437225125 1.36E-05 0.01186722 Dusp6 0.501407306 1.66E-05 0.013857919 Gm3591 -0.072706559 1.77E-05 0.014055497 Gfap 0.418284336 2.12E-05 0.014962328 Gadd45g 0.613683206 2.14E-05 0.014962328 Trp53inp1 0.388230928 2.08E-05 0.014962328

119

Rasl11a 0.679476503 2.36E-05 0.015867901 Heatr6 -0.409448448 2.73E-05 0.017684098 Tsc22d3 0.35867299 3.66E-05 0.022893818 Tle3 0.487546069 4.35E-05 0.02626233 Sox8 0.448010637 5.06E-05 0.02954472 Hps1 -0.498057356 5.79E-05 0.030574672 Mycbp 0.508546951 6.07E-05 0.030574672 Pcgf3 0.337809596 6.11E-05 0.030574672 Dusp5 0.5012408 5.73E-05 0.030574672 Klf10 0.402658594 5.78E-05 0.030574672 Tob2 0.395591401 6.52E-05 0.031694329 Nckap5l 0.48505627 7.46E-05 0.03530243 Egr1 0.418020057 9.21E-05 0.042443904 Errfi1 0.233516791 0.000116803 0.051569216 Fam171a2 -0.185220726 0.000117839 0.051569216 Stard8 -0.326963775 0.000142792 0.0609654 Chrm4 0.230282112 0.00015968 0.066552215 Nemp2 -0.34086457 0.000195967 0.079776783 Fosb 0.307650271 0.000266947 0.097929971 Mycn 0.301224571 0.000257652 0.097929971 Kctd18 -0.146908374 0.000268531 0.097929971 Rps13-ps2 -0.011551793 0.000261168 0.097929971 Gm49496 0.013315828 0.000267613 0.097929971

120

REFERENCES:

Bray, N.L., Pimentel, H., Melsted, P., and Pachter, L. (2016). Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525–527.

Brunet, A., Orr, S.P., Tremblay, J., Robertson, K., Nader, K., and Pitman, R.K. (2008). Effect of post-retrieval propranolol on psychophysiologic responding during subsequent script-driven traumatic imagery in post-traumatic stress disorder. Journal of Psychiatric Research 42, 503–506.

Clem, R.L., and Huganir, R.L. (2010). Calcium-Permeable AMPA Receptor Dynamics Mediate Fear Memory Erasure. Science 330, 1108–1112.

Dębiec, J., and Ledoux, J.E. (2004). Disruption of reconsolidation but not consolidation of auditory fear conditioning by noradrenergic blockade in the amygdala. Neuroscience 129, 267–272.

Delorenzi, A., Pedreira, M.E., Romano, A., Pirola, C.J., Nahmod, V.E., and Maldonado, H. (1995). Acute administration of angiotensin II improves long-term habituation in the crab Chasmagnathus. Neuroscience Letters 196, 193–196.

Gale, G.D. (2004). Role of the Basolateral Amygdala in the Storage of Fear Memories across the Adult Lifetime of Rats. Journal of Neuroscience 24, 3810–3815.

Gazarini, L., Stern, C.A.J., Carobrez, A.P., and Bertoglio, L.J. (2013). Enhanced noradrenergic activity potentiates fear memory consolidation and reconsolidation by differentially recruiting α1- and β-adrenergic receptors. Learn. Mem. 20, 210–219.

Hall, J., Thomas, K.L., and Everitt, B.J. (2001). Fear memory retrieval induces CREB phosphorylation and Fos expression within the amygdala. European Journal of Neuroscience 13, 1453–1458.

Hou, Y., Zhao, L., Zhang, G., and Ding, L. (2015). Effects of oxytocin on the fear memory reconsolidation. Neuroscience Letters 594, 1–5.

121

Kindt, M., Soeter, M., and Vervliet, B. (2009). Beyond extinction: erasing human fear responses and preventing the return of fear. Nature Neuroscience 12, 256–258.

Lazaroni, T.L.N., Raslan, A.C.S., Fontes, W.R.P., de Oliveira, M.L., Bader, M., Alenina, N., Moraes, M.F.D., dos Santos, R.A., and Pereira, G.S. (2012). Angiotensin-(1–7)/Mas axis integrity is required for the expression of object recognition memory. Neurobiology of Learning and Memory 97, 113–123.

Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408.

Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550.

Mamiya, N., Fukushima, H., Suzuki, A., Matsuyama, Z., Homma, S., Frankland, P.W., and Kida, S. (2009). Brain region-specific gene expression activation required for reconsolidation and extinction of contextual fear memory. J. Neurosci. 29, 402–413.

Marinzalda, M. de los A., Pérez, P.A., Gargiulo, P.A., Casarsa, B.S., Bregonzio, C., and Baiardi, G. (2014). Fear-Potentiated Behaviour Is Modulated by Central Amygdala Angiotensin II Receptors Stimulation. BioMed Research International 2014, 1–7.

McGhee, L.L., Maani, C.V., Garza, T.H., Desocio, P.A., Gaylord, K.M., and Black, I.H. (2009). The effect of propranolol on posttraumatic stress disorder in burned service members. J Burn Care Res 30, 92–97.

122

Meloni, E.G., Gillis, T.E., Manoukian, J., and Kaufman, M.J. (2014). Xenon Impairs Reconsolidation of Fear Memories in a Rat Model of Post-Traumatic Stress Disorder (PTSD). PLoS One 9.

Merlo, E., Milton, A.L., Goozee, Z.Y., Theobald, D.E., and Everitt, B.J. (2014). Reconsolidation and Extinction Are Dissociable and Mutually Exclusive Processes: Behavioral and Molecular Evidence. Journal of Neuroscience 34, 2422–2431.

Monfils, M.-H., Cowansage, K.K., Klann, E., and LeDoux, J.E. (2009). Extinction- Reconsolidation Boundaries: Key to Persistent Attenuation of Fear Memories. Science 324, 951–955.

Muravieva, E.V., and Alberini, C.M. (2010). Limited efficacy of propranolol on the reconsolidation of fear memories. Learn. Mem. 17, 306–313.

Nader, K., Schafe, G.E., and Le Doux, J.E. (2000). Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature 406, 722–726.

Nader, K., Hardt, O., and Lanius, R. (2013). Memory as a new therapeutic target. Dialogues Clin Neurosci 15, 475–486.

Nylocks, K.M., Michopoulos, V., Rothbaum, A.O., Almli, L., Gillespie, C.F., Wingo, A., Schwartz, A.C., Habib, L., Gamwell, K.L., Marvar, P.J., et al. (2015). An angiotensin- converting enzyme (ACE) polymorphism may mitigate the effects of angiotensin- pathway medications on posttraumatic stress symptoms. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 168, 307–315.

O’Donovan, K.J., Tourtellotte, W.G., Millbrandt, J., and Baraban, J.M. (1999). The EGR family of transcription-regulatory factors: progress at the interface of molecular and systems neuroscience. Trends in Neurosciences 22, 167–173.

Paxinos, G., and Watson, C. (1998). The Rat Brain in Stereotaxic Coordinates (Deluxe Edition), Fourth Edition (San Diego: Academic Press).

Pitman, R.K., Bolshakov, V.Y., Brunet, A., Gamache, C., and Nader, K. (2013). Developing memory reconsolidation blockers as novel PTSD treatments (MASSACHUSETTS GENERAL HOSPITAL BOSTON).

Reinecke, A., Browning, M., Klein Breteler, J., Kappelmann, N., Ressler, K.J., Harmer, C.J., and Craske, M.G. (2018). Angiotensin Regulation of Amygdala Response to Threat

123

in High-Trait-Anxiety Individuals. Biological Psychiatry: Cognitive Neuroscience and Neuroimaging.

Robinson, M.J.F., and Berridge, K.C. (2013). Instant transformation of learned repulsion into motivational “wanting.” Curr. Biol. 23, 282–289.

Roehr, J.T., Dieterich, C., and Reinert, K. (2017). Flexbar 3.0 – SIMD and multicore parallelization. Bioinformatics 33, 2941–2942.

Sara, S.J. (2000). Retrieval and Reconsolidation: Toward a Neurobiology of Remembering. Learn. Mem. 7, 73–84.

Swiercz, A.P., Seligowski, A.V., Park, J., and Marvar, P.J. (2018). Extinction of Fear Memory Attenuates Conditioned Cardiovascular Fear Reactivity. Frontiers in Behavioral Neuroscience 12.

Taefehshokr, S., Key, Y.A., Khakpour, M., Dadebighlu, P., and Oveisi, A. (2017). Early growth response 2 and Egr3 are unique regulators in immune system. Cent Eur J Immunol 42, 205–209.

Taherian, F., Vafaei, A.A., Vaezi, G.H., Eskandarian, S., Kashef, A., and Rashidy-Pour, A. (2014). Propranolol-induced Impairment of Contextual Fear Memory Reconsolidation in Rats: A similar Effect on Weak and Strong Recent and Remote Memories. Basic Clin Neurosci 5, 231–239.

Thomas, P.D., Campbell, M.J., Kejariwal, A., Mi, H., Karlak, B., Daverman, R., Diemer, K., Muruganujan, A., and Narechania, A. (2003). PANTHER: A Library of Protein Families and Subfamilies Indexed by Function. Genome Res. 13, 2129–2141.

Vetere, G., Piserchia, V., Borreca, A., Novembre, G., Aceti, M., and Ammassari-Teule, M. (2013). Reactivating fear memory under propranolol resets pre-trauma levels of dendritic spines in basolateral amygdala but not dorsal hippocampus neurons. Front. Behav. Neurosci. 7.

Wang, X., Li, M., Zhu, H., Yu, Y., Xu, Y., Zhang, W., and Bian, C. (2018). Transcriptional Regulation Involved in Fear Memory Reconsolidation. Journal of Molecular Neuroscience.

124

Yoshida, T., Tabony, A.M., Galvez, S., Mitch, W.E., Higashi, Y., Sukhanov, S., and Delafontaine, P. (2013). Molecular mechanisms and signaling pathways of angiotensin II-induced muscle wasting: potential therapeutic targets for cardiac cachexia. Int J Biochem Cell Biol 45, 2322–2332.

Yu, Z., Swiercz, A.P., Moshfegh, C.M., Hopkins, L., Wiaderkiewicz, J., Speth, R.C., Park, J., and Marvar, P.J. (2019). Angiotensin II Type 2 Receptor–Expressing Neurons in the Central Amygdala Influence Fear-Related Behavior. Biological Psychiatry S0006322319314441.

Zuccolo, P.F., and Hunziker, M.H.L. (2019). A review of boundary conditions and variables involved in the prevention of return of fear after post-retrieval extinction. Behav. Processes 162, 39–54.

125

CHAPTER 4

Angiotensin II Type 2 Receptor-Expressing Neurons in the Central Amygdala Influence Fear-Related Behavior

*Yu, Z., *Swiercz, A.P., Moshfegh, C.M., Hopkins, L., Wiaderkiewicz, J., Speth, R.C., Park, J., and Marvar, P.J. (2019). Biological Psychiatry S0006322319314441.

(Used with permission)

126

ABSTRACT

Background: The renin-angiotensin system (RAS) has been implicated in posttraumatic stress disorder (PTSD), however the mechanisms responsible for this connection and the therapeutic potential of targeting the RAS in PTSD remains unknown.

Using an angiotensin receptor bacterial artificial chromosome (BAC) reporter mouse combined with neuroanatomical, pharmacological and behavioral approaches we examined the role of angiotensin II type 2 receptor (AT2R) in fear-related behavior.

Methods: Dual immunohistochemistry with retrograde labeling was used to characterize

AT2R-eGFP+ cells in the amygdala of the AT2R-eGFP-BAC reporter mouse. Pavlovian fear conditioning and behavioral pharmacology analyses were used to demonstrate the effects of AT2R activation on fear memory in male C57BL/6 mice.

Results: AT2R-eGFP+ neurons in the amygdala were predominantly expressed in the medial amygdala (MeA) and the medial division of the central amygdala (CeM), with little AT2R-eGFP expression in the basolateral amygdala (BLA) or lateral division of the central amygdala (CeL). Characterization of AT2R-eGFP+ neurons in the CeM demonstrated distinct localization to GABAergic projection neurons. Mice receiving acute intra-central amygdala (CeA) injections of the selective AT2R agonist compound

21 (C21) prior to cued or contextual fear expression tests displayed less freezing.

Retrograde labeling of AT2R-eGFP+ neurons projecting to the periaqueductal gray revealed AT2R-eGFP+ neuronal projections from the CeM to the periaqueductal gray, a key brain structure mediating fear-related freezing. Conclusion: These findings suggest that CeM AT2R-expressing neurons can modulate CeA outputs that play a role in fear

127

expression and provide new evidence for an angiotensinergic circuit and CeM cell type in the regulation of fear.

INTRODUCTION

Current evidence based treatment options for post-traumatic stress disorder

(PTSD), including psychotherapy and pharmacological approaches provide limited benefits in a substantial proportion of individuals (Berger et al., 2009; Lee et al., 2016;

Ravindran and Stein, 2009, 2010), and treatment mechanisms are not fully understood.

An improved neurobiological understanding of this disorder is therefore critical for the development of new treatment and prevention strategies. A growing body of evidence implicates the renin angiotensin system (RAS), a regulator of blood pressure and fluid homeostasis, as a potential therapeutic target for PTSD (Khoury et al., 2012b; Marvar et al., 2014; Nylocks et al., 2015; Reinecke et al., 2018; Terock et al., 2019). Retrospective clinical studies suggest that RAS blockade reduces the severity of PTSD symptoms

(Khoury et al., 2012b; Nylocks et al., 2015) and more recently the angiotensin receptor blocker losartan was shown to affect amygdala activity and emotional processing in high trait anxiety individuals (Pulcu et al., 2019b; Reinecke et al., 2018). Pre-clinical studies confirm and extend these findings by demonstrating that angiotensin type 1 receptor

(AT1R) inhibition (Lazaroni et al., 2016; Marvar et al., 2014; Parrish et al., 2019) or deletion of AT1R from select neuronal populations in the brain facilitates fear extinction

(Hurt et al., 2015). Collectively, these data suggest that the brain RAS may be an important therapeutic target in PTSD, however the underlying mechanisms and the potential roles of other central angiotensin II receptors remain unclear.

128

Angiotensin II is the principal effector peptide of the RAS and mediates its effects by binding to two primary receptor subtypes; the AT1R and the angiotensin type 2 receptor (AT2R) (Chappell, 2015; Inagami et al., 1999). Both AT1R and AT2R subtypes are expressed throughout the brain, in similar regions but on different cellular populations

(Gonzalez et al., 2012; de Kloet et al., 2016b; Lenkei et al., 1996; Lind et al., 1985). In addition, the angiotensin II peptide can have both direct excitatory and inhibitory effects in the brain (und Halbach and Albrecht, 1998; Tchekalarova and Albrecht, 2007). While the AT1R in hypothalamic, forebrain, and brainstem regions mediating cardiovascular and neuroendocrine regulation are widely studied (Coble et al., 2015; Davisson et al.,

2000; Krause et al., 2011), much less is known regarding brain AT2R.

AT2R is considered the protective arm of the RAS (Kloet et al., 2017; Sumners et al., 2013) as it exerts functional effects (e.g. anti-inflammatory, anti-proliferative, antihypertensive) that oppose those produced by AT1R. Pre-clinical studies using the selective non-peptide AT2R agonist Compound 21 (C21) have shown therapeutic potential for the reduction of hypertension, stroke and inflammation (Bennion et al.,

2018; Foulquier et al., 2012; Wan et al., 2004b) and that activation of brain AT2R with

C21 is both neuroprotective (Ahmed et al., 2018; Gelosa et al., 2009; Ishrat et al., 2018) and cognitive enhancing (Jing et al., 2012). Further studies using the AT2R deficient mouse (Agtr2-/-) suggests a role for this receptor in mediating emotional stress and stress- related learning tasks (Bonini et al., 2006; Jan Braszko, 2002; Kerr et al., 2005).

Therefore, to further understand these mechanisms and to build upon growing evidence linking brain angiotensin receptors to fear and anxiety (Hurt et al., 2015; Kerr et al., 2005; Lazaroni et al., 2016; Marinzalda et al., 2014; Marvar et al., 2014), we sought

129

to investigate the role of brain AT2R in fear memory and behavior. Here we examined the distribution, characteristics, and connectivity of AT2R-expressing cells within the brain, testing the hypothesis that site-specific activation of brain AT2R attenuates behavioral and physiological responses to conditioned fear. We propose that endogenous angiotensin II and brain AT1Rs / AT2Rs differentially modulate fear memory through a balance of strengthening excitatory and/or weakening inhibitory synaptic inputs that regulate fear memory. Further understanding the brain angiotensin system in fear learning may provide novel and improved therapeutic strategies for PTSD.

MATERIALS AND METHODS

Animals: All procedures were approved by the Institutional Care and Use

Committee (IACUC) of The George Washington University and were in compliance with

National Institutes of Health guidelines. Adult male C57BL/6J mice (8-10 weeks old) from Jackson Laboratory were housed in a temperature and humidity-controlled room on a 12-hr light/dark cycle with water and food available ad libitum. AT2R-eGFP reporter mice that express eGFP (enhanced green fluorescent protein) in cells that express AT2R

(de Kloet et al., 2016b) were utilized for immunohistochemical (IHC) and tracing studies.

AT2R-eGFP mice were crossed with Gad2-T2A-NLS-mCherry mice (Jackson

Laboratories, Stock #023140) which expresses mCherry in the nuclei of GABAergic neurons.

Immunohistochemistry: To examine the distribution and characteristics of eGFP AT2R-expressing cells within amygdala, mice were anesthetized with Ketamine and Xylazine and perfused transcardially with 4% paraformaldehyde and brains were

130

then post-fixed overnight in the same fixative and transferred into 30% sucrose. After a

2-day dehydration period, brains were embedded in optimal cutting temperature (OCT) compound (Thermo Fisher Scientific, MA) and stored in -80 ℃ until sectioned using a cryostat (CyroStar NX 50, Thermo Fisher Scientific, MA). Additional details are provided in the Supplementary Materials.

Animal Surgery: Mice were anesthetized (i.p) with ketamine/xylazine and stainless steel guide cannulas (0.24mm inner diameter, 0.46 mm outer diameter, Plastics

One, VA) were bilaterally implanted into the CeA at 0.8 mm caudal, ±2.9mm lateral to bregma, and 4.9 mm below the skull surface. Cannulas were fixed in position with dental cement anchored to the skull by a stainless steel screw. Mice were individually housed for a 10-day postoperative period and handled daily for 5 days to familiarize them with the injection procedure.

Drug and Retrograde Tracer Administration: The specific nonpeptide AT2R agonist Compound 21) (Vicore Pharma; Gothenburg, Sweden) (Wan et al., 2004b), with a plasma half-life of approximately 4-6 hr (Gelosa et al., 2009; McCarthy et al., 2014), was infused into the CeA bilaterally (0.06 mg/ml), with an UltraMicroPump III and microprocessor controller (World Precision Instruments, FL) through the internal-cannula

(0.1mm inner diameter, 0.2 mm outer diameter, Plastics One, VA). The intra-CeA C21 concentration was determined based on previous in-vivo application concentration ranges

(Hallberg et al., 2018; Joseph et al., 2014; McCarthy et al., 2014). A total volume of

200nl was injected at a rate of 100 nl/min. For retrograde tracing studies, CTB was injected directly into the periaqueductal grey (PAG) of AT2R-eGFP reporter mice.

Additional details in Supplementary Materials.

131

Pavlovian Fear Conditioning (Cued and Contextual): Cued-fear conditioning was performed to examine the effects of an intra-CeA C21 injection on cue-dependent fear memory as previously described (Marvar et al., 2014; Swiercz et al., 2018). Briefly, mice received 5 CS/US pairings of a 30s auditory cue (6kHz, 75db) co-terminating with a mild footshock (0.5s, 0.5mA) and an inter-trial interval of 5 min. Extinction training was performed 24 hours after fear conditioning. Animals first received bilateral C21 or vehicle injections into the CeA as described above, and 10 min later, were introduced to the extinction chamber. After a 5 min pre-CS period, mice received 20 CS (30s each) with 30s inter-trial intervals. For extinction tests, mice were placed into the same chamber 24 hours after extinction training, and exposed to an additional 20 CS trials.

In a separate group of animals, contextual-fear conditioning was performed to examine the effects of intra-CeA C21 on context-dependent fear memory. Following a 3 min pre-shock period, the animals received a series of 7 electric foot shocks (0.8 mA, 1s) spaced by random intervals varying between 15-120s over 7 minutes. The same procedure was repeated on conditioning day 2. On day 3, animals received bilateral C21 or vehicle injections into the CeA and 10 minutes later re-introduced to the conditioning chamber for a 30 min fear expression test. On day 4, the same procedure was repeated without any drug injection. Percent freezing behavior was recorded and quantified using

Freezeframe 3.32 (Coulbourn Instruments, MA).

Generalized Anxiety Measures: Open field test and elevated plus maze (EPM) tests were used to determine the effect of intra-CeA AT2R activation on basal anxiety levels. Mice were placed in the center of the open field 10 min after C21 or vehicle injection and allowed to freely explore for 30 min. Their activities during this period

132

were recorded and analyzed using the TRU SCAN Activity Monitoring System

(Coulbourn Instruments, PA). For EPM testing, mice were put onto the center area of the maze facing the same closed arm 10 min after C21 or vehicle injection and recorded for 5 min. Total arm entries, open arm entries and the percentage of time spent in the open arm were analyzed using the Top Scan software suite (Clever Sys, VA).

Corticosterone Enzyme-Linked Immunosorbent Assay (ELISA): Plasma corticosterone was measured 30 minutes following contextual fear recall with the commercially available ELISA kit (LDN, Germany). See supplementary materials.

Data Presentation and Statistical Analysis: Data in this study are expressed as mean ± SEM, and p<0.05 were considered statistically significant. All statistical analyses were performed using Prism 6.0 (GraphPad Software Inc., CA). Unpaired two-tailed

Student t tests were used when comparing two groups. When comparing more than two groups a one-way ANOVA followed by Newman-Keuls post hoc test was used.

RESULTS

Intra-Cea C21 Administration Reduces Fear Expression Following Tone-

Dependent and Contextual Fear Conditioning: AT2R-eGFP reporter mice expressed a high level of eGFP in the amygdala, particularly within the central (CeA: 154.5 ± 7.9 cells/mm2) and medial (medial amygdala: 221.2 ± 15.3 cells/mm2) subnuclei, with lower eGFP in the basolateral amygdala (11.7 ± 1.8 cells/mm2) (Fig. 4-1A,B,C). To determine whether CeA-AT2R contribute to expression and extinction of fear memory, we examined the effects of the AT2R agonist (C21) (Wan et al., 2004b) in the CeA (Fig. 4-

1D). Prior to intra-CeA drug injections, both groups exhibited a progressive increase in

133

freezing behavior during the five CS-US pairings (Fig. 4-1F). The next day, animals received CeA microinjections of saline or C21 and exposed to 20 CS trials in a novel context for extinction training (Ext Tr). A reduction in initial fear expression was observed in the C21 group during the first 5 CS block (Vehicle CS 1-5: 75±5 % freezing;

C21 CS 1-5: 55±7 % freezing; F (3,42)=6.52, p<0.05) while during CS 6-20 no differences in percent freezing were found between groups (Vehicle CS 6-20: 52±5; C21

CS 6-20: 38±6; F (3,42)=6.52, p>0.05). Over the course of extinction training (CS1-20) freezing behavior in the vehicle group significantly decreased (Vehicle CS 1-5: 75±5 % freezing, Vehicle CS 6-20: 52±5 % freezing; F (3,42)=6.52, p<0.05), indicating within- session extinction. This within-group reduction did not occur in the C21 animals, likely due to the fact that these animals exhibited significantly lower freezing from the start of the session (Fig. 4-1G).

To determine whether intra-CeA-C21 could modify long-term extinction memory, animals were given a final extinction test (Ext Test) 24 hours later (day 3) in the absence of the drug. The day 3 test reflects extinction recall, and no between-group differences in freezing were observed at this time point (Vehicle CS 1-5: 66±6, vs C21 CS 1-5: 51±9,

F (3,38)=6.27, p>0.05; Vehicle CS 6-20: 35±5, vs C21 CS 6-20: 32±6, F (3,38)=6.27, p>0.05) (Fig. 4-1H). These findings suggest that intra-CeA C21 administration suppresses fear expression to a conditioned auditory cue, while extinction recall twenty- four hours later is unaffected in this model.

Next, to determine whether C21 inhibits the expression of contextual fear memory, a separate group of mice were fear conditioned and 24hr later re-exposed to the conditioned context following intra-amygdala C21 infusions (Fig. 4-1I, J). Animals that

134

received C21 showed a reduced freezing response upon re-exposure to the conditioning context relative to vehicle controls (Vehicle 0-5 min: 69±4 vs C21 0-5 min: 54±4, F

(3,62)=17.56, p<0.05; Vehicle 6-30 min: 43±5 vs C21 6-30 min: 30±3, F (3,38)=6.27, p<0.05) (Fig. 4-1L). 24-hrs later and in the absence of drug, group differences persisted throughout the contextual memory test (Vehicle 0-5 min: 52±7 vs C21 0-5 min: 28±5, F

(3,62)=17.56, p<0.05) (Fig. 4-1M). Together these findings suggest that intra-CeA-C21 has enduring effects following contextual but not cued fear conditioning.

Activation of CeA-AT2R with C21 decreases plasma corticosterone following context re-exposure: Activation of the hypothalamic-pituitary axis (HPA-axis), reflected by an increase in corticosterone levels occurs in response to stress and conditioned fear stimuli (Raio and Phelps, 2015). In a separate animal group, we therefore evaluated the effects of intra-CeA C21 administration during conditioned context re-exposure (Fig 4-

2A,B). In addition to increased freezing behavior, the conditioned context, as expected, elicited a significant rise in plasma corticosterone (Control: 65.8 ±2.6 ng/ml; Context:

98.3 ±10 ng/ml; t=3.14, df=6, p=0.02) (Fig 4-2C) while this response was blunted in mice that received intra-CeA C21 infusions prior to a 5-minute context re-exposure

(Vehicle: 104.2 ±3.9 ng/ml; C21: 76.0 ±8.1 ng/ml; t=3.262, df=13, p=0.006) (Fig 4-2D).

These data suggest HPA-axis modulation by C21 as activation of AT2R in the CeA blunted the release of corticosterone in response to conditioned fear context re-exposure.

Activation of CeA-AT2R with C21 does not alter general anxiety-like behavior or locomotor activity: Treatments that affect anxiety-like behavior or motor activity can confound measurements of freezing behavior. Therefore, to determine whether AT2R activation in the CeA would have off-target effects that might account for

135

the differences observed in our fear conditioning experiments, we injected C21 into the

CeA prior to elevated plus maze (EPM) and open field tests to assess locomotor activity and anxiety-like phenotype (Fig. 4-2E). C21 did not affect the total distance traveled

(Vehicle: 35.7 ±2.6m; C21: 42.1 ±3.6m; t=1.471, df=21, p=0.16) or the number of center entries (Vehicle: 104 ±10; C21: 117 ±9; t=0.975, df=21, p=0.34), although there was a statistical trend for an increase in center time in the open field (Vehicle: 618 ±66s;

C21: 808 ±91s; t=1.741, df=21, p=0.1) was observed (Fig. 4-2F) and as shown in supplemental figure 2. The EPM revealed no group differences in the percentage of time spent in the open arms (Vehicle: 19.7 ±4.7%, n=9; C21: 14.4 ±3.6%; t=0.912, df=17, p=0.37), the number of open arm entries, (Vehicle: 12 ±1, n=9; C21: 11 ±2; t=0.489, df=17, p=0.63) or the total number of open/closed arm entries (Vehicle: 25 ±2; C21: 26

±2; t=0.193, df=17, p=0.85) (Fig. 4-2G). Despite a trend for increased in center time in the open field, there were no group differences in all other parameters measured, suggesting that intra-CeA C21 does not alter locomotor or generalized anxiety behavior.

Distribution and characterization of AT2R+ cells in the amygdala: To further investigate the distribution of AT2R throughout the central amygdala, immunohistochemistry was performed on CeA sections taken from AT2R-eGFP mice

(Fig. 4-3A). As seen in Figure 4-3B,C, AT2R-eGFP+ cells were highly expressed in the medial (CeM) (224.7 ± 9.4 cells/mm2), but not in the lateral (CeL) (3.2 ± 0.8 cells/mm2) regions of the central amygdala. Sections were co-stained with antibodies for PKC-훿, a protein expressed in the lateral but not the medial portion of the CeA (Fig. 4-3E, H)

(Keifer et al., 2015b). The lack of colocalization between eGFP and PKC-훿 (0 out 789

136

cells) in the CeL further suggests that expression of AT2R within the CeA is restricted to the CeM (Fig. 4-3D-I).

To determine whether AT2R in the CeM were of neuronal or glial origin, AT2R- eGFP+ cells were co-stained with a neuronal marker (NeuN), an astrocytic marker (glial fibrillary acidic protein), an oligodendritic marker (APC), and a microglia/macrophage specific protein (ionized calcium binding adapter molecule 1). AT2R-eGFP positive cells were highly expressed with NeuN (457/462 cells, 99%), whereas only 2 out of 266

(0.75%) co-localized with APC (Fig. 4-S1). No co-localization was observed with glial fibrillary acidic protein or ionized calcium binding adapter molecule 1 staining (Fig. 4-

S1). AT2R-eGFP+ mice were then crossed with glutamic acid decarboxylase (GAD)- mCherry animals to determine whether AT2R is expressed on GABAergic neurons (Fig.

4-3J) as identified in other brain regions (Kloet et al., 2017). As seen in Fig. 4-3K-N, the vast majority of eGFP+ cells were co-localized with GAD-mCherry, suggesting that the population of AT2R -eGFP+ cells in the CeM predominately consists of GABAergic neurons.

Further immunohistochemistry was performed to classify AT2R-eGFP+ cells as either projection neurons or interneurons. Sections were stained with 5 interneuronal markers; Calbindin, Calretinin, neuronal nitric oxide synthase (nNOS), parvalbumin (PV) and somatostatin (SOM) (Fig. 4-4A-E). In total, 17% of eGFP+ cells expressed interneuron markers (Fig. 4-4F). Because some cells may express more than one of these markers, it is likely that the actual percentage of interneurons is less than 17% and therefore concluded that the remaining cells that did not stain for any markers are likely

GABAergic projection neurons.

137

CTB retrograde tracing and labelling of medial division of the central amygdala (CeM) to periaquaductal grey (PAG) projection: Given the role of CeA-

PAG connections in defensive responses (e.g. freezing), we next determined whether the

PAG receives input from AT2R-GFP+ projection neurons. Alexa Fluor-conjugated cholera toxin B (CTB) was injected into the PAG of AT2R -eGFP-BAC mice (Fig. 4-

5A,B). After 2 weeks, tissue was collected and coronally sectioned for IHC. CTB injections sites were verified by fluorescence in the PAG (Fig. 4-5C,D). Cell bodies containing CTB were observed in the CeM and assessed for co-localization with eGFP

(Fig. 4-5E-J). The detection of multiple CTB/eGFP+ cells in the CeM suggests that a portion of the eGFP+ GABAergic projection neurons from the CeM may modulate freezing behavior controlled by CeM-PAG projections.

DISCUSSION

Current available treatment options for posttraumatic stress disorder (PTSD) provide limited benefits in a substantial proportion of individuals (Berger et al., 2009; Lee et al.,

2016; Ravindran and Stein, 2009, 2010) and the need for more effective therapeutic strategies remains. Recent clinical studies identify the brain RAS as a potential therapeutic target, and preclinical studies in mice have begun to explore potential neurobiological mechanisms (Fontes et al., 2016; Hurt et al., 2015; Lazaroni et al., 2016,

2012; Marvar et al., 2014). In this study, we determined that AT2R-eGFP+ GABAergic projection neurons are highly expressed in the medial division of the mouse central amygdala (CeM) and that select pharmacological activation of AT2R in the central

138

amygdala (CeA) reduces conditioned fear expression, likely via an AT2R-CeM-PAG projection.

Our previous studies using Pavlovian fear conditioning demonstrated that angiotensin type 1 receptor (AT1R) blockade facilitates fear extinction (Hurt et al., 2015;

Marvar et al., 2014), which is the gradual reduction of a learned response following the withdrawal of reinforcement, and the basis of prolonged exposure treatments for mental health disorders such as PTSD (Milton and Holmes, 2019). Importantly, Pulcu et al., recently showed in healthy volunteer participants that AT1R blockade (losartan) modifies the valence to emotional learning thus supporting a potential therapeutic role in human exposure therapy (Pulcu et al., 2019b). In further understanding the neurobiology, more recent studies in mice have demonstrated that AT1Rs are expressed by neuronal phenotypes associated with stress responses (e.g., corticotropin-releasing factor- expressing neurons in the paraventricular nucleus of the hypothalamus) and limbic brain regions (e.g., the central amygdala), thereby altering the expression and extinction of fear

(Gonzalez et al., 2012). To complement the growing body of evidence linking brain angiotensinergic activity with conditioned fear and anxiety (Hurt et al., 2015; Johnson et al., 2013; Marinzalda et al., 2014; Marvar et al., 2014), here we sought to examine the brain AT2R, about which far less is understood regarding emotional responses to stress

(Hein et al., 1995; Horiuchi et al., 2012; Maul et al., 2008) and which we propose has a modulatory role with AT1R in the regulation of fear learning and recall of an emotional threat memory.

Consistent with previous studies using the AT2R-eGFP+ reporter mouse (de Kloet et al., 2016b), immunohistochemical analysis of brain tissue revealed dense localization

139

of eGFP in two subnuclei of the amygdala; the medial region (MeA), which is generally associated with innate fear and defensive behavior, and the CeA, which is the primary nucleus mediating behavioral and autonomic responses to fear (Davis, 1992; Janak and

Tye, 2015). The CeA is a striatal like structure that can be further divided into capsular, medial (CeM), and lateral (CeL) divisions (Ye and Veinante, 2019). The CeL contains a number of distinct populations (for review see (Keifer et al., 2015b; Sah et al., 2003)), with the two most notable expressing somatostatin (SOM) and protein kinase C delta type

(PKC-훿) (Haubensak et al., 2010; Perumal et al., 2018). PKC-훿 + cells, which are restricted to the CeL, can directly inhibit CeM output neurons and reduce fear responses to conditioned stimuli (McCullough et al., 2018). In order to determine the distribution patterns of AT2R-eGFP+ cells, we identified the CeL/CeM boundary by staining for

PKC-훿, and found negligible overlap between areas positive for PKC-훿 and eGFP expression, confirming that AT2R expression within the CeA occurs primarily in the medial division. While neurons within the CeA are primarily considered inhibitory (Sun and Cassell, 1993), the phenotype of AT2R-eGFP+ neurons in this region is unknown.

Using our AT2R-eGFP+-GAD mCherry mice, we next determined that these AT2R cells are GABAergic projection neurons likely projecting to regions outside of the CeM to mediate their inhibitory effects.

Based on our findings that AT2R-eGFP+ neurons are uniquely positioned to modulate CeM output, we examined the effects of intra-CeA C21 injections prior to conditioned fear responses in two different models of Pavlovian fear conditioning.

Contextual conditioning and auditory fear conditioning vary in their recruitment of areas such as the hippocampus, and therefore reflect different upstream pathways of memory

140

formation and maintenance. Both types of memory, however, require the CeA to affect changes in behavior during fear memory recall (Goosens and Maren, 2001; Phillips and

LeDoux, 1992). Our behavioral experiments indicate that AT2R activation reduced freezing behavior across both contextual and auditory models of fear conditioning. We also observed a sustained reduction in freezing in C21-infused animals during the second day of context re-exposure, indicating a long-lasting effect on memory. Interestingly, this effect was observed only in the contextual model of fear conditioning. While there was a trend for reduced freezing in the C21 group at the start of the cued-extinction test, the difference was not statistically significant. This is likely due to the fact that responses to cue-conditioned stimuli are more resistant to extinction than responses to context- conditioned stimuli (Phillips and LeDoux, 1992). These findings suggest that, like its

AT1R counterpart (Hurt et al., 2015; Marvar et al., 2014), central AT2R activity could modulate the strength of consolidation of extinction memories. This may occur through an AT2R mediated effect on pathways previously implicated in the pathogenesis of trauma-related memory (e.g., PTSD) such as BDNF / TrkB (Diniz et al., 2018; Goel et al., 2018) or pituitary adenylate cyclase-activating polypeptide (PACAP) (Nostramo et al., 2012). Moreover, in the perspective of recent work identifying the complex and tightly controlled CeA micro-circuits that contribute to fear learning (Fadok et al., 2018;

Yu et al., 2017), these results may also indicate a functional role of AT2R-CeM cells that extends beyond that of simply gating the behavioral and endocrine responses during conditioned threat exposure.

Numerous lesioning (Hopkins and Holstege, 1978; Kim et al., 1993; LeDoux et al., 1988) and pharmacological (Bandler et al., 1985; Tomaz et al., 1988; Zhang et al.,

141

1990) studies have identified the periaqueductal grey (PAG) as an essential site of converging signals regulating flight and freezing responses, and input to the PAG from regions such as the CeA has been recently shown to mediate freezing through disinhibition of ventrolateral PAG outputs to the medulla (Tovote et al., 2016). Given the necessity of CeA-PAG connectivity to orchestrate adaptive behavioral responses, we targeted the PAG with the retrograde tracer CTB to determine whether it receives direct

AT2R+-GABAergic input. Our retrograde tracing studies identified an AT2R-eGFP+ projection from the CeM to the PAG. These results suggest AT2R activation in the CeM may modulate fear expression via direct projections to the PAG. Future studies will need to determine the extent to which this particular projection controls freezing behavior, as it is likely that CeM-AT2R+ neurons also project to other regions, such as the hypothalamus and dorsal vagal complex that could simultaneously impact defensive behavior as well as cardiovascular and autonomic adjustments to conditioned fear (Keifer et al., 2015b;

Maddox et al., 2019).

In addition to behavioral adjustments, the CeA also mediates physiological responses to fear stimuli, and lesioning of the CeA is known to attenuate both circulating corticosterone and renin levels to conditioned stress (Van de Kar et al., 1991). Consistent with previously reported sympathoinhibitory effects of central AT2R activation (Gao and

Zucker, 2011; Gao et al., 2011), C21-infused mice exhibited reduced corticosterone responses to contextual fear recall. Importantly, AT2R activation had no significant effect on exploratory behavior or anxiety-like behavior, suggesting that the reductions in conditioned freezing behavior cannot be attributed to non-specific anxiolytic drug-effects,

142

and that AT2R activation in the CeM does not play a significant role in basal anxiety levels.

A potential mechanism through which AT2R activation in the CeM may modulate fear expression is by altering the firing rate of AT2R+ neurons that control freezing behavior. Although we did not examine this in the current study, C21 has been previously shown to facilitate potassium channel function and thereby decrease spontaneous neuronal discharge (Gao et al., 2011; Sumners and Gelband, 1998).

Alternatively, AT2R stimulation could modify freezing behavior by enhancing the inhibitory effects of GABA on signaling pathways that control defensive reactions

(Hopkins and Holstege, 1978). Interestingly, activation of AT2R in GABAergic cells projecting to the paraventricular nucleus of the hypothalamus (PVN) was shown to reduce the activity of target cells within the PVN, inhibiting their release of vasopressin

(de Kloet et al., 2016a). While the effects of AT2R activation on neuronal firing rate and

GABA sensitivity within the amygdala are not currently known, the resulting behavioral changes are likely mediated by downstream effector regions such as the PAG as shown here.

The current study demonstrates that activation of the AT2R in the CeM attenuates freezing behavior in response to conditioned auditory and contextual fear, and simultaneously mitigates fear-induced increases in circulating levels of corticosterone without altering exploratory or anxiety-like behavior. Additionally, we show that the

AT2R+ cell population in the CeM is made up of GABAergic projection neurons, some of which project to the PAG. Together, these results offer functional and anatomical evidence to support a role for brain AT2R in fear expression, expanding the

143

neurobiological understanding of brain angiotensin receptors as a potential therapeutic for

PTSD.

144

Figure 4-1: Intra-central amygdala (CeA) angiotensin II type 2 receptor (AT2R) activation reduces fear expression. A. Representative coronal sections through the amygdala of the AT2R-enhanced green fluorescence protein (eGFP) reporter mice. B. Quantification of AT2R-eGFP+ cells in different amygdala subdivisions. C. Representative rostral-caudal images of AT2R-eGFP+ cells from the amygdala of AT2R- eGFP reporter mice. All staining was performed on four mouse brains (n=4). D-E. Cued fear conditioning (FC) protocol. F. Freezing behavior during cue-dependent fear conditioning. G-H. Freezing behavior during extinction training (Ext Tr) (G) and extinction test (Ext test) (H), (n=11-12. *p<0.05, **p<0.01) I-J. Contextual FC protocol. K-L. Freezing behavior during context test day 1 (K) and day 2 (L), (n=16-17 for test day 1 and n=9-10 for test day 2. *p<0.05, ***p<0.001). CeA central amygdala, BLA basolateral amygdala, MeA medial amygdala; US, unconditioned stimulus.

145

Figure 4-2: The effects of intra-central amygdala (CeA) angiotensin II type 2 receptor (AT2R) activation on basal anxiety measures and plasma corticosterone levels. A-B. Experimental protocol for plasma corticosterone test. C. Plasma corticosterone levels in contextual fear conditioned (FC) and the non-foot shock control mice (n=4 *p<0.05). D. Plasma corticosterone levels in vehicle and C21 injected groups (n=7-8 *p<0.05) E. Experimental protocol for open field and EPM test. F. Total distance traveled (left), time in center (middle) and center entries (right) in vehicle and C21 injected groups (n=10-13). G. Percentage time in open arms (left), open arm entries (middle) and total arm entries of the EPM (right) in vehicle and C21 injected groups (n=9-10). US, unconditioned stimulus.

146

Figure 4-3: AT2R-eGFP expressing GABAergic neurons are present in CeM but not CeL. A. Experimental protocol. B. Representative coronal sections through the amygdala of the AT2R-eGFP reporter mice. The inset shows the enlargement of the CeA area. C. Quantification of AT2R-eGFP+ cells in CeM vs CeL. D-I. Representative images of the amygdala depiction AT2R-eGFP+ cells (D,G), PKC-δ+ cells (E,H) and the merged image (F,I). D-F, low magnification images showing the whole amygdala. G-I, high magnification images only showed the CeA. J. AT2R-eGFP reporter mice were bred with GAD-mCherry mice. K-M. Representative images of the CeA depiction mCherry+ neurons (K), AT2R-eGFP+ neurons (L) and the merged image (M). N. Pie chart depicting the percentage value of AT2R-eGFP+ neurons that co-expressed GAD-mCherry. CeM medial division of central amygdala, CeL lateral division of central amygdala, PKC-δ protein kinase δ, GAD glutamic acid decarboxylase. Images were taken from either the rostral or caudal portions of the CeA, and analysis was performed on 36 brain sections from 6 mice.

147

Figure 4-4: AT2R-eGFP neurons in CeM are predominately projection neurons. A- E. Representative coronal sections through the CeA of the AT2R-eGFP reporter mice with co-staining against interneuron markers calbindin (A), calretinin (B), neuronal nitric oxide synthase (nNOS, C), parvalbumin (PV, D) and somatostatin (SOM, E). The insets show enlargements of the boxed areas. F. Pie charts depicting the percentage value of AT2R-eGFP+ neurons that co-expressed the corresponding interneuron marker. Images were taken from either the rostral or caudal portions of the CeA and 7-10 brain sections from 3-4 mice were used for analysis.

148

Figure 4-5: Angiotensin II type 2 receptor (AT2R)-enhanced green fluorescent protein (eGFP) neurons in medial division of the central amygdala (CeM) project to periaqueductal gray (PAG). (A,B) Experimental protocol for retrograde tracing from CeM using Alexa red fluorescent CTB C. Atlas section through the PAG depicting the localization and spread of CTB injection sites in the three mice used for this study. D. Representative coronal sections through the PAG of the AT2R-eGFP reporter mice with the CTB injection. E-J. Projection images of the CeA depiction AT2R-eGFP+ neurons (E,H), CTB positive PAG-projecting neurons (F,I) and the merged image (G,J). E-G. Low magnification images. H-J. High magnification images. Arrows indicate example double-labeled cells. CTB cholera toxin B, PAG periaqueductal grey. BAC, bacterial artificial chromosome; CeL, lateral division of the central amygdala; dPAG, dorsal PAG; IHC, immunohistochemistry; vl/lPAG, ventrolateral and lateral PAG.

149

Specific Source or Additional Resource Type Reagent or Identifiers Reference Information Resource Antibody Anti-GFP Chicken Abcam Cat# ab13970, RRID:AB_300798 Dilution 1:2000 Anti-PKCδ Mouse BD Biosciences Cat# 610397, RRID:AB_397780 Dilution 1:500 Anti-NeuN Mouse Millipore Cat# MAB377, RRID:AB_2298772 Dilution 1:500 Anti-GFAP Rabbit Abcam Cat# ab7260, RRID:AB_305808 Dilution 1:1000 Anti-Iba1 Rabbit Thermo Fisher Scientific Cat# PA5-27436, Dilution 1:1000 Anti-APC Mouse Millipore Cat#RRID:AB_2544912 MABC200, Dilution 1:500 Anti-Calretinin Rabbit Abcam (ab702) Cat#RRID:AB_11203645 ab702, RRID:AB_305702 Dilution 1:100 Anti- Somatostatin Rabbit Immunostar Cat# 20067, RRID:AB_572264 Dilution 1:2000 Anti- Parvalbumin Rabbit Abcam Cat# ab11427, RRID:AB_298032 Dilution 1:2000 Anti-Calbindin Mouse Abcam Cat# ab75524, RRID:AB_1310017 Dilution 1:1000 Anti-nNOS Rabbit Immunostar Cat# 24287, RRID:AB_572256 Dilution 1:3000 Anti-mCherry Goat LifeSpan Cat# LS-C204207, Dilution 1:500 RRID:AB_2619713 Chemical Compound or Drug Compound 21 (C21) Vicore Pharma Commercial Assay or Kit Corticosterone Mouse/Rat ELISA LDN Cat# AR E-8100 Software; Algorithm ZEN Digital Imaging for Light Carl Zeiss RRID:SCR_013672 Microscopy Table 4-S1: Key resources

150

Supplemental Figure 4-S1: AT2R-eGFP expressing cells are neuronal. A-D. Representative coronal sections through the CeA of the AT2R-eGFP reporter mice with co-staining against NeuN (A), GFAP (B), APC (nNOS, C) and Iba-1 (PV, D). The insets show enlargements of the boxed areas. E. Pie charts depicting the percentage value of AT2R-eGFP+ neurons that co-expressed neuronal marker NeuN. Images were taken from either the rostral or caudal portions of the CeA, and 7-10 brain sections from 3-4 mice were used for analysis. NeuN neuronal nuclei, GFAP Glial fibrillary acidic protein, APC adenomatous polyposis coli, Iba-1 ionized calcium-binding adapter molecule 1.

151

Supplemental Figure 4-S2: The effects of intra CeA AT2R activation on an open field test. Average distance traveled (A), percent time in center (B) and center entries in open field (C) during the 0-5 min and 6-30 min in the open field (n=10-13).

152

Supplementary Methods and Materials

Immunohistochemistry: Dual-immunofluorescence experiments were performed on

30μm free- floating serial brain sections. For each mouse brain, 10-12 coronal sections were collected from Bregma -1.22 to Bregma -1.58. Sections were washed in phosphate buffer saline (PBS) for 10 min and blocked with (5% normal donkey serum, 5% bovine serum albumin and 0.4% Triton-X-100 in PBS) for 1 hr at RT. To examine colocalization of eGFP with other markers, anti-eGFP antibody was used with the antibodies listed in Supplemental Table S1 and the sections and were incubated in the

Ab solution for 48 h at 4°C. Sections were then rinsed (3 x 10 min) with PBS and incubated in the corresponding secondary antibodies (Thermo Fisher Scientific, MA) for

2h at room temperature. After a final series of rinses (3 x 10 min), sections were mounted on Superfrost Plus slides (Thermo Fisher Scientific, MA) and air dried before being cover-slipped with ProLong® Diamond Antifade Mountant (Thermo

Fisher Scientific, MA). After staining, sections were imaged using a 25X oil immersion objective on a Zeiss Spinning Disk Confocal microscope and quantified with Zeiss Microscope Software ZEN 2 (Carl Zeiss, Germany).

Retrograde Tracing: Retrograde tracing studies were performed to investigate

whether periaqueductal grey (PAG) receives AT2R-eGFP+ inputs. AT2R-eGFP

reporter mice received red fluorescent Alexa Fluor-conjugated cholera toxin B

(CTB) (Thermo Fisher Scientific, MA) injections into the left side of the ventral

PAG at the coordinates 3.9mm caudal, 0.6 lateral to bregma, and 2.7mm below the

skull surface. A total of 200nl of 10% CTB in PBS was injected using glass pipets

connected to a microinjector pump (World Precision Instruments, FL) at a rate of 100 153

nl/min. Glass pipets were left in situ for an additional 5 min to allow the CTB to

diffuse. Two weeks after surgery, mice were perfused and brains were processed for

eGFP immunohistochemical staining as described above. A total of 3 mice were used

for this study.

Pavlovian Fear Conditioning (cued): The conditioning chamber consisted of a stainless-steel grid floor with clear front and back walls (Model H10-24T; Coulbourn

Instruments, MA). Mice were habituated to the conditioning chamber for 2 days. The auditory conditioned stimulus (CS) and the foot shock unconditioned stimulus (US) were delivered through the speaker on the wall and the grid floor respectively, and freezing behavior was recorded and quantified using Freezeframe 3.32 (Coulbourn

Instruments, MA). After conditioning, animals were returned to the home cage and the conditioning chamber was cleaned with 70% ethanol. Extinction training and testing were performed in a modified conditioning context, where the shock grid was replaced with a clear Plexiglas floor and the clear chamber walls were covered with colored and patterned paper.

Generalized Anxiety Measures: The lux for the open field center of the apparatus is

80 lux and for the EPM the open arm is 180 lux, while the closed arm is 30 lux. The

open field consisted of a square arena (27 cm X 27 cm) made of clear Plexiglas with a

wall 35 cm high. Mice were placed in the center of the open field 10 min after C21 or

vehicle injection and allowed to freely explore for 30 min.

Corticosterone Enzyme-linked Immunosorbent Assay (ELISA): C57BL/6J mice

154

(n=7- 8/group) received C21 or vehicle as normal but were only placed into the conditioning chamber for 5 min on day 3 (context test day 1), and then were returned to home cages. Thirty minutes after the 5 min context test, mice were decapitated and trunk blood was collected. Another cohort of mice were separated into two groups

(n=4/group), one group received foot shocks as mentioned above and the other group of mice were only exposed to the conditioning chamber for 10 min without any foot shock on context fear conditioning day 1 and day 2. On day 3 (context test day 1), both groups of mice were introduced to the conditioning chamber for 5 min and were sacrificed 30 min later, and their plasma corticosterone levels were also compared. Plasma corticosterone was measured 30 minutes following contextual fear exposure using a commercially available ELISA kit (LDN, Germany). The blood was centrifuged at 1500 rpm at 4°C (Eppendorf Centrifuge, Model 5840R,

Germany) for 10 min. The supernatant plasma was collected and stored in -80°C. The

ELISA was carried out according to the manufacturer’s instructions and plates were read at 450 nm using the Varioskan Flash Multimode Reader (Thermo Fisher

Scientific, MA).

155

REFERENCES

Ahmed, H.A., Ishrat, T., Pillai, B., Fouda, A.Y., Sayed, M.A., Eldahshan, W., Waller, J.L., Ergul, A., and Fagan, S.C. (2018). RAS modulation prevents progressive cognitive impairment after experimental stroke: a randomized, blinded preclinical trial. Journal of Neuroinflammation 15, 229.

Bennion, D.M., Jones, C.H., Dang, A.N., Isenberg, J., Graham, J.T., Lindblad, L., Domenig, O., Waters, M.F., Poglitsch, M., Sumners, C., et al. (2018). Protective effects of the angiotensin II AT2 receptor agonist compound 21 in ischemic stroke: a nose-to- brain delivery approach. Clinical Science 132, 581–593.

Berger, W., Mendlowicz, M.V., Marques-Portella, C., Kinrys, G., Fontenelle, L.F., Marmar, C.R., and Figueira, I. (2009). Pharmacologic alternatives to antidepressants in posttraumatic stress disorder: A systematic review. Progress in Neuro- Psychopharmacology and Biological Psychiatry 33, 169–180.

Chappell, M.C. (2015). Biochemical evaluation of the Renin-Angiotensin System - the Good, Bad, and Absolute? American Journal of Physiology - Heart and Circulatory Physiology ajpheart.00618.2015.

Coble, J.P., Grobe, J.L., Johnson, A.K., and Sigmund, C.D. (2015). Mechanisms of brain renin angiotensin system-induced drinking and blood pressure: importance of the subfornical organ. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 308, R238–R249.

Davis, M. (1992). The role of the amygdala in fear and anxiety. Annu. Rev. Neurosci. 15, 353–375.

Davisson, R.L., Oliverio, M.I., Coffman, T.M., and Sigmund, C.D. (2000). Divergent functions of angiotensin II receptor isoforms in the brain. J Clin Invest 106, 103–106.

Diniz, C.R.A.F., Casarotto, P.C., Fred, S.M., Biojone, C., Castrén, E., and Joca, S.R.L. (2018). Antidepressant-like effect of losartan involves TRKB transactivation from angiotensin receptor type 2 (AGTR2) and recruitment of FYN. Neuropharmacology 135, 163–171.

Fadok, J.P., Markovic, M., Tovote, P., and Lüthi, A. (2018). New perspectives on central amygdala function. Current Opinion in Neurobiology 49, 141–147.

156

Fontes, M.A.P., Martins Lima, A., and Santos, R.A.S. dos (2016). Brain angiotensin-(1- 7)/Mas axis: A new target to reduce the cardiovascular risk to emotional stress. Neuropeptides 56, 9–17.

Foulquier, S., Steckelings, U.M., and Unger, T. (2012). Impact of the AT2 Receptor Agonist C21 on Blood Pressure and Beyond. Curr Hypertens Rep 14, 403–409.

Gao, L., and Zucker, I.H. (2011). AT2 receptor signaling and sympathetic regulation. Current Opinion in Pharmacology 11, 124–130.

Gelosa, P., Pignieri, A., Fändriks, L., de Gasparo, M., Hallberg, A., Banfi, C., Castiglioni, L., Turolo, L., Guerrini, U., Tremoli, E., et al. (2009). Stimulation of AT2 receptor exerts beneficial effects in stroke-prone rats: focus on renal damage: Journal of Hypertension 27, 2444–2451.

Goel, R., Bhat, S.A., Hanif, K., Nath, C., and Shukla, R. (2018). Angiotensin II Receptor Blockers Attenuate Lipopolysaccharide-Induced Memory Impairment by Modulation of NF-κB-Mediated BDNF/CREB Expression and Apoptosis in Spontaneously Hypertensive Rats. Mol. Neurobiol. 55, 1725–1739.

Goosens, K.A., and Maren, S. (2001). Contextual and Auditory Fear Conditioning are Mediated by the Lateral, Basal, and Central Amygdaloid Nuclei in Rats. Learn. Mem. 8, 148–155. und Halbach, O. von B., and Albrecht, D. (1998). Angiotensin II inhibits long-term potentiation within the lateral nucleus of the amygdala through AT 1 receptors. Peptides 19, 1031–1036.

Hallberg, M., Sumners, C., Steckelings, U.M., and Hallberg, A. (2018). Small-molecule AT2 receptor agonists. Med Res Rev 38, 602–624.

Hein, L., Barsh, G.S., Pratt, R.E., Dzau, V.J., and Kobilka, B.K. (1995). Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice. Nature 377, 744–747.

157

Hopkins, D.A., and Holstege, G. (1978). Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat. Exp Brain Res 32, 529–547.

Horiuchi, M., Iwanami, J., and Mogi, M. (2012). Regulation of angiotensin II receptors beyond the classical pathway. Clin. Sci. 123, 193–203.

Inagami, T., Kambayashi, Y., Ichiki, T., Tsuzuki, S., Eguchi, S., and Yamakawa, T. (1999). Angiotensin Receptors: Molecular Biology and Signalling. Clinical and Experimental Pharmacology and Physiology 26, 544–549.

Ishrat, T., Fouda, A.Y., Pillai, B., Eldahshan, W., Ahmed, H., Waller, J.L., Ergul, A., and Fagan, S.C. (2018). Dose–response, therapeutic time-window and tPA-combinatorial efficacy of compound 21: A randomized, blinded preclinical trial in a rat model of thromboembolic stroke. J Cereb Blood Flow Metab 0271678X18764773.

Jan Braszko (2002). AT2 but not AT1 receptor antagonism abolishes Angiotensin II increase of the aquisition of conditioned avoidance responses in rats. BEHAV BRAIN RES 131, 79–86.

Janak, P.H., and Tye, K.M. (2015). From circuits to behaviour in the amygdala. Nature 517, 284–292.

Jing, F., Mogi, M., Sakata, A., Iwanami, J., Tsukuda, K., Ohshima, K., Min, L.-J., Steckelings, U.M., Unger, T., Dahlöf, B., et al. (2012). Direct stimulation of angiotensin II type 2 receptor enhances spatial memory. J. Cereb. Blood Flow Metab. 32, 248–255.

Johnson, P.L., Sajdyk, T.J., Fitz, S.D., Hale, M.W., Lowry, C.A., Hay-Schmidt, A., and Shekhar, A. (2013). Angiotensin II’s role in sodium lactate-induced panic-like responses in rats with repeated urocortin 1 injections into the basolateral amygdala. Progress in Neuro-Psychopharmacology and Biological Psychiatry 44, 248–256.

Joseph, J.P., Mecca, A.P., Regenhardt, R.W., Bennion, D.M., Rodríguez, V., Desland, F., Patel, N.A., Pioquinto, D.J., Unger, T., Katovich, M.J., et al. (2014). The angiotensin type 2 receptor agonist Compound 21 elicits cerebroprotection in endothelin-1 induced ischemic stroke. Neuropharmacology 81, 134–141.

Keifer, O.P., Hurt, R.C., Ressler, K.J., and Marvar, P.J. (2015). The Physiology of Fear: Reconceptualizing the Role of the Central Amygdala in Fear Learning. Physiology 30, 389–401.

158

Kim, J.J., Rison, R.A., and Fanselow, M.S. (1993). Effects of amygdala, hippocampus, and periaqueductal gray lesions on short- and long-term contextual fear. Behavioral Neuroscience 107, 1093–1098.

Kloet, A.D. de, Steckelings, U.M., and Sumners, C. (2017). Protective Angiotensin Type 2 Receptors in the Brain and Hypertension. Curr Hypertens Rep 19, 46. de Kloet, A.D., Wang, L., Ludin, J.A., Smith, J.A., Pioquinto, D.J., Hiller, H., Steckelings, U.M., Scheuer, D.A., Sumners, C., and Krause, E.G. (2016a). Reporter mouse strain provides a novel look at angiotensin type-2 receptor distribution in the central nervous system. Brain Structure and Function 221, 891–912. de Kloet, A.D., Pitra, S., Wang, L., Hiller, H., Pioquinto, D.J., Smith, J.A., Sumners, C., Stern, J.E., and Krause, E.G. (2016b). Angiotensin Type-2 Receptors Influence the Activity of Vasopressin Neurons in the Paraventricular Nucleus of the Hypothalamus in Male Mice. Endocrinology 157, 3167–3180.

Krause, E.G., de Kloet, A.D., Scott, K.A., Flak, J.N., Jones, K., Smeltzer, M.D., Ulrich- Lai, Y.M., Woods, S.C., Wilson, S.P., Reagan, L.P., et al. (2011). Blood-borne angiotensin II acts in the brain to influence behavioral and endocrine responses to psychogenic stress. J. Neurosci. 31, 15009–15015.

Lazaroni, T.L. do N., Bastos, C.P., Moraes, M.F.D., Santos, R.S., and Pereira, G.S. (2016). Angiotensin-(1-7)/Mas axis modulates fear memory and extinction in mice. Neurobiology of Learning and Memory 127, 27–33.

Lee, D.J., Schnitzlein, C.W., Wolf, J.P., Vythilingam, M., Rasmusson, A.M., and Hoge, C.W. (2016). Psychotherapy Versus Pharmacotherapy for Posttraumatic Stress Disorder: Systemic Review and Meta-Analyses to Determine First-Line Treatments. Depression and Anxiety 33, 792–806.

159

Lenkei, Z., Palkovits, M., Corvol, P., and Llorens-Cortes, C. (1996). Distribution of angiotensin II type-2 receptor (AT2) mRNA expression in the adult rat brain. J. Comp. Neurol. 373, 322–339.

Lind, W., Swanson, L.W., and Ganten, D. (1985). Organization of Angiotensin II Immunoreactive Cells and Fibers in the Rat Central Nervous System. NEN 40, 2–24.

Maddox, S.A., Hartmann, J., Ross, R.A., and Ressler, K.J. (2019). Deconstructing the Gestalt: Mechanisms of Fear, Threat, and Trauma Memory Encoding. Neuron 102, 60– 74.

Maul, B., von Bohlen und Halbach, O., Becker, A., Sterner-Kock, A., Voigt, J.-P., Siems, W.-E., Grecksch, G., and Walther, T. (2008). Impaired spatial memory and altered dendritic spine morphology in angiotensin II type 2 receptor-deficient mice. J. Mol. Med. 86, 563–571.

McCarthy, C.A., Vinh, A., Miller, A.A., Hallberg, A., Alterman, M., Callaway, J.K., and Widdop, R.E. (2014). Direct Angiotensin AT2 Receptor Stimulation Using a Novel AT2 Receptor Agonist, Compound 21, Evokes Neuroprotection in Conscious Hypertensive Rats. PLoS ONE 9, e95762.

McCullough, K.M., Morrison, F.G., Hartmann, J., Carlezon, W.A., and Ressler, K.J. (2018). Quantified Coexpression Analysis of Central Amygdala Subpopulations. Eneuro 5, ENEURO.0010-18.2018.

Milton, A.L., and Holmes, A. (2019). Editorial: the psychopharmacology of extinction— from theory to therapy. Psychopharmacology 236, 1–6.

Nostramo, R., Tillinger, A., Saavedra, J.M., Kumar, A., Pandey, V., Serova, L., Kvetnansky, R., and Sabban, E.L. (2012). Regulation of angiotensin II type 2 receptor gene expression in the adrenal medulla by acute and repeated immobilization stress. Journal of Endocrinology 215, 291–301.

160

Perumal, M.B., Lynch, J.W., and Sah, P. (2018). GABAA receptors and inhibitory neurotransmission in the amygdalar complex. Current Opinion in Physiology 2, 58–64.

Phillips, R.G., and LeDoux, J.E. (1992). Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav. Neurosci. 106, 274–285.

Raio, C.M., and Phelps, E.A. (2015). The influence of acute stress on the regulation of conditioned fear. Neurobiology of Stress 1, 134–146.

Ravindran, L.N., and Stein, M.B. (2009). Pharmacotherapy of PTSD: Premises, principles, and priorities. Brain Research 1293, 24–39.

Ravindran, L.N., and Stein, M.B. (2010). The pharmacologic treatment of anxiety disorders: a review of progress. J Clin Psychiatry 71, 839–854.

Sah, P., Faber, E.S.L., Lopez De Armentia, M., and Power, J. (2003). The Amygdaloid Complex: Anatomy and Physiology. Physiological Reviews 83, 803–834.

Sumners, C., and Gelband, C.H. (1998). Neuronal ion channel signalling pathways: modulation by angiotensin II. Cellular Signalling 10, 303–311.

Sun, N., and Cassell, M.D. (1993). Intrinsic GABAergic neurons in the rat central extended amygdala. J. Comp. Neurol. 330, 381–404.

Swiercz, A.P., Seligowski, A.V., Park, J., and Marvar, P.J. (2018). Extinction of Fear Memory Attenuates Conditioned Cardiovascular Fear Reactivity. Frontiers in Behavioral Neuroscience 12.

Tchekalarova, J., and Albrecht, D. (2007). Angiotensin II suppresses long-term depression in the lateral amygdala of mice via L-type calcium channels. Neuroscience Letters 415, 68–72.

161

Terock, J., Hannemann, A., Janowitz, D., Freyberger, H.J., Felix, S.B., Dörr, M., Nauck, M., Völzke, H., and Grabe, H.J. (2019). Associations of trauma exposure and posttraumatic stress disorder with the activity of the renin-angiotensin-aldosterone-system in the general population. Psychol Med 49, 843–851.

Van de Kar, L.D., Piechowski, R.A., Rittenhouse, P.A., and Gray, T.S. (1991). Amygdaloid lesions: differential effect on conditioned stress and immobilization-induced increases in corticosterone and renin secretion. Neuroendocrinology 54, 89–95.

Ye, J., and Veinante, P. (2019). Cell-type specific parallel circuits in the bed nucleus of the stria terminalis and the central nucleus of the amygdala of the mouse. Brain Struct Funct.

Yu, K., Ahrens, S., Zhang, X., Schiff, H., Ramakrishnan, C., Fenno, L., Deisseroth, K., Zhao, F., Luo, M.-H., Gong, L., et al. (2017). The central amygdala controls learning in the lateral amygdala. Nature Neuroscience 20, 1680–1685.

162

CHAPTER 5

General Discussion

163

Study 1 (Chapter 2)

In Chapter 2, we examined how the testing context influences cardiovascular activity during memory recall, and then introduced a novel testing paradigm to assess extinction learning in resting animals. Furthermore, we showed for the first time that blood pressure responses may be a useful index of fear extinction to supplement the current standard of freezing behavior, or as a substitute when freezing behavior is not appropriate.

Freezing behavior, defined as the complete cessation of movement (aside from respiration), is the standard parameter by which associative fear memories are measured in rodents. Techniques for measuring freezing behavior range from hand-scoring to motion-detection systems using automated analyses, each of which has its own threshold of sensitivity. When an animal is placed back into the context it was conditioned in, it will freeze in response to both contextual and auditory cues, making it difficult to discern tone-dependent fear responses. For this reason, auditory cue-dependent freezing responses are typically assessed in novel testing environments which promote a high level of exploratory activity (Bouton and Bolles, 1980; LeDoux et al., 1984). Fear responses are easily identified in novel contexts as exploration is replaced by freezing behavior during CS presentation.

The extinction of conditioned freezing responses to auditory stimuli is also generally measured in novel contexts. However, exposing an animal to a novel context may introduce additional stress that can influence measurements of fear expression and memory. Through the use of radiotelemetry, we examined the physiological responses

164

that occur during common testing procedures, and which are not evident at the behavioral level.

Conditioned cardiovascular responses as markers of learning

Classical aversive conditioning results in a wide range of conditioned responses, including behavioral modifications, startle reflex potentiation, and changes in heart rate and arterial pressure. These behavioral and physiological adjustments are observed together during memory recall, and are integrated within certain structures of the central nervous system. Examples of these central structures include the amygdala and hypothalamus, which both play critical roles in behavioral and cardiovascular regulation

(Abegaz et al., 2013). However, downstream of these integration sites the neuronal projections mediating each aspect of the conditioned response begin to diverge and target individual effector systems (Mccabe et al., 2000). For instance, freezing responses are initiated by central amygdala (CeA) projections to the ventrolateral periaqueductal gray, which is a midbrain region involved in multiple homeostatic processes. Autonomic adjustments, on the other hand, are controlled by amygdaloid projections to the lateral hypothalamus and medullar nuclei (Roelofs, 2017). Furthermore, recent studies have shown that distinct populations of neurons within the medial central amygdala (CeM) can selectively control behavioral and physiological components of the defensive response, suggesting that a simple correlation between freezing and cardiovascular responses cannot always be assumed (Viviani et al., 2011). In order to fully understand the conditioning and extinction of fear responses, it is therefore beneficial to consider

165

physiological markers in addition to purely behavioral readouts such as freezing behavior.

The perception of fear leads to activation of the autonomic nervous system, and changes in autonomic activity cause fluctuations in blood pressure and heart rate that can be used to evaluate fear learning and memory. Because rapid cardiovascular responses are required for survival in the face of threat, they are under high evolutionary selection pressure and can therefore be examined in a wide variety of mammalian species (Stiedl and Hager, 2017).

Heart rate, heart rate variability, and blood pressure are non-invasively measured in human subjects and useful in detecting physiological alterations to various emotional states. These indices of cardiac activity represent important predictors of cardiovascular disease (Böhm et al., 2015), and may also help identify individuals at high risk of developing psychiatric disorders. Low heart rate variability, for instance, is associated with greater PTSD vulnerability (Gillie and Thayer, 2014; Minassian et al., 2015).

However, in small animals like rats and mice, these indirect measures of autonomic activity are more difficult to acquire. Tail-cuff plethysmography is a useful methodology, but it requires temporary restraint and heating of the animal, limiting its practical use during exposure to acute stimuli. We therefore used radio telemetry, which permits the continuous collection of multiple physiological parameters (e.g. blood pressure, heart rate, activity, body temperature, ECG signals) and is well-suited for studies that use Pavlovian conditioning and require the animals to move freely. This technique involves the implantation a small wireless radiotelemetric device under the skin of the animal, and the cannulation of the carotid artery (or abdominal aorta) with a

166

gel-filled pressure sensing catheter (Young and Davisson, 2011). Telemeter implantation is an invasive procedure, however animals exhibit normal movement, behavior, and circadian rhythm after recovery (Butz and Davisson, 2001).

Previous rodent studies show that it is possible to generate conditioned blood pressure and heart rate responses following auditory (Iwata and LeDoux, 1988) and contextual (Carrive, 2000) fear conditioning. Using radiotelemetry in mice, both HR and

MAP have been identified as objective indicators of associative learning (Hsu et al.,

2012; Stiedl and Spiess, 1997). Indeed, it is possible to prevent cardiovascular responses by interfering with memory formation, which suggests that conditioned cardiovascular responses rely upon previously consolidated memories. This has been demonstrated in mice, where administration of the protein synthesis inhibitor cycloheximide (Stiedl et al.,

1999) or the NMDA receptor agonist DL-2-amino-5-phosphonovaleric acid (APV)

(Stiedl et al., 2000) during the consolidation period can prevent the conditioned tachycardia observed after CS-US pairing. The memory-dependent nature of these responses suggests that interventions reducing memory strength may be reflected by a decrease in conditioned cardiovascular activity.

One advantage of using radiotelemetry to study acute fear responses is that conditioned fear leads to rapid changes in cardiovascular activity. However, the high sensitivity of cardiovascular responses to stress does impose some limits on its utility in certain behavioral models. In our attempts to identify conditioned or extinguished responses in a novel context, we found that non-specific elevations in blood pressure and heart rate masked the cardiovascular changes that one would expect in resting animals.

We observed similar elevations in heart rate and blood pressure when animals were

167

transferred from the homecage to a different context, regardless of whether or not they were fear conditioned. Multiple days of animal handling and pre-exposure to the novel context prior to fear conditioning did not eliminate these baseline elevations. The elevated cardiovascular activity was not caused by conditioned fear, but instead was a side effect of the testing protocol. These findings highlight an important, but often overlooked consequence of behavioral testing; the test itself is a stressful experience.

This is especially concerning when studying fear and anxiety, as any type of stress can alter an animal’s behavior and potentially confound freezing results. Stress during testing is equally disruptive to the study of memory, because stress hormones such as catecholamines and glucocorticoids can modulate the strength of memory consolidation.

Handling alone is a stressful event that can alter the exploratory behavior of an animal

(Gouveia and Hurst, 2017) and dramatically alter cardiovascular activity (LeDoux et al.,

1988). Both handling and placement in a novel context lead to rapid heart rate increases and decreased heart rate variability in mice, making it difficult to determine the impact of additional stressors (Liu et al., 2013).

Temporal Dynamics of the conditioned cardiovascular response

Early studies measuring conditioned cardiovascular responses in freely moving animals reveal two important characteristics that our consistent with our data: 1) a biphasic pressor response; and 2) variable heart rate responses reflecting either individual differences or the effects of different conditioning protocols (Sakaguchi et al., 1983).

The pressor response observed by our group and others consists of a transient rise in arterial pressure followed by a second, slower increase (Tovote et al., 2005).

168

Conditioned blood pressure responses are largely mediated by increases in sympathetic activity because unlike the heart, most blood vessels only receive sympathetic innervation

(Gordan et al., 2015). Sympathetic vasomotor neural excitation contributes to both phases of the pressor response because adrenal demedullation fails to alter the response but the adrenergic antagonist phentolamine or sympathectomy completely suppresses it

(Sakaguchi et al., 1983). The rapid initial rise in blood pressure is likely due to an orienting response as opposed to a conditioned memory because it is similar to the response elicited by a startle stimulus in non-conditioned animals (Tovote et al., 2005).

The second phase of the blood pressure response is a result of associative learning and as we show in our homecage experiments, is suppressed during extinction recall.

Overall, the conditioned tachycardic response in our animals was smaller than that reported by other mouse studies (Hager et al., 2014; Stiedl et al., 2000; Tovote et al.,

2005). These differences may stem from the fact that previous studies used a single

CS/US pairing during the fear conditioning protocol, whereas our animals were exposed to five CS/US pairings. The duration and intensity of the conditioning protocol seem to affect the dynamics of the heart rate response. A parasympathetic counter regulation is observed in animals exposed to twenty CS-US pairings (Iwata and LeDoux, 1988), but not in animals exposed to only a single CS-US pairing (Stiedl et al., 1999), suggesting that parasympathetic and sympathetic contributions to the conditioned response may vary according to specific features of the training protocol. While we did not attempt to separate the two branches of the autonomic response, the significant increase in heart rate observed prior to extinction training suggests a net increase in sympathetic activity. This net increase could be due to sympathetic activation, parasympathetic withdrawal, or the

169

combination of both. Future studies might incorporate frequency-domain analysis of heart rate variability to identify the relative contributions of each branch of the autonomic nervous system.

Conditioned cardiovascular responses as a measure of extinction

Extinction of cardiovascular responses has been previously observed in mice as a gradual decay of conditioned heart rate increases over time (Stiedl, 1999), however the impact of condensed multi-trial extinction training sessions on heart rate and blood pressure responses have not been previously examined. We further explored the possibility that conditioned responses might also be used to examine the extinction of fear. Given that extinction training reduces the expression of behavioral defensive responses, we reasoned that it would also lead to a reduction in cue-dependent cardiovascular adjustments, which could then be assessed in resting animals. To address these questions, we examined the effects of massed (30 CS) extinction trials on blood pressure and heart rate response during subsequent tests.

Previous studies have observed extinction of the conditioned heart rate response using single CS presentations over multiple days (Hager et al., 2014; Stiedl, 1999; Stiedl and Hager, 2017). While these studies suggest that the extinction of heart rate responses may provide valuable information about the rate of extinction between various mouse strains, they do not directly identify the effects of repeated extinction trials by comparison with non-extinguished controls. Such a comparison is necessary to determine the utility of cardiovascular responses as a reliable indicator of extinction learning. Furthermore, the extinction of conditioned arterial pressure responses has not

170

been previously reported. Our results indicate that a single extinction session (30 CS in a novel context) can significantly reduce the conditioned blood pressure response 24hr later. Thus, the conditioned blood pressure response reflects previously acquired inhibitory learning, and may be useful to determine the efficacy of extinction-based treatments to reduce the physiological consequences of aberrant fear (Maddox et al.,

2019). These findings are in agreement with the notion that blood pressure-related parameters provide a novel and reliable way to examine the formation and expression of individual components of fear learning and memory.

Future directions

A major challenge of using radiotelemetry to study acute responses to conditioned stimuli is baseline variability. Even in the home cage during the light cycle, slight variations in activity were reflected by differences in heart rate and likely account for the high pre-CS variability observed by us and others (Stiedl and Spiess, 1997). It was not uncommon to see large differences in baseline heart rate (>150bpm) and blood pressure

(>15mmHg) during the pre-CS phase. Future studies will need to address this issue, because the magnitude of the conditioned response depends in part on the initial cardiovascular state of the animal.

One of the limiting factors in the current design is that the CS is presented to a group of four animals simultaneously, regardless of individual heart rate or blood pressure status. To address this issue, we are developing software that can monitor the cardiovascular data generated by the telemetry system in real time, and control the presentation of conditioned stimuli to specific animals depending on their individual

171

average blood pressure and heart rate. In this way, we can normalize the baseline cardiovascular state across all test subjects based on pre-determined criteria. For example, in a conditioned cardiovascular response test, we could initiate the testing protocol only in animals that have had a MAP of 100mm  10mmHG, and a heart rate of

500  50 BPM for a period of at least ten minutes. Initiating CS presentations based on the physiological state of each individual animal will allow for tight control over baseline variability, improve consistency, and reduce the number of animals required for this type of experiment. This approach will be particularly useful in studies that incorporate rodent models of autonomic dysfunction into the examination of threat memory. An investigation of the cardiovascular responses to conditioned stimuli in animal models of genetic hypertension under novel and unpredictable conditions will advance our understanding of the relationship between cardiovascular disease and PTSD. A better understanding of physiological threat responses will likely reveal important characteristics of resilience and susceptibility to the development of maladaptive fear disorders.

Study 2 (Chapter 3)

In addition to extinction learning, reactivation can also initiate the time-dependent process of memory reconsolidation. Interfering with reconsolidation can have amnesic effects on the reactivated memory, and this process could potentially be manipulated to benefit a range of psychiatric conditions, including PTSD, obsessive compulsive disorder, delusions, and hallucinations (Nader et al., 2013). There is evidence that Ang II plays an active role in fear memory reconsolidation in multiple species, and that these

172

effects are mediated by the AT1R (Frenkel et al., 2005; Sierra et al., 2013). We therefore tested the hypothesis that blockade of AT1R during the reconsolidation phase would reduce fear responses in a model of auditory conditioning. Indeed, we found that the acute peripheral administration of losartan resulted in long-term reductions in freezing behavior. Furthermore, this effect was dependent on reconsolidation as no reduction in fear was observed when losartan was given without memory reactivation.

Initial evidence that the RAS contributes to reconsolidation comes from fear conditioning studies in the crab Chasmagnathus, where it was found that water deprivation could lead to memory modifications during the reconsolidation phase. Water deprivation during re-exposure to a training context facilitated reconsolidation, and this was attributed to an increase in endogenous Ang II (Frenkel et al., 2005). Contributions of Ang II to reconsolidation have also been observed in rats, where aversive memory content can be updated during the reconsolidation phase in situations like water deprivation (Sierra et al., 2013). Importantly, the effects of water deprivation reported by

Sierra et al. could be mimicked by the infusion of exogenous Ang II, and these effects were mediated by AT1R receptors in the dorsal hippocampus.

Based on the reductions in freezing behavior of animals given post-retrieval losartan, we anticipated a reduction in the conditioned blood pressure response as well.

Surprisingly, no difference in the conditioned cardiovascular response was observed.

This finding should be interpreted with two important features of the study design in mind. First, the retrieval cue in the telemetry animals was presented in the homecage as opposed to in a novel context. Given that the ability of amnestic agents to disrupt reconsolidation might depend on slight variations in the retrieval procedure (Schroyens et

173

al., 2017), it is possible that the homecage retrieval used in this experiment did not fully engage the process of reconsolidation. The reactivation of a memory is necessary, but not always sufficient to destabilize a memory (Visser et al., 2018). However, because this is the first study to examine reconsolidation effects by assessing the conditioned cardiovascular response, future studies will need to address subtle experimental differences.

Second, it is possible that the conditioned response needs to meet a certain threshold of reduction before changes in the cardiovascular response are observed. While the losartan treatment reduced freezing behavior relative to saline controls, it did not entirely eliminate the conditioned response. Treated animals continued to exhibit a significant increase in freezing during CS exposure in both the 24hr and 1wk tests. It may be necessary to reduce the fear response by a larger degree before it is detectable in the blood pressure or heart rate components of the cardiovascular response. In either case, the inclusion of objective physiological markers allows for a more comprehensive assessment of fear.

Similar to memory consolidation, reconsolidation requires de novo protein synthesis (Nader and Einarsson, 2010). Transcription factor activation is an important part of this process, and certain transcription factors, including Egr1 (also known as

Zif268), Arc, and cAMP response element-binding protein (CREB), are thought be responsible for gene expression changes that occur during learning (Alberini, 2009).

Still, the transcriptional changes that occur during memory reconsolidation, are not fully understood (Wang et al., 2018).

174

Systemic losartan administration has been shown to affect mRNA transcription in brain regions like the posterior hypothalamus, locus coeruleus, and the PVN (Ye et al.,

2002). Furthermore, it has been previously shown that chronic systemic losartan can alter mRNA levels of c-Fos in the BNST and AT1R in the amygdala (Marvar et al.,

2014). Based on evidence that reconsolidation requires de novo mRNA synthesis in the

BLA (Duvarci et al., 2008), we evaluated gene expression following memory retrieval to determine whether systemic losartan during this period could alter the expression of genes relevant to reconsolidation. In agreement with previous studies, we found that

Egr1, c-Fos, and Arc mRNA were all elevated in the BLA following memory retrieval.

This result suggests that the single CS presentation used in our behavioral experiments was sufficient to engage the transcriptional activity required for reconsolidation.

However, there were no significant differences in the expression of these IEGs between losartan and saline-treated animals. Therefore, our data also suggest that losartan did not inhibit the transcription of IEGs known to be required for reconsolidation. Additional

RNA-Seq experiments revealed highly overlapping gene expression profiles between losartan and saline groups. However, pairwise comparisons between losartan vs. NR and saline vs. NR controls suggest that post-retrieval losartan may prevent the upregulation of genes associated with neuronal plasticity (e.g. Egr2, Egr3) and reconsolidation (e.g.

Npas4) (Sun and Lin, 2016) in the BLA. These experiments were conducted on tissue samples collected 40 minutes after the retrieval cue. Given that memory consolidation and reconsolidation are highly dynamic processes involving multiple waves of transcription (Rao-Ruiz et al., 2019), additional studies will need to examine the time- dependent effects of losartan on gene expression during the window of reconsolidation.

175

In conclusion, we found that post-retrieval losartan contributes to long-term reductions in freezing behavior that occur independent of conditioned cardiovascular reactivity and IEG expression associated with reconsolidation. These effects may be mediated by differences in the physiological state of the animals during the reconsolidation, as the injection of losartan acutely lowered blood pressure during the reconsolidation phase. Collectively, these studies add to the growing body of evidence describing RAS contributions to fear learning and memory, and suggest that targeting the

AT1R during reconsolidation may be a valid therapeutic approach in fear-related disorders. The ability of systemic AT1R inhibition to both facilitate extinction (Marvar et al., 2014; Pulcu et al., 2019a), and to modify reconsolidation as shown here, suggest that

AT1R blockade may be unique it is directional effects on fear memory and important to the advancement of novel PTSD treatments.

Future directions

The fact that Ang II contributes to specific cognitive processes like extinction learning and memory reconsolidation may provide opportunities to pharmacologically intervene and reduce symptoms in patients with dysregulated fear. Recent work from our lab and others has shown that interfering with the RAS pathway can facilitate extinction learning. Specifically, inhibition or deletion of the AT1R prior to extinction training results in long-term reduction in freezing responses in rodents (Hurt et al., 2015; Marvar et al., 2014; Parrish et al., 2019). This effect is thought to be the result of improved extinction learning, an idea that is supported by clinical research showing that the AT1R antagonist losartan can improve memory (Mechaeil et al., 2011), enhance learning from

176

positive relative to negative events (Pulcu et al., 2019a), and modulate emotional processing in humans with high trait anxiety (Reinecke et al., 2018). A pivotal role for

Ang II in fear memory is also supported by recent evidence that acute losartan administration in humans can accelerate extinction learning (Zhou et al., 2018). In combination with exposure-based interventions, losartan may aid in the treatment of anxiety disorders.

One of the translational challenges of targeting fear and anxiety disorders by reconsolidation interference is that the boundaries between extinction and reconsolidation are poorly delineated in humans. Many of the pharmacological agents that enhance extinction learning also strengthen memory reconsolidation (Lee et al., 2006).

Treatments that impair extinction, such NMDA receptor antagonists, (Falls et al., 1992) also tend to impair reconsolidation (Torras-Garcia et al., 2005). In a clinical setting where the optimal reactivation procedure is more ambiguous, it is difficult to determine the dominant memory process that is activated by memory recall. This distinction becomes important when medication is involved, because if the reactivation protocol triggers reconsolidation rather than extinction, the administration of a drug that strengthens extinction (e.g. DCS) may inadvertently increase the strength of a traumatic memory (Mataix-Cols et al., 2017). It is therefore critical that future efforts to augment exposure-based therapy also account for potential effects on reconsolidation.

Additionally, future studies examining the role of AT1R in reconsolidation will need to consider the central vs. peripheral actions of the drug. Losartan was chosen for the current studies based on its efficacy in previous in animal and human studies of extinction learning. While peripherally administered losartan can block AT1R within the

177

brain (Song et al., 1991; Wang et al., 2003), other ARBs such as candesartan more readily cross the blood brain barrier and may therefore have greater central effects.

Future studies using transgenic AT1R-Cre and AT1R-floxed mouse models that allow for site-specific activation or deletion of AT1R will aid in determining the role of this receptor throughout the brain.

Study 3 (Chapter 4)

Until recently, most RAS research has focused on the AT1R, as this receptor mediates the prominent physiological effects of Ang II. However, Ang II also binds with similar affinity to the AT2R, which is expressed throughout the brain and plays an important role in the physiological and cognitive effects of RAS signaling. ARB treatments targeting AT1R are associated with a reduced risk of dementia (Chiu et al.,

2014; Mogi and Horiuchi, 2009), decreased brain inflammation (Torika et al., 2018) and protection of the brain against ischemia (Mogi Masaki et al., 2006). Interestingly, the ability of ARBs like valsartan to prevent cognitive decline after cerebral ischemia is decreased in Agtr2- mice (Mogi Masaki et al., 2006), and the neuroprotective effects of irbesartan on ischemia-induced neuronal injury are blocked by antagonism of the AT2R with PD123319 (Li et al., 2005). These studies suggest that the benefits of AT1R blockade may be mediated in part by the increased availability of Ang II to activate

AT2R. In Chapter 4, we examined the function, distribution, and connectivity of cells within the amygdala that express AT2R. We found that AT2R are expressed on inhibitory projection neurons in the CeM, and that activation of these receptors can reduce fear expression.

178

Characteristics of cells expressing AT2R in the CeA

In our immunohistochemical evaluation of AT2R, we used an AT2R-eGFP-BAC reporter mouse (de Kloet et al., 2016a). This strain has been shown to express AT2R in the medial and central regions of the amygdala, however the distribution patterns and characteristics of these cells have not been examined in detail. By crossing the GFP reporter mouse with glutamate acid decarboxylase (GAD)-mCherry mice, we determined that within the CeA, neurons expressing AT2R in the amygdala were GABAergic.

Furthermore, AT2R-eGFP+ cells costained with neuronal markers as opposed to markers for astrocytes, oligodendrocytes, or microglia. We also observed very little costaining with interneuron markers, suggesting that AT2R+ cells in this region are GABAergic projecting neurons.

The locations and cell types expressing AT2R within the CeA provide some insight as to the functions of this receptor in the brain. In agreement with previous reports, we found that eGFP in the amygdala was expressed primarily in the medial and central nuclei (de Kloet et al., 2016a). The flow of information through the amygdala is complex and involves reciprocal connections with the midline and orbital prefrontal cortices as well as the hippocampus. However in a simplified view, information about the external environment is conveyed by the sensory thalamus and sensory cortices to the

LA. Within the BLA, the LA communicates with the basal amygdala, and it also projects to the lateral portion of the nearby CeA (Janak and Tye, 2015). BLA input to the CeL controls an inhibitory microcircuit gating output from the CeM (Huber, 2005). We

179

therefore aimed to determine the role of AT2R+ in this fear learning and expression pathway.

Within the central amygdala, we observed negligible colocalization between

AT2R and PKC-훿, a protein that is abundant in the CeL. AT2R expression in the CeA is therefore restricted mostly to the CeM. The CeM contains output neurons that receive inhibitory input from GABAergic PKC-훿+ neurons in the CeL (Haubensak et al., 2010).

Output neurons in the CeM project to regions like the PAG, the BNST (Ye and Veinante,

2019), and the hypothalamus (Keifer et al., 2015b) which control the behavioral and autonomic responses to fear.

Recent efforts have begun to identify the properties of subregions within the CeA.

It is now known that most of the neurons in the CeM increase in CS responsiveness after fear conditioning, and decrease in responsiveness to the CS following extinction training

(Duvarci et al., 2011). The importance of CeM neurons in the expression of fear is supported by evidence that the direct optogenetic activation of CeM neurons increases fear expression whereas inactivation of the entire CEA (or just the CeM) after fear conditioning with the GABAergic antagonist muscimol leads to a retrieval/expression deficit (Ciocchi et al., 2010). Inactivation of the CeL during fear conditioning leads to freezing deficits, but if the CeL is inactivated 24hr after conditioning it has no effect on freezing levels. Altogether, these findings clearly identify the CeL as an area important to fear learning, and the CeM as a region controlling fear expression. However the exact roles of the medial and lateral divisions of the CeA are not necessarily limited to these functions. CeL neurons have also been shown to directly project to the PAG and

180

paraventricular nucleus of the thalamus. Through these projections the CeL contributes to fear expression independent of CeM output (Penzo et al., 2014).

Based on our understanding of the primary functions of the CeM, the presence of

AT2R in this region suggests that this receptor may be involved in the expression of fear.

We tested this hypothesis by pharmacologically activating AT2R in two separate behavioral models that would expose both short and long-term effects on freezing behavior.

The behavioral effects of AT2R activation were determined by injecting C21 directly into the CeA prior to extinction training. We showed that activation of CeA

AT2R reduces freezing behavior in both auditory and contextual models of fear conditioning. The significant reduction in freezing of C21-treated animals observed at the beginning of each extinction training session demonstrates a direct effect on freezing behavior independent of extinction learning. Such an effect might suggest an inhibitory effect of C21 on neurons in the CeM, as similar results have been reported by application of muscimol in that region (Ciocchi et al., 2010).

In each of our experiments, a second extinction session was performed under drug-free conditions. Group differences in freezing on the second day were observed only in the contextual model, where the C21 group froze significantly less at the start of the test. These findings are in agreement with the emerging view that the central amygdala actively participates in fear learning in addition to simply relaying information from the BLA to the brainstem and hypothalamus (Fadok et al., 2018; Keifer et al.,

2015b; Wilensky et al., 2006; Yu et al., 2017).

181

Numerous efforts to clarify the role of Ang II in learning and memory have produced conflicting results. However, previous studies have implicated AT2R in RAS- mediated effects on memory in various brain regions. For instance, Kerr and colleagues found that Ang II blocks memory consolidation through an AT2R-dependent mechanism, while Bonini et. al., showed that intra hippocampal Ang II hinders avoidance memory retrieval through the AT2R (Bonini et al., 2006; Kerr et al., 2005). Others have reported memory-enhancing effects of central Ang II (Braszko and Wiśniewski, 1988) and suggested that these effects involve both AT1R and AT2R (Braszko, 2002). Although similar behavioral protocols were used in many of these studies, differences in experimental techniques (e.g. ICV vs. direct tissue injections) may have impacted the results. Furthermore, when Ang II is directly injected into the brain while blocking a single type of receptor, it is possible that some of the observed effects could have been caused by excess Ang II binding to other receptor subtypes. The functions of Ang II in learning and memory may also be influenced by the fact that Ang II is a precursor to other neuroactive angiotensin fragments like Ang IV (Ciobica et al., 2009) which can enhance memory and cognition (Chow et al., 2015). These technical discrepancies and alternative RAS signaling pathways may explain some of the conflicting results in the literature regarding the exact influences of Ang II and memory. Now that selective AT2R agonists such as C21 are available, we are able to avoid some of these challenges and target AT2R directly, thereby reducing potential side effects of exogenous Ang II application.

We also found that CeA injections of C21 led to a reduction in corticosterone release during context re-exposure. This result suggests that activation of CeA AT2R+

182

neurons has strong physiological effects on the stress response, in addition to reducing freezing behavior. It has been known for some time that lesioning the CeA can inhibit the release of corticosterone and renin in response to various forms of stress (Van de Kar et al., 1991). Furthermore, the CeM can activate hypothalamic nuclei which regulate sympathetic arousal, activation of the HPA axis, and the release of glucocorticoids and catecholamines into the blood stream (Stockhorst and Antov, 2016). Taken together, these findings are consistent with our behavioral results in that they point to CeA AT2R as having an inhibitory influence on neurons projecting to downstream effector regions that produce fear responses (van den Burg and Stoop, 2019; Huber, 2005).

Drugs that alter stress and anxiety can also affect locomotor activity, and these changes can confound freezing data collected in memory tasks. This was an important factor to consider in our study, as AT2R-deficient mice exhibit a reduction in exploratory behavior (Ichiki et al., 1995), which is commonly used as an index of anxiety. Other groups have found that both noradrenergic and corticotropin-releasing factor (CRF) neuronal systems are involved in the anxiety-like behavior observed in these mice

(Okuyama et al., 1999). Although they are distinct emotional states, fear and anxiety often interact to influence threat responses (Shackman and Fox, 2016). Both are mediated by the extended central amygdala circuit, which consists of two major subdivisions: the CeA and the BNST. These two functionally overlapping regions are anatomically connected by projections from the CeL to the BNST (Oler et al., 2017). A disinhibitory circuit involving SOM+ neurons in the CeL has recently been implicated in excessive anxiety (Ahrens et al., 2018). Importantly, Ang II appears to play a functional role in mediating panic responses caused by activating the amygdala (Shekhar et al.,

183

2003). However, the involvement of CeA-AT2R in anxiety has not been determined. We therefore used elevated plus maze and open field testing to identify any potential anxiolytic or anxiogenic effects of CeA AT2R activation. Injection of C21 did not have any significant effects in either test, suggesting that activation AT2R in the CeM specifically lowers conditioned freezing behavior without generally decreasing anxiety or increasing exploratory behavior. These findings, combined with our IHC results showing minimal co-localization of AT2R and SOM in the CeA, suggest that AT2R may be expressed primarily on neurons that mediate fear as opposed to anxiety.

AT2R may exert its effects by modulating neuronal excitability. In rat brain neuronal culture, AT2R stimulates a delayed rectifier K+ current and a transient K+ current through Gi and the serine threonine phosphatase PP2A (Guimond and Gallo-

Payet, 2012; Kang et al., 1994). In the RVLM, AT2R induce hyperpolarization and decreased firing rate (Matsuura et al., 2005). AT2R stimulation in neuronal cell lines has been shown to increase potassium current activity in a nitric oxide (NO)-dependent pathway (Gao et al., 2011). The AT2R also reduces neuronal excitability in the locus coeruleus (Xiong and Marshall, 1994) and the superior colliculus (Merabet et al., 1997).

The exact mechanisms underlying AT2R effects on neuronal excitability may be region and cell-type specific, however our finding that activation of this receptor in the CeA reduces both freezing behavior and corticosterone release in response to conditioned fear recall are consistent with an inhibitory role for AT2R in this region.

The AT2R+ neurons in the CeM are anatomically positioned to exert the effects on freezing responses that we observed in our behavioral experiments. This was established through retrograde tracing from the vlPAG to the CeM. In combination with our

184

observation that C21 lowered the corticosterone response to contextual fear, these findings suggest that AT2R+ GABAergic projection neurons in the CeM influence various components of the fear response, and are inhibited by local administration of C21. In the case of freezing behavior, the inhibition of these neurons likely prevents the disinhibition of vlPAG outputs to pre-motor targets in the medulla (Tovote et al., 2016).

Future directions

There is a growing interest in the function of the CeA and its role in fear acquisition and threat processing. Numerous neuropeptides are expressed in this region that modulate threat responses to allow for fine control of defensive behaviors (Sanford et al., 2017; Viviani et al., 2011). We have begun to examine AT2R in the CeM, but AT1R is also expressed in the CeA. Future studies will need to identify exactly how Ang II modifies fear learning and behavior within the amygdala, taking into account its multiple receptor subtypes and active peptide fragments, in order to fully understand the function of Ang II in this brain region. Furthermore, we observed AT2R expression in other regions of the brain including the medial amygdala, mPFC, the BNST, hypothalamus, and PAG; all of which are involved in fear learning and expression. Determining the role of AT2R in each of these regions is necessary to gain a complete understanding of the brain RAS and its role in fear learning and expression.

General Summary and Conclusions

The work described in this dissertation leads to three primary conclusions. First, that extinction recall attenuates conditioned blood pressure responses to auditory stimuli.

185

The inhibition of these responses can be remotely measured in undisturbed animals, allowing for the assessment of extinction learning under stress-free conditions. Second, that systemic blockade of AT1R during the window of reconsolidation decreases the long- term behavioral responses to conditioned auditory fear, suggesting that this receptor may be a valuable target in new therapeutic approaches for anxiety disorders like PTSD. And third, that AT2R in the central amygdala can modulate the expression of conditioned fear and may therefore play an important role in brain RAS-mediated fear responses.

Collectively, these findings identify novel functional roles for both Ang II receptor subtypes in fear maintenance and expression, and demonstrate that physiological parameters are particularly useful in the measurement of its contributions to memory strength. Future studies using chemo/optogenetic tools and single-cell sequencing will be needed to identify genetically-defined subsets of neurons on which angiotensin receptors act to influence fear expression and memory. Given our results in pre-clinical models of fear learning, and mounting clinical evidence suggesting that neuroendocrine dysregulation is an important factor in PTSD (Terock et al., 2019), the RAS may prove a valuable target for the treatment of fear-based psychiatric conditions.

186

References:

Abegaz, B., Davern, P.J., Jackson, K.L., Nguyen-Huu, T.-P., Bassi, J.K., Connelly, A., Choong, Y.-T., Allen, A.M., and Head, G.A. (2013). Cardiovascular role of angiotensin type1A receptors in the nucleus of the solitary tract of mice. Cardiovasc Res 100, 181– 191.

Ahrens, S., Wu, M., Furlan, A., Hwang, G.-R., Paik, R., Li, H., Penzo, M.A., Tollkuhn, J., and Li, B. (2018). A central extended amygdala circuit that modulates anxiety. The Journal of Neuroscience 0705–0718.

Alberini, C.M. (2009). Transcription factors in long-term memory and synaptic plasticity. Physiol. Rev. 89, 121–145.

Böhm, M., Reil, J.-C., Deedwania, P., Kim, J.B., and Borer, J.S. (2015). Resting heart rate: risk indicator and emerging risk factor in cardiovascular disease. Am. J. Med. 128, 219–228.

Bouton, M.E., and Bolles, R.C. (1980). Conditioned fear assessed by freezing and by the suppression of three different baselines. Animal Learning & Behavior 8, 429–434.

Braszko, J.J., and Wiśniewski, K. (1988). Effect of angiotensin II and saralasin on motor activity and the passive avoidance behavior of rats. Peptides 9, 475–479. van den Burg, E.H., and Stoop, R. (2019). Neuropeptide signalling in the central nucleus of the amygdala. Cell and Tissue Research 375, 93–101.

Butz, G.M., and Davisson, R.L. (2001). Long-term telemetric measurement of cardiovascular parameters in awake mice: a physiological genomics tool. Physiol. Genomics 5, 89–97.

Carrive, P. (2000). Conditioned fear to environmental context: cardiovascular and behavioral components in the rat. Brain Research 858, 440–445.

Chiu, W.-C., Ho, W.-C., Lin, M.-H., Lee, H.-H., Yeh, Y.-C., Wang, J.-D., Chen, P.-C., and Health Data Analysis in Taiwan (hDATa) Research Group (2014). Angiotension receptor blockers reduce the risk of dementia. J. Hypertens. 32, 938–947.

Chow, L.-H., Tao, P.-L., Chen, Y.-H., Lin, Y.-H., and Huang, E.Y.-K. (2015). Angiotensin IV possibly acts through PKMzeta in the hippocampus to regulate cognitive memory in rats. Neuropeptides 53, 1–10.

187

Ciobica, A., Bild, W., Hritcu, L., and Haulica, I. (2009). Brain renin-angiotensin system in cognitive function: pre-clinical findings and implications for prevention and treatment of dementia. Acta Neurologica Belgica 109, 171.

Duvarci, S., Nader, K., and LeDoux, J.E. (2008). De novo mRNA synthesis is required for both consolidation and reconsolidation of fear memories in the amygdala. Learn. Mem. 15, 747–755.

Duvarci, S., Popa, D., and Paré, D. (2011). Central Amygdala Activity during Fear Conditioning. J Neurosci 31, 289–294.

Fadok, J.P., Markovic, M., Tovote, P., and Lüthi, A. (2018). New perspectives on central amygdala function. Current Opinion in Neurobiology 49, 141–147.

Falls, W.A., Miserendino, M.J., and Davis, M. (1992). Extinction of fear-potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala. The Journal of Neuroscience 12, 854–863.

Gillie, B.L., and Thayer, J.F. (2014). Individual differences in resting heart rate variability and cognitive control in posttraumatic stress disorder. Frontiers in Psychology 5.

Gordan, R., Gwathmey, J.K., and Xie, L.-H. (2015). Autonomic and endocrine control of cardiovascular function. World J Cardiol 7, 204–214.

Gouveia, K., and Hurst, J.L. (2017). Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling. Scientific Reports 7.

Guimond, M.-O., and Gallo-Payet, N. (2012). The Angiotensin II Type 2 Receptor in Brain Functions: An Update. International Journal of Hypertension 2012, 1–18.

Hager, T., Maroteaux, G., Pont, P. du, Julsing, J., van Vliet, R., and Stiedl, O. (2014). Munc18-1 haploinsufficiency results in enhanced anxiety-like behavior as determined by heart rate responses in mice. Behavioural Brain Research 260, 44–52.

188

Hsu, Y.-C., Yu, L., Chen, H., Lee, H.-L., Kuo, Y.-M., and Jen, C.J. (2012). Blood pressure variations real-time reflect the conditioned fear learning and memory. PLoS ONE 7, e32855.

Huber, D. (2005). Vasopressin and Oxytocin Excite Distinct Neuronal Populations in the Central Amygdala. Science 308, 245–248.

Ichiki, T., Labosky, P.A., Shiota, C., Okuyama, S., Imagawa, Y., Fogo, A., Niimura, F., Ichikawa, I., Hogan, B.L.M., and Lnagami, T. (1995). Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature 377, 748– 750.

Iwata, J., and LeDoux, J.E. (1988). Dissociation of associative and nonassociative concomitants of classical fear conditioning in the freely behaving rat. Behavioral Neuroscience 102, 66.

Jan Braszko (2002). AT2 but not AT1 receptor antagonism abolishes Angiotensin II increase of the aquisition of conditioned avoidance responses in rats. BEHAV BRAIN RES 131, 79–86.

Janak, P.H., and Tye, K.M. (2015). From circuits to behaviour in the amygdala. Nature 517, 284–292.

Kang, J., Posner, P., and Sumners, C. (1994). Angiotensin II type 2 receptor stimulation of neuronal K+ currents involves an inhibitory GTP binding protein. Am. J. Physiol. 267, C1389-1397.

Keifer, O.P., Hurt, R.C., Ressler, K.J., and Marvar, P.J. (2015). The Physiology of Fear: Reconceptualizing the Role of the Central Amygdala in Fear Learning. Physiology 30, 389–401.

Kerr, D.S., Bevilaqua, L.R.M., Bonini, J.S., Rossato, J.I., Köhler, C.A., Medina, J.H., Izquierdo, I., and Cammarota, M. (2005). Angiotensin II blocks memory consolidation through an AT2 receptor-dependent mechanism. Psychopharmacology 179, 529–535. de Kloet, A.D., Pitra, S., Wang, L., Hiller, H., Pioquinto, D.J., Smith, J.A., Sumners, C., Stern, J.E., and Krause, E.G. (2016). Angiotensin Type-2 Receptors Influence the Activity of Vasopressin Neurons in the Paraventricular Nucleus of the Hypothalamus in Male Mice. Endocrinology 157, 3167–3180.

189

Lee, J.L.C., Milton, A.L., and Everitt, B.J. (2006). Reconsolidation and Extinction of Conditioned Fear: Inhibition and Potentiation. Journal of Neuroscience 26, 10051–10056.

Li, J., Culman, J., Hörtnagl, H., Zhao, Y., Gerova, N., Timm, M., Blume, A., Zimmermann, M., Seidel, K., Dirnagl, U., et al. (2005). Angiotensin AT2 receptor protects against cerebral ischemia-induced neuronal injury. FASEB J. 19, 617–619.

Liu, J., Wei, W., Kuang, H., Zhao, F., and Tsien, J.Z. (2013). Changes in Heart Rate Variability Are Associated with Expression of Short-Term and Long-Term Contextual and Cued Fear Memories. PLoS One 8.

Maddox, S.A., Hartmann, J., Ross, R.A., and Ressler, K.J. (2019). Deconstructing the Gestalt: Mechanisms of Fear, Threat, and Trauma Memory Encoding. Neuron 102, 60– 74.

Mataix-Cols, D., Cruz, L.F. de la, Monzani, B., Rosenfield, D., Andersson, E., Pérez- Vigil, A., Frumento, P., Kleine, R.A. de, Difede, J., Dunlop, B.W., et al. (2017). D- Cycloserine Augmentation of Exposure-Based Cognitive Behavior Therapy for Anxiety, Obsessive-Compulsive, and Posttraumatic Stress Disorders: A Systematic Review and Meta-analysis of Individual Participant Data. JAMA Psychiatry 74, 501–510.

Matsuura, T., Kumagai, H., Onimaru, H., Kawai, A., Iigaya, K., Onami, T., Sakata, K., Oshima, N., Sugaya, T., and Saruta, T. (2005). Electrophysiological Properties of Rostral Ventrolateral Medulla Neurons in Angiotensin II 1a Receptor Knockout Mice. Hypertension 46, 349–354.

Mccabe, P., Schneiderman, N., Field, T.M., and Wellens, A.R. (2000). Stress, Coping, and Cardiovascular Disease (Psychology Press).

Mechaeil, R., Gard, P., Jackson, A., and Rusted, J. (2011). Cognitive enhancement following acute losartan in normotensive young adults. Psychopharmacology 217, 51–60.

Merabet, L., de Gasparo, M., and Casanova, C. (1997). Dose-dependent inhibitory effects of angiotensin II on visual responses of the rat superior colliculus: AT1 and AT2 receptor contributions. Neuropeptides 31, 469–481.

190

Minassian, A., Maihofer, A.X., Baker, D.G., Nievergelt, C.M., Geyer, M.A., and Risbrough, V.B. (2015). Association of Predeployment Heart Rate Variability With Risk of Postdeployment Posttraumatic Stress Disorder in Active-Duty Marines. JAMA Psychiatry 1.

Mogi, M., and Horiuchi, M. (2009). Effects of angiotensin II receptor blockers on dementia. Hypertens. Res. 32, 738–740.

Mogi Masaki, Li Jian-Mei, Iwanami Jun, Min Li-Juan, Tsukuda Kana, Iwai Masaru, and Horiuchi Masatsugu (2006). Angiotensin II Type-2 Receptor Stimulation Prevents Neural Damage by Transcriptional Activation of Methyl Methanesulfonate Sensitive 2. Hypertension 48, 141–148.

Nader, K., and Einarsson, E.O. (2010). Memory reconsolidation: an update. Ann. N. Y. Acad. Sci. 1191, 27–41.

Nader, K., Hardt, O., and Lanius, R. (2013). Memory as a new therapeutic target. Dialogues Clin Neurosci 15, 475–486.

Okuyama, S., Sakagawa, T., and Inagami, T. (1999). Role of the angiotensin II type-2 receptor in the mouse central nervous system. Jpn. J. Pharmacol. 81, 259–263.

Oler, J.A., Tromp, D.P.M., Fox, A.S., Kovner, R., Davidson, R.J., Alexander, A.L., McFarlin, D.R., Birn, R.M., E Berg, B., deCampo, D.M., et al. (2017). Connectivity between the central nucleus of the amygdala and the bed nucleus of the stria terminalis in the non-human primate: neuronal tract tracing and developmental neuroimaging studies. Brain Struct Funct 222, 21–39.

Rao-Ruiz, P., Couey, J.J., Marcelo, I.M., Bouwkamp, C.G., Slump, D.E., Matos, M.R., Loo, R.J. van der, Martins, G.J., Hout, M. van den, IJcken, W.F. van, et al. (2019). Engram-specific transcriptome profiling of contextual memory consolidation. Nat Commun 10, 1–14.

Reinecke, A., Browning, M., Klein Breteler, J., Kappelmann, N., Ressler, K.J., Harmer, C.J., and Craske, M.G. (2018). Angiotensin Regulation of Amygdala Response to Threat

191

in High-Trait-Anxiety Individuals. Biological Psychiatry: Cognitive Neuroscience and Neuroimaging.

Roelofs, K. (2017). Freeze for action: neurobiological mechanisms in animal and human freezing. Philosophical Transactions of the Royal Society B: Biological Sciences 372, 20160206.

Sakaguchi, A., LeDoux, J.E., and Reis, D.J. (1983). Sympathetic nerves and adrenal medulla: contributions to cardiovascular-conditioned emotional responses in spontaneously hypertensive rats. Hypertension 5, 728–738.

Sanford, C.A., Soden, M.E., Baird, M.A., Miller, S.M., Schulkin, J., Palmiter, R.D., Clark, M., and Zweifel, L.S. (2017). A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats. Neuron 93, 164–178.

Schroyens, N., Beckers, T., and Kindt, M. (2017). In Search for Boundary Conditions of Reconsolidation: A Failure of Fear Memory Interference. Front Behav Neurosci 11, 65.

Shackman, A.J., and Fox, A.S. (2016). Contributions of the Central Extended Amygdala to Fear and AnxietyContributions of the Central Extended Amygdala to Fear and Anxiety. J. Neurosci. 36, 8050–8063.

Shekhar, A., Sajdyk, T.J., Gehlert, D.R., and Rainnie, D.G. (2003). The Amygdala, Panic Disorder, and Cardiovascular Responses. Annals of the New York Academy of Sciences 985, 308–325.

Song, K.F., Zhuo, J.L., and Mendelsohn, F.A. (1991). Access of peripherally administered DuP 753 to rat brain angiotensin II receptors. Br. J. Pharmacol. 104, 771– 772.

Stiedl, O. (1999). Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice. Behavioural Brain Research 104, 1–12.

Stiedl, O., and Hager, T. (2017). Cardiovascular Conditioning: Neural Substrates☆. In Reference Module in Neuroscience and Biobehavioral Psychology, (Elsevier), p.

Stiedl, O., and Spiess, J. (1997). Effect of tone-dependent fear conditioning on heart rate and behavior of C57BL/6N mice. Behavioral Neuroscience 111, 703.

Stiedl, O., Palve, M., Radulovic, J., Birkenfeld, K., and Spiess, J. (1999). Differential impairment of auditory and contextual fear conditioning by protein synthesis inhibition in C57BL/6N mice. Behavioral Neuroscience 113, 496–506.

192

Stiedl, O., Birkenfeld, K., Palve, M., and Spiess, J. (2000). Impairment of conditioned contextual fear of C57BL/6J mice by intracerebral injections of the NMDA receptor antagonist APV. Behavioural Brain Research 116, 157–168.

Stockhorst, U., and Antov, M.I. (2016). Modulation of Fear Extinction by Stress, Stress Hormones and Estradiol: A Review. Front Behav Neurosci 9.

Sun, X., and Lin, Y. (2016). Npas4: Linking Neuronal Activity to Memory. Trends Neurosci 39, 264–275.

Torika, N., Asraf, K., Apte, R.N., and Fleisher‐Berkovich, S. (2018). Candesartan ameliorates brain inflammation associated with Alzheimer’s disease. CNS Neurosci Ther 24, 231–242.

Torras-Garcia, M., Lelong, J., Tronel, S., and Sara, S.J. (2005). Reconsolidation after remembering an odor-reward association requires NMDA receptors. Learn. Mem. 12, 18–22.

Visser, R.M., Lau-Zhu, A., Henson, R.N., and Holmes, E.A. (2018). Multiple memory systems, multiple time points: how science can inform treatment to control the expression of unwanted emotional memories. Philos Trans R Soc Lond B Biol Sci 373.

Viviani, D., Charlet, A., van den Burg, E., Robinet, C., Hurni, N., Abatis, M., Magara, F., and Stoop, R. (2011). Oxytocin Selectively Gates Fear Responses Through Distinct Outputs from the Central Amygdala. Science 333, 104–107.

Wang, J.M., Tan, J., and Leenen, F.H.H. (2003). Central Nervous System Blockade by Peripheral Administration of AT1 Receptor Blockers. Journal of Cardiovascular Pharmacology 41, 593.

193

Wang, X., Li, M., Zhu, H., Yu, Y., Xu, Y., Zhang, W., and Bian, C. (2018). Transcriptional Regulation Involved in Fear Memory Reconsolidation. Journal of Molecular Neuroscience.

Wilensky, A.E., Schafe, G.E., Kristensen, M.P., and LeDoux, J.E. (2006). Rethinking the Fear Circuit: The Central Nucleus of the Amygdala Is Required for the Acquisition, Consolidation, and Expression of Pavlovian Fear Conditioning. Journal of Neuroscience 26, 12387–12396.

Xiong, H., and Marshall, K.C. (1994). Angiotensin II depresses glutamate depolarizations and excitatory postsynaptic potentials in locus coeruleus through angiotensin II subtype 2 receptors. Neuroscience 62, 163–175.

Ye, J., and Veinante, P. (2019). Cell-type specific parallel circuits in the bed nucleus of the stria terminalis and the central nucleus of the amygdala of the mouse. Brain Struct Funct.

Ye, S., Zhong, H., Duong, V.N., and Campese, V.M. (2002). Losartan Reduces Central and Peripheral Sympathetic Nerve Activity in a Rat Model of Neurogenic Hypertension. Hypertension 39, 1101–1106.

Young, C.N., and Davisson, R.L. (2011). In vivo assessment of neurocardiovascular regulation in the mouse: principles, progress, and prospects. AJP: Heart and Circulatory Physiology 301, H654–H662.

Zhou, M., Liu, Z., Melin, M.D., Ng, Y.H., Xu, W., and Südhof, T.C. (2018). A central amygdala to zona incerta projection is required for acquisition and remote recall of conditioned fear memory. Nature Neuroscience 21, 1515–1519.

194