Endocannabinoid Metabolism in Posttraumatic Stress Disorder: Preliminary Results from a Neuroimaging Study with the Novel Fatty Acid Amide Hydrolase Probe [C-11]CURB

by

Erin Victoria Lynn Gaudette

A thesis submitted in conformity with the requirements for the degree of Master of Science

Institute of Medical Science University of Toronto

© Copyright by Erin Gaudette (2020)

Endocannabinoid Metabolism in Posttraumatic Stress Disorder: Preliminary Results from a Neuroimaging Study with the Novel Fatty Acid Amide Hydrolase Probe [C-11]CURB Erin Gaudette Master of Science Institute of Medical Science University of Toronto 2020

Abstract

Background: Preclinical studies suggest that levels of Fatty Acid Amide Hydrolase

(FAAH)—the catabolic enzyme for the endocannabinoid anandamide—may be elevated in the amygdala in posttraumatic stress disorder (PTSD). However, the status of FAAH in vivo in posttraumatic stress disorder remains unknown.

Methods: Healthy subjects (n=29) and individuals with PTSD (n=16) completed a positron emission tomography scan following injection of the FAAH probe [C-11]CURB.

Results: We find no evidence for elevated [C-11]CURB binding in PTSD. Instead, we find marginally lower [C-11]CURB binding in whole brain (-9.38%, p=0.079) and significantly lower [C-11]CURB binding in the amygdala (-14.50%, p=0.020) in PTSD subjects. [C-11]CURB binding did not correlate with PTSD symptomatology.

Conclusion: Our data provide preliminary evidence contrary to preclinical literature, suggesting that FAAH may be lower in people with PTSD. These findings have implications for treatment strategies targeting this enzyme (i.e. FAAH inhibitors) in

PTSD.

ii Acknowledgements

I owe a debt of gratitude to my supervisor, Dr. Isabelle Boileau. Her supervision and guidance on this project have been instrumental in both its success and also in my development as a young scientist. She has been incredibly patient and supportive throughout my degree and of my endeavors outside of the lab. I admire her tremendously, both professionally and personally, and feel so fortunate to have had a supervisor like Isabelle who also served as a mentor and role model to me. It has been a privilege to have completed this work under your supervision. To my advisory committee members, Dr. Benjamin Dunkley and Dr. Richard Bazinet, thank you for your time, patience, and invaluable insights throughout this project. Your feedback and guidance have made this project better, and for that I am very appreciative. I want to also acknowledge the Canadian Institutes for Health Research for funding this work, as well as the participants who generously volunteered their time for this project. Without their contributions, this research would not be possible. I also want to thank the members of our laboratory. In particular, I want to thank Tina McCluskey, whose impeccable eye for details and organizational skills have consistently astounded me and who very often caught my mistakes. To the members of the lab, who are both my colleagues and friends, thank you for your pep talks, encouragement, guidance, and support over the course of this degree. I cannot wait to see the contributions you all will make to the field in the years to come. Finally, I want to thank my friends and family for their support. Thank you especially to my parents and brother, Matthew, Bonni and Alexander Gaudette, for the many ways in which they have supported and believed in me in everything I’ve undertaken, even when I’ve encountered setbacks or been discouraged. Thank you also to Robert Lewis, Yonah Lewis, and Sandra Baumander, whose support and love have been so meaningful to me. Finally, thank you to Lev Lewis, my partner. For your patience with me, your unwavering support and interest in everything I do, and the innumerable ways your presence enriches my life, there is no thank you deep enough.

iii Statement of Contributions

Though I’ve been principally responsible for this project, I have relied on the expertise of the team at the Research Imaging Centre as well as my research team given the extensive, multi-visit nature of this study. All recruitment efforts were undertaken by myself, and I completed phone screens and corresponded with all participants. All PET imaging sessions were conducted by either Alvina Ng, Kayleigh Timmins, or Laura Nguyen, the PET technicians of the Research Imaging Centre. Similarly, all MRI scans were conducted by the MRI technician, Anusha Ravichandran. Myself or another member of my team was present during all study procedures, including scans. Radiochemists within the Research Imaging Centre synthesized the radiotracer for each PET scan. Blood samples taken during the PET scan were processed by either myself or a member of the lab (including Sarah Watling, Laura Best, Jennifer Truong and Nadia Boachie), depending on who was overseeing the scan. I oversaw the majority of study visits (including intake, PET scan, MRI scan), though these visits occasionally were overseen by Duncan Green, and Sarah Watling for certain participants depending on scheduling. All data entry, figure and table design, and statistical analysis have been completed by me.

iv Table of Contents

Abstract ...... ii Acknowledgements ...... iii Statement of Contributions ...... iv Table of Contents ...... v List of Abbreviations ...... viii List of Tables ...... xi List of Figures ...... xii Chapter 1: Introduction...... 1 1.1 Brief History of PTSD ...... 1

1.2 Diagnostic Criteria ...... 1

1.3 Prevalence of PTSD ...... 4

1.4 Public Health Implications of PTSD ...... 5

1.5 Current Therapeutic Options for PTSD ...... 6

1.6 Current Thinking on the Pathophysiology Underlying PTSD ...... 8 1.6.1 General Thinking on the Pathophysiology Underlying PTSD ...... 8 1.6.2 Pathophysiology Underlying Different Symptom Clusters May Vary ...... 11 1.7 The Endocannabinoid System ...... 12 1.7.1 Discovery of the Endocannabinoid System ...... 12 1.7.2 Overview of the Endocannabinoid System ...... 12 1.7.3 Animal Studies of the Endocannabinoid System in Stress and Trauma Models ...... 17 1.7.4 Human Studies of the Endocannabinoid System in Posttraumatic Stress Disorder ...... 31 1.7.5 Summary of the Evidence Suggesting Endocannabinoid System Disruption in PTSD ...... 34 1.8 [C-11]CURB as a FAAH Radiotracer ...... 35 1.8.1 Brief Introduction to PET Imaging ...... 35 1.8.2 Development of [C-11]CURB ...... 35 1.8.3 Radiosynthesis of [C-11]CURB ...... 36 1.8.4 Properties of [C-11]CURB ...... 36 1.9 Summary, Objectives & Hypotheses ...... 39 1.9.1 Summary ...... 39 1.9.2 Objectives ...... 40

v 1.9.3 Hypotheses ...... 40 Chapter 2: Methods ...... 41 2.1 Study Participants ...... 41

2.2 Screening and Inclusion ...... 41

2.3 FAAH Genotyping ...... 46

2.4 MRI Session...... 46

2.5 PET Imaging Session ...... 46

2.6 PET Image Acquisition and Reconstruction ...... 47 2.6.1 Region-Based Image Analysis ...... 49 2.6.2 Quantification of [C-11]CURB ...... 50 2.7 Statistical Plan of Analysis ...... 51

Chapter 3: Results ...... 53 3.1 Demographics and Characteristics...... 53

3.2 [C-11]CURB Binding Between Groups ...... 56

3.3 [C-11]CURB Binding Between Groups in Whole Brain ...... 60

3.4 [C-11]CURB Binding in Relation to PTSD Symptomatology ...... 61

3.5 [C-11]CURB Binding in Relation to BMI ...... 62

Chapter 4: Discussion ...... 64 4.1 Summary of Findings ...... 64

4.2 Interpreting Levels of [C-11]CURB Binding in PTSD ...... 64 4.2.1 Interpreting Unelevated [C-11]CURB Binding in PTSD ...... 64 4.2.2 Interpreting the Possibility of Lowered [C-11]CURB Binding in PTSD...... 69 4.2.3 Interpreting the Lack of Association Between [C-11]CURB Levels and PTSD Symptomatology...... 71 4.3 Strengths ...... 72

4.4 Limitations ...... 72

4.5 Clinical Significance ...... 73

4.7 Future Directions ...... 74

vi 4.6 Conclusions ...... 75

References ...... 76

vii List of Abbreviations

2-AG 2-Arachidonoylglyceral 2TCMi 2-Tissue Compartment Model with Irreversible Trapping in the Second Compartment AEA Anandamide AES Apathy Evaluation Scale ANOVA Analysis of Variance AUDIT Alcohol Use Disorder Identification Test BDI Beck Depression Inventory BIS-11 Barratt Impulsiveness Scale BLA Basolateral Amygdala BMI Body Mass Index CAMH Centre for Addiction and Mental Health cAMP Cyclic Adenosine Monophosphate cDNA Complementary Deoxyribonucleic Acid CB1R Cannabinoid Receptor Type 1 CB2R Cannabinoid Receptor Type 2 CBF Cerebral Blood Flow CES Combat Exposure Scale CRH Corticotropin-Releasing Hormone CRHR1 Corticotropin-Releasing Hormone Type 1 Receptors CNS Central Nervous System CO Carbon Monoxide [C-11]CURB 6-Hydroxy-[1,1′-biphenyl]-3-yl Cyclohexylcarbamate DAGL sn-1-Selective Diacylglycerol Lipases DHEA Docosahexaenoyl Ethanolamide DSM-5 Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition DSPS Dissociative Subtype of PTSD Scale EC Endocannabinoid

viii ECS Endocannabinoid System EEG Electroencephalography FAAH Fatty Acid Amide Hydrolase fMRI Functional Magnetic Resonance Imaging FTND Fagerstrom Test for Nicotine Dependence GABA Gamma-Aminobutyric Acid GAD-7 Generalized Anxiety Disorder-7 GTA Greater Toronto Area HC Healthy Control HPA Hypothalamic-Pituitary-Adrenal HPLC High-Performance Liquid Chromatography HRRT High Resolution Research Tomograph KeV Kiloelectron Volt KO Knock-Out LTP Long-Term Potentiation MAGL Monoacylglycerol Lipase MAPK Mitogen-Activated Protein Kinase MEG Magnetoencephalography MNI Montreal Neurological Institute MRI Magnetic Resonance Imaging mRNA Messenger Ribonucleic Acid mPFC Medial Prefrontal Cortex NAPE-PLD N-Acylphosphatidyl-Ethanolamine-Specific Phospholipase D NAT N-Acyltransferase OEA Oleoylethanolamide OSISS Operational Stress Injury Social Support PCL PTSD Checklist PD Proton Density PEA Palmitoylethanolamide PET Positron Emission Tomography

ix PFC Prefrontal Cortex PHQ-9 Patient Health Questionnaire-9 PKA Protein Kinase A PLC Phospholipase C PPAR Peroxisome Proliferator-Activated Receptor PSQI Pittsburgh Sleep Quality Index PSS PTSD Symptom Scale PTSD Posttraumatic Stress Disorder ROI Region of Interest ROMI Regions of Mental Interest SEA Stearoylethanolamide SHPS Snaith-Hamilton Pleasure Scale SNP Single Nucleotide Polymorphism SPM8 Statistical Parametric Mapping Software, version 8 SSRI Selective Serotonin Reuptake Inhibitor STAI State-Trait Anxiety Inventory TAC Time Activity Curve THC Δ⁹-Tetrahydrocannabinol TLEQ Traumatic Life Events Questionnaire TRPV1 Transient Receptor Potential Cation Channel Subfamily V Member 1 vmPFC Ventromedial Prefrontal Cortex WWI World War One WWII World War Two

x List of Tables

Table 1: Methodological Considerations and Variations in Animal Models of PTSD

Table 2: Summary of Preclinical Studies of the Endocannabinoid System in Relation to

Stress and Anxiety

Table 3: Screening Visit Questionnaires

Table 4: Subject Demographics

Table 5: Characteristics of PTSD Group

Table 6: Partial Correlations Between Amygdala k3 Values and PTSD Symptoms

xi List of Figures

Figure 1: PTSD Symptoms

Figure 2: Timeline of PET Scan

Figure 3: ROMI Process for ROI Delineation

Figure 4: Diagram of 2-Tissue Compartment Model with Irreversible Binding in the

Second Compartment

Figure 5: PTSD Recruitment

Figure 6: k3 in Cortical Regions of Interest

Figure 7: k3 in Subcortical Regions of Interest

Figure 8: k3 Across Whole Brain

Figure 9: Correlations Between Whole Brain k3 and BMI

xii Chapter 1: Introduction

1.1 Brief History of PTSD

Though there are centuries-old descriptions of psychological symptoms after trauma (including early accounts of the symptoms that today are used for diagnosis of

PTSD), these accounts were primarily anecdotal until the eighteenth century.1 During the American Civil War (1861-1865) and early railway crashes of the nineteenth century where people in these contexts experienced similar phenomena, the recognition of these symptoms begins to emerge with terms like “nostalgia” in the American Civil War and

“railway-brain” in the railway crashes.1 These reproduceable post-traumatic experiences began a movement towards scientific recognition of the reliable ways in which people are affected by trauma which continued with the first World War (WWI, 1914-1918). It was at this point that traumatic stress was first considered a clinical entity, and large-scale efforts were made by the scientific community to understand the underpinnings of these phenomena.1 Though WWI did mark a turning point for the evolution of our understanding of traumatic stress reactions, it was not until the second world war (WWII, 1939-1945) that this illness was integrated into mainstream medicine. What was known as “shell- shock” in WWI became replaced with diagnoses of “combat stress reaction” or “combat fatigue”, responsible for many of the military discharges of the war.2 Moreover, the increasing presence of psychiatrists in the military further contributed to the general recognition that environmental stress can affect mental functioning and of the diagnoses that described traumatic stress conditions.3 The experiences of psychiatrists during the second WWII not only greatly impacted the recognition of this particular disorder but psychiatry in general, and eventually led to publication of the first edition of the Diagnostic and Statistical Manual of Mental Disorders as psychiatry moved towards a more standardized clinical approach.3

1.2 Diagnostic Criteria

With the publication of the fifth edition of the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders (DSM-5) came significant changes

1 with respect to how PTSD is classified and diagnosed. Importantly, PTSD was removed from the category of anxiety disorders, and placed in a new diagnostic category called

“Trauma and Stressor-related Disorders”.4,5 This category of disorders requires, in addition to the specific diagnostic criteria, exposure to a stressful event, and it is this condition which unifies the disorders in the category, as opposed to similar symptoms. In addition to PTSD, this category of disorders includes adjustment disorder, acute stress disorder, reactive attachment disorder and disinhibited social engagement disorder.4 Criteria A for diagnosing PTSD according to the DSM-5 is exposure to trauma. The DSM-5 has defined trauma as “actual or threatened death, serious injury or sexual violence”, and this definition represents a significant narrowing of this criteria from earlier versions.4,5 In addition to having experienced the traumatic event, individuals must also have had significant exposure to it. There are four qualifying types of exposure: direct personal exposure, witnessing trauma, indirect exposure through the experiences of a person close to you, and repeated or extreme exposure to aversive details of a traumatic event. The next four criteria (B, C, D and E) address PTSD symptoms. One additional criterion (E) has been newly added with the latest addition of the DSM given the previous edition grouped numbing and avoidance symptoms together, and the number of symptoms has increased from 17 to 20.4 The DSM-5 stipulates that these symptoms must begin or worsen after the traumatic event. Criterion B includes intrusion symptoms like unwanted upsetting memories, nightmares, flashbacks, emotional distress after exposure to reminders of trauma and physical reactivity after exposure to reminders of trauma. Individuals must endorse at least one symptom from this cluster for a diagnosis of PTSD. Criterion C includes avoidance symptoms, like avoidance of trauma-related thoughts or feelings or avoidance of trauma-related external reminders. Individuals must endorse at least one symptom from Criterion C to be diagnosed with PTSD. Criterion D includes negative alterations in cognition and mood including: inability to recall key features of the event; persistent and exaggerated beliefs about oneself, others or the world; persistent negative emotions; diminished interest or pleasure in previously enjoyed activities; feelings of detachment or estrangement from others; persistent distorted cognitions about the cause or consequences of the event; and inability to experience positive emotions.

2 Individuals must endorse at least two of the Criterion D symptoms to be diagnosed with PTSD. Finally, Criterion E includes arousal and hyperreactivity symptoms, like irritable behaviour and angry outbursts, reckless or self-destructive behaviour, hypervigilance, exaggerated startle response, difficulty concentrating, and sleep disturbances. Individuals must endorse two or more symptoms from Criterion E to be diagnosed with PTSD. These symptom clusters are shown in Figure 1: PTSD Symptoms. Criterion F requires that these symptoms have been present at least one month. Criterion G requires that these symptoms cause clinically significant impairment in social, occupation, or other important areas of functioning. Finally, Criterion H stipulates that these symptoms are not attributable to medication, substance use, or other illness. Criteria A-H must all be satisfied to be diagnosed with PTSD.

Figure 1: PTSD Symptoms. There are four clusters of PTSD symptoms according to the DSM-5 criteria for PTSD. Criterion A includes exposure to a traumatic event, while Criteria F requires clinically significant

3 impairment, Criteria G requires symptom duration of at least one month, and Criteria H requires that these symptoms are attributable to medication, substance use, or other illness.

The DSM-5 eliminated the previous edition’s acute and chronic specifiers of

PTSD.5 Instead, there is now a specifier for delayed expression, wherein the diagnostic criteria are not met until 6 or more months after the trauma.4 In addition, there is now a dissociative subtype specifier to note the presence of depersonalization and derealization symptoms.

1.3 Prevalence of PTSD

Several studies have suggested that the lifetime prevalence of PTSD in the United States is around 9-12%, with prevalence in women ranging from approximately 11-13% and prevalence in men ranging from approximately 5-6%.6-8 Prevalence in the general population in Canada appears to be similar. One epidemiological study found a lifetime prevalence of 9.2% in Canada, with lifetime prevalence rates of 5.3% in men and 12.3% in women.9 Another epidemiological study sampling Ontario women found the lifetime prevalence of PTSD was 10.7%.10 The results from the Canadian Community Health Survey completed in 2012 suggest a one-year prevalence rate of 1.7%, while estimates of the one-year PTSD prevalence rate in the United States are around 3.5%, though estimates in other parts of the world tend to be lower.11-13 Given the likelihood of trauma exposure is increased in certain occupations, people working in these occupations are considered to be at a higher risk for developing PTSD (and other mental illnesses more generally). These occupations include first responders, military personnel, correctional officers, among others. One recent study of mental illnesses among public safety personnel in Canada found that 44% of respondents reported symptoms consistent with at least one mental illness, with positive screens for mental illnesses increasing as a function of years of service.14 Moreover, the overall rate of positive screens of PTSD in this sample was found to be 23.2%, with Royal Canadian Mounted Police (30.0%), correctional officers (29.1%) and paramedics (24.5%) having the highest rates of positive PTSD screens when stratified by occupation.14 One study in active duty police officers found that every officer in the sample had experienced one event they considered traumatic, and found that 31.9% of the sample screened positive

4 for PTSD.15 Similarly, another study in paramedics found that the entire sample had experienced at least one traumatic event, with 29.1% of the sample endorsing high or severe levels of PTSD symptoms.16 Another study examining the medical records of Canadian Forces personnel deployed to Afghanistan found that PTSD prevalence was

8% among these veterans.17 While different screening methodologies can make it difficult to compare prevalence rates between studies, these studies provide clear evidence of significantly higher rates of PTSD in high-risk groups. For this reason, and to minimize some of the heterogeneity in our data, we decided to focus on occupational trauma in this project.

1.4 Public Health Implications of PTSD

In addition to the challenges people with PTSD face as a result of their disorder, there are additional health implications as well. For one, the trajectory of PTSD is typically chronic, with one third of patients failing to recover even after intensive treatment.7 There is also a high degree of comorbidity of other psychiatric disorders, suicidality, and substance use disorders.18-20 In terms of physical health, it appears that PTSD increases the risk for other health conditions as well, including musculoskeletal conditions, digestive disorders, and cardiovascular disease.21,22 PTSD has also been shown to predispose people to adverse life course outcomes, including 40% greater risk for high school or college failure, 30% greater risk for teenage childbearing, and 150% greater risk for unemployment.23 These health outcomes may be worsened in developing countries. In addition to the burden to the individual patient, PTSD comes at a significant cost to society. Mental illness has been estimated to cost Canada $20.7 billion dollars annually– about 1.3% of the national Gross Domestic Product–due to lost productivity.11 Moreover, in addition to the actual costs of treating PTSD, particularly since patients with PTSD are known to have greater utilization of healthcare services, absenteeism and unemployment, psychiatric comorbidities, and other physical comorbidities that often come alongside PTSD add to the societal burden, making this disorder a significant area of concern for public health.23,24

5 1.5 Current Therapeutic Options for PTSD

There is a general lack of effective treatment options for PTSD presently. The following paragraphs will briefly survey the present therapeutic evidence for psychotherapy, pharmacotherapy, cannabis and synthetic cannabinoid therapies, and the development of Fatty Acid Amide Hydrolase (FAAH) inhibitors.

Psychotherapy appears to be one of the more effective treatment options.25 Several studies have concluded that of the different psychotherapy approaches, cognitive behavioral therapy appears to be most effective,26,27 though there is some evidence for the efficacy of other approaches including eye movement desensitization and reprocessing and exposure therapy.28,29 However, it bears mentioning that these therapies often require multiple sessions and can be costly, which may pose a barrier to access, particularly for the patients whose workplace-related trauma precludes their ability to work and thus may not have access to stable income and healthcare benefits. Presently, there are two medications approved for the treatment of PTSD by the

Food and Drug Administration: sertraline and paroxetine.28 Both of these drugs belong to the class of medications known as selective serotonin reuptake inhibitors (SSRI). However, a Cochrane review of pharmacotherapies for PTSD found that SSRIs did not perform significantly better than medications of other classes, including tricyclic antidepressants and monoamine oxidase inhibitors.30 Moreover, the same review found that the effect sizes for the medications reviewed failed to exceed the threshold indicative of clinical effectiveness.30 In line with this, treatment with SSRIs have been shown to have generally low response rates, with less than 30% of treated patients reaching full remission, and often take several weeks to for a detectable therapeutic effect.31 Moreover, even if the patient does experience a therapeutic effect, this effect only persists with continuation of the medication,32 suggesting that even in the best case scenario with patients who respond well, the therapeutic efficacy of the drug is only in symptom management. Similarly to these first line treatments, there is little consensus on second line pharmacotherapies, many of which are borrowed from clinical indications for depression and anxiety disorders. Part of the complexity in clinical care may arise from the management of different symptom clusters requiring different drugs, which may have

6 counterindications or may counteract each other. Benzodiazepines may be utilized in reducing the anxiety-like features of PTSD, though there is little clinical consensus on their utility in addition to the fact that there is a significant risk for addiction and misuse with these drugs.31 There is some emerging evidence for the use of Prazosin, an alpha- 1 adrenergic receptor antagonist, to help treat sleep disturbances and nightmares in

PTSD.33-37 Nonetheless, there is a general lack of clinical consensus around which pharmacotherapies are most effective. Anecdotal evidence and case reports have suggested that cannabis may be effective for the management of PTSD, particularly to promote a state of relaxation and to aid in sleep.38,39 Presently, Veterans Affairs Canada will cover the cost of up to three grams of cannabis for medical purposes per day.40 While there has been a general lack of rigorous studies examining the effects of cannabis on PTSD symptoms, there is some evidence from clinical trials for synthetic cannabinoids like Nabilone. Several uncontrolled, open-label pilot studies have shown that Nabilone, both on its own and as an adjunctive therapy, can reduce hyperarousal and nightmares and improve sleep quality in PTSD.41,42 Finally, one small placebo-controlled crossover study demonstrated that Nabilone was able to reduce nightmares and increase well-being.43 While cannabinoid-related therapies may be promising, these results should be interpreted with some caution given the limitations around study design and sample size in study. One challenge of cannabis and cannabinoid therapies is ensuring the correct dose, where an individual experiences a therapeutic effect but the psychoactive effects (i.e. a state of being “high”) are minimized. In this regard, inhibitors of one of the major catabolic enzymes of the endocannabinoid system, FAAH, have been proposed as a novel therapeutic option. Relative to exogenous agonists of the CB1 receptor like cannabis or synthetic cannabinoids, preventing the degradation of anandamide (AEA) with FAAH inhibition is accompanied by comparatively few side effects (given AEA is a partial CB1 receptor agonist produced on-demand), has no abuse potential, and no negative effects on cognition. While exogenous cannabinoids are thought to have biphasic effects on anxiety and chronic agonism of the CB1 receptor may lead to downregulation,44-47 FAAH inhibitors are able to avoid these issues. Indeed, in preclinical studies, FAAH inhibitors have shown promising effects on anxiety and fear learning (surveyed in section 1.7.3.2).

7 Trials of FAAH inhibitors in humans, however, have stalled after clinical trials of BIA10- 2474 resulted in the death of one participant and the hospitalization of five others. However, an inquiry into these deaths concluded that these effects resulted from specific weaknesses of the drug’s low specificity and were not due to the drug’s interaction with

FAAH.48,49 Indeed, many participants have safely completed Phase I trials with FAAH inhibitors from Pfizer, Sanofi, Merck, and Janssen without adverse effects, and the Food and Drug Administration confirmed that other FAAH inhibitors do not pose similar safety risks.50 To this end, a recent randomized placebo-controlled study demonstrated that not only was the FAAH inhibitor PF-04457845 well-tolerated, but it promoted fear extinction and attenuated stress responses.51 Thus, the development of FAAH inhibitors remains a promising route for the development of new therapeutic options for PTSD, and the findings from the present study will inform the rationale for this development by directly surveying in vivo the status of this enzyme in PTSD.

1.6 Current Thinking on the Pathophysiology Underlying PTSD

1.6.1 General Thinking on the Pathophysiology Underlying PTSD

Several neurobiological systems are thought to contribute to the development and maintenance of PTSD. Among the most studied are the hypothalamic-pituitary-adrenal (HPA) axis and the adrenergic system, which are fundamental to physiological stress- responses. For example, it has been suggested that there is dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in response to chronic stress in PTSD, given findings like elevated levels of Corticotropin Releasing Hormone (CRH), reduced corticosterone secretion, increased adrenergic signaling, and prolonged inflammation.52-

56 Relatedly, one prevailing hypothesis suggests that abnormal neurocircuitry— particularly deficits in cortico-limbic circuitry with functions in salience attribution, emotional regulation and cognition—contribute to the development and maintenance of PTSD. Importantly, these hypotheses are not mutually exclusive, and may be complementary, given that the dysregulation of the HPA axis may contribute or exacerbate these changes in key circuits.57

8 Of critical importance is the amygdala, a small group of nuclei found deep in the medial temporal lobe. This structure has functions in detecting threat and mediating threat-related arousal and stress responses, formation and consolidation of emotional memories, and in the generation of anxiety states.58,59 Abnormal signaling and impaired connectivity to other key structures have been demonstrated in PTSD. Indeed, several clinical imaging studies (in addition to findings from animal models of PTSD) have demonstrated that amygdala and several other key structures (including the anterior cingulate, ventromedial prefrontal cortex (vmPFC), and hippocampus) are involved in both processing trauma-related memories and in coordinating the subsequent body’s response.60-63 In PTSD, it appears that the amygdala is hyper-reactive. Functional MRI (fMRI) studies in humans have shown that the amygdala is hyperresponsive in response to “threatening” or fearful stimuli and trauma cues, including threatening faces, personalized traumatic narratives and cues, combat sounds and photos, and trauma- related words.60,61,64-73 In concert with the hyperresponsiveness of the amygdala is the hypo-activation of the vmPFC, such that a greater blunting of the signal in the vmPFC results in greater activity in the amygdala. This structural and function connectivity between the amygdala is known to be critically involved in emotional regulation and emotional memory, and impaired coupling of these structures in PTSD is a well-replicated finding.74,75 It has also been shown that reduced activity of the vmPFC actually inversely correlates with the severity of PTSD symptoms.60 Thus, this evidence suggests a model whereby PTSD symptomatology—in particular increased fear response, intrusive memories, deficits in emotional regulation, and attentional bias towards traumatic memories—may result a failure of the vmPFC to negatively regulate the amygdala. In other words, the hypoactive vmPFC signifies the removal of a regulatory “brake” controlling amygdala activation. Moreover, impairments in the functioning of the hippocampus and insula may also be involved, and may underlie memory deficits and heightened interoceptive awareness, leading to increased startle.76-78 Other modalities have generally paralleled these findings. In particular, electroencephalography (EEG) and magnetoencephalography (MEG) are advantageous over fMRI in that they do not rely on indirect hemodynamic indices of activation. Studies using these modalities to examine the neural networks involved in PTSD have generally

9 found increased synchrony in neural networks related to fear,79,80 which may underlie the attentional bias to threat in PTSD.81 It has been suggested that this hypersynchrony may constrain local signaling dynamics, possibly reflecting relatively inflexible neural configurations that underlie symptoms of PTSD, particularly related to re-experiencing symptoms.79 At a molecular level, the endocannabinoid system (see section 1.7) has been implicated in the processes that prime the amygdala to be hyperresponsive. Given that one of the major endocannabinoid receptors, the CB1 receptor (CB1R), is found on both GABAergic and glutamatergic axon terminals within the amygdala, it appears that endocannabinoid signaling is able to regulate both excitatory and inhibitory neurotransmission in this region.82-84 However, with respect to the actual neuronal firing rates in this region, the net effect of typical endocannabinoid signaling appears to be a reduction in neural firing, indicating that the effect of CB1 receptor signaling at glutamatergic synapses (i.e. a decrease in excitatory signaling) may be more salient than the corresponding effect at GABAergic synapses (i.e. a decrease in inhibitory signaling).83,85,86 Mechanistically, the endocannabinoid system may be involved in the molecular changes that result in amygdala hyperresponsiveness in PTSD and anxiety states after chronic stress. Given that CB1 receptor signaling is known to gate excitatory inputs to pyramidal cells within the basolateral amygdala (BLA), a decrease in AEA (a key endocannabinoid) signaling through these receptors is thought to remove the tonic gating of these excitatory inputs. The resultant increase in excitatory input to pyramidal cells permits increased glutamatergic signaling within the BLA, which promotes dendritic arborization.87-89 The increase in dendritic remodeling towards more dendritic complexity and excitatory contacts along pyramidal neurons in response to chronic stress primes pyramidal neurons to be extremely sensitive to excitation, therefore favouring a state of hyperresponsiveness to stimuli.90 These morphological changes have been found in response to chronic stress and correspond to increased anxiety in rodents.87-89 This evidence provides a putative mechanism for how dysfunction in the endocannabinoid system within the BLA is able to affect neuronal excitability in this key region. Despite the hyperreactivity of the amygdala being well-replicated in humans, the neurochemical basis of this aberrant signaling in humans is unknown. As such, given the involvement of the

10 endocannabinoid system in the aforementioned molecular process underlying amygdala reactivity in rodents and other evidence implicating the endocannabinoid system dysfunction in this disorder (see sections 1.7.3 and 1.7.4), the results from this project will provide important information about the status of this system in vivo in PTSD.

1.6.2 Pathophysiology Underlying Different Symptom Clusters May Vary

While the above model of “classical” presenting PTSD may represent a state of emotional “under-regulation” characterized by hyperarousal, it is thought that the dissociative subtype of PTSD may represent a state of emotional “over-regulation”

(reviewed in Lanius et al., 2010).76 Indeed, patients with more dissociative presentations often display an opposite pattern of activation during functional imaging studies. For example, in one study assessing responses to script-driven trauma imagery, dissociative responses were positively correlated with activation in the medial prefrontal cortex (mPFC) and anterior cingulate cortex and negatively correlated with activation of the insula.91 Moreover, another functional imaging study demonstrated that activation of the mPFC is positively associated with dissociative responses to script-driven trauma imagery while activation of the amygdala is negatively correlated with these dissociative responses.60 Finally, it has also been demonstrated that during consciously perceived fear stimuli, dissociative subtypes of PTSD show greater activation of the ventral prefrontal cortex than non-dissociative subtypes.92 Given the finding of this hyper- inhibition of arousal during conscious threat perception, this may signify an adaptive compensatory mechanism to cope with pathologic hyperarousal in dissociative subtypes of PTSD. Given the neurobiological differences in structural and functional connectivity that may underlie different subtypes of PTSD, it is therefore important to consider PTSD symptoms dimensionally in the current project. For this reason, we will assess our research question (i.e. [C-11]CURB binding) not only between-groups, but will also assess whether [C-11]CURB binding levels are associated with specific symptom clusters of PTSD.

11 1.7 The Endocannabinoid System

1.7.1 Discovery of the Endocannabinoid System

Much of our understanding of the brain’s endogenous cannabinoid system—the “endocannabinoid system”—has come from research into the molecular action of cannabis, and in particular, THC.93 This led to the discovery of cannabinoid-receptors type 1 and 2 (CB1 and CB2, respectively; more on these receptors below in section

1.7.2.3) in the early 1990s94,95 and subsequently the discovery of endogenous ligands for these receptors were coined “endocannabinoids” in 1995.96 Since then, we have come to understand that these ligands are part of a dynamic neuromodulatory lipid system with diverse physiological functions. Of particular interest for this project are the functions served by this system in the brain.

1.7.2 Overview of the Endocannabinoid System

1.7.2.1 Functions of the Endocannabinoid System

The endocannabinoid system (ECS) has been shown to have diverse roles throughout the body. Indeed, this system has been implicated in energy metabolism, regulation of food intake, regulation of lipogenesis, satiety signals, chronic pain, inflammation, immune system function, reproductive regulation, bone remodeling, memory, mood, and addictions, among others. While the diversity of these functions speaks to the ubiquity of endocannabinoids across many physiological systems, the role of the ECS in stress, anxiety, and emotional memory are most relevant to this project.

1.7.2.2 Endocannabinoids

The ligands known as endocannabinoids are structurally derived from polyunsaturated fatty acids, specifically arachidonic acid, and have varying affinities for both cannabinoid receptors as well as other receptors. The two principal and best-studied endocannabinoids are N-arachidonoylethanolamine (anandamide, AEA) and 2- arachidonoylglyceral (2-AG),97-99 though other putative endocannabinoids, which are

12 largely isomers of AEA and 2-AG exist, including virodhamine (an isomer of AEA with an ester linkage instead of an amide link) and noladin (a 2-AG ether).100,101

1.7.2.3 Cannabinoid Receptors

The CB1 receptor (CB1R) was first discovered in 1988 and cloned in 1990.102,103 This receptor is a G-protein coupled receptor, characterized by 7-transmembrane domains and molecular coupling to Gi/o proteins. These receptors tend to be localized to axon terminals, and immunocytochemical and immunohistochemistry studies have demonstrated that CB1R are widely and abundantly expressed throughout the brain, including on GABAergic, glutamatergic, serotonergic, noradrenergic, and dopaminergic neuronal cell types.104-106 Though the greatest density of CB1 receptor expression appears to be in the basal ganglia, cerebellum, hippocampus, and cortex, CB1 receptors are also found peripherally.93 A second cannabinoid receptor, also a Gi/o-protein coupled receptor—the CB2 receptor—was identified in 1993 using sequence homology,95 and this receptor is now known to be distributed peripherally, primarily in immune cells.107 AEA functions as a partial agonist at CB1 receptors,108 binding with increased intrinsic efficacy and affinity to these receptors as compared to CB2 receptors.109 2-AG, by contrast, functions as a full agonist and may slightly preferentially bind to CB1 receptors over CB2 receptors.98,110,111 Both AEA and 2-AG also bind to other targets, including transient receptor potential cation channel subfamily V member 1 (TRPV1), some peroxisome proliferator-activated receptors (PPAR),112,113 and some other G-protein receptors (55,

119).114,115

1.7.2.4 Endocannabinoid Signaling

Endocannabinoids are thought to signal in a retrograde manner. Both 2-AG and AEA are synthesized in an “on-demand” fashion from precursors and released from postsynaptic neurons. This is thought to occur in response to an increase in intracellular levels of Ca2+, given that AEA production can be blocked with Ca2+ chelation and that production can be induced via high-frequency electrical stimulation in rat slices,116,117 though there may also be Ca2+-independent mechanisms for EC release as well).118

13 Broadly speaking, signaling at the CB1R has the function of suppressing, either short- term or in a more sustained manner, neurotransmitter release.

Given both CB1 and CB2 receptors are coupled to Gi/o proteins, there are numerous intracellular signal transduction pathways resulting from their stimulation. However, there are some differences: for example, the CB2 receptor does not interact with ion channels, while the CB1 receptor does. The focus here will be on the latter given its widespread expression in the brain. CB1 receptor activation has been shown to inhibit presynaptic voltage-gated Ca2+ channels and to activate inwardly-rectifying K+ channels.

This reduction in intracellular Ca2+ entry functions to prevent exocytosis of neurotransmitters via synaptic vesicles.

The coupling of cannabinoid receptors to Gi/o proteins means that the signaling cascade that results from their activation results in inhibition of adenylyl cyclase (which modulates the production of intracellular cAMP and thereby inactivates protein kinase A,

PKA).93 As well, stimulation of CB1 receptors have also been shown to result in the phosphorylation and activation of mitogen-activated protein kinase (MAPK). Through both MAPK and PKA, CB1 receptor signaling may control gene expression. Additionally, MAPK has downstream roles in cell proliferation and survival. Finally, stimulation of CB1 receptors may also trigger other protein phosphorylation cascades, including those involving phosphoinositide-3-kinase and protein kinase B.119 Since it has been shown that CB1 receptors are localized primarily to presynaptic axon terminals and are able to suppress presynaptic neurotransmitter release, it has been hypothesized that 2-AG and AEA are thus able to play a role in synapse regulation and maintenance through homeostatic, short-term, and long-term plasticity (including depolarization-induced depression of excitatory and inhibitory neurotransmitter release, long-term potentiation, long-term depression, and long-term depression of inhibition).120,121 Though both endocannabinoids functionally suppress neurotransmitter release, differential subcellular partitioning of both 2-AG and AEA as well as their catabolic enzymes have suggested that AEA may represent a more “tonic” signal to regulate baseline synaptic transmission while 2-AG may represent a more “phasic” signal.122 That endocannabinoid signaling plays a role in synaptic plasticity and maintenance in varied neuronal circuitry across the cortex, hippocampus, amygdala,

14 mesolimbic system and beyond functionally implicates the ECS in processes involved in regulating cognition, emotion, anxiety and stress responses, and memory formation and extinction.

1.7.2.5 Endocannabinoid Synthesis and Degradation

Given that endocannabinoids are synthesized in an “on-demand” manner, both their synthesis and their degradation are processes that must be tightly regulated as a result. 2-AG is synthesized by phospholipase C (PLC)110,123 and sn-1-selective diacylglycerol lipase (DAGL). These enzymes are localized to plasma membranes in postsynaptic neurons.124 2-AG is degraded into glycerol and arachidonic acid by monoacylglycerol lipase (MAGL), which are localized in presynaptic neurons,125 lending further idea to the notion of retrograde signaling. AEA is synthesized by the enzymes N- acyltransferase (NAT)126 and N-acylphosphatidyl-ethanolamine-specific phospholipase D

(NAPE-PLD).127 Of central importance to this project is the enzyme that inactivates AEA by hydrolyzing it to yield arachidonic acid and ethanolamide: Fatty Acid Amide Hydrolase (FAAH), discussed in more detail below. It is thought that a presently unknown membrane transporter facilitates endocannabinoid release and re-uptake from the synapse.128-130

1.7.2.5.1 Discovery and Features of Fatty Acid Amide Hydrolase

Fatty Acid Amide Hydrolase (FAAH), the serine hydrolase enzyme that inactivates and therefore terminates AEA signaling, was initially discovered to hydrolase oleamide to oleic acid in 1995.131 Peptide sequence information of the purified enzyme allowed for the cloning of its cDNA in 1996, and transfection of this cDNA not only demonstrated high levels of oleamide hydrolase activity, but high levels of AEA hydrolase activity as well. The enzyme was aptly named in recognition of the breadth of endogenous fatty acid amides suitable as substrates, including oleoylethanolamide (OEA), palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and docosahexaenoyl ethanolamide (DHEA).132,133 FAAH belongs to a family of enzymes known as amidases, which hydrolyze short-chain fatty acid amides. These amidases share a highly conserved

15 stretch of residues known as the amidase signature sequence.134 Unique among this class of enzymes, FAAH exists as a homodimer integrated within the plasma membrane with a transmembrane domain near the N-terminus region,131,135 though the deletion of this domain alone is insufficient to solubilize and inactivate the enzyme, suggesting that it has other regions associated with the membrane.136 In 2002, x-ray crystallography of

FAAH revealed a core composed of a twisted beta sheet surrounded by alpha helices.137 A helix-turn-helix motif involving α18 and α19 was found to form a hydrophobic cap near the active site of the enzyme. As well, a series of channels were discovered on the crystal structure, which appear to provide access to both the cytosolic and membrane component simultaneously, thought to facilitate substrate binding and release.138 Another channel, called the acyl-binding channel, exists as a hydrophobic channel off of the catalytic core of the enzyme. Given that catalysis occurs in the inner core of each dimer, it is thought that substrates like AEA are able to reach this core through the membrane channel. It has been suggested that the structural flexibility of the substrate in order to move between the acyl-binding and membrane channel may be an important determinant through which FAAH is able to recognize its substrates, which include, in addition to AEA, other fatty acid amides like N-palmitoylethanolamine and N-oleoylethanolamine.139 Mutagenesis studies have revealed Ser241 as the catalytic nucleophile of the enzyme.140 Though serine hydrolases typically use a catalytic triad involving serine, histidine and aspartic acid, the catalytic core of FAAH instead appears to require a specific lysine residue (Lys142), which is able to act as both a base and an acid during catalysis, and an additional serine residue (Ser217).140,141 The catalytic cycle, in brief, is as follows: Lys142 initiates a proton shuttle involving Ser217, which in turn activates Ser241 and forms acyl- enzyme adduct; a second proton transfer step involving Lys142 and Ser217 causes the protonation and leaving of the substrate’s leaving group; finally, an H2O molecule attacks the acyl-enzyme adduct, which deacylates the adduct and allows the release of the catalytic products (arachidonic acid and ethanolamine). Since its initial discovery, FAAH has been conclusively demonstrated to be responsible for AEA hydrolysis (though it has been shown that AEA can also be metabolized by some other enzymes including cyclooxygenases and lipoxygenases).142,143 For example, FAAH knockout mice have 15- fold higher levels of AEA in the brain and lack AEA hydrolytic activity compared to wild-

16 type mice,144 demonstrating that AEA levels and therefore ECS tone in the central nervous system is controlled through FAAH.

1.7.2.5.2 Discovery of a FAAH Gene Polymorphism

In 2002, it was first reported that a single nucleotide polymorphism (SNP) in the human FAAH gene is associated with increased propensity to use drugs and alcohol.145 With this SNP, there is an adenine instead of cytosine base (C385A), which subsequently results in a conserved proline residue at position 129 becoming a threonine residue instead (P129T). Several lines of evidence—including studies from T lymphocytes in human blood, transfected COS-7 cells, and knock-in mice—have shown that this variant results in a significant decrease of both FAAH expression and activity given that proper folding for full function of the enzyme is impaired.145-147 As a result of this decrease in FAAH expression and activity, heterozygotes with one copy of the variant gene and homozygotes with two copies of the variant gene (A/C or A/A) have elevated levels of both AEA and other N-acylethanolamides for which FAAH is the catabolic enzyme. This variant is relatively common in the general population; approximately 38% of individuals with a European ethnic background will have at least one copy of the variant gene.145 The functional consequences of having this variant have been explored through genetic association studies. These studies have shown that decreased levels and activity of FAAH are associated with increased use of cannabis, increased reward reactivity, and increased impulsivity.147-149 Importantly, having at least one copy of the genetic variant has also been associated with decreased amygdala responsiveness to threat during fMRI and self-reported anxiety. This has added support to the notion that inhibiting FAAH in order to improve endocannabinoid system tone may be a useful therapeutic strategy for anxiety disorders.

1.7.3 Animal Studies of the Endocannabinoid System in Stress and Trauma Models

There are several well-replicated findings which implicate disruptions in endocannabinoid signaling (and in particular, of the tonic inhibitory role of AEA) in

17 preclinical models of PTSD and stress. Some of the most commonly used animal models of PTSD are summarized in Table 1: Methodological Considerations and Variations in

Animal Models of PTSD (adapted from Ursano et al. and Deslauriers et al.)150,151. Below, the literature related to both CB1 receptors and FAAH and AEA levels after stress are surveyed. This literature is summarized in Table 2: Summary of Preclinical Studies of the Endocannabinoid System in Relation to Stress and Anxiety, at the end of section of 1.7.3.3.

Table 1: Methodological Considerations and Variations in Animal Models of PTSD

18 1.7.3.1 Evidence from CB1 Receptors in Animal Models of PTSD

CB1R protein and mRNA levels after exposure to stress—There is some evidence from animal models of PTSD that there may be changes in CB1R binding after stress exposure. These effects may differ by region; though many of these findings have implicated a decrease in CB1R binding site density, protein levels and mRNA levels, there have been reports of an increase of CB1R binding in certain regions. Firstly, it has been shown that both chronic stress and chronic doses of exogenous corticosterone decreased

CB1R binding site density and levels of protein in the hippocampus and amygdala.152,153 Further study revealed regional effects within hippocampal subfields, whereby CB1R receptor binding was significantly decreased within the dental gyrus but increased in the

CA3 subfield.154 Other studies have shown that CB1R mRNA, protein levels, and binding site density are reduced in the prefrontal cortex (PFC), amygdala, hippocampus, striatum and cingulate cortex after chronic stress exposure.153,155-160 These observations after exposure to stress have been linked to anxiogenic behavioural effects.159 The hypothalamus seems to be an area where CB1R mRNA levels are increased, though this effect may be sex-dependent, as one study found that females in fact had decreased hypothalamic CB1R mRNA levels.155,161 Another region that may be differentially regulated by sub-region is the PFC, given that increases in CB1R levels have been found in the ventromedial prefrontal cortex (vmPFC) despite findings showing an overall decrease in CB1R levels in this region. One study found increased CB1R binding in this region after chronic stress,162 while another found increased CB1R mRNA after contextual fear conditioning.163 The mechanisms through which CB1R levels are modulated may be epigenetic, given that one study found reduced levels of histone acetylation of the CB1R gene.156 CB1R pharmacological manipulation linked with fear and anxiety—In addition to changes in levels of CB1R in response to stress, pharmacologic antagonism and inverse agonism of the CB1 receptors has been shown to affect animal behaviour and key circuitry in the brain. There are several lines of evidence coming from administration of CB1R inverse agonists and antagonists, such as rimonabant and AM251. Chronic

19 administration of rimonabant has been shown to produce a depressive-like phenotype, increasing rat immobility in the Forced Swim Test and anhedonia.164 Local administration of AM251 into the central nucleus of the amygdala has been shown to increase acute fear responses, while administration of AM251 in the BLA has been shown to increase chronic fear responses after auditory fear conditioning, demonstrating that CB1R antagonism enhances fear responses, particularly later fear responses which appear to be mediated by ECS signaling in the BLA.165 This study also found that blockade of CB1R increased electrophysiological synaptic activity in the central amygdala, demonstrating that signaling through CB1R tonically dampens neurotransmission under normal conditions.165 Both rimonabant and AM251 have also been shown to consistently impair extinction and enhance retention of conditioned and contextual fear.166,167 Additionally, after chronic unpredictable stress, administration of AM251 has been shown to reduce active coping during the Forced Swim Test.168 Moreover, CB1R antagonism has been shown to produce anxiety-like behavioural phenotypes.169-172 Finally, blockade of CB1R signaling in the amygdala and vmPFC increases BLA excitability,169 corticosterone release,173 and activation of the HPA-axis.172,174,175 Administration of pharmacologic agonists (i.e. WIN55,212-2) after chronic stress exposure has been shown to rescue many of the behavioural, fear memory, and circuitry- related responses. For example, in one study, after chronic restraint stress, rats demonstrated impaired long-term potentiation (LTP) in the ventral subiculum-nucleus accumbens pathway, impaired performance on PFC- and hippocampus-dependent cognitive tasks, and increased anxiety.176 Chronic administration of WIN55,212-2 prevented the impairments in LTP and hippocampus-dependent cognition. Co- administration with AM251, a CB1R antagonist, blocked these effects on memory and LTP. Similarly, after stress exposure and situational reminders, administration of WIN55,212-2 has been shown to reverse changes in avoidance, LTP in the hippocampus- nucleus accumbens pathway, anxiety-like behaviours, anhedonia, fear retrieval and extinction, and startle responses.160,177,178 Moreover, after contextual fear conditioning, local administration of AM404 (an endocannabinoid reuptake inhibitor) and AEA to the vmPFC have been shown to attenuate fear conditioned responses by increasing signaling through the CB1R.163 As well, systemic administration of AM404 after fear conditioning

20 produced dose-dependent enhancements in fear memory extinction.166 Co-administration with rimonabant blocked this effect. Studies of THC administration in rats have demonstrated that, in mice, administration of THC (a partial CB1R agonist) appear to have anxiogenic effects, particularly after exposure to stress, though high doses may produce a more anxiolytic effect and response may be influenced by dose.179,180 CB1R genetic knock-out mice have increased anxiety—In addition to the pharmacologic manipulations described above, further evidence for the involvement and dysregulation of CB1R signaling after stress comes from genetic manipulations. In particular, CB1R knockout mice have consistently been shown to have increased anxiety,181-184 displaying a similar behavioural phenotype to mice exposed chronic restraint stress.173 Indeed, this behavioural phenotype seems to be more prevalent under high-stress conditions as opposed to low-stress conditions.182 In addition to displaying anxiety-like phenotypes, mice lacking the CB1R are not able to experience anxiolytic effects of two anti-anxiety drugs, buspirone and bromazepam.181 These findings demonstrate that genetic deletion of the CB1R and the resultant decrease in signaling through this receptor is sufficient to produce an anxiety-like phenotype in mice. Taken together, these findings demonstrate a clear role for CB1R signaling in stress-adaptive responses, such that signaling through the CB1R facilitates these responses and disruption in this signaling impairs these responses. These stress- adaptive responses in which CB1R signaling is implicated include the extinction of fearful memories, anxiety-like behaviours, depressive-like behaviours, changes in key neuronal circuitry, and neuroendocrine responses to stress.

1.7.3.2 Evidence from FAAH in Animal Models of PTSD

FAAH and AEA levels after exposure to stress—In addition to the body of evidence suggesting AEA provides tonic inhibitory regulation at the synapse, there is a body of literature suggesting that a depletion of AEA due to increased AEA catabolism by FAAH underlies the decreased endocannabinoid system tone that has been consistently found after stress. Acute restraint stress seems to reliably produce decreased tissue levels of AEA, as well as a decreased of AEA levels in the amygdala and hippocampus.170,175,185-189 This change in AEA levels appears to be mediated by an

21 increase in FAAH levels, which have been found to be elevated both in the amygdala and hippocampus after acute stress.175,190 The effect of acute stress on levels of AEA in the vmPFC is less clear: though some studies have found a reduction in AEA content after acute stress, others have found no effect on AEA and FAAH levels within this region.173,187,188,190 After chronic repeated stress, levels of AEA are reliably reduced within the amygdala, hippocampus, hypothalamus and PFC.87,185,188 Similarly to acute stress, this reduction appears to be directly linked to an increase in FAAH levels, which have been demonstrated in the amygdala and hippocampus.188,191 Moreover, FAAH knock-out

(KO) mice do not display a decrease in AEA levels after chronic stress.87 The effects of chronic stress exposure appear to be mediated in part by the type of stressor: that is, whether or not the paradigm involves repeated exposure to the same stressor or varied stressors. The increase in FAAH and subsequent decrease in AEA in the former may be mediated in part by chronic corticosterone release, given exogenous corticosterone exposure has produced similar increases in FAAH activity and AEA levels.153 In exposure to chronic unpredictable (i.e. heterotypic) stress, while some studies have found a similar whole-brain decrease in AEA and an increase of FAAH in the hippocampus,191 several other studies have found no changes in AEA or FAAH levels in the striatum, midbrain, hippocampus, and thalamus.152,189 Nevertheless, it appears that both acute and chronic stress do seem to generally result in increased FAAH and decreased levels of AEA. It is thought that this FAAH upregulation may be driven by Corticotropin-Releasing Hormone Type 1 Receptors (CRHR1), particularly at glutamatergic pyramidal neurons in the limbic system, given that CRHR1 knockout mice do not exhibit the resultant increase in FAAH and subsequent decrease in AEA following restraint stress.187 As well, mice who constitutively overexpress CRHR1 display an increase in amygdala FAAH levels and a subsequent decrease in AEA, as well as displaying elevated glutamatergic activity that was found to be mediated by FAAH and an anxiety-like phenotype.192 Interestingly, the differences in findings in paradigms that differ in stressor protocol (i.e. homotypic versus heterotypic) may be due, in part, to differences in CRH signaling. CRH mRNA has been found to be elevated after chronic restraint stress,193 while chronic unpredictable stress has been found to have either little effect on CRH in the amygdala or to downregulate

CRHR1 in the amygdala.194-196 While these differences in CRH after stress have only

22 been reported in the amygdala, it does suggest that different types of stressor protocols may differ in the stress response they elicit. Genetic and pharmacologic modulation of FAAH linked to anxiety and stress responses—Paradigms whereby FAAH is modulated have further been able to link the upregulation of this enzyme with behavioural outcomes. Firstly, pharmacologic inhibition of FAAH (with inhibitors like URB597 and PF3845) has been shown to decrease activation of the HPA-axis and attenuate anxiety-like and depressive-like responses to both acute and chronic stress.85,87,161,171,175,177,185,186,197,198 FAAH knockout mice show reduced behavioural anxiety (without intervention and after stress),87,199 similar to mice who have received a pharmacologic FAAH inhibitor. This effect is prevented by co-administration of rimonabant.199 FAAH knockout mice also do not display the dendritic remodeling in the

BLA after chronic stress that has been linked to anxiety (described above).87 Similarly, overexpression of FAAH using a viral vector within the PFC increases anxiety-like behaviours in rats,200 though one recent report surprisingly found overexpression of FAAH within the BLA produced decreased anxiety and fear responses.201 Of note is that these effects appear most prominent in highly stressful conditions.87,186,198 Knock-in mice bearing the C385A allele with constitutively lower FAAH levels have also displayed better fronto-amygdala connectivity and decreased anxiety-like behaviours,147,202 findings which have been similarly demonstrated in humans (discussed below in section 1.7.4.2).

1.7.3.3 Summary of the Evidence in Animals

Taken together, these findings implicate FAAH in several of the sequelae following stress, including neuroendocrine activation, impairments in fear memory extinction, impairments in neurocircuitry function, and behavioural phenotypes commonly found in PTSD (namely, anxiety and depression). Given the evidence discussed in the preceding section on the CB1R, it appears that these effects are mediated through the CB1R, suggesting a model whereby exposure to stress produces an increase in FAAH, which depletes levels of AEA and thus decreases signaling through the CB1R, removing the tonic synaptic regulation provided by AEA.

23 Table 2: Summary of Preclinical Studies of the Endocannabinoid System in Relation to Stress and Anxiety

24

25

26

27

28

29

30 1.7.4 Human Studies of the Endocannabinoid System in Posttraumatic Stress Disorder

There has been comparatively little work done in humans to gain insight into the status of the endocannabinoid system after stress and trauma. Part of this deficiency, no doubt, is attributable to additional difficulties of directly surveying the status of the ECS in the human brain. However, there are some studies suggesting that this system may be disrupted in humans after chronic exposure to stress and in anxiety conditions.

1.7.4.1 Evidence from Pharmacologic Studies in Humans

Firstly, there is a body of evidence that comes from investigation into the CB1R. Of note, CB1R agonists have shown therapeutic potential for the management of anxiety and treatment-resistant nightmares in PTSD.41,43,203 Initially, there were reports in clinical trials for CB1R antagonists, in trial for the treatment of obesity, of increased risk for neuropsychiatric symptoms, particularly anxiety and often in patients with no history of these symptoms (reviewed in Moreira et al., 2009).204,205 One study found that high doses of the CB1R inverse agonist rimonabant produced an increase in cortisol,206 lending support to the idea that endocannabinoid signaling is able to negatively regulate the neuroendocrine stress response. One very recent report detailed the effects of administering PF-04457845, a FAAH inhibitor, to healthy subjects for 10 days.51 In the report, authors noted that the group receiving the drug had 10-fold increased levels of AEA, enhanced fear memory extinction, attenuated autonomic stress reactivity and attenuated stress-induced negative affect compared to the placebo group. Though preliminary, these results provide promising evidence for the development of FAAH inhibitors in humans, particularly in the treatment of stress disorders like PTSD.

1.7.4.2 Evidence from Genetic Studies in Humans

The C385A FAAH SNP has proven to be instructive for studying the effects of FAAH levels in humans. For example, carriers of the A allele with decreased levels of FAAH demonstrated decreased activation of the amygdala in response to threat, faster habituation to threat, enhanced fear extinction and reduced trait anxiety.62,147,149,207

31 Moreover, carriers of the A allele also show enhanced fronto-amygdala coupling.147 Interestingly, several functional imaging studies in cannabis users or after administration of cannabinoids demonstrate a similar finding wherein the activation of the amygdala is dampened in response to threat cues.208-210 In subjects with PTSD and comorbid alcohol use disorder, carriers of the A allele showed increased basal AEA levels as well as decreased self-reported anxiety during a stress challenge.211 Subjects who carried the SNP also showed significantly decreased hyperarousal symptoms. One interesting case study of a woman in Scotland detailing a woman with a genetic microdeletion downstream of the FAAH gene (in the FAAH-OUT pseudogene) and a copy of the variant C385A A allele reported pain insensitive, extremely low measures of anxiety and depression (scoring 0 on both the GAD-7 and PHQ-9), and

“never panicking, even in dangerous or fearful situations”.212 This case report provides compelling anecdotal evidence for the involvement of FAAH in mediating responses to stress. Taken together, these genetic studies suggest that a decrease in functional FAAH levels results in improved stress-adaptive responses and decreased anxiety.

1.7.4.3 Evidence from Imaging Studies in Humans

There has been relatively little imaging work done in humans to directly survey the status of the ECS in PTSD (in addition to the functional imaging results discussed above). To my knowledge, there are no post-mortem studies of the endocannabinoid system in PTSD. One PET imaging study using the tracer [C-11]OMAR, a CB1R radioligand, found an increase in CB1R in PTSD relative to healthy controls and trauma-exposed subjects without PTSD.213 This may signify a compensatory mechanism to decreased endocannabinoid system tone. Another imaging study that examined the impact of both C385A genetics and CRHR1 genetics found that subjects with high AEA tone and high CRHR1 signaling both exhibited blunted amygdala habituation, and had an increased risk for development of anxiety disorders.214

32 1.7.4.4 Evidence from Peripheral Blood Levels of ECS in PTSD and Stress Conditions

Finally, there is some evidence of disruption in the endocannabinoid system in humans, largely obtained indirectly from circulating levels of endocannabinoids. Firstly, in the CB1R imaging study above, the authors also found a decrease in peripheral levels of AEA in the PTSD group relative to both healthy controls and trauma-exposed controls without PTSD.213 These levels correlated with brain levels of CB1R, suggesting that peripheral levels of ECs may be indicative of central ECs. Two studies have found that levels of peripheral levels of AEA negatively correlate with clinical measures of anxiety both in healthy populations and in those with major depressive disorder.215,216 Another study of first responders to the World Trade Center attacks with PTSD found that 2-AG was decreased among the PTSD sample.217 While no group differences in AEA were found, AEA did negatively correlate with the burden of intrusion symptoms, such that lower AEA was associated with a higher burden of these symptoms. Similarly, another study looking at levels of 2-AG, AEA, and other FAAH substrates in complex PTSD found no group differences in AEA or 2-AG, but did find significantly elevated levels of OEA in the PTSD group.218 Another report found that plasma concentrations of both 2-AG and

AEA were increased in people with PTSD.219 Acute social stress paradigms have also been found to produce elevated levels of 2-AG and AEA.215,220 The reasons for this increase remain unclear, but it is worth considering that circulating levels of AEA and other endocannabinoids may not be reflective of levels in the brain, given both chronic corticosterone treatment and chronic stress in animals have been found to increase levels of AEA peripherally despite a decrease in AEA levels in the brain.153,221,222 Finally, one study investigating hair concentrations—which they hypothesized may be a more reliable assessment of ECs than peripheral blood measurements—of N-acylethanolamides metabolized by FAAH, found that concentrations in hair strongly negatively correlated with PTSD symptom burden.223 The evidence from peripheral blood levels of ECs has been less consistent than the findings in the preclinical literature, but does generally point to dysregulation in the ECS in stress and anxiety conditions.

33 1.7.4.5 Summary of the Evidence in Humans

Overall, the evidence in humans, while generally in line with preclinical evidence of a model of dysfunctional endocannabinoid signaling after chronic stress, provides a less clear and less direct picture of what specifically is dysfunctional within the endocannabinoid system and the chronicity of this dysfunction relative to observable symptomatology. Undoubtedly, more research will be required to fully understand these questions, but this gap in literature, particularly given the strong preclinical evidence for ECS dysfunction in stress and anxiety conditions, provides a strong rationale for the current project.

1.7.5 Summary of the Evidence Suggesting Endocannabinoid System Disruption in PTSD

The above evidence from both preclinical and clinical studies supports a conceptual model whereby the neural dysfunction in PTSD is linked to disruption in the endocannabinoid system. In particular, this disruption appears to be caused by an elevation of FAAH after chronic stress and subsequent depletion of AEA and decreased tonic signaling as a result within the amygdala in particular, and possibly other related circuitry as well. This disturbance of endocannabinoid system tone demonstrated is related to both the neuroendocrine sequelae of the stress response, the behavioural phenotypes that emerge as a response to chronic stress (particularly anxiety), and the extinction of fear learning. The central role of the amygdala in fear processing, which is intricately linked to the core features of PTSD, together with the findings of FAAH disruption in this structure in animal models of PTSD make this structure of particular importance in examining the status of FAAH in people with PTSD. The current project will thus serve to further our understanding of the status of the endocannabinoid system in humans with PTSD.

34 1.8 [C-11]CURB as a FAAH Radiotracer

1.8.1 Brief Introduction to PET Imaging

Positron Emission Tomography (PET) imaging is a technique whereby computerized tomography and radioligand kinetic assay are combined to yield a three- dimensional image.224 By leveraging the radioactive decay of the radioligand, the resultant image allows for the visualization and quantification of the radioligand as a function of time. This radioligand is injected into the subject, typically intravenously. Positrons are emitted from the nucleus of the decaying radiolabeled nuclide, and shortly after emission encounter a free electron.224 The collision of the two particles releases two photons (511 KeV each) 180 apart. The tomograph’s scintillation detectors, on opposite sides of the scanner, detect the origin of emission using coincidence detection.224 Over the course of the scan, the number of times a pair of detectors is hit in coincidence is recorded.224 The angular positions of these coincidence events are grouped together in a sinogram or matrix containing the number of counts at each angle. Reconstruction algorithms are then used to transfer the data in the sinogram to an image depicting the distribution of radioligand in the brain.224

1.8.2 Development of [C-11]CURB

The prototypical and perhaps best characterized FAAH inhibitor is URB597 (3′- carbamoyl-[1,1′- biphenyl]-3-yl cyclohexylcarbamate), a molecule belonging to a class of compounds known as O-arylcarbamates, which have shown promise in development of FAAH inhibitors. In the development of a novel molecular imaging probe for FAAH, this class was thus considered to be a suitable starting point.225 Based on the structure of URB597, a radiolabeled analog—[C-11]URB694 (6-hydroxy-[1,1′-biphenyl]-3-yl cyclohexylcarbamate), otherwise known as [C-11]CURB—was developed here at the

Centre for Addiction and Mental Health by Alan Wilson and colleagues in 2011.226 This analog was developed as a radiotracer given URB694 had shown improved brain penetration compared to URB597.227

35 Based on knowledge of the mechanism of inhibition of other O-arylcarbamates, it was suspected that [C-11]CURB would form a strong covalent bond with FAAH and thus would irreversibly inhibit the enzyme.226 Inhibition of FAAH occurs via attack of the hydroxyl of the serine residue on the radiolabeled carbonyl carbon of the O- arylcarbamates, which causes expulsion of the O-aryl group while the remaining cyclohexyl carbamoyl group is covalently linked to the serine residue. Given that the moiety that becomes linked to the serine residue of FAAH is the radiolabeled carbonyl carbon, the enzyme thus becomes radioactively tagged.

1.8.3 Radiosynthesis of [C-11]CURB

The radiosynthesis of [C-11]CURB uses a novel one-pot reaction [C-11]CO2 fixation technique.228 In brief, a solution of cyclohexylamine and BEMP in acetonitrile at room temperature is prepared. Cyclotron-generated [C-11]-labeled CO2 is then passed through this solution, and then through a solution of POCl3 in acetonitrile. One minute later, a solution of 2-phenyl-1,4-hydroquinone in dry acetonitrile is added. After one additional minute, high-performance liquid chromatography (HPLC) eluent is added to dilute the reaction. The resultant mixture is then purified by HPLC (with purities exceeding 97%), resulting in a final pyrogen-free saline preparation containing 0.5% Tween 80, with a half-life of approximately 20.4 minutes (based on the half-life of the radioisotope [C-

11]).228,229

1.8.4 Properties of [C-11]CURB

1.8.4.1 Safety of [C-11]CURB

[C-11]CURB has demonstrated an excellent safety profile in humans. Based on patient reports, electrocardiogram, blood pressure, heart rate, and oxygen saturation measurements, [C-11]CURB has been shown to cause no physiological disturbances for at least 3.5 hours post injection based on an injected mass of [C-11]CURB ranging from

1.13-2.05 µg.230 Even if the human expression of FAAH was significantly below that of rats, these doses are well below the 5% occupancy of target threshold, commonly cited as a limit for radiotracer dose.230

36 1.8.4.2 Kinetics of [C-11]CURB

Following injection of the tracer, unmetabolized [C-11]CURB in arterial plasma peaks after 84 seconds.230 After 20 minutes post-injection, roughly 48% of the unmetabolized tracer remained in plasma, which declined to 37% 90 minutes post- injection. The free fraction in plasma was estimated to be 0.9%±0.2%.230 Rat experiments have conclusively demonstrated the irreversible binding of [C-

11]CURB.226 Based on this and on the trial of several different compartment models, a two-tissue compartment model with irreversible trapping in the second compartment

(2TCMi) is the preferred model for kinetic modelling of [C-11]CURB.230 This is discussed in more detail in Chapter 2: Methods.

1.8.4.3 Biodistribution of [C-11]CURB

Peak radioactivity in the brain occurs roughly 120-195 seconds after radioactivity arrives to the field of view of the scanner, roughly in frame 6-7.230 After 30 minutes post- injection, the time activity curve in each region of interest remained relatively unchanging. The distribution of [C-11]CURB activity in the brain of humans is widespread and seems to match the known distribution of FAAH in the human brain based on post-mortem studies.231 Thus there is no appropriate region of reference in the brain for the purposes of quantifying [C-11]CURB. In order to demonstrate the specificity of [C-11]CURB and to assess the possibility of off-target binding effects in the proof-of-concept study in rats, [C-11]CURB was administered after initial pre-treatment with increasing doses of both unlabeled URB694 and URB597 to test if FAAH inhibition would saturate the target and thus decrease the amount of radioactivity in the brain.226 Uptake of [C-11]CURB was demonstrated to decrease in a dose-dependent manner, with correspondingly higher levels of radioactivity in plasma. Given that URB597 has been shown to bind exclusively to FAAH in the central nervous system (CNS), the blocking effects of this compound demonstrate that [C- 11]CURB is able to bind selectively to FAAH.

An irreversible radioligand’s capacity to be taken up by tissue (i.e. k3) may potentially be affected by regional blood flow, particularly if the rate constant describing

37 the irreversible trapping is set too high, since the delivery of the radiotracer to tissue then becomes the rate-limiting step in tracer uptake to the second compartment.230 This limits the sensitivity of measurements of binding site density. However, simulations have confirmed that [C-11]CURB is not affected by cerebral blood flow (CBF) and would require an increase of >100% in k3 for this to be the case.230

1.8.4.4 Analysis of [C-11]CURB Binding

A 2TCMi has three parameters that may be used to quantification of [C-11]CURB:

Ki (the net influx constant), λk3 and k3 (see Figure 4 in Chapter 2: Methods for a visual representation of these parameters). k3, the parameter describing the irreversible efflux from the free and nonspecific compartment to the specific compartment, has been shown to be correlated with k2 (the parameter which describes the efflux from the free and nonspecific compartment to the plasma), meaning that this parameter on its own is not suitable for quantification of [C-11]CURB.230 Despite the calculation of Ki depending on k3, changes in Ki may be driven by the confounding effect of CBF if the blood flow is lower than expected.230 By contrast, the composite parameter λk3 has been suggested to reliably index irreversible radioligands.232,233 This parameter is calculated as the product of both k3 and (K1/k2), and has been shown to be independent of regional cerebral blood flow.230 Based on Monte Carlo simulations, it also has excellent identifiability even when k3 is reduced up to 80% and increased up to 100% to simulate a change in FAAH activity.230 Therefore, this measure is both sensitive and independent of regional changes in blood flow.

1.8.4.5 Validation of [C-11]CURB

Several pieces of evidence have validated [C-11]CURB as a radiotracer. Firstly, test-retest studies completed approximately 1-2 months apart showed that [C-11]CURB had an excellent reproducibility, with test-retest variability of 9%, and also had good reliability in most regions of interest.234 This study also confirmed that 2TCMi was indeed the most appropriate kinetic model to use to model [C-11]CURB. In addition, this study used various doses of PF-04457845, one of the most specific FAAH inhibitors, to assess

38 [C-11]CURB binding once FAAH was blocked, and indeed found that blocking of FAAH did correspond to a marked decrease in [C-11]CURB activity.234 Without a reference region, it is difficult to fully characterize the contributions of [C-11]CURB binding in the free and nonspecific compartment and the specific compartment. Therefore, by blocking binding in the specific compartment by inhibiting FAAH, this study was able to characterize [C-11]CURB binding in the free and nonspecific compartment, with λk3 values less than 10% of the values under the baseline condition. Rat studies have previously shown that the FAAH SNP C385A correspond to a decrease in both FAAH expression and activity.147 Using [C-11]CURB as an in vivo probe, a cross-sectional study examining the effect of C385A genotype on FAAH levels was able to demonstrate that having at least one copy of the variant allele (i.e. A/C or A/A) produced measurable differences in [C-11]CURB binding.235 This study was thus able to show that genotype affecting FAAH levels and activity correspondingly affects [C-11]CURB binding.94

1.9 Summary, Objectives & Hypotheses

1.9.1 Summary

The literature summarized above presents an emerging view of PTSD as a disorder whose pathophysiology is, at least partly, tied to dysfunction and/or dysregulation in the endocannabinoid system, particularly within the amygdala. More specifically, the literature suggests that an increase in AEA hydrolysis via increased levels of FAAH contributes to symptom development and maintenance in PTSD. However, much of this literature has been conducted in animal models, leaving open the question of whether and how these findings translate to humans. Both in vivo and post-mortem studies in humans with PTSD are lacking, meaning that inferences about the endocannabinoid system’s status in PTSD have been primarily based on circulating levels of endocannabinoids. Given the diverse set of functions endocannabinoids may have in the CNS and the periphery, these findings, while interesting, are not able to conclusively demonstrate disturbances of endocannabinoid metabolism in the brain or CNS. The aim of the present study is to address this gap by more directly examining the status of the

39 metabolic enzyme FAAH in vivo using PET imaging techniques with the novel radiotracer [C-11]CURB. With these methods, we will address the following questions: 1. Is there an increase in binding of [C-11]CURB, an index for levels of FAAH, in the amygdala in PTSD; 2. Is there an increase in binding of [C-11]CURB across whole brain in PTSD; and 3. Are levels of [C-11]CURB binding correlated with specific symptom clusters and/or overall symptom severity?

1.9.2 Objectives

The primary objective of the present study is to use PET imaging techniques with the novel radioligand [C-11]CURB to provide the first direct measurement of FAAH in PTSD. We will specifically investigate if FAAH levels (indexed through [C-11]CURB binding levels) are elevated in patients with PTSD as compared to healthy controls, both in the amygdala based on preclinical findings details above, as well as in other relevant structures including the hippocampus and PFC, and across the whole-brain based on limited clinical findings and some preclinical findings suggesting widespread changes in endocannabinoid metabolism in PTSD. We will also investigate whether these levels are associated with specific PTSD symptom clusters (including intrusion symptoms and hyperarousal symptoms) and overall PTSD symptom severity.

1.9.3 Hypotheses

Based on the literature above, our primary working hypothesis is that [C-11]CURB levels will be elevated in participants with PTSD. We hypothesize that levels of FAAH will be increased in the amygdala in PTSD. Furthermore, we also hypothesize that levels of FAAH will show an overall whole-brain increase in PTSD. We further hypothesize that levels of FAAH will be positively correlated with overall PTSD symptom severity and with intrusion and hyperarousal symptom severity.

40 Chapter 2: Methods

2.1 Study Participants

All study procedures were approved by The Centre for Addiction and Mental Health (CAMH) Research Ethics Board. Research participants were recruited from the Greater Toronto Area and surrounding areas. Due to the heterogeneity of PTSD symptoms according to the trauma that resulted in the condition and the fact that certain groups are at higher risk for developing PTSD based on their occupation, we sought to specifically recruit participants whose PTSD was occupational in nature (that is, resulting from workplace-related trauma(s)). Participants were recruited through flyers and online ad postings through CAMH clinics and affiliated clinics in London, Hamilton, and Toronto. Significant effort was made to bolster recruitment efforts by expanding our efforts to recruit within the community, including by contacting PTSD support groups such as the Operational Stress Injury Social Support (OSISS) Program, contacting organizations whose mandate includes workplace safety, and promoting our research among colleagues conducting research in similar clinical populations, through the PTSD Research Consortium meetings, among other discussions. As well, several participants who had completed the study offered to distribute information about the study to their networks, and this word-of-mouth recruitment proved to be an effective strategy to recruit additional participants. Study participants underwent a minimum of two study visits: an assessment visit and an imaging visit comprising the PET imaging session, the MRI imaging session, and/or the neurocognitive testing session. Most often study procedures occurred over three study visits. Healthy control data from previous studies in our lab using [C-11]CURB was used for comparison in this study.

2.2 Screening and Inclusion

Participants completed an initial phone screen to determine general eligibility. After successful completion of the phone screen, participants were invited for an initial screening visit to obtain written informed consent and determine their eligibility to participate according to the study inclusion and exclusion criteria. After obtaining informed

41 consent, study staff administered the Structured Clinical Interview for DSM-5 Axis I Disorders to both confirm current diagnosis of PTSD and to ensure that participants do not have any other disorders that would preclude their participation in the study. Study staff also collected information regarding demographics and medical history, for the purposes of ensuring it would be safe for subjects to participants and to characterize the demographics of our sample. Participants were also asked to provide a urine sample; this sample was tested for drugs of abuse with a rapid response panel test, and was thereafter sent to the CAMH Clinical Lab for broad spectrum drug testing. In women under the age of 65, the urine was tested for pregnancy. Following this, several questionnaires were administered which are summarized below in Table 3: Screening Visit Questionnaires. Items denoted with an asterisk were completed as part of a homework package given to participants during the intake visit and collected once completed at a subsequent visit. These questionnaires allow us to broadly characterize trauma exposure, nicotine and alcohol dependence, mood and anxiety, sleep, personality characteristics, PTSD symptomatology in the PTSD group. Inclusion and exclusion criteria for both PTSD and HC follow below:

Inclusion Criteria: Able to sign and date informed consent Willing and able to complete trial as described in protocol >19 years of age, male or female Meet current DSM criteria for PTSD* *applies to PTSD participants only

Exclusion Criteria: Serious, unstable medical condition including but not limited to cerebrovascular, renal, hepatic and coronary heart disease, as determined by self-report and medical records, where applicable Blood clotting disorder (or taking anticoagulants) Past or current neurological illness DSM diagnosis of psychotic disorders, bipolar depression, or current substance use disorder (except nicotine).

42 Pregnancy or lactation Presence of metal objects in the body or implanted electronic devices, that preclude safe MR scanning Claustrophobia Positive drug toxicology Reported history of difficulty with intravenous blood draws or catheter insertions Exposure to radiation in the last 12 month exceeding permissible limit for subjects participating in research Any other problem that in investigators’ opinion would preclude safe participation in the trial

Table 3: Screening Visit Questionnaires

Name of Questionnaire Purpose Format & Scoring Group Administered

Fagerstrom Test for To assess nicotine dependence in 6-item questionnaire where smoking behaviours Smokers in both PTSD and Nicotine Dependence smokers. are rated and summed, with higher scores HC group (FTND)236 indicating greater nicotine dependence.

Alcohol Use Disorder To assess drinking behaviours and 10-item questionnaire that screens for excessive All participants Identification Test dependence. drinking. Each question is rated from 0-4, with total (AUDIT)237 scores ranging from 0-40. Total scores of 8 or more may indicate potentially hazardous and harmful alcohol use.

Patient Health To screen for and assess severity (if 9-item questionnaire which asks about depressive All participants Questionnaire 9 (PHQ- present) of depressive symptoms symptoms in the past two weeks. Questions are 9)238 rated from 0-3, with the total summed score ranging from 0-27. A total score of 10 indicates mild depression.

Generalized Anxiety To screen for and assess severity (if 7-item questionnaire which asks about depressive All participants Disorder Checklist (GAD- present) of anxiety symptoms in the past two weeks. Questions are 7)239 rated from 0-3, with the total summer score ranging from 0-21.

*Beck Depression To evaluate the severity of depressive 21-item questionnaire where subjects rate their All participants Inventory (BDI)240 symptoms agreement with statements relating to negative cognitive distortions in depression. Scores are then summed, ranging from 0-63, with scores of 21-30 indicating moderate depression, with scores above this indicating severe and extreme depression.

*Snaith-Hamilton Pleasure To screen for and assess anhedonia 14-item questionnaire where subjects indicate their All participants Scale (SHPS)241 agreement with statements about pleasure across

43 4 domains. Responses indicating disagreement are scored a 1, with the total score ranging from 0-14.

*Marin’s Apathy Evaluation To screen for and assess apathy 18-item questionnaire where subjects respond All participants Scale (AES)242 according to a 4-point Likert-type scale indicating their agreement with the statement in question. Scores are summed with greater scores indicating greater apathy.

*State-Trait Anxiety To assess both state and trait levels of The STAI consists of two forms, Y-1 and Y-2. Y-1 All participants Inventory (STAI)243 anxiety assess state anxiety, while Y-2 assess trait anxiety. Each form contains 20 items where participants indicate their agreement with statements on a Likert-type scale. Scores are summed, with higher scores indicating greater anxiety.

*Barratt Impulsiveness To assess impulsiveness The BIS-11 consists of 30 items that describe All participants Scale (BIS-11)244 common impulsive or non-impulsive behaviours. Items are scored on a 4-point scale, with non- impulsive behaviours using reverse scoring. These are then summed to obtain both a total score as well as factor scores.

*NEO Personality To assess personality traits according The NEO PI-R is a questionnaire containing 240 All participants Inventory-R245 to the Five Factor Model. This includes statements. Respondents indicate how strongly assessment of a person’s they agree or disagree with each statement on a 5- interpersonal, emotional, experiential, point Likert-type scale. attitudinal and motivational styles.

*SCID II246 To screen for Axis II personality The SCID-II contains 119 items to which All participants disorders according to DSM-IV criteria respondents answer either yes or no. Scoring is based on the frequency of items scored a 3 and whether or not this frequency meets the threshold for a given diagnoses within the range of questions that pertain to that disorder.

Traumatic Life Events To assess history of a variety of The TLEQ is not scored, but does provide All participants Questionnaire (TLEQ)247 traumas information about the extent to which participants have been exposed to various traumas including war, abuse, natural disasters, and others.

Combat Exposure Scale To assess exposure to wartime The CES is a 7-item questionnaire that asks about Participants who have been (CES)248 stressors stressors in combat. Participants respond on a 5- in combat. point frequency scale. Scores are summed, with total scores ranging from 0-41.

44 PTSD Checklist To assess PTSD symptomatology We administer two versions of the PCL: PCL-4 and PTSD group (PCL)249,250 PCL-5, which are named for the DSM version they are based on. PCL-4 is has multiple versions based on whether or not the respondent is a military veteran or not. PCL-4 is a 17-item questionnaire with total summed scores ranging from 17-85. The PCL-5 is a 20-item questionnaire that resembles the PCL-4 but with the addition of 3 questions to assess E cluster symptoms added to the DSM-5 PTSD criteria. PCL-5 total scores range from (0- 80).

PTSD Symptom Scale To assess PTSD symptomatology The PSS is a 17-item scale that asks about PTSD PTSD group (PSS)251 symptoms in the past two weeks. Respondents endorse symptoms on a 4-point scale from 0-3 reflecting symptom severity or frequency. Scores are summed with total scores ranging from 0-51.

Dissociative Subtype of To assess lifetime and current The DSPS is a 15-item questionnaire that asks PTSD group PTSD Scale (DSPS)252 dissociative symptoms associated with about dissociative symptoms. Symptoms present in PTSD the past month are rated for frequency on a 4-point scale and for severity on a 5-point scale. The Derealization/Depersonalization subscale has been shown to clinically differentiate dissociative subtypes of PTSD and is thus the most useful of the three sub-scales to us.

Pittsburgh Sleep Quality To assess sleep quality The PSQI is a 19-item questionnaire and asks All participants Index (PSQI)253 about 7 components related to sleep. Each question is rated from 0-3 depending on frequency and the global PSQI score, ranging from 0-21, is obtained by summing the 7 component scores.

PSQI PTSD Addendum254 To assess disruptive nocturnal The PSQI PTSD Addendum is a 7-item PTSD group behaviours related to PTSD questionnaire whereby the frequency of different disruptive nocturnal behaviours is rated from 0-3 based on the past month. The 7 items are summed for a total score ranging from 0-21, with higher scores indicating greater sleep disturbances.

45 2.3 FAAH Genotyping

In order to account for a known SNP affecting FAAH levels and activity in our population, a sample of venous blood will be used to determine their genotype of endocannabinoid-related genes according to procedures published elsewhere.235 In brief, DNA will be extracted from the samples using a high salt extraction procedure, and the FAAH SNP rs324420 will be genotyped using a TaqMan 5’ nuclease allelic discrimination assay in a 96-well format (Applied Biosystems Inc.). This genotyping will be completed in the laboratory of one of our co-investigators, Dr. Rachel Tyndale (University of Toronto Medical Sciences Building, Toronto, ON, Canada).

2.4 MRI Session

All imaging procedures, including PET imaging procedures, were performed at the Research Imaging Centre at the Centre for Addiction and Mental Health. Proton-density (PD) and T1-weighted images were obtained for each participant during an MRI imaging session lasting roughly 90 minutes using a 3T MR-750 scanner (General Electric, Milwaukee, WI, USA). All participants completed an MRI Safety Screen prior to the session to ensure participant safety. Prior to the MRI scan, a urine sample was collected to analyze drug and/or medication levels. As well, exhaled carbon monoxide (CO) and breath alcohol measurements were used to quantify recent smoking and drinking. Finally, participants completed the PHQ-9, GAD-7, and Y-1 portion of the State Trait Anxiety Inventory to assess depressive symptoms, anxiety symptoms, and state anxiety respectively.

2.5 PET Imaging Session

Participants were asked to refrain from smoking and consuming alcohol and caffeine the night before the scan and were given a standard carbohydrate-based meal prior to the beginning of the PET scan. Prior to the PET scan, participants provided a urine sample which was used to confirm that participants were not pregnant where applicable, and that participants had not consumed drugs of abuse (other than those prescribed to them) using a rapid response drug dipstick. This urine sample was then

46 sent for broad spectrum analysis through the CAMH Clinical Lab to better understand any potential confounds due to medication status. Given this project’s goal is to investigate the endocannabinoid system and the use of cannabis as a pharmacotherapy in PTSD, it was of particular importance to ensure that participants did not test positive for THC. Participants also completed the PHQ-9, GAD-7, and Y-1 portion of the State Trait Anxiety Inventory to assess depressive symptoms, anxiety symptoms, and state anxiety respectively. If the participant was scheduled to complete the MRI and PET imaging session on the same day, these questionnaires were completed only once. PET scans were performed using a 3D high resolution research tomograph (HRRT) (CPS/Siemens, Knoxville, TN, USA). The scanner provides radioactivity measurements in 207 slices with a distance of 1.22 mm between each slice. A thermoplastic mask was custom fitted to each participant and was worn for the duration of the PET scan in order to minimize head movement. Finally, a brief transmission scan was acquired in order to correct PET images for attenuation using a single photon point source 137Cs prior to the 60 minute [C- 11]CURB scan. Radioactivity was captured through sequential frames of increasing duration for the length of the scan.

2.6 PET Image Acquisition and Reconstruction

The radiotracer [C-11]CURB was synthesized in a novel one pot reaction from [C-

11]CO2 as described by Wilson and colleagues.226 Approximately 370±40 MBq of the radiotracer was injected as a bolus intravenously into the antecubital vein of the participant. As well, a respiratory therapist inserted a cannulae into the radial artery of each participant in order to take arterial blood samples throughout the scan. In order to generate the input function for kinetic analysis, manual arterial samples were taken throughout the scan at 3, 7, 12, 20, 30, 45 and 60 minutes after injection. As well, arterial blood was sampled continuously for the first 22.5 minutes after injection at a rate of 2.5mL/min using an automatic blood sampling system (Model #PBS-101 from Veenstra Instruments, Netherlands). Whole blood and plasma aliquots of each sample was measured for radioactivity in a gamma-counter. A Hill function was used to fit the parent plasma fraction. The blood-to-plasma ratios was used to generate a plasma radioactivity curve, with a bi-exponential function used to fit these ratios.230 This allows the generation

47 of a metabolite-corrected plasma curve by multiplying the dispersion-corrected blood curve with the blood-to-plasma ratio and percentage of parent radiotracer. This is then used as the input function for kinetic analysis (CP(t), nCi/mL). After correction for attenuation, PET images were reconstructed from 2D sinograms by a 2D filtered-back projection algorithm using a HANN filter at Nyquist cut-off frequency. A diagram depicting the timeline of the PET imaging session is shown in Figure 2: Timeline of PET Scan.

Figure 2: Timeline of PET Scan. A) A sample plasma radioactivity curve is depicted in red and sample whole brain radioactivity in blue as a function of scan time; B) Depiction of transmission scan and frames (denoted by F) acquired during dynamic emission; and C) Timeline of manual blood sampling (MS) and automatic blood sampling (ABSS).

48 2.6.1 Region-Based Image Analysis

Regions of Mental Interest, an in-house pipeline for delineation of regions of interest, (hereon “ROMI”) was used to conduct a region of interest (ROI) based analysis whereby radioactivity is averaged over an anatomically defined region.255 ROMI fits a standard template of ROIs to each individual proton density high-resolution MRI using probabilities of grey matter, white matter and cerebrospinal fluid. A segmentation step refines these ROIs by using the probability of grey matter in individual voxels in the MR image. The MR image is then co-registered with the dynamical PET image such that the refined ROIs are superimposed onto the PET images, which allows for the generation of a time-activity curve (TAC) for each ROI. Each image was corrected for head motion using Statistical Parametric Mapping software version 8 to realign the frames of the participants’ images (SPM8, Wellcome Trust Centre for Neuroimaging, London, UK). The pipeline used by ROMI for region-based image analysis is shown below in Figure 2: ROMI Process for ROI Delineation.

Figure 3: ROMI Process for ROI Delineation. This process involves five main steps: 1. Normalization: the subject’s PD MRI is moved to template (i.e. MNI) space using a non-linear transformation; 2. Template

49 ROI moved to MRI space: 3-D transformation matrix from MRI to MNI is used to move ROI template to MRI space; 3. Segmentation: Subject’s MRI is segmented and smoothed using SPM (segmentation algorithm and Gaussian smoothing filter), which results in a grey matter mask in which every voxel has >98% probability of grey matter; 4. Refinement: voxels of the ROI template which fall outside the grey matter mask are iteratively removed; 5. Co-registration: PET and MRI images are co-registered using SPM, with the refined ROI template moving from MRI space to PET space.

The ROMI region of interest template covers several cortical and subcortical regions of interest. Subcortical regions of interest include the cerebellum, amygdala, hippocampus, striatum and thalamus. Cortical regions of interest include the insula, occipital lobe, parietal lobe, prefrontal cortex, temporal lobe and anterior cingulate gyrus.

2.6.2 Quantification of [C-11]CURB

The kinetics of [C-11]CURB can be best approximated using a two-tissue compartment model with irreversible binding to the second compartment.230 This model, together with the arterial input function, is used to fit the TACs generated above. There are three parameters that are used under this model to create the composite parameter

λk3. This parameter is calculated as follows: λk3= (K1/k2) ✕ k3, where K1 is the influx of

[C-11]CURB from plasma to the free and non-specific compartment, k2 is the efflux of [C-

11]CURB from the free and non-specific compartment, and k3 is the flux from the free and non-specific compartment to the specifically bound compartment. λ is therefore the equilibrium distribution volume of [C-11]CURB in the free and non-specific compartment.

The composite parameter λk3 is the validated metric used for quantifying [C-11]CURB in each ROI.

50

Figure 4: Diagram of 2-Tissue Compartment Model with Irreversible Binding in the Second

Compartment. CP represents plasma with unmetabolized or parent tracer. CF+NS represents the free and non-specific tissue compartment. CSP represents the specific tissue compartment. Finally, both CSP and

CF+NS together comprise CT, the tissue in which the tracer will bind. Note that since this model assumes irreversible binding to CSP, there is no rate constant describing the flux of tracer from CSP to CF+NS. Adapted from Gunn et al.256

2.7 Statistical Plan of Analysis

To understand the demographics of our population, we will characterize the age, sex, race, body mass index (BMI) and medication status in each group using descriptive statistics and will use independent samples t-tests, Chi square tests, and Mann-Whitney U tests depending on what is most appropriate given the data to assess any group differences in these areas. Descriptive statistics will also be used to characterize PTSD symptom severity in the PTSD group. Further, we will also examine if there are group differences in mood and anxiety between groups using the PHQ-9, GAD-7 and Beck Depression Inventory.

Data will be first tested for normality. As well, visual inspection of histograms will allow the identification of any potential outliers. In order to investigate whether [C- 11]CURB binding was elevated in the amygdala in PTSD, we used an ANCOVA with

51 FAAH genotype entered as a covariate. To investigate whether there are significant group differences in levels of [C-11]CURB binding between groups across other ROIs, we will use a repeated measures ANCOVA with group as the between-subjects factor, ROI as the within-subjects factor, and FAAH genotype as a covariate (Group[2]*ROI[11]). In testing our second hypothesis that PTSD will be associated with higher whole-brain [C- 11]CURB binding, we expect that there will be a main effect of group. Sphericity will be corrected for using the Greenhouse-Geisser method where appropriate. Post-hoc pairwise comparisons will be used to identify where significant effects exist using Tukey’s Honestly Significant Difference. Significant level will be set at p≤0.05.

Given the possibility that certain symptom clusters may be more associated with disturbances in the endocannabinoid system, we will explore whether certain severity of symptom clusters correlation with levels of [C-11]CURB binding across ROIs. To do this, Pearson product-moment correlations and/or Spearman’s rho will be used depending on the normality of data. Partial correlations may also be used to assess these relationships while controlling for potentially confounding factors.

52 Chapter 3: Results

3.1 Demographics and Characteristics

Of the 16 subjects comprising the PTSD group, 7 were cases who completed the study between February 2015 and January 2016. The remaining 9 subjects were recruited and completed the study between March 2019 and February 2020. The recruitment of these 9 subjects is summarized below in Figure 5: PTSD Recruitment. The healthy controls were also recruited as part of another previous project and completed study procedures between November 2013 and October 2015. Healthy controls (HC) were enrolled in the study based on the inclusion criteria described in Chapter 2: Methods. There were 29 healthy controls available who completed the scan, and these controls were entered in the present study irrespective of demographic factors.

Figure 5: PTSD Recruitment. Of the 36 PTSD cases contacted, 32 phone pre-screens were completed. 16 subjects were invited for an in-person screening; 11 participants completed this in-person screening. 10 subjects total completed the study, though one had an unusable scan, and 1 participant is currently enrolled.

There were no statistically significant differences between groups with respect to sex (p=0.36), smoking status (p=0.73), and C385A genotype (p=0.69). Smokers were

53 defined as those who smoked ≥ 5 cigarettes per day. There was a significant difference between groups in age, such that the PTSD sample was older on average than the healthy control sample (p=0.001). As well, there was a significant difference between groups in Body Mass Index (BMI), with the PTSD sample having a greater BMI on average than the healthy controls (p=0.001). Groups also differed significantly in racial composition, since the entirety of the PTSD group was Caucasian (p=0.03). As expected, the PTSD group scored significantly higher on average than the healthy control on the Beck Depression Inventory (BDI; U=354.5, p=0.000). The PTSD group had an average score on the BDI of 15.71, which indicates mild mood disturbance. Finally, there was a slight difference in the amount of radioactivity injected between groups, though this difference was not statistically significant (p=0.09). These results are summarized below in Table 4: Subject Demographics.

Table 4: Subject Demographics (Mean  SD)

54 The PTSD group had an average total score of 21.93 on the PTSD Symptoms Scale (PSS) with a standard deviation of 11.52. Possible scores on the PSS range from 0-51, with greater scores indicating greater overall symptom burden. On the PTSD Checklist (on which possible scores range from 17-85 and a cut-off score of 30-35 is suggested to be indicative of PTSD in the general population, though this cut-off has been suggested to be higher in populations with greater PTSD prevalence), the average total score was 46.27 with a standard deviation of 14.91. On the PSS, subjects endorsed the most symptoms on the D cluster items, followed by C cluster items and then B cluster items (after controlling for the number of questions pertaining to each symptom cluster). Similarly, on the PCL, subjects endorsed the most symptoms on D cluster items, but this was followed by B cluster items and then closely by C cluster items (again, after controlling for the number of questions pertaining to each item). On the State-Trait Anxiety Inventory, the PTSD group had an average total score of 76.75, with an average state anxiety score of 39.43 at screening. A score of 39-40 has been suggested to indicate clinically significant levels of anxiety.257 These subjects were also on a variety of combinations of prescription medications, including selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, benzodiazepines, norepinephrine-dopamine reuptake inhibitors, Z-drugs, tetracyclic antidepressants, alpha blockers, atypical antipsychotics, opiates, antiepileptics and naltrexone. These results are summarized in Table 5: Characteristics of PTSD Group. Finally, no subjects in the PTSD group (nor the HC group) had problematic drinking as assessed by the SCID-IV and/or SCID-5. One subject did have relatively high amount of alcoholic drinks per week (~16, which exceeds the U.S. Department of Health and Human Services’ guideline for excessive drinking) relative the rest of the group (many of whom were infrequent or non- drinkers).258

55

Table 5: Characteristics of PTSD Group (Mean  SD)

3.2 [C-11]CURB Binding Between Groups Across ROIs

A univariate analysis of covariance (ANCOVA) with FAAH genotype as a covariate was conducted to assess differences in [C-11]CURB binding in the amygdala. [C-11]CURB binding was significantly lower in the PTSD group than the HC group

56 (F(1,42)=5.87, p=0.020). This result is highlighted in Figure 7: k3 in Subcortical Regions of Interest. In order to investigate group differences in FAAH binding in other regions of interest, an ANCOVA was conducted with eleven regions of interest: insula, cerebellum, amygdala, hippocampus, occipital lobe, inferior parietal lobe, striatum, prefrontal cortex, temporal lobe, thalamus and cingulate. C385A genotype was included as a covariate.

These data are shown in Figure 6: k3 in Cortical Regions of Interest and Figure 7: k3 in Subcortical Regions of Interest below. The subject who drank heavily did not appear to significantly differ from the PTSD group across ROIs. These data were normally distributed as determined by the Shapiro-Wilk Test. One PTSD subject with wild-type

(i.e. C/C) genotype for the C385A SNP consistently had the lowest k3 values in the PTSD group across all ROIs. Though this subject had values that were greater than 2 standard deviations away from the group mean in 4 ROIs (insula, cerebellum, inferior parietal lobe, and thalamus), the values were never more than 3 standard deviations from the group mean in any ROI and thus they are not considered an outlier for the purposes of these analyses. Mauchly’s Test of Sphericity indicated that the assumption of sphericity was violated, and epsilon (ε)=0.463 (calculated according to the Greenhouse-Geisser method) was used to correct the repeated measures ANOVA. There was no main effect of group, though the effect was trending towards significance (F(1,42)=3.23, p=0.079) such that [C-11]CURB binding tended to be marginally lower in PTSD than in controls (-

9.38%, ηp2=0.071). There was also no Group(2)*ROI(11) interaction effect (F(4.63,194.3)=0.689, p=0.621). There was a significant within-subjects effect of ROI

(F(4.63,194.3)=61.81, p=0.000), such that there were significantly higher k3 values in the thalamus (average k3=0.169), followed by the striatum (average k3=0.156) and insula

(average k3=0.153) while the amygdala had the lowest k3 values (average k3=0.121).

The average k3 in all other ROIs ranged from 0.135-0.148. There was also a main effect of C385A genotype (F(1,42)=17.964, p<0.001), such that those with a copy of the variant allele had lower k3 values. Finally, there was a significant interaction effect between ROI and C385A genotype (F(4.64,194.3)=4.59, p=0.001).

To attempt to mitigate the effect of any confounding variables that may affect k3 values, Analysis of Covariance (ANCOVA) were run with the following covariates in addition

57 to C385A genotype: age, BMI, NIH race, and amount of radioactivity injected. These variables were added as covariates because they both differed between experimental groups and because there is some literature to suggest that race and adiposity in humans may affect the ECS and in animals there is some suggestion that changes in AEA levels may be mediated by age.259,260 Finally, C385A genotype has been shown to modulate levels of [C-

11]CURB binding previously.235 Thus, it was important to control for any possible influence of these factors in the present data. Age was not a significant covariate (F(1,41)=0.030, p=0.864), nor was BMI (F(1,41)=0.092, p=0.763), NIH Race (F(1,41)=0.702, p=0.407), and amount of radioactivity injected (F(1,40)=1.608, p=0.212). The effect of group remained insignificant with each covariate. Moreover, smoking status also did not mediate levels of [C- 11]CURB binding. In the amygdala, controlling for smoking status and sex maintained or increased the significance of observed group differences in [C-11]CURB binding (p’s≤0.021), while entering age and BMI as covariates rendered the finding non-significant (p’s≥0.064). Nearly all of the PTSD group was on a variety of combinations of prescription medications, and as such it was not possible to assess the effects these medications may be having on levels of [C-11]CURB binding.

58 Figure 6: k3 in Cortical Regions of Interest. Line indicates group mean for both groups. A repeated measures ANCOVA revealed no significant group difference in k3 values across 11 ROIs (F(1,42)=3.23, p=0.079). There was also no Group(2)*ROI(11) interaction effect (F(4.63,194.3)=0.689, p=0.621). Six cortical ROIs are graphed here. Percent difference are given for group means adjusted for genotype variance.

Figure 7: k3 in Subcortical Regions of Interest. Line indicates group mean for both groups. [C-11]CURB binding in the amygdala is highlight with a box in the above figure. A repeated measures ANCOVA revealed no significant group difference in k3 values across 11 ROIs (F(1,42)=3.23, p=0.079). There was also no Group(2)*ROI(11) interaction effect (F(4.63,194.3)=0.689, p=0.621). Five subcortical ROIs are graphed here. Percent difference are given for group means adjusted for genotype variance.

59 3.3 [C-11]CURB Binding Between Groups in Whole Brain

In order to assess any overall whole-brain changes in FAAH binding, an average of the k3 value across 11 ROIs was calculated for each subject. A univariate ANOVA was used to assess group differences and C385A genotype was included as a covariate. Across whole brain, group differences in [C-11]CURB were non-significant but trending towards significance (F(1,42)=3.091, p=0.086), such that [C-11]CURB binding was lower on average in the PTSD group than in the HC group. This data is displayed below in Figure 7: k3 Across Whole Brain.

Figure 8: k3 Across Whole Brain. Line indicates group mean for both groups. A univariate ANOVA revealed no significant group difference in averaged whole brain k3 values (F(1,42)=3.091, p=0.086), though PTSD had marginally lower average whole brain k3.

60 3.4 [C-11]CURB Binding in Relation to PTSD Symptomatology

In order to assess whether levels of [C-11]CURB binding were correlated with total symptom burden and symptom burden of specific clusters in the PTSD group, partial correlations were used with C385A genotype as a covariate. [C-11]CURB binding in the amygdala did not significantly correlate with total PSS and PCL scores as well as symptom cluster sub-scores on these two measures (r’s≤-0.27, p’s≥0.41). The results of these partial correlations between amygdala k3 values and scores on the PSS and PCL are shown below in Table 6: Correlations Between Amygdala k3 Values and PTSD Symptoms.

When examining the relationship between k3 values across the other ROIs and total scores on the PSS and PCL, it was found that there was no significant correlation (r’s≤0.180, p≥0.557 and r’s≤0.181 p≥0.623 respectively). Further partial correlations to examine whether a relationship existed between k3 across ROIs and specific symptom clusters on both the PSS and PCL revealed no significant correlations (r’s≤0.547, p’s≥0.053). This nearly significant positive correlation was for PSS D Items (negative alterations in mood and cognition) and k3 values in the thalamus, such that a greater endorsement of PSS D Items correlated to greater k3 values in the thalamus. After controlling for BMI and age (in addition to C385A genotype), this correlation became more significant (r=0.679, p=0.015). This result did not survive after correction for multiple comparisons using the Bonferroni method. Moreover, after repeating this process for both total scores on the State Trait Anxiety Inventory and both state and trait sub-scores, it was found that these measures do not significantly correlate with k3 values (r’s≤0.252, p’s≥0.406). Finally, this process was repeated to assess for correlations between k3 values and scores on the BDI. Again, no significant correlations were found across all 11 ROIs (r’s≤-0.333, p’s≥0.267).

Table 6: Partial Correlations Between Amygdala k3 Values and PTSD Symptoms

61 3.5 [C-11]CURB Binding in Relation to BMI

Finally, in an exploratory analysis, partial correlations were used to determine whether BMI was significantly correlated with k3 values across whole brain in both groups. This exploratory analyses was undertaken for two reasons: first, that endocannabinoid system signaling is known to be differentially regulated in obesity,261-263 and second, that

PTSD has been shown to be associated with a greater risk of being obese or overweight.264-

266 FAAH genotype was entered as a covariate. There was no significant correlation between

k3 values across 11 ROIs and whole brain and BMI in either PTSD (r= 0.232, p=0.405) or HC (r=-0.035, p=0.860). The whole brain correlations are displayed below in Figure 9:

Correlations Between Whole Brain k3 and BMI. Moreover, to see if there were group differences in levels of k3 across 11 ROIs and whole brain in the PTSD group, independent samples t-tests were used to assess group differences in [C-11]CURB binding between those who had a healthy BMI (i.e. BMI<25) and those who were overweight or obese (i.e. BMI>25). There were no significant differences between those who had a healthy BMI and those who had a BMI greater than 25 within the PTSD group across 11 ROIs and whole brain (p’s≥0.367).

62 A)

B)

Figure 9: Correlations Between Whole Brain k3 and BMI. A) Partial correlation controlling for FAAH genotype in HC (r=-0.035, p=0.860). B) Partial correlations controlling for FAAH genotype in PTSD (r= 0.232, p=0.405).

63 Chapter 4: Discussion

4.1 Summary of Findings

This study provides the first direct measurement of FAAH using the radioligand [C- 11]CURB in occupational PTSD in humans. The general objective of this study was to investigate whether levels of this enzyme were upregulated in PTSD. More specifically, we sought to test the hypothesis that [C-11]CURB binding would be higher in PTSD across the whole brain and in the amygdala. We also sought to test the hypothesis that levels of [C-11]CURB binding in PTSD would be associated with PTSD symptomatology. Our results suggest that [C-11]CURB binding is lowered in the amygdala in PTSD compared to healthy controls. In addition, our results suggest that [C-11]CURB binding is not elevated in PTSD, and may actually be slightly lower than binding in healthy controls, across the whole brain. Moreover, we found no evidence that [C-11]CURB levels were correlated with PTSD symptomatology, both total and stratified by symptom cluster. Finally, in an exploratory analysis to examine if a correlation exists between BMI and [C- 11]CURB binding, we found that there was no significant correlation between these parameters in either group. These findings were not in line with our hypotheses, which were largely based on the preclinical literature. The following chapter will discuss potential reasons why these findings were not in line with our hypotheses, and the clinical significance of these results.

4.2 Interpreting Levels of [C-11]CURB Binding in PTSD

4.2.1 Interpreting Unelevated [C-11]CURB Binding in PTSD

These findings are not in line with the general consensus in the preclinical literature, which suggests that FAAH is upregulated after and related to the behavioural effects of chronic stress. While most preclinical studies tend to demonstrate some evidence for FAAH upregulation in animal models of PTSD, this is not the case for all studies. For example, results from some studies have suggested that this upregulation may depend in part on region and sex.152,188,191,267 Moreover, findings around FAAH

64 upregulation and AEA levels tend to be less consistent in animal models using heterotypic stressors (for example, chronic unpredictable stress)152,189 and those that use homotypic stressors (for example, chronic restraint stress).87,185 Nonetheless, it would appear that homotypic stress models have the most face validity in representing our sample of people with occupational PTSD. While regional and sex-specific effects may affect FAAH levels, we did not see elevated FAAH in any regions of interest selected based on the preclinical literature, and sex was not found to have a significant effect on FAAH levels in our sample. In this regard, in our larger cohort of healthy controls, we also do not find an effect of sex

(or age) on FAAH levels.268 Even though we did not find that sex was a moderator of FAAH levels in this analysis, the question of sex affecting FAAH in PTSD should still be addressed in larger studies, particularly since women have higher prevalence rates of

PTSD.9,10 We also considered the possibility that other confounds may exist in our data. There were significant between-groups differences in age, BMI, and NIH race. Of note, there is evidence in humans to suggest that FAAH levels may inversely vary with BMI, such that individuals with higher BMIs (as in our PTSD group) have lower levels of

FAAH,261,269,270 though not all studies have found such an inverse relationship.271 Thus, to control for any effect these variables might have had on the data, we used ANCOVAs to determine the effect of these variables on the data. However, none of these covariates had a significant mediating effect on levels of [C-11]CURB binding, and the finding remained unchanged with the addition of these covariates. Moreover, partial correlations examining BMI and [C-11]CURB binding across ROIs and whole brain in both PTSD and controls revealed no significant correlations between these parameters. However, with respect to the finding in the amygdala, when age and BMI were entered as covariates, the findings became non-significant and instead were trending towards significance. Nonetheless, given these attempts to rule out possibly confounding factors did not reveal any significant effects, interpretation of the results necessitates the challenging task of trying to understand why animal models of PTSD do not seem to translate well to humans in this case. Importantly, the challenge of modeling a complex and heterogenous disorder like PTSD must be noted. While the clear link between the development of PTSD and an initial traumatic event was considered to be an advantage in developing animal models,272

65 both the heterogeneity (I.E hyperarousal versus more dissociative presentations) and the comorbidity of the disorder have meant that it is difficult to develop models that accurately and reliably reflect the clinical complexities of PTSD. For example, operationalizing the symptom clusters of PTSD is challenging because several measurable behavioural indications of these symptoms overlap with those in other disorders (for example, behavioural indices of anxiety), making it a challenge to develop a model with specificity towards PTSD, while other symptom clusters are impossible to assess in rodents (for example, intrusive memories and nightmares).273 Moreover, using typical validation approaches of these models has proven difficult or impossible, given the neurobiology of PTSD in humans is not fully understood (limiting construct validation) and the lack of a gold standard pharmacotherapy for PTSD in humans (limiting pharmacologic validation).272 Another shortcoming of many animal models is that experimental stress exposure is considered to be sufficient to conceptually represent PTSD; in this way, many studies have a stress-exposed group, and a group of non-stressed controls.272,274 However, this is counter to what we know about human stress exposure, which is that exposure to traumatic stress will lead some portion but not all or even most of the exposed group to develop PTSD. Indeed, certain risk factors in humans have been shown to predispose individuals to develop PTSD after trauma, but many of these risk factors are not easy or even possible to incorporate into experimental designs.272 To address these differences in outcomes after stress exposure, some researchers have used a method called cut-off behavioural criteria (CBC),275 whereby the stressed group is stratified according to their behavioural responses after stress exposure.272 While this methodology does allow for more nuanced analysis of variances in stress responses as opposed to the extremes modeled by comparing stressed and non-stressed groups, even this does not perfectly approximate divergent responses to traumatic stress seen in humans, given assessment of the behaviours is relative to the others in the exposed group and even still, it can be difficult to determine whether neurobiology findings signify an adaptive or resilient response or a marker of pathology.272 However, the majority of studies surveyed in section 1.7.3 on the endocannabinoid system in these models have not used this methodology, instead relying on the group differences in stress exposure. Though animal

66 models have clear and important practical investigative advantages, the challenges in modelling a complex disorder like PTSD may limit the translation of this preclinical work. Setting aside these challenges of translating animal models discussed above, there remains the possibility that the timeline in which FAAH levels are examined in animal models varies significantly from a realistic clinical paradigms involving PTSD, and certainly from the paradigm used in the present study.274 Indeed, many of the studies cited in the review of the preclinical literature decapitate the animals immediately or just hours after the last stress exposure. For example, in the portion of Table 2 in section 1.7.3.3 that examines FAAH levels after stress exposure, time from the last stress episode to sacrifice and subsequent measurement ranges from none (i.e. immediate sacrifice after the final stress episode) to a maximum of 24 hours.87,188 This means that although these studies are well-positioned to capture more acute responses to chronic stress, their findings may not be representative of the time course under study in our experiments where participants are far-removed from the initial trauma. Moreover, it is not clear that these acute changes, both behavioural and biological, are clinically relevant in the sense that they may normalize and thus not represent an animal that has developed long-term maladaptive stress responses, which would be most analogous to clinical presentations of PTSD.274 These differences in timelines in the preclinical and clinical literature permit several possibilities. For one, it may be that FAAH is rapidly upregulated in humans after traumatic stress, but that this upregulation is not permanent (hence our finding) and becomes dissociated from the symptoms that persist beyond this upregulation. If this was the case, it may be that the increase in FAAH is involved in the development but not the maintenance of PTSD. To confirm this, both preclinical and clinical studies should attempt to reconcile these differences in timeline: that is, animal studies should examine the course of the enzyme and behavioural effects longitudinally over time after stress exposure, and human studies should attempt to measure changes in the enzyme sooner after trauma exposure and continue these measurements longitudinally. Notwithstanding that the latter may be more challenging practically speaking, this would allow a more informative comparison of the dynamic changes in this system over time after traumatic stress in both humans and animals, and would better position us to determine whether or not these findings are actually as discrepant as they

67 would initially appear. It is a considerable limitation of the current preclinical literature that these longitudinal studies do not exist. An alternative though related explanation for the discrepancy between our finding and the preclinical literature is that in the animal studies (by virtue of their sacrifice very shortly after chronic stress exposure), FAAH measurements are made during an acutely stressed state. In the present study, subjects were not “challenged” (i.e. they were not exposed to a stressor). It is possible that a stress challenge that forced subjects to re- experience the trauma to some extent may have yielded a finding more in line with the upregulation of FAAH seen in the preclinical literature. Again, notwithstanding the practical and possibly ethical concerns around such a design, inducing this state may give more temporal clarity with respect to the enzyme’s levels during episodes of presumed symptom exacerbation versus the relatively more “baseline” measurement taken in this study. Much of the literature that has led to the dysfunctional neurocircuitry hypothesis of PTSD—namely, a hyperreactive amygdala and a hyporeactive vmPFC— has actually come from studies that measure responsiveness not at baseline, but in response to a variety of trauma-related general threat-related cues.60,61,64-72 This adds credibility to the idea that temporal proximity to the sorts of responses that are dysregulated in PTSD, like hyperarousal in response to threat, may affect the study’s ability to capture significant neurobiological differences between groups. While very few clinical studies have been done to examine the endocannabinoid system in PTSD during a stress challenge, one study did find that subjects with lower FAAH activity reported decreased anxiety during the challenge.211 While the fact that this is only a single study wherein subjects had comorbid alcohol use disorder limits the conclusions that can be drawn, it does support the idea that the experimental state of the subjects with PTSD may affect findings. In summary, the exclusion of confounding variables from our data leaves open the question of why findings from animal models do not translate to humans in this case. There are particular weaknesses of animal models of PTSD that may limit the translation of the findings employing these models. There also appears to be a discrepancy both in the timeline of measurement after trauma and the state of subjects during measurement such that the dynamic changes of the endocannabinoid system (and in particular, FAAH)

68 may follow a time course that neither the preclinical literature nor the present study (or other clinical studies) have well-characterized. These important factors may, in part, explain why we did not see the upregulation in FAAH in PTSD that the preclinical literature has suggested. It bears mentioning that the only other PET imaging study to examine the status of the endocannabinoid system in humans with PTSD also found results counter to what the literature would suggest.213 This study used a radioligand to assess levels of CB1R, which have been mostly shown to be downregulated in preclinical models of PTSD

(see Table 2 in section 1.7.3.3).152-156,159,191 However, this study found a significant upregulation in CB1R in PTSD.213 While the authors’ interpretation of that finding—that the upregulation represented a compensatory mechanism for low AEA levels—may not preclude the FAAH upregulation in PTSD that we expected to see in the present study, it does provide additional evidence that the findings from animal models may be limited in their translation, at least until preclinical and clinical paradigms are able to better reconcile the significant ways in which the designs differ.

4.2.2 Interpreting the Possibility of Lowered [C-11]CURB Binding in PTSD

In addition to finding that [C-11]CURB levels were not elevated, there appeared to be a trend towards lower [C-11]CURB binding in the PTSD group (-9.38%, p=0.09) across the whole brain. There was significantly lower [C-11]CURB binding in the amygdala in PTSD compared to healthy controls (-14.5%, p=0.020), with trending significance in the inferior parietal lobe (-10.26%, p=0.056) and the temporal lobe (-9.74%, p=0.056). However, upon visual inspection (see Figures 6–8), it appears that this effect may be driven, at least partially, by one subject in the PTSD group who, despite having the wild- type C385A genotype, had the lowest values in the group across each ROI. Though this subject was not considered an outlier in any ROIs and thus was included in all analyses, it is possible that it is this subject’s data that is driving the effect of marginally lower [C- 11]CURB binding in PTSD. The present study is underpowered to detect a modest decrease in [C-11]CURB binding between groups. For example, to detect a true effect similar to the magnitude suggested by the results in the whole brain (approximately -9%) with α=0.05 and β=0.2

69 with similar group parameters (i.e. group means and standard deviations), we would need 55 participants in each group for a total sample size of 110 to be able to be sufficiently powered to detect an effect of this magnitude. Alternatively, if we were to keep the enrolment ratio in the present study constant to account for the fact that it tends to be easier to recruit HC than PTSD, we would need 43 subjects in the PTSD group and 77 in the HC group, for a total sample size of 120. Put another way, post-hoc power calculations based on our present sample sizes and group means and variability revealed power level of 43.4%. However, post-hoc power calculations reveal that our study is better powered to detect a true effect in the amygdala of the magnitude found in this study, with 75% power, suggesting our study may have been suitably powered to detect an effect of this size in the amygdala. Nonetheless, given the whole-brain power calculation, it appears the present study may be generally underpowered. The clinical literature, mostly consisting of studies assessing endocannabinoid levels in peripheral blood, has been inconsistent on this question. While the aforementioned PET imaging study assessing CB1 receptor levels in PTSD found decreased peripheral levels of AEA in PTSD,213 other studies have reported no group differences in AEA or increased AEA in PTSD.217-219 While it is presently not well known to what extent peripheral levels of endocannabinoids are reflective of levels in the central nervous system, recent work by our group suggests that peripheral levels of FAAH substrates could correlate with FAAH levels in the brain.268 Moreover, unpublished data from a subset of this PTSD sample (n=7) suggest that, at least in this subset, we see no group differences in peripheral levels of AEA (and other FAAH substrates including OEA and DHEA) though there is a significant elevation in 2-AG in PTSD. This is to say that despite inconsistencies in findings, if central levels of endocannabinoids can be at least partially related to peripheral levels of endocannabinoids, then the findings presented here may be in agreement with some of the few studies that have examined this question and found either no group differences in AEA levels or an elevation of these levels in

PTSD.217-219 However, there is likely a somewhat complicated relationship between peripheral and central levels of endocannabinoids and their catabolic enzymes and we should take caution in the absence of well-characterized information about this relationship.

70 Our present data suggests that levels of [C-11]CURB binding may be lower in PTSD. Despite the fact that our study may be underpowered to detect a true effect of this magnitude, we should be cautious in concluding that this is case, particularly due to one subject with very low and somewhat anomalous levels of [C-11]CURB binding in the PTSD group. While a finding of decreased FAAH in the brain would be in line with some clinical studies that have suggested that there may be elevated levels of peripheral AEA, we should avoid drawing this conclusion on the suggestion of this literature particularly because there have been so few studies examining this question and because the relationship between peripheral levels of endocannabinoids and levels in the brain remains poorly understood.

4.2.3 Interpreting the Lack of Association Between [C-11]CURB Levels and PTSD Symptomatology

Moreover, we hypothesized based on the preclinical literature that levels of [C-11]CURB binding would be significantly associated with specific symptom clusters and overall symptom burden. While very few studies have addressed this question in humans, two studies have suggested that peripheral levels of AEA and other FAAH substrates in blood and in hair negatively correlate with PTSD symptomatology (but note again that relationships between peripheral and central levels of ECs remain unclear).213,223 However, we find no evidence for correlations between specific symptom clusters and overall symptom burden and [C-11]CURB binding both across the whole brain and in each ROI. This finding does not preclude the possibility that FAAH levels are related to the clinical features of PTSD. There are many moderating factors that may affect the overall symptom of burden in PTSD. Some of these include variability in time since diagnoses, time since trauma exposure, other common comorbidities (including depression and anxiety), medication usage, levels of social support, and overall level of functioning. Information on many of these considerations was not available for inclusion in this thesis. Nevertheless, given the complex ways in which one’s predisposition towards PTSD and these moderating factors may interact to affect the PTSD trajectory and burden, detecting and fully understanding correlations in light of these complexities becomes challenging. While it is possible that FAAH is not correlated with PTSD

71 symptomatology, it is also possible it does affect symptomatology in concert with a constellation of other mediating factors which may obscure this possible correlation.

4.3 Strengths

The main strength of this study lies in its novelty, given the project provides the first direct, high-resolution measurement of endocannabinoid system tone in people with PTSD. Moreover, another strength of the present study is that our PTSD population was limited to those with occupational trauma. This has the advantage of both reducing some of the heterogeneity within the PTSD population and focusing our research efforts on a group that is known to have a significantly increased risk of developing the disorder. Given the general paucity of human studies of the endocannabinoid system in PTSD and the tendency of these studies towards indirect assessment of the ECS, this study contributes importantly to the existing clinical literature, which has been inconsistent, and to the broader discussion around the development of novel therapeutic options (including FAAH inhibitors) for PTSD. In addition, this study is part of a larger, multimodal effort to better understand PTSD through the integration of PET imaging, functional MRI, peripheral blood measures, and cognitive measures what may provide a more holistic picture of the neurobiological phenomena underlying PTSD.

4.4 Limitations

There are several limitations to the present study. Firstly, the PTSD sample is relatively small (n=16). As mentioned above, this may have resulted in the study not being sufficiently powered to detect a true decrease in [C-11]CURB binding in PTSD, for which we see a trend. Despite our efforts to reduce the heterogeneity in the PTSD group, there was still significant variability among factors like medication use, endorsement of dissociative symptoms, comorbid diagnoses of anxiety disorders or major depressive disorder, time since traumatic event, and time since diagnoses. Some of this information was not available to be included in this thesis due to the COVID-19 pandemic, but future analyses including some of these data may provide additional insight on how these factors may contribute to PTSD symptomatology, for example. Other information, like

72 medication use, was not possible to include in the analyses since all PTSD participants except for one was taking medication, and since each participant on prescription medications was typically on a combination of different drugs. It is not presently well- known whether some of these drugs interact with the endocannabinoid system. For example, there is some evidence to suggest that the SSRI fluoxetine may interact with the endocannabinoid system (though no participants in this study were on this drug),276,277 though the drug citalopram of the same class does not seem to affect the endocannabinoid system.277 However, the relationship between many of these drugs and the endocannabinoid system remains either poorly characterized or uncharacterized. Thus, it is a significant limitation that we were not able to control for any potential effects of medication use in the present study. As well, this study did not include a trauma- exposed control group in addition to healthy controls. This may have provided additional insights into the status of the endocannabinoid system in more resilient phenotypes, particularly since this question remains largely unaddressed by the literature, and potentially could have provided a more instructive group for comparison with the PTSD group. Moreover, while we made significant efforts to ensure our groups were well- matched, the PTSD sample was, on average, older and had a higher BMI than those in our HC group. In a similar vein, another limitation was that our PTSD sample was comprised of all Caucasian subjects. Though statistical analyses have demonstrated that race (in addition to BMI and age) did not have a significant effect on the current dataset, it is nonetheless important to recruit a sample that is appropriately representative, particularly since some (bot not all)278 studies have suggested that, at least in the United

States, the lifetime prevalence of PTSD is increased among black men and women.279,280 Thus, racial disparities in PTSD prevalence underscore the need for a representative sample in this group.

4.5 Clinical Significance

While our study did not find evidence that FAAH is upregulated in PTSD (and may, in fact, be downregulated in this population), the clinical significance of these findings must be interpreted in light of the limitations discussed above. More specifically, on the question of the therapeutic development of FAAH inhibitors, the present study does not

73 preclude the clinical utility of these drugs for PTSD. In light of the recent findings that FAAH inhibitors increased AEA levels, enhanced fear extinction, and attenuated stress responses in a group of healthy controls,51 further research in a larger cohort should be done confirm the status of FAAH in PTSD to better understand if and when these drugs may be clinically valuable in this population.

4.7 Future Directions

Based on the ways in which this study diverges from the majority of the preclinical literature, future studies in humans, in addition to having an increased sample size, should be designed to better capture the dynamic changes of the endocannabinoid system in response to trauma over time. This would provide a more informative picture of the ways in which this enzyme may change in response to traumatic stress, and may therefore inform a better understanding of the ways in which these changes relate to PTSD. For example, one way to do this would be with a longitudinal PET imaging study to measure FAAH levels in military personnel with three time points: before deployment, immediately after returning from deployment, and 6 months after returning. This type of design would have several advantages. First, the initial pre-deployment scan in concert with the exclusion of anyone who had a history of PTSD would allow for a “baseline” measurement of FAAH levels prior to exposure to combat stress. Secondly, a scan relatively shortly after exposure to combat stress means that we may be better positioned to capture more acute changes in FAAH levels that may play a role in determining later outcomes. And by the third time-point, it is likely that some of the group will have been diagnosed with PTSD, while others will not have been. Thus, this type of design would by necessity include a group of trauma-exposed controls without PTSD which may be instructive in understanding how different neurobiological responses at different time points contribute to either resilient outcomes after trauma or to the development of PTSD. It would also be worthwhile to consider using combat-related cues prior to the PET scan within a subsection of those with PTSD at the third time point, to investigate the possibility that temporal states of stress drive acute upregulations in FAAH. Notwithstanding the fact that a study design like this would be resource-intensive and would theoretically involve triple the radiation exposure than in the current design (and thus, greater risk to participants),

74 this would allow a broader understanding of changes in the endocannabinoid system over time. Moreover, it would also be informative to examine the status of 2-AG and its catabolic enzyme, MAGL, in humans with PTSD based on the inconsistent reports in the clinical literature and that 2-AG may also be involved in human responses to trauma.215,218-220 In addition to these studies in humans, longitudinal studies should be done in animal models to confirm whether the general finding of upregulated levels of FAAH after chronic stress exposure persist past the end of this exposure.

4.6 Conclusions

Though we did not find evidence that FAAH is increased in PTSD and instead found that decreased FAAH in the amygdala, the findings from this study do not preclude the possibility that dysregulations in FAAH are involved at some point in the disorder. In this way, if preclinical findings were found to be true in humans such that FAAH is upregulated more acutely after traumatic stress, then this enzyme may contribute more to the development of the disorder rather than its maintenance, and FAAH inhibitors thus may be of greater clinical utility in the more acute stages following trauma exposure. It may also be that this enzyme is acutely upregulated during states of stress. While this study was not positioned to address either of these possibilities, we are hopeful that the findings reported here will stimulate more research into these questions in humans, and that answers to these questions will inform the development of novel therapeutic strategies

75 References 1 Birmes, P., Hatton, L., Brunet, A. & Schmitt, L. Early historical literature for post- traumatic symptomatology. Stress and Health 19, 17-26, doi:10.1002/smi.952 (2003).

2 Crocq, M. A. & Crocq, L. From shell shock and war neurosis to posttraumatic stress disorder: a history of psychotraumatology. Dialogues in clinical neuroscience 2, 47-55 (2000).

3 Grob, G. N. Origins of DSM-I: a study in appearance and reality. Am J Psychiatry 148, 421-431, doi:10.1176/ajp.148.4.421 (1991).

4 Association, A. P. Diagnostic and statistical manual of mental disorders (DSM- 5®). (American Psychiatric Pub, 2013).

5 Pai, A., Suris, A. M. & North, C. S. Posttraumatic Stress Disorder in the DSM-5: Controversy, Change, and Conceptual Considerations. Behav Sci (Basel) 7, 7, doi:10.3390/bs7010007 (2017).

6 Resnick, H. S., Kilpatrick, D. G., Dansky, B. S., Saunders, B. E. & Best, C. L. Prevalence of civilian trauma and posttraumatic stress disorder in a representative national sample of women. J Consult Clin Psychol 61, 984-991, doi:10.1037//0022-006x.61.6.984 (1993).

7 Kessler, R. C., Sonnega, A., Bromet, E., Hughes, M. & Nelson, C. B. Posttraumatic stress disorder in the National Comorbidity Survey. Arch Gen Psychiatry 52, 1048-1060, doi:10.1001/archpsyc.1995.03950240066012 (1995).

8 Breslau, N., Davis, G. C., Andreski, P. & Peterson, E. Traumatic events and posttraumatic stress disorder in an urban population of young adults. Arch Gen Psychiatry 48, 216-222, doi:10.1001/archpsyc.1991.01810270028003 (1991).

9 Van Ameringen, M., Mancini, C., Patterson, B. & Boyle, M. H. Post-traumatic stress disorder in Canada. CNS Neurosci Ther 14, 171-181, doi:10.1111/j.1755- 5949.2008.00049.x (2008).

10 Frise, S., Steingart, A., Sloan, M., Cotterchio, M. & Kreiger, N. Psychiatric Disorders and Use of Mental Health Services by Ontario Women. The Canadian Journal of Psychiatry 47, 849-856, doi:10.1177/070674370204700906 (2002).

11 Wilson, S., Guliani, H. & Boichev, G. On the economics of post-traumatic stress disorder among first responders in Canada. Journal of Community Safety and Well-Being 1, 26-31 (2016).

12 Kessler, R. C., Chiu, W. T., Demler, O., Merikangas, K. R. & Walters, E. E. Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the

76 National Comorbidity Survey Replication. Arch Gen Psychiatry 62, 617-627, doi:10.1001/archpsyc.62.6.617 (2005).

13 Canada, S. Canadian Community Health Survey, 2012: Mental health component [public-use microdata file]. (Statistics Canada, Ottawa, Ontario, 2013).

14 Carleton, R. N. et al. Mental Disorder Symptoms among Public Safety Personnel in Canada. The Canadian Journal of Psychiatry 63, 54-64, doi:10.1177/0706743717723825 (2018).

15 Asmundson, G. J. & Stapleton, J. A. Associations between dimensions of anxiety sensitivity and PTSD symptom clusters in active-duty police officers. Cogn Behav Ther 37, 66-75, doi:10.1080/16506070801969005 (2008).

16 Regehr, C., Goldberg, G., Glancy, G. D. & Knott, T. Posttraumatic symptoms and disability in paramedics. Can J Psychiatry 47, 953-958, doi:10.1177/070674370204701007 (2002).

17 Boulos, D. & Zamorski, M. A. Deployment-related mental disorders among Canadian Forces personnel deployed in support of the mission in Afghanistan, 2001-2008. Cmaj 185, E545-552, doi:10.1503/cmaj.122120 (2013).

18 Saladin, M. E., Brady, K. T., Dansky, B. S. & Kilpatrick, D. G. Understanding comorbidity between PTSD and substance use disorders: two preliminary investigations. Addict Behav 20, 643-655, doi:10.1016/0306-4603(95)00024-7 (1995).

19 Brady, K. T., Killeen, T. K., Brewerton, T. & Lucerini, S. Comorbidity of psychiatric disorders and posttraumatic stress disorder. The Journal of Clinical Psychiatry 61, 22-32 (2000).

20 Calabrese, J. R. et al. PTSD comorbidity and suicidal ideation associated with PTSD within the Ohio Army National Guard. The Journal of Clinical Psychiatry 72, 1072-1078, doi:10.4088/JCP.11m06956 (2011).

21 Boscarino, J. A. A prospective study of PTSD and early-age heart disease mortality among Vietnam veterans: implications for surveillance and prevention. Psychosom Med 70, 668-676, doi:10.1097/PSY.0b013e31817bccaf (2008).

22 Qureshi, S. U., Pyne, J. M., Magruder, K. M., Schulz, P. E. & Kunik, M. E. The Link Between Post-traumatic Stress Disorder and Physical Comorbidities: A Systematic Review. Psychiatric Quarterly 80, 87-97, doi:10.1007/s11126-009- 9096-4 (2009).

23 Kessler, R. C. Posttraumatic stress disorder: the burden to the individual and to society. J Clin Psychiatry 61 Suppl 5, 4-12; discussion 13-14 (2000).

77 24 Richardson, J. D., Elhai, J. D. & Pedlar, D. J. Association of PTSD and depression with medical and specialist care utilization in modern peacekeeping veterans in Canada with health-related disabilities. J Clin Psychiatry 67, 1240- 1245, doi:10.4088/jcp.v67n0810 (2006).

25 National Collaborating Centre for Mental, H. in Post-Traumatic Stress Disorder: The Management of PTSD in Adults and Children in Primary and Secondary Care (Gaskell The Royal College of Psychiatrists & The British Psychological Society., 2005).

26 Bradley, R., Greene, J., Russ, E., Dutra, L. & Westen, D. A multidimensional meta-analysis of psychotherapy for PTSD. Am J Psychiatry 162, 214-227, doi:10.1176/appi.ajp.162.2.214 (2005).

27 Bisson, J. I. et al. Psychological treatments for chronic post-traumatic stress disorder. Systematic review and meta-analysis. Br J Psychiatry 190, 97-104, doi:10.1192/bjp.bp.106.021402 (2007).

28 Cukor, J., Olden, M., Lee, F. & Difede, J. Evidence-based treatments for PTSD, new directions, and special challenges. Ann N Y Acad Sci 1208, 82-89, doi:10.1111/j.1749-6632.2010.05793.x (2010).

29 Foa, E. B., Keane, T. M., Friedman, M. J. & Cohen, J. A. Effective treatments for PTSD: practice guidelines from the International Society for Traumatic Stress Studies. (2008).

30 Stein, D. J., Ipser, J. C. & Seedat, S. Pharmacotherapy for post traumatic stress disorder (PTSD). Cochrane Database Syst Rev 2006, CD002795-CD002795, doi:10.1002/14651858.CD002795.pub2 (2006).

31 Berger, W. et al. Pharmacologic alternatives to antidepressants in posttraumatic stress disorder: a systematic review. Prog Neuropsychopharmacol Biol Psychiatry 33, 169-180, doi:10.1016/j.pnpbp.2008.12.004 (2009).

32 Davidson, J. et al. Efficacy of sertraline in preventing relapse of posttraumatic stress disorder: results of a 28-week double-blind, placebo-controlled study. Am J Psychiatry 158, 1974-1981, doi:10.1176/appi.ajp.158.12.1974 (2001).

33 Raskind, M. A. et al. Trial of Prazosin for Post-Traumatic Stress Disorder in Military Veterans. New England Journal of Medicine 378, 507-517, doi:10.1056/NEJMoa1507598 (2018).

34 Murray A. Raskind, M.D. , et al. A Trial of Prazosin for Combat Trauma PTSD With Nightmares in Active-Duty Soldiers Returned From Iraq and Afghanistan. American Journal of Psychiatry 170, 1003-1010, doi:10.1176/appi.ajp.2013.12081133 (2013).

78 35 Boehnlein, J. K. & Kinzie, J. D. Pharmacologic Reduction of CNS Noradrenergic Activity in PTSD: The Case for Clonidine and Prazosin. Journal of Psychiatric Practice® 13 (2007).

36 Raskind, M. A. et al. Reduction of Nightmares and Other PTSD Symptoms in Combat Veterans by Prazosin: A Placebo-Controlled Study. American Journal of Psychiatry 160, 371-373, doi:10.1176/appi.ajp.160.2.371 (2003).

37 Raskind, M. A. et al. A parallel group placebo controlled study of prazosin for trauma nightmares and sleep disturbance in combat veterans with post-traumatic stress disorder. Biol Psychiatry 61, 928-934, doi:10.1016/j.biopsych.2006.06.032 (2007).

38 Bonn‐Miller, M. O., Vujanovic, A. A., Feldner, M. T., Bernstein, A. & Zvolensky, M. J. Posttraumatic stress symptom severity predicts marijuana use coping motives among traumatic event‐exposed marijuana users. Journal of Traumatic Stress: Official Publication of The International Society for Traumatic Stress Studies 20, 577-586 (2007).

39 Betthauser, K., Pilz, J. & Vollmer, L. E. Use and effects of cannabinoids in military veterans with posttraumatic stress disorder. American Journal of Health- System Pharmacy 72, 1279-1284 (2015).

40 Cannabis for Medical Purposes – Revised Reimbursement Policy, (2019).

41 Fraser, G. A. The use of a synthetic cannabinoid in the management of treatment‐resistant nightmares in posttraumatic stress disorder (PTSD). CNS neuroscience & therapeutics 15, 84-88 (2009).

42 Roitman, P., Mechoulam, R., Cooper-Kazaz, R. & Shalev, A. Preliminary, open- label, pilot study of add-on oral Δ 9-tetrahydrocannabinol in chronic post- traumatic stress disorder. Clinical drug investigation 34, 587-591 (2014).

43 Jetly, R., Heber, A., Fraser, G. & Boisvert, D. The efficacy of nabilone, a synthetic cannabinoid, in the treatment of PTSD-associated nightmares: a preliminary randomized, double-blind, placebo-controlled cross-over design study. Psychoneuroendocrinology 51, 585-588 (2015).

44 Hill, M. N. & Gorzalka, B. B. Enhancement of anxiety-like responsiveness to the cannabinoid CB(1) receptor agonist HU-210 following chronic stress. European journal of pharmacology 499, 291-295, doi:10.1016/j.ejphar.2004.06.069 (2004).

45 Hill, M. N. & Gorzalka, B. B. Increased sensitivity to restraint stress and novelty- induced emotionality following long-term, high dose cannabinoid exposure. Psychoneuroendocrinology 31, 526-536, doi:10.1016/j.psyneuen.2005.11.010 (2006).

79 46 Patel, S. & Hillard, C. J. Pharmacological Evaluation of Cannabinoid Receptor Ligands in a Mouse Model of Anxiety: Further Evidence for an Anxiolytic Role for Endogenous Cannabinoid Signaling. Journal of Pharmacology and Experimental Therapeutics 318, 304, doi:10.1124/jpet.106.101287 (2006).

47 Hirvonen, J. et al. Reversible and regionally selective downregulation of brain cannabinoid CB1 receptors in chronic daily cannabis smokers. Mol Psychiatry 17, 642-649, doi:10.1038/mp.2011.82 (2012).

48 van Esbroeck, A. C. M. et al. Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474. Science 356, 1084-1087, doi:10.1126/science.aaf7497 (2017).

49 Tong, J. et al. Inhibition of fatty acid amide hydrolase by BIA 10-2474 in rat brain. J Cereb Blood Flow Metab 37, 3635-3639, doi:10.1177/0271678x16668890 (2017).

50 FDA finds drugs under investigation in the U.S. related to French BIA 10-2474 drug do not pose similar safety risks, (2016).

51 Mayo, L. M. et al. Elevated Anandamide, Enhanced Recall of Fear Extinction, and Attenuated Stress Responses Following Inhibition of Fatty Acid Amide Hydrolase: A Randomized, Controlled Experimental Medicine Trial. Biol Psychiatry 87, 538-547, doi:10.1016/j.biopsych.2019.07.034 (2020).

52 Dunlop, B. W. & Wong, A. The hypothalamic-pituitary-adrenal axis in PTSD: Pathophysiology and treatment interventions. Prog Neuropsychopharmacol Biol Psychiatry 89, 361-379, doi:10.1016/j.pnpbp.2018.10.010 (2019).

53 Daskalakis, N. P., Bagot, R. C., Parker, K. J., Vinkers, C. H. & de Kloet, E. R. The three-hit concept of vulnerability and resilience: toward understanding adaptation to early-life adversity outcome. Psychoneuroendocrinology 38, 1858- 1873, doi:10.1016/j.psyneuen.2013.06.008 (2013).

54 Southwick, S. M. et al. Neurotransmitter alterations in PTSD: catecholamines and serotonin. Semin Clin Neuropsychiatry 4, 242-248, doi:10.153/scnp00400242 (1999).

55 Dantzer, R., O'Connor, J. C., Freund, G. G., Johnson, R. W. & Kelley, K. W. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci 9, 46-56, doi:10.1038/nrn2297 (2008).

56 Speer, K., Upton, D., Semple, S. & McKune, A. Systemic low-grade inflammation in post-traumatic stress disorder: a systematic review. J Inflamm Res 11, 111- 121, doi:10.2147/jir.S155903 (2018).

80 57 Bremner, J. D. Traumatic stress: effects on the brain. Dialogues Clin Neurosci 8, 445-461 (2006).

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

59 Duvarci, S. & Pare, D. Amygdala microcircuits controlling learned fear. Neuron 82, 966-980 (2014).

60 Shin, L. M. et al. Regional cerebral blood flow in the amygdala and medial prefrontal cortex during traumatic imagery in male and female Vietnam veterans with PTSD. Arch Gen Psychiatry 61, 168-176, doi:10.1001/archpsyc.61.2.168 (2004).

61 Phan, K. L., Britton, J. C., Taylor, S. F., Fig, L. M. & Liberzon, I. Corticolimbic Blood Flow During Nontraumatic Emotional Processing in Posttraumatic Stress Disorder. Archives of General Psychiatry 63, 184-192, doi:10.1001/archpsyc.63.2.184 (2006).

62 Gunduz-Cinar, O. et al. Convergent translational evidence of a role for anandamide in amygdala-mediated fear extinction, threat processing and stress- reactivity. Mol Psychiatry 18, 813-823, doi:10.1038/mp.2012.72 (2013).

63 Milad, M. R., Rauch, S. L., Pitman, R. K. & Quirk, G. J. Fear extinction in rats: implications for human brain imaging and anxiety disorders. Biol Psychol 73, 61- 71, doi:10.1016/j.biopsycho.2006.01.008 (2006).

64 Rauch, S. L. et al. A symptom provocation study of posttraumatic stress disorder using positron emission tomography and script-driven imagery. Arch Gen Psychiatry 53, 380-387, doi:10.1001/archpsyc.1996.01830050014003 (1996).

65 Driessen, M. et al. Posttraumatic stress disorder and fMRI activation patterns of traumatic memory in patients with borderline personality disorder. Biological psychiatry 55, 603-611, doi:10.1016/j.biopsych.2003.08.018 (2004).

66 Liberzon, I. et al. Brain activation in PTSD in response to trauma-related stimuli. Biol Psychiatry 45, 817-826, doi:10.1016/s0006-3223(98)00246-7 (1999).

67 Pissiota, A. et al. Neurofunctional correlates of posttraumatic stress disorder: a PET symptom provocation study. Eur Arch Psychiatry Clin Neurosci 252, 68-75, doi:10.1007/s004060200014 (2002).

68 Hendler, T. et al. Sensing the invisible: differential sensitivity of visual cortex and amygdala to traumatic context. Neuroimage 19, 587-600, doi:10.1016/s1053- 8119(03)00141-1 (2003).

81 69 Shin, L. M. et al. A positron emission tomographic study of symptom provocation in PTSD. Ann N Y Acad Sci 821, 521-523, doi:10.1111/j.1749- 6632.1997.tb48320.x (1997).

70 Protopopescu, X. et al. Differential time courses and specificity of amygdala activity in posttraumatic stress disorder subjects and normal control subjects. Biol Psychiatry 57, 464-473, doi:10.1016/j.biopsych.2004.12.026 (2005).

71 Rauch, S. L. et al. Exaggerated amygdala response to masked facial stimuli in posttraumatic stress disorder: a functional MRI study. Biol Psychiatry 47, 769- 776, doi:10.1016/s0006-3223(00)00828-3 (2000).

72 Shin, L. M. et al. A functional magnetic resonance imaging study of amygdala and medial prefrontal cortex responses to overtly presented fearful faces in posttraumatic stress disorder. Arch Gen Psychiatry 62, 273-281, doi:10.1001/archpsyc.62.3.273 (2005).

73 Williams, L. M. et al. Trauma modulates amygdala and medial prefrontal responses to consciously attended fear. Neuroimage 29, 347-357, doi:10.1016/j.neuroimage.2005.03.047 (2006).

74 Gilmartin, M. R., Balderston, N. L. & Helmstetter, F. J. Prefrontal cortical regulation of fear learning. Trends in neurosciences 37, 455-464 (2014).

75 Likhtik, E. & Paz, R. Amygdala–prefrontal interactions in (mal) adaptive learning. Trends in neurosciences 38, 158-166 (2015).

76 Lanius, R. A. et al. Emotion modulation in PTSD: Clinical and neurobiological evidence for a dissociative subtype. Am J Psychiatry 167, 640-647, doi:10.1176/appi.ajp.2009.09081168 (2010).

77 Lanius, R. A., Frewen, P. A., Tursich, M., Jetly, R. & McKinnon, M. C. Restoring large-scale brain networks in PTSD and related disorders: a proposal for neuroscientifically-informed treatment interventions. Eur J Psychotraumatol 6, 27313-27313, doi:10.3402/ejpt.v6.27313 (2015).

78 Tursich, M. et al. Distinct intrinsic network connectivity patterns of post-traumatic stress disorder symptom clusters. Acta Psychiatrica Scandinavica 132, 29-38, doi:10.1111/acps.12387 (2015).

79 Mišić, B. et al. Post-Traumatic Stress Constrains the Dynamic Repertoire of Neural Activity. The Journal of Neuroscience 36, 419-431, doi:10.1523/jneurosci.1506-15.2016 (2016).

80 Dunkley, B. T. et al. Threatening faces induce fear circuitry hypersynchrony in soldiers with post-traumatic stress disorder. Heliyon 2, e00063, doi:https://doi.org/10.1016/j.heliyon.2015.e00063 (2016).

82 81 Dunkley, B. T., Wong, S. M., Jetly, R., Wong, J. K. & Taylor, M. J. Post-traumatic stress disorder and chronic hyperconnectivity in emotional processing. NeuroImage: Clinical 20, 197-204, doi:https://doi.org/10.1016/j.nicl.2018.07.007 (2018).

82 Domenici, M. R. et al. Cannabinoid Receptor Type 1 Located on Presynaptic Terminals of Principal Neurons in the Forebrain Controls Glutamatergic Synaptic Transmission. The Journal of Neuroscience 26, 5794, doi:10.1523/JNEUROSCI.0372-06.2006 (2006).

83 Azad, S. C. et al. Activation of the cannabinoid receptor type 1 decreases glutamatergic and GABAergic synaptic transmission in the lateral amygdala of the mouse. Learn Mem 10, 116-128, doi:10.1101/lm.53303 (2003).

84 Katona, I. et al. Distribution of CB1 cannabinoid receptors in the amygdala and their role in the control of GABAergic transmission. The Journal of neuroscience : the official journal of the Society for Neuroscience 21, 9506-9518, doi:10.1523/JNEUROSCI.21-23-09506.2001 (2001).

85 Rossi, S. et al. Preservation of striatal cannabinoid CB1 receptor function correlates with the antianxiety effects of fatty acid amide hydrolase inhibition. Molecular pharmacology 78, 260-268 (2010).

86 Pistis, M. et al. Cannabinoids modulate neuronal firing in the rat basolateral amygdala: evidence for CB1- and non-CB1-mediated actions. Neuropharmacology 46, 115-125, doi:10.1016/j.neuropharm.2003.08.003 (2004).

87 Hill, M. N. et al. Disruption of fatty acid amide hydrolase activity prevents the effects of chronic stress on anxiety and amygdalar microstructure. Mol Psychiatry 18, 1125-1135, doi:10.1038/mp.2012.90 (2013).

88 Mitra, R., Jadhav, S., McEwen, B. S., Vyas, A. & Chattarji, S. Stress duration modulates the spatiotemporal patterns of spine formation in the basolateral amygdala. Proceedings of the National Academy of Sciences of the United States of America 102, 9371-9376, doi:10.1073/pnas.0504011102 (2005).

89 Vyas, A., Mitra, R., Shankaranarayana Rao, B. S. & Chattarji, S. Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 22, 6810-6818, doi:10.1523/JNEUROSCI.22-15- 06810.2002 (2002).

90 Padival, M., Quinette, D. & Rosenkranz, J. A. Effects of repeated stress on excitatory drive of basal amygdala neurons in vivo. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 38, 1748-1762, doi:10.1038/npp.2013.74 (2013).

83 91 Hopper, J. W., Frewen, P. A., van der Kolk, B. A. & Lanius, R. A. Neural correlates of reexperiencing, avoidance, and dissociation in PTSD: symptom dimensions and emotion dysregulation in responses to script-driven trauma imagery. J Trauma Stress 20, 713-725, doi:10.1002/jts.20284 (2007).

92 Felmingham, K. et al. Dissociative responses to conscious and non-conscious fear impact underlying brain function in post-traumatic stress disorder. Psychol Med 38, 1771-1780, doi:10.1017/s0033291708002742 (2008).

93 Di Marzo, V., Bifulco, M. & De Petrocellis, L. The endocannabinoid system and its therapeutic exploitation. Nat Rev Drug Discov 3, 771-784, doi:10.1038/nrd1495 (2004).

94 Matsuda, L. A., Lolait, S. J., Brownstein, M. J., Young, A. C. & Bonner, T. I. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561-564, doi:10.1038/346561a0 (1990).

95 Munro, S., Thomas, K. L. & Abu-Shaar, M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 365, 61-65, doi:10.1038/365061a0 (1993).

96 Di Marzo, V. & Fontana, A. Anandamide, an endogenous cannabinomimetic eicosanoid: 'killing two birds with one stone'. Prostaglandins Leukot Essent Fatty Acids 53, 1-11, doi:10.1016/0952-3278(95)90077-2 (1995).

97 Devane, W. A. et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 1946-1949, doi:10.1126/science.1470919 (1992).

98 Mechoulam, R. et al. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 50, 83-90, doi:10.1016/0006-2952(95)00109-d (1995).

99 Sugiura, T. et al. 2-Arachidonoylgylcerol: a possible endogenous cannabinoid receptor ligand in brain. Biochemical and biophysical research communications 215, 89-97 (1995).

100 Porter, A. C. et al. Characterization of a novel endocannabinoid, virodhamine, with antagonist activity at the CB1 receptor. Journal of Pharmacology and Experimental Therapeutics 301, 1020-1024 (2002).

101 Hanuš, L. et al. 2-arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor. Proceedings of the National Academy of Sciences 98, 3662-3665 (2001).

102 Devane, W. A., Dysarz, F. r., Johnson, M. R., Melvin, L. S. & Howlett, A. C. Determination and characterization of a cannabinoid receptor in rat brain. Molecular pharmacology 34, 605-613 (1988).

84 103 Matsuda, L. A., Lolait, S. J., Brownstein, M. J., Young, A. C. & Bonner, T. I. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561-564 (1990).

104 Lau, T. & Schloss, P. The cannabinoid CB1 receptor is expressed on serotonergic and dopaminergic neurons. Eur J Pharmacol 578, 137-141, doi:10.1016/j.ejphar.2007.09.022 (2008).

105 Scavone, J. L., Mackie, K. & Van Bockstaele, E. J. Characterization of cannabinoid-1 receptors in the locus coeruleus: relationship with mu-opioid receptors. Brain Res 1312, 18-31, doi:10.1016/j.brainres.2009.11.023 (2010).

106 Reguero, L. et al. GABAergic and cortical and subcortical glutamatergic axon terminals contain CB1 cannabinoid receptors in the ventromedial nucleus of the hypothalamus. PLoS One 6, e26167-e26167, doi:10.1371/journal.pone.0026167 (2011).

107 Pertwee, R. G. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacology & therapeutics 74, 129-180 (1997).

108 Pertwee, R. G. et al. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB1 and CB2. Pharmacological reviews 62, 588-631 (2010).

109 Mechoulam, R., Hanuš, L. O., Pertwee, R. & Howlett, A. C. Early phytocannabinoid chemistry to endocannabinoids and beyond. Nature Reviews Neuroscience 15, 757-764, doi:10.1038/nrn3811 (2014).

110 Stella, N., Schweitzer, P. & Piomelli, D. A second endogenous cannabinoid that modulates long-term potentiation. Nature 388, 773-778 (1997).

111 Ben-Shabat, S. et al. An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity. European journal of pharmacology 353, 23-31 (1998).

112 Chávez, A. E., Chiu, C. Q. & Castillo, P. E. TRPV1 activation by endogenous anandamide triggers postsynaptic long-term depression in dentate gyrus. Nature neuroscience 13, 1511 (2010).

113 O'Sullivan, S. E. Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors. Br J Pharmacol 152, 576-582, doi:10.1038/sj.bjp.0707423 (2007).

114 Lauckner, J. E. et al. GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current. Proceedings of the National Academy of Sciences of the United States of America 105, 2699-2704, doi:10.1073/pnas.0711278105 (2008).

85 115 Overton, H. A. et al. Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents. Cell metabolism 3, 167-175 (2006).

116 Di Marzo, V. et al. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686-691, doi:10.1038/372686a0 (1994).

117 Di, S. et al. Activity-dependent release and actions of endocannabinoids in the rat hypothalamic supraoptic nucleus. J Physiol 569, 751-760, doi:10.1113/jphysiol.2005.097477 (2005).

118 Jin, X. H. et al. Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners. J Biol Chem 282, 3614-3623, doi:10.1074/jbc.M606369200 (2007).

119 McAllister, S. D. & Glass, M. CB1 and CB2 receptor-mediated signalling: a focus on endocannabinoids. Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA) 66, 161-171 (2002).

120 Wilson, R. I. & Nicoll, R. A. Endocannabinoid signaling in the brain. Science 296, 678-682 (2002).

121 Freund, T. F., Katona, I. & Piomelli, D. Role of endogenous cannabinoids in synaptic signaling. Physiological reviews 83, 1017-1066 (2003).

122 Chevaleyre, V. & Castillo, P. E. Heterosynaptic LTD of hippocampal GABAergic synapses: a novel role of endocannabinoids in regulating excitability. Neuron 38, 461-472 (2003).

123 BISOGNO, T. et al. Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells. Biochemical Journal 322, 671-677 (1997).

124 Bisogno, T. et al. Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. The Journal of cell biology 163, 463-468 (2003).

125 Dinh, T. et al. Brain monoglyceride lipase participating in endocannabinoid inactivation. Proceedings of the national Academy of sciences 99, 10819-10824 (2002).

126 Schmid, P., Reddy, P., Natarajan, V. & Schmid, H. Metabolism of N- acylethanolamine phospholipids by a mammalian phosphodiesterase of the phospholipase D type. Journal of Biological Chemistry 258, 9302-9306 (1983).

86 127 Okamoto, Y., Morishita, J., Tsuboi, K., Tonai, T. & Ueda, N. Molecular characterization of a phospholipase D generating anandamide and its congeners. Journal of Biological Chemistry 279, 5298-5305 (2004).

128 Di Marzo, V. et al. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686-691 (1994).

129 Ligresti, A. et al. Further evidence for the existence of a specific process for the membrane transport of anandamide. Biochemical Journal 380, 265-272 (2004).

130 Hillard, C. J., Edgemond, W. S., Jarrahian, A. & Campbell, W. B. Accumulation of N‐arachidonoylethanolamine (anandamide) into cerebellar granule cells occurs via facilitated diffusion. Journal of neurochemistry 69, 631-638 (1997).

131 Cravatt, B. F. et al. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 384, 83-87, doi:10.1038/384083a0 (1996).

132 Maccarrone, M., Pauselli, R., Di Rienzo, M. & Finazzi-Agro, A. Binding, degradation and apoptotic activity of stearoylethanolamide in rat C6 glioma cells. Biochem J 366, 137-144, doi:10.1042/bj20020438 (2002).

133 Boger, D. L. et al. Fatty acid amide hydrolase substrate specificity. Bioorg Med Chem Lett 10, 2613-2616, doi:10.1016/s0960-894x(00)00528-x (2000).

134 Chebrou, H., Bigey, F., Arnaud, A. & Galzy, P. Study of the amidase signature group. Biochim Biophys Acta 1298, 285-293, doi:10.1016/s0167-4838(96)00145- 8 (1996).

135 Giang, D. K. & Cravatt, B. F. Molecular characterization of human and mouse fatty acid amide hydrolases. Proc Natl Acad Sci U S A 94, 2238-2242, doi:10.1073/pnas.94.6.2238 (1997).

136 Patricelli, M. P., Lashuel, H. A., Giang, D. K., Kelly, J. W. & Cravatt, B. F. Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: identification of the transmembrane domain as a site for oligomerization. Biochemistry 37, 15177-15187, doi:10.1021/bi981733n (1998).

137 Bracey, M. H., Hanson, M. A., Masuda, K. R., Stevens, R. C. & Cravatt, B. F. Structural adaptations in a membrane enzyme that terminates endocannabinoid signaling. Science 298, 1793-1796, doi:10.1126/science.1076535 (2002).

138 McKinney, M. K. & Cravatt, B. F. Structure and function of fatty acid amide hydrolase. Annu Rev Biochem 74, 411-432, doi:10.1146/annurev.biochem.74.082803.133450 (2005).

87 139 Palermo, G. et al. Wagging the Tail: Essential Role of Substrate Flexibility in FAAH Catalysis. Journal of Chemical Theory and Computation 9, 1202-1213, doi:10.1021/ct300611q (2013).

140 Patricelli, M. P. & Cravatt, B. F. Fatty acid amide hydrolase competitively degrades bioactive amides and esters through a nonconventional catalytic mechanism. Biochemistry 38, 14125-14130 (1999).

141 McKinney, M. K. & Cravatt, B. F. Evidence for distinct roles in catalysis for residues of the serine-serine-lysine catalytic triad of fatty acid amide hydrolase. J Biol Chem 278, 37393-37399, doi:10.1074/jbc.M303922200 (2003).

142 Rouzer, C. A. & Marnett, L. J. Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: cross-talk between the eicosanoid and endocannabinoid signaling pathways. Chem Rev 111, 5899- 5921, doi:10.1021/cr2002799 (2011).

143 Van Dross, R. T. Metabolism of anandamide by COX-2 is necessary for endocannabinoid-induced cell death in tumorigenic keratinocytes. Mol Carcinog 48, 724-732, doi:10.1002/mc.20515 (2009).

144 Cravatt, B. F. et al. Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proceedings of the National Academy of Sciences 98, 9371-9376 (2001).

145 Sipe, J. C., Chiang, K., Gerber, A. L., Beutler, E. & Cravatt, B. F. A missense mutation in human fatty acid amide hydrolase associated with problem drug use. Proceedings of the National Academy of Sciences 99, 8394, doi:10.1073/pnas.082235799 (2002).

146 Chiang, K. P., Gerber, A. L., Sipe, J. C. & Cravatt, B. F. Reduced cellular expression and activity of the P129T mutant of human fatty acid amide hydrolase: evidence for a link between defects in the endocannabinoid system and problem drug use. Hum Mol Genet 13, 2113-2119, doi:10.1093/hmg/ddh216 (2004).

147 Dincheva, I. et al. FAAH genetic variation enhances fronto-amygdala function in mouse and human. Nat Commun 6, 6395, doi:10.1038/ncomms7395 (2015).

148 Tyndale, R. F., Payne, J. I., Gerber, A. L. & Sipe, J. C. The fatty acid amide hydrolase C385A (P129T) missense variant in cannabis users: studies of drug use and dependence in Caucasians. Am J Med Genet B Neuropsychiatr Genet 144b, 660-666, doi:10.1002/ajmg.b.30491 (2007).

149 Hariri, A. R. et al. Divergent effects of genetic variation in endocannabinoid signaling on human threat- and reward-related brain function. Biol Psychiatry 66, 9-16, doi:10.1016/j.biopsych.2008.10.047 (2009).

88 150 Ursano, R. J. et al. Models of PTSD and traumatic stress: the importance of research "from bedside to bench to bedside". Prog Brain Res 167, 203-215, doi:10.1016/s0079-6123(07)67014-9 (2008).

151 Deslauriers, J., Toth, M., Der-Avakian, A. & Risbrough, V. B. Current Status of Animal Models of Posttraumatic Stress Disorder: Behavioral and Biological Phenotypes, and Future Challenges in Improving Translation. Biol Psychiatry 83, 895-907, doi:10.1016/j.biopsych.2017.11.019 (2018).

152 Hill, M. N. et al. Downregulation of Endocannabinoid Signaling in the Hippocampus Following Chronic Unpredictable Stress. Neuropsychopharmacology 30, 508-515, doi:10.1038/sj.npp.1300601 (2005).

153 Bowles, N. P. et al. Chronic, noninvasive glucocorticoid administration suppresses limbic endocannabinoid signaling in mice. Neuroscience 204, 83-89, doi:10.1016/j.neuroscience.2011.08.048 (2012).

154 Hill, M. N., Hunter, R. G. & McEwen, B. S. Chronic stress differentially regulates cannabinoid CB1 receptor binding in distinct hippocampal subfields. Eur J Pharmacol 614, 66-69, doi:10.1016/j.ejphar.2009.04.048 (2009).

155 Xing, G. et al. Differential Expression of Brain Cannabinoid Receptors between Repeatedly Stressed Males and Females may Play a Role in Age and Gender- Related Difference in Traumatic Brain Injury: Implications from Animal Studies. Frontiers in Neurology 5, doi:10.3389/fneur.2014.00161 (2014).

156 Lomazzo, E., Konig, F., Abassi, L., Jelinek, R. & Lutz, B. Chronic stress leads to epigenetic dysregulation in the neuropeptide-Y and cannabinoid CB1 receptor genes in the mouse cingulate cortex. Neuropharmacology 113, 301-313, doi:10.1016/j.neuropharm.2016.10.008 (2017).

157 Sabban, E. L., Serova, L. I., Newman, E., Aisenberg, N. & Akirav, I. Changes in Gene Expression in the Locus Coeruleus-Amygdala Circuitry in Inhibitory Avoidance PTSD Model. Cellular and Molecular Neurobiology 38, 273-280, doi:10.1007/s10571-017-0548-3 (2018).

158 Matchynski-Franks, J. J., Susick, L. L., Schneider, B. L., Perrine, S. A. & Conti, A. C. Impaired Ethanol-Induced Sensitization and Decreased Cannabinoid Receptor-1 in a Model of Posttraumatic Stress Disorder. PLoS One 11, e0155759-e0155759, doi:10.1371/journal.pone.0155759 (2016).

159 Campos, A. C., Ferreira, F. R., da Silva, W. A., Jr. & Guimaraes, F. S. Predator threat stress promotes long lasting anxiety-like behaviors and modulates synaptophysin and CB1 receptors expression in brain areas associated with PTSD symptoms. Neurosci Lett 533, 34-38, doi:10.1016/j.neulet.2012.11.016 (2013).

89 160 Korem, N. & Akirav, I. Cannabinoids Prevent the Effects of a Footshock Followed by Situational Reminders on Emotional Processing. Neuropsychopharmacology 39, 2709-2722, doi:10.1038/npp.2014.132 (2014).

161 Surkin, P. N. et al. Pharmacological augmentation of endocannabinoid signaling reduces the neuroendocrine response to stress. Psychoneuroendocrinology 87, 131-140, doi:10.1016/j.psyneuen.2017.10.015 (2018).

162 McLaughlin, R. J. et al. Upregulation of CB(1) receptor binding in the ventromedial prefrontal cortex promotes proactive stress-coping strategies following chronic stress exposure. Behav Brain Res 237, 333-337, doi:10.1016/j.bbr.2012.09.053 (2013).

163 Lisboa, S. F. et al. Cannabinoid CB1 receptors in the medial prefrontal cortex modulate the expression of contextual fear conditioning. International Journal of Neuropsychopharmacology 13, 1163-1173, doi:10.1017/s1461145710000684 (2010).

164 Beyer, C. E. et al. Depression-like phenotype following chronic CB1 receptor antagonism. Neurobiol Dis 39, 148-155, doi:10.1016/j.nbd.2010.03.020 (2010).

165 Kamprath, K. et al. Short-Term Adaptation of Conditioned Fear Responses Through Endocannabinoid Signaling in the Central Amygdala. Neuropsychopharmacology 36, 652-663, doi:10.1038/npp.2010.196 (2011).

166 Chhatwal, J. P., Davis, M., Maguschak, K. A. & Ressler, K. J. Enhancing Cannabinoid Neurotransmission Augments the Extinction of Conditioned Fear. Neuropsychopharmacology 30, 516-524, doi:10.1038/sj.npp.1300655 (2005).

167 Sink, K. S. et al. The CB1 inverse agonist AM251, but not the CB1 antagonist AM4113, enhances retention of contextual fear conditioning in rats. Pharmacol Biochem Behav 95, 479-484, doi:10.1016/j.pbb.2010.03.011 (2010).

168 McLaughlin, R. J., Hill, M. N., Morrish, A. C. & Gorzalka, B. B. Local enhancement of cannabinoid CB1 receptor signalling in the dorsal hippocampus elicits an antidepressant-like effect. Behav Pharmacol 18, 431-438, doi:10.1097/FBP.0b013e3282ee7b44 (2007).

169 Patel, S., Cravatt, B. F. & Hillard, C. J. Synergistic Interactions between Cannabinoids and Environmental Stress in the Activation of the Central Amygdala. Neuropsychopharmacology 30, 497-507, doi:10.1038/sj.npp.1300535 (2005).

170 Patel, S., Roelke, C. T., Rademacher, D. J. & Hillard, C. J. Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signalling. European Journal of Neuroscience 21, 1057-1069, doi:10.1111/j.1460- 9568.2005.03916.x (2005).

90 171 Patel, S., Roelke, C. T., Rademacher, D. J., Cullinan, W. E. & Hillard, C. J. Endocannabinoid Signaling Negatively Modulates Stress-Induced Activation of the Hypothalamic-Pituitary-Adrenal Axis. Endocrinology 145, 5431-5438, doi:10.1210/en.2004-0638 (2004).

172 Dono, L. M. & Currie, P. J. The cannabinoid receptor CB(1) inverse agonist AM251 potentiates the anxiogenic activity of urocortin I in the basolateral amygdala. Neuropharmacology 62, 192-199, doi:10.1016/j.neuropharm.2011.06.019 (2012).

173 Hill, M. N. et al. Recruitment of prefrontal cortical endocannabinoid signaling by glucocorticoids contributes to termination of the stress response. The Journal of neuroscience : the official journal of the Society for Neuroscience 31, 10506- 10515, doi:10.1523/JNEUROSCI.0496-11.2011 (2011).

174 Ganon-Elazar, E. & Akirav, I. Cannabinoid receptor activation in the basolateral amygdala blocks the effects of stress on the conditioning and extinction of inhibitory avoidance. The Journal of neuroscience : the official journal of the Society for Neuroscience 29, 11078-11088, doi:10.1523/JNEUROSCI.1223- 09.2009 (2009).

175 Hill, M. N. et al. Suppression of Amygdalar Endocannabinoid Signaling by Stress Contributes to Activation of the Hypothalamic–Pituitary–Adrenal Axis. Neuropsychopharmacology 34, 2733-2745, doi:10.1038/npp.2009.114 (2009).

176 Abush, H. & Akirav, I. Cannabinoids Ameliorate Impairments Induced by Chronic Stress to Synaptic Plasticity and Short-Term Memory. Neuropsychopharmacology 38, 1521-1534, doi:10.1038/npp.2013.51 (2013).

177 Burstein, O., Shoshan, N., Doron, R. & Akirav, I. Cannabinoids prevent depressive-like symptoms and alterations in BDNF expression in a rat model of PTSD. Prog Neuropsychopharmacol Biol Psychiatry 84, 129-139, doi:10.1016/j.pnpbp.2018.01.026 (2018).

178 Segev, A., Rubin, A. S., Abush, H., Richter-Levin, G. & Akirav, I. Cannabinoid Receptor Activation Prevents the Effects of Chronic Mild Stress on Emotional Learning and LTP in a Rat Model of Depression. Neuropsychopharmacology 39, 919-933, doi:10.1038/npp.2013.292 (2014).

179 Fokos, S. & Panagis, G. Effects of Δ9-tetrahydrocannabinol on reward and anxiety in rats exposed to chronic unpredictable stress. Journal of Psychopharmacology 24, 767-777, doi:10.1177/0269881109104904 (2010).

180 Rock, E. M. et al. Effect of prior foot shock stress and Δ9-tetrahydrocannabinol, cannabidiolic acid, and cannabidiol on anxiety-like responding in the light-dark emergence test in rats. Psychopharmacology 234, 2207-2217, doi:10.1007/s00213-017-4626-5 (2017).

91 181 Uriguen, L., Perez-Rial, S., Ledent, C., Palomo, T. & Manzanares, J. Impaired action of anxiolytic drugs in mice deficient in cannabinoid CB1 receptors. Neuropharmacology 46, 966-973, doi:10.1016/j.neuropharm.2004.01.003 (2004).

182 Haller, J., Varga, B., Ledent, C., Barna, I. & Freund, T. F. Context-dependent effects of CB1 cannabinoid gene disruption on anxiety-like and social behaviour in mice. European Journal of Neuroscience 19, 1906-1912, doi:10.1111/j.1460- 9568.2004.03293.x (2004).

183 Haller, J., Bakos, N., Szirmay, M., Ledent, C. & Freund, T. F. The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety. European Journal of Neuroscience 16, 1395-1398, doi:10.1046/j.1460- 9568.2002.02192.x (2002).

184 Hill, M. N., Hillard, C. J. & McEwen, B. S. Alterations in Corticolimbic Dendritic Morphology and Emotional Behavior in Cannabinoid CB1 Receptor–Deficient Mice Parallel the Effects of Chronic Stress. Cerebral Cortex 21, 2056-2064, doi:10.1093/cercor/bhq280 (2011).

185 Hill, M. N. et al. Endogenous cannabinoid signaling is essential for stress adaptation. Proceedings of the National Academy of Sciences of the United States of America 107, 9406-9411, doi:10.1073/pnas.0914661107 (2010).

186 Bluett, R. J. et al. Central anandamide deficiency predicts stress-induced anxiety: behavioral reversal through endocannabinoid augmentation. Transl Psychiatry 4, e408-e408, doi:10.1038/tp.2014.53 (2014).

187 Gray, J. M. et al. Corticotropin-releasing hormone drives anandamide hydrolysis in the amygdala to promote anxiety. J Neurosci 35, 3879-3892, doi:10.1523/jneurosci.2737-14.2015 (2015).

188 Rademacher, D. J. et al. Effects of acute and repeated restraint stress on endocannabinoid content in the amygdala, ventral striatum, and medial prefrontal cortex in mice. Neuropharmacology 54, 108-116, doi:10.1016/j.neuropharm.2007.06.012 (2008).

189 Wang, W. et al. Deficiency in Endocannabinoid Signaling in the Nucleus Accumbens Induced by Chronic Unpredictable Stress. Neuropsychopharmacology 35, 2249-2261, doi:10.1038/npp.2010.99 (2010).

190 Navarria, A. et al. The dual blocker of FAAH/TRPV1 N-arachidonoylserotonin reverses the behavioral despair induced by stress in rats and modulates the HPA-axis. Pharmacological research 87, 151-159 (2014).

191 Reich, C. G., Taylor, M. E. & McCarthy, M. M. Differential effects of chronic unpredictable stress on hippocampal CB1 receptors in male and female rats. Behav Brain Res 203, 264-269, doi:10.1016/j.bbr.2009.05.013 (2009).

92 192 Natividad, L. A. et al. Constitutive Increases in Amygdalar Corticotropin- Releasing Factor and Fatty Acid Amide Hydrolase Drive an Anxious Phenotype. Biological psychiatry 82, 500-510, doi:10.1016/j.biopsych.2017.01.005 (2017).

193 Gray, M., Bingham, B. & Viau, V. A comparison of two repeated restraint stress paradigms on hypothalamic-pituitary-adrenal axis habituation, gonadal status and central neuropeptide expression in adult male rats. J Neuroendocrinol 22, 92- 101, doi:10.1111/j.1365-2826.2009.01941.x (2010).

194 Sandi, C. et al. Chronic stress-induced alterations in amygdala responsiveness and behavior--modulation by trait anxiety and corticotropin-releasing factor systems. Eur J Neurosci 28, 1836-1848, doi:10.1111/j.1460-9568.2008.06451.x (2008).

195 Kim, S. J. et al. Effects of repeated tianeptine treatment on CRF mRNA expression in non-stressed and chronic mild stress-exposed rats. Neuropharmacology 50, 824-833, doi:10.1016/j.neuropharm.2005.12.003 (2006).

196 Sterrenburg, L. et al. Chronic stress induces sex-specific alterations in methylation and expression of corticotropin-releasing factor gene in the rat. PLoS One 6, e28128, doi:10.1371/journal.pone.0028128 (2011).

197 Bortolato, M. et al. Antidepressant-like Activity of the Fatty Acid Amide Hydrolase Inhibitor URB597 in a Rat Model of Chronic Mild Stress. Biological Psychiatry 62, 1103-1110, doi:10.1016/j.biopsych.2006.12.001 (2007).

198 Haller, J. et al. Interactions between environmental aversiveness and the anxiolytic effects of enhanced cannabinoid signaling by FAAH inhibition in rats. Psychopharmacology (Berl) 204, 607-616, doi:10.1007/s00213-009-1494-7 (2009).

199 Moreira, F. A., Kaiser, N., Monory, K. & Lutz, B. Reduced anxiety-like behaviour induced by genetic and pharmacological inhibition of the endocannabinoid- degrading enzyme fatty acid amide hydrolase (FAAH) is mediated by CB1 receptors. Neuropharmacology 54, 141-150, doi:10.1016/j.neuropharm.2007.07.005 (2008).

200 Rubino, T. et al. Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex. Cereb Cortex 18, 1292-1301, doi:10.1093/cercor/bhm161 (2008).

201 Morena, M. et al. Upregulation of Anandamide Hydrolysis in the Basolateral Complex of Amygdala Reduces Fear Memory Expression and Indices of Stress and Anxiety. The Journal of Neuroscience 39, 1275, doi:10.1523/JNEUROSCI.2251-18.2018 (2019).

202 Gee, D. G. et al. Individual differences in frontolimbic circuitry and anxiety emerge with adolescent changes in endocannabinoid signaling across species.

93 Proceedings of the National Academy of Sciences of the United States of America 113, 4500-4505, doi:10.1073/pnas.1600013113 (2016).

203 Cameron, C., Watson, D. & Robinson, J. Use of a synthetic cannabinoid in a correctional population for posttraumatic stress disorder–related insomnia and nightmares, chronic pain, harm reduction, and other indications: A retrospective evaluation. Journal of clinical psychopharmacology 34, 559 (2014).

204 Christensen, R., Kristensen, P. K., Bartels, E. M., Bliddal, H. & Astrup, A. Efficacy and safety of the weight-loss drug rimonabant: a meta-analysis of randomised trials. The Lancet 370, 1706-1713 (2007).

205 Moreira, F. A., Grieb, M. & Lutz, B. Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression. Best practice & research Clinical endocrinology & metabolism 23, 133-144 (2009).

206 Goodwin, R. S. et al. CB1–cannabinoid receptor antagonist effects on cortisol in cannabis-dependent men. The American journal of drug and alcohol abuse 38, 114-119 (2012).

207 Mayo, L. M. et al. Protective effects of elevated anandamide on stress and fear- related behaviors: translational evidence from humans and mice. Mol Psychiatry, doi:10.1038/s41380-018-0215-1 (2018).

208 Gruber, S. A., Rogowska, J. & Yurgelun-Todd, D. A. Altered affective response in marijuana smokers: an FMRI study. Drug and alcohol dependence 105, 139-153 (2009).

209 Phan, K. L. et al. Cannabinoid modulation of amygdala reactivity to social signals of threat in humans. Journal of neuroscience 28, 2313-2319 (2008).

210 Cornelius, J. R., Aizenstein, H. J. & Hariri, A. R. Amygdala reactivity is inversely related to level of cannabis use in individuals with comorbid cannabis dependence and major depression. Addictive behaviors 35, 644-646 (2010).

211 Spagnolo, P. A. et al. FAAH Gene Variation Moderates Stress Response and Symptom Severity in Patients with Posttraumatic Stress Disorder and Comorbid Alcohol Dependence. Alcoholism: Clinical and Experimental Research 40, 2426- 2434, doi:10.1111/acer.13210 (2016).

212 Habib, A. M. et al. Microdeletion in a FAAH pseudogene identified in a patient with high anandamide concentrations and pain insensitivity. Br J Anaesth 123, e249-e253, doi:10.1016/j.bja.2019.02.019 (2019).

213 Neumeister, A. et al. Elevated brain cannabinoid CB 1 receptor availability in post-traumatic stress disorder: a positron emission tomography study. Mol Psychiatry 18, 1034-1040 (2013).

94 214 Demers, C. H., Drabant Conley, E., Bogdan, R. & Hariri, A. R. Interactions Between Anandamide and Corticotropin-Releasing Factor Signaling Modulate Human Amygdala Function and Risk for Anxiety Disorders: An Imaging Genetics Strategy for Modeling Molecular Interactions. Biol Psychiatry 80, 356-362, doi:10.1016/j.biopsych.2015.12.021 (2016).

215 Dlugos, A., Childs, E., Stuhr, K. L., Hillard, C. J. & de Wit, H. Acute stress increases circulating anandamide and other N-acylethanolamines in healthy humans. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 37, 2416-2427, doi:10.1038/npp.2012.100 (2012).

216 Hill, M. N., Miller, G. E., Ho, W. S., Gorzalka, B. B. & Hillard, C. J. Serum endocannabinoid content is altered in females with depressive disorders: a preliminary report. Pharmacopsychiatry 41, 48-53, doi:10.1055/s-2007-993211 (2008).

217 Hill, M. N. et al. Reductions in circulating endocannabinoid levels in individuals with post-traumatic stress disorder following exposure to the World Trade Center attacks. Psychoneuroendocrinology 38, 2952-2961, doi:10.1016/j.psyneuen.2013.08.004 (2013).

218 Schaefer, C. et al. Fatty acid ethanolamide levels are altered in borderline personality and complex posttraumatic stress disorders. Eur Arch Psychiatry Clin Neurosci 264, 459-463, doi:10.1007/s00406-013-0470-8 (2014).

219 Hauer, D. et al. Plasma concentrations of endocannabinoids and related primary fatty acid amides in patients with post-traumatic stress disorder. PLoS One 8, e62741, doi:10.1371/journal.pone.0062741 (2013).

220 Hill, M. N., Miller, G. E., Carrier, E. J., Gorzalka, B. B. & Hillard, C. J. Circulating endocannabinoids and N-acyl ethanolamines are differentially regulated in major depression and following exposure to social stress. Psychoneuroendocrinology 34, 1257-1262, doi:10.1016/j.psyneuen.2009.03.013 (2009).

221 Hill, M. N. et al. Prolonged glucocorticoid treatment decreases cannabinoid CB1 receptor density in the hippocampus. Hippocampus 18, 221-226, doi:10.1002/hipo.20386 (2008).

222 Bowles, N. P. et al. A peripheral endocannabinoid mechanism contributes to glucocorticoid-mediated metabolic syndrome. Proc Natl Acad Sci U S A 112, 285-290, doi:10.1073/pnas.1421420112 (2015).

223 Wilker, S. et al. Endocannabinoid concentrations in hair are associated with PTSD symptom severity. Psychoneuroendocrinology 67, 198-206, doi:10.1016/j.psyneuen.2016.02.010 (2016).

224 Turkington, T. G. Introduction to PET instrumentation. J Nucl Med Technol 29, 4- 11 (2001).

95 225 Wilson, A. A. et al. Radiosynthesis and evaluation of [(1)(1)C-carbonyl]-labeled carbamates as fatty acid amide hydrolase radiotracers for positron emission tomography. J Med Chem 56, 201-209, doi:10.1021/jm301492y (2013).

226 Wilson, A. A. et al. [11C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography. Nucl Med Biol 38, 247- 253, doi:10.1016/j.nucmedbio.2010.08.001 (2011).

227 Clapper, J. R. et al. A second generation of carbamate-based fatty acid amide hydrolase inhibitors with improved activity in vivo. ChemMedChem 4, 1505-1513, doi:10.1002/cmdc.200900210 (2009).

228 Wilson, A. A., Garcia, A., Houle, S. & Vasdev, N. Direct fixation of [(11)C]-CO(2) by amines: formation of [(11)C-carbonyl]-methylcarbamates. Org Biomol Chem 8, 428-432, doi:10.1039/b916419g (2010).

229 Allard, M., Fouquet, E., James, D. & Szlosek-Pinaud, M. State of art in 11C labelled radiotracers synthesis. Curr Med Chem 15, 235-277, doi:10.2174/092986708783497292 (2008).

230 Rusjan, P. M. et al. Mapping human brain fatty acid amide hydrolase activity with PET. J Cereb Blood Flow Metab 33, 407-414, doi:10.1038/jcbfm.2012.180 (2013).

231 Romero, J., Hillard, C. J., Calero, M. & Rabano, A. Fatty acid amide hydrolase localization in the human central nervous system: an immunohistochemical study. Brain Res Mol Brain Res 100, 85-93, doi:10.1016/s0169-328x(02)00167-5 (2002).

232 Dewey, S. L. et al. Amphetamine induced decreases in (18F)-N- methylspiroperidol binding in the baboon brain using positron emission tomography (PET). Synapse 7, 324-327, doi:10.1002/syn.890070409 (1991).

233 Logan, J. et al. Effects of endogenous dopamine on measures of [18F]N- methylspiroperidol binding in the basal ganglia: comparison of simulations and experimental results from PET studies in baboons. Synapse 9, 195-207, doi:10.1002/syn.890090306 (1991).

234 Boileau, I. et al. Blocking of fatty acid amide hydrolase activity with PF-04457845 in human brain: a positron emission tomography study with the novel radioligand [(11)C]CURB. J Cereb Blood Flow Metab 35, 1827-1835, doi:10.1038/jcbfm.2015.133 (2015).

235 Boileau, I. et al. The fatty acid amide hydrolase C385A variant affects brain binding of the positron emission tomography tracer [11C]CURB. J Cereb Blood Flow Metab 35, 1237-1240, doi:10.1038/jcbfm.2015.119 (2015).

96 236 Heatherton, T. F., Kozlowski, L. T., Frecker, R. C. & Fagerstrom, K. O. The Fagerstrom Test for Nicotine Dependence: a revision of the Fagerstrom Tolerance Questionnaire. Br J Addict 86, 1119-1127, doi:10.1111/j.1360- 0443.1991.tb01879.x (1991).

237 World Health, O. (World Health Organization, Geneva, 2001).

238 Spitzer, R. L., Kroenke, K. & Williams, J. B. Validation and utility of a self-report version of PRIME-MD: the PHQ primary care study. Primary Care Evaluation of Mental Disorders. Patient Health Questionnaire. Jama 282, 1737-1744, doi:10.1001/jama.282.18.1737 (1999).

239 Spitzer, R. L., Kroenke, K., Williams, J. B. & Lowe, B. A brief measure for assessing generalized anxiety disorder: the GAD-7. Arch Intern Med 166, 1092- 1097, doi:10.1001/archinte.166.10.1092 (2006).

240 Beck, A. T., Ward, C. H., Mendelson, M., Mock, J. & Erbaugh, J. An inventory for measuring depression. Arch Gen Psychiatry 4, 561-571, doi:10.1001/archpsyc.1961.01710120031004 (1961).

241 Snaith, R. P. et al. A scale for the assessment of hedonic tone the Snaith- Hamilton Pleasure Scale. Br J Psychiatry 167, 99-103, doi:10.1192/bjp.167.1.99 (1995).

242 Marin, R. S., Biedrzycki, R. C. & Firinciogullari, S. Reliability and validity of the Apathy Evaluation Scale. Psychiatry Res 38, 143-162, doi:10.1016/0165- 1781(91)90040-v (1991).

243 Spielberger, C. Manual for the State-Trait Anxiety Inventory (STAI). (Consulting Psychologists Press, 1983).

244 Patton, J. H., Stanford, M. S. & Barratt, E. S. Factor structure of the Barratt impulsiveness scale. J Clin Psychol 51, 768-774, doi:10.1002/1097- 4679(199511)51:6<768::aid-jclp2270510607>3.0.co;2-1 (1995).

245 Costa, P. T. & McCrae, R. R. The NEO personality inventory. (1985).

246 First, M. B., Spitzer, R. L., Gibbon, M. & Williams, J. B. W. The Structured Clinical Interview for DSM-III-R Personality Disorders (SCID-II). Part I: Description. Journal of Personality Disorders 9, 83-91, doi:10.1521/pedi.1995.9.2.83 (1995).

247 Kubany, E. S. et al. Development and preliminary validation of a brief broad- spectrum measure of trauma exposure: the Traumatic Life Events Questionnaire. Psychological assessment 12, 210 (2000).

97 248 Keane, T. M. et al. Clinical evaluation of a measure to assess combat exposure. Psychological Assessment: A Journal of Consulting and Clinical Psychology 1, 53 (1989).

249 Weathers, F. W., Litz, B. T., Herman, D. S., Huska, J. A. & Keane, T. M. in annual convention of the international society for traumatic stress studies, San Antonio, TX. (San Antonio, TX.).

250 Blevins, C. A., Weathers, F. W., Davis, M. T., Witte, T. K. & Domino, J. L. The posttraumatic stress disorder checklist for DSM‐5 (PCL‐5): Development and initial psychometric evaluation. Journal of traumatic stress 28, 489-498 (2015).

251 Foa, E. B., Riggs, D. S., Dancu, C. V. & Rothbaum, B. O. Reliability and validity of a brief instrument for assessing post‐traumatic stress disorder. Journal of traumatic stress 6, 459-473 (1993).

252 Wolf, E. J. et al. The Dissociative Subtype of PTSD Scale: Initial Evaluation in a National Sample of Trauma-Exposed Veterans. Assessment 24, 503-516, doi:10.1177/1073191115615212 (2017).

253 Buysse, D. J., Reynolds III, C. F., Monk, T. H., Berman, S. R. & Kupfer, D. J. The Pittsburgh Sleep Quality Index: a new instrument for psychiatric practice and research. Psychiatry research 28, 193-213 (1989).

254 Germain, A., Hall, M., Krakow, B., Katherine Shear, M. & Buysse, D. J. A brief sleep scale for Posttraumatic Stress Disorder: Pittsburgh Sleep Quality Index Addendum for PTSD. J Anxiety Disord 19, 233-244, doi:10.1016/j.janxdis.2004.02.001 (2005).

255 Rusjan, P. et al. An automated method for the extraction of regional data from PET images. Psychiatry Res 147, 79-89, doi:10.1016/j.pscychresns.2006.01.011 (2006).

256 Gunn, R. N., Gunn, S. R. & Cunningham, V. J. Positron Emission Tomography Compartmental Models. Journal of Cerebral Blood Flow & Metabolism 21, 635- 652, doi:10.1097/00004647-200106000-00002 (2001).

257 Knight, R. G., Waal-Manning, H. J. & Spears, G. F. Some norms and reliability data for the State--Trait Anxiety Inventory and the Zung Self-Rating Depression scale. Br J Clin Psychol 22 (Pt 4), 245-249, doi:10.1111/j.2044- 8260.1983.tb00610.x (1983).

258 (ed U.S. Department of Health and Human Services) (2015).

259 Maccarrone, M. et al. Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour. Eur J Neurosci 15, 1178-1186, doi:10.1046/j.1460-9568.2002.01957.x (2002).

98 260 Jumpertz, R., Guijarro, A., Pratley, R. E., Piomelli, D. & Krakoff, J. Central and peripheral endocannabinoids and cognate acylethanolamides in humans: association with race, adiposity, and energy expenditure. J Clin Endocrinol Metab 96, 787-791, doi:10.1210/jc.2010-2028 (2011).

261 Sipe, J. C., Waalen, J., Gerber, A. & Beutler, E. Overweight and obesity associated with a missense polymorphism in fatty acid amide hydrolase (FAAH). International Journal of Obesity 29, 755-759, doi:10.1038/sj.ijo.0802954 (2005).

262 Sipe, J. C. et al. Biomarkers of endocannabinoid system activation in severe obesity. PLoS One 5, e8792-e8792, doi:10.1371/journal.pone.0008792 (2010).

263 Martins, C. J. d. M. et al. Circulating Endocannabinoids and the Polymorphism 385C>A in Fatty Acid Amide Hydrolase (FAAH) Gene May Identify the Obesity Phenotype Related to Cardiometabolic Risk: A Study Conducted in a Brazilian Population of Complex Interethnic Admixture. PLoS One 10, e0142728- e0142728, doi:10.1371/journal.pone.0142728 (2015).

264 Farr, O. M. et al. Posttraumatic stress disorder, alone or additively with early life adversity, is associated with obesity and cardiometabolic risk. Nutrition, Metabolism and Cardiovascular Diseases 25, 479-488, doi:https://doi.org/10.1016/j.numecd.2015.01.007 (2015).

265 Vieweg, W. V. R. et al. Posttraumatic stress disorder as a risk factor for obesity among male military veterans. Acta Psychiatrica Scandinavica 116, 483-487, doi:10.1111/j.1600-0447.2007.01071.x (2007).

266 Mitchell, K. S. et al. PTSD and obesity in the Detroit neighborhood health study. General Hospital Psychiatry 35, 671-673, doi:https://doi.org/10.1016/j.genhosppsych.2013.07.015 (2013).

267 Hill, M. N., Karacabeyli, E. S. & Gorzalka, B. B. Estrogen recruits the endocannabinoid system to modulate emotionality. Psychoneuroendocrinology 32, 350-357, doi:10.1016/j.psyneuen.2007.02.003 (2007).

268 Best, L. M. et al. Lower brain fatty acid amide hydrolase in treatment-seeking patients with alcohol use disorder: a positron emission tomography study with [C- 11]CURB. Neuropsychopharmacology, doi:10.1038/s41386-020-0606-2 (2020).

269 Harismendy, O. et al. Population sequencing of two endocannabinoid metabolic genes identifies rare and common regulatory variants associated with extreme obesity and metabolite level. Genome Biology 11, R118, doi:10.1186/gb-2010- 11-11-r118 (2010).

270 Engeli, S. et al. Activation of the Peripheral Endocannabinoid System in Human Obesity. Diabetes 54, 2838-2843, doi:10.2337/diabetes.54.10.2838 (2005).

99 271 Cable, J. C., Tan, G. D., Alexander, S. P. H. & O'Sullivan, S. E. The activity of the endocannabinoid metabolising enzyme fatty acid amide hydrolase in subcutaneous adipocytes correlates with BMI in metabolically healthy humans. Lipids in Health and Disease 10, 129, doi:10.1186/1476-511X-10-129 (2011).

272 Richter-Levin, G., Stork, O. & Schmidt, M. V. Animal models of PTSD: a challenge to be met. Mol Psychiatry 24, 1135-1156, doi:10.1038/s41380-018- 0272-5 (2019).

273 Flandreau, E. I. & Toth, M. Animal Models of PTSD: A Critical Review. Curr Top Behav Neurosci 38, 47-68, doi:10.1007/7854_2016_65 (2018).

274 Yehuda, R. & Antelman, S. M. Criteria for rationally evaluating animal models of posttraumatic stress disorder. Biol Psychiatry 33, 479-486, doi:10.1016/0006- 3223(93)90001-t (1993).

275 Cohen, H., Zohar, J. & Matar, M. The relevance of differential response to trauma in an animal model of posttraumatic stress disorder. Biological psychiatry 53, 463-473 (2003).

276 Umathe, S. N., Manna, S. S. S. & Jain, N. S. Involvement of endocannabinoids in antidepressant and anti-compulsive effect of fluoxetine in mice. Behavioural Brain Research 223, 125-134, doi:https://doi.org/10.1016/j.bbr.2011.04.031 (2011).

277 Gunduz-Cinar, O. et al. Fluoxetine Facilitates Fear Extinction Through Amygdala Endocannabinoids. Neuropsychopharmacology 41, 1598-1609, doi:10.1038/npp.2015.318 (2016).

278 Seng, J. S., Kohn-Wood, L. P. & Odera, L. A. Exploring Racial Disparity in Posttraumatic Stress Disorder Diagnosis: Implications for Care of African American Women. Journal of Obstetric, Gynecologic, & Neonatal Nursing 34, 521-530, doi:10.1177/0884217505278296 (2005).

279 Alegría, M. et al. Prevalence, risk, and correlates of posttraumatic stress disorder across ethnic and racial minority groups in the United States. Med Care 51, 1114-1123, doi:10.1097/MLR.0000000000000007 (2013).

280 Koo, K. H., Hebenstreit, C. L., Madden, E. & Maguen, S. PTSD detection and symptom presentation: Racial/ethnic differences by gender among veterans with PTSD returning from Iraq and Afghanistan. J Affect Disord 189, 10-16, doi:10.1016/j.jad.2015.08.038 (2016).

100