Complete Thesis Printed

Central Amygdala Endocannabinoid Neurotransmission Alters Emotional Memory
Processes in Female Rats
A Thesis
Presented to
The Division of Philosophy, Religion, Psychology, and Linguistics
Reed College
In Partial Fulfillment
of the Requirements for the Degree
Bachelor of Arts
Cloe Beth Moctezuma-Bender
May 2015

Approved for the Division
(Psychology)
Timothy D. Hackenberg
Paul J. Currie

Acknowledgments
To Tim, a supportive and wise advisor. Thank you for guiding me through this process and
for reassuring me when I doubted myself. Every time I left a meeting with you, I was less
nervous than when I had entered, and words cannot express how much that meant.
To Paul, an understanding and patient mentor. I will never forget that you were the one to
plant the seedling of love for neuroscience in my heart. I will forever be grateful. Thank you
for helping me along this journey.
To my parents, I love you both to the moon and back. Thank you for giving me every
opportunity and supporting me every step of the way. To my brother and sister, I love you
guys and I thank you for being the best siblings I could ask for. I feel blessed to call you my
family. And to my Grandma, Grandpa, Abuelita. Aunt Susan and Uncle Chris, Aunt Shari
and Uncle Stephen, to all of my Tíos and Tías, Rose, Joe, Bella, and to Cynthia, Diana,
Jennifer, Tony, Leo and Ana Paula, without you I would not be the person I am today and I
am eternally grateful.
To Laura, my lifeline, thank you for always being there for me. Your love and friendship are
invaluable and without them I would not be where I am today. To the Stein family, I love
you all. Thank you so much for all of the years of love and laughter, and for being my
extended family.
To Daniel, my best friend. Thank you for helping me to grow every day and for helping me
to see the sun.
To my friends back home, Nielsen, Christina, and Josh. Thank you for always having my
back from afar and for always inspiring me. You make life sweeter and more interesting.

Thank you to my wonderful Reed family for keeping me strong. Stewart, Brice, Marisa,
Dylan, Johnathan, and Jesse, you were my rocks in the storm.
And finally, thank you to everyone who participated in making this project a reality. To my
friends, Emma, Joaquín, and Ileana, as well as Greg and Eliotte, without all of you, my life
would have been a miserable juggling act. Thank you for every second you contributed to
making my life easier.

Preface
Un mapa es una manifestación artística del miedo a lo dosconocido.
(A map is an artistic manifestation of the fear of the unknown.)
Alberto Blanco

Table of Contents
Introduction........................................................................................................................1
1.1 Emotional Information Processing: Limbic System Circuitry...................................... 1
1.2 Defining Emotional Memory...................................................................................... 5
1.3 Why the Central Amygdala (CeA)? ........................................................................... 10
Endocannabinoid Signaling and the CeA.................................................................... 12
1.4 The Endocannabinoid System.................................................................................. 13
Cannabis: A History..................................................................................................... 13
The Endocannabinoid Receptors ............................................................................... 15
The CB1 Receptor: Distribution and Function........................................................ 15
The CB2 Receptor: Distribution and Function........................................................ 18
Endogenous Cannabinoid Ligands: Anandamide (AEA) and 2-Arachidonylglycerol (2-
AG)............................................................................................................................ 19
Endocannabinoid Neurotransmission........................................................................ 20
1.5 Fear, Stress, Emotional Memory and the Central Amygdala...................................... 24
Cannabinoid Modulation of the Stress Response: A Role for the Central Amygdala... 25
Cannabinoid Modulation of Emotional Memory in the Central Amygdala: Effects on
Memory Consolidation............................................................................................... 29
The Present Study ........................................................................................................... 35
2.1 Objective.................................................................................................................. 35
2.2 Hypothesis............................................................................................................... 35
Materials & Methods....................................................................................................... 37
3.1 Animals.................................................................................................................... 37
3.2 Stereotaxic Surgeries................................................................................................. 37
3.3 Apparatus................................................................................................................. 38
Inhibitory Avoidance (IA).......................................................................................... 38
Open Field (OF) ........................................................................................................ 39
3.3 AEA Microinjections................................................................................................ 40

3.4 Design...................................................................................................................... 40
Inhibitory Avoidance ................................................................................................. 40
Open Field Test......................................................................................................... 41
3.5 Experimental Procedure........................................................................................... 41
Measure 1: Inhibitory Avoidance................................................................................ 42
Conditioning Phase................................................................................................ 42
Testing Phase......................................................................................................... 43
Measure 2: Open Field............................................................................................... 43
3.6 Histology.................................................................................................................. 43
3.7 Statistical Analysis..................................................................................................... 44
Results.............................................................................................................................. 47
4.1 Inhibitory Avoidance................................................................................................ 47
Conditioning Phase Entrance and Escape Latencies................................................... 47
Testing Phase Entrance and Escape Latencies............................................................ 48
4.2 Open Field............................................................................................................... 48
Time Spent in Center versus Periphery....................................................................... 48
Locomotor Activity: Line Crosses.............................................................................. 49
Discussion.........................................................................................................................51
5.1 Present Findings....................................................................................................... 51
5.2 Potential Mechanisms of Action and Sex Differences............................................... 52
The Stress Response................................................................................................... 53
Amygdala-Hippocampal-Prefrontal Control of the Stress Response: Glucocorticoid
Feedback Mechanisms................................................................................................ 57
5.3 Limitations, Future Studies, and Implications ........................................................... 61
Conclusion ....................................................................................................................... 67
Appendix A: Behavioral Testing Apparatus.................................................................. 69
Appendix B: Statistical Analysis......................................................................................71
Appendix C: Results........................................................................................................ 73
References........................................................................................................................ 77

Tables
Table 1. Inhibitory Avoidance Individual Subject Data....................................................... 73
Table 2. Open Field Individual Subject Data...................................................................... 74

Figures
Figure 1.1. The Limbic System............................................................................................. 2
Figure 1.2. Fear Conditioning............................................................................................... 7
Figure 1.3. Molecular structure of THC and CBD.............................................................. 14
Figure 1.4. Molecular structure of AEA.............................................................................. 20
Figure 4.1. Conditioning Phase Entrance and Escape Latencies ......................................... 47
Figure 4.2. Testing Phase Entrance and Escape Latencies.................................................. 48
Figure 4.3. Percentage of Time Spent in Center of OF....................................................... 49
Figure 4.4. Total Line Crosses............................................................................................ 50
Figure 4.5. Percentage of Line Crosses in Center................................................................ 50
Figure A1. The modified inhibitory avoidance apparatus.................................................... 69
Figure A2. The open field.................................................................................................. 69
Figure B1. R Code used to generate non-parametric randomization tests........................... 72
Figure C1. Overlaid brain slice of rat at the level of the CeA.............................................. 75

Abstract
Evidence for the involvement of the endocannabinoid system in stress-related
disorders, such as anxiety and depression, has been accumulated, providing leads for novel
therapeutic pharmacological approaches. The activation and blockade of CB1 and CB2
receptors by systemic or amygdalar drug or endocannabinoid administration have been
shown to modify emotional memory processing. Evidence has indicated that the central
nucleus of the amygdala (CeA) plays a role in stress-related processes (e.g., emotional
memory), and that cannabinoid signaling within the CeA has observable effects on stress-
related behavioral and physiological responding. In this study, we investigated the effects of
anandamide (AEA) infused into the CeA on the consolidation of a contextual aversive
memory and an active state of stress expression. Female rats received post-training
microinjections of AEA (6 pmol) or vehicle unilaterally into the CeA after exposure to an
inhibitory avoidance apparatus, and were later tested for emotional memory. In addition,
female rats received intra-CeA microinjections of AEA (6 pmol) or vehicle and were
exposed to an open field test. Our results showed that AEA administration into the CeA
post-training prevented consolidation of emotional memory as assessed by memory retrieval
48 h later, whereas intra-CeA infusion of AEA was not found to have an effect on stress-like
behavior, as evaluated by activity in the open field. These data suggest an important role for
endocannabinoid neurotransmission in the CeA in emotional memory formation, and speak
to the relevance of further investigation of the interface between the endocannabinoid
system and neural circuitries which support emotional behavior.

Dedicated to Susan Capanelli, a beautiful soul who inspires not only her loved ones,
but those who were lucky enough to know her for even a moment.

Introduction
1.1 Emotional Information Processing: Limbic System
Circuitry
Anxiety disorders are one of the most prevalent in the United States, according to
the National Institute of Mental Health, who report that, in a given year, approximately 40
million adults and eight percent of teenagers are affected, and that antianxiety drugs are
among the top prescription drugs currently availableon the market. Recently, there have
been estimates that, worldwide, the current prevalence of anxiety disorders within adult
populations is approximately 7.3 percent (Baxter et al., 2013). Various lines of evidence
indicate that stress, which ultimatelycan lead to anxiety, impacts the psychological health of
women more than that of men (Goel et al., 2014). Within the United States, stress-related
mood-disorders, such as general anxiety and major depression, are reported to be two times
more prevalent in women than in men (Kessler et al., 1993; Kessler et al., 2005). Although it
may be suggested that this reflects reporting bias, recent evidence suggests that these
differences may be due to differential neurocircuitry which supports emotional behavior
(Goel et al., 2014). Thus, the investigation of the underlying neurological circuitries which
are associated with both functional and maladaptive developed reactions to stress (clinically
termed ‘anxiety’) is a relevant and critical task, which often constitutes the basis for both
prolific and disastrous pharmacological and pharmaceutical ventures. The neural circuitry
which supports emotionality and emotionally-relevant processes is considerably complex,
but generally is comprised by subcortical structures such as the amygdala, hippocampus,
thalamus, and ventral striatum, as well as cortical structures, including the anterior cingulate
cortex and medial and orbital regions of the prefrontal cortex (PFC) (see Fig. 1.1; Price &
Drevets, 2010). The amygdala and the PFC, along with the hippocampus, have generally
been the focus of research of emotional behavior and memory, largely due to structural and
functional abnormalities within these regions are commonly correlated with mood and
anxiety disorders in clinical populations (Drevets et al., 2008). This corticolimbic circuit
interfaces with autonomic signaling in the hypothalamus and brainstem to regulate

2 Introduction
behavioral and physiological manifestations of emotional expression and modulate activity
of the hypothalamic-pituitary-adrenal(HPA) axis (Price & Drevets, 2010), which is integrally
involved with the stress response and the homeostatic state of an organism. Several neural
circuits have been found to have a prominent role in the regulation of an organism’s
response to stress-related processes, such as the limbic system and the HPA axis. These
brain regions are involved in this evolutionary circuitry. Presently, let us begin with a
discussion of limbic circuitry and its involvement in the production of emotions, emotional
(i.e., “stress-” or “fear-” related) behavior, and emotional memories.
Figure 1.1. The Limbic System
The limbic system supports emotion information processing and is comprised of
regions both forebrain and midbrain regions. (Adapted from www.uab.edu).
It has been repeatedly confirmed that some regions of the telencephalon, the
diencephalon, and the mesencephalon of the brain are structurally and functionally
interrelated. The collection of these regions has come to be known as the limbic system (as
proposed by Paul Maclean; Maclean, 1949) because, as a whole, these areas constitute a
unique functional complex (Maclean, 1952). Specifically, the limbic system is integrally
involved in the production of emotions and emotional behavior, and is capable of
integrating internal and external sensations, which includes both cortical and subcortical
structures (Roxo et al., 2011). The limbic system is critical for both emotional processes,
learning, and for memory (McIntyre et al., 2005), and includes (but is not limited to) the
thalamus, hypothalamus, hippocampus, cingulatecortex, fornix, septal nuclei, stria

Introduction 3
terminalis, olfactory bulb, amygdala (sometimes termed the ‘amygdaloid complex’), septum,
mammillary bodies, and pre-frontal cortex (PFC), although the limbic components of the
cortex may vary according to differing anatomical descriptions (Roxo et al., 2011), and
integrity of this system has been found to be compromised in individuals with histories of
chronic stress exposure (Wang et al., 2013b). In particular, and of interest to our
investigation, the amygdala has been associated with fear, stress, and negativeemotionality. It
is a brain region which has been heavily implicated in various phases on conditioned fear
learning (Pape & Paré, 2010), and is therefore of critical interest in investigations of
pathologies associated with HPA axis dysregulation, such as anxiety and depressive
disorders. It has become increasingly evident that intensely emotional events or chronic
stress exposure can lead to the development of anxiety and mood disorders, including PTSD
and major depression (Roozendaal et al., 2009). Stress has been identified as a predisposing
or aggravating factor in various medical conditions, including psychiatric disorders,
cardiovascular disease, and immune system dysfunction (Arborelius et al., 1999; De Kloet et
al., 2005; Silverman & Sternberg, 2012). Thus, there is a vested interest in understanding the
mechanisms by which the brain supports processes related to stress and memory.
In this study, we investigate the amygdaloid complex – more precisely, the CeA – in
an effort to elucidate how neurotransmission in this region contributes to a dynamic state
caused by acute stress (via interaction with the HPA axis) and to the consolidation of an
emotional memory (i.e., memory of a context-specific stressful event). Stress for biological
organisms is most fundamentally described as a state of strain due to the perception of threat
to homeostasis, which may directly or indirectly disrupt physiological homeostasis, and
requires an adaptive response (De Kloet et al., 2005; Riebe & Wotjak, 2011), which
encompasses the activation of a complex range of responses, involving the neural,
endocrine, nervous, and immune systems. Collectively, the activation of these systems is
known as the stress response (Carrasci & Van de Kar, 2003; Chrousos & Gold, 1992). A
stressor is typically defined as the specific event which induces the stress response, which
can be physical (e.g., thirst, pain) or psychological (e.g., fear, work overload), as well as acute
or chronic (Wolf, 2008). In non-human animal literature which focuses its discussion on
emotions, the terms “stress” and “anxiety” are often conflated. With regard to the present
investigation, we distinguish between these two terms, and have limited our inquiry to the
immediate behavioral expression of and emotional memory for acute stress. All stressors,

4 Introduction
whether physiological or psychological in nature, elicit a generalized stress response in most
typically functioning organisms (Selye, 1936). The activation of the stress response initiates
varying levels (depending on factors, such as personality, personal history, genetics,
contextual environment, etc.) of behavioral and physiological changes, which, in typically
functioning individuals, improve that individual’schance of survival when confronted with
homeostatic challenges (Smith & Vale, 2006).
The CeA is directly involved in the processing of emotional stimuli (stressors), as it
receives efferent projections from the lateral (LA) and basolateral (BLA) nuclei, which
receive highly processed sensory information from various brain regions (LeDoux, 1996),
and evidence indicates its involvement in modulation of HPA axis activity. The HPA axis is
one of two main stress-related neuroendocrine circuits, which is comprised predominantly of
the PVN of the hypothalamus, the anterior lobe of the pituitary gland, and the adrenal gland,
although other neural regions act in concert with the main anatomical loci of the HPA axis
to activate the HPA axis. Whereas the PFC and hippocampus are thought to inhibit HPA
axis activity (via both rapid and delayed glucocorticoid (GC; corticosterone in most rodents,
cortisol in humans) feedback), amygdala neurotransmission is thought to stimulate HPA axis
activity. Amygdalar neuron stimulation has been found to promote GC synthesis and release
into systemic circulation (Matheson et al., 1971; Van de Kar et al., 1999). GCs are an
essential class of stress-related hormones, which are responsible for many components of an
organism’s response to homeostatic threat (Wamsteeker & Bains, 2010). Specifically, the
CeA and the medial nucleus of the amygdala (MeA) are thought to play a pivotal role in
HPA axis activity, as they contribute the majority of afferent projections from the
amygdaloid complex to the cortical, midbrain, and brainstem regions that regulate adaptive
responses to stress (Petrovich et al., 1997). Large amygdaloid lesions or lesions of the CeA or
MeA have been found to decrease adrenocorticotropic hormone (ACTH) and/or
corticosterone secretion following exposure to stress, whereas stimulation has been found to
increase HPA axis activity (Herman et al., 2005). It has been noted that the stimulatory
effects of the amygdaloid complex on the HPA axis are consistent with previously
documented functions of these regions, most notably the activation of autonomic responses
(Gray, 1993) and the involvement in stress-/fear-related behavior (Davis, 1992; Herman et
al., 2005).

Introduction 5
Activation of glucocorticoid receptors (GRs) in the CeA and the bed nucleus of the
stria terminalis (BNST) have been found to promote levels of GC (corticosterone in most
rodents, cortisol in humans) and norepinephrine (NE) mRNA transcription in a distinct
population of PVN neurons that project descending terminals to noradrenergic brainstem
neurons (Makino et al., 1999). Once activated, neurons of the NTS (and to a lesser extent, of
the ventrolateral medulla)relay sensory information to the paraventricular nucleus (PVN) of
the hypothalamus via cranial innervation of several thoracic and abdominal viscera regions
(Smith & Vale, 2006), and receive afferents from limbic circuitry involved in the regulation
of the stress response, such as the medial prefrontal cortex (mPFC) and the central nucleus
of the amygdala (CeA) (Schwaber et al., 1982). In the rat brain, a variety of stressful events
have been shown to lead to marked elevations of noradrenaline release in certain neural
regions, such as the amygdala, hypothalamus, and locus coeruleus (LC) (Tanaka et al., 2000).
Clearly, the CeA is involved in myriad neurocircuitries, of which several are stress-/fear-
related.
The terms “stress”/“fear” and “emotional memory” as they are used in this paper
will be defined in the following sections, as will their operationalization within the
framework of the present study. Although various abbreviations are used to refer to the
various nuclei of the amygdala (see Krettek & Price, 1978; de Olmos et al., 1985), we will use
the abbreviations of Paxton and Watson (2007) for amygdalar nuclei, specifically.
1.2 Defining Emotional Memory
LTM refers to memory that, once encoded in the brain, can remain in the brain
indefinitely, through a series of processes. A functional LTM is dependent on the success of
several processes: memory acquisition (via encoding of raw information from sensory
channels into short-term memory), consolidation, retrieval and the subsequent
reconsolidation of both explicit and implicit aspects of the memory. Memories about
emotional situations and emotionally salient stimuli are often processed and stored by both
types of memory systems (i.e., explicit and implicit memory systems). Explicit aspects of the
memory are processed by explicit memory systems, which support conscious memory (i.e.,
memories about emotions). Likewise, implicit aspectsof the memory are stored by implicit
memory systems, which store information unconsciously (i.e., emotional memory) (Ledoux,

6 Introduction
2007). While “emotional memory” has been characterized in various ways (Akirav, 2011;
Rodrigues et al., 2009), in this study, here we utilize the definition of LeDoux (2007), as it
not only includes memory generated under circumstances of fear and stress, but also of
pleasure. Thus, when mentioning “processes which support stress regulation and emotional
memory,” two distinct concepts are the referent, although the former may be involved in the
latter.
Pavlovian fear conditioning has offered the most widely studied model of emotional
memory, as the involved subcortical neural mechanisms are highly conserved across species
(LaBar & Cabeza, 2006). Many of the tasks which investigate emotional memory utilize
Pavlovian fear conditioning as a behavioral paradigm, as does our present investigation, and
our present understanding of neural systems which process and promote behavioral
responses to emotional stimuli are based on such paradigms. Traditional fear conditioning
procedures typically involvetwo phases (although sometimes a third, initial habituation
phase for to the apparatus is included; see Fig. 1.2). The first, the conditioning phase,
consists of exposing a subject to a neutral conditioned stimulus (CS), such as a light or tone,
followed by an aversive (fear-inducing) unconditioned stimulus(US) (e.g., an electrical foot
shock). This produces an unconditioned emotional response (UR), which would naturally
occur in the presence of frightening stimuli. The UR can take the form of one or a
constellation of behaviors and various autonomic responses, and it is generally assumed that
these responses reflect a central state of fear. These behaviors can then be used to define a
state of fear (Davis, 1997). After repeating one or several pairings of these stimuli, the CS
comes to elicit a conditioned emotional response (CER), the behaviors and autonomic
responses initially present during the conditioning phase without the presence of the US. A
conditioned emotional response refers to changes in autonomic nervous system, behavioral,
and hormonal activity elicited by the CS after conditioning in comparison to before
conditioning occurred, which is measured during the testing phase. Fear conditioning has
been used to investigate the neural mechanisms of learning and memory in both human and
a variety of non-human animals (Davis, 1997; LeDoux, 2007). In humans, autonomic
nervous system (ANS) responses are measurable (oftentimes by measuring galvanic skin
response (GSR)), and are even elicited by the CS even when the CS is masked, so that
participants are unaware of it during either conditioning or testing (Ledoux, 2007; Ohman &
Soares, 1993). This indicates that fear conditioning involves implicit systems of learning and

Introduction 7
memory (i.e., fear conditioning elicits emotional memory), and subliminal fear conditioning
has been influential in characterizing the rise of fear, stress, and downstream anxiety in
absence of the individual’s awareness (LaBar & Cabeza, 2006). Furthermore, the behavioral
effects that are produced in non-human animals in response to the CS have been found to
be similar to the constellation of behaviors that are used to diagnose generalized anxiety
disorder in humans (Davis, 1997). Given the generally acknowledged role of this form of
emotional learning in traumatic memory formation (which can lead to PTSD), anxiety and
depressive disorders (including phobias), and drug addiction, there has been an impetus
towards fully elucidating the psychological and neural mechanisms involved in conditioned
fear behavior (LaBar & Cabeza, 2006).
Figure 1.2. Fear Conditioning
Fear conditioning occurs in multiple phases, the first of which is sometimes omitted,
depending on the paradigm. During habituation, the animal is acclimated to the
chamber and no stimuli are presented. During the conditioning phase, the conditioned
stimulus (CS) (in this figure, a tone) is paired with the unconditioned stimulus (US), the
foot shock. The testing phase involves presentation of the CS without the US.
Typically, the animal exhibits fear responses (CERs, such as freezing) to the CS during
the testing phase. If the CS and US are presented, but their presentation is unpaired,
freezing will not vary as a function of CS presentation. As illustrated, in some fear
conditioning paradigms, the testing phase occurs in a novel chamber, and a unique
stimulus (e.g., an odor such as peppermint) is paired with the CS and the behavioral
index of fear/stress is assessed (adapted from Ledoux, 2007).

8 Introduction
While emotional memory can be assessed in various ways, in the present study, with
regard to emotional memory formation, we focus on a one-time exposure to a modified
inhibitory avoidance (IA) paradigm, which will be fully explicated in following sections. This
paradigm involves the pairing of a typically preferred chamber with an aversive, stressful
event (i.e., an electrical foot shock), and the administration of a chemical compound during
the consolidation phase. Successful interruption of the associative emotional memory
consolidation into LTM is indicated by the obliteration of the negative association in a later
test in which the subject is re-exposed to the chamber. The amygdala, in particular, is a
region repeatedly demonstrated to be involved in fear conditioning (LaBar & Cabeza, 2006;
LaBar et al., 1995), with the BLA having received the vast majority of attention (Maren,
2001). For basic forms of Pavlovian conditioning, the amygdala, together with the thalamic
regions, is essential.
Weiskrantz (1956) was the first who confirmed that the amygdala was a region
heavily involved in emotional processes, by demonstrating that restricted amygdala lesions
could replicate the results of Klüver and Bucy (1937), whose now classic studies
demonstrated the severe emotional side effects caused by medial temporal lobe lesions
monkeys (which included, but were not limited to, the amygdala). The work by Weiskrantz
cemented the fundamental role of the amygdala in the neural machinery related to emotional
processing (Sah et al., 2003), and highlighted the amygdala as an essential component of the
circuitry that assigns emotional valence and promotes adaptive behavioral responses to
salient external stimuli (Gloor, 1960; Aggleton, 1992; LeDoux et al., 2000; Sah et al., 2003).
Accordingly, several studies have led to the conclusion that damage to the amygdala
interferes with the acquisition and expression of conditioned fear (LeDoux, 2000; Maren,
2001). Reflecting the findings of Weiskrantz (1956) in human subjects, it has been found that
patients with amygdala injury fail to recognize fear in presentations of human facial
expressions (Adolphs et al., 1995, 1999; Young et al., 1995). Additionally, it has been found
that bilateral amygdala ablation in rhesus monkeys decreases anxiety, and leads to atypical
comfort in engaging with unfamiliar monkeys (Emery et al., 2001), demonstrating that there
are homologous aspects of emotional processing across species. Furthermore, in human
subjects, functional magnetic resonance imaging (fMRI) studies have shown that among
“anxiety-prone” individuals, the amygdala shows increased activation in response to
emotionally salient stimuli in comparison to controls (Stein et al., 2007). Relatedly, it has

Introduction 9
been repeatedly validated that, in healthy human subjects, amygdala activation is increased
during Pavlovian fear conditioning (Cheng et al., 2006; Cheng et al., 2003; LaBar et al.,
1998), and that studied PTSD patient populations exhibit higher resting state (basal) of
amygdala activation (Semple et al., 2000). Moreover, epileptic patients who have received a
unilateral amygdalectomy treatmentshow deficits in auditory fear conditioning (LaBar et al.,
1995). It has also been demonstrated that amygdala hyperactivityin response to trauma-
related stimuli (Morey et al., 2009) is predictive of symptom severity in patients diagnosed
with PTSD (Dickie et al., 2008; Raunch et al., 2000). Moreover, higher levels of basal
amygdala activation are seen in individuals diagnosed with generalized anxiety (Nitschkeet
al., 2009; Whalen et al., 2008), socialphobia (Tillfors et al., 2001), specificphobia (Straube et
al., 2006; Wright et al., 2003) and panic disorder (Domschke et al., 2008; van den Heuvel et
al., 2005). Additionally, MRIevidence from patients diagnosed with major depressive
disorder has demonstrated that these patients have reduced hippocampal volume dependent
on the duration of their diagnosis (MacQueen et al., 2003; Videbech & Ravnkilde, 2004),
which supports the notion that altered limbic function may have corresponding negative
psychological and behavioral effects.
It has been demonstrated that the amygdala acts as an interface between sensory
inputs and cortical processing (Sah et al., 2003; Shin & Liberzon, 2010). As alluded to,
activation of the amygdala has been demonstrated to be concomitant with the generation of
stress-/fear-related behavior (Sah et al., 2003; Shin and Liberzon, 2010), and, as will be
outlined, to promote action of the HPA axis (see Discussion; Herman et al., 2003). Upon
acquisition, sensory information about the CS and the US converge in the amygdala and
become associated, and translated downstream into behavioral conditioned fear responses
(Davis & Whalen, 2001; LeDoux, 2000), and various studies have confirmed the amygdala’s
role in the extinction learning (Amano et al., 2010; Herry et al., 2008; Pape & Paré, 2010).
Prefrontal regions, particularly the ventromedial PFC (vmPFC), that are interconnected with
the amygdala, are regions critical for consolidation and retrieval of extinction memories
(Phelps et al., 2004; Milad & Quirk, 2002; Paré et al., 2004; Quirk et al., 2006; Quirk et al.,
2003; Quirk & Mueller, 2008; Ochsner & Gross, 2005).
The notion that the amygdala is a central participant in the formation and storage of
CS-US associations during fear conditioning has been supported by evidence obtained from
studies using both permanent and reversible amygdalar lesions. The specific role of the

10 Introduction
amygdala and its nuclei in the activation of the stress response, and in emotional memory,
will be outlined in following sections.
1.3 Why the Central Amygdala (CeA)?
The amygdala is composed of approximately 13 separate, but related nuclei, each
with their own respective subdivisions, which receive input from various brain regions
(Aggleton, 2000; Davis, 1997). These various amygdaloid nuclei differ in cytoarchitecture,
chemoarchitecture, connectionality, as well as functionality(Aggleton, 2000). As has been
discussed, together, they serve as an interface between sensory input and motor output
crucial for learning and memory of fear, stress, and related behavioral responses (Rosen,
2004). These nuclei are traditionally the termed basolateral amygdala (BLA; consisting of the
lateral (LA), basal (BA), and accessory basal, also known as the basomedial (BM), nuclei),
and are surrounded by the amygdalar central, medial, and cortical nuclei. Traditionally, these
are all included when referencing the amygdaloid complex (Davis & Whalen, 2001; Aggleton,
2000). An important advantage of using this nomenclature, originally described by Price et al.
(1987), and further modified by Pitkänen et al. (1997), is that the naming of presumed
homologous portions of the amygdaloid complex have been found to be similar across a
variety of species, including rats, cats (Price et al., 1987), monkeys (Amaral et al., 1992), and
humans (Sorvari et al., 1995). Thus, findings regarding the manner of involvement of the
individual divisions of the amygdala in processes of stress and emotional memory in the rat
brain may help to elucidate the neurocircuitry and neurophysiology of these processes in the
human brain, the dysregulation of which can promote the development of maladaptive
behaviors and psychopathologies (Davis, 1992; LeDoux, 2007b).
Located at the dorsomedial aspect of the rostral half of the amygdala, the CeA
(traditionally subdivided by function into lateral (CeL), and medial (CeM) compartments),
has been recognized to play a central role in the expression of fearful and stressed behaviors
(Davis, 1992; for review of CeA anatomy in the rat, see (Pape & Paré, 2010; Sah et al.,
2003)). In terms of neural input, highly processed sensory information from various cortical
areas reaches the amygdala, first via transmission to the LA and BLA (Aggleton, 2000; Davis,
1997; McDonald, 1998; McIntyre et al., 1996). Subsequently, the information is transmitted
to the CeA (Aggleton, 1985; Pitkänen et al., 1995), both directly, via glutamatergic

Introduction 11
projections and indirectly, by exciting intercalated (ITC) cell masses (non-nuclei, small,
densely packed GABAergic interneuron clusters) that generate feedforward inhibition of
individual CeA neurons (Amano et al., 2010; Duvarci & Paré, 2014; Pitkänen et al., 1997;
Ramikie & Patel, 2012).
It has been hypothesized that the CeA primarily plays a role in attention and arousal
during fear (Gallegher & Holland, 1994; Kapp et al., 1992; Rosen, 2004), and functions to
gate afferent signals from the LA to modulate behavioral expression of learned fear (Davis et
al., 1994). Some lesion studies have suggested that the CeA contributes to the initial
acquisition of a memory for an aversive event (an emotional memory; in the present study,
an inescapable electrical foot shock), but not its retention (Roozendaal et al., 1993). This
claim is overwhelmed by the fact that various groups have shown that the CeA plays an
integral role in the acquisition (or encoding), consolidation, retrieval, reconsolidation,
generalization, and extinction of conditioned fear, which, as described previously, explores
emotional memory (Amano et al., 2010; Ciocchi et al., 2010; Gilpin et al., 2014; Li et al.,
2013; Tye et al., 2011). Briefly, let us define these terms. Memory acquisition refers to the
first encounter with the stimulus. The second phase, consolidation refers to the retention or
storage of the stimulus-associated memory. The consolidation phase is a period of time
during which these memories are fragile and prone to disruption as they are converted into a
LTM form, a process which involves new gene expression and protein synthesis (for review,
see Pape & Paré, 2010). Retrieval is the process of accessing the memory, while
reconsolidation is the process of retrieving the memory traces associated with the specific
memory episode. Generalization refers to the extension of the associativememory to novel
circumstances, and extinction refers to the gradual weakening of the memory. It is important
to note that, in the present investigation, it is not the acquisition, retrieval, reconsolidation,
generalization, or extinction of stress/fear memory that is assessed. Rather, it is the
consolidation of the fear memory that is manipulated, and specific research regarding the
CeA’s involvement in memory consolidation will later be reviewed.
Evidence suggests that the amygdala, and its myriad efferent projections, may
represent a central fear and stress system involved in both the expression and acquisition of
fear and stress behaviors (Davis, 1997; LeDoux, 1988). The amygdala has been found to be a
critical component of the neural circuitry which supports fear learning (including both the
active experience of fear and the associated emotional memory; Davis, 2000, LeDoux, 2000).

12 Introduction
Lesions studies demonstrate that ablation of the CeA produces profound deficits in both the
acquisition and expression of conditioned fear (Hitchcock & Davis, 1986; Iwata et al., 1986;
Kim & Davis 1993; Roozendaal et al., 1991a, b; Young & Leaton 1996), and
pharmacological studies have indicated that this is because of a deficit in the performance of
conditioned fear responses, rather than an associativedeficit (Fanselow & Kim 1994;
Goosens et al., 2000; Maren, 2001). Furthermore, it has been suggested that, due to the
ability of lesions placed in structures efferent to the CeA to produce selective deficits in
either cardiovascular or somatic conditioned fear responses (Amorapanth et al., 1999; De
Oca et al., 1998; LeDoux et al., 1988), the CeA is the terminal common pathway for the
generation of learned fear responses (Maren, 2001).
Electrical stimulation of the CeA produces behavioral and autonomic changes similar
to those evoked by shock-paired stimuli (Applegate et al., 1983; Gelsema et al., 1987; Kapp
et al., 1982; Mogenson & Calaresu, 1973). Thus, it is important to delineate the role of the
various amygdaloid nuclei in the production of the stress response and emotional memory
related to the stress response. Specifically, the CeA is thought to collaborate with the BLA
and the ICT cell clusters to mediate conditioned fear (Duvarci & Paré, 2014).
Endocannabinoid Signaling and the CeA
One emerging field of research in which the CeA has received considerably little
attention in behavioral neuroscience investigations is the literature on the endocannabinoid
system (ECS) and its relation to stress, anxiety, and emotional memory. There are a few
notable exceptions (Zarrindast et al., 2008), which will be discussed in following sections.
CB1 mRNA expression levels have generally been described as low within the CeA but
present, which, in theory, permits endocannabinoid neurotransmission in this region
(Chhatwal et al., 2005; Hermann & Lutz, 2005; Matsuda et al., 1993; Marsicano & Lutz,
1999). Additionally, differences in CB1 mRNA expression within subdivisions of the CeA
have yet to be elucidated (Chhatwal et al., 2005), although this issue will not be discussed in
the present paper. As has been discussed, endocannabinoid neurotransmission is broadly
involved in a host of bioregulatory processes, and the disruption or stimulation of this
system may contribute to altered physiological and psychological functioning, and may thus
influence stress-related behavior and emotional memory. The literature investigating direct

Introduction 13
cannabinergic compound microinjection into the CeA is extremely sparse, and we therefore
find this an important route of inquiry. In the present study, we directly infuse a cannabinoid
compound, anandamide, into the CeA to investigate behavioraleffects on present states of
stress and on emotional memory. To understand the relevance of anandamide to stress
responses and emotional memory, and thus its germaneness to human psychopathologies, it
is necessary to provide a brief review of the ECS, of which anandamide is apart.
1.4 The Endocannabinoid System
Cannabis: A History
The first documented use of the cannabis plant was as a medicinal agent by the
Assyrians in the second millennium BC, from the time its psychoactive and medicinal
properties were first realized (Mechoulam, 1986; Mechoulam & Parker, 2013). According to
Campbell Thompson’s A Dictionary of Assyrian Botany, the cannabis flower was named either
ganzi-gun-nu (“the drugs that takes away the mind”) or azzalu, which was a drug that was used
for “depression of spirits,” for an ailment associated with women (possibly amenorrhea), or
even for annulment of witchcraft (Campbell Thomson 1949, as cited in Mechoulam 2013).
Scientists speculatethat in ancient cannabis preparations, various types of cannabis
plants may have been used, which may have contributed to the varying, and sometimes
contradictory effects experienced by consumers. In general, it has been found that if
endocannabinoid signaling is disrupted, exaggerated neurobehavioral (oftentimes emotional)
responses are observed (Hill & McEwen, 2010). In rodent studies, activation of the CB1
receptor (CB1R), which will be discussed, has been shown to promote stress-mitigating
effects in the EPM (Bortolato et al., 2006; Braida et al., 2007; Hill et al., 2007; Patel &
Hillard, 2006), EZM (Kathuria et al., 2003), and light-dark box (Rutkowska et al., 2006;
Scherma et al., 2008), suggesting that, under various conditions, CB1R activation serves to
promote a neurophysiological protective response to stress, which supports anxiolytic-like
behavior. In human subjects, subjective reports from consumers of recreational cannabis
and other controlled studies on drug effects of both natural and synthetic cannabinoids (e.g.,
THC, Nabilone) support the notion that CB1R-binding ligands promote calmness and/or
reductions in subjective statesof experienced anxiety (such as nervousness; D'Souza et al.,

14 Introduction
2004; Sethi et al., 1986; Wachtel et al., 2002), which helps to validatethis receptor’s role in
emotional information processing. However, these effects are highly dependent on a number
of factors, and thus caution must be maintained when drawing conclusions from human
data.
Recently, a three species were described: Cannabis sativa (tall, branched plants for
fiber, seed for psychoactive use), Cannabis indica (short, broad-leafed plants from
Afghanistan), and Cannabis ruderalis (short, unbranched ‘roadside’ plants usually weak in
cannabinoids) (Schultes et al., 1980). All three species of cannabis can produce various
amounts of phytocannabinoids, which are present in the resin that covers the leaves and
flower clusters of maturing female plants. Phytocannabinoids are cannabinoids formed by
plants, whereas the term ‘endocannabinoids’ refers to intrinsic, endogenously produced lipid
ligands. The first of these phytocannabinoids identified and isolated was ∆9
-
tetrahydrocannabinol (THC) in 1964 by Mechoulam and Gaoni (see Fig. 1.3; Gaoni &
Mechoulam, 1964). Cannabidiol (CBD), another phytocannabinoid, was isolated from
marijuana extract in 1940 (Adams et al., 1940), although its structure was not elucidated until
1963 (see Fig. 1.3; Mechoulam & Shvo, 1963). Due to the apparent anxiolytic effects of
CBD (Fusar-Poli et al., 2009; Zuardi et al., 1993; Zuardi et al., 2006), and its abilityto
attenuate or reverse THC-induced symptoms (Bhattacharyya et al., 2010; Zuardi et al., 1982),
CBD has great therapeutic potential. There have been hundreds of peer-reviewed
publications that have addressed the myriad actions of CBD (for a review, see Mechoulam et
al., 2009). The findings of these studies contribute to the notion that exogenous
pharmacological modulation of cannabinoid receptors is a viable approach to the
investigation of stress-related disorders and viable therapeutic routes of enquiry.
Figure 1.3. Molecular structure of THC and CBD
Chemical structures of ∆9-tetrahydrocannabinol (left) and cannabidiol (right)

Introduction 15
The Endocannabinoid Receptors
Though it was initially assumed the effects of phytocannabinoids were mediated
through nonspecific mechanisms (e.g., modulation of membrane fluidity), data emerged
indicating that, instead, cannabinoids may act through receptors (Howlett et al., 1986). This
group showed that cannabinoids could inhibit adenylate cyclase formation, and the potency
of the cannabinoids examined dose-dependently affected the level of their pharmacological
effects (Howlett et al. 1986). Shortly after, this group demonstrated that binding sites for
cannabinoids were present in neural tissue (Devane et al., 1988). At the neural signaling level,
two cannabinoid receptors have been characterized to date (Howlett, 2002). First identified
and cloned in the early 1990s, CB1 and CB2 receptors have been the focus of cannabinoid
receptor research to date, although emerging research suggests that cannabinoid signaling
engages more than simply these two receptors (Ryberg et al., 2007). Both the CB1 and CB2
receptor are G-protein coupled seven-transmembrane domain receptors (GCPRs;
Basavarajappa et al., 2006; Basavarajappa et al., 2007).
The CB1 Receptor:Distribution and Function
CB1 receptors (CB1Rs) are expressed ubiquitously and are densely located primarily
throughout both human (Mailleux et al., 1992; Westlakeet al., 1994) and non-human brain
(Herkenham et al., 2002; Romero et al., 1997; Tsou et al., 1998), neural tissue, and spinal
cord, although the concentration of CB1Rs may vary depending on the neural region
(Herkenham et al., 1990; Herkenham et al., 1991; Howlett, 2002; Moldrich & Wenger, 2000;
Tsou et al., 1998). The CB1R has also been shown to exhibit certain differential expression
patterns in peripheral tissue, such as immune cells, vascular tissue and adipocytes (Cota et al.,
2003; Hillard, 2000; Parolaro, 1999). CB1Rs have been found to represent the most abundant
class of G-protein-coupled receptors in the central nervous system, their densities being
similar to the levels of γ-aminobutyric acid- (GABA) and glutamate-gated ion channels
(Herkenham et al., 1991; Katona & Freund, 2008). The distribution of CB1Rs is highly
heterogeneous in rats, with the highest density of receptor expression in areas such as the
extended amygdala, cerebral cortex, hippocampus (especially within the dentate gyrus and
the cerebellum’s molecular layer), basal ganglia, ventral striatum, thalamus, hypothalamus,
cerebellum, substantia nigra, pars reticulata, and globus pallidus (Herkenham, 1990;

16 Introduction
Herkenham, 1991; Herkenham, 1992; Katona et al., 2001; Mailleux & Vanderhaechen, 1992;
Witkin et al., 2005), although distribution is relativelylower in the thalamus, hypothalamus,
and midbrain, and is essentially null in the medulla (Herkenham et al., 1990; Tsou et al.,
1998). Importantly, CB1Rs are expressed abundantly in regions involved in the limbic
circuitry (Herkenham et al., 1991; Morena & Campolongo, 2014; Tsou Brown et al., 1998).
Furthermore, a similar distribution of CB1Rs has been found in human populations (Biegon
& Kerman, 2001; Glass et al., 1997), with the highest density found in association with
limbic cortices, with slightly lower, yet still elevated, levelsof expression within the primary
sensory and motor regions (Mechoulam & Parker, 2013). The high degree of
neuroanatomical overlap between the neural networks supporting emotional processes and
the expression of CB1Rs suggests an essential role for CB1Rs in the control of the motor
function and information processing necessary for the perception, response to, and memory
of emotionally salient events (Basavarajappa, 2007; Litvin et al., 2013), and consequently, for
implicit emotional memory processes. Growing evidence suggests that this is the case (Atsak
et al., 2012; Ganon-Elazar & Akirav, 2009; Litvin et al., 2013; Marsicano & Lafenêtre, 2009;
Marsicano et al., 2002; Tan et al., 2011). Importantly, although the CB1R has been primarily
localized to neuronal cells and tissues, this topographical dichotomy has been revised by a
number of studies which report the presence of CB1R receptors in peripheral tissues
(Berdyshev, 2000; Suigiura & Waku, 2000; Wilson, 2001), which further contributes to the
notion that this receptor is involved in an extensive array of physiological processes.
A role for the CB1R in the behavioral expression of stress has been implicated by
studies using CB1R -/- animals, which have reported pronounced stress-like responses in
classical anxiety paradigms, such as the EPM (Haller et al., 2004a, b; Haller et al., 2002; Hill
et al., 2011), socialinteraction test (Martin et al., 2002), and light-dark box (Martin et al.,
2002). Furthermore, it has been reported that CB1R-deficient animals are especially
susceptible to the anhedonic effects of chronic stress exposure (Martin et al., 2002), display
hyperactivation of the HPA axis (Cota et al., 2003; Haller et al., 2004a), are impaired in their
hippocampal neurogenesis (Jin et al., 2004), and respond at lower levels in response to
reinforcing stimuli, such as sucrose and ethanol (Poncelet et al., 2003; Sanchis-Segura et al.,
2004). It has been suggested that CB1R -/- mice exhibit a phenotype that closely resembles
the symptomatic profile of major depression in clinical population (Hill & Gorzalka, 2005).
Clinical trials using CB1R antagonists on individuals with no history of psychiatric diagnoses

Introduction 17
have revealed that chronic blockade of endocannabinoid neurotransmission enhances indices
of anxiety and depression (Christensen et al., 2007; Nissen et al., 2008). However, it is
important to note that preclinical studies have concurrently revealed antidepressant,
anxiolytic (Griebel et al., 2005), anxiogenic (Navarro et al., 1997), and even null effects
(Adamczyk et al., 2008)of CB1R antagonism under certain conditions, which speaks to the
plasticity, bidirectional influence, and general complexity of the ECS. We will touch more on
the biphasic capabilities of endocannabinoid neurotransmission shortly. Further, chronic
treatment with a CB1R antagonist has been shown to mimic the behavioral effects of chronic
stress (i.e., anxiety), such as enhancing passive coping behavior in the forced swim test and
reducing consumption of a sucrose-sweetened water solution (Beyer et al., 2010). On the
other end of the spectrum, CB1R activation has been shown to produce stress-attenuating
effects in similar animal models of anxiety, such as the EPM (Hill et al., 2007; Moreira et al.,
2008; Patel & Hillard, 2006), elevated zero maze (EZM; Kathuria et al., 2003), and light-dark
box (Rutkowska et al., 2006; Scherma et al., 2008), each of which can be used to explore
distinct components of the stress-response and emotional behavior in general, suggesting
that CB1R activation, at least under many conditions, serves to promote anxiolytic-like
responses. Recently, using a battery of behavioral assays, one group demonstrated that both
peripheral and intra-dorsolateral striatum (i.e., directed at the dorsolateral striatum)
microinjection treatments of the CB1R/CB2R agonist, WIN 55,212-2 (WIN), impairs
consolidation of a memory for stimulus-response (Goodman & Packard, 2014), which
implies the ECS as a participant in memory consolidation processes. Nabilone, a synthetic
cannabinoid (also CB1R-selective), has been shown to significantly diminish nervousness in
patients with anxiety in a placebo-controlled study (Fabre & McLendon, 1981). The role of
the CB1R, as well as other endocannabinoid receptors and cannabinergic ligands, in the
regulation of the stress response via HPA axis modulation will be further elucidated in
following sections.
The discovery of endogenous CB1Rs (Herkenham et al., 1990) spurred a
pharmaceutical firm to develop and market a CB1R inverse agonist, SR141716 (named
rimonabant), as a drug capable of fighting obesity, conceptualized in this way due to the
then-limited research on CB1R agonism, which showed effects of enhanced appetite. Clinical
trials targeting obesity demonstrated that anxious and depressive symptoms were a prevalent
response following treatment (van Gaal et al., 2005). Although the drug did indeed affect

18 Introduction
obesity, and even blocked the psychoactive effects of THC, it had to be withdrawn from the
market due to its negative side effects (Mechoulam & Parker, 2013). Despite the
precautionary exclusion criteria, which precluded the involvement of individuals diagnosed
with psychiatric disorders, patients treated with rimonabant developed enhanced anxiety and
stress-related problems, which sometimes manifested in suicidal tendencies (Christensen et
al., 2007). This is expensive yet valuable evidence of the CB1R’s involvement in stress-related
and emotional processes. Studies have since demonstrated that systemic administration of
rimonabant promote stress-related and anxiety-like behavior, as well as anorexia, in rodents
(Blasio et al., 2013).
The CB2 Receptor:Distribution and Function
In contrast to the CB1R, the CB2 receptor (CB2R) is predominantly located in
peripheral immune cells and organs (Munro et al., 1993). While it was originally thought that
CB2Rs were present solely in the peripheral nervous system, recent evidence suggests that
CB2Rs exhibit limited neuronal expression (in comparison to CB1R levels), (Onaiviet al.
2008a), appearing in regions including the amygdala, hypothalamus, hippocampus, VTA,
cerebral cortex, cerebellum, and brainstem (Gong et al., 2006; Zhang et al., 2014). CB2R
mRNA is mainly expressed in immune tissue (Howlett et al., 2002), including white blood
cells, and is also expressed by microglial cells in injured, infected or inflamed CNS tissue
(Benito et al., 2008; Stella 2004). CB2R mRNA has also been found in the spleen, tonsils,
and thymus, which are tissues that are significantly involved in immune cell production
(Cabral & Dove Pettit, 1998). While it has been found that CB2R agonists generally suppress
the functions of cells in these regions, it is possible that both CB1 and CB2 receptors (as well
as other, potentially unrecognized, cannabinoid receptors) contribute to these effects (Cabral
& Dove Pettit, 1998). Enhanced CNS (and other tissue) CB2R expression has been found in
association with some pathological conditions, and it has been widely suggested that the
CB2R is an important component of a general protective biochemical system (Mechoulam &
Parker, 2013; Pacher & Mechoulam 2011). Recently, several studies have indicated that the
CB2R is critically involved in the regulation of mood disorders and emotional responding,
including behavioral responses to stress, by showing that overexpression of CB2R can have
stress-attenuating behavioral consequences(Marco et al., 2011) and, further, that the

Introduction 19
behavioral expression of stress reduction due to pretreatment with endocannabinoid-
modulating compounds can be mediated through CB2Rs (Busquets-Garcia et al., 2011). For
example, in preclinical tests for anxiety, mice lacking CB2Rs have generally been found to
exhibit augmented vulnerability to stressful (aversive)stimuli (Ortega-Alvaro et al., 2011).
Relatedly, in addition to the extensive involvement of exogenous and endogenous
cannabinoids on GABA neurotransmission, it has been reported that in addition to CB1Rs,
CB2Rs are likely involved (Andó et al., 2012). Furthermore, it has recently been
demonstrated that administration of the CB2R-selective phytocannabinoid, β-carophyllene,
promotes alterations in behavior relevant to anxiety and depression (Bahi et al., 2014), and
induces analgesia (Klaukeet al., 2014). This, along with the vast neuronal expression of
CB2R mRNA (Gong et al., 2006; McLaughlin et al., 2014), suggests a role for CB2Rs (and
their ligands) in emotional behavior, emotional memory, and cognitive function. In light of
the CB2R’s perceived protective role, various synthetic CB2-specific receptor agonists have
been developed (Hanuš et al., 1999), which unlike natural endocannabinoids, bind only to
the CB2R (not the CB1R). As CB2R agonists do not appear to cause the psychoactive effects
associated with natural and synthetic CB1R-binding ligands, it seems that targeting the CB2R
offers a viable route for the development of valuable pharmaceutical treatments (Mechoulam
& Parker, 2013). Clearly, the site-specific effects of CB2R manipulation in the context of
stress, emotion-related processes (including emotional memory), and anxiety disorders are of
great value, and thus further research is warranted.
Endogenous Cannabinoid Ligands: Anandamide (AEA) and 2-
Arachidonylglycerol (2-AG)
The discovery of endogenous cannabinoid receptors and further pharmacological
characterization suggested that endogenous molecules, which serve to stimulate or inhibit
the receptors, were present in the mammalian body, and thus initiated the search for
naturally occurring, endogenously produced cannabinoid ligands (endocannabinoids). The
phytocannabinoid, THC, which binds to these receptors, is a lipid compound. It was
therefore assumed that potential endocannabinoids would, similarly, havea lipid structure
(Mechoulam & Parker, 2013). The pursuit for endogenously produced cannabinoid ligands
was realized upon the discovery of the arachidonate-derived lipophilic molecules N-

20 Introduction
arachidonylethanolamide (anandamide (AEA), based on the Sanskrit word ananda (“supreme
joy”); see Fig. 1.4) in the brain and 2-arachidonylglycerol (2-AG) in peripheral tissues
(Devane et al., 1992; Mechoulam et al. 1995). While several other potential
endocannabinoids have been isolated, research has focused on AEA and 2-AG due to their
potent activity at the CB1R (Katona & Freund, 2008; McLaughlin et al., 2014). For example,
AEA demonstrates high affinity for the CB1R (approximately 50 - 100 nM), but given its
partial agonist properties, has poor efficacy at inducing intracellular signal transduction. In
contrast, 2-AG has a slightly lower receptor affinity (approximately 1 - 10 nM), but produces
a robust intracellular response (Hillard, 2000). Consequently, AEA is thought to evoke tonic,
mild CB1R stimulation, which may help to regulate and maintain adaptive homeostatic
functioning (Ahn et al., 2008; Gorzalka et al., 2008).
Figure 1.4. Molecular structure of AEA
Endocannabinoid Neurotransmission
Thus, it seems reasonable to suspect that the ECS, especially endocannabinoid
neurotransmission, is not only a dynamic and critical component of an organism’s active
homeostatic state, but that it also has bearing on emotional memory processes. The various
functions of endocannabinoid neurotransmission have been recently and thoroughly
reviewed (Katona & Freund, 2012). The ECS modifies synaptic branching, plays a role in
growth and development, and modulates locomotor, feeding, pain-related, and emotional
behavior (Katona & Freund, 2012). This system has been described as one which primarily
supports activity related to inter- and intracellular signaling, metabolic functioning, and the
organization of cellular regulation (Alger & Kim, 2011). It has been suggested that tonic
endocannabinoid signaling plays a role in constraining HPA axis (stress-related) activity

Introduction 21
(Riebe & Wotjak, 2011), and that tonic AEA levels may serve as a “gatekeepers” of HPA
axis functioning, which must be lowered to allow for the stress-induced activation of the
HPA axis response (Hill et al., 2009; Patel et al., 2004). Due to the ECS’ central physiological
role in pain modulation, memory processes, cancer, appetite, circadian rhythms,
cardiovascular diseases, immune response, neuroprotection, and energy homeostasis in
mammals and vertebrates, since its discovery it has been the target of a substantial amount
of research (Battista et al., 2012).
As mentioned, the CB1 and CB2 receptors are G-protein coupled seven-
transmembrane domain receptors. Depending on the brain region, these receptors may be
co-localized on neurons expressing GABA, glutamate, or cholecystokinin (CCK) (Katona &
Freund, 2012), or uniformly distributed on inhibitory and excitatory terminals. As lipids,
endocannabinoids cannot be stored in vesicles, and so they have traditionally been thought
to be synthesized ‘on-demand’ in the postsynaptic cell membrane, prior to their release into
the extracellular synapticcleft (Maejima et al., 2001). The biosynthesis of 2-AG appears to be
mediated by the conversion of phosphatidylinositol by phospholipase C (PLC) into
diacylglycerol, which is subsequentlyconverted to 2-AG by diacylglycerol lipase(DGL;
Hillard, 2000).
The understanding of the pathways which support AEA synthesis is more obscured.
Three distinct and independent mechanisms by which AEA is synthesized have thus far
been characterized (for putative pharmacokineticdetails, see Ahn et al., 2008). Post-
synthesis, endocannabinoids are released into the synaptic cleft, where they act retrogradely
on presynaptic exon terminals to suppress co-localized neurotransmitter release at central
synapses, specificallyby prompting the inhibition of adenylylcyclase (AC) activity, which
subsequently causes a reduction in the cyclic adenosine monophosphate (cAMP) cascade
(the activation of which typically leads to the stimulation of neural events), downstream
inhibition of cAMP-dependent protein kinase (PKA), attenuation of presynaptic cell calcium
influx via voltage-gated calcium (Ca2+
) channels (Basavarajappa, 2007), and has various other
presynaptic effects (Howlett, 1995; Mechoulam & Parker, 2013). In this way,
endocannabinoids can interface with various neurotransmitter systems via GABA, glutamate,
acetylcholine, serotonin, opioid, dopamine (DA), and norepinephrine receptors and
interneurons (Freund et al., 2003; Macguire et al., 2013; Witkin, 2005). Activation of
cannabinoid neurotransmission has generally been found to inhibit an array of excitatory and

22 Introduction
inhibitory neurotransmitters, both in the central and peripheral nervous systems
(Mechoulam & Parker, 2013).
Specifically, CB1Rs have been shown to be localized presynaptically on GABAergic
interneurons and glutamatergic neurons (Howlett et al., 2002). Recently, it has been
demonstrated that CB2Rs expressed in the brain, like CB1Rs, exert their effects at via a
similar presynaptic binding mechanism of action (Atwood et al., 2012), as cannabinergic
compounds often bind to presynaptic receptors to hyperpolarize the presynaptic cell. These
data are consistent with the proposed role of endocannabinoid compounds in the
modulation of regulatory neurotransmission (Basavarajappa, 2007). Increased levelsof
postsynaptic intracellular Ca2+
due to excitatory activity causes phospholipids within the
postsynaptic cell to synthesize endocannabinoids, such as AEA and 2-AG, which can occur
with or without the co-activation of G-protein-coupled and other receptors (Piomelli, 2003).
One study in particular demonstrated that electrophysiological stimulation of amygdala
afferents induces a postsynaptic release of endocannabinoids, and thereby prompts LTD of
inhibitory GABAergic synaptic transmission via a presynaptic mechanism (Azad, 2004). This
unique ability to modulate diverse and opposing types of neurotransmission (by acting on
both GABA- and glutamate-expressing neurons) helps to explain some of the behavioral,
psychological, and physiological effects. Of note, not all of these actions have been
demonstrated in various amygdalar nuclei, which may be an important consideration for
future work, given the differences in CB1R signaling mechanisms across neural regions
(Bosier et al., 2010). Although a recent study has demonstrated the presence of diverse
effects within the extended amygdala (specifically, the BNST, the major amygdalar output
pathway) precise endocannabinoid signaling within other regions of the amygdala, such as
the CeA and its subnuclei, remain largely uncharacterized (Gunduz-Cinar et al., 2013).
Importantly, retrograde signaling permits the ECS’ widespread participation in
bioregulatory processes. Numerous studies have utilized CB1R -/- mice and CB1R
antagonists demonstrate that the ECS is critical for adaptive and functional responding
(Riebe & Wotjak, 2011). Blocking or ablating huge components of the ECS, such as the
CB1R, have been found to lead to the suppression of extinction of aversive memories
(Ratano et al., 2014), learning of a water maze test (Varvel & Lichtman, 2002),
neurobehavioral recovery after brain damage (Panikashvili et al., 2005), feeding behaviors
(Pagotto et al., 2006), eye-blink conditioning (Maldonado et al., 2006), and analgesia induced

Introduction 23
by stress exposure (Hohmann et al., 2005). As endocannabinoids are sensitive to neuronal
excitability, they are ideal candidatesas mediators of synaptic homeostatic plasticity(Alger &
Kim, 2011). Notably, CB1R activation leads to stimulation of mitogen-activated protein
(MAP) kinase activity, which is a synaptic mechanism by which cannabinoids affect cellular
plasticity, cellmigration, and potentially neuronal proliferation (Howlett et al., 2002), and
thus their absence has an impressive impact upon these processes. Furthermore,
electrophysiological studies havedemonstrated that various forms of retrograde
endocannabinoid signaling lead to endocannabinoid-mediated short-term depression (STD)
and long-term depression (LTD) in different brain regions (Masanobu, 2014), which helps to
elucidate how endocannabinoids influence the underlying the plasticity mechanisms which
support memory processes. Recently, endocannabinoid-based pharmacologicalapproaches
have received great attention, and it therefore becomes increasingly relevant and necessary to
assess potential side effects, such as the loss, inhibition, or amplification of emotionally-
valenced memories (Rabinak & Phan, 2014), as well as dysregulation of adaptive responses
to stress, which can result in anxiogenic-like responses under certain conditions (Blasio et al.,
2013).
Curiously, ligands that interact similarly with CB1Rs may have vastly distinct
pharmacokinetic profiles (Mechoulam & Parker, 2013), which may be partially due to the
ability of CB1Rs to form heteromeric complexes (a combination of two or more individual
GPCR subunits) with other GPCRs (Pertwee et al., 2010). Recently, a novel form of
cannabinoid-mediated modulation of synaptic transmission has been demonstrated
(Hofmann et al., 2011), although to date this has been shown only in the dentate gyrus. This
group reported that AEA action, under certain conditions, was not found to be mediated by
CB1Rs, CB2Rs, or the non-selective cation channel type-1 vanilloid receptor (transient
receptor potential vanilloid 1 (TRPV1); activated by capsaicin, noxious stimuli, and AEA),
and that AEA effects persisted in CB1R -/- animals. This unique pathway, which may
involve the formation of heteromeric complexes, has yet to be fully explored and is, at
present, far from being characterized. It should be noted that, between AEA and 2-AG,
there exist slight pharmacokinetic differences which may promote distinct patterns of
signaling (McLaughlin et al., 2014), and it is therefore unwise to extend pharmacological
models developed from studies utilizing AEA to those which use 2-AG. Thus, as AEA is the

24 Introduction
chosen cannabinoid compound of present investigation, the literature on AEA
neurotransmission will be presented.
Slightly more complex than stress, anxiety describes the state of the organism’s
developed reaction to stress. Anxiety is associated with non-sensory activation of the HPA
axis (Davis, 1992; Davis, 1997). At present, we intend to directly stimulate the CeA with a
compound related to fear and stress (i.e., the endocannabinoid, AEA), as well as use a
modified IA paradigm and an open field (OF) test as behavioral measures, which exploit
sensory pathways (via an electrical foot shock and exposure to a novel environment), and
thus align well with traditional descriptions of acute stress/fear. Hence, to frame our
discussion in terms of fear and stress seems appropriate. In the present study, we will not
distinguish fear from stress, as it has been demonstrated that both emotional processes share
a fundamental pathway, so the discussion of the expression of stress responses includes
anything that can also be defined as a response to fear (an aversive stimulus).
1.5 Fear, Stress, Emotional Memory and the Central
Amygdala
First, let us summarize the literature on the role of the CeA in mediating the effects
of stress-/fear-related processes, which have been equated functionallywith relation to HPA
axis modulation for the purposes of this investigation, and processes which support the
formation of emotional memory, with a focus on the consolidation phase of these implicit
processes. First, we review the intersection of the ECS, circuitries which promote regulation
of the stress response, and the amygdala. Then, the overlap between endocannabinoid
neurotransmission, the neural mechanisms which support emotional memory, and amygdala
activity will be outlined. In each case, referencing the existing literature, the role of the CeA
will be detailed when possible.

Introduction 25
Cannabinoid Modulation of the Stress Response: A Role for the
Central Amygdala
With respect to the ECS, as mentioned, there is high degree of neuroanatomical
intersection between the expression of endocannabinoid receptors (CB1R, in particular) and
the neural circuitries which support emotional (including stress-related) processes (Witkin,
2005), indicating that endocannabinoids play a significant role in both the regulation of
locomotor function and the information processing required for the perception, response to,
and memory of emotionally salient events. Notably, this includes implicit emotional memory
processes, the dysregulation of which appears to contribute to anxiety and mood disorders.
The first studies which indicated that endocannabinoid signaling may be regulated by stress
and thus, GC signaling, came from a sophisticated series of in vitro experiments which
demonstrated that, within the hypothalamic PVN (as well as the supraoptic nucleus), GCs
evoked a rapid induction of endocannabinoid synthesis and release (Hill & McEwen, 2010).
This paraventricular GC-mediated releaseof endocannabinoids was found to result in the
inhibition of incoming excitatory neurotransmission to CRF neurosecretory cells, providing
the explanation for a fast mechanism by which GCs could terminate HPA axis activity (Di et
al., 2003). As noted by Hill and McEwen (2010), the first implication of these studies was
that endocannabinoid neurotransmission was capable of dampening the HPA axis activation.
The second was that the hypothalamus was a locus for the interaction of endocannabinoids,
stress, and GC signaling (Hill & McEwen, 2010). The first in vivo study examining stress and
endocannabinoid signaling found that exposing mice to acute stress resulted in a reduction in
hypothalamic 2-AG content, while tissue levels of AEA were unaffected (Patel et al., 2004).
The present collection of knowledge suggests that exposure to acute stress (such as an open-
field or restraint apparatus) mobilizes 2-AG levels, and simultaneouslydecreasesAEA
content, in limbic regions (Hill & McEwen, 2010). Further examination of the effects of
acute stress (using a restraint apparatus) revealed no effects of on endocannabinoid ligand
content in the forebrain or cerebellum.
Of relevance to the present study, individuals diagnosed with anxiety and mood
disorders characterized by GC hypersecretion usually exhibit elevated basal
neurotransmission within the amygdala and have exaggerated hemodynamic responses to
stressors (Price & Drevets, 2010). It has been found that CB1Rs are highly expressed in the

26 Introduction
human amygdala, as well as other regions of the limbic system (Killgore & Yurgelun-Todd,
2004), which suggests their involvement in emotional processing in humans. Furthermore,
recent studies on individuals diagnosed with PTSD have supported this notion. In vivo
imaging studies utilizing MRI and positron emission tomography (PET) in populations with
PTSD have indicated that CB1R availability is enhanced (in comparison to healthy and
trauma-exposed controls), and that this effect is especially pronounced in women
(Neumeister et al., 2013). In addition to other regions, this effect was notably found in the
amygdala-hippocampal-cortico-striatal circuitthat has been implicated in PTSD and other
anxiety and mood disorders. Moreover, peripheral concentrations of AEA were found to be
reduced in the PTSD group, and cortisol levels were lower in the PTSD and trauma-exposed
group relative to the healthy control (Neumeister et al., 2013). Another group examined
peripheral levels of circulating endocannabinoid levels in individuals with PTSD that
developed following exposure to the World Trade Center attacks in the United States in
2001 (Hill et al., 2013). This study revealed that, after controlling for various other factors,
circulating peripheral 2-AG content was significantly reduced among individuals meeting the
diagnostic criteria for PTSD (in comparison to those who had not). While no differences
were revealed with respect to AEA and cortisol levels, it is important to consider that
measurements were taken from the periphery, and thus conjectures about neural differences
in 2-AG, AEA, and cortisol signaling cannot be drawn. Interestingly, across the entire PTSD
sample, AEA levels were found to correlate negatively with the degree of intrusive symptom
presentation (Hill et al., 2013). Comparatively, it has been confirmed that in both nonclinical
individuals and individuals living with major depression, circulating AEA concentrations
negatively correlate with levels of subjectively-experienced anxiety (Dlugos et al., 2012; Hill
et al., 2008). While these studies are far from thoroughly elucidating the role of
endocannabinoid signaling stress-related processes, they support the hypothesis that
deficient endocannabinoid signaling may be an important element of the glucocorticoid
(GC) dysregulation associated with PTSD (Hill et al., 2013) and suggest the ECS’
involvement in a host of other anxiety and mood disorders.
A rodent study demonstrated that, within the amygdala, acute stress resulted in a
reduction of AEA content, without influencing levels of 2-AG (Patel et al., 2005), suggesting
that diminished amygdalar AEA content may be associated with stress-inducing situations
and, likewise, that an elevated levelof AEA content may have stress-attenuating effects.

Introduction 27
Subsequent studies (from this same group) confirmed these results, by reproducing a
reduction in amygdalar AEA content without affecting levels of 2-AG (Rademacher et al.,
2008), although they were generally inconsistent with the findings of the first in vitro studies.
Greater consistency was later accomplished using acute restraint stress models in rats, which
was found to produce an increase in 2-AG levels in the PFC, hippocampus, and
hypothalamus (Hill et al., 2007), but not within the amygdala (Hill et al., 2009c). In these
experiments, stress was found to attenuate AEA content within the amygdala (Hill et al.,
2009c), the PFC, and the hippocampus, but not the hypothalamus (Hill et al., 2007). At least
within the amygdala, this reduction in AEA content appears to be partially caused by a rapid
induction of fatty acid amide hydrolase (FAAH) activity, as activity of FAAH was found to
increase 3-fold in this region (Hill et al., 2009c). Once within the postsynaptic cell, AEA is
undergoes enzymatic hydrolysis primarily by FAAH into arachidonic acid and ethanolamine
or glycerol (Ahn et al., 2008; Mechoulam & Parker, 2013). Furthermore, AEA content
becomes significantly diminished within the amygdala in animals exposed to chronic
unpredictable stress (CUS), while neither 2-AG or CB1R binding is affected (Hill et al.,
2008b). Likewise, AEA content is decreased in the amygdala of rats subjected to olfactory
bulbectomy (OBX), with no alterations to levels of 2-AG (Eisenstein et al., 2010). Further,
AEA signaling has been shown to be critically involved in the habituation of the HPA axis to
restraint stress, such that repeated stress produced a decrease in amygdalar AEA content,
while inhibition of AEA hydrolysis attenuated the development of basal corticosterone
hypersecretion in comparison to rats treated with a vehicle compound (Hill et al., 2010).
Finally, this group reported that intra-BLA administration of a CB1R antagonist (AM251)
prior to a final stress exposure prevented the effects of repeated stress-induced decline in
corticosterone (Hill et al., 2010). In addition, studies which have employed genetic deletion
of FAAH have suggested that augmented AEA signaling can promote anxiolytic- and
antidepressant-likeresponses, while disruption of this system may bring about impairments
in adaptive homeostatic behavior (Gunduz-Cinar et al., 2013; Moreira et al., 2008).
Collectively, this literature suggests that the amygdala is a region of emotional processing
which is crucially supported by endocannabinoid neurotransmission, and that functional
alterations in AEA signaling may have particular relevance for the experience of and
habituation to stress, as well as the manifestation of stress-related emotional disorders.

28 Introduction
Along with the ECS, the CeA has been broadly demonstrated to generally play a role
in the amygdala’s modulation of the HPA axis. Ablation of the CeA has been shown to
significantly reduce the secretion of two hormones typically produced in reaction to stress,
adrenocorticotropic hormone (ACTH) (Beaulieu et al., 1987), and corticosterone (Van de
Ker et al., 1991), in response to immobilization stress. Electricalstimulation of the CeA has
been shown to elicit a pattern of behavioral and autonomic changes that constitute a state
highly resembling fear (Roozendaal et al., 1992). Optical stimulation and inhibition of BLA-
CeA synapses bidirectionally has been found to modulate anxiety-like behavior in mice (Tye
et al., 2011). Recently, Ventura-Silva et al., (2013)demonstrated that lesioning the CeA
attenuates anxiety-likebehavior as measured in the elevated-plus maze (EPM), which was
accompanied by a decrease of stress-induced corticosterone levels. Additionally, this group
showed that CeA lesions precluded the appearance of fear behavior in a fear-potentiated
startle paradigm in both non-stress and stressed rats (Ventura-Silva et al., 2013).
With respect to cannabinoids, research on stress and endocannabinoid signaling in
the BLA is vast, while the CeA, in contrast, has received less attention with a few notable
exceptions (Zarrindast et al., 2008). This may be due to the fact that CB1R are expressed
abundantly in the BLA, whereas lower levels of CB1R mRNA have been detected in the CeA
(Marsicano & Lutz, 1999). It has been found that the combination of restraint stress and
CB1R agonist (2.5 mg/kg of THC or 0.3 mg/kg of CP55940) administration produces
robust c-fos induction within the CeA, indicating a synergistic interaction between
environmental stress and CB1R activation (Patelet al., 2005). The early gene c-fos is used as
a marker of neuronal activation following stress exposure and c-fos analysis permits the
identification of the neural regions that play a role in tonic stress activation and reflects the
degree to which they are activated. In addition, this group found that treatment a relatively
high dose of a CB1R antagonist (10 mg/kg of rimonabant) produced elevated levelsof the
early gene c-fos expression. In contrast to CB1R agonism, these levels were not affected by
exposure to restraint stress. A non-CB1R site of action has been suggested for rimonabant
and it is possible that this mechanism is involved (Haller et al., 2002). Furthermore, one
group, using CB1R antagonist AM251, found that CB1Rs localized to the CeA mediate
anxiety-like behavior as measured by the EPM, via interaction with the opioid system
(Zarrindast et al., 2008). Direct stimulation of CeA-localized CB1Rs (with
arachidonylcyclopropylamide (ACPA), an agonist shown to selectivelyactivateCB1Rs; 1.25

Introduction 29
and 5 ng/rat) was found to increase some anxiolytic-like responses, whereas CB1R
antagonism in this region (using AM251; 2.5-100 ng/rat) was found to have no effect on
open arm time and open arm entries, although the two higher doses (25 and 100 ng/rat)
reduced locomotor activity. In line with these findings, a recent study found that intra-CeA
administration of rimonabant (0.5-1.5 μg/side) precipitated stress-related behavior and
anorexia (Blasio et al., 2013). Thus, it seems reasonable to hypothesize that endocannabinoid
signaling within the CeA affects neurotransmission involved in the expression of present
stress states. Further, given the HPA axis’ involvement in memory processes, it follows that
endocannabinoid neurotransmission within the CeA may have downstream effects on
emotional memory.
Cannabinoid Modulation of Emotional Memory in the Central
Amygdala: Effects on Memory Consolidation
Emotional arousal, whether positively-valenced or negatively-valenced, can
significantly increase memory processes, such as consolidation (Roozendaal et al., 2009). In
addition to observed hippocampal involvement, the amygdala is integrally involved in the
consolidation, recall, and extinction of emotionally salient memories (LeDoux et al., 2007).
At present, evidence has accumulated for several purported mechanisms by which the
amygdala mediates stress-related memory enhancement, particularly with regard to the
consolidation phase. Modulatory effects of noradrenergic signaling, GCs (stress-related
hormones), emotional arousal, as well as interactions with other brain regions have all been
implicated (Roozendaal et al., 2009). While an abundance of literature exists on BLA
participation in endocannabinoid signaling, stress, and emotional memory, few studies have
investigated the role of the CeA in these processes, despite the fact that these systems are
involved in functionally overlapping mechanisms. As mentioned, a number of studies have
demonstrated that the CeA is involved in various aspects of emotional memory (Gilpin et al.,
2014), but the literature on endocannabinoid contribution to these processes is sparse.
As noted, the focus here will be on the consolidation phase of emotional memory
processes, for which inconsistent data has been found. To isolate the consolidation phase of
memory, drugs may be administered after a learning event. This excludes any influence on
memory acquisition, as well as influence of any sensory, locomotor, or motivational

30 Introduction
processes which may indirectly affect memory processes (McGaugh, 1966).
Endocannabinoids have been found to affect various forms of memory processes. Systemic
post-training administration of cannabinoid receptor agonists has been found to impair
several forms of memory consolidation (Yim et al., 2008), whereas systemic post-training
injection of cannabinoid receptor antagonists (and inverse agonists) have been reported to
enhance memory under a variety of conditions (Wise et al., 2008; Wolff & Leander, 2003).
To control for the potential confounding factor of non-specificity in studies utilizing
systemic cannabinoid receptor agonist manipulations, specific endocannabinoid degradation
enzyme activity (such as FAAH) can be pharmacologically inhibited. For instance, URB597,
an FAAH inhibitor which increases AEA content in brain regions in which it is
endogenously released, was found to impair memory consolidation in an object recognition
test when it was administered systemicallypost-training (Busquets-Garcia et al., 2011).
Conflicting data have been reported regarding the effects of local infusion of
cannabinoid compounds in discrete neural regions on the consolidation of emotional
memory. For instance, post-training activation of hippocampal CB1Rs (with WIN; 0.25 - 10
μg/rat) has been shown to disrupt LTM consolidation on several behavioral tasks, such as
IA and the Morris water maze (Jamali-Raeufy et al., 2011; Yim et al., 2008; Zarrindast et al.,
2011). In contrast, others have reported enhancing effects of hippocampal AEA
administration (0.17 ng/side) (de Oliveira Alvares et al., 2008). Similarly, evidence has been
provided that emotionally-arousing training (a foot shock) promotes elevated AEA content
within prefrontal-limbic circuitry (specifically, the hippocampus, mPFC, and BLA), and that
enhancement of AEA levels in these regions enhances emotional memory consolidation,
suggesting that endogenously released AEA can modulate emotional arousal effects on the
consolidation of memory (Morena & Campolongo, 2014). In line with this, it has been
shown that hippocampal administration of CB1R antagonist, AM251, impairs the
consolidation of memory for an aversive event (de Oliveira Alvares et al., 2005).
The amygdala has also been noted as another primary neuromodulator of the
consolidation of emotional memory. When microinjected into the BLA, WIN (50 ng/side)
has been demonstrated to enhance emotional memory in an IA paradigm, an effect which
was attenuated by co-administration of AM251 (Campolongo et al., 2009b). Furthermore,
administration of AM251 blocked the typical consolidation-enhancing effect caused by
systemic corticosterone treatment, suggesting that endocannabinoids may not simply

Introduction 31
modulate emotional memory directly, but also indirectly via the modification of GC
neurotransmission (Campolongo et al., 2009b). Similarly, another later study found that
intra-hippocampal AM251 infusion prevented memory enhancement typically induced by
the synthetic GC dexamethasone (de Oliveira Alvares et al., 2010). Thus, it has been
suggested that endocannabinoid neurotransmission is required for mediating GC effects on
memory consolidation (Atsak et al., 2012). Another group demonstrated that blockade of
BLA CB1Rs had no effect on the consolidation of an emotional memory, while intra-BLA
activation of CB1R transmission (or blockade of endocannabinoid reuptake) potentiated the
emotional salience of normally subthreshold fear-conditioning stimuli (Tan et al., 2011). This
was, however, in contrast to an earlier study, which reported that infusion of AM251 into the
BLA disrupted the consolidation of LTM (Bucherelli et al., 2006). Again, differences in
handling procedures, experimental conditions, behavioral tasks, doses (which may contribute
to interactions with various neurotransmitter systems which have robust effects on memory
consolidation; McGaugh, 2000), drug administered, and factors related to arousal, stress, and
emotional state at the time of training may influence cannabinoid effects on emotional
memory and may account for the range of findings reported.
Perhaps most well-documented is the modulatory role of the CeA in paradigms of
unconditioned and conditioned fear (Ciocchi et al., 2010; Tye et al., 2011), fear extinction,
conditioned inhibition (Amano et al., 2010), and of conditioned orienting behavior to
emotionally salient stimuli (El-Amamy & Holland, 2007). It appears that the CeA is a locus
for stress and emotional memory consolidation. As mentioned, various groups have
demonstrated that the CeA plays an integral role in the acquisition, expression,
generalization, consolidation, retrieval, and extinction of conditioned fear (Amano et al.,
2010; Ciocchi et al., 2010; Gilpin et al., 2014; Tye et al., 2011). There is an abundance of
research demonstrating that the CeA is critical for the acquisition and expression of
conditioned fear (Campeau & Davis, 1995b; Davis, 1992; Helmstetter 1992; LeDoux et al.,
1988). It has been found that ablation of the CeA prior to fear conditioning acquisition
prevents the occurrence of the conditioned autonomic responses (e.g., blood pressure, heart
rate, blood flow changes) that typically accompany physiological fear and stress (LeDoux et
al., 1988), which again highlights the CeA’s position as a locus for interaction between the
neurological processes underlying stress and emotional memory. Furthermore, the plasticity
of the lateral subdivision of the CeA (CeL) has been found to contribute to the acquisition

32 Introduction
of conditioned fear, while the efferent projects of the medial subdivision (CeM) have been
found to be excited by emotional stimuli in a manner which decays with extinction (Gilpin et
al., 2014). It has been found that both pre-training and post-training lesions of the CeA
block a number of measures of stress/fear, including fear-conditioned freezing, fear-
potentiated startle, as well as heart rate and blood pressure changes (Rosen, 2004). In a study
using mice, local infusion of AM251 into the CeA resulted in an enhanced fear response in
an auditory fear conditioning paradigm (Kamprath et al., 2011). Although it did not result in
alterations to stress-related behavior as measured by an EPM, this may be due to a variety of
reasons, such as the species used or the experimental conditions employed (Kamprath et al.,
2011). Moreover, another group found that intra-CeA administration of ACPA (2 ng/rat)
immediately post-training decreased IA memory (i.e., emotional memory) consolidation
(Ghisvand et al., 2011), suggesting that, under some aversive conditions, stimulation of
CB1Rs in the CeA may lead to a weakening of emotional memory consolidation.
Furthermore, it has been suggested that the CeA is specifically involved in conditioned, but
not unconditioned fear (Rosen, 2004), which may indicate that the CeA is differentially
involved in various components of the fear response, and that conditioned fear paradigms
may be more appropriate for investigating the role of the CeA in stress and memory (for a
review of the amygdalar cellular specifics involved in fear conditioning, see Rosen, 2004).
This, in combination with fear conditioning studies which manipulate endocannabinoid
activity in the CeA, justifies the choice of using IA to assess emotional memory in this
region.
Thus, cannabinoid signaling in the CeA appears to play a prominent role in the
expression of stress states and the encoding of emotionally salient stimuli into LTM. Despite
the prominent role of endocannabinoid neurotransmission in the modulation of stress-/fear-
related responses (Hill et al., 2010; Ramikie& Patel, 2012), the role of endocannabinoid
signaling in the regulation of CeA stress circuitry has been highly under-investigated, most
likely due to anatomical studies demonstrating weak CB1R immunoreactivity within the CeA
(Kamprath et al., 2011). However, recent studies that utilizenew agents have revealed
abundant expression of endocannabinoid signaling elements at CeA glutamatergic synapses
(for more on mechanistically and temporally distinct modes of postsynaptic
endocannabinoid mobilization in CeL neurons, see Ramikie et al., 2014). Investigation into

Introduction 33
endocannabinoid action within the CeA is therefore necessary to fully clarify stress-related
neurotransmission.

The Present Study
2.1 Objective
The neural circuitry, particularly limbic structures, which support the inhibitory
effects of the ECS on HPA axis activation is not well understood. While there has been an
abundance of research into the involvement of the BLA on stress and emotional memory
consolidation, the literature on the CeA has been much more limited. Although one study
did demonstrate that intra-CeA CB1R agonism (using 50 ng of WIN) failed to affect memory
consolidation (Campolongo et al., 2009b), this study was conducted with male rats. It has
recently been highlighted that significant sex differences exist with regard to the HPA axis
and processing of emotional material (Goel et al., 2014). For example, it has been found that,
following stress exposure, the HPA axis in female organisms produces a greater output of
stress hormones (GCs) and initiates its response more rapidly than within male organisms
(Goel et al., 2014). The clear sex differences, along with the evidence that stress has a greater
negative impact on the psychological well-being of women in comparison to men (Kessler et
al., 1993; Kessler et al., 2005), makes the investigation of endocannabinoid signaling within
the amygdala of female organisms even more crucial, as it is a key component of stress
neurocircuitry. The main objective of this study is to further elucidate the effects of
endocannabinoid signaling in the CeA, specifically with regard to whether AEA infusion into
the CeA of female rats will have an anxiolytic effect as assessed by the OF test, and whether
AEA infusion into the CeA will modulate emotional memory, such that the memory trace of
the aversive context-paired experience is impaired in AEA-treated animals, as assessed by an
IA paradigm.
2.2 Hypothesis
Given the CeA’s putative role in both the behavioral expression of a present stress
state and the modulation of emotional memory, it seems likely that treatment with
endocannabinoid compounds will result in alterations to behavioral expression when

36 The Present
Study
```````````
treatment is combined with stress-related paradigms, such as IA and the OF test.
Specifically, because it has been found that acute stress results in a reduction of amygdalar
AEA content (Patel et al., 2005), it seems that heightened amygdalar content would have, in
contrast, a stress-attenuating effect. Thus, in the OF test, subjects should demonstrate
anxiolytic-like effects, which would support a model in which endocannabinoid signaling in
the CeA participates in the immediate modulation of stress states. Further, while the
information on the CeA and consolidation of emotional memory is scarce, the present
literature on endocannabinoids, the CeA, and emotional memory, suggests that the CeA
serves as a major locus of interaction. As intra-CeA CB1R agonism has been demonstrated to
diminish emotional memory in IA paradigms in studies using rats (Ghisvand et al., 2011), it
is expected that AEA treatment targeted at the CeA will lead to a reduction in the emotional
memory trace, which will be measured by the testing phase of IA.

Materials & Methods
3.1 Animals
Young adult female Sprague-Dawley rats (n = 18), approximately 9 weeks old at time
of stereotaxic surgery, were group-housed in propylene cages and maintained in a
temperature controlled room (22 ± 2°C) on a 12:12-hour light/dark cycle (lights off at
15:00) with ad libitum access to standard rat chow (LabDiet) and water. Rats were handled in
the colony room approximately 1 - 3 times per week by both male and female experimenters.
The purpose of this was to reduce any stress associated with experimenter exposure, as well
as to ensure that differences would not occur due to the sex of the handler. Similarly, during
experimental procedures, both male and female experimenters were present. Rats weighed
200 - 250 g at the time of stereotaxic surgery, and were housed individually following
surgery. All experiments were conducted in accordance with the Institutional Animals Care
and Use Committee (IACUC).
3.2 Stereotaxic Surgeries
All rats were stereotaxically implanted with unilateralchronic indwelling guide
cannulae (stainless steel22-gauge; Plastics One, Roanoke, VA). Cannulae were aimed 4 mm
above the target site, the lateral division of the central amygdala (CeA). Left and right
cannulae placement was counterbalanced across animals. Rats were towel-wrapped and
anesthetized prior to surgery using a combination of xylazine (5 mg/kg intraperitoneally (IP);
Sigma-Aldrich Co., St. Louis, MO) and ketamine (60 mg/kg IP; Sigma-Aldrich Co., St.
Louis, MO). Rats were placed in a stereotaxic frame (Kopf, Tujunga, CA) with the incisor
bar set at -3.5 mm below the ears. If additional anesthetic was required at any point during
the surgery, a supplemental dose of ketamine (0.1 - 0.3 ml IP) was administered. CeA
coordinates relative to Bregma were as follows: posterior 2.3 mm, lateral ± 4.0 mm, and
ventral 3.6 mm. Implants were secured to the skull with stainless steel screws, which were
embedded in the skull (but did not touch the brain), and approximately 4 layers of dental

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