Coallocation of Appetitive and Aversive Memories in the Lateral Amygdala

by

Alexander Jacob

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

Department of Psychology University of Toronto

© Copyright by Alexander Jacob 2016

ii

Coallocation of Appetitive and Aversive Memories in the Lateral

Amygdala

Alexander Jacob

Master of Arts

Department of Psychology

University of Toronto

2016

Abstract

The amygdala plays a key role in representing memories for both fear and reward. However, it

is currently not understood how neurons in this structure differentially code memory traces for

such strikingly different emotions. Amygdala neurons might be valence-specific, able to encode

only fear or reward. Alternatively, these neurons may be equipotent – able to encode memories

of any valence. We examined these two alternatives by allocating a fearful and rewarding

memory to a single population of neurons in the lateral amygdala (LA). Here we show that co-

allocation of these memories leads to overwriting: the fear memory formed first is erased and

replaced by the reward memory formed second. These results provide evidence that LA neurons

are equipotent and capable of switching the valence they encode. Together, these findings

establish support for a dynamic, activity-dependent view of valence allocation in the lateral

amygdala.

iii

Acknowledgements

I would like to acknowledge my supervisor, Dr. Sheena Josselyn for her support and

encouragement throughout the course of my Master’s degree. Her mentorship and assistance

were invaluable in the completion of this thesis.

I would also like to thank my committee members Dr. Morgan Barense and Dr. Junchul Kim for

the excellent discussion, consideration and feedback they contributed to this project.

Thanks also to all members of the Josselyn and Frankland labs, especially Dr. Asim Rashid,

Chen Yan and Liz Hsiang for their continuous help and guidance.

iv

Table of Contents

Table of Contents ........................................................................................................................... iv

List of Figures ................................................................................................................................ vi

Chapter 1: Background and Literature Review ................................................. 1

1.1 Introduction ......................................................................................................................... 1

1.2 Literature Review................................................................................................................ 3

1.2.1 Engram Theory ............................................................................................................... 3

1.2.2 Emotional Memory ......................................................................................................... 6

1.2.3 The Amygdala and Valence ............................................................................................ 7

1.2.3.1 Associative Fear ...................................................................................................... 8

1.2.3.2 Reward Processing ................................................................................................ 10

1.2.4 Engrams in the Amygdala ............................................................................................. 12

1.2.4.1 The Structural View .............................................................................................. 12

1.2.4.2 The Functional View............................................................................................. 15

1.3 Aims .................................................................................................................................. 18

Chapter 2: Materials and Methods .................................................................... 19

2.1 Experimental Design ......................................................................................................... 19

2.2 Mice .................................................................................................................................. 19

2.3 Virus & Surgery ................................................................................................................ 20

2.4 Behavioural Measures ....................................................................................................... 20

2.4.1 Auditory Fear Conditioning .......................................................................................... 20

2.4.1.1 Training ................................................................................................................. 20

2.4.1.2 Testing................................................................................................................... 21

v

2.4.2 Conditioned Place Preference ....................................................................................... 21

2.4.2.1 Habituation ............................................................................................................ 21

2.4.2.2 Conditioning ......................................................................................................... 21

2.4.2.3 Testing................................................................................................................... 22

2.5 Verification of Viral Expression ....................................................................................... 22

2.6 Statistical Analysis ............................................................................................................ 22

Chapter 3: Results ............................................................................................... 23

3.1 Wild-type Mice Successfully Learn Valenced Associations ............................................ 23

3.2 CREB Expression Successfully Localized to the LA ....................................................... 24

3.3 CREB-Overexpressing Animals do not Successfully Learn a Fearful Association ......... 24

3.4 CREB-Overexpressing Animals Successfully Lean an Appetitive Task ......................... 25

Chapter 4: Discussion .......................................................................................... 27

References ............................................................................................................... 30

Copyright Acknowledgements...................................................................................................... 40

vi

List of Figures

Figure 1: Multiple levels of the engram. ......................................................................................... 4

Figure 2: Schematic of amygdala anatomy and connectivity. ........................................................ 8

Figure 3: Schematic of experimental design ................................................................................. 19

Figure 4: Wild type animals form fear and reward memories. ..................................................... 23

Figure 5: Expression of CREB in the LA. .................................................................................... 24

Figure 6: CREB animals show impaired fear memory. ................................................................ 25

Figure 7: CREB animals display normal reward memory. ........................................................... 26

1

Chapter 1 Background and Literature Review

1.1 Introduction

Emotional memories play a critical role in the survival of humans and other animals. The ability

to recall emotionally relevant situations allows an organism to determine what kinds of foods are

safe to eat, which sounds signal danger, and how to distinguish between safe and unsafe

locations. Beyond this, the emotional content of a memory enhances its ability for later recall

(Hamann, 2001), making emotional memories a particularly salient and effective means of

storing information in the brain. Given the biological significance of emotional memory, much

work has been done in neuroscience to characterize this memory system and elucidate the

mechanisms underlying emotional memory formation.

Emotion and emotional memory encompass a strikingly diverse range of internal states,

extending from the strongly unpleasant to the highly pleasurable. This pleasantness-

unpleasantness spectrum – termed emotional valence – is commonly used to describe emotion

across many domains of psychology (Pizarro & Levine, 2006; Russell, 1980). Within

neuroscience, especially the neuroscience of learning and memory, the continuous spectrum of

emotional valence is often broken into two discrete categories – fear and reward. These

emotional states are particularly amenable to neuroscientific study: they are highly evolutionarily

conserved, have clear behavioural readouts in non-human animals, and can be used to generate

long-lasting memories from a single training episode (Izquierdo, Furini, & Myskiw, 2016;

LeDoux, 2012). For these reasons, much work has been done to characterize the brain circuits

underlying representations of fear and reward in the brain.

Fear and reward produce markedly different behavioural responses in animals. Fear is usually

characterized by fleeing behaviours or the engagement of species-typical defense responses, such

as freezing. By contrast, reward is often associated with approach behaviours or increases in

exploration. Given these distinctive behavioural outputs, it is perhaps unsurprising that much of

the early work on emotional memory investigated the fear and reward systems separately. These

studies suggested, for example, that the amygdala was essential for the encoding of fear, while

the ventral tegmental area was an area primarily important in reward (Goddard, 1964; Olds &

2

Olds, 1963; Wise & Bozarth, 1984). However, more recent work has revealed that the roles of

these structures are not so clearly delineated. Fear and reward circuits have increasingly been

shown to overlap, and brain regions once thought to be exclusively involved in one type of

processing are now being shown to have involvement across the spectrum of valence (Ungless,

Magill, & Bolam, 2004; Wassum & Izquierdo, 2015). In particular, a number of recent studies

have highlighted the role of the amygdala in processing memories of both fear and reward

(Beyeler et al., 2016; Namburi, Al-Hasani, Calhoon, Bruchas, & Tye, 2015; Namburi, Beyeler, et

al., 2015). Given the distinctive behaviours associated with these differently valenced memories,

this overlap raises an interesting question: what mechanisms determine how emotionally

valenced information is encoded in the amygdala? How does the amygdala segregate incoming

inputs and ensure the correct behavioural outputs for such strikingly different emotional

memories?

From a theoretical standpoint, there are two possible answers to this question. The first is a

structural or architectural answer –the amygdala is structured such that positively valenced

memories are allocated to one population of neurons, while negatively valenced memories are

allocated to a separate population. This separation is anatomical, with structurally distinct, fixed

fear and reward circuits in the amygdala. These circuits receive different, valence-specific inputs

and have distinct anatomical outputs to downstream regions associated with fear or reward

behaviours.

The second possibility is a functional answer – that the amygdala contains a population of

valence-nonspecific neurons capable of encoding fear or reward depending on activity at the

time of encoding. Under this view, circuits for fear and reward are not anatomically fixed

structures in the amygdala. Rather, these circuits consist of neurons which come to represent

fear or reward memories dynamically as the result of activity in the population during encoding.

A key prediction of this model is that there exists a population of amygdala neurons which are

equipotent – initially capable of encoding memories of either emotional valence.

At present, it is unclear which of these possibilities most accurately reflects the role of the

amygdala in encoding emotional valence. Several recent reviews have advocated for the

structural view (Janak & Tye, 2015; Namburi, Al-Hasani, et al., 2015), and recent evidence

3

indicates that anatomically distinct populations of neurons may code for valence in the

basolateral nucleus of the amygdala (BLA) (Beyeler et al., 2016; Namburi, Beyeler, et al., 2015).

However, findings from several studies of the lateral nucleus of the amygdala (LA, a subregion

of the BLA) lend support to the functional hypothesis. In these studies which separately

examined fear and reward, researchers found that the same molecular mechanisms were

responsible for the allocation of LA neurons into memory traces for both fear and reward (Han et

al., 2009; Hsiang et al., 2014; Yiu et al., 2014). This evidence suggests that the LA might be a

site containing equipotent neurons in the amygdala, and therefore may be a region suited to

examining the functional hypothesis.

The current study formally examines this functional hypothesis. By manipulating principal

neurons in the LA during the encoding of fearful and rewarding memories, we provide direct

evidence that this region contains a population of equipotent neurons. This finding supports a

functional view of the LA and argues against the presence of valence-specific circuits in this

nucleus. More broadly, these findings help to clarify how emotionally valenced memories are

encoded and processed by the amygdala.

1.2 Literature Review

1.2.1 Engram Theory

In order to understand how lateral amygdala neurons encode valence, this study necessarily

makes a series of assumption about memory and its physical substrate in the brain. Engram

theory is a conceptual framework which underpins these core assumptions.

Engram theory seeks to explain how memories are physically represented in the brain. Its

fundamental claim is that individual memories are represented by the activity of a sparse

population of neurons distributed in different brain areas. This sparse population is called the

engram, and activity in this population is both necessary and sufficient to cause recall of a

particular memory.

Josselyn, Köhler, & Frankland (2015) recently put forward four criteria for defining an engram:

4

1. PERSISTENCE – An engram is a physical change in the brain formed in response to a

specific experience. This change is stable and persists over time.

2. ECPHORY – An engram can be expressed behaviourally in response to a retrieval cue. In

other words, it can be recalled or activated by outside stimuli. The process by which an

engram generates a behavioural response is called ecphory.

3. CONTENT – An engram reflects the content of the experience it encodes, and predicts the

activity that will be seen at recall. The pattern of cells present in the engram partially

recapitulates the pattern of activity present at encoding, and is similar to brain activity

during retrieval.

4. DORMANCY – An engram is dormant but present in brain circuits between periods of

encoding and retrieval.

Figure 1: Multiple levels of the engram (adapted from Josselyn et al., 2015). (A) An

engram at the network level. Memory relevant brain areas (pictured in red) share functional

and anatomical with each other and with non-memory structures (cyan). The engram is the

pattern of regions active during encoding and retrieval. (B) Engram population within a single

brain area. The engram is the population of active neurons (red) representing a memory.

5

In addition to these four properties, the engram is theorized to exist at multiple levels of

organization in the brain (Figure 1). Convergent evidence from human fMRI studies and whole

brain mapping in rodents has shown that memory retrieval is accompanied by brain-wide

changes in activity, with some brain regions becoming more active during recall relative to a

baseline state (Schacter & Wagner, 1999; Wheeler et al., 2013). Furthermore, certain brain

regions are known to be more critical for certain types of memory (for example, the

hippocampus for contextual memories). These specialized regions can be thought of as hubs for

a particular engram, and damage or inactivation of such hub regions can impair or abolish

ecphory (Han et al., 2009; Sano et al., 2014; Tanaka et al., 2014). Thus the engram can be

viewed from a network level, where patterns of activity between brain regions characterize the

physical trace of a memory.

On a more local scale, the engram can also be viewed as activity of a population of neurons in a

particular structure. Indeed, sparse populations of engram cells have been found throughout the

brain in areas such as the hippocampus, cortex and amygdala (Chawla et al., 2005; Reijmers,

Perkins, Matsuo, & Mayford, 2007; Sano et al., 2014). By tagging and manipulating these

sparse populations within a single structure, researchers have shown that their activity is both

necessary and sufficient to produce a behavioural recall of memory even in the absence of other

retrieval cues (Tonegawa, Liu, Ramirez, & Redondo, 2015). For example, reactivation of only

2-4% of dentate gyrus neurons active during the encoding of a fearful episode is sufficient to

induce freezing in a novel context not previously associated with the aversive stimulus (Ramirez

et al., 2013). Silencing of this population leads to an attenuation of freezing even in the initially

conditioned context (Denny et al., 2014).

Importantly, this work demonstrates that although the engram is broadly distributed throughout a

network of brain areas, activity of a small population in a single hub region is capable of driving

recall of the entire memory. The present study focusses on such a population of neurons in the

lateral amygdala, and the role of these neurons in representing emotionally valenced memories.

6

1.2.2 Emotional Memory

As introduced above, the emotional memory system offers a means for storing particularly

salient episodes over long periods of time. Philosophers and psychologists have long recognized

that the emotional content of a memory can improve its chances of retention. As early as 80

B.C.E, Cicero discussed the effectiveness of “exceptional beauty or singular ugliness” in

enhancing the memorability of a scene (Cicero, trans. 1954). Francis Bacon similarly

acknowledged that “things which make their impression by way of a strong affection … assist

the memory” (Bacon, 1857). More recently, evidence from cognitive neuroscience has clarified

the mechanisms that underpin this memory-enhancing effect of emotional content (LaBar &

Cabeza, 2006).

In the case of human declarative memory, research has shown that emotional content can

improve recall both through processes of increased attention and improved retention.

Emotionally charged words have been shown to distract human participants from colour-naming

in a Stroop test, indicating that emotional content is capable of strongly capturing attention

(MacKay et al., 2004). This attentional capture takes place across a number of modalities,

including visual search (Eastwood, Smilek, & Merikle, 2001; Frischen, Eastwood, & Smilek,

2008) and dichotic listening (Conway, Cowan, & Bunting, 2001; Nielsen & Sarason, 1981). In

all these cases, emotionally valenced stimuli (e.g. angry faces, taboo words) are seen to interfere

and take priority over the processing of neutral stimuli (Yiend, 2010). Even when attentional

resources are controlled during encoding, emotional stimuli are still encoded more readily than

neutral stimuli (Sharot & Phelps, 2004).

The attentional capture facilitated by emotional content translates into improved retention for

these valenced items (LaBar & Cabeza, 2006). Even when emotional content is irrelevant to the

task at hand, experimental participants have been found to encode this information and recall it

more readily than task-relevant information during a later surprise recall test (MacKay et al.,

2004). This facilitated recall is persistent; emotionally arousing stimuli are remembered more

readily than neutral stimuli up to one year after encoding (Bradley, Greenwald, Petry, & Lang,

1992). A possible reason for this improved retention is that emotional content is preferentially

consolidated. Following sleep, a period theorized to be important in the memory consolidation

process (Rasch & Born, 2013), emotionally charged texts are remembered more readily than

7

neutral texts (Wagner, Gais, & Born, 2011). Stress hormones have also been shown to play a

role in memory consolidation, and release of these hormones often accompanies negatively

valenced episodes (Cahill & McGaugh, 1998).

Together, this evidence illustrates how emotional content may improve related memories.

Emotionally charged stimuli capture attention, are encoded more readily, and enjoy preferential

consolidation. Together, these factors result in greatly improved retention relative to neutral

stimuli. The ability of emotional stimuli to enhance memory performance also finds support

when considered from an evolutionary perspective; strong emotional responses often come from

situations particularly relevant to survival (the reward of consuming food, the fear associated

with a predator encounter). As such, the emotional memory system can be thought of as a

repository for highly salient experiences with strong bearing on survival (Hamann, 2001;

LeDoux, 2012).

1.2.3 The Amygdala and Valence

The distinct and important role of emotional memory is subserved by several specialized brain

regions. Key among these is the amygdala, a temporal lobe structure with strong connections to

the hippocampus and other memory sites (Pitkänen, Pikkarainen, Nurminen, & Ylinen, 2000).

Convergent evidence from human and animal literature has long implicated the amygdala as a

critical region for emotional processing (Janak & Tye, 2015; LaBar & Cabeza, 2006; Phelps &

LeDoux, 2005). The earliest evidence for the amygdala’s role as an emotional centre came in the

late 19th century with reports that damage to the temporal lobe – including the amygdala –

resulted in a loss of some motivated behaviours (Brown & Schafer, 1888). Nearly 50 years later,

Klüver & Bucy (1937) reported that amygdalar lesioning led to a loss of emotional inhibition, as

well as reduced anger and fear responses. Since that time, numerous studies in rodents and other

animals have confirmed the central role of the amygdala in emotional processing (Cahill,

Babinsky, Markowitsch, & McGaugh, 1995; Hamann, 2001; LaBar & Cabeza, 2006; Ledoux,

2000). In humans, lesions of the amygdala result in impaired recall for emotionally arousing

words and images (Adolphs, Cahill, Schul, & Babinsky, 1997; Markowitsch et al., 1994) while

in healthy controls, imaging studies have revealed increased amygdala activity during encoding

of emotionally arousing stimuli (Cahill et al., 1996). From these lines of evidence, it is clear that

the amygdala plays a crucial role in the processing of emotional information.

8

1.2.3.1 Associative Fear

An extensive body of evidence for the amygdala’s role in emotional memory comes from studies

of Pavlovian auditory fear conditioning (Ledoux, 2000; Maren, 2001). In this paradigm, animals

are trained to associate an initially neutral tone (the conditioned stimulus, CS) with an aversive

footshock (unconditioned stimulus, US). Over time, the animal learns to produce the same

species-specific fear responses (for example, freezing) originally elicited by the US in response

to the CS alone. The amygdala is necessary for learning the pairing of CS and US which

underpins auditory fear conditioning; selective lesioning of the amygdala impairs acquisition and

expression of the conditioned fear response (Cousens & Otto, 1998; Maren, Aharonov, &

Fanselow, 1996). Importantly, lesioning produces deficits only in memory for learned CS-US

pairs and not for baseline footshock sensitivity or other motor correlates of fear behaviour

Figure 2: Schematic of amygdala anatomy and connectivity (adapted from Maren, 2001).

The BLA (green), consisting of the basal amygdala (BA) and lateral amygdala (LA). The

central nucleus of the amygdala (CeA) is pictured in brown. The LA receives inputs from the

insular cortex (INS) and medial geniculate nucleus of the thalamus (MGN). The BA also

receives input from the MGN, as well as from hippocampal formation structures such as CA1,

the subiculum (SUB) and entorhinal cortex (EC). The CeA outputs to the dorsomedial

nucleus of the medulla (DMN). Outputs from the BLA to the reward system are pictured in

blue, including the nucleus accumbens (NAc), dorsomedial striatum (DMS) and orbitofrontal

cortex (OFC).

9

(Maren, 1998), highlighting the amygdala’s role as an emotional memory structure and not

simply an emotional output centre.

Much work has focused on amygdalar neuroanatomy and the microcircuits within the amygdala

that control the auditory fear response. In order to understand this microcircuity, we must briefly

examine some of the relevant nuclei which together comprise the amygdaloid complex.

Although the structure of the amygdala is complex and heterogeneous (see Sah, Faber, Armentia,

& Power, 2009 for a detailed review), this paper will limit itself to three main anatomical

regions: the lateral nucleus (LA), basal nucleus (BA) and central nucleus (CeA) of the amygdala

(Figure 2). The LA and BA together make up a structure called the basolateral amygdala (BLA),

but there are important differences in function and connectivity between these two substructures.

The LA receives inputs from a variety of sensory areas, including processed information from

the thalamus and projections from associative cortical areas (Farb, Ruggiero, & LeDoux, 1988;

LeDoux, Farb, & Ruggiero, 1990; Linke, Braune, & Schwegler, 2000; McDonald, 1998). This

makes it a prime candidate site for storing the association between thalamic auditory signals and

somatosensory footshock (Ledoux, 2000). The BA also receives thalamic inputs, but its

connectivity is distinguished by a relatively higher concentration of projections from the ventral

hippocampus (Canteras & Swanson, 1992) which are thought to provide contextual information.

Indeed, lesions to the BA lead to impairment in contextual fear conditioning, in which an animal

is trained to associate an aversive footshock with a particular context, but not to auditory fear

conditioning (Majidishad, Pelli, & LeDoux, 1996). Together, this anatomical evidence suggests

the BLA as a site of where associative representations are formed, with the LA playing a larger

role in auditory association and the BA playing a larger role in contextual association (Ledoux,

2000; Maren, 2001). Both the BA and LA make extensive projections back to the cortex,

including projections to important modulatory centres such as the prefrontal cortex (Duvarci &

Pare, 2014). In this way, the BLA is thought to both hold associative representations and

modulate the activity of diverse neural circuits throughout the brain.

The inputs and outputs of the CeA are varied and complex (Sah et al., 2003), but for the

simplified purposes of the present study, the CeA will be discussed primarily as an output

structure of the amygdala. It receives dense input projections from the BLA and sends afferents

to several brainstem nuclei which effect visceral responses to fear and species-specific defense

behaviours including freezing (Hopkins & Holstege, 1978). Electrical stimulation of the CeA

10

leads heart-rate increases similar to those seen in the auditory fear response (Kapp, Gallagher,

Underwood, McNall, & Whitehorn, 1982). Thus, the CeA can be thought of as the effector

nucleus of the amygdala to downstream targets controlling the physical response to fear.

Together, the amygdalar nuclei described above interact in response to an aversive event to

create a fear memory. Prior to conditioning, the BLA receives signals from auditory areas, but

these signals are not sufficient to drive strong activity in the CeA and the initiation of a fear

response. During auditory fear conditioning, the auditory CS causes activation of thalamic

neurons while the footshock US concurrently activates somatosensory areas. Projections from

these brain regions terminate on principal neurons in the LA, and the concurrent excitement from

these two sources is sufficient to activate LA neurons. This activity propagates through the

amygdala to the CeA, which leads to the initiation of a fear response. Importantly, this CS-US

pairing also results in the potentiation of thalamic-to-LA synapses (Rogan, Stäubli, & LeDoux,

1997). After conditioning, activation of these thalamic projectors alone is sufficient to activate

LA neurons, leading to a recapitulation of the fear response. In this way, pairing of an initially

neutral CS with an aversive US leads to the development of a conditioned response in which the

animal displays fearful behaviour when presented with the CS alone.

1.2.3.2 Reward Processing

Although the amygdala has classically been viewed as a structure responsible for producing fear

responses, in the past decade increasing evidence has implicated it in the representation of

reward as well. From an anatomical standpoint, the amygdala is well positioned to influence

important reward circuits elsewhere in the brain (Figure 2). The BLA sends afferents to the

dorsomedial striatum and orbitofrontal cortex, two regions which play an important role in

controlling motivation, learning, and appetitive behaviours (Corbit, Leung, & Balleine, 2013;

Schoenbaum, Chiba, & Gallagher, 2000; Stalnaker, Franz, Singh, & Schoenbaum, 2007). The

BLA also projects to the nucleus accumbens (NAc), a key site mediating feelings of pleasure and

reward (Britt et al., 2012; Friedman, Aggleton, & Saunders, 2002; Phillipson & Griffiths, 1985).

While it is tempting to consider the amygdala’s role in reward processing as analogous to its role

in the fear system, several findings complicate this interpretation. Stimulation of the BLA does

not result in the direct behavioural correlates of reward in the same way that stimulation of the

CeA produces the behavioural correlates of fear. Furthermore, the BLA is not necessary for the

11

formation of a Pavlovian reward association, as amygdalar lesions do not prevent this form of

learning from taking place (Parkinson, Robbins, & Everitt, 2000). Together, these data suggest a

different role for the amygdala in reward processing. In a recent review, Wassum & Izquierdo

(2015) argue that instead of low-level associations between a predictor stimulus with another

emotionally valenced stimulus (as in the case of fear, in which a neutral tone is associated with

an aversive shock), the amygdala instead holds higher-order associations between stimuli and

more abstract features such as predicted outcomes or reward values. For example, several

studies have reported that lesions of the BLA prevent animals from updating their perceived

value of a reinforcer (Balleine, Killcross, & Dickinson, 2003; Johnson, Gallagher, & Holland,

2009). In these experiments, a food-deprived animal is presented with two rewarding choices

(for example, two differently flavoured food pellets). One of these choices, food A, is presented

ad libitum for the animal to consume; the other choice, food B, is presented briefly only at the

beginning of training. Following several days of this protocol, animals are presented with a

choice to select either food A or B. Mice with an intact BLA select food B, the food which has

not been freely available to them. This behaviour implies that animals have learned some value

associated with each of the food choices, and that continuous exposure to food A has devalued it,

rendering it less appealing than the comparatively rare food B. However, when mice receive

BLA lesions prior to ad lib access to food A, they do not show this devaluation response; during

test they prefer foods A and B equally. From this evidence, researchers have concluded that the

BLA is necessary for updating the appetitive values of various stimuli. Lesions to the BLA also

produce deficits in activities in which an animal must remember the costs and benefits associated

with a given stimulus, or how that stimulus has been rewarded in the past (Salinas, Packard, &

McGaugh, 1993; Wassum & Izquierdo, 2015). All these behaviours rely on associations

between appetitive stimuli and complex reward contingencies and values.

Together, this evidence implies that the BLA as a whole is responsible for higher-order reward

processing. However, when specific populations of cells within the BLA are targeted, we begin

to see evidence for the BLA playing a part in more low-level, direct reward seeking as well.

Several studies have demonstrated that selective activation of BLA neurons projecting to the

NAc can induce appetitive behaviours (Ambroggi, Ishikawa, Fields, & Nicola, 2008; Namburi,

Beyeler, et al., 2015; Stuber et al., 2011). Both Stuber et al. (2011) and Namburi et al. (2015)

targeted and transfected BLA cells projecting to the NAc with channelrhodopsin (ChR2, an

12

excitatory opsin), allowing for optical control of this circuit. They found that mice readily

learned to nose-poke for photostimulation of BLA-to-NAc projecting neurons, indicating that

these afferents are sufficient to reinforce reward-seeking activity. This finding does not conform

to Wassum & Izquierdo’s view of the amygdala as a higher-order reward centre, although

perhaps a distinction must be drawn between whole-BLA lesioning studies cited by Wassum &

Izquierdo and the circuit-selective targeted activation studies done by Stuber and Namburi.

Despite this apparent disagreement, it is clear that the amygdala is capable of influencing

rewarded behaviours both directly and through higher-order processing.

1.2.4 Engrams in the Amygdala

The evidence summarized in the previous sections strongly suggests that the amygdala,

specifically the BLA, plays a role in processing memories for both fear and reward. From the

perspective of engram theory, this implies that there is some ensemble of neurons within the

BLA which form the physical substrate of these valenced memories. Indeed, the BLA satisfies

several of the criteria put forward by Josselyn, Köhler & Frankland for holding an engram. Its

anatomical inputs from various sensory and association areas allow it to respond to experiences,

while its outputs to brainstem nuclei and various reward centres allow it to cause behavioural

changes in response to a retrieval cue. Lesions to the BLA impair the expression of a learned

auditory fear association even weeks after initial learning (Maren et al., 1996), indicating that

some persistent change in the amygdala underlies this association. This finding also provides

indirect evidence that the engram is present but dormant within the BLA.

Given the BLA’s suitability as a site for engram storage, a body of research has investigated how

manipulations of the neuronal ensemble in the BLA can affect memories for fear and reward.

This literature is divided on the issue of valence processing in the BLA. As detailed above, there

are two main theoretical viewpoints on this topic: the structural view and the functional view.

We will examine the evidence for these two viewpoints in turn.

1.2.4.1 The Structural View

The structural view of valence processing holds that fearful and rewarding engrams are

segregated based on the architecture and structural organization of the BLA. This theory argues

that there are distinct populations of BLA neurons which encode either fear or reward. These

13

neurons are defined primarily by their anatomical projections to downstream effector regions, as

well as their responsiveness to valenced stimuli (Namburi, Al-Hasani, et al., 2015).

Early evidence for the structural view came from electrophysiological recordings of BLA

neurons during emotionally valenced tasks (Paton, Belova, Morrison, & Salzman, 2006;

Salzman, Paton, Belova, & Morrison, 2007). In these studies, animals were trained to associate

visual stimuli with either an appetitive or aversive outcome. Following initial training, these

outcomes were reversed such that a stimulus which initially predicted an aversive event now

predicted an appetitive one. Researchers recorded BLA neurons before and after this switch, and

noted that a number of these neurons responded specifically to the valence of the association.

For example, a positive-valence neuron would respond strongly to the visual stimulus when it

was paired with rewarding sucrose delivery, but ceased responding to this same stimulus when it

became paired with an aversive air puff. From these findings, the authors concluded that

subpopulations of BLA neurons were selectively responsive to differently valenced events.

Although this work indicated some valence specificity in the BLA, it left several questions

unanswered. Valence-responsive neurons were not clearly delineated in any way; instead it

seemed that these two populations of neurons were anatomically intermingled in the BLA.

Furthermore, it was unclear whether activity in these neurons had any causal link to fear or

reward memory. To clarify these issues, Namburi, Beyeler, et al. (2015) first sought out features

that might distinguish positive-valence responsive neurons from negative valence responsive

ones. They chose to trace projections backwards from anatomical regions known to be related to

fear and reward behaviours, reasoning that BLA neurons projecting to these regions might be

involved in specific valenced representations. The authors selected the CeA as the terminus of

putative negative-valence BLA neurons, and the NAc as the terminus for positive valence

neurons. Using retrograde tracers, they identified populations of BLA neurons that projected to

these two areas and tagged these populations for further study.

To begin, Namburi, Beyeler et al. examined changes in synaptic strength onto these theorized

fear and reward neurons following emotionally valenced learning. Mice were first trained to

associate an auditory cue with either an aversive footshock or appetitive delivery of sucrose.

Following this training session, the authors examined the ratio of AMPA (α-amino-3-hydroxy-5-

methyl-4-isoxazolepropionic acid) to NMDA (N-methyl-D-aspartate) receptors, a proxy of

14

synaptic strength, at synapses onto their BLA neurons of interest. The researchers noted that fear

training led to an increase in synaptic strength onto CeA projecting, but not NAc projecting

neurons. Reward training had the opposite effect, with synaptic strength increasing for NAc but

not CeA projectors. From this finding, the authors concluded that their two populations of BLA

neurons showed differential synaptic plasticity to differently valenced training episodes, and

therefore might be the sites for differential fear and reward engrams.

To test the causal involvement of NAc and CeA projecting BLA neurons in valenced memories,

the authors next turned to optogenetics. By transfecting these populations with the excitatory

ChR2, Namburi and colleagues gained precise temporal control of their fear and reward circuits.

Following transfection, animals received optogenetic stimulation of BLA neurons projecting to

the CeA or NAc in response to a nose-poking behaviour. The authors found that

photostimulation of NAc projecting neurons in this task was sufficient to cause reinforcement of

nose poking, an appetitive response. Stimulation of CeA projecting neurons did not lead to an

increase in nose-poking, but in a subsequent task, researchers found that stimulation of these

CeA projectors in a particular spatial location led to the avoidance of this location. This

evidence, taken together, suggests a causal role for NAc and CeA projecting BLA neurons in the

acquisition of appetitive and aversive memories respectively.

Although this work demonstrates a role for two distinct BLA populations in acquiring

emotionally valenced memories, it does not demonstrate any persistent responsiveness in these

neurons following a learning event. Given that persistence is a necessary prerequisite of the

engram, Beyeler, Namburi, Glober, Simonnet, et al. (2016) conducted another study in which

they recorded from BLA neurons during the recall of emotionally valenced memories. To first

establish these memories, the authors trained mice to associate one auditory tone with the

delivery of an appetitive sucrose solution, and a second tone with the delivery of an aversive

quinine solution. Once animals had learned to discriminate between these tones and display the

appropriate behavioural responses, the researchers recorded from NAc and CeA projecting

neurons in the BLA. They found that compared to the population of the BLA as a whole, NAc

projecting neurons were significantly more likely to increase their firing in response to the

reward-predictive tone or decrease their firing in response to the tone predicting the aversive

stimulus. Thus, BLA neurons projecting to the NAc were more responsive to cues associated

with reward. However, this relationship did not hold in the opposite direction. CeA projecting

15

neurons were not significantly more likely to increase firing in response to the aversive cue or

decrease firing in response to the appetitive cue, compared with the aggregate BLA population.

It therefore appears that although some populations of BLA neurons show persistent

responsiveness to valenced memories, this relationship alone is not adequate to explain how

emotionally valenced engrams are formed in the amygdala.

In addition to their anatomically distinct circuits, Beyeler, Namburi et al. also identified

approximately 13% of BLA neurons which responded equally to both positive and negatively-

reinforced cues. This finding is consistent with earlier reports of valence-nonspecific neurons in

the BLA (Shabel & Janak, 2009). Together, these findings indicate that the structural,

architecturally separated view of valence processing in the BLA may not capture the full

complexity of emotional memory encoding. Instead, there is a role for valence-nonspecific

neurons in this system.

1.2.4.2 The Functional View

Rather than anatomically distinct, structurally separated circuits for fear and reward, the

functional view holds that valence processing occurs in a single population of valence non-

specific neurons. These neurons are predicted to be equipotent, capable of forming memories of

any valence. Instead of an architectural distinction, the functional view holds that neurons are

allocated to valenced memory traces based on their activity at the time of encoding. In this view,

fear and reward circuits emerge dynamically as the result of activity in the equipotent population

over time.

While the existence of a valence-nonspecific population of BLA neurons cited above is a

necessary prerequisite for the functional view, this observation alone is insufficient support for

the functional view. Instead, these neurons must also be shown to have a causal role in both fear

and reward memories, and that single neurons within this population are capable of encoding

both fear and reward memories. As well, there must be some mechanism by which this

equipotent population determines which neurons will be allocated into a given memory trace.

Several papers have worked toward establishing these criteria.

The first evidence for functional allocation of valence in the BLA emerged in 2007 when Han et

al. reported that the transcription factor CREB (cyclic adenosine monophosphate response

16

element binding protein) influenced which neurons in the LA were included in a fear memory

engram. The authors found that by overexpressing CREB in a random 20% of LA neurons, they

could bias these neurons to become preferentially recruited into a subsequently formed fear

memory trace. Han et al. quantified this bias by examining neurons positive for the immediate

early gene Arc (activity-regulated cytoskeleton-associated protein), a marker of recent neuronal

activity (Guzowski, McNaughton, Barnes, & Worley, 1999) shortly after recall of an aversive

memory. Neurons expressing Arc at this timepoint must have been active during fear memory

retrieval and were therefore considered part of the memory trace. Han et al. observed that

CREB-overexpressing neurons were significantly more likely to be Arc-positive as compared

with non-infected cells. This finding suggests that CREB-expressing neurons are

disproportionately represented in a valenced memory trace, and thus that CREB is initiating

some intracellular change in LA neurons which is biasing their inclusion in the engram. Further

research revealed that CREB was exerting this intracellular effect by increasing neuronal

excitability (Yiu et al., 2014; Zhou et al., 2009), the propensity of a neuron to fire an action

potential. Other methods of increasing excitability, either through pharmacogenetic or

optogenetic means, were shown to reproduce this same preferential allocation of LA neurons into

the engram.

This excitability-dependent allocation of LA neurons into the engram offers a possible

mechanism by which the amygdala might dynamically form valenced memories. At any given

time, a subset of LA neurons are more excitable than their neighbours due to stochastic changes

in membrane properties or gene expression. This population is allocated into an engram formed

at that time. As neuronal excitability continues to fluctuate in the LA population, different

neurons will be allocated to subsequently formed memories.

While this potential mechanism for valence allocation is a requirement for the functional

hypothesis, it is also necessary to show a causal link between LA neurons and emotional memory

engrams. Two studies, using similar techniques, have established that LA neurons are necessary

for the persistent representation of differently valenced emotional memories.

In the first of these studies, Han et al. (2009) demonstrated the necessity of CREB-expressing

LA neurons to a fear memory. The authors first overexpressed CREB in a random 10% of LA

neurons, then trained mice on an auditory fear conditioning task. Animals were then tested to

17

assess successful formation of the fear memory engram, and following this test Han et al. utilized

a transgenic inducible diptheriatoxin (iDTR) system to selectively ablate CREB-overexpressing

neurons. They then tested animals’ fear memory a second time, and noted that ablation of CREB

neurons led to a significant decrease in freezing. Behaviourally, it was as if animals had

forgotten the tone-fear association following ablation. This memory erasure effect occurred only

when CREB-expressing neurons (and not an equally-sized random population of LA neurons)

were ablated, indicating that these CREB neurons were necessary for the representation of the

fear memory.

Hsiang et al. (2014) performed a similar experiment examining an appetitive memory formed in

a conditioned place preference (CPP) task. In CPP, an animal is placed in a two-chambered box

and trained to associate one chamber with a rewarding cocaine injection. During a subsequent

test phase, the animal is allowed to roam freely between the two chambers and choose where to

spend its time. Animals exhibiting a place preference will stay in the chamber where they

received a cocaine injection, showing behavioural evidence for an appetitive memory formed by

this rewarding experience. Hsiang et al. were able to show, using similar CREB-overexpression

and iDTR techniques described above, that ablation of LA CREB neurons could erase a cocaine

reward memory. This finding provides evidence that the LA plays a critical role in holding

engrams for reward, and does so using the same CREB-mediated excitability-dependent

mechanisms as are seen in fear.

From these studies, it is apparent that LA neurons are necessary for storing and maintaining

emotional memories. Furthermore, these neurons use an identical, excitability-dependent

mechanism to determine inclusion into both fear and reward engrams. Given this evidence, it is

reasonable to conclude that LA neurons may be equipotent, able to hold memories of any

emotional valence. As such, the LA is a site uniquely suited to examine the functional

hypothesis of valence processing. The present study provides the first direct support for this

hypothesis by allocating a fear memory and reward memory to the same population of LA

neurons.

18

1.3 Aims

The primary aim of this paper is to determine whether LA neurons are equipotent. In order to

achieve this aim, this study examines a specific question: what is the effect on memory recall

when a fear memory (formed by auditory fear conditioning) and a reward memory (formed by

cocaine-cued conditioned place preference) are artificially allocated to the same ensemble of LA

neurons? Do mice successfully express both fear and reward memories during a subsequent

memory test?

We hypothesize that mice will not successfully express both fear and reward memories following

co-allocation. Instead, there will be an overwriting effect: the fear memory formed first will be

erased and replaced by the reward memory formed second. Such an overwriting effect would

provide evidence that LA neurons are equipotent. For overwriting to occur, neurons must

change the valence they encode from negative to positive, implying that they are initially capable

of coding either valence.

Chapter 2 Materials and Methods

2.1 Experimental Design

This study was divided into three phases: pre-training, training and testing (Figure 3).

During pre-training, animals were first habituated to the CPP conditioning chamber. On the

following day, mice received viral infusion of CREB bilaterally into the lateral amygdala.

Following two days of recovery, animals entered the training phase.

In this phase, animals were first given a session of auditory fear conditioning. Four hours after

this training, animals were handled individually for 10 minutes each. One day later, animals

received CPP conditioning (see section 2.4.2.2 below). Following an additional rest day, animals

entered the test phase

During testing, animals were first placed back into the CPP chamber and their place preference

was assessed. One day later, animals were re-exposed to the auditory CS in a new context, and

freezing was measured. Following the last testing session, animals sacrificed and their brains

were sectioned and imaged to confirm the extent of viral infection.

2.2 Mice

This experiment utilized wild-type male and female F1 hybrid mice (C57BL/6NTac ×

129S6/SvEvTac) aged between 7-10 weeks. These animals were bred at the Hospital for Sick

Children and were group housed (4 animals per cage) on a 12-hour light/dark cycle. Food and

water were available to the animals ad libitum. All experiments took place during the light phase

Figure 3: Schematic of experimental design

20

of the cycle. Experiments were carried out in accordance with the policies of the Hospital for

Sick Children Animal Care and Use Committee and the standards set forth by the Canadian

Council on Animal Care.

2.3 Virus & Surgery

A replication-defective herpes simplex viral vector was used to infect a sparse, random

population of LA principal neurons in this study. This vector expressed either GFP or the

experimental construct – a GFP-CREB fusion protein – under the IE4/5 promoter. HSV

expression peaks at 3 days post-infusion and persists for 10-14 days (Barrot et al., 2002;

Carlezon et al., 1997; Josselyn et al., 2001; Vetere et al., 2011), making it an appropriate tool for

overexpressing CREB in this study.

Prior to the training phase, experimental animals received viral infusions bilaterally. Mice were

anesthetized with chloral hydrate (400mg/kg, i.p.) and placed in a stereotaxic frame.

Craniometies were opened bilaterally above the LA (AP = -1.25, ML = ±3.4, V = -5.0 mm from

bregma) according to (Paxinos & Franklin, 2001). A glass micropipette attached to a

microsyringe was lowered into the brain and virus was infused (1.5µL per side) at a rate of

0.1µL/minute. Following infusion, the micropipette was left in place for an additional 5 minutes

to allow for diffusion of the virus. The micropipette was then retracted and the incision was

closed. Mice were treated with analgesic (ketoprofen, 5mg/kg subcutaneously) and allowed to

recover in their home cage.

2.4 Behavioural Measures

2.4.1 Auditory Fear Conditioning

2.4.1.1 Training

An aversive memory was formed through the use of auditory Pavlovian fear conditioning.

Animals were placed in a Plexiglas conditioning chamber capable of delivering footshocks

through floor mounted shock bars. Animals were recorded as they freely explored the chamber

21

for 2 minutes. Following this period, a tone (2800 Hz, 85dB, 30 seconds) was played and co-

terminated with a footshock (2 sec, 0.7 mA). Animals remained in the chamber for an additional

30 seconds following the termination of the footshock.

2.4.1.2 Testing

Memory for the association between tone and aversive shock was assessed by presenting the

previously trained tone CS to mice placed in a novel context. To generate this new context,

opaque plastic sheets were used to cover the floor and change the layout of the conditioning

chamber used during training. As well, the chamber was illuminated by near-infrared light and

sprayed with 70% ethanol to change the visual and olfactory properties of the environment.

Mice were placed into this new context and their movements were recorded by a front-mounted

digital camera within the chamber. Mice were allowed to explore the chamber for 2 minutes,

then the auditory tone was presented for 1 minute. Freezing behaviour, defined as immobility

and a hunched posture lasting for bouts of more than one second, was quantified using automated

scoring software (Video Freeze version 2.7.1, Med Associates).

2.4.2 Conditioned Place Preference

2.4.2.1 Habituation

Cocaine-cued conditioned place preference (CPP) was used to create an appetitive memory. In

this paradigm, animals were first exposed to the conditioning apparatus: a pair of 15 × 20 cm

Plexiglas chambers connected by a guillotine door. Each chamber was distinguished by a unique

combination of visual, tactile and olfactory cues: one chamber had white walls and a clear, rough

plastic floor while the other chamber had dark walls and a smooth white plastic floor smelling of

acetic acid. Mice were allowed to explore the apparatus, moving freely between the chambers

for 10 minutes.

2.4.2.2 Conditioning

The conditioning phase of CPP consisted of two sessions: morning and afternoon. During the

morning session, mice were injected with 0.2mL of saline and placed in one chamber of the

conditioning apparatus (placement will be counterbalanced between light and dark chambers).

22

The guillotine door was closed and animals were confined to the chamber for 15 minutes.

Following this, animals were returned to their home cage.

During the afternoon session, which began 4 hours after the end of the saline session, animals

were injected with cocaine (30 mg/kg dose) and placed into the opposite conditioning chamber

for 15 minutes.

2.4.2.3 Testing

During testing, animals were returned to the apparatus. The guillotine door was opened and

mice were allowed to freely move between chambers. Their movement was tracked by an

overhead digital camera, and time spent in each chamber was automatically quantified using

scoring software (LimeLight version 3.4.05, ActiMetrics).

2.5 Verification of Viral Expression

Following the final testing session, animals were transcardially perfused first with 40mL of 0.1M

phosphate buffered solution (PBS), then with 40mL of 4% paraformaldehyde (PFA). Brains

were collected and stored in PFA for an additional 24 hours before being transferred to PBS.

Specimens were then mounted in an adhesive compound and 50µm coronal sections were

collected using a vibratome. Sections containing the LA were then imaged on an upright

epifluorescence microscope to qualitatively determine successful expression of the HSV vector.

2.6 Statistical Analysis

Statistica software (Statsoft, version 13) was used to perform one-way and two-way repeated

measures ANOVAs. Post hoc tests were conducted using the Newman-Keuls method.

23

Chapter 3 Results

3.1 Wild-type Mice Successfully Learn Valenced Associations

To first assess the validity of our experimental design, we trained wild-type mice on an aversive

(tone-fear conditioning) and appetitive (conditioned place preference) task. Mice successfully

learned both these associations. Following tone-fear conditioning, mice spent a significantly

greater proportion of time freezing in response to the tone CS (M = 64.86%, SD = 8.26, n = 8) as

compared with the pre-tone period (M = 16.66%, SD = 8.26, n = 8) [one way ANOVA (pre-tone

freezing, tone freezing), F(1,14) = 88.74, p < 0.001]. As well, mice had significantly higher CPP

scores (defined as time spent in the cocaine-paired chamber minus time spent in the saline-paired

chamber) after the cocaine conditioning session (M = 494.30, SD = 230.91, n = 8) as compared

with prior to conditioning (M = 57.14, SD = 116.49, n = 8) [one way ANOVA (pre-conditioning,

post-conditioning), F(1,14) = 22.85, p < 0.001].

Figure 4: Wild type animals form fear and reward memories. (A) Wild type animals (n=8)

displayed significantly more freezing during the tone-on period compared with the tone-off

period [p<0.001]. (B) Wild type animals spent significantly more time in the cocaine-paired

chamber following CPP conditioning compared with the pre-conditioning session [p < 0.001]

24

3.2 CREB Expression Successfully Localized to the LA

To confirm the successful expression of CREB in LA neurons, the brains of experimental

animals were sectioned and imaged. GFP signal was used as a proxy measure for CREB

expression. Collected images were compared to the atlas of Paxinos & Franklin (2001), and all

animals were confirmed to have strong HSV expression localized to the LA

3.3 CREB-Overexpressing Animals do not Successfully Learn a Fearful Association

We next examined the ability of CREB-overexpressing mice to learn fearful and rewarding tasks.

Unlike their WT counterparts, CREB mice were unable to successfully form a tone-fear

association. Comparing these CREB animals to controls expressing GFP only, a repeated

measures ANOVA revealed a significant effect of Treatment [(GFP, CREB), F(1,6) = 18.47, p <

0.001], and a significant effect of Session [(pre-tone, tone), F(16) = 12.92, p < 0.05].

Figure 5: Expression of CREB in the LA. Representative image of GFP signal localized to the

LA.

25

Post-hoc Newman-Keuls testing indicated significantly greater freezing in GFP animals after

tone presentation (M = 44.32, SD = 16.64, n = 4) compared with the pre-tone period (M = 15.41,

SD = 10.82, n = 4) [p < 0.05], but no such difference between pre-tone (M = 5.38, SD = 2.84, n =

4) and tone (M = 20.12, SD = 4.47, n = 4) freezing in CREB animals [p > 0.05]. As well, GFP

controls froze significantly more than CREB animals when the tone was presented [p < 0.01].

These data indicate that GFP-infused animals learned the fearful association, but CREB-

overexpressing animals did not.

3.4 CREB-Overexpressing Animals Successfully Lean an Appetitive Task

One day after animals’ fear memories were tested, their memory for reward was assessed. In this

test, CREB animals performed comparably to controls. A repeated measures ANOVA failed to

reveal a significant effect of Treatment [(GFP, CREB), F(1,6) = 0.88, p > 0.05] or a Treatment ×

Session interaction [F(1,6) = 3.38, p > 0.05], but indicated a significant effect of Session [(pre-

conditioning, conditioning), F(1,6) = 483.31, p < 0.001].

Figure 6: CREB animals show impaired fear memory. Compared to GFP-expressing controls

(n=4), CREB-overexpressing animals (n=4) show significantly lower freezing during the tone-on

period [p < 0.01].

26

Newman-Keuls post-hoc tests showed that after conditioning, GFP animals (M = 508.70, SD =

76.43, n = 4) had significantly higher CPP scores compared to prior to conditioning (M = 25.17,

SD = 95.90, n = 4) [p < 0.001]. The same was true of CREB animals, whose post-conditioning

scores (M = 420.88, SD = 92.45, n = 4) were significantly higher than those recorded before

conditioning (M = 11.95, SD = 52.77, n = 4) [p < 0.001]. There was no significant difference

between GFP and CREB animals either before or after conditioning. These findings indicate that

both experimental and control animals learned the rewarding task equally well.

Figure 7: CREB animals display normal reward memory. Both CREB animals (n=4)

and GFP controls (n=4) spend significantly more time in the cocaine-paired chamber

following conditioning as compared with the pre-conditioning session [p < 0.001].

27

Chapter 4 Discussion

This study investigated the effect of co-allocating two differently valenced emotional memories

to the same population of lateral amygdala neurons. Co-allocation was achieved by virally

overexpressing the transcription factor CREB in a random subset of LA neurons, which biased

these neurons to be preferentially included in subsequently formed memory traces. We took

advantage of this privileged status of CREB neurons and sequentially trained experimental

animals on a fearful task followed by a rewarding task, ensuring that both these memories would

be allocated to the same CREB-expressing neurons. This co-allocation had divergent effects on

the retrieval of the two memories. Behaviourally, we observed no recall of the fear memory

formed first, but robust and preserved memory for the reward memory formed second. This

pattern of results implies that the reward memory overwrote and erased the fear memory.

This overwriting effect supports the functional view of valence processing, and provides

evidence that LA neurons are equipotent. We theorize that during training, CREB-expressing

neurons first came to represent the fear memory formed on day 5 (Figure 3). On day 6, these

neurons were again recruited into a new memory trace, this time of the opposite valence. The

fact that this second reward memory was successfully expressed at test indicates that LA neurons

were able to switch the valence they encoded, moving from their initial aversive representation

to an appetitive one. The structural view, which argues that individual LA neurons are unable to

change the valence they represent, is unable to account for this pattern of findings. By contrast,

the functional view characterizes our results well. Under this view, populations representing fear

and reward emerge dynamically as a result of neuronal activity at the time of encoding. Here,

we have shown that when a population of LA neurons is pre-selected to store emotional

memories, these neurons can change the valence they represent in order to capture the most

recently memory episode. Thus, our observation of an overwriting effect provides the first

evidence that LA neurons are equipotent.

While our evidence lends support to the functional hypothesis, an alternative explanation from

the functional camp could be that our CREB infection targeted only reward-circuit neurons.

Under this view, our lack of fear memory is unsurprising as we are allocating this memory to a

population of neurons unable to effect a fearful behavioural response. While this explanation is a

28

plausible interpretation of our evidence taken in isolation, it fails to account for a large body of

previous literature. Several studies have shown, using methods identical to those described here,

that CREB overexpression in the LA is sufficient to bias infected neurons to a fear memory

formed shortly after infusion (Han et al., 2007, 2009; Zhou et al., 2009). This is precisely what

occurs on day 5 of our protocol when animals are trained on an auditory fear task. It is difficult

to reconcile why this intervention should result in allocation to the fear circuit in those previous

studies, but not in this case. Instead, the more parsimonious explanation is that the initial fear

memory is allocated to CREB-expressing neurons, and is overwritten and erased by the

subsequent fear memory.

Another possible explanation of these data is simply that the behavioural tasks used to establish

fear and reward memories interfere with one another. Auditory fear conditioning and CPP are

strikingly different tasks that assay different behavioural outputs and use different modalities to

establish our memories of interest. These complicating factors could make the tasks difficult for

animals to learn, and therefore produce the pattern of results described above. This interference

explanation finds some support from the low levels of freezing shown in our GFP control group.

This finding could be interpreted to mean that even when activity in the LA is not manipulated

by CREB, animals still display impaired freezing. However, interference fails to account for our

preserved and robust levels of both fear and reward memory in wild type animals. Given that

these animals were trained on the same paradigm, it stands to reason that they too should show

the fear memory impairment seen in the other groups. Since this is not the case, it is unlikely

that our behavioural measures alone have produced the results seen here. As well, GFP animals

show a significant increase in freezing during the tone period which is not observed in the CREB

group, indicating that these animals have successfully formed a fearful association, even if its

magnitude is smaller than in the wild type group.

In all, these results provide the first evidence that neurons in the lateral amygdala are not

segregated into valence-specific circuits. Rather, they are equipotent and come to represent

particular valenced memories as the result of a dynamic, excitability-mediated selection process

occurring during encoding. Our results further indicate that LA neurons are only capable of

holding a single valence of memory at any given time. The overwriting effect we have observed

in these experiments shows that one population of neurons cannot effectively represent both fear

and reward simultaneously. Rather, it seems that the memory formed most recently

29

predominates and overwrites a previous valenced engram stored in the same population.

However in a natural setting where CREB has not been manipulated, it seems unlikely that

differently valenced memories would be allocated to the same neuronal population and interfere

with one another. Indeed, in this study we observed strong memories for both fear and reward in

our wild-type control group. It is therefore interesting to consider how activity in the LA might

regulate the allocation of differently valenced memories in such a way that they do not overlap or

interfere with one another under normal conditions. This question plays directly into a growing

literature within engram research which examines how multiple memories interact. For example,

recent work has shown that two memories of the same valence formed close together in time

become allocated to the same population of amygdala neurons (Rashid et al., 2016). Considered

in this light, the present study represents the first steps into investigating how different emotional

engrams are allocated in the amygdala. Future work will be required to elucidate the

mechanisms underlying the differential processing of emotional engrams, and ultimately how the

memory system performs the critical task of distinguishing between distinct memory episodes.

References

Adolphs, R., Cahill, L., Schul, R., & Babinsky, R. (1997). Impaired declarative memory for

emotional material following bilateral amygdala damage in humans. Learning & Memory

(Cold Spring Harbor, N.Y.), 4(3), 291–300. http://doi.org/10.1101/lm.4.3.291

Ambroggi, F., Ishikawa, A., Fields, H. L., & Nicola, S. M. (2008). Basolateral amygdala neurons

facilitate reward-seeking behavior by exciting nucleus accumbens neurons. Neuron, 59(4),

648–661. http://doi.org/10.1016/j.neuron.2008.07.004

Bacon, F. (1857). The New Organon. In S. and Ellis (Ed.), The Works of Francis Bacon.

London: Longman.

Balleine, B. W., Killcross, A. S., & Dickinson, A. (2003). The effect of lesions of the basolateral

amygdala on instrumental conditioning. The Journal of Neuroscience : The Official Journal

of the Society for Neuroscience, 23(2), 666–75.

http://doi.org/http://www.jneurosci.org/content/23/2/666.short

Barrot, M., Olivier, J. D. A., Perrotti, L. I., DiLeone, R. J., Berton, O., Eisch, A. J., … Nestler, E.

J. (2002). CREB activity in the nucleus accumbens shell controls gating of behavioral

responses to emotional stimuli. Proceedings of the National Academy of Sciences of the

United States of America, 99(17), 11435–40. http://doi.org/10.1073/pnas.172091899

Beyeler, A., Namburi, P., Glober, G. F., Luck, R., Wildes, C. P., Tye, K. M., … Tye, K. M.

(2016). Divergent Routing of Positive and Negative Information from the Amygdala during

Memory Retrieval. Neuron, 90(2), 348–361. http://doi.org/10.1016/j.neuron.2016.03.004

Bradley, M. M., Greenwald, M. K., Petry, M. C., & Lang, P. J. (1992). Remembering pictures:

pleasure and arousal in memory. Journal of Experimental Psychology: Learning, Memory,

and Cognition, 18(2), 379–390. http://doi.org/10.1037/0278-7393.18.2.379

Britt, J. P., Benaliouad, F., McDevitt, R. a., Stuber, G. D., Wise, R. a., & Bonci, A. (2012).

Synaptic and Behavioral Profile of Multiple Glutamatergic Inputs to the Nucleus

Accumbens. Neuron, 76(4), 790–803. http://doi.org/10.1016/j.neuron.2012.09.040

31

Brown, S., & Schafer, E. A. (1888). An Investigation into the Functions of the Occipital and

Temporal Lobes of the Monkey’s Brain. Philosophical Transactions of the Royal Society of

London, 179, 303–327.

Cahill, L., Babinsky, R., Markowitsch, H. J., & McGaugh, J. L. (1995). The amygdala and

emotional memory. Nature, 377(6547), 295–296. http://doi.org/10.1038/377295a0

Cahill, L., Haier, R. J., Fallon, J., Alkire, M. T., Tang, C., Keator, D., … McGaugh, J. L. (1996).

Amygdala activity at encoding correlated with long-term, free recall of emotional

information. Proceedings of the National Academy of Sciences of the United States of

America, 93(15), 8016–8021. http://doi.org/10.1073/pnas.93.15.8016

Cahill, L., & McGaugh, J. L. (1998). Mechanisms of emotional arousal and lasting declarative

memory. Trends in Neurosciences, 21(7), 294–299. http://doi.org/10.1016/S0166-

2236(97)01214-9

Canteras, N. S., & Swanson, L. W. (1992). Projections of the ventral subiculum to the amygdala,

septum, and hypothalamus: a PHAL anterograde tract-tracing study in the rat. The Journal

of Comparative Neurology, 324(2), 180–94. http://doi.org/10.1002/cne.903240204

Carlezon, W. A., Boundy, V. A., Haile, C. N., Lane, S. B., Kalb, R. G., Neve, R. L., … White, F.

J. (1997). Sensitization to morphine induced by viral-mediated gene transfer. Science (New

York, N.Y.), 277(5327), 812–4. http://doi.org/10.1126/science.277.5327.812

Chawla, M. K., Guzowski, J. F., Ramirez-Amaya, V., Lipa, P., Hoffman, K. L., Marriott, L. K.,

… Barnes, C. A. (2005). Sparse, environmentally selective expression ofArc RNA in the

upper blade of the rodent fascia dentata by brief spatial experience. Hippocampus, 15(5),

579–586. http://doi.org/10.1002/hipo.20091

Cicero. (trans. 1954). Rhetorica ad Herennium. , III (p. 221) (H. Caplan trans.). Cambridge,

Mass: Harvard University Press.

Conway, A. R. A., Cowan, N., & Bunting, M. F. (2001). The cocktail party phenomenon

revisited: The importance of working memory capacity. Psychonomic Bulletin & Review,

8(2), 331–335. http://doi.org/10.3758/BF03196169

32

Corbit, L. H., Leung, B. K., & Balleine, B. W. (2013). The Role of the Amygdala-Striatal

Pathway in the Acquisition and Performance of Goal-Directed Instrumental Actions.

Journal of Neuroscience, 33(45), 17682–17690. http://doi.org/10.1523/JNEUROSCI.3271-

13.2013

Cousens, G., & Otto, T. (1998). Both pre- and posttraining excitotoxic lesions of the basolateral

amygdala abolish the expression of olfactory and contextual fear conditioning. Behavioral

Neuroscience, 112(5), 1092–1103. http://doi.org/10.1037/0735-7044.112.5.1092

Denny, C. A., Kheirbek, M. A., Alba, E. L., Tanaka, K. F., Brachman, R. A., Laughman, K. B.,

… Hen, R. (2014). Hippocampal memory traces are differentially modulated by experience,

time, and adult neurogenesis. Neuron, 83(1), 189–201.

http://doi.org/10.1016/j.neuron.2014.05.018

Duvarci, S., & Pare, D. (2014). Amygdala Microcircuits Controlling Learned Fear. Neuron,

82(5), 966–980. http://doi.org/10.1016/j.neuron.2014.04.042

Eastwood, J. D., Smilek, D., & Merikle, P. M. (2001). Differential attentional guidance by

unattended faces expressing positive and negative emotion. Perception & Psychophysics,

63(6), 1004–1013. http://doi.org/10.3758/BF03194519

Farb, C. F., Ruggiero, D. A., & LeDoux, J. E. (1988). Projections from the acoustic thalamus

terminate in the lateral but not central amygdala. Soc. Neurosci. Abstr., 14, 1227.

Friedman, D. P., Aggleton, J. P., & Saunders, R. C. (2002). Comparison of hippocampal,

amygdala, and perirhinal projections to the nucleus accumbens: combined anterograde and

retrograde tracing study in the Macaque brain. The Journal of Comparative Neurology,

450(4), 345–365. http://doi.org/10.1002/cne.10336

Frischen, A., Eastwood, J. D., & Smilek, D. (2008). Visual search for faces with emotional

expressions. Psychological Bulletin, 134(5), 662–676. http://doi.org/10.1037/0033-

2909.134.5.662

Goddard, G. V. (1964). Functions of the Amygdala. Psychological Bulletin, 62(2), 89–109.

http://doi.org/10.1037/h0044853

33

Guzowski, J. F. F., McNaughton, B. L., Barnes, C. A., & Worley, P. F. (1999). Environment-

specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles.

Nature Neuroscience, 2(12), 1120–1124. http://doi.org/10.1038/16046

Hamann, S. (2001). Cognitive and neural mechanisms of emotional memory. Trends in

Cognitive Sciences, 5(9), 394–400. http://doi.org/10.1016/S1364-6613(00)01707-1

Han, J., Kushner, S. a, Yiu, A. P., Cole, C. J., Matynia, A., Brown, R. a, … Josselyn, S. A.

(2007). Neuronal competition and selection during memory formation. Science, 316, 457–

460.

Han, J., Kushner, S. A., Yiu, A. P., Hsiang, H.-L. L., Buch, T., Waisman, A., … Josselyn, S. A.

(2009). Selective erasure of a fear memory. Science, 323(5920), 1492–6.

http://doi.org/10.1126/science.1164139

Hopkins, D. A., & Holstege, G. (1978). Amygdaloid projections to the mesencephalon, pons and

medulla oblongata in the cat. Experimental Brain Research. Experimentelle Hirnforschung.

Experimentation Cerebrale, 32(4), 529–547. http://doi.org/10.1007/BF00239551

Hsiang, H.-L., Epp, J. R., van den Oever, M. C., Yan, C., Rashid, a. J., Insel, N., … Josselyn, S.

A. (2014). Manipulating a “Cocaine Engram” in Mice. Journal of Neuroscience, 34(42),

14115–14127. http://doi.org/10.1523/JNEUROSCI.3327-14.2014

Izquierdo, I., Furini, C. R. G., & Myskiw, J. C. (2016). Fear Memory. Physiological Reviews,

96(2), 695–750. http://doi.org/10.1152/physrev.00018.2015

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

517(7534), 284–292. http://doi.org/10.1038/nature14188

Johnson, A. W., Gallagher, M., & Holland, P. C. (2009). The Basolateral Amygdala Is Critical to

the Expression of Pavlovian and Instrumental Outcome-Specific Reinforcer Devaluation

Effects. Journal of Neuroscience, 29(3), 696–704.

http://doi.org/10.1523/JNEUROSCI.3758-08.2009

Josselyn, S. A., Köhler, S., & Frankland, P. W. (2015). Finding the engram. Nature Reviews

Neuroscience, 16(9), 521–534. http://doi.org/10.1038/nrn4000

34

Josselyn, S. A., Shi, C., Carlezon, W. A., Neve, R. L., Nestler, E. J., & Davis, M. (2001). Long-

term memory is facilitated by cAMP response element-binding protein overexpression in

the amygdala. The Journal of Neuroscience : The Official Journal of the Society for

Neuroscience, 21(7), 2404–12. http://doi.org/21/7/2404 [pii]

Kapp, B. S., Gallagher, M., Underwood, M. D., McNall, C. L., & Whitehorn, D. (1982).

Cardiovascular responses elicited by electrical stimulation of the amygdala central nucleus

in the rabbit. Brain Research, 234(2), 251–262. http://doi.org/10.1016/0006-

8993(82)90866-6

Klüver, H., & Bucy, P. C. (1937). “Psychic blindness” and other symptoms following bilateral

temporal lobectomy in Rhesus monkeys. American Journal of Physiology, 119, 352–353.

LaBar, K. S., & Cabeza, R. (2006). Cognitive neuroscience of emotional memory. Nature

Reviews Neuroscience, 7(1), 54–64. http://doi.org/10.1038/nrn1825

Ledoux, J. E. (2000). Emotion Circuits in the Brain. Annu. Rev. Neurosci., 23, 155–184.

LeDoux, J. E. (2012). Rethinking the Emotional Brain. Neuron, 73(4), 653–676.

http://doi.org/10.1016/j.neuron.2012.02.004

LeDoux, J. E., Farb, C., & Ruggiero, D. a. (1990). Topographic organization of neurons in the

acoustic thalamus that project to the amygdala. The Journal of Neuroscience : The Official

Journal of the Society for Neuroscience, 10(4), 1043–1054.

Linke, R., Braune, G., & Schwegler, H. (2000). Differential projection of the posterior

paralaminar thalamic nuclei to the amygdaloid complex in the rat. Exp Brain Res, 134(4),

520–532. http://doi.org/10.1007/s002210000475

MacKay, D. G., Shafto, M., Taylor, J. K., Marian, D. E., Abrams, L., & Dyer, J. R. (2004).

Relations between emotion, memory, and attention: Evidence from taboo stroop, lexical

decision, and immediate memory tasks. Memory & Cognition, 32(3), 474–488.

http://doi.org/10.3758/BF03195840

Majidishad, P., Pelli, D., & LeDoux, J. E. (1996). Disruption of fear conditioning to contextual

stimuli but not to a tone by lesions of the accessory basal nucleus of the amygdala. Soc.

35

Neurosci. Abstr., 22, 1116.

Maren, S. (1998). Overtraining does not mitigate contextual fear conditioning deficits produced

by neurotoxic lesions of the basolateral amygdala. The Journal of Neuroscience : The

Official Journal of the Society for Neuroscience, 18(8), 3088–3097.

Maren, S. (2001). Neurobiology of Pavlovian fear conditioning. Annu. Rev. Neurosci., 24, 897–

931. http://doi.org/10.1146/annurev.psych.55.090902.141409

Maren, S., Aharonov, G., & Fanselow, M. S. (1996). Retrograde abolition of conditional fear

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

Behavioral Neuroscience, 110(4), 718–726. http://doi.org/10.1037/0735-7044.110.4.718

Markowitsch, H. J., Calabrese, P., Würker, M., Durwen, H. F., Kessler, J., Babinsky, R., …

Gehlen, W. (1994). The amygdala’s contribution to memory--a study on two patients with

Urbach-Wiethe disease. Neuroreport. http://doi.org/10.1097/00001756-199406270-00013

McDonald, A. J. (1998). Cortical pathways to the mammalian amygdala. Progress in

Neurobiology, 55(3), 257–332. http://doi.org/10.1016/S0301-0082(98)00003-3

Namburi, P., Al-Hasani, R., Calhoon, G. G., Bruchas, M. R., & Tye, K. M. (2015). Architectural

Representation of Valence in the Limbic System. Neuropsychopharmacology, (September),

1–60. http://doi.org/10.1038/npp.2015.358

Namburi, P., Beyeler, A., Yorozu, S., Calhoon, G. G., Halbert, S. a., Wichmann, R., … Tye, K.

M. (2015). A circuit mechanism for differentiating positive and negative associations.

Nature, 520(7549), 675–678. http://doi.org/10.1038/nature14366

Nielsen, S. L., & Sarason, I. G. (1981). Emotion, personality, and selective attention. Journal of

Personality and Social Psychology, 41(5), 945–960. http://doi.org/10.1037/0022-

3514.41.5.945

Olds, M. E., & Olds, J. (1963). Approach-avoidance analysis of rat diencephalon. The Journal of

Comparative Neurology, 120(2), 259–295. http://doi.org/10.1002/cne.901200206

Parkinson, J. A., Robbins, T. W., & Everitt, B. J. (2000). Dissociable roles of the central and

36

basolateral amygdala in appetitive emotional learning. European Journal of Neuroscience,

12(1), 405–413. http://doi.org/10.1046/j.1460-9568.2000.00960.x

Paton, J. J., Belova, M. a, Morrison, S. E., & Salzman, C. D. (2006). The primate amygdala

represents the positive and negative value of visual stimuli during learning. Nature,

439(7078), 865–870. http://doi.org/10.1038/nature04490

Paxinos, G., & Franklin, K. B. J. (2001). Paxinos and Franklin’s the Mouse Brain in Stereotaxic

Coordinates. Academic Press, 2, 360.

Phelps, E. A., & LeDoux, J. E. (2005). Contributions of the Amygdala to Emotion Processing:

From Animal Models to Human Behavior. Neuron, 48(2), 175–187.

http://doi.org/10.1016/j.neuron.2005.09.025

Phillipson, O. T., & Griffiths, A. C. (1985). The topographic order of inputs to nucleus

accumbens in the rat. Neuroscience, 16(2), 275–296. http://doi.org/10.1016/0306-

4522(85)90002-8

Pitkänen, A., Pikkarainen, M., Nurminen, N., & Ylinen, A. (2000). Reciprocal connections

between the amygdala and the hippocampal formation, perirhinal cortex, and postrhinal

cortex in rat. A review. Annals of the New York Academy of Sciences, 911, 369–391.

http://doi.org/10.1111/j.1749-6632.2000.tb06738.x

Pizarro, L. J., & Levine, D. A. (2006). Emotional Valence, Discrete Emotions, and Memory. In

B. Uttl, N. Ohta, & A. Siegenthaler (Eds.), Memory and emotion: interdisciplinary

perspectives (pp. 37–58). Oxford: Blackwell Publishing.

Ramirez, S., Liu, X., Lin, P.-A., Suh, J., Pignatelli, M., Redondo, R. L., … Tonegawa, S. (2013).

Creating a false memory in the hippocampus. Science (New York, NY), 341(6144), 387–391.

http://doi.org/10.1126/science.1239073

Rasch, B., & Born, J. (2013, April). About Sleep’s Role in Memory. Physiological Reviews.

Bethesda, MD. http://doi.org/10.1152/physrev.00032.2012

Rashid, A. J., Yan, C., Mercaldo, V., Hsiang, H.-L. L., Park, S., Cole, C. J., … Gordon, J. A.

(2016). Competition between engrams influences fear memory formation and recall.

37

Science (New York, N.Y.), 353(6297), 383–7. http://doi.org/10.1126/science.aaf0594

Reijmers, L. G., Perkins, B. L., Matsuo, N., & Mayford, M. (2007). Localization of a stable

neural correlate of associative memory. Science (New York, N.Y.), 317(5842), 1230–1233.

http://doi.org/10.1126/science.1143839

Rogan, M. T., Stäubli, U. V, & LeDoux, J. E. (1997). Fear conditioning induces associative long-

term potentiation in the amygdala. Nature, 390(6660), 604–7. http://doi.org/10.1038/37601

Russell, J. A. (1980). A circumplex model of affect. Journal of Personality & Social Psychology,

39(6), 1161–1178. http://doi.org/10.1037/h0077714

Sah, P., Faber, E. S. L., Lopez De Armentia, M., & Power, J. (2003). The Amygdaloid Complex:

Anatomy and Physiology. Physiological Reviews, 83(3), 803–834.

http://doi.org/10.1152/physrev.00002.2003

Salinas, J. A., Packard, M. G., & McGaugh, J. L. (1993). Amygdala modulates memory for

changes in reward magnitude: reversible post-training inactivation with lidocaine attenuates

the response to a reduction in reward. Behav.Brain Res., 59(1-2), 153–159.

http://doi.org/10.1016/0166-4328(93)90162-J

Salzman, C. D., Paton, J. J., Belova, M. A., & Morrison, S. E. (2007). Flexible Neural

Representations of Value in the Primate Brain. Annals of the New York Academy of

Sciences, 1121(1), 336–354. http://doi.org/10.1196/annals.1401.034

Sano, Y., Shobe, J. L., Zhou, M., Huang, S., Shuman, T., Cai, D. J., … Silva, A. J. (2014).

CREB regulates memory allocation in the insular cortex. Current Biology : CB, 24(23),

2833–7. http://doi.org/10.1016/j.cub.2014.10.018

Schacter, D. L., & Wagner, A. D. (1999). Medial temporal lobe activations in fMRI and PET

studies of episodic encoding and retrieval. Hippocampus, 9(1), 7–24.

Schoenbaum, G., Chiba, A. A., & Gallagher, M. (2000). Changes in functional connectivity in

orbitofrontal cortex and basolateral amygdala during learning and reversal training. The

Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 20(13),

5179–89. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10864975

38

Shabel, S. J., & Janak, P. H. (2009). Substantial similarity in amygdala neuronal activity during

conditioned appetitive and aversive emotional arousal. Proceedings of the National

Academy of Sciences of the United States of America, 106(35), 15031–15036.

http://doi.org/10.1073/pnas.0905580106

Sharot, T., & Phelps, E. a. (2004). How arousal modulates memory: disentangling the effects of

attention and retention. Cognitive, Affective & Behavioral Neuroscience, 4(3), 294–306.

http://doi.org/10.3758/CABN.4.3.294

Stalnaker, T. A., Franz, T. M., Singh, T., & Schoenbaum, G. (2007). Basolateral amygdala

lesions abolish orbitofrontal-dependent reversal impairments. Neuron, 54(1), 51–58.

http://doi.org/10.1016/j.neuron.2007.02.014

Stuber, G. D., Sparta, D. R., Stamatakis, A. M., van Leeuwen, W. A., Hardjoprajitno, J. E., Cho,

S., … Bonci, A. (2011). Excitatory transmission from the amygdala to nucleus accumbens

facilitates reward seeking. Nature, 475(7356), 377–380. http://doi.org/10.1038/nature10194

Tanaka, K. Z., Pevzner, A., Hamidi, A. B., Nakazawa, Y., Graham, J., & Wiltgen, B. J. (2014).

Cortical representations are reinstated by the hippocampus during memory retrieval.

Neuron, 84(2), 347–354. http://doi.org/10.1016/j.neuron.2014.09.037

Tonegawa, S., Liu, X., Ramirez, S., & Redondo, R. (2015). Memory Engram Cells Have Come

of Age. Neuron, 87(5), 918–931. http://doi.org/10.1016/j.neuron.2015.08.002

Ungless, M. A., Magill, P. J., & Bolam, J. P. (2004). Uniform inhibition of dopamine neurons in

the ventral tegmental area by aversive stimuli. Science (New York, N.Y.), 303(5666), 2040–

2. http://doi.org/10.1126/science.1093360

Vetere, G., Restivo, L., Cole, C. J., Ross, P. J., Ammassari-Teule, M., Josselyn, S. A., &

Frankland, P. W. (2011). Spine growth in the anterior cingulate cortex is necessary for the

consolidation of contextual fear memory. Proceedings of the National Academy of Sciences

of the United States of America, 108(20), 8456–60. http://doi.org/10.1073/pnas.1016275108

Wagner, U., Gais, S., & Born, J. (2011). Emotional Memory Formation Is Enhanced across

Sleep Intervals with High Amounts of Rapid Eye Movement Sleep. Cold Spring Harbor

39

Laboratory Press, 8, 112–119. http://doi.org/10.1101/lm.36801

Wassum, K. M., & Izquierdo, A. (2015). The basolateral amygdala in reward learning and

addiction. Neuroscience & Biobehavioral Reviews, 57, 271–283.

http://doi.org/10.1016/j.neubiorev.2015.08.017

Wheeler, A. L., Teixeira, C. M., Wang, A. H., Xiong, X., Kovacevic, N., Lerch, J. P., …

Frankland, P. W. (2013). Identification of a Functional Connectome for Long-Term Fear

Memory in Mice. PLoS Computational Biology, 9(1).

http://doi.org/10.1371/journal.pcbi.1002853

Wise, R. A., & Bozarth, M. A. (1984). Brain reward circuitry: Four circuit elements “wired” in

apparent series. Brain Research Bulletin, 12(2), 203–208. http://doi.org/10.1016/0361-

9230(84)90190-4

Yiend, J. (2010). The effects of emotion on attention: A review of attentional processing of

emotional information. Cognition and Emotion: Reviews of Current Research and Theories,

9931(July), 211–275. http://doi.org/10.1080/02699930903205698

Yiu, A. P., Mercaldo, V., Yan, C., Richards, B., Rashid, A. J., Hsiang, H.-L. L., … Josselyn, S.

A. (2014). Neurons Are Recruited to a Memory Trace Based on Relative Neuronal

Excitability Immediately before Training. Neuron, 83(3), 722–735.

http://doi.org/10.1016/j.neuron.2014.07.017

Zhou, Y., Won, J., Karlsson, M. G., Zhou, M., Rogerson, T., Balaji, J., … Silva, A. J. (2009).

CREB regulates excitability and the allocation of memory to subsets of neurons in the

amygdala. Nature Neuroscience, 12(11), 1438–43. http://doi.org/10.1038/nn.2405

Copyright Acknowledgements

Figure 1 of this paper was adapted with permission from figure 2 of Josselyn, S. A., Köhler, S.,

& Frankland, P. W. (2015). Finding the engram. Nature Reviews Neuroscience, 16(9), 521–534.

This figure was licensed for reuse by the Nature Publishing Group (licensing number

3910240080799).