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Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Social Sciences 81
Erasing Fear
Effect of Disrupting Fear Memory
Reconsolidation on Central and Peripheral
Nervous System Activity
THOMAS ÅGREN
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2012
ISSN 1652-9030
ISBN 978-91-554-8455-2
urn:nbn:se:uu:diva-180202
Dissertation presented at Uppsala University to be publicly examined in Universitetshuset, Sal
IX, Biskopsgatan 3, Uppsala, Friday, October 12, 2012 at 13:15 for the degree of Doctor of
Philosophy. The examination will be conducted in Swedish.
Abstract
Ågren, T. 2012. Erasing Fear: Effect of Disrupting Fear Memory Reconsolidation on
Central and Peripheral Nervous System Activity. Acta Universitatis Upsaliensis. Digital
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Social Sciences 81.
62 pp. Uppsala. ISBN 978-91-554-8455-2.
Fear memories, here defined as learned associations between a stimulus and a physiological fear
reaction, are formed through fear conditioning. In animals, fear memories, present in the lateral
amygdala, undergo reconsolidation after recall. Moreover, this reconsolidation process can be
disrupted both pharmacologically and behaviourally, resulting in a reduced fear response to the
stimulus. This thesis examines the attenuation of fear memories by disrupting reconsolidation in
humans, using measures of both the central and peripheral nervous system activity. Serotonergic
and dopaminergic genes have previously been tied to both fear conditioning and anxiety
disorders, where fear conditioning mechanisms are important. In order to evaluate the possible
role of fear memory reconsolidation mechanims in the effect on fear and anxiety by these genes,
this thesis also compare the reconsolidation disruption effect between different serotonergic and
dopaminergic genotypes.
Study I examined the attentuation of fear memories by disrupting reconsolidation in humans
using reacquisition as a measure of the return of fear. Moreover, study I investigated the impact
of differences in serotonergic and dopaminergic alleles on this process.
Study II examined the attentuation of fear memories by disrupting reconsolidation in
humans using reinstatement as a measure of the return of fear. Study II also investigated the
impact of differences in serotonergic and dopaminergic alleles on the process of fear memory
reconsolidation.
Study III used psychophysiology and fMRI to localize the functional neural activity mediating
the fear memory reconsolidation disruption effect.
In summary, this thesis provides evidence that fear memories are attenuated by
reconsolidation disruption in humans and that serotonergic and dopaminergic alleles influence
this process. Moreover, this thesis support that human fear memory reconsolidation is amygdaladependent, suggesting an evolutionary shared memory mechanism.
Keywords: Memory reconsolidation; fear conditioning; serotonin; dopamine; polymorphism;
gene; exposure therapy; fMRI; amygdala
Thomas Ågren, Uppsala University, Department of Psychology, Box 1225,
SE-751 42 Uppsala, Sweden.
© Thomas Ågren 2012
ISSN 1652-9030
ISBN 978-91-554-8455-2
urn:nbn:se:uu:diva-180202 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-180202)
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
II
III
Agren, T., Furmark, T., Eriksson, E., & Fredrikson, M. (2012)
Human fear reconsolidation and differences in serotonergic and
dopaminergic genes. Translational Psychiatry, 2(2):e76
Agren, T., Furmark, T., Eriksson, E., & Fredrikson, M. Serotonergic and dopaminergic modulation of the prevention of return of fear using reconsolidation. (manuscript in preparation)
Agren, T., Engman, J., Frick, A., Björkstrand, J., Larsson, EM., Furmark, T., & Fredrikson, M. Disruption of reconsolidation erases a fear memory trace in the human amygdala. (accepted for publication in Science)
Reprints were made with permission from the respective publishers.
Contents
Abbreviations .................................................................................................. 7
Populärvetenskaplig sammanfattning på svenska ........................................... 9
Introduction ................................................................................................... 11
Fear conditioning .......................................................................................... 12
Spontaneous recovery .......................................................................... 12
Reinstatement ...................................................................................... 13
Renewal ............................................................................................... 13
Reacquisition ....................................................................................... 13
Extinction - new learning vs. unlearning.................................................. 13
Neurobiology of fear conditioning ................................................................ 15
Consolidation and reconsolidation ................................................................ 18
Consolidation ........................................................................................... 18
Reconsolidation. ....................................................................................... 20
Brain imaging of fear conditioning ............................................................... 23
Acquisition ............................................................................................... 23
Extinction ................................................................................................. 24
Extinction recall ....................................................................................... 25
Anxiety disorders and fear conditioning ....................................................... 27
Anxiety disorders and consolidation/reconsolidation ................................... 29
Genetics of fear conditioning ........................................................................ 30
5-HTTLPR ............................................................................................... 30
COMT ...................................................................................................... 31
Methods ........................................................................................................ 33
Psychophysiology- skin conductance ....................................................... 33
Functional Magnetic Resonance Imaging (fMRI).................................... 34
Aim ............................................................................................................... 35
Study I: .......................................................................................................... 36
Aim and background ................................................................................ 36
Methods .................................................................................................... 36
Results ...................................................................................................... 37
Discussion ................................................................................................ 37
Study II: ........................................................................................................ 38
Methods .................................................................................................... 38
Results ...................................................................................................... 39
Discussion ................................................................................................ 39
Study III: ....................................................................................................... 40
Methods .................................................................................................... 40
Results ...................................................................................................... 41
Discussion ................................................................................................ 41
Results overview (study I, II & III)............................................................... 42
General Discussion ....................................................................................... 47
Main findings ........................................................................................... 47
Discussion ................................................................................................ 47
Limitations ............................................................................................... 50
Future directions ....................................................................................... 51
Acknowledgment .......................................................................................... 53
References ..................................................................................................... 54
Abbreviations
5-HTTLPR
ACC
BLA
BOLD
Ca
CBT
CE
COMT
CS
CS+
CSDA
EDA
EPI
GABA
LA
LTP
NE
NMDA
NS
MAPk
OCD
PET
PTSD
REM
SCR
SSRI
UCS
vlPAG
vmPFC
Serotonin transporter gene linked polymorphic region
Anterior cingulate cortex
Basolateral Amygdala
Blood oxygen-level dependent
Calcium
Cognitive behaviour therapy
Central amygdala
Catechol-O-methyltransferase
Conditioned stimulus
Conditioned stimulus, reinforced
Conditioned stimulus, not reinforced
Dopamine
Electrodermal activity
Echo planar imaging
Gamma-Aminobutyric acid
Lateral Amygdala
Long term potentiation
Norepinephrine
N-methyl d-aspartate
Neutral stimulus
Mitogenactivated protein kinase
Obsessive compulsive disorder
Positron emission tomography
Post-traumatic stress syndrome
Rapid eye movement
Skin conductance response
Selective serotonin reuptake inhibitor
Unconditioned stimulus
ventrolateral periaqueductal grey
ventromedial prefreontal cortex
Populärvetenskaplig sammanfattning på
svenska
Rädslominnen har en central roll i alla ångeststörningar. Vid postraumatiskt
stressyndrom kan t ex traumatiska minnen ge upphov till ångest och hos
patienter med specifik fobi framkallar minnen av bestämda föremål eller
situationer rädsla. Det går att skapa rädslominnen i experiment, genom att
para ett neutralt med ett obehagligt stimulus. Sådan rädslobetingning kan
användas för att studera under vilka förhållanden vi skapar, upprätthåller och
förlorar minnen. Man talar förenklat om att vi först lagrar en upplevelse i
korttidsminnet, som försvinner ganska snabbt, och efter en tid kodar den i ett
bestående långtidsminne. Den process som gör minnen bestående över tid
kallas för konsolidering. Redan sedan 1800-talet har det varit känt att patienter med skallskador ibland drabbas av minnesförlust av vad som hände några
timmar innan skadan. Man tänker sig att anledningen till minnesförlusten är
att konsolideringsprocessen blivit störd och därmed förhindrat att upplevelsen lagrats i långtidsminnet. Ett sätt att utnyttja detta i behandling av personer som varit med om traumatiska upplevelser vore att försöka störa ut konsolideringen av rädslan som är förknippad med händelsen för att på så sätt
göra ett traumatiskt minne mer neutralt
Då man drar sig något till minnes blir minnet återigen instabilt och genomgår en ny inkodningsprocess kallad rekonsolidering. Vi kan alltså efter att vi
har framkallat ett minne under en period påverka det minnet innan det åter
hamnar i långtidsminnet. Ett exempel på detta kan vara samtalsterapi där
terapeuten får patienten att minnas något och sedan försöker påverka känsloinnehållet i minnet som sedan kodas om som neutralt innan det åter lagras i
långtidsminnet.
Den här avhandlingen har, med hjälp av experimentella rädslominnen, undersökt hur återbildning av minnen kan påverkas. Studierna visar att det är
möjligt att påverka rädsloinnehållet i minnen genom att störa minnets återbildande. Genvarianter som styr serotonin och dopamin i hjärnan och är viktiga vid ångest har här relaterats till hur rädslominnen bildas och glöms bort.
I en studie användes hjärnavbildning för att lokalisera var i hjärnan de funktionella förändringar sker, som leder till att rädslan minskar. I likhet med
studier på andra djur framstod amygdala som en funktionellt central punkt i
9
nätverket som upprätthåller rädsla. Det nya är att även människor kan omforma rädslominnen till neutrala minnen och att rädslominnet i amygdala då
försvinner. Rekonsolidering av rädslominnen är alltså en evolutionärt bevarad minnesmekanism som vi delar med många arter.
10
Introduction
Fear memories cause problems for people suffering from situationally elicited fear reactions like phobias and post traumatic stress disorder (PTSD).
Hence, it is possible that individual differences in the encoding and retrieval
processes of fear memories are part of the etiology of these disorders. Memories are made stable and permanent through a process called consolidation
that stretches on for hours after the initial encoding. Consolidation is dependent on protein synthesis in building the endurable structures of memories. When a memory is recalled it is again rendered unstable and becomes
the subject of a new stabilization process termed reconsolidation, also dependent on protein synthesis. During both the consolidation and reconsolidation processes some memories can be modified by the disruption of these
processes.
Fear memories, here defined as a learned association between a stimulus and
a physiological fear reaction, can be formed through fear conditioning. In
animals, these fear memories, present in the lateral amygdala, undergo reconsolidation after recall. Moreover, this reconsolidation process can be
disrupted both pharmacologically and behaviourally, resulting in a reduced
fear response to the stimulus. This attenuation of fear memories by means of
disrupted reconsolidation has recently been demonstrated in humans and
may represent a novel treatment strategy for fear and anxiety. This thesis
examines the attenuation of fear memories in humans by means of reconsolidation disruption, using measures of both the central and peripheral nervous
system. Also, a repeated cycle of recall and reconsolidation of fear memories
represent a kind of fear memory maintenance. It is conceivable that individual differences in fear memory maintenance present a vulnerability trait for
anxiety disorders. Therefore this thesis also compare the reconsolidation
disruption effect between different genotypes of serotonergic and dopaminergic genes previously associated with both fear conditioning, which may be
etiologically relevant in situationally elicited anxiety disorders, and anxiety.
11
Fear conditioning
Conditioning is a process in which a neutral stimulus (NS), through pairing
with a stimulus that elicits a physiological reaction (Unconditioned Stimuli;
UCS), becomes a conditioned stimulus (CS) that in itself produces the physiological reaction. The process of conditioning was first reported by Ivan
Pavlov in 1907 and is sometimes simply called Pavlovian conditioning
(Pavlov, 1927). When the UCS involved elicits a physiological fear reaction,
the process is called fear conditioning. Fear conditioning enables an organism to identify and remember what is dangerous and should be avoided, thus
representing a crucial mechanism for an organism’s survival.
The learning of an association between a CS and a UCS is called acquisition.
Specifically, acquisition is the process in which a fear reaction to a CS is
learned and a fear memory is formed. In single cue conditioning, a single NS
is transformed to a CS. In differential conditioning, two CS are used, one
that is paired with UCS and one that is not. The one that is paired with UCS
is called CS+ and the other one, used as control for CS+, is called CS-. The
use of CS- enables researchers to rule out the possibility that a subjects fear
reactions are unspecific and not solely caused by the presence of CS+.
A learned fear reaction to a CS can be reduced using extinction, a process in
which the CS is repeatedly shown without pairing with the UCS. This leads
to a weakened fear response to the CS and with a sufficient amount of
presentations, no fear response at all. However, extinction does not permanently erase the fear reactions to a CS, because it is known that fear can return after extinction. Listed below are circumstances in which return of fear
has been noted and can be measured.
Spontaneous recovery
Since Pavlov’s seminal work (Pavlov, 1927), it has been known that the fear
reaction associated with a CS returns, simply with the passage of time. Spontaneous recovery of fear is tested by presenting the subject with the CS after
a suitable passage of time and measure the fear reaction.
12
Reinstatement
Reinstatement refers to the return of fear to a CS that occurs if subjects are
presented with unpaired presentations of the UCS, for example if a subject
who has acquired a fear association between a stimulus and shock is presented with only the shock. This has the ability to somehow strengthen or activate the dormant fear memory. In experiments using reinstatement, unpaired
UCS presentations are presented before, or intermixed with CS presentations
and the return of fear is measured by the reactions to the CS (Rescorla &
Heth, 1975).
Renewal
Renewal refers to the return of fear to a CS that occurs if subjects are moved
to a new context, different from the one in which acquisition or extinction
took place. For example, if a subject has acquired a fear reaction to a CS and
then had it extinguished in a certain room, the fear can return when presenting the CS in another room. The return of fear is measured by the reactions
to the CS in the new context.
Reacquisition
Reacquisition is the relearning of an already learned association of a CS to
UCS. It is noted that an acquired and extinguished fear association is relearned faster than the original acquisition, probably as a result of a remaining memory of the original learning (Bouton, 2002). Using reacquisition,
return of fear is measured as the readiness for relearning, examining how
lingering fear memories may affect future vulnerability to fear renewal
(Kehoe & Macrae, 1998).
Extinction - new learning vs. unlearning
The return of fear observed in spontaneous recovery, reinstatement, renewal
and reacquisition, strongly suggest that extinction does not completely remove a learned fear association, but simply inhibits it for a period of time.
This is why it is generally believed that extinction is based on new learning
consisting of an association between CS and the absence of UCS (Bouton,
2002; Myers & Davis, 2007). This new association will represent a kind of
‘safety memory’, signaling that the CS will be followed by the absence of
UCS. This memory is thought to compete with the original fear memory that
signals that CS will be followed by UCS. In these terms, spontaneous recovery is explained as follows: After extinction the safety memory dominates,
but the fear memory will remain even after the safety memory fade, possibly
13
because the safety memory is more fragile (Pavlov, 1927) or context specific
(Bouton, 2002). A competing theory to the one claiming new learning of a
‘safety memory’ is that the original association is simply unlearned. The
main weakness of this theory is that it can’t easily account for the phenomena of ‘return of fear’ described above. However, recent advances in both
neurobiological and behaviour studies provide evidence for such an unlearning mechanism (Kwapis, Jarome, Schiff, & Helmstetter, 2011; Monfils,
Cowansage, Klann, & LeDoux, 2009; Myers, Ressler, & Davis, 2006;
Schiller et al., 2010), which is the subject of study in this thesis, and suggests
that extinction may consist of both unlearning and new learning (Pape &
Pare, 2010).
14
Neurobiology of fear conditioning
The literature concerning the neurobiology of fear conditioning is dominated
by the amygdala, an almond shaped structure in the limbic cortex. Amygdala
was tied to fear behaviour in the 1950s when it was established that amygdala lesions were crucial for the disappearance of several fear behaviours in
monkeys (Weiskrantz, 1956) previously reported after temporal lobe lesions
(Klüver & Bucy, 1939). Following this discovery, the dependence of amygdala in different inhibitory avoidance tasks, tasks in which an animal learns
to avoid something that is paired with an aversive stimuli, were explored in
several species of animals (Horvath, 1963; Robinson, 1963). In 1972, it was
established that amygdala was necessary for the acquisition of fear conditioning, demonstrating that amygdala not only was vital for fear expression,
but also for fear learning (Blanchard & Blanchard, 1972). Finally, in the
1990s it was shown in patients with lesions of the amygdala that also in humans, amygdala is a necessary structure for acquiring fear conditioning
(Bechara et al., 1995; LaBar, LeDoux, Spencer, & Phelps, 1995). Furthermore, brain imaging has demonstrated amygdala activations to emotional
stimuli, such as facial expressions of fear (Morris, Frith, Perrett, & Rowland,
1996), as well as fear conditioning (LaBar, Gatenby, Gore, LeDoux, &
Phelps, 1998).
In more detail, transmission of sensory information converges in the amygdala, particularly the lateral amygdala (LA), where inputs from auditory
cortex, auditory thalamus, thalamic areas that receive information from the
spinal-thalamic tract, cortical areas processing somatosensory stimuli, olfactory cortex and visual cortex provide all the information needed for associations between CS and UCS to be formed (LeDoux, 2000; Maren, 2001;
McDonald, 1998). Lesions of LA as well as pharmacological inactivation of
LA neurons prevent fear acquisition. Moreover, single cell recordings show
that LA neurons fire in response to both CS and US stimuli, demonstrating
that an association is present in the LA. For reviews, see (Johansen, Cain,
Ostroff, & LeDoux, 2011; Maren, 2001). From amygdala, particularly from
the central nucleus (CE) of amygdala, projections go to brainstem areas controlling expression of fear responses. These connections, together with the
fact that lesions of the central nucleus prevent fear responses, make a strong
case that the CE is necessary for fear responses. CE receives input from the
LA and is generally believed to mediate and execute fear responses learned
15
in the LA (LeDoux, 2000; Maren, 2001). However, recent research argues
that its role in fear conditioning also involves learning (Wilensky, Schafe,
Kristensen, & LeDoux, 2006).
On a molecular level, Hebbian mechanisms, mechanisms in which synaptic
activity between two neurons increase the strength of the coupling between
those two neurons, are important for fear learning in the LA. Important molecular functions for Hebbian mechanisms in the amygdala with a direct
impact on fear conditioning are NMDA-type ionotropic glutamate receptors;
a receptor known to detect pre- and postsynaptic synchronized depolarization, autophosphorylation of Ca2+/calmudulin-dependent protein kinase II; a
process vital for memory formation in several types of learning, and inhibitory GABA-ergic neurotransmission (Johansen et al., 2011). Moreover, there
is evidence that monoamine transmitters released in emotional situations, for
example norepinephrine (NE) and dopamine (DA), regulate glutamatergic
transmission and Hebbian plasticity (Rosenkranz & Grace, 1999; Tully, Li,
Tsvetkov, & Bolshakov, 2007; de Oliveira et al., 2011). In addition to the
above direct evidence of memory formation in LA there is also much indirect evidence simply showing that long term potentiation (LTP), an important function in forming memories, is taking place in LA (Clugnet &
LeDoux, 1990; Maren, Fanselow, & Angeles, 1995; Rogan & LeDoux,
1995). In conclusion, there is much evidence that acquisition of fear conditioning takes place within the amygdala.
The neurobiological underpinnings of extinction training are not as wellknown as those involved in acquisition. It is likely that extinction memory is
distributed among a network of structures where extinction-related plasticity
in different regions have different functions (Quirk & Mueller, 2008). As
with acquisition, the amygdala is implicated also in extinction learning.
Blocking mitogenactivated protein kinase (MAPk) activity, a way of measuring signaling activity, in basolateral amygdala (BLA) completely prevented extinction learning. Also, a higher level of MAPk activity was noted in
BLA during extinction training. Moreover, blockade of glutamate receptors
in BLA impair extinction learning (Pape & Pare, 2010; Quirk & Mueller,
2008). In addition to amygdala, blocking opioid receptors in the ventrolateral
periaqueductal gray (vlPAG), a midbrain structure important for fear expression (De Oca, DeCola, Maren, & Fanselow, 1998) prevents extinction learning, implicating this structure in extinction (De Oca et al., 1998).
There is much evidence that extinction is context-dependent and that this is
the reason for the renewal effect (Bouton, 2002). It is generally believed that
this context-dependency relies on hippocampus and its connection to amygdala (Kim & Fanselow, 1992; Phillips & LeDoux, 1992). Finally, the ventromedial prefrontal cortex (vmPFC) is often implicated in extinction train16
ing; albeit most in the consolidation of extinction training (consolidation will
be discussed in the next section). Lesions of the vmPFC, as well as pharmacological inhibition of vmPFC activity, either by blocking glutamate or NE
activity or by blocking protein synthesis, do not hinder extinction training,
but impairs retrieval of extinction on following days (Quirk & Mueller,
2008). The vmPFC is also implicated in human brain imaging of extinction
training (see Brain imaging of fear conditioning) providing evidence for at
least a partly evolutionary shared mechanism of extinction in the mammalian
brain. In conclusion, both fear and extinction learning appear to be amygdala
dependent, but extinction is also dependent on other areas that modulate the
fear response.
17
Consolidation and reconsolidation
Consolidation
Already in the 1800s it was noted that brain injuries could lead to a loss of
recent memories. To explain these findings, it was proposed that memories
became stable over time, that they consolidated, and that injuries to the brain
would disrupt ongoing consolidation and hence, erase memories acquired
shortly before the injury. In 1949, this kind of retrograde amnesia was experimentally induced in rats using electroconvulsive shocks (Duncan, 1949).
These findings led to the development of a dual-trace memory theory in
which one neural system or process was thought to underlie short term
memory and that short term memory subsequently was transformed by consolidation to long term memory, possibly relying on another neural system
or process (Hebb, 1949). Today, consolidation is a well-accepted concept,
referring to a time-dependent stabilization process that fixates short term
memory into a more permanent one. A wealth of research during the last 40
years have yielded much knowledge of the neurobiological underpinnings of
consolidation, but it is still the target of intense study (McGaugh, 2000).
Why is a consolidation process necessary when we know the brain almost
instantly can form short term memory? It is argued that the consolidation
process adds an adaptive function by enabling other systems to modulate the
strength and character of the memory. For instance, adrenal stress hormones
released in emotionally arousing situations facilitate long term memory by
affecting consolidation (Liang, Juler, & McGaugh, 1986). There is a lot of
evidence that this modulation is largely depending on the basolateral amygdala (BLA). Amygdala lesions block the effect of well-known modulators of
consolidation, like the adrenal stress hormones. Moreover, blocking the activity of adrenal stress hormones in the BLA also block the modulating effect adrenal stress hormones have on consolidation. However, the BLA interacts with other brain systems to perform this modulation. For example,
lesions of stria terminalis, a bundle of fibers containing many of the projections from amygdala, also block the modulating effects of intra-amygdala
drug infusions (McGaugh, 2004). Moreover, consolidation following fear
conditioning activates synaptic plasticity in brain areas other than the amygdala, notably, the hippocampus, striatum and cortex (Ressler, Paschall, Zhou,
& Davis, 2002). Why would fear conditioning induce synaptic plasticity in
18
several brain areas if the memory is located in LA? The answer could be that
LA stores a certain aspect of memory, the arousal components, and that other components, for example context memory or episodic memory, are situated elsewhere, and that all components together form a complete memory
representation.
Memory representations are in general thought to be dependent on long term
potentiation (LTP). LTP, in short, is a mechanism by which increased firing
between two neurons induces a long lasting enhancement in signal transmission between the active neurons. LTP, corresponding with the notion of
memory consolidation, also have different temporal phases. There is an early
phase of LTP, that lasts for minutes, and a late phase, that depend on protein
synthesis, that can last for hours (Milner, Squire, & Kandel, 1998). As previously noted, rodent studies strongly suggest that the locus for fear conditioning memory is the lateral amygdala (LA) (see Neurobiology of fear conditioning). Consistently, inputs to amygdala, crucial for fear conditioning,
induces LTP in the LA, further implicating the LA as a the site for fear conditioning memory, and if late phase LTP is tied to consolidation, also fear
memory consolidation (Clugnet & LeDoux, 1990; Maren et al., 1995; Rogan
& LeDoux, 1995).
In animals, memories induced by fear conditioning have successfully been
attenuated by disrupting consolidation following fear learning. This has been
done both pharmacologically by preventing protein synthesis in the amygdala (Kwapis et al., 2011; Wilensky et al., 2006) and behaviourally by use of
extinction training (Myers et al., 2006). Studies using protein synthesis inhibition suggest that the consolidation interval during which disruption is possible roughly starts a few minutes after training, the time it takes to synthesize new protein and transport it to a location where it can affect interneuronal communication, and ends around 3-6 hours after training. Also, protein
synthesis inhibition must be initiated within 90 min after training to make an
impact on memory retention, suggesting that consolidation disruption initiated later than 90 min after training probably stem from the disruption of other
mechanisms than protein synthesis (Davis & Squire, 1984), for example the
modulation of consolidation by adrenal stress hormones mentioned above.
The pharmacological manipulations used on animals to disrupt consolidation are not safe for humans and the disruption of consolidation
using behavioural manipulations have not been readily replicated in
humans (Huff, Hernandez, Blanding, & LaBar, 2009; Norrholm et al.,
2008). Still, consolidation of episodic memories are strengthened by
arousal (Cahill & Alkire, 2003; Cahill, Gorski, & Le, 2003) and diminished by activity similar in character to the memory (Holmes,
19
James, Kilford, & Deeprose, 2010), suggesting that it may be possible
to use consolidation disruption as a novel procedure in reducing fear.
Reconsolidation.
After recall, fear memories appear to again become destabilized and the target of a renewed consolidation process termed reconsolidation. This phenomenon was noted in rodent research in the late 1960s when a fear conditioned memory was reduced by inducing retrograde amnesia with an electroconvulsive shock during the reconsolidation interval after a fear memory
reminder (Misanin, Miller, & Lewis, 1968). Several interesting studies examined what kind of cue that was needed for reactivation and during what
circumstances reactivation of a memory occurred (Spear, Lewis, Mcgaugh,
& Ralph, 1973). In essence, a memory can be reactivated by a presentation
of at least one element of it, for example the stimuli itself, the context in
which the stimuli was presented, or even an internal context like that of
arousal. During the reconsolidation interval, but not after, memories can be
enhanced (Rodriguez, Horne, & Padilla, 1999), impaired (Nader, Schafe, &
Le Doux, 2000) or interfered with conflicting learning (Spear et al., 1973).
Moreover, a reactivation with a following undisturbed reconsolidation seems
to strengthen the memory (Inda, Muravieva, & Alberini, 2011).
The term reconsolidation suggests that the process would consist of a second
consolidation. However, research show that for certain memories, different
brain areas are involved in consolidation and reconsolidation, although both
processes seem to use the same molecules and mechanisms (Alberini, 2005).
Because of the initial belief that reconsolidation was the same process as
consolidation, research on reconsolidation have considered the reconsolidation interval to have the same duration as the consolidation interval. Considering that the neurobiological mechanisms underlying consolidation and
reconsolidation seem to be the same, this may be true, but it is up to future
research to examine possible differences in the duration of the reconsolidation interval and the consolidation interval.
Fear conditioning is a well suited paradigm in which to study retrograde
amnesia induced by reconsolidation disruption. The neural mechanisms of
fear conditioning are well known in comparison with other types of learning
and it has clinical relevance suggesting novel treatments for anxiety disorders (see section Anxiety disorders and consolidation). In rodents, fear
memories can be reduced by disrupting the reconsolidation process following a fear memory reminder. Like consolidation, reconsolidation of fear
conditioning memories has been tied to the BLA. Pharmacologically, reconsolidation processes can be disrupted, and thus, fear memories attenuated, by
20
inhibiting protein synthesis in BLA with anisomycin (Nader et al., 2000),
possibly reflecting the late phase LTP dependency on protein synthesis.
Moreover, blocking noradrenergic activity in the amygdala disrupt reconsolidation (Debiec & Ledoux, 2004) and enhancing noradrenergic activity in
the amygdala enhances the reconsolidation process and lead to a stronger
fear memory (Dębiec, Bush, & LeDoux, 2011). Recently, fear memories
were successfully reduced using behavioural means, that is, by using extinction training during the reconsolidation interval (Monfils et al., 2009). New
conflicting learning had been used for reconsolidation disruption earlier, but
not by using extinction training (Gordon, Spear, 1973).
Also in humans, recent studies report successfully modulated memories by
disrupting reconsolidation. The first study to show an effect of disrupted
reconsolidation in humans used a procedural finger-tapping task (Walker,
Brakefield, & Hobson, 2003), but fear conditioned memories have also been
successfully modulated. Kindt et al. (2009) used propranolol, a drug that
reduces noradrenergic activity in both the peripheral and the central nervous
system, to disrupt consolidation of human fear conditioning memory (Kindt,
Soeter, & Vervliet, 2009). The same research group also demonstrated that
enhancement of noradrenergic activity during reconsolidation enhanced the
strength of the memory, although only for startle responses and not for skin
conductance responses (Soeter & Kindt, 2011). In the same vein as the research on rodents by Monfils et al. (2009), fear conditioning reconsolidation
has also been successfully disrupted by extinction training. In a study by
Schiller et al. (2010), subjects underwent fear conditioning to induce an experimental fear memory. Twenty-four hours after the initial training, they
received a fear memory reminder consisting of a presentation of the CS. One
group of subjects then received extinction training 10 min later, hence during
the reconsolidation interval, and one group received extinction training 6 hrs
later, after reconsolidation was completed. Spontaneous recovery was tested
another twenty-four hrs later using a second session of extinction. Return of
fear was noted in the group that received extinction training outside of the
reconsolidation interval and hence, after completed reconsolidation, while no
return of fear was noted in the group which received reconsolidation disruption, that is extinction training during the reconsolidation interval (Schiller et
al., 2010).
Not all memories seem to be open for modulation through manipulation of
the reconsolidation process. In the 1970s, Electroconvulsive therapy was
used in an attempt to disrupt the reconsolidation of several declarative memories after a memory reminder, but no effect could be noted (Squire, Slater,
& Chace, 1976). The authors of the study suggest that the memories that can
be reduced with reconsolidation disruption may have to contain an element
of arousal, since all rodent studies that successfully produced the phenome21
non had used fear or hunger memories. However, the reconsolidation disruption effect have also been produced using procedural memories with no apparent element of arousal (Walker et al., 2003). In conclusion, the disruption
of consolidation and reconsolidation processes show some promise into attenuating the arousal component of memories, but without removing the
episodic memory component of the events.
22
Brain imaging of fear conditioning
Acquisition
The first fear conditioning imaging studies was published in the middle of
the 90s and used PET. The first study to image acquisition with the contrast
CS+>CS- found heightened activity in the pulvinar and the thalamus
(Morris, Friston, & Dolan, 1997). Amygdala was implicated only as an area
that was positively correlated to the pulvinar activity. The advent of eventrelated fMRI studies introduced a way to image and compare the same time
intervals of CS+ and CS- presentations that were traditionally analysed in
psychophysiology. The two first event-related fMRI studies using the contrast CS+>CS- both noted stronger BOLD signal to CS+ in amygdala and the
anterior cingulate cortex. Moreover, both studies noted that reactions to CS+
diminished over time (Büchel, Morris, Dolan, & Friston, 1998; LaBar et al.,
1998). Since then, over fifty imaging studies of fear acquisition have been
made and the gathered knowledge is starting to reveal a pattern. A recent
review (Sehlmeyer et al., 2009) reports that the most common finding in fear
acquisition is the amygdala, the ACC and the insular cortex. Drawing from
rodent research on the neurobiological underpinnings of fear conditioning,
amygdala is vital for acquisition (see Neurobiology of fear conditioning)
which makes it a primary target for imaging of fear conditioning. However,
fear memory activity in amygdala is no easy target for imaging. Animal experiments, using single cell recordings, note that the associative memory in
amygdala is stored in a small number of sparsely distributed neurons which
make them hard to register with neuroimaging methods (Bach, Weiskopf, &
Dolan, 2011; Reijmers, Perkins, Matsuo, & Mayford, 2007). However, new
methods of imaging and analyses are stepping up to the task (Bach et al.,
2011; Visser, Scholte, & Kindt, 2011).
In contrast to the amygdala, the function of the ACC and the insular cortex
in fear conditioning are less known. The anterior cingulate cortex is known
to be involved in attentional processes of emotional stimuli and may reflect
the attention to the CS as an emotional stimuli (Lane, Fink, Chau, & Dolan,
1997). Both the anterior cingulate cortex and the insular cortex are known to
be involved in control of cardiovascular (Oppenheimer, Gelb, Girvin, &
Hachinsky, 1992) and electrodermal responses (Critchley, 2002), so their
activation may reflect a control over activations of the sympathetic nervous
23
system. Moreover, both areas have connections to the amygdala and may
modulate fear learning and expression. It is a task for combined connectivity
and activation studies of fear conditioning to further substantiate the function
of all implicated areas of the brain.
Extinction
Extinction is not nearly as common in imaging studies as acquisition. In
spite of this, the very first imaging studies of fear conditioning were of extinction. These early studies, using PET, reported activations in the thalamus, hypothalamus and midbrain (Fredrikson, Wik, Fischer, & Andersson,
1995) consistent with the presence of fear reactions, as well as mainly rightsided activations in frontal and temporal cortices (Hugdahl et al., 1995). The
first event-related fMRI study revealed amygdala activity, predominantly on
the right side, along with activations in prefrontal cortex. Amygdala activity
was notable only in the early phase of extinction and habituated over time
(LaBar et al., 1998). Since then, studies have noted increased amygdala activity (Barrett & Armony, 2009; Gottfried & Dolan, 2004; Knight, Smith,
Cheng, Stein, & Helmstetter, 2004; Milad et al., 2007), decreased amygdala
activity (Phelps, Delgado, Nearing, & LeDoux, 2004) and no change in
amygdala activity (Delgado, Nearing, Ledoux, & Phelps, 2008; Kalisch et
al., 2006; Lang et al., 2009; Spoormaker et al., 2011; Yágüez et al., 2005).
Research into the neurobiology of fear conditioning suggest that fear extinction is dependent on frontal areas to inhibit the fear response generated by
the amygdala. Implicated from rodent research are especially the vmPFC
(Milad & Quirk, 2002), although the translation from rodent brain areas to
human brain areas is not trivial making the implications from rodent studies
on human anatomy uncertain. Consistent with this theory the vmPFC has
been implicated in several studies of extinction. Activity in the vmPFC have
been found to be negatively correlated to SCRs reflecting extinction (Phelps
et al., 2004) and to reflect decreasing aversive value of the CS+ (Gottfried &
Dolan, 2004).
Both the ACC (Barrett & Armony, 2009; Lang et al., 2009; Phelps et al.,
2004; Spoormaker et al., 2011) and the insular cortex (Delgado et al., 2008;
Gottfried & Dolan, 2004; Phelps et al., 2004; Spoormaker et al., 2011;
Yágüez et al., 2005) are also frequently found activated during extinction.
They both lack the evidence, as compared to the vmPFC, to be considered as
inhibiting areas, but reflect perhaps attentional processes, cortical control
over the sympathetic nervous system, or nodes in a distributed memory or
control system in the human fear circuitry. These results taken together, imply that fear extinction involves a complex network of areas in humans, per24
haps reflecting the complexity of our cognitive abilities. Only two studies
noted involvement of hippocampus (Knight et al., 2004; Phelps et al., 2004),
a structure frequently implied in memory research (Eichenbaum, 1999). This
implies that fear conditioning may be hippocampus independent, at least as
long as it doesn’t involve contextual cues (Phillips & LeDoux, 1992).
One could argue that there is reason to differentiate between extinction of
fear memories that had a complete consolidation after acquisition and fear
memories that were extinguished immediately upon acquisition. In the latter
case, consolidation of acquisition and extinction training is simultaneous
which may affect subsequent extinction, return of fear and extinction recall.
The only study, to this authors knowledge, of brain correlates of extinction
of fear memories that consolidated before the first extinction session found
activations in thalamus, middle cingulate cortex and bilateral insular cortex
(Spoormaker et al., 2011). The only indication of involvement of the amygdala was a group×stimulus interaction comparing two conditioned CS, one
that was extinguished and one that was not extinguished, between REM
sleep deprived and controls. Sleep deprived had “more” activity in the right
amygdala as compared to controls.
Extinction recall
To study the lingering effect of extinction some time after the extinction
training is called extinction recall. The term ‘extinction recall’ implies that a
new ‘safety memory’ is learned during extinction and that this ‘safety
memory’ can be recalled at a later time. Another way of putting it is that
extinction recall studies the return of fear, possibly inhibited by a memory
from the preceding extinction. There are both similarities and differences in
findings between studies of extinction and extinction recall. As for extinction, extinction recall studies note activity in the vmPFC (Kalisch et al.,
2006; Milad et al., 2007). Moreover, correlations of both vmPFC activity
and vmPFC thickness with SCRs reflecting return of fear have been reported
(Milad et al., 2005, 2007). Also, as in both acquisition and extinction, the
ACC and insula are activated during extinction recall (Phelps et al., 2004). In
contrast to studies of extinction, studies of extinction recall often implicate
hippocampus with increased hippocampal activity correlating with both
SCRs reflecting return of fear and the vmPFC (Kalisch et al., 2006; Milad et
al., 2007). This may indicate that hippocampus is vital only for consolidated
memories and/or the process of consolidation. Interestingly, amygdala activations have not been found during extinction recall. SCRs indicate a return
of fear in subjects so an amygdala response would be expected (Phelps et al.,
2004). Maybe, there is a fast degradation of the amygdala response, as have
been found in both acquisition and extinction (Büchel et al., 1998; LaBar et
25
al., 1998), meaning that the amygdala mainly reacts to the first few trials. On
the other hand, amygdala activity could be decreased or not involved at all.
The studies cited above reflect extinction recall of fear memories that were
extinguished immediately following acquisition, which as stated earlier may
differ from fear memories with undisturbed consolidations. The one study
using extinction of fear memories with an undisturbed consolidation process
also studied extinction recall. It noted involvement of the same areas (thalamus, midcingulate cortex, supplementary motor area and insula) in extinction recall as in extinction (Spoormaker et al., 2011).
26
Anxiety disorders and fear conditioning
Anxiety disorders have long been considered as reflecting possible deficits
in the processes governing fear conditioning. Indeed, Pavlov himself, proposed that the development of anxiety occurred by means of fear conditioning (Pavlov, 1927). Today, there are several theories proposing a link between fear conditioning mechanisms and anxiety disorders. For instance, it
has been proposed that anxiety disorders are caused by a failure to extinguish acquired fear (Eysenck, 1979), increased conditionability (Orr et al.,
2000), and that they are a result of the inability to inhibit fear responses in
the presence of ‘safety cues’ (Davis, Falls, & Gewirtz, 2000).
Theories aside, the most convincing evidence that fear conditioning processes are tied to anxiety disorders come from clinical studies. Exposure therapy,
a treatment based on the process of extinction, is highly effective in treating
anxiety disorders, including PTSD and obsessive compulsive disorder
(OCD) (Foa, 2011; Franklin & Foa, 2011; Ougrin, 2011). A recent metaanalysis comparing cognitive behaviour therapy (CBT) and exposure treatment found the two treatments equally effective in most anxiety disorders,
with the exception of social phobia, where CBT was more effective (Ougrin,
2011). Moreover, phenomena that follow extinction in fear conditioning
experiments are also noticed in clinical populations after treatment with exposure therapy. For instance, fear reoccur after treatment as an effect of both
spontaneous recovery (Rachman, 1989) and renewal (Mineka, Mystkowski,
& Hladek, 1999; Mystkowski, Mineka, Vernon, & Zinbarg, 2003; B.
Rodriguez, Craske, & Mineka, 1999). A meta-analysis, concentrating on
studies of fear conditioning conducted on clinical populations, reported
modest increases in fear acquisition and fear expression during extinction in
anxiety patients. However, this difference was mainly a result of studies
using single cue conditioning, that is, studies where no CS- was used. In
studies of differential conditioning, where the difference of the reactions to
CS+ and CS- is used as a measure of fear, the meta-analysis found no difference between patients and healthy controls (Lissek et al., 2005). Thus, it
cannot be ruled out that the increased responsiveness of patients found in
single cue conditioning studies reflect an increased sensitization to the situation as a whole and perhaps not specifically to the CS+, hence a fear memory
process may not be involved. In line with this, and based on the results of
several studies in the meta-analysis, Lissek and coworkers (2005) argued
27
that the lack of differences between patients and controls regarding differential conditioning may be because the patients reacted more to both CS+ and
CS- and that this could indicate an inability in patients to inhibit their reaction to the CS- (Lissek et al., 2005). In addition, brain imaging of anxiety
disorders often report elevated activations of the amygdala (Holzschneider &
Mulert, 2011), a vital structure for fear conditioning (LeDoux, 2000).
However, equating fear conditioning with anxiety is problematic. For instance, many patients suffering from specific phobia do not remember a specific learning instance from which the phobia originated, which would be
expected from fear conditioning theory; see (Mineka & Oehlberg, 2008) for
a discussion. Moreover, most studies find no link between anxiety-related
traits (Chambers, Power, & Durham, 2004; Gershuny & Sher, 1998; Jorm et
al., 2000) and fear conditioning (Davidson, Payne, & Sloane, 1964; Otto et
al., 2007; Pineles, Vogt, & Orr, 2009). That is, there is no strong evidence
that people at risk for developing anxiety disorders behave differently in fear
conditioning as compared to people who are not. Recently it has been argued
that perhaps the traditional experimental fear conditioning paradigm needs to
incorporate other measures of fear than physiological, such as avoidance
behavior, to expose the possible differences between anxiety patients and
controls (Beckers, Krypotos, Boddez, Effting, & Kindt, 2012). Also, since
phobias generally concerns a limited number of stimuli and situations it has
been suggested that organisms are evolutionary prepared to fear certain
stimuli (Seligman, 1972) and that it is in fear conditioning processes regarding these specific stimuli that anxiety patients differ from controls. This theory has gained some support in social phobia (Lissek et al., 2008), but have
some obvious theoretical problems that must be dealt with. For instance,
either fear conditioning to prepared stimuli is a qualitatively different neural
process, which lacks evidence, or fear conditioning is modulated by an evolutionary old fear detection system other than the amygdala, still unknown to
us.
Fear conditioning and its relation to anxiety disorders is still, a hundred years
after its discovery, a hot topic for study. As reported previously in this thesis,
research has taken great strides to understand the neural processes underpinning fear conditioning. However, how these neural processes are ultimately
involved in the development and maintenance of anxiety disorders and how
they could be targeted for treatment remains largely unknown.
28
Anxiety disorders and
consolidation/reconsolidation
Treating anxiety by disrupting a memory’s consolidation processes is possible only if treatment follows within hours of the trauma leading to PTSD or
phobia. Still, a few clever experimental studies show promising results. Arguing that humans have limited capacity for processing and consolidation of
visual information, subjects performed different cognitive tasks after watching a stressful movie. Subjects then documented the frequency of involuntary memory recalls of the movie during the following week. Subjects that
performed a visuo-spatial task had fewer episodes of involuntary memory
recall than subjects who performed a verbal task, or no task (Deeprose,
Zhang, Dejong, Dalgleish, & Holmes, 2012; Holmes, James, Coode-Bate, &
Deeprose, 2009; Holmes et al., 2010). A more direct study tried to disrupt
the consolidation process of fear memories in trauma patients by treating
them with the noradrenergic antagonist propranolol shortly after initial trauma with some success (Pitman et al., 2002).
Treating anxiety by disrupting reconsolidation processes have potentially a
much wider clinical use, as compared to preventing consolidation, since the
treatment does not have to follow immediately after the trauma. Also, if reconsolidation disruption can be used to influence fear and anxiety, other
anxiety disorders that not necessarily are trauma dependent can be treatment
candidates. In essence, many forms of therapy consist of re-imagining and
re-framing memories by means of cognitive analysis of the events, leading to
new perspectives, and less anxiety, in subsequent remembrance of the previous events. The first clinical study to use disruption of reconsolidation used a
novel treatment approach with PTSD patients. Patients provided a written
account of the event that triggered the PTSD, producing the written report
served as a reactivation of the memory, and were administered propranolol
to disrupt the following reconsolidation. Patients who were treated with propranolol reported large symptom improvements (Brunet et al., 2011).
In conclusion, disruption of consolidation and reconsolidation processes holds clinical promise and hopefully, as we learn more of the
phenomenon, this will lead to novel treatment strategies.
29
Genetics of fear conditioning
This section is limited to the functional polymorphisms and genotypes of
direct interest for the thesis.
5-HTTLPR
The serotonin transporter gene linked polymorphic region (5-HTTLPR) is a
functional polymorphism where different alleles have different efficacy of
the serotonin reuptake mechanism. This polymorphism directly impacts the
mechanism that is targeted by selective serotonin reuptake inhibitors
(SSRIs); arguably the most widely used medical treatment of anxiety. The
polymorphism comes in two variants, simply called short (s) or long (l) allele, creating the possible genotypes, ss, sl, or ll. The short allele carriers
have a more efficient reuptake mechanism leaving less serotonin in the synaptic cleft in vitro (Lesch et al., 1996). However, studies examining the
availability of serotonin in vivo have yielded mixed results (Bah et al., 2008;
Dyck & Malison, 2004; Shioe et al., 2003). Carriers of the short allele of 5HTTLPR have been reported to have elevated anxiety-related personality
traits (Lesch et al., 1996) and increased risk for development of psychopathology (Caspi et al., 2003). Moreover, short allele carriers have enhanced
reactivity of the amygdala (Hariri et al., 2002; Munafò, Brown, & Hariri,
2008), a key structure for fear conditioning (LeDoux, 2003).
There is also evidence that 5-HTTLPR is associated more directly with fear
conditioning. Short allele carriers have been associated with stronger acquisition and slower extinction as compared to long allele homozygotes (Crişan
et al., 2009; Garpenstrand, Annas, Ekblom, Oreland, & Fredrikson, 2001;
Lonsdorf et al., 2009).
The target function of SSRIs is to block serotonin reuptake from the synaptic
cleft and this is believed to increase the effectiveness of released serotonin.
In rodents, a single dose of SSRIs facilitate fear acquisition (Burghardt,
Sullivan, McEwen, Gorman, & LeDoux, 2004) and expression of earlier
acquired fear memories (Burghardt, Bush, McEwen, & LeDoux, 2007).
Moreover, a single dose SSRI treatment facilitate memory consolidation in
30
humans (Harmer, Bhagwagar, Cowen, & Goodwin, 2002). These results
indicate that more serotonin means better fear conditioning, which is in line
with the results reported above that implicate short allele carriers as having
stronger acquisition and slower extinction. However, other differences between carriers of the long and short allele of 5-HTTLPR may be equally
important. Short allele carriers, as compared to ll homozygotes, have been
found to have altered functional connectivity between areas involved in
emotion (Pezawas et al., 2005). It is still too early to draw any far reaching
conclusions from this study, but serotonin is used as a neurotrophic factor
during neural development, providing a plausible avenue for structural
changes to depend on serotonergic genes (Nordquist & Oreland, 2010). In
any case, the studies that reveal changes in fear conditioning after a single
dose of SSRIs, further implicate serotonergic mechanisms as having an impact on fear memory learning and persistence. In conclusion, the study of 5HTTLPR’s impact on fear conditioning offer a possible link between fear
memory processes, serotonergic neurotransmission and SSRI treatment.
COMT
The functional polymorphism val158met (rs4680) of the catechol-Omethyltransferease (COMT) enzyme has two alleles, valine and methionine,
(val and met) that differ in the efficacy of the COMT enzyme, which is vital
for the elimination of extracellular dopamine. The COMT enzyme is present
throughout most of the brain (Hong, Shu-leong, Tao, & Lap-Ping, 1998).
Functional analysis of the polymorphism report that val carriers have a
higher activity of the COMT enzyme leading to the presence of less extracellular dopamine (Chen et al., 2004) particularly in val/val homozygotes, as
reported by an in vivo imaging study (Slifstein et al., 2008). There are also
reports claiming val158met COMT has an impact on D1-receptors and not
D2-receptors, suggesting a possible receptor-specific mechanism of the polymorphism (Hirvonen et al., 2010; Slifstein et al., 2008).
Functional polymorphisms affecting dopamine in the brain are interesting for
a number of reasons. In anxiety research, this is mainly because of: 1) Dopamine is necessary in the amygdala for fear conditioning to take place
(Darvas, Fadok, & Palmiter, 2011; de Oliveira et al., 2011). 2) Dopamine is
necessary in BLA for long term fear memories to form (Fadok, Darvas,
Dickerson, & Palmiter, 2010; Rossato, Bevilaqua, Izquierdo, Medina, &
Cammarota, 2009) and basolateral amygdala is vital for the modulation of
arousal memory consolidation (McGaugh, 2004). 3) Dopamine is a part of
the arousal reaction and a candidate element of the mechanisms that enables
the amygdala to form an association between CS and UCS (Fadok et al.,
2010; Marinelli & Piazza, 2002; Rodrigues, LeDoux, & Sapolsky, 2009). 4)
31
Research in rodents demonstrate that dopamine affect the BLA by increasing
the firing in inhibitory interneurons and decreasing the firing in projection
neurons. Also dopamine receptor activity in the BLA reduce the impact of
prefrontal and thalamic inputs to the amygdala (Rosenkranz & Grace, 1999).
This means that dopaminergic signalling provide a possible mechanism for
the modulatory role on other areas that the amygdala have in arousal
memory consolidation (McGaugh, 2004). Hence, the val158met polymorphism is widely researched as a candidate gene for psychiatric disorders.
Brain imaging studies have associated the val allele with psychiatric disorders (Domschke & Dannlowski, 2010; Lelli-Chiesa et al., 2011). However,
association studies on large samples did not find any obvious link between
COMT and anxiety disorders (Stein, Fallin, Schork, & Gelernter, 2005;
Wray et al., 2008).
Considering the above reported importance of dopamine in amygdala
memory functions, it is likely that there should be some association between
the COMT val158met polymorphism and fear conditioning. Two studies
have examined fear conditioning and its relation to the COMT val158met
genotype in humans, but with mixed findings. One, using startle response,
reported slower extinction in met homozygotes (Lonsdorf et al., 2009) and
the other, using learning rates based on a skin conductance signal model,
reported stronger reacquisition in a val/met-group, but only in the presence
of a certain dopamine transporter (DAT1) polymorphism (Raczka et al.,
2011). More research is needed to evaluate these results and fit them into a
bigger picture of how the COMT val158met polymorphism affect fear conditioning processes.
32
Methods
Psychophysiology- skin conductance
The arousal reaction is vital both for forming fear memories and fear expression in general. As part of the arousal reaction, the sympathetic subdivision
of the autonomic nervous system trigger defensive responses such as an increase in heart rate, blood pressure and sweating as well as diversion of
blood from digestion to limb musculature. The induced sweating lead to
changes in skin conductance which can be measured. The study of skin conductance, or electrodermal activity (EDA), has been around since the 1840s,
but the use for it in psychophysiology arose in the 1880s when two independent scientists, Féré in France and Tarchanoff in Russia, measured
changes in skin conductance in response to sensory stimuli, imagination and
expectation. During the second half of the 1900s, technological progress
provided better equipment and the use of the skin conductance measurement
became more widely used. In short, skin conductance uses a weak current
that flows between two electrodes fastened on the skin using a conductive
gel. When sweat glands open, for instance as a result of a fear reaction, this
changes the conductivity of the skin, and a change in current can be noted
(Boucsein, 1992). The measurement of skin conductance reactions is inherently event-related because there is no real baseline that can easily be measured.
Simply put, the brainstem controls the activation of the sympathetic nervous
system, but there is evidence that cortical areas in turn control the brainstem.
For instance, direct stimulation of the amygdala, hippocampus, anterior cingulate cortex, lateral prefrontal cortex and middle temporal gyrus elicit
changes in skin conductance. Moreover, an imaging study reported activity
in the insular cortex and the ventromedial prefrontal cortex to vary with skin
conductance (Critchley, 2002). In essence, all structures that we consider
part of the emotion circuit of the brain (LeDoux, 2000) can produce or covariate with the production of skin conductance responses, which is encouraging for its use as a measurement of emotion reactions.
33
Functional Magnetic Resonance Imaging (fMRI)
FMRI is presently the most widely used functional brain imaging method.
An MRI-scanner can produce different signals (sequences) by shifting the
magnetic fields in different ways. The most widely used MRI-sequence in
psychological research is an echo planar imaging (EPI)-sequence measuring
a blood oxygen level dependent signal, in short, the BOLD response. The
functional principle behind a MRI-scanner is beyond the scope of this thesis
and requires extensive knowledge of physics. Simply put, a magnetic field
changes direction many times per second and measures a signal produced by
the induced movement in blood molecules. It then uses the change in magnetization between oxygen-rich and oxygen-poor blood to produce the
BOLD signal.
Brain activation results in fMRI research are presented as contrasts between
conditions and/or groups, that is, as differences between two conditions
and/or groups. For example, the condition of watching a picture can be contrasted with the condition of looking at a blank screen. This contrast can then
itself be contrasted between a group of patients and healthy controls. An
experiment’s timeline will consist of many scans, for example one scan per
three seconds. Every point in the brain is then usually modeled in a regression (y=β1x1+β2x2+…+βnXn+C), using all the scans as data points, where
every condition is a separate regressor (β) and the scans between conditions
become an additive element that serves as a baseline (C). A contrast between
conditions is performed by subtracting the beta value for one condition from
the beta value of another condition. These beta value differences are themselves normally distributed so it is possible to make a statistical test for each
measured point in the brain. It is then possible to search for clusters of points
where the activation differ with statistical significance between conditions or
groups. These areas are often projected onto pictures of brains when presented as results (Logothetis, 2008).
34
Aim
The general aim of this thesis was to examine the modulation of fear memories, achieved by disrupting the reconsolidation process that follow upon fear
memory reactivation, using measures of peripheral (study I, II and III) and
central nervous system activity (study III).
The aim of the first study was to examine the attenuation of fear memories
by reconsolidation disruption and to explore the impact of two functional
polymorphisms previously associated with fear conditioning, the serotonin
transporter gene linked polymorphic region and the val158met (rs4680) of
the catechol-O-methyltransferease enzyme, on fear memory reconsolidation,
using reacquisition as the memory probe. The second study aimed at attenuating fear memories by reconsolidation disruption and, using reinstatement
as a memory probe, explore the impact of the 5-HTTLPR and val158met
COMT polymorphisms on fear memory processes. Finally, study III aimed
at testing the hypothesis that a fear memory trace localized in the amygdala
is erased through reconsolidation disruption.
35
Study I:
Aim and background
Attenuation of fear memories by means of extinction during ongoing reconsolidation, that disrupts the reconsolidation processes, have recently been
shown in rodents (Monfils et al., 2009) and humans (Schiller et al., 2010).
Study I aimed to replicate this phenomenon by installing a fear memory with
fear conditioning and then to disrupt the reconsolidation by means of extinction after a fear memory reminder. The strength of the fear memory was then
tested with reinstatement and reacquisition. In addition, study I explored if
the functional polymorphisms 5-HTTLPR and val158met COMT, implicated
in fear conditioning (Garpenstrand et al., 2001; Lonsdorf et al., 2009) also
affect fear memory reconsolidation processes.
Methods
Sixty-six participants (38 females) were recruited from public billboards
around campus. Subjects visited the lab on three consecutive days. On day 1
they completed a session of fear conditioning. The subjects watched a computer screen with a photo of a lamp in a neutral environment. The lamp
could be lit in either blue or red. One of the colours was paired with an electric shock and became CS+ and the other colour became CS-. Colours were
counterbalanced between subjects. The session consisted of 16 presentations
of CS+ and CS- each. CS+ was always paired with an unpleasant shock to
the lower dorsal right arm. On day 2, twenty-four hours later, subjects returned for a fear memory activation. Following memory activation, one
group underwent an extinction session 10 min after the memory activation
(the 10 min group), and thus, during the ongoing reconsolidation. The other
group underwent extinction 6 h after the memory activation (the 6 h group),
and thus, after completed reconsolidation. Return of fear was tested on day
3, another twenty-four hours later, using reinstatement and reacquisition.
After the final testing on day 3, subjects left a saliva sample that was used
for gene analysis. Skin conductance responses were measured during all
segments of the experiment.
36
Results
The 6 h group discriminated between CS+ and CS- during reacquisition,
demonstrating relearning, while the 10 min group did not. Moreover, reactions to CS+ were stronger in the 6 h group than in the 10 min group, while
reactions to CS- did not differ between groups, demonstrating that the
groups differed mainly in their reactions to the stimulus previously associated with shock. Thus, this study conceptually replicated the previously reported results (Schiller et al., 2010). In addition, gene analysis revealed that
this effect was mainly found in short allele carriers of 5-HTTLPR and
val/val homozygotes of the val158met COMT functional polymorphisms.
Due to a programming error, most of the reinstatement data was corrupted.
However, the data that was not corrupted indicated a trend towards stronger
fear reactions in the 6 h group than the 10 min group during reinstatement.
Discussion
The present study demonstrates attenuation of fear memories by means of
disrupting their reconsolidation and suggests that this effect is mainly found
in certain genotypes. Reacquisition was mainly found in s carriers of 5HTTLPR and val/val homozygotes of the val158met COMT polymorphism
and only when extinction was performed after an undisturbed reconsolidation. L/L homozygotes of 5-HTTLPR and met-carriers of COMT did not
display reacquisition regardless of whether reconsolidation was disrupted or
not, possibly because the reconsolidation process lasts longer in these genotypes making both groups reconsolidation processes disrupted. Also, due to
technical reasons, most of the reinstatement data was corrupted making the
replication of the reconsolidation effect measured by reinstatement unclear.
37
Study II:
Aim and background
The aim of Study II was to attenuate a fear memory by disrupting memory
reconsolidation. Specifically, the aim was to install an experimental fear
memory using fear conditioning and then disrupt the reconsolidation process
after a memory activation using extinction training and finally, to test the
strength of the fear memory using reinstatement. It could be argued that both
reinstatement and reacquisition activate a previously formed, but now passive and dormant memory, and that reacquisition in addition restores and
possibly creates an active memory. Thus, reinstatement examines fear expression of a previously acquired memory without the possible confound of
new memory formation. In addition, study II investigated the effect of the
functional polymorphisms 5-HTTLPR and val158met COMT enzyme on
fear conditioning and fear memory reconsolidation processes. A cold pressor
test was used prior to acquisition with the intention to boost initial memory
encoding.
Methods
Fifty participants (32 females) were recruited by public advertisements. Subjects visited the lab on three consecutive days. On day 1, subjects went
through a session of fear conditioning. Subjects watched a computer screen
with a photo of a lamp in a neutral environment. The lamp was lit in either
blue or red. One of the colours became CS+ and the other CS-, counterbalanced between subjects. The session consisted of 16 presentations of CS+
and CS- each. CS+ was always paired with an unpleasant shock to the lower
dorsal right arm. On day 2, twenty-four hours later, subjects returned for a
fear memory activation, consisting of a CS+ presentation. Following this
memory activation, one group underwent an extinction session 10 min after
the memory activation (the 10 min group), and thus, during the ongoing reconsolidation. The other group underwent extinction 6 h after the memory
activation (the 6 h group), and thus, after completed reconsolidation. Return
of fear was tested on day 3, another twenty-four hours later, using reinstatement. Skin conductance responses were measured during all segments of the
38
experiment. After the final testing on day 3, subjects left a saliva sample for
gene analysis.
Results
This study partially replicated study I in that an effect of reconsolidation
disruption was found, but only in s carriers of 5-HTTLPR and val/val homozygotes of the val158met COMT polymorphism. In addition, the effect was
weaker, displaying a trend in val/val homozygotes and a significant difference in s carriers only in reactions to CS+. No main effect of reconsolidation
disruption averaged over all genotypes was found.
Discussion
The group with disrupted reconsolidation (the 10 min group) showed weaker
reactions to reinstatement than the group with no disrupted reconsolidation
(the 6 h group). However, this was only noted as a trend in val/val homozygotes of the val158met COMT polymorphism and a significant difference in
reactions to CS+ in s carriers of 5-HTTLPR. Unlike study I, no main effect
of reconsolidation disruption irrespective of genotype was found. Numerically, it appears as the effect is there, but large variance between individuals
contributes to the lack of statistical significance. Possibly the phenomenon is
fragile or maybe the cold pressor test produced the larger variation between
individuals.
39
Study III:
Aim and background
The attenuation of fear memories by disruption of reconsolidation has been
localized to the amygdala in animals (Nader et al., 2000) using protein synthesis inhibition, but no such studies have been made in humans because the
process of inhibiting protein synthesis used in animals are not safe. The aim
of study III was to localize the processes of reconsolidation in humans by
performing fMRI neuroimaging of BOLD signal changes reflecting fear
memory strength linked to fear expression comparing subjects who had a
disrupted reconsolidation with those who had not.
Methods
Thirty subjects (13 females) were recruited by public billboards and on underwent a session of fear conditioning (day 1). Subjects watched a computer
screen projecting a picture of a lamp in a neutral environment that could be
lit in either red or blue. One of the colours was always paired with a shock to
the lower dorsal right arm and became the CS+ while the other colour became CS-. CS+ colour was balanced between subjects. The session consisted
of 16 presentations of CS+ and CS- each. Twenty-four hours later (day 2),
they returned for a two minute CS+ presentation serving as a fear memory
activation. One group then got extinction after 10 minutes (the 10 min
group) and hence, within the reconsolidation interval, and one group got
extinction after 6 hours (the 6 h group) and hence, outside of the reconsolidation interval. Another twenty-four hours later (day 3) the subjects returned for a second extinction session, in order to image the renewal of fear
in a fMRI scanner. Two days later (day 5), subjects returned for another test
of return of fear using reinstatement. Skin conductance responses were
measured in all phases of the experiment except during the extinction session
performed in fMRI because of technical reasons.
40
Results
The 6 h group displayed stronger return of fear, as defined as the difference
between the last extinction and the first reinstatement trial, than the 10 min
group. BOLD signal changes indicated the presence of a memory trace in the
amygdala in the 6 h, but not the 10 min group. Moreover, BOLD activity in
the 6 h group predicted return of fear the subsequent day and was correlated
with fear expression the previous day. In addition, the area in amygdala in
which the groups differed in BOLD activity overlapped with the area predicting return of fear and the area that correlated with fear expression in the
6 h group, further implicating the presence of a memory trace in the 6 h
group. Also, fear circuit activity was more tightly linked to amygdala activity in the 6 h group as BOLD signal changes in the amygdala covaried with
activity in the insula, ACC and hippocampus to a greater extent in the 6 h as
compared to the 10 min group.
Discussion
The present study demonstrated attenuation of fear memories by reconsolidation disruption after a fear memory reminder. Also, using fMRI, it showed
that the group with undisrupted reconsolidation, and hence, a more complete
memory, had significantly more amygdala activation than the group with
disrupted reconsolidation. Because the amygdala activation, indicating a
difference in memory strength between groups, was functionally associated
with return of fear and fear memory recall in the 6 h group, this suggests the
presence of a spared memory in the 6 h group and in contrast, a disrupted or
abolished memory in the 10 min group. Hence, there is reason to believe that
in humans, as in rodents, fear memory reconsolidation is in part amygdala
dependent.
41
Results overview (study I, II & III)
Included here are figures intended to give an easily accessible overview of
the results included in this thesis. Figure 1 presents the skin conductance
results of study I, II and III. Figure 2 presents the skin conductance results of
study I and study II regarding the examined genotypes. Figure 3 gives an
overview of the sample sizes and the partitioning of genotypes on experimental group for study I and study II. Table 1 and 2 demonstrates the overlap between genotypes in the samples of study I and II.
42
Day 2
0
0,1
0,2
0,3
0,4
-0,1
-0,2
-0,1
-0,2
0,1
0,2
0,3
0,4
6h
10 min
Reconsolidation interval
-0,2
-0,1
0
0,1
0,2
0,3
0,4
-0,2
-0,2
0
0,1
0,2
0,3
0,4
-0,2
-0,1
0
0,1
0,2
0,3
0,4
-0,1
6h
10 min
6h
10 min
Extinction
Day 2
-0,1
0
0,1
0,2
0,3
0,4
-0,2
-0,1
Memory
activation
0
6h
10 min
6h
10 min
6h
10 min
Fear conditioning
Day 1
0
0,1
0,2
0,3
0,4
-0,2
-0,1
0
0,1
0,2
0,3
0,4
-0,2
-0,1
0
0,1
0,2
0,3
0,4
A
SCR
SCR
SCR
SCR
SCR
SCR
SCR
SCR
SCR
10 min
6h
6h
10 min
6h
10 min
Return of fear
Day 3
Experimental design and
timeline (top). Mean differential skin conductance
delta scores (CS+ minus
CS-) for acquisition (Fear
Conditioning), extinction
and return of fear for study
I (top panels), study II
(middle panels) and study
III (bottom panels) after
extinction inside (10 min)
and outside (6 h) the reconsolidation interval. Error
bars are standard error of
means.
Title: Extinction during
reconsolidation attenuates
return of fear.
Overview Figure 1.
SCR
SCR
10 min
6h
0
0,1
0,2
0,3
-0,2
met
ll
-0,2
Genotype
Genotype
-0,2
-0,1
0
0,1
0,2
0,3
-0,1
val/val
s
10 min
6h
-0,1
0
0,1
0,2
0,3
-0,2
-0,1
0
0,1
0,2
0,3
Study I
SCR
SCR
val/val
s
Genotype
Genotype
Study II
met
ll
10 min
6h
10 min
6h
Mean differential skin conductance delta scores (CS+ minus
CS-) reflecting return of fear as
measured by reacquisition (Study
I, left panels) and reinstatement
(Study II, right panels) after extinction inside (10 min) and outside (6 h) the reconsolidation
interval in short allele carriers (s)
and ll homozygotes of the 5HTTLPR polymorphism (top
panels) and val/val homozygotes
and met carriers of the val158met
COMT polymorphism (bottom
panels). Error bars are standard
error of means.
Title: The effect of reconsolidation disruption is modulated by
genes.
Overview Figure 2.
met
N = 10
val/val
N=3
met
N = 14
COMT
COMT
val/val
N=5
5-HTTLPR
ll
s
N=5
N = 13
5-HTTLPR
ll
s
N=4
N = 10
val/val
N=4
met
N=9
COMT
5-HTTLPR
ll
s
N=9
N=6
6 h:
N = 16
val/val
N=6
met
N=9
COMT
5-HTTLPR
ll
s
N=7
N=8
10 min:
N = 15
Conditioned:
N = 31
Conditioned:
N = 33
10 min:
N = 18
All subjects:
N = 50
All subjects:
N = 66
6 h:
N = 15
Study II
Study I
Sample sizes and the partitioning of genotypes for
conditioned subjects over
experimental groups in
study I and II. In study I,
5-HTTLPR genotyping
failed for one of the subjects and COMT genotyping failed for another of
the subjects. In study II,
genotyping failed for two
subjects regarding 5HTTLPR and for three
subjects regarding COMT.
Title: Sample sizes and the
partitioning of genotypes
over experimental groups.
Overview Figure 3.
Table 1: Genotype partitioning of conditioned subjects in study I
5HTTLPR
COMT
val/val
COMT
val/met
COMT
met/met
fail
5-HTTLPR
all
SS
1
3
4
0
8
SL
6
7
1
1
15
LL
1
5
3
0
9
fail
0
0
1
0
1
COMT all
8
15
9
1
33
Table 2: Genotype partition of conditioned subjects in study II
5HTTLPR
COMT
val/val
COMT
val/met
COMT
met/met
fail
5-HTTLPR
all
SS
2
1
2
0
5
SL
4
5
1
0
10
LL
4
9
3
0
16
fail
0
0
0
0
0
COMT all
10
15
6
0
31
46
General Discussion
Main findings
The main findings of the thesis is that attenuation of fear memories by reconsolidation disruption is a reproducible phenomenon and that the effect
may be driven mainly by subjects with certain genotypes. Specifically, the
phenomenon is present in short allele carriers of the 5-HTTLPR and val/val
homozygotes of the val158met COMT polymorphisms. Moreover, fear reconsolidation disruption alters the activity and functionality of the amygdala
at subsequent fear memory recall suggesting that reconsolidation processes
are amygdala-dependent.
Discussion
All three studies demonstrate the attenuation of fear memories by reconsolidation disruption, although in varying degree. Taken together with previous
results (Kindt et al., 2009; Schiller et al., 2010), this supports that the reconsolidation of fear memories occurs also in humans and present an evolutionary shared mechanism with other mammals such as rodents (Monfils et al.,
2009). The evolutionary tie is strongly implicated by study III which finds
functional differences depending on reconsolidation conditions in the amygdala, where reconsolidation also takes place in rodents (Nader et al., 2000).
The finding that s carriers of 5-HTTLPR showed more return of fear is in
line with other findings in fear conditioning implicating s carrier as having
stronger acquisition and slower extinction (Garpenstrand et al., 2001;
Lonsdorf et al., 2009). Moreover, since fear conditioning is an amygdaladependent process, this is also in line with the reported results of increased
amygdala reactivity in s carriers (Hariri et al., 2002).
The specific neurobiological mechanisms underlying these results
(Garpenstrand et al., 2001; Hariri et al., 2002; Lonsdorf et al., 2009) are unknown, but studies using acute SSRI treatments produce stronger reactions
in fear conditioning, implicating that an increased presence of extracellular
serotonin in the synaptic cleft is involved (Burghardt et al., 2007; Harmer et
al., 2002). However, although a stronger return of fear was noted in short
47
carriers in both study I and study II, neither study I nor study II found evidence for stronger acquisition in s carriers.
The findings implicating the val/val homozygotes of the val158met COMT
polymorphism as having increased return of fear doesn’t fit as easily with
previous results in fear conditioning. It has been reported that met-carries
have slower extinction (Lonsdorf et al., 2009) and that a val/met-group had
stronger reacquisition (Raczka et al., 2011), which are results that are somewhat conflicting with the present results. However, imaging of emotional
aversive stimuli have reported increased amygdala activity in val-carriers
(Domschke et al., 2008, 2012), possibly affecting the amygdala-dependent
fear conditioning processes. Study I and II of this thesis both implicate
val/val homozygotes as functionally different from met-carriers in fear
memory reconsolidation. This corresponds to results from in vivo imaging
where val/val homozygotes differed from met-carriers in the amount of dopamine in cortico-limbic areas (Slifstein et al., 2008), validating the partioning of val/val homozygotes from met-carriers in the study of dopamine functionality. Val/val homozygotes had a higher activity of the COMT enzyme
leading to the presence of less extracellular dopamine (Chen et al., 2004)
throughout most of the brain (Hong et al., 1998). Basolateral amygdala appear vital for fear conditioning processes (LeDoux, 2000; Maren, 2001;
Quirk & Mueller, 2008) and rodent studies suggest that dopamine mainly
affect the BLA by increasing the firing in inhibitory interneurons and decreasing the firing in neurons projecting to the nucleus accumbens. In addition, dopamine receptor activity in the BLA seem to reduce the impact of
prefrontal and thalamic inputs to the amygdala (Rosenkranz & Grace, 1999).
It is possible that less dopamine leads to less inhibition and that this would
lead to increased fear responses, in turn leading to better acquisition of fear
memories and increased return of fear. However, one could also argue the
reverse, since dopamine is necessary in the BLA for fear memory consolidation (Fadok et al., 2010) and that less dopamine could lead to weaker long
term memories of fear. Although the specific mechanisms remain unclear,
the result of this thesis suggests that val/val homozygotes maintain and update fear memories more effectively than met carriers.
What do the results from study I and study II that fear memory reconsolidation disruption only seems to take place for specific genotypes mean? Met
carriers of the val158met COMT polymorphism and the ll homozygotes of
5-HTTLPR displayed no return of fear, neither when extinction was performed after 10 min or 6 h after the memory reminder. Possibly, reconsolidation of fear memory arousal is simply weaker, or less effective, in these
genotypes, so that the memory strength is comparable with the memories of
disrupted reconsolidation in val/val homozygotes and s carriers. However, if
48
this was true, fear memories would have a more transient character in met
carriers of COMT and ll homozygotes of 5-HTTLPR which would possibly
be reflected in a lower propensity to anxiety disorders in these genotypes.
Such a direct connection between these alleles and anxiety disorders has not
been found (Samochowiec et al., 2004; Stein et al., 2005; Wray et al., 2008).
An alternative is that the reconsolidation of met carriers of val158met
COMT and ll homozygotes of 5-HTTLPR is more easily disrupted, in fact,
disrupted by the learning of a new contingency. One could reason that the
reminder is not only a reminder of the stimulus-shock contingency, but also
learning of a new contingency of stimulus-no shock. This thesis have discussed that the removal of conditioned fear responses consist of both inhibition resulting from new learning (extinction) and unlearning of the original
memory (reconsolidation disruption). It is possible that there in met carriers
of COMT and ll homozygotes in 5-HTTLPR exist a direct interplay between
new learning and reconsolidation disruption. However, a number of arguments can be raised against this view. It is well known from animal studies
that a presentation consisting of a single element of a stimulus-shock contingency is enough to serve as a reminder of the stimulus-schock contingency
without extinguishing the learned fear response, on the contrary, it strengthens the response. Also, the new contingency is supposed to be learned instantly, which our extinction data speak against. Moreover, the protein synthesis of reconsolidation need a few minutes to start, requiring the subjects to
keep the presumably instantly learned new contingency in short term
memory to subsequently affect reconsolidation. Furthermore, if an instant
coupling between new learning and reconsolidation disruption exist in these
genotypes, they would arguably have problems with generalizing fears from
one situation to another, something that lends itself easily to experimentation.
Perhaps a better alternative is that the met carriers of val158met COMT and
ll homozygotes of 5-HTTLPR have a reconsolidation window that is open
longer than val/val homozygotes and s carriers, rendering also the extinction
that took place 6 h after the reminder as disruptive. However, it appears that
protein synthesis inhibitors must be initiated 90 min after training to disrupt
consolidation (Davis & Squire, 1984). This suggests that if a reconsolidation
disruption can be initiated more than 6 h after a reminder, the disrupted
mechanism is probably not protein synthesis. Indeed, injecting catecholamine synthesis inhibitors produces consolidation disruption in a different
time-course and in different brain structures than protein synthesis inhibitors,
suggesting a different reconsolidation interval for the modulation of reconsolidation by catecholamines than by protein synthesis inhibition (Davis &
Squire, 1984). Future studies could establish if the reconsolidation interval
differ between means of disruption or modulation such as protein synthesis
49
inhibitors or norepinephrine antagonists, both in timeframe, brain structures
involved, and in different relevant genotypes.
The demonstration of amygdala involvement in study III is in line with animal data on reconsolidation disruption (Nader et al., 2000). Molecular mechanisms are most easily studied in animals, but human studies give an important opportunity to study task-dependent whole brain connectivity in vivo
using neuroimaging. Although the amygdala localized fear memory presence
and reconsolidation may be an evolutionary shared mechanism the tighter
coupling between amygdala and the so called fear circuitry (including hippocampus, insula and ACC) found in study III may represent speciesspecific memory- and cognitive processes. It is conceivable that what we
colloquially mean with a fear memory consist of several aspects (such as
cognitive, episodic and emotional) with different representations in our
brains and that amygdala is responsible, through consolidation mechanisms,
to update several of these representations with regards to emotional content
(McGaugh, 2004). However, if the amygdala modulates memories in other
brain structures with emotional content during the consolidation and reconsolidation process, it would not be seen in the imaging data of study III. Instead, the synchronized activation of insula, ACC and hippocampus may
reflect different aspects of the memory activating in concert in their respective parts of the brain. On the other hand, these areas may be involved in fear
expression or cognitive evaluation of the present fear memory. For instance,
insular activity correlate with spontaneous fluctuation in skin conductance
(Critchley, 2002) which may reflect a function in fear expression and the
association between ACC and attentional processes (Lane et al., 1997) suggest that the activity in ACC may be the result of selective attention to the
stimulus. However, with the present methodology it is almost impossible to
separate the activation of fear memories from the production of linked fear
expression without accompanying knowledge of the neurobiological processes involved. Study III, together with neurobiological studies on rodents
placing both fear memories (Reijmers et al., 2007) and reconsolidation of
fear memories (Nader et al., 2000) in the amygdala, strongly suggest that the
disruption of reconsolidation reduces an amygdala localized fear memory
also in humans.
Limitations
The studies contained in this thesis have some limitations. First, the programming error that corrupted most of the reinstatement data in study I. Second, study II would have benefited from an extra control group in order to
be able to evaluate the evaluation of the stress manipulation thoroughly.
Also, the sample sizes for the behavioural genetics results are to be consid50
ered small. However, the results are strengthened by the fact that the results
were highly similar in the two different cohorts. Still, replications with larger
sample sizes are warranted, especially considering the results that did not
agree between study I and II. Moreover, the small sample sizes make it difficult to dissociate the effect of one polymorphism from another since subjects
possess different combinations of the polymorphisms involved. The studies
contained in this thesis have used ANCOVAs in an attempt to dissociate the
effects of the different polymorphisms examined. Finally, technical limitations stopped us from using skin conductance measurements in the fMRIscanner, which would have provided an extra manipulation check.
Future directions
This thesis lends further support to the notion that attenuation of fear memories by reconsolidation disruption is possible in humans. It would prove interesting to further specify what kind of manipulations that can disrupt reconsolidation. For instance, there is some evidence that conflicting learning
can be used to disrupt consolidation in humans (Holmes et al., 2010) and
reconsolidation in rodents (Spear et al., 1973). To identify the methods that
can be used for reconsolidation disruption limits the set of possible underlying mechanisms and also has clinical implications. Also, the timing conditions that enable reconsolidation disruption should be further investigated.
For instance, is there a critical time window in which reconsolidation is most
effective? Furthermore, what kind of memories can be modulated by reconsolidation disruption? Is it true that the memories must have an arousal component as previously speculated (Squire et al., 1976) ?
Moreover, further imaging studies, using fMRI and PET, could perhaps clarify the findings implicating certain genotypes in this phenomenon by providing a picture of neural activity, neural structure, and neurochemical differences. For instance, do s carriers and ll homozygotes of the 5-HTTLPR polymorphism differ in serotonin receptor availability during encoding, extinction or recall?
At the present time, the empirical justification for using fear conditioning as
a model for anxiety disorders is mainly based on the presence of fear conditioning-related renewal phenomena in clinical patients (Mineka et al., 1999;
Mystkowski et al., 2003; Rachman, 1989; Rodriguez, Craske, et al., 1999),
modest increases in fear acquisition and extinction (Lissek et al., 2005) and
the effectiveness of extinction therapy (Ougrin, 2011). It would be interesting, using the methodology proposed in this thesis, to investigate the fear
memory processes of clinical populations to see if they differ from healthy
controls. If a clinical population would differ from healthy controls in fear
51
memory maintenance, this would provide evidence to further justify fear
conditioning as a model for anxiety disorders. The existing somewhat poor
results associating anxiety disorders with enhanced fear conditioning may be
a result of investigating elements of fear conditioning in which patients and
controls do not differ, i.e. acquisition and extinction. Perhaps an investigation regarding the fear memory processes involved in fear conditioning
would provide stronger results. If differences in fear memory processes between anxiety patients and controls exist, they could provide new target
mechanisms for treatment. Indeed, the attenuation of fear memories using
consolidation and reconsolidation disruption have already been addressed as
a possible avenue for novel treatment strategies (Brunet et al., 2011; Holmes
et al., 2010; Pitman et al., 2002). Finally, on a grand scale, the attenuation of
fear memories using reconsolidation may provide a future neurobiological
framework for many forms of therapy. Because, in essence, many forms of
therapy consist of the re-imagining and re-framing of memories by means of
cognitive analysis of an event, hopefully leading to less anxiety in subsequent remembrance.
In summary, future research should clarify what manipulations that can disrupt reconsolidation, under what conditions reconsolidation can be disrupted,
the neural mechanisms underlying reconsolidation disruption, and the possible clinical implications of the phenomenon.
52
Acknowledgment
This work was supported by the Swedish Research Council, the Swedish
Council for Working Life and Social Research, the Swedish Brain Foundation and Heumanska stiftelsen.
I would also like to show my gratitude towards my supervisors,
Mats Fredrikson and Tomas Furmark, for their generosity, time, immense
knowledge, and patience. I learned so much from you about the value of
good writing, simple designs, and scientific rigor. You are very talented and
I am forever in your debt.
Thanks to my fellow PhD-students in the group, from former titans to new recruits: Fredrik Åhs, perhaps the most generous man in science,
Clas Linnman, perhaps the most conscientious man in science, Vanda Faria,
perhaps the shortest and most violent woman in science, Åsa Michelsgård,
perhaps the trickiest roborally player in science, Malin Gingngnell, perhaps
the hardest woman to spell to in science, Jonas Engman, perhaps the neatest
man in science, Andreas Frick, containing perhaps the best gui in science,
Johannes Björkstrand, perhaps the most cross-training man in science and
Iman Alaie, perhaps the most literate man in science. Thanks also to all the
friends I made among other PhD-students through the years: the old gang,
Simon L, Jessika K, Vanda (again, and yes, you are not only violent, but
sweet) and Hällena, who introduced me to the champagne evenings, and so
many more, too many to name. Thanks all for being such a fun-loving and
friendly lot. Thank you Mats G for the brain gymnastics. We’ll get there.
Thanks to mom and dad for telling us kids to go for it. Thank you Krister,
Karin, Måns and Max. Thank you to all friends, old and new, who have endured the specific breed of spiritual taint that I bring to every situation. I
don’t know where to start. I usually start with a salad or a soup before moving on to the main course, and this is no exception. Thank you students, audiences, writer friends, hedgehogs, moomins, consciousness, thank you for
the music, the songs I’m singing, the whispers, the dreams, the madness, the
colours out of space and time, H. P. Lovecraft, popcorn, but to make a long
story unbareable…
…finally there is, the Hanna, who makes this otherwise so wonderful life, so
enjoyable.
53
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62
Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Social Sciences 81
Editor: The Dean of the Faculty of Social Sciences
A doctoral dissertation from the Faculty of Social Sciences,
Uppsala University, is usually a summary of a number of
papers. A few copies of the complete dissertation are kept
at major Swedish research libraries, while the summary
alone is distributed internationally through the series Digital
Comprehensive Summaries of Uppsala Dissertations from the
Faculty of Social Sciences.
Distribution: publications.uu.se
urn:nbn:se:uu:diva-180202
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2012

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