Yosif (Joe) Zaki

February 28, 2018

P2D1; Models of Fear Relapse: Different Animal or Same Beast?
Word Count: 2906
Citation Method: AMA

Abstract
[to come]

Introduction
Fear is a defensive behavior that can be adaptive and promote an animal’s survival;
however, uncontrollable levels of fear can be maladaptive and detrimental to an animal’s health.
In the clinic, patients with these symptoms are often diagnosed with anxiety-related disorders or
Post-Traumatic Stress Disorder (PTSD). In the lab, fear conditioning is a model of acquired fear
that has been largely studied in rodents for decades1, and serves as a model of Post-Traumatic
Stress Disorder (PTSD) and other trauma-related disorders. The behavioral paradigm involves
co-presentation of an unconditioned stimulus and a conditioned stimulus, where the
unconditioned stimulus represents the innately aversive experience and the conditioned stimulus
is the stimulus that was initially neutral and, through association with the unconditioned
stimulus, gained negative valence. The subsequent conditioned response is freezing behavior.
Freezing behavior is characterized by total immobility of the animal, and it is accompanied by
autonomic responses such as increased heart rate2 and increased levels of stress hormones such
as corticosterone3. This behavioral response is a proxy for “fear,” and this behavioral paradigm
has become the dominant model for studying trauma in rodent models of fear. Figure 1
exemplifies this behavioral paradigm.

Figure 1: (A) Schematic of contextual fear conditioning paradigm. CS-US pairing (fear conditioning; first
box) leads to expression of fear expression (fear retrieval; second box). Sequential placement in the
conditioned context leads to reduced fear expression (fear extinction; third box). The level of fear
expression can be assessed by placing the subject back in the conditioned context after fear extinction
(second fear retrieval; fourth box). (B) Schematic for cued fear conditioning paradigm. The steps are the
same as they are above; however, an additional cue for the expression of fear in this case is an auditory tone
played during fear conditioning22.
 For the past few decades, efforts have been aimed at understanding the neural
underpinnings of fear conditioning, in the hopes that this may uncover the etiologies of trauma-
related disorders such as PTSD. Regions of the brain including the hippocampus and the
amygdala have been implicated in the acquisition, storage, and retrieval of fear memories; for
example, modulation of activity in these brain regions during fear conditioning or fear retrieval
has disrupted normal fear memory formation or expression, respectively4. These two brain
regions have become the major players in the “fear circuit,” and several other brain regions such
as the bed nucleus of the stria terminalis (BNST) and the periaqueductal gray (PAG) have also
entered as modulatory forces of fear behavior. The fear circuit refers to the collection of brain
regions that seem to collectively process fear-related information. See figure 2 for a
comprehensive circuit schematic.

Figure 2: Schematic for the fear circuit, the brain regions involved, their structural connectivity, and their
putative functional properties of information processing
Abbreviations: AC, auditory cortex; BA, basal nuclei of the amygdala; BNST, bed nuclei of the stria
terminalis; CEl, lateral division of central nucleus of the amygdala; CEm, medial division of the central
nucleus of the amygdala; HIP, hippocampus; IC, inferior colliculus; IL, infralimbic division of the medial
prefrontal cortex; ITC, intercalated cells of the amygdala; LA, lateral nucleus of the amygdala; LH, lateral
hypothalamus; MGdv, dorsal and ventral divisions of the thalamic medial geniculate nucleus; MGm,
medial division of the thalamic medial geniculate nucleus; NAcc, nucleus accumbens, dlPAG, dorsolateral
division of the periaqueductal gray; vPAG, ventral division of the periaqueductal gray; PIN, posterior
intralaminar nucleus of the thalamus; PL, prelimbic division of the medial prefrontal cortex; PRh,
perirhinal cortex; PVN, paraventricular nucleus of the hypothalamus23.

In addition to the abundance of studies surrounding fear conditioning, there has been
significant attention given to the behavioral model of suppression of fear—namely, fear
extinction learning. Fear extinction involves repeated exposure to the conditioned stimulus in the
absence of the unconditioned stimulus, and the subsequent behavioral response is a reduction in
freezing behavior5. This serves as a laboratory model for exposure therapy, which similarly aims
to mitigate fear-related behavior in human patients with excessive fear.
The neural circuitry underlying fear extinction learning has also been extensively studied,
and the same brain regions involved in fear conditioning have also been found to be involved in
fear extinction learning—whether for acquisition, expression, retention, or a combination of
those functions6. While the hippocampus7 and amygdala8 seem to play roles in both fear
conditioning and fear extinction learning, the prefrontal cortex was a new player that seemed to
modulate fear expression after extinction learning and control fear extinction retention over
time9.
While extinction learning (and analogously, exposure therapy in the clinic) is generally
successful, people and rodents are prone to relapse under several different conditions10. The three
most well characterized forms of relapse are termed fear renewal, fear reinstatement, and
spontaneous recovery. Fear renewal is triggered by a shift in context. Fear reinstatement is
triggered by presentation of the unconditioned stimulus in a novel context. And spontaneous
recovery describes the unforeseen re-emergence of fear triggered by the passage of time. These
models are visualized in Figure 3. The fact that fear memories are prone to relapse has led the
learning and memory field to question what these behavioral methods of suppressing fear are
doing: do these interventions really abolish the fear memory and a new fear memory is formed
during relapse, or is this behavioral intervention merely a new form of learning that inhibits the
original fear memory11? If the latter case were true, it would be possible that relapse phenomena
are the result of a disinhibition of the fear memory. Indeed, it has been shown that, in the
amygdala, cells that seem to store the original fear memory are directly inhibited by a structural
reorganization of inhibitory interneurons after extinction and this inhibition is necessary for fear
extinction expression12.

Figure 3: Behavioral paradigms for relapse of fear memory expression; these manipulations all lead to a
relapse in freezing behavior in rodents6.
 While fear relapse has been behaviorally acknowledged in both humans and rodents—
potentially indicating an evolutionarily conserved neural phenomenon—little work has been
done to resolve the neural constituents of fear relapse13. A deeper understanding of fear relapse
may lend to therapeutic treatments that could more robustly combat excessive fear and related
disorders. Thus, here I review recent work that has been done to elucidate the neurobiology of
fear relapse, and discuss future directions that the field may take to further our understanding of
these behaviors.

Methods
Search Criteria
A literature search was conducted on NCBI PubMed database with the following search
terms: fear reinstatement, fear renewal, fear relapse. Literature was excluded if the paper did not
utilize fear conditioning as their model for the onset of trauma and also if the employed model
organism was not a rodent (excluded papers mainly used human models). Literature was also
excluded if studies involved drug addiction; addiction disorders has been shown to be comorbid
with anxiety- and fear-related disorders, and thus many studies have investigated the relationship
between addiction and fear. That work is outside the scope of this review. The resulting list of
papers is a collection of work studying fear relapse on several levels of complexity—from
neuromodulation, to functional connectivity, to behavioral manipulation. The list was
purposefully made to comprise work from several groups at different institutions; few labs study
fear relapse and so it is easy to fall into the pigeonhole of evaluating work from one laboratory or
institution. A similar diversity is noted for the journal in which each paper was published.

Research Design Comparison

None of these studies address all models of fear relapse, assess the same brain regions,
use the same behavioral parameters, or use the same manipulation techniques. Thus, a table
comparing these measures across all the papers would help draw connections and potential
disparities between papers. Below is this table:

Author Species Model of Fear Brain Regions Method of Power Tone or

Relapse Manipulation Contextual Fear
Conditioning
1 Goode, 201514 Rat Reinstatement, BNST Intracranial 6-11 rats Tone
Renewal muscimol per group
2 Hitora- Mouse Reinstatement PFC, CeA, Intracranial 4-14 mice Contextual
Imamura, VTA muscimol, per group
201515 SCH23390, or
PBS (Control)
3 Kim, 200716 Rat Reinstatement None Only behavior 7-10 rats Tone
per group
4 Marek, 201817 Rat Renewal vHipp, PFC Optogenetics, 1-16 rats Tone
DREADD, per group
intracranial
muscimol or
picrotoxin
5 Yoshii, 201618 Mouse Artificial Whole Brain DREADD 3-12 mice Contextual
Relapse per group
6 Jingji, 201519 Rat Renewal vHipp, Only 26 rats total Tone
Amygdala, PFC histological
quantification
Results
Behavior
In all of the above studies, the researchers were able to elicit re-emergence of freezing
behavior in their studies. The one exception to this was in one experiment from Kim, 2007. They
found that, in 16-day old rats, reinstatement could not be induced, whereas in 23-day old rats it
could. They concluded that there must be two different mechanisms by which animals early in
development represent and process fearful memories, as compared to animals later on in
development. This is in line with work done by previous work showing that BLA à PFC
connectivity emerges later in development than many other PFC pathways, and this connection
seems to be crucial for the modulation of fear states in rodents20. In the other five studies where
relapse was successfully elicited, adult animals were exclusively used.

Structural and Functional Connectivity

The functional connectivity of brain regions that seem to be important for the encoding,
expression, and modulation of fear has been well characterized (see Figure 2). Similarly, the
brain regions that seem to regulate extinction have been and continue to be dissected. Gaining a
similar map of different models of relapse of fear would be immensely valuable: connections
could be made between circuitry processing fear learning, extinction, and relapse. Additionally,
relationships could be drawn between different models of relapse. Renewal and reinstatement
may be regulated by overlapping circuits; however, it is also possible that different systems in
the brain control different forms of relapse. The above studies provide preliminary insights into
the circuits behind these phenomena.

Fear Renewal
It has been appreciated that there is structural and functional interconnectivity between the
vHipp, PFC, and amygdala and that this is important for fear- and anxiety-related behaviors.
Jingji, 2015 showed that following fear renewal, there is an upregulation of activity in vHipp
neurons that send dual projections to both the PFC and the amygdala. Similar patterns of activity
were not observed in vHipp neurons that sent projections to just one of those targets. Several
years later, another group found that not only is there a structural connection between the vHipp
and the PFC, but that vHipp neurons that innervate the PFC provide feed-forward inhibition to
the PFC, and the functional consequence of this connection is fear renewal. Thus, it seems that
inhibition in the PFC is crucial for retention of extinction, and disruption of this inhibition is a
potential mechanism by which fear renewal occurs. Alongside these pieces of work, Goode,
2015 demonstrated that pharmacological inactivation of the BNST does not prevent fear renewal,
indicating that perhaps the BNST is not a crucial node that regulates fear renewal. Of course, the
dynamics of pharmacological inactivation is dissimilar from optogenetic inactivation. It is
possible that a different mode of manipulation would lead to a different behavioral outcome.

Fear Reinstatement
While Goode, 2015 did not find a relationship between BNST activity and fear renewal, they
did find that pharmacological inactivation of the BNST was able to prevent fear reinstatement,
indicating that the BNST is necessary for fear reinstatement to occur. Additionally, Hitora-
Imamura, 2015 introduced the PFC, CeA, and VTA to the story. They showed that following
fear reinstatement, there is decreased activity in the PFC and concurrent increased activity in the
CeA, and that pharmacological inactivation of the PFC leads to an enhancement of freezing.
They found that a reinstating shock was accompanied by increased levels of activity in VTA
neurons projecting to the PFC. They reasoned that since the VTA provides dopaminergic inputs
to the PFC, and since there is an inverse relationship of activity between the PFC and CeA as a
result of reinstatement, perhaps the VTAàPFCàCeA connectivity plays a role in reinstatement.
They showed that inactivation of dopaminergic signaling in the PFC using a D1R antagonist led
to a restoration of PFC activity, a decrease in CeA activity, and a disruption of fear reinstatement
behavior. Whether the BNST and CeA play a combinatorial role in the expression of fear
reinstatement is an open question.

Artificial Relapse
Yoshii, 2016 employed a recent technique whereby neurons active during a discrete
experience are genetically modified and thereafter liable to artificial manipulation. They refer to
this label an “engram,” or, physical representation of a memory trace in the brain, and they use
this technique to label cells active during fear conditioning. After extinction learning, they
artificially activate the brain-wide fear engram to see whether this set of cells is sufficient to
drive a re-emergence of fear in rodents. Indeed, this is what they found; however, how this
relates to one of the established models of fear relapse is unclear. Moreover, it cannot be
resolved what elements of this network hold exceptional importance in this phenomenon; it is
likely that some portion of this network is crucial for this behavioral outcome while other regions
play no role at all.

Similarities
Few studies have systematically assessed the potentially differential consequences of
neural manipulations in different models of fear relapse. It is possible that while different sorts of
manipulations (e.g. pharmacological vs optogenetic) do not agree across studies of different
models of relapse (e.g. renewal vs reinstatement), similar manipulations across relapse models
may lead to similar outcomes.
Furthermore, there is initial evidence that similar brain regions and potentially similar
mechanisms may be regulating both fear reinstatement and fear renewal. For instance,
dysregulation of inhibitory signaling in the PFC may be at the heart of both fear renewal (Marek,
2018) and fear reinstatement (Hitora-Imamura, 2015). While Yoshii, 2016 did not provide high
enough resolution to tease out the differential contributions that different brain regions may be
playing in this behavior, it is possible that the pharmacogenetic stimulation that they
administered provided the appropriate dysregulation of signaling in the PFC that is necessary to
elicit re-emergence of fear behavior. These, of course, are questions open for neuroscientists to
answer.

Future Directions
Here, I have reviewed six recent papers regarding several different models of fear relapse
and the implications they may have for uncovering the neural bases for these behavioral
phenomena.
While these studies have begun to tie structural connectivity in the brain with function,
there are still many crucial questions that can be tackled based on the knowledge that these
studies have introduced. For example, Marek, 2018 and Hitora-Imamura, 2015 both exemplified
a role that the PFC may have in regulating renewal and reinstatement, respectively. However, it
is striking that there is a convergence point that both these papers share in the inhibitory
networks of the PFC. A next logical question would be whether these two behaviors share the
same functional circuitry, or if they share any overlapping information processing.
The BNST is another region of the brain that has, until recently, gone largely unstudied.
In fact, attention has more recently been drawn to the BNST as a potential regulator of stress-
related behaviors21. This is an exciting time for research in fear learning, since tools such as
optogenetics, genetic manipulations, and in vivo imaging tools such as 1-photon calcium imaging
have provided us with unprecedented spatiotemporal and genetic control over observation and
manipulation in the brain.
It is important to note that this review has largely focused on fear renewal and
reinstatement because literature on spontaneous recovery is scarce. This is likely due to the
technical limitation that spontaneous recovery generally takes several weeks to wait for in
rodents, whereas renewal and reinstatement can be triggered by the experimenter directly after
extinction learning. Despite this limitation, spontaneous recovery of fear should be actively
explored to discern the similarities and differences between this and other models of relapse—
especially since spontaneous recovery is not triggered by a salient external stimulus and thus
makes it the most peculiar model of fear relapse.
Lastly, the developmental disparity in the ability to induce reinstatement in young versus
adult rats may provide novel insights into how aberrant fear circuitry develops in organisms with
early life trauma. Perhaps even more interesting is that another group found that female juvenile
rats were prone to all three models of fear relapse whereas male juvenile rats were prone to none
of them22, which is in line with Kim, 2007. This speaks to a broader issue in neuroscience
research which is that sex differences are rarely acknowledged; that is, most neuroscience
research involves male organisms, despite the overwhelming understanding that there are major
differences between male and female rodent brains in characteristics as global as distribution of
inhibitory networks throughout the brain23.
In the past few decades, an abundance of research has been aimed at discovering how the
brain processes, stores, and expresses fear and the subsequent suppression of fear. While strides
have been made in answering these questions, the question of why fear relapse continues to
plague both rodents and humans remains largely enigmatic. I argue that we have the tools and
conceptual basis to begin to answer these questions on many levels, from molecular to circuit to
behavioral. And considering the innovative work discussed here to begin to open these doors, the
horizon for this question looks fruitful.

Acknowledgements
I would like to acknowledge Jake Campolo and Katie Kerr for always listening to me
ramble on about my science. I would like to thank Drs. Patrick Davis and Leon Reijmers for
introducing me to the field of learning and memory, and additionally to Dr. Steve Ramirez for
supporting me as I navigate through the trenches of science research, both financially and—
much more importantly—morally. Thank you Dr. Cecelia Musselman, for being available to
give me feedback, and for being understanding about my weird schedule the last few weeks.
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