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A window of vulnerability: Impaired fear extinction in adolescence

Kathryn D. Baker*, Miriam L. Den, Bronwyn M. Graham, & Rick Richardson

School of Psychology, The University of New South Wales, Sydney 2052, Australia

*Corresponding author

School of Psychology

The University of New South Wales

Sydney 2052, Australia

Email: [email protected]

Phone: 61-2-93850552

Fax: 61-2-93853641

Author email addresses: Kathryn D. Baker, [email protected]; Miriam L. Den, [email protected]; Bronwyn M. Graham, [email protected]; Rick Richardson, [email protected].

Article Type: Review

Keywords: Extinction, Fear, Adolescence, Stress, Functional Connectivity

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Abstract

There have been significant advances made towards understanding the processes mediating extinction of learned fear. However, despite being of clear theoretical and clinical significance, very few studies have examined fear extinction in adolescents, which is often described as a developmental window of vulnerability to psychological disorders. This paper

reviews the relatively small body of research examining fear extinction in adolescence. A prominent finding of this work is that adolescents, both humans and rodents, exhibit a marked impairment in extinction relative to both younger (e.g., juvenile) and older (e.g., adult) groups. We then review some potential mechanisms that could produce the striking extinction deficit observed in adolescence. For example, one neurobiological candidate mechanism for impaired extinction in adolescence involves changes in the functional connectivity within the fear extinction circuit, particularly between prefrontal cortical regions and the amygdala. In addition, we review research on emotion regulation and attention processes that suggests that developmental changes in attention bias to threatening cues may be a cognitive mechanism that mediates age-related differences in extinction learning. We also examine how a differential reaction to chronic stress in adolescence impacts upon extinction retention during

adolescence as well as in later life. Finally, we consider the findings of several studies illustrating promising approaches that overcome the typically-observed extinction impairments in adolescent rodents and that could be translated to human adolescents.

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Adolescence is often described as a developmental window of vulnerability in which

the majority of psychological disorders emerge (Paus, Keshavan, & Giedd, 2008; Spear,

2000). Anxiety disorders are the most common class of psychological disorders in

adolescence (Kessler et al., 2012). Further, it has been estimated that approximately 75% of

adults with fear-related disorders met diagnostic criteria as children or adolescents (Kim-

Cohen et al., 2003). As noted by McNally (2007), exposure-based treatments for anxiety

disorders have been an undeniable success within psychology. An important component of

these therapies is the process of extinction, which involves repeatedly exposing the individual

to the feared stimulus/situation in the absence of any danger. As noted in several recent

reviews (e.g., Milad & Quirk, 2012), substantial progress has been made in the past decade

on understanding the processes mediating extinction of learned fear. Although there are over

a thousand publications on fear extinction in animals and humans since 2000 (Milad & Quirk,

2012), very few of these studies have examined fear extinction during development. There have been a few recent studies in infants (for review see Kim & Richardson, 2010), but scarcely any in adolescents. In this paper, we first review this relatively small body of research on fear extinction in adolescent rodents and humans. A major finding of this work has been that adolescents, both humans and rodents, exhibit a marked impairment in extinction compared to both younger (e.g., juvenile) and older (e.g., adult) groups. We then move on to a consideration of various factors/mechanisms that could mediate this pronounced impairment in extinction in adolescence. We conclude that changes in the functional connectivity within the fear extinction circuit, particularly between prefrontal cortical regions and the amygdala, may be the neurobiological basis for the impaired extinction observed during adolescence. We then describe work examining the impact of chronic stress on fear extinction in adolescence; this research shows that stress may increase the likelihood of resistance to extinction earlier or later in life, depending on the age at which the stressor is

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experienced. We also briefly describe another body of research – on emotional regulation and

attentional processes – for additional clues as to potential cognitive mechanisms that may mediate the observed impairments in extinction in adolescence. Finally, we describe several recent studies that provide evidence for approaches that overcome extinction impairments in adolescent rodents, and that could be translated to treating adolescent humans with an anxiety disorder.

1.1 Fear extinction in adolescent rodents

Those studies that have examined extinction of fear in adolescence have all used

Pavlovian procedures to first condition fear. This involves pairing an initially neutral

conditioned stimulus (CS; e.g., a tone or a light) with a naturally aversive unconditioned

stimulus (US; e.g., a shock or a loud noise). At some point following this, the CS is presented

by itself, and over repeated trials the fear elicited by the CS diminishes. Before describing

those studies, however, it is important to define adolescence given that there are some disagreements about exactly when adolescence begins and ends, in both rodents and humans

(Spear, 2000). In this review we will be very inclusive and define adolescence in rodents as being between postnatal (P) day 28 to P50, and in humans as being 12-17 years of age.

In perhaps the first study on fear extinction in adolescent rodents, Hefner and Holmes

(2007) examined differences in conditioned fear acquisition and extinction between early adolescent (P28), mid-adolescent (P42), and young adult (P56) mice. Early adolescent mice showed enhanced fear acquisition as well as higher levels of freezing during extinction training compared to mid-adolescent and young adult mice. However, there were no age differences in the rate of within-session extinction. Further, extinction retention was not tested in that study. Two subsequent studies replicated this finding in rats, demonstrating that adolescents (P35) did not differ in rates of within-session extinction compared to juvenile

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(P24) and adult (P70) rats, and these studies also found a marked impairment in extinction

retention in the adolescents (McCallum, Kim, & Richardson, 2010; Kim, Li, & Richardson,

2011). That is, although adolescent rats expressed the same low-level of CS-elicited freezing at the end of the extinction training session as did juvenile and adult rats, when tested the following day they exhibited a striking return of fear, relative to the other two age groups (see

Figure 1A; redrawn from McCallum et al., 2010). A more recent study did not detect any age-related differences in extinction retention between adolescent and adult rats (Broadwater

& Spear, 2013). Although adolescent (~P35) rats in that study showed a remarkable recovery of fear when extinction retention was tested, so too did adult (~P71) rats (i.e., rats of both ages exhibited ~80% CS-elicited freezing at test). This high level of fear at test makes it nearly impossible to detect any potential extinction retention impairment in the adolescents.

In contrast to those results, another study reported impaired extinction learning and retention in adolescent mice (Pattwell et al., 2012). Specifically, when extinction training was spread over several days (with 5 trials per day over 4 days), adolescent mice (P29) displayed impaired extinction learning and retention compared to juvenile (P23) and adult (P70) mice.

These deficits in fear extinction in adolescent rodents do not seem to be due to differences in fear conditioning. Although Hefner and Holmes (2007) observed enhanced fear acquisition

(as reflected by higher levels of CS-elicited freezing) in adolescents, such a difference was not observed in the three studies that reported impaired extinction retention in adolescents. In addition, there are other studies that have reported similar acquisition of fear in adolescent compared to adult rats (Brasser & Spear, 2004). Taken together, these studies demonstrate a marked deficit in fear regulation in adolescence, but clearly more research needs to be done in this area.

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1.2 The study of fear extinction in adolescent humans

Neumann, Waters, and Westbury (2008) gave 13-17 year old participants pairings of a

geometric shape (CS+) with the unpleasant sound of metal scraping on a slate (US). Across a

number of dependent variables (including potentiated startle, skin conductance, and self- report measures), robust fear conditioning and within-session extinction was observed.

Contrary to this finding of robust within-session extinction, Haddad, Lissek, Pine, and Lau

(2011) reported that adolescents were resistant to extinction. A social threat cue task was

used in that study; this type of task was chosen because negative social relationships are

highly salient during adolescence and may contribute to the etiology and maintenance of

anxiety disorders. Of course, the level of conditioned fear in this type of study is going to be

much less than what occurs in studies where painful stimulation (e.g., shock or loud noise) is

used as the US. Nonetheless, the participants in the study by Haddad and colleagues were 12-

15 years of age and were given pairings of three different neutral face CSs with three

different USs: (1) CS-positiveUS (i.e., a neutral face CS was paired with a US that was the same face displaying a positive facial expression and a positive comment), (2) CS-

negativeUS (i.e., a neutral face CS was paired with a US that was the same face with a

negative facial expression and negative comment), and (3) CS-neutralUS (i.e., a neutral face

CS was paired with the same neutral face and a neutral comment). After conditioning, participants rated the CS that was paired with the negative expression and comment as more unpleasant and scary than both of the other two CSs. More importantly, this difference persisted after extinction trials in which the CS that had been paired with the negative expression was repeatedly presented alone. This finding supports the claim that adolescents show impaired within-session extinction. In both of the abovementioned studies, conclusions about age-related differences in within-session extinction were not possible as only adolescent participants were tested. However, a study by Pattwell et al. (2012) compared

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extinction in children, adolescents, and adults. The results showed that adolescents exhibited

a marked deficit in within-session extinction compared to both children and adults.

Specifically, in a discriminative fear conditioning procedure one CS (a colored square

presented on a computer screen) was followed by an aversive noise US 50% of the time (i.e.,

CS+) and a second CS (a different colored square) was not followed by anything aversive

(i.e., CS-). A differential skin conductance response to the CS+ versus CS- was used to

compare within-session extinction across age groups; the average differential skin

conductance response from the last two extinction trials was subtracted from that of the first

two trials. Adolescents (12-17 years old) showed attenuated fear-extinction learning compared with children (5-11 year olds), and a trend (p = .078) towards poorer extinction learning compared to adults (18-28 years). These differences were not attributable to age differences in fear acquisition, sex differences in extinction learning, or trait anxiety. These studies suggest that healthy adolescents are impaired at extinction learning, but it is not known whether human adolescents also exhibit the same deficits in extinction retention as do adolescent rodents (Kim, et al., 2011; McCallum, Kim, & Richardson, 2010; Pattwell, et al.,

2012) because extinction retention was not assessed in any of the above studies.

In another study, fear extinction in anxious versus non-anxious adolescents was compared. Lau et al. (2008) employed a differential fear conditioning procedure in which the

CS+ (a neutral face) was paired with an aversive outcome (a loud scream and fearful facial expression) whereas the CS- (a different neutral face) was never followed by the aversive outcome. Extinction training occurred over two sessions, with the first session immediately after conditioning (3 presentations of each CS by itself), and the second session approximately 16 days after conditioning (12 presentations of each CS by itself). Following conditioning, both anxious and healthy adolescents rated the CS+ as more fear-provoking than the CS-, and the size of this difference was comparable across the two groups (a finding

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similarly observed in adults; for a review see Lissek et al., 2005). Participants, whether anxious or not, showed a resistance to extinction because higher fear ratings to the CS+ relative to the CS- remained at the end of the second extinction session. However, given that only adolescents were tested in that study, no developmental comparisons in either extinction learning or extinction retention can be made.

Although this body of research is very small at this point, the findings strongly suggest that there are impairments in extinction of fear during adolescence, in both rodents and humans. Given that this is the period of development during which many anxiety disorders first emerge, it will be important to determine what emotional and cognitive processes may mediate this impairment as well as any contribution made by neural changes occurring in this period of development. Therefore, in the subsequent sections of this review

we examine how (1) significant structural and functional neural changes in the fear extinction

circuit during adolescence may lead to impaired extinction, (2) fear extinction in adolescence

is altered by adverse experiences, and (3) emotional regulation and attentional biases may

contribute to the impaired extinction observed in adolescence.

1.3 Neural changes in the fear extinction circuit during adolescence

Significant structural and functional neural changes occur during adolescence. Of

most relevance to fear extinction, several studies have documented changes within the

prefrontal cortex (PFC) and amygdala during adolescence. To date, no studies have examined

functional neural activity in these regions in human adolescents during fear extinction tasks.

However, findings from research that has examined neural activity in adolescents during

other laboratory tasks (e.g., decision making and emotional processing) provide a potential

framework that can be used to understand the reported behavioral deficits in fear extinction in

adolescents, and to make novel hypotheses about the neural mechanisms that mediate such

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deficits.

1.3.1 Structural Changes in the Prefrontal Cortex and Amygdala during Adolescence

Human adolescents exhibit a reduction in gray matter in the PFC. This reduction may be due to a decline in the number of synapses in cortical regions (“synaptic pruning”), as has been demonstrated in non-human animal studies, or alternatively due to an increase in white matter (Paus, et al., 2008; Sturman & Moghaddam, 2011). In addition, both human and non- human animal studies have demonstrated that maturation of the PFC is much more protracted than is the maturation of sub-cortical regions (reviewed in Casey, Duhoux, & Cohen, 2010;

Casey, Getz, & Galvan, 2008).

Relative to the PFC, the amygdala is an earlier maturing structure that undergoes much less structural change during adolescence (Giedd et al., 1996). However, recent raw volumetric analyses of amygdala size in 4-18 year olds revealed that amygdala volume was greatest during early adolescence (at around the age of 14 years for female participants, around 15-16 years for male participants) and was smallest in the youngest and oldest participants in that cohort (Hu, Pruessner, Coupé, & Collins, 2013). This suggests that while

PFC volume decreases during adolescence (Paus, et al., 2008; Sturman & Moghaddam,

2011), amygdala volume transiently increases during adolescence. It is also known that adolescence is a period of relatively active cell proliferation in the amygdala compared to young adulthood. In fact, cell proliferation in the amygdala of adolescent rats occurs at a rate four-five times higher than in young adults (Saul, Helmreich, Callahan, & Fudge, 2013), consistent with previous stereological studies showing active amygdala growth in adolescence relative to adulthood (Rubinow & Juraska, 2009). The changes in amygdala growth may be driven by increases in sex hormones, as other findings have demonstrated that adolescent boys in later stages of pubertal development exhibit larger amygdala volumes

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Smyth, & Delgado, 2013) and that growth of the amygdala during puberty is associated with circulating testosterone levels (Neufang et al., 2009).

1.3.2 Functional Changes in the Prefrontal Cortex and Amygdala during Adolescence

In addition to structural changes, numerous changes in the functional activation of the

PFC and amygdala have been documented in adolescence. In general, studies have reported

PFC hypoactivation and amygdala hyperactivation in adolescents during a range of tasks. For example, adolescents recruit the medial PFC (mPFC) to a lesser extent than adults when viewing emotional faces (Hare et al., 2008) and when tracking changes in emotional state

(Monk et al., 2003). In contrast, amygdala activation is augmented in adolescents compared to children (Hare, et al., 2008) and adults (Guyer et al., 2008; Hare, et al., 2008) when viewing emotional faces. Again, these functional changes in amygdala activity may be mediated by sex hormones, as a positive correlation has been reported between pubertal stage and amygdala hyperactivation during face presentations (Moore et al., 2012). However, it should be noted that there are some inconsistencies in the literature with respect to patterns of functional PFC and amygdala activation during adolescence, where, in some cases, hyperactivity of the amygdala and hypoactivity of the PFC are not observed (Somerville,

Jones, & Casey, 2010). A more consistent picture of the neurological characteristics of adolescence may be gained through the study of neural circuits as opposed to discrete structures, as outlined next.

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1.3.3 Functional Connectivity between the Prefrontal Cortex and Amygdala during

Adolescence

Recent neuroimaging research has moved towards the study of neural circuits rather

than the morphology and function of single neural structures in isolation. Such an approach

takes into account that the strength of coupling between two or more regions may be more important in determining behavioral outcomes than the overall activation of discrete neural regions. For example, it may be that strong inverse functional connectivity between the PFC and amygdala (where PFC activation is associated with amygdala inhibition) is necessary for fear extinction to occur. If so, then strong PFC activity would be insufficient to support fear extinction in the absence of functional connectivity with the amygdala. From this perspective,

it makes more sense to examine the functional connectivity within the fear extinction circuit,

rather than the activity within individual structures, to determine the neurobiological basis for

impaired extinction during adolescence.

Only a few studies have investigated functional connectivity of these structures across

development. Qin, Young, Supekar, Uddin and Menon (2012) reported that resting-state

connectivity between the amygdala and PFC was reduced during childhood relative to

adulthood. Another study reported that effective connectivity (i.e., the degree to which

activity in one region impacts activity in another region) between the anterior cingulate and

the amygdala during the presentation of emotional faces increased with age across childhood

(Perlman & Pelphrey, 2011). However, adolescent participants were not examined in either

of these studies.

A recent study by Roy et al. (2013) examined intrinsic (resting state) functional

connectivity in adolescents with and without generalized anxiety disorder (GAD). They

reported decreased connectivity between the amygdala and ventromedial PFC in adolescents

with GAD relative to adolescents without GAD, suggesting that reduced connectivity

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14-17 year olds; i.e., connectivity between these regions was weaker in these age groups). It is interesting to note that the pattern of increased amygdala-mPFC connectivity from adolescence to adulthood mirrors the observed improvement in extinction ability from adolescence to adulthood, suggesting that low connectivity between these regions during early-/mid-adolescence, combined with amygdala hyperactivation, may underlie the deficits in fear extinction during adolescence.

1.3.4 Current Neurobiological Models of Adolescent Behavior

Findings like those reviewed above have led to the emergence of several neurobiological models that are designed to account for the myriad of affective and behavioral changes observed during adolescence (Casey, et al., 2008; Nelson, Leibenluft,

McClure, & Pine, 2005; Steinberg, 2008). These models posit that as the PFC is a late- maturing structure, connectivity between it and sub-cortical structures does not develop until late adolescence or early adulthood. Thus, the PFC does not have the structural and/or

13 functional connections to drive the inhibition of behaviors mediated by sub-cortical structures until later in life. During childhood, because sub-cortical structures are still developing and are often hypo-responsive, a balance is maintained between brain systems that regulate emotional and reward-driven behavior and those that exert inhibitory control over such behavior. However, as sub-cortical structures develop more rapidly than the PFC and are often hyper-responsive during adolescence, at this stage of development there is an imbalance in activity due to an excess of activity within sub-cortical structures (that mediate emotional/reward driven behavior) that is not inhibited by the late-maturing PFC due to the low level of connectivity between these neural regions.

While no studies have investigated whether these neurobiological models can account for adolescent deficits in fear extinction, extensive research has been conducted in applying these models to adolescent reward-driven behavior, with the aim of understanding why adolescents engage in risky behavior. Just as the amygdala is more active during processing of fearful faces in adolescence, so too is the ventral striatum more active in adolescents during reward-focused tasks. Furthermore, frontal-striatal connections increase both structurally and functionally across development, and the increased connectivity has been associated with increased capacity to recruit top-down inhibitory control during reward- focused tasks (reviewed in Casey, Duhoux, et al., 2010). Thus, increased risky behavior during adolescence can be accounted for by reduced prefrontal inhibitory control over excessive striatal activation in the presence of appetitive stimuli. Similarly, impairments in extinction during adolescence may be due to reduced prefrontal inhibitory control over excessive amygdala activation in the presence of fearful stimuli.

This sort of neurobiological model where there is an imbalance in activity between prefrontal inhibitory regions and sub-cortical structures mediating emotional behavior (i.e., the amygdala) provides a nice account for why extinction is impaired in adolescence.

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Critically, this type of model needs to be explicitly tested in future research. However, it is worth noting that in other populations in which extinction deficits are observed (e.g., adults with PTSD or schizophrenia), a reduction in ventromedial PFC activity combined with hyperactivity in the amygdala at the time of extinction training/recall has been documented

(Etkin & Wager, 2007; Holt, Coombs, Zeidan, Goff, & Milad, 2012; Jovanovic et al., 2013), which lends support to the possibility that similar mechanisms may be underlying the deficits observed in adolescents.

Some preliminary support for the above hypothesis comes from several studies in rodents that have documented differences in the functional activity in the neural circuitry supporting fear extinction during adolescence versus adulthood. Notably, these studies demonstrated that the mPFC may not be recruited as efficiently during fear extinction in adolescent rodents as in younger and older animals. For example, immunohistochemical analyses using phosphorylated mitogen-activated protein kinase (pMAPK) as a marker of neuronal activity have shown that adolescent rats do not exhibit the same extinction-induced increases in neuronal activity in the infralimbic mPFC that are observed in juvenile and adult rats (Kim, et al., 2011). Consistent with this observation, there is a lack of synaptic plasticity associated with extinction in the infralimbic mPFC of adolescent mice compared with what is observed in the juvenile and adult mPFC (Pattwell, et al., 2012). The adolescent mPFC also shows elevated basal excitatory synaptic transmission compared to juveniles and adults, which may contribute to a lack of prefrontal synaptic plasticity and impaired extinction in adolescence (Pattwell, et al., 2012). Furthermore, during adolescence there is a dramatic increase in projections from the basolateral amygdala to ventromedial PFC GABAergic inhibitory interneurons which could destablize and impair mPFC function (Cunningham,

Bhattacharyya, & Benes, 2008). Together, these findings suggest that adolescents may be less efficient in utilizing prefrontal regions to maintain the inhibition of fear following extinction.

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1.4 Fear extinction in adolescence is altered by adverse experiences

1.4.1 Adolescence is a vulnerable developmental period to the effects of stress

Adolescence is often characterized as a period of “storm and stress” (Casey et al.,

2010; Romeo, 2013). One factor that may contribute to adolescent vulnerability is that the hypothalamic-pituitary-adrenal (HPA) axis, which mediates the mammalian response to stress, undergoes subtle changes during adolescence (reviewed in McCormick, Mathews,

Thomas, & Waters, 2010; Romeo, 2013). In adults, the HPA stress response involves the protracted release of several hormones, including corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH), the latter of which stimulates the synthesis and secretion of glucocorticoids by the adrenal glands. The primary neural targets of the glucocorticoids include the hypothalamus, hippocampus, and PFC, and their overall function is to reduce the production and release of CRH and ACTH via negative feedback, effectively terminating the stress response. Dysfunctions in adult HPA-axis activity and reactivity have been implicated in a host of mental health disorders, including anxiety and depression

(Holsboer & Ising, 2010).

Although the consequences of stress during adulthood for HPA-axis function has received great attention (McEwen, 2007), fewer studies have examined the impact of stress on HPA-axis function during adolescence. The few studies that have examined this issue have demonstrated that under non-stressed conditions basal levels of ACTH and corticosterone (a major glucocorticoid in rats) are comparable between adolescent and adult rats (Foilb, Lui, & Romeo, 2011; Lui et al., 2012; Romeo, Lee, Chhua, McPherson, &

McEwen, 2004; Romeo, Lee, & McEwen, 2004). When acutely stressed, however, the amount of ACTH and corticosterone released is significantly greater in adolescent than adult rats (Foilb, et al., 2011; Lui, et al., 2012). Moreover, the stress-induced release of these hormones is significantly more protracted in adolescent rats, resulting in a much slower

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return to baseline levels of stress hormones compared to adult rats (Foilb, et al., 2011;

Romeo, Lee, Chhua, et al., 2004; Romeo, Lee, & McEwen, 2004). Adolescent rats also respond differently to chronic stress than do adults. For example, the HPA-axis response in adult rats habituates with chronic exposure to the same stressor, whereas adolescent rats fail to exhibit such habituation (i.e., they continue to exhibit robust release of ACTH and corticosterone whereas adult rats exhibit attenuated release of these hormones; Lui et al.,

2012).

Data from humans resemble the rodent findings, with one study reporting that while there were no differences in baseline cortisol levels between children and adolescents pre- stressor, adolescents exhibited greater cortisol responses during a performance stressor compared to children, and these differences in cortisol levels persisted after stressor termination during a recovery period (Stroud et al., 2009). Another study demonstrated increased baseline cortisol (the major glucocorticoid in humans) under non-stressed conditions in 15 year olds compared to 11 and 13 year olds; cortisol levels were also significantly higher amongst 15 year olds during a social stressor task (Gunnar, Wewerka,

Frenn, Long, & Griggs, 2009). It should be noted that a limitation in the current literature is that no studies using human participants to date have compared HPA-axis activity in

adolescents versus adults (baseline or under stress conditions). Nevertheless, taking these

human and rodent findings together, it appears that both the sensitivity of the HPA-axis and

the consequences of chronic stress are different in adolescence than other stages of

development, where adolescence is characterized by an over-active, protracted stress

response. It is thought that due to the continued maturation of the limbic and prefrontal

cortical regions in adolescence, these structures are particularly vulnerable to the effects of

stress (Giedd & Rapoport, 2010; Romeo, 2013). Adolescence would therefore be a vulnerable

developmental period to stressors, which would consequently increase sensitivity to the onset

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of stress-related mental disorders (Kessler et al., 2007; Lupien, McEwen, Gunnar, & Heim,

2009; Malter Cohen, Tottenham, & Casey, 2013). The following section reviews the impact of chronic stress on fear extinction in adolescent rodents. Consistent with neurobiological models suggesting that exposure to stress has different outcomes depending on the age at which it is experienced (Lupien, et al., 2009), the effect of stress on fear extinction differs depending on whether it is experienced in early life, adolescence, or adulthood.

1.4.2 The impact of chronic stress on fear extinction in adolescent rodents

The results of several recent studies have suggested that exposure to chronic stress alters the nature of the extinction system in adolescence. The first example is that early-life stress has been shown to accelerate the transition into and out of the adolescent profile of fear extinction (Callaghan & Richardson, 2012). In that study, infant rats were exposed to repeated bouts of maternal separation (3 hours per day from P2-14). Animals exposed to early-life stress showed an early emergence of poor extinction retention during pre- adolescence (at P27) and good extinction retention during adolescence (at P35). One explanation of these findings is that exposure to early-life stress accelerated the maturation of neural structures, such as the PFC, required for adult-like extinction behavior (Callaghan &

Richardson, 2012).

In contrast to early-life stress which causes an earlier emergence of the adolescent profile of extinction, chronic stress in early adolescence appears to cause deficits of extinction acquisition in adolescence as well as impaired extinction retention in adulthood.

Although one study did not find an effect of chronic social instability stress in adolescence on fear extinction in adolescence (P46) or adulthood (Morrissey, Mathews, & McCormick,

2011), two studies showed a disruption of extinction acquisition with chronic stress in adolescent male rats, and one of these also demonstrated impaired extinction retention in

18 adult male rats. Zhang and Rosenkranz (2013) examined the effect of chronic restraint stress

(7 out of 9 days from either P29 or P65) on the acquisition and extinction of learned fear in male adolescent (P39) and adult (P76) rats. At both ages, restraint stress enhanced fear conditioning. However, repeated restraint led to impaired acquisition of fear extinction only in adolescence. The effect of restraint stress on extinction retention was not tested in that study. A disruption of extinction acquisition by chronic stress in early adolescence in male rats was also found in an earlier study by Toledo-Rodriguez and Sandi (2007), but this disruption was sex-dependent as female rats were unaffected. Male adolescent rats (P41) which experienced stress (exposure to predator odor followed by placement on an elevated platform on P28–P30) exhibited increased conditioned fear and failed to show the same reduction in freezing across a 5 min test that was observed in unstressed animals. In contrast, female rats, regardless of whether they received exposure to stress or not, exhibited within- session extinction. The detrimental effects of stress were long-lasting in male rats because when the same animals were tested in adulthood, those animals exposed to chronic stress during adolescence exhibited impaired extinction retention. One limitation of this study was that it did not include an adult-stressed group to examine whether adolescent animals were more susceptible to the effects of stress than adults. However, the findings do show that chronic stress during adolescence increases the likelihood of impaired extinction retention later in life.

Exactly how chronic stress in adolescence contributes to deficits in fear extinction is unclear at this stage. One possibility is that stress in adolescence leads to a decrease in synaptic plasticity in the neural regions that support fear extinction, and an increase in synaptic plasticity in the regions that support fear expression. For example, chronic stress in male and female adolescent rats leads to dendritic retraction in the hippocampus and PFC, and dendritic hypertrophy in the amygdala (Eiland, Ramroop, Hill, Manley, & McEwen,

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2012). It is possible that this stress-induced dendritic remodeling tips the balance between prefrontal inhibition and amygdala excitation, leading to a disruption in the extinction of fear.

Future research is needed to determine whether such changes to the functional connectivity between the PFC and amygdala contribute to the impaired extinction retention observed in adulthood following stress encountered during adolescence.

In addition, stress-induced changes to the hippocampus are likely to impact extinction in adolescence given the importance of this structure in the contextual modulation of fear extinction as well as the consolidation of extinction memories (reviewed in Quirk & Mueller,

2008). The hippocampus, implicated in the negative feedback regulation of the HPA-axis, is particularly vulnerable to the effects of stress during adolescence and adulthood, due to the high density of glucocorticoid receptors in that structure (reviewed in McCormick et al.,

2010). Chronic stress has been shown to impact hippocampal structure and function by causing dendritic retraction (McLaughlin, Gomez, Baran, & Conrad, 2007; Watanabe et al.,

1992), reducing long-term potentiation (Foy, Stanton, Levine, & Thompson, 1987), and decreasing neurogenesis (Barha, Brummelte, Lieblich, & Galea, 2011). Interestingly, while research suggests that in adulthood these stress-induced changes are transient and reversible

(Luine, Villegas, Martinez, & McEwen, 1994; Sousa, Lukoyanov, Madeira, Almeida, &

Paula-Barbosa, 2000), converging animal and human studies have demonstrated that stress exposure during adolescence can have a profound and long-lasting impact on the neural circuitry underlying fear extinction, given that dramatic neuronal reorganization is taking place (reviewed in Koenig, Walker, Romeo, & Lupien, 2011). It is therefore likely that stress- induced dendritic remodeling during adolescence would lead to impaired fear extinction both later in adolescence as well as in adulthood. Although studies of this kind have not yet been conducted, they would have clear practical implications for the treatment of both adolescents with anxiety disorders as well as adults exposed to stress during their adolescent years.

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1.5 Attentional bias to threatening cues may influence fear extinction in adolescence

Despite recent advances in our understanding of the neural circuitry mediating

extinction, and how this circuitry may change during adolescence, few studies have examined how cognitive processes such as attention contribute to impaired extinction during adolescence. During conditioning, repeated pairings of a CS with an aversive US results in

anticipatory fear and attention biases towards the CS+ (Mackintosh, 1975). Then, subsequent

extinction training trials (i.e., CS+ alone presentations) reduce attention to the CS+ (e.g., Van

Damme, Crombez, Hermans, Koster, & Eccleston, 2006). Persistent attention biases towards

the CS+ during extinction would be expected to result in poorer extinction learning, and this

has been observed in adult patients with posttraumatic stress disorder (Fani et al., 2012).

Unfortunately, studies have not yet looked at whether developmental changes in

attention bias to threatening cues contribute to age-related differences in extinction learning.

However, age-related differences in the response to threatening stimuli have been the focus of

a number of recent studies. For example, Hare et al. (2008) demonstrated that adolescents

show an exaggerated amygdala response to cues that signal possible threat (i.e., fearful

faces), relative to both children and adults, and this was largely due to inefficiency in

recruiting prefrontal regions involved in dampening the fear response. In another example,

Lau et al. (2011) showed that adolescents exhibit poorer threat-safety discriminations compared to adults, perceiving both the CS+ (i.e., the threat cue that actually predicted the aversive US) and the CS- (i.e., the safety cue that did not predict the aversive US) as threatening. Given these findings, adolescents might be expected to show a heightened sensitivity to learning fearful associations, relative to both children and adults. While this has not yet been demonstrated in humans, a recent study by Den and Richardson (2013)

demonstrated that adolescent (~P35), but not juvenile (P23) or adult (P90) rats, exhibit high levels of fear after being trained on a trace conditioning procedure (i.e., where long, stimulus-

21

free intervals of up to 40 s separated the CS and US). These studies raise a number of

interesting questions for future research on extinction in adolescence regarding (1) whether

attention bias to the CS+ persists following extinction learning, (2) whether such bias predicts

a poorer response to extinction learning, and (3) whether attention bias differs in anxious

versus healthy adolescent and adult populations. Research of this kind in humans would

ultimately indicate whether current treatments need to be tailored so that they optimally target

potential underlying attentional dysfunctions mediating anxiety during adolescence,

compared to adulthood.

1.6 Development of the fear extinction circuit: an imbalance during adolescence

The fear extinction system undergoes many changes across development. Figure 2 shows a schematic of how a balance within the fear circuit is important for appropriate fear regulation across development. Juveniles and adults can express and inhibit fear appropriately when various situations are encountered in their environment. However, the reason behind this comparable balance in fear regulation may be different between these two age groups.

For example, juveniles may have reduced connectivity between the amygdala and mPFC relative to adults (Gee et al., 2013), but the amygdala is less active/underdeveloped at this point of development so a balance is maintained. Adults have a fully developed amygdala and mPFC, and have strong connectivity between these regions relative to younger ages, and

so a balance in fear regulation is maintained. However, maturational changes in the brain during adolescence have a striking impact on fear expression and fear inhibition. As reviewed above, during adolescence there is growth in amygdala volume and reduction in gray matter in the PFC (see Casey et al., 2008; Hu et al., 2013; Saul et al., 2013), as well as changes in

the functional connectivity between these regions from childhood, through adolescence and

into adulthood (Gee et al., 2013). In addition, age-related differences in extinction learning

22

may also be mediated by cognitive mechanisms such as developmental changes in attention

bias to threatening cues. We propose that the combination of brain maturation and cognitive

changes disrupts the balance between PFC and amygdala activity and tips the balance towards poor fear inhibition (Figure 2). Specifically, a hypoactivation of the PFC and hyperactivation of the amygdala would result in the adolescent being primed to express more fear compared to juveniles or adults. This imbalance model is well supported by findings that adolescent rodents show heightened sensitivity to learning fearful associations (Den &

Richardson, 2013) and impaired fear inhibition (Kim et al, 2011; McCallum et al., 2010;

Pattwell et al., 2012) compared to younger and older animals. That adolescents also show increased fear generalization to safety cues (i.e., poor threat-safety discrimination; Lau et al.,

2011) and a combination of an exaggerated amygdala response and dampened prefrontal activity to fearful faces compared to children and adults (Hare et al., 2008) is also consistent with an imbalance between top-down prefrontal inhibitory control of the amygdala in response to fearful stimuli. This imbalance in fear regulation may contribute to an increased

likelihood of anxiety symptoms emerging during adolescence, consistent with the finding that anxious adolescents have decreased connectivity between the amygdala and ventromedial

PFC relative to non-anxious adolescents (Roy et al., 2013). Once the maturation of the brain is complete during the transition out of adolescence into adulthood and adult cognitive

processes have developed, the balance between prefrontal and amygdala activity returns and

appropriate fear regulation is restored.

1.7 Manipulations which overcome deficits in fear extinction during adolescence

There are no studies to date which have examined ways to improve fear extinction in

human adolescents. Fortunately, preclinical studies have suggested several promising

pharmacological and non-pharmacological (e.g., behavioral) manipulations that are effective

23

at reducing the impaired extinction retention observed in adolescent rats. Understanding how to reduce the recovery of extinguished fear in adolescence may provide novel insights into therapeutic interventions for anxiety disorders.

One of the most effective pharmacological adjuncts for enhancing extinction is the

NMDA receptor partial agonist D-cycloserine (DCS). Numerous studies have shown that

DCS enhances the effectiveness of fear extinction in adult rats (e.g., Baker, McNally, &

Richardson, 2012; Langton & Richardson, 2008; Ledgerwood, Richardson, & Cranney, 2003;

Walker, Ressler, Lu, & Davis, 2002; reviewed in Graham, Langton, & Richardson, 2011).

Importantly, DCS improves extinction retention in adolescent rats when administered immediately after extinction. DCS was as effective as giving double the amount of extinction training (see Figure 1B; re-drawn from McCallum, et al., 2010). There is considerable translational value of DCS as a treatment for human anxiety disorders given that it has been shown to have positive therapeutic effects for several anxiety disorders in adults (e.g., de

Kleine, Hendriks, Kusters, Broekman, & van Minnen, 2012; Guastella et al., 2008; Hofmann et al., 2006; Ressler et al., 2004; reviewed in Hofmann, Wu, & Boettcher, 2013). With particular relevance to fear inhibition in adolescence, one preliminary study in youth (aged 8-

17) with obsessive-compulsive disorder detected some promising treatment effects of DCS

(Storch et al., 2010). Small to moderate treatment effects (d = .31-.47) were reported with cognitive-behavioral therapy sessions paired with DCS compared to placebo, suggesting the need for more extensive follow-up studies in adolescent populations.

With respect to the neural mechanisms underlying how DCS facilitates fear extinction, it may produce treatment gains by enhancing activity in the amygdala or PFC during extinction. Although the neural effects of DCS in adolescent animals or humans is unknown, there is some evidence in juvenile rats (24-28 days old) that DCS upregulates pMAPK activity in the mPFC (in both the infralimbic and prelimbic subregions) and

24

amygdala (Gupta et al., 2013). Consistent with these findings, functional studies in adults

with a snake phobia using fMRI have shown that DCS can produce long-lasting changes to prefrontal activity. One week after treatment, individuals that were given DCS before a single session of exposure therapy exhibited different activation of the ventromedial PFC and other prefrontal regions (i.e., increased medial orbitofrontal, subgenual anterior cingulate, and left dorsolateral PFC activation) in response to fearful stimuli compared to those given placebo

(Nave, Tolin, & Stevens, 2012).

Considering that adolescence is a time of substantial brain maturation, non- pharmacological alternatives may be a preferable approach provided that similar beneficial

effects are obtained. One example of an effective behavioral procedure is that doubling the

amount of extinction training leads to adult-like extinction retention in adolescent rats (see

Figure 1B; redrawn from McCallum, et al., 2010) and adolescent mice (Clem & Huganir,

2010). In the latter study, adolescent mice given a retrieval trial before extensive extinction

showed reduced fear relapse when tested one week later (Clem & Huganir, 2010).

Unfortunately, there are several reasons why extensive extinction training may be impractical

in adolescent humans with an anxiety disorder (e.g., administering extensive exposure-based

therapy would be both time consuming and costly, and also increase the likelihood of patients

not completing treatment). A recent study by Baker, McNally, and Richardson (2013) demonstrated a reliable, but less time-consuming, manipulation which prevented the recovery of extinguished fear in adolescent rats. In that study it was found that a single retrieval trial

(rather than 30 extra trials) given 10 min before extinction training reduced fear in adolescent rats at test the following day (see Figure 1C; redrawn from Baker et al., 2013). The retrieval- extinction procedure not only overcame the impairment of extinction retention typically observed in adolescent rats, but it also attenuated renewal of fear when animals were tested in the training, rather than the extinction, context. The loss of fear reported following retrieval-

25

extinction manipulations in adult rats and humans is typically interpreted as a disruption of

reconsolidation of the original fear memory (e.g., Flavell, Barber, & Lee, 2011; Monfils,

Cowansage, Klann, & LeDoux, 2009; Schiller et al., 2010). However, a key result in the

study by Baker et al. was that a single retrieval trial given soon after extinction produced a

similar loss of fear as that produced by a retrieval trial before extinction (see Figure 1C), a

finding which is inconsistent with the disruption of reconsolidation perspective. As an

alternative explanation, it has been suggested that a retrieval trial before or after extinction

may promote better discrimination at test between the competing training and extinction

memories (Baker, et al., 2013; Millan, Milligan-Saville, & McNally, 2013). An important implication from this study is that a single retrieval trial around the time of extinction training may offer a simple, effective way of enhancing long-term treatment gains for human populations, including adolescents, when extinction deficits are seen.

1.8 Future directions

As noted above, the improvement in fear extinction retention during the transition

from adolescence to adulthood may arise from increased prefrontal inhibitory control over the

amygdala. There are a number of structural changes which occur during the maturation of the

PFC across adolescence. One example is the formation of perineuronal nets (PNNs). PNNs

are extracellular matrix structures (containing chondroitin sulphate proteoglycans) which

enwrap many neurons in the brain, particularly the inhibitory neurons that contain the

calcium-binding protein parvalbumin, as well as excitatory pyramidal neurons (Brückner et

al., 1993; Celio & Blumcke, 1994; Giamanco & Matthews, 2012). Past studies have

demonstrated that the formation of PNNs in the brain is important in limiting neural plasticity

at the conclusion of several developmental ‘critical periods’ (e.g., the critical period for

ocular dominance plasticity in the visual cortex; Pizzorusso et al., 2002). There are also

26

recent post-mortem studies in humans suggesting that PNNs increase in the PFC across

adolescence into adulthood and that aberrant PNN formation in humans may contribute to

psychological disorders (e.g., schizophrenia; Mauney et al., 2013). Given these findings, it is

tempting to speculate that the formation of PNNs in the PFC at the conclusion of adolescence

may help to support the adult fear extinction system. A corollary of this would be that

abnormal formation of PNNs in the prefrontal brain circuitry regulating fear during

adolescence may contribute to the etiology of anxiety disorders. There is already evidence

that PNNs are involved in the developmental regulation of fear extinction in young rodents.

Specifically, the maturation of PNNs in the mouse amygdala coincides with the transition

from a relapse-resistant extinction system in infants to relapse-prone extinction in juveniles

(Gogolla, Caroni, Lüthi, & Herry, 2009). Thus, an exciting direction for future research will be to examine whether the formation of PNNs in the later maturing PFC modulates the transition from impaired extinction in adolescence into good extinction retention in the adult.

Another important issue that requires greater attention is determining the unique contribution of developmental age versus puberty (and the associated increase in fluctuations in sex hormones), as well as the interaction between the two, in mediating the impairment in extinction observed during adolescence. There are many examples in which the mere exposure to sex hormones during pre-adolescence is not sufficient to induce adolescent-like behavior in rodents, suggesting that some changes in adolescence are more to do with age (or the combination of age plus exposure to sex hormones) than with puberty stage per se. For example, the pre-pubertal removal of ovaries does not prevent the protracted HPA-axis reactivity to stress in adolescent female rats (Romeo, Lee, & McEwen, 2004). This suggests that the alteration in HPA-axis reactivity observed during adolescence is mediated by age rather than sex hormones. Preclinical research in rodents, which allows for the systematic manipulation of sex hormones, is required in order to determine whether the deficits in fear

27

extinction observed during adolescence are a consequence of age, sex hormone exposure, or

an interaction between the two. This will be particularly important in light of recent research

suggesting that sex hormones play a significant role in the consolidation of fear extinction

memories in adult rodents and humans (Graham & Milad, 2013).

Another interesting issue to explore involves how the interaction of individual genetic

variation and development may predispose some individuals to anxiety disorders. There are

many examples of polymorphisms in human genes which have been associated with deficits

in fear extinction. For example, both adult humans and adult inbred genetic knock-in mice

that express the variant BDNF allele (Val66Met), which is associated with reduced activity-

dependent release of BDNF, exhibit a slower rate of fear extinction learning (Soliman et al,

2010). Functional MRI imaging has demonstrated that adult human Met allele carriers showed less ventromedial PFC activity, and greater amygdala activation, during extinction compared to noncarriers, indicating alterations to activity within the fear extinction circuit in

Met allele carriers (Soliman et al, 2010). However, it is unknown when these genotype

differences in extinction learning and functional brain activity first emerge; it is possible that

such deficits may already be evident in adolescents. Related to this, Bath and colleagues

(2012) argued that anxiety-like behaviors measured in the elevated plus maze increased over

the transition from adolescence to adulthood in female Met/Met mice but not wild-type

Val/Val mice, however, the differences between genotypes were primarily observed in adults

over 100 days of age, not in adolescents. Thus, the effect of BDNF gene polymorphisms on

fear extinction in adolescent animals or humans has not yet been investigated. Interestingly though there is some evidence that variation in the expression of mRNA coding for the serotonin transporter may modulate extinction learning and retention in adolescent mice.

There are two alternative mRNA forms coding for the serotonin transporter. Human carriers

of a common polymorphism which reduces expression of the form containing the distal

28

polyadenylation sequence exhibit impaired fear extinction retention as adults and increased

anxiety (Hartley et al., 2012) and also have an increased risk for panic disorder (Gyawali et

al., 2010). Variation in serotonin transporter genes may confer a risk for impaired fear inhibition and increased anxiety in adolescence, but this has not been directly tested.

However, Riddle et al. (2013) demonstrated that two manipulations which enhanced extinction learning and retention in adolescent female mice (specifically, caloric restriction

and chronic fluoxetine treatment) were associated with increased expression of the particular

distal polyadenylation mRNA form. Further research examining the effect of gene polymorphisms on fear extinction in adolescent animals and humans may provide insight into whether individuals with a particular genetic makeup may be more likely to develop symptoms of anxiety across the transition from adolescence to adulthood.

Finally, a further area of interest is the neural basis for how manipulations such as

DCS and memory retrieval around the time of extinction improve the extinction of fear in adolescence. For example, extensive extinction training has been demonstrated to increase neuronal activation in the infralimbic and prelimbic regions of the mPFC in adolescent rats

(Kim et al., 2011); is a similar effect observed following retrieval-extinction manipulations?

This work may lead to the determination of whether manipulations that improve extinction retention in adolescents also enhance the functional coupling between the amygdala and PFC.

Different types of interventions may not necessarily work through the same mechanisms, which would be both theoretically and practically important, in that such results would suggest that there are several different approaches by which fear inhibition in adolescence

can be enhanced.

In conclusion, there is emerging research suggesting that there is a striking

impairment in extinction in adolescence. This impairment in extinction during adolescence is

likely to arise from reduced prefrontal inhibitory control over excessive amygdala activation

29 in the presence of fearful stimuli. In addition to the functional changes in the neural circuitry supporting fear extinction, developmental changes in attention bias to threatening cues may also contribute to age-related differences in extinction learning. A combination of such changes may result in an imbalance in fear regulation such that there is poor fear inhibition and heightened fear expression during adolescence (see Figure 2). Whatever is causing the deficits in extinction during adolescence, there are several effective manipulations for improving the inhibition of fear in adolescent rodents, providing promising translational interventions for human adolescents. Given that anxiety during adolescence is a strong predictor of adult anxiety and other psychological disorders (Kessler, et al., 2012), continued research into fear extinction in adolescence will improve our ability to develop early and effective interventions for anxiety.

Acknowledgements

Preparation of this manuscript was supported by an Australian Postgraduate Award (MLD), and grants from the Australian Research Council (DP120104925) and the National Health and Medical Research Council (APP1031688) to RR. KDB is a National Health and Medical

Research Council Peter Doherty Early Career Fellow (APP1054642).

30

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Figure Captions

Figure 1. Adolescent rats show impaired extinction retention at test compared to juvenile and adult rats (Panel A). D-cycloserine (DCS) and extended extinction training improved extinction retention in adolescent rats (Panel B). Memory retrieval 10 min before or after extinction augmented extinction retention in adolescent rats (Panel C). The data shown in

Panels A and B were taken from McCallum, Kim, and Richardson (2010) and the data shown in Panel C were taken from Baker, McNally, and Richardson (2013).

Figure 2. A schematic of fear regulation across development. The circles with – and + symbols denote a variety of contributing factors to fear inhibition and expression, including changes to the volume, activity, and functional connectivity between brain regions within the fear circuit, as well as cognitive changes. Although juveniles and adults exhibit a comparable balance between fear inhibition and fear expression, the underlying reasons behind this balance may be different between the two age groups. In contrast, the balance is tipped towards poor fear inhibition and heightened fear expression during adolescence.

A B C 100 100 100

80 80 80 g g g n n n i i 60 60 i 60 z z z e e e e e e r r r

F 40 F 40 40 F % % % 20 20 20

0 0 0 Juvenile Adolescent Adult Saline DCS Extended No Retrieval- Extinction- Extinction Retrieval Extinction Retrieval Group Group Group Fear Fear Fear Fear Fear Fear inhibition expression inhibition expression inhibition expression

- - - - + + + + + - - - + + + + + +

Juvenile Adolescent Adult