Learning About Safety: Conditioned Inhibition as a Novel Approach to Fear Reduction Targeting the Developing Brain

Difficulty regulating fear is a central feature of anxiety disorders (16, 17). Fear learning is an adaptive function that allows an organism to predict potentially aversive events from environmental cues (see Table 1 for a glossary). When a stimulus no longer signals threat, fear expression gradually diminishes, allowing individuals to override the previously learned association (18). In classical conditioning, fear extinction (19) is operationalized by repeated exposure of the previously learned threat cue (i.e., conditioned stimulus, CS) without any aversive outcome (i.e., unconditioned stimulus, US) and is tested using threat and safety discrimination during extinction (i.e., extinction learning) or after a delay (i.e., extinction retention or recall). Deficits in extinction learning or extinction retention can result in unremitting fear that persists even after threat has subsided (20).

The primary evidence-based psychosocial treatment for anxiety disorders is exposure-based cognitive-behavioral therapy (CBT), which was initially based on models such as corrective information processing (21, 22) and is thought to rely on principles of fear extinction (22, 23). During exposures, patients repeatedly and systematically confront fear-provoking stimuli with the goal of reducing anxiety (9, 24). Such extinction-based interventions may be especially vulnerable to the return of extinguished fear responses, as the formation of a new extinction memory does not overwrite the initial fear association. Rather, the extinction memory competes with the original threat memory, and previously extinguished fear responses can return via the mere passage of time (spontaneous recovery), exposure to a stressor (fear reinstatement), or return to a fear-associated context (fear renewal) (25).

The challenges of sustained efficacy with CBT may be further compounded for youths with anxiety disorders. A high proportion of clinically anxious youths do not remit after gold-standard treatments (i.e., CBT and selective serotonin reuptake inhibitors [SSRIs]), with estimates ranging from 25% to 50% of youths still meeting criteria for a principal anxiety disorder after treatment (11, 26). A large-scale study of pediatric anxiety (12) found that only approximately 22% of youths achieved stable remission (i.e., did not meet diagnostic criteria for any anxiety disorder) across 4 years of annual follow-up that began 4–12 years after initial randomization to one of four 12-week treatment options (CBT, an SSRI, combined CBT and SSRI, or pill placebo). Within the group randomized to CBT alone, remission rates ranged from 40% to 60% in each follow-up year, highlighting the need to optimize treatments for pediatric anxiety.

Although response rates for CBT are similar across childhood, adolescence, and adulthood (11, 12, 14, 26), the factors contributing to low response rates may differ across age groups. In youths, one hypothesis is that some children and adolescents do not benefit sufficiently from current treatments because these interventions are largely based on principles that have been studied and implemented in adults. Delineating the biological state of the developing brain—that is, the nature of neural structure, function, and connectivity at a given developmental stage—and applying this knowledge to intervention approaches may be critical to optimizing treatment for anxious youths (27). Given evidence for diminished fear extinction learning during adolescence (28, 29), as well as altered ventromedial prefrontal cortex (vmPFC) and amygdala involvement during extinction learning in healthy and anxious adolescents (30), adolescents with anxiety may particularly benefit from efforts to optimize exposure-based therapies with approaches to fear reduction that either augment the strength of fronto-amygdala connections or bypass prefrontally mediated extinction processes.

Identifying the neural mechanisms underlying core features of anxiety disorders is essential for the discovery of novel therapeutics and optimized treatments. Extensive research across species has shown that cortical-subcortical interactions primarily involving the amygdala, vmPFC, and hippocampus are central to fear learning and extinction learning and retention (23, 31–34). The amygdala is critically involved in fear learning and fear expression (35, 36), as well as extinction learning (37). Bidirectional connections between the amygdala and vmPFC modulate fear expression (38). Whereas the prelimbic (PL) region of the rodent vmPFC is involved in fear maintenance (39), the infralimbic (IL) region of the rodent vmPFC inhibits fear expression and stores and retrieves extinction memory (40). Consistent with these findings, the IL cortex has been implicated in both extinction learning and retention (for reviews, see 38, 41, 42). The hippocampus has also been highlighted as a central region involved in fear learning and extinction and supplies critical information about the degree of threat or safety in the environment (43–45) via projections from ventral CA1 hippocampus to the basolateral amygdala (BLA) (46, 47). Additionally, recent molecular evidence may link learning about safety in the environment (i.e., safety learning) and extinction learning, including evidence that administering a cannabinoid receptor 1 antagonist in the hippocampus in adult rodents prevents extinction of avoidance behavior via safety learning (48). Furthermore, recent evidence suggests that glucocorticoid regulation enhances fear extinction learning and safety learning in individuals with posttraumatic stress disorder (PTSD) (49), further linking these processes at the molecular level with the endocannabinoid system and glucocorticoid hormones.

Neuroimaging studies are providing increasing insight into the neural circuitry supporting fear learning and extinction learning and recall in humans. Consistent with findings in rodents, evidence suggests that the amygdala plays a central role in fear learning in humans (23, 50–52) and that the dorsal anterior cingulate cortex (dACC) may modulate fear expression (53). Also in line with findings in rodents, human neuroimaging studies indicate that the amygdala also contributes to extinction learning (23, 52), whereas the vmPFC and hippocampus are involved in extinction retention (23, 54). Importantly, human neuroimaging studies must be considered in the context of the challenges and limitations that the field currently faces. Low reproducibility, low statistical power (e.g., stemming from a large number of dependent variables but a relatively small number of observations [subjects]), small effect sizes, and flexibility in data analyses have all been noted as limitations on the conclusions that can be drawn from neuroimaging studies (55–57). Thus, inferences about the neural circuitry supporting fear reduction in humans that are drawn from neuroimaging studies should be interpreted with caution, and it will be important that future studies employ evolving guidelines for best practices for reproducible science (57, 58). While studies are needed to replicate human neuroimaging findings with larger samples and greater statistical power, the existing evidence suggests involvement of frontolimbic circuitry in fear learning and extinction learning and retention, consistent with evidence in rodents.

Disruption in fear learning and extinction is a key etiological feature of anxiety disorders (59). Across studies, adults with anxiety disorders show increased subjective anxiety, skin conductance response, and startle response to safety cues during fear acquisition and increased fear responding to threat cues during extinction learning relative to individuals without anxiety (60, 61). Furthermore, both normative and clinically impairing anxiety involve alterations in the frontolimbic circuitry that underlies fear extinction (62). In human neuroimaging studies, adults with anxiety disorders display diminished prefrontal control of the amygdala, amygdala hyperreactivity, and weaker connectivity between the amygdala and various regions of prefrontal cortex, along with some evidence of altered hippocampal activation in studies of affective stimuli (62–68). Consistent with these disruptions, evidence has demonstrated alterations in this circuitry during extinction retention in anxiety disorders. Findings suggest impaired extinction retention (69) and functional alterations in the vmPFC, hippocampus, and dACC during extinction recall (63) among individuals with anxiety disorders.

Fear learning and extinction undergo dynamic changes during childhood and adolescence (29, 70; for a review, see 71). Neuroimaging studies in humans have begun to delineate age-related patterns of functional activation and connectivity during fear learning (72–74), extinction learning (30), and extinction recall (75–77). These findings suggest that changes in frontolimbic circuitry with age may contribute to changes in fear learning and extinction across development (78, 79). Cross-species evidence has suggested that fear extinction learning is diminished during adolescence (28, 29), relative to children and adults. This reduced extinction learning during adolescence has been associated with altered neuroplasticity in the vmPFC, namely, an absence of extinction-learning-induced plasticity within the rodent IL cortex (29). Furthermore, a recent study in healthy adolescents and young adults showed altered age-related involvement of the amygdala and vmPFC during extinction learning, such that younger adolescents showed higher amygdala activity and later engagement of the vmPFC to threat versus safety cues during extinction learning (30). These findings may be consistent with broader evidence of age-related changes in fronto-amygdala circuitry during childhood and adolescence (e.g., 80–85).

Although less is known about anxiety-related alterations to developmental trajectories, evidence suggests that youths with anxiety disorders may exhibit alterations in fear and extinction learning (86). Relative to nonanxious youths, anxious youths display increased self-reported fear (87) and skin conductance (88, 89) to both threat and safety cues and are more resistant to fear extinction, measured using startle response (90) and skin conductance (89). However, consistent evidence that anxious youths discriminate between threat and safety differently than nonanxious youths during conditioning is lacking (87, 91; for a review, see 92). For example, a recent meta-analysis of seven fear conditioning studies found that anxious youths exhibit stronger fear responses (i.e., self-reported fear, skin conductance response, or fear-potentiated startle) to individual threat and safety cues than nonanxious youths, but that differential fear acquisition and extinction (i.e., responding to threat versus safety cues) are similar between anxious and nonanxious youths (93). Given the small number of studies and participants in studies of fear and extinction learning in anxious youths, these results should be interpreted with caution, and further research is needed to understand these processes in pediatric anxiety disorders.

Building on behavioral and physiological studies in youths with anxiety, a small but growing body of research has investigated neurobiological alterations during fear learning and extinction learning and retention in anxious youths. Relatively few of these studies have conducted fear conditioning during fMRI scanning. However, initial results suggest that anxious adolescents show lower activation in the medial prefrontal cortex (PFC)/paracingulate gyrus in response to the safety cue (72). In addition to this alteration that was consistent across age, relative to nonanxious youths, anxious youths exhibited a stronger pattern of age-related decrease in prefrontal activation to the safety cue during fear conditioning (72). Neuroimaging studies have provided growing insight into neural processes related to retention of fear extinction in anxious youths. Anxious adolescents show lower subgenual anterior cingulate cortex activation, relative to healthy counterparts, when asked to indicate whether or not they were afraid of the stimuli (i.e., threat appraisal) during extinction recall (75). Furthermore, adolescents with anxiety also show higher vmPFC activation to the most prototypical threat and safety cues along a continuum, compared with anxious adults and nonanxious youths, when engaging in threat appraisal during extinction recall (75). One additional study using the same paradigm found that anxious youths showed higher negative amygdala-PFC functional connectivity when engaged in both threat appraisal and explicit threat memory during extinction recall, relative to anxious adults and nonanxious youths (76). Building on this work, a recent study from the same group (77) used the same extinction recall paradigm but increased the number of stimulus replicates and sample size (N=200) to increase statistical power. Findings showed that differences between anxious and nonanxious individuals were dependent on age. Specifically, anxious individuals showed an age-related decrease in amygdala-vmPFC functional connectivity compared with nonanxious individuals during extinction recall as stimuli increasingly resembled safety cues. The directionality of these results differed from the previous finding of higher amygdala-vmPFC connectivity in anxious youths (76); however, both findings highlight the relevance of this circuitry for extinction recall in anxious adolescents. While further human neuroimaging studies (e.g., with larger sample sizes) are needed to replicate these findings in anxious adolescents and to reconcile inconsistent findings, these studies suggest that youths with anxiety exhibit alterations in neural regions involved in fear learning and extinction recall and that these anxiety-related alterations may be developmental in nature.

Several approaches to enhancing fear reduction have been proposed, including reconsolidation update, in which the fear memory is recalled prior to extinction with the aim of updating the original fear memory trace (94, 95); combined cue and contextual fear extinction, in which extinction occurs in the original conditioning context (96); and safety signal learning, which is the process through which an organism learns about safety (or the lack of threat) in the environment (97). In this review, we focus on safety signal learning because of its potential capacity for translation that aims to optimize exposure-based therapies for fear reduction in a neurodevelopmentally informed manner. We aim to bridge the gap between cross-species research on basic neuroscience and its application to clinical treatment. The material that follows below comprises three sections: first, we review the literature on safety signal learning through conditioned inhibition and its potential neural correlates in rodents, nonhuman primates, and humans; next, we critically examine the current empirical literature on safety cues in treatment for anxiety, including the mechanisms and conditions under which safety cues may enhance or interfere with treatment; and, finally, we propose the application of safety signal learning to inform clinical translation that will augment evidence-based treatments for anxious youths.

Our proposed model highlights two particular aspects of translational research—translation between animal models and human research, and between basic neuroscience and clinical practice. Both of these levels of translation have critical importance for guiding future research and the treatment of anxiety disorders. For example, human neuroimaging findings in anxious populations can lead to circuitry-focused work in animal models; reciprocally, circuitry-focused work in experimental organisms is essential to delineating mechanisms of anxiety and can guide the selection of regions of interest in human neuroimaging research. Furthermore, neuroscientific findings across species can identify treatment targets or provide key insights that inform novel therapeutic techniques. Methodical implementation of interventions with neuroimaging can be used to evaluate the effectiveness of treatment and the utility of biomarkers. Taken together, findings from the basic science of safety signal learning across species and from treatment studies can reciprocally inform one another to guide the optimization of interventions for anxious youths by targeting the biological state of the developing brain. Throughout the review, we propose hypotheses based on the existing cross-species and clinical literature and provide recommendations for future translational research on safety signal learning.

During safety signal learning, a cue that is overly trained to signal the absence of threat (i.e., the safety cue) is used to reduce fear in the presence of a threatening cue (97–99). Safety signal learning is a special class of conditioned inhibition in which the safety cue must inhibit the conditioned response (CR) as a result of learning (as opposed to the process by which stimuli inhibit the CR without training, called external inhibition) (97). Conditioned inhibition via safety signal learning is thereby a process in which a stimulus is trained, through Pavlovian conditioning, to signal safety (or the absence of threat), and as a result this safety cue can inhibit the conditioned fear response. During the acquisition phase of conditioned inhibition, the threat cue (CS+) is paired with the US, and the safety cue (CS−) is never paired with the US. Research in rodents and nonhuman primates has relied on two procedures to test whether a stimulus acts as a conditioned inhibitor. During the “summation test,” the threat and safety cues are presented simultaneously as a compound stimulus (safety compound), yielding a reduction in fear-related behavior. During the “retardation test,” the safety cue is paired with the US (100). If a safety cue has been effectively learned, fear responding should be slower to emerge (relative to initial conditioning).

Safety signal learning via conditioned inhibition differs in several important ways from extinction learning or safety discrimination, which have been traditionally examined in relation to fear learning and anxiety disorders. Safety cues in Pavlovian conditioning are typically operationalized using a CS− that predicts the absence of a threatening stimulus (101). Safety discrimination can be measured during extinction learning, extinction recall, or reversal by comparing reactivity to the CS+ (threat cue) and the CS− (safety cue) (101). A large body of research in rodents (102) and humans (16, 103, 104) has investigated discrimination between threat and safety cues in extinction recall during development. However, these safety cues were not trained as conditioned inhibitors (i.e., the safety and threat cues were not paired or evaluated with a summation or retardation test). In contrast, safety signal learning via conditioned inhibition must include the pairing of the CS+ and CS− in order to test for the active inhibition of threat in the presence of safety. In extinction learning, a new and competing association forms when a cue that was previously associated with threat is repeatedly presented in the absence of threat. In contrast, conditioned inhibition involves associating distinct environmental stimuli (i.e., safety cues) with the nonoccurrence of aversive events (97). Thus, safety cues have the potential to inhibit the expression of fear-related behaviors to cues that signal threat without the competing association involved in extinction (see Table 2 for a comparison of safety signal learning and extinction learning). “Conditioned inhibition” is used hereafter to refer to the process by which a safety cue inhibits fear in the presence of threat, which is tested via summation and retardation. The relation between conditioned inhibition and general inhibitory control, a core executive function traditionally measured using tasks such as the AX continuous performance task (AX-CPT), stop-signal, go/no-go, and antisaccade tasks in neutral contexts (105), has yet to be investigated. Future research will be important in determining the extent to which these processes are related or distinct.

Safety signal learning has been shown to be effective for reducing fear responding in behavioral studies with rodents (98), nonhuman primates (99), and healthy adult humans (106). In humans, the presence of a safety cue has been shown to reduce physiological correlates of fear, as measured by fear-potentiated startle (106). Most recently, reduced fear-related reactivity in the presence of a safety cue was observed in a cross-species study in adult mice and healthy adult humans using freezing behavior and skin conductance response, respectively (107). Evidence has shown disruption in safety signal learning in adults with PTSD (108; for a review, see 109), suggesting that this process may also be disrupted in anxious individuals relative to nonanxious individuals, although safety signal learning has been explored less in adults with anxiety disorders or following stress. Nevertheless, recent behavioral evidence in rodents suggests that conditioned inhibition of fear via a safety cue may not be as susceptible to the effects of prior stress (e.g., unsignaled foot shocks) as fear extinction (110). These studies suggest that safety cues may effectively reduce fear, even in the presence of a threat stimulus, and may be even more beneficial than typical extinction for individuals with anxiety or a history of stress exposure.

Although rarely integrated with the neuroscience literature, a rich and growing literature on safety signal learning and safety behaviors (i.e., behaviors that employ a safety cue) exists in clinical science. Patients with anxiety disorders often use safety cues with the goal of reducing fear. Whereas safety cues are often studied in basic science both behaviorally and neurally using standardized paradigms with neutral stimuli (e.g., shapes, sounds, and odors), safety cues outside of the laboratory can take many forms, including people, objects, actions, or mental acts. For example, an individual with panic disorder might carry antianxiety medication, or an individual with social anxiety disorder might enter a new situation only in the presence of their partner. Although conceptualized in basic science research as reducing fear (97), safety cues likely have more complex effects in the everyday lives of individuals with anxiety. Safety cues may reduce anxiety in the short term (94); however, anxious individuals may come to rely on safety cues to function or engage with anxiety-provoking stimuli, such that the cues impede learning that they can tolerate a feared situation. Thus, evidence-based treatments, including CBT, often focus on eliminating patients’ reliance on safety cues (24). In this section, we summarize the literature on clinical research, conducted with adults, on the impacts of safety behaviors in exposure-based therapy, with a particular focus on the potential mechanisms by which safety cues might influence treatment and potential moderators that could guide an understanding of when safety cues might be more helpful rather than detrimental.

Throughout this review, we attempt to draw connections between the literature on neural and clinical mechanisms leading to fear reduction (Figure 1). In the neurobiological literature, evidence includes recent findings on the involvement of the ventral hippocampal-PL pathway—as an alternative to the canonical amygdala-IL pathway involved in fear extinction—as well as the potential involvement of regions including the amygdala, vmPFC, and NAc (97, 107). In the clinical literature, proposed mechanisms for fear reduction via the inclusion of safety cues in treatment include enhancing treatment acceptability, facilitating approach behavior, and altering inhibitory learning (for reviews, see 24, 146, 166). Further research is necessary to disentangle these proposed clinical mechanisms in carefully controlled translational treatment studies. In particular, delineating the conditions under which safety cues may enhance or interfere with inhibitory learning will be essential to informing interventions.

Here we propose that these neural and clinical mechanisms may interact to enhance or interfere with fear reduction via safety signal learning. Based on the existing clinical literature, safety signal type (i.e., preventive versus restorative safety cues [166]) may moderate the effect of safety signal learning on physiological indices of fear reduction (as in 106, 107), either directly or through the clinical or neural mechanisms. In parallel, based on the existing neurobiological literature, developmental stage, specifically adolescence versus adulthood, may also moderate the effect of safety signal learning on fear reduction. Our conceptual model integrates key clinical and neurobiological factors and highlights the importance of future translational research to test these relations across basic science, including circuitry-focused work with animal models and brain imaging studies with humans, and treatment research, including clinical treatment in tandem with brain imaging to evaluate neural targets identified from basic neuroscience.

Anxiety disorders often emerge during childhood and adolescence, yet not all youths benefit sufficiently from current evidence-based treatments. A key feature of anxiety disorders is difficulty regulating fear (16), which may stem from difficulty in learning about or implementing cues that signal safety (99). Behavioral and neuroscientific studies using conditioned inhibition paradigms have shown that safety cues can effectively reduce fear and prevent the onset of new fears in animals (98, 99), and behavioral and neuroimaging studies suggest that safety cues are effective for actively inhibiting fear in humans (106, 107). The hippocampus, vmPFC, amygdala, OFC, NAc, and insula have been proposed as candidate regions involved in conditioned inhibition in animal models. Recent cross-species evidence in humans and rodent models suggests that conditioned inhibition via learned safety may involve connections between the ventral hippocampus and the PL cortex (and connections between the hippocampus and dACC in humans), a pathway that may be strengthened during adolescence (96). Translating these findings on safety cues into the clinical domain may provide a novel approach to reducing fear in youths by targeting the biological state of the developing brain. Here, we propose a neurodevelopmental model of safety signal learning in which the hippocampus plays a central role in down-regulating fear responses through up-regulation of the PL cortex during adolescence.

Existing evidence in the clinical literature suggests that judicious use of safety cues may help enhance treatment efficacy under certain conditions, including careful selection of the stages in treatment during which to incorporate and to eliminate the use of safety cues. Evidence suggests that the use of restorative safety cues may be particularly effective given that they allow for full confrontation with a core threat and active inhibition of the threat representation. We propose theoretical examples of how safety cues could be differentially integrated for children, such as systematically incorporating parental presence in treatment, and for adolescents, such as the use of music or social support figures. Furthermore, we propose a conceptual model for integrating clinical and neural mechanisms, as well as proposed moderators, for fear reduction via safety signal learning. Although this conceptual model remains largely untested, in this review we formulate hypotheses and recommendations for future translational research in a manner that is consistent with theories and predictions being recorded prior to empirical testing.

Further elucidating the neural mechanisms that support the effective use of safety cues to inhibit fear and how they change across development will be essential to translating research on conditioned inhibition for clinical applications. Integrating empirical findings across these literatures suggests that conditioned inhibition may be especially relevant for children and adolescents with anxiety. Particularly during adolescence, when frontolimbic connections that support traditional fear extinction are still developing (29, 80, 83) and connections between the hippocampus and PL cortex are strengthened (96), the use of safety signal learning may be especially beneficial for optimizing treatment of anxiety based on the biological state of the developing brain. Finally, we call for developmentally informed translational investigations that judiciously incorporate knowledge about the type and timing of safety cues into exposure-based treatments, which have the potential to identify novel ways to maximize fear reduction and optimize treatment outcomes for anxious youths.

Supported by the NIH Director’s Early Independence Award (DP5OD021370) to Dr. Gee, a Brain and Behavior Research Foundation Young Investigator Grant to Dr. Gee, a Jacobs Foundation Early Career Research Fellowship to Dr. Gee, and a National Science Foundation Graduate Research Fellowship Program award (DGE1122492) to Ms. Odriozola.