Jan Haaker

Don’t fear ‘fear conditioning’: Methodological considerations for the design and analysis of studies on human fear acquisition, extinction, and return of fear

HighlightsOriginates from discussions on replicability and researchers degrees of freedom.Aims at stimulating discussions on methods applied in fear conditioning research.Addresses critical issues on terminology, design, methods, analysis.Serves as comprehensive compendium and critical evaluation of read‐out measures.Highlights methodological considerations when studying individual differences. ABSTRACT The so‐called ‘replicability crisis’ has sparked methodological discussions in many areas of science in general, and in psychology in particular. This has led to recent endeavours to promote the transparency, rigour, and ultimately, replicability of research. Originating from this zeitgeist, the challenge to discuss critical issues on terminology, design, methods, and analysis considerations in fear conditioning research is taken up by this work, which involved representatives from fourteen of the major human fear conditioning laboratories in Europe. This compendium is intended to provide a basis for the development of a common procedural and terminology framework for the field of human fear conditioning. Whenever possible, we give general recommendations. When this is not feasible, we provide evidence‐based guidance for methodological decisions on study design, outcome measures, and analyses. Importantly, this work is also intended to raise awareness and initiate discussions on crucial questions with respect to data collection, processing, statistical analyses, the impact of subtle procedural changes, and data reporting specifically tailored to the research on fear conditioning.

A review on human reinstatement studies: an overview and methodological challenges

In human research, studies of return of fear (ROF) phenomena, and reinstatement in particular, began only a decade ago and recently are more widely used, e.g., as outcome measures for fear/extinction memory manipulations (e.g., reconsolidation). As reinstatement research in humans is still in its infancy, providing an overview of its stability and boundary conditions and summarizing methodological challenges is timely to foster fruitful future research. As a translational endeavor, clarifying the circumstances under which (experimental) reinstatement occurs may offer a first step toward understanding relapse as a clinical phenomenon and pave the way for the development of new pharmacological or behavioral ways to prevent ROF. The current state of research does not yet allow pinpointing these circumstances in detail and we hope this review will aid the research field to advance in this direction. As an introduction, we begin with a synopsis of rodent work on reinstatement and theories that have been proposed to explain the findings. The review however mainly focuses on reinstatement in humans. We first describe details and variations of the experimental setup in reinstatement studies in humans and give a general overview of results. We continue with a compilation of possible experimental boundary conditions and end with the role of individual differences and behavioral and/or pharmacological manipulations. Furthermore, we compile important methodological and design details on the published studies in humans and end with open research questions and some important methodological and design recommendations as a guide for future research.

Higher anxiety and larger amygdala volumes in carriers of a TMEM132D risk variant for panic disorder

Recent case–control genome-wide association studies have linked common variants of TMEM132D (KIAA1944, MOLT) with panic disorder (PD), anxiety comorbidity in depression, and anxiety symptom severity in healthy and diseased subjects.1 One risk genotype (rs11060369 AA) is associated with enhanced TMEM132D mRNA expression in the brain; brain mRNA expression is also higher in mice bred for extreme anxiety-like behavior.1 The current study demonstrates enhanced amygdala gray matter volumetric estimates and an anxiety-related (but not panic-specific) personality profile in healthy normal carriers of the rs11060369 AA genotype. Our data suggest a role for TMEM132D in shaping threat processing. TMEM132D is a transmembrane protein expressed in neurons and colocalized with actin filaments2 that putatively functions as a cell-surface marker for oligodendrocyte differentiation.3 The TMEM132D single-nucleotide polymorphisms related so far to PD in patients of European ancestry or to anxiety in general are intronic and presumably tag yet unknown intronic functional regulatory variants.1,4 Next to common variants, rare TMEM132D variants have also been linked with pathological anxiety.5 We interrogated an independent sample of 315 healthy normal subjects (99 female) of Caucasian descent, of which 132 (22 female) underwent structural magnetic resonance imaging, for TMEM132D genotype effects on personality and brain morphology (see Supplementary Methods and Supplementary Table 1 for details). The variant rs11060369 was the only one that showed a significant omnibus effect on a battery of anxiety-related personality questionnaires (F(8,32)=3.34, P=0.0008, η2=0.07). A homozygotes had higher scores than C carriers on general measures of trait anxiety (F(1,302)=12.72, P 0.17; see Supplementary Table 2). Figure 1 Higher anxiety-related personality scores in rs11060369 A homozygotes (N=164) as compared to C carriers (N=151). (a) Higher gray matter volumetric estimates in rs11060369 A homozygotes (N=68) as compared to C carriers (N=65) in the left amygdala (bars ... rs11060369 A homozygotes also had higher gray matter volumetric estimates in the left amygdala (Z=3.39, P=0.014, small volume corrected for multiple comparisons (SVC); right side: Z=2.87, P(SVC)=0.089, trend; Figure 1b). Including trait anxiety, behavioral inhibition and negative affect scores into the volumetric analysis as covariates of no interest did not change the results (data not shown). An exploratory whole-brain analysis at an uncorrected threshold of P<0.001 additionally yielded higher estimates in A homozygotes in the left hippocampus extending into the amygdala and the cerebellum (Supplementary Table 3; reported descriptively only). There were no supra-threshold voxels in the inverse contrast. In a further exploratory analysis of the other common TMEM132D risk variants, carriers of the T/A rs11060369/7309727 combination showed a strong trend for higher estimates in the left amygdala (Z=3.17, P(SVC)=0.026 and Z=3.15, P(SVC)=0.027) than carriers of the C/A and C/C combinations (Supplementary Table 3, Supplementary Figure 1). At an uncorrected threshold, differences were also observed in the hippocampus, the insula and the other regions (Supplementary Table 3). At the same exploratory threshold (P<0.001), we observed higher volumetric estimates in the right hippocampus and right caudate in T Carrier compared to C homozygotes in an additional common TMEM132D risk variant, rs7309727 (Supplementary Table 3). There were minor effects of variant rs7309727 and no effects of variants rs900256 and rs879560 (Supplementary Table 3). rs11060369, rs7309727 and their combination have so far been more closely linked to a diagnosis of PD than to anxiety disorders in general or to non-disease-specific dimensional anxiety phenotypes.1,4 A correlation with dimensional anxiety measures for rs900256 and rs879560 and the cited mouse TMEM132D expression data, however, have been taken to support a more generic role for the protein in threat processing.1 Our observation in a sample of healthy normal volunteers of extended, genotype-dependent volumetric differences in a key neural structure associated with fear and anxiety6, 7, 8 can be interpreted as pointing toward a generic role in threat processing. This conclusion is further supported by our personality data. Our results therefore highlight TMEM132D as having an important molecular role in fear and anxiety.

Deficient inhibitory processing in trait anxiety: Evidence from context-dependent fear learning, extinction recall and renewal

BACKGROUND Impaired fear inhibition has been described as a hallmark of pathological anxiety. We aimed at further characterizing the relation between fear inhibition and anxiety by extending previous work to contextual safety stimuli as well as to dimensional scores of trait anxiety in a large sample. METHODS We employed a validated paradigm for context-dependent fear acquisition/extinction (day 1) and retrieval/expression (day 2) in 377 healthy individuals. This large sample size allowed the employment of a dimensional rather than binary approach with respect to individual differences in trait anxiety. RESULTS We observed a positive correlation on day 1 between trait anxiety with all CSs that possess an inherent inhibitory component, conveyed either by reliable non-reinforcement of a specific CS in a dangerous context (safe cue) or by the context itself (i.e., safe context). No correlation however was observed for a CS that possesses excitatory (threatening) properties only. These results were observed during fear learning (day 1) for US expectancy and fear ratings but not for SCRs. No such pattern was evident during fear and extinction retrieval/expression (day 2). CONCLUSION We provide further evidence that high trait anxiety is associated with the inability to take immediate advantage of environmental safety cues (cued and contextual), which might represent a promising trans-diagnostic marker for different anxiety disorders. Consequently, the incorporation of methods to optimize inhibitory learning in current cognitive behavioral therapy (CBT) treatments might open up a promising avenue for precision medicine in anxiety disorders. LIMITATIONS We did not include patients diagnosed with anxiety disorders.

Effects of post-extinction l-DOPA administration on the spontaneous recovery and reinstatement of fear in a human fMRI study

Relapse is a pertinent problem in the treatment of anxiety disorders. In the laboratory, relapse is modeled as return of conditioned fear responses after successful fear extinction and is explained by insufficient retrieval and/or expression of the fear-inhibitory extinction memory that is generated during extinction learning. We have shown in mice and humans that return of fear can be prevented by administration of a single dose of the dopamine precursor l-3,4-dihydroxyphenylalanine (l-DOPA) immediately after extinction. In mice, this effect could be attributed to an enhancement of extinction memory consolidation. In our human study, we could not exclude that l-DOPA might have acted by interfering with the consolidation of the original fear memory. In the present study, we therefore used a combined differential cue and context conditioning paradigm where initial fear conditioning and extinction were conducted one day apart, in analogy to previous mouse studies. l-DOPA (N=21) or placebo (N=19) were administered after extinction, precluding any action on fear memory consolidation. In the return-of-fear test conducted one week later, drug effects on conditioned skin conductance responses were absent. However, we found evidence indicative of reduced neural activity, measured with functional magnetic resonance imaging (fMRI), in the l-DOPA group in areas related to conditioned fear and return of fear (amygdala, posterior hippocampus) and enhanced activity in a key area of extinction retrieval/expression (ventromedial prefrontal cortex), relative to placebo controls. These findings require further corroboration in additional experiments. Implications for further investigations on the role of the dopamine system in extinction and on the neuropharmacological augmentation of extinction-based therapies are discussed.

Mismatch or allostatic load? Timing of life adversity differentially shapes gray matter volume and anxious temperament

Traditionally, adversity was defined as the accumulation of environmental events (allostatic load). Recently however, a mismatch between the early and the later (adult) environment (mismatch) has been hypothesized to be critical for disease development, a hypothesis that has not yet been tested explicitly in humans. We explored the impact of timing of life adversity (childhood and past year) on anxiety and depression levels (N = 833) and brain morphology (N = 129). Both remote (childhood) and proximal (recent) adversities were differentially mirrored in morphometric changes in areas critically involved in emotional processing (i.e. amygdala/hippocampus, dorsal anterior cingulate cortex, respectively). The effect of adversity on affect acted in an additive way with no evidence for interactions (mismatch). Structural equation modeling demonstrated a direct effect of adversity on morphometric estimates and anxiety/depression without evidence of brain morphology functioning as a mediator. Our results highlight that adversity manifests as pronounced changes in brain morphometric and affective temperament even though these seem to represent distinct mechanistic pathways. A major goal of future studies should be to define critical time periods for the impact of adversity and strategies for intervening to prevent or reverse the effects of adverse childhood life experiences.

Endogenous opioids regulate social threat learning in humans

Many fearful expectations are shaped by observation of aversive outcomes to others. Yet, the neurochemistry regulating social learning is unknown. Previous research has shown that during direct (Pavlovian) threat learning, information about personally experienced outcomes is regulated by the release of endogenous opioids, and activity within the amygdala and periaqueductal gray (PAG). Here we report that blockade of this opioidergic circuit enhances social threat learning through observation in humans involving activity within the amygdala, midline thalamus and the PAG. In particular, anticipatory responses to learned threat cues (CS) were associated with temporal dynamics in the PAG, coding the observed aversive outcomes to other (observational US). In addition, pharmacological challenge of the opioid receptor function is classified by distinct brain activity patterns during the expression of conditioned threats. Our results reveal an opioidergic circuit that codes the observed aversive outcomes to others into threat responses and long-term memory in the observer.

Neural signals of vicarious extinction learning

Social transmission of both threat and safety is ubiquitous, but little is known about the neural circuitry underlying vicarious safety learning. This is surprising given that these processes are critical to flexibly adapt to a changeable environment. To address how the expression of previously learned fears can be modified by the transmission of social information, two conditioned stimuli (CS + s) were paired with shock and the third was not. During extinction, we held constant the amount of direct, non-reinforced, exposure to the CSs (i.e. direct extinction), and critically varied whether another individual-acting as a demonstrator-experienced safety (CS + vic safety) or aversive reinforcement (CS + vic reinf). During extinction, ventromedial prefrontal cortex (vmPFC) responses to the CS + vic reinf increased but decreased to the CS + vic safety This pattern of vmPFC activity was reversed during a subsequent fear reinstatement test, suggesting a temporal shift in the involvement of the vmPFC. Moreover, only the CS + vic reinf association recovered. Our data suggest that vicarious extinction prevents the return of conditioned fear responses, and that this efficacy is reflected by diminished vmPFC involvement during extinction learning. The present findings may have important implications for understanding how social information influences the persistence of fear memories in individuals suffering from emotional disorders.

Assessment of social transmission of threats in humans using observational fear conditioning

Across the human life span, fear is often acquired indirectly by observation of the emotional expressions of others. The observational fear conditioning protocol was previously developed as a laboratory model for investigating socially acquired threat responses. This protocol serves as a suitable alternative to the widely used Pavlovian fear conditioning, in which threat responses are acquired through direct experiences. In the observational fear conditioning protocol, the participant (observer) watches a demonstrator being presented with a conditioned stimulus (CS) paired with an aversive unconditioned stimulus (US). The expression of threat learning is measured as the conditioned response (CR) expressed by the observer in the absence of the demonstrator. CRs are commonly measured as skin conductance responses, but behavioral and neural measures have also been implemented. The experimental procedure is suitable for divergent populations, can be administered by a graduate student and takes ∼40 min. Similar protocols are used in animals, emphasizing its value as a translational tool for studying socioemotional learning.

Converging evidence for an impact of a functional NOS gene variation on anxiety-related processes

Being a complex phenotype with substantial heritability, anxiety and related phenotypes are characterized by a complex polygenic basis. Thereby, one candidate pathway is neuronal nitric oxide (NO) signaling, and accordingly, rodent studies have identified NO synthase (NOS-I), encoded by NOS1, as a strong molecular candidate for modulating anxiety and hippocampus-dependent learning processes. Using a multi-dimensional and -methodological replication approach, we investigated the impact of a functional promoter polymorphism (NOS1-ex1f-VNTR) on human anxiety-related phenotypes in a total of 1019 healthy controls in five different studies. Homozygous carriers of the NOS1-ex1f short-allele displayed enhanced trait anxiety, worrying and depression scores. Furthermore, short-allele carriers were characterized by increased anxious apprehension during contextual fear conditioning. While autonomous measures (fear-potentiated startle) provided only suggestive evidence for a modulatory role of NOS1-ex1f-VNTR on (contextual) fear conditioning processes, neural activation at the amygdala/anterior hippocampus junction was significantly increased in short-allele carriers during context conditioning. Notably, this could not be attributed to morphological differences. In accordance with data from a plethora of rodent studies, we here provide converging evidence from behavioral, subjective, psychophysiological and neuroimaging studies in large human cohorts that NOS-I plays an important role in anxious apprehension but provide only limited evidence for a role in (contextual) fear conditioning.