Evelyn Glotzbach-Schoon

Contextual fear conditioning in virtual reality is affected by 5HTTLPR and NPSR1 polymorphisms: effects on fear-potentiated startle

The serotonin (5-HT) and neuropeptide S (NPS) systems are discussed as important genetic modulators of fear and sustained anxiety contributing to the etiology of anxiety disorders. Sustained anxiety is a crucial characteristic of most anxiety disorders which likely develops through contextual fear conditioning. This study investigated if and how genetic alterations of the 5-HT and the NPS systems as well as their interaction modulate contextual fear conditioning; specifically, function polymorphic variants in the genes coding for the 5-HT transporter (5HTT) and the NPS receptor (NPSR1) were studied. A large group of healthy volunteers was therefore stratified for 5HTTLPR (S+ vs. LL carriers) and NPSR1 rs324981 (T+ vs. AA carriers) polymorphisms resulting in four genotype groups (S+/T+, S+/AA, LL/T+, LL/AA) of 20 participants each. All participants underwent contextual fear conditioning and extinction using a virtual reality (VR) paradigm. During acquisition, one virtual office room (anxiety context, CXT+) was paired with an unpredictable electric stimulus (unconditioned stimulus, US), whereas another virtual office room was not paired with any US (safety context, CXT−). During extinction no US was administered. Anxiety responses were quantified by fear-potentiated startle and ratings. Most importantly, we found a gene × gene interaction on fear-potentiated startle. Only carriers of both risk alleles (S+/T+) exhibited higher startle responses in CXT+ compared to CXT−. In contrast, anxiety ratings were only influenced by the NPSR1 polymorphism with AA carriers showing higher anxiety ratings in CXT+ as compared to CXT−. Our results speak in favor of a two level account of fear conditioning with diverging effects on implicit vs. explicit fear responses. Enhanced contextual fear conditioning as reflected in potentiated startle responses may be an endophenotype for anxiety disorders.

Enhanced discrimination between threatening and safe contexts in high-anxious individuals.

Trait anxiety, a stable personality trait associated with increased fear responses to threat, is regarded as a risk factor for the development and maintenance of anxiety disorders. Although the effect of trait anxiety has been examined with regard to explicit threat cues, little is known about the effect of trait anxiety on contextual threat learning. To assess this issue, extreme groups of low and high trait anxiety underwent a contextual fear conditioning protocol using virtual reality. Two virtual office rooms served as the conditioned contexts. One virtual office room (CXT+) was paired with unpredictable electrical stimuli. In the other virtual office room, no electrical stimuli were delivered (CXT-). High-anxious participants tended to show faster acquisition of startle potentiation in the CXT+ versus the CXT- than low-anxious participants. This enhanced contextual fear learning might function as a risk factor for anxiety disorders that are characterized by sustained anxiety.

The BDNF Val66Met polymorphism modulates the generalization of cued fear responses to a novel context.

Brain-derived neurotrophic factor (BDNF) has a crucial role in activity-dependent synaptic plasticity and learning and memory. The human functional single-nucleotide BDNF rs6265 (Val66Met) polymorphism has been found to be associated with alteration in neural BDNF release and function correlating with altered emotional behavior. Here, we investigated for the first time the hypothesis that this polymorphism in humans modulates the context dependency of conditioned fear responses. Applying a new paradigm examining generalization of cued fear across contexts, 70 participants stratified for BDNF Val66Met polymorphism were guided through two virtual offices (context) in which briefly illuminated blue and yellow lights served as cues. In the fear context, one light (conditioned stimulus, CS+) but not the other light (CS−) was associated with an electric shock (unconditioned stimulus, US). In the safety context, both lights were presented too, but no US was delivered. During the test phase, lights were presented again both in learning contexts and in a novel generalization context without any US. All participants showed clear fear conditioning to the CS+ in the fear context as indicated by potentiation of startle responses and reports of fear. No fear reactions were found for the CS+ in the safety context. Importantly, generalization of fear responses indicated by the potentiation of startle response to the CS+ compared with the CS− in the novel context was evident only in the Met-carrying group. These are the first results to provide evidence in humans that BDNF modulates the generalization of fear responses. Such context-dependent generalization processes might predispose Met carriers for affective and anxiety disorders.

GLRB allelic variation associated with agoraphobic cognitions, increased startle response and fear network activation : a potential neurogenetic pathway to panic disorder

The molecular genetics of panic disorder (PD) with and without agoraphobia (AG) are still largely unknown and progress is hampered by small sample sizes. We therefore performed a genome-wide association study with a dimensional, PD/AG-related anxiety phenotype based on the Agoraphobia Cognition Questionnaire (ACQ) in a sample of 1370 healthy German volunteers of the CRC TRR58 MEGA study wave 1. A genome-wide significant association was found between ACQ and single non-coding nucleotide variants of the GLRB gene (rs78726293, P=3.3 × 10−8; rs191260602, P=3.9 × 10−8). We followed up on this finding in a larger dimensional ACQ sample (N=2547) and in independent samples with a dichotomous AG phenotype based on the Symptoms Checklist (SCL-90; N=3845) and a case–control sample with the categorical phenotype PD/AG (Ncombined =1012) obtaining highly significant P-values also for GLRB single-nucleotide variants rs17035816 (P=3.8 × 10−4) and rs7688285 (P=7.6 × 10−5). GLRB gene expression was found to be modulated by rs7688285 in brain tissue, as well as cell culture. Analyses of intermediate PD/AG phenotypes demonstrated increased startle reflex and increased fear network, as well as general sensory activation by GLRB risk gene variants rs78726293, rs191260602, rs17035816 and rs7688285. Partial Glrb knockout mice demonstrated an agoraphobic phenotype. In conjunction with the clinical observation that rare coding GLRB gene mutations are associated with the neurological disorder hyperekplexia characterized by a generalized startle reaction and agoraphobic behavior, our data provide evidence that non-coding, although functional GLRB gene polymorphisms may predispose to PD by increasing startle response and agoraphobic cognitions.

Pain predictability reverses valence ratings of a relief-associated stimulus

Relief from pain is positively valenced and entails reward-like properties. Notably, stimuli that became associated with pain relief elicit reward-like implicit responses too, but are explicitly evaluated by humans as aversive. Since the unpredictability of pain makes pain more aversive, this study examined the hypotheses that the predictability of pain also modulates the valence of relief-associated stimuli. In two studies, we presented one conditioned stimulus (FORWARDCS+) before a painful unconditioned stimulus (US), another stimulus (BACKWARDCS+) after the painful US, and a third stimulus (CS−) was never associated with the US. In Study 1, FORWARDCS+ predicted half of the USs while the other half was delivered unwarned and followed by BACKWARDCS+. In Study 2, all USs were predicted by FORWARDCS+ and followed by BACKWARDCS+. In Study 1 both FORWARDCS+ and BACKWARDCS+ were rated as negatively valenced and high arousing after conditioning, while BACKWARDCS+ in Study 2 acquired positive valence and low arousal. Startle amplitude was significantly attenuated to BACKWARDCS+ compared to FORWARDCS+ in Study 2, but did not differ among CSs in Study 1. In summary, predictability of aversive events reverses the explicit valence of a relief-associated stimulus.

Generalization of contextual fear in humans

Enhanced fear responses to cues, which were not associated with the threat but share perceptual characteristics with the threat signal, indicate generalization of conditioned fear. Here, we investigated for the first time generalization processes in contextual fear conditioning. Thirty-two participants were guided through two virtual offices (acquisition phases). Mildly painful electric shocks (unconditioned stimulus, US) were unpredictably delivered in one office (anxiety context, CTX+), but never in the other office (safety context, CTX-). During the generalization test, participants were guided through CTX+, CTX-, and the generalization context (G-CTX), which contained features of both the CTX+ and the CTX-, but no US was delivered. We found successful contextual fear conditioning (i.e., the CTX+ compared to the CTX- elicited potentiated startle responses and was rated with more negative valence, higher arousal and higher anxiety). Importantly, implicit and explicit responses dissociated in the generalization test. Thus, participants rated the G-CTX as more arousing and anxiogenic than the CTX- indicating anxiety generalization, but they showed enhanced startle responses to the CTX+ only, while the G-CTX and the CTX- did not differ. In summary, healthy participants on an explicit level responded to the generalization context like to the anxiety context, but on an implicit level responded to the generalization context like to the safety context. Possibly, this dissociation suggests distinct and specific generalization processes underlying contextual fear.

Delay and trace fear conditioning in a complex virtual learning environment—neural substrates of extinction

Extinction is an important mechanism to inhibit initially acquired fear responses. There is growing evidence that the ventromedial prefrontal cortex (vmPFC) inhibits the amygdala and therefore plays an important role in the extinction of delay fear conditioning. To our knowledge, there is no evidence on the role of the prefrontal cortex in the extinction of trace conditioning up to now. Thus, we compared brain structures involved in the extinction of human delay and trace fear conditioning in a between-subjects-design in an fMRI study. Participants were passively guided through a virtual environment during learning and extinction of conditioned fear. Two different lights served as conditioned stimuli (CS); as unconditioned stimulus (US) a mildly painful electric stimulus was delivered. In the delay conditioning group (DCG) the US was administered with offset of one light (CS+), whereas in the trace conditioning group (TCG) the US was presented 4 s after CS+ offset. Both groups showed insular and striatal activation during early extinction, but differed in their prefrontal activation. The vmPFC was mainly activated in the DCG, whereas the TCG showed activation of the dorsolateral prefrontal cortex (dlPFC) during extinction. These results point to different extinction processes in delay and trace conditioning. VmPFC activation during extinction of delay conditioning might reflect the inhibition of the fear response. In contrast, dlPFC activation during extinction of trace conditioning may reflect modulation of working memory processes which are involved in bridging the trace interval and hold information in short term memory.

Brain activity associated with illusory correlations in animal phobia

Anxiety disorder patients were repeatedly found to overestimate the association between disorder-relevant stimuli and aversive outcomes despite random contingencies. Such an illusory correlation (IC) might play an important role in the return of fear after extinction learning; yet, little is known about how this cognitive bias emerges in the brain. In a functional magnetic resonance imaging study, 18 female patients with spider phobia and 18 healthy controls were exposed to pictures of spiders, mushrooms and puppies followed randomly by either a painful electrical shock or nothing. In advance, both patients and healthy controls expected more shocks after spider pictures. Importantly, only patients with spider phobia continued to overestimate this association after the experiment. The strength of this IC was predicted by increased outcome aversiveness ratings and primary sensory motor cortex activity in response to the shock after spider pictures. Moreover, increased activation of the left dorsolateral prefrontal cortex (dlPFC) to spider pictures predicted the IC. These results support the theory that phobia-relevant stimuli amplify unpleasantness and sensory motor representations of aversive stimuli, which in turn may promote their overestimation. Hyper-activity in dlPFC possibly reflects a pre-occupation of executive resources with phobia-relevant stimuli, thus complicating the accurate monitoring of objective contingencies and the unlearning of fear.

Effects of context preexposure and delay until anxiety retrieval on generalization of contextual anxiety

Animal studies suggest that time delay between acquisition and retrieval of contextual anxiety increases generalization. Moreover, such generalization is prevented by preexposure to the context (CTX), presumably due to an improved representation of such context. We investigated whether preexposure and time-passing modulate generalization of contextual anxiety, in humans. On Day 1, 42 participants (preexposure group) explored two virtual offices, while 41 participants (no-preexposure group) explored a virtual stadium. On Day 2 (24 h later), all participants learned to associate one office (CTX+) with unpredictable unconditioned stimuli (USs), and another office (CTX-) with safety. On Day 3, either 24 h (recent test) or 2 wk (remote test) later, participants revisited CTX- and CTX+ without USs, as well as a generalization context (G-CTX). Results revealed successfully conditioned anxiety and anxiety generalization for ratings (G-CTX was as aversive as CTX+ was), while safety generalization was found for startle responses (G-CTX elicited startle attenuation as CTX- did). Time between learning and testing enhanced generalization as reflected by comparable startle responses to all three offices in the remote test. Contextual preexposure facilitated extinction of explicit conditioned anxiety assessed with ratings. These results suggest that memory trace of a context degrades with passage of time in humans like in animals and, consequently, anxiety generalization enhances. After context preexposure, high cognitive processes seem to be crucially involved in facilitating extinction (or safety) learning.

Reinstatement of contextual anxiety in humans: Effects of state anxiety

After successful extinction of conditioned fear, the presentation of an unsignaled unconditioned stimulus (US) leads to return of fear, thus, the previously extinguished conditioned stimulus (CS) triggers fear responses again. Human studies on such reinstatement processes are still inconclusive. Some revealed a general increase of fear reactions, both to the fear (CS+) and the safety stimulus (CS-), whereas other studies discovered a differential return of fear with enhanced fear responses to the CS+ only. Moreover, we know little about reinstatement of contextual anxiety, a state of general anxious apprehension and chronic worry. Therefore, the present study investigated reinstatement of contextual anxiety with an ecological valid virtual reality (VR) design. Additionally, we examined whether the current state anxiety might modulate the reinstatement of contextual anxiety. To this end, two groups underwent context conditioning on Day 1, i.e., one context (CXT+) became paired with unpredictable USs, but not the other context (CXT-), and an extinction training on Day 2. On Day 3 a reinstatement test was conducted, i.e., one group (reinstatement group, n=21) received one unsignaled US before testing, whereas the control group (n=21) did not. Only the reinstatement group showed a differential return of contextual anxiety as measured by fear-potentiated startle and anxiety ratings. Interestingly, the reinstatement of fear-potentiated startle was additionally influenced by state anxiety. Conclusively, an anxious state before an unsignaled aversive event might favor a return of contextual anxiety.