Aversive Unconditioned Stimulus Research Articles

The ventromedial prefrontal cortex in response to threat omission is associated with subsequent explicit safety memory

In order to memorize and discriminate threatening and safe stimuli, the processing of the actual absence of threat seems crucial. Here, we measured brain activity with fMRI in response to both threat conditioned stimuli and their outcomes by combining threat learning with a subsequent memory paradigm. Participants (N = 38) repeatedly saw a variety of faces, half of which (CS+) were associated with an aversive unconditioned stimulus (US) and half of which were not (CS-). When an association was later remembered, the hippocampus had been more active (than when forgotten). However, the ventromedial prefrontal cortex predicted subsequent memory specifically during safe associations (CS- and US omission responses) and the left dorsolateral prefrontal cortex during outcomes in general (US and US omissions). In exploratory analyses of the theoretically important US omission, we found extended involvement of the medial prefrontal cortex and an enhanced functional connectivity to visual and somatosensory cortices, suggesting a possible function in sustaining sensory information for an integration with semantic memory. Activity in visual and somatosensory cortices together with the inferior frontal gyrus also predicted memory performance one week after learning. The findings imply the importance of a close interplay between prefrontal and sensory areas during the processing of safe outcomes—or ‘nothing’—to establish declarative safety memory.

Observational learning of threat-related attentional bias

ABSTRACT Attentional bias to threat has been almost exclusively examined after participants experienced repeated pairings between a conditioned stimulus (CS) and an aversive unconditioned stimulus (US). This study aimed to determine whether threat-related attentional capture can result from observational learning, when participants acquire knowledge of the aversive qualities of a stimulus without themselves experiencing aversive outcomes. Non-clinical young-adult participants (N = 38) first watched a video of an individual (the demonstrator) performing a Pavlovian conditioning task in which one colour was paired with shock (CS+) and another colour was neutral (CS−). They then carried out visual search for a shape-defined target. Oculomotor measures evidenced an attentional bias toward the CS+ colour, suggesting that threat-related attentional capture can ensue from observational learning. Exploratory analyses also revealed that this effect was positively correlated with empathy for the demonstrator. Our findings extend empirical and theoretical knowledge about threat-driven attention and provide valuable insights to better understand the formation of anxiety disorders.

Unconditioned stimulus devaluation decreases the generalization of costly safety behaviors

Safety behaviors are often maladaptive in clinical anxiety as they typically persist without realistic threat and cause various impairments. In the laboratory, safety behaviors are modelled by responses to a conditioned stimulus (CS) that reduce the occurrence of an expected aversive unconditioned stimulus (US). Preliminary evidence suggests that US devaluation, a procedure that decreases US aversiveness, devalues the threat value of the CS and thus diminishes safety behaviors to the CS. This study (n = 78) aimed to extend this finding and examined whether US-devaluation can reduce the generalization of safety behaviors to various stimuli. After acquiring safety behaviors to CSs of different categories, the US predicted by one CS category was devalued. In test, participants showed a selective reduction in safety behaviors to novel stimuli of the devalued CS category, reflecting a decrease in generalization of safety behaviors. Trait anxiety was associated with persistent generalized safety behaviors to novel stimuli of the devalued category. We discuss how US devaluation may improve treatment outcome but also the challenges of clinical translation.

Rapamycin attenuates reconsolidation of a backwards-conditioned aversive stimuli in female mice.

The mammalian target of rapamycin (mTOR) kinase is known to mediate consolidation and reconsolidation of aversive memories. Most studies in this area use a forward conditioning paradigm in which the conditioned stimulus (CS) precedes the unconditioned stimulus (US). Little is known, however, about the neurobiological underpinnings of backwards (BW) conditioning paradigms, particularly in female mice. In BW conditioning, the CS does not become directly associated with the US; it instead evokes conditioned fear by reactivating a memory of the conditioning context and indirectly retrieving a memory of the aversive US. We sought to examine BW conditioned fear memory processes in female mice. First, we examined whether freezing to a BW CS is mediated by fear to the conditioning context. Second, we tested whether blocking consolidation of a BW CS attenuated memory of the CS and conditioning context. Finally, we tested whether blocking reconsolidation of a BW CS attenuated memory of the conditioning context. We show that conditioned freezing to a BW CS is mediated by fear to the conditioning context. Furthermore, rapamycin-an mTOR inhibitor, when given immediately following BW conditioning, impairsconsolidation of both cued and contextual fear memory. Similarly, rapamycin given following retrieval of a BW CS blocks context recall. Rapamycin is acting on reconsolidation as CS retrieval is necessary to see the effects of rapamycin on context memory recall. Our study provides novel evidence that indirect retrieval cues are sensitive to rapamycin in female mice. The capacity to indirectly reactivate memories and render them susceptible to disruption is critical in the translation of reconsolidation-based approaches to the clinic.

Far from the threatening crowd: Generalisation of conditioned threat expectancy and fear in COVID-19 lockdown.

Fear and anxiety are rarely confined to specific stimuli or situations. In fear generalisation, there is a spread of fear responses elicited by physically dissimilar generalisation stimuli (GS) along a continuum between danger and safety. The current study investigated fear generalisation with a novel online task using COVID-19-relevant stimuli (i.e., busy or quiet shopping street/mall scenes) during pandemic lockdown restrictions in the United Kingdom. Participants (N = 50) first completed clinically relevant trait measures before commencing a habituation phase, where two conditioned stimuli (CSs; i.e., a busy or quiet high street/mall scene) were presented. Participants then underwent fear conditioning where one conditioned stimulus (CS+) was followed by an aversive unconditioned stimulus (US; a loud female scream accompanied by a facial photograph of a female displaying a fearful emotion) and another (CS-) was not. In a test phase, six generalisation stimuli were presented where the US was withheld, and participants provided threat expectancy and fear ratings for all stimuli. Following successful conditioning, fear generalization was observed for both threat expectancy and fear ratings. Trait worry partially predicted generalised threat expectancy and COVID-19 fear strongly predicted generalised fear. In conclusion, a generalisation gradient was evident using an online remote generalisation task with images of busy/quiet streets during the pandemic. Worry and fear of COVID-19 predicted fear generalisation.

Excitatory subtypes of the lateral amygdala neurons are differentially involved in regulation of synaptic plasticity and excitation/inhibition balance in aversive learning in mice.

The amygdala plays a crucial role in aversive learning. In Pavlovian fear conditioning, sensory information about an emotionally neutral conditioned stimulus (CS) and an innately aversive unconditioned stimulus is associated with the lateral amygdala (LA), and the CS acquires the ability to elicit conditioned responses. Aversive learning induces synaptic plasticity in LA excitatory neurons from CS pathways, such as the medial geniculate nucleus (MGN) of the thalamus. Although LA excitatory cells have traditionally been classified based on their firing patterns, the relationship between the subtypes and functional properties remains largely unknown. In this study, we classified excitatory cells into two subtypes based on whether the after-depolarized potential (ADP) amplitude is expressed in non-ADP cells and ADP cells. Their electrophysiological properties were significantly different. We examined subtype-specific synaptic plasticity in the MGN-LA pathway following aversive learning using optogenetics and found significant experience-dependent plasticity in feed-forward inhibitory responses in fear-conditioned mice compared with control mice. Following aversive learning, the inhibition/excitation (I/E) balance in ADP cells drastically changed, whereas that in non-ADP cells tended to change in the reverse direction. These results suggest that the two LA subtypes are differentially regulated in relation to synaptic plasticity and I/E balance during aversive learning.

Manipulating reinforcement probability changes the temporal overestimation of anticipated aversive stimuli.

The interval timing can be distorted by emotions. Previous studies indicated that anticipated fear stimuli can lead to temporal overestimation, similar to the effects observed from direct exposure to fear stimuli. However, this time distortion may not always manifest when anticipated. This study aimed to systematically examine the effect of the reinforcement probability of anticipated fear stimuli on time perception in predictable emotional scenarios. The experiment established 100% fear conditioning by associating a conditioned stimulus (CS+) with an aversive unconditioned stimulus (US), electrical stimulation. Participants completed a temporal bisection task under different cues (threat CS+ and safe CS-) expectations. Participants were explicitly informed that an aversive electrical stimulus would always follow the threat cues (CS+) with 100% probability, though in reality, different blocks presented the threat cue with probability manipulation of 50 and 100%. Results showed that only in the 50% reinforcement probability, participants overestimated the duration when anticipating aversive electrical stimulation, while no significant differences were observed in the 100% reinforcement probability. Additionally, the effect of anxiety on temporal judgment failed to capture the overall trend as fixed effects but only contributed to the individual variations as random effects. The findings suggest that the anticipated aversive electrical stimulation may lead to temporal overestimation. Furthermore, the results indicate that a reliable approach for manipulating the effect of anticipated aversive electrical stimulation on temporal overestimation is to establish 100% fear conditioning and use a reinforcement probability like 50%.

The influence of instructions on reversing the generalization of valence, US expectancy, and electrodermal responding in fear conditioning.

Pairing a conditional stimulus (CS) with an aversive unconditional stimulus (US) causes negative valence and US expectancy to generalize to stimuli that are perceptually and/or conceptually similar to the CS. Past research has shown that instructing participants that the US is more likely to follow stimuli that are dissimilar to the CS reversed the generalization of US expectancy but left generalized valence unchanged. Here, we examined whether instructions about the relationship between stimuli that are perceptually similar would affect the generalization of valence. A picture of an alien (CS+) was paired with an electric stimulus, while a perceptually different alien stimulus (CS-) was presented alone. After conditioning, valence, US expectancy, and electrodermal responses generalized to different aliens that were perceptually similar (by color and shape) to the CS+ and CS-. Participants were then instructed that aliens perceptually similar to the CS+ belonged to the same group as the CS- and that aliens perceptually similar to the CS- belonged to the same group as the CS+. The instructions caused an elimination (but not a reversal) of generalized expectancy and valence but did not affect generalized electrodermal responses. This suggests that evaluations of generalization stimuli are sensitive to instructions about their relationship to the CS and that dissociations reported in the literature between valence and expectancy after instructions may occur due to the type of instruction used.

To remember or not to remember: Neural oscillations and ERPs as predictors of intentional associative fear learning

It is widely accepted that impaired safety learning to a safe stimulus is a pathological feature of anxiety disorders. Safety learning refers to learning that a stimulus is associated with the absence of threat. Cognitive mechanisms that underlie successful threat and safety learning are, however, poorly understood. This study aimed to identify various physiological markers, including neural oscillations and event-related potentials (ERPs) that predict successful threat and safety learning. Therefore, to detect potential differences in these markers, we measured EEG in a fear learning framework combined with a subsequent memory paradigm. Thirty-seven participants were asked to memorize a series of associations between faces and an aversive unconditioned stimulus (US) or its omission. We found a decrease of power in the alpha band in occipital brain regions during learning for both threatening (conditioned stimuli, CS+) and safe faces (control stimuli, CS−) that were subsequently remembered to be associated with a US or not. No effects in theta band were found. In regard to ERPs, a late positive potential (LPP) and a P300 component were larger for remembered than for forgotten CS-US associations. The P300 was also enhanced to remembered US and US omissions, thus replicating previous findings. These results point to the importance of cognitive resource allocation as an underlying mechanism of fear learning and electrophysiological measurements as potential biomarkers for successful threat and safety learning.

Effects of Experiencing CS-US Pairings on Instructed Fear Reversal.

Fear learning allows us to identify and anticipate aversive events and adapt our behavior accordingly. This is often thought to rely on associative learning mechanisms where an initially neutral conditioned stimulus (CS) is repeatedly paired with an aversive unconditioned stimulus (US), eventually leading to the CS also being perceived as aversive and threatening. Importantly, however, humans also show verbal fear learning. Namely, they have the ability to change their responses to stimuli rapidly through verbal instructions about CS-US pairings. Past research on the link between experience-based and verbal fear learning indicated that verbal instructions about a reversal of CS-US pairings can fully override the effects of previously experienced CS-US pairings, as measured through fear ratings, skin conductance, and fear-potentiated startle. However, it remains an open question whether such instructions can also annul learned CS representations in the brain. Here, we used a fear reversal paradigm (female and male participants) in conjunction with representational similarity analysis of fMRI data to test whether verbal instructions fully override the effects of experienced CS-US pairings in fear-related brain regions or not. Previous research suggests that only the right amygdala should show lingering representations of previously experienced threat ("pavlovian trace"). Unexpectedly, we found evidence for the residual effect of prior CS-US experience to be much more widespread than anticipated, in the amygdala but also cortical regions like the dorsal anterior cingulate or dorsolateral prefrontal cortex. This finding shines a new light on the interaction of different fear learning mechanisms, at times with unexpected consequences.SIGNIFICANCE STATEMENT Humans are able to learn about aversive stimuli both from experience (i.e., repeated pairings of conditioned stimulus (CS) and unconditioned stimulus (US; pavlovian conditioning), and from verbal instructions about stimulus pairings. Understanding how experience-based and verbal learning processes interact is key for understanding the cognitive and neural underpinnings of fear learning. We tested whether prior aversive experiences (CS-US pairings) affected subsequent verbal learning, searching for lingering threat signals after verbal instructions reversed a CS from being threatening to being safe. While past research suggested such threat signals can only be found in the amygdala, we found evidence to be much more widespread, including the medial and lateral PFC. This highlights how experience-based and verbal learning processes interact to support adaptive behavior.

Npas4-mediated dopaminergic regulation of safety memory consolidation

Amygdala circuitry encodes associations between conditioned stimuli and aversive unconditioned stimuli and also controls fear expression. However, whether and how non-threatening information for unpaired conditioned stimuli (CS-) is discretely processed remains unknown. The fear expression toward CS- is robust immediately after fear conditioning but then becomes negligible after memory consolidation. The synaptic plasticity of the neural pathway from the lateral to the anterior basal amygdala gates the fear expression of CS-, depending upon neuronal PAS domain protein 4 (Npas4)-mediated dopamine receptor D4 (Drd4) synthesis, which is precluded by stress exposure or corticosterone injection. Herein, we show cellular and molecular mechanisms that regulate the non-threatening (safety) memory consolidation, supporting the fear discrimination.

The cerebellum and fear extinction: evidence from rodent and human studies.

The role of the cerebellum in emotional control has gained increasing interest, with studies showing it is involved in fear learning and memory in both humans and rodents. This review will focus on the contributions of the cerebellum to the extinction of learned fear responses. Extinction of fearful memories is critical for adaptive behaviour, and is clinically relevant to anxiety disorders such as post-traumatic stress disorder, in which deficits in extinction processes are thought to occur. We present evidence that supports cerebellar involvement in fear extinction, from rodent studies that investigate molecular mechanisms and functional connectivity with other brain regions of the known fear extinction network, to fMRI studies in humans. This evidence is considered in relation to the theoretical framework that the cerebellum is involved in the formation and updating of internal models of the inner and outer world by detecting errors between predicted and actual outcomes. In the case of fear conditioning, these internal models are thought to predict the occurrence of an aversive unconditioned stimulus (US), and when the aversive US is unexpectedly omitted during extinction learning the cerebellum uses prediction errors to update the internal model. Differences between human and rodent studies are highlighted to help inform future work.

Evaluating the effects of counterconditioning, novelty-facilitated, and standard extinction on the spontaneous recovery of threat expectancy and conditioned stimulus valence.

Extinction training has proved effective to diminish the expectancy of the aversive unconditioned stimulus (US). However, the negative valence of the conditioned stimulus (CS) may still stay intact. In fact, several studies have suggested that the CS negative valence may be a factor that promotes the return of fear. Our study focuses on the role of changes in the CS valence as a potential mechanism to reduce the spontaneous recovery of threat expectancies. To do that, we evaluated counterconditioning (CC), a technique aimed to reduce the CS negative valence by paring it with a positive stimulus and compared its efficacy to that of a novelty-facilitated extinction (NFE) and a standard extinction interventions. Using a 2-day protocol, participants first learned the relationship between a figure and an aversive sound, using a differential conditioning paradigm, and were then randomly assigned to one of three different groups. For the CC group, CS+ or cue A was paired with a positive US. The standard extinction group was exposed to cue A alone. For a third NFE group, cue A was followed by a neutral US. Finally, on the second day, spontaneous recovery was tested. Our findings did not provide evidence to suggest that CC could be more effective to prevent or reduce the return of threat expectancies or influence valence ratings when compared with NFE and standard extinction.

The influence of cross unconditional stimulus reinstatement on electrodermal responding and conditional stimulus valence in differential fear conditioning.

We examined whether the inhibitory Conditional Stimulus (CS)-no Unconditional Stimulus (US) association formed during extinction can be triggered by a novel US during the reinstatement of conditional electrodermal responding and self-reported CS valence in human differential fear conditioning. Participants were trained with either a shock or an aversive scream US before undergoing extinction. Participants then received either the same (i.e., shock_shock or scream_scream) or a different US during reinstatement (i.e., shock_scream, scream_shock). Differential conditioning across all indices was stronger when a shock US was used during acquisition. After reinstatement, electrodermal responding to both the CS+ and the CS- increased regardless of the type of US used during reinstatement (non-differential reinstatement). Differential CS valence evaluations were larger after reinstatement in the groups that received the same US during acquisition and reinstatement (differential reinstatement), but differential evaluations did not increase in the groups receiving a different US at reinstatement. This dissociation suggests that the reinstatement of negative stimulus valence and the reinstatement of expectancy learning may differ.

Effects of phase synchronization and frequency specificity in the encoding of conditioned fear-a web-based fear conditioning study.

Oscillatory synchronization in the theta-frequency band was found to play a causal role in binding information of different modalities in declarative memory. Moreover, there is first evidence from a laboratory study that theta-synchronized (vs. asynchronized) multimodal input in a classical fear conditioning paradigm resulted in better discrimination of a threat-associated stimulus when compared to perceptually similar stimuli never associated with the aversive unconditioned stimulus (US). Effects manifested in affective ratings and ratings of contingency knowledge. However, theta-specificity was not addressed so far. Thus, in the present pre-registered web-based fear conditioning study, we compared synchronized (vs. asynchronized) input in a theta-frequency band vs. the same synchronization manipulation in a delta frequency. Based on our previous laboratory design, five visual gratings of different orientations (25°, 35°, 45°, 55°, 65°) served as conditioned stimuli (CS) with only one (CS+) paired with the auditory aversive US. Both CS and US were luminance or amplitude modulated, respectively, in a theta (4 Hz) or delta (1.7 Hz) frequency. In both frequencies, CS-US pairings were presented either in-phase (0° phase lag) or out-of-phase (90°, 180°, 270°), resulting in four independent groups (each group N = 40). Phase synchronization augmented the discrimination of CSs in CS-US contingency knowledge but did not affect valence and arousal ratings. Interestingly, this effect occurred independent of frequency. In sum, the current study proves the ability to successfully conduct complex generalization fear conditioning in an online setting. Based on this prerequisite, our data supports a causal role of phase synchronization in the declarative CS-US associations for low frequencies rather than in the specific theta-frequency band.

Hippocampus and neocortex send and receive predictive information.

A recent paper published in Nature by Yadav and colleagues (Nature 608 (7921): 153-160, 2022) illustrates a fundamental aspect on how different brain areas participate in memory storage and retrieval. After identifying neuron activity in the hippocampus CA1 region specific to multi-modal stimuli that predicted appetitive and aversive unconditioned stimuli (conjunctive stimulus encoding), the authors showed that neurons located in the anterior cingulate cortex dynamically encode the discrete sensory features of the outcome-predictive stimuli during training, and these highly specific feature-based projections can excite or inhibit conjunctive-coding neurons in the hippocampus during retrieval.

Age- and sex-specific effects of maternal separation on the acoustic startle reflex in rats: early baseline enhancement in females and blunted response to ambiguous threat.

Early life adversity (ELA) increases the incidence of later-life anxiety disorders. Dysregulated threat processing, including responsivity to ambiguous threats, is an indicator of anxiety disorders and can be influenced by childhood experiences. The acoustic startle response is a defensive reflex displayed by mammals when exposed to sudden intense stimuli reflecting individual variations in vigilance. These measures can be altered by previous experience and experimental modifications, including the introduction of unconditioned aversive stimuli. Rats emit ultrasonic vocalizations (USVs) in the 22 KHz range in negative contexts. As such, 22 KHz USVs are an ethologically relevant social cue of environmental threat shown to induce anxiety-like behavior in recipient rats. Because the timing of symptom manifestation after early life adversity can differ between sexes, the current study sought to identify the age- and sex-specific effects of daily maternal separation (MS) on responsivity to ambiguous threat in rats. In Experiment 1, rat pups underwent MS or control rearing from postnatal day (P) 2-20, then underwent behavioral testing beginning on P24, 34, or 54 to determine whether MS modified the baseline startle response or the modulation of startle by 22 KHz USVs. In Experiment 2, rats were tested in a light-enhanced startle paradigm at P54 after MS or control rearing to determine whether MS influenced light-enhanced startle. Results show an enhancement of the baseline startle magnitude by MS in females at P34. At P54, MS reduced the modulation of the startle response by 22 KHz USVs and prevented light-enhanced startle, indicating an MS-induced deficit in defensive responsivity when exposed to potential threat.

Fear learning and extinction predicts anxiety in daily life: a study of Pavlovian conditioning and ecological momentary assessment.

The association between anxious mood and aberrant fear learning mechanisms has not been fully elucidated. Studying how fear conditioning and extinction constructs relate to anxiety symptoms and reactivity to stressful and benign moments in everyday life provides a powerful addition to experimental paradigms. Fifty-one young adults completed laboratory-based differential conditioning and extinction tasks with (CS + ) and without (CS-) an aversive unconditional stimulus (US). Electrodermal skin conductance responses were measured during each phase, followed by ecological momentary assessment (EMA) tapping anxiety and stressors six times daily for seven days (2, 142 moments). Conditioned electrodermal reactivity to the CS + and overgeneralisation to the CS- were associated with greater change in anxiety (measured via EMA), across non-stressful situations, remaining the same across stressful situations. Likewise, during extinction when the CS + is now safe, more electrodermal reactivity to the CS + was associated with more anxiety change across non-stressful situations and remained the same across stressful situations. Also, during extinction when threat is absent, more electrodermal reactivity at the late stage of the CS- was associated with less momentary anxiety change in response to stressful situations; more electrodermal activity at the late stage of the CS + was associated with more anxiety change across non-stressful situations and remained the same across stressful situations. Sampling 'in vivo' emotion and stress experiences, study findings revealed links between conditioned electrodermal reactivity and overgeneralisation to safe stimuli and heightened anxious reactivity during non-stressful (i.e. safe) moments in daily life, coupled with less change in response to actual stressors.

Prefrontal projections to the nucleus reuniens signal behavioral relevance of stimuli during associative learning

The nucleus reuniens (RE) is necessary for memories dependent on the interaction between the medial prefrontal cortex (mPFC) and hippocampus (HPC). One example is trace eyeblink conditioning, in which the mPFC exhibits differential activity to neutral conditioned stimuli (CS) depending on their contingency with an aversive unconditioned stimulus (US). To test if this relevancy signal is routed to the RE, we photometrically recorded mPFC axon terminals within the RE and tracked their changes with learning. As a comparison, we measured prefrontal terminal activity in the mediodorsal thalamus (MD), which lacks connectivity with the HPC. In naïve male rats, prefrontal terminals within the RE were not strongly activated by tone or light. As the rats associated one of the stimuli (CS+) with the US, terminals gradually increased their response to the CS+ but not the other stimulus (CS-). In contrast, stimulus-evoked responses of prefrontal terminals within the MD were strong even before conditioning. They also became augmented only to the CS+ in the first conditioning session; however, the degree of activity differentiation did not improve with learning. These findings suggest that associative learning selectively increased mPFC output to the RE, signaling the behavioral relevance of sensory stimuli.

Intolerance of uncertainty predicts altered threat expectancy ratings during reinstatement

Intolerance of uncertainty (IU) is a transdiagnostic risk factor for psychiatric disorders, and plays an important role in fear and threat learning under uncertainty. The ‘reinstatement of fear/threat’ is an understudied phenomenon thought to represent clinical symptom relapse. Reinstatement of conditioned responding can be captured in the laboratory by presenting unsignaled presentations of an aversive unconditional stimulus. The present study investigated IU as a predictor of reinstatement effects, such that individuals higher in IU, relative to lower IU, would show larger reinstatement. Sixty-two participants completed the Intolerance of Uncertainty Scale and provided threat expectancy ratings (i.e. certainty of receiving a shock) during a differential threat conditioning and extinction paradigm with reinstatement. Findings suggested a differential increase in threat expectancy ratings to both the threat and safety cue following reinstatement, although this effect was small and did not survive follow-up tests. Nevertheless, IU was a significant predictor of reinstatement to the threat cue but not the safety cue, although this effect was not in the expected direction. Specifically, higher IU was associated with reduced threat expectancy ratings post-reinstatement. These findings provide support for the limited literature demonstrating an important role for IU in reinstatement effects and should be investigated further.