Are fear memories erasable? An attempt to extend the reconsolidation paradigm in humans

Transcription

1 Institutionen för klinisk neurovetenskap Psykologprogrammet, termin 6 Huvudämne: Psykologi Examensarbete (C-nivå) i psykologi (2PS013), 15 poäng Vårterminen 2010 Are fear memories erasable? An attempt to extend the reconsolidation paradigm in humans Författare: Martin Bellander Handledare: Armita Golkar, Institution för klinisk neurovetenskap Andreas Olsson, Institution för klinisk neurovetenskap Examinator: Professor Petter Gustavsson, Institutionen för klinisk neurovetenskap

2 Institutionen för klinisk neurovetenskap Psykologprogrammet, termin 6 Huvudämne: Psykologi Examensarbete (C-nivå) i psykologi (2PS013), 15 poäng Vårterminen 2010 Are fear memories erasable? An attempt to extend the reconsolidation paradigm in humans Sammanfattning/ abstract Nya fynd i experimentella paradigm för rädslobetingning har gett hopp om en förbättrad behandling av ångestsjukdomar. Studier har visat att genom att aktivera ett redan konsoliderat rädslominne blir det instabilt, och det är då möjligt att ändra eller radera minnet genom en intervention (t.ex. utsläckning), innan minnet åter blir stabilt. Denna studie syftade till att utvidga dessa resultat genom användandet av rädslorelevanta stimuli och en kombination av två mått på rädsla: hudkonduktansresponsen och den rädslopotentierade startle-reflexen. Undersökningsdeltagarna genomgick betingning, utsläckning och s.k. reinstatement under tre på varandra följande dagar, där ett av två förstärkta betingade stimulus reaktiverades 10 minuter före utsläckning. Undersökningsdeltagarna visade betingning och utsläckning i båda rädslomåtten, men ingen effekt konstaterades för reaktivering. Resultatet står i motsats till tidigare studier och de nya stimuli som användes kan vara en förklaring till detta. Uppföljande studier behövs för att reda ut de nödvändiga villkoren för en reaktiveringseffekt. Nyckelord: Klassisk rädslobetingning, hudkonduktans respons, rädslopotentierad startle, rädslorelevant stimuli, rekonsolidering Recent findings in experimental fear conditioning paradigms have given hope of an improved treatment of anxiety disorders. It has been shown that reactivating an already consolidated fear memory renders it instable and it is thereby possible to alter or erase the memory through an intervention (e.g. extinction), before the memory once again becomes stable. The present study aimed at extending these findings by using fear relevant stimuli and the combination of two measures of fear: the skin conductance response and the fear potentiated startle reflex. Participants went through conditioning, extinction and reinstatement on three consecutive days, with one of two reinforced conditioned stimuli being reactivated 10 minutes prior to extinction. Successful conditioning and extinction in both measures were achieved, but no effect was found for the reactivation. The result stands in contrast to previous findings, and the use of fear-relevant stimuli might account for this discrepancy. Follow up studies are needed to disentangle the necessary conditions for the reactivation effect. Keywords: classical fear conditioning, fear potentiated startle, fear relevant stimuli, reconsolidation, skin conductance response

3 Are fear memories erasable? An attempt to extend the reconsolidation paradigm in humans Martin Bellander Anxiety disorders affect a large number of people and cause both suffering and hindrances in daily chores. According to Kessler et al. (2005), 28.8% of the US population will sometime during their lifetime meet the criteria for an anxiety disorder. The corresponding percentage in the Netherlands is 19.3% (Bijl, Ravelli, & Van Zessen, 1998). A similar estimate has also been reported in a Norwegian sample (Kringlen, Torgersen, & Cramer, 2001). Thus, anxiety disorders are a serious problem both on the individual and on the societal level, with economic cost estimates as high as million US-$-PPP per 1 million inhabitants (Konnopka, Leichsenring, Leibing, & König, 2009), and these disorders are a great challenge for researchers and clinicians alike. It is therefore not surprising that a great deal of research has been put into developing an experimental model for these disorders. One of the models most favoured by the scientific community is based on classical fear conditioning (Anderson & Insel, 2006; Mineka & Zinbarg, 2006). Through this model, researchers hope to gain knowledge of the processes underlying anxiety disorders and their aetiology. Classical fear conditioning is the process by which a neutral stimulus is repeatedly paired with a naturally aversive stimulus (unconditioned stimulus, US) capable of eliciting a fear response (unconditioned response, UR), so that the previously neutral stimulus (now a conditioned stimulus, CS) acquires the ability to elicit a fear response (conditioned response, CR). The process of pairing the CS with the US until a CR is elicited by the CS alone is called acquisition. The acquisition of a conditioned fear response is dependent on the integrity of the amygdala, a subcortical brain structure consisting of a number of nuclei in the medial temporal lobe. This has been shown mainly in non-human animals through lesion studies and pharmacological interference with protein synthesis (LeDoux, 2003). Human evidence derives from studies of patients with lesions, and to some extent from functional Magnetic Resonance Imaging (fmri), (LeDoux, 2000). Sensory input reaches the lateral nucleus (LA) of the amygdala, both directly from thalamus, but also via different sensory cortices. The LA in turn, projects to basal (B), accessory basal (AB), and central nuclei (CE). B and BA have connections to both CE and hippocampus. The CE projects to areas controlling physiological responses, e.g. blood pressure, heart rate, hormone release, and freezing behaviour (LeDoux, 2000). As LA is the location of CS-US input convergence, LA has been suggested to be the substrate of conditioning (LeDoux, 2003). Conditioned fear is commonly viewed as an important mechanism in the aetiology of anxiety disorders. In the treatment of these disorders, cognitive behavioural therapy has shown to be effective. An important part of this treatment is exposure therapy, where the patient s fear is reduced by systematically approaching the feared object, situation or thought under controlled circumstances, i.e. in the absence of negative outcome. The analogue of exposure therapy in the experimental classical fear conditioning paradigm is the process called extinction. This is the process by which, following acquisition, exposure to the CS alone repeatedly reduces and eventually extinguishes the CR. If this analogy holds, understanding and facilitating experimental extinction could have major implications for the improvement of exposure therapy. Less is known about the neural underpinnings of extinction learning, but some preliminaries can be stated. Extinction also seems to involve amygdala, and in addition probably also recruits medial prefrontal cortex, which has inhibitory connections to the amygdala (Delgado, Olsson, & Phelps, 2006; Myers & Davis, 2007).

4 2 Extinction is a somewhat misleading name for this process, as the acquired fear memory 1 is not erased. Rather, extinction is viewed as a new learning process which results in a second, inhibitory memory, opposite to the original fear memory. That the initially acquired fear memory is not really erased by extinction is evident from the fact that the initial fear response can reappear, a process known as recovery. The recovery of the CR can be attained in different ways. One is simply to let time pass (spontaneous recovery). A second form of recovery is dependent on the fact that extinction is context specific, that is, if subjects are extinguished in a context different from the one in which they acquired the fear, a retention test in the original acquisition context, but not the extinction context, will elicit the CR (renewal). The CR can also reappear if, after extinction, the US are presented alone followed by a retention test (reinstatement) (Myers & Davis, 2002, 2007). The inhibitory learning account of extinction has recently been challenged (Myers, Ressler, & Davis, 2006). These authors suggest that extinction immediately following acquisition was able to erase the original fear memory, whereas delayed extinction did not. This view is based on results from studies in rat where immediate, but not delayed, extinction resulted in no recovery of fear (neither as reinstatement, renewal or spontaneous recovery). These results can be understood in relation to the theoretical framework of memory consolidation. The consolidation of a memory is the time dependent process by which a new instable memory trace becomes stable and lasting as time passes. The mechanism of consolidation is well understood at the synaptic level, and it has been noted in all studied species and memory systems when long-term memory is the product (Dudai, 2004). If a memory is fragile when new, then immediate extinction of a conditioned response may be able to erase the memory altogether. The results of Myers et al. (2006) would then be explained by the fact that in immediate extinction, the memory has not yet consolidated and is still fragile. But this view has been disputed by others (Schiller et al., 2008). In recent years there has been a renewed interest in the field of reconsolidation (Dudai, 2006). Reconsolidation is the process by which an already consolidated and stable memory, on being activated by a reminder, again becomes fragile and susceptible to interference for a period (the reconsolidation window). Some authors claims that the memory undergoes a new consolidation process before becoming stable again (Hartley & Phelps, 2009), while others interpret data more cautiously (Dudai, 2006). Reconsolidation studies often consist of three phases, separated in time. First, there is a conditioning phase. Second, when enough time has passed for the conditioning memory to be consolidated, a reminder of the conditioned memory is presented (usually a presentation of the CS), followed by an intervention (or no intervention for the control group) aiming at altering the now reactivated memory trace (this can be an extinction phase, a systemic drug administration, or an infusion of a drug to target a specific neural structure, e.g. the amygdala). Third, a recovery test is administered, where the effect of the manipulation is measured. Nader, Schafer and LeDoux (2000) showed that the reconsolidation process is dependent on new protein synthesis. In this study, rats were conditioned and the next day a reminder was presented (the CS) directly after which a protein synthesis inhibitor (anisomycin) was infused into the lateral and basal nuclei of the amygdala (LBA). This infusion greatly reduced fear response (percent freezing) at the retention test. The same protocol but without the reminder administered to a control group did not result in a reduction 1 The reader should note the use of the word memory in the conditioning literature. Although the term will here be used naïvely in accordance with this literature to avoid confusion, in the proper sense of the word, conditioned responses are not memories, as one need not retain any knowledge in order to have acquired one. Exactly how to interpret the term in this literature is problematic and outside the scope of the current essay, but see Bennett and Hacker (2003) for a clarifying analysis.

5 3 of the fear response. Neither was the fear response reduced when the anisomycin infusion was postponed for six hours. A later study (Duvarci & Nader, 2004) demonstrated that the fear memory does not show any spontaneous recovery (24 days), renewal (context change) or reinstatement. Furthermore, a systemic injection of the β-adrenergic receptor antagonist propranolol had the same reconsolidation blocking effect as anisomycin infusion into the LBA (Debiec & LeDoux, 2004). The reactivated memory can also be disrupted by extinction training (Monfils, Cowansage, Klann, & LeDoux, 2009). This study, analogous to the above (Duvarci & Nader, 2004; Nader et al., 2000) except that drug infusion was replaced by extinction training, showed that if extinction training took place within the reconsolidation window, the fear response showed less spontaneous recovery, renewal and reinstatement, compared with a non-reminded control group. Reconsolidation of fear memories has mainly been studied in animals, but a limited number of studies in humans exist. Following the animal results of Debiec and LeDoux (2004), Kindt, Soeter and Vervliet (2009) used a between subject design and showed that the administration of propranolol before a single reminder presentation of the reinforced CS reduced the fear response (as measured by the fear potentiated startle reflex, FPS) at a later retrieval test, compared to only reactivation or only propranolol administration. The results were interpreted as evidence that administration of propranolol before memory reactivation disrupted the reconsolidation process of the fear memory, not allowing it to consolidate, and thereby interfering with it. A recent study by Schiller et al. (2010) showed an effect of extinction training in the reconsolidation window in humans. Here the reminded group showed both less spontaneous recovery and less reinstatement a year later. In an additional experiment, Schiller et al. used a within subject design where two stimuli were paired with an electric shock, while a third was not. Twenty-four hours later, only one reinforced CS and the unreinforced CS was reactivated 10 minutes prior to extinction. An additional 24 hours later a reinstatement test showed a reduced response (as measured by the skin conductance response, SCR) to the reactivated CS compared to the non-reactivated. This is taken to mean that a reactivation of the fear memory prior to extinction renders it labile and therefore able to be erased or updated by extinction training. If extinction following the reactivation of a fear memory can not only facilitate a new learning but also cause an erasure of the fear memory, the clinical implications for treatment of anxiety disorders could be profound. The wished-for analogue of these experimental results in the treatment of disorders would be a more efficient exposure therapy with more stable results and less relapses. The theory has yet to be properly tested in the clinical setting, but a study by Brunet et al. (2008) gave some preliminary support. In this study, PTSD-patients were administered propranolol or placebo during mental imagery of a past traumatic event. One week later the patients again engaged in mental imagery of the event, while physiological measures were recorded. The propranolol group showed lower SCRs and lower heart rates than the placebo group. The findings were discussed in terms of reconsolidation effects. However, the study lacked a control group receiving propranolol without reactivating the traumatic memory. Well-designed clinical studies are still needed to confirm the usefulness of the reconsolidation theory for treatment. The purpose of the present study was to extend the previous fear conditioning reconsolidation findings on humans (Schiller et al., 2010) to fear relevant stimuli (namely pictures of faces with fearful facial expressions) and to use a more direct measure of fear response, the FPS reflex. The difference between fear relevant and non-relevant stimuli in conditioning paradigms is well documented; responses to fear relevant stimuli are both easier to condition and harder to extinguish (Öhman & Mineka, 2001). In addition, the objects of phobias are more often fear relevant than non-fear relevant (Öhman & Mineka, 2001). Furthermore, fearful faces activate amygdala even more than do angry faces, a possible

6 4 explanation being that they are more ambiguous in their signal of threat, as the source of the danger might not be known (Whalen et al., 2001). For a well formulated theory of reconsolidation it is important that the results generalize to different types of stimuli, especially fear relevant ones, as these stimuli have a closer resemblance to the objects people usually acquire fears to (i.e. they are more ecologically valid). In the context of fear learning, previous research has established a dissociation between fear potentiated startle and skin conductance responses. Thus, whereas fear potentiated startle was shown to reflect an affective level of learning, skin conductance responses relied more heavily on cognitive awareness of stimulus contingencies (Hamm & Vaitl, 1996; Hamm & Weike, 2005). Neurally, the FPS involves both direct and indirect connections from the amygdala to the brainstem (Davis & Whalen, 2001) and is a validated measure of conditioned fear across species. 2 Method Participants Nineteen participants, age (M = 27.2, SD = 9.55) were recruited through poster advertising on Karolinska Institutet campus. All participants gave written consent for their participation and were given three cinema tickets. Due to technical problems, one participant was excluded from the final SCR analysis and three from the FPS analysis. Stimulus Material Three different pictures depicting fearful male faces from the Karolinska Directed Emotional Faces (Lundqvist, Flykt, & Öhman, 1998) served as CSs. For each picture, the background was removed and colour was converted to grey-scale. A white fixation cross was shown on a black background during the inter-trial intervals (ITIs), the duration of which varied between 10 and 18 s (M = 14) throughout all experimental sessions. The experiment was run in a sound-attenuated chamber on a desktop PC with a standard 21-inch cathode ray tube (CRT) monitor. Screen resolution was 800 x 600 pixels and the refresh rate was set to 60 Hz. The experiment was programmed in Presentation 13.1 (Neurobehavioral Systems, Participants viewed pictures at a distance of about 1 m. The US was a monopolar DC-pulse electric stimulation (STM200; Biopac Systems Inc, applied to the participant s right wrist. Startle probes were 50-ms bursts of 95-dB[A] white noise with a near instantaneous rise time (<1 ms). Startle probes were presented binaurally through headphones (Sennheiser HD202). Measurements and Recordings The eyeblink component of the startle response was measured through electromyographic (EMG) recordings of the left orbicularis oculi muscle using two miniature Ag/AgCl electrodes prepared with electrolyte gel. A third ground electrode was placed behind the left ear over the mastoid. The raw EMG signal was amplified and filtered through a Hz bandpass, which was rectified and integrated with a time constant of 20 ms. The skin conductance response (SCR) was assessed using two Ag/AgCl electrodes connected to the index and middle finger of the left hand. The raw signal was low pass 2 A point relating to the measurement of fear needs to be made here. One should not take for granted that by measuring the intensity of the emotional perturbations, one is measuring the emotion itself. This is simply assumed here and in much of the conditioning literature, and is something the reader should keep in mind. See Hacker (2004) and Bennett and Hacker (2005) for useful discussions of this matter.

7 5 filtered with a cut off frequency of 1 Hz, the number of coefficients being The signal was then converted from DC to AC using a high pass filter with frequency set to Procedure The experiment used a within subject design with one unreinforced CS (CS-) and two reinforced CSs (CS+), that co-terminated with a shock. The experiment was divided into three sessions: acquisition (ACQ), extinction (EXT) and reinstatement (REI), taking place on three consecutive days. Each session started with a presentation of 6 noise-alone trials to habituate to the startle sound. The shock was adjusted individually for each participant by starting on a low level and gradually increasing the intensity up to a level they found uncomfortable but not painful. Before the ACQ started, participants were told that two of the faces sometimes could give the participant a shock, while one face would never give them a shock. They were also instructed to pay attention to the relation between the faces and the shock. The ACQ session started with a habituation phase where the three CSs were shown twice each without reinforcement, whereof one of each was startled. During ACQ each CS was presented twelve times, and for the two CS+s 50% of the presentations were followed by an electric shock. After acquisition, participants were asked to try to remember the following day what they had learnt during the session. The EXT session began with one CS+ being reminded (CSa+), that is, presented once without reinforcement or startle sound (the non reminded CS+ are referred to as CSb+). After the reminder there was a 10 minute pause, during which participants watched a video clip. After the pause the extinction took place by unreinforced presentation of all the CSs 13 times each (12 times for CSa+). The REI session began with 4 unsignaled shocks during the presentation of a black screen. After this there was a 10 minute pause, just as in the previous session. The pause was followed by a re-extinction phase where each CS was presented 9 times unreinforced. The experimental design is illustrated in Figure 1. Stimuli were presented in a pseudo randomized order, with the criterion that there could be no more than two trials of the same CS in a row. In each trial the CS was presented for 6 seconds, the startle probe after 4-5 seconds following CS onset, and on reinforced trials the shock was administered 5.5 seconds into the CS presentation (see figure 2). Startle probes were presented on 75% of the trials of each CS and the same number of ITIs, throughout the whole experiment. The same trial order was used for all participants, but which CS+ was being reactivated was balanced over participants to avoid an effect of stimulus presentation order. Figure 1. Schematic display of the study design.

8 6 Figure 2. The course of events during a stimulus trial. Statistical Analyses As the magnitude of the FPS differs greatly between participants, t-standardization (M = 50, SD = 10) was used to ensure equal contribution of all participants in the analyses. The standardization was performed separately for each participant over all recorded values. To reduce noise, for each CS and the ITI, averages for the acquisition data were computed for early, middle, and late acquisition, respectively (each consisting of three trials). The same was done for extinction, but with only early and late phase (consisting of the first three and last three trials respectively). To be able to analyze CS differentiation, difference scores were calculated, taking the averages for each phase and subtracting the corresponding ITI average. The same was done for the last extinction trial alone, and the first reinstatement trial alone. The SCR were square root transformed to reduce the skewness of their distribution, and then normalized by dividing each participant s responses by their maximum response to the shock. Just like for the FPS, SCR data from the ACQ and the EXT were blocked into early and late phase (each consisting of three trials). Data were analyzed using t-tests and repeated measure ANOVAs. Results SCR Acquisition and extinction were analyzed using a 2-way repeated measures ANOVA, Stimulus(CSa+, CSb+, CS-) x Time(early, late). For acquisition there were significant main effects of Time (F(1, 17) = 51.6, p <.01) as well as Stimulus (F(2, 34) = 12.8, p <.01). There was no significant interaction. For extinction there was a significant main effect of Time (F(1, 17) = 48.2, p <.01) but not of Stimulus (F(2, 34) = 1.4, n.s.). There was no significant interaction. Follow-up t-tests were performed to assess acquisition and extinction and that there were no differences between the CS+s before reinstatement. Acquisition did occur, as there were significant differences between the CS- and the CSa+ (t(17) = -4.1, p <.01) and the CS- and the CSb+ (t(17) = -3.8, p <.01) during the last three trials of the acquisition phase. Extinction training was successful as there were no significant differences between the CS- and the CSa+ (t(17) = -.3, n.s.) or the CS- and the CSb+ (t(17) = -.3, n.s.) during the last three trials of the extinction phase. Also, the CSa+ and the CSb+ did not differ significantly after either acquisition (t(17) =.9, n.s.) or extinction (t(17) =.0, n.s.).

9 7 The changes in fear response from acquisition to extinction were examined with a t-test, showing a significant decrease in the CSb+ response (t(17) = 2.4, p =.03), a trend for CSa+ (t(17) = 1.8, p =.08), but no significant change in the CS- response (t(17) = -.2, n.s.). Finally, the effect of the reactivation was examined by comparing the last trial of the extinction phase with the first trial of the reinstatement phase with a t-test. There were significant increases for all three CSs, CSa+ (t(17) = -3.6, p <.01), CSb+ (t(15) = -4.3, p <.01), CS- (t(16) = -3.2, p <.01). This implies that even though the CSa+ was reactivated, participants still showed a reinstated fear response to it, indicating a failure to interfere with the fear memory during the reconsolidation period. The SCR data are presented in Figure 3. Startle Acquisition and extinction were analyzed using a 2-way repeated measures ANOVA, for acquisition Stimulus(CSa+, CSb+, CS-) x Time(early, middle, late), and for extinction Stimulus(CSa+, CSb+, CS-) x Time(early, late). For acquisition there was no significant main effect for Time (F(1, 14) = 3.1, n.s.) but a significant one for Stimuli (F(2, 28) = 9.8, p <.01); there was no significant interaction between Time and Stimuli (F(2, 28) = 1.4, n.s.). For extinction there were significant main effects for Time (F(2, 30) = 57.5, p <.01) and Stimuli (F(1, 15) = 4.4, p <.05), but no significant interaction (F(2, 30) = 2.0, n.s.). Follow-up tests were performed to assess acquisition and extinction and that there were no differences between the CS+s in the phases before reinstatement. Simple contrasts revealed that the main effect of Stimuli was driven by a significant difference between the CS+s and the CS- (F(1, 14) = 15.3, p <.01), and that there was no significant difference between the CS+s (F(1, 14) = 1.8, n.s), implying that acquisition was successful. Participants showed extinction as there were no significant differences between the CS- and the CSa+ (t(15) = -1.4, n.s.) or the CS- and the CSb+ (t(15) = -1.0, n.s.) during the last three trials of the extinction phase. Also, the CSa+ and the CSb+ did not differ significantly after either acquisition (t(14) =.7, n.s.) or extinction (t(15) = -.4, n.s.). The changes in fear response from acquisition to extinction were checked with a t-test, showing a significant decrease in the response to the CSa+ (t(14) = 2.5, p <.05) and the CSb+ (t(14) = 2.7, p <.05), but no significant change in the CS- response (t(14) = 1.8, n.s.). Finally, the effect of the reactivation was examined by comparing the last trial of the extinction phase with the first trial of the re-extinction phase. There was a significant increase only for the CSa+ (t(9) = -2.3, p <.05), and not for the CSb+ (t(10) = -1.9, n.s.) or the CS- (t(10) = -1.5, n.s.). Thus, the increase in fear response from extinction to re-extinction testing was only significant for the CS+ that was reactivated. The FPS data are presented in Figure 4.

10 8 Figure 3. Trial by trial means of the SCR for the three phases of the experiment. Error bars represent standard errors. Figure 4. Trial by trial means of the FPS for the three phases of the experiment. Error bars represent standard errors. Discussion The aim of the present study was to extend current knowledge of the workings of reconsolidation of fear memories in humans. This was done by using fear relevant stimuli, depicting fearful faces, and using both the SCR and the FPS as measurements. Participants showed successful conditioning, extinction on both SCR and FPS. However, there was no effect of the reminder on reinstatement; that is, the reactivation of the CSa+ did not result in a reduced fear response. Rather, for the SCR all CSs showed a reinstatement, whereas for FPS, the reinstatement effect was small and only significant for the CSa+. Although only the CSa+ had a significant increase in the reinstatement test for FPS, the CSb+ was just above conventional significance. Moreover, as evident from Figure 4, only on the first re-extinction trial was the CSa+ higher than CSb+; on the second trial the order was reversed, so one should be cautious when interpreting these results. The reason for small or no

11 9 reinstatement effects in the FPS are partly due to the high ITI responses in the first reinstatement trial (see Figure 4), as the difference scores were used for these analyses. The results of the present study accordingly differ from those of Schiller et al. (2010), who found a significantly lower reinstatement for the reactivated CS+. This is probably not a power problem, as the study by Schiller et al. had 18 participants, while this study had 18 participants in the SCR and 16 in the PFS analysis. On the other hand, Schiller et al. used an exclusion criterion in their analyses, excluding all participants who either did not acquire a conditioned response (CS- > CS+, for any CS+ during the second half of acquisition) or did not extinguish the acquired response (any CS+ - CS- >0.1 μs during the second half of extinction). Therefore one might suspect that this could be the reason for the failure to replicate. However, when these exclusion criteria were applied to the present study, 8 of the participants were excluded, but this did not alter the results of the analyses. There are some obvious differences between the studies that might explain the difference in outcome. The aim of this study was to extend the reconsolidation results of previous work to a new stimulus category, that is, fear relevant stimuli. Therefore a possible cause of the failure to replicate could be the difference in stimulus material. This could also be seen in the light of previously demonstrated differences in conditioning properties between fear relevant and non-relevant stimuli (Öhman & Mineka, 2001). Nevertheless, it is probably not reasonable to ascribe the failure to replicate to the difference in stimuli, as others have found successful interference with reconsolidation using other fear relevant stimuli (Kindt et al., 2009), although propranolol was used instead of extinction training in that study. Furthermore, a pilot study in our laboratory has shown similar results to those presented here, but with non-fear relevant stimuli. This pilot study used exactly the same procedure as in this study, but the face stimuli were replaced by coloured squares. The reinstatement difference was still absent, although this sample was too small to perform statistical analyses on (data not shown). Another possible explanation for the non-replication in the SCR might be the simultaneous measurement of FPS used. The SCR is probably influenced by the use of FPS because the startle probe is both aversive and indicative of the time left to the US, if the trial is to be reinforced. There is a marked SCR to the startle probe, and participants often report the sound as aversive. One should therefore not exclude the possibility that the SCR to the startle sound is interfering with the SCR to the stimulus, as they are adjacent in time. This, however, does not explain why the reactivation effect was absent, or even reversed in the FPS measure. As Schiller et al. (2010) did not use FPS, one cannot be sure if one should expect an effect of reactivation in the FPS measure or not. On the other hand, Kindt et al. (2009) did use FPS and had clear effects, although they used a between subject design and a systemic drug administration to interfere with the reactivated memory. The present study s design is closer to that of Schiller et al. and it is not feasible to a priori assume that there should be congruence in the results between the present study design and that of Kindt et al. The deviant result in this study points to a general problem in experimental psychology, namely the difficulties in replicating findings across labs. The non-replication is to some extent informative of how robust the reconsolidation effect is, as the experimental design of this study closely followed that of Schiller et al (2010). A more open scientific community, where protocol and data are openly shared, would make replication easier and facilitate the process of finding confounding differences between experiments. It would also help in understanding to what extent findings can be generalized; a small difference in protocol should not result in a deviant result if the effect is robust enough to be interesting. To determine exactly what experimental conditions are necessary for obtaining an effect of reactivation, follow up studies are needed. For example, adding a group using non fear

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