480 Learning and memory

Transcranial direct current stimulation of the prefrontal cortex: a means to modulate fear memories Aditya Mungeea,b, Philipp Kazzera,b, Melanie Feeserb, Michael A. Nitsched, Daniela Schillere and Malek Bajbouja,b,c Targeting memory processes by noninvasive interventions is a potential gateway to modulate fear memories as shown by animal and human studies in recent years. Modulation of fear memories by noninvasive brain stimulation techniques might be an attractive approach, which, however, has not been examined so far. We investigated the effect of transcranial direct current stimulation (tDCS) applied to the right dorsolateral prefrontal cortex and left supraorbital region on fear memories in humans. Seventy-four young, healthy individuals were assigned randomly to two groups, which underwent fear conditioning with mild electric stimuli paired with a visual stimulus. Twenty-four hours later, both groups were shown a reminder of the conditioned fearful stimulus. Shortly thereafter, they received either tDCS (right prefrontal – anodal, left supraorbital – cathodal) for 20 min at 1 mA current intensity or sham stimulation. A day later, fear responses of both groups were compared by monitoring skin conductance. On day 3, during fear response assessment, the tDCS group had a significantly (P < 0.05) higher mean skin conductance in comparison

with the sham group. These results suggest that tDCS (right prefrontal – anodal, left supraorbital – cathodal) enhanced fear memories, possibly by influencing the prefrontal cortex–amygdala circuit underlying the memory c 2014 Wolters Kluwer for fear. NeuroReport 25:480–484 Health | Lippincott Williams & Wilkins.

Introduction

enhances or reduces spontaneous cortical activity and excitability, and the resulting behavioural effects depend not only on this primary area of stimulation but also on other connected or distant areas [4–6]. Anodal tDCS results in depolarization of neurons, leading to an excitatory effect, whereas cathodal tDCS results in hyperpolarization, and thus inhibition of cortical neurons [7]. On the basis of these antagonistic effects on cortex physiology, effects have been shown on various forms of memory and learning. The aim of our study was to study the effects of prefrontal tDCS as a potential means to modulate conditioned fear memories. With respect to recall of fear memories, we hypothesized that anodal tDCS of the right dorsolateral prefrontal cortex (DLPFC) could induce and enhance facilitatory plasticity in the cortex, which in turn should result in a stronger fear memory trace as compared with sham stimulation, We chose to stimulate the right DLPFC with anodal tDCS because the valence lateralization theory proposes that the right prefrontal cortex is activated during negative affect [8]. The cathodal reference electrode was positioned over the left supraorbital area, close to the ventromedial prefrontal cortex (vmPFC), which also plays a major role in the neurocircuitry of fear, and possibly has an inverse relationship with the amygdala [9]. Disruption of these inhibitory connections from the vmPFC to the

Fear and the process of acquiring fear serve as an important adaptive function necessary for survival under adverse circumstances. When learned fear responses become maladaptive, as in the case of psychiatric disorders, it becomes necessary to modify fear memories. Many different techniques have been explored, both in animals and in humans, to change learned fear responses. One way to modify learned fear is by targeting fear memories not immediately after learning, but at a later time point through a reminder, which makes retrieved memories labile, and thus more susceptible to interventions [1]. Pharmacological interventions during this phase using protein synthesis inhibitors in animal studies have resulted in persistent effects on fear memories [2]. Such invasive interventions have an obvious limitation, however; they cannot be used safely in humans. Schiller et al. [3] showed that it is possible to rewrite emotional memory in humans using a noninvasive method. They showed that safety learning after reactivation of a conditioned fear memory interfered with the reconsolidation of the memory and prevented return of fear. Another approach for modulation of fear memories (as an alternative to behavioural interference) could be noninvasive brain stimulation techniques, such as transcranial direct current stimulation (tDCS). tDCS polarity dependently c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965

NeuroReport 2014, 25:480–484 Keywords: direct current stimulation, fear conditioning, memory, prefrontal cortex a Department of Psychiatry, Campus Benjamin Franklin, Charite´ University of Medicine, bCluster of Excellence ‘Languages of Emotion’, Freie Universita¨t, c Dahlem Institute for Neuroimaging of Emotion, Freie Universita¨t, Berlin, d Department of Clinical Neurophysiology, University of Medicine, Goettingen, Germany and eDepartment of Psychiatry, Mount Sinai Hospital New York, New York, USA

Correspondence to Aditya Mungee, Affective Neuroscience and Emotion Modulation, Department of Psychiatry, Campus Benjamin Franklin, Charite´ University of Medicine, 14050 Berlin, Germany Tel: + 493084458658; fax: + 493084458233; e-mail: [email protected] Received 30 November 2013 accepted 9 December 2013

DOI: 10.1097/WNR.0000000000000119

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Effect of tDCS on fear memories Mungee et al.

amygdala could also result in stronger fear memory. Hence, we propose that excitatory stimulation of the right DLPFC together with inhibitory stimulation of the left vmPFC would result in fear memory enhancement.

Methods and materials Participants

Seventy-four healthy individuals were recruited by poster advertisements for the study. Individuals with metal implants inside the skull or eye, severe scalp skin lesions, cranial bone fractures, known history of epilepsy or previous seizures, pregnant or breast-feeding women and patients with a known psychiatric disorder or on CNS-acting medications were excluded from the study. The Charite´ institutional ethics committee approved the protocol, which was performed in accordance with the Declaration of Helsinki. All participants were provided a complete oral and written description of the study and informed consent was obtained from each participant before participation. Procedure

The experiment was conducted over 3 days, with a gap of 24 h between the sessions. The protocol for all 3 days is summarized in Table 1. The experiment was conducted using Presentation software (Version 0.70, http://www.neurobs.com).

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4 s, with a 10–12 s gap between stimuli. The duration between stimuli presentation was randomized to avoid false responses because of habituation. Skin conductance responses (SCR) were measured from the left ring finger using the Schuhfried Biofeedback X-pert 2000 device (Schuhfried, Moedling, Austria). The electrode was connected to the ring finger of the left hand. Day 2: tDCS

On the second day, all participants were reminded of the CS + using a single presentation of the coloured square paired with the shock on day 1 (CS +). Immediately after this, the participants in the real tDCS group were stimulated for a total duration of 20 min by two salinesoaked surface sponge electrodes (15 cm2). The participants in the sham group received only a brief current for the first 30 s to mimic the itching associated with real stimulation [10]. The anodal electrode was placed over the right DLPFC with electrodes (5  3 cm) placed at the right frontolateral location (F4 of the international 10 : 20 EEG system [11]) and the cathode over the contralateral supraorbital area (see Fig. 1). We used a constant current battery-driven stimulator (CX6650; Rolf Schneider Electronics, Gleichen, Germany). The current was ramped up to 1 mA over a period of 30 s to minimize side effects. Day 3: fear response assessment

Day 1: fear acquisition

Two randomly assigned groups (tDCS and sham) underwent a Pavlovian fear conditioning paradigm with partial reinforcement. The conditioned stimuli (CS) were blue and yellow squares and the unconditioned stimulus (US) was a low-intensity electric shock to the right wrist. One stimulus was paired with the US in 38% of the trials (CS +) and the other was never paired with a shock (CS –). A Grass Medical Instruments stimulator (Grass Medical Instruments, Quincy, Massachusetts, USA) was used to deliver 50 pulses/s for a duration of 200 ms. The intensity of the electric shock was adjusted to every individual participant, the threshold stimulus being uncomfortable but not painful. We used a starting stimulus of 10 V and went up to a maximum intensity of 60 V. CS + and CS – were each presented 10 times in a randomized order; six additional CS + presentations were associated with a shock (US). The order of appearance of the colour paired with the shock was randomized to avoid bias. We presented the stimuli for Table 1 Day 1

To test whether the conditioned fear response was influenced by stimulation, we presented both groups Fig. 1

Right DLPFC

Left vmPFC

Overview and timeline of the experiment Day 2

Day 3

Fear response assessment Fear acquisition Group 1a-tDCS (real) (F4) Group 2b-tDCS (sham) (F4) a Anodal stimulation of the right dorsolateral prefrontal cortex (DLPFC) [F4] and combined with cathodal transcranial direct current stimulation (tDCS) the contralateral supraorbital area. b Sham stimulation with identical electrode positions.

Electrode positions for transcranial direct current stimulation (right dorsolateral prefrontal – anodal, left supraorbital – cathodal), prepared using the navigated brain stimulation, NBS System (eXimia; Nexstim Ltd, Helsinki, Finland). DLPFC, dorsolateral prefrontal cortex; vmPFC, ventromedial prefrontal cortex.

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NeuroReport 2014, Vol 25 No 7

with the conditioned stimulus without the stimulus to assess their fear responses. We presented the participants with 10 CS + and 11 CS – presentations. The order of appearance was randomized to prevent bias. As the participants were shown a reminder of CS + on the second day, we used one extra presentation of CS – on day 3 to maintain the total number of trials on all 3 days equally. Data analysis

The response to the last trial during fear acquisition on day 1 was used as a criterion to decide whether the participants had successfully acquired fear conditioning as we expected the learning effect to be at its peak here. We first assessed fear conditioning in all participants (n = 74) before discarding any data. Participants (n = 24) who showed responses to CS + equal or less than CS – during the last trial of acquisition were excluded from further analysis. This is in line with data from previous studies [12] because the effects of an intervention on fear memory cannot be assessed in participants who fail to acquire fear responses. Hence, 50 participants were included in the final sample [tDCS = 28 (male = 13, female = 15); sham = 22 (male = 15, female = 7)]. We used Ledalab (a MATLAB-based software; http://www.ledalab.de/), more specifically, the continuous decomposition analysis method to analyse the skin conductance data. This method extracts the phasic information underlying the SCR and aims at retrieving the signal characteristics of the underlying sudomotor nerve activity [13]. The first trial at the start of the experiment was excluded from the analysis to avoid any effects induced by an orienting response. As we expected the fear responses to be most pronounced in the early phase on day 3, we restricted our analysis to the first three presentations of the CS + and CS – . As approximately one-third of the CS + trials were paired with a shock (US) on the first day, we expected the conditioned participants to show a fear response to at least the first three trials of CS + on the third day. However, as no shocks were actually administered on the third day, a gradual learning effect and thus reduction in the fear responses is expected after the early phase. We compared the mean differential SCR between tDCS and the sham groups in the 0.5–4.5-s time window after stimulus onset (CS + minus CS –). Square root transformation of the raw data was performed to normalize distributions. Each participant’s normalized score was then divided by the mean square-root-transformed US response of that participant. Statistical analysis was carried out using SPSS 19 (SPSS Inc., Chicago, Illinois, USA).

fear responses (CS + minus CS –) at the end of fear conditioning. Responses for the last three trials were significantly different from zero in each group, and both the sham [t(21) = 2.28, P < 0.05] and the tDCS [t(27) = 4.23, P < 0.0001] groups showed higher responses to the CS + versus the CS – trials. Fear acquisition did not differ between groups, as shown by the results of an independent t-test [real tDCS (mean = 0.37, SD = 0.46), sham tDCS (mean = 0.77, SD = 1.57); t(48) = 1.28, P = 0.21]. Day 3: fear memory test

Next, we compared these two groups during the early phase on day 3. An independent-samples t-test was used to compare the mean differential SCR in the tDCS and sham group for the first three trials on day 3. The scores between the tDCS (mean = 0.06, SD = 0.31) and the sham group [mean = 0.17, SD = 0.46; t(48) = 2.05, P < 0.05] differed significantly with a moderate effect size (Cohen’s d = 0.59). These results suggest an enhancement effect of tDCS on fear memory because the participants in the sham group had a significantly lower mean differential SCR compared with the participants in the real tDCS group (Fig. 2).

Discussion Our study examined whether tDCS can be a potential tool to influence fear memories. In accordance with this hypothesis, tDCS (right prefrontal – anodal, left supraorbital – cathodal) after memory retrieval enhanced fear memory. Previous studies have shown that prefrontal Fig. 2

0.20 Mean differential SCR (μS)

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0.10

0.00 −0.10 −0.20 −0.30 Sham

Results No adverse affects such as skin burns, headaches, etc. were reported after tDCS stimulation. Day 1: fear acquisition

We first analysed the SCR data for the tDCS and sham groups at the end of day 1 to check for differences in the

Real Group

Mean differential skin conductance response (SCR) in microSiemens (CS + minus CS –) for the sham and real transcranial direct current stimulation (tDCS) groups in the early phase on day 3. The participants in the sham group have a significantly lower mean differential SCR (P < 0.05) compared with the participants in the tDCS group (see the results section). Error bars represent SEM.

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Effect of tDCS on fear memories Mungee et al.

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tDCS can affect various types of memory, for example working [14] and declarative memory [15]. Anodal tDCS of the DLPFC has also been shown to affect emotional memory [16]. However, despite a number of studies exploring various forms of memory and tDCS, the effects of tDCS on fear memory processes had not been investigated yet. A possible explanation for this could lie in technical difficulties involved in stimulating deep brain areas such as the amygdala, which play an important role in the neural circuit underlying fear [14]. We propose an approach to overcome these difficulties by choosing electrode sites above superficial brain areas of the prefrontal cortex using these as a potential window to influence fear memories.

that anatomically and functionally, the vmPFC and amygdala are connected bidirectionally [17]. Further studies with additional control groups using alternate reference electrode sites are needed to differentiate between these possible origins of these effects and to explain the effects of tDCS on the neural circuit underlying fear memories in larger detail.

Two explanations for the finding that right prefrontal – anodal and supraorbital – cathodal stimulation enhanced the fear memory, which are not necessarily exclusive, might apply. Anodal tDCS of the right DLPFC could have resulted in a strengthening of the memory trace coding for conditioned fear memories. This is in line with previous studies [8], which have found an association between the DLPFC and negative affect with lateralization towards the right. DLPFC plays an important role in cognitive regulation of fear specifically with respect to the interpretation of the stimulus [14]. Imaging studies [17] show correlations between DLPFC activity and neutral or fearful stimuli. In our study, it appears that the participants who underwent tDCS showed higher fear responses most probably because of an increase in cortical activity in the right DLPFC accomplished by anodal tDCS, which might result in an interpretation of the stimulus as ‘more fearful’ (Fig. 2).

Our results further suggest the interesting possibility that emotionally positive stimuli might be enhanced in a similar manner. This may provide a new therapeutic venue for diseases where fear plays a critical role (e.g. anxiety disorders, phobia and PTSD). Future studies should explore alternative stimulation sites and the effects of reverse stimulation protocols on conditioned fear. Our findings are in line with previous rodent and human studies that described a critical reconsolidation window for interventions to modulate fear [3,22]. This should, however, be interpreted with caution because we did not include a control group that was stimulated outside the reconsolidation window or had tDCS without reactivation of the fear memory. Moreover, we applied a relatively abstract laboratory fear-inducing protocol. Further studies are needed to test more complex memory traces before these results can be applied to fear memories in general. Because of the relatively large electrode sizes used for tDCS, we cannot rule out the possibility of having stimulated other cortical areas not directly under the stimulation electrodes but also involved in the neural circuit modulating fear. We used the 10–20 international system for EEG electrode placement as opposed to stereotaxic approaches or neuronavigation to determine the stimulation sites, which offers only a medium degree of precision with respect to localization of stimulation sites [23]. We did not record changes in regional brain metabolism to confirm the effects of our stimulation in specific brain areas. Finally, because of our bipolar stimulation design, we could not discern between the effects of the left and the right prefrontal electrodes.

Moreover, the cathode positioned over the left orbit might have inhibited the left vmPFC (refer to Fig. 1). vmPFC is considered to play an important role in extinction, retention and recall of fear memories [12,14]. Furthermore, Milad et al. [18] found a correlation between vmPFC thickness and extinction/retention of fear memories in humans. Clinically, diseases where fear plays a critical role, for example anxiety disorders and post-traumatic stress disorder (PTSD) are also related to smaller vmPFC volumes as shown by structural and functional neuroimaging studies [19]. Interestingly, PTSD patients, when subjected to traumatic stimuli from the past, show decreased medial prefrontal cortex activity in comparison with healthy controls [20]. We showed a reminder cue of the unpleasant stimulus just before tDCS, perhaps augmenting the impact of tDCS on fear memory. Specifically, excitability diminution of the vmPFC might have prevented ‘unlearning’ by diminishing stimulus-associated activation of this area. Anatomically, the second explanation is more likely as the DLPFC does not project directly to the amygdala [21]. An indirect inhibitory relationship is nevertheless possible [14]. Evidence from animal and human studies shows

The results of our study show an enhancing effect of tDCS on fear memory, indicating that tDCS could be an effective tool in modulating emotional memories, either by enhancing (as in this study) or by diminishing (using opposite patterns of stimulation) them. The latter assumption, however, has to be substantiated by future studies.

Conclusion To summarize, we show that it is possible to influence fear memories in humans using noninvasive brain stimulation techniques, namely, tDCS. The results of our study support the findings of Schiller et al. [3], who also showed that fear memories can be persistently modified in humans using noninvasive methods. Given the limited efficacy of present therapeutic approaches to modulate fear memories in clinical syndromes, these

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findings might provide a valuable new therapeutic window. Further research with noninvasive brain stimulation methods, for example tDCS and transcranial magnetic stimulation, is necessary to evaluate the clinical efficacy of this new approach.

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Acknowledgements The authors thank the German Academic Exchange Service (DAAD), Charite Medical Neuroscience Program and the cluster ‘Languages of Emotion’ for supporting this project. They also thank their colleagues Anne Weigand, Torsten Eggert and Marie Molitor for technical assistance.

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This work was supported by the Cluster of Excellence ‘Languages of Emotion’ at the Free University, Berlin.

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Conflicts of interest

A.M. is supported by the German Academic Exchange Service (DAAD). M.A.N. is a member of the advisory board of Neuroelectronics, Eisai and UCB. M.B. received an unrestricted research grant from Medtronic. For the remaining authors there are no conflicts of interest.

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Transcranial direct current stimulation of the prefrontal cortex: a means to modulate fear memories.

Targeting memory processes by noninvasive interventions is a potential gateway to modulate fear memories as shown by animal and human studies in recen...
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