Human Fear Conditioning and Extinction in Neuroimaging: A Systematic Review

Fear conditioning and extinction are basic forms of associative learning that have gained considerable clinical relevance in enhancing our understanding of anxiety disorders and facilitating their treatment. Modern neuroimaging techniques have significantly aided the identification of anatomical structures and networks involved in fear conditioning. On closer inspection, there is considerable variation in methodology and results between studies. This systematic review provides an overview of the current neuroimaging literature on fear conditioning and extinction on healthy subjects, taking into account methodological issues such as the conditioning paradigm. A Pubmed search, as of December 2008, was performed and supplemented by manual searches of bibliographies of key articles. Two independent reviewers made the final study selection and data extraction. A total of 46 studies on cued fear conditioning and/or extinction on healthy volunteers using positron emission tomography or functional magnetic resonance imaging were reviewed. The influence of specific experimental factors, such as contingency and timing parameters, assessment of conditioned responses, and characteristics of conditioned and unconditioned stimuli, on cerebral activation patterns was examined. Results were summarized descriptively. A network consisting of fear-related brain areas, such as amygdala, insula, and anterior cingulate cortex, is activated independently of design parameters. However, some neuroimaging studies do not report these findings in the presence of methodological heterogeneities. Furthermore, other brain areas are differentially activated, depending on specific design parameters. These include stronger hippocampal activation in trace conditioning and tactile stimulation. Furthermore, tactile unconditioned stimuli enhance activation of pain related, motor, and somatosensory areas. Differences concerning experimental factors may partly explain the variance between neuroimaging investigations on human fear conditioning and extinction and should, therefore, be taken into serious consideration in the planning and the interpretation of research projects.


Introduction
Fear conditioning is an ability that is vital for the detection of danger, initiation of self-protection mechanisms, and for survival of a species. Disorders in humans associated with increased anxiety and fear levels, such as posttraumatic stress disorder, phobias, or panic disorder, exemplify how misguided fear conditioning might render originally innocuous stimuli fear-inducing and threatening. In addition, extinction of these associations is also hampered in these disorders. A life time prevalence of anxiety disorders of about 16,6% [1] highlights the substantial clinical and socioeconomic relevance of fear conditioning and extinction.
The term conditioning refers to the process of learning the association between two previously unrelated stimuli [2]. In a typical differential fear conditioning design, a previously neutral conditioned stimulus (CS+) is associated with an aversive and fearinducing unconditioned stimulus (US) and becomes intrinsically aversive, while another neutral stimulus remains unpaired (CS-) [3]. Two main types of conditioning designs can be distinguished, which differ in the temporal relationship between CS+ and US, hence in the temporal contiguity. In trace conditioning, a time interval ranging from for example 500 milliseconds [4] to 10 seconds [5] separates the presentation of the CS+ from presentation of the US. The expression ''trace conditioning'' stems from the idea that a memory trace needs to bridge the gap between CS+ and the delayed US to form an association, therefore working-memory processes are more strongly involved in trace conditioning. In contrast, in delay conditioning the CS+ overlaps or is immediately followed by the US. A repeated exposure of the originally neutral stimulus without presenting the aversive stimulus gradually eliminates the fear reaction and is defined as extinction. In the past, extinction was regarded as a process of forgetting this association. However, the phenomena of spontaneous recovery, renewal, rapid acquisition, and reinstatement after extinction, suggest that fear extinction is ''an active learning process that is distinct from acquisition and requires additional training to develop'' [6].
While the literature on animals has been summarized in several review articles (see e.g. [3,6]), there has been no such approach in the current functional neuroimaging literature on human fear conditioning. So far, only a few reviews have been published and they focus on special topics such as extinction of conditioned fear [19,20], or socio-cultural and cognitive influences on learning [21]. Büchel et al.'s (2000) review compared event-related fear conditioning studies to block-design studies and positron emission tomography (PET) studies. This important review was one of the first to identify a common core network for human aversive conditioning, including the amygdala and anterior cingulate cortex (ACC) [22]. Other reviews concentrated on cellular and synaptic mechanisms, or on plasticity within this neuroanatomical circuitry [3,23].
Even though a core network for fear conditioning has consistently been reported in most imaging studies, results obtained from modern neuroimaging techniques differ in many respects, for example, in the number or the type of activated areas.
Therefore, the main aim of this review is to identify consistent and common findings on aversive conditioning and extinction in humans, as assessed by PET and functional magnetic resonance imaging (fMRI), and to present them in a structured manner. The second aim is to look at the differences between neuroimaging studies with respect to neuroimaging results and design parameters. We therefore identify and evaluate typical experimental factors that may influence brain activation patterns and may thereby contribute to the heterogeneity of neuroimaging results. Overall, this review is intended to facilitate the interpretation of seemingly contradictory neuroimaging findings, as well as the selection of an appropriate conditioning design for specific research purposes. Therefore, this review is relevant both to clinicians seeking for a state-of-the-art overview and to researchers investigating fear conditioning or extinction by means of neuroimaging.
The main results of the reviewed studies will be briefly summarized first, followed by an evaluation of specific consequences on activation patterns of critical factors concerning conditioning paradigms, measures of conditioning success, stimuli, and their timing. The review concludes with a critical discussion of these factors and an evaluation of their impact on past and future research.

Literature Search
To identify relevant neuroimaging studies on human fear conditioning and extinction, a computerized database search of journal articles via Pubmed was conducted for the years 1994-2008. This Pubmed search, as of December 2008, used combinations of the keywords ''conditioning'', ''extinction'', ''aversive'', ''fear'', ''fMRI'', ''neuroimaging'', ''PET'' and ''humans''. No truncations and language restrictions were applied. We screened the abstracts for relevant literature based on the literature search criteria and additionally examined the references sections of articles and reviews for potentially useful studies.

Selection criteria
Studies were included if they were: (1) PET or fMRI studies, (2) performed on healthy volunteers, (3) focused on cued fear conditioning and/or extinction. Furthermore, exclusion criteria were: (1) pharmacological modulation, (2) subliminal or masked presentation, (3) context conditioning, (4) combination of fear conditioning with other experimental tasks, such as cognitivedemanding working-memory tasks. Inclusion criteria were applied independently by two reviewers. Specific experimental designs for fear conditioning in fMRI and PET were compared, focusing on the impact of critical experimental variables, such as timing parameters, the contingency rate, or characteristics of the stimuli, on neuroimaging results.

Data Extraction
Data were extracted by the first author (CS) and doublechecked independently by the second author (SS). The discrepancies were resolved by consensus and the senior author (CK) was consulted if needed. The following variables were extracted and presented in Table 1: 1) demographic characteristics (number of participants, gender, and age), 2) study design (delay, trace, and extinction), 3) neuroimaging technique (fMRI, PET), 4) characteristics of the stimuli (modality of CS and US), 5) independent assessments of the conditioning process (e.g. heart rate), and 6) neuroimaging results. In the data analysis, the outcomes of interest were brain areas activated during conditioning and extinction. Therefore, we extracted the neuroimaging data presented by each study as the main results. Finally, we extracted those contrasts of interest that represent the conditioning or extinction effect (e.g. CS+.CS-).

Data Analysis
The review provides a qualitative summary of neuroimaging findings on fear conditioning and extinction of the included empirical studies. These studies were classified according to the type of study design (delay, trace, and extinction), the modality of the CS and US, the contingency rate, and the independent assessment of the conditioned response. For each category, we extracted the absolute frequency of activated brain areas for the contrasts of interest. Moreover, we attempted to identify common and divergent activations across individual study results. Studies, reporting additional or different activation from those described in the core fear network, were examined for the following variables to shed light on reasons for the discrepant findings: conditioning design (delay, trace, and extinction), contingency rate, and characteristics of the CS and US. We refrained from statistically combining results from the studies due to the differences in their design.

Included studies
Based on the literature search strategies, 147 citations were retrieved from the Pubmed database. Among these, we identified 33 relevant studies. Additionally, we examined the references of relevant articles and reviews. Thirteen citations met the selection criteria. As a whole, we reviewed 46 articles on human fear conditioning and/or extinction. Figure 1 shows the search and selection process. Forty studies exclusively used a delay conditioning paradigm during the acquisition phase (Table 1; No. 1-3, 5, 7, 9, 11-18, 20-23, 26, 28-29, 31-33, 35-36, 38-40, 42-45) [4,5,. Only two studies investigated solely trace conditioning during acquisition (

Summary of Findings
Brain regions involved in delay fear conditioning. As a major and stable result, the amygdala, the ACC and the insular cortex turned out to be crucial structures in the acquisition of aversive delay conditioning, independent of general design characteristics. Twenty-five of the 44 delay conditioning studies reported amygdala activation, with results varying with respect to the laterality of activation. While nine studies reported bilateral amygdala activation (e.g. [31,49,64]), eight studies detected leftlateralized (e.g. [27,61,65]), and eight right-lateralized activations (e.g. [53,62]). Methodologically, nineteen of the 25 studies additionally tested for temporal interactions of amygdala activation or split up the acquisition phase into an early and late phase, in order to assess the temporal gradation in the signal intensity of the amygdala. Seventeen of these studies reported learning-related responses of the amygdala (e.g. [45,48,57,66]): fourteen studies found initial increase and rapid decrease of activation during repeated exposure to unpleasant stimuli (e.g. [33,44]), whereas three studies only reported increases of amygdala activation during the acquisition phase [40,51,58]. The remaining 19 delay conditioning studies did not report activation of the amygdala. Seventeen of them did not test for temporal aspects of amygdala activation (e.g. [39]). Sixteen delay conditioning studies found activation of the ACC (e.g. [25,26]), five of the posterior cingulate cortex (PCC) (e.g. [30]), and two reported activation of the cingulate cortex [33,57]. Sixteen studies detected insular activities (e.g [39,54,65]). These areas are all part of the classical key fear network as described previously [22,23]. Activation of brain areas such as the hippocampus, the cerebellum, the thalamus, the striatum or the sensory cortices has been reported by fewer delay conditioning studies, underlining the considerable variability in neuroimaging findings. Hippocampal activity, mostly lateralized, was found for example by ten studies (e.g. [34]). Twelve studies showed activation of the striatum (including putamen, accumbens nucleus, caudate nucleus) (e.g. [39,61]), whereas thalamic activity (including pulvinar, geniculate nucleus) was reported by twelve delay conditioning studies (e.g. [48]) (for details, see Figure 2). As argued below, we believe such differences in results to be methodological in origin [14].
Brain regions involved in trace fear conditioning. So far, only two fMRI studies have employed solely trace conditioning [66,67] and four fMRI studies were conducted on both delay and trace conditioning [4,5,27,28] (for details, see Table 1), all with either auditory, visual or tactile aversive stimulation. Again, the amygdala and the medial temporal lobe (MTL) were predominantly activated during the acquisition of trace conditioning in five studies (e.g. [4,27,66]). Activation of the ACC was apparent in three studies [5,66,67] and of the PCC in one study [5]. The hippocampus was bilaterally activated in three trace conditioning studies [5,27,66], and two studies showed additional activation of the insula [66,67]. These fear-related structures such as the amygdala, the hippocampus, the ACC, the insula and the MTL were active independently of US-modality.  Table 1. cont.
(e.g. [66]) or the middle frontal gyrus [5,27] in four trace conditioning studies. Activation of other brain areas, such as the cerebellum, was reported in one study, of the motor cortices in three studies (e.g. [4,5]) (for details, see Figure 2). Again, this variability in study results may be due to critical design characteristics, which will be discussed below. Brain areas involved in fear extinction. Although extinction is very relevant in therapeutic settings, only seven studies with focus on extinction met criteria for our review [37,42,44,47,51,56,60]. All seven used a classical delay conditioning design during acquisition. Six studies used a tactile US (e.g. [51]), and one an olfactory US [37]. Three of the seven studies reported major activation foci in the amygdala [37,42,44], two in the ACC [51,60], one study in the PCC [47] and three in the insula (e.g. [37,47]), whereas four studies observed activation in frontal regions such as the prefrontal cortex (PFC), and the ventromedial prefrontal cortex (VMPFC) (e.g. [60]). Activation of the hippocampus was found in only one study [42] (for details, see Figure 2).
Although consensus exists that the amygdala again plays an important role in extinction, a closer look reveals that the details about amygdala activation vary. As with acquisition, four of the seven studies reported habituation of the amygdala response during extinction [37,42,44,51]. To assess the temporal gradation in signal intensity of the amygdala, two of them split up the extinction phase into an early and late phase [44,51], and one study tested for time6condition interaction [37]. Knight and coworkers (2004) reported an increase of right amygdala and a decrease of left amygdala activation during extinction, by t-test comparison [42]. Three other studies that did not analyze temporal activation patterns failed to find amygdala activation [47,56,60].
The influence of CS-US-contingency. Contingency describes the rate of pairing between the previously neutral CS+ and the aversive US, and therefore the predictability of the US in relation to the CS. In some cases, the CS is paired with the US on every trial (continuous pairing), whereas in other conditioning designs, CS and US are paired intermittently.
Awareness about this CS-US-contingency, mediated by conscious US-expectancies or by explicit instruction about the CS-US-contingency, also influences brain activation. Participants were explicitly informed about the CS-US pairing before the experiment in some studies (e.g. [33]), but not in others (e.g. [44]).
Finally, the choice of contingency rates is related to a problem specific to neuroimaging studies: the choice of contrasts between conditions. In a continuous pairing paradigm where the CS+ is always presented with the US, contrasts may be calculated between CS+ and CS-(e.g. [58]), between paired und unpaired subjects (e.g. [42]), or between conditioned and pseudo-conditioned phases -in which CS and US are not correlated in time (e.g. [25]). In a partial-reinforcement design, CS+ may be paired or unpaired with the US. Here, contrasts are mainly calculated between CS+unpaired and CS-(e.g. [26]).
Again, activation of the fear network was observed to be independent of US-modality. In spite of different USs, activations of the amygdala, ACC and insula were reported for every stimulus type. Of the 33 studies with tactile stimulation, fifteen found activation of the amygdala (e.g. [29]), ten of the ACC (e.g. [35]), and ten of the insular cortex (e.g. [39]). Other main activation foci for tactile stimuli concern the thalamus in seven (e.g. [46]), and the striatum in ten studies (e.g. [52]). Other regions such as the occipital cortex, motor or somatosensory cortices are also activated during tactile conditioning in 16 studies (e.g. [27,35]). By contrast, the nine studies on auditory fear conditioning mainly report activation of the fear network, with emphasis on amygdala in Figure 2. Brain areas involved in aversive conditioning and/or extinction. Different brain areas (with at least unilateral activation during aversive conditioning and/or extinction) are plotted against the x-axis. The number of studies out of 46 studies per brain region is plotted against the y-axis, taking into account the conditioning design which is delay conditioning in 40, trace conditioning in two, delay and trace conditioning in four, and extinction in seven studies. doi:10.1371/journal.pone.0005865.g002 seven (e.g. [31]), on ACC in five (e.g. [48]), and on insula in five studies (e.g. [41]). Moreover, activations of the motor or sensory cortices (e.g. auditory, occipital) are also apparent in five studies (e.g. [26]). The one study on olfactory conditioning mainly reports activations in amygdala, insula and orbitofrontal cortex (OFC) [37], areas that are also associated with the perception of disgust [70,71]. All three studies on visual aversive conditioning reported activation of key fear areas such as the amygdala and ACC or the PCC [30,40,67]. Activation of the insula was found in two of the studies (e.g. [67]). Furthermore, activations of the DLPFC, OFC, thalamus, nucleus accumbens and the occipital cortex are apparent in these visual conditioning studies (e.g. [40]) (for details, see Figure 3).
Our review reveals that 38 of the reviewed studies employed different modalities of US and CS. Only five studies chose an auditory CS paired with an auditory US (e.g. [31,41,66]), and three were conducted on visual CS and US (e.g. [40]). Again, research is needed to quantify this effect of common CS-USmodality on neuroimaging results.
Independent assessment of the conditioning process. A control procedure to ensure that a physiological response towards the CS+ has actually occurred, with data from dependent variables other than brain activation, was used in 41 of the 46 studies cited here. Autonomous, endocrine, or behavioral responses, such as skin-conductance responses, heart rate, verbal responses (ratings of the CS, US-expectancy ratings, or CS-US-contingency assessment), reaction times, or eye-blink reflex qualify as parameters of successful conditioning. The majority of the studies employed independent measures online during scanning. Autonomous measures, such as heart rate, were applied in two [35,63], skin-conductance responses in 26 (e.g. [4,32,50,62,64,65]), and eye-blink startle response in eight studies (e.g. [24,25,46,56]). Only three studies used SCR outside the scanner: before and after conditioning [38] or in an additional experiment [44,57]. Online assessments of verbal responses, such as CS-ratings, were used in one study [30], and US-expectancy ratings in seven studies (e.g. [28,31,62]). Two studies compared ratings of the CS before and after scanning [57,64]. Twelve studies employed CS-US-contingency ratings and three studies CS-ratings post experimentally (e.g. [27,40]). To conclude, twenty-three studies combined different measurements of the conditioned response (e.g. [27,44]) (for details, see Table 1, Figure 4). To summarize, objective measurements are necessary when studying conditioning, to verify that conditioned learning has indeed occurred.

Discussion
This review deals with the neural correlates of human fear conditioning in current fMRI and PET studies. Our analysis indicates that neuroimaging studies on human fear conditioning and extinction activate a common core fear network which is in accordance with evidence from other sources (e.g. [17]). Some neuroimaging studies do not find these activations. This heterogeneity is not surprising taking into account the large methodological variety in imaging and design parameters. Methodological differences were found a) in the conditioning protocol (delay, trace), b) in the contingency rate (100%, 80%, 50% or less) and awareness, c) in the modality of CS and US (tactile, auditory, visual, olfactory), and d) with respect to the Figure 3. Brain areas involved in aversive conditioning according to the modality of the US. Different brain areas (with at least unilateral activation during aversive conditioning) are plotted against the x-axis. The number of studies out of 46 studies per brain region is plotted against the y-axis, taking into account US modality, which is tactile in 33 studies (such as electrical shocks), auditory in nine studies (such as noise), olfactory in one study (such as odors), or visual in three studies (such as aversive pictures). doi:10.1371/journal.pone.0005865.g003 further assessment of the conditioned response (e.g. psychophysiological measurements, verbal ratings).
Neuroimaging studies have substantially extended our understanding of fear conditioning and extinction, adding in vivo evidence from humans to previous electrophysiological and lesion studies from animals [3,72]. Consistent with comparative animal data, neuroimaging investigations have corroborated the finding of a neural fear network activated during fear conditioning. Within this core fear network, key structures for the acquisition and the extinction of conditioned fear have been identified, although there is considerable methodological heterogeneity between studies, with some of them not reporting these activations. Furthermore, it turned out that anatomical regions relevant in fear conditioning are also involved in the extinction of fear memories. In conformity with animal and lesion data, our review indicates that the amygdala, as one principal structure of the limbic system, is one of the key regions involved in fear conditioning and extinction. Amygdala activation occurs in response to emotional stimuli and is therefore regarded as the gate keeper funnelling emotionally relevant information into different processing channels. This region is activated during conditioned-fear acquisition as well as during the expression of learned fear (see for an overview [23]). Furthermore, amygdala activation undergoes rapid habituation during acquisition and extinction that should be taken into account in neuroimaging studies (e.g. [26]). This typical response profile of the amygdala may not be detected by categorical comparisons of e.g. CS+ and CS-, as this contrast reflects timeinvariant neural responses. Consequently, some studies carried out an analysis that tested for this type of time-dependent response profile. They set up a statistical model that allows characterizing the activation of the amygdala by a time by condition interaction. Therefore, we suppose that testing for interactions between conditions and time may reveal conditioning results that otherwise remain hidden, such as amygdala activation.
Furthermore, some brain regions, especially the MTL, are difficult to assess using echo-planar imaging (EPI) because they are highly vulnerable to susceptibility artifacts [73]. These differences may cause image distortion and signal dropout [73][74][75]. This might be another reason why some studies did not find amygdala activation during conditioning. Activation of the insula, another central structure for emotion processing, was also shown in 40% of the neuroimaging studies. Phelps and co-workers (2001) assume that the insula cortex conveys a cortical representation of fear to the amygdala [52], and that uncertainty about the advent of the aversive stimulus during intermittent pairing is reflected by insula and dorsal prefrontal cortex activation [31,32,76]. Another region belonging to the core fear conditioning and extinction network described by the majority of the cited neuroimaging studies is the ACC (for an overview, see [17,26]). The ACC plays an important role in approach and avoidance learning [77] as well as in fear learning [78]. The frontal cortex is particularly crucial for emotional regulation and therefore for the extinction of conditioned fear. Although extinction is the essential process in therapeutic settings, only seven studies have so far focused on extinction. From both animal data and theoretical considerations, it is evident that fear extinction involves mainly interactions between cortical and subcortical structures, such as the PFC and the amygdala or the hippocampus (see for an overview [79]). As one of the principal structures of the brain's extinction circuitry, the PFC regulates the expression of fear by inhibiting the amygdala, such that the fear-conditioned stimulus is prevented from causing a conditioned fear response [79][80][81]. In this review, only one study reported hippocampal activation during extinction. This is surprising, because from other studies is known that the hippocampus and the VMPFC seem specifically important during late phases of extinction, and therefore for the retention of extinction [51,82].
There are, however, considerable variances and discrepancies between studies. Whereas some studies only report activation of the core network, others do not find these activations or observe activation within additional brain regions, such as the hippocampus, striatum, sensory cortices or thalamus. The choice of conditioning protocol, CS-US contingency, and modality of the US seem to be very important factors modifying brain-activation patterns in fear conditioning studies.
Our review indicates that of these factors, the conditioning protocol has great impact on brain activation. Delay conditioning leads to more rapid learning of the CS-US association than trace conditioning [83][84][85]. Thus, from the experimental point of view, delay conditioning has the advantage of a shorter acquisition time, fewer trials, and a more rapid conditioning process than trace conditioning. Additionally, delay conditioning designs are known to extinguish associations faster than those established during trace conditioning [86]. Therefore, all studies that investigated extinction employed delay conditioning in advance. By contrast, in trace conditioning, CS is separated from the US by a temporal gap, resulting in prolonged acquisition times and a larger number of trials being required to form an association. The length of the temporal gap and its distance to the subsequent stimulus also exerts a strong influence. When the US is followed immediately by the next CS, backward conditioning (US-CS associations) or contextual conditioning can occur. In backward conditioning, the US is associated with the next CS, so that no conditioned response is established [87]. Contextual conditioning describes the association of the CS with contextual cues [88,89]. Hence, there is no contiguity in trace conditioning. While in general, delay and trace conditioning involve comparable fear-related networks, activation of the hippocampus is typical of trace conditioning. In trace conditioning, hippocampal activation is required to bridge the gap between CS and US, retaining a memory trace which is needed to form an association between CS and US [90]. The hippocampus is involved in trace conditioning irrespective of the length of trace interval. However, animal data show that some neurons in the hippocampus encode the duration of trace interval [91]. Thus we assume that the level of hippocampal activation may be enhanced by increasing the length of trace interval.
Another important variable contributing to heterogeneity of neuroimaging results is the CS-US pairing or contingency rate. Effects of CS-US-contingency on conditioning have been repeatedly described in the psychological and behavioral literature [31,32,[92][93][94]. Contingency rates determine how fast conditioned responses are acquired, and regulate extinction processes. Our review reveals that the activation of the core fear network consisting of amygdala, insula and ACC is independent of pairing rate, but the time courses of neural responses and the degree of activation may be influenced by contingency. In general, a predictable US is less aversive than an unpredictable US. Therefore, the continuous (100%) pairing of CS+ and US reduces fear responses and activity in fear-related brain areas [31,32], and promotes the habituation of the amygdala [26,44,57,58], relative to intermittent pairing. Nevertheless, the majority of the studies cited in this review employed a continuous pairing paradigm. In intermittent procedures, US expectancy and response frequency is decreased, which slows conditioning and prolongs the extinction phase [31,32,51]. The choice of these pairing parameters has important implications for analysis of imaging data. First, in the light of habituation processes, analysis of time by condition interactions may well improve the detection of amygdala activation. Second, the choice of the contingency rate influences the definition of contrasts of interest between test and control conditions. The cited studies differ in their contrasts of interest which may also influence resulting activation and complicate comparing studies even further. For example, in a 100% pairing design resulting differences in neural responses may be confounded by US-induced BOLD changes. In contrast, in a partial reinforcement design, differences in neural responses are only due to the anticipation of the US.
Our review also illustrates that there is an ongoing controversy on the role of contingency awareness. It seems clear that awareness of the CS-US contingency bridges the CS-US gap in trace conditioning [95,96]. Therefore, it may be very important for trace conditioning, but less so for delay conditioning. Still, this topic requires further investigation. While some researchers found autonomic fear reactions only in contingency-aware subjects, others reported activation of the fear-network independently of contingency awareness [40,97,98]. For example, Phelps et al. (2001) showed that instructions alone can induce fear and that activation of the amygdala can occur without direct experience of the aversive event [52]. Tabbert et al. (2006) explicitly investigated the effect of contingency awareness. They either informed their subjects about the relationship of CS and US or prevented contingency detection by employing a distracter figure or a working-memory task. Amygdala and the OFC were only activated in the unaware group [98], but Klucken et al. (2008) found activation of fear-related areas independent of awareness [40]. However, robust conditioned skin-conductance responses have been observed only in aware participants who acquired a cognitive representation of CS-US-contingencies, and who were able to recall the correct contingency [97]. At this moment, concrete advice as to whether participants should be informed about contingency to obtain faster conditioning responses, is premature.
Concerning the modality of the US and CS, 33 of the 46 studies employed a tactile US, making it the most frequently applied US. Only nine studies used auditory aversive stimuli which may be due to the surrounding and interfering scanner noise. To the best of our knowledge, the problem of scanner noise as being aversive itself has not been discussed so far. The activation of the key fear network including amygdala, ACC and insula seems to be independent of the applied stimuli (auditory, olfactory, tactile, and visual). Nevertheless, many studies do not show activation of the key fear network or observe modality-specific activations. In fear conditioning with tactile US, activation of the thalamus, the striatum, somatosensory and of motor cortices is often reported. These areas are also associated with the nociceptive system, pain anticipation and perception (e.g. [99][100][101][102]). The nociceptive system includes the somatosensory cortices, ACC, insula, prefrontal and parietal cortices [103]. Koyama et al. (2005) showed that ACC activation increases with the magnitude of expected pain, and pain-intensity [104]. The thalamus, a major relay site for nociceptive inputs to cortical and subcortical structures, is thought to be responsible for the onset plasticity in the amygdala during fear conditioning [105]. Therefore, we suggest that a ''pain-fear network'' may be activated during tactile fear conditioning. The one study on olfactory conditioning reported mainly activations of amygdala, ACC and OFC [37]. Odour perception is more often related to disgust than to fear. Disgust and fear are basic emotions with different elicitors and expressions, and appear to be mediated by different neuronal circuits [70,71,106,107]. Therefore, further research is needed to clarify if olfactory conditioning activates a ''disgust-fear-network'' rather than a mere ''fear-network''. To conclude, it seems likely that odours, visual or acoustic stimuli may weaken conditioning effects and may cause activations in different brain regions than electrical stimuli. But to the best of our knowledge, this has never been tested directly in neuroimaging studies. Again, research is needed to quantify the effect of common CS-US-modality on neuroimaging results.
Concerning the modality of the CS, the majority of the studies used visual stimuli as CS, especially photographs of human faces. Faces as CS might be more emotionally relevant to human subjects than tones or coloured lights [108]. However, there seems to be a gender-related effect that needs to be considered in neuroimaging studies. For example, in women, the presentation of faces leads to stronger and persisting amygdala activation, while amygdala activation in men decreases rapidly [109]. Moreover, it is known that the amount of preexposure influences the outcome of aversive learning. These phenomena, so called ''latent inhibition'' and ''US-preexposure effect'', emphasize that novel and unknown CS and US produce more robust conditioning effects than familiar stimuli [31,110]. The disadvantage of unfamiliar stimuli is the mixing of novelty effects and conditioning effects.
Finally, it is very important to ensure that conditioning really takes place by sampling a second psycho-physiological or behavioral measure to avoid contamination of successful conditioning with unsuccessful trials. Skin-conductance responses as measures of autonomic responses have been widely investigated and are well validated [41]. Classifying subjects as ''responders'' and ''non-responders'', or classifying single trials as ''successful'' or ''not successful'' conditioning based on autonomous measures has proven extremely useful, to exclude erroneous trials or subjects from further analysis (e.g. [28,51]). However, technical issues in the scanner environment have to be solved. Measurement of skinconductance responses may well prolong the experiment beyond critical time values for such experimental designs. On the other hand, verbal ratings may easily be influenced and consciously manipulated. Alternatives are the assessment of heart rate, or of the startle reflex, which is an elegant measure if an eye-tracker or electromyography is available. In all, the combination of different psycho-physiological and behavioral methods has proven valuable to assure that conditioning has really taken place.

Strengths and Limitations
To the best of our knowledge, this review is the first summarizing current literature on neuroimaging fear conditioning and extinction and providing an overview on similarities and heterogeneities between study results. In this review, we focused on discussing experimental factors that are typical for conditioning paradigms, such as the design (delay, trace), the contingency rate, the contrasts of interests, or the stimuli (CS, US), and that may contribute to the reported heterogeneity in neuroimaging results. Other experimental factors that may influence fear conditioning and fMRI-studies are, for example, the MR-sequence (e.g. [74,75]), the sample size, gender of participants (e.g. [111,112]), genetic variables (e.g. [17,[113][114][115]), or personality factors (e.g. [116][117][118][119][120][121][122][123][124]). These variables may also contribute to the diversity of neuroimaging results. Another limitation is that our search did not include conditioning studies that were conducted on context conditioning, on patients, on pharmacological interventions, or that included another experimental task. However, we excluded these studies to limit the number of potential influencing variables.

Conclusion
This review provides an overview of 46 current neuroimaging studies on fear conditioning and extinction. Neuroimaging yields new in-vivo evidence with respect to humans revealing and corroborating a consistent pattern of key areas in aversive conditioning and extinction. These structures encompass the amygdala, ACC, and insular cortex for both associative conditioning and extinction. This confirms previous electrophysiological or lesion studies on animals. The key fear-related brain areas, such as amygdala, ACC and insula, are activated independently of specific design parameters. However, some studies still do not report these findings or observe additional modality-specific activations. We pinpointed a number of methodological differences between the functional imaging studies and conclude that these may contribute to the observed variance between results. Prime candidate factors for modifying brain activation patterns are the choice of conditioning protocol, CS-US contingency, and modality of the US. Thus, the contingency and timing parameters, the modality of the CS and US, as well as the assessment of conditioned responses are important for conducting and interpreting neuroimaging studies on fear conditioning and extinction.