1. Introduction {#s0005}
===============

Social anxiety disorder (SAD) is marked by persistent fear or anxiety regarding social or performance situations in which the individual is exposed to possible negative evaluation by others, e.g. meeting unfamiliar people, being observed while eating or giving a public speech ([@bb0010]; [@bb0180]). Furthermore, the mere anticipation of such situations can lead to restrictions in everyday life, avoidance behavior and impairment in social or occupational areas of functioning ([@bb0010]; [@bb0155]). Because of the key role of maladaptive anticipation processes in the maintenance of the pathophysiology of SAD ([@bb0195]), insight into its underlying neural correlates is essential for a better understanding of this disorder and future directions in therapeutic interventions ([@bb0020]; [@bb0170]).

Individuals with SAD seem to be characterized by a hyperactive fear circuitry (amygdala, insula, anterior cingulate \[ACC\] and prefrontal cortex \[PFC\]) and decoupled medial parietal and occipital regions (posterior cingulate cortex \[PCC\], precuneus, cuneus) during threat processing ([@bb0065]; [@bb0230]). This neural pattern suggests a dysbalance between heightened threat processing on the one hand and disturbed cognitive control and emotion regulation on the other hand ([@bb0065]; [@bb0085]).

Only few studies investigated the neural correlates of anticipatory anxiety in SAD patients. Although partially afflicted with methodological limitations, findings of these studies point towards a maladaptive involvement of similar emotion processing and emotion regulation regions as during threat processing. An early positron emission tomography study compared SAD patients within a 2.5 min private speech, either performed before (anticipation condition) or after (control condition) a public speech. Enhanced activity was reported in the dorsolateral prefrontal cortex (dlPFC), inferior temporal cortex and in the amygdaloid-hippocampal region ([@bb0300]). However, conclusions drawn from this study are limited due to a small sample size (n = 9), the lack of a control group and the presence of actual speaking during data acquisition. Lorberbaum and colleagues ([@bb0210]) used functional magnetic resonance imaging (fMRI) to compare brain activity in SAD patients to that of HC during a 12 s public speech anticipation period relative to rest. Enhanced activity was found in the striatum, pons, amygdala/uncus/parahippocampus, insula, temporal pole and reduced activity in the dorsal ACC and PFC. Again, the small sample size (n = 8) and the usage of a rest period instead of a neutral anticipation control condition hamper firm conclusions. Another fMRI - study with a larger sample size (n = 17) investigated the anticipation of a public speech during a 40 s anticipation period compared to the anticipation of a "computer test" control condition ([@bb0035]). SAD patients relative to HC showed increased activation of the insula and decreased activation in the ventral striatum. Solely for the first half (20s) of the anticipation period, increased amygdala activation was reported. The most recent fMRI study examining anticipation processes in SAD ([@bb0090]) used a 90 s public speech anticipation period compared to a 90 s control anticipation period. SAD patients relative to HC showed less variability in amygdala activity during speech anticipation but no mean differences in amygdala, insula, ventral striatum and dorsal ACC.

Besides these disorder-specific paradigms, another study ([@bb0060]) investigated the anticipation (7 s) of disorder-unspecific negative, positive and neutral pictures in SAD patients. In addition to predictable positive, negative and neutral conditions announced by matching anticipation cues, an unpredictable "unknown" condition was introduced, in which the cue did not indicate the emotional valence of the following picture. Anticipation of negative compared to neutral pictures revealed higher activations for SAD patients relative to HC in medial and dorsolateral PFC and in occipitotemporal regions. When comparing the anticipation of unknown to neutral pictures, SAD patients showed higher activations in occipitotemporal regions.

To date, none of the existing studies investigating anticipatory processes in SAD patients varied the temporal predictability of threat. However, maladaptive and heightened responses under conditions of uncertainty about upcoming threat were highlighted as a key feature across anxiety disorders ([@bb0155]). Unpredictability (or uncertainty) of threat is related to higher subjective and physiological stress responses ([@bb0105]), increased threat attention, hypervigilance, heightened reactivity and biased expectancies of aversion ([@bb0150]; [@bb0255]). On a neural level, the amygdala ([@bb0190]; [@bb0330]) and more specifically the central nucleus of the amygdala (CeA) and the BNST, as parts of the so-called extended amygdala ([@bb0005]; [@bb0100]; [@bb0155]; [@bb0260]) are supposed to be central in the response to threat uncertainty. Several recent studies provide concurrent evidence for hyperactivation of amygdala and BNST during anticipation of temporally unpredictable threat in healthy volunteers ([@bb0160]; [@bb0185]), generalized anxiety disorder ([@bb0070]), panic disorder ([@bb0045]) and post-traumatic stress disorder ([@bb0050]). These findings strongly support the assumption of heightened responding to temporally unpredictable aversiveness for patients with anxiety- and stress-related disorders. Moreover, these studies revealed that both, the amygdala and the BNST, appear to follow a temporal dissociation of activity during threat anticipation, such that they consistently reported transient amygdala activity immediately after threat anticipation cue onset and sustained BNST activation over the course of the threat anticipation interval ([@bb0045], [@bb0050]; [@bb0070]; [@bb0160]; [@bb0185]). These findings were interpreted as transient vigilance and threat detection response through amygdala activity ([@bb0155]; [@bb0250]) as a reaction to the threat-indicating cue, turning into a longer-lasting state of sustained fear reflected by enhanced BNST activity ([@bb0100]). Especially CeA and BNST are supposed to be highly interconnected ([@bb0075]; [@bb0100]; [@bb0130]; [@bb0165]; [@bb0260]; [@bb0305]; [@bb0315]). However, recent investigations question a strict temporal dissociation between phasic amygdala and sustained BNST responding and suggest that the BNST is equally involved in phasic threat processing ([@bb0055]; [@bb0125]; [@bb0165]; [@bb0260]).

Given this growing and highly relevant research field, studies investigating BNST responding especially during temporal unpredictability of threat and the temporal dissociation of amygdala and BNST during threat anticipation also in SAD patients are highly warranted. The present functional magnetic resonance imaging (fMRI) study investigated CeA and BNST activation during anticipation of temporally unpredictable aversive events in SAD patients compared to healthy controls (HC). The SAD-specific threat induction was operationalized via camera observation, a method known to induce anxiety, alertness and tension in socially anxious individuals and SAD patients ([@bb0135]; [@bb0140]). This passive method was favored over a speech anticipation paradigm ([@bb0035]; [@bb0090]; [@bb0145]; [@bb0210]; [@bb0300]) to avoid active motion throughout the experiment and thus to establish a better comparability to equally passive picture- or sound-based stimuli used in threat anticipation paradigms investigating other anxiety disorders ([@bb0045], [@bb0050]; [@bb0070]). In order to create uncertainty about stimulus occurrences (also referred to as "manipulation of threat imminence", ([@bb0125])), cue-stimulus-intervals in the present instructed threat anticipation paradigm varied in length. Following the idea of temporally dissociable roles for amygdala and BNST ([@bb0160]; [@bb0185]) we modeled a phasic anticipation blood oxygenation level dependent (BOLD) signal time course immediately after cue onset and a sustained anticipation time course over the whole anticipation period. According to research in other anxiety disorders, we expected increased phasic activation in the CeA as well as increased sustained activation in the BNST for SAD versus healthy controls during anticipation of aversive relative to neutral events.

2. Methods {#s0010}
==========

2.1. Subjects {#s0015}
-------------

In total 22 SAD patients and 22 HC were recruited via public advertisement and from a collaborating outpatient clinic. Three SAD patients with a comorbid major depression and a score \> 30 on the Beck Depression Inventory ([@bb0175]) indicating a severe depression as well as one HC with elevated scores in all SAD questionnaires (\> *M* + 2 *SD*) were excluded from the study. Our final sample comprised 19 SAD patients (12 female) and 21 healthy controls (13 female) matched for gender (*χ*^2^(1) = 0.007, *p* = 1), age and education ([Table 1](#t0005){ref-type="table"}). All participants were right-handed and had normal or corrected-to-normal vision and no history of neurological diseases or psychotic disorders. Participants meeting the general MRI requirements were screened by an experienced psychologist via standardized clinical interview (German version of the SCID; [@bb0340]). Included HC were free of any diagnosis. SAD patients met the criteria of a social anxiety disorder according to DSM-IV-R as main diagnosis.Table 1Demographic and questionnaire data.Table 1SAD (*M* ± *SD*)HC (*M* ± *SD*)*t*-value*p*-valueAge29.47 ± 8.8927.14 ± 5.930.98.33Education (years in school)12.83 ± 1.112.90 ± 0.77−0.24.81LSAS71.26 ± 13.199.48 ± 7.9517.71≤ .001SPS33.68 ± 11.642.62 ± 3.4911.19≤ .001SIAS47.32 ± 12.629.9 ± 5.9211.80≤ .001BDI12.05 ± 7.620.55 ± 0.896.54≤ .001STAI-T52.63 ± 9.6428.75 ± 4.0110.00≤ .001[^1]

Comorbid diagnoses (multiple entries possible) comprised current major depression (n = 5, BDI-score \< 30), obsessive-compulsive disorder (n = 1), general anxiety disorder (n = 1) and specific phobia (n = 1). Three SAD patients took antidepressant medication. Liebowitz-Social-Anxiety-Scale ([@bb0275]), Social Interaction Anxiety Scale ([@bb0280]), Social Phobia Scale ([@bb0280]), and the State-Trait-Anxiety-Inventory ([@bb0270]) supported the diagnosis of SAD. Assessment of depression severity by the Beck Depression Inventory ([@bb0175]) indicated minimal depressive symptoms in the patients sample. SAD patients scored higher than HC in all questionnaires listed ([Table 1](#t0005){ref-type="table"}). Written informed consent was obtained from each participant prior to the experiment. The study was approved by the local ethics committee and conducted in compliance with the Declaration of Helsinki.

2.2. Experimental paradigm {#s0020}
--------------------------

The experiment consisted of 11 aversive and 11 neutral trials. Each trial started with the presentation of a cue (either "Δ" or "Ο", counterbalanced across participants) either signaling that at any time in the following period the camera would switch on (aversive condition, e.g. signaled by "Δ") or stay switched off (control condition, e.g. signaled by "Ο"). To induce temporal unpredictability, anticipatory intervals had variable durations (2 × 3 s, 1 × 5 s, 1 × 10 s, 7 × 16s). After the anticipatory interval, a symbol was presented to inform the participants that the camera was now switched on (camera symbol, 10 s) or that it remained switched off (crossed camera symbol, 10 s). Participants were briefed about the assignment of cues to conditions via standardized screen instructions before the experiment. Furthermore, they were informed that their faces would be recorded by a video camera when the camera symbol was presented and that two experimenters were going to judge their facial rubescence ([@bb0025]; [@bb0265]). For that purpose a camera dummy was installed on the MRI head coil.

2.3. Behavioral data {#s0025}
--------------------

After fMRI scanning participants were requested to rate their feelings associated to both stimuli pairs shown during scanning procedure ("Δ" vs. "Ο" and "camera on" vs. "camera off") on three nine-point Self-Assessment Manikin Scales ([@bb0040]) regarding valence, arousal and anxiety (valence: 1 = very negative, 5 = neutral, 9 = very positive; arousal: 1 = not arousing, 9 = very arousing; anxiety: 1 = not anxious, 9 = very anxious). Behavioral data were analyzed by two-way mixed measures analysis of variance (ANOVA) and post-hoc *t*-tests including Welch\'s correction for unequal variances and Bonferroni correction for multiple comparisons (corrected significance level *p* \< .004) using SPSS software (Version 23.0, IBM Corp., Armonk, N.Y., USA).

2.4. fMRI data {#s0030}
--------------

Anatomical and functional MRI was performed on a 3 T scanner ("Magnetom PRISMA", Siemens Medical Systems, Erlangen, Germany) using a 20-channel head-neck coil. Following a high resolution T1-weighted anatomical scan with 192 slices, functional data were acquired with a T2\*-weighted echo-planar sequence (TE = 30 ms, flip angle = 90°, matrix = 92 × 92, FOV = 208 mm^2^, TR = 2080 ms) consisting of 410 volumes with 36 axial slices (thickness = 3 mm, gap = 0.3 mm, in plane resolution = 2.26 × 2.26 mm).

Functional data were preprocessed and analyzed using BrainVoyager QX (Version 2.8.4; Brain Innovation, Maastricht, the Netherlands). The first four volumes were skipped from each run to allow for magnetization equilibrium before image acquisition. Data preprocessing comprised correction for slice time errors and movement artifacts, co-registration of anatomical and functional data, normalization to fit Talairach space ([@bb0290]) and spatial smoothing (6 mm full-width at half maximum \[FWHM\] Gaussian kernel) as well as temporal filtering (high pass filter: 10 cycles per run; low pass filter: 2.8 s; linear trend removal).

The preprocessed data were analyzed using multiple linear regression of the signal time course at each voxel. The expected BOLD signal change for each condition was modeled with a canonical double-gamma hemodynamic response function (HRF). We calculated a general linear model (GLM) including two temporally distinct regressors for the anticipation interval. The HRF of the "phasic" regressor was limited to the onset (first second), whereas the "sustained" regressor was modeled over the whole anticipation interval. Additionally, the six movement parameters and the presentation of camera symbols were modeled as predictors of no interest. %-standardized predictor estimates based on voxel-wise statistical maps for each participant were calculated. Random effects analysis with adjustment for autocorrelation following a global AR(2) model across the individual predictor estimates for planned contrasts was performed. Average beta values were estimated for each subject within a-priori defined regions of interest (ROIs).

The bilateral centromedial amygdala ROI was defined on the basis of cytoarchitectonic probability maps ([@bb0015]) provided by the Anatomy toolbox ([@bb0115]). For the bilateral BNST ROI, we used a BNST mask based on a multisubject manual BNST segmentation method elaborated and provided on [neurovault.org](http://neurovault.org){#ir0005} by Theiss and colleagues ([@bb0295]). MNI-coordinates for both ROIs were transformed into Talairach space with ICBM2tal ([@bb0205]).

We calculated two-factor analysis of variance (ANOVA) with repeated measures including group affiliation (SAD patients/HC) and condition (aversive / neutral anticipation) separately for each ROI and each regressor of interest using SPSS software (Version 25.0, IBM Corp., Armonk, N.Y., USA). An alpha-level \< .05 was considered statistically significant.

For subsequent whole brain analysis, we used a cluster-based permutation (CBF) approach based on an in-house Matlab script (Release 2014a; The MathWorks, Inc., Natick, Massachusetts) to correct for multiple comparisons. This non-parametric method is assumed to result in adequate control over the familywise alpha level ([@bb0120]). We analyzed the interaction effect of group (SAD, HC) by anticipation condition (aversive, neutral) separately for phasic and sustained regressors. Voxel-level threshold was set to *p* \< .005. Testing was performed with 1000 permutations. For each permutation, individual beta maps were randomly assigned without replacement to one of the groups. Cluster mass was assessed by adding the absolute values of all *t*-values in neighboring significant voxels. The observed cluster mass was compared with the distribution of the maximum cluster mass observed in each of the 1000 permutations. Cluster masses larger or equal to the 95th percentile of the permutation distribution were considered as statistically significant (*p* \< .05). For SAD patients pearson correlation coefficients were calculated for the differences between aversive minus neutral condition mean beta estimates and the differences between aversive minus neutral ratings on a familywise error level of *p* \< .05 using SPSS software (Version 25.0, IBM Corp., Armonk, N.Y., USA).

3. Results {#s0035}
==========

3.1. Behavioral data {#s0040}
--------------------

### 3.1.1. Anticipation of "camera on" vs. "camera off" {#s0045}

Analyses of arousal, valence and anxiety ratings revealed significant main effects of group (arousal: *F*\[1,38\] = 21.03, *p* \< .001; anxiety: *F*\[1,38\] = 23.36, *p* \< .001) and condition (arousal: *F*\[1,38\] = 25.72, *p* \< .001; valence: *F*\[1,38\] = 4.83, *p* = .034; anxiety: *F*\[1,38\] = 16.77, *p* \< .001). Interaction effects of Group by Condition were found regarding valence (*F*\[1,38\] = 4.83, *p* = .034), arousal (*F*\[1,38\] = 14.00, *p* = .001) and anxiety (*F*\[1,38\] = 16.77, *p* \< .001). Post-hoc independent *t*-tests showed that the aversive anticipation cue was rated as more arousing (*t*\[27\] = 4.84, *p* \< .001) and more anxiety-inducing (*t*\[18\] = 4.78, *p* \< .001) by SAD patients compared to HC. There were no significant group differences for the neutral anticipation cue. Within-subject effects were analyzed by post-hoc paired *t*-tests revealing that SAD patients rated the aversive relative to the neutral anticipation cue as more arousing (*t*\[18\] = 4.87, *p* \< .001) and more anxiety-inducing (*t*\[18\] = 3.89, *p* = .001), whereas HC showed no differences ([Fig. 1](#f0005){ref-type="fig"}).Fig. 1Mean ratings on a 9-point Self-assessment manikin scale ([@bb0040]). Ratings for valence (1 = very unpleasant, 9 = very pleasant, with 5 = neutral), arousal (1 = not arousing, 9 = highly arousing), and anxiety (1 = not anxiety-inducing, 9 = highly anxiety-inducing) for anticipation cues (aversive = symbol associated with "camera on" condition; neutral = symbol associated with "camera off" condition) and camera condition (aversive = camera on; neutral = camera off). \*p \< .004.Fig. 1

### 3.1.2. Camera on vs. camera off {#s0050}

Analyses of arousal, valence and anxiety ratings each revealed significant main effects of group (arousal: *F*\[1,38\] = 25.28, *p* \< .001; anxiety: *F*\[1,38\] = 38.47, *p* \< .001) and condition (arousal: *F*\[1,38\] = 32.38, *p* \< .001; valence: *F*\[1,38\] = 17.46, *p* \< .001; anxiety: *F*\[1,38\] = 18.01, *p* \< .001). Interaction effects of Group by Condition were found regarding valence (*F*\[1,38\] = 8.22, *p* = .007), arousal (*F*\[1,38\] = 15.04, *p* \< .001) and anxiety (*F*\[1,38\] = 13.72, *p* = .001). Post-hoc independent *t*-tests showed that SAD patients compared to HC rated the "Camera on" symbol as more negative (*t*\[38\] = −3.40, *p* = .002), more arousing (*t*\[28\] = 5.19, *p* \< .001) and more anxiety-inducing (*t*\[21\] = 5.27, *p* \< .001). There were no significant group differences for the "Camera off" symbol. Furthermore, SAD patients rated the "Camera on" symbol relative to the "Camera off" symbol as more negative (*t*\[18\] = −3.77, *p* = .001), more arousing (*t*\[18\] = 4.94, *p* \< .001) and more anxiety-inducing (*t*\[18\] = 3.94, *p* = .001). HC showed no differences ([Fig. 1](#f0005){ref-type="fig"}).

3.2. fMRI data {#s0055}
--------------

### 3.2.1. Phasic response average ROI analysis {#s0065}

A significant group by condition interaction was revealed for the left CeA (*F*\[1,38\] = 5.67, *p* = .022) and the left BNST (*F*\[1,38\] = 8.27, *p* = .007), indicating higher activation for SAD patients than HC for aversive relative to neutral threat anticipation. All other comparisons were not significant ([Table 2](#t0010){ref-type="table"} and [Fig. 2](#f0010){ref-type="fig"}).Table 2Phasic response two-way repeated measures ROI ANOVA.Table 2ROIdfmean square*F*-value*p*-valueCeA leftcondition10.771.82.186group10.873.43.072condition x group12.405.67.022[⁎](#tf0005){ref-type="table-fn"}CeA rightcondition10.070.20.655group10.411.28.265condition x group10.120.36.552BNST leftcondition10.060.18.676group10.490.58.451condition x group12.908.27.007[⁎](#tf0005){ref-type="table-fn"}BNST rightcondition10.040.15.704group10.040.08.777condition x group10.752.53.120[^2][^3]Fig. 2Phasic and sustained responses during anticipation.Social anxiety disorder (SAD) patients compared to healthy controls (HC) showed increased phasic activation for anticipation of "camera on" relative to "camera off" in the left central nucleus of the amygdala (CeA) and the left bed nucleus of the stria terminalis (BNST). Participants across both groups showed higher sustained responses for anticipation of "camera on" relative to "camera off" in the right and left BNST. In the left column CeA and BNST ROIs are overlaid on an averaged T1 scan. Bar graphs in the second column display contrasts of parameter estimates for phasic, bar graphs in the third column for sustained anticipation (anticipation of "Camera on" vs. "Camera off"; mean ± *SE*; \*p ≤ .05; n.s., not significant). Line charts in the right column represent time courses of the percentage blood oxygenation level dependent (BOLD) signal change for anticipation of "Camera on" vs. "Camera off" averaged across all 16 s-trials and across participants per group. Shorter trials were excluded for the analysis of time course data.Fig. 2

### 3.2.2. Sustained response average ROI analysis {#s0070}

For the left BNST (*F*\[1,38\] = 4.67, *p* = .037) and the right BNST (*F*\[1,38\] = 12.79, *p* = .001) significant main effects of condition were revealed, indicating higher activation for SAD patients as well as HC for aversive compared to neutral antcipation. All other comparisons were not significant ([Table 3](#t0015){ref-type="table"} and [Fig. 2](#f0010){ref-type="fig"}).Table 3Sustained response two-way repeated measures ROI ANOVA.Table 3ROIdfMean square*F*-value*p*-valueCeA leftCondition1\< 0.0010.002.961Group10.043.00.091Condition × group10.012.07.158CeA rightCondition10.011.15.289Group10.042.22.144Condition × group10.0020.33.567BNST leftCondition10.054.67.037[⁎](#tf0010){ref-type="table-fn"}Group10.010.49.490Condition × group10.021.66.206BNST rightCondition10.0912.79.001[⁎](#tf0010){ref-type="table-fn"}Group10.0010.46.831Condition × group10.0050.73.398[^4][^5]

### 3.2.3. Whole brain analysis and correlations of fMRI and behavioral data {#s0075}

Whole brain analysis of phasic and sustained responses did not reveal any significant clusters when comparing SAD patients to HC for the contrast anticipation of aversive minus neutral events.

For SAD patients there were no significant correlations between aversive minus neutral condition mean beta estimates and the differences between aversive minus neutral ratings concerning valence, anxiety and arousal.

4. Discussion {#s0080}
=============

In the present study, we investigated BNST and CeA activity in SAD patients compared to HC during the anticipation of temporally unpredictable aversive (camera monitoring) versus neutral events. For phasic anticipatory response modeling we used the first second after cue onset and found hyperactivation of the CeA as well as the BNST for SAD patients compared to HC. Contrary to our hypothesis, there was no differential activity in the BNST during sustained threat anticipation in SAD patients compared to HC. However, we detected increased sustained activation in the BNST for aversive relative to neutral anticipation across groups.

We revealed an elevated phasic CeA response for SAD patients compared to HC when anticipating social observation. The amygdala is thought to function as a hub region ([@bb0245]) relevant in vigilance, arousal, threat detection and interpretation processes ([@bb0075]; [@bb0240]; [@bb0325]). In SAD patients, increased threat attention and vigilance ([@bb0155]), reflected by a hyperresponsive emotion processing network including the amygdala, are supposed to be essential in the presence of potentially threatening social situations ([@bb0065]; [@bb0085]; [@bb0110]; [@bb0230]). Thus, the present result might be interpreted as SAD patients perceiving the signal that a video camera is going to be switched on as more salient and arousing than HC, resulting in an initial hypervigilant reaction following cue onset. To date, none of the existing studies on threat anticipation in SAD examined a phasic anticipatory time interval as modeled in our paradigm. However, the present finding of elevated phasic amygdala activity to threat anticipation in SAD patients is in line with previous studies investigating panic disorder ([@bb0045]), post-traumatic stress disorder ([@bb0050]) and generalized anxiety disorder ([@bb0070]) pointing towards a transdiagnostically shared neural response to threatening stimuli in anxiety disorders.

Interestingly, we additionally revealed a higher phasic BNST activation in SAD patients compared to HC during anticipation of aversive compared to neutral events. Among other functions, enhanced BNST activity is supposed to reflect heightened reactivity to threat uncertainty associated with hypervigilance and increased arousal ([@bb0020]; [@bb0155]). Thus, SAD patients seem to be more affected by states of uncertainty than HC, especially when confronted with unpredictable aversive events. This is in line with theoretical models for SAD suggesting a higher Intolerance of Uncertainty (IU) level for such patients ([@bb0080]; [@bb0155]; [@bb0335]) paired with heightened vigilance and increased processing of social threat ([@bb0065]; [@bb0085]; [@bb0110]; [@bb0230]). Originally, the function of the BNST was assumed to be restricted to longer lasting, more diffuse and unpredictable threatening situations ([@bb0095]) and to be more relevant later in time course ([@bb0225]; [@bb0320]). However, recent reviews ([@bb0020]; [@bb0125]; [@bb0165]; [@bb0260]) questioned the strict temporal dissociation of amygdala and BNST and suggest instead an involvement of the BNST in both, longer lasting responses during sustained threat, and transient responses during immediate threat. In line with this suggested role of the BNST in short-term responses to threat, a recent study found phasic BNST activation during processing of short threat stimuli in a large sample of healthy volunteers ([@bb0055]). Additionally, phasic BNST activation has been found during onset of an aversive anticipation context ([@bb0005]). In the present study, SAD patients compared to HC showed an elevated phasic BNST response in the first second following anticipation onset for the aversive relative to neutral condition. This finding strongly supports the hypothesis of an early, short-term BNST involvement in threat processing and suggests that SAD patients might be hyperresponsive regarding cue onset during anticipation of aversive events.

During sustained threat anticipation, contrary to our hypothesis we did not reveal a BNST activation difference for individuals with SAD compared to HC. Across groups however, we found a higher BNST response during aversive relative to neutral anticipation. These findings suggest that both, SAD and HC respond similarly with BNST activation to threat uncertainty when regarding the whole anticipation interval. Previous studies investigating other anxiety disorders consistently reported an elevated sustained BNST activation for patients compared to HC in aversive relative to neutral situations for spider phobia ([@bb0235]; [@bb0285]), generalized anxiety disorder ([@bb0070]), post-traumatic stress disorder ([@bb0050]) and panic disorder ([@bb0045]). Thus, the present findings provide preliminary evidence for SAD specific BNST responding to threat anticipation suggesting a fast salience detection reaction ([@bb0030]; [@bb0200]) mediated by phasic BNST and CeA responses following threat-indicating cues but a less pronounced sustained response compared to HC.

To summarize, we investigated BNST and CeA involvement during anticipation of temporally unpredictable aversive (camera switched on) relative to neutral (camera switched off) events. Compared to HC, SAD patients showed elevated phasic CeA as well as BNST responses in the first second after cue onset. However, both groups did not differ regarding a more sustained time period. For the first time, we could reveal a BNST involvement during threat anticipation in SAD patients. Additionally, present results point towards a SAD-specific threat processing marked by elevated phasic but not sustained CeA and BNST responses when compared to HC.

Conflict of interest {#s0085}
====================

The authors report no conflicts of interest and no financial relationships with commercial interests.

Ethical standards {#s0090}
=================

The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.

This work was supported by the German Research Foundation (DFG: SFB/TRR 58: C07).

[^1]: Note: SAD, social anxiety disorder; HC, healthy controls; LSAS, Liebowitz Social Anxiety Scale ([@bb0275]); SPS, Social Phobia Scale ([@bb0280]); SIAS, Social Interaction Anxiety Scale ([@bb0280]); BDI, Beck Depression Inventory II ([@bb0175]); STAI-T, State-Trait Anxiety Inventory ([@bb0270]).

[^2]: Note: CeA, central nucleus of the amygdala; BNST, bed nucleus of the stria terminalis; Factor condition includes two levels (aversive and neutral anticipation); Factor group includes two levels (SAD and HC).

[^3]: p \< 0.05.

[^4]: Note: CeA, central nucleus of the amygdala; BNST, bed nucleus of the stria terminalis; Factor condition includes two levels (aversive and neutral anticipation); Factor group includes two levels (SAD and HC).

[^5]: p \< 0.05.
