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Author’s Accepted Manuscript Unsmoothed functional MRI of the human amygdala and bed nucleus of the stria terminalis during processing of emotional faces Ronald Sladky, Nicole Geissberger, Daniela M Pfabigan, Christoph Kraus, Martin Tik, Michael Woletz, Katharina Paul, Thomas Vanicek, Bastian Auer, Georg S Kranz, Claus Lamm, Rupert Lanzenberger, Christian Windischberger PII: DOI: Reference: www.elsevier.com S1053-8119(16)30746-7 http://dx.doi.org/10.1016/j.neuroimage.2016.12.024 YNIMG13645 To appear in: NeuroImage Received date: 30 July 2016 Revised date: 2 December 2016 Accepted date: 9 December 2016 Cite this article as: Ronald Sladky, Nicole Geissberger, Daniela M Pfabigan, Christoph Kraus, Martin Tik, Michael Woletz, Katharina Paul, Thomas Vanicek, Bastian Auer, Georg S Kranz, Claus Lamm, Rupert Lanzenberger and Christian Windischberger, Unsmoothed functional MRI of the human amygdala and bed nucleus of the stria terminalis during processing of emotional faces, NeuroImage, http://dx.doi.org/10.1016/j.neuroimage.2016.12.024 This is a PDF file of an unedited manuscript that has been accepted for publication. 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Sladky et al., NeuroImage Unsmoothed functional MRI of the human amygdala and bed nucleus of the stria terminalis during processing of emotional faces Ronald Sladky 1 1,2 1 3 4 1 , Nicole Geissberger , Daniela M Pfabigan , Christoph Kraus , Martin Tik , Michael 3 4 3 4 3 Woletz , Katharina Paul , Thomas Vanicek , Bastian Auer , Georg S Kranz , Claus Lamm , Rupert 4 Lanzenberger , Christian Windischberger 1 1,* MR Center of Excellence, Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Austria 2 Department of Psychiatry, Psychotherapy and Psychosomatics, Psychiatric Hospital, University of Zürich, Switzerland 3 Social, Cognitive and Affective Neuroscience Unit, Department of Basic Psychological Research and Research Methods, Faculty of Psychology, University of Vienna, Austria 4 Department of Psychiatry and Psychotherapy, Medical University of Vienna, Austria *Correspondence to: Assoc. Prof. PD DI Dr. Christian Windischberger MR Center of Excellence, Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Austria, Lazarettgasse 14, 1090 Vienna, AUSTRIA. Tel: +43 1 40400 64630. Email: [email protected] 1/27 Sladky et al., NeuroImage Abstract Functional neuroimaging of the human amygdala has been of great interest to uncover the neural underpinnings of emotions, mood, motivation, social cognition, and decision making, as well as their dysfunction in psychiatric disorders. Yet, several factors limit in vivo imaging of amygdalar function, most importantly its location deep within the temporal lobe adjacent to air-filled cavities that cause magnetic field inhomogeneities entailing signal dropouts. Additionally, the amygdala and the extended amygdalar region consist of several substructures, which have been assigned different functions and have important implications for functional and effective connectivity studies. Here we show that high-resolution ultra-high field fMRI at 7T can be used to overcome these fundamental challenges for acquisition and can meet some of the demands posed by the complex neuroanatomy and -physiology in this region. Utilizing the inherently high SNR, we use an optimized preprocessing and data analysis strategy to demonstrate that imaging of the (extended) amygdala is highly reliable and robust. Using unsmoothed single-subject data allowed us to differentiate brain activation during processing of emotional faces in the central and basolateral amygdala and, for the first time, in the bed nucleus of the stria terminalis (BNST), which is critically involved in the neural mechanisms of anxiety and threat monitoring. We also provide a quantitative assessment of single subject sensitivity, which is relevant for connectivity studies that rely on time course extraction of functionally-defined volumes of interest. Keywords (max 6). Amygdala, BNST, faces, emotions, fMRI, 7 Tesla 2/27 Sladky et al., NeuroImage 1. Introduction The amygdala is one of the most prominently researched brain structures in affective neuroscience due to its pivotal role in the neural mechanisms of emotions and affectivity (Fitzgerald et al., 2006; Ochsner et al., 2002), social cognition (Adolphs, 2010; Morris et al., 2002), fear conditioning (Rogan et al., 1997; Tovote et al., 2015), and even complex decision making in ambiguous and uncertain situations (Hsu et al., 2005; Ousdal et al., 2014). On a more general level, the amygdala can be conceptualized as a fast-acting detector for relevance and significance latent in the environment (Pessoa and Adolphs, 2010; Sander et al., 2003). In addition, the amygdala is of utmost importance for clinical neuroscience and psychiatry due to its observed dysfunctions in mood, anxiety, and a variety of psychiatric disorders (Leppanen, 2006). Unfortunately, there are three major challenges associated with in vivo investigations into human amygdalar functions and mechanisms: (1) its particular localization in the ventral brain, (2) the functional segregation of amygdalar sub nuclei and (3) the functional relevance of small subcortical structures surrounding the amygdala. However, we argue that ultra-high field fMRI at 7 Tesla provides ways to overcome some of the present limitations, offering novel research perspectives for improving our understanding of the functions and dysfunctions of the human amygdala. The aim of this study is to demonstrate 7T data quality and assess analysis results on single-subject and group level. In addition, we aim at providing a benchmark for future high and ultra-high field studies of this particular brain region to allow for feasibility evaluations and establishing an empirical foundation for the selection of the appropriate imaging method and acquisition strategy. In contrast to cortical regions, such as the visual or motor cortex, the amygdalae are located deep within the temporal lobe and thus the application of non-invasive electrophysiological methods is strongly limited. Among invasive approaches, intracranial EEG has provided 3/27 Sladky et al., NeuroImage invaluable evidence for amygdala’s role during face perception (Huijgen et al., 2015; KrolakSalmon et al., 2004; Pourtois et al., 2010). The main drawback of these studies lies, however, in the fact that they were performed only in small samples taken from clinical populations undergoing brain surgery due to medical indications (e.g., epilepsy). Generalization of these intracranial findings to an overall population is limited, as is the application of this method to healthy subjects due to its invasive nature. As a fully noninvasive alternative, functional MRI enables high-resolution imaging of cortical and subcortical regions and has become one of the most widely used methods for studying human amygdala function in healthy subjects. Nevertheless, fMRI in this brain region poses several methodological challenges. Blood oxygen level dependent (BOLD) fMRI is an imaging method that is primarily based on the contrast of susceptibility differences between oxygenated and deoxygenated hemoglobin, which serves as a well-established proxy for clustered neural activity (Logothetis, 2002). The amygdala and other ventral brain areas, however, are located in a part of the brain that is strongly affected by inhomogeneities of the MR scanner’s static magnetic field B0 due to its vicinity to air-filled cavities (i.e., sinuses) and bone structures. This leads to considerable local image distortions and signal dropouts due to intra-voxel dephasing in gradient echo planar imaging (Merboldt et al., 2001), as commonly used in fMRI. The negative effects of intra-voxel dephasing can be efficiently compensated by reducing voxel sizes, i.e. increased in-plane resolution and/or thinner slices (Morawetz et al., 2008; Robinson et al., 2004). A number of fMRI studies have used such optimized acquisition protocols to obtain high quality activation maps from the amygdala at a field strength of 3 Tesla (Derntl et al., 2009a; Derntl et al., 2009b; Habel et al., 2007; Hahn et al., 2011; Karlsson et al., 2010; Morawetz et al., 2010; Morawetz et al., 2016; Sladky et al., 2013; Sladky et al., 2012; Sladky et al., 2015a; Sladky et al., 2015b). A second challenge for successful amygdala fMRI is the fact that 4/27 Sladky et al., NeuroImage activation amplitudes in the amygdalae are considerably smaller than in their cortical counterparts. As such, moving to ultra-high magnetic fields (7T and above) provides the fundamental benefit that signal-to-noise ratio (SNR) scales approximately linear with field strength when moving from 3T to 7T, as shown repeatedly (Triantafyllou et al., 2005; van der Zwaag et al., 2009). There is, however, also an increase of artifacts originating from magnetic field inhomogeneities and physiological noise (i.e., respiratory and cardiac signal) (Hutton et al., 2011; Van de Moortele et al., 2002; Windischberger et al., 2010) and sophisticated acquisition and analysis approaches are required to achieve high-quality fMRI data. In a previous study, we have shown that the BOLD response in the amygdala is almost linearly increased at 7 Tesla compared to 3 Tesla (left amygdala: +130%, right amygdala: +80%) in a facial emotion discrimination task (Sladky et al., 2013). This increased overall sensitivity even allows for detection of small signal differences in the amygdala such as those induced by directed or averted gaze of fearful faces (van der Zwaag et al., 2012). Similar benefits of ultra-high field fMRI have been observed for the visual cortex (Hoffmann et al., 2009), the periaqueductal gray (Hahn et al., 2013), the hippocampus (Theysohn et al., 2013), and ventral parts of the language network (Geissler et al., 2014). The other major methodological challenge for non-invasive neuroscientific research on the human amygdala is its anatomical and functional segregation into subareas or nuclei. Basal and lateral amygdalar nuclei (BLA) receive neural input from unimodal and multimodal sensory cortices, thalamus, and hippocampus and project back to multimodal sensory cortices, the ventral striatum, and prefrontal regions (LeDoux, 2007). A second major nucleus is the central amygdalar region (CeA) that integrates inputs from viscero-sensory cortex and the sensory brain stem, which is involved in the processing of taste, pain, and visceral information. Output of the CeA is considered to be important for physiological changes associated with affective processes and includes arousal (via neuromodulatory systems), 5/27 Sladky et al., NeuroImage freezing behavior (via the periaqueductal gray), activation of the sympathetic and parasympathetic nervous systems and endocrine system (via the hypothalamus and nervus vagus) (LeDoux, 2007). Additionally, small nuclei surrounding the amygdala play an important role in amygdala regulation. For example, a small region of particular interest within this extended amygdala network is the bed nucleus of the stria terminalis (BNST) (Avery et al., 2014; Torrisi et al., 2015), which entertains direct anatomical connections to the CeA. Its involvement in sustained anxiety as well as reward-processing and addiction (Avery et al., 2016), which has also been observed in animal models, has important implications for clinical neuroscience and psychiatric basic research in humans (Avery et al., 2016; Lebow and Chen, 2016). In humans, however, most fMRI findings are mainly at the level of the whole amygdalar region rather than its individual nuclei (LeDoux, 2007). The reason for this is to a large degree the limited attainable spatial resolution of fMRI at field strengths of 3T and below and, more importantly, the limited signal-to-noise ratio (SNR). Moreover, a common preprocessing technique to increase SNR of fMRI data is the use of spatial smoothing (e.g., using Gaussian kernels with sizes of 6-12 mm FWHM), which additionally reduces spatial specificity (Tabelow et al., 2009). Without the required spatial accuracy and sufficient SNR, it is not possible to properly differentiate individual contributions of amygdala subareas or reliably detect activation within extra amygdalar structures as small as the BNST. The motivation of the present study was to investigate and quantify the potential of ultra-high field, high-resolution fMRI of the human amygdala sub nuclei. We assessed the feasibility of fMRI group analyses using unsmoothed single subject data for a complex task that involves processing of emotional faces. Using independent anatomical masks, we quantified individual signal changes within the BLA and CeA. Additionally, we explored whether the acquisition setup presented herein also allows for the detection of task activation in the 6/27 Sladky et al., NeuroImage BNST. Finally, we assessed the single-subject reliability of the observed task-dependent activations within our volumes of interest, which could be crucial for feasibility evaluation and power estimation required for future functional and effective connectivity studies in healthy subjects and also clinical populations. 7/27 Sladky et al., NeuroImage 2. Participants and Methods 2.1. Study population 38 healthy volunteers (18f/20m, mean age ± SD: 27.5±6.8 years) were recruited for participation in the fMRI study from the general public via billboard advertising. All subjects underwent medical examination and an interview with an experienced psychiatrist including the Structural Clinical Interview for DSM-IV (SCID) to rule out any psychiatric, neurological and physical disorders. Further exclusion criteria were metal implants, past or present substance abuse, intake of psychopharmacological medication within the last three months and pregnancy (as assessed with a drug-urine and a urine human chorionic gonadotropin pregnancy test, respectively). Written informed consent was obtained from each subject after detailed explanation of the study protocol and subjects received reimbursement for their participation. The study was approved by the ethics committee of the Medical University of Vienna and the General Hospital of Vienna, Austria, and was performed in accordance with the Declaration of Helsinki (1964) including current revisions. 2.2. 7T functional magnetic resonance imaging protocol Measurements were performed on a MAGNETOM 7 Tesla whole-body MR scanner (Siemens Medical, Erlangen, Germany) at the MR Centre of Excellence, Medical University of Vienna. A 32-channel head coil (Nova Medical, USA) and the CMRR multiband EPI sequences (Moeller et al., 2010) were used for data acquisition (245 whole-brain volumes, repetition time TR=1.4s, echo time TE=23ms, flip angle 62°). Signal losses in the amygdala region and orbitofrontal cortex were minimized by acquiring fMRI data with very high spatial resolution: 78 slices with 1mm slice thickness (0.25mm gap), in plane resolution 1.5×1.5mm2 (Ganger et al., 2015). 2.3. Functional MRI task 8/27 Sladky et al., NeuroImage Subjects were presented with a well-established emotion discrimination task (Hariri et al., 2002). When presented with a triplet of emotional faces, subjects were instructed to indicate by pressing a button whether the left or the right face matches the emotional valence of the reference face presented in the middle. The presented faces were either neutral or expressed anger, disgust, fear, happiness, sadness, surprise, and contempt. All trials were self-paced. As a control condition, an object discrimination task was used, where subjects had to match polygons that were superimposed on a skin-colored, circular background image (Figure 1). All trials were presented in blocks of 20 second duration, alternating faces and object blocks, with 20 second baseline (crosshair) blocks in-between. Each condition was repeated four times, resulting in a total task duration of 5 minutes, 40 seconds. Each trial was shown for at least 2 seconds and until a response was given via button press. Face stimuli were obtained from the Radboud Faces Database (Langner et al., 2010) and object stimuli were created in house. Figure 1. Emotion and object discrimination task. Each task condition was presented four times in alternating blocks (duration 20s). Subjects had to decide by button press if the left or right face/object matches the one in the center. 2.4. Preprocessing and general linear model (GLM) analysis of fMRI data 9/27 Sladky et al., NeuroImage Data were slice-timing corrected (FSL, http://fsl.fmrib.ox.ac.uk/fsl) (Sladky et al., 2011), biasfield corrected (ANTs, (Avants et al., 2011)), realigned (FSL), and non-linearly normalized to MNI space (ANTs, final resolution 1.5×1.5×1.5 mm3) using a custom pre-processing pipeline. Importantly, except the resampling to a marginally lower resolution during normalization, we did not additionally smooth the data to preserve the spatial specificity in the main analysis. Additionally, to demonstrate the effect of smoothing, VOI analyses were repeated using data that has been smoothed with 3mm and 6mm FWHM Gaussian kernels. Data analyses were performed in SPM (SPM12, build 6225, http://www.fil.ion.ucl.ac.uk/spm/software/spm12/) using general linear model analysis. Single-subject GLM analyses included boxcar functions convolved with SPM’s canonical hemodynamic response function to model the effects of emotional faces and objects. Additionally, realignment parameter estimates and the first 6 principal components of white matter and ventricle time courses were added as nuisance regressors (Sladky et al., 2013; Weissenbacher et al., 2009) to model for signal variability unrelated to neural activity. Second-level random effects group analysis consisted of a onesample t-test on the individual contrasts between face and object discrimination (FACES>OBJECTS). Significance threshold for the resulting statistical parametric map was set to p<0.001 (voxel-wise threshold) and p<0.05 FWE cluster-level corrected, resulting in a minimum cluster size of k=18 voxels. 2.5. Volume of interest analysis To allow for an unbiased analysis, independent masks from the literature were used for volume of interest (VOI) analysis. For definition of the BLA and CeA, we created masks based on a high resolution amygdala parcellation by Tyszka and Pauli (2016), where the amygdala was segmented into 9 sub nuclei. For the main analysis, the individual sub nuclei were grouped into three clusters, given that some of the sub region masks could be too small to be actually relevant for current fMRI studies, given the spatial extent of the BOLD 10/27 Sladky et al., NeuroImage response and the typical voxel sizes that are used for image acquisition. In accordance with the descriptions and definitions in their publication, we grouped the sub nuclei into: (1) Basolateral amygdala (BLA) comprising lateral, basolateral, paralaminar, accessory basal nuclei, (2) Central amygdala (CeA) comprising medial and cortical area, the nucleus of the lateral olfactory tract and amygdalohippocampal area, as well as the periamygdaloid cortex, and (3) the remaining amygdalar nuclei (OTH) comprising central, anterior amygdaloid area, and amygdalostriatal transition area. However, to also report the detailed fMRI effects within the small individual amygdalar sub structures, all 9 masks were used in a subsequent VOI analysis. To localize the BNST, we used an anatomically-defined mask by Torrisi et al. (2015). 11/27 Sladky et al., NeuroImage 3. Results 3.1. Behavioral data On average, each subject solved 39.41 (SD=1.40) FACES and 39.49 (SD=1.52) OBJECTS trials. Between conditions there was a moderate but significant difference in accuracy rates (t(37)=3.28, Cohen’s d=0.53, p=0.006), 92.93% (SD=4.61%) for FACES and 95.23% (SD=5.72%) for OBJECTS, and response times (t(37)=6.3, Cohen’s d=1.02, p<0.001), 1381ms (SD=323ms) for FACES and 1160ms (SD=287ms) for OBJECTS. 3.2. fMRI whole-brain data For the target contrast FACES>OBJECTS highest activation was observed within the extended amygdalar region (k=7579 voxels) with peak voxels in the left (T-20, -10, -16 =15.41) and the right amygdala (T21, -8, -14 = 14.75). Note that voxel numbers are considerably high compared to standard analyses because of the very high spatial resolution. The clusters with the second and third strongest activation were found in the right fusiform gyrus (k=1169 voxels) with a peak at T48 -50 -30 = 7.03 and superior temporal lobe (k=4283 voxels) with a peak at T48 -44 14 = 6.43. Large clustered activations were also observed in the left fusiform gyrus and superior temporal cortex, additionally in other areas in the visual cortex, dorsoand ventrolateral PFC, as well as dorso- and ventromedial PFC. For a detailed report see Table 1, Figure 2 for axial slices, and Figure 3 for coronal slices. Importantly, we also observed significant brain activation within a cluster (k=60 voxel) with peaks at T-4, -1, -2 =6.32 and T6, 0, -1 =5.56 that encompasses the BNST region, as defined by the anatomical mask. Bilateral activations within the amygdala masks for BLA, CeA and OTH were also reliably observable on group level (Figure 2, 3). 12/27 Sladky et al., NeuroImage Figure 2. Mosaic of selected axial slices (z=-29.5 to +3.5 mm [MNI]) of the SPM t-map for FACES > OBJECTS. Results are p<0.05 FWE cluster-level corrected (k=18 voxels minimum cluster size, p<0.001 whole-brain threshold). Overlay of anatomical masks for basolateral amygdala (BLA, blue), central amygdala (CeA, green), and other amygdalar regions (OTH, orange). 13/27 Sladky et al., NeuroImage Figure 3. Detailed view of coronal slices of the SPM t-map for FACES > OBJECTS. Results are p<0.05 FWE cluster-level corrected (k=18 voxels minimum cluster size, p<0.001 whole-brain threshold). Overlay of anatomical masks for basolateral amygdala (BLA, blue), central amygdala (CeA, green), other amygdalar nuclei (OTH, orange) and bed nucleus of the stria terminalis (BNST, yellow). 3.3. Volume of interest analyses To quantify the effects of face in contrast to object discrimination within bilateral BLA, CeA, OTH and BNST we conducted an additional VOI analysis. First, we assessed the average percent signal change within the individual anatomical regions and found significant brain activation in all VOIs. Median, interquartile range and distribution of the individual singlesubject mean percent signal changes are displayed in Figure 4 for unsmoothed data and data that has been spatially smoothed with a Gaussian kernel of 3 mm and 6 mm FWHM. The most prominent effect of smoothing was a reduction of effect sizes within all VOIs. Using the individual amygdala sub nuclei masks, revealed that the strongest group activation was found in the left and right paralaminar amygdala nucleus (BLV). After smoothing this effect is no longer observed. A detailed visualization of the sub nuclear activation is provided in the supplementary material (Supplementary Figure S1). 14/27 Sladky et al., NeuroImage Figure 4. Violin plots of percent signal changes in left and right VOIs for FACES>OBJECTS. Results are displayed for different levels of spatial smoothing (i.e., unsmoothed, 3 mm and 6 mm FWHM). Complete distribution of single subject results (averaged within anatomical VOI) is plotted in the background, white dot indicates median, and black bars represents the interquartile range. Left and right basolateral amygdala (BLA, blue), central amygdala (CeA, green), other amygdalar nuclei (OTH, orange), and bed nucleus of the stria terminalis (BNST, yellow). A more detailed report of the activation within the amygdala sub nuclei is provided in Supplementary Figure S1. In addition, methods that use functional localizers, such as a variety of real-time fMRI neurofeedback setups and DCM and PPI data analysis strategies usually rely on extracting 15/27 Sladky et al., NeuroImage time courses from activated voxels within a designated brain region. To provide this information for future studies, the spatial extent of the activation was determined by quantifying the percentage of significantly activated voxels within the VOIs per subject relative to the thresholds of p<0.999, 0.500, 0.100, 0.050, 0.010, 0.005, and 0.001 (Figure 5). The most robust activations were observed in the CeA, followed by BLA, and BNST. A crucial question for methods that rely on extracting time courses from task-active voxels (e.g., dynamic causal modeling) is the number of individual subjects from a study population that exhibit significant activations within a given VOI. Using a common threshold of p<0.05, significant voxels were observed in all 38 subjects for bilateral CeA, and the left BLA, in 37 subjects for the right BLA and the left BNST, and 36 subjects in the right BNST. With a more liberal threshold of p<0.1, significant voxels for FACES>OBJECTS were observed in every subject in all VOIs, except the right amygdala nuclei that are not part of the BLN or CMN clusters (Figure 5). Figure 5. Percentage of significantly activated voxels for basolateral amygdala (BLA), central amygdala (CeA), other amygdalar nuclei (OTH), and bed nucleus of the stria terminalis (BNST). Color coding indicates the percentage of significantly activated voxels within the respective VOI. Single-subject sensitivity was consistently high within each VOI. E.g., for a typical threshold of p<0.05 significant voxels were detected in all BLA, CeA, and BNST VOIs in 36 out of 38 subjects, X-axis shows typical single-subject thresholds and Y-axis indicates number of subjects. 16/27 Sladky et al., NeuroImage 4. Discussion Using 7 Tesla ultra-high field MR we performed high-resolution functional MRI of small nuclei within the (extended) amygdalar region in 38 healthy subjects performing an emotion discrimination task. This is the first study that robustly detected activation in the amygdalar region using unsmoothed fMRI data at 7T. Using this dataset with high spatial accuracy, we were able to differentiate the BOLD response towards emotional faces (in contrast to object discrimination) within the CeA and BLA. In addition, we show for the first time in humans BNST activation that is related to emotional faces. Beyond the ability to detect BOLD responses in small anatomical structures, analysis of unsmoothed data entails two additional fundamental advantages that are particularly relevant for functional and effective connectivity studies. First, from a methodological perspective, unsmoothed data with sufficient SNR allows for a more accurate (e.g., anatomical or functional) definition of task-active regions or voxels that can be used for time course extraction. There is striking evidence that inappropriate definition of VOIs introduces biases in the time series that are ‘extremely damaging to network estimation’ (Smith et al., 2011). Importantly, these biases do not only degrade the sensitivity in connectivity analysis to some degree. They also introduce the risk of incorrect estimations of connectivity strength, direction, and network architecture. Second, there are important anatomical differences between the nuclei in the amygdalar region that also reflect fundamental functional differences as research investigating intraamygdala neural mechanisms in rodents has shown (LeDoux, 2007; Pitkanen et al., 1997). Recent studies were able to uncover a microcircuit of GABA-erg neurons within CeA playing an important role in encoding and gating of fear responses towards conditioned stimuli (Ciocchi, 2010; Haubensak et al., 2010). These findings have important implications for future research on the mechanisms of fear learning and reversal in humans. 17/27 Sladky et al., NeuroImage The BNST, on the other hand, has shown to mediate sustained anxiety processes in rodents (Davis et al., 1997; Sullivan et al., 2004; Waddell et al., 2006) and non-human primates (Kalin et al., 2005). Increasing evidence for a similar role in humans is currently being established (Avery et al., 2016) and supported by functional connectivity studies (Avery et al., 2014; Torrisi et al., 2015). BNST BOLD response to emotional faces was lower than in CeA and BLA. There are methodological explanations such as the smaller size of this brain structure and that the employed masks encompassed to a larger degree the lateral portions of the BNST (Torrisi et al., 2015), while the focus of our activation was more located in the medial part. On the other hand, the paradigm that we used is not particularly designed to induce anxiety, sustained uncertainty, and long durations of threat, which are typically associated with BNST function. However, the fact that we observed significant BNST activation indicates that emotional faces can be used to induce BNST activation. This is important for clinical studies on, e.g., anxiety and affective disorders, where emotional faces are frequently used. Considering BNST’s role in anxiety, we could expect increased activation towards emotional faces, e.g., in patients with social anxiety disorders. On the other hand, our results demonstrate the feasibility to investigate task-related BOLD effects in the BNST in future studies where induction of anxiety or longer phases of threat could be used as experimental paradigms. Regarding the neurobiological mechanisms relevant for clinical research, BNST catecholaminergic mechanisms, involving dopamine and norepinephrine, are assumed to be influenced by a large variety of commonly administered antidepressants (Cadeddu et al., 2014). Previous studies have already shown that, e.g., the selective serotonin reuptake inhibitor citalopram can increase downregulation of the amygdala by the orbitofrontal cortex (Sladky et al., 2015b), which is highly relevant, e.g., in social anxiety disorders where this down regulation is significantly decreased compared to healthy participants (Sladky et al., 18/27 Sladky et al., NeuroImage 2015a). By using high-resolution fMRI data, future studies on effective connectivity can entertain more refined network models that allow for a more detailed estimation of neuronal function and dysfunction. These models with improved biological plausibility would then allow for a better translation of findings from animal models to non-invasive human (clinical) neuroscience. There are some limitations to the present study that can motivate future research directions. First, the stimulation protocol that was used in this study is a very robust and reliable paradigm to induce BOLD response in the amygdala region. However, it would be interesting to use more complex designs to illuminate the individual functional contributions of the different amygdala sub regions and the BNST in emotional face processing. Additionally, we were not able to investigate the effects of explicit and implicit emotion regulation strategies that were used by the participants using this simple paradigm. Next, including psychiatric patients into the study population would be of high interest, as functional deviations that might be characteristic for certain disorders can be expected in amygdala sub nuclei and BNST. Then, regarding data acquisition, it has to be noted that we decided to use wholebrain coverage. Studies that focus only the amygdala and its sub nuclei could opt for partial coverage in order to achieve even higher spatial and temporal resolution. Finally, there is an increasing number of connectivity studies that demonstrate the benefits of high resolution 7T data for the non-invasive in vivo investigation of neurobiological mechanisms in subcortical regions (e.g., Metzger et al., 2013; Torrisi et al., 2015; Walter et al., 2008), which clearly motivates additional studies on functional and effective connectivity within this network. In summary, this study has demonstrated that ultra-high field fMRI in combination with optimized data acquisition and preprocessing protocols allows for robust group analysis based on unsmoothed, high-resolution single-subject data. We were able to discriminate the individual contributions of CeA and BLA to the amygdalar BOLD signal in response to 19/27 Sladky et al., NeuroImage emotional faces. Additionally, we observed face-related neural activation in the BNST, which has important implications for future human in vivo studies on amygdala regulation, in general, and specifically its role in anxiety and addiction disorders. Finally, we have demonstrated that single-subject sensitivity of unsmoothed data is sufficient to allow for reliable detection of task-activated voxels within our VOIs. These findings can be used as a motivation for and justification of the feasibility of future time-series-based connectivity studies within the extended amygdala. Disclosure and Conflict of Interest The authors declare no financial conflict of interest in the context of this study. This study was supported by the research cluster MMI-CNS, funded by the Medical University of Vienna and University of Vienna, Austria (FA103FC001) and by the CREAM project by the European Commission under Grant Agreement (FP7-ICT-612022). This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. 20/27 Sladky et al., NeuroImage References Adolphs, R., 2010. What does the amygdala contribute to social cognition? Ann N Y Acad Sci 1191, 42-61. Avants, B.B., Tustison, N.J., Song, G., Cook, P.A., Klein, A., Gee, J.C., 2011. A reproducible evaluation of ANTs similarity metric performance in brain image registration. Neuroimage 54, 2033-2044. Avery, S.N., Clauss, J.A., Blackford, J.U., 2016. The Human BNST: Functional Role in Anxiety and Addiction. 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T x y <0.001 <0.001 7579 <0.001 <0.001 15.41 -20 -10 -16 Left Amygdala <0.001 <0.001 14.75 21 -8 -14 Right Amygdala <0.001 <0.001 10.39 15 -6 -25 Right Parahippocampal Gyrus <0.001 <0.001 10.30 48 -50 -30 Right Fusiform Gyrus <0.001 <0.001 10.03 45 -60 -24 Right Fusiform Gyrus <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 1169 4283 2670 3346 809 543 322 784 248 2650 674 z Talairach-Tournoux Atlas / VOI masks 0.008 0.034 6.53 39 -64 -19 Right Fusiform Gyrus <0.001 <0.001 8.82 48 -44 14 Right Superior Temporal Gyrus 0.001 <0.001 8.07 48 -68 18 Right Middle Temporal Gyrus 0.001 0.001 7.69 58 -46 16 Right Superior Temporal Gyrus <0.001 <0.001 8.79 -2 -76 4 Left Cuneus 0.003 0.007 7.05 3 -68 12 Right PCC 0.004 0.007 7.02 -21 -58 -7 Left Parahippocampal Gyrus <0.001 <0.001 8.48 -42 18 26 Left Middle Frontal Gyrus 0.001 0.001 7.74 -48 24 23 Left Inferior Frontal Gyrus 0.002 0.001 7.56 -33 11 28 Left Middle Frontal Gyrus 0.002 0.002 7.54 -14 65 30 Left Superior Frontal Gyrus 0.006 0.017 6.76 -8 50 50 Left Superior Frontal Gyrus 0.011 0.062 6.33 -8 59 38 Left Superior Frontal Gyrus 0.002 0.002 7.42 -50 -49 -30 Left Fusiform Gyrus 0.011 0.054 6.37 -48 -56 -26 Left Fusiform Gyrus 0.014 0.111 6.15 -45 -64 -19 Left Fusiform Gyrus 0.006 0.020 6.70 48 5 56 Right Middle Frontal Gyrus 0.014 0.113 6.14 38 2 42 Right Middle Frontal Gyrus 0.036 0.733 5.54 40 -1 65 Right Middle Frontal Gyrus 0.006 0.020 6.70 0 -60 35 Left Precuneus 0.027 0.415 5.72 4 -58 26 Right Precuneus 0.031 0.560 5.63 3 -60 46 Right Precuneus 0.006 0.022 6.66 6 20 62 Right Superior Frontal Gyrus 0.021 0.258 5.88 4 8 60 Right Superior Frontal Gyrus 0.105 1.000 4.88 4 18 71 Right Superior Frontal Gyrus 0.007 0.023 6.65 -64 -52 10 Left Superior Temporal Gyrus 0.008 0.029 6.57 -45 -56 20 Left Superior Temporal Gyrus 0.010 0.046 6.43 -48 -36 4 Left Superior Temporal Gyrus 0.010 0.050 6.40 3 41 25/27 -22 Right Orbital Gyru Sladky et al., NeuroImage <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 43 259 60 0.013 0.093 6.20 -8 26 -24 Left Rectal Gyrus 0.014 0.123 6.11 -8 34 -19 Left Medial Frontal Gyrus 0.011 0.052 6.39 -8 -10 6 Left Thalamus 0.545 1.000 3.80 -3 -18 5 Left Thalamus 0.011 0.060 6.34 -38 5 44 Left Middle Frontal Gyrus 0.044 0.997 5.44 -45 10 52 Left Middle Frontal Gyrus 0.168 1.000 4.59 -36 0 36 Left Inferior Frontal Gyrus 0.011 0.064 6.32 -4 -1 -2 Left BNST 0.035 0.692 5.56 6 0 -1 Right BNST 0.115 1.000 4.82 3 -2 -12 Right Hypothalamus 0.039 <0.001 18 0.014 0.120 6.12 30 28 -2 <0.001 <0.001 85 0.023 0.313 5.81 -52 2 -22 Left Middle Temporal Gyrus 0.106 1.000 4.88 -52 -7 -18 Left Middle Temporal Gyrus 0.425 1.000 3.99 -54 0 -13 Left Middle Temporal Gyrus Right Inferior Frontal Gyrus <0.001 <0.001 76 0.023 0.326 5.80 0 -13 38 Left Cingulate Gyrus <0.001 <0.001 422 0.024 0.348 5.78 -2 29 50 Left Superior Frontal Gyrus 0.030 0.496 5.67 -9 29 65 Left Superior Frontal Gyrus 0.040 0.872 5.48 -3 17 58 Left Superior Frontal Gyrus 0.028 0.451 5.70 8 46 40 Right Medial Frontal Gyrus 0.049 0.999 5.37 3 52 35 Right Superior Frontal Gyrus 0.236 1.000 4.37 4 60 35 Right Superior Frontal Gyrus 0.045 0.998 5.41 2 62 -12 Right Medial Frontal Gyrus 0.153 1.000 4.65 3 62 -20 Right Superior Frontal Gyrus 0.049 0.999 5.37 57 -52 -8 Right Middle Temporal Gyrus 0.242 1.000 4.35 64 -54 -6 Right Middle Temporal Gyrus 0.049 0.999 5.36 -44 18 -38 Left Superior Temporal Gyrus 0.201 1.000 4.47 -44 10 -40 Left Middle Temporal Gyrus -36 Left Superior Temporal Gyrus <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 98 55 77 129 0.323 1.000 4.18 -38 14 <0.001 <0.001 63 0.088 1.000 5.00 15 -90 0.039 <0.001 18 0.110 1.000 4.85 0 -62 -36 Cerebellum 0.001 <0.001 31 0.118 1.000 4.81 18 -74 24 Right Precuneus 0.001 <0.001 28 0.143 1.000 4.69 -18 -73 22 Left Precuneus 0.646 1.000 3.67 -18 -79 30 Left Cuneus -9 -94 0 Left Cuneus 5 Right Cuneus <0.001 <0.001 33 0.149 1.000 4.66 0.005 <0.001 24 0.204 1.000 4.47 10 -14 8 Right Thalamus 0.001 <0.001 29 0.242 1.000 4.35 -18 -46 -6 Left Parahippocampal Gyrus 0.004 <0.001 25 0.435 1.000 3.97 16 -70 29 Right Precuneus 0.449 1.000 3.94 18 -80 34 Right Precuneus 26/27 Sladky et al., NeuroImage Highlights (3-5. max 85 characters). 7 Tesla functional MRI of emotional face discrimination task in 38 healthy subjects Unsmoothed single-subject data applicable for whole-brain group analysis Differentiation of activation in central and basolateral amygdala Significant effects in bed nucleus of stria terminalis, a key region for anxiety Robust effects also on single-subject level, relevant for connectivity analyses 27/27