Accepted Manuscript

Title: Networks of phobic fear: Functional connectivity shifts

in two subtypes of specific phobia

Authors: Maria R. Stefanescu, Ralph J. Endres, Kevin Hilbert,

Hans-Ulrich Wittchen, Ulrike Lueken

PII: S0304-3940(17)30857-1
DOI: https://doi.org/10.1016/j.neulet.2017.10.031
Reference: NSL 33175

To appear in: Neuroscience Letters

Received date: 10-7-2017

Revised date: 16-10-2017
Accepted date: 17-10-2017

Please cite this article as: Maria R.Stefanescu, Ralph J.Endres, Kevin
Hilbert, Hans-Ulrich Wittchen, Ulrike Lueken, Networks of phobic fear:
Functional connectivity shifts in two subtypes of specific phobia, Neuroscience
Letters https://doi.org/10.1016/j.neulet.2017.10.031

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 1

Networks of phobic fear:

Functional connectivity shifts in two subtypes of specific phobia

Authors: Maria R. Stefanescu1*, Ralph J. Endres1*, Kevin Hilbert2, 3, Hans-Ulrich

Wittchen3,4, and Ulrike Lueken1,3,4

1
Center of Mental Health, Department of Psychiatry, Psychosomatics and Psychotherapy,
University Hospital of Würzburg, Margarete-Höppel-Platz 1, 97080 Würzburg, Germany
2
Behavioral Epidemiology, Institute of Clinical Psychology and Psychotherapy, Technische
Universität Dresden, Chemnitzer Straße 46, 01187 Dresden, Germany
3
Neuroimaging Center, Technische Universität Dresden, Chemnitzer Straße 46a, 01187
Dresden, Germany
4
Institute of Clinical Psychology and Psychotherapy, Technische Universität Dresden,
Chemnitzer Straße 46, 01187 Dresden, Germany

*Shared first Co-Authorship

Running title: Functional connectivity in two subtypes of specific phobia

Corresponding author:
Maria R. Stefanescu, PhD
Department of Psychiatry, Psychosomatics, and Psychotherapy
University Hospital of Würzburg
Margarete-Höppel-Platz 1
D-97080 Würzburg, Germany
Phone: +49 (931) 201-46433
Fax: +49 (931) 201-646391
E-Mail: Stefanescu_M@ukw.de

Ralph Julian Endres: ralph.j.endres@stud-mail.uni-wuerzburg.de

Kevin Hilbert: kevin.hilbert@tu-dresden.de
Hans-Ulrich Wittchen: hans-ulrich.wittchen@tu-dresden.de
Ulrike Lueken: Lueken_U@ukw.de
 2

Highlights:

 Phobic fear may be associated with connectivity in emotion-processing circuits.

 We found a positive coupling between para-limbic regions in phobia groups only.

 The observed negative connectivity in controls might represent fear inhibition.

 Stimulus modality may alter functional brain connectivity in dental phobia.

Abstract

Anxiety disorders can be conceptualized by an abnormal interplay of emotion-processing brain

circuits; however, knowledge of brain connectivity measures in specific phobia is still limited.

To explore functional interactions within selected fear-circuitry structures (anterior cingulate

cortex (ACC), amygdala, insula), we re-examined three task-based fMRI studies using a

symptom provocation approach (n = 94 subjects in total) on two different phobia subtypes

(animal subtype as represented by snake phobia (SP) and blood-injection-injury subtype as

represented by dental phobia (DP)), and a non-phobic healthy control group (HC). Functional

connectivity (FC) analyses detected a negative coupling between the amygdala and the ACC in

HC for both classes of phobic stimuli, while SP and DP lacked this inhibitory relationship

during visual stimulus presentation. However, a negative FC between the insula and the

amygdala was observed in DP during visual symptom provocation, which reversed to a positive

FC under auditory symptom provocation pointing to effects depending on stimulus modality in

DP. SP showed significantly higher FC towards snake-anxiety eliciting stimuli than HC on an

average measure of FC, while DP showed a similar pattern under auditory stimulation only.

These findings altogether indicate FC shifts during symptom provocation in specific phobia

possibly reflecting impaired emotion regulation processes within fear-circuitry networks. FC

 3

hence could represent a prime target for neuroscience-informed augmentation strategies when

treating pathological forms of fear.

Keywords: specific phobia; functional connectivity; fear regulation; insula; amygdala; anterior

cingulate cortex

1. Introduction

Sufficient fear inhibition requires a well-balanced functional interplay of emotion-processing

regions such as the amygdala, insula and the anterior cingulate cortex (ACC) [1,2]. These fear-

circuitry regions also play crucial roles in specific phobia [3–5]. Altered brain connectivity

within these structures has been previously shown to feature a variety of anxiety disorders (e.g.

generalized anxiety disorder [6] and social anxiety disorder [7]) whereas findings for specific

phobia as a model disorder for fear are rare and inconsistent [8,9].

Due to various findings describing altered ACC, insula and amygdala activity during phobic

stimulus processing [3,4] deficiencies in top-down emotion regulation might contribute to

specific phobia´s pathophysiology [10] and have been hypothesized as a principal target for

behavioral interventions such as exposure therapy [11] . Particularly, the ACC/ the medial

prefrontal cortex has been suggested to exert an inhibitory influence on amygdala activity

[12,13]. One study so far observed a negative functional connectivity (FC) between the

amygdala and the ACC in spider and snake phobics (SP) only during a neutral condition

suggesting a functional decoupling during phobogenic symptom provocation [10]. Regarding

dental phobia (DP), one study [9] revealed a positive coupling of the ACC and the amygdala in

healty controls (HC) compared to phobics during visual symptom provocation.

 4

The amygdala and insula are associated with clinical forms of anxiety [4]. Both are

simultaneously active during states of anxiety in healthy subjects [14], in which case their

connectivity contributes to the normative expression of state and trait anxiety [15].

Additionally, the insula might represent a functional relay transmitting interoceptive

information to the amygdala and ACC to modulate behavioral responses [16–18].

The ACC – insula circuitry is considered to modulate the integration of interoceptive, emotional

salient information in order to generate a subjective representation of the body [17,19].

Moreover, this circuitry might contribute toward an integrated awareness of cognitive, affective

and physical aspects to prepare for responses to a given stimulus including the initiation of

autonomic arousal [20,21]. Both regions are linked to attentional direction, salience processing

and anticipatory anxiety [17,22–24]. It is noteworthy that both structures are involved in disgust

processing [25,26] as well as in pain anticipation and modulation [27], which are key features

of DP [28].

The present study aimed to further investigate the role of functional brain networks during the

processing of phobogenic stimuli by exploring FC in two subtypes of specific phobia (DP and

SP) as well as in non-phobic controls. We re-analyzed three prior studies examining

neurofunctional correlates of specific phobia using task designs with a symptom provocation

approach and conducted ROI-to-ROI FC analyses between the bilateral ACC, amygdala and

insula as key hubs within the fear-circuitry. We hypothesized heightened FC when processing

phobic stimuli in phobic groups, particularly in SP due to a characteristically strong activation

of fear-circuitry structures in this subtype [29,30]. While these regions are less active in DP

during visual symptom provocation, auditory stimuli might alter the neural response [31]. Thus,

we expected a positive FC during auditory, but not during visual dental stimulus presentation

in DP.
 5

2. Methods

2.1 Participants

The used material is based on three independent studies: dataset 1 (D1) from Lueken et al. [29],

dataset 2 (D2) from Lueken et al. [30], and dataset 3 (D3) from Hilbert et al. [31]. D1 included

data of 41 subjects (12 DP, 12 SP, 17 HC), D2 included data of 39 subjects (13 DP, 13 SP, 13

HC) and D3 of 26 subjects (13 DP, 13 HC) of whom the HC group was overlapping with D2.

The total available sample size was n = 94.

Subjects were recruited using an online screening tool. Established cutoffs of 20 or more points

in the Snake Anxiety Questionnaire (indicating clinically relevant SP [32]) and of 76 points in

the Dental Fear Survey (indicating severe DP [33]) were applied to select phobic subjects.

Subjects scoring in the lower quartiles of both questionnaires were selected as HC. Subjects

scoring above cut-offs for both questionnaires were excluded. Further exclusion criteria were

either MRI-related or neurological diseases, psychotropic medication, a diagnosis according to

DSM-IV-TR criteria [34] of psychotic, bipolar, or obsessive-compulsive disorders, post-

traumatic stress disorder, severe depressive disorders or substance dependence (except

nicotine). After obtaining written informed consent, psychiatric exclusion criteria were assessed

by the Composite International Diagnostic Interview (CIDI) [35]. Additionally, subjects were

characterized via questionnaires on depressive symptoms (Beck Depression Inventory, [38])

and anxiety sensitivity (Anxiety Sensitivity Index, [39]). The study protocols of the original

studies were approved by the ethics committee of the Technische Universität Dresden.
 6

2.2 Experimental procedure

The experimental procedure is briefly described below, for additional information, please refer

to D1 [29], D2 [30] and D3 [31].

D1: The fMRI task consisted of 40 videos with a length of 15 seconds that were presented in a

block-design. The sequences were based on a validated video set by Lueken et al. [38]. It

depicted ecologically valid scenarios of first-person encounters with phobic stimuli such as a

dental treatment and respective neutral conditions providing a baseline for non-anxiety specific

processes. Altogether four stimuli conditions (snake anxiety: SA; snake neutral: SN; dental

anxiety: DA; dental neutral: DN) with 10 sequences each were used. Videos were shown in a

pseudo-randomized order in which no condition was presented more than two times after one

another.

D2: An event-related design to investigate anticipation and perception processes using a cued

presentation of phobic stimuli was applied. Two pictures per condition from the videos

mentioned for D1 were extracted and presented for a perception phase (PP) of 1.250

milliseconds. Preceding the PP, an anticipation phase (AP) was inserted that announced the

following stimulus content with a letter (“Z” for dental stimulus (German: “Zahnarzt”), “S” for

snake stimulus and “N” for neutral stimuli). An AP interval between 5 and 10 seconds and a

jittered inter-stimulus interval (5.6, 9.3, 11.9 s) was applied to avoid prediction of PP onset.

Eight conditions were presented in randomized order (AP: snake neutral (AP-SN), snake

anxiety, (AP-SA), dental neutral (AP-DN) and dental anxiety (AP-DA) stimuli; PP: snake

neutral (PP-SN), snake anxiety (PP-SA), dental neutral (PP-DN) and dental anxiety (PP-DA)

stimuli). In the present analysis, we only focused on data from the PP.

D3: The paradigm was conducted in DP and HC and aimed at comparing different stimulus
 7

modalities (audio and video stimuli) in a block-design. Visual stimuli were based on the video

set by Lueken et al. [38] while auditory stimuli were taken from a publicly available databank

and included sounds such as a dental drill. The task comprised four conditions of 15 seconds

length each (dental audio neutral (DAN), dental audio anxiety (DAA), dental video neutral

(DVN) and dental video anxiety (DVA)).

2.3 fMRI data acquisition and analysis

MRI data were collected on a 3-Tesla Trio-TIM MRI whole-body scanner (Siemens, Erlangen,

Germany); a 12-channel head coil, goggles for visual presentation and standard headphones

were used. Functional images were obtained via a T2*-weighted gradient (EPI) sequence that

covered the whole brain (487 volumes in D1, 560 in D2 and D3, repetition time (TR) 2.500 ms,

echo time (TE) 25 ms, field of view (FOV) 192 x 192 mm and matrix 64 x 64). Axial slices

(D1: 43, D2 and D3: 44) were recorded with a slice thickness of 3.5 mm in D1 and 3 mm in D2

and D3 (interleaved acquisition, no gap, inplane resolution 3 x 3 mm). Slices were recorded in

tilted angle to reduce susceptibility in inferior brain areas [39]. The first four volumes were

discarded with regard to T1 equilibration effects. Structural images were acquired via

MPRAGE (176 sagittal slices, slice thickness 1mm, TE 2.26 ms, TR 1900 ms, flip angle 9°,

FOV 256 x 256 mm and matrix 256 x 256).

FC analyses [40] were carried out using the Matlab-based CONN 15.h connectivity toolbox

(http://www.nitrc.org/projects/conn) and Statistical Parametric Mapping (SPM8,

http://www.fil.ion.ucl.ac.uk/spm/). The pre-processing steps (realignment, segmentation,

normalization and co-registration) were performed with the toolbox mentioned above, to

exclude any variances amongst them. The smoothing kernel for the functional volumes was set

out to 8-mm full width at half maximum (FWHM). Spatial resolution was selected to 2 mm

isotropic, the same as the template. Slice-time correction was performed with the Artifact
 8

Detection Tool (http://www.nitrc.org/projects/artifact_detect/). The noise detection was

accomplished by identifying the prominent spontaneous neural activity or inconsistent general

image intensity by initiating a z-threshold = 3 and the identified outliers were regressed out

later on. Other regressors counted in the noise reduction were estimated using the anatomical

CompCor algorithm, for which WM and CSF time series indicated a compelling reason to

correct the physiological fluctuations, hence excluding the effect on the neuronal signal [41,42].

Simultaneously with the regression, a band-pass filter of frequency between 0.008 and 0.09 Hz

was added. Six unilateral ROIs were selected: the ACC, the amygdala and the insula (15 FC

measures-per-condition). The Automated Anatomical Labeling software ([43],

http://www.gin.cnrs.fr/AAL) was used for the creation of the ROIs. Across all ROIs, FC was

explored within groups for dental and snake stimuli respectively. Matched neutral conditions

served as a baseline to mask non-anxiety specific contrasts. Thus, contrasts of interest were:

D1: HC (SA > SN), HC (DA > DN), SP (SA > SN), DP (DA > DN); D2: PP: HC (SA > SN),

PP: HC (DA > DN), PP: SP (SA > SN), PP: DP (DA > DN); D3: HC (DAA > DAN), HC (DVA

> DVN), HC: ((DAA > DAN) > (DVA > DVN)), and DP (DAA > DAN), DP (DVA > DVN)

and DP: ((DAA > DAN) > (DVA > DVN). Multiple comparison correction was performed

using the false discovery rate (FDR) with a corrected threshold of p (FDR) < 0.05. Each dataset

was analyzed independently. In addition to the single FC measures, we computed an average

FC index across all single FC measures per condition on which we compared the groups using

a two-way Analysis of Variance (ANOVA; group*condition) for study 1 and a three-way

ANOVA (group*condition*modality) for study 3. Greenhouse-Geisser corrections were used

in case sphericity assumptions were not met. Demographic and clinical data were evaluated

using chi2-tests and ANOVAS in SPSS 22 (IBM SPSS Statistics for Windows, Version 22.0.

Armonk, NY: IBM Corp.). The level of significance was set to p < 0.05.

3. Results
 9

3.1. Demographic and clinical data

Across all datasets, subgroups were comparable with regards to demographic characteristics

(see supplementary Table S1; for detailed sample descriptions see [29–31]). Phobia groups did

not differ in the number of subjects with comorbid disorders (DP: 12, SP: 6; chi2= 0.424, p =

0.515).

3.1 Functional Connectivity Analyses

See Figure 1 for a visualization of the main findings from all three datasets. Additionally, a list

of significant results from all ROI-to-ROI analyses is given in the supplementary information

(Table S2).

D1: HC exhibited a significantly negative FC between the right ACC and the left amygdala

both for dental (DA > DN) (t = -3.49, p = 0.0152) and snake (SA > SN) stimuli (t = -3.31, p =

0.0220). A negative FC was observed between the left ACC and left amygdala in HC for snake

stimuli (t = -2.75, p = 0.0358). DP showed a negative connectivity for dental stimuli between

the right amygdala and the left insula (t = -3.54, p = 0.0232). In contrast, SP revealed a positive

connectivity of the left ACC with the left insula (t = 4.15, p = 0.0067) and the right insula (t =

3.14, p = 0.0474), and of the right ACC with the left insula (t = 3.85, p = 0.0067) during the

contrast (SA > SN). Group comparisons on the average FC indicated a significant

group*condition interaction effect for snake stimuli (F (1, 27) = 5.442, p = 0.027; SA condition:

SP > HC; p = 0.07), while the interaction effect for dental stimuli failed to show significance

(F (1. 27) = 2.356, p = 0.136).

 10

D2: No significant FC was observed in D2.

D3: HC did not exhibit any significant FC in D3. The contrast (DAA > DAN) in DP revealed

a positive FC between the right insula and both right (t = 3.06, p = 0.0248) and left amygdala

(t = 2.68, p = 0.0333). The left amygdala showed a positive FC to both left (t = 3.67, p = 0.092)

and right insula (t = 3.59, p = 0.0092) and to the right amygdala (t = 2.68, p = 0.0333). No

significant FC was observed for the contrast (DVA > DVN). The comparison between stimulus

modalities ((DAA > DAN) > (DVA > DVN)) yielded a positive connectivity of the right ACC

with the right insula in DP (t = 2.89, p = 0.0404). Group comparisons on the average FC yielded

a significant three-way interaction effect on group*condition*modality (F (1, 24) = 5.709, p =

0.025, DP during auditory stimulation: DVA > DVN, p = 0.004).

Insert Figure 1

4. Discussion

We aimed to investigate FC alterations possibly contributing to impaired emotion regulation

within fear-processing structures in two subtypes of specific phobia under different forms of

phobogenic stimulus processing, including visual and auditory stimuli. Main findings are: a)

HC showed an inhibitory FC particularly between the ACC and the amygdala during

phobogenic stimulus processing; b) phobic subjects showed a positive FC in fear-circuitry

structures; and c) in DP, the connectivity effect was specific for auditory, but not visual

stimulation, possibly reflecting their prime phobogenic stimulus modality.

In D1, a negative FC for dental and snake stimuli was observed between the ACC and the

amygdala only in HC indicating that effective fear inhibition in controls may be achieved by an

inhibitory ACC - amygdala coupling. Although an expected positive connectivity between the
 11

amygdala and the ACC in phobics, particularly in SP due to the often-reported amygdala

overactivation, was not observed, SP showed significantly positive FC between the left ACC

and the bilateral insula, while a negative FC was observed in DP. Notably, D1 only applied

visual stimuli. These findings were also reflected by significant group differences on the

average measure of FC where SP compared to HC showed increased average FC across the

selected ROIs when processing snake-anxiety stimuli. These findings are in line with our

hypothesis stating increased positive FC as a pathophysiological feature of SP during

phobogenic stimulus processing.

D3 included DP as a phobic group comparing only visual to auditory stimuli. Under auditory

symptom provocation, a positive right insula – right ACC FC was observed in DP mirroring

SP´s results from D1 for DP although it should be noted that this finding was evident on a trend

level only (p (FDR) = 0.0533). DP exhibited a positive connectivity between the amygdala and

the insula, both highly associated with fear and arousal. Notably, these regions showed a

negative FC in D1 in DP under visual stimulation. Directly comparing stimulus modalities

((DAA > DAN) > (DVA > DVN)) even augmented FC values between the insula and ACC

pointing to a connectivity shift under auditory symptom provocation. These findings are in line

with the group comparisons in the average measure of FC showing that in DP a shift towards

increased FC is observed during auditory stimulus processing.

Although both modalities from the present datasets were rated as fear inducing in DP [29,31],

auditory stimuli may induce stronger effects on a neural processing level. Hence, it might be

assumed that the auditory channel may be the pivotal feared sensation quality in DP [28,44].

Thus, applying auditory stimuli may contribute to different FC possibly reflecting inappropriate

fear regulation efforts. The findings are consistent with the study by Hilbert et al. [31] proposing

more similarities between the neural activation pattern in DP under auditory stimulus
 12

provocation and the animal subtype under visual stimulus provocation. D3 further reflects the

relatively sparse pattern under visual symptom provocation in DP in D1. Particularly with

regards to DP, it should be considered that connectivity might be mediated through further

regions. Since various studies have reported increased involvement of pre- and orbitofrontal

regions in DP and the BII subtype in general [38,45,46], emotion regulation could primarily

take place on superordinated, cognitive levels. In turn, this could affect top-down controlled

regions like the insula and amygdala over functional relays such as the ACC [47] possibly

contributing to the observed connectivity shift.

Beyond specific phobia, generalized anxiety disorder has been reported to exhibit a similar task-

based pattern of heightened amygdala-insula, amygdala – ACC, and insula – ACC FC [6,48,49].

Moreover, increased resting-state FC between the amygdala and the insula was observed in

posttraumatic stress disorder [50,51]. Findings regarding social anxiety disorder are mixed, yet

two studies revealed increased connectivity of the amygdala and the ACC [7,49]. Worthy of

note, inhibitory amygdala – ACC coupling during a fear-conditioning task was associated with

treatment response in panic disorder with agoraphobia [52]. Hence, an increasing body of

evidence is emerging that altered (para-)limbic connectivity might be a common feature of

pathological stimulus-induced fear across a variety of anxiety disorders that may moderate

response to treatments aiming at fear inhibitory learning.

Several limitations need to be considered when interpreting these findings. Even though we

analyzed three different studies to provide replicated evidence, the single samples were

relatively small and might limit the ability to detect small-scale effects. The contribution of

comorbidity cannot be excluded, although the comorbidity load was comparable across the

sample. The task design possibly influenced the FC findings due to differences in immediate
 13

and prolonged fear processing. Whereas block-design and long stimulus presentation periods

seem to be more appropriate for regions exhibiting longer activation such as the insula [29,31],

event-related designs appear to be more suitable to detect activation within regions with short-

time duration like the amygdala [30]. However, no significant FC was observed in D2 maybe

because event-related paradigms by design provide less statistical power to detect neural

activity compared to block-designs [53] as a precondition to measure FC.

In conclusion, based on previous evidence conferring the relevance of ACC-amygdala circuits

for emotion regulation, we expanded these results to the model disorder of specific phobia,

comprising two phobia subtypes from three different datasets. Current results underscore the

relevance of (para)limbic circuit connectivity for emotion dysregulation in pathological forms

of fear and thus could contribute to a better understanding of connectivity shifts in this disorder.

These findings may bear the potential to inform novel treatment approaches such as brain

stimulation [54] or neurofeedback [55] to augment exposure-based interventions in this highly

prevalent disorder.

Disclosure Statement

The following authors report no conflicts of interest concerning the content of this paper: MS,

RE, KH, UL. HU Wittchen has been member and served as a general consultant (non-product

related) for Pfizer, Organon, Servier and EssexPharma. He has received travel support and grant

funding from Pfizer, Lundbeck, Novatis, Sanofi Aventis, Essex Pharma, Servier and Wyeth.

These cooperations have no relevance to the work that is covered in the manuscript.

Authors’ contributions
 14

UL, KH and HUW designed these studies. UL, KH conducted the data acquisition. MRS, RE

and UL conducted the statistical analyses, and interpreted the data. MRS, RE and UL drafted

the manuscript. MRS, RE, UL, KH, HUW critically revised and approved the final manuscript.

Acknowledgements

We thank Michael Gottschalk, PhD for his help, suggestions and writing assistance. The study

was funded by an internal grant from the TU Dresden given to UL. The funding source had no

involvement in the study design; in the collection, analysis and interpretation of data; in the

writing of the report; and in the decision to submit the article for publication.
 15

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Figure 1. Visualization of functional connectivity (FC) results for study 1 and 3. Study 1, left

side: significant FC at a level of p (FDR) < 0.05 for controls (HC), snake phobics (SP) and

dental phobics (DP) for the contrasts “Snake Anxiety” (SA) > “Snake Neutral” (SN) and

“Dental Anxiety” (DA) > “Dental Neutral” (DN), respectively. Positive connectivity is

displayed in red, negative connectivity in blue. Study 1, right side: group differences on the

average FC for the interaction effect of group*condition for snake stimuli in SP and dental

stimuli in DP. Study 3, left side: significant FC at a level of p (FDR) < 0.05 for DP for the

contrast “Dental Audio Anxiety” (DAA) > “Dental Audio Neutral” (DAN). Study 3, right

side: group differences on the average FC for the interaction effect of

group*condition*modality for auditory and visual dental stimuli in DP. Error bars indicate the

standard error of mean. FC, functional connectivity; AMY, amygdala; INS, insula; ACC,

anterior cingulate cortex; l., left; r., right. Tp < 0.10, **p < 0.01
23