Nx4 Modulated Resting-State Functional Connectivity Between Amygdala and Prefrontal Cortex in a Placebo-Controlled, Crossover Trial

Abstract

Background: The basic functional organization of the resting brain, assessed as resting-state functional connectivity (rsFC), can be affected by previous stress experience and it represents the basis on which subsequent stress experience develops. Notably, the rsFC between the amygdala and the cortical regions associated with emotion regulation and anxiety are affected during stress. The multicomponent drug Neurexan® (Nx4) has previously demonstrated a reduction in amygdala activation in an emotional face matching task and it ameliorated stress-related symptoms. We, thus, investigated the effect of Nx4 on rsFC of the amygdala before stress induction compared with baseline in mildly to moderately stressed participants. Methods: In a randomized, placebo-controlled, double-blind, crossover trial 39 participants received a single dose of placebo or Nx4. Resting-state functional magnetic resonance imaging scans were performed pre-dose and 40 to 60 min post-dose, before any stress induction. First, highly connected functional hubs were identified by global functional connectivity density (gFCD) analysis. Second, by using a seed-based approach, rsFC maps of the left centromedial amygdala (CeMA) were created. The effect of Nx4 on both was evaluated. Results: The medial prefrontal cortex was identified as a relevant functional hub affected by Nx4 in an explorative whole brain gFCD analysis. Using the seed-based approach, we then demonstrated that Nx4 significantly enhanced the negative connectivity between the left CeMA and two cortical regions: the dorsolateral and medial prefrontal cortices. Conclusions: In a resting-state condition, Nx4 reduced the prefrontal cortex gFCD and strengthened the functional coupling between the amygdala and the prefrontal cortex that is relevant for emotion regulation and the stress response. Further studies should elaborate whether this mechanism represents enhanced regulatory control of the amygdala at rest and, consequently, to a diminished susceptibility to stress. ClinicalTrials.gov ID: NCT02602275.

Keywords: amygdala; brain networks; complementary therapies; fMRI; resting-state functional connectivity.

Conflict of interest statement

M.W. received institutional research support from Heel paid to his institution for this study, and from BrainWaveBank and H. Lundbeck A/S outside the submitted work. The University of Tübingen received institutional fees for advisory services by Prof. Walter from Heel GmbH, Servier Deutschland GmbH, Bayer AG, and Janssen-Cilag GmbH. The University of Tübingen received financial support for conference attendance of L.H. and M.L. from Heel for this study.

J.C.V is a senior biometric consultant of idv Datenanalyse & Versuchsplanung (conceptualization, methodology, formal analysis, writing—original draft, writing—review and editing) and received personal fees for biometric services from the Foundation of the Society for the Study of Neuroprotection and Neuroplasticity (SSNN) outside the submitted work, and idv Datenanalyse & Versuchsplanung received payments for biometric services from Heel, University Medical Center Göttigen, IgNova GmbH, Abnoba GmbH, AOP Orphan Pharmaceuticals AG, IDEA AG, PBB Entrepreneur Ltd., Tillots Pharma AG, STORZ Medical AG, EVER Neuro Pharma GmbH, MUCOS Pharma GmbH & Co. KG, and Steigerwald Arzneimittelwerk GmbH outside the submitted work.

M.S. and B.N. are employed by Heel (project administration, validation, writing, review and editing, supervision).

T.C. S.A., M.L, Y.F., H.J., L.D., M.N.Z., L.H., and J.V.M. were part of M.W. team for this study and declare no other conflict of interest outside the submitted work. All investigators followed the institutional guidelines for COI management in full compliance with the regulations of the Otto v. Guericke University, Magdeburg.

Figures

FIG. 1.

Study flow for primary end-points 2 and 3 evaluating rsFC without prior stress induction. On both study days, baseline rsfMRI scans were acquired immediately before dosing (RS0) and another rsfMRI about 1-h post-dose (RS1) without any stress-related tasks in between. Participants of the crossover trial received Nx4 on day 1 and placebo on day 2 or vice versa. A total of four rsfMRI scans per participant were performed for primary end-points 2 and 3. Nx4, Neurexan®; rsFC, resting-state functional connectivity; rsfMRI, resting-state functional magnetic resonance imaging.

FIG. 2.

Definition of left CeMA (shown in red) for seed-based FC (MNI coordinates −22 − 5 −14) provided by the Anatomy Toolbox (Eickhoff et al., 2005). CeMA, centromedial amygdala; FC, functional connectivity; MNI, Montreal Neurological Institute.

FIG. 3.

Number of participants in the two sequences of the cross-over trial, placebo first and Nx4 first, analyzed for primary end-points 2 and 3 evaluating rsFC without prior stress induction.

FIG. 4.

Statistical parametric mapping of prominent gFCD hubs (voxel-wise FWE-corrected p < 0.05) using baseline resting-state data (RS0) at day 1. Orange-yellow colorbar indicates the T values. Highly connected functional hubs were identified mainly in the cortex. FWE, family-wise error rate; gFCD, global functional connectivity density.

FIG. 5.

(A) Whole-brain analysis of FCD showed a significant interaction between drug and time for FCD in the mPFC. Results were reported at uncorrected p < 0.005, cluster size >10. (B) Strength of FCD in the mPFC at baseline (RS0) and 1-h post-dose (RS1) under Nx4 or placebo. Data are given as mean with standard error of mean. Significant differences are indicated as **p < 0.01 and *p < 0.05. mPFC, medial prefrontal cortex.

FIG. 6.

Whole-brain functional connectivity map from left CeMA using baseline resting-state data (RS0) at day 1. All the results were reported at p < 0.05, corrected for multiple comparisons using FDR correction. Colorbar indicates T values. FDR, false discovery rate.

FIG. 7.

Brain regions showing significant (cluster-wise FWE-corrected at p < 0.05) drug × time interaction on seed-based rsFC for left CeMA. Significant effects of Nx4 versus placebo were observed for the rsFC between left CeMA and two cortical regions, the dlPFC and the mPFC. Colorbar indicates T values. dlPFC, dorsolateral prefrontal cortex.

FIG. 8.

Strength of rsFC between left CeMA and (A) mPFC and (B) dlPFC at baseline (RS0) and 1 h post-dose (RS1) under Nx4 or placebo. Data are given as mean with standard error of mean. Significant differences are indicated as ***p < 0.001, **p < 0.01, and *p < 0.05; ns, not significant.