New Results

Human extinction learning is accelerated by an angiotensin antagonist via ventromedial prefrontal cortex and its connections with basolateral amygdala

Feng Zhou, Yayuan Geng, Fei Xin, Jialin Li, Pan Feng, Congcong Liu, Weihua Zhao, Tingyong Feng, Adam J. Guastella, Richard P. Ebstein, Keith M. Kendrick, Benjamin Becker
doi: https://doi.org/10.1101/512657

Abstract

Extinction is considered a core mechanism underlying exposure-based therapy in anxiety-related disorders. However, marked impairments in threat extinction learning coupled with impaired neuroplasticity in patients strongly impede the efficacy of exposure-based interventions. Recent translational research suggests a role of the renin-angiotensin (RA) system in both these processes. However, the efficacy of pharmacological modulation of the RA system to enhance threat extinction in humans and the underlying neural mechanisms remain unclear. The present pre-registered, randomized placebo-controlled pharmacological neuroimaging trial demonstrates that pre-extinction administration of the angiotensin II type 1 receptor antagonist losartan accelerated attenuation of the psychophysiological threat response during extinction. On the neural level the acceleration of extinction was accompanied by threat-signal specific enhanced ventromedial prefrontal cortex (vmPFC) activation and its coupling with the basolateral amygdala. Multivoxel pattern analysis and voxel-wise mediation analysis further revealed that that losartan reduced the neural threat expression, particularly in the vmPFC, and confirmed that acceleration of extinction critically involved treatment-induced modulation of vmPFC activation. Overall the results provide the first evidence for a pivotal role of the RA system in extinction learning in humans and suggest that adjunct losartan administration can be leveraged to facilitate the efficacy of extinction-based therapies.

Introduction

Extinction learning refers to the attenuation of a previously learned defensive response when the threat predictive stimulus is repeatedly encountered in the absence of adverse consequences. Exposure-based interventions capitalize on extinction learning mechanisms to reduce excessive fear in patients with anxiety-related disorders and are considered an efficient therapy in these illnesses [1]. A significant number of patients however does not adequately respond to exposure therapy [2] and impaired extinction processes are considered one key mechanism underpinning the lack of therapeutic efficacy [3]. Anxiety-related disorders are highly prevalent and associated with significant psychosocial impairments and societal costs [4]. As such, innovative strategies to improve the efficacy or shorten the duration of exposure therapies are urgently needed.

Whereas the behavioral and neural pathomechanisms underlying anxiety disorders are increasing understood, translation into efficacious clinical interventions remains inadequate. Notably, the neural mechanisms mediating extinction are extremely well-conserved over the course of evolutionary time; hence, the identification in animal models of receptor targets sensitive to pharmacological modulation that facilitate neural plasticity in pathways supporting extinction learning, can feasibly augment the efficacy of exposure-based interventions [5].

Animal models and human neuroimaging research have demonstrated a crucial role of the infra-limbic cortex (IL), which is homologous to the human ventromedial prefrontal cortex, vmPFC, and its interactions with the amygdala in extinction learning [6-12]. The vmPFC is critically engaged in the reduction of threat expression during extinction [3, 9, 13] and governs the amygdala inhibition of conditioned threat response [8, 10]. Translational models suggest that dysfunctions in amygdala-prefrontal neuroplasticity contribute to extinction-failure in anxiety disorders [14]. Converging evidence from clinical research suggests that anxiety disorders are characterized by deficient extinction, hypoactivation within the vmPFC and attenuated vmPFC-amygdala functional connectivity [6, 8, 11, 13, 15].

Intriguingly, recent evidence suggests that the renin-angiotensin (RA) system, primarily known for its role as a blood pressure and renal absorption regulator, represents a promising target to facilitate extinction [5]. Central angiotensin receptors are densely expressed in limbic and prefrontal brain regions and are critical to changes in neuroplasticity and extinction [16-18]. Initial studies in rodents have demonstrated the potential of pharmacological modulation of RA signaling towards facilitating extinction using the selective competitive angiotensin II type 1 antagonist losartan (LT) [19, 20]. LT is an approved treatment for high blood pressure with an excellent safety record [21, 22]. Notably, initial clinical observations suggested unexpected beneficial effects on memory and anxiety-symptomatology [5, 21]. Taken together, accumulating evidence suggests that LT-modulation of central RA signaling represents a promising target to enhance extinction learning.

Against this background, we conducted a pre-registered randomized placebo-controlled pharmacological experiment to determine whether targeting the RA system can facilitate extinction in humans. To uncover the underlying neural mechanisms functional magnetic resonance imaging (fMRI) and psychophysiological threat responses (skin conductance, SCR) were simultaneously acquired. The specific goal was to determine the potential of LT (50mg, single-dose, p.o.) as a therapeutic candidate for the clinical augmentation of extinction learning. Based on previous translational research we expected that LT would (1) accelerate attenuation of the psychophysiological threat responses, and that enhanced extinction would be mediated by two neural processes: (2) increased activation in the vmPFC and attenuation of its threat expression in the context of (3) stronger functional interaction of the vmPFC with the amygdala.

Materials and Methods

Participants and experimental protocols

Seventy healthy males underwent a validated Pavlovian threat acquisition and extinction procedure with simultaneous fMRI and SCR acquisition. To reduce variance related to hormonal and menstrual-cycle-associated variation in extinction [23-25] only male participants were included in the present proof-of-concept experiment [for similar approach see ref. 26]. Due to technical issues (SCR recording failure n = 3) or absence of threat acquisition (n = 8) data from 11 subjects were excluded leading to n = 30 LT- and n = 29 PLC-treated subjects for the evaluation of the primary study hypotheses. For exclusion criteria and a description of the study sample see Supplementary methods.

The experiment consisted of three sequential stages: (1) acquisition, (2) treatment administration and, (3) extinction. 20-min after acquisition, participants were administered either a single 50mg (p.o.) dose of the selective, competitive angiotensin II type 1 receptor antagonist losartan (LT, Cozaar; Merck, USA) or placebo (PLC), packed in identical capsules. Consistent with the pharmacodynamic profile of LT [27] extinction was observed 90min post-treatment. Although previous studies reported no effects of single-dose LT on cardiovascular activity or mood [18, 27, 28], these indices were monitored to primarily control for unspecific effects of treatment and not focused on behavior per se. The experimental time-line is provided in Fig. 1a (for the pharmacodynamic profile and assessment of confounders see Supplementary Methods).

Fig 1.
Fig 1. Experimental timeline and losartan effects on psychophysiological threat responses.

(a) Experimental timeline and schematic synopsis of fMRI tasks. (b) Psychophysiological threat responses (CS+ - CS-) during acquisition demonstrating successful CS discrimination with enhanced SCR to the CS+ relative to the CS- in both groups. (c) Mean SCR for CS- presentations during acquisition. (d) Psychophysiological threat responses (CS+ - CS-) during extinction learning. Psychophysiological threat responses during early extinction (across two runs) are presented in the inset. (e) Mean SCR for CS- presentations during extinction learning. †P < 0.05, one-tailed; *P < 0.05, two-tailed, error bars represent standard errors. The filled curve indicates the null-hypothesis distribution of the difference of means (Δ) and the 95% confidence interval of Δ is illustrated by the black line.

The study was approved by the local institutional ethics committee and adhered to the Declaration of Helsinki and was a pre-registered (ClinicalTrials.gov, NCT03396523).

Experimental Paradigm

During the acquisition stage, participants were repeatedly presented with two different colored squares, the CS (conditioned stimulus). One CS (CS+, 4s) was pseudo-randomly paired with a mild electric shock (US, 2ms) with 43% contingency, whereas the other CS (CS-, 4s) was never paired with a US. Acquisition was followed by extinction, where the same cues were presented without US (Supplementary methods). To enhance threat memory acquisition as well as to increase the statistical power to determine treatment effects, both learning phases included two subsequent runs of the task [for a similar approach see ref. 29]). Prior to each run, subjects were informed that “the experimental runs are independent and you may or may not receive the electric shock” thus subjects were unable to predcit the presence or absence of the US at the beginning of the respective run.

Skin Conductance Response Analysis

Skin conductance responses were computed as done in previous studies [9, 30-32]. Psychophysiological threat responses were defined as baseline-corrected CS+ by subtracting the mean responses to the CS- (see Supplementary Methods). Treatment effects were determined employing a phase (early, late) × run (run1, run2) × treatment (LT, PLC) 3-way mixed ANOVA with psychophysiological threat responses as dependent variable.

MRI Acquisition and Analysis

MRI data were acquired using a Siemens TRIO 3-Tesla system with a 12-channel head coil. Functional time-series were processed using SPM12 (Statistical Parametric Mapping, https://www.fil.ion.ucl.ac.uk/spm/software/spm12/). On the first-level, the CS+ and CS- stimuli were modeled and condition-specific regressors for early (first half) and late (second half) of extinction were defined (details see Supplementary Methods).

Whole-Brain Analyses

Effects of LT on extinction were assessed using a whole-brain phase (early, late) × run (run1, run2) × treatment (LT, PLC) 3-way mixed ANOVA with the CS+ > CS-contrasts as dependent variable. Significant interaction effects were further disentangled by two independent post hoc approaches to warrant both high regional-specificity (whole-brain voxel-wise post hoc t-tests) and high robustness (leave-one-subject-out cross-validation (LOSO-CV) procedure [33]). Group-level analyses (including LOSO-CV) were conducted using FSL Randomise (FMRIB Software Library, http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/) with permutation-based inferences (10,000 permutations). Significant clusters were determined using a height threshold of P < 0.001 (two-tailed) and an extent threshold of P < 0.05 (two-tailed) with cluster-based family-wise error (FWE) correction.

Region-of-interest - vmPFC contributions during early extinction

Due to the critical role of the vmPFC in extinction [8, 34-37] and our a priori regional hypothesis, we explored LT effects on stimulus-specific vmPFC activation during early extinction. To this end, activity estimates were extracted from an anatomically-defined vmPFC region of interest (ROI) (Supplementary Methods) and subjected to a stimulus (CS+, CS-) × run (run1, run2) × treatment (LT, PLC) 3-way mixed ANOVA.

Neural Threat Expression - Multi-Voxel Pattern Analysis (MVPA)

Following Reddan, et al. [36], a neural pattern of threat was developed to differentiate CS+ versus CS- (trained on the acquisition data) and subsequently applied to early extinction activation to determine treatment effects on the neural threat expression (Supplementary Methods).

Voxel-wise Mediation Analyses

To determine whether treatment effects on vmPFC activation (CS+ > CS-) during early extinction critically contributed to the accelerated attenuation of the psychophysiological threat responses (SCR, CS+ > CS-), voxel-wise mediation analyses were conducted (Mediation Toolbox, https://github.com/canlab/MediationToolbox) [38, 39]. Mediation effects were inferred using bootstrapping (10,000 replacements) and false discovery rate (FDR) correction.

Network-level Effects - Functional Connectivity Analysis

Given that both human and animal studies strongly implicate vmPFC-mediated inhibition of the amygdala as a key extinction mechanism [10-12, 34], a functional connectivity analysis [40] was employed to determine treatment effects on the vmPFC-amygdala coupling during early extinction. We hypothesized that LT-induced extinction enhancement would be accompanied by stronger functional interaction between these regions (one-sided).

Results

Participants

Consistent with previous studies [18, 27, 28], no effects of drug or placebo on cardiovascular and affective indices were observed, which together with the chance level guesses for treatment, argues against unspecific confounding effects of treatment (Supplementary Table 1). During the pre-treatment acquisition phase, both groups exhibited successful threat acquisition on the psychophysiological (Figs. 1b,c) as well as neural level (Supplementary results and supplementary Fig. 1). Importantly, two-sample t-tests did not reveal between-group activation differences during this stage.

View this table:
Supplemental Table 1

Participant demographics and neuropsychological performance STAI, Spielberger Trait State Anxiety Inventory; PANAS, Positive Affect Negative Affect Scale; BMI, Body Mass Index

Supplemental Fig.1.
Supplemental Fig.1. Non-reinforced CS+ versus CS- BOLD responses during acquisition, across both experimental groups.

Images displayed at P < 0.05, cluster-level family-wise error (FWE)-correction with a cluster-forming threshold of P < 0.001, two-tailed. Hot color indicates CS+ > CS-, whereas cold color indicates CS+ < CS-.

Reduced Psychophysiological Threat Responses during Early Extinction

A mixed ANOVA model using the psychophysiological threat response during extinction learning as a dependent variable, demonstrated a significant main effect of run (F(1, 57) = 17.063, P < 0.001, partial η2 = 0.230; η2 indicates effect size in terms of eta squared) reflecting decreased psychophysiological threat responses in run2 compared to run1 and a marginally significant main effect of phase (F(1, 57) = 3.387, P = 0.071, partial η2 = 0.056) reflecting decreased psychophysiological threat responses during late extinction. Together these results demonstrated successful extinction learning. Moreover, a significant treatment × phase interaction effect (F(1, 57) = 5.017, P = 0.029, partial η2 = 0.081) was observed, with post hoc two-sample t-tests indicating that relative to the PLC group, the LT group exhibited decreased psychophysiological threat responses during early extinction learning across both runs (t(57) = −2.179, P = 0.034, d = −0.567, d indicates effect size in terms of Cohen’s d, see insert Fig. in Fig. 1d), suggesting accelerated extinction learning. Exploratory run-specific analyses confirmed that LT-treatment enhanced early extinction learning during both initial as well as repeated extinction learning (run1 t(57) = −1.805, P = 0.038, d = −0.470; run2 t(57)= −2.012, P = 0.024, d = −0.525; two-sample t-tests comparing the treatment groups, one-tailed, Fig. 1d). No further significant main or interaction effects were observed on the psychophysiological threat responses (all Ps > 0.4). Nor were any effects of treatment observed on the safety signal (CS-) per se (all Ps > 0.15, Fig. 1e) confirming the threat-specific effects of LT and the absence of unspecific treatment effects on the SCR psychophysiological signal.

Increased Threat-specific vmPFC Engagement during Early Extinction

Whole-brain analysis revealed a significant treatment × phase interaction effect on extinction-related neural activity (CS+ > CS-) in the vmPFC (peak MNIxyz = [-3, 27,-12], F(1,57) = 23.582, PclusterFWE = 0.011, k = 187) (Fig. 2a). Regional-specificity of treatment effects was demonstrated by voxel-wise whole-brain post-hoc comparisons demonstrating increased vmPFC activity following LT relative to PLC during early (peak MNIxyz = [6, 48, −3], t(57) = 4.505, PclusterFWE = 0.013, two-tailed, k = 139), but not late extinction (Fig. 2b). Results from the LOSO-CV procedure further demonstrated that LT - relative to PLC - increased vmPFC activation during early (t(57) = 3.417, P = 0.001, d = 0.890), but not late (t(57) = −1.556, P = 0.125, d = −0.405), extinction (Fig. 2c, Fig. 2d displays an overlay of all leave-one-subject-out-ROIs).

Fig 2.
Fig 2. Losartan treatment effects on brain activity (CS+ - CS-) during extinction learning.

(a) vmPFC activity showed significant treatment by phase interaction effect. (b) Losartan specifically increased vmPFC activity during early extinction learning. (c) Mean vmPFC activity (CS+ - CS-) extracted from the ROIs depicted in (d) showed that losartan increased vmPFC activity during early, but not late, extinction learning. (d) Overlay of all 59 leave-one-subject-out (LOSO) cross-validation (CV) ROIs. All ROIs were created leaving out one subject at the group-level statistic (cluster-level family-wise error (FWE)-corrected). Statistical images were thresholded at P < 0.05 (two-tailed), cluster-level FWE-corrected with a cluster-forming threshold of P < 0.001 (two-tailed). Examples of unthresholded patterns are presented in the insets; small squares indicate voxel statistical weight; red-outlined squares indicate significance at PclusterFWE < 0.05. n.s. represents not significant; *P < 0.05. The filled curve indicates the null-hypothesis distribution of the difference of means (Δ) and the 95% confidence interval of Δ is illustrated by the black line. vmPFC, ventromedial prefrontal cortex. vmPFC, ventromedial prefrontal cortex.

Further exploring vmPFC contributions during early extinction by means of extraction of beta estimates, revealed significant main effects of stimulus type and run, as well as a significant treatment × stimulus type interaction effect (details see Supplementary Results). Exploratory post-hoc two-sample t-tests demonstrated that LT enhanced threat-specific vmPFC reactivity during both the initial (run1 t(57) = 2.141, P = 0.037, d = 0.557) and the repeated extinction (run2 t(57) = 2.126, P = 0.038, d = 0.554), in the absence of effects on the safety signal (CS-, Ps > 0.29) (Fig. 3a).

Fig 3.
Fig 3. Losartan treatment specifically increased vmPFC activity to threat stimulus (CS+) during early extinction learning.

(a) Losartan increased vmPFC activity to CS+, but not CS-, in the early extinction phase in both runs. n.s. represents not significant; *P < 0.05; and ***P < 0.001, error bars represent standard errors. (b) Single trial analysis confirmed that losartan increased vmPFC activity to CS+ in early trials during extinction learning. *q < 0.05, FDR corrected. Data are represented as group mean ± SEM. (c) vmPFC ROI. vmPFC, ventromedial prefrontal cortex.

Consistent with our hypothesis on accelerated extinction by LT, an exploratory single trial analysis (Supplementary Methods) revealed that LT specifically increased vmPFC activation during initial trials of re-exposure to the threat stimulus (q < 0.05, FDR corrected) (Fig. 3b).

Reduced Neural Threat Expression during Early Extinction

The threat-predictive pattern reliably evoked neural threat reactivity during acquisition (comparable to [ref. 36], see Supplementary Results and supplementary Figs. 2a,b). Applying the threat-predictive pattern to early extinction activation (CS+ > CS-) using a LOSO-CV procedure, demonstrated that first, in the entire sample higher neural threat expression was associated with stronger psychophysiological threat reactivity (r(57) = 0.571, P < 0.001) and confirmed functional relevance of the neural expression of threat [36]. Second, relative to PLC, LT significantly decreased the magnitude of the threat-predictive pattern expression (t(57) = −2.091, P = 0.041, d = −0.544, two-sample t-test), confirming attenuated neural threat expression during early extinction. Based on our a priori regional hypothesis, and the key role of the vmPFC in extinction [3, 8, 9, 13], a vmPFC-focused partial threat expression analysis was conducted.In concordance with the whole-brain results, LT significantly attenuated the vmPFC partial threat pattern expression (CS+ > CS-) during early extinction (t(57) = −3.410, P = 0.001, d = −0.888) (see Supplementary Results and supplementary Fig. 2c).

Supplemental Fig.2.
Supplemental Fig.2. Multivariate neural threat-predictive pattern results.

(a) Neural threat-predictive pattern, consisting of voxels in which activity reliably predicted threatening (unreinforced CS+) versus non-threatening (CS-) stimuli during threat acquisition. The map shows weights that exceed a threshold (q < 0.05, FDR corrected based on bootstrapped 10,000 samples) for display only. dACC, dorsal anterior cingulate cortex; vmPFC, ventromedial prefrontal cortex. Hot color indicates positive weights and cold color indicates negative weights. (b) ROC plot. The neural threat-predictive pattern yielded a classification accuracy of 87.93% in a leave-one-subject-out cross-validation (LOSO CV) procedure. (c) Losartan treatment reduced the partial threat pattern expression of the vmPFC. **P < 0.01. The filled curve indicates the null-hypothesis distribution of the difference of means (Δ) and the 95% confidence interval of Δ is illustrated by the black line.

vmPFC Activation Drives LT-induced Accelerated Extinction

The voxel-wise mediation analysis aimed at further determining the relationship between treatment, psychophysiological threat attenuation and the underlying neural basis. Conjunction effects (paths a, b and a × b) were observed in a vmPFC cluster (peak MNIxyz = [-3, 45, −15], Z = −3.692, q < 0.05, FDR-SVC corrected in the anatomical vmPFC, k = 138), demonstrating that LT increased vmPFC activation (path a), while activation in this region was associated with stronger suppression of psychophysiological threat independent of treatment (path b). Importantly the a × b mediation effect reached significance, indicating that vmPFC activation critically mediated the effects of LT on extinction acceleration (Fig. 4a, details see Supplementary Results). To test the robustness and visualize the mediation effect, an independent vmPFC-focused mediation analysis was conducted which confirmed the critical contribution of the vmPFC (Fig. 4B; a × b effect, bootstrapped P value = 0.016; each path is shown in Fig. 4C).

Fig 4.
Fig 4. vmPFC activity mediated losartan treatment effect on accelerated extinction learning.

(a) Sagittal slice showing regions whose activity increased response to the losartan treatment in yellow (path a), regions whose activity significant negative correlated with psychophysiological threat responses while controlling for the treatment effect in green (path b), and regions whose activity showed significant mediation (a × b) effect in blue. All images were thresholded at q < 0.05, FDR corrected within the vmPFC mask. (b) Mediation path diagram with the brain activity in the vmPFC ROI. (c) Examples of each path in the mediation path diagram. *P < 0.05; **P < 0.01; ***P < 0.001. The filled curve indicates the null-hypothesis distribution of the difference of means (Δ) and the 95% confidence interval of Δ is illustrated by the black line. vmPFC, ventromedial prefrontal cortex.

Enhanced vmPFC-Amygdala Coupling

During early extinction LT-induced enhanced functional coupling between the vmPFC and the right amygdala (peak MNIxyz = [24, −3, −24], t(57) = 3.557, q < 0.05, one-tailed, FDR-SVC corrected in the amygdala, k = 13. This was cytoarchitectonically [41] mapped to the basolateral amygdala BLA, Fig. 5a). Subsequent examination of stimulus-specific connectivity estimates from the vmPFC-bilateral BLA pathway, confirmed specific effects of LT on threat signal (CS+) processing (t(57) = 2.147, P = 0.036, d = 0.559) (Fig. 5b).

Fig 5.
Fig 5. Losartan treatment effect on vmPFC-amygdala functional coupling.

(a) Sagittal slice showing that losartan increased functional connectivity between vmPFC and BLA. The image was thresholded at q < 0.05, FDR corrected within the amygdala mask. (b) Extracted gPPI parametric estimates in the bilateral BLA showed that losartan treatment specifically enhanced vmPFC-bilateral BLA functional pathway during fear-associated stimulus presentation. n.s. represents not significant; *P < 0.05. The filled curve indicates the null-hypothesis distribution of the difference of means (Δ) and the 95% confidence interval of Δ is illustrated by the black line. vmPFC, ventromedial prefrontal cortex; BLA, basolateral amygdala.

Discussion

The present study demonstrated that LT treatment accelerated the attenuation of a previously acquired psychophysiological threat response, indicating its potential to facilitate threat extinction learning in humans. During early extinction, the acceleration was was critically mediated by enhanced threat-signal specific vmPFC activity and stronger functional coupling of the vmPFC with the BLA. These findings were further paralleled by a pattern classification approach showing that LT-treatment accelerated attenuation of the neural threat expression, particularly in the vmPFC. Overall, the present findings provide first evidence for an important contribution of the RA system to fear extinction in humans and the potential of LT to accelerate extinction through effects on the vmPFC and its inhibitory connections with the BLA.

In humans, successful extinction is accompanied by decreased psychophysiological threat reactivity and concomitantly increased vmPFC activation in response to the threat signal [35, 37, 42]. In the present study LT-treatment reduced the psychophysiological threat responses and selectively enhanced vmPFC activation in response to the threat signal (CS+) during early extinction, indicating its potential to accelerate extinction learning in humans. Moreover, the acceleration effects were found not only during the initial extinction learning (i.e., run1), but also in the following “new” learning process (for extinction run2, participants were told that the two extinction runs were independent, and thus they could not predict the absence of the US during the extinction run2). The findings resemble previously observed LT-enhanced extinction learning in rodents [19, 20] and further confirm the important contribution of the vmPFC to successful extinction. Exploring the stimulus-specific effects of LT-treatment on vmPFC activation revealed that reactivity to the safety signal (CS-) remained unaffected. Decoding the temporal pattern of LT-effects further suggested that LT specifically attenuated vmPFC reactivity during early re-exposure towards the previously conditioned threat signal. Non-pharmacological stimulation of the vmPFC homologous infra-limbic cortex accelerates extinction learning in rodents [43-45], whereas inactivation or lesion of this region critically impede threat reduction during extinction ([for reviews see refs. 8, 13]). Compatible with the present findings, previous studies demonstrated that non-pharmacological stimulation of the vmPFC can enhance early extinction learning in humans [46], albeit unspecific effects on CS- reactivity have also been reported [47].

Converging evidence from different research focuses suggests that the vmPFC, or the homologous IL in rodents, critically contributes to the reduction of threat expression during extinction learning [3, 8, 13, 34] and regulates amygdala output to inhibit the conditioned threat response [8, 10]. Consonant with the proposed contribution of the vmPFC to extinction learning, LT-attenuated psychophysiological threat responses during early extinction were accompanied by an attenuated neural threat expression, particularly in the anatomically defined vmPFC. Compatible with previous studies that showed critical contributions of the vmPFC to extinction enhancement [43-45] as well as associations between activity in this region and psychophysiological threat reactivity during extinction [48], an additional mediation analysis was carried out. It revealed that higher vmPFC activation was associated with stronger suppression of the psychophysiological threat response. Notably, LT-facilitated suppression of the psychophysiological threat response crucially involved enhanced vmPFC activation (for convergent mediation effects of vmPFC threat expression see Supplementary Methods and Results) further emphasizing the key role of this region in extinction enhancement.

On the network level, LT-accelerated threat reduction during early extinction was paralleled by stronger functional communication between the vmPFC and the amygdala, specifically the basolateral subregion. Previous lesion studies in humans demonstrated a critical role of the BLA in threat processing [49] and of the vmPFC in inhibiting amygdala threat responses by exerting top-down control over this region [50]. Animal models further confirmed the importance of pathway-specific neuroplastic changes in the vmPFC-amygdala circuitry during extinction memory formation [10-12] and suggest that vmPFC inputs to the amygdala, instruct threat memory formation and/or gate the expression of conditioned threat [10] during early extinction [12]. The present findings of CS+-specific increased vmPFC-BLA functional connectivity following LT-treatment likely reflects an important modulatory role of angiotensin signaling on vmPFC regulation of the amygdala. Previously, animal models demonstrated that stimulation of vmPFC inputs to the amygdala promotes the formation of extinction memories [10]. We suggest the notion that enhanced transmission in this pathway may possibly reflect a core mechanism underlying angiotensin regulation of extinction learning. Angiotensin receptors are densely expressed in limbic and prefrontal regions critically engaged in extinction [16-18] and are considered to modulate learning-related neuroplasticity. LT is a selective competitive antagonist of the angiotensin II type 1 receptor but also increases availability of angiotensin II-converted angiotensin IV-an agonist at the AT4 receptor subtype. The AT4 system is thought to play a role in neuroplasticity and learning and memory [16, 17, 28, 51], a mechanism that is suggested to likely contribute to LT-induced extinction enhancement.

Consistent with previous animal models demonstrating the potential of LT to enhance extinction in rodents [19, 20], the present study successfully demonstrated the potential of a single, low-dose administration of LT to facilitate extinction learning in humans. In the context of recent findings suggesting a direct association between extinction-related vmPFC functioning and exposure therapy success [52], the current results indicate that LT represents a highly promising candidate to augment the efficacy of exposure-based interventions in therapeutic settings. On the neural level the effects of LT were mediated by circuits consistently involved in anxiety disorders with exaggerated threat reactivity and deficient extinction being associated with decreased vmPFC activation and dysfunction in the vmPFC-BLA circuit [6, 8, 11, 13]. Importantly, dysregulations in this circuitry normalize during the course of successful treatment [53] suggesting that they represent treatment-responsive elements rather than stable - markers and consequently promising targets for innovative therapeutic interventions. A previous human neuroimaging study reported that LT improves threat discrimination in high anxious individuals [18] and together with LT’s excellent safety record in clinical applications [22], the currently observed extinction enhancing potential and selective effects on vmPFC-BLA threat signaling, make this drug an attractive candidate for augmenting the effects of exposure therapy.

However, despite these initial promising results subsequent studies need to (1) examine effects of LT on subsequent extinction consolidation and recall in humans [as previously demonstrated in rodents 19], (2) determine the generalization of the effects to female subjects and, finally, (3) evaluate its potential to enhance exposure-based interventions in clinical trials.

Overall, the present results indicate an important regulatory role of the RA system in fear extinction learning in humans that are mediated by modulatory effects on vmPFC threat processing and its interaction with the amygdala. From a clinical perspective adjunct LT-treatment may represent an innovative strategy to enhance the efficacy of exposure-based interventions.

Author contributions

F.Z. and B.B. designed the study, analyzed the data and wrote the manuscript. F.Z., Y.G., F.X., J.L., P.F., C.L, and W.Z. conducted the experiment. P.F., T.F., A.G., R.E. and K.K. revised the manuscript draft.

Conflict of Interest

The authors declare that they have no conflict of interest.

Data and materials availability

Unthresholded group-level statistical maps are available on NeuroVault (https://neurovault.org/collections/4722/) and code that supports the findings of this study is available from the corresponding author upon reasonable request.

Supplementary Results

Acquisition Results

During the pre-treatment acquisition phase, both groups exhibited CS discrimination with enhanced SCR to the CS+ relative to the CS- (Fig. 1b), confirming successful threat acquisition. On the neural level acquisition of threat was accompanied by stronger CS+ versus CS- responses in the threat acquisition networks [see e.g. refs. 1, 29] (supplementary Fig. 1).

Losartan Treatment Enhanced Threat-signal Specific vmPFC Activity

A more detailed examination of the treatment effects by means of extraction of the condition-specific neural signal from the independently (structurally) defined vmPFC ROI additionally revealed significant main effects of stimulus type (F(1, 57) = 15.630, P < 0.001, partial η2 = 0.215, vmPFC activity was decreased to CS+ compared to CS-) and run (F(1, 57) = 16.848, P < 0.001, partial η2 = 0.228 with increased vmPFC activity in run2 in relative to run1), as well as a significant treatment × stimulus type interaction effect (F(1, 57) = 17.353, P < 0.001, partial η2 = 0.233) during early extinction. Post hoc comparisons between the treatment groups demonstrated that the interaction effect during early extinction was driven by a LT-induced selective increase of vmPFC responses to the CS+ (t(57) = 2.777, P = 0.007, d = 0.723), in the absence of significant effects on the CS- (t(57) = −1.098, P = 0.277, d = −0.286). Post hoc comparisons between the stimulus types showed that in the PLC group the BOLD signal in response to the CS+ was decreased relative to the CS- (t(28) = −5.685, P < 0.001, d = −1.056). In contrast, LT-treated subjects exhibited a comparable vmPFC BOLD response to the CS+ compared to the CS- (t(29) = 0.152, P = 0.881, d = 0.028). In addition, in line with previous studies [30, 31] exploratory post-hoc paired sample t-tests showed conditioned threat responses with a decrease in BOLD signal for the CS+ relative to the CS- in early extinction in PLC-treated subjects in both run1 (t(28) = −3.917, P < 0.001, d = −0.727) and run2 (t(28) = −4.829, P < 0.001, d = −0.897), reflecting persistence of the acquired conditioning memory. In contrast LT-treated subjects exhibited comparable responses to the CS+ and CS- in both runs (Ps > 0.6), suggesting that following LT-treatment differential vmPFC responsivity was already normalized during early extinction.

MVPA Results

To test whether LT-treatment reduces neural threat response during early extinction, a whole-brain multivariate threat-predictive pattern was decoded [24]. Results showed a classification accuracy of 87.93% (P < 0.001) and sensitivity and specificity to threat were 0.862 (CI: 0.766-0.944) and 0.897 (CI: 0.811-0.967) respectively suggesting that the neural threat-predictive pattern could effectively distinguish neural threat versus non-threat processing (before treatment). When thresholded (bootstrapped 10,000 samples) and corrected for multiple comparisons (q < 0.05, FDR corrected), the neural threat-predictive pattern encompassed a distributed threat representation network including the vmPFC, insula, dorsal anterior cingulate cortex, thalamus and hippocampus resembling findings from mass-univariate analyses of threat acquisition in the present as well as previous studies [overview see ref. 29] (supplementary Figs. 2a,b).

Based on our a priori regional hypothesis and the key role of the vmPFC in the reduction of threat expression during extinction [15, 25-27] an additional analysis specifically focused on the effects of LT on the vmPFC partial threat expression. Briefly, the unthresholded neural threat-predictive pattern was masked by the anatomical vmPFC mask and next applied to early extinction activation maps with the higher partial pattern expression chosen as the threat stimulus (i.e., a forced-choice test). Results revealed that only the PLC group (accuracy = 89.66%, P < 0.001) but not the LT group (accuracy = 56.67%, P = 0.585) exhibited neural threat experience during early extinction.

Mediation Results

A key question is how LT-treatment leads to accelerated extinction learning as reflected by reduced psychophysiological threat responses in early extinction. Based on previous studies indicating crucial contributions of the vmPFC to extinction and the facilitation of extinction [26, 27, 32-34] we hypothesized that treatment-induced effects on vmPFC activation would critically mediate accelerated extinction. We found that LT-treatment significantly contributed to both the reduced psychophysiological threat responses (path c; t(57) = −2.179, P = 0.034) and enhanced vmPFC activation (path a; peak MNIxyz = [6, 36, −21], Z = 4.616, q < 0.05, SVC-FDR corrected in pre-defined vmPFC mask, k = 544, Figure 4A in yellow). Furthermore, a significant negative correlation between vmPFC activity and the psychophysiological threat responses (path b; controlled for treatments, peak MNIxyz = [-6, 48, −15], Z = −4.882, q < 0.05, SVC-FDR corrected, k = 221, Figure 4A in green) as well as a significant negative mediation effect in the vmPFC (peak MNIxyz = [-3, 45, −18], Z = −3.795, q < 0.05, SVC-FDR corrected, k = 139, Figure 4A in blue) were observed. Importantly, a vmPFC cluster exhibited conjunction effects of both LT-treatment (path a) and fear expression (path b) as well as a mediation effect (peak MNIxyz = [-3, 45, −15], Z = −3.692, q < 0.05, SVC-FDR corrected, k = 138), suggesting that activation in this region significantly mediated LT-induced attenuation of psychophysiological threat responses.

In parallel, an independent vmPFC-focused (ROI) mediation analysis demonstrated that LT-treatment significantly reduced the vmPFC partial threat expression (path a, b = −0.007, Z = −3.41, P < 0.001) and that lower vmPFC neural threat expression led to reduced psychophysiological threat expression with treatment as an adjustor (path b, b = 9.511, Z = 4.323, P < 0.001). More important, a significant mediation effect was found (a × b, b = −0.066, Z = −3.715, P < 0.001). In summary, these results suggested that LT enhanced early extinction learning through vmPFC processing.

Visualization

Statistical maps were visualized using the Connectome Workbench (https://www.humanconnectome.org/software/connectome-workbench) and Mango (http://ric.uthscsa.edu/mango/). Behavioral data were plotted using Seaborn (https://seaborn.pydata.org/) and Dabest (https://github.com/ACCLAB/DABEST-python) [35].

Acknowledgements

We would like to thank Marianne Cumella Reddan for assistance with the threat expression analysis. This work was supported by grants from National Natural Science Foundation of China (NSFC) [91632117; 31530032]; Fundamental Research Funds for the Central Universities [ZYGX2015Z002]; Science, Innovation and Technology Department of the Sichuan Province [2018JY0001].

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Posted April 12, 2019.
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Human extinction learning is accelerated by an angiotensin antagonist via ventromedial prefrontal cortex and its connections with basolateral amygdala
Feng Zhou, Yayuan Geng, Fei Xin, Jialin Li, Pan Feng, Congcong Liu, Weihua Zhao, Tingyong Feng, Adam J. Guastella, Richard P. Ebstein, Keith M. Kendrick, Benjamin Becker
bioRxiv 512657; doi: https://doi.org/10.1101/512657
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