Aberrant associations between neuronal resting-state fluctuations and working memory-induced activity in major depressive disorder.

Previous investigations have revealed performance deficits and altered neural processes during working-memory (WM) tasks in major depressive disorder (MDD). While most of these studies used task-based functional magnetic resonance imaging (fMRI), there is an increasing interest in resting-state fMRI to characterize aberrant network dynamics involved in this and other MDD-associated symptoms. It has been proposed that activity during the resting-state represents characteristics of brain-wide functional organization, which could be highly relevant for the efficient execution of cognitive tasks. However, the dynamics linking resting-state properties and task-evoked activity remain poorly understood. Therefore, the present study investigated the association between spontaneous activity as indicated by the amplitude of low frequency fluctuations (ALFF) at rest and activity during an emotional n-back task. 60 patients diagnosed with an acute MDD episode, and 52 healthy controls underwent the fMRI scanning procedure. Within both groups, positive correlations between spontaneous activity at rest and task-activation were found in core regions of the central-executive network (CEN), whereas spontaneous activity correlated negatively with task-deactivation in regions of the default mode network (DMN). Compared to healthy controls, patients showed a decreased rest-task correlation in the left prefrontal cortex (CEN) and an increased negative correlation in the precuneus/posterior cingulate cortex (DMN). Interestingly, no significant group-differences within those regions were found solely at rest or during the task. The results underpin the potential value and importance of resting-state markers for the understanding of dysfunctional network dynamics and neural substrates of cognitive processing.

Investigation of Neurofunctional Changes Over the Course of Electroconvulsive Therapy.

BACKGROUND: Electroconvulsive therapy (ECT) is an effective treatment for patients suffering from depression. Yet the exact neurobiological mechanisms underlying the efficacy of ECT and indicators of who might respond best to it remain to be elucidated. Identifying neural markers that can inform about an individual's response to ECT would enable more optimal treatment strategies and increase clinical efficacy. METHODS: Twenty-one acutely depressed inpatients completed an emotional working memory task during functional magnetic resonance imaging before and after receiving treatment with ECT. Neural activity was assessed in 5 key regions associated with the pathophysiology of depression: bilateral dorsolateral prefrontal cortex and pregenual, subgenual, and dorsal anterior cingulate cortex. Associations between brain activation and clinical improvement, as reflected by Montgomery-Åsberg Depression Rating Scale scores, were computed using linear regression models, t tests, and Pearson correlational analyses. RESULTS: Significant neurobiological prognostic markers or changes in neural activity from pre- to post ECT did not emerge. CONCLUSIONS: We could not confirm normalization effects and did not find significant neural markers related to treatment response. These results demonstrate that the search for reliable and clinically useful biomarkers for ECT treatment remains in its initial stages and still faces challenges.

Modulatory Effects of Ketamine and Lamotrigine on Cognition: Emotion Interaction in the Brain.

INTRODUCTION: Cognition and emotion are fundamentally integrated in the brain and mutually contribute to behavior. The relation between working memory (WM) and emotion is particularly suited to investigate cognition-emotion interaction since WM is an essential component of many higher cognitive functions. Ketamine affects not only WM but also has a profound impact on emotional processing. Effects of acute ketamine challenge are sensitive to modulation by pretreatment with lamotrigine, which inhibits glutamate release. Accordingly, a combination of these approaches should be particularly suited to investigate cognition-emotion interaction. METHODS: Seventy five healthy subjects were investigated in a double-blind, placebo-controlled, randomized, single-dose, parallel-group study with three treatment conditions. All subjects underwent two scanning sessions (acute/post 24 h). RESULTS: Compared to placebo, acute ketamine administration induced significant dissociative, psychotomimetic, and cognitive effects, as well as an increase in neural activity during WM for positive stimuli. Inhibition of glutamate release by pretreatment with lamotrigine did not influence ketamine's subjective effects, but significantly attenuated its impact on emotional WM and associated neural activity. There was no effect on these measures 24 h after ketamine administration. CONCLUSION: Our results demonstrate differential acute effects of modulated glutamate release and a swift restoration of disturbed neurobehavioral homeostasis in healthy subjects.

Functional connectivity changes between amygdala and prefrontal cortex after ECT are associated with improvement in distinct depressive symptoms.

Electroconvulsive therapy (ECT) is one of the most effective treatments for treatment-resistant depression. However, the underlying mechanisms of action are not yet fully understood. The investigation of depression-specific networks using resting-state fMRI and the relation to differential symptom improvement might be an innovative approach providing new insights into the underlying processes. In this naturalistic study, we investigated the relationship between changes in resting-state functional connectivity (rsFC) and symptom improvement after ECT in 21 patients with treatment-resistant depression. We investigated rsFC before and after ECT and focused our analyses on FC changes directly related to symptom reduction and on FC at baseline to identify neural targets that might predict individual clinical responses to ECT. Additional analyses were performed to identify the direct relationship between rsFC change and symptom dimensions such as sadness, negative thoughts, detachment, and neurovegetative symptoms. An increase in rsFC between the left amygdala and left dorsolateral prefrontal cortex (DLPFC) after ECT was related to overall symptom reduction (Bonferroni-corrected p = 0.033) as well as to a reduction in specific symptoms such as sadness (r = 0.524, uncorrected p = 0.014), negative thoughts (r = 0.700, Bonferroni-corrected p = 0.002) and detachment (r = 0.663, p = 0.004), but not in neurovegetative symptoms. Furthermore, high baseline rsFC between the left amygdala and the right frontal pole (FP) predicted treatment outcome (uncorrected p = 0.039). We conclude that changes in FC in regions of the limbic-prefrontal network are associated with symptom improvement, particularly in affective and cognitive dimensions. Frontal-limbic connectivity has the potential to predict symptom improvement after ECT. Further research combining functional imaging biomarkers and a symptom-based approach might be promising.

The influence of childhood emotional maltreatment on cognitive symptoms, rumination, and hopelessness in adulthood depression.

Childhood emotional maltreatment (CEM) is a risk factor for the pathogenesis of depressive disorders. However, it is not clear whether CEM is more strongly related to specific symptoms of depression and whether specific traits or cognitive states may mediate the association between CEM and depressive symptoms. In our cross-sectional study, including 72 patients with a current depressive episode, we investigated if CEM is specifically related to cognitive symptoms of depression. In addition, we evaluated whether CEM also influences the extent of rumination and hopelessness in adult depression. Using multiple regression analyses, we tested if CEM and rumination could predict cognitive symptoms and hopelessness. A structural equation model (SEM) was used to examine if rumination mediates the relationship between CEM and cognitive symptoms. Correlational analyses revealed that CEM was related to cognitive symptoms, rumination, and hopelessness. The regression analyses showed that only rumination was a significant predictor for cognitive symptoms and hopelessness, whereas CEM could not significantly predict the two constructs. SEM revealed that the association between CEM and cognitive symptoms in adult depression was mediated by rumination. Our results thereby suggest that CEM is a risk factor particularly for the development of cognitive symptoms as well as rumination and hopelessness in adult depression. However, the influence on cognitive symptomatology seems to be indirectly regulated by rumination. These findings may contribute to a better understanding of processes that promote depression, as well as provide guidance for more targeted treatment options.

Predicting Antidepressant Effects of Ketamine: the Role of the Pregenual Anterior Cingulate Cortex as a Multimodal Neuroimaging Biomarker.

BACKGROUND: Growing evidence underscores the utility of ketamine as an effective and rapid-acting treatment option for major depressive disorder (MDD). However, clinical outcomes vary between patients. Predicting successful response may enable personalized treatment decisions and increase clinical efficacy. METHODS: We here explored the potential of pregenual anterior cingulate cortex (pgACC) activity to predict antidepressant effects of ketamine in relation to ketamine-induced changes in glutamatergic metabolism. Prior to a single i.v. infusion of ketamine, 24 patients with MDD underwent functional magnetic resonance imaging during an emotional picture-viewing task and magnetic resonance spectroscopy. Changes in depressive symptoms were evaluated using the Beck Depression Inventory measured 24 hours pre- and post-intervention. A subsample of 17 patients underwent a follow-up magnetic resonance spectroscopy scan. RESULTS: Antidepressant efficacy of ketamine was predicted by pgACC activity during emotional stimulation. In addition, pgACC activity was associated with glutamate increase 24 hours after the ketamine infusion, which was in turn related to better clinical outcome. CONCLUSIONS: Our results add to the growing literature implicating a key role of the pgACC in mediating antidepressant effects and highlighting its potential as a multimodal neuroimaging biomarker of early treatment response to ketamine.

Acute effects of ketamine on the pregenual anterior cingulate: linking spontaneous activation, functional connectivity, and glutamate metabolism.

Ketamine exerts its rapid antidepressant effects via modulation of the glutamatergic system. While numerous imaging studies have investigated the effects of ketamine on a functional macroscopic brain level, it remains unclear how altered glutamate metabolism and changes in brain function are linked. To shed light on this topic we here conducted a multimodal imaging study in healthy volunteers (N = 23) using resting state fMRI and proton (1H) magnetic resonance spectroscopy (MRS) to investigate linkage between metabolic and functional brain changes induced by ketamine. Subjects were investigated before and during an intravenous ketamine infusion. The MRS voxel was placed in the pregenual anterior cingulate cortex (pgACC), as this region has been repeatedly shown to be involved in ketamine's effects. Our results showed functional connectivity changes from the pgACC to the right frontal pole and anterior mid cingulate cortex (aMCC). Absolute glutamate and glutamine concentrations in the pgACC did not differ significantly from baseline. However, we found that stronger pgACC activation during ketamine was linked to lower glutamine concentration in this region. Furthermore, reduced functional connectivity between pgACC and aMCC was related to increased pgACC activation and reduced glutamine. Our results thereby demonstrate how multimodal investigations in a single brain region could help to advance our understanding of the association between metabolic and functional changes.

The role of comorbid depressive symptoms on long-range temporal correlations in resting EEG in adults with ADHD.

Attention deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder, characterized by core symptoms of inattention, hyperactivity and impulsivity. Comorbid depression is commonly observed in ADHD-patients. Psychostimulants are recommended as first-line treatment for ADHD. Aberrant long-range temporal correlations (LRTCs) of neuronal activities in resting-state are known to be associated with disorganized thinking and concentrating difficulties (typical in ADHD) and with maladaptive thinking (typical in depression). It has yet to be examined whether (1) LRTC occur in ADHD-patients, and if so, (2) whether LRTC might be a competent biomarker in ADHD comorbid with current depression and (3) how depression affects psychostimulant therapy of ADHD symptoms. The present study registered and compared LRTCs in different EEG frequency bands in 85 adults with ADHD between groups with (n = 28) and without (n = 57) additional depressive symptoms at baseline. Treatment-related changes in ADHD, depressive symptoms and LRTC were investigated in the whole population and within each group. Our results revealed significant LRTCs existed in all investigated frequency bands. There were, however, no significant LRTC-differences between ADHD-patients with and without depressive symptoms at baseline and no LRTC-changes following treatment. However, depressed ADHD patients did seem to benefit more from the therapy with psychostimulant based on self-report.

Effects of Mindfulness Training on Emotion Regulation in Patients With Depression: Reduced Dorsolateral Prefrontal Cortex Activation Indexes Early Beneficial Changes.

OBJECTIVE: Mindfulness-based interventions (MBIs) have been found to be a promising approach for the treatment of recurrent courses of depression. However, little is known about their neural mechanisms. This functional magnetic resonance imaging study set out to investigate activation changes in corticolimbic regions during implicit emotion regulation. METHODS: Depressed patients with a recurrent lifetime history were randomized to receive a 2-week MBI (n = 16 completers) or psychoeducation and resting (PER; n = 22 completers). Before and after, patients underwent functional magnetic resonance imaging while labeling the affect of angry, happy, and neutral facial expressions and completed questionnaires assessing ruminative brooding, the ability to decenter from such thinking, and depressive symptoms. RESULTS: Activation decreased in the right dorsolateral prefrontal cortex (dlPFC) in response to angry faces after MBI (p < .01, voxel-wise family-wise error rate correction, T > 3.282; 56 mm3; Montreal Neurological Institute peak coordinate: 32, 24, 40), but not after PER. This change was highly correlated with increased decentring (r = -0.52, p = .033), decreased brooding (r = 0.60, p = .010), and decreased symptoms (r = 0.82, p = .005). Amygdala activation in response to happy faces decreased after PER (p < .01, family-wise error rate corrected; 392 mm3; Montreal Neurological Institute peak coordinate: 28, -4, -16), whereas the MBI group showed no significant change. CONCLUSIONS: The dlPFC is involved in emotion regulation, namely, reappraisal or suppression of negative emotions. Decreased right dlPFC activation might indicate that, after the MBI, patients abstained from engaging in elaboration or suppression of negative affective stimuli; a putatively important mechanism for preventing the escalation of negative mood.Trial Registration: The study is registered at ClinicalTrials.gov (NCT02801513; 16/06/2016).

A symptom-based approach in predicting ECT outcome in depressed patients employing MADRS single items.

Establishing symptom-based predictors of electroconvulsive therapy (ECT) outcome seems promising, however, findings concerning the predictive value of distinct depressive symptoms or subtypes are limited; previous factor-analytic approaches based on the Montgomery-Åsberg Depression Rating Scale (MADRS) remained inconclusive, as proposed factors varied across samples. In this naturalistic study, we refrained from these previous factor-analytic approaches and examined the predictive value of MADRS single items and their change during the course of ECT concerning ECT outcome. We used logistic and linear regression models to analyze MADRS data routinely assessed at three time points in 96 depressed psychiatric inpatients over the course of ECT. Mean age was 53 years (SD 14.79), gender ratio was 58:38 (F:M), baseline MADRS score was M = 30.20 (SD 5.42). MADRS single items were strong predictors of ECT response, remission and overall symptom reduction, especially items 1 (apparent sadness), 2 (reported sadness) and 8 (inability to feel), assessing affective symptoms. Strongest effects were found for regression models including item 2 (reported sadness) with up to 80% correct prediction of ECT outcome. ROC analyses were performed to estimate the optimal cut-point for treatment response. MADRS single items during the course of ECT might pose simple, reliable, time- and cost-effective predictors of ECT outcome. More severe affective symptoms of depression at baseline and a stronger reduction of these affective symptoms during the course of ECT seem to be positively associated with ECT outcome. Precise cut-off values for clinical use were proposed. Generally, these findings underline the benefits of a symptom-based approach in depression research and treatment in addition to depression sum-scores and generalized diagnoses.

Correction to: Brief training in mindfulness may normalize a blunted error-related negativity in chronically depressed patients.

Maria Fissler and Emilia Winnebeck also are affiliated at Department of Psychiatry and Psychotherapy, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.

Echoes of Affective Stimulation in Brain connectivity Networks.

Affective experience has effects on subjective feelings, physiological indices, entails immediate activity changes in the brain, and even influences brain networks in a protracted manner. However, it is still unclear, how the functional connectivity (FC) interplay between major intrinsic connectivity networks upon affective stimulation depends on affective valence, and whether this is specific for affective experience, i.e., can be distinguished from cognitive task execution. Our study included fMRI scans during and after affective stimulation with sad and neutral movies and a working memory task complemented with measures of cardiovascular activity and mood. Via parcellation of the brain into default mode network (DMN), central executive network (CEN), and dorsal attention network, and application of network-based statistics, we identified subnetworks associated with changing psychological contexts. Specific effects for affective stimulation with negative valence were both reduced heart rate variability and mood, and upregulated FC of inter-CEN-DMN connections while intra-DMN connections were downregulated. Furthermore, results demonstrated a valence-specific dynamic carry-over effect in nodes of the CEN, which temporarily increased their FC strength after affective stimulation with negative valence and exhibited distinct temporal profiles. The reported effects were clearly distinguishable from those of a cognitive task and further elucidate the trajectory of affective experience.

The interplay of genetic and environmental factors in shaping well-being across the lifespan: Evidence from the serotonin transporter gene.

BACKGROUND: Converging evidence suggests that well-being plays an important role in promoting and maintaining mental health across the life span. It has been shown that well-being has a considerable heritable component, but little is known about the specific genes involved. METHODS: In this study, we investigated a healthy sample (N = 298) that was genotyped for the serotonin transporter-linked polymorphic region (5-HTTLPR). We hypothesized that 5-HTTLPR gene variation would influence well-being, and additionally investigated interaction effects with age and the environmental influence of early life stress (ELS). RESULTS: Using multiple regression, our results showed a significant three-way interaction between genotype, ELS, and age. Exploration of this interaction showed that young subjects had decreased levels of well-being if they were exposed to ELS and homozygous for the short variant of 5-HTTLPR. This relationship was reversed in old age: subjects that were exposed to ELS and carried the long variant of 5-HTTLPR had decreased levels of well-being. CONCLUSION: Our results indicate that genetic and environmental factors have joint effects on well-being that are susceptible to profound changes across the life span.

Brief training in mindfulness may normalize a blunted error-related negativity in chronically depressed patients.

The error-related negativity (ERN), an evoked-potential that arises in response to the commission of errors, is an important early indicator of self-regulatory capacities. In this study we investigated whether brief mindfulness training can reverse ERN deficits in chronically depressed patients. The ERN was assessed in a sustained attention task. Chronically depressed patients (n = 59) showed significantly blunted expression of the ERN in frontocentral and frontal regions, relative to healthy controls (n = 18). Following two weeks of training, the patients (n = 24) in the mindfulness condition showed a significantly increased ERN magnitude in the frontal region, but there were no significant changes in patients who had received a resting control (n = 22). The findings suggest that brief training in mindfulness may help normalize aberrations in the ERN in chronically depressed patients, providing preliminary evidence for the responsiveness of this parameter to mental training.

Negative emotionality shapes the modulatory effects of ketamine and lamotrigine in subregions of the anterior cingulate cortex.

Neuroimaging studies have identified the anterior cingulate cortex (ACC) as one of the major targets of ketamine in the human brain, which may be related to ketamine's antidepressant (AD) mechanisms of action. However, due to different methodological approaches, different investigated populations, and varying measurement timepoints, results are not consistent, and the functional significance of the observed brain changes remains a matter of open debate. Inhibition of glutamate release during acute ketamine administration by lamotrigine provides the opportunity to gain additional insight into the functional significance of ketamine-induced brain changes. Furthermore, the assessment of trait negative emotionality holds promise to link findings in healthy participants to potential AD mechanisms of ketamine. In this double-blind, placebo-controlled, randomized, single dose, parallel-group study, we collected resting-state fMRI data before, during, and 24 h after ketamine administration in a sample of 75 healthy male and female participants who were randomly allocated to one of three treatment conditions (ketamine, ketamine with lamotrigine pre- treatment, placebo). Spontaneous brain activity was extracted from two ventral and one dorsal subregions of the ACC. Our results showed activity decreases during the administration of ketamine in all three ACC subregions. However, only in the ventral subregions of the ACC this effect was attenuated by lamotrigine. 24 h after administration, ACC activity returned to baseline levels, but group differences were observed between the lamotrigine and the ketamine group. Trait negative emotionality was closely linked to activity changes in the subgenual ACC after ketamine administration. These results contribute to an understanding of the functional significance of ketamine effects in different subregions of the ACC by combining an approach to modulate glutamate release with the assessment of multiple timepoints and associations with trait negative emotionality in healthy participants.

Investigating the impact of rumination and adverse childhood experiences on resting-state neural activity and connectivity in depression.

BACKGROUND: Both ruminative thought processes and adverse childhood experiences (ACEs) are well-established risk factors for the emergence and maintenance of depression. However, the neurobiological mechanisms underlying these associations remain poorly understood. METHODS: We examined resting-state functional magnetic resonance imaging data (3 T Tim Trio MR scanner; Siemens, Erlangen) of 44 individuals diagnosed with an acute depressive episode. Specifically, we focused on investigating functional brain activity and connectivity within and between three large-scale neural networks associated with processes affected in depression: the default mode network (DMN), the salience network (SN), and the central executive network (CEN). Correlational and regression-based analyses were performed. RESULTS: Our regions of interest analyses revealed that region-specific spontaneous neural activity in the anterior DMN was associated with self-reported trait rumination, specifically, the pregenual anterior cingulate cortex (pgACC). Furthermore, using a liberal statistical threshold, we found that spontaneous neural activity of the ventromedial prefrontal cortex and the pgACC were associated with depression symptom severity. Neither spontaneous neural activity in the SN and CEN nor functional connectivity within and across the investigated networks was associated with depression severity or rumination. Furthermore, there was no association between ACEs and brain activity and connectivity. LIMITATIONS: Lack of a formal control group or low-risk group for comparison. CONCLUSIONS: Overall, our results indicate network-specific changes in spontaneous brain activity, that are linked to both depression severity and rumination. Findings underscore the crucial role of the pgACC in depression and contribute to a dimensional and symptom-based understanding of depression-related network imbalances.

Neural underpinnings of open-label placebo effects in emotional distress.

While placebo effects are well-known, research in the last decade revealed intriguing effects that placebos may have beneficial effects even when given without deception. At first glance, this seems paradoxical, but several studies have reported improvements in pain, depression, or anxiety. However, it still remains unclear whether these results represent objective biological effects or simply a bias in response and what neural underpinnings are associated with the open-label placebo effects. In two studies, we address this gap by demonstrating that open-label placebos reduce self-reported emotional distress when viewing highly arousing negative pictures. This reduced emotional distress was associated with an activation of brain areas known to modulate affective states such as the periaqueductal gray, the bilateral anterior hippocampi, and the anterior cingulate cortex. We did not find any prefrontal brain activation. Furthermore, brain activation was not associated with expectation of effects. In contrast, we found that brain responses were linked to general belief in placebos. The results demonstrate that the neural mechanisms of open-label placebo effects are partly identical to the neurobiological underpinnings of conventional placebos, but our study also highlights important differences with respect to a missing engagement of prefrontal brain regions, suggesting that expectation of effects may play a less prominent role in open-label placebos.

Experiencing sweet taste is associated with an increase in prosocial behavior.

Taste may be the first sense that emerged in evolution. Taste is also a very important sense since it signals potential beneficial or dangerous effects of foods. Given this fundamental role of taste in our lives, it is not surprising that taste also affects our psychological perception and thinking. For example, previous research demonstrated remarkable psychological effects of sweet taste experiences, suggesting that sweetness may be a source domain for prosocial functioning. Recent research reports that briefly experiencing sweet taste made participants more helpful in their intentions and behavior. The current study aims to test this hypothesis and to examine the neural underpinnings of this effect by using an fMRI approach. Participants were asked to taste sweet, salty, and neutral taste while lying in the fMRI scanner. Subsequently their prosocial behavior was tested by playing the dictator game, a measure of prosocial behavior. Results showed that sweet taste was associated with an increase in prosocial behavior compared with previously experiencing salty taste but did not affect control stimuli ratings. FMRI results revealed a modulation of the dorsal anterior cingulate cortex associated with this sweetness effect. This brain area is known to play a central role for monitoring conflicts and decisions and has been directly linked to selfish and prosocial economic decisions. The results demonstrate that sweet taste has complex psychological effects including positive and socially desirable outcomes. We discuss the results with other studies on psychological sweetness effects and suggest possible implications of these findings.

Region- and time- specific effects of ketamine on cerebral blood flow: a randomized controlled trial.

There is intriguing evidence suggesting that ketamine might have distinct acute and delayed neurofunctional effects, as its acute administration transiently induces schizophrenia-like symptoms, while antidepressant effects slowly emerge and are most pronounced 24 h after administration. Studies attempting to characterize ketamine's mechanism of action by using blood oxygen level dependent (BOLD) imaging have yielded inconsistent results regarding implicated brain regions and direction of effects. This may be due to intrinsic properties of the BOLD contrast, while cerebral blood flow (CBF), as measured with arterial spin labeling, is a single physiological marker more directly related to neural activity. As effects of acute ketamine challenge are sensitive to modulation by pretreatment with lamotrigine, which inhibits glutamate release, a combination of these approaches should be particularly suited to offer novel insights. In total, 75 healthy participants were investigated in a double blind, placebo-controlled, randomized, parallel-group study and underwent two scanning sessions (acute/post 24 h.). Acute ketamine administration was associated with higher perfusion in interior frontal gyrus (IFG) and dorsolateral prefrontal cortex (DLPFC), but no other investigated brain region. Inhibition of glutamate release by pretreatment with lamotrigine abolished ketamine's effect on perfusion. At the delayed time point, pretreatment with lamotrigine was associated with lower perfusion in IFG. These findings underscore the idea that regionally selective patterns of CBF changes reflect proximate effects of modulated glutamate release on neuronal activity. Furthermore, region- specific sustained effects indicate both a swift restoration of disturbed homeostasis in DLPFC as well changes occurring beyond the immediate effects on glutamate signaling in IFG.