Neural Sensitivity following Stress Predicts Anhedonia Symptoms: A 2-Year Multi-wave, Longitudinal Study

Animal models of depression show that acute stress negatively impacts functioning in neural regions sensitive to reward and punishment, often manifesting as anhedonic behaviors. However, few human studies have probed stress-induced neural activation changes in relation to anhedonia, which is critical for clarifying risk for affective disorders. Participants (N=85, 12–14-years-old, 53 female), oversampled for risk of depression, were administered clinical assessments and completed an fMRI guessing task to probe neural response to receipt of rewards and losses. After the initial task run, participants received an acute stressor and then, were re-administered the guessing task. Including baseline, participants provided up to 10 self-report assessments of life stress and symptoms over a 2-year period. Linear mixed-effects models estimated whether change in neural activation (post- vs. pre-acute stressor) moderated the longitudinal associations between life stress and symptoms over time. Primary analyses indicated that adolescents with stress-related reductions in right ventral striatum response to rewards exhibited stronger longitudinal associations between life stress and anhedonia severity pFDR=.048. Secondary analyses showed that longitudinal associations among life stress and depression severity were moderated by stress-related increases in dorsal striatum response to rewards pFDR<.002. Additionally, longitudinal associations among life stress and anxiety severity were moderated by stress-related reductions in dorsal anterior cingulate cortex and right anterior insula response to loss pFDR≤.012. All results held when adjusting for comorbid symptoms. Results show convergence with animal models, highlighting mechanisms that may facilitate stress-induced anhedonia as well as a separable pathway for the emergence of depressive and anxiety symptoms.

Dopamine is released from the ventral tegmental area (VTA) and projects to the ventral striatum, (e.g., the nucleus accumbens; NAcc) and medial prefrontal cortex, which then feeds back to the dorsal striatum (caudate, putamen) [11]. Prolonged stress can reduce dopamine availability [10,12], and over time, reduces motivation, incentive-based learning, and social interactions [9,13,14]. Building on extensive animal research demonstrating that stress negatively affects neural pathways [8], the current study aimed to investigate whether neural sensitivity following acute stress exacerbated the longitudinal association between life stress and anhedonia among adolescents.
Human research shows that stressful life events impact reward functioning, which can contribute to anhedonia [15]. Research in adolescents has primarily focused on early life adversity, which associates with blunted reward-related striatal activation [16][17][18][19] and increases insula activation following reward [20]. However, insula ndings may vary depending on the type of adversity [21]. The effects of acute stress on reward activation among adolescents are less clear. In adults, acute stress reduces rewardrelated activation in the dorsal striatum [22,23], orbitofrontal cortex [23], and increases medial prefrontal cortex activation [24]. Additionally, we previously observed reduced ventral striatal, dorsal anterior cingulate cortex (dACC), and anterior insula activation during reward processing post-stress [25]. Overall, there is evidence that acute stress impacts cortico-striatal activation during reward processing, but further research is needed to test how this contributes to the emergence of psychiatric symptoms during a peak adolescent period of risk.
Stress also impacts neural pathways that contribute to depressive and anxiety symptoms. In depression, research consistently shows striatal blunting to rewarding stimuli, but results are less consistent regarding loss or punishment [26][27][28][29][30]. In the context of stress, early life stress in humans relates to blunted striatal activation to reward [19], a pattern of activity that also associates with depression severity [31]. Moreover, blunted striatal reward activation is an important potential mediator of the association between early life stress and depressive symptoms [32]. Additionally, activation in the anterior insula [33] and dACC [34] is typically increased in depression, implicating possible roles in pain, salience monitoring, and tracking of loss magnitude [35][36][37]. Interestingly, we also have found that increased anterior insula and dACC activation in response to social rejection exacerbates the longitudinal association between peer stress and depression severity [38].
However, during acute stress, research shows increased ventral but decreased dorsal striatum activation in relation to anxiety [43]. Increased insula and dACC activation in response to rejection or loss have been associated with anxiety [44,45] and uncertainty [46]. Yet, ndings are mixed regarding dACC responses to rejection in relation to early life stress [47,48]. Together, reward-related striatal responses tend to be decreased in depression and increased in anxiety; though, this may vary between the ventral and dorsal striatum in anxiety. Moreover, both depression and anxiety tend to increase dACC and insula activation.
However, the effects of acute stress on reward processing in these regions in relation to symptom severity remains unclear.
The current study oversampled adolescents at risk of depression (by virtue of a maternal history of depression) to increase variability of clinical and stress symptoms. At the initial assessment, participants completed a reward processing task both prior to and following an acute social stressor. Additionally, life stress exposure and psychiatric symptoms were assessed at regular intervals over a 2-year period. Our primary hypothesis was that an acute stress-related decrease in striatal response to monetary rewards would strengthen the longitudinal association between life stress and anhedonia. Our secondary hypotheses parsed associations with depression and anxiety severity. We expected that an acute stressrelated decrease in striatal response to monetary rewards as well as increased dACC and anterior insula response to monetary loss would strengthen the longitudinal association between life stress and depression severity. Moreover, acute stress-related increase in striatal response to monetary rewards as well as increased dACC and anterior insula response to loss would strengthen the longitudinal association between life stress and anxiety symptom severity.

Participants and Procedure
Adolescents (N = 149, ages 12-14-years-old) and their birth mothers were enrolled from the Boston metro area [25,38]. Adolescents were enrolled as either high-risk for depression if their mothers had experienced at least one MDD episode or low-risk for depression if their mothers had no lifetime history of MDD.
Inclusion criteria for adolescent participants included right-handedness and uency in English. Adolescent participants were excluded at baseline if they endorsed any lifetime psychiatric disorder, current psychotropic medication, neurological illnesses, or MRI contraindication.
The Partners Institutional Review Board approved study procedures. Adolescents assented and legal guardians provided written consent. At baseline, participants were administered clinical interviews and self-report assessments. Then, 1-2 weeks later, fMRI data were acquired. In the scanner, participants completed one run of the Guessing Task (no-stress condition), were exposed to an acute social stressor, and then completed a second run of the Guessing Task (stress condition). Including baseline assessments at the scan, participants completed up to ten self-reported stress and symptom assessments over a two-year period.
Following the initial clinical assessment, participants were excluded: (a) based on child and parent diagnoses (n = 20), (b) if they did not complete baseline clinical measures (n = 2), and (c) if they did not complete the MRI scan (n = 5). Additionally, participants were excluded if they did not nish both runs of the Guessing Task (n = 9), one or both scan runs contained excessive head movement (i.e., > 30% of volumes with FD > .3mm; n = 17), or if they did not complete at least two follow-up assessments (n = 11). The nal sample included 85 participants (high-risk, n = 21).
Adolescent Self-report Measures. Participants rated their developmental stage using the Tanner Staging Questionnaire [51], which measures developmental status on a scale from 1 (no pubertal development) to 5 (adult level of pubertal development). Additionally, participants completed self-reported questionnaires of symptoms and stress. The Snaith-Hamilton Pleasure Scale (SHAPS) [52] is a 14-item questionnaire designed to measure hedonic capacity. Items were reverse scored, and thus, higher total scores, ranging from 14-56, re ected greater anhedonia severity (Cronbach's =0.85-0.92). The Mood and Feelings Questionnaire (MFQ) [53] is a 33-item questionnaire (score range = 0-66) assessing depression symptom severity in the past two weeks. Higher scores indicate greater depression severity (Cronbach's =0.86-0.93). The Multidimensional Anxiety Scale for Children (MASC) [54] is a 39-item questionnaire (score range = 0-117) that measures recent anxiety symptom severity. Higher scores indicate greater anxiety severity (Cronbach's =0.83-0.91). The Adolescent Life Events Questionnaire (ALEQ) [55] is a 57-item questionnaire (score range = 0-228) that measures past-month stress across family, peer, romantic, and academic domains. Higher scores indicate greater stress severity.

fMRI Task
The Guessing Task [56] probes brain activation following the receipt of monetary reward and loss feedback. For each trial, there was a jittered inter-trial interval, which presented a xation cross for 1300-9100 ms. Then, participants viewed two identical doors side-by-side and were instructed to select the door they thought contained a reward as quickly as possible by pressing the left or right button on the button box, respectively. Participants were instructed that for each trial, there was an option to win $0.50 behind one of the doors or lose $0.25 behind the other door. The doors were presented for up to 3900 ms, after which the doors disappeared. After a brief xation cue, feedback was displayed: either a green '↑' indicating a correct guess (Reward Feedback) or a red '↓' re ecting an incorrect guess (Loss Feedback). Participants completed 48 trials (a total of 12 minutes). Unbeknownst to participants, the outcome was xed, as participants received equivalent win and loss feedback in pseudorandom order. This task was administered twice, which occurred both before (no-stress condition) and after (post-stress condition) the acute stressor.

Acute Stress Manipulation
Prior to entering the scanner, participants rated their positive (i.e., happy, joyful) and negative (i.e., upset, discouraged) affect on a visual analog scale from 0 (not very true of me) to 100 (very true of me). After the rst run of the Guessing Task, participants completed the Chatroom Task [40,57], which is designed to probe neural processes related to social feedback. Brie y, participants completed an online pro le, rated pro les of age-and gender-matched peers, and then, were informed that peers from collaborating institutions would review their pro les and indicate whether they were interested (i.e., peer acceptance) or not interested (i.e., peer rejection) in chatting online with them [38]. The Chatroom MRI Task was then used as an acute stressor. Speci cally, following the completion of the task, a screen was displayed with the following feedback, "Individual Performance: Peer Acceptance: 38%, Peer Rejection: 62%; Average Participant Performance: Peer Acceptance: 64%, Peer Rejection: 36%." Study staff explained the feedback with the following, "Based on the breakdown from today, it seems like you're accepted by fewer teens compared to other teens completing the task. Additionally, you are being rejected more than other teens that have completed the selection process." The second part of the stressor included the rationale for redoing The Guessing Task. For this part, study staff read the following statement to participants, "Unfortunately, your performance in the Guessing Task was below average. Remember, you earned only $12 out of a possible $24. For the data to be usable, a participant needs to earn more than $14. Thus, we're going to need to redo this task. Please try to focus." After study staff read these statements aloud, α α participants rated how they felt on the same visual analog scale that was administered prior to entering the scanner, which was followed by completing the second run of The Guessing Task.

fMRI Analysis
We used AFNI [62, 63] to postprocess data and perform rst-level GLMs using 3dDeconvolve, separately for Pre-stress and Post-stress conditions. First, we calculated the number of voxel outliers at each volume of the timeseries using 3dToutcount. Second, we spatially smoothed the data using 3dBlurInMask with a 4mm FWHM kernel. Next, a functional mask was created per participant using 3dAutomask to remove areas with signal dropout. Third, functional data were rescaled (Mean = 100, range = 0-200). Last, we constructed GLMs (3dDeconvolve), which regressed the 32 motion confounds and volumes denoted as outliers. The GLMs also included regressors for the doors trials with standard gamma HRF (GAM) or a gamma function convolved with a variable duration boxcar (dmBLOCK): (1) Cue plus Anticipation, (2) Decision; variable duration, (3) Loss Feedback, (4) Win Feedback, and (5) Non-Response. We t models with restricted maximum likelihood estimation of temporal auto-correlation structure using 3dREML.

Motion Correction
Several steps were undertaken to reduce the effects of motion artifact. Based on Fair et al. (2020), we applied a notch lter with minimum and maximum respiratory rates of 0.31Hz and 0.43Hz, respectively [64] to the 6-parameter head motion estimates to remove respiration-related effects. TRs exhibiting large motion (≥ 0.3mm) between successive TRs were regressed out (i.e., denoted as an outlier) in addition to TRs where at least 5% of brain voxels were computed as timeseries outliers.

ROI Selection
Nine a priori ROIs (Supplementary Fig. 1) were selected based prior literature and on our previous work with a subset of the current sample [25]. Reward-related ROIs included the bilateral nucleus accumbens, caudate, and putamen extracted from the probabilistic Harvard-Oxford subcortical atlas at a 50% threshold. Loss-related ROIs included the dACC and bilateral anterior insula. We used neurosynth (www.neurosynth.org; Yarkoni et al., 2011) to obtain a reward mask (uniformity test: p < 0.01 FDR corrected), which included the dorsal anterior cingulate cortex (dACC) and anterior insula. All ROIs were resampled to 2mm isotropic voxels and grey-matter masked (MNI152Nlin2009cAsym at 25% probability threshold). Authors visually con rmed ROI coverage of each participant's signal (3dAutomask) across pre-and post-stress conditions. Sensitivity analyses were conducted to exclude participants who did not exhibit full coverage of ROIs. Finally, the mean activation for all non-zero voxels within each ROI was extracted for each participant using 3dROIstats. To obtain estimates of change, we regressed post-stress activation onto pre-stress activation, which yields standardized residuals for each region.

Data Analytic Approach
Analyses were conducted in Rv4.2.2 [66]. Linear mixed-effects models were rst performed to estimate the effect of repeated measures of clinical symptoms across baseline and follow-up assessments without brain variables to determine covariates for subsequent models, including life stress (ALEQ), age, sex, pubertal stage (Tanner), risk group, and visit number. Separate linear effects models were conducted with anhedonia (SHAPS), depression (MFQ), and anxiety (MASC) symptoms as the dependent variables. Clinical outcomes were Winsorized if values exceeded Q3 ± 3*IQR. To disaggregate the between-and within-person effects of life stress, all models included a level-2 xed effect for sample-centered mean stress score (i.e., between-person, or time-invariant) and a level-1 person-mean-centered stress score (i.e., within-person stress, or time-varying). We included a random intercept of person and random slope of visit number, and 95% con dence intervals were bootstrapped.
Following determination of covariates for each outcome, linear mixed-effects models estimated whether change in activation (post-vs. pre-acute stressor) moderated the longitudinal associations between within-person life stress and symptoms over time. False discovery rates (FDR) corrected for six multiple tests probing reward-related regions (bilateral NAcc, bilateral caudate, and bilateral putamen) and three multiple tests probing loss-related regions (dACC, bilateral anterior insula). After determining signi cant interaction effects, separate models were conducted to covary longitudinal (i.e., baseline through followup) clinical symptom data. Code is available at: https://github.com/jackieschwartz/neural_sensitivity_to_stress.

Preliminary Analyses
Participant characteristics are summarized in Table 1. There were no differences in sociodemographic or clinical characteristics among retained and excluded participants (Supplementary Table 1). Correlations among stress and clinical symptoms are included in Table 2 Supplementary Fig. 4), suggesting the acute stress manipulation achieved the desired effect.

Primary Analysis: Predicting Anhedonia Symptoms
An acute stress-related reduction in the right NAcc activation to win signi cantly moderated the association between life stress exposure and anhedonia severity over time, adjusting for age, Risk Group, sex, and between-person stress (β=-.06, 95%CI[-0.11, -0.02], p = .008, p FDR =.048). Importantly, after separately covarying longitudinal depression and anxiety symptoms, the interactions remained signi cant (ps ≤ .01, see Fig. 1). Simple slopes analyses indicated that the association between life stress and anhedonia was blunted among those with stress-related increase in NAcc activation. Results remained signi cant in sensitivity analyses excluding three participants missing up to 10 voxels of NAcc coverage (ps < .018, see Supplementary analyses indicated that participants with higher stress-related increase in brain response to rewards showed the strongest associations between life stress and depression severity. These models remained signi cant when removing three potential outlier observations (ps ≤ .002), one participant missing 2 voxels of putamen coverage (ps < .001, see Supplementary Table 3 Table 6) or within the loss condition emerged (Supplementary Table 7).
Stress-related change in dACC activation (β=-.07, 95%CI[-0.12,-0.02], p = .008, p FDR =0.012) and right anterior insula activation (β=-.07, 95%CI[-0.12,-0.02], p = .002, p FDR =.006) to loss moderated the association between follow-up stress and anxiety, adjusting for sex and between-person stress. Importantly, after adjusting for longitudinal depression and anhedonia symptoms during the follow-up periods, the interactions remained signi cant (ps ≤ .008; Fig. 3). Contrary to our hypotheses, simple β β β β β slopes analyses indicated that participants with a greater stress-related decrease in brain response to loss showed the strongest associations between life stress and anxiety severity. There also were no signi cant interactions between the dorsal or ventral striatum and stress within the win condition predicting follow-up anxiety severity (Supplementary Table 8) or between the left anterior insula and stress in the loss condition (Supplementary Table 9).

DISCUSSION
Stress strongly impacts reward functioning and, for some, alters incentive processing [14,67,68]. Stress exposure increases risk for psychiatric symptoms [69,70], but the mechanisms through which stress leads to these symptoms remains unclear. Animal models have implicated dysfunction of dopaminergic system, and although challenging in humans, it is possible to indirectly probe dopaminergic neural processes during stress-related change. Accordingly, we implemented an acute stress manipulation to test whether change in neural activation to monetary rewards and losses pre-to-post stress moderated the longitudinal association between life stress exposure and psychiatric symptoms.
In line with our hypothesis, decreased activation of the ventral striatum (right NAcc) to rewards pre-topost-stress moderated the longitudinal association between life stress and anhedonia in adolescents. Speci cally, individuals with stress-related reduction in striatal response to rewards showed stronger association between life stress and anhedonia, whereas those exhibiting stress-related increases showed blunted association between life stress and anhedonia. Acute stress temporarily recruits dopamine to engage in adaptive learning and coping mechanisms [9,71]. Thus, a reduced response to rewards following acute stress may indicate greater sensitivity to the effects of future life stressors and a more anhedonic phenotype (i.e., diminished pleasure or motivation). Although this association was identi ed in the right NAcc, we did not observe a signi cant effect in the left NAcc. Consistent with the lateralization of our ventral striatum ndings, Webb and colleagues [72] found that greater pre-treatment right, but not left, striatal response to wins predicted greater improvement in anhedonia post-treatment in adolescents. However, Eckstrand et al., (2019) reported the left, but not right, activation of the ventral striatum to rewards associated with improved longitudinal severity in young adults, and there are also reports of bilateral activation in the ventral striatum in relation to anhedonia [30]. It is possible that developmental changes in lateralization of the ventral striatum in relation to stress and anhedonia occur throughout adolescence. Contrary to our hypotheses and to previous research [74][75][76][77], we did not nd that the dorsal striatum moderated the association between stress and anhedonia. Although the dorsal and ventral striatum have shown blunted reactivity to rewards, these regions are functionally distinct in their reward processing roles. Whereas the ventral striatum is involved mainly in reward valuation [35,78], the dorsal striatum is often involved in response inhibition and action-dependent decision making [23,79]. Further research comparing ventral and dorsal activation probing different aspects of reward processing (e.g., reward learning) may clarify speci city of the striatum as it relates to stress and risk for the unfolding of anhedonia symptoms.
The striatum is part of a larger cortico-striatal circuit that includes the insula and anterior cingulate cortex [80], which are regions typically recruited during stressful situations to help guide attention and shift goaldirected behaviors [81]. In our study, we did not nd that the change in activation to loss in the dACC and anterior insula following acute stress moderated the association between life stress and anhedonia.
Given evidence of dACC and anterior insula activation relating to anhedonia particularly in the context of uncertain reward cues [82] and estimation of effort associated with rewards and costs [83], it may be that change in activation to loss in these regions following stress relates more to apprehension dimensions of anhedonia [84], rather than general anhedonia.
Our secondary aim was to test whether neural sensitivity to stress moderates the association between life stress and depression and anxiety symptom severity. Contrary to our hypotheses, increased dorsal striatal (putamen and caudate) activation to rewards post-stress moderated the longitudinal association between life stress and depression severity. Reductions in dorsal striatal activation during reward processing have been implicated in depression [75,85], and dorsal striatal activity to rewards typically decreases following acute stress [23]. Twenty-ve percent of our sample had mothers with a history of depression, which commonly co-occurs with other disorders (e.g., addiction, anxiety, eating disorders) [86,87]. It is possible that risk for these comorbidities may be re ected in striatal activation patterns observed.
Contrary to our hypothesis, reduced dACC and right anterior insula activation to loss post-stress moderated the longitudinal association between life stress and anxiety severity. The dACC and anterior insula are key regions of the salience network, which are connected to subcortical regions of the striatum, and implicated in response to uncertainty and salient environmental cues [88]. Our ndings con ict with previous research linking heightened activation of salience network regions with anxiety [89]. Although, one study found that that stress-induced activation of the dorsal ACC and anterior insula was not related to anxiety in adolescents [43], other studies have found activation of the ACC and insula is negatively associated with anxiety in youth [90,91], possibly re ecting in exibility responding to change or error, particularly from childhood to adolescence. Given the dACC and insula are regions that also help to engage cognitive control [92], it is possible that a stress-induced decrease in dACC and anterior insula activation to loss in our study re ects a di culty to exibly respond to future life stress.
Although our study has important strengths (e.g., within-scanner stress manipulation, longitudinal psychiatric and stress data), this study has some limitations. First, although adolescents repeatedly reported on their psychiatric symptoms and life stress for up to ten times over the course of two years, which allowed us to detect within-person effects, our sample size was relatively small to detect betweenperson individual differences. Second, the reward paradigm was repeated within a single session to gauge neural responses to acute stress, but it was not repeated during the follow-up. Future research may explore whether developmental differences in neural sensitivity to stress impact the association between psychiatric symptoms and life stress. Third, although our sample included a portion of adolescents who were at high risk for depression given their maternal history, adolescents were psychiatrically healthy at the time of the scan. Results may differ in a more clinically acute sample of adolescents. Additionally, our sample were mostly White with a high socioeconomic status limiting the generalizability of our results to minoritized individuals.
Our study expanded on animal studies examining the effects of acute stress by investigating stressrelated change in neural activation within an fMRI paradigm in relation to several assessments of anhedonia and life stress symptoms. Similar to other research [38], our ndings can be interpreted in the context of diathesis-stress models, as stress-related changes in the ventral striatum may serve as a vulnerability marker that increases risk for heightened anhedonia severity when life stress occurs. Additionally, stress-related changes in the striatum, dACC, and anterior insula differentially moderate the association between life stress and other related clinical symptoms, such as depression and anxiety. As adolescents experience new life stressors and increases in internalizing problems, our ndings may shed light on potential neurobiological mechanisms that link the long-established associations between life stress and internalizing symptoms.

Declarations
Author Contributions: DAP and RPA designed research; JSK and DP conducted analyses; JSK wrote rst draft; JSK, DP, DAP, and RPA reviewed and edited drafts.