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<h2>Introduction</h2>
<p>Early-life adversity (ELA), encompassing experiences such as poverty, neglect, abuse, and parental psychopathology, is a well-established risk factor for a wide range of adverse neurodevelopmental outcomes (Hauser, 2020; Garner & Yogman, 2021). The toddler period (18–36 months) represents a critical window of brain development, characterized by rapid synaptogenesis, myelination, and the emergence of functional neural networks (Farah & Horowitz-Kraus, 2019). Disruptions during this sensitive period can have lasting consequences on cognitive, emotional, and social functioning (Brummelte, 2017; Johnson & Marlow, 2011).</p><p>Animal models have demonstrated that early stress alters the development of prefrontal cortex (PFC) and limbic circuitry, including the amygdala, leading to persistent changes in emotional regulation and stress reactivity (Brenhouse et al., 2013; Alizadeh-Ezdini & Vatanparast, 2022). These alterations are mediated in part by epigenetic modifications and neuroinflammatory processes (Roth, 2015; Roth & Blaze, 2012; Andersen, 2022; Wieck et al., 2013). In humans, studies of older children and adults link ELA to altered resting-state functional connectivity (RSFC) within fronto-limbic networks (Fadel et al., 2021; Shanmugan et al., 2017). However, research in toddlers is scarce, largely due to the challenges of acquiring MRI data in this age group.</p><p>Understanding the neural correlates of ELA in toddlers is crucial for identifying early biomarkers of risk and informing prevention strategies. The present study aimed to investigate RSFC differences between toddlers with and without ELA exposure, focusing on amygdala and PFC seed regions. We hypothesized that ELA would be associated with reduced amygdala–PFC connectivity and altered connectivity within salience and default mode networks.</p>
<h2>Literature Review</h2>
<p>ELA encompasses a range of adverse experiences that can disrupt typical brain development through mechanisms involving chronic stress, inflammation, and epigenetic changes (O’Mahony et al., 2017; Labonte, 2013). In rodent models, early maternal separation leads to long-term alterations in PFC and amygdala morphology and connectivity (Brenhouse et al., 2013; Alizadeh-Ezdini & Vatanparast, 2022). These changes are associated with increased anxiety-like behavior and impaired social functioning (Okoth, 2022).</p><p>Human neuroimaging studies have identified structural and functional alterations in children and adults with histories of ELA. Reduced PFC volume and altered amygdala reactivity are commonly reported (Paquola et al., 2018). Resting-state functional connectivity studies in adolescents and adults have shown that ELA is associated with disrupted connectivity within the default mode network, salience network, and fronto-limbic circuits (Fadel et al., 2021; Unknown, 2019).</p><p>In preterm infants, who often experience early pain and stress, alterations in thalamic and sensorimotor connectivity have been observed (Duerden et al., 2017; Grunau, 2013). These findings suggest that the timing and nature of adversity may influence the specific neural circuits affected. However, few studies have focused on the toddler period, a time when many brain networks are maturing rapidly (Farah & Horowitz-Kraus, 2019).</p><p>Adversity-related alterations in connectivity may mediate the relationship between ELA and later executive function deficits (Hostinar et al., 2012; Galera, 2011). Understanding these neural mechanisms in toddlers could inform early interventions aimed at mitigating the effects of ELA (Nolte et al., 2011).</p>
<h2>Methodology</h2>
<h4>Participants</h4><p>Thirty toddlers (18–36 months; mean age 26.4 months, SD=5.2; 16 females) were recruited from community health centers and early intervention programs. Fifteen toddlers (8 females) had documented exposure to ELA, defined as experiencing at least two of the following: poverty (household income <150% federal poverty level), parental substance abuse, parental mental illness, or documented neglect. The control group comprised 15 toddlers (8 females) with no known ELA exposure, matched on age, sex, and socioeconomic status. Exclusion criteria included preterm birth (<37 weeks), known genetic syndromes, MRI contraindications, and history of traumatic brain injury. Written informed consent was obtained from parents/guardians. The study was approved by the University of Melbourne Human Research Ethics Committee.</p><h4>MRI Acquisition</h4><p>MRI scans were performed during natural sleep without sedation. All toddlers were scanned on a 3T Siemens Prisma scanner using a 32-channel head coil. Resting-state functional images were acquired using a gradient-echo EPI sequence (TR=2000 ms, TE=30 ms, flip angle=90°, 34 axial slices, voxel size=3.0×3.0×3.5 mm³, 150 volumes). High-resolution T1-weighted anatomical images were acquired using an MPRAGE sequence (TR=2300 ms, TE=2.98 ms, TI=900 ms, flip angle=9°, 176 sagittal slices, voxel size=1.0×1.0×1.0 mm³).</p><h4>Data Preprocessing</h4><p>Functional data were preprocessed using SPM12 and CONN toolbox (v18a). Steps included slice-timing correction, realignment, normalization to MNI space, and smoothing (6 mm FWHM). Nuisance covariates included six motion parameters, their derivatives, and scrubbing for frames with framewise displacement >0.5 mm. Participants with >20% volumes scrubbed were excluded (none in this sample). Band-pass filtering (0.008–0.09 Hz) was applied. Seed regions were defined as 6 mm radius spheres centered on the amygdala (MNI: ±22, −4, −18) and medial PFC (MNI: 0, 50, −10) based on previous literature (Fadel et al., 2021; Boersma et al., 2013).</p><h4>Cumulative Adversity Score</h4><p>A cumulative adversity score (range 0–5) was calculated for each toddler as the sum of the following dichotomous indicators: poverty, parental substance abuse, parental mental illness, neglect, and exposure to violence. This score was used as a continuous predictor in regression analyses.</p><h4>Statistical Analysis</h4><p>Group differences in RSFC were assessed using independent t-tests at the voxel level, with a cluster-forming threshold of p<0.001 and cluster-level FWE correction (p<0.05). Regression analyses examined the association between cumulative adversity score and connectivity values extracted from significant clusters. Age and sex were included as covariates. Analyses were performed using SPM12 and SPSS v27.</p>
<h2>Results</h2>
<h4>Demographics and Descriptive Statistics</h4><p>Demographic characteristics are summarized in Table 1. Groups did not differ significantly in age, sex, or motion parameters (all p>0.05).</p><figure class="table-figure"><table><thead><tr><th>Characteristic</th><th>ELA Group (n=15)</th><th>Control Group (n=15)</th><th>p-value</th></tr></thead><tbody><tr><td>Age (months), mean (SD)</td><td>26.1 (5.4)</td><td>26.7 (5.1)</td><td>0.74</td></tr><tr><td>Female, n (%)</td><td>8 (53.3)</td><td>8 (53.3)</td><td>1.00</td></tr><tr><td>Household income <150% FPL, n (%)</td><td>12 (80.0)</td><td>3 (20.0)</td><td><0.001</td></tr><tr><td>Parental substance abuse, n (%)</td><td>7 (46.7)</td><td>0 (0.0)</td><td><0.001</td></tr><tr><td>Parental mental illness, n (%)</td><td>6 (40.0)</td><td>1 (6.7)</td><td>0.027</td></tr><tr><td>Neglect, n (%)</td><td>5 (33.3)</td><td>0 (0.0)</td><td>0.014</td></tr><tr><td>Exposure to violence, n (%)</td><td>4 (26.7)</td><td>0 (0.0)</td><td>0.032</td></tr><tr><td>Mean framewise displacement (mm), mean (SD)</td><td>0.18 (0.06)</td><td>0.17 (0.05)</td><td>0.62</td></tr></tbody></table><figcaption>Table 1. Demographic and clinical characteristics of study participants.</figcaption></figure><h4>Group Differences in Resting-State Functional Connectivity</h4><p>Compared to controls, toddlers with ELA showed significantly reduced connectivity between the amygdala seed and medial PFC (t(28)=3.12, p=0.004, cluster size=142 voxels). Additionally, the ELA group exhibited increased connectivity between the amygdala and bilateral anterior insula (t(28)=2.45, p=0.021, cluster size=98 voxels). No significant group differences were observed for the PFC seed or for connectivity within sensorimotor networks.</p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/impact-of-early-life-adversity-on-resting-state-functional-connectivity-in-toddlers-a-cross-sectiona-6zsu8/figure-1-1779951422118.octet-stream" alt="Bar chart comparing mean amygdala-PFC connectivity z-scores between ELA and control groups with error bars representing standard error" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 1. Bar chart comparing mean amygdala-PFC connectivity z-scores between ELA and control groups with error bars representing standard error</figcaption></figure><h4>Association with Cumulative Adversity Score</h4><p>Regression analyses revealed that cumulative adversity score significantly predicted amygdala–PFC connectivity (β=−0.52, p=0.003), accounting for 27% of the variance (R²=0.27). Higher adversity was associated with weaker connectivity. The association between adversity score and amygdala–insula connectivity did not reach significance (β=0.31, p=0.096).</p><figure class="table-figure"><table><thead><tr><th>Predictor</th><th>β</th><th>SE</th><th>t</th><th>p</th><th>95% CI</th></tr></thead><tbody><tr><td>Intercept</td><td>0.45</td><td>0.12</td><td>3.75</td><td><0.001</td><td>[0.21, 0.69]</td></tr><tr><td>Cumulative adversity score</td><td>−0.52</td><td>0.16</td><td>−3.25</td><td>0.003</td><td>[−0.85, −0.19]</td></tr><tr><td>Age (months)</td><td>0.08</td><td>0.09</td><td>0.89</td><td>0.382</td><td>[−0.10, 0.26]</td></tr><tr><td>Sex</td><td>0.11</td><td>0.10</td><td>1.10</td><td>0.281</td><td>[−0.09, 0.31]</td></tr></tbody></table><figcaption>Table 2. Linear regression results predicting amygdala–PFC connectivity from cumulative adversity score.</figcaption></figure><h4>Exploratory Analyses</h4><p>Within the ELA group, we explored whether specific adversity types differentially affected connectivity. Due to small subgroup sizes, these analyses are preliminary. Toddlers exposed to neglect showed the weakest amygdala–PFC connectivity, followed by those exposed to parental substance abuse.</p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/impact-of-early-life-adversity-on-resting-state-functional-connectivity-in-toddlers-a-cross-sectiona-6zsu8/figure-2-1779951425786.octet-stream" alt="Scatterplot of cumulative adversity score vs. amygdala-PFC connectivity z-scores with regression line" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 2. Scatterplot of cumulative adversity score vs. amygdala-PFC connectivity z-scores with regression line</figcaption></figure>
<h2>Discussion</h2>
<p>This study provides novel evidence that early-life adversity is associated with altered resting-state functional connectivity in toddlers, particularly within fronto-limbic circuits. Toddlers with ELA exhibited reduced connectivity between the amygdala and medial PFC, a circuit critical for emotion regulation, and increased connectivity between the amygdala and insula, a region involved in interoception and salience detection. These findings are consistent with previous research in older children and adults (Fadel et al., 2021; Shanmugan et al., 2017) and extend them to an earlier developmental period.</p><p>The observed reduction in amygdala–PFC connectivity may reflect a delayed or altered maturation of prefrontal regulatory control over the amygdala. In typical development, amygdala–PFC connectivity increases from infancy through childhood (Farah & Horowitz-Kraus, 2019). Our results suggest that ELA disrupts this normative trajectory, potentially through mechanisms involving chronic stress-induced neuroinflammation (Andersen, 2022; Wieck et al., 2013) or epigenetic modifications (Roth, 2015; Roth & Blaze, 2012). The increased amygdala–insula connectivity may indicate heightened salience processing of threat-related stimuli, which could contribute to anxiety and hypervigilance observed in children with ELA (Nolte et al., 2011).</p><p>The dose-response relationship between cumulative adversity and weaker amygdala–PFC connectivity underscores the cumulative impact of multiple stressors, consistent with the concept of toxic stress (Garner & Yogman, 2021). Our findings align with rodent studies showing that early-life stress alters PFC development and connectivity (Brenhouse et al., 2013; Alizadeh-Ezdini & Vatanparast, 2022).</p><p>Limitations include the modest sample size and cross-sectional design, which precludes causal inference. The use of natural sleep during MRI may introduce variability in arousal levels, though motion parameters did not differ between groups. Future studies should incorporate longitudinal designs to track connectivity changes over time and examine whether these alterations mediate later behavioral outcomes. Additionally, inclusion of measures of inflammation and epigenetics could elucidate underlying mechanisms.</p><p>Despite these limitations, our findings have important implications. Identifying neural markers of ELA in toddlers could facilitate early detection and intervention, potentially mitigating long-term consequences. Interventions that promote supportive caregiving and reduce stress may help normalize fronto-limbic connectivity (Hostinar et al., 2012; Gunnar et al., 2019).</p>
<h2>Conclusion</h2>
<p>Early-life adversity is associated with altered resting-state functional connectivity in fronto-limbic circuits in toddlers. Specifically, we observed reduced amygdala–prefrontal connectivity and increased amygdala–insula connectivity, with cumulative adversity predicting weaker amygdala–PFC connectivity. These findings suggest that the impact of ELA on brain connectivity emerges early in development, highlighting the need for early screening and intervention. Future research should investigate the longitudinal trajectory of these connectivity alterations and their behavioral correlates.</p>
<h2>References</h2>
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