In the present study, we investigated neural system habituation to eye gaze in individuals with fragile X syndrome compared with individuals group-matched for sex, intellectual functioning, autism symptoms, and adaptive behavior. Our primary result reveals less neural habituation in individuals with fragile X syndrome in response to all facial stimuli (direct gaze + averted gaze), and the lack of group difference in activation in run 1 indicates that initial activation differences did not drive this result. There was no evidence for differential habituation to direct gaze compared with averted gaze within or between groups. Less habituation and neural system sensitization was found in the fragile X syndrome group (Figure 2). This effect was evidenced in widespread cortical regions, including those involved in lower- and higher-level visual processing (the fusiform gyrus and bilateral occipital cortex) and emotion processing (the cingulate), as well as executive functioning regions (frontal). This suggests a deficit in habituation to face/eye gaze that is distributed across multiple levels of neural processing. The regions affected are consistent with findings of abnormal morphology and/or function in fragile X syndrome (12, 14, 16). Significant correlations in female participants with fragile X syndrome suggest that habituation is related to individual differences in FMRP levels and autism symptoms.

The habituation observed in our comparison group is consistent with that seen in typically developing individuals: decreased activation in response to repeated stimuli (21, 32) and evidence of neural plasticity reflecting more efficient neural processing (33). Conversely, the sensitization displayed in the fragile X syndrome group indicates deficient modulation of neural responses to repeated gaze stimuli. Sensitization was seen in regions involved in emotion and social cognition (the cingulate, fusiform, and frontal cortex). One possible interpretation of this result is that neural system sensitization to gaze is related to the inability to modulate arousal (11). While the link between sensitization and physiological hyperarousal is not directly supported by our data, previous studies suggest support for this hypothesis. Neuroimaging in individuals with fragile X syndrome demonstrates that both social anxiety and gaze durations are related to brain activation in regions supporting social cognition (34). Atypical eye-gaze behavior has also been linked to aberrant physiology in individuals with fragile X syndrome, including changes in skin conductance (8), cortisol reactivity (9), and pupillary reactivity (10). However, additional factors, such as impaired attention and social cognition, may also contribute to habituation deficits. Furthermore, our design did not allow us to address the specificity of habituation deficits because we did not include nonsocial stimuli. Future investigations of neural system habituation to social and nonsocial stimuli with simultaneous measurement of physiological arousal may help clarify the nature and specificity of habituation deficits.

Further study of habituation in fragile X syndrome could significantly enhance our understanding of associated social avoidance behaviors and provide a useful quantitative measure for evaluating the efficacy of treatments for gaze aversion in fragile X syndrome. Exposure therapy is one treatment technique with the potential to help normalize habituation to eye gaze and reduce gaze aversion. Exposure therapy involves repeated presentations of an aversive stimulus with the goal of decreasing fear or anxiety (35). Exposure therapy in the form of systematic desensitization has been shown to attenuate symptoms of auditory sensitivity in individuals with autism spectrum disorder (36). Furthermore, a previous study by our research group indicated that behavioral shaping reduces gaze aversion in fragile X syndrome (19). These findings suggest that repeated exposure combined with a well-designed behavioral program may facilitate direct gaze. Our present results show increased neural system response to repeated presentations over the course of our relatively brief fMRI experiment, demonstrating plasticity but in the direction of sensitization rather than habituation. Sensitization may be, in part, a result of the social anxiety present in individuals with fragile X syndrome (17), suggesting that adjunctive pharmacological intervention may prove to be useful in reducing anxiety and physiological arousal, thus enhancing the effectiveness of any behavioral (e.g., desensitization) treatment. In this regard, exposure therapy with graded desensitization to eye gaze may attenuate gaze-aversion behavior, and neural system habituation may be a useful outcome measure.

We attribute deficits in habituation primarily to fragile X syndrome and not to general cognitive ability, autism symptoms, or adaptive behavior level because our comparison group was matched to the fragile X syndrome group on IQ and scores on the Autism Diagnostic Observation Schedule and Vineland Adaptive Behavior Scales. Our finding of differential habituation between groups matched on autism symptoms indicates that the neurobiological mechanisms underlying gaze aversion in fragile X syndrome are different from those underlying similar symptoms in other disorders. These findings underscore the importance of designing disease-specific treatments for fragile X syndrome.

Moreover, we revealed a correlation between the degree of habituation and FMRP levels, suggesting a dose-response relationship with the basic biomolecular component underlying fragile X syndrome. Although correlations with habituation were only significant among female participants with fragile X syndrome, potentially as a result of the skewed distribution of FMRP in males, we note that among the few male participants with FMRP levels >20%, FMRP levels appear to fall along the regression line generated for FMRP levels among female participants (Figure 3A). Previously, FMRP levels have been correlated with brain morphology in a group of male and female subjects (13) and with brain function in a study that examined only female subjects (37). Future studies with larger sample sizes for each sex, and that include male and female subjects with similar FMRP levels, will be important for determining the specificity of relationships between FMRP and neurobiological outcomes. The correlation between habituation deficits and autism symptoms in females with fragile X syndrome supports the theory that habituation deficits underlie gaze aversion and impaired social functioning. Studies directly comparing habituation between individuals with fragile X syndrome and those with similar social deficits, such as autism spectrum disorder and social anxiety, would also be beneficial for determining the specificity of these relationships. Furthermore, examining developmental trajectories of habituation, hyperarousal, and autism symptoms will contribute to our understanding of causal pathways among these related deficits. In the present study, the relationships between habituation and clinical measures (FMRP levels and autism symptoms) were significant only within the fusiform gyrus, a face-processing region important for social functioning and known to have abnormal function (14, 16) and morphology (12, 13) in fragile X syndrome.

Interestingly, we did not find significant group differences for habituation in the amygdala, despite its key role in emotion processing. We also did not find a group-by-gaze interaction or any within-group differences in habituation to direct gaze compared with averted gaze in this region. It is possible that our task demands resulted in sustained amygdala activation, and thus habituation or sensitization between runs was not present. This finding is not consistent with those from a previous study by our group, which revealed significant within- and between-group differences in amygdala activation related to gaze direction (15). However, differences in the experimental design and sampling frame may have contributed to the inconsistent results. Most notably, the present study examined habituation and thus presented a smaller number of unique faces (four compared with 120) for longer durations each (4 seconds compared with 1.75 seconds) over a longer experimental period (approximately 14 minutes compared with approximately 9 minutes). Video clips have been shown to elicit differential responses to gaze direction in individuals with autism (38) and may have elicited group differences in amygdala habituation and/or a significant effect of gaze direction in individuals with fragile X syndrome.

Our estimate of eye-movement frequency, BOLD signal change in the eye region (see the online data supplement), indicates that there was no significant group-by-run interaction and no within-group differences in eye movements in run 1 compared with run 2. Thus, a group difference in eye movements did not drive the difference in habituation. We conclude that it is unlikely that group differences in interest or attention were responsible for the differential habituation we observed because such circumstances would be accompanied by changes in eye movements. We observed more eye movements in the fragile X syndrome group than in the comparison group for direct gaze, suggesting atypical responses to direct gaze, perhaps in an effort to avoid direct gaze. More eye movements during direct gaze may have contributed to the lack of difference between habituation to direct gaze compared with averted gaze in the fragile X syndrome group and/or to lack of a group difference within the direct gaze condition for run 1. Future studies using an eye tracker could be used to determine where a participant is fixating and to further delineate face/gaze processing in fragile X syndrome.

Inclusion of individuals receiving medications was a necessary limitation because medication use is high among the populations we studied. Importantly, the fragile X syndrome and comparison groups did not differ with regard to the number of medications used (in general and by class [Table 1]). Therefore, it is unlikely that the group differences were driven by medication use.

In summary, individuals with fragile X syndrome displayed a deficit in neural habituation and demonstrated sensitization to face/gaze that is distributed across multiple levels of processing. Sensitization in regions involved in emotion and social cognition may be related to an inability to modulate social anxiety, given previous results demonstrating social anxiety and hyperarousal related to gaze processing. Correlation results suggest that habituation is related to individual differences in FMRP levels and autism symptoms assessed outside the scanner. Although the relationships were only significant among female participants, they provide preliminary evidence supporting the relevance of neural system habituation as a biomarker for designing and assessing treatment trials, such as exposure therapy, for gaze aversion in fragile X syndrome.