Altered behavioral and amygdala habituation in high-functioning adults with autism spectrum disorder: an fMRI study

Habituation to repeatedly presented stimuli is an important adaptive property of the nervous system. Autism spectrum disorder (ASD) has been associated with reduced neural habituation, for example in the amygdala, which may be related to social impairments. The main focus of this study was to investigate habituation effects on the level of behavioral responses as well as amygdala responses in adults with ASD during a working memory task flanked by task-irrelevant face stimuli. Twenty-two patients with high-functioning autism and 24 healthy controls (HC) were included in this functional magnetic resonance imaging (fMRI) study. We employed an established habituation index to investigate habituation effects. Suggestive of altered habituation, the habituation index showed a decrement of reaction time over the course of the experiment in the HC but not in the ASD group. Similarly, an expected pattern of habituation was evident in amygdala activation in HC but absent in ASD participants. These results provide evidence that habituation may be altered not only on a neural, but also on a behavioral level in ASD. While more research is needed to develop a better understanding of the underlying mechanisms, the current findings support the possibility that deficient habituation may be a biomarker of ASD.


Participants
Participants of both groups were excluded if they had a history of any of the following diagnoses: organic brain syndrome, epilepsy, dementia, schizophrenia, psychosis NOS, bipolar disorder or substance dependence. Further exclusion criteria for all participants were intelligence quotient (IQ) <85, current substance abuse (including alcohol), current moderate to severe depression, current acute inflammatory, neurologic or metabolic illness, pregnancy or breast feeding. Healthy controls (HC) were excluded if they had any history of psychiatric illness. Regarding psychiatric comorbidities in the autism spectrum disorder (ASD) group, 10/22 participants reported symptoms of depression in their lifetime (one patient was treated with sertraline and one with agomelatine at the time of the study), 1/22 reported symptoms of anxiety and 3/22 had been diagnosed with attention deficit hyperactivity disorder or attention deficit disorder (two patients were treated with methylphenidate at the time of the study).
None of the participants had been diagnosed with obsessive-compulsive disorder. Supplementary

Behavioral measures
For all behavioral measures, the first trial in a block was excluded and only valid button presses, defined by a reaction time (RT) between 150ms and 1500ms after stimulus presentation, were included. The hit rate was defined as the number of hits divided by the number of targets. The false-alarm rate was defined as the number of false alarms divided by the number of non-targets.
Tam et al., Supplementary Information for Altered behavioral and amygdala habituation in highfunctioning adults with autism spectrum disorder

Behavioral data
To investigate if the emotional content of the face stimuli had an impact on behavioral measures, we conducted an ANOVA including all face conditions (happy faces, angry faces, neutral faces) as within-subjects factors and group as between-subjects factor.
For RT, we found no significant main effect for condition and no significant interaction between group and condition but a trend for group (F(1,1)=3.762, p=0.059) with participants of the ASD group having longer RTs than the HC group. There were no significant effects for hit rate or false alarm rate. Supplementary

Neuroimaging data
Exploratory whole-brain analyses (one-sample t-tests) of working memory load (contrast 2-back with scrambled images > 0-back with scrambled images) and face processing (contrast 2-back with all face conditions > 2-back with scrambled images) confirmed that our task elicited expected activation patterns in commonly associated brain regions (Supplementary Figure S1). However, no group differences (two-sample t-tests) were evident anywhere in the brain at a family-wise error (FWE)-corrected threshold of p<0.05. Additionally, we extracted parameter estimates from the bilateral amygdala region of interest (ROI) for the five different conditions included in our basic General Linear Model (GLM) (2-back task with neutral faces, 2-back task with happy faces, 2-back task with angry faces, 2-back task with scrambled images, 0-back task with scrambled images). Two-sample t-tests did not reveal group differences for any condition (2-back task with neutral faces, 2-back task with happy faces, 2-back task with angry faces, 2-back task with scrambled images), for the working memory load  Figure S1. Results of exploratory whole-brain analyses designed to confirm that the employed task elicited expected activation patterns associated with working memory load (Panel A) and face processing (Panel B). Panel A: Activation map for the contrast 2-back with scrambled images > 0-back (p=0.001, cluster size=50, uncorrected) over all participants with activation in the typical working memory-related brain regions DLPFC, VLPFC and mediofrontal wall 1 , no group differences between ASD group and HC group emerged (correction with FWE at p=0.05). Slices are located at the following MNI coordinates (-4,20,42). Panel B: Activation map for the contrast 2-back with all face conditions > 2-back with scrambled images (p=0.001, uncorrected) over all participants with bilateral activation in face-sensitive areas 2 , no group differences between ASD group and HC group emerged (correction with FWE at p=0.05). Slices are located at the following MNI coordinates (44,-50,-18). Results were visualized using xjView toolbox (http://www.alivelearn.net/xjview). Supplementary Table S3 provides more details.  Figure S3: Box plot for amygdala activation (z-standardized parameter estimates), showing the median, first and third quartile and outliers (asterisks, defined as 1.5 interquartile ranges below the first quartile or above the third quartile). Abbreviations: ASD=autism spectrum disorder, HC=healthy controls.
Supplementary Figure S4: Box plot for b' for reaction time, showing the median, first and third quartile and outliers (asterisks, defined as 1.5 interquartile ranges below the first quartile or above the third quartile). Abbreviations: b'RT= b' for reaction time, ASD=autism spectrum disorder, HC=healthy controls.