Frequency-Specific Regional Homogeneity Alterations in Tourette Syndrome

Tourette syndrome (TS) is a developmental neuropsychiatric disorder with onset during childhood. Because of its complex spectrum of phenotypes, the underlying pathophysiology of TS is still unclear. Resting-state functional magnetic resonance imaging demonstrated aberrant spontaneous neural synchronization in conventional frequency band (0.01–0.08 Hz) in TS. No published studies have reported abnormalities of local synchronization across different frequency bands. We estimated the alterations of local synchronization across five bands ranging from 0 to 0.25 Hz. Seventy-nine children with TS and 63 age-, sex-, and handedness-matched healthy children were recruited. Frequency-specific regional homogeneity (ReHo) and independent component analysis were used to identify functional alterations between TS and healthy children. TS patients showed significantly increased ReHo in the left precentral gyrus and decreased ReHo in the right operculum. Abnormal ReHo alterations of the superior frontal gyrus, superior parietal gyrus, anterior cingulate gyrus, putamen, superior temporal gyrus, and operculum were observed in different frequency bands. TS patients showed increased connectivity of the right superior frontal gyrus within the left executive control network. In addition, a significantly negative correlation was found between Yale Global Tic Severity Scale (YGTSS) vocal score and ReHo values of the right operculum in the highest frequency bands (0.198–0.25 Hz), while a significant positive correlation was found between YGTSS motor score and altered connectivity of the right superior frontal gyrus. The present study revealed frequency-specific abnormal alterations of ReHo in the whole brain and altered connectivity within the executive control network of TS children. Its neural importance and clinical practicability require further investigation.


INTRODUCTION
Tourette syndrome (TS) is a developmental neuropsychiatric disorder with an onset during childhood, defined by the co-occurrence of motor and phonic tics lasting at least 12 months. Tics typically show a waxing and waning pattern of severity, intensity, and frequency (1). The prevalence of TS for children in China is 1.7% (2). Previous studies showed that ∼90% of TS children have comorbid conditions, including obsessivecompulsive disorder (OCD), attention-deficit hyperactivity disorder (ADHD), rage attacks, sleep disturbances, and depressive symptoms (3), which might have a negative impact on their quality of life (4). Moreover, the underlying pathophysiology of TS is controversial and poorly understood because of the complex spectrum of phenotypes involved in TS (5).
Advanced neuroimaging methods have been used to investigate the neural basis of TS over the last few decades. Many neuroimaging studies using structural magnetic resonance imaging (MRI), positron emission tomography (PET), and diffusion tensor imaging techniques have reported abnormalities in the basal ganglia and associated thalamic and frontal cortical regions (6), supporting a hypothesis of dysfunction in the cortico-striato-thalamo-cortical (CSTC) networks in TS (7). A recent meta-analysis of task-based neuroimaging studies showed that TS patients had abnormal activation in the prefrontal, anterior cingulate, motor preparation cortices, and sensory and temporo-parietal association cortices (8). A previous study using functional MRI (fMRI) showed abnormal functional connections in the executive control network, especially in the fronto-parietal network in TS patients (9). This suggested that the broadly distributed involvement of multiple cortical regions or brain networks, in addition to the CSTC network, might play a critical role in TS pathophysiology.
Because TS children cannot perform specified tasks well, there have been few task-related fMRI studies in TS children. Resting-state fMRI (RS-fMRI) has the advantage of measuring spontaneous neural activity such as regional homogeneity (ReHo) in the TS population (10,11). A previous study of TS with a small sample size (n = 21) showed decreased ReHo in the right cerebellum that was positively correlated with disease duration (11); however, no cortical regions were identified with abnormal ReHo. That study only observed the local synchronization of spontaneous RS-fMRI signals at a low frequency band (0.01-0.08 Hz), and therefore information about other lower-or higherfrequency realms is unknown (12). In contrast, a broader band of 0.01-0.08 Hz, containing several mixed physiological alterations of potentially specific frequencies, may lead to negative or inexact results. Zuo et al. discovered that even in the conventional lowfrequency band (<0.1 Hz), blood oxygenation level-dependent (BOLD) fluctuations were stronger at 0.01-0.027 Hz in the subcortical region and at 0.027-0.073 Hz in the cortical region (13). Frequency characteristic analysis demonstrated that ReHo oscillations lower than 0.02 Hz mainly occurred in the putamen and that higher-frequency oscillations mainly occurred in limbic areas (>0.08 Hz) (14). Thus, a frequency-specific approach may provide more information than that of the conventional band (<0.1 Hz) to help understand the local BOLD activity of the cortical regions in TS.
To discover frequency-specific functional alterations that might have an important role in the pathophysiology of TS, we used RS-fMRI in a frequency-specific manner (0-0.01 Hz, 0.01-0.027 Hz, 0.027-0.073 Hz, 0.073-0.198 Hz, and 0.198-0.25 Hz) to compare TS patients with healthy controls (HC) (13,15). Independent component analysis (ICA) was used to detect connectivity differences within brain networks between TS and HC groups. In the current study, we included subjects with comorbid symptoms who are most typical of TS [as "pure" TS without any comorbidity occurs in only 10% of patients (3)] to obtain generalizable results for clinical practice.

Participants
Ninety-four children with TS were recruited from outpatient clinics of the Second Affiliated Hospital, Zhejiang University School of Medicine. All patients met the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, text revision (DSM-IV-TR) criteria. Sixty-three age-, sex-, and handednessmatched healthy children without any neurological or psychiatric disorders were recruited as HC. Participants were excluded if they had (i) any neurological (other than tics) or psychiatric diseases (other than ADHD and OCD), (ii) structural abnormalities on visual inspection of structural imaging, and (iii) head motion exceeding 3 mm in translation or 3 • rotation in any direction. After head-motion control, fifteen patients were excluded. Seventy-nine TS patients were eligible to take part in the current study. All clinical evaluations were performed on the day of acquisition of MRI scans by an experienced pediatric neurologist ( For ICA, fMRI data were spatially smoothed with 6 mm full width half maximum Gaussian kernel after normalization. Subsequently, Group ICA of the fMRI Toolbox v3.0b (GIFT, http://icatb.sourceforge.net) was used to calculate spatially independent components (ICs). Dimension estimation of data from both groups was conducted using the minimum description length (MDL) criterion to determine the number of ICs (18). Then, individual maps in each group were conjoined and the temporal dimension of the convergent data was reduced via principal component analysis, followed by IC estimation. For all subjects, the spatial component maps were converted into z-score maps.
The number of ICs in the two groups (TS and HC) was 43. These components were selected based on the largest spatial correlation with a specific resting-state network template (http:// findlab.stanford.edu/functional_ROIs.html) for further analysis. The brain networks included basal ganglia, sensorimotor, and the left and right executive control networks (one sample t-test, multiple-comparison correction based on Gaussian random field theory, single voxel p < 0.001, cluster level p < 0.05, Figure 1).

Statistical Analysis
Two-way repeated-measures analysis of variance (ANOVA) was performed to examine the effects of group and frequency band on the ReHo maps. Group (TS and HC) served as a betweensubject factor and frequency band (0-0.01 Hz, 0.01-0.027 Hz, 0.027-0.073 Hz, 0.073-0.198 Hz, and 0.198-0.25 Hz) served as a within-subject factor. An F-map of the "frequency by group" interaction effect and an F-map of the group main effect were obtained. Multiple-comparison correction was conducted based on Gaussian random field (GRF) theory (single voxel p < 0.001, cluster level p < 0.05). The peak ReHo values of the clusters surviving from the group main effect were extracted and then entered SPSS software for two-sample t-tests in each frequency band The peak ReHo values of TS patients were correlated against the clinical measurements, including YGTSS scores and SNAP-IV scores.
To explore connectivity differences between the two groups, two sample t-tests were performed within each network. Multiple-comparison correction was conducted based on GRF theory (single voxel p < 0.001, cluster level p < 0.05).

RESULTS
There were no significant differences in age (p = 0.599), sex (p = 0.066), and handedness (p = 0.370) between the two groups. The detailed demographic variables and clinical characteristics of participants are shown in Table 1.

Differences in Regional Homogeneity
ANOVA showed a significant main effect of group and a significant interaction effect (Figure 2, Table 2). For the main effect of group, the brain regions included the left precentral gyrus and the right operculum (frontal and temporal lobes). For the interaction effect, the brain regions included the right anterior cingulate gyrus (ACC)/supplementary motor cortex (SMA), right superior frontal gyrus, right Rolandic operculum, right putamen, right superior parietal gyrus, left superior temporal gyrus, and left white matter. Generally, in TS patients, the right superior frontal, and superior parietal gyrus showed an increased ReHo in lower bands and turned to normal in the higher bands, while the right   Rolandic operculum and ACC/SMA showed a decreased ReHo in lower bands and turned to normal in higher bands. The right putamen and the left superior temporal gyrus showed a trend of decreased ReHo in lower frequency bands and increased ReHo in higher frequency bands (Figure 3, Table 3).
The two-sample t-tests showed significantly increased ReHo in full bands of the left precentral gyrus and significantly decreased ReHo in full bands of the right operculum (frontal and temporal lobes) ( Table 4).
The ReHo value of the right operculum (frontal and temporal lobes) showed significant negative correlations with vocal scores of YGTSS in the highest frequency bands (0.198-0.25 Hz) (Bonferroni correction, 0.05/15 = 0.0033; Figure 4, Table 5). No significant correlation was found between the ReHo value and any rating score in the left precentral gyrus.

Differences of Connectivity Within Networks
Compared with HC, TS patients showed increased connectivity of the right superior frontal gyrus (BA 6, MNI coordinates X = 21, Y = 9, Z = 63, T = 4.132, Cluster size = 945 mm 3 ) within the left executive control network (Figure 5). No significant group difference was found in the basal ganglia, sensorimotor, and right executive control network. The peak Z-value of the right superior frontal gyrus was extracted to perform correlation analysis against clinical assessment scores. Only the motor score of YGTSS showed a positive correlation with abnormal connectivity (Figure 6).

Reduced Synchronization Within the CSTC Circuit in TS
The present study found decreased ReHo in the right putamen and increased ReHo in the left precentral gyrus of TS children. The putamen, a key node of the basal ganglia, is involved in motor loop circuits (19). The precentral gyrus responds to visual input and codes information during movement preparation (20), which involves cognitive processing for movements such as motor preparation and execution (21). The putamen and precentral gyrus are both parts of the CSTC circuit.
Functional deficits in the CSTC circuit may contribute to the motor symptoms of TS (7). The putamen, the center of the motor portion of the CSTC in the genesis of tics, receives abundant projections from the motor and somatosensory cortices (22). Defects in the modulation of activity in the structures of the motor CSTC network may underlie the failure to inhibit unwanted impulses in patients with TS (7). Previous studies demonstrated the aberrant structure, metabolism, and activity of the putamen and primary motor cortices in TS patients (23)(24)(25). Microstructural changes in the white matter of the left precentral gyrus and putamen in unmedicated and pure TS, and the regional apparent diffusion coefficient values were positively correlated with tic severity (25). A proton magnetic resonance spectroscopy study of children with TS showed significant reductions of N-acetylaspartate and choline in the putamen and reduced N-acetylaspartate in the frontal cortical (24). TS children were shown to have increased spontaneous local brain activity in the putamen and bilateral thalamus during RS-fMRI scans (26). Two event-related functional neuroimaging studies support the idea that the primary motor cortex processes preparatory signals related to motor tic behavior in TS (23,27). One study showed significant fMRI activity in the primary motor cortex before tic onset (23). Another study found that cortical structures, including the primary motor cortex, preceded subcortical activation prior to tic onset (27). Decreased ReHo in the putamen and increased ReHo in the precentral gyrus in the current study indicated reduced synchronization of spontaneous neural activity in the putamen might inhibit the output nuclei of the basal ganglia, which in turn excite the precentral cortex to produce tics.

Abnormal Synchronization Within Executive Control Networks in TS
Executive control networks are thought to include the dorsolateral prefrontal, anterior cingulate cortices, and the orbitofrontal cortex (28). Many studies provided evidence of possible executive deficits in TS during various executive tasks such as working memory, fluency, shifting, and especially inhibition (29). TS patients could not suppress unwanted movements, and mainly comorbid with ADHD (3). The present study found that ReHo was decreased in the frontal and temporal    operculum and ACC, but increased in the superior frontal and parietal cortexes. These regions were reported to be involved in two principal attention control networks. The cingulo-opercular network showed sustained activation across all tasks or nearly all tasks and was hypothesized to maintain task sets (30). The fronto-parietal network, which flexibly segregated or integrated in different aspects of control such as focused proactive control and episodic memory (31), has a crucial role in rapidly adaptive online control (32).
The present results showed reduced synchronization within the cingulo-opercular network (frontal operculum and ACC). Task-maintenance processes may be affected resulting in unwanted breakthroughs of normally suppressed behaviors, such as tics. However, the hyperactive frontal-parietal network (superior frontal and parietal cortexes) motivated initiate and adjust change in task control, again leading to unwanted movements and vocalizations (9). In previous fMRI studies of TS, activation of the operculum, ACC, insular, SMA, primary motor, and somatosensory cortex were found prior to tic onset (23), while decreased amplitude of low-frequency fluctuation (ALFF) and fractional ALFF in the ACC, frontal and parietal gyrus were detected during the resting state (26). In addition, fewer functional connections were found in the fronto-parietal and cingulo-opercular networks in TS children vs. adults and adolescents (9). Abnormalities of metabolism, anatomy, and function in the ACC, an important component of the cinguloopercular network, have been reported in many previous TS studies (33)(34)(35)(36)(37). For instance, significantly decreased gray matter volumes were found in the ACC of TS patients (36). A singlephoton emission tomography study reported the hypoperfusion of the ACC during the resting state, which was related to the severity of tics (35), while another study found increased ACC activation during voluntary tic suppression (34,37). A recent proton magnetic resonance spectroscopy study of TS patients also found that concentrations of glutamine, an important excitatory neurotransmitter, negatively correlated with tic severity scores in ACC (33). Furthermore, we observed a negative correlation between tic severity and ReHoin the operculum. The greater the reduction of synchronization in the operculum, the more serious the disease is thought to become. Above all, altered ReHo of the cingulo-opercular and frontoparietal networks, especially in the operculum and ACC in the current study, suggested that these brain areas may have an important role in the progress of tic generation mainly by breaking task set maintenance and enhancing adaptive tasks.
In addition to local synchronization, the present study used ICA to detect connectivity differences within the basal ganglia, sensorimotor, and executive control networks. We found increased connectivity of the right superior frontal gyrus within the left executive control network. The executive control network shares major nodes with the frontal-parietal network, including the dorsolateral prefrontal cortex and posterior parietal cortex. It is involved in the control of higher-order cognitive neural functions such as attention, planning, decision-making, and working memory (38). Recent years transcranial magnetic stimulation (TMS) studies attempted to indirectly impact deep or remote brain areas via functional connectivity. Some researchers have successfully observed TMS-induced functional connectivity alterations by stimulating the superficial cortex (39,40). Our previous work also found increased ReHo in cerebellum after high frequency TMS on precentral gyrus (41). In addition, there was a significant positive correlation between functional connectivity of the right superior frontal gyrus and YGTSS motor score. The right superior frontal gyrus might be a direct or indirect target of TMS for TS treatment. This also suggested that Tics may be aggravated by enhanced functional connectivity within the executive control network, supporting the putative role of the control network in the pathogenesis of TS.

Frequency-Specific Altered Synchronization in TS
To the best of our knowledge, this is the first study to examine abnormalities of regional synchronization in a relatively large and typical pediatric TS population using the frequency specificity of ReHo. Several studies demonstrated the frequency specificity of ReHo changes in neurologic and psychiatric disorders, such as paroxysmal kinesigenic dyskinesia (42), schizophrenia (43), and major depression (44). In the present study, significantly increased ReHoin the superior frontal gyrus and superior parietal gyrus of TS children indicated compensatory functions of the frontal-parietal network in the lower frequency bands. These findings are consistent with previous reports that the lower frequency band (0.01-0.027 Hz) had a higher BOLD fluctuation in the cortical regions (13). We also found decreased ReHo in the putamen, ACC, and superior temporal gyrus in the lower-frequency bands, which was normal or reversed in the higher frequency bands. Previous RS-fMRI studies indicated that the higher band (0.027-0.073 Hz) exhibited increased ALFF compared with the lower band (0.01-0.027 Hz) in the subcortical regions (13,45). The interaction of frequency band × group on ReHo indicated a significant difference in frequency-specific alterations (Figure 3, Table 3). The results of the current study suggested that the local intrinsic brain activity of TS was sensitive to specific frequency bands. More frequency-specific investigations are needed in the future.
The significant negative correlation between ReHo and YGTSS scores was found only in the highest frequency bands (0.198-0.25 Hz) (Figure 4, Table 5), suggesting that the ultrahigh frequency band might be of clinical importance. These findings indicate that abnormal ReHoin TS is frequencydependent and might be missed when using routine frequency band. Although the nature of these frequency-specific alterations of local neuronal homogeneity is still unclear, different frequency bands should be considered when measuring the ReHo of TS children in future studies to further understand the pathology of TS.

CONCLUSION
In summary, the present RS-fMRI study revealed frequencyspecific abnormal regional homogeneity and altered connectivity in children with TS. The regions of frequency-specific abnormalities included the frontal-parietal network, ACC, and putamen, as well as an altered connectivity region in the executive control network. These brain regions are involved in multiple cognitive dysfunctions characteristic of TS. All these aberrant regions might be direct treatment targets or indirect targets for brain stimulation, especially those regions with frequency-specific abnormalities, which might be sensitive to specific stimulation frequencies for TMS. These targets should be tested and verified in TS populations in the future.

LIMITATIONS
This study had several limitations. The drugs used for TS treatment may affect brain function. The dose and duration of drug information were not recorded completely in the current study. The variability of the doses and types of drugs used makes it difficult to analyze the contribution of each drug on ReHo. Therefore, drug-naïve status and type of medication used should be taken into consideration in the future.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/supplementary material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Center for Cognition and Brain Disorders, Hangzhou Normal University, China. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.
Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
Y-TL: design the study, statistical analysis, and writing of the first draft. X-LL: fMRI data processing and statistical analysis. YW: neurological evaluation and psychiatric evaluation. G-JJ: fMRI data acquisition. Y-FZ: revised the article and developed the research concept. JW: fMRI data processing and statistical analysis, revised the article, and developed the research concept. J-HF: research project conception and organization, neurological evaluation, revised the article, and developed the research concept. All authors: contributed to the article and approved the submitted version.