Functional connectivity-based parcellation of the human sensorimotor cortex

Page 1 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 1 Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation Yang Fan1, 2, Lisa D. Nickerson3, 4, Huanjie Li1, 2, Yajun Ma1, 2, Bingjiang Lyu1, 2, Xinyuan Miao5, Yan Zhuo5, Jianqiao Ge1, Qihong Zou1, *, Jia-Hong Gao1, 2, 6, * 1 2 Center for MRI Research, Peking University, Beijing, China Beijing City Key Lab for Medical Physics and Engineering, School of Physics, Peking University, Beijing, China 3 4 5 McLean Imaging Center, McLean Hospital, Belmont, MA, USA Harvard Medical School, Harvard University, Boston, MA, USA State Key Laboratory of Brain and Cognitive Science, Beijing MRI Center for Brain Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 6 McGovern Institute for Brain Research, Peking University, Beijing, China *Please send correspondences to: Jia-Hong Gao, Ph.D. and Qihong Zou, Ph.D. Center for MRI Research, Peking University, Beijing, China, 100871 Emails: jgao@pku.edu.cn and zouqihong@pku.edu.cn Phone: +86-10-62752918 Running head: Functional connectivity-based parcellation of the thalamus Key Words: Thalamus, Parcellation, Resting-state fMRI, Functional connectivity, K-means, Visuomotor task. 1 Page 2 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 2 Abstract Node definition, or delineating how the brain is parcellated into individual functionally-related regions, is the first step to accurately map the human connectome. As a result, parcellation of the human brain has drawn considerable attention in the field of neuroscience. The thalamus is known as a relay in the human brain, with its nuclei sending fibers to cortical and subcortical regions. Functional MRI techniques offer a way to parcellate the thalamus in vivo based on its connectivity properties. However, the parcellations from previous studies show that both the number and the distribution of thalamic subdivisions vary with different cortical segmentation methods. Here, we used an unsupervised clustering method that does not rely on a priori information of the cortical segmentation to parcellate the thalamus. Instead, this approach is based on the intrinsic resting-state functional connectivity profiles of the thalamus with the whole brain. A series of cluster solutions were obtained, and an optimal solution was determined. Furthermore, the validity of our parcellation was investigated through: 1) identifying specific resting-state connectivity patterns of thalamic parcels with different brain networks; and 2) investigating the task activation and psychophysiological interactions of specific thalamic clusters during 8-Hz flashing checkerboard stimulation with simultaneous finger tapping. Together, the current study provides a reliable parcellation of the thalamus and enhances our understating of thalamic. Furthermore, the current study provides a framework for parcellation that could be potentially extended to other subcortical and cortical regions. 2 Page 3 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 3 Acronyms AAL: Automated Anatomical Labeling DC: Dice’s coefficient fMRI: functional MRI FWHM: full-width at half-maximum FWE: family-wise error GLM: general linear model ICA: independent component analysis LFP: local field potential MNI: Montreal Neurological Institute MPRAGE: magnetization-prepared rapid gradient echo MRI: magnetic resonance imaging MD: mediodorsal nucleus PPI: psychophysiological interactions ROI: region of interest VI: variation of information 3 Page 4 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 4 1. Introduction The human brain is a complex system with densely interconnected regions and mapping of this “human connectome” has become a major focus of neuroscience research (Fornito et al., 2013; Sporns, 2013; Wig et al., 2014). The first crucial step to delineate the topological properties of the human brain is node definition, or delineating how the brain is parcellated into individual subunits that can be assessed for functional or structural connections with each other (Fornito et al., 2013). As a result, more and more researchers have lent their attention to parcellation of the human brain, in both cortical and subcortical areas (Bzdok et al., 2013; Craddock et al., 2012; Kim et al., 2013; Shen et al., 2013; Wig et al., 2013). The thalamus acts as a “gateway” between subcortical areas and cerebral cortex (Liang et al., 2013; Sherman and Guillery, 2002), with nearly all areas of neocortex receiving afferents from the thalamus (Guillery and Sherman, 2002). Moreover, the thalamus plays an important role in regulating states of sleep and wakefulness (Steriade and Llinas, 1988) and thalamo-cortical connectivity is reported to be fundamental for the establishment of oscillatory brain waves (Jones, 2001). Dysfunction of the thalamus is implicated in the pathophysiology of neurological and neuropsychiatric disorders (Cauda et al., 2009; Welsh et al., 2010; Woodward et al., 2012). Based on its cytoarchitecture, the thalamus can be divided into several nuclei (Morel et al., 1997) that show distinct anatomical connectivity with different cortical and subcortical regions (Jones, 2007; Sherman and Guillery, 2002). In vivo 4 Page 5 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 5 parcellation of the thalamus into different subdivisions would provide locations of specific thalamic nuclei and thus be of great importance for understanding thalamic functioning and revealing thalamic involvement in the pathophysiology of brain disorders. Magnetic resonance imaging (MRI) techniques are powerful methods for parcellating brain structures in vivo based on either anatomical or functional connectivity to provide insight beyond cytoarchitecture information. For example, there are a few reports that have used a diffusion tensor MRI-based fiber tractography technique (Basser et al., 1994) to parcellate the thalamus into subdivisions according to its anatomical connectivity with seven cortical areas (Behrens et al., 2003; Johansen-Berg et al., 2005; Mastropasqua et al., 2014). Those studies (Behrens et al., 2003; Johansen-Berg et al., 2005)(Mastropasqua et al., 2014) were limited to the investigation of connections between thalamus and large cortical regions. However, as noted by Behrens et al. (2003), the non-human primate literature provides support for a finer-grained topographic mapping between subregions of, e.g., the mediodorsal nucleus of the thalamus and smaller regions in the prefrontal cortex (Goldman‐ Rakic and Porrino, 1985; Kievit and Kuypers, 1977). Furthermore, while anatomical structure is thought to be the foundation of brain function, studies have found a discrepancy between anatomical and functional connectivity in human brain (Honey et al., 2009; Honey et al., 2010). Thus, for 5 Page 6 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 6 functional MRI (fMRI) studies it is equally essential to parcellate the brain based on its functional connectivity. Over the last decade, resting-state fMRI has emerged as a powerful tool for mapping the spontaneous or intrinsic functional connectivity of the human brain with high sensitivity in a completely non-invasive manner that is easy to implement (Biswal et al., 1995; Kim et al., 2013; Zhang et al., 2008; Zhang et al., 2010). Kim and his colleagues (Kim et al., 2013) used an ICA based approach to parcellate thalamus and basal ganglia into several sub-divisions and Zhang et al. used seed-based resting-state fMRI to parcellate the thalamus into five sub-divisions with a Winner-Take-All method to label each thalamic voxel (Zhang et al., 2008; Zhang et al., 2010). The number of thalamic sub-divisions in ICA-based approach is directly determined by the number of independent components. Different definition of independent components will result in varied thalamic parcellation results. By definition, the approach utilized by Zhang et al. is based on a priori knowledge of how the cerebral cortex is segmented. As a result, parcellation of the thalamus with different templates of cortical areas, for example, using the Automated Anatomical Labeling (AAL) template (Tzourio-Mazoyer et al., 2002) or the Brodmann Area template (Brodmann, 1909), will result in a different parcellation of thalamic nuclei, both in the number of parcels and in the spatial distribution of each parcel. Previous study have shown that using functionally inaccurate ROIs, for example, anatomically defined ROIs, for network analysis is extremely damaging to network estimation (Smith et al., 2011). 6 Page 7 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 7 To obviate those mentioned issues, in the present study, a recently proposed approach for parcellating brain regions based on the similarity of their functional profiles with other regions of the brain (Craddock et al., 2012; Kahnt et al., 2012; Kelly et al., 2012; van den Heuvel et al., 2008) was adopted to parcellate the thalamus into different spatially continuous subdivisions without any spatial restrictions. An optimal solution of the number of thalamic subdivisions was obtained by evaluating the stability of diverse parcellation results. The validity of this approach was investigated by assessing: 1) specific functional connectivity fingerprints of the thalamic parcels with known brain networks; 2) brain activation and psychophysiological interactions (PPI) of specific thalamic nuclei during a visuomotor task (8-Hz flashing checkerboard stimulation with simultaneous finger tapping). The visuomotor task allows us to examine the validity of our parcellation because it activates specific thalamic subdivisions (i.e. lateral dorsal and ventral posterior nuclei (Purushothaman et al., 2012; Stepniewska et al., 2007; Strick, 1976)) and it can be used to assess PPIs, which characterize direct connections between thalamic nuclei and other regions during task performance. For example, Purushothaman and his colleagues found that the posterior part of thalamus, which sends fibers to primary visual areas, was involved in visual signal processing. 2. Materials and Methods 2.1. Data Acquisition Both resting-state fMRI and visuomotor task fMRI datasets were included in this 7 Page 8 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 8 study. Resting-state fMRI dataset: Resting-state fMRI data that were acquired at Beijing Normal University and have been made freely available on the website of the 1000 functional Connectomes Project (www.nitrc.org/projects/fcon_1000) were used for this study. The data from 60 subjects (30 females/30 males, 20.8  2.6 years) with complete coverage of cortical regions were selected from the available scans. All subjects provided written informed consent before the experiment, and the study was approved by the institutional review board of Beijing Normal University. MR Images from this dataset were acquired using a Siemens 3T Trio MRI scanner. Resting-state functional images were obtained using a T2*-weighted echo-planar imaging sequence with the following parameters: 33 axial slices, thickness/gap = 3.0/0.6 mm, FOV = 200  200 mm2 with in-plane matrix = 64  64, TR/TE/  = 2000 ms/30 ms/90o, and scanning duration = 7 minutes and 30 seconds with 225 functional volumes. A high-resolution structural MRI 3D image was acquired using a T1-weighted magnetization-prepared rapid gradient echo (MPRAGE) imaging sequence (spatial resolution = 1 mm  1 mm  1.33 mm, TR/TE/  = 2530 ms/3.39 ms/7o, inversion time (TI) = 1100 ms). During the resting-state scanning session, subjects were asked to keep their eyes closed but not fall asleep. Visuomotor task fMRI dataset: Visuomotor task data were acquired in eighteen 8 Page 9 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 9 healthy subjects (9 females/9 males; 22.8  1.8 years) at Chinese Academy of Science. This study was approved by the institutional review board of Chinese Academy of Science. MR images of this dataset were also collected on a Siemens 3T Trio scanner. Task fMRI image parameters of this dataset are as follows: 22 axial slices without gap, slice thickness = 5 mm, FOV = 220  220 mm2 with in-plane matrix = 64  64, and TR/TE/  = 2500 ms/30 ms/90o. A high-resolution structural MRI 3D image was acquired using a T1-weighted MPRAGE sequence (spatial resolution = 1 mm3 isotropic, TR/TE/  = 2530 ms/ 3.39 ms/7o, TI = 1100 ms). In the visuomotor task scans, a block-design was used. It began with a 30-s “off” block and consisted of twelve cycles of alternated 20-s “on” and 20-s “off” blocks. During the “on” block, subjects were asked to view a screen with 8-Hz flashing checkerboard stimulation and tap their finger simultaneously. 2.2. Resting-state data processing The resting-state fMRI images were processed using SPM8 (www.fil.ion.ucl.ac.uk/spm/) and REST (Song et al., 2011; Yan and Zang, 2010) after discarding the first ten functional volumes of each subject to avoid transient signal changes before the longitudinal magnetization reached a steady state. Pre-processing steps are shown in a flow chart (Figure 1). Steps in SPM8 included slice timing correction, head motion correction and spatial normalization to Montreal Neurological 9 Page 10 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 10 Institute (MNI) space (Collins et al., 1994). The voxel size of the normalized functional images was 3 mm isotropic. No subject had head motion larger than 2 mm in translation and/or 2 degree in rotation. As a result, no subject was excluded for large head motion. Linear trend removal and temporal band-pass filtering (0.01-0.08 Hz) (Biswal et al., 1995) were then performed using REST. Spatial smoothing with a Gaussian kernel of 4 mm full-width at half-maximum (FWHM) was applied to our imaging data. To reduce to potential contamination of non-neuronal sources of noise, the anatomical CompCor (aCompCor) method was adopted for noise estimation and removal of noise signals (Behzadi et al., 2007). To further reduce head motion effects, a “temporal scrubbing” method was applied to the pre-processed fMRI data with the threshold for frame-wise displacement (FD) set to 0.5 mm (Power et al., 2012). After the “scrubbing” procedure, most subjects (44/60) had no volumes scrubbed and the most volumes removed were 35 (in one subject). Scrubbing has been shown to improve functional connectivity maps, and in this study, this procedure will have minimal impact from slight differences in degrees of freedom from subject to subject (since most subjects did not have any volumes scrubbed and this is a single-group study). If the "scrubbed" data is less than 5 minutes, then exclude that data. Here, no subjects are excluded in this procedure. 2.3. Resting-state functional connectivity-based parcellation The first goal of this study was to segment the entire thalamus (both left and right 10 Page 11 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 11 parts together) into distinct subdivisions based on their resting-state functional connectivity patterns with the whole brain. The template of the thalamus was extracted from the AAL template (Tzourio-Mazoyer et al., 2002). The parcellation approach used here was modified from previous studies (Craddock et al., 2012; Kelly et al., 2012; van den Heuvel et al., 2008). A flow chart of parcellation procedure is shown in Figure 1. For each subject, functional connectivity between each thalamic voxel and every voxel of the whole brain was calculated (Figure 1). Then, an individual similarity matrix was computed using Pearson correlation coefficient to evaluate similarity between those spatial functional connectivity maps of each pair of thalamic voxels. A K-means clustering algorithm was applied to this similarity matrix to obtain individual thalamic parcellations, generating an individual adjacency matrix (Craddock et al., 2012). The group adjacency matrix was obtained by averaging all individual adjacency matrixes. Then, the group adjacency matrix was input to the K-means clustering algorithm to obtain the group level clustering result. For each clustering procedure (both single-subject and group levels), the number of clusters was chosen from two to fifteen. 2.4. Determination of the optimal solution Since the cluster number, K, is a freely chosen parameter, we further assessed whether an optimal solution, i.e., stable solution, existed. The variation of information (VI) index (Kahnt et al., 2012; Meilă, 2007) and Dice’s coefficient (DC) (Craddock et al., 2012; Zhang et al., 2014b) were calculated here to assess the stability of parcellation 11 Page 12 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 12 procedure and determine an optimal solution. The computation of VI and DC is shown in Supplementary Materials. To obtain those indices, a split-half comparison approach was used, which divided subjects randomly into two subgroups (30 subjects in each group) for 100 instances. For each instance, the above-mentioned parcellation approach was applied to each subgroup and VI and DC values were computed according to the parcellation results of the two subgroups. The K value whose values of VI was statistically indistinguishable from that of the K - 1 solution (Kahnt et al., 2012; Kelly et al., 2010) or with local maximum DC value are defined as a stable solution. Combining the values of VI and DC, we can obtain the optimal solution. 2.5. Functional connectivity fingerprints of thalamic subdivisions To define the functional connectivity fingerprints of the estimated thalamic subdivisions from the optimal solution, we calculated the Pearson correlation coefficient between mean time courses of each thalamic subdivision and those of several common resting-state brain networks (Smith et al., 2009). Nine of ten functional networks were selected, including three visual networks, default mode network, motor network, auditory network, executive control network, left and right frontal-parietal networks (Figure S1). The tenth network reported by Smith et al. (2009), which covered cerebellum, was not assessed in the current study because the fMRI data from resting-state fMRI dataset did not fully cover the cerebellum. The Pearson correlation coefficients were then transformed to Fisher Z-scores, and then a series of one-sample t-tests were used to assess the statistical significance of each 12 Page 13 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 13 correlation. The statistical threshold was FWE corrected at p < 0.05. 2.6. Specific functional response of the thalamic subdivisions to visuomotor task For the task fMRI dataset, preprocessing steps were as follows: 1) slice timing; 2) head motion correction; 3) spatial normalization; 4) linear trend removal and 5) spatial smoothing with 4 mm FWHM. For the first-level analyses, a general linear model (GLM) was constructed to detect task-based activation areas in each subject. Specifically, the block design convolved with a hemodynamic response function and six head motion parameters were used as predictors in the GLM. The spatial maps of regression coefficients for the block-design predictor were used as contrast maps. Group activation maps were generated from a higher-level GLM analysis implementing a one-sample t-test of the contrast maps from the first-level GLM. Whole-brain results were corrected for multiple tests to control for family-wise error (FWE) using Monte Carlo simulations (FWE corrected at p < 0.01, with uncorrected p < 0.001 and minimum cluster size = 270 mm3). To investigate whether specific thalamic clusters responded during the visuomotor task, we averaged the raw regression coefficients in each cluster from the optimal solution. Then a one-sample t-test of average activation amplitude (computed from raw regression coefficients) for each thalamic cluster was conducted across subjects with a significance threshold of p < 0.05. To investigate the functional connectivity profiles of specific clusters during task performance, each thalamic cluster was used 13 Page 14 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 14 as seed region to perform whole brain voxel-wise PPI analysis (Friston et al., 1997). As running in SPM, the centered block design convolved with hemodynamics response function, the first eigenvalue of BOLD signals of a given thalamic subdivisions and their interactions were used as regressors in PPI analysis. Only the element for beta value of interactions tern was set to one in contrast matrix. A series of two-tailed one-sample t-tests were then conducted to test for significance of previous results with a whole-brain multiple comparison correction based on Monte Carlo simulations (corrected p < 0.01 with uncorrected p < 0.001 and minimum cluster size was 270 mm3). In the case of a simple first-level GLM the averaged activation amplitudes can be used to test hypotheses related to the simple question of “what regions in the brain have activity that is being modulated by the task”. In the case of PPI, the hypotheses being tested are asking the question “what regions of the brain show increased connectivity with the thalamic nucleus during the visuomotor stimulation itself (e.g., relative to the control condition).” These are very different hypotheses that inform different aspects of thalamic nuclei behavior during visuomotor stimulation. Thus, a PPI analysis provides strong evidence for testing our parcellation scheme as direct connections of thalamic nuclei with visual and motor regions should be separately disentangled (whereas the GLM activation results will just show nuclei involved in task response, but not their direct connections during the task). 3. Results 14 Page 15 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 15 3.1. Thalamus parcellation results Parcellation results with different cluster solutions obtained from the resting state data are shown in Figure 2. The K = 2 cluster solution revealed two spatially symmetric subdivisions of the thalamus. The larger cluster (Cluster 1) stretched from the ventral anterior part to the medial and almost covered the complete dorsal part of the thalamus. The other cluster (Cluster 2) covered only the ventral posterior and ventral lateral part of the thalamus. Interestingly, the two subdivisions appeared in both the left and right hemispheres and showed excellent symmetry. As we parcellated the entire thalamus together, the symmetric results indicated that the parcellation was dependent on the functional connectivity profiles, and not constrained by the distance relationship (Mars et al., 2012; Tomassini et al., 2007). Compared with the K = 2 cluster solution, Cluster 3 from the K = 3 solution mostly stemmed from Cluster 2 (the ventral posterior cluster) of the K = 2 solution. Cluster 4 from the K = 4 solution was spilt from the medial cluster from K = 3, and it covered the posterior part of the medial thalamus. Cluster solutions with K = 5 to K = 8 resulted in further splittings of these clusters into subdivisions to show a clear hierarchical structure of the parcellations. As it is shown in Figure 2, Cluster 9 from the K = 9 solution was lateralized to the left part of the thalamus. Similarly, when K > 9, one or more clusters were lateralized to a single hemisphere, thus those results are not shown here. 3.2. Determination of the optimal cluster solution To determine the optimal solution, a split-half procedure with 100 randomly generated 15 Page 16 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 16 split-half groups was used and VI and DC values were computed (Figure 3) to assess the stability of the parcellation and the similarity between the adjacency matrices calculated for each group in the split-half procedure. VI increased as a function of K, indicating that the stability decreased as K increased (Figure 3A). After performing a series of paired t-test statistics, VI was found to be significantly different with K (p < 0.001), except for K = 7. VI indices for the K = 6 and K = 7 parcellation were statistically undistinguishable (p = 0.988), Moreover, the parcellation with K = 7 corresponded to a local maximum of the DC values (Figure 3B), thus K = 7 was selected as the optimal solution. 3.3. Functional connectivity fingerprints between the thalamic parcellation and resting state brain networks The functional connectivity fingerprints of the thalamic subdivisions from the optimal thalamic parcellation (K = 7) are shown in Figure 4 and the connectivity strength computed as values of the group averaged correlation coefficients between mean time courses of each thalamic subdivision and each resting-state brain network are listed in Table 1. It can be seen that different subdivisions of the thalamus showed distinct functional connectivity fingerprints. Table 1 shows that all statistically significant connections are positive. Cluster 1 was significantly correlated with default mode network, motor network, auditory network, executive control network and bilateral frontal parietal networks. (Figure 4). Cluster 2 demonstrated significant functional connectivity with all networks, except for bilateral frontal parietal networks. Among 16 Page 17 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 17 these connections, the one with motor network has the greatest connectivity strength (r = 0.341). Cluster 3 showed significant but weak correlations with motor, auditory and executive control networks. Cluster 4 and Cluster 6, which are almost the same cluster in the K = 5 solution, showed a similar connectivity fingerprint, with very significant connections with default mode network and executive control network. Cluster 5 and Cluster 7, though with distinct connectivity patterns, both showed significant connectivity with almost all networks except for occipital pole visual and lateral visual networks. 3.4. Activations and PPI of the thalamic subdivisions during the visuomotor task Visuomotor task activation of thalamic regions is shown in Figure 5A. The activated regions are located mainly in the bilateral ventral posterior part and left ventral lateral part of the thalamus, which overlap primarily with Clusters 2, Cluster 3 and Cluster 5 for the K = 7 solution. The percentage overlap between visuomotor activated regions and the thalamic subdivisions are listed in Table 2. The activation amplitude of each thalamic cluster is shown in Figure 5B. Clusters 2, 3 and 5 showed statistically significant responses to visuomotor stimulation. Psychophysiological interactions during the visuomotor task condition relative to baseline were investigated between every subdivisions of the optimal solution (K = 7) and the whole brain. Only clusters (Cluster 2, Cluster 3 and Cluster 5) activated by the visuomotor task showed significant negative PPIs with visual cortex or/and motor 17 Page 18 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 18 cortex (Figure 5C). Clusters 2 and 5 showed negative PPIs with visual and motor cortices, while Cluster 3 only showed a negative PPI with visual cortex. There were no significant positive PPIs of thalamic subdivisions with other regions of the brain. 4. Discussion In the present study, we parcellated the thalamus into different subdivisions using an unsupervised approach based on their resting-state functional connectivity profiles with the whole brain. The validity of our thalamic parcellation approach was tested by assessing: 1) the functional connectivity fingerprints of our thalamic parcels with known brain networks; and 2) the thalamic responses and PPIs with visual and motor regions during visuomotor stimulation. 4.1. Connectivity-based parcellation of the thalamus Behrens and colleagues proposed a novel method to parcellate the thalamus based on its anatomical connectivity with a priori segmented cortical areas (Behrens et al., 2003; Johansen-Berg et al., 2005; Mastropasqua et al., 2014). Depending on the number of cortical regions used for the parcellation, they obtained different parcellation results, i.e., parcellation of the thalamus into five or seven sub-divisions. In the human brain, the anatomical structure is assumed to be the foundation of its function. With equal importance, the underlying anatomical structure is continually modulated by function in relation to experience (Damoiseaux and Greicius, 2009; Zhang et al., 2010). It is commonly assumed that functional connectivity between 18 Page 19 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 19 brain regions reflects the anatomical connectivity between them (Damoiseaux and Greicius, 2009; Honey et al., 2009; Honey et al., 2010). However, the relationship between functional and structural connectivity appears not be so straightforward. Studies have shown that the strength of functional and structural connectivity is positively correlated when structural connectivity is present, functional connectivity is also observed between brain regions without structural connectivity (Honey et al., 2009). Thus, a thalamic parcellation based on functional connectivity properties is needed for functional MRI studies. To that end, a few studies have defined thalamic subdivisions based on functional connectivity with a template of brain cortical regions (Zhang et al., 2008; Zhang et al., 2010). Kim and colleagues proposed a multi-level independent component analysis (ICA) technique to parcellate the thalamus and basal ganglia (Kim et al., 2013). However, ICA has some intrinsic methodological limitations (Hyvarinen, 2011), specifically, ICA-based schemes will depend on the choice of dimensionality of the ICA. In the current study, we adopted a technique based on the intrinsic functional connectivity profiles of thalamus. Compared to previous seed-based connectivity parcellation methods, it does not rely on a priori definition of cortical areas. Compared to ICA-based techniques, our approach is more hypothesis-driven in the sense of the determination of number of nuclei. Moreover, the ICA will separate out the connectivity of each parcel into different components according to networks they belong to. This makes interpretation a little trickier as one have to worry about 19 Page 20 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 20 “mixing” in many different components. However, seed-based connectivity approach, as what we do, can assess the whole connectivity profile of each parcel. Thus, the results of the proposed methods is perhaps easier to interpret. 4.2. Resting-state functional connecitivity with specific brain networks Distinct and anatomically relevant functional connectivity fingerprints of each subdivision with brain networks provided evidence for the validity of our parcellation approach. For instance, the Cluster 6 mainly overlapped with the mediodorsal part of the thalamus. Significant functional connectivity was observed between this sub-division and the executive control and default mode networks (Figure 4). Previous studies have shown that the mediodorsal nucleus anatomically projects to prefrontal cortex and posterior cingulate cortex (Jones, 2007), which are key regions in the executive control network and the default mode network, respectively. Thus, our findings suggest a close correspondence between the functional and anatomical connectivity of Cluster 6. Notably, Clusters 2 and 3 are primarily located in the ventral lateral and ventral posterior parts of the thalamus, which sends fibers to motor and sensory cortices. Figure 4 shows that these subdivisions correlated with motor network. Moreover, Cluster 4 mainly overlapped with the medial part of the pulvinar nucleus. This part of thalamus is anatomically connected with prefrontal cortex, cingulate cortex, and visual and auditory cortices (Jones, 2007). Thus, the significant functional connectivity observed between Cluster 4 and these cortical areas (Figure 4, Table 1) is consistent with the known anatomical connections of the pulvinar. 20 Page 21 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 21 4.3. Response of specific thalamic clusters to visuomotor stimulation 4.3.1. Visuomotor task activation Activity of the thalamic clusters from the K = 7 parcellation showed responses in certain clusters to visuomotor stimulation that are consistent with previous reports of visuomotor task activation (Calhoun et al., 2001; Miao et al., 2014; Witt et al., 2008), providing validity of our thalamic parcellation results. Specifically, Clusters 2, Cluster 3 and Cluster 5 were activated during the visuomotor task. Cluster 2 was primarily comprised of the posterior lateral part of the thalamus, a region that has been shown to project to both primary and higher order visual areas and to contribute to visual perception and attention (Fischer and Whitney, 2012; Purushothaman et al., 2012). The ventral lateral and ventral posterior parts of the thalamus, which were in Cluster 3 and Cluster 5, relay somatosensory and motor information to and among the brain (Morel et al., 1997; Stepniewska et al., 2007; Strick, 1976) and these regions were activated during the finger-tapping task when the flashing checkerboard stimulation was on. 4.3.2. PPI during the visuomotor task Interestingly, only those thalamic clusters which were activated by the visuomotor task, showed a significant PPI with visual and motor cortices. However, the PPI analysis revealed that in fact Clusters 2, 3 and 5 showed strong negative connectivity with visual and/or motor cortices. Although this may seem counterintuitive, 21 Page 22 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 22 theoretically, it can be explained. The statistical model used to detect brain activation (which is a GLM with a predictor that corresponds to the task plus any nuisance predictors) detects regions with increased or decreased BOLD signal responses to a given task (e.g., a main effect of the task), while a PPI analysis, which is also a GLM, but includes a region of interest (ROI) time course and an interaction regressor, investigates the interaction between the seed ROI and other regions of the brain during task performance. In other words, the PPI helps distinguish whether two regions activated by a task become more strongly functionally connected during task performance (e.g., have a stronger positive or negative correlation), or whether the regions act independently with both becoming more active during the task. Several previous studies have reported negative PPIs of thalamus with other brain regions during task performance. For example, Chang et al. (2011) reported a negative PPI between the ventral lateral thalamus and primary auditory cortex during a speech production task and Zhang et al. (2014a) reported a negative PPI between the thalamus and medial prefrontal cortex during a stop signal task. Second, a recent animal electrophysiology study found that the increases in thalamic action potential firing rate during sensory stimulation correlated with decreases in the amplitude of low frequency local field potential (LFP) fluctuations of neurons from barrel cortex (Poulet et al., 2012). BOLD fMRI signals are closely related to LFP signals (Logothetis et al., 2001), so an increase in thalamic action potential firing leading to a decrease in the amplitude of the LFP could be one possible mechanism for the strong negative PPIs we observed between these two regions during visuomotor stimulation, 22 Page 23 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 23 although clearly this is speculative. Thus, the presence of a strong PPI effect between those activated clusters and visual and motor cortices provides further evidence to the validity of our parcellation scheme. E.g., it is consistent that thalamic nuclei would interact with visual and motor cortices during a visuomotor task. Our findings reveal that this occurs via negative PPIs with the thalamic nuclei, which sheds insight into mechanisms underlying thalamocortical interactions. Future work to understand the exact processes giving rise to these negative PPIs is warranted. 4.5. Limitations There are potential limitations of our parcellation procedure that should be considered. First, the parcellation results were performed on group-averaged data. More work is needed to obtain stable individual parcellation results. Second, considering the convergence and divergence between functional connectivity and structural connectivity (Buckner et al., 2008; Honey et al., 2010), a multi-modal diffusion-based anatomical scan (e.g., Diffusion Spectrum Imaging) to assess structural connectivity combined with functional connectivity measurements would shed great light on the correspondence between structural and functional connectivity and perhaps result in a more robust thalamic parcellation. Third, improvements in signal noise ratio and spatial resolution would result in a parcellation of the thalamus with higher precision. Fourth, considering that thalamus is surrounded by the ventricular system, spatial smoothing might be a potential source of contamination of thalamic signals. In this study, to minimize the effect of smoothing, a 4-mm FWHM smooth kernel was used, 23 Page 24 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 24 with 3 mm voxel size of normalized functional images. In addition, the seed-based functional connectivity analysis method has intrinsic shortcomings. For example, it is less sensitive in detecting inter-individual variability than ICA-based analyses (Smith et al., 2014) and one must contend with the global signal regression problem. Here, we used the aCompCor approach for removing physiological noise signals that contribute to global signals and thus obviated the issue of GSR. 5. Conclusion In the current study, we used an unsupervised clustering approach to parcellate the thalamus based on resting-state functional connectivity profiles. Our optimal thalamic parcellation scheme resulting in five symmetric thalamic clusters was validated by assessing the distinct fingerprints of functional connectivity profiles of the resulting thalamic clusters with brain networks and by assessing the thalamic responses and PPIs during a visuomotor task. Acknowledgements This work was supported by China’s National Strategic Basic Research Program (973) (2012CB720700 and 2015CB856400) and the Natural Science Foundation of China (81227003, 81430037, 31421003, 31200761 and 81201142). 24 Page 25 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 25 References Basser PJ, Mattiello J, LeBihan D, 1994. MR diffusion tensor spectroscopy and imaging. Biophys J 66, 259-267. Behrens TE, Johansen-Berg H, Woolrich MW, Smith SM, Wheeler-Kingshott CA, Boulby PA, et al., 2003. Non-invasive mapping of connections between human thalamus and cortex using diffusion imaging. Nat Neurosci 6, 750-757. Behzadi Y, Restom K, Liau J, Liu TT, 2007. 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Figure Legends 29 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 30 Figure 1. Flow chart. Procedures of resting-state fMRI data analysis and thalamic parcellation. 30 Page 30 of 41 Page 31 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 31 31 Page 32 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 32 Figure 2. The resting-state functional connectivity-based thalamic parcellation. Resting-state functional connectivity-based parcellation of the thalamus with different cluster solutions (K = 2, 3 … 9). Seven slices (Z values from -1 mm to 17 mm, slice thickness: 3 mm) covering the entire thalamus are shown. Different colors represent different thalamic subdivisions from the parcellation. L and R denote the left and right hemispheres, respectively. 32 Page 33 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 33 Figure 3. VI and DC values for each cluster solution (K). (A) Values of VI as a function of K. Values of VI for each K cluster solution are significantly different from the K+1 solution (p < 0.001) except for the difference between K = 6 and K = 7 (p = 0.988), (B) Values of DC as a function of K. K = 7 is a local maximum and thus considered to be a relatively stable solution. 33 Page 34 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 34 Figure 4. Functional fingerprints of thalamic subdivisions from the optimal solution (K = 7). Fingerprints of Clusters 1-7 from K = 7 solution. Each value of connectivity strength in fingerprint is the group averaged correlation coefficient between mean time course of each thalamic subdivision and each functional network. The MNI axial slice for the overlay corresponds to Z = 11 mm. Abbreviations: VN1 – Medial Visual Network, VN2 – Occipital Pole Visual Network, VN3 – Lateral Visual Network, DMN – Default Mode Network, MN – Motor Network, AN – Auditory Network, ECN – Executive Control Network, rFPN – right Frontal-Parietal Network, lFPN – left Frontal Parietal Network. 34 Page 35 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 35 35 Page 36 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 36 Figure 5. Response of specific thalamic clusters from the optimal solution (K = 7) to visuomotor stimulation. (A) Task activation results are shown in the upper panel (corrected p < 0.01) with the corresponding thalamic parcellation at the same Z locations. (B) Bar and error bar plots of activation amplitude for each thalamic subdivision, averaged across subjects. Clusters 2, 3, and 5 show significant activation during the task. (C) Regions showing significant PPI effects with thalamic clusters during the visuomotor task period relative to the control period. 36 Page 37 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 37 Table 1 - Mean functional connectivity strength between thalamic subdivisions and brain networks Cluster Number Brain Networks 1 2 3 4 5 6 7 VN1 0.034 0.196 0.011 0.117 0.134 0.209 0.150 VN2 -0.006 0.139 0.000 0.125 0.087 0.206 0.101 VN3 -0.036 0.175 0.030 -0.026 0.093 0.018 0.030 DMN 0.172 0.176 0.013 0.335 0.163 0.392 0.310 MN 0.127 0.341 0.157 0.117 0.294 0.063 0.151 AN 0.164 0.225 0.134 0.009 0.313 0.153 0.249 ECN 0.355 0.136 0.141 0.299 0.289 0.543 0.459 rFPN 0.278 0.073 0.067 0.171 0.160 0.228 0.283 lFPN 0.261 0.069 0.048 0.147 0.120 0.192 0.247 Thalamic subdivisions are those from the optimal solution (K = 7) using the resting state FMRI data. Functional connectivity values in bold indicate statistically significant connectivity across subjects (corrected p < 0.05). VN1: Medial Visual Network, VN2: Occipital Pole Visual Network, VN3: Lateral Visual Network, DMN: Default Mode Network, MN: Motor Network, AN: Auditory Network, ECN: Executive Control Network, rFPN: right Frontal-Parietal Network, lFPN: left Frontal Parietal Network. 37 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 38 of 41 38 Table 2 - Percentage overlap between thalamic subdivisions and visuomotor task activations Cluster Number Task 1 2 3 4 5 0% 50.8% 15.9% 3.4% 25.9% data (with global signal regression). 38 6 7 Activations 0% 4.0% Thalamic subdivisions are those from K = 7 parcellation using the resting state FMRI Page 39 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 39 Supplementary Materials Computation of VI and DC The VI index was used to evaluate the stability of various parcellation results (Kahnt et al., 2012; Kelly et al., 2012; Meilă, 2007). VI values for each K were computed as follows: VIk C1, C2   Hk C1   Hk C2   2Ik C1, C2  (1) where C1 and C2 represent clustering results from group 1 and group 2, respectively. Hk (C) is the entropy of clustering results of a subgroup and Ik (C1, C2 ) mutual information between clustering results of the two subgroups. is the H k and I k can be computed as: H k  C   P(k )  log  P  k   K k 1 I K  C1 , C2   P  k1 , k2   log K K k1 1k2 1 (2) P  k1 , k2  P  k1  P  k2  (3) where P(k) is the probability that a voxel belongs to cluster k; P(k1,k2) is the probability that a voxel belongs to cluster k1 in clustering result C1 and cluster k2 in C2. Low VI indicates that clustering results estimated from the two subgroups share more information, i.e., they are more similar. In contrast, high VI values indicate low similarity. We defined a stable solution as the K value whose VI was statistically indistinguishable from that of the K - 1 solution (Kahnt et al., 2012; Kelly et al., 2010). Dice’s Coefficient (DC; (Craddock et al., 2012; Zhang et al., 2014) was used to evaluate the similarity between the results from the two subgroups during the 39 Page 40 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 40 split-half procedure. DC is computed as follows: DC  2  C1  C2 C1  C2 (4) This, DC values are between zero and one, where a value of one indicates perfect correspondence, and zero indicates no similarity. The higher the DC values, the more similar the parcellation results from the two subgroups, and the more stable the cluster solution. 40 Page 41 of 41 Brain Connectivity Functional connectivity-based parcellation of the thalamus: An unsupervised clustering method and its validity investigation (doi: 10.1089/brain.2015.0338) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 41 Figure S1. Resting-state brain networks. These brain networks are generated using ICA (Smith et al., 2009). Nine networks out of ten were selected in the present study. We did not investigate the cerebellar network because the cerebellum was not fully covered in our data. Abbreviations: VN1 – Medial Visual Network, VN2 – Occipital Pole Visual Network, VN3 – Lateral Visual Network, DMN – Default Mode Network, MN – Motor Network, AN – Auditory Network, ECN – Executive Control Network, rFPN – right Frontal-Parietal Network, lFPN – left Frontal Parietal Network. 41