Brain connectivity with functional magnetic resonance imaging (fMRI) is a popular approach for detecting differences between healthy and clinical populations. Before creating a functional brain network, the fMRI time series must undergo several preprocessing steps to control for artifacts and to improve data quality. However, preprocessing may affect the results in an undesirable way. Spatial smoothing, for example, is known to alter functional network structure. Yet, its effects on group-level network differences remain unknown. Here, we investigate the effects of spatial smoothing on the difference between patients and controls for two clinical conditions: autism spectrum disorder and bipolar disorder, considering fMRI data smoothed with Gaussian kernels (0–32 mm). We find that smoothing affects network differences between groups. For weighted networks, incrementing the smoothing kernel makes networks more different. For thresholded networks, larger smoothing kernels lead to more similar networks, although this depends on the network density. Smoothing also alters the effect sizes of the individual link differences. This is independent of the region of interest (ROI) size, but varies with link length. The effects of spatial smoothing are diverse, nontrivial, and difficult to predict. This has important consequences: The choice of smoothing kernel affects the observed network differences. Author Summary Spatial smoothing is a preprocessing tool commonly applied to reduce the amount of noise in functional magnetic resonance imaging (fMRI) data. However, smoothing is known to affect the outcomes of functional brain network analysis at the level of individual subjects in undesired ways. Here, we investigate how spatial smoothing affects the observed differences in brain network structure between subject groups. Using fMRI data from two clinical populations and healthy controls, we show that the between-group differences in network structure depend on the amount of spatial smoothing applied during preprocessing in a nontrivial way. The optimal level of spatial smoothing is difficult to define and probably depends on a set of analysis parameters. Therefore, we recommend applying spatial smoothing only after careful consideration.

The properties of functional brain networks strongly depend on how their nodes are chosen. Commonly, nodes are defined by Regions of Interest (ROIs), predetermined groupings of fMRI measurement voxels. Earlier, we demonstrated that the functional homogeneity of ROIs, captured by their spatial consistency , varies widely across ROIs in commonly used brain atlases. Here, we ask how ROIs behave as nodes of dynamic brain networks. To this end, we use two measures: spatiotemporal consistency measures changes in spatial consistency across time and network turnover quantifies the changes in the local network structure around an ROI. We find that spatial consistency varies non-uniformly in space and time, which is reflected in the variation of spatiotemporal consistency across ROIs. Furthermore, we see time-dependent changes in the network neighborhoods of the ROIs, reflected in high network turnover. Network turnover is nonuniformly distributed across ROIs: ROIs with high spatiotemporal consistency have low network turnover. Finally, we reveal that there is rich voxel-level correlation structure inside ROIs. Because the internal structure and the connectivity of ROIs vary in time, the common approach of using static node definitions may be surprisingly inaccurate. Therefore, network neuroscience would greatly benefit from node definition strategies tailored for dynamical networks. Author Summary Regions of Interest (ROIs) are often used as the nodes of functional brain networks. ROIs consist of several fMRI measurement voxels that are assumed to be functionally homogeneous, that is, behave similarly. Earlier, we showed that the assumption of similar voxel dynamics is not always true: functional homogeneity varies widely across ROIs. In this paper, we demonstrate that functional homogeneity changes in time. These changes are connected to changes in local network structure around ROIs, which suggests that an ROI’s functional homogeneity may reflect its role in the network. Finally, we show that there is rich, time-dependent structure of voxel-level connectivity inside ROIs. This leads us to ask if the dynamic brain networks can be described by any set of static ROIs.

Author Summary Network methods have opened new insights on structure and functional dynamics of the human brain. However, constructing functional brain networks is far from trivial—the neuroscientific community still lacks a standard definition of the nodes of brain networks. In the present article, we consider the two most commonly used approaches: using either imaging voxels or predefined Regions of Interest (ROIs) as nodes of the network. We investigate what happens when voxel-level signals are averaged for obtaining ROI-level networks. We introduce the concept of ROI consistency to characterize the similarity of the dynamics of voxels in an ROI. With the help of consistency, we show that although voxels in an ROI are assumed to behave similarly, this assumption does not hold for all ROIs. Abstract The functional network approach, where fMRI BOLD time series are mapped to networks depicting functional relationships between brain areas, has opened new insights into the function of the human brain. In this approach, the choice of network nodes is of crucial importance. One option is to consider fMRI voxels as nodes. This results in a large number of nodes, making network analysis and interpretation of results challenging. A common alternative is to use predefined clusters of anatomically close voxels, Regions of Interest (ROIs). This approach assumes that voxels within ROIs are functionally similar. Because these two approaches result in different network structures, it is crucial to understand what happens to network connectivity when moving from the voxel level to the ROI level. We show that the consistency of ROIs, defined as the mean Pearson correlation coefficient between the time series of their voxels, varies widely in resting-state experimental data. Therefore the assumption of similar voxel dynamics within each ROI does not generally hold. Further, the time series of low-consistency ROIs may be highly correlated, resulting in spurious links in ROI-level networks. Based on these results, we recommend that averaging BOLD signals over anatomically defined ROIs should be carefully considered.