Images were adjusted for the slice intensity differences introduced by contiguous interleaved slice acquisition. Next, a 6-parameter rigid body realignment process was used to minimize movement-induced noise across all frames in all runs for each subject. Images were resliced by 3-dimensional cubic spline interpolation. Data were transformed into a common stereotactic space based on Talairach and Tournoux (1988) but using an in-house atlas composed of the average anatomy of 12 healthy young adults (ages 21-29 years) (see Lancaster et al; Snyder for methods).[38, 39] As part of the

atlas transformation, the data were resampled isotropically at 3 mm × 3 mm × 3 mm. Registration was accomplished via a 12-parameter affine warping of each individual’s MP-RAGE to the atlas target, using difference image variance Decitabine purchase minimization as the objective

function. INK 128 cell line Subjects’ T2-weighted images were used as intermediate targets for transforming the BOLD images. The atlas-transformed images were checked against a reference average to ensure appropriate registration. Rs-fc preprocessing included removal of the linear trend, temporal band-pass filtering (0.009 Hz < f < 0.08 Hz), Gaussian blur of 2 voxels full-width half maximum, as well as regression of several “noise” parameters (6 motion parameters and signals from whole brain, white matter, and ventricles) and their time-based derivatives.[16, 40] Data volumes (ie, MR frames) likely to be contaminated with motion-related artifact that was not addressed by standard movement regression routines were identified and eliminated using a volume-censoring technique.[41] Data volumes with a frame-by-frame movement >0.5 mm or a whole brain change >0.5% were identified and eliminated. Rs-fc analyses (methods summarized in Fig. 1 —) employed a region of interest (ROI)-based approach using 5 a priori selected regions that participate in affective pain processing. Rs-fc maps were derived using 10 mm diameter spherical ROIs centered on: left anterior insula (Montreal Neurological Institute coordinates −35, 18, −1), right anterior insula (36, 19, −2), left amygdala (−21, −3,

−27), right amygdala (20, −3, −28), and anterior cingulate cortex (−1, 10, 32). Coordinates were selected based upon those reported in the pain MCE and headache literature.[8, 42-45] For each seed, a resting-state time series was extracted separately for each subject by computing the mean of the BOLD intensity of all voxels enclosed by the seed region boundaries at each MR frame (time point). Correlations with this time series were calculated for each voxel in the brain, then Fisher Z-transformed to produce a functional connectivity map for each seed in each subject. To determine the rs-fc of the 5 affective pain ROIs, t-tests were used to identify functional connections with the 5 pain ROIs that differed from zero (P ≤ .01, uncorrected).