Two cohorts of subjects were used in this study. The first cohort has been described previously (Scheinost et al., 2013) and consisted of 10 subjects without any clinical diagnosis of OCD, but with high levels of contamination anxiety. Only the 10 subjects who received true neurofeedback in our previous study—not the 10 who received sham neurofeedback in the control condition—are included in the present analysis. The second cohort consisted of five OCD patients with moderate symptom severity (Table 1) and prominent contamination obsessions.

Healthy subjects and OCD patients received neurofeedback training following a previously detailed protocol (Hampson et al., 2012a). Of the five OCD patients, the first two underwent only a single neurofeedback session, without pre- or post-neurofeedback resting-state scans. All other individuals participated in four separate MRI scanning sessions, spaced several days apart. In the first session, rs-fMRI data were collected and a functional localizer was used to identify the target area of the OFC/BA 10 region to be used for neurofeedback. The second and third sessions involved rt-fMRI neurofeedback training based on the target OFC/BA 10 region. A final session (not of relevance to this work) involved collecting post-intervention rs-fMRI data. The rs-fMRI data were always collected before any other functional scans in a given session to avoid possible effects of previous task on the rs-fMRI data.

The overlap of the target area for feedback for all 15 subjects is shown in Figure 1. Overlap was calculated by (1) smoothing the target region of each individual with a 6 mm Gaussian smoothing kernel to account for differences in functional anatomy and registration errors; (2) warping the target regions to a common reference; and (3) averaging across subjects the likelihood of a voxel being included in the target region.

Behavioral measures of control over contamination anxiety (for the first cohort) and clinical measures (for the second cohort) were collected before and after neurofeedback training. The pre-intervention assessment data was collected before the first neurofeedback session (second overall imaging session), immediately prior to the start of neurofeedback training. The post-intervention assessment data was collected several days after the completion of neurofeedback training, either in a separate session with no imaging (for the first two OCD patients) or in conjunction with the fourth fMRI session (for all other patients). Finally, midpoint assessment data was collected in between the first and second neurofeedback sessions for the subjects who received two sessions of neurofeedback.

For the healthy subjects, with subclinical contamination anxiety we used a behavioral measure designed to assess the subjects’ ability to control their anxiety. Subjects were instructed to try to control their anxiety while viewing 25 contamination-related images and to indicate their experienced anxiety for each image on a 1–5 scale. A rating of one indicated the least anxiety and a rating of five indicated the most anxiety. The ratings for the 25 contamination-related images were then averaged yielding a single measure of anxiety. Different sets of images were used before and after the intervention, but the sets were designed to induce similar levels of contamination related anxiety and piloted to verify that they were balanced in this respect (Hampson et al., 2012a).

For the patients, we administered a modified version of the Yale–Brown Obsessive Compulsive Scale (Y-BOCS), in which they were instructed to report on their symptoms over the last 3 days, rather than over the past week as in the traditional Y-BOCS (Goodman et al., 1989a,b). The Y-BOCS ranges from 0–40, with higher scores representing more severe symptoms, and measures the frequency, intrusiveness, and distress associated with obsessions and compulsions. Scores in the mid-twenties, as these patients had (Table 1), correspond to moderate to severe disease.

For both groups, change in behavior measures were calculated as score prior to neurofeedback minus score after neurofeedback, such that a positive change indicates an improvement in anxiety.

All imaging was done on a 1.5-T Siemens Sonata scanner (Siemens Medical Systems, Erlangen, Germany). A sequence designed to optimize signal in the OFC was used for all functional data collection (repetition time = 2000 ms, echo time = 30 ms, flip angle = 80, bandwidth = 2604, 200 mm field of view for 3.1 mm isotropic voxels, 31 axial-oblique slices covering almost the whole cerebrum and most of the cerebellum). Two 5 min resting data runs were collected.

Images were preprocessed using a previously detailed pipeline (Hampson et al., 2012b). All images were slice time and motion corrected using SPM.¹ Unless otherwise specified, all further analysis was performed using BioImage Suite (Joshi et al., 2011). Several covariates of no interest were regressed from the data including linear and quadratic drift, six rigid-body motion parameters, mean cerebrospinal fluid (CSF) signal, mean white-matter signal, and mean global signal. The data were low-pass filtered via temporal smoothing with a 0 mean unit variance Gaussian filter (approximate cutoff frequency = 0.12 Hz). Finally, a gray matter mask was applied to the preprocessed data so that only voxels in the gray matter were used in subsequent calculations. After preprocessing, all resting-state runs were concatenated and the connectivity for each voxel was then calculated in each subject’s individual brain space.

The gray and white matter and CSF masks were defined on a template brain (Holmes et al., 1998), and warped to individual subject space using a series of transformations, described below. The gray matter mask was dilated to ensure full coverage of the gray matter after warping into individual subject space. Regions that were not included in all subjects’ data (for e.g., the bottom of the cerebellum) were excluded from analysis. Likewise, the white matter and CSF masks were eroded to ensure only pure white matter or CSF signal were regressed from the data.

Global functional connectivity of each voxel was measured from rs-fMRI data using the network theory measure degree (Bullmore and Sporns, 2009) as previously described (Martuzzi et al., 2011). The BOLD time course for each voxel was correlated with every other voxel in the gray matter. Two voxels were considered connected if correlation of their timecourses was greater than r = 0.25; the degree of each voxel was defined as the number of such connections. The process was repeated for every voxel in the gray matter. Each subject’s degree map was normalized by subtracting the mean across all voxels and dividing by the standard deviation across all voxels. This normalization has been shown to reduce the impact of confounds related to motion (Yan et al., 2013).

To facilitate comparisons of imaging data, all degree maps were spatially smoothed with a 6 mm Gaussian filter and then warped to a common template space through the concatenation of a series of linear and non-linear registrations, as previously described (Scheinost et al., 2013). All transformations were computed using the intensity-based registration algorithms in BioImage Suite (Papademetris et al., 2004).

To identify which brain regions predicted response to neurofeedback training, we related the rs-fMRI data acquired before any neurofeedback training with changes in the behavioral measure of control over contamination anxiety (for the healthy subjects) and changes in clinical severity (for the patients). For the healthy subjects, we performed a data-driven, whole-brain analysis by correlating the change in control of anxiety with the degree maps in a voxel-wise manner. Significance was assessed at a p < 0.05 level after correcting for multiple comparisons across the gray matter via AFNI’s AlphaSim program. From this voxel-wise analysis, we defined a region of interest (ROI) that showed significant effects in the healthy subjects to explore whether this finding translated to the smaller cohort of OCD patients. For the three OCD patients on whom pre-neurofeedback rs-fMRI was collected, degree averaged over all voxels in this ROI was extracted and related to changes in Y-BOCS scores.

As reported previously (Scheinost et al., 2013), healthy subjects with subclinical contamination anxiety showed a significant (p < 0.05) increase in control over anxiety after neurofeedback training. Whole-brain connectivity analysis revealed a single significant cluster (p < 0.05 corrected; MNI coordinate of peak voxel: 0, 66, −4, max t-value = 5.84, cluster size = 5857 mm³) in which degree prior to neurofeedback training was significantly correlated with improved control over anxiety (Figure 2A). This cluster was located in the OFC/BA 10 target region. Subjects with the highest connectivity in this region prior to neurofeedback training exhibited the most improvement in post-treatment anxiety. A scatterplot of the average connectivity change in this region vs. the change in control of anxiety is shown in Figure 2B. As the choice of threshold used to consider whether two voxels are connected can impact connectivity results (Scheinost et al., 2012), we repeated this analysis over a range of thresholds (0.10 < r < 0.65). This produced no qualitative change in the findings. Additionally, as motion has been shown to confound functional connectivity results, average frame to frame displacement was calculated for each group (Van Dijk et al., 2012). Motion was not correlated with improved control of anxiety (r = 0.18, p > 0.60) and adding motion as a covariate in the group analysis did not change the presented results.

Five patients with moderate-to-severe OCD and prominent contamination symptoms underwent one or two sessions of neurofeedback (Table 1). All five tolerated the procedure well and exhibited reduced symptoms, as evaluated by the Y-BOCS several days after the last neurofeedback session. Average symptom improvement was 20%. Of the OCD patients, the three with the greatest symptom improvements also had a co-diagnosis or a history of major depressive disorder (MDD). Demographic and clinical details are given in Table 1.

Next, we tested whether a similar relationship between connectivity and behavioral improvements would be found in OCD patients. Pre-neurofeedback rs-fMRI was not measured on the first two subjects; this analysis was therefore performed only on the three subjects who underwent the full two-session neurofeedback protocol. To maximize power in this very limited dataset, we used the OFC/BA 10 region defined in the first cohort as an a priori ROI, Average degree in this ROI prior to neurofeedback training was related to clinical improvement for the three patients. Consistent with the pattern seen in the healthy subjects, a strong linear relationship was observed (r = 0.99). Thus, in both groups, increased connectivity in the OFC/BA 10 measured from rs-fMRI data collected prior to neurofeedback training was associated with greater behavioral improvements.