Thirty-eight healthy individuals (mean age 27 ± 3.7 y) participated in the study. Two separate groups of 19 subjects were assigned to either the Coherent-Narrative or to the Incoherent-Narrative group (8 and 9 females respectively in the coherent and incoherent narrative groups). All participants had normal visual acuity (without glasses or corrective lenses, enabling eye tracking). None of the subjects were familiar with the movie presented during the scan. The experimental procedures were approved by a Institutional review board of Tel-Aviv University and the Ethics committee of the Chaim Sheba Medical Center, Tel-Hashmoer, Israel, as required by Israeli law. All subjects provided written informed consent prior to the experiment, and all methods were performed in accordance with the relevant guidelines and regulations.
Stimuli and experimental design
In the MRI scanner, participants watched either the coherent or incoherent narrative (experimental conditions), which consisted of 22 scenes (mean scene length: 55.15 ± 23 sec) edited from the movie "Bicycle Thieves" (Italy, 1948), interleaved by 10 sec blank screens. Briefly, the movie portrays a young father, who manages to obtain a pair of bicycles that are required for his employment. On his first day at work, his bicycles are stolen (the plot's turning point), and from that moment on the main protagonist tries to retrieve them with his son. The unfortunate events that follow feed his desperation, which culminate in a failed attempt to steal someone else's bicycles, just to be caught by the police and shamed in front of his son. The experiment lasted 25 minutes, wherein the scenes were identical across groups and differed only in their order of presentation, such that in the coherent-narrative group, scenes were presented in their correct order - from first to last, and in the incoherent narrative the same scenes were presented in reversed order, from last scene to first. The movie was edited using "Movie-Maker" software (Microsoft ©), and were presented using Presentation Version 20.1 software (www.neurobs.com).
Immediately following scanning, comprehension of the story was assessed using a questionnaire targeting narrative comprehension and personal ratings regarding the movie. Participants were asked whether or not they felt they understood the movie’s plot and how confident they were about their answer. They were also asked to briefly describe the main characters and the general story line. Their accounts were subsequently analyzed according to their mentioning of key plot features; namely, the main characters, the main protagonist’s goal, and the main dramatic development in terms of causality. Given that the plot includes a protagonist that aims at achieving a specific goal, we examined the participants' understanding regarding this process. In addition, they were asked more specifically about the casual connection between two specific successive scenes that occurred in the middle of the movie; the first depicted the main protagonist slapping his child, and the second showed a compensation scene wherein the father took the kid to a fancy restaurant. This assessment was designed to probe the participants’ understanding of the event’s causation - a central component in the definition of coherent narrative.
MRI data acquisition and preprocessing
Whole-brain imaging was performed in a 3T Siemens Magnetom MRI system (Siemens Medical Systems, Erlangen, Germany) using a 16-channel head coil. Blood-oxygenated-level-dependent (BOLD)-sensitive T2*-weighted functional images were acquired using a single shot gradient-echo EPI pulse sequence (TR = 2000 ms, TE = 30 ms, flip angle = 82°, 64 axial slices, 2mm3, FoV = 192 × 192 mm, interleaved slice ordering) and corrected online for head motion. Participants completed the task in two fMRI runs, each lasting ~ 12.5 minutes. The first two volumes were discarded to allow for equilibration effects. Visual stimuli were presented on a screen behind the scanner using Presentation software (www.neurobs.com), and were viewed through a mirror attached to the head coil. Following functional imaging, a high-resolution T1 scan was acquired for anatomic normalization.
Imaging data were slice-time corrected and realigned using the SPM12 package (Wellcome Institute of Cognitive Neurology, London). Functional volumes were co-registered, normalized to the Montreal Neurological Institute (MNI) template brain, and smoothed with an 8 mm3 isotropic Gaussian kernel. We assessed task-related functional connectivity using the CONN toolbox (v17) 64. The implemented CompCor routine was carried out for each participant, aimed at identifying principal components associated with white matter (WM) and cerebrospinal fluid (CSF), which were segmented. These components, as well as realignment correction information were entered as covariates in the first-level model.
Functional connectivity analysis
In order to detect brain regions that differed in their functional connectivity between the two conditions (i.e., the two groups), we carried out the following analysis steps: 1. A first-level multivariate pattern analysis (MVPA) of pairwise connections between all measured voxels separately for film and blank segments (voxel-to-voxel analysis); 2. Second-level between-group analyses that tested for differences in connectivity between the groups using F-tests; and 3. A seed-based correlation analysis (SCA), using the regions that the MVPA analysis yielded as seeds. Correlations were computed between these regions-of-interest (ROIs) and the rest of the brain (seed-to-voxel analysis). These correlations served to form statistical maps that revealed differences between the coherent and incoherent narrative groups, and served for characterizing the temporal dynamics of narrative related processes between the groups.
Multivariate pattern analysis
Multivariate pattern analysis (MVPA) enables to differentiate among cognitive states based on activation / co-activation patterns across voxels rather than within specific voxels, hence avoiding the exclusion of sub-threshold voxels that may still contain meaningful information as part of a wider pattern 65. Pertinent to functional connectivity, MVPA allows for detection of group differences without asserting presumptions concerning a-priori seed regions. Using the MVPA routine, voxel-to-voxel functional correlation matrices were computed across the entire dataset of each subject. Subsequently, the connectivity matrices were reduced into three principle components, which explained the most variance of the matrix 64,66. The measures of this analysis were then entered into a second-level general linear model (GLM) to assess group differences between the functional connectivity components. This analysis was separately performed for film and blank epochs.
Inter-subject correlation analysis
The continuous nature of movie-sequences does not lend itself to the application of standard fMRI analyses that require multiple repetitions of discrete event types. For exploring the neural dynamics of coherent-narrative processing, we therefore assumed a model-free inter-subject correlation (ISC) approach, which has previously been utilized to examine neural processes associated with naturalistic experimental designs 67,68. The ISC analysis was performed on the ROIs delineated by the MVPA results (Table 1), such that for each ROI, we calculated the correlations between the mean time-series of all subject-pairs within each group, yielding a correlation matrix for each ROI for each group. We subsequently compared the mean correlation values for each ROI between the two conditions.
Seed-based correlation analysis
Since MVPA is an omnibus test that informs on differences between groups but not on the source of the differences 69, we performed a complementary post-hoc exploration to characterize the MVPA effects and delineate brain networks that support coherent narrative formation. To characterize the differences between the coherent and incoherent conditions, we performed seed correlation analyses (SCA), using the resulting MVPA regions as seeds 70,71. The SCA was carried out for each subject by computing Fisher-transformed correlation coefficients between the mean seed time courses and all other voxels. Following this, a second-level analysis was performed, where between-group t-tests were computed on the resulting correlation values (using a height threshold of P < 0.005 and cluster level of P < 0.05, FDR corrected).
In addition to the explorative MVPA approach detailed above, we performed a seed correlation analysis using the left and right hippocampi as anatomically-delineated seed regions. For each participant, we calculated correlations between the mean time-series of each hippocampus with the rest of the brain, based on CONN's integrated atlas. Following this, we compared the correlations between the groups and sought for significant differences during 'movie' and 'blank' parts separately. To explore the relationship between hippocampus and key regions that showed differential functional connectivity between the conditions, and considering that the unfolding over time is inherent to the concept of narrative, we tested hippocampal correlations with the posterior precuneus, anterior and posterior cuneus, and fusiform gyrus across bins of 100 sec (15 segments in total) for each subject and for each group. We then performed a mixed-design analysis of variance for each condition separately using ROI and time as repeated-measures factors and condition as a between-group factor.
Eye-tracking was performed during scanning using an EyeLink 1000 system (SR Research, Canada) installed at the rear end of the scanner. Gaze positions and pupil diameter were sampled with a frequency of 500 Hz. Calibration was performed prior to each scan. Due to technical issues of data quality, eye tracking was successful in 26 participants (13 from each group). For the purpose of this study, pupil diameter (PD) was analyzed throughout the experiment by calculating for each participant the percent PD change from baseline, which was defined as the average PD of the 1.5 minutes prior to the onset of the first scene of each run. Percent PD changes were subsequently averaged within each condition, and plotted to assess physiological responses during the unfolding of the experiment in both groups.