Participants
We recruited 36 young participants from southern Taiwan through advertisements on the Internet and bulletin boards. We conducted a power analysis (G*Power 3.1.9.712, power = 0.95, effect size f = 0.25, α = 0.05, within-between subjects’ design, correlation among repeat measures = 0.5). The analysis indicated that a sample size of 36 participants would be sufficient to detect an estimated medium effect size. All participants were right-handed and without evidence of neurological or psychiatric disorders based on self-reports. This study was reviewed and approved by the Human Research Ethics Committee at National Cheng Kung University (NCKU), Tainan, Taiwan, R.O.C., authorized by the Ministry of Education, Taiwan. All experimental procedures and the informed consent were obtained from all the participants and were approved under Approval No. NCKU HREC-E-112-120-2. All research was performed in accordance with the relevant guidelines and regulations, including the Declaration of Helsinki. Upon completion of all experiments — including computerized span tasks conducted outside the MRI scanner and a visual discrimination task performed inside the MRI scanner — participants received compensation of 1,500 New Taiwan Dollars (NTD). Detailed demographic characteristics are presented in Table 1.
Span tasks outside an MRI scanner
We assessed participants’ span capacity (individual differences) using computerized span tasks developed by Stone and Towse (2015) in JAVA13. The tasks included three complex span tasks and corresponding simple span tasks in both the verbal and visuo-spatial domains.
Verbal domain
Operation span task (complex span)
The Operation Span task was chosen as the complex span task for the verbal domain (see Supplementary Figure S1). This task involved a repetitive sequence of memory and processing components. In each trial, participants were presented with an integer to memorize and recall in its original serial position at the end of the trial. Following each memory element (the integer), there was a processing phase where participants encountered a mathematical operation, such as ‘9 + 9 = 27’. They had to determine whether the presented answer was correct. Digits and operations were generated randomly for each trial, with digits ranging from 1 to 99. Each operation had an equal 50% chance of being correct, and the types of operations (multiplication, division, addition, subtraction) each had a 25% probability, ensuring a diverse range of operation types requiring both correct and incorrect responses. Digit span (simple span). Digit span corresponded to the simple span task for Operation Span. Essentially, it was Operation Span without the processing phase. Participants only needed to remember the digits and recall them in sequence at the end of the trial.
Visuo-spatial domain
Symmetry span task (complex span)
The Symmetry Span task is a type of visuo-spatial complex span task where participants were required to recall grid locations in a 4 × 4 grid in the correct serial order (see Supplementary Figure S2). Following the presentation of each To-Be-Remembered (TBR) grid, participants engaged in a processing operation where they judged whether the presented pattern was symmetrical along the vertical axis, using the left/right arrow keys. Patterns were displayed on an 8 × 8 grid for this assessment. The recall phase began once the required number of storage-processing elements had been completed for a trial. Participants were prompted to recall by presenting them with the 4 × 4 grid, allowing them to click on the boxes in the order they remembered seeing them. Upon selection, a box turned blue, helping participants keep track of their responses. Matrix span (simple span). The matrix span task served as the simple span counterpart to the symmetry span task. Its procedure closely mirrored that of the symmetry span task, except for omitting the processing element.
Rotation span task (complex span)
The Rotation Span task was another visuo-spatial complex span task (see Supplementary Figure S3). The To-Be-Remembered (TBR) stimuli consisted of arrows characterized by two features: length (long or short) and angle of rotation (0°, 45°, 90°, 135°, 180°, 225°, 270°, or 315°). Participants were tasked with remembering these arrows presented in their correct serial order during the storage phase. In this complex span task, the processing operation involved presenting participants with a letter (F, G, or R) that could appear in its standard form or as a mirror image, and it could also be rotated at one of the 45-degree angles. Participants had to mentally rotate the image to determine whether the letter was presented normally or as a mirror image, using the left/right keys for their judgment. During the recall phase, participants were shown a 2 × 8 grid displaying all 16 possible arrows. The top row displayed all the long arrows, while the bottom row displayed all the short arrows. Participants used the mouse to select the arrows they recalled seeing in the correct sequential order. Arrow span (simple span). The processing phase was omitted in the arrow span task, which served as the memory span equivalent to the rotation span task. Consequently, the arrow span task focused on the participant’s ability to remember the arrows in their correct serial positions.
Visual discrimination tasks with difficulty manipulation in an MRI scanner
Participants engaged in a visual discrimination task featuring four distinct conditions, as outlined in Fig. 1. The task was programmed using OpenSesame14. Inside the MRI scanner, the stimulus display was projected onto a mirror affixed to the head coil. The task design was adapted from difficulty manipulations reported by Crittenden and Duncan (2014). These tasks varied in cognitive demands, ranging from simple perceptual discrimination to complex rule-based tasks11.
Each trial began with a uniform gray screen in the baseline 4-line (4 L) condition. Participants were required to select the one odd line out of four based on length, considered the baseline or least demanding condition involving simple perceptual discrimination. At the start of each trial, a small fixation cross appeared at the center of the screen for 200 ms. Subsequently, four vertical lines were briefly presented (100 ms), aligned along the middle of the screen with their midpoints distributed symmetrically on either side of the fixation cross (total width 8.3° visual angle). Among these lines, three were equal in length (13.4°), while the fourth was consistently 50% shorter (6.7°). Participants indicated the position of the shorter line by pressing the corresponding key on an 8-button response box (e.g., Fig. 1, leftmost line shortest, response with the left middle finger). Responses were recorded only within the time window of the fixation cross, which persisted for 1000 ms after the lines disappeared from the display. Following a response, there was a jittered interstimulus interval of 500 to 1500 ms before the onset of the subsequent trial.
The remaining conditions exhibited similarities with some alterations as described below. In the 8 L condition, similar to the 4 L condition but with eight lines instead of four, two additional vertical lines were presented on either side of the original four lines (total display width 16.7°; see Fig. 1). This required participants to use each hand’s little and ring fingers for a response. The 8 L condition increased the perceptual load by doubling the number of lines, thereby raising the cognitive demand compared to the 4 L condition.
In the fine discrimination (FD) condition, similar to the 4 L condition, there were still four lines, but the shortest line was reduced to only 10% shorter than the other three lines (see Fig. 1). Participants had to discriminate between lines of very similar lengths, necessitating fine perceptual judgments. This condition heightened the cognitive demand further by requiring precise discrimination of line lengths, adding to the perceptual load.
In the mapping switch (MS) condition, the stimulus-response mapping was modified from the natural one to the alternative illustrated in Fig. 1. Participants were still required to discriminate between lines of very similar lengths, making fine perceptual judgments. This condition was considered the most demanding, as participants had to select the right-most odd line but press the left-most button. This complex rule introduced higher-order cognitive processing involving working memory and response inhibition. It placed significant demands on executive functions and was generally regarded as the most challenging15,16. Participants practiced this condition until their accuracy rate reached 70% or higher.
Trials were grouped into blocks, each dedicated to one task condition. Each block displayed a schematic similar to Fig. 1 indicating the upcoming condition in the middle of the screen for 2,000 ms. Following the cue, there was a 3,800 ms pause before the onset of the first trial. Each block consisted of 8 trials, with a total duration of 18,400 milliseconds. There was a 10-second interval between blocks. To maintain task engagement, the accuracy of each block was displayed at the end.
The experiment was divided into three scanning sessions, each separated by a 30-second break. Within each session, there were 20 task blocks, comprising five blocks for each of the four conditions (4 L, 8 L, FD, and MS). The sequence of blocks was arranged in a pseudorandom order.
Behavioral data analysis
Span task performance
For each span task, we calculated their Full-Trial Accuracy (FTA) score. In the case of simple span tasks, points were awarded only when all Target-to-Be-Remembered (TBR) stimuli within a trial were correctly recalled. The score for each trial was determined by the loading of that particular trial, and the sum of scores across all trials constituted the FTA score for that task. In the case of complex span tasks, the calculation method was similar to that of simple span tasks, with the distinction that points were awarded only for trials where the response to the processing component was correct.
Grouping participants based on a median split of the FTA score
Our study employed the complex span task total scores to perform a median split, grouping participants into high-span and low-span groups based on individual differences in span task performances. This decision was based on the higher complex span scores compared to simple span scores, making them more effective for distinguishing individual differences in span capacity. However, we acknowledge that this approach may inadvertently capture differences in perceptual abilities, as evidenced by significant differences in simple span performance between groups (p <.005; see Results). Complex span tasks, which involve both storage and processing components, are known to be more sensitive measures of working memory capacity compared to simple span tasks that only require storage17. Given that our participant sample consisted of young adults, the complex span tasks were particularly effective in capturing individual differences in working memory capacity, which is critical for examining PFC activation patterns under varying cognitive demands18,19.
Additionally, using the median split method allowed for a clear and balanced division of participants into two groups, facilitating the analysis of how these differences associate with neural activation patterns during task performance20. Specifically, using a median split ensured an equal distribution of participants into high- and low-span groups, facilitating balanced and well-built statistical comparisons. This approach also mitigated potential biases that could arise from uneven group sizes, enhancing the validity and reliability of our findings21. By employing a median split on complex span scores, we aimed to provide a clear delineation of how varying levels of individual differences impact neural activation patterns and cognitive control processes. However, we acknowledge that treating individual differences as a continuous covariate in a regression analysis might offer greater statistical power and a more profound understanding of its relationship with PFC activation. To address this, we conducted an additional regression analysis using complex span FTA scores as a continuous covariate, confirming that the observed patterns of PFC activation remained significant (p <.001; see Figure S4 in Supplementary Information).
Visual discrimination task performance
Behavioral performance (reaction time [RT] and accuracy) was measured separately for each of the four conditions (4 L, 8 L, FD, MS). Subsequently, we conducted a one-way analysis of variance (ANOVA) on the four conditions to determine if the behavioral performance replicated previous findings reported by Duncan and colleagues11. To test the hypothesis regarding whether individual differences would be associated with primary contrasts between conditions of interest in task performance, we initially compared the 8 L, FD, and MS conditions with the 4 L condition to identify any increase in reaction time (RT) and/or decrease in accuracy resulting from manipulation difficulty. Tasks with higher task demands, such as increased perceptual load in the 8 L condition, finer discrimination in the FD condition, or complex rule mapping in the MS condition, require greater working memory resources. High-span individuals typically exhibit superior cognitive control in these contexts22. In contrast, low-span individuals exhibited broader neural recruitment, which may reflect the additional engagement of brain regions under increased task demands23.
Subsequently, we contrasted the conditions of interest. We used the 4 L condition as a baseline to evaluate how the 8 L, FD, and MS conditions demanded greater performance costs. The three contrast pairs were as follows:
8 L–4 L (Perceptual Load): The primary difference lies in the number of items to be processed. The 8 L condition has double the items of the 4 L condition, increasing perceptual load and cognitive demand.
FD-4 L (Precision of Discrimination): While both conditions involve selecting an odd line, the FD condition requires finer perceptual judgments, thereby increasing cognitive load compared to the broader discrimination required in the 4 L condition.
MS-4 L (Complexity of Rule Application): The MS condition introduces a complex rule that reverses the typical response mapping, significantly increasing cognitive demands compared to the straightforward perceptual discrimination in the 4 L condition.
We then employed mixed-design repeated-measures 2 (high- and low-span groups) x 3 (paired-contrasts: 8 L–4 L, FD-4 L, MS-4 L) ANOVAs on RT and accuracy, respectively, to examine the effects of the three contrasted conditions and determine if they interacted across two groups of individuals with high versus low span performances. Following the initial statistical testing, post hoc analyses were conducted using the Holm correction to further explore and compare the significant differences identified among the conditions.
Imaging acquisition and analysis for the visual discrimination task
The imaging data was gathered utilizing a General Electric (GE) Discovery MR750 3 Tesla scanner (General Electric Medical Systems, Milwaukee, USA) equipped with a 32-channel receive-only phased-array head coil at the Mind Research Imaging Center, National Cheng Kung University. High-resolution structural images were obtained using a fast-SPGR sequence comprising 166 axial slices (TR/TE/flip angle 7.6 ms/3.3 ms/12°; field of view (FOV) 22.4 × 22.4 cm2; matrix size 224 × 224; slice thickness 1 mm). Functional EPI images were acquired through an interleaved T2* weighted gradient-echo planar imaging (EPI) pulse sequence (TR/TE/flip angle, 2000 ms/30 ms/78°; matrix size, 64 × 64; FOV, 22 × 22 cm2; slice thickness, 3 mm; voxel size, 3.4375 × 3.4375 × 3 mm). Each run comprised 368 volumes, with the initial eight being dummy scans discarded to mitigate T1 equilibrium effects.
fMRI imaging preprocessing
Functional imaging data were analyzed using the FMRIB Software Library (FSL)24 software. The analysis process comprised several specific steps at the 1 st level: Initially, preprocessing involved correcting head motion artifacts using the Motion Correction FMRIB’s Linear Image Registration Tool (MCFLIRT)25,26,27. Subsequently, the brain extraction tool eliminated non-brain tissue from the preprocessed MR images (BET27. The FSL Motion Outliers tool25,27 accessible at https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSLMotionOutliers, was then employed to detect outlier volumes based on frame displacement between volumes (exceeding the 75th percentile + 1.5 times the interquartile range). The results of this process were utilized to reduce the influence of those volumes in subsequent analyses. Individual brain functional images underwent registration to the high-resolution T1 structural image via linear transformation, followed by registration of the individual structural image to the standard MNI152 template via linear transformation28. The first-level General Linear Model (GLM) in FEAT tool29,30,31 was then established, incorporating a 9 mm full-width half-maximum (FWHM) Gaussian kernel for spatial smoothing.
Statistical analysis: fMRI blocked analyses for the visual discrimination task
A general linear model (GLM) was used to estimate parameter values reflecting the mean difference between experimental conditions of the visual discrimination task. Contrasts were performed to identify the regions recruited more for the 8 L, FD, and MS conditions relative to the 4 L condition.
The start time of each condition block’s stimulus to the endpoint of the block was captured and used to generate the onset file. The onset files for the four conditions were incorporated into the model as EVs (explanatory variables) and convolved with the double gamma hemodynamic response function. The six head motion parameters and the motion outlier data obtained in the previous step were included as covariates in the model for control.
The second-level analysis integrated data from the three runs, and the results of the three paired-contrasts (8 L–4 L, FD-4 L, MS-4 L) obtained at the first level were averaged separately.
To investigate our hypotheses regarding the high- and low-span groups, the subject-level files of the two groups were compared by averaging them separately according to the three paired-contrasts. We further examined the significant clusters for the combinations with simple main effects based on the ANOVA results of behavioral data, separately contrasting high span > low span and low span > high span.
Whole-brain univariate analysis
All group-level analyses involved computing the activation level across the whole brain region for each participant and submitting each of those to a group-level t-test, treating the participant as a random effect. We identified clusters of activity that were significant at a cluster-level rate of 0.01, using a 3.1 z-threshold to define contiguous clusters32. Subsequently, the estimated significance level of each cluster (derived from Gaussian Random Field theory) was compared with the probability threshold33. To account for potential confounding factors, weperformed a partial regression analysis to control for gender effects between the high- and low-span groups. This step aimed to isolate the specific contributions of individual differences observed in neural activation patterns between the two groups.
fMRI preprocessing and GLM for multivoxel pattern analysis (MVPA)
The fMRI data were preprocessed using Statistical Parametric Mapping (SPM) 1234,35 implemented in MATLAB (The MathWorks, Inc., Natick, MA). The preprocessing steps included slice-time correction and realignment to correct head motion using a rigid-body transformation36. The T1 image was co-registered to the mean EPI image, and then both the T1 image and functional volumes were normalized to the MNI template. All images were resliced to a 2 × 2 × 2 mm voxel size, resulting in a data cube of 79 × 95 × 79 voxels. The onset files, marked with the start times for three runs and four conditions, were input into the GLM model. Head motion parameters obtained from realignment were included as regressors. After preprocessing, we obtained the beta maps and SPM.mat files for subsequent MVPA analysis.
MVPA
MVPA was conducted using the Decoding Toolbox (TDT, version 3.999 F) implemented in MATLAB, following standard preprocessing procedures. A whole-brain searchlight analysis was performed to identify regions whose activation patterns allowed classification between higher difficulty conditions and the 4 L condition used as the baseline37. The input data for the classifier were the beta maps obtained from preprocessing and GLM analysis, normalized to MNI standard space. For every voxel in the brain, a sphere with a radius of 5 voxels centered on that voxel was used to train and test a linear support vector machine (SVM) using leave-one-run-out cross-validation within each participant38,39. The classification accuracy of each sphere was assigned to the center voxel, resulting in a subject-level accuracy map. Accuracy maps were then entered into group-level analysis to identify regions where decoding accuracy was significantly above chance (50%). To ensure independence between training and testing phases, cross-validation was performed within each participant’s functional space. This method avoids assumptions of voxel-wise anatomical correspondence across participants38,40.