Twenty volunteers (7 females) aged 20-40 years participated in the study, which was approved by the Institutional Review Board of both Brown University and Memorial Hospital of Rhode Island. All subjects were in good health with no past history of psychiatric or neurological disease and gave informed consent. Subjects had normal or corrected-to-normal vision.
Stimuli and Procedure
There were five experimental conditions: sex task, bar-orientation task at easy, medium, and hard difficulty levels, and the bars-only condition. During sex and bar-orientation conditions, central faces with neutral or fearful expression were presented centrally along with peripheral bars (). The experimental conditions were presented in a blocked fashion and were separated from each other by a fixation condition lasting approximately 15 s (14900 ms). For the sex and bar-orientation conditions, blocks lasted approximately 45 s (44700 ms) and contained an initial instruction screen and 14 trials (7 fearful faces and 7 neutral ones); blocks for the bars-only condition were shorter and lasted approximately 21 s (20860 ms) and contained an initial instruction screen and 6 trials. Individual trials within a block lasted 2980 ms. During trials of the sex and bar-orientation tasks, an initial green fixation cross was shown for 450 ms and followed by a 200-ms display containing a central neutral or fearful face (approximately 4 deg vertically) and two peripheral bars to the right and left of fixation (presented at 6 deg eccentricity). After this stimulus display, a white fixation cross was shown for 2330 ms. Subjects were instructed to respond both rapidly and accurately. Subjects were explicitly told that fixation should be maintained during the presentation of the main stimulus display. The brief presentation of the stimulus, as well as the symmetrical positioning of the bars to the left/right of fixation, were aimed at essentially eliminating the occurrence of deliberate saccades; indeed, the timing was insufficient to allow subjects to gaze to one side and then the other to successfully perform the bar-orientation task. During sex-task trials subjects indicated whether faces were male or female. During bar-orientation trials, subjects indicated whether the orientation of the bars was the same or not; for such trials, 50% were matches and 50% were non matches. For the easy, medium, and hard blocks, task difficulty was manipulated via a staircase procedure that adjusted the angular difference of the bars during non-match trials such that performance was maintained at the desired difficulty level. That such control of task difficulty was successful was evidenced by the performance levels obtained (averaged across participants): 92%, 84%, and 67%, for the easy, medium, and hard conditions, respectively. For the bars-only condition, only the right and left bars were shown, together with a central fixation cross, and task difficulty was targeted to be equivalent to that of the hard level of the bar-orientation condition (average across participants: 68%).
Figure 1 Experimental design. The experiment had a blocked component involving five experimental conditions: sex task, bar-orientation task at easy, medium, and hard difficulty levels, and bars-only task. During the sex task, subjects attended the faces and determined (more ...)
Each block of trials was cued by an instruction display that indicated the type of trial as well as the difficulty of the task for bar-orientation trials. Blocks contained trials in which face type (neutral or fearful) and bar orientations were randomly chosen, but visual stimuli were identical for both sex and bar-orientation conditions (in different random orders), such that only the focus of attention alternated between faces and bars. Overall our design was hybrid, containing a general block structure and an event-related structure (facial expression) within each block. Events of interest were repeated 63 times or more, depending on the total number of runs performed by each subject (range: 9-12).
Face stimuli were obtained from the Ekman set (Ekman and Friesen, 1976
), a set recently developed by Ohman and colleagues (KDEF, Lundqvist, D., Flykt, A., and Ohman, A.; Karolinska Hospital, Stockholm, Sweden), as well as a set developed and validated by Alumit Ishai (Ishai et al., 2004
) at NIMH (Bethesda, USA).
fMRI data acquisition and analysis
fMRI data were collected using a Siemens 1.5 Tesla scanner. Each scanning session began with the acquisition of a high-resolution MPRAGE anatomical sequence (TR = 1900 ms, TE = 4.15 ms, TI = 1100 ms, 1-mm isotropic voxels, 256 mm field of view). Gradient echo echo-planar images were acquired with a TE of 38 ms and a TR of 2980 ms. Each volume consisted of 37 axial slices with slice thickness of 3 mm and in-plane resolution of 3 mm × 3 mm.
fMRI data analysis
fMRI data were analyzed using AFNI tools (Cox, 1996
), unless indicated otherwise. Initially, both anatomical and functional data were normalized to the standard space defined by the Montreal Neurological Institute by using the BET and FLIRT tools from the FSL package (http://www.fmrib.ox.ac.uk/fsl/
). For the functional data, the first 3 volumes of each run were discarded to allow for equilibration effects. The remaining volumes were then spatially registered to the first functional volume (i.e., volume acquired closest in time to the particular subject's high-resolution anatomy). Next, each volume was spatially smoothed with an 8-mm Gaussian filter (FWHM). Each subject's data were then analyzed with standard multiple regression methods (Friston et al., 1995
). The linear models included constant and linear terms (for each run) that served as covariates of no interest (these terms controlled for drifts of MR signal). We optimized our design to allow for adequate separation of responses to different trial types, which amounted to choosing an experimental sequence that minimized the standard error associated with the statistical test of interest (e.g., comparing two experimental conditions); see (Birn et al., 2002
). Such “optimal” experimental sequence was obtained by randomly generating a large number (i.e., 105
) of experimental sequences and choosing the best 12 (each sequence was used for a separate run).
The main goal of the present study was to determine the effects of attentional load and cognitive modulation on amygdala responses. However, we also performed a whole-brain voxel-wise analysis to investigate general task-related activations, as well as to further investigate amygdala responses. A standard two-stage mixed-effects analysis was performed. The first (fixed) level involved determining the regression coefficients of regressors of interest, which modeled the effects of each experimental condition and facial expression (e.g., fearful faces during the hard bar-orientation task). The second level treated subject as a random factor and tested for task-related differences via paired t
tests. As random-effects analysis may be fairly conservative in the context of fMRI data (Worsley et al., 2002
), we employed a threshold of p < 0.001 (uncorrected), although activations survived stricter thresholds (e.g., see ).
Figure 2 (A) Group map of the contrast of the sex-task vs. bar-orientation task. Responses in the amygdala were stronger during the sex-task when the faces were attended. (B) Group map of the contrast of the bars-only condition (when no faces were shown) relative (more ...)
For the region of interest (ROI) analysis, for every individual, a site in the amygdala was chosen based on the contrast of fearful vs. neutral faces when they were attended (i.e., during the sex task). This condition was employed as the selection criterion because the associated differential responses were also observed at the individual level; see Anderson et al. (2003)
for a similar strategy. Because we smoothed individual data with an 8-mm filter, the regression coefficients estimated via linear regression were taken from the peak voxel of the above selection contrast as representative coefficients for the ROI. We then interrogated the ROI at the group level in a random-effects manner for effects of valence and attentional load during the bar-orientation task when faces were unattended by performing pre-planned paired t
To determine “blocked” average time courses for the amygdala (, middle and bottom rows), we treated our experiment as a blocked design and averaged the responses for each of the five block types (sex task, easy/medium/hard bar-orientation task, and bars-only), and expressed responses in terms of percent increase relative to responses during fixation. Note that for the blocks involving faces, the order of the fearful and neutral faces were not fixed across blocks, but instead were randomized. Although responses due to fearful and neutral faces were thus mixed together, averaging was used to summarize blocked activity for the associated condition.