Five lean ((mean ± s.d.) BMI 22.0 ± 2.9 kg/m2, age 23.4 ± 1.1 years) and five obese (BMI 41.6 ± 5.0 kg/m2, age 31.6 ± 8.8 years) women were recruited from the community. There was no difference in years of education between the groups (16.2 ± 1.3 vs. 14.4 ± 2.6). No subject had evidence of Axis-I psychiatric disorders or neurological disorders of the brain. All performed normally on a 20-item version of the Smell Identification Test ((25); range 16–19), with no between-group differences. Nine of the ten subjects were nonsmokers, with the 10th indicating only occasional smoking (average 0.10 cigarettes/day). Individuals were excluded if they were pregnant within the past 6 months, breast feeding, or if their food preferences were inconsistent with the food aromas used. All voluntarily signed informed consent statements approved by the institutional review board at the Indiana University School of Medicine.
Subjects were admitted to the General Clinical Research Center the day before imaging. At their arrival on day 1 (12 pm), weight and height were recorded and the subjects were provided a standard lunch meal (sandwich or pasta) providing one-third of the daily kilocalories required for weight maintenance (range ∼60% carbohydrate, ∼30% fat). After eating lunch, subjects were not allowed to eat (water ad libitum was allowed) before the imaging session on day 2 (2 pm), resulting in an ∼24-h fast before imaging.
Olfactory stimuli. Odorants were delivered using an eight-channel air-dilution olfactometer as previously described (26,27), with air delivered to the subject's nose via a small polytetrafluoroethylene tube at 2.0 l/min. Three classes of odorants (International Flavors & Fragrances, Union Beach, NJ) were used: (i) fat food-related odors (F-FRO), with each subject choosing two of three F-FRO from potato chips, roast beef, and pasta, (ii) sweet food-related odors (S-FRO), with each subject choosing two of three from strawberry shortcake, chocolate cake, and caramel ice cream, and (iii) NApO, with subjects choosing from four of the seven odors of grass, leather, lilac, Douglas fir, lily of the valley, balsam, and patchouli. By allowing subjects to choose odors, unpleasant or aversive odors could be excluded. Small, porous polyethylene disks were used to absorb the NApO, F-FRO and S-FRO, and then placed at the bottom of a glass vial, over which the olfactometer airstream passed before being delivered to the subject.
Stimulus training. Before entering the scanner room, subjects were familiarized with the odorants by smelling each (grouped by the stimulus classes of FRO and NApO) through the olfactometer while simultaneously viewing representative images on a computer monitor (e.g., roast beef odor presented with an appealing photograph of a plate of roast beef).
Craving/mood questionnaire and preferred food-related odors (P-FRO). Just before the combined odor/picture cue-exposure, subjects answered questions probing craving/desire to eat and mood. Subjects rated food craving by responding to three questions (“I want to eat right now,” “Right now I crave something sweet,” “Right now I crave something salty”) on visual analogue scale (1 = strongly disagree, 7 = strongly agree). Subjects also rated their desire to eat each of the four specific foods corresponding to the four FRO they had chosen (e.g., “If given the opportunity to eat (roast beef) right now, I would,” 1 = be uninterested, 7 = definitely eat it). Subjects similarly rated mood (“Right now, I feel angry, grouchy, annoyed, bad-tempered,” “Right now, I feel happy, energetic, full of pep, cheerful, vigorous”). Craving/desire to eat scores were then used to define each subject's preferred type of food at the time of imaging. These preferred odors were used in the analysis as P-FRO, as preliminary analyses showed these to provide the most robust results.
Activation paradigm. Six functional imaging scans of olfactory stimulation per subject were performed, for a total of 36 odor events per class (Figure 1). However, due to two subjects' inability to complete the full functional imaging protocol and excessive motion in one subject, only four scans could be analyzed for three obese subjects. Compliance was established by prompting (via a tone) the report of an odorant's presence (button 1) or absence (button 2) on a two button response-box. Unlike the odor familiarization period, no pictures were presented during imaging, and subjects were instructed to close their eyes during image acquisition.
Figure 1. Odor stimulation paradigm. During 40 s epochs, subjects sniffed either fat food-related odors (F-FRO; e.g., roast beef, potato chips), sweet food-related odors (S-FRO; e.g., strawberry shortcake, chocolate cake), or nonappetitive odors (NApO; e.g., grass, Douglas fir), as well as odorless air (sham stimuli) over 20 s epochs. Each epoch consisted of 2 s odor valve openings, with auditory commands (yellow inset) instructing subjects to sniff. Each odorant in a given class was delivered twice in alternate order (e.g., roast beef, potato chips, roast beef, potato chips) over the course of three 40 s periods, with a 10-s stimulus onset asynchrony (SOA) between single-odorant pulses. Different stimulation sequences were randomized across subjects such that no stimulus class was repeated without another intervening class, and any one odor class was always followed by an odorless baseline period. Within each scan, food-related odors were always presented in alternate order with NApO. Periods 1 and 2 correspond to either F-FRO and NApO, or S-FRO and NApO, depending on odor presentation randomization.
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Odor ratings. In a post-imaging evaluation session outside the scanner room, subjects rated on a linear 9-point visual analog scale the odors' intensity, pleasantness, and representativeness (how well the odor represented its intended source).
Subjects were imaged on a Siemens 3T Magnetom Trio-Tim scanner (Siemens). A whole-brain high-resolution anatomical image volume (1.0 × 1.0 × 1.2 mm voxels) was first collected using a 3D magnetization prepared rapid gradient echo sequence to enable anatomic registration of the functional volumes. Six functional scans were then performed with a blood oxygenation level dependent (BOLD) contrast sensitive gradient echo, echo-planar imaging sequence (repetition time 2,250 ms, echo time 30 ms, flip angle 78°, 37 3-mm thick interleaved axial slices without a gap, in-plane voxel dimension 2.5 × 2.5 mm, field of view 220 × 220 mm) using a 12-channel head coil array and incorporating a 3D prospective acquisition correction that minimizes motion effects by adjusting the acquisition of functional volumes in real time.
Imaging processing and data analysis
Data were analyzed using SPM5 (Welcome Department of Imaging Neuroscience, University College, London, UK). Functional volumes were corrected for slice acquisition timing differences and rigid-body realigned to the initial volume of the first functional imaging scan (used as a reference volume), correcting for residual movement after prospective correction. Each subject's high-resolution anatomic image was coregistered to subject's reference volume and segmented into gray, white, and cerebrospinal fluid tissue components producing nonlinear spatial transformation parameters for converting functional volumes to the Montreal Neurological Institute coordinate space (isotropic 2 mm voxels). Normalized functional volumes were smoothed by a 6 mm full-width at half-maximum isotropic Gaussian kernel.
Within-subject effects were first estimated. Discrete 2 s periods of odorant (or sham) valve openings (Figure 1) were modeled in the general linear model using as basis functions SPM's canonical hemodynamic response function and its time and dispersion derivatives to account for variations in response onsets and durations. Individual odorant pulses were modeled as initial investigation showed that convolving the hemodynamic response function with the individual 2-s odor pulses provided better olfactory sensory system (piriform and orbitofrontal cortex) responses than convolution with the entire 40 s blocks (Figure 1). Movement parameters from realignment were included as regressors to control for residual movement-induced effects. A high-pass filter with a cut-off of 1/128 Hz was applied to each voxel's time series to remove low-frequency noise but autoregression was not used given the long interstimulus interval. This first level, within-subject model, yielded contrast images representing activation within an odorant condition ((P-FRO derived from either F-FRO or S-FRO and NApO), as well as the differences between conditions (P-FRO > NApO)). These contrast images were then used in second level (group) random effects analyses. Statistical significance was inferred using corrected cluster statistics with a height threshold of P < 0.05, false discovery rate (FDR) corrected.