Data Acquisition

Subjects were placed supine in a Signa LX (model 3.0T; General Electric Medical Systems, Waukesha, WI) equipped with an eight-channel receiver head coil and body quadrature transmit radiofrequency coil. Subjects were trained outside the scanner to maintain the lips and lower jaw relaxed and closed, and to swallow in a single motion of tongue pressing backward and upward against palate without rolling or a side-to-side motion resulting in a single swift laryngeal elevation. Subjects were instructed to avoid repetitive swallowing, and conscious nodding head motion was discouraged. Cardiorespiratory monitoring was performed at a sampling rate of 40 Hz with a pulse oximeter, and respiratory bellows equipment was provided as part of the MRI system. Paradigm-driven functional MRI data were acquired during two distinct 540-s runs while subjects performed 21 random single trial swallows across two sessions. A rear projection screen was placed at the head of the scanner bed to display visual cues to swallow and a fixation crosshair between swallows. Visual cue was a simple verbal command of “please swallow” shown on the screen replacing the fixation crosshair for 3 s. Timing of the visual cue was not locked to repetition times (TRs). These two scans were designed as a part of larger protocol and were 30 min apart across two different scanning sessions. Two anatomic scans were also acquired during each session using the high-resolution spoiled gradient recalled acquisition technique, consisting of 140 sagittal whole brain 1-mm thick slices over a 240-mm field of view and 256 × 224 within slice voxel resolution. These high-resolution anatomical images were used for subsequent superposition of cortical activity regions derived from the lower resolution echo planar BOLD contrast image data in each subject. Echo planar images (EPI) were acquired as 34 contiguous 4-mm thick sagittal slices over the whole brain volume in an interleaved fashion without any gap or overlap. EPI images were acquired with a slice-wise resolution of 64 × 64 voxels over a 240-mm field of view yielding a within-slice resolution of 3.75 × 3.75 mm, captured with an echo time (TE) of 23.4 ms and a TR of 2,000 ms in the first session, and TE = 17.5 ms, TR = 1,700 ms during the second session.

Regions of Interest Identification

The studied regions of interest (ROI) were all defined anatomically a priori on individual original space reference high-resolution anatomic scan according to the literature and statistical probabilistic maps included in Analysis of Functional Neuroimages (AFNI) software suite (9). We marked ROIs manually and consistently by one investigator (S. Ahmad) on both hemispheres. These areas were chosen based on earlier reports of swallowing fMRI activity as well as negative BOLD response to nonswallow motor tasks reported in the literature.

The cingulate cortex was defined as the portion of the cortex on the medial surface of cerebral hemispheres extending above corpus callosum and confined by the cingulate sulcus (15, 63). Cingulate sulcus is the most prominent sulcus on the medial surface and runs in parallel to corpus callosum. Paracingulate sulcus, if present, is noted to be superior and parallel to anterior cingulate sulcus and was included in cingulate cortex (14). Two imaginary vertical lines passing through anterior and posterior commissure divided cingulate gyrus to anterior, middle, and posterior regions. Anterior cingulate cortex primarily consisted of Brodmann areas (BAs) 24, 25, and 32; middle cingulate cortex mostly covered BAs 23, 24, and 33; posterior cingulate cortex mainly contained BAs 23, 29, 30, and 31 (63). The prefrontal area is the granular cortex, comprising majority of the frontal lobe (50), which, with electrical stimulation, does not evoke any visible bodily movement (13). Prefrontal cortex traditionally is divided into anterior, dorsolateral, and ventrolateral areas. Anterior (or frontopolar) prefrontal cortex extends from the most anterior (rostral) end of frontal lobe on the medial surface to cingulate sulcus posteriorly and superiorly to supplementary motor area. In axial sections, as we move inferiorly, the prefrontal cortex extends laterally and inferiorly on to the orbital surface of frontal lobe. It contains mostly BA 10 with minimal overlapping of BAs 11 and 12, inferiorly (45). The dorsolateral prefrontal cortex laterally occupies most of the middle frontal gyrus containing BAs 8, 9, and 46 extending posteriorly to premotor cortex. It includes small areas of inferior gyrus laterally and superior frontal gyrus medially just anterior to supplementary motor area. Ventrolateral prefrontal cortex mainly includes BAs 44 and 45 within inferior frontal gyrus, lateral orbital gyrus, and frontal operculum adjacent to insular lobe (43). Insula was defined as the region folded deep within the lateral sulcus buried underneath frontal and temporal lobes. Insula on sagittal view is divided by a prominent oblique central sulcus into anterior and posterior compartments. Insula covered BA 13, and short 3–5 gyri located rostral/anterior and long 2–3 gyri located caudal/ posterior to the central sulcus comprise the anterior and posterior insula, respectively (39). On axial section, insula was surrounded laterally by claustrum and capsule extrema and medially by opercular region. The sensory motor cortex consisted of BAs 1, 2, 3, and 4 and is defined as the immediate band of the precentral and postcentral gyrus adjacent to central sulcus on the lateral surface of the cortex, extending medially into the longitudinal cerebral fissure and laterally above axial plane of intersection of lateral and central sulcus (65, 69). Stimulation of the most caudolateral segment of motor cortex that has been associated with oropharyngeal deglutitive muscles (47, 48). We identified and marked the most caudolateral section of precentral and postcentral gyrus as rolandic operculum that included BAs 4, 40, and 43. Rolandic operculum extended 15 mm above the axial plane of intersection of lateral and central sulcus superiorly (to border sensory motor cortex) and 15 mm medially. This ROI also included areas commonly referred to as secondary and ventral somatosensory area. We marked supplementary motor and premotor areas with a more inclusive definition that mainly covered BA 6 and overlapped with BA 8. On sagittal section of the medial surface of hemisphere we marked all grey matter between prefrontal cortex anteriorly to sensory motor cortex posteriorly extending 15 mm from longitudinal hemispheric fissure as supplementary motor area. The remaining lateral section of frontal lobe anterior to sensory motor cortex and precentral sulcus covering BAs 6 and 8 was marked as secondary motor cortex bordering anteriorly with dorsolateral and ventrolateral prefrontal cortex. The precuneus region was defined as the medial surface (15 mm) of superior parietal cortex bordering sensory motor and posterior cingulate cortex ventrally. Precuneus was inferiorly demarcated by parietooccipital and anteriorly by marginal ramus of the cingulate sulcus and primarily contained BA 7 with overlap of BA 31 (5, 31). The remainder of the lateral parietal cortex including superior and inferior parietal lobule covering BAs 7, 39, and 40 was subdivided into two parts. First, we marked rostrolaterally and just posterior to the sensory motor and rolandic operculum region supramarginal gyrus and anterior section of inferior parietal lobule covering a small section of parietal operculum. This area covered primarily BAs 40 and 5 and was inferiorly limited by superior temporal gyrus/sylvian fissure and superiorly by intraparietal sulcus and was identified as inferior parietal lobule ROI. Two, further dorsolaterally angular gyrus and posterior section of inferior and superior parietal lobule that covered BAs 40, 39, and 7 was identified as angular ROI. Parahippocampal gyrus was identified on sagittal and coronal sections as the convoluted grey matter just superior and lateral to hippocampus deep in temporal lobe. It was located above fusiform gyrus and anterior to lingual gyrus of the occipital lobe and mainly covered BA 30. Subcortical regions with known strong cortical connections were also identified and masked. Dorsal striatum was easily identified on axial and sagittal sections and included putamen and caudate nucleus that were marked as a single ROI. Putamen (the outermost portion of basal ganglia) was identified as triangular-shaped grey matter nucleus just medial to claustrum and external capsule and lateral to globus pallidus posterior to internal capsule. Caudate nucleus was identified as the C-shaped grey matter nucleus adjacent to the lateral ventricles. Head of caudate nucleus forms part of the floor of anterior horn of the lateral ventricle and is located anterior to internal capsule that separates it from the putamen. Body and tail of caudate nucleus travels on the floor of lateral ventricle backward and then curves anteriorly to form the roof of inferior horn of lateral ventricle. Thalamus was identified as the midline large ovoid grey matter structure that is situated medial/superior to internal capsule and lateral to third ventricle. Red nucleus was a small region marked inferior to the thalamus and dorsorostral to substantia nigra. Cerebellum was identified and marked as the most inferior and posterior large mass of heavily folded grey matter infratentorial structure located posterior to the pons and brain stem. None of the ROIs had any overlapping areas (Fig. 1). Total volume of all ROIs bilaterally covered 177 of 193 cm³ of the group brain.

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Fig. 1.

A priori manual masking of the 40 regions of interest (ROI) on a reference high-resolution anatomical scan is displayed. This example is from subject 14 and all ROIs are color coded. All ROIs were drawn based on standard stereotaxic statistical probabilistic maps available in the Analysis of Functional Neuroimages (AFNI) suite and were modified based on individualized known gyral and sulcal patterns described in the literature.

Image Analysis

All fMRI signal analyses were carried out using the AFNI package (9). We processed the data according to accepted fMRI analysis pipeline and took some additional steps to minimize inherent noise associated with oropharyngeal movement and performed confirmatory analysis to verify subjects' compliance to swallow commands. All participants maintained a peak-to-peak global (EPI volume) head motion of < 2 mm, and only two subjects showed > 1 mm global head motion that was swallow related. Physiologic signal (cardiac and respiratory)-related signal changes were retrospectively corrected (17). Brain in a structural anatomical data set was extracted from surrounding tissue (55). We then computed an alignment matrix to a mean anatomical dataset from the two acquired anatomic scans in each of the sessions and registered both anatomic scans to this mean dataset. We used the resultant dataset as the reference anatomic scan for alignment of all EPI datasets to remove the bias of either session. Reference anatomical scan was spatially normalized to match the standard Talaraich-Tournoux stereotaxic template (60). We performed slice timing correction and modeled head motion using 12 degrees of freedom indexed by time, which was used to interpolate the time series back to the original acquisition grid (51). We computed the alignment matrix between EPI datasets and reference anatomical scan, and applied that to register and align all datasets to the reference anatomic scan grid. The data was transformed and resampled into standard Talaraich-Tounoux space with voxel size of 3.75 × 3.75 × 3.75 mm in one interpolation within AFNI package. Voxel-wise extreme fluctuations of the signal were replaced by a fitted smooth curve to the time series. fMRI BOLD signal was then normalized voxel-wise to the average signal over the course of time series to yield percent signal change at the end of analysis pipeline. White matter and cerebrospinal fluid containing voxels were identified automatically based on their signal intensity and manually verified within each subject. The average time course within the white matter and cerebrospinal fluid voxels were extracted. General linear modeling techniques were used to remove undesirable signal contamination correlated with 12 motion parameters, white matter, and cerebrospinal fluid temporal components out of the BOLD signal as covariates of no interest. We utilized functionally acquired pharyngeal signal and identified each swallow with its associated susceptibility changes to extract timing of actual swallows. We used actual swallow timing as our reference time course and the main signal of interest. Analysis was performed with multiple linear regression using generalized least squares time series fit with partially nonlinear restricted maximum likelihood estimation of serial temporal autocorrelation structure. Results were then spatially smoothed to a full width at half maximum of 5 mm. A second-level group, random, two-sample, mixed-effects, multilevel analysis was then performed to identify active cortical regions (6). Clusters with P < 0.05 corrected for multiple comparisons (using Monte Carlo simulation) were considered to be significant. A clustered activity map exceeding the significance threshold was overlapped by a priori identified group mask of each ROI to determine activated clusters within each ROI.

Positive BOLD Network

Group analysis showed significant clustered-positive BOLD activity within the targeted ROIs of the deglutition network during both scan sessions: sensorimotor cortex, premotor area, rolandic operculum, middle cingulate, insula and adjacent prefrontal operculum, and inferior parietal lobule (Fig. 3). Total volume of significant clustered-positive BOLD activity within our ROIs was 7.2 and 5.9 cm³ across first and second sessions, respectively, which covered 7.4 and 6.1% of their respective ROIs. There was significant bilateral activity within cuneus or occipital cortex (BAs 18 and 19) and superior and transverse temporal (Heschl's) cortex (BAs 38, 41, and 42) that we did not further analyze. This activity is presumably related to the processing of visual commands that triggered the swallow (28).

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Fig. 3.

Positive BOLD cortical swallow-related activity displayed across 2 sessions. Sagittal, axial, and coronal images are displayed to demonstrate the extent and location of activity throughout the whole brain.

Sensory motor region activity was the largest cluster of activity that was located in lateral segment of the pericentral sulcus. During the first session, left and right sensory motor clusters' volumes of activity were 1.5 and 1.8 cm³, which remained the largest clusters during the second session with volume of 1.3 and 0.9 cm³, respectively. Rolandic operculum bilaterally showed activity that compared with all of other ROIs and occupied the largest percentage of a priori identified ROI (55 and 31% of the ROI for left hemisphere, 79 and 52% of the ROI for right hemisphere, during the first and second sessions, respectively). Rolandic operculum also carried the highest magnitude of percent signal increase (1.04 and 1.08% for the left and right hemispheres, respectively) during the first session, and (0.80 and 0.82% for the left and right hemisphere, respectively) during the second session.

Insular region clustered activity was almost located in middle of the insula crossing the superior insular sulcus into adjacent frontal operculum and was divided by oblique middle insular sulcus into anterior and posterior clusters, respectively. Right precuneus and right supplementary motor clusters did not reach the statistically significant threshold in one of the sessions. They were both midline structures, and the clustered activity was divided into hemispheric subclusters that may have not reached statistical significance on one side with family-wise error correction. Subcortical activity appeared to be unilateral in our study involving right dorsal striatum, right thalamus, and left cerebellum. Thalamic activity was located in medial dorsal nucleus, and striatal activity was centered at the junction of posterior part of right putamen to lateral globus pallidus. Red nucleus activity was not observed in the first session of scanning; however, it showed significant clustered activity during the second session. Details of volume, magnitude, and coordinates of peak and centroid (geometric center of the mass) of positive BOLD activity are shown in Table 1.

Reproducibility of positive BOLD activity.

Scatter plot and linear regression analysis of volume of activity (y = 0.59x + 73) and magnitude of percent signal change (y = 0.50x + 0.24) across both sessions demonstrated significant correlation across sessions (Fig. 4). As demonstrated, the first session revealed higher volume and magnitude of clustered activity compared with the second session. The decrease in activation volume and percent change in the second session, considering reduced echo and repetition times, is consistent with a true BOLD response instead of motion artifact. Overlap of activity maps showed that coordinates of the centeroid of the activated clusters were within 6 ± 5 mm across sessions.

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Fig. 4.

Reproducibility of the volume and magnitude of swallow-related positive BOLD signal change across two functional MRI sessions.

Swallowing and Negative BOLD fMRI Activity

Functional brain mapping operates on the assumption that regions of cortex showing a positive temporal correlation between the signal and a particular stimulus are involved in processing that stimulus. However, both PET and fMRI studies have shown certain areas of the cortex with responses that are anticorrelated with stimulus. The ambiguous origin of observed anticorrelated cortical responses (negative BOLD, in case of fMRI) with stimuli is poorly understood. Three theoretical mechanisms have been proposed for negative BOLD response (64): 1) vascular steal that is a hemodynamic effect of vascular diversion of oxygenated hemoglobin resulting in negative BOLD signal of neighboring areas (10, 24); 2) active neuronal suppression suggesting reduced functional neuronal activity of the regions below baseline due to active neuronal inhibition (1, 20, 54); and 3) extended initial dip that actually represents an increased level of neuronal activity during the stimulus period that was not matched by a corresponding boost in blood flow, depleting local oxygen levels. (25). There is accumulating evidence to suggest that decreased neuronal activity is the underlying origin of the negative BOLD signal (18, 52, 53); however, nature of this neuronal suppression is still debatable.

The present study documents, for the first time, the extent and magnitude of negative BOLD activity during swallowing. These negative BOLD-activated regions were comparable to the regions that comprise the default-mode network described in the literature (44). The default-mode network has been identified in other functional brain studies, including motor tasks by reduced activity compared with baseline resting state. Distinct regions of the brain have been regularly observed to decrease their activity during cognitively engaging tasks and are thought to mediate brain processes during the resting state that is suspended during goal-directed behaviors (16, 44). These regions have been called task-negative or default-mode network (19, 44). Studies have also shown that attention-demanding tasks, regardless of the nature of the task (sensory, motor, cognitive, visual, or auditory), consistently activate a group of brain regions (8). These regions have been collectively called task-positive or task-control networks (8, 11). The results of our study are in accordance with these previous reports, in that, among 40 distinct regions involved in swallowing, nearly half showed negative BOLD activity quite similar to regions of default-mode network (19), and the other half that showed positive BOLD activity displayed overlap to the task-positive network previously described in literature (11).