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Animal preparation. All procedures were in compliance with and approved by the Institutional Animal Care and Use Committee of Vanderbilt University and followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Three squirrel monkeys were anesthetized with ketamine hydrochloride (10 mg/kg)/atropine (0.03 mg/kg) and maintained with isoflurane (0.5 to 3%) delivered in a 70:30 O2/N2O mixture. Animals were intubated and artificially ventilated. End-tidal CO2, respiration rate, SpO2, heart rate, electrocardiogram, and rectal temperature were monitored by MR compatible capnography, fiber-optic pulse oximetry, and small animal monitoring system. The end-tidal CO2 was kept around 4% by adjusting the rate and volume of the ventilator, and the rectal temperature of 37.5° to 38.5°C was maintained by an animal warming system. Under general anesthesia (1 to 2% isoflurane), two monkeys were implanted with custom-made imaging chambers, with a transparent artificial dura over the primary somatosensory cortex, secured by ceramic screws. After placement in an MRI-compatible stereotaxic tube, laser stimulation (Lockheed Martin Aculight, Bothell, WA, USA) was delivered via a 400-μm-diameter optic fiber through the imaging chamber.

Stimulus protocol. Laser stimuli consisted of a train of laser pulses (wavelength, 1875 ± 10 nm; radiant energy, 0.1 to 1.0 J/cm2 per pulse, calibrated with a power meter) via a 400-μm-diameter optical fiber positioned normal to the brain surface. Each train consisted of 0.5-s duration bursts of 250 μs per pulse at 200 Hz. In a single trial, 12 pulse trains were repeated once every 2.5 s for 30 s (interstimulus interval between pulse trains was 2.0 s), followed by 30-s blank. Blocks of 10 to 15 trials (300 imaging volumes) were acquired within each scanning block of the same laser energy.

MRI methods. All MRI scans were performed on a 9.4-T (Varian Medical Systems, Palo Alto, CA, USA) horizontal bore magnet with an inner bore size of 21 cm and an actively shielded gradient (400 mT/m and 3000 T/m per second). A custom 3-cm-diameter transmit-receive surface coil was used in all MR scans, with injection of MIONs at 10 to 20 mg of Fe per kilogram of animal weight. The signal of all functional data with MION injection was inverted to facilitate comparison with BOLD data. T2-weighted oblique structural images at 68 μm by 68 μm in-plane resolution were acquired to identify venous structure on the cortical surface for locating the central sulcus and lateral sulcus and for coregistration of fMRI maps across imaging sessions. fMRI data were acquired from the same slices using a two-shot or four-shot EPI with a TE of 10 ms and a TR of 750 ms at voxel sizes of 270 μm by 270 μm by 2000 μm (tangential slices) or 270 μm by 270 μm by 1500 μm (orthogonal slices).

MR data processing. Functional EPI data were preprocessed for motion correction and then analyzed in MATLAB (Mathworks, Natick MA, USA) codes [for details, see (44)]. EPI data were collected as a 128 × 128 matrix. Raw EPIs were interpolated to a 512 × 512 matrix and overlaid on anatomical images. Time courses were drift-corrected using a linear model and temporally smoothed with a low-pass filter (cutoff frequency set above the second harmonic of task function). The correlation of each functional time course to a reference waveform was calculated, and functional maps were generated by identifying regions of clustered voxels whose correlation with the reference waveform was significant to P < 10−4. Choice of this threshold was guided by area, amplitude, and centroid of activation determined via optical imaging and electrophysiology in the same animals. Activations were overlaid on corresponding anatomical images and then compared with known topography.

Optical imaging/fMRI image registration. Somatosensory areal borders in squirrel monkeys were estimated by collecting optical imaging and by distinguishing receptive field preferences by electrophysiological single and multi-unit recording. Digit maps were obtained by vibrotactile stimulation of each digit. For coregistration, we first identified corresponding anatomical and blood vessel landmarks in surface blood vessel images and structural images, such as visible surface vessels and transcortical veins. These coordinates were then put into a point-based registration algorithm [implemented in MATLAB; for details, see (47, 48)]. We have used these mapping methods in a number of publications [e.g., (39, 40, 44)].

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