• Breath-Holding Task
  This task is used to measure subjects’ global vascular reactivity.
  The task consists of a block design with alternating on/off blocks of 15-second periods of breath holding and normal breathing. There are 8 complete on/off cycles for a scan time of 240s. During the on-block, the subject is shown a picture of a circle that becomes smaller as time progresses. This serves as a general indication of time before breathing can resume. During the off-block, the subject breathes normally and fixates on a cross. No psychophysical data are recorded.
• Reaction Time Task
  The Reaction Time (RT) task measures the effects of keypad ergonomics on subjects’ RTs. Because motor tasks are believed to demonstrate good reproducibility, differences in a subject’s RT between scan sites will be attributed to differences in keypad ergonomics.
  Stimuli presentation, developed with E-Prime programming software consists of random numbers ranging from 1 to 4. Subjects press one of the 4 keypad buttons when they see the corresponding visual cue. RTs are measured from the button box. 32 total stimuli appear at 1s intervals. The stimulus remains on the screen until a response is given or 2s if no button is pressed. RT is measured in milliseconds, i.e., 1000 milliseconds is 1 second. Psychophysical data includes the subject response and reaction time. No scan data are
  recorded.
• Sensory Task
  Subjects perform finger apposition task in time with 3Hz audio cue and watch 3Hz flashing checkerboard square. Tones are 333ms long. The task consists of a block design with block durations of 15s on/off. There are 8 complete on/off cycles for a scan time of 240s. The off-block is fixation/silence/no tapping. Volume balance is specified by the experimenter at the start of E-Prime, and is determined by subjects’ response in the audio setup task. Task instruction and stimuli are presented visually with E-Prime programming software. No psychophysical data are recorded.

All procedures are performed under Duke IACUCC protocol #A022 00 01 1. This is a conventional perfusion fixation with inflow to left ventricle and outflow from right atrium. The syringe pump is set at 9 ml/min. The pressure at this setting and for the size of the tubing and stub adapter, is about 75 mmHg. When setting up the pump, make sure there are no bubbles in the line.

MR Microscopy
All scans are performed at 9.4 T using a 1 cm solenoid rf coil.
All scans are acquired in a single setting so the data sets are registered.
All scans are acquired with a fixed field of view of 11x11x22 mm with a matrix of 256x256x512 yielding voxels of 0.043x0.043x0.043 mm i.e. 8 x 10-5 mm3.
We are acquiring three registered data sets using standard 3DFT spin warp encoding. The TR and NEX have been adjusted so that each set requires 14.56 hrs:
Data set A: T1 weighted @ TR=100 ms, TE=5.5 ms, Bandwidth ±31.25 kHz, NEX=8
Data set B: T2 weighted @ TR=200 ms, TE- 20 ms, Bandwidth ±15.62 kHz,NEX=4
Data set C: Spin density @ T2 weighted @ TR=200 ms, TE- 5.5 ms, Bandwidth ±15.62 kHz,NEX=4

Shipping
When the Duke MRM is finished, the specimen is removed from the fomblin, rinsed in 10% buffered formalin and placed in a sealed container with formalin. The container is placed on chipped (wet) ice and shipped overnight to Cal Tech where the diffusion tensor imaging takes place. When the diffusion tensor imaging is completed, the specimen is forwarded to UCLA.

Diffusion Tensor Imaging
Magnetic resonance imaging is done at 37° C in an 11.7 Tesla, vertical bore (89 mm) Bruker AMX500 microimaging system (Bruker Instruments). We use an Acustar shielded gradient set (max 100 gauss/cm gradient strength) with home-built RF probes and low-noise preamplifier. Images are recorded using 3D multispin echo protocols (one to eight echoes) with a data matrix of 256 x 128 x 128 points. Typical spatial resolution is approximately 60 µm3 per voxel. The images are padded with zeros to double the number of time domain points in each dimension, the Fourier transformed to yield a matrix of 512 x 256 x 256. This procedure is commonly called "zero-filling" and is a well known interpolation method
Blockface and Histology
After post-fixation the brains are dipped in a mixture of india ink (Pelikan) and 5% gelatin (Sigma) to simplify segmentation of tissue from background later. The brains are then embedded in OCT compound (Sakura) at 4° C and snap-frozen at -70° C in a 2-methylbutane/dry ice bath.

Once the blockface and histological samples are prepared, the brains are attached to a chuck with OCT compound (Sakura), and sections are cut serially in 50µm thick coronal sections on a modified CM3050S cryostat (Leica). A DMCIe digital camera (Polaroid) captures images of the blockface prior to each section at a resolution of 1600 x 1200 (approximately 6.7µm/pixel) in 24-bit color. Sections 200µm apart are Nissl-stained (Thionin) and alternating sections 200µm apart are myelin-stained using a modified myelin impregnation stain. Stained preparations are digitized using a 0.5X objective on an AX70 microscope (Olympus) with a DMCIe digital camera (Polaroid) at a resolution of 1600 x 1200 (approximately 6.1µm/pixel) in 24-bit color. The images are acquired using a macro imaging system that provides undistorted high-resolution images with even illumination across the entire field of view.

Image Processing
The method we use to extract mouse brain tissue from the embedding medium used during cryosectioning and the slide background for histologically stained sections takes advantage of the rich textural information of the images, for example, the texture of the frozen OCT compound is quite different from that of the brain tissue. A previously trained neural net classifies a region within the image as either tissue or background based on the textural descriptors and output a segmented image. Combined with color information, it provides sufficient discriminating power to perform the segmentation. The two dimensional digital images of the stained sections are brought roughly into register with their corresponding blockface images acquired during sectioning using automated software tools produced at LONI. The preregistration program produces an initialization file for Automated Image Registration (AIR). Once the registered images are reconstructed into a three-dimensional volume, the resulting volume is brought into register with an inherently three-dimensional uMRI in a defined and common coordinate system. All image processing is done on a 32-processor Onyx 200 supercomputer (SGI).