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Both in vivo and ex vivo images of the rat brains were obtained using the Inveon microPET scanner and were analyzed using the Acquisition Sinogram Image Processing (ASIPRO, Siemens Medical Solutions USA, Inc., Knoxville, TN, USA) and Pixelwise Modeling Software (PMOD Technologies, Zurich, Switzerland).
Synaptosomes from ASMko and wt mice brains were obtained using Percoll gradients [29].
The fluorescent images of whole brains were obtained using a digital camera attached to a fluorescence stereoscopic microscope (Olympus, SZX10).
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Thirty-two axial slices (field of view = 225 mm × 225 mm, matrix = 64 × 64, thickness = 3.5 mm) parallel to the anterior-posterior commissure plane and covering the whole brain were obtained using a T2-weighted single-shot, gradient-recalled echo planar imaging sequence (repetition time = 2000 ms, echo time = 30 ms, flip angle = 90°).
MRI scans of the brain were obtained using a 3 Tesla Philips MRI system with either the 8-channel SENSE head coil for 1H MRS, or the phosphorus surface coil (diameter 14 cm) for 31P MRS. 1H MRS was preceded by the acquisition of a transverse 3D T1-weighted scan, which was reformatted to obtain coronal and sagittal series.
High-resolution anatomical images of the entire brain were obtained using a 3D spoiled gradient echo sequence ("SPGR"; GE Medical Systems), with 0.86 × 0.86 × 1 mm voxel dimensions.
Serial coronal sections (300 μm thick) of the brain were obtained using a cryostat (Slee Mainz, London, UK) maintained at −10°C.
T2-weighted MR images and T1-weighted contrast-enhanced MR images of the brain were obtained using a Bruker Biospec imager with a 7 T/21 cm magnet and a quadrature volume coil 2 cm in diameter.
First, anatomical images of each dog's brain were obtained using a T1 weighted sequence with repetition time (TR) = 11 ms and echo time (TE) = 5.2 ms with a field of view (FOV) of 220 mm and 0.7 mm × 0.7 mm × 0.7 mm isotropic voxels.
Eight T2-weighted MR images spanning the brain were obtained using a multi-slice multi-echo (TE = 26.8 ms, TR = 2500 ms, matrix size = 256×256, FOV = 2.5×2.5 cm, slice thickness = 0.75 mm, interslice gap = 0.25 mm, 8 echoes, 2 averages) sequence.
One millimeter-thick fresh brain slices were obtained using a mouse brain matrix and 1.2 mm diameter punches were sampled from the GFP-positive area under a fluorescent microscope and were quickly frozen in dry ice and stored at −80°C.
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