Computational protocol: Differential Dopamine Receptor Occupancy Underlies L-DOPA-Induced Dyskinesia in a Rat Model of Parkinson's Disease

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Protocol publication

[…] Dynamic emission scans were acquired in list-mode format over 120 min. The data files were displayed as 3D sinograms with a maximum ring difference of 47 and a span of 3. The acquired data were then sorted into 31 time-frames [1*15 s, 5*30 s, 1*45 s, 6*1 min, 1*1.5 min, 4*2 min, 1*3.5 min, 5*5 min, 1*7.5 min, 6*10 min]. Finally, each emission sinogram was normalized, corrected for attenuation and radioactivity decay, and reconstructed using Fourier rebinning and 2-dimensional ordered-subsets expectation maximization (16 subsets, 4 iterations).Time frames collected were summed to create an integrated image. In order to define volumes of interest (VOIs) using anatomical landmarks, rats underwent as well a T2-weighted MR imaging that was used for PET/MRI co-registration. To this aim, rats were placed on a 7 Tesla MR system (Varian-Agilent Technologies, USA) equipped with a gradient coil reaching 600 mT/m (120 µs rise time), a radiofrequency birdcage 1H coil for transmission, and a 4-channel surface receive coil and T2-weighted images were acquired over a total acquisition time of 9 minutes. PET/MRI co-registration was then performed using the in-house image processing software Anatomist (http://www.brainvisa.info). Striatal VOI were delineated using the microPET Data analysis software ASIPro (ASIPro VM, Siemens) and ROI drawn over 13 to 15 continuous planes.To measure in vivo the fraction of [18F]fallypride non-specific binding, a 10-voxel-diameter spherical region of interest (voxel size of 0.47; 0.47; 0.796 mm) was drawn over the cerebellum, in a region devoid of D2R. The mean activity concentration values in all VOIs (left and right striata, cerebellum) were then calculated and plotted over time yielding regional time-activity curves. These curves were then normalized to the injected dose and body weight and expressed as standardized uptake values (SUVs).Kinetic modelling analysis was performed using the PMOD software package (version 2.95; PMOD Technologies). D2R occupancy by the released DA in each set of experiments was estimated by calculating the distribution volume ratio (DVR) using the Logan noninvasive method . This method which yields an estimate of specific binding (BP) through the relationship DVR = BP+1, assuming the existence of a reference region, such as the cerebellum, which is almost devoid of dopamine receptor and can be used to assess the pharmacokinetics of the radiotracer in the absence of a specific compartment. For each PET experiment, BP was calculated based on 9 regression points, starting after an equilibrium time of 42.5 min post-injection. [...] The numbers of FosB/δFosB immunoreactive cells in the striatum were analyzed on images captured using a 10x Plan-Fluor objective on a Nikon BXA microscope equipped with a Olympus DX72 camera using cellSens standard 1.5 software (Olympus Corporation). The images were captured from the striatal level corresponding to AP +1.20 relative to bregma according to the rat brain atlas of Paxinos and Watson . The quantification was carried out using TIFF formatted images analyzed by using the ImageJ software (Version 1.42i, NIH). After background subtraction, the total numbers of profiles specifically labeled with FosB/δFosB were counted using the particle analysis tool. The density of immunopositive FosB/δFosB profiles was expressed as number of immunoreactive cellular profiles per mm2. […]

Pipeline specifications

Software tools cellSens, ImageJ
Application Microscopic phenotype analysis
Organisms Rattus norvegicus, Homo sapiens
Diseases Parkinson Disease
Chemicals Dopamine, Levodopa, Serotonin, Oxidopamine