Male and female rats (2 per condition) were injected (IV, 1μCi/g) with radiolabeled [3H]-(S)-ketamine or [3H]-(R)-ketamine and euthanized at 10, 30, 40, or 60 min post injection and brain, blood, and tissues were collected for radiometric analyses. The brains were flash frozen in 2-methylbutane and stored at −80ºC until use. The blood was centrifuged (13,000 rpm, 10 min at RT) and serum was collected. The tissues were bleached with hydrogen peroxide and solubilized (Solvable, Perkin Elmer). Serum and tissue samples (were dissolved in scintillation cocktail (2.5 mL) and radioactivity counts were determined using a liquid scintillation counter.
The brains were sectioned (20 μm) on a cryostat (Leica), mounted into glass microscope slides and air-dried overnight at RT. The day after slides were placed into a Hypercassette™ covered by a BAS-TR2025 phosphor screen (FujiFilm; GE Healthcare). The slides were exposed to the phosphor screen for 10–15 days and imaged using a phosphor imager (Typhoon FLA 7000; GE Healthcare). The digitized images were calibrated using C-14 standard slides (American Radiolabeled Chemicals). ROIs were hand-drawn based on anatomical landmarks and radioactivity was quantified using ImageJ. The activity in 6 to 10 different sections was averaged per each animal and brain region.
The brains were sectioned (20 μm) on a cryostat (Leica), mounted into glass microscope slides and air-dried overnight at RT. The day after slides were placed into a Hypercassette™ covered by a BAS-TR2025 phosphor screen (FujiFilm; GE Healthcare). The slides were exposed to the phosphor screen for 10–15 days and imaged using a phosphor imager (Typhoon FLA 7000; GE Healthcare). The digitized images were calibrated using C-14 standard slides (American Radiolabeled Chemicals). ROIs were hand-drawn based on anatomical landmarks and radioactivity was quantified using ImageJ. The activity in 6 to 10 different sections was averaged per each animal and brain region.