All chemicals and solvents were purchased from Aldrich Chemical (Aldrich Chemical Company, Wilwaukee, WI, USA) and Fisher Scientific (Fisher Scientific UK Ltd., Leicestershire, UK). Deionized water was acquired from Millipore Milli-Q Water Purification System (Millipore, Billerica, MA, USA). Gilson high-performance liquid chromatography (HPLC) was used for the semipreparative reverse phase column chromatography. Fluorine-18 fluoride was produced via MC-17 cyclotron using oxygen-18-enriched water. Radioactivity was counted using a Capintec dose calibrator while low level counting was done using a well counter. Inveon preclinical Dedicated PET (Siemen's Inc., Munich, Germany) was used for the microPET studies which has a resolution of 1.45 mm . 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). Slices of the rat brain were prepared at 10 to 40-μm thick using the Leica 1850 cryotome (Leica Instruments, Nussloch, Germany). In vitro- or ex vivo-labeled brain sections were exposed to phosphor films (Perkin Elmer Multisensitive, Medium MS) and were read using the Cyclone Phosphor Imaging System (Packard Instruments, Meriden, CT, USA). An analysis of in vitro or ex vivo autoradiographs was done using the Optiquant Acquisition and Analysis software (Packard Instruments, Meriden, CT, USA). All animal studies have been approved by the Institutional Animal Health Care and Use Committee of the University of California, Irvine.
A synthesis of 18F-nifene was carried out following reported procedures (Pichika et al. 2006). The automated radiosynthesis of 18F-nifene was carried out in the chemistry processing control unit box. An Alltech C18 column (10 μm, 250 × 10 mm2) was used for reverse phase HPLC purification and specific activity of 18F-nifene was approximately 2,000 Ci/mmol.
MicroPET 18F-nifene studies
Male Sprague-Dawley rats were fasted 24 h prior to the time of scan. On the day of the study, rats were anesthetized using 4.0% isoflurane. The rat was then positioned on the scanner bed by placing it on a warm water circulating heating pad, and anesthesia was applied using a nose cone. A transmission scan was subsequently acquired. The preparation of the dose injection was as follows: 0.7-1.0 mCi of 18F-nifene was drawn into a 1-mL syringe with a 25-gauge needle and was diluted with sterile saline to a final volume of 0.3 mL. The dose was injected intravenously into the tail vein of the rat. Isoflurane was reduced and maintained at 2.5% following the injection. The scans were carried out for 90 min and were acquired by the Inveon microPET in full list mode. The list mode data were collected dynamically which were rebinned using a Fourier rebinning algorithm. The images were reconstructed using a two-dimensional Filter Back Projection using a Hanning Filter with a Nyquist cutoff at 0.5, and were corrected for attenuation using the Co-57 attenuation scan data. A calibration was conducted to Becquerel per cubic centimeter units using a germanium-68 phantom which was scanned in the Inveon microPET and was reconstructed under the same parameters as the subjects. Analyses of all data were carried out using the Acquisition Sinogram Image Processing IDL's virtual machine (ASIPRO VM) and Pixelwise Modeling software (PMOD 3.0). The test and retest microPET studies on the same animal were carried out within an interval of approximately 2 weeks.
Blood was collected at four different time points (5, 15, 60, and 90 min) after the injection of 18F-nifene. The blood was centrifuged for 5 min at 3,000 g. The plasma was separated and counted. Acetonitrile was added to the blood samples, and the organic layer was spotted on the analytical thin layer chromatography (TLC) plates (silica-coated plates, Baker-Flex, Phillipsburg, NJ, USA) and was developed in 15% methanol in dichloromethane. A sample of the plasma was also collected prior to the injection of 18F-nifene and was spiked with the tracer and was used as a standard.
Male Sprague-Dawley rats were injected intravenously (IV) with 0.5 mCi of 18F-nifene in a total volume of 0.3 mL and were sacrificed 40 min after injection. The brain was extracted and dissected into two hemispheres. The sagittal sections of 40-μm thickness were obtained from the left hemisphere using the Leica 1850 cryotome and were exposed to phosphor films overnight. The films were read using the Cyclone Phosphor Imaging System and were analyzed using the Optiquant software. The right hemisphere was homogenized with 1.15% KCl (2 mL), and this homogenized mixture was vortexed with 2% acetic acid in methanol (2 mL). This mixture was centrifuged for 10 min at 10,000 g, and the supernatant was removed for analysis. RadioTLC (9:1, dichloromethane and methanol) was obtained for both 18F-nifene standard and the brain extract.
Ex vivo microPET
In order to ascertain the brain uptake of 18F-nifene, after completion of the in vivo microPET scans, the rats were sacrificed and the brain was extracted for ex vivo microPET imaging. The whole brain was placed in a hexagonal polystyrene weighing boat (top edge side length, 4.5 cm; bottom edge side length, 3 cm) and was covered with powdered dry ice. This boat was placed securely on the scanner bed, and a transmission scan was acquired. Subsequently, a 60-min emission scan was acquired by the Inveon microPET scanner in full list mode. The list mode was collected in a single frame, and a reconstruction of the images was similar to the procedure described previously in the section "MicroPET 18F-nifene studies." The images were analyzed using the ASIPRO VM and PMOD 3.0 software.
Ex vivo autoradiography
The brain after the ex vivo microPET acquisition in the section "Ex vivo microPET" was removed from the dry ice and was rapidly prepared for sectioning. Horizontal sections (40-μm thick) containing brain regions of the thalamus, subiculum, cortex, striatum, hippocampus, and cerebellum were cut using the Leica CM1850 cryotome. The sections were air-dried and exposed to phosphor films overnight. The films were read using the Cyclone Phosphor Imaging System. The regions of interest of the same size were drawn and analyzed on the brain regions rich in α4β2 nicotinic receptors using the OptiQuant software, and the binding of 18F-nifene was measured in digital light units per square millimeter.
MicroPET studies of nicotine challenge
Nicotine challenge experiments were of two types. In order to demonstrate reversibility of bound 18F-nifene and to measure the off-rate, the postinjection nicotine effects were first measured. Sprague-Dawley rats were injected with 18F-nifene (0.2 to 0.5 mCi, IV) and at approximately 30 min postinjection of the 18F-nifene, 0.3 mg/kg of nicotine free base (administered as a ditartarate salt from Sigma Chemical Company, St. Louis, MO, USA) was administered intravenously. The total time of scan was 90 min and was acquired in full list mode, similar to the protocol for the control scans described in "MicroPET 18F-nifene studies." Before and after images were analyzed using the PMOD 3.0 software, and a time-activity curve was generated.
The second set of nicotine challenge experiments were designed to measure α4β2 nAChR receptor occupancy (nAChROCC) by nicotine. Male Sprague-Dawley rats were preinjected intravenously with nicotine using saline for baseline, and four different doses of nicotine (0.02, 0.1, 0.25, and 0.5 mg/kg free base, administered as a ditartarate salt) were diluted in a total volume of 0.3 mL sterile saline. Nicotine was injected 15 min prior to intravenous injection of 18F-nifene (0.8-1.0 mCi). Once anesthetized, the rats were scanned for 90 min using the Inveon microPET scanner in full list mode. Dynamic data were reconstructed and analyzed as described in the section "MicroPET 18F-nifene studies." Time-activity curves were measured and analyzed using the ASIPRO VM and PMOD 3.0 software. Percent occupancy was calculated from: (Thalcont - Thalnic/Thalcont]) × 100, where Thalcont is the percent injected dose of 18F-nifene in the brain regions of the control study, and Thalnic is the percent injected dose of 18F-nifene in the brain regions of the nicotine study at 60 min postinjection of 18F-nifene.