Animals and Radiopharmaceuticals
Male Sprague Dawley (SD) rats were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and housed in an animal housing facility with free access to food and drinking water. 18F-FDG and 11C-CFT was prepared at the PET Center, Huashan Hospital, Fudan University according previous report and under GMP condition . All small animal experiments were conducted according to a protocol approved by Fudan University, Shanghai, China.
Conditioned place preference (CPP) apparatus
The CPP apparatus consisted of two large compartments (30 cm × 30 cm × 40 cm) separated by a small compartment (10 cm × 30 cm × 40 cm). The two large compartments were distinguishable by distinct visual and tactile cues: one compartment had a white wall and a floor with many quadrate holes, and the other had a black wall and a floor with many hollow longitudinal lines. The middle section contained a transitional compartment with a grey wall and a solid grey polyvinyl chloride floor. Between the three compartments were two guillotine doors (8 cm × 8 cm) patterned to match the outer compartments, which were closed on the conditioning days and opened on the adaptation and testing days. In this manner, the rats were able to identify the black and white compartments not only by visual but also touch perception. Infrared cameras in each compartment were used to record time spent in each of the three compartments during CPP testing.
The CPP procedure was similar to that described previously . Behavioral subjects were individually habituated to the investigator by handling for 5 min and placed in the experimental environment for adaptation for 3 days before the CPP session. A standard CPP protocol consisted of four consecutive phases: adaptation, pre-test, conditioning, and post-test phases. During the adaptation phase, the rats were put into the CPP apparatus for 30 min to allow them to adapt to the testing environment. During this phase, the doors were always open so that the rats could freely access all three compartments. During the pre-test phase, the rats were placed in the CPP apparatus for 15 min, and the time that the rats spent in each compartment was recorded. The rats were removed when the time spent in any of the three compartments exceeded 540 s. For all experimental rats, the large compartment that was occupied for the shorter time was designated as the drug-paired compartment, and the other large compartment was designated as the drug-unpaired compartment. Then, the conditioning sessions were conducted: during this phase, each group received alternating injections of either saline or morphine every day at 6-h intervals. Briefly, for the CPP group, during the morning of the first training day (at 08:00), the rats were intraperitoneally injected with morphine (20 mg/kg) and immediately confined to the drug-paired compartment. After the rats stayed quietly in the drug-paired compartment for 45 min with the doors closed, they were returned to their feeding cage. Similarly, 6 h later, in the afternoon (at 14:00), the rats were confined to the drug-unpaired compartment for 45 min after the injection of saline (2 mL/kg, i.p.). On the next day, the procedure was performed in reverse order in the morning and the afternoon. Additionally, the rats in the the addiction group received only morphine or saline injection but without conditioning in CPP apparatus. After the final conditioning session, the post-test phase was carried out 24 h later. For the post-test, the doors were raised, and the rats were permitted to access the entire apparatus for 15 min. CPP score, defined as the time spent in the drug-unpaired compartment minus the time spent in the drug-paired compartment, was calculated. From our perspective, a post-test CPP score ＜ 0 indicated successful establishment of the CPP model.
Micro-PET/CT imaging and Analysis
Micro-PET/CT imaging in these rats was conducted at the designed time point according to a method described previously [24, 21]. Briefly, the rats were anesthetized with 2% isoflurane in 100% oxygen (1 L/min) at room temperature using an isoflurane vaporizer (Molecular Imaging Products Company, USA). The rats were positioned in a spread-supine position on the imaging bed and subjected to 2% isoflurane in 100% oxygen (1 L/min) via inhalation during the PET/CT procedure. After the intravenous administration of 18F-FDG (~10 μCi/g body weight) and 11C-CFT (~10 μCi/g body weight), rats were allowed to move freely in 10 min, then these rats were permitted to access the CPP apparatus for 15 min and Static PET/CT imaging was obtained for 20 min at 60 min post-injection of 18F-FDG or 20 min at 40 min post-injection of 11C-CFT. PET/CT images were reconstructed using the ordered subsets expectation maximization 3D algorithm (OSEM3D), and the data were reviewed and processed using the IRW. Image processing was performed in PMOD software (version 3.4, PMOD Technologies Ltd., Zurich, Switzerland). Imaging was manually fused with the PMOD rat template, and 58 brain regions of interest (ROIs) were collected (Supplementary Fig. 1). 18F-FDG and 11C-CFT uptakes were quantified as the SUV, and the SUV ratio (SUVR) was calculated by comparing the SUV uptake in the ROIs to that of the cerebellum.
Voxel-wise analysis was performed using spmratIHEP  based on Statistical parametric mapping 8 (SPM) software (Wellcome Department of Cognitive Neurology, Institute of Neurology, London, UK). Firstly, the images were spatially normalized to the standard FDG-PET template in Paxinos & Watson space; then a Gaussian kernel of [2 2 4] in full width at half maximum was used to smooth the data for voxel-wise analysis. After that, two sample t test was performed to compare the group difference between addiction rats with CPP and baseline. The statistical level was set at P < 0.001 with uncorrected multiple comparisons and a minimum cluster extent ke ≥20 voxels.
A longitudinal, sequential PET/CT study was performed to assess the glucose metabolism and DAT change in rat brains as shown in Fig. 1. All rats conducted CPP pre-test and the results were recorded. 18F-FDG and 11C-CFT imaging were performed at the following 2 days after pre-test. After PET imaging, chronic morphine or saline treatment was added on these rats at the following 5 days. At the same time, one group of rats had CPP procedure as CPP group, and the other rats only had chronic morphine treatment without CPP procedure as additive group. 18F-FDG and 11C-CFT imaging were performed one day after these treatments as short term addiction and short term CPP, also as cue-induced craving. All of these rats were housed with food and water available ad libitum without any training or drug administration in the next 14 days. post-test procedure was conducted and the results were recorded, which is to reflect environmental cue-induced retrieval of withdrawal memory. And then, 18F-FDG and 11C-CFT imaging were performed as same at last time as long term addiction and long term CPP, long term CPP group also reflected memory of retrieval.
Statistical analyse was performed using Prism7 (GraphPad Software, CA, USA) software. All data are presented as mean ± standard deviation (SD). To determine whether the difference of the between the CPP score in these 2 group of rats, we applied two-way analysis of variance (ANOVA). For data of SUV and the SUVr in the ROIs in these rats differed among different groups, we applied two-way ANOVA, followed by Bonferroni's corrections for multiple comparisons test. A P value of less than 0.05 was considered statistically significant.