Animals and housing

B6 ob/ob mice (strain name: B6.Cg-Lepob/J) were obtained from the Model Animal Research Center of Nanjing University. Leprflox/flox mice, in which exon 1 of the Lepr gene is floxed, were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) (strain name: B6.129P2-Leprtm1Rck/J). All mice were bred onto the C57BL/6J background. Leprflox/flox mice and their wild-type (WT) littermates were obtained from self-crossing of Leprflox/+ (heterozygous) mice. Mice were housed in groups on a 12 h light/dark cycle with food and water available ad libitum except for in the special case noted below. Genotypes were determined by PCR of mouse tail DNA samples. All animal treatments were strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the School of Basic Medical Sciences, Fudan University. Male mice, 8–10 weeks old, were used for behavioral tests.

Cannula implantation and microinjection

Mice anesthetized with choral hydrate were placed in a mouse stereotactic instrument (Stoelting, Kiel, WI, USA) and implanted with cannula guides (Plastics one, Roanoke, VA, USA) over the lateral ventricles, VTA, or NAc core. The cannulas were secured to the mice with dental cement. The intended stereotaxic coordinates were as follows: lateral ventricles: anterior–posterior (AP) −0.2 mm; medial–lateral (ML) ±1.0 mm; dorsal–ventral (DV) −2.2 mm; VTA: AP −3.2 mm; ML ±0.5 mm; DV −4.4 mm; and NAc core: AP +1.4 mm; ML ±1.2 mm; DV −4.3 mm. All mice were given at least 7 days to recover before behavioral experiments.

SMLA (BioSource, San Diego, CA, USA) mimics the Asp-23 mutation of leptin that exhibits a high affinity for the leptin receptor and antagonistic activity in vitro and in vivo. SMLA was dissolved in artificial cerebrospinal fluid (ACSF) (126 mM NaCl, 26 mM NaHCO 3 , 1.2 mM NaH 2 PO 4 , 3 mM KCl, 2.4 mM CaCl 2 , 1.3 mM MgCl 2 , 10 mM d-glucose) to 250 ng μl−1, and intracerebroventricular (i.c.v.) administration of SMLA through the cannula was performed at a total volume of 2 μl at a slow rate (0.2 μl min−1). Mice received microinjection of SMLA (500 ng) or ACSF as the vehicle (2 μl) 30 min before or after cocaine conditioning. D2-dopamine receptor (D2R) agonist bromocriptine (Tocris Bioscience, Bristol, UK) was dissolved in ACSF to 250 ng μl−1, and mice received a bilateral microinjection of 2 μl bromocriptine solution into the NAc core at a slow rate (0.2 μl min−1) 30 min before cocaine conditioning.

Viral infection

Male adult Leprflox/flox mice and their WT littermates anesthetized with choral hydrate were placed in a mouse stereotactic instrument. Microinjections were performed using custom-made injection needles (33-gauge) connected to a 10 μl Hamilton syringe. Each brain nucleus was injected with 0.5 μl of purified and concentrated AAV2/5 (5 × 1012 IU ml−1) encoding CAG-eGFP-T2A-Cre at a slow injection rate (0.1 μl min−1). The intended stereotaxic coordinates were as follows: NAc core: AP +1.4 mm; ML ±1.2 mm; DV −4.3 mm; VTA: AP −3.2 mm; ML ±0.5 mm; DV −4.4 mm; and CeA: AP −1.3 mm; ML ±2.7 mm; DV −4.6 mm. All mice were given at least 14 days to recover before behavioral experiments, and the virus infections and the knockdown efficiency were checked 14 days after the injection by immunostaining. The histology slides were examined blindly to quantify the expression of LepR in EGFP positive cells.

High-performance liquid chromatography

The levels of monoamine neurotransmitters, including norepinephrine (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT), and their respective metabolites, 3,4-hydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), were detected 30 min after cocaine (15 mg kg−1 i.p.) injection using high-performance liquid chromatography (HPLC) as previously described.21 Within 5 min of the cocaine injection, the NAc and amygdala of mice were dissected in iced phosphate-buffered saline by a vibratome (Microm HM 650V, Thermo Fisher Scientific, Waltham, MA, USA), according to the stereotaxic coordinates listed below: NAc: from Bregma 1.54 to 0.54 mm and amygdala (AMG): from Bregma −1.06 to −2.06 mm. The NAc and AMG were then homogenized in 100 μl ice-cold 0.1 M perchloric acid containing 10 μM ascorbic acid, 0.1 mM EDTA disodium salt and 0.02 μM 3,4-dihydroxybenzylamine. The homogenates were centrifuged at 20 000 g for 10 min at 4 °C, and the supernatants were collected for analysis. An HPLC system with electrochemical detection (UltiMate 3000 system; Thermo Fisher Scientific) was used, and the supernatants were injected onto an Acclaim C18 column (2.2 μm, 2.1 × 100 mm; Thermo Fisher Scientific) at 38 °C. Separations were performed at a flow rate of 0.2 ml min−1 using a mobile phase of phosphate buffer, containing 0.05 mM EDTA, 1.7 mM orthosilicic acid, 90 mM Na 2 HPO 4 , 50 mM citric acid and 5% acetonitrile. The data were collected and analyzed by Chromeleon chromatography workstation (Thermo Fisher Scientific).

Cocaine- or palatable-food-induced CPP

CPP induced by cocaine hydrochloride (Qinghai Pharmaceutical Firm, Qinghai, China) was blindly performed by an investigator using a two-chamber apparatus (Med-Associates, St Albans, VT, USA) with distinct tactile environments to maximize contextual differences. One chamber of the box had a wire mesh floor, while the other chamber had a grid rod floor. A manual guillotine door (7 × 5 cm) separated the two chambers. On Day 1, mice were placed in one of the chambers and allowed to freely explore the entire apparatus for 20 min (pre-test). The mice staying in one chamber for more than 13 min were excluded from the experiment. Mice were randomized into two groups by tossing a coin to receive vehicle or drugs. On days 2, 3 and 4, mice were given an intraperitoneal injection of cocaine (15 mg kg−1, i.p.) in the morning and confined to one of the chambers (drug-paired) for 30 min, and in the afternoon, they received an i.p. injection of saline (an equivalent volume to that of cocaine) and were confined to the other chamber for 30 min (conditioning). The distance the mice traveled within the 30 min was recorded. On Day 5, mice were allowed to freely explore the entire apparatus for 20 min (test). The time spent in each chamber was recorded during the pre-test and test sessions. The CPP score was defined as the time (in seconds) spent in the cocaine-paired chamber minus the time spent in the saline-paired chamber. All mice used in the cocaine-CPP were satiated.

CPP induced by palatable food was assessed using an approach similar to that previously reported.22, 23 All mice were food-restricted for 6 h before being conditioned to chocolate-flavored pellets (20 mg, 5.5% fat, 18.4% protein, 59.1% carbohydrate, 3.6 kcal g−1, #F05301 Bio-serv, Flemington, NJ, USA) or chow food (6% fat, 18% protein, 58% carbohydrate, Xietong Organism, Jiangsu, China). Plates containing chocolate-flavored pellets and chow food were placed in the home-cages at least 1 week before behavior tests for adaptation. A two-chamber apparatus (each chamber was 13 × 26 cm) with distinct tactile environments to maximize contextual differences was used. A manual guillotine door separated the two chambers. One chamber had a white rhombus-patterned wall and black plastic floor, and the other consisted of a black striped wall and white floor with three 3 cm abrasive sticks. On Day 1, mice were placed in one of the chambers and allowed to freely explore the entire apparatus for 20 min (pre-test). On Days 2, 4, 6, 8 and 10, mice were confined to one chamber in the corner of which was placed a plate of chocolate-flavored pellets available ad libitum for 30 min, and on Days 3, 5, 7, 9 and 11, they were confined to the other chamber, which contained a plate of chow food for 30 min. On Day 12, mice were allowed to freely explore the entire apparatus for 20 min (test). The food intake was calculated as the weight of the palatable food or chow food before each session minus the weight recorded after the session. The time spent in each chamber was recorded during the pre-test and test sessions. The CPP score was defined as the time (in seconds) spent in the palatable food-paired chamber minus the time spent in the chow food-paired chamber.

Saccharin operant task

Each operant behavior apparatus (21.6 cm length × 17.8 cm width × 12.7 cm height, Med-Associates) was equipped with a dim light source and a nose-poke hole (3.8 cm in diameter, 0.64 cm deep, 1.0 cm from the grid floor, Med-Associates) equipped with infrared photo-beams connected to a computer. In the center of the wall was a trough situated 2 cm from the grid floor from which liquid (0.1% saccharin (w/v) or water) was delivered when the photo-beam of the nose-poke hole was interrupted for at least 500 ms. A fixed-ratio 1 reinforcement schedule was initially applied for 12 consecutive days; that is, one nose-poke resulted in one delivery of liquid. Mice were divided into three groups by generating a random digit: water, acute saccharin and chronic saccharin, and each group of mice were confined to the apparatus for a 1-h test daily. The water group received a delivery of water after a nose-poke during the 1-h session for 12 days; the mice in the acute saccharin group received a delivery of water after a nose-poke for 11 days and, on Day 12, received a delivery of 0.1% saccharin as a reward after a nose-poke; the chronic saccharin group received a delivery of 0.1% saccharin after a nose-poke for 12 days. Cocaine CPP was blindly performed by an investigator.

Statistical analysis

Seven to twelve mice per group were used for behavior tests, and 4–6 mice per group were used for HPLC and biochemistry studies. All data are presented as the mean±s.e.m. Student’s t-test, two-way analysis of variance (ANOVA), or two-way repeated-measures analysis of variance (RM ANOVA) was used for statistical analysis. Tukey’s post hoc analysis was subsequently performed after a two-way ANOVA or two-way RM ANOVA. *P<0.05, **P<0.01 and ***P<0.001.