Male Long-Evans rats (n = 5–8/group) (Harlan, IN) weighing 250–300 g were housed individually in a vivarium with a 12:12 light/dark schedule and maintained at 25° C. All animals had ad libitum access to food and water throughout the study.
Rats receiving bilateral leptin injections were maintained on chow (Teklad, 3.41 kcal/g, 0.51 kcal/g from fat). Animals receiving viral injections had ad libitum access to one of two diets: chow (Chow) or high-fat diet (HFD) (Research Diets, New Brunswick, NJ, 4.41 kcal/g, 1.71 kcal/g from fat).
Operant procedures were conducted in identical conditioning chambers constructed of aluminum walls and Plexiglas sides measuring 21.6 × 21.6 × 27.9 cm. A grid of 0.48-cm diameter stainless steel bars, spaced 1.9 cm apart, served as the floor of each chamber. A food cup was located on one wall of each chamber inside a 5 × 5-cm recessed opening. Two levers were located approximately 3 cm to the left and right of the food cup. Only the right lever was active during this experiment. All experimental events were controlled and recorded by computers running ABET software (Lafayette Instruments; Lafayette, IN).
In both the LH and midbrain studies, four groups of rats (n = 8/group) were injected with scrambled control virus or LepR virus (LepR) and maintained on either Chow or HF diet for 4 weeks prior to the beginning of the operant training. All training was conducted in the dark and the animals were not food restricted during any phase of operant training. The reinforcer was a single 45-mg sucrose pellet (TestDiet, Richmond, IN). Operant training was carried out over 8 consecutive days with one 1-h trial per day. During the first two days of training, a fixed ratio (FR) 1 autoshaping procedure was employed, in which each lever press earned a single reinforcer. In addition, whenever 600 sec elapsed with no reinforcer delivery, a “free” sucrose pellet was dispensed into the food cup. All animals were then trained for 2 days using an FR1 schedule with no autoshaping component, followed by 2 days of FR3 training. At the conclusion of the 6-day operant training regimen, animals were given a single trial to lever press for sucrose under an incremental progressive ratio schedule of reinforcement where the lever press requirement for each subsequent reinforcer increased incrementally. The response requirements of the PR schedule increased through the following series: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 693, 737, 901. The breakpoint for each animal was defined as the final completed requirement that preceded a 20-min period without responding.
Design and Construction of shRNA
We used shRNA sequences identical to those described by (Hommel et al., 2006). Lentivirus vector construction and packaging were performed by America Pharma Source, LLC (Rockville, MD). Briefly, scrambled (SCR) shRNA (Top, 5′-GATCCAGCACCATTTCCGCTTCAATATTCAAGAGATATTGAAGCGGAAATGGTGCTTTTTGG-3′-Bottom, 5′ AATTCCAAAAAGCACCATTTCCGCTTCAATATCTCTTGAATATTGAAGCGGAAATGGTGCTG-3′) or shRNA directed against the rat leptin receptor (LepR) (Top, 5′-GATCCAGAAATCTTTAAATTACCATCATCTTTCGAGATGATGGTAATTTAAAGATTTATTTTTGG -3′ Bottom, 5′-AATTCCAAAAATAAATCTTTAAATTACCATCATCTCGAAAGATGATGGTAATTTAAAGATTTCTG-3′) were used. Both strands of oligonucleotides including linker and restriction sites were synthesized by Invitrogen. After annealing, the double strand shRNA DNA insert was ligated into lentivector (pHR-U6-EF-GFP) at BamHI and EcoRI sites directly, followed by DNA sequencing confirmation. GFP was used as selection marker driven by EF-1α promoter. Lentiviruses were packaged by transfecting 293T cells, with vsv-g as envelope protein. The titers of the packaged virus for further experiments were in the range of 2–5 × 109 IU/mL. Cells were infected with the prepared lentivirus according to the protocol provided by America Pharma Source. Following in-vitro confirmation of gene knockdown, the virus was concentrated to approximately 1–2 × 109 infectious particles/ml.
Rats were sacrificed via CO2 asphyxiation, and brains were rapidly removed, frozen and stored at −80° C until processing. The LH and midbrain from each animal were microdissected using a AHP-1200CPV freezing plane (Thermoelectric Cooling America, Chicago, Il) which maintained a constant temperature of 12° C throughout the dissection process. High-quality mRNA was isolated by Trizol (Invitrogen, Carlsbad, CA) and chloroform (Sigma, St. Louis, MO) extraction. Complementary DNA was synthesized from 300 ng of mRNA by oligo DT priming (Invitrogen, Carlsbad, CA). cDNA were amplified in triplicate using 8 pg of each specific primer with quantification of the product by SYBR green fluorescence (Applied Biosystems, Foster City, CA). Leptin receptor was normalized to L32 control gene expression. All dissected tissue was further normalized using the viral reporter gene EGFP. EGFP mRNA expression was used as an internal positive control for both LH and midbrain infected tissue. Animals that did not express EGFP in the LH or midbrain were excluded from any further analysis.
The following primer sequences (IDT, San Diego, CA) were used to probe for rat leptin receptor, L32 and GFP, respectively: Leptin: 5′ - AAT TGG AGC AGT CCA GCC TA - 3′, 5′ - TTT CCC ACA TCT TGT GAC CA - 3′ and L32: 5′ - CAG ACG CAC CAT CGA AGT TA - 3′, 5′ - AGC CAC AAA GGA CGT GTT TC - 3′, GFP: 5′ -GAC GTA AAC GGC CAC AAG TT- 3′, 5′ –AAG TCG TGC TGC TTC ATG TG- 3′.
Following viral delivery into the LH or midbrain animals were injected with an overdose of pentobarbital and perfused transcardially with ice cold saline for 1 min followed by 4% paraformaldehyde in 1× PBS for 20 min. Cryprotected brains were frozen and sectioned at 35 mm intervals. A full series of sections was double-labeled for pStat3 and GFP immunohistoreactivity. The sections were incubated in the following solutions with the appropriate washes between solutions: 1% sodium hydroxide + 1% hydrogen peroxide/PBS, 0.3% glycine/PBS; 0.03% sodium dodecyl sulfate/PBS; Sections were then blocked by incubation in 4% horse serum with 0.4% Triton X-100. Rabbit anti-pStat3 (Cell Signalling Cat # 9145S) antibody diluted 1:100 in 4% horse serum with 0.4% Triton X-100 was applied to the sections for overnight incubation. The secondary antibody, biotinylated goat anti-rabbit IgG (1:250; Vector Laboratories; Burlingame, CA), was applied followed by incubation in avidin-biotin complex (1:500; Vectastain ABC; Vector Laboratories; Burlingame, CA) and an incubation in biotinyl tyramide signal amplification solution (1:250; PerkinElmer; Boston, MA). Cyanine 3 Strepavidin (Cy3; 1:200; Jackson Immuno Research; West Grove, PA) was then applied to the sections. The sections were then washed with PBS and incubated in 4% goat serum + 0.4% Triton X-100 blocking solution followed by rabbit anti GFP (Invitrogen Cat# A11122), formulated in the same blocking solution, overnight at RT followed by incubation in Alexa-488 goat anti-mouse IgG diluted (1:200; Invitrogen, Carlsbad CA, catalog # A11001). Slides were cover slipped with gelvatol mounting media containing DABCO antifade agent.
Bilateral micropunches from the nucleus accumbens (NAcc) were dissected from each animal (Scrambled-Chow, Scrambled-HFD, LepR-Chow, LepR-HFD) under basal conditions. For high-performance liquid chromatography (HPLC) analysis, an antioxidant solution (0.4 N perchlorate, 1.343 mM ethylenediaminetetraacetic acid (EDTA) and 0.526 mM sodium metabisulfite) was added to the samples followed by homogenization using an ultrasonic tissue homogenizer (Biologics, Gainesville, VA). A small portion of the tissue homogenate was dissolved in 2% sodium dodecyl sulfate (SDS) (w/v) for protein determination (Pierce BCA Protein Reagent Kit, Rockford, IL). The remaining suspension was spun at 14,000g for 20 min in a refrigerated centrifuge. The supernatant was reserved for HPLC. Samples were separated on a Microsorb MV C-18 column (5 Am, 4.6×250 mm, Varian, Walnut Creek, CA) and simultaneously examined for DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), the latter two being markers of dopamine degradation, and for 5-HT and 5-HIAA to assess serotonin activity. Compounds were detected using a 12-channel coulometric array detector (CoulArray 5200, ESA, Chelmsford, MA) attached to a Waters 2695 Solvent Delivery System (Waters, Milford, MA) under the following conditions: flow rate of 1 ml/min; detection potentials of 50, 175, 350, 400 and 525 mV, and scrubbing potential of 650 mV. The mobile phase consisted of a 10% methanol solution in distilled H2O containing 21 g/l (0.1 M) citric acid, 10.65g/l (0.075 M) Na2HPO4, 176 mg/l (0.8 M) heptanesulfonic acid and 36 mg/l (0.097 mM) EDTA at a pH of 4.1. Experimental samples were quantified against a 6-point standard curve with a minimum R2 of 0.97. Quality control samples were interspersed with each run to ensure HPLC calibration.
Midbrain Cannulation Surgery
Rats (n = 5/group) were allowed to acclimate for 1–2 weeks before surgery. Under deep anesthesia, guide cannulae (Plastics One, Roanoke, Virginia) were implanted bilaterally in the midbrain. Coordinates for placement of the guide cannula in the midbrain were based on (Hommel et al., 2006) as follows: 5.7 mm posterior to bregma, 0.75 mm lateral from the midline and 7.8 mm ventral from the surface of the skull with lambda and bregma at the same vertical coordinate. Holes were drilled into the skull and self-tapping stainless steel screws were inserted. After placement, the 26-gauge guides were attached to the skull using cranioplastic cement (Plastics One). Once the surgery was complete sterile obturators (33gauge; 0.8 mm longer than the guide cannula) were inserted into the guides to reduce the potential for brain infection. For LH viral delivery, Hamilton syringe needles were targeted to 2.3 mm posterior from bregma, 2.0 mm lateral from the midline and 8.2 mm ventral to the skull surface. For midbrain viral delivery, syringe needles were targeted to 5.6 mm posterior from bregma, 2.2 mm lateral from the midline and 8.6 mm ventral from the surface of the skull. A total of 2.0 (LH) or 1.0 μl (midbrain) of purified virus (1–2 × 109 infectious particles/ml) was delivered per side over a 5-min period.
Leptin Microinjection Procedure
After surgery, the rats were allowed to recover for two weeks before operant training began. Once PR was stable, injectors were inserted into the guides and the rats were placed into an open cage and allowed to move freely during the infusion. The infusion pumps were operated for 4 min, delivering 125 ng of leptin in 0.0125 μl or an equivalent volume of vehicle per min, per side. Leptin (R&D Systems, Torrance, CA) was dissolved according to manufacturer’s protocol. The injectors were left in the brain for an additional minute after the end of the injection before being removed and replaced with obturators.
Determination of Cannula Placement
At the completion of the infusion experiments, animals were injected with leptin (500 ng/side) directly into the VTA and sacrificed 45 min later via transcardial infusion of 4% paraformaldehyde. Brain sections were then analyzed by histology for phospho-STAT3 (p-STAT3) staining to evaluate the exact placement relative to tyrosine hydroxylase (TH) staining, the rate limiting enzyme regulating dopamine production. Brains from rats injected with leptin that did not display p-STAT3 staining within the midbrain region: bregma-5.0 through bregma-6.0 were not included in further analyses; this resulted in the rejection of two animals from the behavioral studies.
Data were analyzed using STATISTICA version 6.0 for PCs. Operant responding, leptin receptor expression and mesolimbic dopamine levels were analyzed using analysis of variance (ANOVA). A least significant differences (LSD) post-hoc comparison was used to assess the source of significant main effects and interactions.