PPARγ activation attenuates opioid consumption and modulates mesolimbic dopamine transmission.

Supplemental Information

Supplemental Materials and Methods

Animals

Male Wistar rats (Charles River, Kisslegg, GE) weighing 150-200 g at the beginning of the experiments were used. Pairs of rats were housed in a room with artificial 12:12h light/dark cycle (lights off at 9 AM), at constant temperature (20–22°C) and humidity (45–55%). All training and experimental sessions were conducted during the nocturnal phase of the light/dark cycle.

For the electrophysiological studies we used mice with neuron-specific PPARγ deletion that was achieved by crossing mice with a floxed PPARγ allele [TgH(PPARγ lox)1Mgn, TgH(PPARγ del)2Mgn] with Nestin-Cre mice [B6.Cg-Tg(Nes-cre)1Kln/J] as previously described (Jones et al, 2002). These mice were obtained from the original colony developed at Vanderbilt University School of Medicine (Nashville, TN) and now breed in our laboratory.

Animals were given ad libitum access to food and water throughout except during experimental test sessions. All of the procedures were conducted in adherence with the European Community Council Directive for Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Intravenous and Intracranial Surgeries

Chronic jugular intravenous catheter implantation was conducted as previously described (de Guglielmo et al, 2012; Kallupi et al, 2010). Briefly, animals were anesthetized by intramuscular injection of 100-150 µl of a solution containing tiletamine chloridrate (58.17mg/ml) and zolazepam cloridrate (57.5 mg/ml). For IV surgery, incisions were made to expose the right jugular vein and a catheter made from silicon tubing (I.D. =0.020 inches, O.D. =0.037 inches) was subcutaneously positioned. After insertion into the vein, the proximal end of the catheter was anchored to the muscles underlying the vein with surgical silk. The distal end of the catheter was attached to a stainless-steel cannula bent at a 90° angle. The cannula was inserted in a support made by dental cement on the scull of the animals, fixed with screws and covered with a plastic cap. For one week after surgery, rats were daily treated with 0.2 ml of the antibiotic Sodium Cefotaxime (262 mg/ml). For the duration of the experiments catheters were daily flushed with 0.2-0.3 ml of heparinised saline solution. Body weights were monitored every day and catheter patency was confirmed approximately every 3 days with an injection of 0.2-0.3 ml of thiopental sodium (250 mg/ml) solution. Patency of the catheter was assumed if there was an immediate loss of reflexes. Self-administration experiments began 1 week after surgery.

For the intracranial surgery, the animals underwent stereotaxic surgery in which bilateral cannulae were implanted and aimed at the RMTg or at the VTA. Animals were anesthetized by intramuscular injection of 100-150 µl of a solution containing tiletamine chloridrate (58.17mg/ml) and zolazepam cloridrate (57.5 mg/ml) and placed in a Kopf stereotaxic frame (Kopf Instrument, Tujunga, Calif., USA). Guide cannulae were implanted in the RMTg or in the VTA with the following coordinates (Paxinos and Watson, 1998) with reference to Bregma: RMTg anterior-posterior (AP), −6.8; lateral (L), ±3.0; ventral (V), -7.4; angle 14°; VTA anterior-posterior (AP), −6.0; lateral (L), ±3.0 ; ventral (V), -7.4 ; angle 14°. Cannulae were implanted bilaterally. Drugs were administered through an injector protruding beyond the cannula tip 1.75 mm.

Drugs

Heroin and morphine were purchased by SALARS. (Como, Italy), and dissolved in sterile saline solution (0.9% NaCl). Pioglitazone for OS injections was prepared from Actos (30 mg) tablets, and dissolved in distilled water. Pioglitazone for intracranial injections was purchased from Molcan Corporation (Ontario, Canada) and dissolved in a vehicle made with 20% DMSO and 80% distilled water. GW 9662, saccharine was purchased by Sigma-Aldrich (Milano, Italy) and dissolved in a vehicle made with 5% DMSO, 5% Tween 80 and 90% distilled water. CNQX, strychnine and eticlopride were purchased from Sigma-Aldrich (Italy), whereas AP-5 was purchased from Tocris (UK). Drugs were applied in known concentrations to the superfusion medium. CNQX and AP5 were dissolved in distilled water, whereas all the other drugs were dissolved in DMSO. The final concentration of DMSO was < 0.01%.

Operant Training

The self-administration stations consisted of operant conditioning chambers (Med Associates, Inc.) enclosed in lit, sound attenuating, ventilated environmental cubicles. Each chamber was equipped with two retractable levers located in the front panel. Heroin was delivered through a plastic tube that was connected to the catheter before the beginning of the session. An infusion pump was activated by responses on the right or “active” lever, while responses on the left or “inactive” lever were recorded but did not result in any programmed consequences. Activation of the pump resulted in a delivery of 0.1 ml of fluid. An IBM compatible computer controlled the delivery of fluids and the recording of the behavioral data. For the saccharine self-administration study, each chamber was equipped with a drinking reservoir positioned 4 cm above the grid floor in the center of the front panel of the chamber.

Immunohistochemistry

Brain tissue processing

In brief, rats were transcardially perfused, first with 100 ml normal saline followed immediately with 4% paraformaldehyde (PFA) in 1X PBS. Brains were removed and post-fixed two hours in 4% PFA followed by dehydration in 30% sucrose in 1xPBS for 48 hours, snap frozen in powdered dry ice, and stored at -80°C until further use. Coronal sections of the mPFC, NAcC and NACsh (30um, mPFC - Bregma: +2.70 to +3.70; NAcC and NAcSh – Bregma: +0.70 to 0.70 mm) were taken by a cryostat (Leica CM3050), collected in cryoprotectant solution and stored at -20 °C.

Immunohistochemistry

pDARPP-32 staining was adapted from a previous published studies (Randall et al, 2012; Segovia et al, 2012). In brief, free-floating sections were washed with 0.01 M PBS (pH 7.4) and then incubated in 0.3% H2O2 for 30 min. On rotating platform prior to primary antibody, non-specific binding sites were blocked in 0.01 M PBS containing 1% of bovine serum albumin (Sigma, MO, USA) and 0.1% Triton X-100 for 30 minutes. Sections were subsequently incubated with a primary rabbit anti-pDARPP32(Thr34) (1:1000, sc-21601-R, Santa Cruz Biotechnology, CA, USA) in 0.01 M PBS containing 1% BSA/PBS for 48 hours on a shaker at 4°C. Negative controls were performed using BSA without primary antibody. The tissue was then washed with 0.01 PBS (3x for 15 min) and incubated with a secondary anti-rabbit HRP-conjugated envision plus (DAKO, Carpinteria, CA, USA) for 2 hours on a shaker at room temperature. The immunohistochemical reaction was developed using diaminobenzidine (DAB) as the chromagen (Sigma, MO, USA) for 3 min. The tissue was then rinsed in 0.01 PBS, mounted on gelatine-coated slides and processed the next day through alcohol-xylene for light microscopy examination. Some of the sections were stained with cresyl violet in order to verify basic histology.

Quantification of pDARPP-32 immunoreactivity.

Pictures were taken on a Leica light microscope DM6000CS (Leica Microsystem Inc., Bannockbern, IL, USA) at 40X magnification and BioQuant imaging software (R&M Biometrics, Nashville, TN) was used for cell density analysis. Two coronal sections (two levels) containing the PFC and NAc, both left and right side, were analyzed for pDARPP-32 positive cells (coordinates: mPFC - Bregma: +2.70 to +3.70; NAcC and NAcSh – Bregma: +0.70 to 0.70 mm (Paxinos and Watson, 2005). Results are depicted as the positively stained cells per square mm.

Peroxidase immunohistochemistry

Immunohistochemistry was performed on free-floating coronal brain sections according to standard procedures. In brief, sections were reacted with 0.3% H2O2 (in PBS; 30 min) to block endogenous peroxidase, rinsed with PBS and incubated in a 3% blocking solution with 0.2% of Triton X100 (in PBS; 60 min). Then, they were incubated with the primary antibody against PPARγ (rabbit monoclonal from Cell Signaling Technology Inc., Beverly, MA, USA, cat # 2435, dilution 1:1000) (in PBS, overnight at 4°C). After a thorough rinse in PBS, sections were incubated in a 1:200 v/v solution of biotinylated anti-rabbit IgG secondary antibody (Vector, Burlingame, CA) (in PBS, for 30 min), rinsed in PBS and incubated in avidin-biotin peroxidase complex (ABC Elite PK6100, Vector), washed several times in PBS and incubated in 3,3 diaminobenzidine tetrahydrochloride (0.05% in 0.05 M Tris with 0.03% H2O2; 5 min). After immunohistochemical staining, the sections were mounted on slides, air-dried, dehydrated, cleared with xylene and covered with Entellan. Staining was never observed when the primary antibody was omitted and in PPARγ knock out mice.

Immunofluorescence and confocal microscopy

For double-labeling experiments, free-floating sections were processed as described above until the primary antibody incubation step. Then, they were incubated overnight in a mixture of two or three primary antibodies: anti-PPARγ antibody (dilution 1:800); anti-NeuN monoclonal mouse antibody (Chemicon, Temecula, CA, USA, cat # MAB377, dilution 1:100); anti-HuC/D monoclonal mouse antibody (Molecular Probes, Eugene, OR, USA, cat # A21271, dilution 1:50); anti-glial fibrillary acidic protein (GFAP) monoclonal mouse antibody (Sigma, St Louis, MO, USA, cat # G3893, dilution 1:1000); anti-APC monoclonal mouse antibody (Calbiochem, La Jolla, CA, USA, cat # OP80, dilution 1:400); anti-tyrosine hydroxylase (TH) polyclonal sheep antibody (Chemicon, cat # AB1542, dilution 1:500); anti-GAD67 monoclonal mouse (Chemicon, cat # MAB5406, dilution 1:800). On the second day, sections were washed twice with PB and incubated in a cocktail of fluorophore-linked secondary antibodies (DyLightTM549-anti-rabbit, DyLightTM488-anti-mouse, DyLightTM649-anti-sheep; Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:100 v/v in PB for 1 h at room temperature. They were subsequently washed twice with PB, mounted on standard glass slides, air-dried and coverslipped using Vectashield mounting medium (Vector). Sections were viewed under a motorized Leica DM6000 microscope at various magnifications. Fluorescence was detected with a Leica TCS-SL confocal microscope equipped with Argon and He/Ne mixed gas lasers. Fluorophores were excited and imaged separately. Images (1024 x 1024 pixels) were obtained sequentially from two channels using a confocal pinhole of 1.1200 and stored as TIFF files. Brightness and contrast of the final images were adjusted using Photoshop 6 software (Adobe Systems; Mountain View, CA, USA).

Microdialysis

Probe preparation. Concentric dialysis probes with a dialysing portion of 1.5 mm were prepared with AN69 (acrylonitrile sodium methallyl sulfonate copolymer) dialysis fiber (310 µm o.d. 220 µm i.d. Hospal, Dasco, Italy), a previously reported (De Luca et al, 2007).

Implant of microdialysis probe. Rats were anaesthetized with chloral hydrate (400 mg/kg i.p.) and placed in a stereotaxic apparatus. The scalps were incised and the skull leveled between lambda and bregma. A two small holes were drilled to expose the dura and two probes were inserted vertically at the level of the NAc shell and core or NAc shell according to the atlas by Paxinos and Watson (1998) (Paxinos et al, 1998) (coordinates, shell: A: 2.2, L: 1.0 from bregma, V: -7.8 from dura; core: A, 1.4; L,1.6 from bregma; V −7.6 from dura;). Probes were fixed to the skull with glasionomeric cement (CX-Plus, Shofu Inc., Japan). Experiments were performed on freely moving rats 24 hours after probe implant. A Ringer's solution (147 mM NaCl, 4 mM KCl, 2.2 mM CaCl2) were pumped through the dialysis probe at a constant rate of 1 µl/min. Samples from the NAc shell and core were taken every 10 min and analyzed.

Analytical procedure. Dialysate samples (10 µl) were injected into an HPLC equipped with a reverse phase column (C8 3.5 um, Waters, Mildford, MA, USA) and a coulometric detector (ESA, Coulochem II, Bedford, MA) to quantify DA. The first electrode was set at +125 mV and the second electrode at -175 mV. The following solution were utilized as mobile phase: 50 mM NaH2PO4, 0.1 mM Na2-EDTA, 0.5 mM n-octyl sodium sulfate, 15% (v/v) methanol, pH 5.5. The mobile phase were pumped with a Jasco pump with a flow rate of 0.6 ml/min. The sensitivity of the assay for DA were 5 fmol per sample.

Histology. At the end of the experiment, animals were transcardially perfused with 100 ml of saline (0.9% NaCl) and 100 ml of a formaldehyde (10%). The probes were removed and the brains cut on a vibratome in serial coronal slices to locate the position of the microdialysis probes in the shell and core of NAc Only animals with correct probe placement were used for statistical purposes.

Electrophysiology

Whole cell patch-clamp recordings ex vivo. The preparation of the ventral tegmental area (VTA) slices was performed as described elsewhere (Melis et al, 2010). Briefly, male Wistar rats (10-18 d) and PPARg -/- and PPARg +/+ mice (30-40 d) were anesthetized with halothane in a vapor chamber and euthanized by decapitation. A block of tissue containing the midbrain was rapidly dissected and sliced in the horizontal plane (300 and 280 µm for rats and mice respectively) with a vibratome (Leica VT1000S) in ice-cold low-Ca2+ solution containing (in mM): 126 NaCl, 1.6 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 0.625 CaCl2, 18 NaHCO3, and 11 glucose. Slices were transferred to a holding chamber with artificial cerebrospinal fluid (ACSF, 37° C) saturated with 95% O2 and 5% CO2 containing (in mM): 126 NaCl, 1.6 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 18 NaHCO3, and 11 glucose. They were allowed to recover for at least 1 hr before being placed in the recording chamber and superfused with the ACSF (37° C) saturated with 95% O2 and 5% CO2. Neurons were visualized with an upright microscope with infrared illumination (Axioskop FS 2 plus, Zeiss), and whole-cell patch-clamp recordings were performed by using an Axopatch 200B amplifier (Molecular Devices, CA). Current-clamp recordings were made with electrodes filled with a solution containing the following (in mM): 117 KCl 144, 10 HEPES, BAPTA 3.45, CaCl 1, 2.5 Mg2ATP, and 0.25 Mg2GTP (pH 7.2-7.4, 275-285 mOsm). Experiments began only after series resistance was stabilized (typically 15-40MW). Data were filtered at 2 kHz, digitized at 10 kHz, and collected on-line with acquisition software (pClamp 8.2, Molecular Devices, CA). DA neurons were identified according to the already published criteria(Melis et al, 2013): cell morphology and anatomical location (i.e., medial to the medial terminal nucleus of the accessoryoptic tract), large hyperpolarization activated current (Ih100 pA), slow pacemaker-like firing rate (5 Hz), and long action potential duration (> 2ms). Each slice received only a single drug exposure. Action potential frequency was analyzed off-line with MiniAnalysis software (Synaptosoft). The averaged action potential frequency within the first minute immediately before drug administration was taken as baseline, and the averaged frequency for the 1 min period centered on the peak response was taken for drug effect. Each slice received only one single drug exposure. All GABAA IPSCs were recorded in the presence of 2-amino-5-phosphonopentanoic acid (100 mM), 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (10 mM), strychnine (1 mM), and eticlopride (100 nM) to block N-methyl-D-aspartate, a-amino-3-hydroxy-5-methyl-isoxazolepropionic acid, glycine, and DA D2-mediated synaptic currents, respectively. As already described (Bonci and Williams, 1997), there was no effect of this solution on the holding current of the DA cells.