Supplemental Material and Methods Animals

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Supplemental Material and Methods


Experiments were performed on male Sprague Dawley rats (Janvier, Le Genest-Saint-Isle, France) weighing 200 g (6 weeks old) at the time of surgery. Animals were housed four per cage until the second week after surgery, and then transferred to individual cages until the end of the study, under standard laboratory conditions (12 h light/dark cycle, with lights on at 7 a.m.), with food and water supplied ad libitum. Protocols used were in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC) for the care of laboratory animals, French Ministry of Agriculture regulations (authorization no. 38-09-17) and French guidelines for the use of living animals in scientific investigations, and were approved by the Grenoble Institut des Neurosciences ethics committee, under agreement number 129.

Drugs and reagents

Sumanirole maleate was generously provided by Pfizer Inc. SKF-38393, SKF-82958 (chloro-APB hydrobromide), desipramine hydrochloride, and 6-OHDA hydrobromide were purchased from Sigma (St Quentin-Fallavier, France), while PD-128907 and L-741,626 from Tocris (Bioscience, Ellisville, Missouri) and SB-277011A from Santa Cruz Biotechnology (Heidelberg, Germany). Mouse monoclonal anti-TH antibody was purchased from Chemicon (Temecula, USA) and biotinylated goat anti-mouse IgG antibody BA-9200, avidin-peroxidase conjugate Vectastain ABC Elite and Peroxidase substrate detection kits were purchased from Vector Laboratories, (Burlingame, CA, USA).

Bilateral 6-hydroxydopamine (6-OHDA) lesions

As previously described1, rats were anesthetized with a mixture of xylazine (15 mg/kg i.p.) and ketamine (100 mg/kg, i.p.) and treated with desipramine hydrochloride (25 mg/kg s.c.; Sigma, St Quentin-Fallavier, France) to protect noradrenergic neurons2, 30 min before 6-OHDA injection. Rats were secured in a Kopf stereotaxic apparatus (Phymep, Paris, France) and 6 µg of 6-OHDA dissolved in 2.3 µl of sterile 0.9% NaCl or 2.3 µl of sterile 0.9% NaCl (sham conditions), were injected bilaterally, at a flow rate of 0.5 µl/min. at the following coordinates relative to bregma3: AP, -5.4 mm; ML, ±1.8 mm and DV, -8.1 mm. After each injection, the cannula was left in position for 5 min to allow the injected solution to be absorbed and to minimize the spread of the toxin along the needle tract. After recovery from anesthesia, animals were returned to the animal facility for three weeks, to allow the 6-OHDA lesion to develop and stabilize2, before the beginning of the behavioral experiments. A subset of animals (around 20%) received supplementation with a high-caloric liquid diet and palatable food for 1 to 2 weeks, as transient starvation states may occur during the development of the DA SNc lesions1.

TH-immunohistochemistry and quantification of striatal DA denervation

Immunohistochemical analysis was carried out as previously described1,4. Rats were killed under chloral hydrate anesthesia at the end of the behavioral experiments, perfused intracardially with paraformaldehyde and brains removed. Free-floating 30 µm-thick coronal sections from the mesencephalon and the striatum were incubated with an anti-TH antibody, and then with a biotinylated goat anti-mouse IgG antibody. Immunoreactivity was visualized with avidin-peroxidase conjugate.

TH immunoreactivity (TH-IR) was visualized and optical density was measured with the ICS FrameWork computerized image analysis system (TRIBVN, 2.9.2 version, Châtillon, France) coupled to a light microscope (Nikon, Eclipse 80i) and a Pike F-421C camera (ALLIED Vision Technologies, Stadtroda, Germany) for digitalization, in the VTA, the SNc (-5.2 to -5.6 from bregma), the dorsal striatum and the NAcc (+1.6 to +1.0 from bregma), taking into account the topography of DA innervation5. Masks from these different striatal subregions at a section localized around +1.2 mm anterior to bregma were drawn with the computer analysis system to ensure that appropriate comparisons were made between homologous anatomical regions. Optical densities (OD) were measured for each striatal and mesencephalic subregion, and the mean OD was calculated with ICS FrameWork software (TRIBVN, 2.9.2 version, Châtillon, France). OD were expressed as percentages relative to the mean optical density values obtained for the homologous regions of sham-operated animals. The OD value obtained for an unlabeled area (the corpus callosum) was used as the background and was subtracted from each of the OD values measured.

Only animals with a reduction of TH-IR less than 30% in the NAc were included in the analysis. Bilateral loss of TH-IR within the dorsal striatum was comprised between 50 % and 85%.

Behavioral procedures

Operant sucrose self-administration. Rats were first habituated to voluntary consume a 2% sucrose solution in their home cage, under free-access conditions over a period of six days to avoid neophobia. Rats were then trained to self-administer the 2% sucrose solution in operant chambers (Med Associates, St. Albans, VT, USA), as previously described1,6, under a fixed ratio 1 schedule of reinforcement (FR1), with an active, reinforced, lever, for which presses resulted in the delivery of 0.2 ml of the sucrose solution, and an inactive, non-reinforced, lever. Once their performance had stabilized (less than 20% performance variation over three consecutive sessions), rats were subjected to a progressive ratio schedule session (PR), in which the number of active lever presses required for a reward increased exponentially after each reward, as described in1. The session ended when the rat failed to complete a response requirement (i.e., a ratio) within one hour. The breakpoint was defined as the final ratio completed by the animal 1.

Elevated plus-maze (EPM). The EPM (Viewpoint S.A., Champagne au Mont d'Or, France) consisted of two opposing open arms and two opposing arms enclosed by 40 cm-high walls, placed in a dimly lit room. Each arm was 50 cm long and 10 cm wide and made of black Perspex™, suspended 55 cm above the floor. The rats were placed in the center of the maze and their behavior was recorded for 5 minutes with a video-tracking system (Viewpoint S.A., Lyon, France). The total time spent in the open and closed arms, the central area and the end of the open arms was quantified by the video-tracking system. Head dippings in and stretched attend postures toward non-protected areas were scored by two experimenters blind to the experimental conditions. A non-protected head dip was defined as an exploratory movement of head/shoulders over sides of the arms7, when the animal has its four paws in an open arm. Head dips were counted in the proximal and distal area of the open arms, but were eventually pooled as the data obtained in these two parts were similar (data not shown). Stretched attend postures were defined as exploratory postures in the front of an open arm, in which the rat stretches forward and retracts to original position without locomoting forward7. Stretched attend postures toward the closed arms were also scored but the frequency of this discrete behavior was very low and unaffected by the different treatments (data not shown).

Forced swim test (FST). Rats were placed in a cylinder 40 cm high and 20 cm in diameter, filled with water (24±1°C) to a depth of 30 cm, for 10 min. The rats were then dried, returned to their home cages and warmed under an infrared light for 15 to 30 min. Animal activity in the cylinder was detected and recorded with a video-tracking system (Viewpoint S.A., Lyon, France).

Locomotor activity. Rats were placed in a dimly lit white Perspex™ open arena (50 × 50 × 40 cm) and horizontal distances traveled were recorded with a video-tracking system (Viewpoint S.A., Champagne au Mont d'Or, France), over a 1 h period.

Data and statistical analysis

All data are expressed as mean  SEM. Data were analyzed by one-, two or three-way ANOVAs, depending on the subject design. Post-hoc analyses were carried out with the Student-Newman Keuls procedure or the method of contrasts when indicated1.

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2. Schwarting RK, Huston JP. Unilateral 6-hydroxydopamine lesions of meso-striatal dopamine neurons and their physiological sequelae. Prog Neurobiol 1996; 49: 215-266.

3. Paxinos G, Watson C. The rat brain in stereotaxic coordinates, fith edn. Elesvier Academic Press: San Diego, 2005.

4. Boulet S, Lacombe E, Carcenac C, Feuerstein C, Sgambato-Faure V, Poupard A, et al. Subthalamic stimulation-induced forelimb dyskinesias are linked to an increase in glutamate levels in the substantia nigra pars reticulata. J Neurosci 2006; 26: 10768-10776.

5. Bjorklund A, Dunnett SB. Dopamine neuron systems in the brain: an update. Trends Neurosci 2007; 30: 194-202.

6. Carnicella S, Kharazia V, Jeanblanc J, Janak PH, Ron D. GDNF is a fast-acting potent inhibitor of alcohol consumption and relapse. Proc Natl Acad Sci U S A 2008; 105: 8114-8119.

7. Rodgers RJ, Johnsono NJT. Factor analysis of spatiotemporal and ethological measures in the murine elevated plus-maze test of anxiety. Pharm Bioch Behav 1995; 52: 297-303.

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