Supplementary Material and Methods (doc 66K)

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 (chloroAPB 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
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, nonreinforced, 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,
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
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