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Temporal dissociation of striatum and prefrontal cortex uncouples anhedonia and
defense behaviors relevant to depression in 6-OHDA-lesioned rats
Supplemental Information
Supplemental Experimental Procedures
Animals
Male Wistar rats were housed in groups of four to five per cage (50 x 30 x 15 cm)
in a temperature-controlled room (22±1 °C), with free access to food and water, and
with a 12 h light/12 h dark cycle (lights on at 7:00 AM).
Stereotaxic surgery and 6-OHDA infusion
To produce a PD model with partial lesion of the nigrostriatal dopaminergic
pathway, 6-OHDA (10 µg per injection) was bilaterally injected into the DLS in a
volume of 3 µL at the rate of 1.0 µL/min. For the stereotaxic infusion was used a
Hamilton 10 µL syringe with a 26-gauge needle connected to a 30-gauge cannula.
Following injection, the cannula was left in place for 5 min before being retracted, to
allow complete diffusion of the drug.
Behavioral tests
Assessment of motor function
Open field. Rats were placed in the center of a wooden arena (100 x 100 cm, grey
walls and grey floor) and allowed to freely explore it during 15 min. To avoid urine and
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odor impregnation, the open field apparatus was carefully cleaned up with a 10%
ethanol solution and then wiped with dry paper.
Rotarod. Before the surgery, the animals were tested to confirm their basal ability
to perform the task. The latency to fall was measured as an indicator of balance and
motor coordination.
Assessment of emotional parameters
Sucrose consumption. Animals were transferred into single housing cages with
free access to food. Each rat was provided with two water bottles on the extreme sides
of the cage during the 24 h training phase used for adaptation to the two bottles. After
training, one bottle was randomly switched to contain 0.8% sucrose solution and 24 h
later, the bottles were reversed to avoid perseveration effects. The consumption of water
and sucrose solution was estimated simultaneously in sham and 6-OHDA groups by
weighing the bottles.
Forced swimming. Rats were placed in individual glass cylinders (55 cm in height
and 25 cm in diameter) containing water (water depth was 45 cm, kept at 25±1 °C).
Two swimming sessions were conducted (an initial 15-min pre-test followed 24 h later
by a 5-min test). The total duration of immobility and swimming were manually scored
during the test session (5 min). A rat was regarded as immobile when floating
motionless or making only those movements necessary to keep its head above the water.
The swimming behavior is defined as movement (usually horizontal) throughout the
swimming chamber.
Social interaction. The rats subjected to social interaction experiment were tested
only once and rats within a given cage received identical treatment either with 6-OHDA
or vehicle. In an attempt to maximize social behavior in animals during the test, an
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intense adaption to the testing arena was carried out by allowing rats to explore the
apparatus for 10 min for 2 consecutive days. On the test day, two rats that had received
identical treatment and were unfamiliar to each other were placed simultaneously into
the apparatus in two opposite corners. The difference in body weight between the paired
rats was within 10 g. Their behavior was monitored during 10 min and then they were
returned to their home cage. To avoid the presence of olfactory cues, the apparatus was
thoroughly cleaned with 10% ethanol solution and then wiped with dry paper after each
trial. The amount of time spent by each rat in a pair in described behaviors in the main
text was summed to produce a single social interaction score. Experiments were
performed under a red light (230 V, 15 W).
Elevated plus-maze. It consisted of two open arms (50 x 10 cm) and two enclosed
arms (50 x 10 x 40 cm), arranged such that two pairs of identical arms were opposite to
each other. Arms emerged from a central platform (10 x 10 cm), and the entire
apparatus was raised to a height of 50 cm above floor level. Experiments were
performed under a red light (230 V, 15 W). At the beginning of the test, the rat was
placed on the central platform facing an enclosed arm. After each 5 min test, the maze
was carefully cleaned up with a 10% ethanol solution and then wiped with dry paper.
Anxiety-like responses were evaluated through the following behavioral parameters:
percentage of open arm entries (% open arm entries: open entries/total entries x 100)
and percentage of time spent on open arms (% open time: open time/300 x 100). Since
anxiolytic or anxiogenic effects can be confounded by changes in motor activity, the
locomotion of the rats was also evaluated on the basis of enclosed arm entries.
Operational criterion for entry was whole body and four paws.
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Immunohistochemistry
The free-floating sections were rinsed (3 times, 5 min each) with washing buffer
containing 0.15% Triton-X100 in phosphate buffered saline (PBS; 0.1 M NaH2PO4, pH
7.4) and then pre-incubated for 30 min with 1% hydrogen peroxide in PBS to remove
endogenous peroxidase activity. To avoid unspecific activity, free-floating sections
were also incubated in a solution containing 5% bovine serum albumin, washing buffer
and 5% normal chicken serum for 1 h. The sections were incubated overnight at 21 ºC
with rabbit anti-TH polyclonal antibody (1:1500, ab-112 Abcam) and, after washing
(washing buffer: 3 times, 5 min each), were incubated for 2 h with the goat anti-rabbit
secondary antibody (1:2000; ab60317, ChromeoTM 546 Abcam). After washing, the
sections were mounted on slides and covered with glass coverslips.
Western blotting
The striatum and PFC were rapidly dissected on dry ice and stored at -70 ºC until
processing. Tissues were gently homogenized in ice-cold HEPES solution (10 mM pH
7.4) containing 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride
(PMSF), 0.5 mM dithiothreitol and 10 µg/mL of each protease inhibitor (aprotinin,
soybean trypsin inhibitor, pepstatin, leupeptin). The homogenates were chilled on ice
for 15 min and then shaken for 15 min in the presence of 0.1% Nonidet P-40. The
homogenates were centrifuged at 10.000 x g for 30 min, and the resulting supernatant
was collected and stored at -70 ºC until use. Protein concentration was determined using
the Bio-Rad protein assay kit (Bio-Rad, USA). Equivalent amounts of proteins were
mixed in loading buffer (200 mM Tris, 10% glycerol, 2% SDS, 2.75 mM mercaptoethanol and 0.04% bromophenol blue) and boiled for 20 min at 70 ºC. Proteins
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(40 µg) were separated by SDS-PAGE in 10% gels. Proteins were detected
immunologically following electrotransfer onto nitrocellulose membranes (AmershamPharmacia Biotechnology, USA). Protein and molecular weight markers (BioRad,
Mississauga, Canada) were revealed by Ponceau Red staining. Membranes were
blocked in PBS containing 5% powdered milk and 0.05% Tween-20 for 1 h at 25 ºC.
Membranes were then incubated overnight at 4 ºC with anti-D1R antibody (sc-14001;
Santa Cruz Biotechnology, USA), anti-D2R antibody (sc-5303; Santa Cruz
Biotechnology) and anti-DAT antibody (MAB369; Millipore, Germany) in blocking
solution and, after washing, with horseradish peroxidase-conjugated anti-rabbit, antimouse and anti-rat antibodies for 2 h. Blots were visualized using the PerkinElmer ECL
system.
Extracellular electrophysiological recordings
Animals were anesthetized under halothane atmosphere, decapitated and the brain
rapidly removed and submerged in ice-cold artificial cerebrospinal fluid (aCSF)
solution of the following composition (mM): NaCl 125, KCl 3, MgSO4 1, CaCl2 2,
Na2HPO4 1.25 NaHCO3 25 and glucose 11, pH 7.4 (osmolality, 290-310 mOsmol/kg)
and bubbled with a 95% O2 + 5% CO2 mixture. Coronal slices containing the prelimbic
medial prefrontal cortex (mPFC) (250-300 µm thick) or containing the dorsolateral
striatum (DLS) (400 µm thick) were cut with a Vibratome 1500 sectioning system
(Vibratome, Germany). The slices were maintained in a pre-chamber containing aCSF
under continuous oxygenation at 32ºC for at least 90 min, to allow for their metabolic
recovery.
One individual slice was then transferred to a submerged recording chamber and
continuously superfused at a rate of 2-3 mL/min with oxygenated aCSF at 32 ºC.
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Extracellular field recordings were obtained with micropipettes (2–4 MΩ) filled with a
4 M NaCl solution. In the PFC, population spikes were recorded with an amplifier
AxonPatch 200B (Axon instruments, U.S.A.) upon positioning the recording electrode
in the layer V of the prelimbic mPFC, and the bipolar concentric stimulation electrode
(SNE-100, Kopf, Germany) in layer II/III of the prelimbic mPFC and delivering
rectangular pulses (60 – 150 µA) of 0.1 ms duration applied with a Digitimer DS3
stimulator (Digitimer LTD, U.K.) once every 20 s. Population spikes in the DLS were
obtained with an ISO-80 amplifier (World Precision Instruments, Hertfordshire, UK)
and digitized using an ADC-42 board (Pico Technologies, Pelham, NY, USA) upon
positioning the recording electrode in the DLS and the bipolar concentric stimulation
electrode in the white matter above the DLS and delivering stimuli (60 – 150 µA) of 0.1
ms duration a frequency of 0.05 Hz (S44 stimulator, Grass Instruments, West Warwick,
RI). Averages of 4 consecutive responses were continuously monitored on a personal
computer, digitalized at 10 kHz with the WinLTP 1.1 program (Anderson and
Collingridge, 2001), and quantified as their amplitude. After obtaining stable responses,
an input/output curve was carried out to choose an intensity of stimulation yielding 4050% of the maximum response.
The effect of dopamine on synaptic transmission was assessed by testing three
concentrations of dopamine (25, 50 and 100 µM), cumulatively added through the
superfusion solution, and their effect estimated by the change of population spike
amplitude.
Paired-pulse stimulation consisted of 2 stimuli delivered with an inter-stimuli
interval of 50 or 250 ms for mPFC slices and 20 or 160 ms for DLS slices. The pairedpulse ratio (PPR) was calculated as the ratio of the second response to the first response.
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Experimental protocol
Rats were first challenged with 6-OHDA stereotaxically administered in the DLS
(Supplement 3) and independent groups of animals were tested only once in the
behavioral tasks after 7 or 21 days: motor function was first characterized using the
rotarod, the open field and the grip force tests; then the rats were tested in the non-motor
behavioral tasks such as sucrose preference and splash tests (anhedonia), forced
swimming and social interaction (defense behaviors relevant to depression) and elevate
plus-maze (anxiety-like behaviors). Some rats were then transcardiacally perfused and
their brains were processed for immunohistochemical quantification of tyrosine
hydroxylase (TH), a marker of dopaminergic innervation, in the DLS and SN. Other rats
were sacrificed and their brains were dissected for Western blotting quantification of
dopamine D1 (D1R) receptors, D2 receptors (D2R) and dopamine transporter (DAT) in
the striatum and PFC. A last set of rats were sacrificed to carry out extracellular
electrophysiological recordings in DLS and mPFC slices. Finally, independent
experiments were carried out following the same protocol, to test the impact of the
antidepressant drugs fluoxetine (10 mg/kg, i.p.) or bupropion (10 mg/kg, i.p.) on the
anhedonic-like and defense behaviors relevant to depression 7 and 21 days after 6OHDA lesion.
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Figure
S1.
Two
doses
of
6-hydroxydopamine
(6-OHDA)
decreased
immunohistochemical quantification of TH optical density in DLS and TH positive cells
in SN in the same amount in both hemispheres left (L) and right (R). (A) Quantification
of TH optical density of DLS. (B) Representative coronal sections of DLS. (C)
Quantification of TH positive cells of SN. (D) Representative coronal sections of SN. (*
p< 0.05 vs. control group; # p<0.05 vs. 6-OHDA group 7 days after injection, two-way
ANOVA followed by the Newman-Keuls post-hoc tests). N=5 animals/group.
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Figure S2. Analysis of different kinds of motor and non-motor functions 7 or 21 days
after 6-hydroxydopamine (6-OHDA, doses of 5 and 20 µg). (A) Total distance travelled
during 15 min in the open field apparatus. (B) Average speed in open field. (C) Latency
to fall in the accelerated rotarod test. (D) Grip force test to evaluate the strength of the
forelimbs. (E) Sucrose preference test 7 days after de injection of 6-OHDA. (F) Sucrose
preference test 21 days after de injection of 6-OHDA. (G) Forced swimming test in
both periods of time. (* p< 0.05 vs. control group, two-way ANOVA followed by the
Newman-Keuls post-hoc tests). N=8-10 animals/group.
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Figure S3. Experimental protocol. Male Wistar rats with 3 months old underwent to the
bilateral stereotaxic surgery for the injection of 6-OHDA in the DLS. After 7 or 21 days
we tested the animals in behavioral tasks described above. In the first moment we
characterized the motor function. After that we tested the rats in the non-motor
behavioral tasks. The animals were sacrificed and the brains collected for the ex vivo
experiments.
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Figure S4. Anxiety-like behaviors were analyzed 7 and 21 days after the injection of 6OHDA in elevated plus-maze test (EPM). (A) Percentage of open-arms time in EPM.
(B) Percentage of open-arms entries in EPM. (C) Enclosed-arm entries in EPM. Twoway ANOVA followed by the Newman-Keuls post-hoc tests. N=8-10 animals/group.
Figure S5. 6-OHDA injection did not alter stimulus-sensitivity curve in the DLS and in
mPFC slices in both periods of time. (A) Diagram showing the positioning of
stimulating and recording electrodes in the DLS. (B) Stimulus-sensitivity curve in the
DLS slices evaluated 7 days after the induction of the lesion. (C) Stimulus-sensitivity
curve in the DLS slices evaluated 21 days after the induction of the lesion. (D) Diagram
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showing the positioning of stimulating and recording electrodes in the mPFC. (E)
Stimulus-sensitivity curve in the mPFC slices evaluated 7 days after the induction of the
lesion. (F) Stimulus-sensitivity curve in the mPFC slices evaluated 21 days after the
induction of the lesion. N= 4 animals/group.