**7.9 Preparation of synaptosomes**

264 Neuroscience – Dealing with Frontiers

purified by high performance liquid chromatography (HPLC) on a C18 Microbondapak column (Millipore-Waters, France) using H3PO4 (1 mmol/l, pH 3) as a mobile phase. [3H]TYR was added to artificial CSF (80 µCi/ml) 1 hour after implantation and superfusates

Dialysates collected for measuring catecholamines were protected with 5 µl perchloric acid (0.05 M) and immediately underwent HPLC analysis. Dialysates collected for amino-acid

The catecholamines DA, DOPAC and HVA were measured in the collected fractions by electrochemical detection with the potential of the working electrode maintained at 0.7 V (Antec-Decade) after HPLC separation (column C18 Brownlee RP18, 5 µm, 2.1 x 220 mm, maintained at 32 °C; Mobile phase: 50 mM KH2PO4, 0.1 mM EDTA-Na2, 0.28 mM sodium octyl sulfate, 6% methanol, adjusted to pH 4.5; flow rate 0.25 ml/min). Catecholamine peaks were identified based on their retention times (Olivier et al., 1995; Dzahini et al., 2010). Extracellular catecholamine concentrations were estimated by evaluating the peak areas of each substance and their respective external standard (analytical software AZUR, Datalys France). The running time for each determination was 25 min. When superfusion was conducted with 3,5-[3H]TYR added to the CSF, the radioactivity corresponding to each HPLC peak was counted using a continuous flow scintillation detector (Packard-

The concentrations of the amino acids GLU, GABA and ASP in the dialysates were determined after HPLC, via laser-induced fluorescence detection (Dzahini et al., 2010). Briefly, 2 µl of sample or standard was derivatized with naphthalene-2,3-dicarboxaldehyde. The resulting mixture was automatically loaded onto a Symmetry Shield-C18 reverse-phase column (100\_2.1 mm, 3.5 \_m particle size; Waters, Milford, MA), using a refrigerated Triathlon auto injector (Polymer Laboratories, Marseille, France). The mobile phase consisted of 0.04 M NaH2PO4, pH6, in a 3–50% acetonitrile gradient. The flow rate was 0.35 ml/min, maintained using two Shimadzu (Kyoto, Japan) LC 10AT pumps. Amino acid peaks were identified based on their retention time. Extracellular amino acid concentrations were estimated by evaluating the peak areas of each amino acid and their respective external standard (analytical software class LC10; Shimadzu). The running time for each

At the end of each experiment, to verify the location of the cannula, the animals were intracardially perfused with a 4% formaldehyde solution, after which the brain was

The homogenate (20 µl) was mixed with 20 µl of 0.3 N perchloric acid containing 0.8 mM EDTA, added to an internal standard (3,4-dihydroxybenzylamine) and centrifuged (10,000 g, 10 min). The supernatant of each sample (10 µl) was injected into a C18 reverse-phase microcolumn (Spheri5, RP-18, 220 2.1 mm, Browlee labs). The mobile phase consisted of 40 mM KH2PO4 (pH 4.5), 0.26 mM octane sulfonic-acid, 15 mg/l EDTA and 11% methanol

Protein weights were measured using the micro BCA Protein Assay (Pierce, Biorad).

were collected thereafter as successive 20 minute fractions.

analysis were maintained at -80 °C and kept frozen until analysis.

Radiomatic, Flo-One B A250)( Leviel et al., 1989, 1990, 1991).

**7.7 Biochemical analysis** 

determination was 12 min.

**7.8 DA and DOPAC tissue concentration** 

removed, sliced (50 µm) and stained with cresyl violet.

The method for synaptosomal preparation and measurement of [3H]DA uptake was modified from Masserano et al. (1994). Rat brains were rapidly removed and chopped into 1.6-mm-thick slices, in the sagittal plane corresponding to the *anterior commissura*. The median part of the CP, ipsilateral to the injection site, was pooled for all rats of each group and homogenized in 25 ml cold 0.32 M sucrose (10 up and down strokes, at 850 rpm, in a glass-teflon homogenizer). The homogenate was centrifuged at 800 g for 10 min at 4 °C and the pellet was discarded. The supernatant (S1) was kept on ice until it was resuspended for the [3H]DA uptake or [3H]GBR12935 binding protocols.

*[3H]DA uptake:* The supernatant (S1, 15 ml) was centrifuged (20,000 × g for 10 min at 4 °C), and the resulting pellet (P2) was resuspended in 20 ml of ice-cold incubation buffer (in mM): NaCl, 125; K2HPO4, 1.5; MgSO4, 1.5; CaCl2, 1.25; *d*-glucose, 10; HEPES, 25; ascorbic acid, 0.1; pargyline, 1 and EDTA, 0.1, pH 7.4. The buffer was oxygenated with 100% O2 for 10 min before use. The assays were performed in triplicate with 400 µl of pellet resuspended in 1 ml of incubation buffer. After preincubation of 3 min at 37 °C, the assays were initiated by adding 10 µl of increasing concentrations of [3H]DA (10, 20, 40, 50, 100, 200, 400 nM) for 7 min at 37 °C. Nonspecific values were determined in the presence of Mazindol (1 µM) for 7 min at 4 °C. Assays were terminated by immediate filtration using Whatman GF/B filters soaked in 0.32 M ice-cold sucrose containing 0.05% polyethylenimine. Filters were washed three times with 3 ml of 0.32 M ice-cold sucrose and radioactivity was measured using a Packard TRI-CARB 2100 TR. The apparent *K*m and *V*max of DA uptake were calculated by linear regression of the reciprocal plot (1/*v* vs. 1/*S*). The protein content in the resuspended pellet P2 was measured using the micro BCA kit (Pierce, Biorad).

#### **7.10 [<sup>3</sup> H]GBR12935 binding**

The [3H]GBR12935 binding protocol was modified from Berger et al. (1985). The supernatant (S1, 10 ml) was centrifuged (20,000 g for 10 min at 4 °C), and the resulting pellet (P2) was resuspended in 13 ml of 50 mM ice-cold Tris-HCl buffer, pH 7.7, containing 120 mM NaCl. Assays were performed in triplicate with 500 µl of pellet suspension and 50 mM Tris-HCl buffer, pH 7.7, containing 120 mM NaCl and 0.01% of Bovine Serum Albumin in a final volume of 2 ml. Incubation was initiated by the addition of 100 µl of [3H]GBR12935 (5 µM) for 45 min at 25 °C. Non-specific binding was determined in the presence of 1 µM Mazindol. Filters were washed three times with 4 ml ice-cold Tris-HCl 50 mM buffer pH 7.7 and radioactivity was counted using a Packard TRI-CARB 2100 TR. The protein content in the resuspended pellet P2 was measured using the micro BCA kit (Pierce, Biorad).

### **7.11 Determination of TH protein content**

TH protein content was measured using a semi quantitative immunoblotting technique as previously described by Garcia et al. (1994). Briefly, after centrifugation of the homogenate

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(10,000 g, 30 min), 1.5 µl of supernatant was placed onto a nitrocellulose membrane (Bio-rad 162-147, 0.2 µm). Non-specific binding sites were saturated with 50 mM Tris buffer containing 2% bovine serum albumin. The protein was revealed using, in succession, a mouse monoclonal antibody to TH (10 ng/ml; Boehringer Mannheim), a 125I-labeled protein A (S.A: 1.11x 10-9 Bq/mg, 1850 Bq/ml, Amersham) and 3H-hyperfilms (Amersham). The radioimmunochemical labeling was calibrated using a scale of standard TH protein (extracted from adult rat adrenals and diluted in homogenates of cerebellum). One U of TH (UTH) is defined as the mean TH protein content of 10 µg (wet weight) of adult rat adrenal gland. Optical density measurements were converted into UTH/mg tissue by reference to the standards. Each reading provided the surface area (mm2) of the dot and the TH tissue concentration (UTH/mg tissue). Using the surface area and the TH tissue concentration, the amount of TH (UTH) in the region of interest was calculated and normalized by expressing the amount of TH as a percentage of values in the control rats.

#### **7.12 Immunoprecipitation of TH enzyme**

Rats were perfused transcardially with NaCl 9°/°° and PFA 4%. The brain was then removed and stored in PBS buffer containing 0.1% sodium azide pH 7.4 (PBSA). To evaluate the extent of the lesion in the SN and the extent of CPc denervation, TH immuno precipitation was performed on coronally sectioned slices (40–50 µm thickness). Brains were cut on a cryostat (HM440E, Microm); free-floating slices were collected and stored in PBSA. Sections were incubated in 3% H2O2 in PBS to block endogenous peroxidase activity and were then incubated overnight with the primary rabbit anti-TH antibody (Chemicon, Temecula, CA; 1/2500) in 10% normal swine serum, TBS, pH 7.4, containing 2% NGS and 0.2%Triton X-100 for 24 h at 4 °C on a platform shaker. After rinsing in PBS 0.1% plus triton X100, sections were incubated with biotinylated swine anti-rabbit secondary antibody for 30 min at room temperature. This was followed by incubation with Strept-ABC-HRP complex (Dako, Glostrup, Denmark). TH immunoreactive neurons were visualized using 3.3 diaminobenzidine (DAB) in N2+ 0.5% and H2O2. To test the specificity of the staining, control sections were processed in an identical manner but with omission of the primary or secondary antibody. All sections were then washed for 10 min in PBS, mounted on slides, dried, dehydrated in increasing grades of ethanol, cleared in toluene, and mounted with DPX and cover slipped. These sections were scanned and analyzed immediately using Imagemaster Labscan V-3.00 (Pharmacia Biotech, Dzahini et al., 2010).
