**7.5 Voltammetric investigations**

262 Neuroscience – Dealing with Frontiers

and DA terminals; 3) indirect activation through GLU afferents to DA terminals of tonic DA release, which initially constitutes autotherapy allowing the behavioral compensation observed in Parkinson's disease. However the toxic effect of DA on the DA terminal (production of H2O2 when present in excess) could constitute a secondary pathological agent, maintaining the degenerative process. Each of the three steps mentioned here require further elucidation and demonstration, but a large set of data in each case is consistent with

DA homeostasis constitutes one of the functions of the DAT via DAT-RT. This is under the control of many regulatory processes including tonic GLU neurotransmission. Experiments regarding the consequences of partial lesions seem to indicate a role for this GLU-dependent regulation in the tonic DA overflow and in maintaining a permanent excess of extracellular DA. It remains to be determined how local GLU can induce the presynaptic and tonic activation of DA release. This could be achieved directly through GLU receptors located on DA terminals or indirectly through heterologous neurotransmission systems such as GABA

Male Wistar rats (Iffa-Credo France) weighing 250 g were used in the studies reported here. They were maintained and sacrificed in accordance with the European Communities Council Directive (86/609/EEC). The animals were anesthetized with 1.5% isoflurane in pure oxygen in air or were injected with chloral hydrate (400 mg/kg) and spontaneously inspired (Leviel et al., 1989; Olivier et al., 1995). When necessary they were placed in a stereotaxic device. For the short interventions (less than 1 hour), a mixture of ketamine/rompun was used (150/10 mg/Kg). Rectal temperature was maintained at 38 °C.

The lateral part of the SN was unilaterally injected with a solution of 6-OHDA (3 µg free base in 1 µl isotonic saline containing 0.2% ascorbic acid, pH 4). The stereotaxic coordinates were, in mm: Ant.: 3.5, Lat.: 2.5 and Ht.: 3.4. The horizontal plane passed through the interaural axis and incisor bar. Unlesioned rats were only submitted to nigral injection of the

Animals were anesthetized with ketamine (150 mg/Kg; Panpharma), maintained in a stereotaxic device and implanted with a cannula (0.71 mm o.d. with a removable mandrel) in the anterior part of the left lateral ventricle. The head of the cannula was sealed with acrylic cement, embedding screws set in the cranial bone. The stereotaxic coordinates were the following (in mm): AP: 0.48; LM: 1; H: 6 from the bregma (following Paxinos and Watson, 1986). Rats were allowed to recover for 2 weeks. In order to inject 6-OHDA (30 min after administering 25 mg/kg imipramine i.p. to protect noradrenergic neurons), the mandrel of the cannula was removed and replaced by an injector connected, through a thin

Cardiac and respiratory rhythms were continuously monitored.

**7.3 Surgery and procedures for recurrent ventricular injections** 

vehicle (Dentresangle et al., 2001; Dzahini et al., 2010).

this hypothesis.

or ACH.

**7.1 Animals** 

**7. Technical procedures** 

**7.2 Lesions of the lateral SN** 

Difference Normal Pulse Voltammetry (DNPV) and Differential Pulse Amperometry (DPA) were performed using treated carbon fiber electrodes produced as described previously (Olivier et al., 1995; Dentresangle et al., 2001). Their active part was the surface of one pyrolytic carbon fiber (SOFICAR, France), 250 µm long and 8 µm in diameter. A stainless steel tweezer fixed on the interaural bar was used as an auxilliary electrode and the Ag/AgCl reference electrode was a silver wire coated with AgCl. This was maintained in contact with the skull by means of a sponge moistened with Phosphate Buffer Saline (PBS) solution (PBS, pH 7.4). The three electrodes were connected to a pulse voltammetric system (Biopulse, SOLEA Tacussel, France). Carbon-fiber electrodes were electrochemically treated as previously described (Gonon et al., 1984). To calibrate the electrodes, voltammograms were recorded *in vitro* in a standard solution of DOPAC or DA and ascorbic acid in PBS. The values of the oxidation potentials and the amplitude of the peaks were stabilized after 4–5 successive scans (10 to 15 min). Their values were 60 mV for ascorbic acid, 60 mV for DOPAC and 90 mV for DA. It had previously been verified that the amplitude of the oxidation peak for these substances is linearly correlated with their concentrations. The oxidation potentials for DA and DOPAC were however too close to be properly differentiated *in vivo* and DA was three orders of magnitude lower than DOPAC. Thus, for adequate detection of DA, DOPAC formation was inhibited by pretreatment with pargyline (75 mg/kg, i.p.), an inhibitor of monoamine oxidase.

#### **7.6 Superfusion procedures (microdialysis & push-pull cannula)**

Animals were submitted to superfusion of the striatum to analyze extracellular DA, DOPA, HVA, GLU, GABA and ASP (Dzahini et al., 2011). They were implanted with a dialysate probe (250 µm in diameter and 4 mm length, cut off 6000 dalton, CMA, Sweden) in the anterior part of the caudate nucleus (Ant.: 8.4 mm; Lat.: 2.5 mm; H.: 6 mm, same stereotaxic references). The cannula was supplied (1 µl/min) with an artificial CSF (in mM, 145 NaCl, 2.7 KCl, 1.0 MgCl2, 1.2 CaCl2, 0.45 NaH2PO4, 2.3 Na2HPO4, adjusted to pH 7.4).

Push-pull cannulae were used in an experiment devoted to measuring the radioactive form of DA and DOPAC (Leviel et al., 1989, 1990, 1991). The cannulae (1.0 mm outer diameter) were supplied (flow rate: 12.5 µl/min) with an artificial cerebrospinal fluid (CSF) adjusted to pH 7.4 with an O2-CO2 (95:5 v/v) mixture. 3,5-[3H]TYR (50 Ci/mmole, Dositek, France) was

Normal and Physio-Pathological Striatal Dopamine Homeostasis 265

(vol/vol), and the flow rate was 0.20 ml/min. The liquid chromatography system (TSP) was coupled to an electrochemical detector (Millipore) with a working electrode set at 0.8 V. The concentrations of DA and its metabolites in CP were calculated for each sample (in ng/mg prot). The results were normalized by expressing the changes in DA and DOPAC levels as

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

*[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

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

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

resuspended pellet P2 was measured using the micro BCA kit (Pierce, Biorad).

percentages of the values obtained in control animals (Dentresangle et al., 2001).

**7.9 Preparation of synaptosomes** 

**H]GBR12935 binding** 

**7.11 Determination of TH protein content** 

**7.10 [<sup>3</sup>**

the [3H]DA uptake or [3H]GBR12935 binding protocols.

pellet P2 was measured using the micro BCA kit (Pierce, Biorad).

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 were collected thereafter as successive 20 minute fractions.

### **7.7 Biochemical analysis**

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

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

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 determination was 12 min.

#### **7.8 DA and DOPAC tissue concentration**

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

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

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 percentages of the values obtained in control animals (Dentresangle et al., 2001).
