Materials used for cell cultures and Reverse Transcription-PCR were purchased from Invitrogen-Gibco BRL (Burlington, Ontario, Canada) and from Sigma Chemical Co. (Oakville, On, Canada). Haloperidol, (-)-eticlopride, raclopride, chlorpromazine and risperidone were obtained from Sigma Chemical Co. (Oakville, On, Canada). U 99194 maleate and L-741,742 hydrochloride were obtained from Tocris (Ellisville, MO, USA). The ginkgo biloba extract EGb 761 was kindly provided by IPSEN laboratories (Paris, France). Unless stated otherwise, other chemicals were purchased from Sigma-RBI (Natik, MA, USA). All drugs were freshly prepared on the day of the experiment in a final concentration of ethanol or DMSO that does not exceed 0.01%.
Neuronal hippocampal cell cultures
Enriched neuronal hippocampal cells were prepared from E19 fetuses obtained from Sprague-Dawley rats (Charles River Canada, St-Constant, Québec, Canada) as described previously . Animal care was according to protocols and guidelines of the McGill University Animal Care Committee and the Canadian Council for Animal Care.
Hippocampal cells were plated at day 0 at a density of approximately 12 × 104 viable cells per well in 96-well plates. They were grown in Dulbecco's modified Eagles medium (D-MEM) medium supplemented with 20 mM KCl, 15 mM HEPES and 1% (v/v) serum-free growth medium N2 (final composition: 5 μ g/ml insulin, 100 μM putrescine, 20 nM progesterone, 100 μg/ml transferrin, 30 nM selenium), and maintained at 37°C in a 95% air/5% CO2 humidified atmosphere during 3 days.
On day 0, hippocampal neurons were plated on poly d-lysine (25 μg/mL)-coated 12 mm glass coverslips (Fisher, Nepean, On, Canada) placed in multiwell plates and grown in the same medium as described above. On day 3, the medium was removed, the cells rinsed with PBS and fixed with 4% paraformaldehyde at room temperature (RT) for 15 min. Cells were pre-treated with 0.1% Triton X-100 for 20 min followed by a blocking step with 5% normal donkey serum (NDS)/bovine serum albumine (BSA) 5%/0.1% Triton X-100 in PBS for 20 min at RT. The cells were then incubated overnight at 4°C with a mouse anti- NeuN monoclonal antibody (1:250; Chemicon, Temecula, CA, USA) in PBS supplemented with 0.1% Triton X-100, NDS (5%) and BSA (0.5%). After several washes in PBS, the secondary antibody (Alexa Fluor 568 goat anti-mouse IgG1, 1:200; Invitrogen) diluted in the same buffer as the primary antibody was added and incubation proceeded for 2 hrs at RT. The coverslips were washed several times then mounted on slides with DAPI-containing Vectashield (Vector Laboratories, Burlington, On, Canada). Hippocampal cells were examined using conventional immunofluorescence microscopy and counted from three 40× magnification fields on one slide for each experimental condition. Each experiment was repeated using a different culture preparation.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
RT-PCR was performed using a sensitive two-step PCR protocol according to  with some minor modifications. Total RNA was isolated from 3-day-old rat primary cultured hippocampal neurons (from two different experiments) and from rat striatum (P14) by using the Qiagen (Mississauga, On, Canada) RNeasy midi-kit in conjunction with the RNase-free DNase set according to the manufacturer's protocol. First strand cDNA was generated from 1 μg total RNA in a 20 μl reaction containing: 2.5 μM random hexamers (Applied Biosystems, Foster City, CA, USA), 10 mM DTT (Sigma), 20 U Ribonuclease Inhibitor (Takara Biomedicals, Otsu, Japan), 0.5 mM dNTP, 1X First strand buffer, and 100 U SuperScript II RNase H- Reverse Transcriptase (all from Invitrogen). Following an overnight incubation at 42°C, the enzyme was denatured at 70°C and the RNA complementary to the cDNA was hydrolysed with 2U RNaseH (Takara Biomedicals) for 20 min at 37°C. Reactions in which the reverse transcriptase was omitted were run in parallel as controls for any residual genomic DNA.
In the first step PCR, cDNAs for dopamine receptor subtypes D1 to D5 were amplified simultaneously from 2 μl of each reverse transcription reactions in 20 cycle multiplex reactions (mCPR). This was followed by a second round of 35 cycles PCR in which individual cDNAs (D1 to D5) were amplified separately in reactions using 2% of the first round products as substrate. All PCR amplifications (94°C, 30 s; 60°C, 30 s; 72°C, 35 s) were performed in a 96-well thermocycler (GeneAmp 9700, Applied Biosystems). The final reaction volume for each amplification reaction was 100 μl and contained 1× PCR buffer, 2 mM MgCl2, 200 μM dNTP, 1 U Platinum Taq DNA polymerase (all from Invitrogen), and 10 pmoles of each selected forward and reverse primers. Primer pairs (custom-synthesized by Invitrogen) for D2-like dopamine receptor subtypes D2, D3, and D4 were designed to flank at least one intron according to the NCBI GenBank sequence database and to lie outside regions of significant homology. Likewise, primer pairs amplifying sequences from intronless coding regions of D1-like (D1 and D5) receptor subtypes were derived from regions of low homology. Primer positions for D2 or D3 were chosen in the vicinity of those used by  to detect possible alternative splicing isoforms.
The following oligonucleotide primers were used (the predicted size for PCR products are given in parentheses): receptor D1, forward 5'-CATCACCTTCGATGTGTTTGTGTG-3' and reverse 5'-GCTATTCCACCAGCCTCTTCCTT-3' (300 bp); receptor D2, forward 5'-GCCAACCCTGCCTTTGTGGT-3' and reverse 5'-GCTTTCTGCGGCTCATCGTCT-3' (538 bp and 451 bp); receptor D3, forward 5'-GCCTGGTATGTGCTGCTGTGCT-3' and reverse 5'-CGTTTTCTTTGCCTTTGCCTCA-3' (523 bp and 410 bp); receptor D4, forward 5'-TCTACTCCGAGGGTGGCGTGT-3' and reverse 5'-GCAGGAAGAAGGAACAAATGGATG-3' (324 bp); receptor D5, forward 5'-GGAGGAAGGCTGGGAGCTAGAA-3' and reverse 5'-GCTGACACAAGGGAAGCCAGTC-3' (403 bp).
Fifteen μl of each second round PCR were analyzed on a 2% agarose gel with 1 μg of molecular size standards (Invitrogen). Discrimination between potential amplification of genomic DNA sequences and RT-PCR on mRNA was based on the size of the PCR product (in the case of D2, D3, and D4 receptors) and on the absence of a PCR product when reverse transcriptase was omitted (for all 5 subtypes). PCR products of the anticipated sizes were then purified with the QIAquick PCR purification kit (Qiagen), and sequenced at Laval University's Service d'Analyse et de Synthèse SCF Facility (Québec, Canada) to ensure they matched the respective known cDNA sequences.
Toxicity induced by growth medium deprivation
At day 3 of plating, the medium was removed and cells were incubated at 37°C in D-MEM medium supplemented with 15 mM HEPES and 5 μg/ml insulin and devoid of putrescine, progesterone, transferrin, selenium and KCl. Cells were then treated with either vehicle or different drugs. Neuronal viability was determined 3 days later using the MTT and neutral red (NR) colorimetric assays (see below).
Assessment of neuronal survival
Neuronal survival was estimated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and NR [3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride] dyes, which are respectively indicators of mitochondrial activity and lysosomal uptake of living cells. Cell survival was spectrophotometrically determined at 570 nm (for MTT assay) and 540 nm (for NR assay) using a micro-plate reader (Bio-Tek Instruments® Inc., Ville St-Laurent, Québec, Canada) .
Assessment of intracellular reactive oxygen species
Dichlorofluorescein (DCF) fluorescence assay was used to determine the intracellular production of reactive oxygen species . Briefly, cells were treated with the cell permeable 2,7-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes Inc., Eugene, OR) which is converted into 2',7'-dichlorofluorescein. 2',7'-dichlorofluorescein is then able to interact with intracellular peroxides to form the highly fluorescent compound DCF. The medium was removed 3 days after plating and replaced with fresh medium containing 15 mM HEPES, 5 μg/ml insulin and 5 μM DCFH-DA in the presence of absence of either haloperidol (1 μM) or EGb 761 (50 μg/ml). DCF fluorescence was quantified (excitation = 485 nm, emission = 530 nm) the day after using a fluorescence multiwell plate reader (Bio-Tek Instruments® Inc., Ville St-Laurent, Québec, Canada).
Optical density (OD) reflecting MTT reduction and NR intake into intact cells, was proportional to the number of viable cells. The OD of the control group (CT, i.e. the group of non-treated cells deprived during 3 days with growth medium) was regarded as 100%. The rate of surviving cells treated with various drugs during 3 days was expressed as percent of control groups. Statistical analysis was performed using one-way ANOVA followed by a Newman Keuls' multiple comparison test with p < 0.05 being considered statistically significant. An unpaired t-test was used to compare reactive oxygen species production (as estimated by the DCF assay) between control group and groups treated with drugs, survival of cells treated with clozapine alone and cells treated with raclopride and clozapine (Table 2), and survival of non-treated cells and cells treated with caspases (Table 3).