Effect of P2X
receptor antagonists on cell viability after rotenone and MPTP pretreatment in primary substantia nigra (SN) culture. Non-treated SN cultures contained large tyrosine hydroxylase-positive (arrow) neurons that were identified by MAP2 (red; A) and TH (green; B) immunoreactivity (merge; C). Rotenone treatment disrupted the neurons (phase contrast morphology; D and MAP2 immunostaining; E). Scale bar: 20 μm. MAP2+ and TH+ neurons represented 26.28 ± 10.6% and 7.32 ± 2.81% of the total cell number. Total cell number was determined by counting DAPI-positive nuclei on each of 40 microscopic fields. Cells were counted at 250 × (2 × 10 fields) and 500 × (2 × 10 fields) magnification (independent determinations: n = 4). F. and G. Effects of BBG and AZ10606120 on changes in cell viability induced by rotenone (F) and MPTP (G) measured in the MTT assay. Cells were pretreated with L-deprenyl and with the P2X7 antagonists, BBG and AZ10606120, in 10 and 100 nM concentrations for 1 h before treatment with 1 μM rotenone or MPTP for 20 h. Data are expressed as the percentage of values of control cultures and are the means ± S.E.M. of four experiments. *P < 0.05, ** P < 0.01, significance vs. controls using an ANOVA followed by the Dunnett test. Note that the concentration of rotenone and MPTP are indicated in μM, whereas the concentration of other drugs in nM.