Figure 5

The effect of in vitro rotenone pretreatment on the mRNA expression of P2X ₇ and P2X ₄ receptors and the P2X ₇ (k) splice variant, ATP, ADP and AMP contents and on the energy charge in the substantia nigra and striatal slices of wild-type (WT) and P2X ₇ receptor knockout (KO) mice. Striatal slices were preincubated with in vitro rotenone (10 μM) for 60 min. (A) Quantitative SYBR Green real-time PCR was performed using specific primers. The experiments were repeated twice with similar results. The expression level of the P2X receptors was normalized to the expression level of the distinct housekeeping gene, 18S rRNA. Data are displayed as the means ± S.E.M. Asterisks indicate significant differences from the corresponding control (*P < 0.05, Student's t-test). B. Confirmation of the expression of the P2X₇(k) variant using RT-PCR with specific forward primers for P2X₇(k) isoforms and a common reverse primer. RT-PCR analysis showed that P2X₇(k)-specific primer pairs produced amplicons are present in striatal slices. The gel is 1.5%, 1× TBE buffer. A 100-bp DNA ladder (Fermentas, Vilnius, Lithuania) was used to identify PCR fragment sizes, and the amplification of 18s RNA was used as an internal control. C. Changes in mRNA expression levels of P2X₇(k) variant in the striatum obtained from P2X₇ receptor wild-type mice after in vitro rotenone pretreatment. Quantitative SYBR Green real-time PCR was performed using specific primers. The experiments were repeated twice with similar results. The expression level of the P2X₇(k) isoform was normalized to the expression level of the distinct housekeeping gene 18S rRNA. Data are displayed as the means ± S.E.M. Asterisks indicate significant differences from the corresponding control (**P < 0.01, Student's t-test). D, E, F. Tissue contents of ATP, ADP and AMP, expressed in nmol/mg protein (n = 5-8). G. For the calculation of energy charge (EC) see Materials and Methods. * P < 0.05, ** P < 0.01, significance vs. control.