Fig. 1. Activity and expression of nNOS in cerebellar development of control and L-NAME-treated rat pups. Pups were s.c. injected with L-NAME (60 mg/kg/day, divided in two daily administrations) for various neonatal intervals (P1-P3, P3-P5, P5-P7) while control pups received vehicle. Animals were killed on the last day of treatment. (A) Maturation of calcium-dependent NOS activity in developing cerebella (white bars) and almost complete inhibition obtained by L-NAME treatment (black bars). Values are the mean ± s.e.m. of at least three experiments for each time point. ^*P<0.05, compared with the control at P3, #P<0.001 compared with the corresponding control (Bonferroni's test after ANOVA). (B) Total RNA was extracted from cerebella of control and L-NAME-treated rat pups and equal amounts of RNA were used for real-time PCR performed with specific primers for nNOS RNA as described in the Materials and Methods section. These results, expressed as a percentage of control at P3 are the mean ± s.e.m. of four different experiments performed in triplicate. ^*P<0.05, ^**P<0.01 compared to P3 (Bonferroni's test after ANOVA). (C) In western blot experiments, cerebellar extracts from control and L-NAME-treated rat pups were probed with an antibody specific for nNOS. Bands were quantified by optical densitometry and normalized for the amount of ß-actin. These results are the mean ± s.e.m. of four different experiments. ^*P<0.05, ^**P<0.01 compared with P3 (Bonferroni's test after ANOVA).