cultured neurons. Rat hippocampal neurons at 7 days in vitro (DIV), were exposed to various concentrations of IL-1β (1 to 100 ng/ml, prepared in Krebs buffer; pH 7.4) for different times (5, 10, 15, 20, 25, 30, 60 and 180 minutes), after which cells were either lysed on ice in radio-immunoprecipitation assay (RIPA) buffer for (A–D) western blotting analysis or fixed for (E–F) immunocytochemical analysis of the pattern of activation (phosphorylation) of the various MAPKs. (A) IL-1β (10 ng/ml) activated JNK in a time-dependent manner, reaching significance at 10 to 15 minutes of incubation with IL-1β. (B) Activation of JNK increased in line with increasing concentrations of IL-1β. (C) After 15 minutes of exposure to 100 ng/ml IL-1β, p38 MAPK was also activated. (D) However, under similar experimental conditions, IL-1β failed to significantly affect the amount of phosphorylated p42 ERK. In each panel, the bar graphs display the mean ± SEM of four to eight cultures, and the blots below illustrate a representative experiment. Membranes were first used to detect the phosphorylated MAPK, and then reprobed for the total amount of MAPK. MAPK activation was calculated as a ratio between the phosphorylated and total immunoreactivities, expressed as a percentage of the control values (that is, in the absence of IL-1β). The values are mean ± SEM of four to eight experiments, *P < 0.05, **P < 0.01 and ***P < 0.001. (E) Representative immunocytochemistry images of hippocampal neurons (left) in the absence and (right) after 15 minutes in the presence of 100 ng/ml IL-1β, probed for the phosphorylated form of JNK (green fluorescence) which co-localized with the class III β-tubulin, a constitutive protein exclusively of neuronal microtubules (red fluorescence). (F) Representative immunocytochemistry images of hippocampal neurons (left) in the absence and (right) after 15 minutes in the presence of 100 ng/ml IL-1β, probed for the phosphorylated form of p38 (red fluorescence) which co-localized with synaptophysin (green fluorescence). (E,F) The neuronal nuclei were stained with DAPI (blue fluorescence).