Fig. 1.

E2-mediated calpain activation in primary hippocampal neuronal cultures. (A and B) Cultured hippocampal neurons were preloaded with the FRET substrate as described in Materials and Methods. E2 (10 nM) was applied at time 0 (A), and confocal images were recorded at various times after application. (B) The image shows increased fluorescence after 4 min of E2 application. Arrows indicate dendritic spine-like structures. (C) Kinetic analysis of the FRET signal analyzed by spectrofluorometry. Cortical neurons were preloaded with the FRET probe and were treated with E2 (10 nM) for various periods of times. Maximum increase in fluorescence was observed after 5 min. Fluorescence intensity was normalized by subtraction of values measured in untreated controls and the results represent means of ± SEM of eight experiments. *, P < 0.05 (Student's t test).