Time course of FRET changes in neurons treated with TgCM analyzed from endogenous CaNA/CaNB and CaNA/CaM FRET pairs. Cultured wild-type primary neurons at 24 DIV were treated with TgCM, and then fixed with 4% paraformaldehyde at indicated time points and immunolabeled with endogenous CaNA-Alexa 488 and either CaNB-Cy3 or CaM-Cy3. A, Time-dependent changes of the FRET based on interaction between endogenous CaNA and CaNB. Within 60 min, application of TgCM caused no significant difference in FRET levels of CaNA and CaNB in cytosolic and nuclear compartments (n ≥ 45 cells). However, significantly increased FRET signal between endogenous CaNA and CaNB in spines was detected as early as 15 min after TgCM application and increased with time. **p < 0.01. B, Gaussian distributions show FRET levels of endogenous CaNA and CaNB in spines in resting condition and changes with time in response to TgCM. C, Histograms of FRET values of CaNA and CaNB in response to TgCM application at indicated time points. In resting condition (0 min), the FRET distribution in spine is dominated by a low FRET peak (0.36; top left panel). The FRET distribution was changed from basal to high level with time after application of TgCM. The FRET peak was 0.45 at 15 min (top right panel), 0.48 at 30 min (bottom left panel), and 0.48 at 60 min after TgCM application (bottom right panel). D, Similarly, time-dependent changes of the normalized FRET levels between CaN and CaM show an early increase in spines after 15 min, whereas no difference was observed in both the cytoplasmic and nuclear compartments up to 60 min. E, Gaussian distribution shows CaNA/CaM FRET levels in spines in resting condition and changes with time in response to TgCM. F, Histograms of CaNA/CaM FRET values after 15 min (peak at 0.38; top right panel), 30 min (peak at 0.39; bottom left panel), and 60 min (peak at 0.4; bottom right panel) of TgCM exposure. In both C and F, inset top panels show CaNA-Alexa 488 fluorescent in dendrites and spines and bottom panels show FRET images of dendritic spines. n = 200 spines for each indicated time point; *p < 0.05. Data represent mean ± SEM.