Fig. 5

Additional validations of GCaMP-X. a Nuclear accumulation was substantially relieved for cortical neurons transfected with GCaMP-X. N/C fluorescence ratios indicative of GCaMP distributions in cortical neurons (DIV 5) were quantified 2 days after being transfected with GCaMP3, GCaMP3-X_O or GCaMP3-X_C. b GCaMP-X sensors no longer perturbed pCREB signals. For cortical neurons transfected with YFP, GCaMP3-X_O or GCaMP3-X_C, fluorescence of pCREB immunostaining (left) was normalized and compared (right). Dotted lines represent the mean values from nuclear-excluded GCaMP3 (pink) or nuclear-filled GCaMP3 (cyan) neurons, respectively (adopted from Fig. 3c). c Dysregulations of neurite outgrowth were diminished with GCaMP-X. Based on neurite tracing images (left), Sholl analysis (middle) and measurement of neurite length (right) were performed for cortical neurons transfected with YFP, GCaMP3-X_O or GCaMP3-X_C. Similar to b, dotted lines depict mean values of neurite length for nuclear-excluded GCaMP3 (pink) or nuclear-filled GCaMP3 (cyan) neurons (adopted from Fig. 1h). d Problematic nuclear accumulations were strongly attenuated for long-term viral expression of GCaMP6m-X_C. Fluorescence indicative of sensor localization was mostly restricted to the cytosol of neurons infected with AAV-Syn-GCaMP6m-X_C (GCaMP6-X_C), as shown by representative images at different time points (upper panel). The temporal trend of N/C ratio for GCaMP6-X_C (red) is distinct from that for GCaMP6f (black, GCaMP6 data adopted from Fig. 1b). e Apoptotic damages arising from GCaMP were strongly attenuated in GCaMP-X_C expressing neurons. Fluorescence signals of apoptosis (red) were detected by Annexin V assay, to correlate with the expression of sensors (green). AAV-Syn-GCaMP6m-X_C (GCaMP6-X_C), EGFP and GCaMP6f (data adopted from Fig. 1c) were compared in a similar fashion to Fig. 1c. Standard error of the mean (S.E.M.) and Student’s t-test (two-tailed unpaired with criteria of significance: *p < 0.05; **p < 0.01, and ***p < 0.001) were calculated when applicable, and n.s. denotes “not significant”