Dynamics of CREB hot spots in the nuclei of living cortical neurons.

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Dynamics of CREB hot spots in the nuclei of living cortical neurons. Dynamics of CREB hot spots in the nuclei of living cortical neurons. A–C, The number of TMR-CREB spots for the entire observation time (60 s) was counted in each subarea. For the nucleus depicted here, eight and 11 of 157 spots (>5%) accumulated in the two different subnuclear areas defined as hot spots (red), indicated by the arrows. A, The area covering the whole nucleus in a cortical neuron was divided into 625 (25 × 25) equal subareas (0.8 × 0.8 μm). Scale bar, 10 μm. Color contour maps represent the spatial distribution of the frequency of appearance of TMR-CREB spots having long residence times (>1 s) in the living neuron shown in A before (pre, B) and after (post, C)photostimulation. The dashed line marks the perimeter of the nucleus. D, The distribution histogram shows the number of hot spots before (pre) and after (post) photostimulation (0.1 Hz, 1 s duration; n = 26 cells). The average number of hot spots was significantly different before (0.1 ± 0.1) and after stimulation (1.4 ± 0.2; Mann–Whitney U test, p < 0.001). E, The distribution histogram shows the number of hot spots of TMR-CREB before (pre) and after (post) photostimulation (2 Hz, 50 ms duration; n = 19 cells) for 5 min. The average number of hot spots was significantly different before (0.1 ± 0.1) and after stimulation (1.6 ± 0.4; Mann–Whitney U test, p < 0.001). F, The histogram shows the number of TMR-CREB spots with long-residence times (>1 s) observed in hot spots (n = 37) or other sites (n = 32) in 26 cells. Bars represent the mean ± SEM. The asterisk indicates a significant difference between before (pre) and after (post) photostimulation (Mann–Whitney U test, *p < 0.001). G, The distribution histogram shows the number of hot spots in two successive control measurements without photostimulation (n = 16 cells). The average number of hot spots in the first (1st; 0.1 ± 0.1) and the second (2nd; 0.4 ± 0.2) measurement was not significantly different (Mann–Whitney U test, p = 0.686). H, The distribution histogram shows the number of hot spots of mutant CREB (R301L) before (pre) and after (post) photostimulation (0.1 Hz, 1 s duration; n = 6 cells) for 5 min. The average number of hot spots was not significantly different before (0.2 ± 0.2) and after stimulation (0.2 ± 0.2; Mann–Whitney U test, p = 1.000). Hironobu Kitagawa et al. J. Neurosci. 2017;37:1-10 ©2017 by Society for Neuroscience