Fig. 3. DA_opto effects on STDP stimulation-induced increases in [Ca²⁺]_i and CaMKII activities.

(A and B) Increases in Fluo4-FF fluorescence, representing [Ca²⁺]_i increases, within single spines in response to a train of STDP stimulation in the absence [(A), 20 spines, 15 dendrites] and presence of DA_opto with a 0.6-s delay [(B), 15 spines, 8 dendrites]. Blue laser irradiation during DA_opto is blanked by the blue bar. (C) STDP and DA_opto protocols for Ca²⁺ and CaMKII imaging. Unlike plasticity induction (Fig. 1N), only one train was applied. (D) No effect of DA_opto on the peak values of [Ca²⁺]_i (fig. S6, A to C). P = 0.91 with Kruskal-Wallis test. (E and F) Ratiometric imaging with Camuiα-CR during STDP stimulation in the absence [(E), 33 spines, 14 dendrites] or presence of DA_opto with a 0.6-s delay [(F), 42 spines, 14 dendrites]. Relative increases in the ratio are shown as pseudocolor coding in (E). (Bottom) Time courses of FRET ratios in the spines stimulated with glutamate uncaging or the neighboring spines. Scale bars, 1 μm. (G) Dependence of the peak Camuiα-CR FRET ratios on the DA_opto delay (fig. S6, D to F). P = 1.3 × 10−5 with Kruskal-Wallis test and ***P = 8.4 × 10−5 with Steel test versus those without DA_opto. (H) Normalized increases in Camuiα-CR ratios by STDP + DA_opto with a 0.6-s delay in the stimulated (42 spines, 14 dendrites) and neighboring spines (42 spines, 14 dendrites), and in the presence of DARPP-32 inhibitory peptide in the stimulated spines (43 spines, 10 dendrites) (fig. S6G). Data are presented as mean ± SEM. P = 8.8 × 10⁻⁹ with Kruskal-Wallis test and *** P = 2.6 × 10⁻⁹ and 1.1 × 10⁻⁶ with Steel test.