Fig. 1. A temporal profile of dopamine actions on spine enlargement.

(A) Injection of AAV vectors for ChR2 and the D1R-MSN marker (PPTA-mCherry) in 3-week-old DAT-Cre mice. (B) Selective stimulation of dopaminergic and glutamatergic inputs by means of blue laser field irradiation to ChR2 and two-photon uncaging of caged-glutamate at a single spine, respectively, in acute slices of NAc obtained from 5- to 7- week-old mice. (C) An amperometric measurement of dopamine (top) by carbon-fiber electrode and whole-cell recording of glutamate-induced current (bottom, 2pEPSP) in identified D1R-MSNs. (D) An STDP protocol with dopamine puff application. (E) Images of the dendritic spine (red arrowhead) that received STDP stimulation in the presence of dopamine (100 μM). (F and G) Time courses of spine enlargement in the presence [(F), 13 spines, 4 dendrites] and absence of dopamine [(G), 58 spines, 14 dendrites]. (H) Amplitudes of spine enlargements with or without dopamine. **P = 0.0041 by Mann-Whitney U test. (I) STDP with repetitive activation of dopaminergic fibers containing ChR2 (blue lines) at 30 Hz, 10 times (DA_opto). (J) Images of the dendritic spine (arrowhead) that received STDP + DA_opto with a delay of 1 s. (K to M) Time courses of spine enlargement induced by STDP + DA_opto at 1 s [(K), 48 spines, 14 dendrites], −1 s [(L), 20 spines, 5 dendrites] and 5 s [(M), 28 spines, 7 dendrites] after STDP onset. (N) Timings of DA_opto application. (O) Increases in spine volumes by STDP + DA_opto plotted versus DA_opto delay (fig. S2, A to C). Data are presented as mean ± SEM. P = 4.2 × 10⁻⁶ with Kruskal-Wallis and **P = 0.0018 (0.6 s) and 0.0027 (1 s) by Steel test in comparison with STDP in the absence of DA_opto. Scale bars, 1 μm.