Extended Data Fig. 8: Transient change in DAN activity is sufficient to modulate PKA activity in SPNs in the NAc.

a, Left, dLight responses in DAT-IRES-cre mice (n = 3 mice) to DAN activation (20 Hz, 2-ms pulse width, 14.3 mW illumination) for different durations of illumination (red = 0.5 s, orange = 1 s, green = 5 s, blue = 10 s, one-way repeated-measures ANOVA, F(1.064, 2.127) = 35.89, P = 0.023). Middle, D2R-SPN FLIM-AKAR responses in DAT-IRES-cre;Adora2a-cre mice (n = 4 mice) plotted in the same way as the left (one-way repeated-measures ANOVA, F(1.353, 4.059) = 4.140, P = 0.109). Right, D2R-SPN FLIM-AKAR responses in DAT-IRES-cre;Adora2a-cre mice (n = 4 mice) to 10 s illumination without (black) and with (red) intraperitoneal injection of D2R antagonist at least 10 mins before recording (paired t-test, valley: P = 0.338, peak: P = 0.033). Statistics performed on mean dLight (0–10 s) and AKAR (end of illumination to ending time + 20 s except for valley estimation (0–10 s) for D2R antagonist experiment) signal. To test whether D2R-SPN PKA activity can respond to DAN activation at all, we activated DAN for 10 s, which increases DA levels far more than does a natural food reward response (b, left). This nonphysiological level of DA release results in a bidirectional modulation of D2R-SPN net PKA activity (b, right) with net PKA activity slightly decreasing and then increasing. However, given that D2R antagonist does not significantly affect the initial reduction in PKA activity, this reduction is unlikely to be D2R-mediated. On the other hand, the delayed activation of PKA was blunted by D2R antagonist, suggesting a contribution of indirect circuit mechanisms, such as modulation of the activity of D2R-expressing cholinergic interneurons in the NAc. b, dLight and D1R-SPN FLIM-AKAR responses to DAN terminal stimulation (20 Hz, 2-ms pulse width) in the NAc (red = VTA DAN stimulation for 1 s and 10.5 mW, orange = DAN terminal stimulation for 1 s and 7.7 mW). Left, dLight responses in DAT-IRES-cre mice (n = 3 mice, paired t-test, P = 0.429). Right, D1R-SPN FLIM-AKAR responses in DAT-IRES-cre;Drd1a-cre mice (n = 6 mice, paired t-test, P = 0.597). Statistics performed on mean dLight (0–3 s) and AKAR (end of illumination to ending time + 20 s) signal. c, Optogenetic induction of ramping DA level change and consequent PKA activity change in SPNs to different illumination duration. Left, dLight responses in DAT-IRES-cre mice (n = 3 mice) to ramping DAN activation for different ramping durations (red = 3 s, orange = 5 s, green = 7 s ramping activation) and to food reward (blue) (one-way repeated-measures ANOVA, F(1.123, 2.246) = 4.260, P = 0.163). To induce ramping DA level change, the frequency of stimulation was gradually increased from 24 Hz to 34 Hz for 3 s (at 10.5 mW), 16 Hz to 34 Hz for 5 s (at 6.1 mW), and 4 Hz to 30 Hz for 7 s (at 10.5 mW). Middle, D1R-SPN FLIM-AKAR responses in DAT-IRES-cre;Drd1a-cre mice (n = 4 mice) plotted in the same way as the left (one sample t-test on 7-s ramp, P = 0.038). Right, D2R-SPN FLIM-AKAR responses in DAT-IRES-cre;Adora2a-cre mice (n = 4 mice) plotted in the same way as the left (one sample t-test on 7-s ramp, P = 0.779). d, D1R-SPN PKA activation versus DA release analysis. Left, mean of D1R-SPN AKAR (0–80 s) versus mean of dLight (0–20 s) for different stimulations (reward, 3-s ramp, 5-s ramp and 7-s ramp). Right, peak of D1R-SPN AKAR (0–80 s) versus mean of dLight (0–20 s) for different stimulations. Each data point represents the average and the s.e. across mice (n = 3 mice for dLight, n = 4 mice for AKAR). All t-tests are two-sided. All graphs are plotted as mean ± s.e.m. (if shaded) across mice. Dashed vertical line = illumination onset. The average response of each mouse was calculated from 10 trials. Blue bars indicate the periods of laser illumination (NAc) for ChrimsonR during which accurate FLIM-AKAR measurements were not possible. Pellet response for dLight was aligned to the peak after receptacle entry and time-shifted so that the upward slope starts near 0 s. Pellet response for D1R-SPN AKAR was aligned to the receptacle entry. Statistics for c were performed on mean dLight signal (0–20 s) and mean AKAR signal (0–80 s).