Figure 1. PDE10A inhibition increases cAMP levels in both in D1 and D2 MSNs, and PKA-dependent phosphorylation only in D2 MSNs. A, MSNs in a neostriatal mouse brain slice expressing the cAMP biosensor Epac-SH150 were imaged with two-photon microscopy during the application of PQ-10 (100 nm). Images (vertical projection of the image stack) show the raw fluorescence at 535 nm (left, in grayscale) and the ratio (in pseudocolor) indicating intracellular cAMP concentrations, at the times indicated by the arrows on the graph below. The calibration square in A indicates the spatial scale (the size of the square is indicated in micrometers), and shows the ranges of intensity (horizontally) and ratio (vertically). Each trace on the graph indicates the F480/F535 emission ratio measured in regions indicated by the color contour drawn on the raw image. Traces in gray correspond to regions that are not visible on these images. Traces are plotted in two groups according to their response to either CGS 21680, an adenosine A2A receptor agonist (CGS, 1 µm), or SKF-38393, a D1-like receptor agonist (SKF, 1 µm). The thick black line represents the average of all the traces in a group. FSK (13 µm) and IBMX (200 µm) were applied at the end of the recording to determine the maximal response. B, The same experiment was repeated for every PQ-10 concentration tested. No significant difference was found between D1 and D2 MSNs (two-way ANOVA: dose effect, F(6,54) = 40.91, p < 10
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4; D1/D2 effect, F(1,54) = 2.56, p = 0.115; dose × D1/D2 interaction, F(6,54) = 0.625, p = 0.709). Error bars indicate the SEM. C, Same as A, except that the AKAR3 biosensor was used to monitor PKA-dependent phosphorylation, and the ratio was calculated as F535/F480. D, Same as B for AKAR3 measurements. Data were analyzed with two-way ANOVA: dose effect, F(6,38) = 28.31, p < 10
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4; D1/D2 effect, F(1,38) = 143.73, p < 10
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4; dose × D1/D2 interaction, F(6,38) = 9.23, p < 10
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4 Bonferroni’s post hoc test, ***p < 0.001 .