Figure 3. Regulation of dopamine (DA)-mediated activation of the Golgi-localized D1DR in striatal neurons by OCT2.

(a) Representative medium spiny neurons (MSN) expressing Snap-D1DR and Nb6B9-GFP at indicated times after 10 µM DA addition. DA stimulates D1DR activation at the Golgi in MSNs (n = 22 cells, Pearson’s coefficient = 0.67, six biological replicates). Arrow indicates active D1DR at plasma membrane; Arrowhead indicates active D1DR at Golgi membrane; Scale bar = 10 µm. (b) Representative MSN cell expressing Snap-D1DR and Nb6B9-GFP, pretreated with 100 μM imipramine for 15 min, before and after 10 µM DA addition. Inhibition of OCT2 blocks Golgi-localized D1DR activation at MSN n = 18 cells, Pearson’s coefficient = 0.38, six biological replicates but SKF81297 can still reach the Golgi membranes and activate D1DR Golgi pool (n = 6 cells, Pearson’s coefficient = 0.75, four biological replicates). Arrow indicates active D1DR at plasma membrane; Arrowhead indicates active D1DR at Golgi membrane; scale bar = 10 μm. (c) Quantification of Nb6B9-GFP recruitment at Golgi upon 10 μM DA stimulation in MSNs cells pretreated with OCT2 inhibitor; normalized fluorescence intensity of Nb6B9-GFP relative to Snap D1DR at Golgi (n = 12 and 7, respectively, five biological replicates). (d) Representative MSN expressing Snap-D1DR and Nb37-GFP before and after 10 µM DA addition. DA stimulates G protein activation at the Golgi in D1DR-expressing MSNs (n = 10 cells, Pearson’s coefficient = 0.34 and 0.62 before and after 10 µM DA stimulation, six biological replicates). Arrowhead indicates active Gs at Golgi membrane; right panels show zoomed images of insets for Snap-D1DR, Nb37-GFP, and the Golgi marker (GM130). Scale bar = 10 μm.

Figure 3—figure supplement 1. Medium spiny neurons endogenously express OCT2 and D1DR at the plasma membrane and the Golgi.

(a) Detection of OCT2 expression in different tissue extracts and brain slices by Western blot. (b) Top panel: colocalization of Nb6B9-GFP at the Golgi after 10 μM dopamine (DA) stimulation for 10 min. Lower panel: endogenous localization of D1DR at the plasma membrane and the Golgi membranes. Medium spiny neuron (MSN) cells were labeled with D1DR-specific antibody and the Golgi antibody (GM130). Arrowhead indicates Golgi localizations. (c) Endogenous localization of D1DR at the plasma membrane and the Golgi membranes in the presence or absence of blocking peptide. MSN cells were labeled with D1DR-specific antibody and the Golgi antibody (GM130) (n = 36 cells, Pearson’s coefficient = 0.67, three biological replicates). Arrowhead indicates active D1DR at Golgi membrane; scale bar = 10 µm. (d) Endogenous localization of OCT2 at the plasma membrane and the Golgi membranes. MSN cells were labeled with OCT2-specific antibody and the Golgi antibody (GM130) (n = 34 cells, Pearson’s coefficient = 0.6, three biological replicates).

Figure 3—video 1. Confocal image series of D1DR-expressing medium spiny neurons (MSNs) (magenta) and Nb6B9-GFP (cyan), incubated with 10 μM dopamine.

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Figure 3—video 2. Confocal image series of D1DR-expressing medium spiny neurons (MSNs) (magenta) and Nb6B9-GFP (cyan), pretreated with 100 μM imipramine and incubated with 10 μM dopamine and SKF81297.

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