Development and characterization of GPR52 sensors in HEK293T cells.

(A) Schematic drawing shows the strategy of developing GRAB GPR52 sensors to identify endogenous ligands of GPR52. (B) Screening and optimization steps of GRAB GPR52 sensors and the fluorescence response to 10 μM synthetic agonist; the black arrow indicates the best candidate GPR52-1.0. (C) Example images (left), traces (middle) and quantification (right) of the change in GPR52-1.0 fluorescence in response to 10 μM agonist with (blue) or without (green) antagonist pretreatment; n = 17–20 cells from 3 coverslips; ***p < 0.001; Two-Sample t-test. (D) Normalized dose–response curves measured in HEK293T cells expressing GPR52-1.0, with the corresponding EC50 value for agonist shown; n = 3 repeats. (E) Normalized dose–response curves measured in HEK293T cells expressing GPR52-1.0, with the corresponding IC50 value for antagonist in the presence of 10 μM agonist shown; n = 3 repeats. (F) Top, representative fluorescence changes in GPR52-1.0-expressing cells in response to the local perfusion (100 μM agonist in pipette with normal bath solution). Bottom, group data summarizes on time constants measured upon application of agonist; n = 10 cells from 3 coverslips. (G) Normalized fluorescence change in response to the indicated compounds (each at 10 μM) measured in cells expressing GPR52-1.0. DA, dopamine; L-Dopa, levodopa; NE, norepinephrine; 5-HT, 5-hydroxytryptamine; GABA, γ-aminobutyric acid; His, histamine; ACh, acetylcholine; n = 3 repeats; ***p < 0.001; One-Way ANOVA. In this and subsequent figures, unless indicated otherwise summary data are presented as the mean±SEM.

Characterization of GPR52-1.0 in cultured neurons and acute mouse brain slices.

(A) In GPR52-1.0-expressing neurons, example images (left), traces (middle) and quantification (right) of the change in GPR52-1.0 fluorescence in response to 10 μM agonist with (blue) or without (green) antagonist pretreatment; n = 35–36 neurons from 3 coverslips; ***p < 0.001; Two-Sample t-test. (B) Normalized dose–response curves measured in neurons expressing GPR52-1.0, with the corresponding EC50 value for agonist shown; n = 3 repeats. (C) Normalized fluorescence change in response to the indicated compounds (each at 10 μM) measured in neurons expressing GPR52-1.0. When indicated, the antagonist was also added. Glu, glutamate; n = 3 repeats; ***p < 0.001; One-Way ANOVA. (D) Left: top, schematic illustration depicting the experimental design of virally expressing GPR52-1.0 in the striatum; bottom, representative pseudo-color images of the fluorescence change in GPR52-1.0-expressing acute mouse brain slices; where indicated, 10 μM agonist and 10 μM antagonist were applied. Middle, a representative trace; where indicated, agonist and antagonist were added. Right, summary data; n = 3 slices; s; *p < 0.05; Paired t-test.

GPR52-1.0 can report the release of GPR52 endogenous ligands ex vivo.

(A) Left, schematic illustration depicting the experimental design in the striatum for (B1–B3); right, a representative image of GPR52-1.0-expressing brain slices. (B1) Representative pseudo-color images of GPR52-1.0-expressing brain slices at baseline (top) and in response to the indicated stimuli (bottom) in the presence of artificial cerebrospinal fluid (ACSF). The dashed circles indicate the ROI used to calculate the response, and the approximate location of the stimulating electrode is indicated. (B2–B3) Representative traces (B2) and summary data (B3) for the change in GPR52-1.0 fluorescence in response to the indicated stimuli in ACSF or antagonist; n = 9 slices.