Journal: bioRxiv

Article Title: In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors

doi: 10.1101/2024.12.22.629999

Figure Lengend Snippet: ( A ) (Left) Schematic diagram illustrating the principle of the far-red dopamine (DA) sensor (top left) and L-Z equilibrium of rhodamine derivatives (bottom left). (Right) Idealized traces depicting the emission spectra of current GFP-and RFP-based sensors, alongside the new far-red and near-infrared (NIR) sensors. ( B ) Optimization of far-red DA sensor variants in response to 100 μM DA application, with stepwise changes in the insertion sites, linker, cpHaloTag and GPCR optimization. The variants in step 1 were screened using the dye JF635, while the variants in steps 2, 3, and 4 were screened using the dye JF646. ( C ) Representative images of HEK293T cells expressing HaloDA1.0 and labeled with JF646, before and after application of 100 μM DA. Scale bar, 20 μm. ( D ) Dose-response curves of HaloDA1.0 and HaloDAmut labeled with JF646 in HEK293T cells; n = 3 wells with 300–500 cells per well. ( E ) One-photon excitation (Ex) and emission (Em) spectra of HaloDA1.0 labeled with JF646 in the presence of 100 μM DA (solid lines) or saline (dashed lines). F.I., fluorescence intensity. ( F ) Maximum ΔF/F 0 (left) and normalized dose-response curves (right) for HaloDA1.0 labeled with the indicated dyes in HEK293T cells; n = 3 wells with 300–500 cells per well for each dye. ( G ) Representative images of cultured rat cortical neurons expressing HaloDA1.0 and labeled with the indicated dyes (top row) and fluorescence response to 100 μM DA (bottom row). Scale bar, 50 μm. ( H ) Dose-response curves (left), maximum ΔF/F 0 (top right), and signal-to-noise ratio (SNR) relative to JF635 (bottom right) for cultured rat cortical neurons expressing HaloDA1.0 and labeled with the indicated dyes; n = 120 regions of interest (ROIs) from 4 coverslips for each dye. ( I ) Normalized ΔF/F 0 (relative to DA) for HaloDA1.0 expressed in cultured neurons and labeled with JF646. SCH, SCH-23390 (D1R antagonist); Etic, eticlopride (D2R antagonist); SKF, SKF-81297 (D1R agonist); Quin, quinpirole (D2R agonist); 5-HT, serotonin; HA, histamine; OA, octopamine; TA, tyramine; ACh, acetylcholine, GABA, γ-aminobutyric acid; Glu, glutamate; L-Dopa, levodopa. All chemicals were applied at 1 μM; n = 3 wells with an average of 50 neurons per well. The inset shows the dose-response curves for DA and norepinephrine (NE); n = 3–4 coverslips with 30 ROIs per coverslip. ( J ) Luciferase complementation assay to measure G protein coupling. Cells expressing miniGs-LgBit alone served as a negative control; n = 3 wells per group. WT, wild-type. ( K ) Tango assay to measure β-arrestin coupling. Non-transfected cells served as a negative control; n = 3 wells per group. ( L ) Schematic diagram depicting the strategy for multiplex imaging (left) and representative images (right) of cultured neurons co-expressing the far-red DA sensor (JF646-labeled HaloDA1.0), the red fluorescent 5-HT sensor (r5-HT1.0), and the green fluorescent NE sensor (NE2m). Scale bar, 50 μm. ( M ) Fluorescence responses of JF646-labeled HaloDA1.0 (magenta), r5-HT1.0 (red), and NE2m (green). Where indicated, DA (1 μM), 5-HT (1 μM), NE (1 μM), yohimbine (YO, 2 μM), RS23597-190 (20 μM), and SCH (10 μM) were applied; n = 40 ROIs from 3 coverslips.

Article Snippet: During imaging, the following compounds were applied via bath application at the indicated concentrations: DA (Sigma-Aldrich), SCH-23390 (MedChemExpress), eticlopride (Tocris), SKF-81297 (Tocris), quinpirole (Tocris), serotonin (Tocris), histamine (Tocris), octopamine (Tocris), tyramine (Sigma-Aldrich), ACh (Solarbio), γ-aminobutyric acid (Tocris), glutamate (Sigma-Aldrich), levodopa (Abcam), and NE (Tocris).

Techniques: Expressing, Labeling, Saline, Fluorescence, Cell Culture, Luciferase, Negative Control, Transfection, Multiplex Assay, Imaging