Fig. 2: HypnoS reveals distinct iAdo dynamics in neurons and astrocytes in cultures. | Nature Communications

Fig. 2: HypnoS reveals distinct iAdo dynamics in neurons and astrocytes in cultures.

From: A high-performance fluorescent sensor spatiotemporally reveals cell-type specific regulation of intracellular adenosine in vivo

Fig. 2

a Schematic diagram illustrates the experimental protocol. DIV refers to days in vitro. b Representative pseudocolor images of HypnoS- or PercevalHR-expressing neurons before and after treatment with 10 mM 2-DG. c Left panel shows example traces of the fluorescence response for both sensors. Right panel summarizes the peak ΔR/R0 change. The HypnoS and PercevalHR fluorescence response is presented as a pixel-by-pixel ratio between the 488 nm excitation image and the 405 nm excitation image (F488/F405). ΔR/R0 denotes the change in the fluorescence ratio excited at 488 nm and 405 nm. n = 112 region of interest (ROIs) from 3 cultures. Two-tailed Student’s t-test: for iAdo, P = 3.1 × 10−83 between baseline and 2-DG, P = 5.0 × 10−146 between baseline and Ado; for ATP/ADP, P = 4.4 × 10−106 between baseline and 2-DG. Scale bar: 100 μm. d Representative pseudocolor images of HypnoS- or PercevalHR-expressing neurons before and after electrical field stimulation (10 Hz). e Left panel shows example traces of the fluorescence response for both sensors. Right panel summarizes the peak ΔF/F0 and ΔR/R0 change. For HypnoS, ΔF/F0 denotes the change in fluorescence intensity excited at 405 nm. For PercevalHR, ΔR/R0 denotes the change in the fluorescence ratio excited at 488 nm and 405 nm. Data are from n = 110 ROIs from 3 cultures. Two-tailed Student’s t-test: for iAdo, P = 1.1 × 10−68 between baseline and 600 pulses; for ATP/ADP, P = 3.4 × 10−104 between baseline and 600 pulses. Scale bar: 100 μm. f, g Same to b, c except HypnoS was expressed in astrocytes. Data are from n = 90 ROIs from 3 cultures. Two-tailed Student’s t-test: for iAdo, P = 1.1 × 10−22 between baseline and 2-DG, P = 2.9 × 10−80 between baseline and Ado; for ATP/ADP, P = 3.4 × 10−30 between baseline and 2-DG. Scale bar: 100 μm. h Representative pseudocolor images of cultured hippocampal astrocytes co-expressing HypnoS (upper panel) and jRGECO1a (calcium indicator, lower panel) in response to 100 μM ATP or 30 nM Thrombin. i Left panel shows example traces of the fluorescence response for HypnoS and jRGECO1a. Right panel summarizes the peak ΔF/F0 change for HypnoS. Data are from n = 102 cells from 3 cultures. Scale bar: 100 μm. All the data are shown as mean ± s.e.m. with the error bars or shaded regions indicating the s.e.m. ***P < 0.001. Source data are provided as a Source Data file.

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