Skip to main content
. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Nat Chem Biol. 2020 Sep 28;17(1):39–46. doi: 10.1038/s41589-020-00660-y

Fig. 5 |. ExRai-AKAR2 enables rapid and sensitive kinase activity imaging in vivo.

Fig. 5 |

a, Mice were head-fixed on a circular treadmill and subjected to pupillometry together with two-photon imaging of L2/3 neurons expressing ExRai-AKAR2. b, Average pupillometry timecourses show relative pupil area in mice subjected to forced running before (−CPX, green; n = 7 trials across four animals) and after (+CPX, orange; n = 4 trials across four animals) injection of CPX (5 mgkg−1; highly potent D1R, D2R, D3R, H1R, 5-HT2R, mAchR, α1 adrenergic receptor antagonist). c, Representative traces from a forced locomotion experiment with pupillometry and head-fixed two-photon imaging. Forced locomotion (5cms−1, pink trace) induced robust pupil dilation (mm2, blue trace) and diverse ExRai-AKAR2 responses (ΔF/F, green trace) in V1 L2/3 neurons. Insets, high- (middle) and low-contrast (right) images of a representative neuron expressing ExRai-AKAR2 (F930 nm) and the mean-subtracted response to forced locomotion (μF, right). A three-dimensional region of interest encompassing the neuronal soma was selected to quantify ExRai-AKAR2 responses (dashed circle). Scale bar, 20 μm. Representative images of eye pupil monitoring during baseline and forced running are also shown to the left. Back illumination from scattered two-photon excitation light provided clear pupil segmentation for dilation measurements. d,e, Averaged ExRai-AKAR2 responses for a representative neuron (d) (n = 7 repeats from a single neuron) and average response across the whole population of neurons (e). The average population response was positive before (green trace, P = 0.0140, one-sample t-test, n = 10 neurons from two mice, 0.88± 0.29%) and negative after (orange trace, P= 0.0019, unpaired t-test; n = 4 neurons from two mice, −1.28±0.54%) CPX injection. f, The ExRai-AKAR2 phosphomutant (T/A) showed minimal responses to forced locomotion both before (gray trace, P=0.933, n = 11 neurons from two mice, 0.04±0.40%) and after CPX injections (orange trace, P= 0.895, n = 7 neurons from two mice, 0.05±0.42%). Solid lines in b,d-f indicate mean responses; shaded areas, s.d.