Fig. 3

The same orexin neuron responded to both itch and pain stimulation. A Schematic diagram representing the temporally controlled expression of the GCaMP6 protein under c-Fos promoter using the Tet-off system in cFos-tTA; TetO-GCaMP6 mice. B Diagram showing the experimental procedure, doxycycline timing, and schedule. C Expression of GCaMP6 (green) and c-Fos (blue) in orexin neurons (red) after the injection of vehicle or stimulants (capsaicin and chloroquine). Yellow arrowheads indicate the ORX cells overlapping with GCaMP6 and c-Fos expression. D The quantification of GCaMP6 and c-Fos expression in orexin neurons revealed a significant increase of GCaMP6 and c-Fos double-positive cells in orexin neurons (%) after pain and itch stimulation compared with the vehicle control. n = 5 for each group. The data represent the mean ± SEM. ***P < 0.001 (unpaired t-test). E Venn diagram showing the response ratio of orexin neurons to pain and itch stimulation. Left, the proportion of orexin neurons that responded to vehicle injection. Right, the proportion of orexin neurons that responded to pain (GCaMP6; green) and itch (c-Fos; red) stimulation. More than half of the orexin neurons responded to both pain and itch stimulation