Fig. 1

Acute pruriceptive stimulus activates Glu^LHb neurons. A Representative images of c-Fos immunostaining in pruritogen-treated or saline-treated mice. Scale bars, 200 μm and 50 μm. B Quantification of c-Fos staining in pruritogen-treated or saline-treated mice in the LHb (n = 4 mice per group). C Schematic depicting the fiber photometry apparatus, viral injection and optical fiber implantation for GCaMP6s expression used to measure Ca²⁺ signals in LHb CaMKIIα neurons. D Schematic diagram of the experimental design. E Left: representative immunofluorescence staining images showing viral expression and position of the fiber track in a C57 mouse. MHb: the medial habenula. Right: Quantification (pie chart) show double staining of anti-Glutamate and GCaMP6s in the LHb (n = 3 or 4 sections per animal from 4 mice). Scale bars, 200 μm (left) and 50 μm (right). F Left: Peri-event plots of the average change of Ca²⁺ fluorescence intensity when airing puff to the body. Vertical gray dashed line: onset of air puff stimulus; red, GCaMP6s virus channel recording; green, EGFP control virus channel recording; Middle: the heat map of Ca²⁺ transients aligned to the beginning of air puff in 40 trials from 5 mice (8 trials per mouse). Vertical white dashed line, onset of the air puff stimulus. Color scale indicates △F/F; Right: Quantification analysis of area under the curve (AUC) during 0–5 s of average Ca²⁺ fluorescence induced by air puff stimulus in the GCaMP6s group compared with the EGFP group. G The average Ca²⁺ fluorescence during histamine-evoked scratching. 25 trials from 5 mice were statistically analyzed (5 trials per mouse). Vertical gray and white dashed line: onset of the histamine-evoked scratching. H The average Ca²⁺ fluorescence during chloroquine-evoked scratching. 40 trials from 5 mice were statistically analyzed (8 trials per mouse). Vertical gray and white dashed lines: onset of chloroquine-evoked scratching. Significance was assessed by two-tailed unpaired Student’s t-test in (B, F-H), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data were shown as mean ± SEM.