Figure 2.

lPBN^NK1R neurons respond to sustained noxious stimuli

A: Characterization of NK1R-CreGFP mouse by immunostaining of GFP reporter (green) and in situ hybridization of NK1R mRNA (purple) in lPBN. Representative images show vast majority of GFP⁺ cells are NK1R⁺ (arrows). Right panels show quantification of percentage of GFP/NK1R co-expressing cells in GFP⁺ or NK1R⁺ populations, respectively (n=4 mice per group, including two males and two females). Scale bars: 200 μm for lower magnification and 20 μm for higher magnification, respectively. B, C: Triple-in situ hybridization of NK1R mRNA (purple) with vGlut2 mRNA (green) and VGAT mRNA (red). Right panel in C shows quantification of percentage of NK1R⁺ cells co-expressing vGlut2 or VGAT (n=3 mice per group, including two males and one female). Scale bars: 200 μm for lower magnification and 10 μm for higher magnification. D: Left, schematic of viral injection and fiber photometry recordings for lPBN^NK1R neurons. Right, representative image of AAV-DIO-GCaMP6s expression in slPBN. Scale bars: 200 μm for lower magnification and 50 μm for inset. E, F: No observed increase in average fluorescent Ca²⁺ signals in response to brush (E) and von Frey (F) stimulation (n=3 mice per test, including two males and one female). G–J: Observed increase of average fluorescent Ca²⁺ signals in response to hot plate exposure (G), skin pinching (H), toe clipping (I), and acetic acid injection (J) (n=3 mice per test, including two males and one female). K–M: Representative images of immunostaining of GFP reporter and c-Fos protein expression induced by skin pinching (K), toe clipping (L), and acetic acid injection (M). Arrows indicate co-expression of GFP and c-Fos (n=4 mice per test, including two males and two females). Scale bars: 200 μm for lower magnification, 20 μm for higher magnification, respectively. Green and red circles represent male and female mice, respectively. Data are mean±SEM, unpaired t-test. ^***: P<0.001.