The procedures were described previously37,62. Deeply anesthetized mice (ketamine, 90 mg/kg and Xylazine, 10 mg/kg) were perfused transcardially with 0.01 M PBS (PH 7.4) and paraformaldehyde (PFA) (4% in PBS). Spinal cord and brain were removed and post-fixed in 4% PFA for 2–4 h. The tissues were then cryoprotected in 20% sucrose overnight at 4 °C. Free-floating frozen sections were incubated with 2% donkey serum and 0.3% Triton X-100 for 1 h at room temperature followed by incubation with primary antibodies overnight at 4 °C. The sections were then washed and incubated with secondary antibodies for 2 h at room temperature. The following primary antibodies were used: chicken anti-GFP (1:500, Aves Labs, GFP-1020), guinea-pig anti-NK1R (1:500, AB15810, EMD Millipore), rabbit anti-FG (1:5000, AB153, Millipore). The following secondary antibodies were used: Alexa-Fluor 488 conjugated donkey anti-chicken (1:1000, Jackson ImmunoResearch, 703–545–155), Cy3-conjugated donkey anti-rabbit (1:1000, Jackson ImmunoResearch, 711–165–152) and Cy5-conjugated donkey anti-guinea pig 1(:1000, Jackson ImmunoResearch, 703–175–148). Fluorescent Images were taken using a Nikon C2+ confocal microscope system (Nikon Instruments, Inc.).
To observe the connections between GRPR+ neurons and FG retrograde labelled PBN projection neurons in the spinal dorsal horn, immuno-electron microscopic studies were performed as previously described37. Briefly, for GRPR/FG double staining, cross sections of lumbar spinal cord of adult GRPR-eGFP mice were double immune-labeled by chicken anti-GFP antibody (1:500; Aves Labs) and rabbit anti-FG (1:5000, AB153, Millipore) using immunogold-silver method and immunoperoxidase method, respectively. Further, 50-nm-thick ultrathin sections were cut and examined with a JEM-1400 electron microscope (JEM, Tokyo, Japan). The digital micrographs were captured by VELETA (Olympus,Tokyo, Japan).
Spinal cord slice preparation
Slice preparation and electrophysiological recordings were performed as previously described45. Briefly, GRPR-eGFP mice (P16-P25) were anesthetized with isoflurane and decapitated, the spinal cord and vertebrae were rapidly removed and placed in ice-cold dissecting Krebs’ solution (composition in mM:125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 25 glucose, 6 MgCl2, 1.5 CaCl2, and 1 kynurenic acid, pH 7.4, 320 mOsm), bubbled with 95% O2, 5% CO2. The lumbar spinal cord was isolated, embedded in an agarose block (low melting point agarose 3%, Thermo Fisher Scientific, Waltham, USA), and transverse slices (500 µm thick) were obtained using a vibrating microtome (WPI, Sarasota, USA). Slices were incubated in oxygenated incubation Krebs’ solution (same as dissecting but without kynurenic acid) at 35 °C for 30 min and then used for recording.