Gene expression of and was determined by real-time PCR (StepOnePlus, Applied Biosystems)
Gene expression of and was determined by real-time PCR (StepOnePlus, Applied Biosystems). itch, GRPR+ neurons are likely to act downstream of NMBR+ neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions. Together, we define the respective function of NMBR and GRPR in itch transmission, and reveal an unexpected relationship not only between the two receptors but also between the two populations of interneurons in itch signaling. KO mice (Hampton et al., 1998), NMB-eGFP mice (MMRRC), NMBR-eGFP mice (MMRRC), and their wild-type (WT) littermates were used. double-knock-out mice were generated by crossing KO mice. We validated NMBR-eGFP mice using single-cell reverse transcription-PCR Exicorilant (RT-PCR). All eGFP+ neurons picked from spinal sections showed expression of mRNA, but not mRNA (= 9; data not shown). All mice were housed under a 12 h light/dark cycle with food and water provided Gpr124 hybridization. Immunohistochemistry (IHC) staining was performed as described previously (Chen et al., 2001; Zhao et al., 2006). Briefly, mice were anesthetized with an overdose of a ketamine/xylazine cocktail and fixed by intracardiac perfusion of cold 0.01 m PBS, pH 7.4, and 4% paraformaldehyde. Tissues were immediately removed, postfixed in the same fixative overnight at 4C, and cryoprotected in 30% sucrose solution. Tissues were frozen and sectioned at 20 m thickness on a cryostat. Free-floating sections were blocked in a solution containing 2% donkey serum and 0.3% Triton X-100 in PBS for 1 h at room temperature. The sections were incubated with primary antibodies or fluorescein isothiocyanate (FITC)-conjugated Isolectin B4 (IB4) overnight at 4C followed by secondary antibodies. The secondary antibodies were purchased from Jackson ImmunoResearch Laboratories including Cy3- or FITC-conjugated donkey anti-rabbit or anti-mouse IgG (Cy3, 0.5 g/ml; FITC, 1.25 g/ml), biotin-SP (long-spacer)-conjugated donkey anti-chicken or anti-rabbit IgG (1 g/ml) and Alex Fluor 488-avidin (0.33 g/ml). hybridization (ISH) was performed using digoxigenin-labeled cRNA probe as previously described (Chen et al., 2001). For antibody staining after ISH of KO mice (Liu et al., 2009; Zhao et al., 2013), demonstrating that the anti-GRP antibody does not recognize other proteins. Preadsorption with GRP also resulted in a complete loss of immunofluorescence in mouse DRGs (Fleming et al., 2012). Rabbit antiserum against Fluoro-Gold (FG; AB153, Millipore) was used at a concentration of 1 1:5000 (Bernstein et al., 2006). Control mice that did not Exicorilant receive injections of FG did not produce any immunostaining. FITC-conjugated IB4 (L2895, Sigma-Aldrich) was used at a concentration of 5 g/ml (Reisfeld et al., 1967). Retrograde tracing. A total of 32 adult NMBR-eGFP male mice were anesthetized with an intraperitoneal injection of a ketamine/xylazine cocktail and were fixed in a stereotaxic frame (Stoelting). An incision was made along the midline of the skull, and a small hole was drilled through the bone over the approximate location of the injection sites. A pulled borosilicate glass pipette with a tip that was 20 m in diameter was backfilled with mineral oil and attached to a Nanoject II auto-nanoliter injector. The injector was attached to a manipulator and moved to the coordinates. A solution of 4% FG (Biotium) was pulled into the pipette. FG (0.150.25 l) was injected into each injection site in different brain areas. For thalamus (ventral posterolateral thalamic nucleus; ventral posteromedial thalamic nucleus; posterior thalamic nuclear group; and posterior thalamic nuclear group, triangular part), 0.15 l of FG was injected into Exicorilant site a [anteroposterior (AP), ?0.94; mediolateral (ML), 1.00; dorsoventral (DV), ?3.45], 0.25 l into site b (AP, ?1.82; ML, 1.10; DV, ?3.20), and 0.15 l into site c (AP, ?2.18; ML, 1.25; DV, ?3.25). For parabrachial nucleus (PBN; lateral PBN, medial PBN, superior cerebellar peduncle, and K?lliker-Fuse nucleus), 0.25 l of FG was injected into this site (AP, ?5.20; ML, 1.25; DV, ?2.40). For ventrolateral periaqueductal gray matter (VLPAG), 0.20 l of FG was injected into this site (AP, ?4.84; ML, 0.70;.