Identification of a receptor for OCN in the brain. (A) Strategy used to identify an OCN brain receptor. (B) In situ hybridization of Gpr158 in E14.5 WT mouse embryos. Bar, 0.5 mm. (C) In situ hybridization of Gpr158, Gpr156, Gpr179, Gprc5a, Gprc5b, Gprc5c, and Gprc5d in the brain of 10-d-old WT mice. Bar, 200 µm. (D) Expression of Gpr158 and Gprc6a in various tissues of 3-mo-old WT mice (Student’s t test, n = 4 mice per group). In the brain-derived tissues, expression is relative to Gpr158 expression, and in peripheral tissues, expression is relative to Gprc6a. (E) In situ hybridization of Gpr158 in the brain of 3-mo-old WT mice. For the ventral tegmental area (VTA), Th was used as a positive control. Bar, 200 µm. (F) Immunofluorescence of Lacz and Map2 in brain slices of 3-mo-old Gpr158−/− mice. The right-most panel is a 40× magnification of the region indicated in the middle panel. Bar, 50 µm. (G) Immunofluorescence of Gpr158, Map2, and Gfap in primary hippocampal neuronal preparation. Bar, 50 µm. (H) Gpr158 and serotonin receptor 2C (SR2C) accumulation in Ocn−/− and WT hippocampi. Na,K ATPase channel was used as a loading control (Student’s t test, n = 4 mice per group). (I) Bioactive OCN content in the serum of 3-mo-old Gpr158−/− (n = 4) and WT (n = 5 mice; Student’s t test). (J) Immunofluorescence of c-Fos 1 h after stereotaxic injection of either saline or OCN (10 ng) into the anterior hippocampus of 3-mo-old Gpr158−/− and WT mice. Bar, 50 µm. (K) Pull-down assay using biotinylated-OCN on solubilized Ocn−/− or Gpr158−/− hippocampal membranes. Purified proteins were subjected to Western blot analysis using anti-Gpr158 and anti-Gαq antibodies. (L) IP1 accumulation in Gpr158−/− and WT hippocampal neurons treated with saline, OCN, or glutamate as a positive control for 1 h (Student’s t test, n = 12). Results are given as mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.