Fluo gel

All mice after behavioral test were perfused and checked for virus expression and implanted fiber or cannula location. For immunofluorescent staining, mice were transcardially perfused with 20 ml PBS followed by 20 ml 4% paraformaldehyde in PBS. For cryosection staining, brains were dissected out and immersed in 15% sucrose overnight. Cryosections of 30 μm thickness were processed. For the vibratome sectioning, brains were removed and postfixed in 4% paraformaldehyde overnight, then the section were cut with a vibratome (Leica, VT1000S) at 100 μm thickness. Sections were stained with primary antibody at 4 °C overnight, in a blocking solution contains 1% BSA or 5% donkey serum and 0.5% Triton X-100. After 3 × 10 min wash in PBS, standard Alexa Fluor secondary antibodies (Invitrogen, 1:250) were used at room temperature for 1 hour. Sections were then washed 3 × 10 min in PBS and mounted in Fluo Gel (17985-10; Electron Microscopy Sciences, with DAPI) and viewed under an Olympus confocal microscope. Primary antibodies used: mouse anti-PKC-δ (BD Biosciences, 610398, 1:500), rabbit anti-CGRP (Bachem, T4032, 1:500), goat anti-c-Fos (Santa Cruz Biotech, sc-52-G, 1:250).