Figure 1. SNAP induces CGRP expression and production in TGNs. Primary TGNs isolated from 3-day-old Wistar rats (n = 9) were cultured as described in Materials and Methods and subjected to the experiments as follows. (A) Cells were treated with 1.0 mM SNAP for 2 h, followed by culture in NB medium without SNAP for an additional 24 h, after which cells were fixed with 4% paraformaldehyde. Double immunofluorescent staining was then performed using primary antibodies against CGRP (red) and Tuj1 (green, a marker of neurons). Cells were counterstained with Heochst33342 (blue) for nuclei. Scale bar = 20 μm. Arrows indicate representative neurons with increased expression of CGRP. (B) Cells were treated with the indicated concentrations of SNAP for 2 h, followed by culture in NB medium without SNAP for an additional 24 h, after which qPCR was performed to monitor the mRNA levels of CGRP. (C) In parallel, ELISA was conducted to determine the amount of CGRP protein in culture medium.