Fig. 4. BoNT/A differentially inhibits Ca²⁺-dependent CGRP release evoked from rat TGNs by three stimuli and cleaves SNAP25: receptors of BoNT/A occur on VR1-positive cells. TGNs were exposed to BoNT/A and release of CGRP was assayed over 30 minutes. Cells were then solubilised in SDS-sample buffer and equal volumes subjected to SDS-PAGE and western blotting, using an antibody that recognises intact and truncated SNAP25. The proportion of remaining substrate was calculated relative to an internal syntaxin control, using digital images of the gels. (A) Immunoblot showing the cleavage by the neurotoxin of SNAP25 but not syntaxin I. (B). Dose-response curve for BoNT/A-induced blockade of CGRP release evoked by 60 mM K⁺ (), which correlates with the percent of remaining SNAP25 (). Lesser extents of inhibition by BoNT/A were observed for release evoked by 0.1 µM bradykinin () and, especially, 1 µM capsaicin (). Data plotted are the mean ± s.e.m.; n=5. (C) Western blot of TGNs visualised with antibodies specific against syntaxin I or SNAP23 (^*). (D) Representative micrographs demonstrating VR1 and SV2A, SV2B and SV2C in rat TGNs. Fluorescent images were obtained after labelling the cells with antibodies raised in guinea pig specific for VR 1 (1:1000) and in rabbit for SV2A or SV2B (1:1000) or in goat for SV2C (1:100). The controls were treated similarly except in the absence of primary antibodies but incubated with fluorescently labelled secondary IgGs against rabbit (as in Fig. 1) and guinea pig (goat anti-guinea-pig Alexa Fluor-488, 1:200) (1) or goat (donkey anti-goat Cy3, 1:800) and guinea pig (donkey anti-guinea-pig Cy2, 1:200) (2). Bars, 20 µm. Note that all the SV2 isoforms are present in VR1-positive neurons.