Heat nociception is dependent on TRPV1 neurons and the transmitters glutamate and CGRP but not substance P. A, R26DTA/wt;Trpv1–Cre mice are less sensitive to thermal pain compared with control mice assessed by the Hargreaves test (n = 12–13 per genotype). Similarly Vglut2f/f;Trpv1–Cre mice displayed decreased sensitivity to Hargreaves stimulation (n = 10 per genotype). Pretreatment with Win51708 (intraperitoneally) did not alter the phenotype and still resulted in decreased sensitivity to heat in Vglut2f/f;Trpv1–Cre mice (n = 10 per genotype). Conversely, intraperitoneal pretreatment with BIBN4096BS led to decreased sensitivity to heat in both control littermates and Vglut2f/f;Trpv1–Cre mice compared with untreated mice of corresponding genotype (n = 6 per genotype). Intrathecal injections of BIBN4096BS also led to a decreased heat sensitivity in control mice compared with untreated control mice (n = 6 per genotype). B, Schematic illustration of the segment of spinal cord with the highest expression of c-Fos induced by Hargreaves stimulation, which was involving mostly segment L2 with some additional sections from the end of segment L1 and beginning of segment L3. C, Ablation of the Trpv1–Cre population resulted in a reduction of c-Fos-expressing spinal cord neurons after Hargreaves stimulation throughout laminae I–III at spinal segment L2 in R26DTA/wt;Trpv1–Cre mice compared with control mice (n = 2 per genotype, 65 sections for controls, 66 sections for R26DTA/wt;Trpv1–Cre). D, Likewise, genetic ablation of Vglut2 from Trpv1–Cre-positive neurons resulted in decreased reduction of c-Fos expression in Vglut2f/f;Trpv1–Cre mice compared with controls (n = 2 per genotype, 58 sections for controls, 65 sections for Vglut2f/f;Trpv1–Cre). E, Win51708 treatment resulted in a reduction of c-Fos-activated neurons in laminae I–III in Vglut2f/f;Trpv1–Cre compared with controls (n = 2 per genotype, 60 sections for controls and 77 sections for Vglut2f/f;Trpv1–Cre). F, Pretreatment with BIBN4096BS led to a drastic reduction of c-Fos-positive cells in laminae I–III in both Vglut2f/f;Trpv1–Cre mice and control littermates (n = 2 per genotype, 58 sections for controls and 65 sections for Vglut2f/f;Trpv1–Cre), representing no statistical difference between the genotypes. Data represent means ± SEMs. Shown are Vglut2f/f;Trpv1–Cre (black), R26DTA/wt;Trpv1–Cre (gray), and respective controls (white). **p < 0.01, ***p < 0.001. Mann–Whitney two-tailed test (A, C–F). Scale bars: B, 72 μm; C–F, 120 μm.