J Physiol 588.10 Pain modulation by tethered toxins 1703 Tg-t-MrVIa: 111.6 ± 19.2 pA pF−1, n = 19) (Fig. 3E; Supplemental Table 2). TTX-R peak current densities were indeed higher in IB4+ve neurones (Fig. 3E) consistent with the reported higher content of Nav1.8 in this cell population (Stucky & Lewin, 1999; Snape et al. 2010). We hypothesized that the t-toxin might have a stronger effect on IB4+ve nociceptors since the Scn10a gene promoter was used to genetically drive expression of the t-toxin. Therefore, we measured AP amplitudes in IB4+ve and IB4–ve nociceptors (Fig. 3F). Current clamp assays revealed that IB4+ve but not IB4–ve nociceptors from Tg-t-MrVIa mice had signiﬁcantly reduced AP amplitudes ( = 14.9 mV) (Fig. 3F; Supplemental Table 2). No signiﬁcant changes in the number of myelinated or unmyelinated ﬁbres (A-ﬁbres: Wt 711.8 ± 3.1, Tg-t-MrVIa 714.8 ± 50.7; C-ﬁbres: Wt 2920.6 ± 197.0, Tg-t-MrVIa 2660.7 ± 173.1; n = 3 mice; online Supplemental Fig. 3), and in the distribution of IB4+ve and IB4–ve nociceptor populations (n = 2 mice, online Supplemental Fig. 4) were observed. The central terminations in the outer laminae of the dorsal horn were also unchanged (Fig. 3G). In summary, t-MrVIa selectively reduces TTX-R VGSC currents in nociceptors in the absence of detectable morphological changes. A battery of behavioural tests was performed on Tg-t-MrVIa mice. Motor activity and baseline sensitivities to noxious thermal and mechanical stimuli, as well as thermal hyperalgesia after inﬂammation, were normal (Supplemental Fig. 5; Fig. 4A). In contrast, mechanical hyperalgesia after inﬂammation, shown as the relative threshold for paw withdrawal before and after inﬂammation, was signiﬁcantly reduced in Tg mice with respect to wild-type mice (n = 10 mice) (Fig. 4B), as was the nocifensive response to noxious cold (number of licks and jumps during 90 s on a 0◦C plate: Wt 3.3 ± 0.9, Tg-t-MrVIa 1.2 ± 0.4) (n = 10 mice) (Fig. 4C; Supplemental Movies 1 and 2). No differences in behavioural thermoreception were observed (Fig. 4D). Thus, our data demonstrate that Tg-t-MrVIa mice have strong and speciﬁc deﬁcits in nociception. We next asked whether the behavioural deﬁcits we observed can be explained by changes in the stimulus–response properties of identiﬁed cutaneous nociceptors. We thus used the skin–nerve preparation to measure the ﬁring frequencies of single cold-sensitive C-ﬁbres. Two types of cold-activated ﬁbres were recorded when subjected to a cooling gradient. A low-threshold population that started ﬁring at innocuous temperatures (∼25◦C, Fig. 4E) and a high-threshold population that started to respond only below 10◦C (Fig. 4F). While the ﬁring frequency in the low-threshold population did not differ between wild-type and Tg-t-MrVIa mice (Fig. 4E), the high-threshold population had signiﬁcantly decreased ﬁring rates during the cold ramp (Fig. 4F). Maximal differences were observed around 0◦C in agreement with C 2010 The Authors. Journal compilation C 2010 The Physiological Society our observations on noxious cold-evoked behavioural responses. The ﬁring rates of C-ﬁbres to noxious cold were signiﬁcantly different in Tg-t-MrVIa mice, and were 4.5 ± 1.2 spikes s−1 (n = 8) in wild-type and 1.9 ± 0.5 spikes s−1 (n = 4) in Tg-t-MrVIa mice when the cold ramp reached 0◦C (Fig. 4F). Thus our data indicate that TTX-R VGSC currents blocked by t-MrVIa in nociceptors are required both for nociception in the cold and for detection of noxious cold per se. Discussion This study establishes the utility of genetically encoded, cell membrane-anchored peptide toxins to dissect the contribution of VGSC conductances in a deﬁned neuro- nal type in vivo, and thereby establish a causal link between cellular physiology and behaviour. We show that VGSC currents in nociceptors are cell-autonomously and selectively inactivated by t-MrVIa toxin and that this block can be reversed by enzymatic cleavage of the tether (Fig. 3). Within the targeted neuronal population, the t-toxin shows a high degree of speciﬁcity for TTX-R VGSCs and there was no evidence for compensatory changes in the activity of other ion channels present in the same neurones such as TTX-S VGSCs, VGCCs and TRP channels. Detailed studies of Nav1.8 and Nav1.7 ion channel mutations have demonstrated the distinct roles of these channels in vivo, and compensatory mechanisms that are activated in abnormal physiological states (Akopian et al. 1999; Nassar et al. 2004; Rush et al. 2006). For example, analyses of Nav1.8 knockout mice have shown that functional inactivation of Nav1.8 is accompanied by a signiﬁcant upregulation (about twofold) of TTX-S currents and a compensatory shift of the steady-state inactivation due to over-expression of Scn9a transcripts encoding the TTX-S VGSC Nav1.7 (Akopian et al. 1999; Matsutomi et al. 2006). Here we asked whether a similar compensatory mechanism operates in Tg-t-MrVIa mice. As described above, partial inhibition of TTX-R currents in Tg-t-MrVIa was observed with no evidence of compensation by TTX-S currents. Interestingly the selective, but partial, block of TTX-R currents in the Tg-t-MrVIa mice described here resulted in pain-related phenotypes such as reduced mechanical hyperalgesia and insensitivity to noxious cold, which have been previously observed in Nav1.8 mutant mice and in in vivo models with selective inactivation of Nav1.8 (Akopian et al. 1999; Jarvis et al. 2007; Zimmermann et al. 2007). However, while cutaneous ﬁbres of Nav1.8 mutants show a signiﬁcant reduction of menthol-sensitized cold responses (Zimmermann et al., their Fig. 3G) and reduced ﬁring rates when stimulated by constant mechanical pressure at low temperatures (Zimmermann et al., their Fig. 3E and F) they do not show differences in cold-induced discharge