1698 A. S. St u¨rzebecher and others J Physiol 588.10 non-absorbable sterile surgical thread (Catgut GmbH) and removed at the next surgery. After surgery frogs were kept for one day of observation in the laboratory before being returned to the main aquarium. The preparation of Xenopus laevis oocytes, cRNA trans- cripts and two-electrode voltage clamp recordings were conducted as described previously (Iban˜ez-Tallon et al. 2002). VGSC subunit cRNAs alone or with tethered toxins (ratio channel:toxin was 1:4) were injected into oocytes and recorded 3–5 days later. Electron microscopy Mice (n = 2 per genotype) were anaesthetized by intra-peritoneal injection of 500 μl of 5 mg Ketavet/200 μg Rompun in saline. Reﬂex responses such as limb withdrawal to paw pressure were used to indicate the need for a further dose of anaesthetic. Saphenous nerves were isolated from mice which had been perfused with 4% paraformaldehyde and postﬁxed in 4% paraformaldehyde/2.5% glutaraldehyde in PBS. Following treatment with 1% OsO4 in 0.1 M phosphate buffer for 2 h for osmication, tissue samples were dehydrated in a graded ethanol series and propylene oxide and embedded in Poly/BedR 812 (Polysciences, Inc., Eppelheim, Germany). Semi-thin sections (1 μm) were stained with toluidine blue. Ultra-thin sections (70 nm) were contrasted with uranyl acetate and lead citrate. The numbers of non-myelinated and myelinated axons in the saphenous nerve of wild-type and transgenic mice were determined by the counting of 12 random ﬁelds under the electron microscope and normalising to total cross-sectional nerve area observed in semi-thin sections. Behavioural experiments Mice were backcrossed to C57/BL6/J for at least ﬁve generations. Motor coordination: motor coordination was assessed by placing the mice on an accelerating rotarod apparatus (Ugo Basile, Italy). Animals were scored for time to fall. Thermal nociception: thermal latency was determined using the Plantar test (Ugo Basile) (Hargreaves et al. 1988). Fifteen minutes after placing the mice in the Plexiglas chamber a high intensity light beam was directed at the plantar surface of the hindpaw. Movement of the paw stopped the light beam and the latency was indicated on a digital screen. Measurements were made ﬁve times on each paw and mean values were used for statistical analysis. Mechanical nociception: mechanical sensitivity was determined using a dynamic plantar aesthesiometer (Ugo Basile, Milan, Italy). All animals were acclimatised to the behavioural testing apparatus for 15 min. An increasing mechanical force (1 g s−1) was applied to the plantar surface of the mouse hindpaw. Each paw was measured ﬁve times and mean values of the applied forces were determined and used for statistical analysis. Cold nociception: mice were placed onto a Peltier-cooled plate maintained at 0 ± 1◦C. The number of lickings and jumpings were determined during 90 s. Preferential heat test : mice had free access to two plates kept at 10◦C and 30◦C. The time spent on each plate was measured. Inﬂammatory pain: mice (n = 10 per genotype) were anaesthetized by isoﬂurane inhalation. Thirty microlitres of 2% carrageenan in saline (Fluka) was injected into the plantar surface of the mouse right hindpaw to induce inﬂammatory hyperalgesia. On the following 3 days mice were tested for responses to heat and mechanical stimuli as described for the acute pain studies. Mice were killed by placing in a box ﬁlled with 100% CO2 at the termination of the tests. Statistical analysis Results are presented as means ± S.E.M. Unpaired t tests or two-way ANOVAs were used when indicated. P < 0.05 was considered statistically signiﬁcant. Results To generate a membrane-tethered isoform of MrVIa (t-MrVIa) we inserted a nucleotide sequence coding for the mature toxin between the sequences coding for a secretion signal and a glycophosphatidylinositol (GPI) anchor signal (Iban˜ez-Tallon et al. 2004) (Fig. 1A). Tethered toxin expression in nociceptors was achieved by (BAC) trans- genic methodology (Gong et al. 2002) using genomic DNA that contains the Scn10a nociceptor-speciﬁc gene encoding for the Nav1.8 VGSC α-subunit. Figure 1B shows the diagram of the BAC containing the Scn10a gene and the homology boxes for recombination with the modiﬁed shuttle vector containing the t-toxin cassette. Four transgenic founder lines were identiﬁed by Southern blotting (Fig. 1C). Comparison of the endogenous Scn10a allele to the transgene band indicated that transgenic lines 2 and 27 had the highest BAC transgene copy number. We encountered breeding problems with trans- genic line 27 in that very few transgenic mice were born, which may have been a non-speciﬁc effect due to the size or the site of transgene insertion. We thus used line 2 (Tg(Scn10a-t-MrVIa)2Iit referred to hereafter as Tg-t-MrVIa mice) for all further experiments. Tg-t-MrVIa mice express t-MrVIa and Scn10a in DRGs, but not in other tissues along the nociceptive pathway whereas wild-type littermates showed comparable levels of Scn10a transcripts in DRGs, but lacked t-MrVIa expression (Fig. 2A). As expected from the developmental pattern of Scn10a (Benn et al. 2001), in situ hybridisation C 2010 The Authors. Journal compilation C 2010 The Physiological Society