J Physiol 588.10 Pain modulation by tethered toxins 1697 perfused with extracellular solution containing (in mM): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 4 glucose, 10 Hepes (pH 7.4) and pipettes were ﬁlled with solution containing (in mM): 122 KCl, 10 NaCl, 1 MgCl2, 1 EGTA, 10 Hepes (pH 7.3). PI-PLC (Prozyme) was applied with 10 μg ml−1 for 1 h at 37◦C. TTX experiments were performed with 0.5 μM TTX. Membrane potentials were kept at −60 mV, prepulsed to −120 mV for 150 ms and depolarised from −50 to +40 mV for 50 ms in increments of 10 mV. For current clamp analyses, neurones were held at resting membrane potential and current injection was increased from 80 pA in 80 pA increments. For VGCC recordings, the bath solution was replaced with (mM): 140 TEA-Cl, 2 CaCl2, 10 Hepes, 10 glucose and 100 nM TTX, pH 7.4 with TEA-OH. The pipette solution contained (mM): 120 CsCl, 4 MgCl2, 10 Hepes, 10 EGTA, pH 7.3 with CsOH. Currents were activated by 100 ms depolarisation pulses from a holding potential of −80 to 0 mV with 10 s inter- vals. Recordings were performed using an EPC10 ampliﬁer (HEKA) at a sampling rate of 50 kHz. The series resistance was compensated for 40–60%. Data acquisition and analysis were performed with Patchmaster and Fitmaster software (HEKA Electronics, Darmstadt, Germany). Skin–nerve assays Single afferent units of the saphenous nerve from adult mice were identiﬁed using a mechanical stimulus (Milenkovic et al. 2008). C-ﬁbres were subsequently examined for responsiveness to a cold ramp in which the temperature decreased from 30 to 0◦C. Experiments were carried out blind, i.e. the genotypes of the mice were not known to the experimenter. Cold stimuli were delivered with a computer-controlled Peltier device in contact with the skin receptive ﬁeld. Electroporation and immunostaining of DRG cultures DRG neurones were isolated from an adult mouse and cultured as previously described (Hu & Lewin, 2006). Two hundred micrograms per millilitre of biotinylated isolectin-B4 (IB4; Vector Labs) were used for IB4 live-stainings. DRGs were electroporated (Amaxa) with 5 μg of the pCS2+ eukaryotic expression plasmid containing t-MrVIA (CS2-t-MrVIA) and 0.5 μg of pCS2+ plasmid encoding the green ﬂuorescent protein (CS2-EGFP) and plated on poly-L-lysine–laminin-coated coverslips. One day later, cells were ﬁxed with 4% paraformaldehyde (PFA) and washed in PBS. Three per cent goat serum was used for blocking. The following anti- bodies and markers were used: mouse anti-FLAG (Sigma) at 1:1000, wheat germ agglutinin (Molecular Probes) at 1:200 (5 μg ml−1), goat anti-mouse-Alexa 633 (Molecular Probes) at 1:400. Pictures were taken on a Zeiss LSM confocal microscope. C 2010 The Authors. Journal compilation C 2010 The Physiological Society Transfection and immunoprecipitation in HEK293-T cells HEK293-T cells were transfected with the CS2-t-MrVIa expression vector using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Immuno- precipitation with FLAG agarose beads (Sigma) and Western blot assays were performed as described (Iban˜ez-Tallon et al. 2002). Immunostaining of spinal cord sections Mice (n = 1 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. Mice were then perfused with 4% PFA and cryosections were cut with 14 μm thickness. Mouse sections were blocked with 3% goat serum in PBS. The following antibodies and marker were used for immunostaining: rabbit anti-subP (Zymed Laboratories) at 1:1000, isolectin-B4–Alexa 488 (Vector Labs) at 1:500 and horse anti-rabbit–Alexa 594 (Molecular Probes) at 1:1000. Calcium imaging Standard fura-2-based ratiometric calcium imaging was used to record responses of DRG neurones cultivated from either wild-type or Tg-t-MrVIa mice (2–5 weeks of age) to application of different TRP channel agonists: capsaicin (TRPV1, 100 nM, 30 s), menthol (TRPM8, 100 mM, 30 s) and cinnamaldehyde (TRPA1, 100 mM, 30 s). KCl (40 mM, 10 s) was applied at the end of experiments and only cells exhibiting a robust response were analysed. An inverted microscope (Zeiss Observer A1) equipped with a MetaFluor photonics imaging system, including Poly- chromator V, and a CoolSNAP ES camera (Visitron) was used for cell imaging. Mouse genotype was unknown to the experimenter. Frog surgery and oocyte recordings Xenopus laevis female frogs (n = 5) were anaesthetized by immersion for 30 min in a 0.35% tricaine (Roth, Karlsruhe, Germany) solution until the animals were completely immobile and no further reﬂex limb withdrawal was detected. Frogs were rinsed in water before surgery and kept on ice during the surgery procedure. A relatively small piece of ovary was taken after performing a 1–2 cm-long abdominal incision through the overlying skin and a second incision through the muscles. The muscular incision was closed with DS16-5USP re-absorbable sterile surgical thread (Catgut GmbH). The skin incision was closed with DS16-5USP