A tarantula peptide against pain via ASIC1a channels and opioid mechanisms Michel Mazzuca 1,4 , Catherine Heurteaux 1,4 , Abdelkrim Alloui 2,4 , Sylvie Diochot 1 , Anne Baron 1 , Nicolas Voilley 1 , Nicolas Blondeau 1 , Pierre Escoubas 1 , Agne `s Ge ´lot 2 , Anny Cupo 1 , Andreas Zimmer 3 , Anne M Zimmer 3 , Alain Eschalier 2 & Michel Lazdunski 1 Psalmotoxin 1, a peptide extracted from the South American tarantula Psalmopoeus cambridgei, has very potent analgesic properties against thermal, mechanical, chemical, inflammatory and neuropathic pain in rodents. It exerts its action by blocking acid-sensing ion channel 1a, and this blockade results in an activation of the endogenous enkephalin pathway. The analgesic properties of the peptide are suppressed by antagonists of the l and d-opioid receptors and are lost in Penk1 –/– mice. Improving the treatment and the prevention of acute and chronic pain is an important goal for modern medicine. Ion channels have recently become very attractive targets in the search of new analgesics 1 . Acid-sensing ion channels (ASICs) are expressed throughout the central and peripheral nervous systems 1–5 . ASIC1a, ASIC1b, ASIC2b and ASIC3 are abundantly expressed in small DRG neurons, where ASIC3 is predominently implicated in a variety of pain sensations 6–8 . ASIC1a, ASIC2a and ASIC2b are abundant in brain and spinal cord neurons 2–8 . The most potent pharmacological agent for ASIC channels is psalmo- toxin 1 (PcTx1). This peptide, which was isolated from a tarantula venom 9 , potently and specifically inhibits homomeric ASIC1a channels. PcTx1 is not lethal, even at high concentrations, and could be a candidate for therapeutic applications. PcTx1 blocks ASIC1a channels in brain neurons 9 , as well as in nociceptors 10 . It also blocks ASIC1a channels in spinal neurons (see Supplementary Results and Supple- mentary Fig. 1 online), which permits the use of intrathecal injections. We first evaluated the effects of PcTx1 on behavioral reactions in acute pain models (see Supplementary Methods online). In tail immersion and hot plate tests, a large antinoceptive effect was observed following both intrathecal and intracerebroventricular (ICV) injections of PcTx1 (Fig. 1). Intraperitoneal or subcutaneous injections had no effect, which was expected, as PcTx1 is a peptide. Morphine induced an antinociceptive effect very similar to that of PcTx1 (Fig. 1a,b,d). A similar antinociceptive effect was also produced by intrathecal injection of specific antisense nucleotides (Fig. 1c) that have previously been used successfully 11 to eliminate ASIC1a expression in the retina. We used a formalin test to investigate the PcTx1 effects (intrathecal) in *** *** *** *** *** ** *** *** *** *** *** *** PcTx1 PcTx1 PcTx1 PcTx1 Morphine Morphine Morphine Morphine Vehicle Vehicle Vehicle Vehicle 0 20 40 60 80 100 Licking time (s) Phase II Phase I Formalin 0 5 10 15 20 25 Hot plate latency (s) ICV Intrathecal Hot plate 5 4 3 2 1 0 Veh PcTx1 Time after antisense injection (d) Vehicle Mismatch Antisense INJECTION Tail immersion 25 20 15 10 5 0 Tail-flick latency (s) Tail-flick latency (s) 70 60 50 40 30 20 10 0 Time after administration (min) 70 60 50 40 30 20 10 0 Time after administration (min) 0 5 10 15 20 25 30 35 Tail-flick latency (s) 0 5 10 15 20 25 30 35 * * * * * Tail immersion Tail immersion ICV injection Intrathecal injection Vehicle Morphine PcTx1 * * a b c d e Figure 1 PcTx1, ASIC 1a knockdown and acute pain. (a,b) Time course of the effect of PcTx1 on thermal nociception in healthy mice. Mice (n ¼ 15 per group) received an intrathecal (a) or ICV (b) injection of PcTx1 (0.1 nmol per mouse), morphine (15 nmol in a) or vehicle. (c) Effect of intrathecal injection of specific antisense directed against ASIC1a on thermal nociception in healthy mice. Mice (n ¼ 10 per group) were intrathecally injected twice daily for 4 d with either 10 mg of ASIC1a antisense oligodeoxy- nucleotide or of the mismatch oligodeoxynucleotide, or with the vehicle alone. Results are given as mean ± s.e.m. (d,e) Time course of the effect of PcTx1 on thermal (d) and chemical (e) nociception in healthy mice. Mice received PcTx1, morphine or vehicle as in a and b, but 31 nmol per mouse of morphine were used. *P o 0.05 versus morphine in a and b, ***P o 0.001 versus vehicle in c–e. In conditions used for the pain assays, PcTx1, unlike morphine, had no effect on motor behavior (Supplementary Fig. 7). Received 4 June; accepted 22 June; published online 15 July 2007; doi:10.1038/nn1940 1 Institut de Pharmacologie Mole ´culaire et Cellulaire, Centre National de la Recherche Scientifique (CNRS), Institut Paul Hamel, Universite ´ de Nice-Sophia-Antipolis, 660 Route des Lucioles 06560 Valbonne, France. 2 Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ Mixte de Recherche 766, Faculte ´ de Me ´decine, CHU, Clermont-Ferrand, France. 3 University of Bonn, Department of Molecular Psychiatry, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. 4 These authors contributed equally to this work. Correspondence should be addressed to M.L. (ipmc@ipmc.cnrs.fr). NATURE NEUROSCIENCE VOLUME 10 [ NUMBER 8 [ AUGUST 2007 943 BRIEF COMMUNICATIONS © 2007 Nature Publishing Group http://www.nature.com/natureneuroscience