1 Scorpion venom increases acetylcholine release by prolonging the duration 2 of somatic nerve action potentials. 3 4 Rita de Cássia O Collaçoac, Stephen Hyslopa, Valquíria AC Dorceb, Edson Antunesa, 5 Edward G. Rowanc* 6 7 aDepartment of Pharmacology, Faculty of Medical Sciences, State University of Campinas 8 (UNICAMP). Rua Tessália Vieira de Camargo, 126, Cidade Universitária Zeferino Vaz, 9 13083-887, Campinas, SP, Brazil 10 11 bLaboratory of Pharmacology, Division for Scientific Development, Butantan Institute. Avenida 12 Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil. 13 14 cStrathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde. 15 Robertson Trust Wing Building, Room 601, 161 Cathedral Street, G4 0RE, Glasgow, UK. 16 17 *Send correspondence to: 18 Edward G. Rowan PhD. Tel: (+44) 141 548 2689; Fax: (+44) 141 552 2562; E‐ mail: 19 [email protected]. 20 21 Highlights 22 - Low concentrations of T. bahiensis venom enhanced spontaneous and evoked ACh release. 23 - At high concentrations, the venom blocked nerve conduction; 24 - Partial membrane depolarization could account for the enhanced spontaneous release; 25 - A conduction delay increased the quantal content by causing longer nerve depolarization; 26 - Tetrodotoxin-sensitive voltage-gated sodium channels as a major target of the venom. 27 28 29 30 31 Abstract 32 Scorpionism is frequently accompanied by a massive release of catecholamines and 33 acetylcholine from peripheral nerves caused by neurotoxic peptides present in these venoms, 34 which have high specificity and affinity for ion channels. Tityus bahiensis (T. bahiensis) is the 35 second most medically important scorpion species in Brazil but, despite this, its venom remains 36 scarcely studied, especially with regard to its pharmacology on peripheral (somatic and 37 autonomic) nervous system. Here, we evaluated the activity of T. bahiensis venom on somatic 38 neurotransmission using myographic (chick and mouse neuromuscular preparations), 39 electrophysiological (MEPP, EPP, resting membrane potentials, perineural waveforms, 40 compound action potentials) and calcium imaging (on DRG neurons and muscle fibres) 41 techniques. Our results show that the major toxic effects of T. bahiensis venom on 42 neuromuscular function are presynaptically driven by the increase in evoked and spontaneous 43 neurotransmitter release. Low venom concentrations prolong the axonal action potential leading 44 to a longer depolarization of the nerve terminals that enhances neurotransmitter release and 45 facilitates nerve-evoked muscle contraction. The venom also stimulates the spontaneous release 46 of neurotransmitters, probably through partial neuronal depolarization that allows the calcium 47 influx. Higher venom concentrations block the generation of action potentials and resulting 48 muscle twitches. The venom pharmacology was reversed by low concentrations of TTX, 49 indicating voltage-gated sodium channels as the primary target of the venom toxins. These 50 results suggest that the major neuromuscular toxicity promoted by T. bahiensis venom is 51 probably mediated mainly by α- and β-toxins interacting with presynaptic TTX-sensitive ion 52 channels on both axons and nerve terminals. 53 54 Key-words: Tityus bahiensis; somatic nervous system; electrophysiology; ion channels; nerve 55 conduction. 56 57 Abbreviations 58 BC: chick biventer cervicis preparation; 59 CAP: compound action potential; 60 EDL: extensor digitorum longus preparation; 61 PND: phrenic nerve-diaphragm preparation; 62 RPM: resting membrane potential. 63 64 1. Introduction 65 Scorpion envenomation is a major health problem in tropical and subtropical regions 66 worldwide with >1.2 million cases reported every year (Chippaux and Goyffon, 2008). In 67 Brazil, scorpionism has assumed epidemic proportions, with data from the Brazilian Ministry 68 of Health showing that the number of registered cases in this country has increased by 332 % 69 over the last 10 years, with >124,000 cases reported in 2017; scorpion stings are now the 70 primary cause of human envenomation countrywide (Brasil, 2018). Almost all cases of 71 scorpionism in Brazil are caused by the genus Tityus, with the two most commonly implicated 72 species being T. bahiensis (brown scorpion) and T. serrulatus (yellow scorpion), the latter being 73 clinically the most important (Bucaretchi et al., 2014), particularly in the states of Minas Gerais 74 and São Paulo in southeastern Brazil (von Eickstedt et al., 1996). Other species involved include 75 T. obscurus (Pardal et al., 2014; Torrez et al., 2015) and T. silvestris in the eastern Brazilian 76 Amazon (Monteiro et al., 2016) and T. stigmurus in northeastern Brazil (Albuquerque et al., 77 2013). To date, the most studied and best characterized venom of the various Tityus spp. is that 78 of T. serrulatus (Bordon et al., 2015; Pucca et al., 2015; Nencioni et al., 2018). 79 The clinical manifestations of envenomation by Tityus spp. include local and systemic 80 effects. The local effects consist of immediate severe pain (described as an excruciating and 81 burning sensation along the affected area) and varying degrees of erythema, swelling (edema) 82 and paraesthesia; the intense pain can trigger transient bradycardia and hypertension can be 83 present (Chippaux and Goyffon, 2008). The systemic effects, in turn, are mediated 84 predominantly by the massive release of sympathetic and parasympathetic neurotransmitters 85 that contribute to central and peripheral neurotoxicity, as well as cardiac and hemodynamic 86 alterations (arrhythmias, bradycardia, tachycardia, hypertension and hypotension) that can lead 87 to cardiogenic shock and pulmonary edema, the primary causes of death (Bucaretchi et al., 88 2014, 2016; Pucca et al., 2015; Housley et al., 2017). Additional systemic effects such as acute 89 kidney injury and pancreatitis have also been observed (Pucca et al., 2015). 90 Scorpionism by Tityus spp. can be life-threatening, with children being more 91 susceptible to fatalities than adults (Bucaretchi et al., 1995, 2014; Chippaux and Goyffon, 2008; 92 Albuquerque et al., 2013). 93 Transcriptomic and proteomic analyses have shown that Tityus venoms contain a variety 94 of toxins, most of which have molecular masses <10 kDa (Barona et al., 2006; Batista et al., 95 2006, 2007; Dias et al., 2018); toxin classes identified include metalloproteinases (the 96 predominant toxin class), Na+ and K+ ion channel toxins, hyaluronidase, hypotensin and a 97 variety of peptides (Pucca et al., 2015; Oliveira et al., 2015, 2018). The modulation of ion 98 channel activity by scorpion toxins is believed to contribute to most of the sympathetic and 99 parasympathetic actions of these venoms (Lourenço et al., 2002; Pucca et al., 2015). Whereas 100 the sympathetic and parasympathetic effects of Tityus venoms have been extensively 101 investigated in laboratory animals (Pucca et al., 2015; Nencioni et al., 2018) and clinically 102 (Bucaretchi et al., 2014; Torrez et al., 2015), much less is known about the effects of these 103 venoms on neurotransmission in skeletal muscle, although the uncoordinated neuromuscular 104 activity may contribute to respiratory failure (Bucaretchi et al., 1995; Torrez, et al., 2015). 105 Envenomation by the Amazonian species T. obscurus in the western Amazon (northern 106 Brazilian state of Pará) is characterized by electric shock-like sensations throughout the body 107 associated with myoclonus, ataxia, dysarthria, dysmetria, dyslalia and muscle fasciculations 108 that are not observed in individuals stung by this species in the eastern part of this state (Pardal 109 et al., 2014; Torrez et al., 2015) or in patients stung by T. bahiensis or T. serrulatus (Bucaretchi 110 et al., 2014). These alterations have been associated with acute cerebellar dysfunction rather 111 than with direct interference in peripheral neurotransmission by venom toxins (Pardal et al., 112 2014; Torrez et al., 2015). Monteiro et al. (2016) described a case of envenomation by T. 113 silvestris in the Brazilian Amazon that involved recurrent generalized severe muscle spasms for 114 three days post-envenomation. As with T. obscurus, the spasms were of central rather than 115 peripheral origin as they were progressively attenuated by diazepam, phenytoin and 116 promethazine; antivenom was ineffective in treating these spasms. 117 Few experimental studies have examined the effects of Tityus venoms on 118 neurotransmission in skeletal muscle, and those that have, have generally used T. serrulatus 119 venom. Vital Brazil et al. (1973) reported that T. serrulatus venom evoked spontaneous muscle 120 twitches in innervated rat diaphragm muscle without electrical stimulation and caused sustained 121 muscle contraction in low Ca2+ solution. The venom also enhanced the twitch responses to 122 supramaximal stimulation and delayed subsequent muscle relaxation. These effects were 123 attributed to the presynaptic release of acetylcholine (ACh) by venom since they were abolished 124 by d-tubocurarine (d-Tc), muscle denervation, the absence of Ca2+ or the presence of Mg2+, 125 neomycin (aminoglycoside antibiotic) or lidocaine (local anaesthetic) in the physiological 126 solution. The venom contained no antiacetylcholinesterase activity that could have contributed 127 to these effects. The venom also had no effect on muscle contractility in directly stimulated 128 curarized preparations although a slight initial transitory increase in baseline tension