Research Article: New Research | Disorders of the Nervous System Characterisation of seizure induction methods in Drosophila https://doi.org/10.1523/ENEURO.0079-21.2021 Cite as: eNeuro 2021; 10.1523/ENEURO.0079-21.2021 Received: 2 March 2021 Revised: 2 June 2021 Accepted: 10 June 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 Mituzaite et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Characterisation of seizure induction methods in Drosophila 2 Abbreviated title: Characterisation of seizure induction in Drosophila 3 Jurga Mituzaite1,2, Rasmus Petersen1, Adam Claridge-Chang2,3,4 and Richard A. Baines1 4 1Division of Neuroscience and Experimental Psychology, School of Biological Sciences, 5 Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic 6 Health Science Centre, Manchester, M13 9PL, UK 7 2Institute for Molecular and Cell Biology, A*STAR, Singapore 8 3Department of Physiology, National University of Singapore, Singapore 9 4Program in Neuroscience and Behavioral Disorders, Duke-NUS Medical School, Singapore 10 11 Correspondence should be addressed to Dr. Richard Baines at 12 [email protected] 13 14 Author contributions: JM, ACC and RAB designed research; JM performed research; JM and 15 RP analyzed data; JM, ACC, and RAB wrote the paper. 16 17 6. Number of Figures 5 18 7. Number of Tables 1 19 8. Number of Multimedia 0 20 9. Number of words for Abstract 194 21 10. Number of words for Significance Statement 120 22 11. Number of words for Introduction 868 23 12. Number of words for Discussion 1498 1 24 25 Conflict of Interest 26 The authors report no conflict of interest. 27 28 Funding sources 29 JM was supported by funding from the University of Manchester, UK and the A*STAR 30 Graduate Academy, Singapore. Work on this project was also supported by funding from 31 Biotechnology and Biological Sciences Research Council (BBSRC, BB/N/014561/1). Work 32 on this project benefited from the Manchester Fly Facility, established through funds from 33 the University of Manchester and the Wellcome Trust (087742/Z/08/Z). 34 35 Acknowledgements 36 The authors would like to thank Prof Diane O’Dowd (UC Irvine) for providing DS, DS-C, 37 GEFS+ and GEFS-C fly lines and Dr Joses Ho (A*STAR) for help with Python scripts. 38 Schematics were created with BioRender.com. 39 40 41 42 43 44 45 46 47 48 49 2 50 Abstract 51 Epilepsy is one of the most common neurological disorders. Around one third of patients do 52 not respond to current medications. This lack of treatment indicates a need for better 53 understanding of the underlying mechanisms and, importantly, the identification of novel 54 targets for drug manipulation. The fruitfly Drosophila melanogaster has a fast reproduction 55 time, powerful genetics, and facilitates large sample sizes, making it a strong model of 56 seizure mechanisms. To better understand behavioural and physiological phenotypes 57 across major fly seizure genotypes we systematically measured seizure severity and 58 secondary behavioral phenotypes at both the larval and adult stage. Comparison of several 59 seizure-induction methods; specifically electrical, mechanical and heat-induction, show 60 that larval electroshock is the most effective at inducing seizures across a wide range of 61 seizure-prone mutants tested. Locomotion in adults and larvae was found to be non- 62 predictive of seizure susceptibility. Recording activity in identified larval motor neurons 63 revealed variations in action potential patterns, across different genotypes, but these 64 patterns did not correlate with seizure susceptibility. To conclude, while there is wide 65 variation in mechanical induction, heat induction, and secondary phenotypes, electroshock 66 is the most consistent method of seizure induction across known major seizure genotypes 67 in Drosophila. 68 69 3 70 Significance Statement 71 Epilepsy is a neurological disorder affecting 1 in 130 people globally, with a significant 72 impact on patients, families, and society. Approximately one third of epileptics do not 73 respond to currently available medication. Thus, better insights into underlying disease 74 mechanisms and identification of new drugs are needed. Fruit flies (Drosophila 75 melanogaster) are a powerful genetic model: a number of single gene mutant flies exhibit 76 seizures, phenotypes that have been shown to respond to established antiepileptic drugs. 77 We compare methods of seizure induction and their utility, to establish which induction 78 method is the most consistent across a range of different seizure-inducing genetic 79 backgrounds. Adopting a common method for seizure analysis in this model will, we 80 predict, speed identification of novel anti-convulsive treatments. 4 81 Introduction 82 Epilepsy is one of the most common neurological disorders, affecting ~60 million people 83 worldwide (Chen et al., 2018). While a variety of causes contribute to epilepsy, including 84 traumatic brain injury and brain infections, the major contribution is from underlying 85 genetic mutations (Poduri and Lowenstein, 2011). It has been estimated that ~70% of 86 epilepsies do not have a single known cause; of these, 60% have been associated with 87 genetic mutations (Heron et al., 2007; Poduri and Lowenstein, 2011). There are around 700 88 identified gene mutations currently associated with epilepsy. These include genes 89 contributing to planar cell polarity and the noncanonical WNT signaling pathway (e.g. 90 PRICKLE1), autism spectrum associated genes (e.g. AUTS2) and mTOR signaling pathway 91 genes (e.g. mTOR and TSC1) (Bassuk et al., 2008; Citraro et al., 2016; Wang et al., 2017). 92 However, a majority of epilepsy genes directly influence ion-channel function, specifically 93 mutations in voltage-gated sodium, potassium and calcium channels. 94 Clinicians have access to over 25 antiepileptic drugs (AEDs) to minimize epileptic seizures 95 (Löscher and Schmidt, 2011). A majority of these drugs target ion channels or 96 neurotransmitter signaling, in an attempt to re-establish an appropriate balance between 97 excitatory and inhibitory signaling in the brain (Löscher and Schmidt, 2011; Vezzani et al., 98 2011). Although many new AEDs have been approved in recent years, many new drugs 99 have proven no more effective in treating drug-resistant epilepsies than older compounds 100 (Chen et al., 2018; Moshé et al., 2015; Schmidt and Schachter, 2014). It seems likely that 101 treating drug-refractory epilepsy will require novel drug targets and therefore a deeper 102 understanding of epilepsy at the level of basic mechanism(s). 103 In the genetic model Drosophila, both wild-type and mutant animals exhibit seizure-like 104 behaviors; mutants undergo seizures with greatly reduced stimulus thresholds and/or 105 seizure-like activity (SLA) lasts far longer (Baines et al., 2017; Ganetzky and Wu, 1982; 106 Kuebler and Tanouye, 2000; Parker et al., 2011a; Pavlidis et al., 1994). In the adult fly, SLA 107 includes repetitive proboscis extension, wing buzzing and loss of posture (Parker et al., 108 2011a; Tan et al., 2004). These SLAs have formed the basis of a variety of seizure-severity 109 assays (Pavlidis and Tanouye, 1995). Several reviews have previously characterised fly 110 electrophysiological and behavioural responses to specific seizure assays (Kuebler and 111 Tanouye, 2000; Lee et al., 2019; Parker et al., 2011a; Pavlidis and Tanouye, 1995). In adult 5 112 flies, seizure-susceptible mutants fall into two main categories based on seizure induction: 113 mechanical (termed Bang-sensitive, BS)- and temperature-induced (Burg and Wu, 2012; 114 Ganetzky and Wu, 1982; Kasbekar et al., 1987; Pavlidis and Tanouye, 1995). Mechanical 115 induction has been standardized with the use of a laboratory vortexer to hyper-stimulate 116 sensory inputs and is termed the ‘vortex assay’ (Kuebler and Tanouye, 2000) (Fig. 1A). By 117 contrast, the heat assay exploits temperature change to induce seizure (Burg and Wu, 118 2012; Saras and Tanouye, 2016) (Fig. 1B). There is a third, and more involved seizure 119 induction method in adult flies, known as high-frequency stimulation of the giant fibre (GF) 120 pathway (Pavlidis and Tanouye, 1995). This method induces seizures in all bang-sensitive 121 and other seizure genotypes like shaker (Lee and Wu, 2006). Whilst this assay allows 122 investigation of synaptic transmission during seizures, stimulation of the GF pathway 123 requires a more complex set up than either the vortex or temperature-shock assays and is 124 not suitable for medium- or high-throughput screening. 125 In larvae, seizures have been induced using a simplified electroshock assay, during which 126 the whole body is subjected to electroshock (Marley and Baines, 2011) (Fig. 1C). A 127 particular advantage of larvae is that they are well-suited to drug screening and, moreover, 128 provide unparalleled understanding of CNS structure and function (Choi et al., 2004; Kadas 129 et al., 2015; Lemon et al., 2015; Marley and Baines, 2011; Worrell and Levine, 2008). 130 Genetic epilepsies include syndromes characterized by febrile (heat-‘fever’-induced) 131 seizures that often present in children. Genetic epilepsy with febrile seizures plus (GEFS+) 132 is commonly caused by sodium channel mutations (Camfield and Camfield, 2015). Some 133 extreme cases of GEFS+ are classified as Dravet Syndrome (DS). This often affects children 134 in their first year of life , and has additional comorbidities including motor and mental 135 impairments (Ziobro et al., 2018).