Abstract
PTZ is a convulsive agent that acts via selective blockage of GABAA receptor channels, whereas 4-AP leads to a convulsive episode via blockage of K+ channels. However, the mechanism(s) by which pentylenetetrazole (PTZ) and 4-aminopyridine (4-AP) cause toxicity to Drosophila melanogaster needs to be properly explored, once it will help in establishing an alternative model for development of proper therapeutic strategies and also to counteract the changes associated with exposure to both epileptic drugs. For the purpose, we investigated the effects of exposure (48 h) to PTZ (60 mM) and/or 4-AP (20 mM) on survival, locomotor performance, and biochemical markers in the body and/or head of flies. 4-AP-fed flies presented a higher incidence of mortality and a worse performance in the open field test as compared to non-treated flies. 4-AP also caused a significant increase in the reactive species (RS) and protein carbonyl (PC) content in the body and head. Also a significant increase in catalase and acetylcholinesterase (AChE) activities was observed in the body. In the same vein, PTZ exposure resulted in a significant increase in RS, thiobarbituric acid reactive substances (TBARS), PC content, and catalase activity in the body. PTZ exposure also caused a significant increase in AChE activity both in body and head. It is important to note that PTZ-treated flies also down-regulated the NRF2 expression. Moreover, both 4AP- and PTZ-fed flies presented a significant decrease in MTT reduction, down-regulation, and inhibition of SOD in body. However, SOD was significantly more active in the head of both 4-AP and PTZ-treated flies. Our findings provide evidence regarding the toxicological potential of both PTZ and/or 4-AP to flies. This model will help in decoding the underlying toxicological mechanisms of the stated drugs. It will also help to properly investigate the therapeutic strategies and to counteract the drastic changes associated with both epileptogenic drugs.
Similar content being viewed by others
References
Martinc B, Grabnar I, Vovk T (2014) Antioxidants as a preventive treatment for epileptic process: a review of the current status. Curr Neuropharmacol 12:527–550. doi:10.2174/1570159X12666140923205715
Mehla J, Reeta KH, Gupta P, Gupta YK (2010) Protective effect of curcumin against seizures and cognitive impairment in a pentylenetetrazole-kindled epileptic rat model. Life Sci 87:596–603. doi:10.1016/j.lfs.2010.09.006
Stewart AM, Desmond D, Kyzar E, Gaikwad S, Roth A, Riehl R, Collins C, Monnig L, Green J, Kalueff AV (2012) Perspectives of zebrafish models of epilepsy: what, how and where next? Brain Res Bull 87:135–143. doi:10.1016/j.brainresbull.2011.11.020
White HS (2002) Animal models of epileptogenesis. Neurology 59:S7–S14
Takechi K, Suemaru K, Kawasaki H, Araki H (2012) Impaired memory following repeated pentylenetetrazol treatments in kindled mice. Yakugaku Zasshi 132:179–182
Ferando I, Mody I (2012) GABAA receptor modulation by neurosteroids in models of temporal lobe epilepsies. Epilepsia 53(Suppl 9):89–101. doi:10.1111/epi.12038
Eloqayli H, Dahl CB, Gotestam KG, Unsgard G, Hadidi H, Sonnewald U (2003) Pentylenetetrazole decreases metabolic glutamate turnover in rat brain. J Neurochem 85:1200–1207
Yudkoff M, Daikhin Y, Nissim I, Horyn O, Lazarow A, Nissim I (2003) Metabolism of brain amino acids following pentylenetetrazole treatment. Epilepsy Res 53:151–162
Buckingham SD, Hosie AM, Roush RL, Sattelle DB (1994) Actions of agonists and convulsant antagonists on a Drosophila melanogaster GABA receptor (Rdl) homo-oligomer expressed in Xenopus oocytes. Neurosci Lett 181:137–140
Stilwell GE, Saraswati S, Littleton JT, Chouinard SW (2006) Development of a Drosophila seizure model for in vivo high-throughput drug screening. Eur J Neurosci 24:2211–2222. doi:10.1111/j.1460-9568.2006.05075.x
Gonzalez-Sulser A, Wang J, Queenan BN, Avoli M, Vicini S, Dzakpasu R (2012) Hippocampal neuron firing and local field potentials in the in vitro 4-aminopyridine epilepsy model. J Neurophysiol 108:2568–2580. doi:10.1152/jn.00363.2012
Laura MC, Xochitl FP, Anne S, Alberto MV (2015) Analysis of connexin expression during seizures induced by 4-aminopyridine in the rat hippocampus. J Biomed Sci 22:69. doi:10.1186/s12929-015-0176-5
Wang JW, Wu CF (1996) In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels. Biophys J 71:3167–3176. doi:10.1016/S0006-3495(96)79510-3
Zhao ML, Sable EO, Iverson LE, Wu CF (1995) Functional expression of Shaker K+ channels in cultured Drosophila “giant” neurons derived from Sh cDNA transformants: distinct properties, distribution, and turnover. J Neurosci 15:1406–1418
Yao WD, Wu CF (1999) Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons. J Neurophysiol 81:2472–2484
Pena F, Tapia R (2000) Seizures and neurodegeneration induced by 4-aminopyridine in rat hippocampus in vivo: role of glutamate- and GABA-mediated neurotransmission and of ion channels. Neuroscience 101:547–561
Tapia R, Sitges M, Morales E (1985) Mechanism of the calcium-dependent stimulation of transmitter release by 4-aminopyridine in synaptosomes. Brain Res 361:373–382
Brito VB, Rocha JB, Folmer V, Erthal F (2009) Diphenyl diselenide and diphenyl ditelluride increase the latency for 4-aminopyridine-induced chemical seizure and prevent death in mice. Acta Biochim Pol 56:125–134
Folbergrova J (2013) Oxidative stress in immature brain following experimentally-induced seizures. Physiol Res 62(Suppl 1):S39–S48
Folbergrova J, Kunz WS (2012) Mitochondrial dysfunction in epilepsy. Mitochondrion 12:35–40. doi:10.1016/j.mito.2011.04.004
Ikonomidou C, Kaindl AM (2011) Neuronal death and oxidative stress in the developing brain. Antioxid Redox Signal 14:1535–1550. doi:10.1089/ars.2010.3581
Lin JJ, Mula M, Hermann BP (2012) Uncovering the neurobehavioural comorbidities of epilepsy over the lifespan. Lancet 380:1180–1192. doi:10.1016/S0140-6736(12)61455-X
Patel M (2004) Mitochondrial dysfunction and oxidative stress: cause and consequence of epileptic seizures. Free Radic Biol Med 37:1951–1962. doi:10.1016/j.freeradbiomed.2004.08.021
Waldbaum S, Patel M (2010) Mitochondria, oxidative stress, and temporal lobe epilepsy. Epilepsy Res 88:23–45. doi:10.1016/j.eplepsyres.2009.09.020
Loscher W (2011) Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs. Seizure 20:359–368. doi:10.1016/j.seizure.2011.01.003
Mohammad F, Singh P, Sharma A (2009) A Drosophila systems model of pentylenetetrazole induced locomotor plasticity responsive to antiepileptic drugs. BMC Syst Biol 3:11. doi:10.1186/1752-0509-3-11
Benton R (2008) Chemical sensing in Drosophila. Curr Opin Neurobiol 18:357–363. doi:10.1016/j.conb.2008.08.012
Hirth F (2010) Drosophila melanogaster in the study of human neurodegeneration. CNS Neurol Disord 9:504–523
Marley R, Baines RA (2011) Increased persistent Na+ current contributes to seizure in the slamdance bang-sensitive Drosophila mutant. J Neurophysiol 106:18–29. doi:10.1152/jn.00808.2010
Jeibmann A, Paulus W (2009) Drosophila melanogaster as a model organism of brain diseases. Int J Mol Sci 10:407–440. doi:10.3390/ijms10020407
Parker L, Howlett IC, Rusan ZM, Tanouye MA (2011) Seizure and epilepsy: studies of seizure disorders in Drosophila. Int Rev Neurobiol 99:1–21. doi:10.1016/B978-0-12-387003-2.00001-X
Adedara IA, Abolaji AO, Rocha JB, Farombi EO (2016) Diphenyl diselenide protects against mortality, locomotor deficits and oxidative stress in Drosophila melanogaster model of manganese-induced neurotoxicity. Neurochem Res 41:1430–1438. doi:10.1007/s11064-016-1852-x
Bianchini MC, Gularte CO, Escoto DF, Pereira G, Gayer MC, Roehrs R, Soares FA, Puntel RL (2016) Peumus boldus (Boldo) aqueous extract present better protective effect than boldine against manganese-induced toxicity in D. melanogaster. Neurochem Res 41:2699–2707. doi:10.1007/s11064-016-1984-z
Feany MB, Bender WW (2000) A Drosophila model of Parkinson’s disease. Nature 404:394–398. doi:10.1038/35006074
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358
Levine RL, Wehr N, Williams JA, Stadtman ER, Shacter E (2000) Determination of carbonyl groups in oxidized proteins. Methods Mol Biol 99:15–24. doi:10.1385/1-59259-054-3:15
Perez-Severiano F, Santamaria A, Pedraza-Chaverri J, Medina-Campos ON, Rios C, Segovia J (2004) Increased formation of reactive oxygen species, but no changes in glutathione peroxidase activity, in striata of mice transgenic for the Huntington’s disease mutation. Neurochem Res 29:729–733
Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–77
Lushchak VI, Bagnyukova TV, Husak VV, Luzhna LI, Lushchak OV, Storey KB (2005) Hyperoxia results in transient oxidative stress and an adaptive response by antioxidant enzymes in goldfish tissues. Int J Biochem Cell Biol 37:1670–1680. doi:10.1016/j.biocel.2005.02.024
Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126
Ellman GL, Courtney KD, Andres V, Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95
Hosamani R (2009) Neuroprotective efficacy of Bacopa monnieri against rotenone induced oxidative stress and neurotoxicity in Drosophila melanogaster. Neurotoxicology 30:977–985. doi:10.1016/j.neuro.2009.08.012
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408. doi:10.1006/meth.2001.1262
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Littleton JT, Ganetzky B (2000) Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron 26:35–43
Pandey UB, Nichols CD (2011) Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmacol Rev 63:411–436. doi:10.1124/pr.110.003293
Epel ES, Lithgow GJ (2014) Stress biology and aging mechanisms: toward understanding the deep connection between adaptation to stress and longevity. J Gerontol A 69(Suppl 1):S10–S16. doi:10.1093/gerona/glu055
Martin JR (2004) A portrait of locomotor behaviour in Drosophila determined by a video-tracking paradigm. Behav Processes 67:207–219. doi:10.1016/j.beproc.2004.04.003
Wang P, Saraswati S, Guan Z, Watkins CJ, Wurtman RJ, Littleton JT (2004) A Drosophila temperature-sensitive seizure mutant in phosphoglycerate kinase disrupts ATP generation and alters synaptic function. J Neurosci 24:4518–4529. doi:10.1523/JNEUROSCI.0542-04.2004
Chang HY, Grygoruk A, Brooks ES, Ackerson LC, Maidment NT, Bainton RJ, Krantz DE (2006) Overexpression of the Drosophila vesicular monoamine transporter increases motor activity and courtship but decreases the behavioral response to cocaine. Mol Psychiatry 11:99–113. doi:10.1038/sj.mp.4001742
Caughlan A, Newhouse K, Namgung U, Xia Z (2004) Chlorpyrifos induces apoptosis in rat cortical neurons that is regulated by a balance between p38 and ERK/JNK MAP kinases. Toxicol Sci 78:125–134. doi:10.1093/toxsci/kfh038
Sudati JH, Vieira FA, Pavin SS, Dias GR, Seeger RL, Golombieski R, Athayde ML, Soares FA, Rocha JB, Barbosa NV (2013) Valeriana officinalis attenuates the rotenone-induced toxicity in Drosophila melanogaster. Neurotoxicology 37:118–126. doi:10.1016/j.neuro.2013.04.006
Takahashi S, Abe T, Gotoh J, Fukuuchi Y (2002) Substrate-dependence of reduction of MTT: a tetrazolium dye differs in cultured astroglia and neurons. Neurochem Int 40:441–448
Wang S, Yu H, Wickliffe JK (2011) Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2. Toxicol In Vitro 25:2147–2151. doi:10.1016/j.tiv.2011.07.007
Bibi F, Ullah I, Kim MO, Naseer MI (2017) Metformin attenuate PTZ-induced apoptotic neurodegeneration in human cortical neuronal cells. Pak J Med Sci 33:581–585. doi:10.12669/pjms.333.11996
Fergestad T, Bostwick B, Ganetzky B (2006) Metabolic disruption in Drosophila bang-sensitive seizure mutants. Genetics 173:1357–1364. doi:10.1534/genetics.106.057463
Boiko N, Kucher V, Eaton BA, Stockand JD (2013) Inhibition of neuronal degenerin/epithelial Na+ channels by the multiple sclerosis drug 4-aminopyridine. J Biol Chem 288:9418–9427. doi:10.1074/jbc.M112.449413
Zhang Y, Du Y, Jiang D, Behnke C, Nomura Y, Zhorov BS, Dong K (2016) The receptor site and mechanism of action of sodium channel blocker insecticides. J Biol Chem 291:20113–20124. doi:10.1074/jbc.M116.742056
Ilhan A, Gurel A, Armutcu F, Kamisli S, Iraz M (2005) Antiepileptogenic and antioxidant effects of Nigella sativa oil against pentylenetetrazol-induced kindling in mice. Neuropharmacology 49:456–464. doi:10.1016/j.neuropharm.2005.04.004
de Oliveira CC, de Oliveira CV, Grigoletto J, Ribeiro LR, Funck VR, Grauncke AC, de Souza TL, Souto NS, Furian AF, Menezes IR, Oliveira MS (2016) Anticonvulsant activity of beta-caryophyllene against pentylenetetrazol-induced seizures. Epilepsy Behav 56:26–31. doi:10.1016/j.yebeh.2015.12.040
Golechha M, Bhatia J, Arya DS (2010) Hydroalcoholic extract of Emblica officinalis Gaertn. affords protection against PTZ-induced seizures, oxidative stress and cognitive impairment in rats. Indian J Exp Biol 48:474–478
Ribeiro MC, de Avila DS, Schneider CY, Hermes FS, Furian AF, Oliveira MS, Rubin MA, Lehmann M, Krieglstein J, Mello CF (2005) alpha-Tocopherol protects against pentylenetetrazol- and methylmalonate-induced convulsions. Epilepsy Res 66:185–194. doi:10.1016/j.eplepsyres.2005.08.005
Sharma V, Nehru B, Munshi A, Jyothy A (2010) Antioxidant potential of curcumin against oxidative insult induced by pentylenetetrazol in epileptic rats. Methods Find Exp Clin Pharmacol 32:227–232. doi:10.1358/mf.2010.32.4.1452090
Xie T, Wang WP, Mao ZF, Qu ZZ, Luan SQ, Jia LJ, Kan MC (2012) Effects of epigallocatechin-3-gallate on pentylenetetrazole-induced kindling, cognitive impairment and oxidative stress in rats. Neurosci Lett 516:237–241. doi:10.1016/j.neulet.2012.04.001
Zhen JL, Chang YN, Qu ZZ, Fu T, Liu JQ, Wang WP (2016) Luteolin rescues pentylenetetrazole-induced cognitive impairment in epileptic rats by reducing oxidative stress and activating PKA/CREB/BDNF signaling. Epilepsy Behav 57:177–184. doi:10.1016/j.yebeh.2016.02.001
Ternes AP, Zemolin AP, da Cruz LC, da Silva GF, Saidelles AP, de Paula MT, Wagner C, Golombieski RM, Flores EM, Picoloto RS, Pereira AB, Franco JL, Posser T (2014) Drosophila melanogaster—an embryonic model for studying behavioral and biochemical effects of manganese exposure. EXCLI J 13:1239–1253
Cruz LC, Ecker A, Dias RS, Seeger RL, Braga MM, Boligon AA, Martins IK, Costa-Silva DG, Barbosa NV, Canedo AD, Posser T, Franco JL (2016) Brazilian pampa biome honey protects against mortality, locomotor deficits and oxidative stress induced by hypoxia/reperfusion in adult Drosophila melanogaster. Neurochem Res 41:116–129. doi:10.1007/s11064-015-1744-5
Nguyen T, Nioi P, Pickett CB (2009) The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J Biol Chem 284:13291–13295. doi:10.1074/jbc.R900010200
Patsoukis N, Zervoudakis G, Panagopoulos NT, Georgiou CD, Angelatou F, Matsokis NA (2004) Thiol redox state (TRS) and oxidative stress in the mouse hippocampus after pentylenetetrazol-induced epileptic seizure. Neurosci Lett 357:83–86. doi:10.1016/j.neulet.2003.10.080
Shin EJ, Jeong JH, Chung YH, Kim WK, Ko KH, Bach JH, Hong JS, Yoneda Y, Kim HC (2011) Role of oxidative stress in epileptic seizures. Neurochem Int 59:122–137. doi:10.1016/j.neuint.2011.03.025
Taiwe GS, Moto FC, Ayissi ER, Ngoupaye GT, Njapdounke JS, Nkantchoua GC, Kouemou N, Omam JP, Kandeda AK, Pale S, Pahaye D, Ngo Bum E (2015) Effects of a lyophilized aqueous extract of Feretia apodanthera Del. (Rubiaceae) on pentylenetetrazole-induced kindling, oxidative stress, and cognitive impairment in mice. Epilepsy Behav 43:100–108. doi:10.1016/j.yebeh.2014.11.022
Pahuja M, Mehla J, Kumar Gupta Y (2012) Anticonvulsant and antioxidative activity of hydroalcoholic extract of tuber of Orchis mascula in pentylenetetrazole and maximal electroshock induced seizures in rats. J Ethnopharmacol 142:23–27. doi:10.1016/j.jep.2012.04.006
Choudhary KM, Mishra A, Poroikov VV, Goel RK (2013) Ameliorative effect of Curcumin on seizure severity, depression like behavior, learning and memory deficit in post-pentylenetetrazole-kindled mice. Eur J Pharmacol 704:33–40. doi:10.1016/j.ejphar.2013.02.012
Visweswari G, Prasad KS, Chetan PS, Lokanatha V, Rajendra W (2010) Evaluation of the anticonvulsant effect of Centella asiatica (gotu kola) in pentylenetetrazol-induced seizures with respect to cholinergic neurotransmission. Epilepsy Behav 17:332–335. doi:10.1016/j.yebeh.2010.01.002
Serra M, Dazzi L, Cagetti E, Chessa MF, Pisu MG, Sanna A, Biggio G (1997) Effect of pentylenetetrazole-induced kindling on acetylcholine release in the hippocampus of freely moving rats. J Neurochem 68:313–318
Meilleur S, Aznavour N, Descarries L, Carmant L, Mamer OA, Psarropoulou C (2003) Pentylenetetrazol-induced seizures in immature rats provoke long-term changes in adult hippocampal cholinergic excitability. Epilepsia 44:507–517
Zhang X, Lu L, Liu S, Ye W, Wu J, Zhang X (2013) Acetylcholinesterase deficiency decreases apoptosis in dopaminergic neurons in the neurotoxin model of Parkinson’s disease. Int J Biochem Cell Biol 45:265–272. doi:10.1016/j.biocel.2012.11.015
Craig LA, Hong NS, McDonald RJ (2011) Revisiting the cholinergic hypothesis in the development of Alzheimer’s disease. Neurosci Biobehav Rev 35:1397–1409. doi:10.1016/j.neubiorev.2011.03.001
Greenspan RJ, Finn JA Jr, Hall JC (1980) Acetylcholinesterase mutants in Drosophila and their effects on the structure and function of the central nervous system. J Comp Neurol 189:741–774. doi:10.1002/cne.901890409
Krishna G (2016) Aqueous extract of tomato seeds attenuates rotenone-induced oxidative stress and neurotoxicity in Drosophila melanogaster. J Sci Food Agric 96:1745–1755. doi:10.1002/jsfa.7281
Acknowledgements
The authors are grateful to FAPERGS, CAPES, CNPq, FINEP, INCT-EN, and UNIPAMPA. Additional support was provided by CNPq/FAPERGS/DECIT/SCTIE-MS/PRONEM #11/2029-1 and CNPq (Universal) research grants # 449428/2014-1 and # 456207/2014-7. DCSS and JLP are grateful to CAPES for the scholarship.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that there is no conflict of interest.
Rights and permissions
About this article
Cite this article
Soares, D.C.S., Portela, J.L.R., Roos, D.H. et al. Treatment with pentylenetetrazole (PTZ) and 4-aminopyridine (4-AP) differently affects survival, locomotor activity, and biochemical markers in Drosophila melanogaster . Mol Cell Biochem 442, 129–142 (2018). https://doi.org/10.1007/s11010-017-3198-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-017-3198-3