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Nicotine-Induced Neuroprotection in Rotenone In Vivo and In Vitro Models of Parkinson’s Disease: Evidences for the Involvement of the Labile Iron Pool Level as the Underlying Mechanism

  • Camila Mouhape
  • Gustavo Costa
  • Margot Ferreira
  • Juan Andrés Abin-Carriquiry
  • Federico Dajas
  • Giselle Prunell
ORIGINAL ARTICLE
  • 1 Downloads

Abstract

Parkinson’s disease (PD) is characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Clinical and experimental evidence suggest that the activation of the nicotinic acetylcholine receptor (nAChR) could be protective for PD. In this study, we investigated the neuroprotective capacity of nicotine in a rat PD model. Considering that iron metabolism has been implicated in PD pathophysiology and nicotine has been described to chelate this metal, we also studied the effect of nicotine on the cellular labile iron pool (LIP) levels. Rotenone (1 μg) was unilaterally injected into the median forebrain bundle to induce the degeneration of the nigrostriatal pathway. Nicotine administration (1 mg/K, s.c. daily injection, starting 5 days before rotenone and continuing for 30 days) attenuated the dopaminergic cell loss in the SNpc and the degeneration of the dopaminergic terminals provoked by rotenone, as assessed by immunohistochemistry. Furthermore, nicotine partially prevented the reduction on dopamine levels in the striatum and improved the motor deficits, as determined by HPLC-ED and the forelimb use asymmetry test, respectively. Studies in primary mesencephalic cultures showed that pretreatment with nicotine (50 μM) improved the survival of tyrosine hydroxylase-positive neurons after rotenone (20 nM) exposure. Besides, nicotine induced a reduction in the LIP levels assessed by the calcein dequenching method only at the neuroprotective dose. These effects were prevented by addition of the nAChRs antagonist mecamylamine (100 μM). Overall, we demonstrate a neuroprotective effect of nicotine in a model of PD in rats and that a reduction in iron availability could be an underlying mechanism.

Keywords

Parkinson’s disease Rotenone Nicotine Labile iron pool Neuroprotection 

Notes

Acknowledgments

We thank to Prof. Prem Ponka for providing us with the iron quelator salicylaldehyde isonicotinoyl hydrazone. We also thank Prof. Cecilia Scorza and MSc. José P Prieto for helping with the behavioral experiments, Andrés Di Paolo for his technical assistance with the confocal microscopy, and Dr. Federico Dajas-Bailador for revising the manuscript.

Funding Information

This work was partially supported by the Agencia Nacional de Investigación e Innovación (ANII), Uruguay (FCE2007-517) and Programa de Desarrollo de las Ciencias Básicas (PEDECIBA), Uruguay.

Compliance with Ethical Standards

This study was approved by the Committee on Ethical Care and Use of Laboratory Animals of the IIBCE.

References

  1. Abin-Carriquiry JA, Costa G, Urbanavicius J, Cassels BK, Rebolledo-Fuentes M, Wonnacott S, Dajas F (2008) In vivo modulation of dopaminergic nigrostriatal pathways by cytisine derivatives: implications for Parkinson’s disease. Eur J Pharmacol 589:80–84.  https://doi.org/10.1016/j.ejphar.2008.05.013 CrossRefPubMedGoogle Scholar
  2. Andresen JH, Saugstad OD (2008) Effects of nicotine infusion on striatal glutamate and cortical non-protein-bound iron in hypoxic newborn piglets. Neonatology 94:284–292.  https://doi.org/10.1159/000151648 CrossRefPubMedGoogle Scholar
  3. Beckstead RM, Domesick VB, Nauta WJH (1979) Efferent connections of the substantia nigra and ventral tegmental area in the rat. Brain Res 175:191–217.  https://doi.org/10.1016/0006-8993(79)91001-1 CrossRefPubMedGoogle Scholar
  4. Belluardo N, Olsson PA, Mudo’ G, Sommer WH, Amato G, Fuxe K (2005) Transcription factor gene expression profiling after acute intermittent nicotine treatment in the rat cerebral cortex. Neuroscience 133:787–796.  https://doi.org/10.1016/j.neuroscience.2005.01.061 CrossRefPubMedGoogle Scholar
  5. Blesa J, Phani S, Jackson-Lewis V, Przedborski S (2012) Classic and new animal models of Parkinson’s disease. J Biomed Biotechnol 2012:1–10CrossRefGoogle Scholar
  6. Breuer W, Shvartsman M, Cabantchik ZI (2008) Intracellular labile iron. Int J Biochem Cell Biol 40:350–354CrossRefPubMedGoogle Scholar
  7. Bridge MH, Williams E, Lyons MEG, Tipton KF, Linert W (2004) Electrochemical investigation into the redox activity of Fe(II)/Fe(III) in the presence of nicotine and possible relations to neurodegenerative diseases. Biochim Biophys Acta - Mol Basis Dis 1690:77–84.  https://doi.org/10.1016/j.bbadis.2004.05.007 CrossRefGoogle Scholar
  8. Connolly BS, Lang AE (2014) Pharmacological treatment of Parkinson disease: a review. JAMA 311:1670–1683.  https://doi.org/10.1001/jama.2014.3654 CrossRefPubMedGoogle Scholar
  9. Costa G, Abin-Carriquiry JA, Dajas F (2001) Nicotine prevents striatal dopamine loss produced by 6-hydroxydopamine lesion in the substantia nigra. Brain Res 888:336–342.  https://doi.org/10.1016/S0006-8993(00)03087-0 CrossRefPubMedGoogle Scholar
  10. Dauer W, Przedborski S (2003) Parkinson’s disease. Neuron 39:889–909.  https://doi.org/10.1016/S0896-6273(03)00568-3 CrossRefPubMedGoogle Scholar
  11. Epsztejn S, Kakhlon O, Glickstein H, Breuer W, Cabantchik ZI (1997) Fluorescence analysis of the labile iron pool of mammalian cells. Anal Biochem 248:31–40.  https://doi.org/10.1006/abio.1997.2126 CrossRefPubMedGoogle Scholar
  12. Fine JM, Forsberg AC, Renner DB, Faltesek KA, Mohan KG, Wong JC, Arneson LC, Crow JM, Frey WH 2nd, Hanson LR (2014) Intranasally-administered deferoxamine mitigates toxicity of 6-OHDA in a rat model of Parkinson’s disease. Brain Res 1574:96–104.  https://doi.org/10.1016/j.brainres.2014.05.048 CrossRefPubMedGoogle Scholar
  13. Finkelstein DI, Billings JL, Adlard PA, Ayton S, Sedjahtera A, Masters CL, Wilkins S, Shackleford DM, Charman SA, Bal W, Zawisza IA, Kurowska E, Gundlach AL, Ma S, Bush AI, Hare DJ, Doble PA, Crawford S, Gautier ECL, Parsons J, Huggins P, Barnham KJ, Cherny RA (2017) The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson’s disease. Acta Neuropathol Commun 5:53.  https://doi.org/10.1186/s40478-017-0456-2 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Fossom LH, Sterling C, Tank AW (1991) Activation of tyrosine hydroxylase by nicotine in rat adrenal gland. J Neurochem 57:2070–2077CrossRefPubMedGoogle Scholar
  15. Friedman L, Mytilineou C (1987) The toxicity of MPTP to dopamine neurons in culture is reduced at high concentrations. Neurosci Lett 79:65–72.  https://doi.org/10.1016/0304-3940(87)90673-2 CrossRefPubMedGoogle Scholar
  16. Giniatullin R, Nistri A, Yakel JL (2005) Desensitization of nicotinic ACh receptors: shaping cholinergic signaling. Trends Neurosci 28:371–378CrossRefPubMedGoogle Scholar
  17. Giorguieff-Chesselet MF, Kemel ML, Wandscheer D, Glowinski J (1979) Regulation of dopamine release by presynaptic nicotinic receptors in rat striatal slices: effect of nicotine in a low concentration. Life Sci 25:1257–1261.  https://doi.org/10.1016/0024-3205(79)90469-7 CrossRefPubMedGoogle Scholar
  18. Guo C, Hao LJ, Yang ZH, Chai R, Zhang S, Gu Y, Gao HL, Zhong ML, Wang T, Li JY, Wang ZY (2016) Deferoxamine-mediated up-regulation of HIF-1α prevents dopaminergic neuronal death via the activation of MAPK family proteins in MPTP-treated mice. Exp Neurol 280:13–23.  https://doi.org/10.1016/j.expneurol.2016.03.016 CrossRefPubMedGoogle Scholar
  19. Hare DJ, Double KL (2016) Iron and dopamine: a toxic couple. Brain 139:1026–1035.  https://doi.org/10.1093/brain/aww022 CrossRefPubMedGoogle Scholar
  20. He Y, Thong PSP, Lee T, Leong SK, Shi CY, Wong PTH, Yuan SY, Watt F (1996) Increased iron in the substantia nigra of 6-OHDA induced parkinsonian rats: a nuclear microscopic study. Brain Res 735:149–153.  https://doi.org/10.1016/0006-8993(96)00313-7 CrossRefPubMedGoogle Scholar
  21. He Y, Lee T, Leong SK (1999) Time course of dopaminergic cell death and changes in iron, ferritin and transferrin levels in the rat substantia nigra after 6-hydroxydopamine (6-OHDA) lesioning. Free RadicRes 31:103–112.  https://doi.org/10.1080/10715769900301611 CrossRefGoogle Scholar
  22. Hernán MA, Takkouche B, Caamaño-Isorna F, Gestal-Otero JJ (2002) A meta-analysis of coffee drinking, cigarette smoking, and the risk of Parkinson’s disease. Ann Neurol 52:276–284.  https://doi.org/10.1002/ana.10277 CrossRefPubMedGoogle Scholar
  23. Huang LZ, Parameswaran N, Bordia T, Michael McIntosh J, Quik M (2009) Nicotine is neuroprotective when administered before but not after nigrostriatal damage in rats and monkeys. J Neurochem 109:826–837.  https://doi.org/10.1111/j.1471-4159.2009.06011.x CrossRefPubMedPubMedCentralGoogle Scholar
  24. Jiang H, Qian ZM, Xie JX (2003) Increased DMT1 expression and iron content in MPTP-treated C57BL/6 mice. Sheng Li Xue Bao 55:571–576PubMedGoogle Scholar
  25. Johnson ME, Bobrovskaya L (2015) An update on the rotenone models of Parkinson’s disease: their ability to reproduce the features of clinical disease and model gene–environment interactions. Neurotoxicology 46:101–116.  https://doi.org/10.1016/j.neuro.2014.12.002 CrossRefPubMedGoogle Scholar
  26. Kanlikilicer P, Zhang D, Dragomir A, Akay YM, Akay M (2017) Gene expression profiling of midbrain dopamine neurons upon gestational nicotine exposure. Med Biol Eng Comput 55:467–482.  https://doi.org/10.1007/s11517-016-1531-8 CrossRefPubMedGoogle Scholar
  27. Kaur D, Yantiri F, Rajagopalan S, Kumar J, Mo JQ, Boonplueang R, Viswanath V, Jacobs R, Yang L, Beal MF, DiMonte D, Volitaskis I, Ellerby L, Cherny RA, Bush AI, Andersen JK (2003) Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson’s disease. Neuron 37:899–909.  https://doi.org/10.1016/S0896-6273(03)00126-0 CrossRefPubMedGoogle Scholar
  28. Keller RF, Kanlikilicer P, Dragomir A, Fan Y, Akay YM, Akay M (2017) Investigating the effect of perinatal nicotine exposure on dopaminergic neurons in the VTA using miRNA expression profiles. IEEE Trans Nanobiosci 16:843–849.  https://doi.org/10.1109/TNB.2017.2776841 CrossRefGoogle Scholar
  29. Kosta P, Argyropoulou MI, Markoula S, Konitsiotis S (2006) MRI evaluation of the basal ganglia size and iron content in patients with Parkinson’s disease. J Neurol 253:26–32.  https://doi.org/10.1007/s00415-005-0914-9 CrossRefPubMedGoogle Scholar
  30. Lee S, Woo J, Kim YS, Im HI (2015) Integrated miRNA-mRNA analysis in the habenula nuclei of mice intravenously self-administering nicotine. Sci Rep 5.  https://doi.org/10.1038/srep12909
  31. Linert W, Bridge MH, Huber M, Bjugstad KB, Grossman S, Arendash GW (1999) In vitro and in vivo studies investigating possible antioxidant actions of nicotine: relevance to Parkinson’s and Alzheimer’s diseases. Biochim Biophys Acta - Mol Basis Dis 1454:143–152.  https://doi.org/10.1016/S0925-4439(99)00029-0 CrossRefGoogle Scholar
  32. Liu Q, Zhao B (2004) Nicotine attenuates beta-amyloid peptide-induced neurotoxicity, free radical and calcium accumulation in hippocampal neuronal cultures. Br J Pharmacol 141:746–754.  https://doi.org/10.1038/sj.bjp.0705653 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lu JYD, Su P, Barber JEM, Nash JE, le AD, Liu F, Wong AHC (2017) The neuroprotective effect of nicotine in Parkinson’s disease models is associated with inhibiting PARP-1 and caspase-3 cleavage. PeerJ 5:e3933.  https://doi.org/10.7717/peerj.3933 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Malczewska-Jaskóła K, Jasiewicz B, Mrówczyńska L (2016) Nicotine alkaloids as antioxidant and potential protective agents against in vitro oxidative haemolysis. Chem Biol Interact 243:62–71.  https://doi.org/10.1016/j.cbi.2015.11.030 CrossRefPubMedGoogle Scholar
  35. Marshall DL, Redfern PH, Wonnacott S (1997) Presynaptic nicotinic modulation of dopamine release in the three ascending pathways studied by in vivo microdialysis: comparison of naive and chronic nicotine-treated rats. J Neurochem 68:1511–1519.  https://doi.org/10.1046/j.1471-4159.1997.68041511.x CrossRefPubMedGoogle Scholar
  36. Napolitano A, Pezzella A, Prota G (1999) New reaction pathways of dopamine under oxidative stress conditions: nonenzymatic iron-assisted conversion to norepinephrine and the neurotoxins 6-hydroxydopamine and 6,7-dihydroxytetrahydroisoquinoline. Chem Res Toxicol 12:1090–1097.  https://doi.org/10.1021/tx990079p CrossRefPubMedGoogle Scholar
  37. Newman MB, Arendash GW, Shytle RD, Bickford PC, Tighe T, Sanberg PR (2002) Nicotine’s oxidative and antioxidant properties in CNS. Life Sci 71:2807–2820CrossRefPubMedGoogle Scholar
  38. Nieuwenhuys R, Geeraedts LM, Veening JG (1982) The medial forebrain bundle of the rat. I. General introduction. J Comp Neurol 206(1):49–81CrossRefPubMedGoogle Scholar
  39. Norazit A, Meedeniya ACB, Nguyen MN, MacKay-Sim A (2010) Progressive loss of dopaminergic neurons induced by unilateral rotenone infusion into the medial forebrain bundle. Brain Res 1360:119–129.  https://doi.org/10.1016/j.brainres.2010.08.070 CrossRefPubMedGoogle Scholar
  40. Oakley AE, Collingwood JF, Dobson J, Love G, Perrott HR, Edwardson JA, Elstner M, Morris CM (2007) Individual dopaminergic neurons show raised iron levels in Parkinson disease. Neurology 68:1820–1825.  https://doi.org/10.1212/01.wnl.0000262033.01945.9a CrossRefPubMedGoogle Scholar
  41. Ortega R, Cloetens P, Devès G, Carmona A, Bohic S (2007) Iron storage within dopamine neurovesicles revealed by chemical nano-imaging. PLoS One 2:e925.  https://doi.org/10.1371/journal.pone.0000925 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Papanikolaou G, Pantopoulos K (2005) Iron metabolism and toxicity. Toxicol Appl Pharmacol 202:199–211CrossRefPubMedGoogle Scholar
  43. Parain K, Marchand V, Dumery B, Hirsch E (2001) Nicotine, but not cotinine, partially protects dopaminergic neurons against MPTP-induced degeneration in mice. Brain Res 890:347–350.  https://doi.org/10.1016/S0006-8993(00)03198-X CrossRefPubMedGoogle Scholar
  44. Parain K, Hapdey C, Rousselet E, Marchand V, Dumery B, Hirsch EC (2003) Cigarette smoke and nicotine protect dopaminergic neurons against the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Parkinsonian toxin. Brain Res 984:224–232.  https://doi.org/10.1016/S0006-8993(03)03195-0 CrossRefPubMedGoogle Scholar
  45. Paris I, Martinez-Alvarado P, Perez-Pastene C, Vieira MNN, Olea-Azar C, Raisman-Vozari R, Cardenas S, Graumann R, Caviedes P, Segura-Aguilar J (2005) Monoamine transporter inhibitors and norepinephrine reduce dopamine-dependent iron toxicity in cells derived from the substantia nigra. J Neurochem 92:1021–1032.  https://doi.org/10.1111/j.1471-4159.2004.02931.x CrossRefPubMedGoogle Scholar
  46. Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates, 3rd (edn). Academic Press, San DiegoGoogle Scholar
  47. Picciotto MR (2008) Neuroprotection via nAChRs: the role of nAChRs in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Front Biosci 13:492.  https://doi.org/10.2741/2695 CrossRefPubMedGoogle Scholar
  48. Pringsheim T, Jette N, Frolkis A, Steeves TDL (2014) The prevalence of Parkinson’s disease: a systematic review and meta-analysis. Mov Disord 0:1–8.  https://doi.org/10.1002/mds.25945 Google Scholar
  49. Quik M, Parameswaran N, McCallum SE et al (2006) Chronic oral nicotine treatment protects against striatal degeneration in MPTP-treated primates. J Neurochem 98:1866–1875.  https://doi.org/10.1111/j.1471-4159.2006.04078.x CrossRefPubMedGoogle Scholar
  50. Ren Y, Liu W, Jiang H, Jiang Q, Feng J (2005) Selective vulnerability of dopaminergic neurons to microtubule depolymerization. J Biol Chem 280:34105–34112.  https://doi.org/10.1074/jbc.M503483200 CrossRefPubMedGoogle Scholar
  51. Ritz B, Ascherio A, Checkoway H, Marder KS, Nelson LM, Rocca WA, Ross GW, Strickland D, van den Eeden SK, Gorell J (2007) Pooled analysis of tobacco use and risk of Parkinson disease. Arch Neurol 64:990–997CrossRefPubMedGoogle Scholar
  52. Riveles K, Huang LZ, Quik M (2008) Cigarette smoke, nicotine and cotinine protect against 6-hydroxydopamine-induced toxicity in SH-SY5Y cells. Neurotoxicology 29:421–427.  https://doi.org/10.1016/j.neuro.2008.02.001 CrossRefPubMedPubMedCentralGoogle Scholar
  53. Ryan RE, Ross SA, Drago J, Loiacono RE (2001) Dose-related neuroprotective effects of chronic nicotine in 6-hydroxydopamine treated rats, and loss of neuroprotection in alpha4 nicotinic receptor subunit knockout mice. Br J Pharmacol 132:1650–1656.  https://doi.org/10.1038/sj.bjp.0703989 CrossRefPubMedPubMedCentralGoogle Scholar
  54. Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST (2000) CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology 39:777–787.  https://doi.org/10.1016/S0028-3908(00)00005-8 CrossRefPubMedGoogle Scholar
  55. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedPubMedCentralGoogle Scholar
  56. Singh K, Singh S, Singhal NK, Sharma A, Parmar D, Singh MP (2010) Nicotine- and caffeine-mediated changes in gene expression patterns of MPTP-lesioned mouse striatum: implications in neuroprotection mechanism. Chem Biol Interact 185:81–93.  https://doi.org/10.1016/j.cbi.2010.03.015 CrossRefPubMedGoogle Scholar
  57. Soto-Otero R, Méndez-Álvarez E, Hermida-Ameijeiras Á et al (2002) Effects of (-)-nicotine and (-)-cotinine on 6-hydroxydopamine-induced oxidative stress and neurotoxicity: relevance for Parkinson’s disease. Biochem Pharmacol 64:125–135.  https://doi.org/10.1016/S0006-2952(02)01070-5 CrossRefPubMedGoogle Scholar
  58. Srinivasan R, Henderson BJ, Lester HA, Richards CI (2014) Pharmacological chaperoning of nAChRs: a therapeutic target for Parkinson’s disease. Pharmacol Res 83:20–29CrossRefPubMedGoogle Scholar
  59. Takeuchi H, Yanagida T, Inden M, Takata K, Kitamura Y, Yamakawa K, Sawada H, Izumi Y, Yamamoto N, Kihara T, Uemura K, Inoue H, Taniguchi T, Akaike A, Takahashi R, Shimohama S (2009) Nicotinic receptor stimulation protects nigral dopaminergic neurons in rotenone-induced Parkinson’s disease models. J Neurosci Res 87:576–585.  https://doi.org/10.1002/jnr.21869 CrossRefPubMedGoogle Scholar
  60. Tiwari MN, Agarwal S, Bhatnagar P, Singhal NK, Tiwari SK, Kumar P, Chauhan LKS, Patel DK, Chaturvedi RK, Singh MP, Gupta KC (2013) Nicotine-encapsulated poly(lactic-co-glycolic) acid nanoparticles improve neuroprotective efficacy against MPTP-induced parkinsonism. Free Radic Biol Med 65:704–718.  https://doi.org/10.1016/j.freeradbiomed.2013.07.042 CrossRefPubMedGoogle Scholar
  61. Toulorge D, Guerreiro S, Hild A, Maskos U, Hirsch EC, Michel PP (2011) Neuroprotection of midbrain dopamine neurons by nicotine is gated by cytoplasmic Ca2+. FASEB J 25:2563–2573.  https://doi.org/10.1096/fj.11-182824 CrossRefPubMedGoogle Scholar
  62. Urbanavicius J, Ferreira M, Costa G, Abin-Carriquiry JA, Wonnacott S, Dajas F (2007) Nicotine induces tyrosine hydroxylase plasticity in the neurodegenerating striatum. J Neurochem 102:723–730.  https://doi.org/10.1111/j.1471-4159.2007.04560.x CrossRefPubMedGoogle Scholar
  63. Visanji NP, O’Neill MJ, Duty S (2006) Nicotine, but neither the α4β2 ligand RJR2403 nor an α7 nAChR subtype selective agonist, protects against a partial 6-hydroxydopamine lesion of the rat median forebrain bundle. Neuropharmacology 51:506–516.  https://doi.org/10.1016/j.neuropharm.2006.04.015 CrossRefPubMedGoogle Scholar
  64. Wonnacott S, Kaiser S, Mogg A, Soliakov L, Jones IW (2000) Presynaptic nicotinic receptors modulating dopamine release in the rat striatum. Eur J Pharmacol 393:51–58.  https://doi.org/10.1016/S0014-2999(00)00005-4 CrossRefPubMedGoogle Scholar
  65. Xie YX, Bezard E, Zhao BL (2005) Investigating the receptor-independent neuroprotective mechanisms of nicotine in mitochondria. J Biol Chem 280:32405–32412.  https://doi.org/10.1074/jbc.M504664200 CrossRefPubMedGoogle Scholar
  66. Zhang L, Yagnik G, Jiang D, Shi S, Chang P, Zhou F (2012) Separation of intermediates of iron-catalyzed dopamine oxidation reactions using reversed-phase ion-pairing chromatography coupled in tandem with UV-visible and ESI-MS detections. J Chromatogr B Anal Technol Biomed Life Sci 911:55–58.  https://doi.org/10.1016/j.jchromb.2012.10.026 CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Departamento de NeuroquímicaInstituto de Investigaciones Biológicas Clemente EstableMontevideoUruguay

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