Abstract
The purpose of this study was to clarify the relationship between neuron cells and astrocyte cells in regulating glutamate toxicity on the 10th and 20th day in vitro. A mixed primary culture system from newborn rats that contain cerebral cortex neurons cells was employed to investigate the glutamate toxicity. All cultures were incubated with various glutamate concentrations, then viability tests and histological analyses were performed. The activities of glutamate transporters were determined by using in vitro voltammetry technique. Viable cell number was decreased significantly on the 10th day at 10−7 M and at 10−6 M glutamate applications, however, viable cell number was not decreased at 20th day. Astrocyte number was increased nearly six times on the 20th day as compared to the 10th day. The peak point of glutamate reuptake capacity was about 2 × 10−4 M on the 10th day and 10−3 M on the 20th day. According to our results, we suggested that astrocyte age was important to maintain neuronal survival against glutamate toxicity. Thus, we revealed activation or a trigger point of glutamate transporters on astrocytes due to time since more glutamate was taken up by astrocytes when glutamate transporters on the astrocyte were triggered with high exogenous glutamate concentrations. In conclusion, the present investigation is the first voltammetric study on the reuptake parameters of glutamate in vitro.
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References
Adams RN (1990) In vivo electrochemical measurements in the CNS. Prog Neurobiol 35:297–311
Aizenman E, White WF, Loring RH, Rosenberg PA (1990) A 3,4-dihydroxyphenylalanine oxidation product is a non-N-methyl-D-aspartate glutamatergic agonist in rat cortical neurons. Neurosci Lett 116:168–171
Araque A, Parpura V, Sanzgiri RP, Haydon PG (1998) Glutamate-dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons. Eur J Neurosci 10:2129–2142
Bortz DM, Mikkelsen JD, Bruno JP (2013) Localized infusions of the partial alpha 7 nicotinic receptor agonist SSR180711 evoke rapid and transient increases in prefrontal glutamate release. Neuroscience 255:55–67
Brown DR (1999) Neurons depend on astrocytes in a coculture system for protection from glutamate toxicity. Mol Cell Neurosci 13:379–389
Burmeister JJ, Moxon K, Gerhardt GA (2000) Ceramic-based multisite microelectrodes for electrochemical recordings. Anal Chem 72:187–192
Burmeister JJ, Pomerleau F, Palmer M, Day BK, Huettl P, Gerhardt GA (2002) Improved ceramic-based multisite microelectrode for rapid measurements of L-glutamate in the CNS. J Neurosci Methods 119:163–171
Choi DW, Maulucci-Gedde M, Kriegstein AR (1987) Glutamate neurotoxicity in cortical cell culture. J Neurosci 7:357–368
Coyle JT, Bird SJ, Evans RH, Gulley RL, Nadler JV, Nicklas WJ, Olney JW (1981) Excitatory amino acid neurotoxins: selectivity, specificity, and mechanisms of action. Based on an NRP one-day conference held June 30, 1980. Neurosci Res Program Bull 19:1–427
Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65:1–105
Day BK, Pomerleau F, Burmeister JJ, Huettl P, Gerhardt GA (2006) Microelectrode array studies of basal and potassium-evoked release of L-glutamate in the anesthetized rat brain. J Neurochem 96:1626–1635
Domoki F, Kis B, Gáspár T, Snipes JA, Parks JS, Bari F, Busija DW (2010) Rosuvastatin induces delayed preconditioning against L-glutamate excitotoxicity in cultured cortical neurons. Neurochem Int 56:404–409
Fairman WA, Amara SG (1999) Functional diversity of excitatory amino acid transporters: ion channel and transport modes. Am J Physiol 277:F481–F486
Genoud C, Quairiaux C, Steiner P, Hirling H, Welker E, Knott GW (2006) Plasticity of astrocytic coverage and glutamate transporter expression in adult mouse cortex. PLoS Biol 4:e343
Gepdiremen A, Düzenli S, Hacimüftüoglu A, Bulucu D, Süleyman H (2000a) The effects of melatonin in glutamate-induced neurotoxicity of rat cerebellar granular cell culture. Jpn J Pharmacol 84:467–469
Gepdiremen A, Hacimüftüoglu A, Düzenli S, Oztaş S, Süleyman H (2000b) Effects of salicylic acid in glutamate- and kainic acid-induced neurotoxicity in cerebellar granular cell culture of rats. Pharmacol Res 42:547–551
Gepdiremen A, Hacimuftuoglu A, Buyukokuroglu ME, Suleyman H (2002) Nitric oxide donor sodium nitroprusside induces neurotoxicity in cerebellar granular cell culture in rats by an independent mechanism from L-type or dantrolene-sensitive calcium channels. Biol Pharm Bull 25:1295–1297
Goudriaan A, Camargo N, Carney KE, Oliet SH, Smit AB, Verheijen MH (2014) Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures. Front Cell Neurosci 8:12
Hascup KN, Rutherford EC, Quintero JE, Day BK, Nickell JR, Pomerleau F, Huettl P, Burmeister JJ, Gerhardt GA (2007) Second-by-second measures of L-glutamate and other neurotransmitters using enzyme-based microelectrode arrays. In: Michael AC, Borland LM (eds) Electrochemical methods for neuroscience. CRC Press, Boca Raton, FL, pp 407–450
Kasasbeh A, Lee K, Bieber A, Bennet K, Chang SY (2013) Wireless neurochemical monitoring in humans. Stereotact Funct Neurosurg 91:141–147
Kim K, Lee SG, Kegelman TP, Su ZZ, Das SK, Dash R, Dasgupta S, Barral PM, Hedvat M, Diaz P, Reed JC, Stebbins JL, Pellecchia M, Sarkar D, Fisher PB (2011) Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics. J Cell Physiol 226:2484–2493
Kugler P, Schleyer V (2004) Developmental expression of glutamate transporters and glutamate dehydrogenase in astrocytes of the postnatal rat hippocampus. Hippocampus 14:975–985
Martínez-Contreras A, Huerta M, Lopez-Perez S, García-Estrada J, Luquín S, Beas Zárate C (2002) Astrocytic and microglia cells reactivity induced by neonatal administration of glutamate in cerebral cortex of the adult rats J Neurosci Res 67:200–210
Mattson MP, Zhang Y, Bose S (1993) Growth factors prevent mitochondrial dysfunction, loss of calcium homeostasis, and cell injury, but not ATP depletion in hippocampal neurons deprived of glucose. Exp Neurol 121:1–13
McKenna MC (2013) Glutamate pays its own way in astrocytes. Front Endocrinol (Lausanne) 4:191
Nicholls D, Attwell D (1990) The release and uptake of excitatory amino acids. Trends Pharmacol Sci 11:462–468
Pertusa M, García-Matas S, Rodríguez-Farré E, Sanfeliu C, Cristòfol R (2007) Astrocytes aged in vitro show a decreased neuroprotective capacity. J Neurochem 101:794–805
Pines G, Danbolt NC, Bjørås M, Zhang Y, Bendahan A, Eide L, Koepsell H, Storm-Mathisen J, Seeberg E, Kanner BI (1992) Cloning and expression of a rat brain L-glutamate transporter. Nature 360:464–467
Robert LS Jr (2011) Glutamate transporter activators as anti-nociceptive agents. Eurasian J Med 43:182–185
Robinson DL, Hermans A, Seipel AT, Wightman RM (2008) Monitoring rapid chemical communication in the brain. Chem Rev 108:2554–2584
Rothman SM, Olney JW (1986) Glutamate and the pathophysiology of hypoxic–ischemic brain damage. Ann Neurol 19:105–111
Rothstein JD, Martin L, Levey AI, Dykes-Hoberg M, Jin L, Wu D, Nash N, Kuncl RW (1994) Localization of neuronal and glial glutamate transporters. Neuron 13:713–725
Rothstein JD, Van Kammen M, Levey AI, Martin LJ, Kuncl RW (1995) Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann Neurol 38:73–84
Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, Welty DF (1996) Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16:675–686
Sanchez-Prieto J, Herrero I, Miras-Portugal MT, Mora F (1994) Unchanged exocytotic release of glutamic acid in cortex and neostriatum of the rat during aging. Brain Res Bull 33:357–359
Schmitt A, Asan E, Puschel B, Jons T, Kugler P (1996) Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry. Neuroscience 71:989–1004
Sinor JD, Du S, Venneti S, Blitzblau RC, Leszkiewicz DN, Rosenberg PA, Aizenman E (2000) NMDA and glutamate evoke excitotoxicity at distinct cellular locations in rat cortical neurons in vitro. J Neurosci 20:8831–8837
Storck T, Schulte S, Hofmann K, Stoffel W (1992) Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci U S A 89:10955–10959
Swanson RA, Liu J, Miller JW, Rothstein JD, Farrell K, Stein BA, Longuemare MC (1997) Neuronal regulation of glutamate transporter subtype expression in astrocytes. J Cogn Neurosci 17:932–940
Takasaki C, Okada R, Mitani A, Fukaya M, Yamasaki M, Fujihara Y, Shirakawa T, Tanaka K, Watanabe M (2008) Glutamate transporters regulate lesion-induced plasticity in the developing somatosensory cortex. J Cogn Neurosci 28:4995–5006
Wierda KD, Toonen RF, de Wit H, Brussaard AB, Verhage M (2007) Interdependence of PKC-dependent and PKC-independent pathways for presynaptic plasticity. Neuron 54:275–290
Zhang Y, Bhavnani BR (2006) Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF) and this process is inhibited by equine estrogens. BMC Neurosci 7:49
Acknowledgments
This study was supported by grants from The Scientific and Technological Research Council of Turkey (TUBİTAK-Project No: 107S067) and Atatürk University BAP (Project No: 2005/160 and 2011/270-271)
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The authors declared that there are no conflicts of interest.
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Hacimuftuoglu, A., Tatar, A., Cetin, D. et al. Astrocyte/neuron ratio and its importance on glutamate toxicity: an in vitro voltammetric study. Cytotechnology 68, 1425–1433 (2016). https://doi.org/10.1007/s10616-015-9902-9
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DOI: https://doi.org/10.1007/s10616-015-9902-9