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In the spinal cord high extracellular glutamate evokes excitotoxic damage with neuronal loss and severe locomotor impairment. During the cell dysfunction process, extracellular pH becomes acid and may activate acid-sensing ion channels (ASICs) which could be important contributors to neurodegenerative pathologies. Our previous studies have shown that transient application of the glutamate analog kainate (KA) evokes delayed excitotoxic death of spinal neurons, while white matter is mainly spared. The present goal was to enquire if ASIC channels modulated KA damage in relation to locomotor network function and cell death. Mouse spinal cord slices were treated with KA (0.01 or 0.1 mM) for 1 h, and then washed out for 24 h prior to analysis. RT-PCR results showed that KA (at 0.01 mM concentration that is near-threshold for damage) increased mRNA expression of ASIC1a, ASIC1b, ASIC2 and ASIC3, an effect reversed by the ASIC inhibitor 4′,6-diamidino-2-phenylindole (DAPI). A KA neurotoxic dose (0.1 mM) reduced ASIC1a and ASIC2 expression. Cell viability assays demonstrated KA-induced large damage in spinal slices from mice with ASIC1a gene ablation. Likewise, immunohistochemistry indicated significant neuronal loss when KA was followed by the ASIC inhibitors DAPI or amiloride. Electrophysiological recording from ventral roots of isolated spinal cords showed that alternating oscillatory cycles were slowed down by 0.01 mM KA, and intensely inhibited by subsequently applied DAPI or amiloride. Our data suggest that early rise in ASIC expression and function counteracted deleterious effects on spinal networks by raising the excitotoxicity threshold, a result with potential implications for improving neuroprotection. © 2016 IBRO


Documento: Artículo
Título:ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury
Autor:Mazzone, G.L.; Veeraraghavan, P.; Gonzalez-Inchauspe, C.; Nistri, A.; Uchitel, O.D.
Filiación:Laboratorios de Investigación aplicada en Neurociencias (LIAN) – Fundación para la Lucha conntra las Enfermedades Neurológicas de la Infancia (FLENI), CONICET, Buenos Aires, Argentina
Neuroscience Department, International School for Advanced Studies (SISSA), Trieste, Italy
Instituto de Fisiología, Biología molecular y Neurociencias, CONICET, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina
Spinal Person Injury Neurorehabilitation Applied Laboratory (SPINAL), Istituto di Medicina Fisica e Riabilitazione, Udine, Italy
Palabras clave:acid sensing ion channels (ASICs); fictive locomotion; kainic acid; neuroprotection; pH; spinal cord injury; 4',6 diamidino 2 phenylindole; acid sensing ion channel; acid sensing ion channel 1a; acid sensing ion channel 1b; acid sensing ion channel 2; acid sensing ion channel 3; kainic acid; unclassified drug; acid sensing ion channel; acid sensing ion channel blocking agent; glutamic acid; indole derivative; kainic acid; messenger RNA; proton; animal cell; animal experiment; animal model; animal tissue; Article; cell counting; cell death; cell viability assay; concentration response; controlled study; excitotoxicity; gene expression; immunohistochemistry; mouse; nerve cell; nerve cell network; nervous system electrophysiology; neuroprotection; neurotoxicity; newborn; nonhuman; oscillatory potential; pH measurement; priority journal; protein expression; reverse transcription polymerase chain reaction; spinal cord injury; staining; animal; cell death; cell survival; disease model; dose response; drug effects; genetics; glia; metabolism; nerve cell; pathology; physiology; spinal cord; spinal cord injury; synaptic transmission; tissue culture technique; Acid Sensing Ion Channel Blockers; Acid Sensing Ion Channels; Animals; Cell Death; Cell Survival; Disease Models, Animal; Dose-Response Relationship, Drug; Glutamic Acid; Indoles; Kainic Acid; Mice; Neuroglia; Neurons; Protons; RNA, Messenger; Spinal Cord; Spinal Cord Injuries; Synaptic Transmission; Tissue Culture Techniques
Página de inicio:398
Página de fin:410
Título revista:Neuroscience
Título revista abreviado:Neuroscience
CAS:4',6 diamidino 2 phenylindole, 47165-04-8; kainic acid, 487-79-6; glutamic acid, 11070-68-1, 138-15-8, 56-86-0, 6899-05-4; proton, 12408-02-5, 12586-59-3; Acid Sensing Ion Channel Blockers; Acid Sensing Ion Channels; DAPI; Glutamic Acid; Indoles; Kainic Acid; Protons; RNA, Messenger


  • Alexander, S.P.H., Mathie, A., Peters, J.A., Guide to receptors and channels (GRAC), 5th edition (2011) Br J Pharmacol, 164, pp. S1-S324
  • Andreeva, N., Khodorov, B., Stelmashook, E., Sokolova, S., Cragoe, E., Victorov, I., 5-(N-ethyl-N-isopropyl)amiloride and mild acidosis protect cultured cerebellar granule cells against glutamate-induced delayed neuronal death (1992) Neuroscience, 49, pp. 175-181
  • Anwar, M.A., Al Shehabi, T.S., Eid, A.H., Inflammogenesis of secondary spinal cord injury (2016) Front Cell Neurosci, p. 10
  • Arun, T., Tomassini, V., Sbardella, E., de Ruiter, M.B., Matthews, L., Leite, M.I., Gelineau-Morel, R., Palace, J., Targeting ASIC1 in primary progressive multiple sclerosis: evidence of neuroprotection with amiloride (2013) Brain, 136, pp. 106-115
  • Ballerini, L., Galante, M., Grandolfo, M., Nistri, A., Generation of rhythmic patterns of activity by ventral interneurones in rat organotypic spinal slice culture (1999) J Physiol, 517, pp. 459-475
  • Baraldi, P.G., Bovero, A., Fruttarolo, F., Preti, D., Tabrizi, M.A., Pavani, M.G., Romagnoli, R., DNA minor groove binders as potential antitumor and antimicrobial agents (2004) Med Res Rev, 24, pp. 475-528
  • Baranauskas, G., Nistri, A., NMDA receptor-independent mechanisms responsible for the rate of rise of cumulative depolarization evoked by trains of dorsal root stimuli on rat spinal motoneurones (1996) Brain Res, 738, pp. 329-332
  • Barbieri, M., Nistri, A., Depression of windup of spinal neurons in the neonatal rat spinal cord in vitro by an NK3 tachykinin receptor antagonist (2001) J Neurophysiol, 85, pp. 1502-1511
  • Baron, A., Voilley, N., Lazdunski, M., Lingueglia, E., Acid sensing ion channels in dorsal spinal cord neurons (2008) J Neurosci, 28, pp. 1498-1508
  • Beato, M., Bracci, E., Nistri, A., Contribution of NMDA and non-NMDA glutamate receptors to locomotor pattern generation in the neonatal rat spinal cord (1997) Proc Roy Soc Biol Sci, 264, pp. 877-884
  • Borgens, R.B., Liu-Snyder, P., Understanding secondary injury (2012) Q Rev Biol, 87, pp. 89-127
  • Boscardin, E., Alijevic, O., Hummler, E., Frateschi, S., Kellenberger, S., (2016), () International union of basic and clinical pharmacology review acid-sensing ion channel and epithelial Na+ channel nomenclature review: IUPHAR Review. Br J Pharmacol. doi: 10.1111/bph.13533. [Epub ahead of print]; Brana, C., Benham, C., Sundstrom, L., A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry (2002) Brain Res Protoc, 10, pp. 109-114
  • Chen, X., Qiu, L., Li, M., Dürrnagel, S., Orser, B.A., Xiong, Z.-G., MacDonald, J.F., Diarylamidines: High potency inhibitors of acid-sensing ion channels (2010) Neuropharmacology, 58, pp. 1045-1053
  • Chesler, M., Regulation and modulation of pH in the brain (2003) Physiol Rev, 83, pp. 1183-1221
  • Chu, X.-P., Xiong, Z.-G., Physiological and pathological functions of acid-sensing ion channels in the central nervous system (2012) Curr Drug Targets, 13, pp. 263-271
  • Cifra, A., Mazzone, G.L., Nani, F., Nistri, A., Mladinic, M., Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures (2012) Dev Neurobiol, 72, pp. 1140-1160
  • Deval, E., Lingueglia, E., Acid-sensing Ion channels and nociception in the peripheral and central nervous systems (2015) Neuropharmacology, 94, pp. 49-57
  • Dietz, V., Behavior of spinal neurons deprived of supraspinal input (2010) Nat Rev Neurol, 6, pp. 167-174
  • Duan, B., Wu, L.-J., Yu, Y.-Q., Ding, Y., Jing, L., Xu, L., Chen, J., Xu, T.-L., Upregulation of acid-sensing ion channel ASIC1a in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity (2007) J Neurosci, 27, pp. 11139-11148
  • Gahwiler, B.H., Organotypic monolayer cultures of nervous tissue (1981) J Neurosci Methods, 4, pp. 329-342
  • Giffard, R.G., Monyer, H., Christine, C.W., Choi, D.W., Acidosis reduces NMDA receptor activation, glutamate neurotoxicity, and oxygen-glucose deprivation neuronal injury in cortical cultures (1990) Brain Res, 506, pp. 339-342
  • Gründer, S., Pusch, M., Biophysical properties of acid-sensing ion channels (ASICs) (2015) Neuropharmacology, 94, pp. 9-18
  • Hnasko, T.S., Edwards, R.H., Neurotransmitter co-release: mechanism and physiological role (2012) Annu Rev Physiol, 74, pp. 225-243
  • Holzer, P., Acid-sensitive ion channels and receptors (2009) Handb Exp Pharmacol, 194, pp. 283-332
  • Hu, R., Duan, B., Wang, D., Yu, Y., Li, W., Luo, H., Lu, P., Feng, H., Role of acid-sensing ion channel 1a in the secondary damage of traumatic spinal cord injury (2011) Ann Surg, 254, pp. 353-362
  • Huang, L., Zhao, S., Lu, W., Guan, S., Zhu, Y., Wang, J.-H., Acidosis-induced dysfunction of cortical GABAergic neurons through astrocyte-related excitotoxicity (2015) PLoS ONE, 10, p. e0140324
  • Huang, Y., Jiang, N., Li, J., Ji, Y.-H., Xiong, Z.-G., Zha, X., Two aspects of ASIC function: synaptic plasticity and neuronal injury (2015) Neuropharmacology, 94, pp. 42-48
  • Jalalvand, E., Robertson, B., Wallén, P., Grillner, S., Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3 (2016) Nat Commun, 7, p. 10002
  • Jalalvand, E., Robertson, B., Tostivint, H., Wallén, P., Grillner, S., The spinal cord has an intrinsic system for the control of pH (2016) Curr Biol, 26, pp. 1346-1351
  • Kiehn, O., Locomotor circuits in the mammalian spinal cord (2006) Annu Rev Neurosci, 29, pp. 279-306
  • Kiehn, O., Decoding the organization of spinal circuits that control locomotion (2016) Nat Rev Neurosci, 17, pp. 224-238
  • Kiehn, O., Kjaerulff, O., Distribution of central pattern generators for rhythmic motor outputs in the spinal cord of limbed vertebrates (1998) Ann New York Acad Sci, 860, pp. 110-129
  • Kjaerulff, O., Kiehn, O., Distribution of networks generating and coordinating locomotor activity in the neonatal rat spinal cord in vitro: a lesion study (1996) J Neurosci, 16, pp. 5777-5794
  • Krishtal, O., The ASICs: signaling molecules? Modulators? (2003) Trends Neurosci, 26, pp. 477-483
  • Leng, T.-D., Si, H.-F., Li, J., Yang, T., Zhu, M., Wang, B., Simon, R.P., Xiong, Z.-G., Amiloride analogs as ASIC1a inhibitors (2016) CNS Neurosci Ther, 22, pp. 468-476
  • Li, T., Yang, Y., Canessa, C.M., A method for activation of endogenous acid-sensing ion channel 1a (ASIC1a) in the nervous system with high spatial and temporal precision (2014) J Biol Chem, 289, pp. 15441-15448
  • Llaudet, E., Hatz, S., Droniou, M., Dale, N., Microelectrode biosensor for real-time measurement of ATP in biological tissue (2005) Anal Chem, 77, pp. 3267-3273
  • Luo, P., Chen, T., Zhao, Y., Zhang, L., Yang, Y., Liu, W., Li, S., Fei, Z., Postsynaptic scaffold protein Homer 1a protects against traumatic brain injury via regulating group I metabotropic glutamate receptors (2014) Cell Death Dis, 5, p. e1174
  • Ma, L., Zhang, X., Zhou, M., Chen, H., Acid-sensitive TWIK and TASK two-pore domain potassium channels change ion selectivity and become permeable to sodium in extracellular acidification (2012) J Biol Chem, 287, pp. 37145-37153
  • Marchetti, C., Beato, M., Nistri, A., Alternating rhythmic activity induced by dorsal root stimulation in the neonatal rat spinal cord in vitro (2001) J Physiol (Lond), 530, pp. 105-112
  • Mazzone, G.L., Margaryan, G., Kuzhandaivel, A., Nasrabady, S.E., Mladinic, M., Nistri, A., Kainate-induced delayed onset of excitotoxicity with functional loss unrelated to the extent of neuronal damage in the in vitro spinal cord (2010) Neuroscience, 168, pp. 451-462
  • Mazzone, G.L., Mladinic, M., Nistri, A., Excitotoxic cell death induces delayed proliferation of endogenous neuroprogenitor cells in organotypic slice cultures of the rat spinal cord (2013) Cell Death Dis, 4, p. e902
  • Mazzone, G.L., Nistri, A., Effect of the PARP-1 inhibitor PJ 34 on excitotoxic damage evoked by kainate on rat spinal cord organotypic slices (2011) Cell Mol Neurobiol, 31, pp. 469-478
  • Mazzone, G.L., Nistri, A., Electrochemical detection of endogenous glutamate release from rat spinal cord organotypic slices as a real-time method to monitor excitotoxicity (2011) J Neurosci Meth, 197, pp. 128-132
  • Mazzone, G.L., Rigato, I., Ostrow, J.D., Tiribelli, C., Bilirubin effect on endothelial adhesion molecules expression is mediated by the NF-kappaB signaling pathway (2009) Biosci Trends, 3, pp. 151-157
  • Miletic, G., Driver, A.M., Miyabe-Nishiwaki, T., Miletic, V., Early changes in Homer1 proteinsin the spinal dorsal horn are associated with loose ligation of the rat sciatic nerve (2009) Anesth Analg, 109, pp. 2000-2007
  • Mitra, P., Brownstone, R.M., An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse (2012) J Neurophysiol, 107, pp. 728-741
  • Osmakov, D.I., Andreev, Y.A., Kozlov, S.A., Acid-sensing ion channels and their modulators (2014) Biochemistry (Moscow), 79, pp. 1528-1545
  • Oyinbo, C.A., Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade (2011) Acta Neurobiol Exp (Wars), 71, pp. 281-299
  • Park, E., Velumian, A.A., Fehlings, M.G., The role of excitotoxicity in secondary mechanisms of spinal cord injury: a review with an emphasis on the implications for white matter degeneration (2004) J Neurotrauma, 21, pp. 754-774
  • Petroff, E.Y., Price, M.P., Snitsarev, V., Gong, H., Korovkina, V., Abboud, F.M., Welsh, M.J., Acid-sensing ion channels interact with and inhibit BK K+ channels (2008) Proc Natl Acad Sci USA, 105, pp. 3140-3144
  • Pfaffl, M.W., A new mathematical model for relative quantification in real-time RT-PCR (2001) Nucleic Acids Res, 29, p. e45
  • Price, M.P., McIlwrath, S.L., Xie, J., Cheng, C., Qiao, J., Tarr, D.E., Sluka, K.A., Welsh, M.J., The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice (2001) Neuron, 32, pp. 1071-1083
  • Quintana, P., Soto, D., Poirot, O., Zonouzi, M., Kellenberger, S., Muller, D., Chrast, R., Cull-Candy, S.G., Acid-sensing ion channel 1a drives AMPA receptor plasticity following ischaemia and acidosis in hippocampal CA1 neurons (2015) J Physiol (Lond), 593, pp. 4373-4386
  • Robinson, D.L., Hermans, A., Seipel, A.T., Wightman, R.M., Monitoring rapid chemical communication in the brain (2008) Chem Rev, 108, pp. 2554-2584
  • Ruffin, V.A., Salameh, A.I., Boron, W.F., Parker, M.D., Intracellular pH regulation by acid-base transporters in mammalian neurons (2014) Front Physiol, 5, p. 43
  • Shabbir, A., Bianchetti, E., Cargonja, R., Petrovic, A., Mladinic, M., Pilipović, K., Nistri, A., Role of HSP70 in motoneuron survival after excitotoxic stress in a rat spinal cord injury model in vitro (2015) Eur J Neurosci, 42, pp. 3054-3065
  • Shabbir, A., Bianchetti, E., Nistri, A., The volatile anesthetic methoxyflurane protects motoneurons against excitotoxicity in an in vitro model of rat spinal cord injury (2015) Neuroscience, 285, pp. 269-280
  • Sinning, A., Hübner, C.A., Minireview: pH and synaptic transmission (2013) FEBS Lett, 587, pp. 1923-1928
  • Sivilotti, L.G., Thompson, S.W., Woolf, C.J., Rate of rise of the cumulative depolarization evoked by repetitive stimulation of small-caliber afferents is a predictor of action potential windup in rat spinal neurons in vitro (1993) J Neurophysiol, 69, pp. 1621-1631
  • Su, J.-J., Pan, H., Zhou, H.-G., Tang, Y.-P., Dong, Q., Liu, J.-R., Acid-sensing ion channels activation and hypoxia upregulate Homer1a expression (2014) CNS Neurosci Ther, 20, pp. 264-274
  • Swain, S.M., Parameswaran, S., Sahu, G., Verma, R.S., Bera, A.K., Proton-gated ion channels in mouse bone marrow stromal cells (2012) Stem Cell Research, 9, pp. 59-68
  • Taccola, G., Margaryan, G., Mladinic, M., Nistri, A., Kainate and metabolic perturbation mimicking spinal injury differentially contribute to early damage of locomotor networks in the in vitro neonatal rat spinal cord (2008) Neuroscience, 155, pp. 538-555
  • Tang, C.M., Dichter, M., Morad, M., Modulation of the N-methyl-D-aspartate channel by extracellular H+ (1990) Proc Natl Acad Sci USA, 87, pp. 6445-6449
  • Vukicevic, M., Kellenberger, S., Modulatory effects of acid-sensing ion channels on action potential generation in hippocampal neurons (2004) Am J Physiol – Cell Physiol, 287, pp. C682-C690
  • Wemmie, J.A., Chen, J., Askwith, C.C., Hruska-Hageman, A.M., Price, M.P., Nolan, B.C., Yoder, P.G., Welsh, M.J., The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory (2002) Neuron, 34, pp. 463-477
  • Xiong, Z.-G., Zhu, X.-M., Chu, X.-P., Minami, M., Hey, J., Wei, W.-L., MacDonald, J.F., Simon, R.P., Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels (2004) Cell, 118, pp. 687-698
  • Yang, L., Palmer, L.G., Ion conduction and selectivity in acid-sensing ion channel 1 (2014) J Gen Physiol, 144, pp. 245-255
  • Zhong, G., Shevtsova, N.A., Rybak, I.A., Harris-Warrick, R.M., Neuronal activity in the isolated mouse spinal cord during spontaneous deletions in fictive locomotion: insights into locomotor central pattern generator organization (2012) J Physiol (Lond), 590, pp. 4735-4759


---------- APA ----------
Mazzone, G.L., Veeraraghavan, P., Gonzalez-Inchauspe, C., Nistri, A. & Uchitel, O.D. (2017) . ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury. Neuroscience, 343, 398-410.
---------- CHICAGO ----------
Mazzone, G.L., Veeraraghavan, P., Gonzalez-Inchauspe, C., Nistri, A., Uchitel, O.D. "ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury" . Neuroscience 343 (2017) : 398-410.
---------- MLA ----------
Mazzone, G.L., Veeraraghavan, P., Gonzalez-Inchauspe, C., Nistri, A., Uchitel, O.D. "ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury" . Neuroscience, vol. 343, 2017, pp. 398-410.
---------- VANCOUVER ----------
Mazzone, G.L., Veeraraghavan, P., Gonzalez-Inchauspe, C., Nistri, A., Uchitel, O.D. ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury. Neuroscience. 2017;343:398-410.