KISEP Esperimental Researchs J Korean Neurosurg Soc 291429-1436, 2000 일차배양해마신경세포에서 NMDA- 및 Glutamate- 유도전류의특성 김일만 손은익 김동원 김인홍 임만빈송대규 * 박원균 * 배재훈 * 최하영 ** = Abstract = Characteristics of NMDA- and Glutamate-Induced Currents in Primary Cultured Rat Hippocampal Neurons Il-Man Kim, M.D., Eun-Ik Son, M.D., Dong-Won Kim, M.D., In-Hong Kim, M.D., Man-in Yim, M.D., Dae-Kyu Song, M.D.,* Won-Kyun Park, M.D.,* Jae-Hun ae, M.D.,* Ha-Young Choi, M.D.** Departments of Neurosurgery and Physiology,* Keimyung University School of Medicine, Taegu, Korea Department of Neurosurgery, Chunbuk National University Medical School, Chunju, Korea bjectives Method Results Conclusion KEY WORDS 서 론 J Korean Neurosurg SocVolume 29November, 2000 1429
재료및방법 1. 해마신경세포의분리와배양 1430 2. 전기생리학적측정 J Korean Neurosurg SocVolume 29November, 2000
3. 약물처리에따른시냅스전류의기록 결 1. 세포외액에 Mg 2+ 존재시 Glutamate 및 NMDA 유도전류 2. Mg 2+ 의 NMDA 수용체전류에대한영향 Fig. 1. Inward currents activated by glutamate receptor agonists in a cultured rat hippocampal neuron. NMDA 10mol did not elicit a noticable current but glutamate10mol induced an inward current in the presence of 1mmol Mg 2. Whole-cell configuration was clamped at 60mV. Vertical bars are on time when ramp pulse from 80mV to 60mV was injected. 과 J Korean Neurosurg SocVolume 29November, 2000 1431
A Fig. 2. oth NMDA10mol and glycine5mol-induced current witha or without 1mmol Mg 2 in a cultured rat hippocampal neuron. Whole-cell configu- ration was clamped at 60mV. Vertical bars in are on time when ramp pulse was injected. A Fig. 3. oth NMDA10mol and glycine5mol-induced current in the presence of 50mol Mg in a cultured rat hippocampal neuron. AInward or outward currents according to the change of membrane potentials elicited by NMDA and glycine application. I-V plot for NMDA and glycine-induced current with Mg 2. 1432 3. 세포막전압변화에의한 Mg 2+ 의 NMDA 수용체전류의차단 Fig. 4. Effect of 50mol Mg 2 on both NMDA10mol and glycine5mol-induced current. AI-V plot for NMDA and glycine-current without Mg 2. I-V plot for the amount of Mg 2 -sensitive NMDA current. J Korean Neurosurg SocVolume 29November, 2000 A
4. Non-NMDA 수용체전류의특성 A A Fig. 5. Effect of glutamate receptor antagonists and nifedipine on glutamate-induced current. AGlutamateinduced inward current was partially eliminated by its antagonistsap550mol, CNQX10mol and nifedipine10mol were applied subsequently. Vertical bars are on time when ramp pulse was injected. Wholecell configuration was clamped at 60mV. I-V plots recorded from ramp pulse during the ramp pulses in serial drug application. Fig. 6. Effect of glutamate receptor antagonists and nifedipine on glutamate-induced current. ACurrent-voltage relationships representing the block currents by each drug. Percentage values of CNQX-dependent current in overall glutamate current. J Korean Neurosurg SocVolume 29November, 2000 1433
고찰 1434 J Korean Neurosurg SocVolume 29November, 2000
결 론 References 1) Akaike A, Tamura Y, Terada K, Nakata NRegulation by neuroprotective factors of NMDA receptor mediated nitric oxide synthesis in the brain and retina. rain Res 103391-401, 1994 2) Alford S, Frenguelli G, Schofield JG, Collingridge GL Chracterization of Ca signals induced in hippocampal CA1 neurons by the synaptic activation of NMDA receptors. J Physiol 469693-716, 1993 3) Artola A, Singer WLong-term potentiation and NMDA receptors in rat visual cortex. Nature 330:649-652, 1987 4) Aszteley F, Gustafsson Ionotropic glutamate receptors. Mol Neurobiol 121-11, 1996 5) ading H, Gunty DD, Greenberg MERegulation of gene expression in hippocampal neurons by distinct calcium signalling pathways. Science 260181-186, 1996 J Korean Neurosurg SocVolume 29November, 2000 1435
6) owie D, Smart TGInterplay between expressed non-nmda receptors and endogenous calcium-activated chloride currents in Xenopus laevis oocytes. Neurosci Lett 1514-8, 1993 7) rewer GJ, Torricelli JR, Evege EK, Price PJOptimized survival of hippocampal neurons in 27 supplemented neurobasal TM a new serum-free medium combination. J Neurosci Res 35567-576, 1995 8) Castillo PE, Malenka RC, Nicoli RAKainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons. Nature 388182-186, 1997 9) Collingridge GL, Singer WExcitatory amino acid receptors and synaptic plasticity. Trends Pharmacol Sci 11290-296, 1990 10) Golshani P, Warren RA, Jones EGProgression of change in NMDA, non-nmda, and metabotropic glutamate receptor function at the developing corticothalamic synapse. J Neurophysiol 80143-54, 1998 11) Hamill OP, Marty A, Neher E, Sakmann, Sigworth FJImproved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflgers Arch 39185-100, 1981 12) Jahr CE, Westbrook GLPhysiological approaches to the study of glutamate receptors. In Chad J, Wheal H, Molecular Neurobiologya practical approach. 1st ed. Oxford, Oxford University Press, 1991, pp49-73 13) Johnson JW, Ascher PGlycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325529-531, 1997 14) Joels M, Yool AJ, Gruol DLUnique properties of non-nmda excitatory responses in cultured purkinje neurons. Proc Natl Acad Sci USA 863404-3408, 1989 15) Karlsson U, Sundgren AK, Nasstrom J, Johansson SGlutamateevoked currents in acutely dissociated neurons from the rat medial preoptic nucleus. rain Res 759270-276, 1998 16) Kleckner NW, Dingledine RRequirment for glycine in activation of NMDA-receptors expressed in Xenopus Oocytes. Science 241835-837, 1988 17) MacDoanld JF, artlett MC, Mody I, Pahapill P, Reynolds JN, Salter MW, et alactions of ketamine, phencyclidine and MK 801 on NMDA receptor currents in cultured mouse hippocampal neurones. J Physiol 432483-508, 1991 18) Mayer ML, Vyklicky Jr LConcanavalin A selectively reduces desensitization of mammalian neuronal quisqualate receptors. Proc Natl Acad Sci USA 861411-1415, 1989 19) Mayer ML, Westbrook GL, Vyklicky Jr LSites of antagonist action on N-methyl-D-aspartic acid receptors studied using fluctuation analysis and a rapid perfusion technique. J Neurophysiol 60645-663, 1988 20) Monaghan DT, ridges RJ, Cotman CWThe excitatory amino acid receptorstheir classes, pharmacology, and distinct properties in the function of the central nervous system. Annu Rev Pharmacol Toxicol 29:365-402, 1989 21) Nowak L, regestovski P, Ascher P, Herbert A, Prochiantz A Magnesium gates glutamate-activated channels in mouse neurones. Nature 307462-465, 1984 22) Simonian NA, Coyle JTOxidative stress in neurodegenerative diseases. Annu Rev Pharmacol 3683-106, 1996 23) Tang C-M, Dichter M, Morad MQuisqualate activates a rapidly inactivating high conductance ionic channel in hippocampal neurons. Science 2431474-1477, 1989 24) Ying WA new hypothesis of neurodegenerative disease the deliterious network hypothesis. Med Hypothesis 47307-313, 1996 25) Zafra F, Hengerer, Leibrock J, Thoenen H, Lindholm D Activity dependent regulation of DNF and NGK mrnas in the rat hippocampus is mediated by non-nmda glutamate receptors. EMO J 93545-3550, 1990 1436 J Korean Neurosurg SocVolume 29November, 2000