Applied Microscopy

Korean Society of Microscopy (한국현미경학회)
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Immunohistochemical Localization of NMDA Receptor in the Auditory Brain Stem of Postnatal 7, 16 Circling Mouse

생후 7일, 16일된 circling mouse 청각 뇌줄기에서 N-메틸-D 아스파르트산염 수용체(NMDA receptor)에 대한 면역염색학적 분포

  • Choi, In-Young (Department of Anatomy, Dankook University College of Medicine) ;
  • Park, Ki-Sup (Department of Anatomy, Dankook University College of Medicine) ;
  • Kim, Hye-Jin (Department of Anatomy, Dankook University College of Medicine) ;
  • Maskey, Dhiraj (Department of Anatomy, Dankook University College of Medicine) ;
  • Kim, Myeung-Ju (Department of Anatomy, Dankook University College of Medicine)
  • 최인영 (단국대학교 의과대학 해부학교실) ;
  • 박기섭 (단국대학교 의과대학 해부학교실) ;
  • 김혜진 (단국대학교 의과대학 해부학교실) ;
  • 디라즈 마스키 (단국대학교 의과대학 해부학교실) ;
  • 김명주 (단국대학교 의과대학 해부학교실)
  • Received : 2010.03.26
  • Accepted : 2010.06.19
  • Published : 2010.06.30
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Abstract

Glutamate receptors may play a critical role in the refinement of developing synapses. The lateral superior olivary nucleus (LSO)-medial nucleus of trapezoid body (MNTB) synaptic transmission in the mammalian auditory brain stem mediate many excitatory transmitters such as glutamate, which is a useful model to study excitatory synaptic development. Hearing deficits are often accompanied by changes in the synaptic organization such as excitatory or inhibitory circuits as well as anatomical changes. Owing to this, circling mouse whose cochlea degenerates spontaneously after birth, is an excellent animal model to study deafness pathophysiology. However, little is known about the development regulation of the subunits composing these receptors in circling mouse. Thus, we used immunohistochemical method to compare the N-Methyl-D-aspartate receptor (NMDA receptor) NR1, NR2A, NR2B distribution in the LSO which project glutamergic excitatory input into the auditory brainstem, in circling mouse of postnatal (p) 7 and 16, which have spontaneous mutation in the inner ear, with wild-type mouse. The relative NMDAR1 immunoreactive density of the LSO in circling mouse p7 was $128.67\pm8.87$ in wild-type, $111.06\pm8.04$ in heterozygote, and $108.09\pm5.94$ in homozygote. The density of p16 circling mouse was $43.83\pm10.49$ in wild-type, $40\pm13.88$ in heterozygote, and $55.96\pm17.35$ in homozygote. The relative NMDAR2A immunoreactive density of LSO in circling mouse p7 was $97.97\pm9.71$ in wild-type, $102.87\pm9.30$ in heterozygote, and $106.85\pm5.79$ in homozygote. The density of LSO in p16 circling was $47.4\pm20.6$ in wild-type, $43.9\pm17.5$ in heterozygote, and $49.2\pm20.1$ in homozygote. The relative NMDAR2B immunoreactive density of LSO in circling mouse p7 was $109.04\pm6.77$ in wild-type, $106.43\pm10.24$ in heterozygote, and $105.98\pm4.10$ in homozygote. the density of LSO in p16 circling mouse was $101.47\pm11.5$ in wild-type, $91.47\pm14.81$ in heterozygote, and $93.93\pm15.71$ in homozygote. These results reveal alteration of NMDAR immunoreactivity in LSO of p7 and p16 circling mouse. The results of the present study are likely to be relevant to understand the central change underlying human hereditary deafness.

글루타메이트 수용체는 신경연접의 발달에서 신경연접과 정제 등에 중요한 역할을 한다. 포유류 청각뇌줄기의 가쪽위올리브 핵-안쪽마름섬유체핵 신경연접에서의 흥분성 신경전달물질의 전달은 글루타메이트와 같은 많은 흥분성 신경전달물질의 전달을 조절하므로 발생학적 연구에 매우 유용한 모델이다. 귀먹음은 흥분성 회로 또는 억제성 회로의 신경연접에서 신경전달물질들의 변화뿐 아니라 형태학적 변화도 초래하게 된다. 이런 이유로 선천적으로 태어나면서 달팽이 기관이 퇴화되는 선회생쥐는 귀먹음의 병태생리를 연구하는데 가장 좋은 동물모델이다. 그러나, 선회생쥐에서 이런 NMDA 수용체 각각의 아형들이 발달에 따라 어떻게 나타나는지에 관해서는 거의 알려진 사실이 없다. 따라서, 본 연구에서는 선천적으로 속귀에 돌연변이를 가진 선회생쥐를 이용하여 면역조직화학염색법으로 생후 7일과 16일의 쥐를 정상쥐와 비교해서 글루타메이트성의 흥분성 신호가 전달되는 청각뇌줄기의 가쪽위올리브핵(LSO)에서 N-메틸-D-아스파르 트산염(NMDA) 수용체의 아형인 NR1, NR2A, NR2B의 분포를 각각 조사하였다. 생후 7일째 선회생쥐의 NR1에 대한 면역반응성의 세기는 정상군에서 $128.67\pm8.87$로 나타났고 이형접합체에서는 $111.06\pm8.04$, 동형접합체에서는 $108.09\pm5.94$으로 나타났다. 그리고 생후 16일째 선회생쥐에서는 정상군에서 $43.83\pm10.49$, 이형접합체에서는 $40\pm13.88$, 동형접합체에서는 $55.96\pm17.35$으로 나타났다. 생후 7일째 선회생쥐의 NR2A에 대한 면역반응성의 세기는 정상군에서 $97.97\pm9.71$, 이형접합체에서 $102.87\pm9.30$, 동형접합체에서 $106.85\pm5.79$로 나타났다. 생후 16일 선회생쥐의 가쪽위올리브핵에서의 NR2A에 대한 면역반응성 세기는 정상군에서 $47.4\pm20.6$, 이형접합체에서 $43.9\pm17.5$, 동형접합체에서 $49.2\pm20.1$로 나타났다. 생후 7일의 선회생쥐의 NR2B의 면역반응성 세기는 정상군에서 $109.04\pm6.77$, 이형접합체에서 $106.43\pm10.24$, 동형접합체에서 $105.98\pm4.10$으로 나타났다. 생후 16일째 선회생쥐의 NR2B의 면역반응성의 세기는 정상군에서 $101.47\pm11.50$, 이형접합체에서 $91.47\pm14.81$, 동형접합체에서 $93.93\pm15.71$으로 나타났다. 이 결과들은 생후 7일과 16일의 선회생쥐의 가쪽위올리브핵에서 NMDA 수용체의 면역반응성의 변화를 나타낸 것으로 인간 귀먹음의 주요한 병태생리를 파악하는 데 기본적인 자료를 제시할 수 있을 것이다.

Keywords

References

  1. Avraham KB, Hasson T, Sobe T, Balsara B, Testa JR, Skvorak AB, Morton CC, Copeland NG, Jenkins NA: Characterization of unconventional MYO6, the human homologue of the gene responsible for deafness in Snell's waltzer mice. Hum Mol Genet 6(8) : 1225-1231, 1997. https://doi.org/10.1093/hmg/6.8.1225
  2. Bi H, Sze CI: N-methyl-D-aspartate receptor subunit NR2A and NR2B messengerRNA levels are altered in the hippocampus and entorhin ancortex in Aazheimer's disease. J Neurol Sci 200(1-2) : 11-18, 2002. https://doi.org/10.1016/S0022-510X(02)00087-4
  3. Bilak MM, Bilak SR, Morest DK: Differential expression of Nmethyl-D-aspartate receptor in the cochlear nucleus of the mouse. Neuroscience 75(4) : 1075-1097, 1996. https://doi.org/10.1016/0306-4522(96)00197-2
  4. Blanton SH, Liang CY, Cai MW, Pandya A, Du LL, Landa B, Mummalanni S, Li KS, Chen ZY, Qin XN, Liu YF, Balkany T, Nance WE, Liu XZ: A novel locus for autosomal dominant nonsyndromic deafness (DFNA41) maps to chromosome 12q24-qter. J Med Genet 39(8) : 567-570, 2002. https://doi.org/10.1136/jmg.39.8.567
  5. Bliss TV, Collingridge G: A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361(6407) : 31-39, 1993. https://doi.org/10.1038/361031a0
  6. Caicedo A, Eybalin M: Glutamate receptor phenotypes in the auditory brainstem and mid-brain of the developing rat. Eur J Neurosci 11(1) : 51-74, 1999. https://doi.org/10.1046/j.1460-9568.1999.00410.x
  7. Colwell CS, Cepeda C, Crawford C, Levine MS: Postnatal development of glutamate receptor-mediated responses in the neostriatum. Dev Neurosci 20(2-3) : 154-163, 1998. https://doi.org/10.1159/000017310
  8. Cull-Candy S, Brickley S, Farrant M: NMDA receptor subunits: diversity, development and disease. Curr Opin Neurobiol 11(3) : 327-335, 2001. https://doi.org/10.1016/S0959-4388(00)00215-4
  9. Dickson KS, Kind PC: NMDA receptors: neural map designers and refiners? Curr Biol 2;13(23) : R920-922, 2003.
  10. Dunah AW, Yasuda RP, Luo J, Wang Y, Prybylowski KL, Wolfe BB: Biochemical studies of the structure and function of the Nmethyl-D-aspartate subtype of glutamate receptors. Mol Neurobiol 19(2) : 151-179, 1999. https://doi.org/10.1007/BF02743658
  11. Ene FA, Kullmann PH, Gillespie DC, Kandler K: Glutamatergic calcium responses in the developing lateral superior olive: receptor types and their specific activation by synaptic activity patterns. J Neurophysiol 90(4) : 2581-2591, 2003. https://doi.org/10.1152/jn.00238.2003
  12. Gaiarsa JL, Caillard O, Ben-Ari Y: Long-term plasticity at GABAergic and glycinergic synapses: mechanisms and functional significance. Trends Neurosci 25(11) : 564-570, 2002. https://doi.org/10.1016/S0166-2236(02)02269-5
  13. Gillespie DC, Kim G, Kandler K: Inhibitory synapses in the developing auditory system are glutamatergic. Nat Neurosci 8(3) : 332-338, 2005. https://doi.org/10.1038/nn1397
  14. Grant ER, Bacskai BJ, Pleasure DE, Pritchett DB, Gallagher MJ, Kendrick SJ, Kricka LJ, Lynch DR: N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunitspecific increases in free intracellular calcium. J Biol Chem 272(1) : 647-656, 1997. https://doi.org/10.1074/jbc.272.1.647
  15. Haberny KA, Paule MG, Scallet AC, Sistare FD, Lester DS, Hanig JP, Slikker W Jr: Ontogeny of the N-methyl-D-aspartate (NMDA) receptor system and susceptibility to neurotoxicity. Toxicol Sci 68(1) : 9-17, 2002. https://doi.org/10.1093/toxsci/68.1.9
  16. Hofer M, Prusky GT, Constantine-Paton M: Regulation of NMDA receptor mRNAduring visual map formation and after receptor blockade. J Neurochem 62(6) : 2300-2307, 1994.
  17. Hong SH, Kim MJ, Ahn SC: Glutamatergic transmission is sustained at later period of development of medial nucleus of the trapezoid body-lateral superior olive synapses in circling mice. J Neurosci 28(48) : 13003-13007, 2008. https://doi.org/10.1523/JNEUROSCI.3002-08.2008
  18. Hunter C, Petralia RS, Vu T, Wenthold RJ: Expression of AMPAselective glutamate receptor subunits in morphologically defined neurons of the mammalian cochlear nucleus. J Neurosci 13(5) : 1932-1946, 1993.
  19. Ikonomidou C, Bittigau P, Koch C, Genz K, Hoerster F, Felderhoff-Mueser U, Tenkova T, Dikranian K, Olney JW: Neurotransmitters and apoptosis in the developing brain. Biochem Pharmacol 15;62(4) : 401-405, 2001.
  20. Joelson D, Schwartz IR: Development of N-methyl-D-aspartate receptor subunit immunoreactivity in the neonatal gerbil cochlear nucleus. Microsc Res Tech 1;41(3) : 246-262, 1998.
  21. Kandler K, Friauf E: Development of glycinergic and glutamatergic synaptic transmission in the auditory brainstem of perinatal rats. J Neurosci 15(10) : 6890-6904, PubMed PMID: 7472446. 1995.
  22. Kandler K, Kullmann PH, Ene FA, Kim G: Excitatory action of an immature glycinergic/GABAergic sound localization pathway. Physiol Behav 77(4-5) : 583-587, 2002. https://doi.org/10.1016/S0031-9384(02)00905-8
  23. Kaye CI, Martin AO, Rollnick BR, Nagatoshi K, Israel J, Hermanoff M, Tropea B, Richtsmeier JT, Morton NE: Oculoauri-culovertebral anomaly; segregation analysis. Am J Med Genet 1;43(6) : 913-917, 1992. https://doi.org/10.1002/ajmg.1320430602
  24. Kotak VC, Sanes DH: Developmental influence of glycinergic transmission: regulation of NMDA receptor-mediated EPSPs. J Neurosci 1;16(5) : 1836-1843, PubMed PMID: 8774451. 1996.
  25. Kotak VC, Sanes DH: Deafferentation weakens excitatory synapses in the developing central auditory system. Eur J Neurosci 9(11) : 2340-2347, 1997. https://doi.org/10.1111/j.1460-9568.1997.tb01651.x
  26. Kotak VC, Korada S, Schwartz IR, Sanes DH: A developmental shift from GABAergic to glycinergic transmission in the central auditory system. J Neurosci 18(12) : 4646-4655, 1998.
  27. Lee JW, Lee EJ, Hong SH, Chung WH, Lee HT, Lee TW, Lee JR, Kim HT, Suh JG, Kim TY, Ryoo ZY: Circling mouse; possible animal model for deafness. Comp Med 51(6) : 550-554, 2001.
  28. Liang Y, Wang A, Probst FJ, Arhya IN, Barber TD, Chen KS, Deshmukh D, Dolan DF, Hinnant JT, Carter LE, Jain PK, Lalwani AK, Li XC, Lupski JR, Moeljopawiro S, Morell R, Negrini C, Wilcox ER, Winata S, Camper SA, Friedman TB: Genetic mapping refines DFNB3 to 17p11.2, suggests multiple alleles of DFNB3, and supports homology to the mouse model shaker-2. Am J Hum Genet 62(4) : 904-915, 1998. https://doi.org/10.1086/301786
  29. McDonald JW, Johnston MV: Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res Rev 15(1) : 41-70, 1990. https://doi.org/10.1016/0165-0173(90)90011-C
  30. Moore DR: Trophic influences of excitatory and inhibitory synapses on neurons in the auditory brain stem. Neuroreport 3(3) : 269-272, 1992. https://doi.org/10.1097/00001756-199203000-00014
  31. Nakagawa H, Sato K, Shiraishi Y, Kuriyama H, Altschuler RA: NMDAR1 isoforms in the rat superior olivary complex and changes after unilateral cochlear ablation. Brain Res Mol Brain Res 77(2) : 246-257, 2000. https://doi.org/10.1016/S0169-328X(00)00059-0
  32. Nakanishi S, Masu M, Bessho Y, Nakajima Y, Hayashi Y, Shigemoto R: Molecular diversity of glutamate receptors and their physiological functions. EXS 71 : 71-80, 1994.
  33. Noben-Trauth K, Zheng QY, Johnson KR, Nishina PM: A deafnesssusceptibility locus that interacts with deaf waddler (dfw). Genomics 15;44(3) : 266-272, 1997.
  34. Rao H, Jean A, Kessler JP: Postnatal ontogeny of glutamate receptors in the rat nucleus tractus solitarii and ventrolateral medulla. J Auton Nerv Syst 14;65(1) : 25-32, 1997. https://doi.org/10.1016/S0165-1838(97)00031-3
  35. Ritter LM, Unis AS, Meador-Woodruff JH: Ontogeny of ionotropic glutamatereceptor expression in human fetal brain. Brain Res Dev Brain Res 127(2) : 123-133, 2001. https://doi.org/10.1016/S0165-3806(01)00126-2
  36. Sanes DH, Friauf E: Development and influence of inhibition in the lateral superior olivary nucleus. Hear Res 147(1-2) : 46-58, 2000. https://doi.org/10.1016/S0378-5955(00)00119-2
  37. Sato K, Nakagawa H, Kuriyama H, Altschuler RA: Differential distribution of N-methyl-D-aspartate receptor-2 subunit messenger RNA in the rat superior olivary complex. Neuroscience 89(3) : 839-853, 1999. https://doi.org/10.1016/S0306-4522(98)00350-9
  38. Scheetz AJ, Constantine-Paton M: Modulation of NMDA receptor function; implications for vertebrate neural development. FASEB J 8(10) : 745-752, 1994. https://doi.org/10.1096/fasebj.8.10.8050674
  39. Sheng M, Cummings J, Roldan LA, Jan YN, Jan LY: Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368(6467) : 144-147, 1994. https://doi.org/10.1038/368144a0
  40. Suneja SK, Potashner SJ, Benson CG: Plastic changes in glycine and GABA release and uptake in adult brain stem auditory nuclei after unilateral mm dle ear ossicle removglycinecochlear ablation. Exp Neurol 151(2) : 273-288, 1998. https://doi.org/10.1006/exnr.1998.6812
  41. Thompson AM, Schofield BR: Afferent projections of the superior olivarycomplex. Microsc Res Tech 15;51(4) : 330-354, 2000. https://doi.org/10.1002/1097-0029(20001115)51:4<330::AID-JEMT4>3.0.CO;2-X