Ionotropic Glutamate Receptor Expression in the Hypothalamus: An Immunohistochemical Localization Study

Öz

major excitatory amino acid neurotransmitter in the hypothalamus. Glutamate performs its regulatory function on neurons by binding to its receptors, which are classified in two subfamilies as ionotropic and metabotropic. It has been determined by electrophysiological and pharmacological studies that glutamate is effective on some neuroendocrine neurons. Due to the lack of studies supporting glutamatergic innervation histomorphologically, our study focused on hypothalamic glutamate receptor subunits. The distributions of AMPA (GluA1, GluA2, GluA3, GluA4), kainic acid (GluK5, GluK1, GluK2, GluK3) and NMDA (GluN1, GluN2A) in the hypothalamus, which are ionotropic glutamate receptor subunits, were investigated using the immunohistochemistry method. Sections taken by vibratome from brain tissues of female rats were evaluated for the presence and density of glutamate receptor subunits in hypothalamic neurons. It was determined that the AMPA receptors subunits GluA1 and GluA2 are highly expressed in the hypothalamus while the synthesis of GluA3 and GluA4 is too limited. Very high number of GluK5-immunoreactive neurons was detected, where the expression of GluK1/2/3 was moderate. GluN1A, one of the NMDA receptor subunits, was found to be densely localized in the hypothalamus. In conclusion, with this study, many hypothalamic neurons can express ion-specific iGluR channels in different amounts and intensities, which would allow a differential regulation of the target neurons by glutamate.

Anahtar Kelimeler

• Glutamate
• Kainic Acid
• AMPA
• NMDA
• Hypothalamus

Hipotalamusta İyonotropik Glutamat Reseptör Ekspresyonu: İmmunohistokimyasal Lokalizasyon Çalışması

Öz

Merkezi sinir sisteminde (MSS) nöron fonksiyonlarının düzenlenmesinde çok çeşitli nörotransmitter madde rol oynar. Glutamat, hipotalamusta en önemli eksitatör amino asit nörotransmitterlerden biri olarak yer alır ve nöronlar üzerindeki düzenleyici işlevini iyonotropik ve metabotropik olarak iki alt ailede sınıflandırılan reseptörlerine bağlanarak gerçekleştirir. Glutamatın bazı nöroendokrin nöronlar üzerinde etkili olduğu yapılan elektrofizyolojik ve farmakolojik çalışmalarla belirlenmiştir. Çalışmamız kapsamında, glutamaterjik innervasyonun histomorfolojik olarak desteklendiği çalışmaların bulunmaması sebebiyle hipotalamik glutamat reseptör alt birimlerine odaklanılmıştır. İyonotropik glutamat reseptör alt birimlerinden AMPA (GluA1, GluA2, GluA3, GluA4), Kainik asit (GluK5, GluK1, GluK2, GluK3) ve NMDA’nın (GluN1, GluN2A) hipotalamustaki dağılımları immünohistokimya yöntemi kullanılarak araştırılmıştır. Dişi sıçanların beyin dokularından vibratom aracılığıyla alınan kesitler, hipotalamik nöronlardaki glutamat reseptör alt birimlerinin varlığı ve yoğunluğu açısından değerlendirilmiştir. Çalışmada, AMPA reseptörlerinden GluA1 ve GluA2’nin yaygın olarak hipotalamusta lokalize olduğu, GluA3 ve GluA4 immünoreaksiyonunun ise çok sınırlı olduğu; kainat reseptör alt birimlerinden GluK5’in çok yaygın olduğu ve GluK1/2/3 içeren nöronların daha az sayıda olduğu, NMDA reseptör alt birimlerinden GluN1’in ise hipotalamusta yoğun olarak bulunduğu saptanmıştır. Sonuç olarak, bu çalışma ile hipotalamik birçok nöronun iyon spesifik iGluR kanallarını farklı miktar ve yoğunlukta eksprese edebildiği, bunun da hedef nöronların glutamat tarafından farklı şekilde düzenlenmesine izin verebileceği gösterilmiştir.

Anahtar Kelimeler

• Glutamat
• Kainik Asit
• AMPA
• NMDA
• Hipotalamus

Kaynakça

1. Niciu MJ, Kelmendi B, Sanacora G. Overview of glutamatergic neurotransmission in the nervous system. Pharmacol Biochem Behav 2012;100:656-64.
2. Nakanishi S. Molecular Diversity of Glutamate Receptors and Implications for Brain Functions. Science 1992;258:597-603.
3. Danbolt NC. Glutamate Uptake. Prog Neurobiol 2001;65:1-105.
4. Meldrum BS. Glutamate As a Neurotransmitter in the Brain: Review of Physiology and Pathology. J Nutr 2000;130:1007S-15S.
5. Van Den Pol AN, Wuarin JP, Dudek FE. Glutamate Neurotransmission in the Neuroendocrine Hypothalamus. Brann DW, Mahesh VB (eds). Excitatory Amino Acids: Their Role in Neuroendocrine Function. 1st edition. Boca Raton: CRC Press; 1996. 1-54.
6. Hollmann M, Heinemann S. Cloned glutamate receptors. Annu Rev Neurosci 1994;17:31-108.
7. Ozawa S, Kamiya H, Tsuzuki K. Glutamate receptors in the mammalian central nervous system. Prog Neurobiol 1998;54:581-618.
8. Koles L, Wirkner K, Illes P. Modulation of Ionotropic Glutamate Receptor Channels. Neurochem Res 2001;26:925-32.

9. Bettler B, Mulle C. Neurotransmitter receptors II. AMPA and kainate receptors. Neuropharmacology 1995;34:123-139.
10. Kew JN, Kemp JA. Ionotropic and metabotropic glutamate receptor structure and pharmacology. Psychopharmacology 2005;179:4-29.
11. Mori H, Mishina M. Structure and function of the NMDA receptor channel. Neuropharmacology 1995;34:1219-37.
12. Alt A, Weiss B, Ogden AM et al. Pharmacological characterization of glutamatergic agonists and antagonists at recombinant human homomeric and heteromeric kainate receptors in vitro. Neuropharmacology 2004;46:793-806.
13. Howe JR. Homomeric and heteromeric ion channels formed from the kainate type subunits GluR6 and KA2 have very small, but different, unitary conductances. J Neurophysiol 1996;76: 510-19.
14. Monyer H, Sprengel R, Herb A, et al. Heteromeric NMDA Receptors: Molecular and Functional Distinction of Subtypes. Science 1992;256:1217-21.
15. Lerma J, Paternain AV, Naranjo J R, Mellstrom B. Functional kainate-selective glutamate receptors in cultured hippocampal neurons. Proc Natl Acad Sci U S A 1993;90:11688-92.
16. Paternain AV, Herrera MT, Nieto MA, Lerma J. GluR5 and GluR6 kainate receptor subunits coexist in hippocampal neurons and coassemble to form functional receptors. J Neurosci 2000;20:196-205.
17. Molina PE, (eds). Endocrine Physiology. 4th edition. New York: Mc Graw-Hill; 2004.
18. Van Den Pol AN, Wuarin JP, Dudek FE. Glutamate, the Dominant Excitatory Transmitter in Neuroendocrine Regulation. Science 1990;250:1276-8.
19. Paxinos G, Watson C, (eds). The rat brain in stereotaxic coor-dinates. 6th edition. Elsevier Academic Press: Amsterdam; 2009.
20. Herman JP, Eyigor O, Ziegler DR, Jennes L. Expression of Ionotropic Glutamate Receptor Subunit mRNAs in the Hypothalamic Paraventricular Nucleus of the Rat. J Comp Neurol 2000;422:352-62.
21. Van Den Pol AN, Hermans-Borgmeyer I, Hofer M, Ghosh P, Heinemann S. Ionotropic Glutamate-Receptor Gene Expression in Hypothalamus: Localization of AMPA, Kainate, and NMDA Receptor RNA with in Situ Hybridization. J Comp Neurol 1994;343:428-44.
22. Sato K, Kiyama H, Tohyama M. The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4) in the rat brain. Neuroscience 1993;52:515-39.
23. Eyigor O, Centers A, Jennes L. Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus. J Comp Neurol 2001;431:101-24.
24. Brann DW, Mahesh VB. Excitatory Amino Acids: Function and Significance in Reproduction and Neuroendocrine Regulation. Front Neuroendocrinol 1994;15:3-49.
25. Van Den Pol AN. Glutamate and Aspartate Immunoreactivity in Hypothalamic Presynaptic Axons. J. Neurosci 1991;11:2087-101.
26. Brann DW, Zamorano PL, Chorich LP, Mahesh VB. Steroid Hormone Effects on NMDA Receptor Binding and NMDA Receptor mRNA Levels in the Hypothalamus and Cerebral Cortex of the Adult Rat. Neuroendocrinology 1993;58:666-72.
27. Cotman CW, Monaghan DT, Ottersen OP, Storm-Mathisen J. Anatomical Organization of Excitatory Amino Acid Receptors and Their Pathways. Trends Neurosci 1987;10:273-80.
28. Kus L, Handa RJ, Sanderson JJ, Kerr JE, Beitz AJ. Distribution of NMDAR1 Receptor Subunit mRNA and [125I]MK-801 Binding in the Hypothalamus of Intact, Castrate and Castrate-DHTP Treated Male Rats. Mol Brain Res 1995;28:55-60.
29. Goldsmith PC, Thind KK, Perera AD, Plant TM. Glutamate-Immunoreactive Neurons and Their Gonadotropin-Releasing Hormone-Neuronal Interactions in the Monkey Hypothalamus. Endocrinology 1994;134:858-68.
30. Gu G, Varoqueaux F, Simerly RB. Hormonal Regulation of Glutamate Receptor Gene Expression in the Anteroventral Periventricular Nucleus of the Hypothalamus. J Neurosci 1999;19:3213-22.
31. Kawakami S. Glial and Neuronal Localization of Ionotropic Glutamate Receptor Subunit-Immunoreactivities in the Median Eminence of Female Rats: GluR2/3 and GluR6/7 Colocalize With Vimentin, Not With Glial Fibrillary Acidic Protein (GFAP). Brain Res 2000;858:198-204.
32. Michel S, Itri J, Colwell CS. Excitatory Mechanisms in the Suprachiasmatic Nucleus: the Role of AMPA/KA Glutamate Receptors. J Neurophysiol 2002;88;817-28.
33. Petralia RS, Wenthold RJ. Light and Electron Immunocytochemical Localization of AMPA-Selective Glutamate Receptors in the Rat Brain. J Comp Neurol 1992;318:329-54.
34. Petralia RS, Wang YX, Wenthold RJ. Histological and Ultrastructural Localization of the Kainate Receptor Subunits, KA2 and GluR6/7, in the Rat Nervous System Using Selective Antipeptide Antibodies. J Comp Neurol 1994;349:85-110.
35. Warembourg M, Leroy D. AMPA Glutamate Receptor Subunits in the Guinea Pig Hypothalamus: Distribution and Colocalization with Progesterone Receptor. J Comp Neurol 2002;453:305-21.
36. Minbay FZ, Serter S, Gok Yurtseven D, Eyigor O. Immunohistochemical localization of ionotropic glutamate receptors in the rat red nucleus. Bosnian J Basic Med 2017;17:29–37.
37. Doherty FC, Sladek CD. NMDA receptor subunit expression in the supraoptic nucleus of adult rats: dominance of NR2B and NR2D. Brain Res 2011;1388:89-99.
38. Portera-Cailliau C, Price DL, Martin LJ. N-methyl-D-aspartate receptor proteins NR2A and NR2B are differentially distributed in the developing rat central nervous system as revealed by subunit-specific antibodies. J Neurochem 1996;66:692-700.
39. Wenzel A, Fritschy JM, Mohler H, Benke D. NMDA receptor heterogeneity during postnatal development of the rat brain: differential expression of the NR2A, NR2B, and NR2C subunit proteins. J Neurochem 1997;68:469-78.
40. Eyigor O, Minbay Z, Cavusoglu I, Jennes L. Localization of Kainate Receptor Subunit GluR5-Immunoreactive Cells in the Rat Hypothalamus. Brain Res Mol Brain Res 2005;136: 38-44.
41. Minbay FZ, Eyigor O, Oksitosin Nöronlarında Kainat Reseptör Alt Birimlerinin Sentezi: İmmünohistokimyasal Çalışma. Uludağ Üniversitesi Tıp Fakültesi Dergisi 2020;46:357-363.
42. Kim JH, Marton J, Ametamey SM, Cumming P. A Review of Molecular Imaging of Glutamate Receptors. Molecules 2020;25:47-9.