Abstract
Transient expression of different NMDA receptors (NMDARs) plays a role in development of the cerebellum. Whether similar processes undergo during neuronal differentiation in culture is not clearly understood. We studied NMDARs in cerebellar neurons in cultures of 7 and 21 days in vitro (DIV) using immunocytochemical and electrophysiological approaches. Whereas at 7 DIV, the vast majority of neurons were immunopositive for GluN2 subunits, further synaptoginesis was accompanied by the time-dependent loss of NMDARs. In contrast to GluN2B- and GluN2C-containing NMDARs, which at 7 DIV exhibited homogenous distribution in extrasynaptic regions, GluN2A-containing receptors were aggregated in spots both in cell bodies and dendrites. Double staining for GluN2A subunits and synaptophysin, a widely used marker for presynaptic terminals, revealed their co-localization in about 75% of dendrite GluN2A fluorescent spots, suggesting postsynaptic origin of GluN2A subunits. In agreement, diheteromeric GluN2A-containing NMDARs contributed to postsynaptic currents recorded in neurons throughout the timescale under study. Diheteromeric GluN2B-containing NMDARs escaped postsynaptic regions during differentiation. Finally, the developmental switch favored the expression of triheteromeric NMDARs assembled of 2 GluN1/1 GluN2B/1 GluN2C or GluN2D subunits in extrasynaptic regions. At 21 DIV, these receptors represented over 60% of the NMDAR population. Thus, cerebellar neurons in primary culture undergo transformations with respect to the expression of di- and triheteromeric NMDARs that should be taken into account when studying cellular aspects of their pharmacology and functions.
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References
Akazawa C, Shigemoto R, Bessho Y, Nakanishi S, Mizuno N (1994) Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats. J Comp Neurol 347(1):150–160. https://doi.org/10.1002/cne.903470112
Andreescu CE, Prestori F, Brandalise F, D’Errico A, De Jeu MT, Rossi P, Botta L, Kohr G, Perin P, D’Angelo E, De Zeeuw CI (2011) NR2A subunit of the N-methyl D-aspartate receptors are required for potentiation at the mossy fiber to granule cell synapse and vestibulo-cerebellar motor learning. Neuroscience 176:274–283. https://doi.org/10.1016/j.neuroscience.2010.12.024
Bartschat S, Fieguth A, Könemann J, Schmidt A, Bode-Jänisch S (2012) Indicators for acute hypoxia—an immunohistochemical investigation in cerebellar Purkinje-cells. Forensic Sci Int 223(1-3):165–170. https://doi.org/10.1016/j.forsciint.2012.08.023
Bidoret C, Bouvier G, Ayon A, Szapiro G, Casado M (2015) Properties and molecular identity of NMDA receptors at synaptic and non-synaptic inputs in cerebellar molecular layer interneurons. Front Synaptic Neurosci 7. https://doi.org/10.3389/fnsyn.2015.00001
Brickley SG, Misra C, Mok MHS, Mishina M, Cull-Candy SG (2003) NR2B and NR2D subunits coassemble in cerebellar Golgi cells to form a distinct NMDA receptor subtype restricted to extrasynaptic sites. J Neurosci 23(12):4958–4966
Burnashev N, Szepetowski P (2015) NMDA receptor subunit mutations in neurodevelopmental disorders. Curr Opin Pharmacol 20:73–82. https://doi.org/10.1016/j.coph.2014.11.008
Cathala L, Misra C, Cull-Candy S (2000) Developmental profile of the changing properties of NMDA receptors at cerebellar mossy fiber-granule cell synapses. J Neurosci 20(16):5899–5905
Frizelle PA, Chen PE, Wyllie DJ (2006) Equilibrium constants for (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl-phosphonic acid (NVP-AAM077) acting at recombinant NR1/NR2A and NR1/NR2B N-methyl-D-aspartate receptors: implications for studies of synaptic transmission. Mol Pharmacol 70(3):1022–1032. https://doi.org/10.1124/mol.106.024042
Fu Z, Logan SM, Vicini S (2005) Deletion of the NR2A subunit prevents developmental changes of NMDA-mEPSCs in cultured mouse cerebellar granule neurones. J Physiol 563(3):867–881. https://doi.org/10.1113/jphysiol.2004.079467
Glasgow NG, Siegler Retchless B, Johnson JW (2014) Molecular bases of NMDA receptor subtype-dependent properties. J Physiol 593(1):83–95. https://doi.org/10.1113/jphysiol.2014.273763
Hansen KB, Ogden KK, Yuan H, Traynelis SF (2014) Distinct functional and pharmacological properties of triheteromeric GluN1/GluN2A/GluN2B NMDA receptors. Neuron 81(5):1084–1096. https://doi.org/10.1016/j.neuron.2014.01.035
Hedegaard M, Hansen KB, Andersen KT, Bräuner-Osborne H, Traynelis SF (2012) Molecular pharmacology of human NMDA receptors. Neurochem Int 61(4):601–609. https://doi.org/10.1016/j.neuint.2011.11.016
Hirano T, Ohmori H (1986) Voltage-gated and synaptic currents in rat Purkinje cells in dissociated cultures. Proc Natl Acad Sci U S A 83(6):1945–1949. https://doi.org/10.1073/pnas.83.6.1945
Hockberger PE, Tseng HY, Connor JA (1989) Development of rat cerebellar Purkinje cells, electrophysiological properties following acute isolation and in long-term culture. J Neurosci 9(7):2258–2271
Huang Z, Gibb AJ (2014) Mg2+ block properties of triheteromeric GluN1-GluN2B-GluN2D NMDA receptors on neonatal rat substantia nigra pars compacta dopaminergic neurons. J Physiol 592(10):2059–2078. https://doi.org/10.1113/jphysiol.2013.267864
Iijima K, Abe H, Okazawa M, Moriyoshi K, Nakanishi S (2008) Dual regulation of NR2B and NR2C expression by NMDA receptor activation in mouse cerebellar granule cell cultures. Proc Natl Acad Sci U S A 105(33):12010–12015. https://doi.org/10.1073/pnas.0805574105
Jones S, Gibb AJ (2005) Functional NR2B- and NR2D-containing NMDA receptor channels in rat substantia nigra dopaminergic neurones. J Physiol 569(1):209–221. https://doi.org/10.1113/jphysiol.2005.095554
Misra C, Brickley S, Wyllie D, Cull-Candy S (2000a) Slow deactivation kinetics of NMDA receptors containing NR1 and NR2D subunits in rat cerebellar Purkinje cells. J Physiol 525(2):299–305. https://doi.org/10.1111/j.1469-7793.2000.t01-1-00299.x
Misra C, Brickley SG, Farrant M, Cull-Candy SG (2000b) Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in Golgi cells of the rat cerebellum. J Physiol 524(1):147–162. https://doi.org/10.1111/j.1469-7793.2000.00147.x
Mosley CA, Acker TM, Hansen KB, Mullasseril P, Andersen KT, Le P, Vellano KM, Bräuner-Osborne H, Liotta DC, Traynelis SF (2010) Quinazolin-4-one derivatives, a novel class of noncompetitive NR2C/D subunit-selective N-methyl-D-aspartate receptor antagonists. J Med Chem 53(15):5476–5490. https://doi.org/10.1021/jm100027p
Neyton J, Paoletti P (2006) Relating NMDA receptor function to receptor subunit composition, limitations of the pharmacological approach. J Neurosci 26(5):1331–1333. https://doi.org/10.1523/JNEUROSCI.5242-05.2006
Paoletti P, Ascher P, Neyton J (1997) High-affinity zinc inhibition of NMDA NR1-NR2A receptors. J Neurosci 17(15):5711–5725
Paoletti P, Bellone C, Zhou Q (2013) NMDA receptor subunit diversity, impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci 14(6):383–400. https://doi.org/10.1038/nrn3504
Papa M, Bundman MC, Greenberger V, Segal M (1995) Morphological analysis of dendritic spine development in primary cultures of hippocampal neurons. J Neurosci 15(1 Pt 1):1–11
Piochon C, Irinopoulou T, Brusciano D, Bailly Y, Mariani J, Levenes C (2007) NMDA receptor contribution to the climbing fiber response in the adult mouse Purkinje cell. J Neurosci 27(40):10797–10809. https://doi.org/10.1523/JNEUROSCI.2422-07.2007
Renzi M, Farrant M, Cull-Candy SG (2007) Climbing-fibre activation of NMDA receptors in Purkinje cells of adult mice. J Physiol 585(1):91–101. https://doi.org/10.1113/jphysiol.2007.141531
Reynolds IJ, Miller RJ (1989) Ifenprodil is a novel type of N-methyl-D-aspartate receptor antagonist, interaction with polyamines. Mol Pharmacol 36(5):758–765
Ridler TW, Calvard S (1978) Picture thresholding using an iterative selection method. IEEE Transactions on Systems, Man, and Cybernetics 8(8):630–632. https://doi.org/10.1109/TSMC.1978.4310039
Sibarov DA, Bruneau N, Antonov SM, Szepetowski P, Burnashev N, Giniatullin R (2017) Functional properties of human NMDA receptors associated with epilepsy-related mutations of GluN2A subunit. Front Cell Neurosci 11:155. https://doi.org/10.3389/fncel.2017.00155
Thompson CL, Drewery DL, Atkins HD, Stephenson FA, Chazot PL (2000) Immunohistochemical localization of N-methyl-D-aspartate receptor NR1, NR2A, NR2B and NR2C/D subunits in the adult mammalian cerebellum. Neurosci Lett 283(2):85–88. https://doi.org/10.1016/S0304-3940(00)00930-7
Tovar KR, Westbrook GL (1999) The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci 19(10):4180–4188
Weber A, Schachner M (1984) Maintenance of immunologically identified Purkinje cells from mouse cerebellum in monolayer culture. Brain Res 311(1):119–130. https://doi.org/10.1016/0006-8993(84)91404-5
Wiedenmann B, Franke WW (1985) Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell 41(3):1017–1028. https://doi.org/10.1016/S0092-8674(85)80082-9
Yuzaki M, Forrest D, Verselis LM, Sun SC, Curran T, Connor JA (1996) Functional NMDA receptors are transiently active and support the survival of Purkinje cells in culture. J Neurosci 16(15):4651–4661
Funding
The work was supported by Russian Science Foundation grant (no. 16-15-10192). Purkinje cells imaging experiments were supported by RFBR grant (no. 15-04-08283).
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Experimental work and data acquisition: D.A.S., Y.D.S., P.A.A., and T.V.K. Data analysis and preparation of figures: D.A.S. and Y.D.S. Study design/interpretation and drafting of the manuscript: S.M.A. and D.A.S.
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All procedures using animals were in accordance with recommendations of the Federation for Laboratory Animal Science Associations and approved by the Bioethics Committee of Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (IEPhB RAS).
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Sibarov, D.A., Stepanenko, Y.D., Silantiev, I.V. et al. Developmental Changes of Synaptic and Extrasynaptic NMDA Receptor Expression in Rat Cerebellar Neurons In Vitro. J Mol Neurosci 64, 300–311 (2018). https://doi.org/10.1007/s12031-017-1021-y
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DOI: https://doi.org/10.1007/s12031-017-1021-y