Developmental Changes of Synaptic and Extrasynaptic NMDA Receptor Expression in Rat Cerebellar Neurons In Vitro
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.
KeywordsCerebellum NMDA receptors Purkinje cells Triheteromeric GluN2A GluN2B GluN2C
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.
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).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
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).
- 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 CrossRefPubMedGoogle Scholar
- 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
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar