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Journal of Molecular Neuroscience

, Volume 64, Issue 2, pp 300–311 | Cite as

Developmental Changes of Synaptic and Extrasynaptic NMDA Receptor Expression in Rat Cerebellar Neurons In Vitro

  • Dmitry A. Sibarov
  • Yulia D. Stepanenko
  • Ivan V. Silantiev
  • Polina A. Abushik
  • Tatiana V. Karelina
  • Sergei M. Antonov
Article

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.

Keywords

Cerebellum NMDA receptors Purkinje cells Triheteromeric GluN2A GluN2B GluN2C 

Notes

Author Contributions

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.

Funding Information

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.

Ethical Statement

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  • Dmitry A. Sibarov
    • 1
  • Yulia D. Stepanenko
    • 1
  • Ivan V. Silantiev
    • 1
  • Polina A. Abushik
    • 1
  • Tatiana V. Karelina
    • 1
  • Sergei M. Antonov
    • 1
  1. 1.Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, Laboratory of Comparative NeurophysiologySaint PetersburgRussia

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