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Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons

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NMDA Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2799))

Abstract

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

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Acknowledgments

Work on this chapter was supported by the MEYS CR: Program LT – INTER-EXCELLENCE LTAUSA19122 (AB), GACR 23-04922S (LV), TACR TN02000109, Research Project of the CAS CR (RVO: 67985823), and GAUK 376221 (VK). Supported also by project num. LX22NPO5107 (MEYS CR): Financed by EU – Next Generation EU (MH).

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Authors and Affiliations

  1. Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic

    Viktor Kuchtiak, Tereza Smejkalova, Ladislav Vyklicky & Ales Balik

  2. Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic

    Martin Horak

Authors
  1. Viktor Kuchtiak
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  2. Tereza Smejkalova
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  3. Martin Horak
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  4. Ladislav Vyklicky
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  5. Ales Balik
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Correspondence to Ales Balik .

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Editors and Affiliations

  1. INSERM, Institut de Neurobiologie de la Méditerranée (INMED), Marseille, France

    Nail Burnashev

  2. INSERM, Institut de Neurobiologie de la Méditerranée (INMED), Marseille, France

    Pierre Szepetowski

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Kuchtiak, V., Smejkalova, T., Horak, M., Vyklicky, L., Balik, A. (2024). Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons. In: Burnashev, N., Szepetowski, P. (eds) NMDA Receptors. Methods in Molecular Biology, vol 2799. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3830-9_3

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  • DOI: https://doi.org/10.1007/978-1-0716-3830-9_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3829-3

  • Online ISBN: 978-1-0716-3830-9

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