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.

• Klíčová slova
• Colocalization, Fluorescence immunostaining, Fluorescence microscopy, Glutamate receptor, ImageJ analysis, Lentivirus, Primary hippocampal neurons, Surface expression,
• MeSH
• exprese genu MeSH
• hipokampus * metabolismus   cytologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• Lentivirus genetika   MeSH
• lidé MeSH
• neurony * metabolismus   MeSH
• podjednotky proteinů metabolismus   genetika   MeSH
• primární buněčná kultura metody   MeSH
• receptory N-methyl-D-aspartátu * metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• N-methyl D-aspartate receptor subtype 2A MeSH Prohlížeč
• podjednotky proteinů MeSH
• receptory N-methyl-D-aspartátu * MeSH