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

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.

Spatial Learning and Memory Impairment in Growing Mice Induced by Major Oxidized Tyrosine Product Dityrosine

“…In general, the behavioral tests (TFT, EPM, and MWM) reflected brain damage and behavioral abnormalities caused by pure DT and OTPs from different macroscopic perspectives, confirming our hypothesis, which was corroborated at the molecular level by differential gene expression analysis. NMDAR are ionotropic glutamate receptors that are crucial for memory development. , They function as ion channels permeable to sodium, potassium, and calcium ions and localize to the presynaptic and postsynaptic membranes as well as the protruding outer neuron membrane . Downregulation of the Nr2b subunit suppresses the Ca 2+ influx, reducing the intracellular Ca 2+ concentration .…”

“…NMDAR are ionotropic glutamate receptors that are crucial for memory development. 26,27 They function as ion channels permeable to sodium, potassium, and calcium ions and localize to the presynaptic and postsynaptic membranes as well as the protruding outer neuron membrane. 27 Downregulation of the Nr2b subunit suppresses the Ca 2+ influx, reducing the intracellular Ca 2+ concentration.…”

“…26,27 They function as ion channels permeable to sodium, potassium, and calcium ions and localize to the presynaptic and postsynaptic membranes as well as the protruding outer neuron membrane. 27 Downregulation of the Nr2b subunit suppresses the Ca 2+ influx, reducing the intracellular Ca 2+ concentration. 28 Ca 2+ is an important intracellular signaling molecule that controls synaptic plasticity, differentiation, and excitotoxicity, and is necessary for longterm potentiation (LTP) and long-term depression (LTD) of synaptic transmission.…”

NMDA receptor antagonists as potential therapy in cerebellar degenerative disorders

“…Имеются противоречивые данные об экспрессии субъединиц GluN2 во взрослых КП: в одних исследованиях мРНК GluN2A была обнаружена как у крыс, так и у людей [28,39,40], тогда как в других работах мРНК GluN2 в КП взрослых грызунов не выявлена [35,41].…”

“…В период эмбрионального и постнатального развития мозга отмечается изменение субъединичного состава NMDAR [28]. На всех этапах ген GluN1 экспрессируется практически во всех нейронах, в частности, в КП эта субъединица экспрессируется уже в самом начале жизни у мышей и в течение всей взрослой жизни [20,29,30].…”