Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro‐induced neurogenesis

Abstract

NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-de®cient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluR11 and GluR14 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluR13 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluR12 gene were detected only after neural network formation. The presence of the GluRz1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)-and exon 22 (C2)-containing GluRz1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca 21 -signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our ®ndings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication. Glutamic acid, one of the most abundant small molecular neurotransmitters, has multiple roles during the early stages of neural differentiation, well before synapses become functionally active (Mattson and Hauser 1991;Meier et al. 1991). In the early phases of neural tissue formation, neural progenitors and young neurons express various glutamate receptors and display low vulnerability to glutamate toxicity (LoTurco et al. 1991(LoTurco et al. , 1995Scherer and Gallo 1998).The physiological effects of glutamate are mediated by metabotropic and ionotropic glutamate receptors (Nakanishi 1992; Seeburg 1993). Ionotropic glutamate receptor channels have been classi®ed into three structurally and pharmacologically distinct subtypes: the a-amino-3-hydroxy-5-methylisoxazole-4-propionate, kainate and NMDA receptors.Cloning the genes encoding NMDA receptor subunits has provided molecular evidence for the existence of heteromeric receptors (Monyer et al. 1992;Nakanishi 1992). Functional NMDA receptors contain the GluRz1-type subunit (GluRz1 in mouse; NR1 in rat) and any of the four GluR1-type subunits (GluR11±4 in mouse, NR2A±D in rat; Moriyoshi et al. 1991;Meguro et al. 1992). The presence of the GluRz1 subunit is necessary for the assembly of functional channels (Schoepfer et al. 1994). Several properties of NMDA receptors, however, appear to be modulated by particular GluR1 subunits (Monyer et al. 1992. Abbreviations used: ATRA, all-trans retinoic acid; d,l-AP5, dl-2-amino-5-phosphonopentanoic acid; FCS, fetal calf serum; FITC, uorescein isothiocyanate; GAPDH, glycerol-aldehyde-3-phosphate dehydrogenase; GFAP, glial ®brillary acidic protein; GluR1...

Glutamate as a Modulator of Embryonic and Adult Neurogenesis

“…Despite the fact that NR1 and some of the NR2 subunits were detected in the proliferative zones of the embryonic and perinatal brain [10,[104][105][106][107] or in dividing striatal progenitor cells in vitro [8], electrophysiological and Ca 2+ imaging experiments showed functional NMDA receptors only in young, postmitotic neurons just before or during the onset of their migratory processes [5,6,9,59,[108][109][110]. Similar delay between subunit expression and the formation of functional NMDA receptors was observed in ES cells and several neuronal progenitor cell lines during their in vitro neural differentiation [48][49][50][51][52][53][54][56][57][58]. These data suggest that proper intracellular trafficking and assembly of the subunits leading to the formation of functional heteromeric NMDA receptors requires a certain level of neuronal maturation, which is not achieved by dividing stem cells or neural precursors.…”

“…By contrast, neuroectodermal stem cell lines have more limited ways of differentiation and generate only those cell types which are normally formed in the ventricular zone during development of the CNS. Using adequate protocols to induce neuronal differentiation in the cell lines, the role of Glu and their receptors during the proliferation, early development, neuronal maturation and lineage commitment has been investigated extensively [48][49][50][51][52][53][54][55][56][57][58]. Last, but not least, electrophysiological and/or histological observations in freshly isolated CNS tissue have also provided valuable experimental data on the direct and short-term effects of Glu on progenitor cell activity and metabolism [5,41,[59][60][61].…”

Changes in the regulation of heat shock gene expression in neuronal cell differentiation

“…Previous studies showed that NMDARs are heteromeric composed of NR1 subunits, which binds glycine, and NR2 subunit, which binds glutamate; both NR1 and NR2 subunits are required to create a functional receptor (Waxman and Lynch 2005). Importantly, expression and function of the NMDAR protein appeared to be modulated in neural differentiation: (1) Neurogenesis is correlated with the expression of various NMDAR subunits (Varju et al 2001;Pizzi et al 2002), and (2) differentiation of the NG108-15 cells is associated with an increase in the NMDAR mRNA level (Beczkowska et al 1996(Beczkowska et al , 1997. Therefore, it is most likely that the selective vulnerability of the differentiated NG108-15 cells toward glutamate plus glycine, shown in Fig.…”

Glutamate and Regulation of Proliferation in the Developing Mammalian Telencephalon

“…In our previous work, we have noted NMDA receptors in proliferating neuroblasts from the ventral telencephalon germinal zones [Luk et al, 2003]. Interestingly, mRNA for the NR1 subunit of the NMDA receptor has been detected in multipotent forebrain precursors in vitro [Maric et al, 2000;Varju et al, 2001;Jelitai et al, 2002]. The observation that glutamate fails to influence proliferation and/or survival in early stem cell populations while influencing more differentiated neuroblasts could indicate that nonresponsive subpopulations lack functional receptor expression or downstream cellular pathways which couple receptor activation with proliferation [Maric et al, 2000].…”