Regulation of neuronal differentiation by N‐methyl‐D‐aspartate receptors expressed in neural progenitor cells isolated from adult mouse hippocampus

Abstract

In vitro culture of neural progenitor cells isolated from adult murine hippocampus according to the Percoll density gradient method resulted in formation of round spheres not immunoreactive to microtubule-associated protein-2 (MAP-2) or glial fibrillary acidic protein in the presence of basic fibroblast growth factor within 12 days in vitro (DIV). Reverse-transcription PCR analysis revealed constitutive expression in these neurospheres of different subunits required for assembly of functional heteromeric N-methyl-D-aspartate (NMDA) receptor channels. Immunocytochemical analysis confirmed expression of NR1, NR2A, and NR2B subunits in neurospheres cultured for 4-12 DIV. Brief (5 min) exposure to NMDA induced marked expression of c-Fos, Fos-B, Fra-2, and c-Jun proteins in neurospheres cultured for 12 DIV 2 hr later. The NMDA receptor antagonist dizocilpine markedly inhibited expression of both c-Jun and c-Fos proteins in NMDA-exposed neurospheres. Sustained exposure to NMDA not only markedly inhibited neurosphere formation by 12 DIV when exposed from 4-12 DIV, but also resulted in facilitation of subsequent differentiation of neurospheres exposed to all-trans retinoic acid to cells immunoreactive to both neuron-specific enolase and neuronal nuclei, in addition to MAP-2, as revealed by Western blot and immunocytochemistry analyses. These results suggest that functional heteromeric NMDA receptors may be expressed constitutively in neural progenitor cells before differentiation to play a crucial role in commitment and differentiation to neurons in adult murine hippocampus.

Neural stem cells: mechanisms and modeling

“…Evidence based on cultured NSCs supports the finding that nNOS gene expression is involved in the feedback modulation of NSC differentiation by NMDARs (Hu et al, 2008). Interestingly, several other studies using NSC cultures have suggested that NMDA activation could promote the neuronal fate determination of NSCs (Deisseroth et al, 2004;Kitayama et al, 2004;Yoneyama et al, 2008). Hence, the differentiation and proliferation of NSCs is likely subtly regulated by activity within a range; too-high or too-low levels of neuronal activity can both delay the process of neurogenesis.…”

“…First of all, the generation of new neurons by NSCs is definitely influenced by the surrounding newly differentiated neurons. Due to the lack of AMPAR-mediated glutamatergic transmission at the early developmental stage, NMDAR has been proposed to be the major type of glutamate receptor that has an effect on the physiology of NSCs (Kitayama et al, 2004;Nácher et al, 2007;Wegner et al, 2009). This hypothesis is also based on the important role of NMDARs in long-term learning and memory.…”

The role of the N-methyl-d-aspartate receptor in the proliferation of adult hippocampal neural stem and precursor cells

“…When NSPCs isolated from young adult mice were cultured in the sustained presence of NMDA, the formation of neurospheres was markedly inhibited. When these neurospheres were transferred to a differentiationpromoting medium (containing all-trans retinoic acid, ATRA), the prior NMDA treatment promoted commitment to a neuronal fate [70]. Deisseroth and colleagues [71] have also investigated whether manipulations that increase NMDA-mediated neuronal activity can influence NSPC differentiation in vitro.…”

“…Using neurospheres cultivated from NSPCs, Kitayama and colleagues [70] performed real-time polymerase chain reaction (RT-PCR) to detect NMDAR subunit mRNA, and immunohistochemistry to label NMDAR subunit proteins. Cells showed marked GluN1 subunit mRNA levels throughout the culturing period of 210 d, which peaked and plateaued at day 6.…”

“…GluN1, GluN2A and GluN2B expression was detected by immunohistochemistry from the fourth day in vitro. To confirm the GluN subunits formed functional NMDARs, 12-day-old neurospheres were briefly exposed to NMDA, which induced the marked expression of activity-dependent Jun and Fos family transcription factors, in an MK801-sensitive manner [70]. In another study, NSPCs isolated from the P0 rat hippocampus were cultured for 5 d, and shown to express GluN1 and GluN2B mRNA (as determined by RT-PCR and Western blotting), as well as GluN1 and GluN2B proteins (visualised by immunohistochemistry) [72].…”