Study On NMDA Stimulates Migration Of Oligodendrocyte Precursor Cells

During development of central nervous system (CNS), the presence of functional myelin is dependent on accurate position of Oligodendrocyte Precursor Cells (OPCs). OPCs were also termed oligodendrocyte-type-II astrocyte progenitor cells (O-2A progenitor cells). However, subsequent studies show that O-2A progenitor cells have no ability to differentiate into type-II astrocyte in vivo as in differentiation medium for cultured purified OPCs in vitro. Thus, they are believed to be ancestors of oligodendrocytes (OLs) now. Most of them would eventually form myelin following the processes of proliferation, migration and differentiation, though some would always keep the state of precursor for their distinguishing functions in variety areas of CNS. It is generally believed that they are derived just from some given restrict ventricular zones. As one of the indispensable premises for myelin, OPCs migration has gradually been widely concerned. And what's more, remyelination is also partly dependent on the migrated and accurate positioned OPCs which are derived from subventricular zone (SVZ). The interaction between neurons and glial cells relies partially on neurotransmitters. Indeed, OPCs express many neurotransmitter receptors which are invovled in the regulation of cellular migration.Glutamate, as an important neurotransmitter, is widely distributed in whole brain and regulates numerous neurophysiological activities. During cortical development of Rats, glutamate is present in the cortex with the gradually increased concentration from VZ to dorsal cortex. Correspondingly, the first wave of OPCs in the telencephalon origins from SVZ at around E13 and then migrates into dorsal cortex, suggesting a critical role of glutamate in regulating OPCs migration. Two classes of ion-channel type glutamate receptors involve in NMDA receptors and AMPA/KA receptors. Actually, it is known that glutamate promotes the migration of neurons and cerebellum granular cells through NMDA receptors activation. And recently, it is domenstrated that AMPA/KA can mediate the migration of OPCs, but it remains unknown whether NMDA receptors participate in regulating OPCs migration.This study involves two parts, the first part is to identify whether NMDA receptors are expressed in OPCs and mediate cellular migration in Costar-transwell model with the cultured OPCs in vitro, and we manipulated the brain slices to further confirm the phenomenon in vivo. Secondly, we explored the tentative molecular mechanisms on NMDA induced OPCs migration.The results are as follows:1. Establishment of purified OPCs cultured system in vitro. Our cultured OPCs are identical with the others previous studies. OPCs are bipolar with small cell bodies when observed with light microscope. They are NG2, A2B5 and Nestin positive with immunocytochemistry labeled. Besides, cultured OPCs can also differentiate into mature oligodendrocytes in vitro.2. NMDA stimulates OPCs migration in a dose-dependent way. NMDA has the ability to promote OPCs migration in a dose-dependent way with peaks at 10uM NMDA in Costar-Transwell migration assay in vitro. Besides that, NMDA induced OPCs migration is partially dependent on characteristics of substrates.3. NR2A expressed in OPCs and mediates cellular migration. We adopted RT-PCR, western blotting and immunocytochemistry to identify the subunits of NMDA receptors expressed in cultured OPCs. It shows that NR1, NR2A, NR2B, NR2D and NR3A could be detectable with RT-PCR. We further confirmed the expression of NR1, NR2A and R2B with western blotting and immunocytechemistry, respectively. NMDA induced OPCs migration was inhibited when pretreated with noncompetitive antagonist of NMDARs or receptor-specific antagonist of NR2A. No obvious blocking was detectable when pretreated with antagonist of NR2B.4. NMDA receptors mediate OPCs migration in brain cortex slices. Antagonist of either NMDA receptors or NR2A can inhibit OPCs migration from SVZ into dorsal cortex.5. NMDA receptors mediated OPCs migration involves in an ERK signaling dependent pathway. All varying concentrations (from 0.1uM to 1mM) of NMDA could activate ERK signaling pathway. The lost ability of OPCs migration is present when ERK activation is inhibited.