Roles of Ionotropic Glutamate Receptors in Early Developing Neurons Derived from the P19 Mouse Cell Line

Abstract

We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the α-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75^NTR expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.

Tra2βl regulates P19 neuronal differentiation and the splicing of FGF‐2R and GluR‐B minigenes

“…The neuronal cells differentiated from P19 cells 9 d after RA induction were immunocytochemically analyzed using b-III tubulin antibodies. In the present study, the yield of the b-III tubulin positive neurons in the control cells was lower relative to that of other reports (Lee et al, 2003;Chou et al, 2003). However, this low yield reduces the high yield-caused limiting effect on determining the promoting effect of Tra2bl on neuronal differentiation of P19 cells.…”

Neurotransmitter receptor expression and activity during neuronal differentiation of embryonal carcinoma and stem cells: from basic research towards clinical applications

“…In undifferentiated P19 cells, transcripts encoding the NMDA 2A and 2B receptor units were identified, whereas the NMDA R1 subunit, critical to receptor function, was expressed together with the 2A and 2B subtypes, only following induction of the neuronal phenotype (McPherson et al 1997). Glutamate receptor channels are highly permeable for calcium ions, and differentiating P19 cells are known to express NMDA and non-NMDA receptors (AMPA and kainate receptors) (Canzoniero et al 1996;Lee et al 2003b). Glutamate-induced ion flux into the cell depolarizes the cell membrane and results in activation of voltage-dependent calcium channels (Morley et al 1995;Canzoniero et al 1996).…”