Regulation of neuronal differentiation by N-methyl-D-aspartate receptors expressed in neural progenitor cells isolated from adult mouse hippocampus.
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.
Kitayama
Tomoya
T
Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Japan.
Yoneyama
Masanori
M
Tamaki
Keisuke
K
Yoneda
Yukio
Y
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002452:Cell Count / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D020934:Ciliary Neurotrophic Factor / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004334:Drug Administration Schedule; D004347:Drug Interactions; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D015870:Gene Expression; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology*; D007150:Immunohistochemistry / Q000379:methods; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D016759:Oncogene Protein p65(gag-jun) / Q000378:metabolism; D016761:Oncogene Proteins v-fos / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology
D020934:Ciliary Neurotrophic Factor; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D016759:Oncogene Protein p65(gag-jun); D016761:Oncogene Proteins v-fos; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D014212:Tretinoin; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D010751:Phosphopyruvate Hydratase