Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation

Increasing evidence has shown that some neurotransmitters act as growth-reg ulatory signals during brain development. Here we report a role for the cla ssical neurotransmitter acetylcholine (ACh) to stimulate proliferation of n eural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the mu scarinic receptor. Neural precursor cells dissociated from the embryonic ra t cortical neuroepithelium were expanded in culture with basic fibroblast g rowth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PC R) revealed the presence of m2, m3 and m4 muscarinic receptor subtype trans cripts, while immunocytochemistry demonstrated m2 protein. ACh and carbacho l induced an increase in cytosolic Ca2+ and membrane currents in proliferat ing (BrdU(+)) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased bot h cell number and DNA synthesis, but also enhanced differentiation of neuro ns. These effects were blocked by atropine, indicating the involvement of m uscarinic ACh receptors. The growth-stimulating effects were also antagoniz ed by a panel of inhibitors of second messengers, including 1,2-bis-(O-amin ophenoxy)-ethane-N, N, N',N'-tetraacetic acid (BAPTA-AM) to chelate cytosol ic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncoupl es certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-ac tivated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activa ted MARK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors tr ansduce a growth-regulatory signal during neurogenesis via pathways involvi ng pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.