The activation of M ₁, muscarinic receptor signaling induces neuronal differentiation in pyramidal hippocampal neurons

Muscarinic receptors have been proposed to play an important role during brain development by regulating cell survival, proliferation, and differentiation. This study investigated the effect of muscarinic receptor activation on prenatal rat hippocampal pyramidal neuron differentiation and the signal transduction pathways involved in this effect. The cholinergic agonist carbachol, after 24 h in vitro, increased the length of the axon, without affecting the length of minor neurites. Carbachol-induced axonal growth was also observed in pyramidal neurons from the neocortex but not in granule neurons from the cerebellum. The effect of carbachol was mediated by the M ₁ subtype of muscarinic receptors. The Ca ²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, the two protein kinase C (PKC) inhibitors 3-[1-[3-(dimethylaminopropyl]-1H- indol-3-yI]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X) and 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl}-3- (1-methylindol-3-yl)maleim-ide (Ro-32-0432), and the extracellular signal-regulated kinase (ERK)1/2 inhibitors 2′-amino-3′- methoxyflavone (PD98O59) and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126) all blocked carbachol-induced axonal outgrowth. In addition, down-regulation of ERK1/2 with small interfering RNA abolished the neuritogenic effect of carbachol. These data suggest an involvement of Ca ²⁺, PKC, and ERK1/2 in carbachol-induced axonal growth. Carbachol indeed increased the release of Ca ²⁺ from intracellular stores and induced PKC and ERK1/2 activation. Additional experiments showed that PKC, but not Ca ²⁺, is involved in carbachol-induced ERK1/2 activation. Together, these results show that cholinergic stimulation of prenatal hippocampal pyramidal neurons accelerates axonal growth through the induction of Ca ²⁺ mobilization and the activation of PKC and especially of ERK1/2.