P19 neurons, obtained by neuronal differentiation from P19 embryonal carcinoma cells in vitro, contain nicotinic and muscarinic acetylcholine (ACh) receptors which are characteristic for functional neurons.  However, little is known about the assembly of embryonic nicotinic receptor subunits into functional receptors or whether they play an active role during neuronal differentiation before synapses are functional.
Here we report evidence for functional nicotinic acetylcholine receptor channels and G-protein coupled muscarinic receptors along neuronal differentiation of mouse embryonic carcinoma cells. Three different cholinergic agonists, ACh, nicotine, and muscarine, evoked cytosolic Ca²⁺-signals in P19 cells. Ca²⁺-signals due to nicotinic receptor activation were found in more than 70% of all embryonic cells. The Ca²⁺-response following nicotine application was markedly prolonged in cells from early embryonic stages compared to cells from later stages of differentiation. M₃ muscarinic receptors and β2, α3, α4, α7 nicotinic receptor subunit expression increased during the course of neuronal differentiation, whereas α2 and α5 nicotinic receptor expression decreased when cells differentiated to neurons. Gene and protein expression of nicotinic receptor subunits β2, β4 and α6 augmented when differentiated P19 cells expressed proteins specific for neural-progenitor cells, and subsequently decreased when cells undergo final neuronal maturation. The incidence of subunits of nicotinic receptors declining with embryonic age, suggests a role of these receptors in early development.