Cholinergic properties of neurons differentiated from an embryonal carcinoma cell-line (P19)

P19 is a mouse-derived embryonal carcinoma cell-line capable of differentiation toward ectodermal, mesodermal and endodermal lineages. Following treatment with retinoic acid these cells differentiated into neurons, astrocytes and fibroblast-like cells. We induced P19 differentiation under conditions which lead to a homogeneous neuronal culture (> 95% neurons). Under these conditions, most cells (∼85%) express high levels of the cholinergic markers acetyl cholinesterase and choline acetyltransferase while ∼10% of cells express the GABAergic marker glutamic acid decarboxylase. While the proportion of the GABAergic neurons is constant at different culture conditions, the cholinergic phenotype is suppressed at high cell densities. The cholinergic nature of P19 neurons is also evident in their ability to form contacts with a muscle cell-line - C2. At day 10 of differentiation cells are capable of depolarization-dependent acetylcholine release. The release is Ca²⁺ dependent, and drops to baseline levels at 0.5 mM Ca²⁺. The cells also respond to sub-nM levels of α-latrotoxin by acetylcholine release. All major proteins implicated in synapse functionality are expressed prior to day 10 at both at RNA and protein levels. However, the expression pattern of each gene is unique. The genes include cytoskeletal proteins, synaptic vesicle proteins and terminal specific proteins. We suggest that this cell-line can serve as an in-vitro model system for the study of neuronal phenotype acquisition. Under our conditions, the P19 cells can also provide a system in which to study the differentiation of functional cholinergic neurons.