SYNAPSE FORMATION AND ESTABLISHMENT OF NEURONAL POLARITY BY P19 EMBRYONIC CARCINOMA-CELLS AND EMBRYONIC STEM-CELLS

A number of different cell lines that exhibit a partial neuronal pheno type have been identified, but in many cases the full extent of their neuronal differentiation has not been directly addressed by functional studies. We have used electrophysiology and immunofluorescence to exa mine the formation of synapses and the development of neuronal polarit y by murine embryonic stem (ES) cells and the mouse P19 embryonic carc inoma cell line. Within 2-3 weeks after induction by retinoic acid, su bsets of P19 and ES cells formed excitatory synapses, mediated by glut amate receptors, or inhibitory synapses, mediated by receptors for GAB A or glycine. In ES-cell cultures, both NMDA and non-NMDA receptors co ntributed to the excitatory postsynaptic response. Staining with antib odies to growth-associated protein-43 and microtubule-associated prote in-2 revealed segregation of immunoreactivity into separate axonal and somato-dendritic compartments, respectively. Consistent with our phys iological evidence for synapse formation, intense punctate staining wa s observed with antibodies to the synaptic vesicle proteins synapsin, SV2, and synaptophysin, These results demonstrate the in vitro acquisi tion by pluripotent cell lines of neuronal polarity and functional syn aptic transmission that is characteristic of CNS neurons.