GABA(A) RECEPTORS MODULATE EARLY SPONTANEOUS EXCITATORY ACTIVITY IN DIFFERENTIATING P19 NEURONS

P19 embryonic carcinoma (EC) stem cells are pluripotent and are effici ently induced to differentiate into neurons and glia with retinoic aci d (RA) treatment. Within 5 days, a substantial number of differentiati ng P19 cells express gene products that are characteristic of a neuron al phenotype. P19 neurons were used as a model to explore the relation ship between neuronal ''differentiation'' in vitro and the acquisition of gamma-aminobutyric acid (GABA(A)) receptors and functional GABA re sponses. Pulse-labeling experiments using bromodeoxyuridine indicated that all neurons had become postmitotic within 3-4 days after treatmen t with RA. This was confirmed by a reduction in the immunocytochemical detection of the undifferentiated stem cell antigen SSEA-1. Subsequen tly, a transient expression of nestin was observed during the first 5 days in vitro (DIV) after exposure to RA. By 5-10 DIV after RA, a sign ificant number of neurons (similar to 80-90%) expressed immunocytochem ically detectable glutamate decarboxylase and GABA coincident with the acquisition of membrane binding sites for tetanus toxin. These phenot ypic markers were maintained for >30 DIV after RA. Under current-clamp conditions, random, low-amplitude, spontaneous electrical activity ap peared in neurons within the first few days after RA treatment and thi s was blocked by the specific GABA(A) receptor antagonist bicuculline. Thereafter, the appearance and progressive increases in the frequency of spontaneous action potentials in P19 neurons were observed that we re similarly attenuated by bicuculline. In neurons > 5 DIV after RA, e xogenous application of GABA elicited similar action potentials, The o nset of excitatory responses to GABA or muscimol in voltage-clamped ne urons appeared immediately after the cessation of neuronal mitosis and before the previously reported acquisition of responses to glutamate. In fura-e imaging studies, the exogenous application of GABA resulted in neuron-specific increases in intracellular Ca2+. Thus, P19 neurons provide an in vitro model for the study of the early acquisition and properties of electrical excitability to GABA and the expression of fu nctional GABA(A) receptors.