We have used pluripotent P19 embryonic carcinoma stem cells as an in vitro system to study the early neuronal expression and function of GABA and subunit mRNAs of the GABA(A) receptor. These cells are differentiated with retinoic acid (RA) into GABAergic neurons and glia and maintained in culture for 30-40 days in vitro; DIV. We have shown that the differentiation of P19 cells into neurons occurs in a synchronous manner both by the immuncytochemical detection of sequential expression of stage-specific markers and an analysis of neuronal mitosis using BrdU pulse-labeling. The acquisition of functional responses to GABA coincided with the differentiation of P19 cells from neuroprogenitors into post-mitotic """"""""embryonic-like"""""""" neurons. Moreover, electrophysiological analyses revealed that GABA, acts in an autocrine manner to produce excitatory membrane responses as early as 3-4 DIV post- RA. These responses mature into repetitive, all-or-none action potentials by 8-10 DIV post-RA. Using single cell Fura-2 image analysis, we demonstrated that GABA-elicited action potentials result in neuron- specific increases in free intracellular calcium, in contrast to the hyperpolarizing action of GABA in adult neurons. We then used RT-PCR to determine the temporal (5-30 DIV) expression of 13 subunit mRNAs of the GABA(A) receptor in P19 cells during in vitro differentiation. This in vitro pattern of expression of subunit mRNAs (alpha3/5>alpha1/2/4 >>>alpha6; beta3> beta2>>beta1; gamma1>gamma2/gamma3) closely resembled the pattern of expression revealed by ISHH studies in vivo in the mammalian CNS shortly after neurogenesis and prior to mature synaptogenesis and histogenesis. This model system will allow us to examine to what degree the expression of subunits of the GABA(A) receptor in developing neurons is determined by an intrinsic program of gene expression or directed by local environmental cues such as growth and differentiation factors or GABA itself. We are currently developing single-cell mRNA/PCR detection techniques to examine the combinational subunit heterogeneity within P19 neurons (and possible glial progenitors) and to correlate subunit composition to functional properties of the receptor. In addition, we are utilizing molecular biological approaches to apply antisense oligonucleotides, and construct mammalian expression vectors capable of sustained cDNA expression in precursor and/or terminally differentiated neurons, to knockout or alter GABA(A) receptor function in these cells. These approaches may provide both an understanding and permit manipulation of the earliest expression of subunit genes of the GABA(A) receptor in order to better define a developmental role for GABA and its receptor in the mammalian CNS.
