We have employed in situ hybridization histochemistry ( ISHH) studies to investigate the embryonic and early postnatal expression of subunit mRNAs of the GABAA receptor in the developing rat CNS. The results demonstrate the early (E14-E15) and restricted expression of specific (alpha2/3/5>alphal/4 >>>alpha6; beta3> beta2>> beta1; gamma 2S>>gamma 1/gamma 2L/gamma 3) subunit mRNAs in certain brain regions concurrent with or shortly after neurogenesis. Little data exist as to the nature of the developmental cues that direct these patterns of expression. In addition, while there appears to be compelling evidence for a developmental role for GABA and its receptor, there is very little fundamental information about the functions, either individually or in combination, of specific subunits in embryonic neurons. We have investigated two in vitro systems: pluripotent stem cells, that differentiate under controlled conditions into neurons and cultured embryonic neurons of defined developmental stage and anatomic location that express GABA synthetic capability and subunits of the GABAA receptor. One such in vitro model, murine P19 embryonic carcinoma stem cells, undergo synchronous differentiation upon exposure to retinoic acid to produce GABAergic neurons (approximately 50-60%), glia (20-30%) and some fibroblast-like cells with 72 hr. We have used PCR to determine the temporal expression (0~30 DIV) of 13 subunit mRNAs and initial characterizations parallel ISHH studies in vivo. Moreover, between 3-10 days after RA-induced differentiation, P19 neurons exhibit GABA-mediated membrane depolarization and increases in intracellular Ca2+. Additional studies are to be carried out on differentiating embryonic neurons isolated from olfactory bulb and cerebellum. We are currently developing single cell mRNA/PCR techniques to correlate subunit composition in embryonic neurons with electrophysiological and other functional characterizations of channel activation. Our long-term goal is to use molecular biological approaches in particular, antisense phosphorothioate oligodeoxynucleotides and the expression of antisense episomal vectors, to alter the expression of subunits of the GABAA receptor in embryonic and terminally differentiated neurons. To this end, we have constructed alternative expression vectors and delivery systems capable of sustained expression in neurons. We hope that these approaches may permit manipulation of the earliest expression of subunit genes of the GABAA receptor under conditions of more defined environmental and cellular interactions to further eludicate a role for the expression of the GABAergic system during CNS development.
