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; gamma2S>>gamma1~gamma21~gamma3) subunit mRNAs in certain brain regions concurrent with or shortly after neuro~genesis. Little data exist as to the nature of the developmental cues, either environmental or intrinsically programed, 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 chosen to investigate two in vitro systems: pluripotent stem cells, of both cell line and primary origin, that differentiate under controlled conditions into neurons and express GABA synthetic capability and subunits of the GABAA receptor. Preliminary studies on retinoic acid~induced differentiation of murine P19 embryonic carcinoma cells indicate differentiation into neurons (50%), glia (20~30%) and fibroblast~like cells within 72 hr. We have used PCR to determine the temporal expression of subunit mRNAs and initial characterizations appear to be in close agreement with ISHH studies in vivo. Parallel studies are to be carried out on differentiating neuroprogenitors isolated from olfactory bulb and cerebellum. These results will be correlated with electrophysiological and other functional characterizations of channel activation. In vitro and in vivo models of neuronal~glial interactions will be explored to provide insight into the developmental role of the embryonic expression of the GABAergic system. Molecular biological approaches in particular, the use of antisense phosphorothioate oligodeoxynucleo~tides and the expression of antisense episomal vectors, aimed at altering the expression of components of the GABAergic system in embryonic and postmitotic neurons are currently under development. To this end, we are constructing alternative expression vectors capable of sustained expression in precursor and post~differentiated neurons to effect changes in the GABAergic phenotype. It is hoped that these approaches may provide both a view and permit manipulation of the earliest expression of subunit genes of the GABAA receptor under conditions of more defined cellular interactions.
