Flow cytometry, discontinuous-gradient cell isolation, dissociated cell culture, immunoblots, cell migration, immunochemistry and in situ hybridization methods are applied to embryonic-early postnatal rat CNS tissues to study the development, differentiation and cellular distribution of transmitter, transmitter-related enzymes and their corresponding receptors. During the past several years, we have focussed primarily on GABA, which is transiently expressed in a widespread manner during CNS development before it becomes relatively restricted to fast- transmitting synapses in the adult where it often functions in an inhibitory manner. In FY 94 we investigated the following: 1) transcripts encoding three GABA-synthesizing GAD enzymes and those encoding most GABA receptor subunit proteins have been detected by in situ in progressively more regions of the developing CNS; 2) they appear to be expressed at all levels of the embryonic neuraxis, even during the period of intense neuroblast proliferation; 3) several distinct patterns of GAD and GABA transcript co-expressions in CNS development are apparent: one almost exclusively in cells of the neuroepithelial proliferative zone, one in many, if not most differentiating cells during embryogenesis and one differentiating during the postnatal period; 4) it is clear that some transcripts are only transiently detected for variable periods while others persist, becoming restricted to subpopulations; 5) taken together, the data show that transcripts encoding GAD and GABA receptors are more abundant and widely distributed during embryogenesis than after differentiation is completed; 6) chemotropic effects of GABA on embryonic spinal cord and cortical cells have been discovered with postmitotic neurons migrating to fM, pM and microM concentrations, in an age- and-or region-dependent manner; 7) both chemotactic (gradient- dependent) and chemokinetic (gradient-independent) effects of GABA have been recorded, which can be mimicked by GABAmimetics active at GABA/A and GABA/B receptors suggesting novel GABA receptor structure-activity relations; 8) pertussis toxin inhibits GABA-induced chemokinesis, but not chemotaxis induced by NGF, implicating G protein-mediated signal transduction in these novel effects of GABA's; 9) staurosporine inhibits both effects of GABA on cortical dissociates, implicating protein kinase activity in the motility and migration signals. 10) 10-18-10-8 M GABA transiently increases cytoplasmic free Ca2+ in postmitotic progenitors and neuroblasts during the embryonic period of cortical development in an all-or-none manner; 11) this effect involves discharge of Cai2+ from an intracellular store that is sensitive to ryanodine; 12) 10-7-10-5 M GABA depolarizes progressively more embryonic cortical cells in a Cl-- dependent manner and elevates Ca2+ by promoting its entry.
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