Flow cytometry and immunochemistry and in situ hybridization methods are applied to embryonic/early postnatal rat spinal and supraspinal regions to study the development, differentiation and cellular distribution of transmitters and their corresponding receptors. During the past several years we have begun to focus on the development of the GABA phenotype, which in the adult rat and chicken is widespread and thought to be involved in many CNS functions at all levels of the neuraxis. Principal observations this year include: 1) in situ hybridization of GAD-related genes and immunocytochemistry of GAD gene projects show that they appear in the midembryonic period (E12/13) in the rat spinal cord; 2) truncated GAD gene and predicted low molecular weight protein products emerge in many neurons before immunocytochemical detection of GABA, the product of GAD enzyme activity; 3) GAD* signals first appear in the neuroepithelium, then colocalize with GABA as neurons migrate; 4) GABA+ fibers descending in the ventral funiculus parallel the emergence of GAD+ GABA + motoneurons and interneurons along a rostrocaudal gradient in the ventral region while GABA + fibers in the dorsal funiculus parallel the expression of GABAergic neurons emerging in the dorsal region; 5) in situ hybridization signals of specific GABA-A receptor subunit messages at E15 in spinal and supraspinal regions coincide with the advent of GABAergic neurons; 6) flow cytometric study of excitatory GABA-A receptors in suspension of intact cells shows that they have similar efficacies in cervical and lumbosacral metabolites excite the majority of newly differentiating embryonic spinal and supraspinal cells apparently via GABA-A receptors; 7) FACS analysis of embryonic chick CNS tissues suggests a developmental schedule for GABA-A/steroid receptor expression qualitatively similar to that observed in the rat; 8) depolarized embryonic rat cortical neurons release GABA well above baseline levels; 9) one-step buoyant density gradient separation of cellular from subcellular material identifies the various subpopulations of characteristically complex fluorescence/forward light scatter signals recorded in FACS experiments on embryonic CNS elements; 10) cell cycle analysis during embryogenesis of the rat CNS reveals a developmental map and calendar of terminal cell division occurring during the mid- and late embryonic periods beginning in the region of the cervical spinal cord/rhombencephalon; 11) there is a relative disappearance of GABA immunoreactivity in the early postnatal period, indicating that much of the embryonically expressed GABA in cells of the ventral regions of the cord is transient.
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