Flow cytometry and immunochemical methods are applied to embryonic rat spinal and supraspinal regions to study the development, differentiation and cellular distribution of transmitters and their corresponding receptor- coupled alterations in membrane excitability. At present this multidisciplinary investigation is focused on elucidation of a developmental calendar outlining the expression of specific transmitters and their functional receptors with a primary focus on the development of the GABA phenotype and a secondary focus on the glutamate phenotype. Principal observations this year include: 1) immunological detection of GAD-related proteins during embryonic development of the rat spinal cord with low molecular weight proteins apparent in many neurons before the advent of AB:,the product of GAD enzyme activity; 2) progressive appearance of GABA+ fibers, then GABA+ neurons during embryogenesis along rostrocaudal and ventrodorsal axes in the spinal cord coincident with the appearance of high molecular weight GAD proteins; 3) postnatal emergence of a second set of GAD-related proteins with a subcellular distribution and colchicine sensitivity characteristic of vesicular transport in neurons also immunoreactive for embryonic GAD proteins; 4) biochemical dejection of GABA transiently derived from ornithine and increasingly synthesized by GAD in embryonic rat cortex; 5) sizable secretion of GABA in vitro under resting conditions from embryonic cortical neurons; 6) early embryonic appearance of functional voltage-gated Na+ channels and depolarizing GABA-A progesterone metabolite receptors in rat cortex, cerebellum and thalamus; 7) in situ hybridization evidence of GABA-A receptor subunit beta receptor message preceding a subunit message throughout the embryonic rat CNS; 8) emergence of depolarizing GABA-A receptors along a rostrocaudal gradient throughout the early embryonic chick CNS; 9) critical requirement of GABA-A receptors for chick spinal cord neuron adherence in vitro and process formation; 10) arrival of spinothalamic tract projections in the thalamus about the time of birth in the rat.
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