The immediate and continuing aim of this line of investigation involves the implementation of strategies to probe the development and differentiation of phenotypic properties expressed by functionally distinct types of nerve, glial and endocrine cells, especially those properties involved in signal transduction mechanisms. The primary strategy involves dual-laser fluorescence- activated cell analysis and sorting (FACS). Biochemical and immunocytochemical studies of cultured CNS elements are also utilized. Principal observations this year include: 1) further refinement of a voltage-sensitive indicator dye protocol on the FACS for assaying membrane potential distributions in heterogenous suspensions of embryonic and early postnatal cells from the mammalian and avian CNS; 2) characterization of the birth dates and ontogenetic expressions of important forms of membrane excitability resident in developing rat and chick CNS; 3) in rat card the development of voltage-dependent NA+ channel expression coincides with C1- ion channel regulated by the inhibitory transmitter GABA and both of these precede NA+- dependent responses to excitatory aminoacids; 4) in rat cord the appearance of functional NA+ and C1- channels correlates anatomically with the emergence of post- mitotic cells; 5) in chick cord functional NA+ channels may precede both inhibitory and excitatory transmitter responses; 5) embryonic hippocampal cells respond to the GABA analogue muscimol beginning at low nM concentrations in a dose-dependent manner while striatal and spinal cord cells require about 50nM; 6) these responses are blocked by bicuculline and picrotoxin, but not altered by benzodiazepines or barbiturates; 7) cells from postnatal, spinal and mesencephalic regions can be back-labelled with fluorescent beads (and therefore identifiable with fluorescence microscopy) and cultured for days to weeks for multi-disciplinary study.
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