The immediate and continuing aim of this line of investigation involves the development of strategies to probe the development and differentiation of phenotypic properties expressed by functionally distinct types of nerve, endocrine and immune cells. The strategies in development include dual-laser fluorescence-activated cell analysis and sorting (FACS), light-microscopic characterization of cells in monolayer culture using immunoreagents reacting with cytoplasmic and surface antigens and semi-quantitative analysis of optically detectable fluorescence signals emanating from cells in monolayer culture. Principal observations this year include: 1) implementation of voltage-sensitive indicator dye technology for use with the FACS, application to cell suspensions of embryonic rat spinal cord, initial characterization of a complex distribution of membrane potentials arising in vital elements, and discovery of functional Na+ channel expression developing earlier on spinal elements than sensory cells; 2) implementation of surface-immunoreactions for quantitative analysis of antigen expression in nerve, endocrine, immune and renal cells on the FACS; 3) routine isolation of nerve and endocrine cells based on surface-immunoreaction signals in the FACS; 4) days-long culture of adult pituitary prolactin and growth hormone cells; 5) implementation of Ca2+-indicator dye technology to monitor intracellular Ca2+ levels in single clonal pituitary cells with initial observations on spontaneously arising fluctuations in Ca2+ levels and hormone-induced changes in Ca2+ levels; 6) cell-cycle analysis on the FACS of cells co-transfected with senescent DNA and the gene encoding for interleukin-2; and 7) preliminary data showing that natural killer-type lymphocyte cytotoxicity and mitogen-stimulated lymphocyte proliferation are compromised in a behavioral model of fear-conditioning.
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