The long term objectives of this project are to gain an understanding of the cellular and molecular mechanisms responsible for the development and function of the mammalian visual system. Knowledge of these mechanisms is essential to any understanding of congenital abnormalities in visual system development and the wide variety of degenerative diseases that can affect both the visual system and other areas of the central nervous system. To achieve these objectives it is necessary to have methods that allow the identification of the cell types and the individual molecules of the visual system. Two recent developments of molecular biology, namely the introduction of monoclonal antibodies and recombinant DNA molecules, have provided these methods. Antibodies that can identify the major subclasses of cells in the rat retina have already been produced. The objectives of this proposal are to produce further antibodies that recognise more discrete subclasses of cells of retina and visual cortex, and to use cloned cDNA fragments to study the heterogeneity and development of cell types in the visual system by using in situ hybridisation. To prepare the monoclonal antibodies, cells or membranes enriched in the cell type of interest will be used to immunise mice. Spleen cells will be taken from mice mounting an adequate immune response and fused with a mouse plasmacytoma. In addition spleen cells will be immunised by in vitro incubation with antigen and then fused. Hybrids will be selected by standard techniques and those secreting antibodies detected by a combination of radioactive binding assays and immunocytochemical assays. The antibodies will be used to study the in vivo development of cells of the visual system by immunocytochemical analysis of tissue from animals of various ages. The antibodies will also be used to purify defined populations of cells by combinations of affinity separations and fluorescence-activated cell sorting. Purified cells will be used both for further immunisations and for studies in tissue culture. Monolayer cultures of cells from retina or visual cortex will be prepared and the cell types identified by labelling with specific antibodies. By using anatomical and electrophysiological methods the nature and specificity of synaptic interactions between identified cells will be studied.
