Functions of neuronal membrane-associated proteins for neuronal connection and synapase formation.

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1. Many parts of brain have laminated structures, and each lamina receives afferents from specific sets of neuron groups, then make physiologically relevant synapses. In the chick tectum, sixteen layers can be distinguished, and retinal inputs make arborizations and synapses only in the three of those lamina (retinorecipient laminae; RRL) in its superficial region. N-cadherin is concentrated specifically in RRL, and remained in adult, while SC1/JC7/DM-GRASP/BEN, an immunoglobulin superfamily cell adhesion molecule appeared to be expressed in deeper two RRL with its peak at embryonic day 14-16, then gradually decreased. A coculture system was developed to analyze the lamina-specificity and retinal neuron terminal behavior. When antibodies against N-cadherin were applied, more overshooting of neurites beyond RRL border, and reduced terminal arborization were observed. SC1 antibodies had similar but less effects on the overshooting to N-cadherin, however arborization was not affected. Tho … More se results suggest that some of cell-surface molecules with spatially-restricted expression patterns may regulate the development of lamina-specific retinotectal neuronal connectivity in the chick, and may also be applicable to other laminated brain portions and/or other species.2. We developed an hippocampal slice culture system from neonatal mice of which organotypic structures are well maintained for at least three weeks. In order to obtain neuron-specific expression of exogenous genes, we prepared the slice cultures from a transgenic line with Cre recombinase under control of CaMKII gene promoter, the the cultures are infected with adenoviruses bearing a promoter and EGFP with loxP-STOP-loxP cassette in between. Adenoviruses are chosen, because of their low cytotoxicity to mammalian cultures. High resolutional observation of synaptic structures in living neurons can be achieved using a two-photon laser microscope. We also analyzed dynamics of PSD-95-GFP and GFP-Zip45, showing that they behave in similar way to that of dissociation cultures which we previously reported. To study the function of N-cadherin in synapse formation and plastic change, we are now preparing several systems for suppressing N-cadherin gene expression with anti-sense oligonucleotides as well as direct inhibition of its adhesion activity with inhibitory peptides or proteins. Less