|Budget Amount *help
¥6,700,000 (Direct Cost : ¥6,700,000)
Fiscal Year 1993 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1992 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1991 : ¥3,400,000 (Direct Cost : ¥3,400,000)
Synapse formation between cultured cerebral cortical neurons was observed by multi-site Ca^<2+> fluorometry(Muramoto, K., et al., Proc.Japan Acad.Ser.B, 64,319,1988). Most neurons in the culture showed synchronous oscillations of the fluorescence intensity which correspond to spontaneous synaptic activity. Continuous application of K-252b, a protein kinase inhibitor, blocked the synchronous firing and significantly decreased the number of synapses identified morphologically using electron microscopy (Kuroda, Y., et al., Neurosci.Lett., 135,255,1992). Since K-252b does not permeate the cell membrane, the data strongly suggest that phosphorylation of cell surface by an ecto-protein kinase (Ehrlich, Y.H., et al., Nature, 320,67,1986) is inhibited by K-252b. Since it has been reported that an ecto-protein kinase activity is regulated by a specific type of gangliosides (Tsuji, S., et al., J.Biochem., 104,498,1988), we applied purified endoglycoceramidase (EGCase) which can remove all carbohydrate moieties from any ganglioside but not from proteins and others (Ito, M., et al., J.Biol.Chem., 264,9510,1989) to the cortical culture. Continuous presence of EGCase together with its activator protein inhibited the occurrence of synchronous oscillation after 8 days in culture, when control culture showed oscillation which represents a significant amount of synapse formation between cerebral cortical neurons. The inhibition was dose-dependent and without any significant morphological changes of neurons and their neurites which was observed by phase-contrast microscopy and immunostaining of MAP 2 and neurofilament.