Shigeo Okabe

Hippocampal pyramidal neurons in culture showed a developmental shift in synapse distribution from dendritic shafts to spines. Using dual wavelength time-lapse fluorescence microscopy, we analyzed the morphogenesis of three synaptic components: dendritic spines, postsynaptic densities (PSDs), and presynaptic vesicles. Local assembly of a major PSD protein, PSD-95, was spatially and temporally correlated with spine morphogenesis. Clustering of postsynaptic PSD-95 and that of a predominant synapti

Subcellular localization of the NMDA receptor NR1 splice forms was studied by expressing individual splice variants and their epitope-tagged derivatives in mouse fibroblasts and in hippocampal neurons. When NR1 splice variants were expressed in fibroblasts, the amount of NR1 molecules expressed on the cell surface varied among forms with different C-terminal cytoplasmic domains. The splice forms with the longest C-terminal cytoplasmic tail (NR1-1a and NR1-1b) showed the lowest amount of cell sur

The mechanism of actin incorporation and turnover in the nerve growth cone was examined by immunoelectron microscopy and low-light-level video microscopy of cultured neurons injected with biotin-labeled actin or fluorescently labeled actin. We first determined the sites of actin incorporation into the cytoskeleton of growth cones by immunoelectron microscopy of cultured neurons injected with biotin-labeled actin and reacted with an anti-biotin antibody and a gold-labeled secondary antibody. Shor

To study microtubule (MT) dynamics in nerve cells, we microinjected biotin-labeled tubulin into the cell body of chemically fused and differentiated PC12 cells and performed the immunofluorescence or immunogold procedure using an anti-biotin antibody followed by secondary antibodies coupled to fluorescent dye or colloidal gold. Incorporation of labeled subunits into the cytoskeleton of neurites was observed within minutes after microinjection. Serial electron microscopic reconstruction revealed

PSD-Zip45 (Homer 1c) and PSD-95 are postsynaptic density (PSD) proteins containing distinct protein-interacting motifs. Green fluorescent protein (GFP)-tagged PSD-Zip45 and PSD-95 molecules were targeted to the PSD in hippocampal neurons. We analyzed dynamic behavior of these GFP-tagged PSD proteins by using time-lapse confocal microscopy. In contrast to the less dynamic properties of PSD-95, PSD-Zip45 showed rapid redistribution and a higher steady-state turnover rate. Differential stimulation

To characterize the behavior of axonal microtubules in vivo, we analyzed the movement of tubulin labeled with caged fluorescein after activation to be fluorescent by irradiation of 365-nm light. When mouse sensory neurons were microinjected with caged fluorescein-labeled tubulin and then a narrow region of the axon was illuminated with a 365-nm microbeam, photoactivated tubulin was stationary regardless of the position of photoactivation. We next introduced caged fluorescein-labeled tubulin into

The effects of changing NMDA receptor subunit composition on synaptic plasticity in the hippocampus were analyzed by creating transgenic mice overexpressing NR2D, a predominantly embryonic NMDA receptor subunit. NMDA-evoked currents in the transgenic mice had smaller amplitudes and slower kinetics. The transgenics also displayed age-dependent deficits in synaptic plasticity in area CA1 of the hippocampus. Long-term depression was selectively impaired in juvenile mice when NR2D overexpression was

We have analyzed the dynamics of neuronal intermediate filaments in living neurons by using the method of photobleaching of fluorescently-labeled neurofilament L protein and immunoelectron microscopy of incorporation sites of biotinylated neurofilament L protein. Low-light-level imaging and photobleaching of growing axons of mouse sensory neurons did not affect the rate of either axonal growth or the addition of intermediate filament structures at the axon terminal, suggesting that any perturbat

We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin