Figure 1.
NCAM regulates accumulation of spectrin at immature axodendritic contacts. (A and B) NCAM+/+ and NCAM−/− neurons maintained in homogenotypic cultures (A) or heterogenotypic coculture (B) for 4 d were labeled with antibodies against spectrin and MAP2. Immunolabeling for NCAM was included for heterogenotypic cultures to identify the genotype of neurons. Immunofluorescence signals were inverted to accentuate the difference in immunolabeling intensities between genotypes. Axodendritic contacts were identified as points of cross section between spectrin- and MAP2-positive dendrites and spectrin-positive and MAP2-negative axons (arrows). Note that spectrin accumulates more efficiently at NCAM+/+ contacts. Bar, 5 μm. Graphs show averaged values of spectrin and MAP2 immunolabeling intensity ± the SEM in arbitrary units (AU). Profiles of spectrin and MAP2 immunolabeling intensity were measured along dendrites with zero on the abscissa, corresponding to the center of the contact and profiles measured along neurites 2.5 μm away from the contact center in both directions. Note that spectrin accumulates at NCAM-positive contacts with a characteristic bell-shaped distribution around the contact center. Accumulation of spectrin at contact sites in which one or both neurites were NCAM negative is reduced. (C) Histograms show R spectrin and R MAP2 for NCAM+/+ and NCAM−/− contacts and contacts formed by axons (a) and dendrites (d) of different genotypes. R is defined as the ratio of labeling intensities at the center of a contact and 2.5 μm away from the contact center. Mean values ± the SEM are shown. The number of analyzed contacts (n) is shown for each bar. Asterisks show statistically significant differences when compared with NCAM+/+ contacts; P < 0.05, t test.