FIGURE 6:

Neurons expressing MycKalSolo or MycKal4 form spines with aberrant morphology. (A) Spine-like protrusions along dendrites (at least 10 neurons in each group) within 100 μm of the cell soma were quantified using MetaMorph as described; one-way analysis of variance followed by Dunnett's test; *p < 0.05, **p < 0.01, ^#p < 0.05, compared with GFP and ΔKal7. MycΔKal7 did not differ from GFP in its ability to induce spine formation. Myc-positive and Vglut1-positive clusters on the dendritic shaft were included in the measurements for hippocampal neurons reported in Figure 3; for the cortical neurons shown here, Myc-positive clusters on the dendritic shaft were excluded. Synaptic (Myc/Vglut1 cluster) area (B) and spine length (C) were quantified using MetaMorph; data are displayed as a Kolmogorov–Smirnov distribution. (B) Kal7 larger than KalSolo and KalSolo larger than Kal4 (all **p < 0.01, Kolmogorov–Smirnov). (C) Kal7 larger than KalSolo and KalSolo larger than Kal4 (all **p < 0.01, Kolmogorov–Smirnov). (D) DIV20 cortical neurons expressing MycKalSolo or MycKal4 (as described in Figure 5) were stained simultaneously with antibodies to Myc (red) and NR1 (green); antibody specific to MAP2 was included to verify the identity of dendrites (not shown). Antisera to PSD95 and GluR1 produced staining patterns similar to those shown for NR1 (data not shown). (E, F) Cortical neurons prepared from E20 total Kalirin-knockout embryos were nucleofected with vectors encoding MycKal7 (not shown), MycKalSolo, or MycKal4; Myc (red), Vglut1 (green), and MAP2 (blue) were visualized simultaneously in cultures fixed on DIV19. As observed in primary neurons from wild-type rats, MycKalSolo and MycKal4 localized to spine-like structures; Vglut1-positive endings were juxtaposed to the small puncta formed by MycKalSolo and MycKal4.