Figure 3.

The expression of mGlu7 shRNA or pathologic variants inhibits axon outgrowth during early neuronal development. A, Cortical neurons (DIV1) were transfected with lentiviral vector harboring control (Ctl) shRNA or mGlu7 shRNA. The longest neurite was confirmed to be an axonal branch by immunostaining with SMI312, a specific marker for axons. The length of the longest axons was measured using NeuronJ software. Scale bar: 50 μm. Scatter plots represent the mean ± SEM. *p < 0.0001. B, Hippocampal neurons (DIV1) were transfected with the rescue constructs in which mGlu7 shRNA and myc-mGlu7 WT-IRES-EGFP or myc-mGlu7 variant-IRES-EGFP were expressed in the same vector. Because the GFP signal in a bicistronic IRES-EGFP construct was weak, neurons (DIV3) were stained with anti-GFP and then Alexa Fluor 488 secondary antibodies. Representative images of these GFP signals are displayed in black. Scale bar: 50 μm. Scatter plots represent the mean ± SEM. *p = 0.0012, **p = 0.0302, ***p = 0.0034, #p = 0.0011, ##p = 0.0075.