Figure 6.

Recombinant GluA1-NTD inhibits NPR-mediated induction of postsynaptic excitatory, but not inhibitory, specializations during heterologous synapse formation. A, Addition of recombinant GluA1-NTD (added as a purified Fc-fusion protein) inhibits NPR-induced heterologous synapse formation as visualized by staining for the postsynaptic density marker PSD95. Control or NPR-expressing HEK293 cells were cocultured with hippocampal neurons (Fig. 1). HEK/neuron cocultures were performed in the absence and presence of 100 μg/ml GluA1-NTD for 24 h. The recombinant GluA1-NTD was added immediately before plating HEK cell on neurons at DIV16. Induced postsynaptic specializations on the HEK293 cells were assessed by staining for PSD95 and binding of the GluA1-NTD to the cells by staining for the Fc-domain that was fused to GluA1-NTD. Left, Representative images. Right, Quantifications of the PSD95 (left) and GluA1-NTD staining intensities (right). Expanded picture below representative images shows merged PSD95 and GluA1-NTD labeling under the NPR + GluA1-NTD condition to illustrate that two molecules are mutually exclusive, suggesting that the remaining PSD95 induction in the presence of GluA1-NTD is due to incomplete occupation of all NPRs on the cells. B, Same as A, except measuring the concentration of GluA1 receptors induced by NPR during heterologous synapse formation. C, Same as A, except measuring the induction of inhibitory synapses by staining for gephyrin. In addition, HEK293 cells expressing neurexin-1β were included as a positive control. Scale bars, 10 μm. Graphs represent mean ± SEM. Numbers in bars indicate number of HEK293 cells/experiments analyzed. *p < 0.05 (one-way ANOVA test with Tukey's post hoc test). **p < 0.01 (one-way ANOVA test with Tukey's post hoc test). ***p < 0.001 (one-way ANOVA test with Tukey's post hoc test).