Figure 3.

NETO2 alters the distribution of GluK1 receptors in hippocampal neurons. A, Representative images from cultured hippocampal neurons transfected with myc-GluK1–2a alone or with either NETO1 or NETO2. Plasma membrane (red) and intracellular (green) myc-GluK1 proteins were differentially labeled before and after permeabilization, respectively. Expanded images on the bottom of each panel are taken from the area indicated by white rectangles. Scale bars: 20 μm; inset, 4 μm. B, Quantitation of relative plasma membrane (PM) expression as calculated from the ratio of red to total (red + green) fluorescence intensity. Ratios were calculated for entire neurons (first two columns) or for the somatic and dendritic compartments separately (second and third pairs of columns). Values shown are mean ± SEM. Statistical significance is denoted as *p < 0.05. C, Representative image from a neuron transfected with myc-GluK1–2a (surface expressed myc labeled in red) and YFP-PSD-95 (green) showing patchy distribution of the receptor on dendritic shafts rather than within spines. D, Representative images of dendrites from neurons transfected with myc-GluK1–2a, YFP-PSD-95, and either NETO1 or NETO2. Anti-myc labeling of neurons before permeabilization localizes GluK1 receptors on the plasma membrane (red), YFP-PSD-95 labels postsynaptic densities (green), and anti-bassoon labeling identifies presynaptic terminals. GluK1–2a is colocalized to synaptic sites positive for PSD-95 and bassoon only in the presence of NETO2. Scale bar, 1 μm. E, Quantification of the percentage of YFP-PSD-95 puncta that also contain myc-GluK1–2a fluorescence in neurons transfected with GluK1–2a alone or with either NETO1 or NETO2. Values shown are mean ± SEM. Statistical significance is denoted as ***p < 0.001.