Figure 5.

Transient increase in PICK1 localization at endosomal compartments after glycine stimulation. A, Colocalization between GluA2 and PICK1 is transiently increased at endosomal compartments after stimulation with glycine. Dissociated hippocampal neurons were preloaded with Alexa Fluor–transferrin to label endosomes (red channel). Neurons were exposed to elevated glycine for 3 min, returned to normal medium for the specified time, fixed at 0, 5, 10, or 20 min after stimulus, and stained using anti-GluA2 (green channel) and anti-PICK1 (blue channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of GluA2 colocalized with PICK1 within the transferrin compartment normalized to control. *p < 0.0125, ***p < 0.001. n = 3 independent experiments, with 10–15 cells in each. Scale bar, 5 μm. B, Colocalization between PICK1 and EEA1 is transiently enhanced in dendrites at 5 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in A and stained using anti-EEA1 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with EEA1 normalized to control. **p < 0.01. n = 4 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. C, Colocalization between PICK1 and the recycling endosomal marker Rab11 is transiently enhanced in dendrites at 5 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in A and stained using anti-Rab11 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with Rab11 normalized to control condition. **p < 0.01, ***p < 0.001. n = 4 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. D, Colocalization between PICK1 and the synaptic marker PSD95 is unchanged at 5 min after glycine stimulation but increases at 20 min. Dissociated hippocampal neurons were glycine stimulated as in A and returned to normal medium for the specified time, fixed at 5 or 20 min after stimulus, and stained using anti-PSD95 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with PSD95 normalized to control condition. *p < 0.025. n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. E, Association between PICK1 and Rab11 analyzed by live imaging. Dissociated hippocampal neurons expressing GFP–Rab11 and mCherry–PICK1 were exposed to elevated glycine for 3 min, followed by washout with normal medium. Images were collected every minute, and fluorescence intensity for mCherry–PICK1 was measured within a mask generated from the GFP–Rab11 signal. Images show representative images from selected time points, and graph shows fluorescence normalized to baseline (before glycine application). n = 7. Scale bar, 2 μm.