Receptor internalization assay and confocal microscopy

Primary hippocampal neurons plated on glass coverslips were transfected with myc-mGlu7 at 10-day in vitro (DIV). At DIV14, the neurons were incubated with 2 μg/mL anti-c-myc antibody for 10 min at RT to label surface-expressed mGlu7. The neurons were returned to the conditioned medium in the absence or presence of 400 μM L-AP4 at 37 °C for 15 min to allow receptor internalization. The neurons were fixed with 4% paraformaldehyde/4% sucrose in PBS for 20 min and blocked with 10% normal goat serum for 1 h. The surface receptors were labeled with Alexa Fluor 647 anti-mouse IgG (converted to red). The neurons were then permeabilized with 0.25% Triton X-100 for 5 min and blocked with 10% normal goat serum for 1 h. The internalized intracellular receptors were then labeled with Alexa Fluor 568 anti-mouse IgG (converted to green). The amount of internalization was quantified from the z-stacked maximum-projection images using MetaMorph software (RRID:SCR_002368, Molecular Devices, Sunnyvale, CA, USA) following the manufacturer’s instructions. The images were separated into red (surface-bound receptors after internalization) and green (internalized receptors) channels. A region of interest (ROI) per neuron was selected along the neurites, but the soma regions were excluded due to signal saturation. The upper and lower thresholds were set to remove saturated and background signals, respectively. The integrated intensities from each channel were calculated from the selected ROIs. The internalization rate was obtained by dividing the internalized signal intensity by the total signal intensity (the sum of the signal intensities of surface-bound receptors plus those of internalized receptors). The relative internalization rate was normalized to the WT internalization rate. For endogenous protein staining, the fixed and permeabilized neurons were incubated overnight with primary antibodies at 4 °C, washed with PBS three times, and incubated with Alexa Fluor secondary antibodies for 1 h at RT. Z-stacked maximum projection images were obtained using a Zeiss LSM 800 confocal microscope (RRID:SCR_015963, Carl Zeiss, Oberkochen, Germany).

To quantify synaptic maturation, Z-stack images were analyzed with Imaris software (RRID:SCR_007370, Bitplane, Zurich, Switzerland) using the FilamentTracer tool in autodepth mode. VGLUT1- or VGAT-positive puncta within 30 μm dendrites were masked using the Surface tool. The surface algorithm was used to calculate the number, area (size), and intensity of each punctum.