Immunocytochemistry

COS cells were plated in 6-well plates containing coverslips coated with poly-L-lysine (Sigma-Aldrich). Cells were plated at a density of 300,000 cells per well and cultured for 24 h prior to transfection. All transient transfection experiments contained a total of 2 μg of DNA (1.75 μg receptor and 250 ng of either SD4 or pRK5 empty vector) using Lipofectamine 2000 (Invitrogen) and cells were cultured for an additional 24 h. For live labeling, cells were first incubated at 4^°C for 10 min. Cells were washed once with cold PBS and incubated in rat anti-HA antibody diluted in COS media for 20 min at 4^°C. After primary staining, cells were washed three times with cold PBS and incubated in donkey Alexa 594-conjugated anti-rat secondary antibody diluted in COS media for 20 min. Cells were washed three times with cold PBS and then with warm COS media. Plates were transferred back to 37^°C incubator for 30 min. Cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) for 10 min.

For staining of total SD1 or total SD4, coverslips were incubated in 0.1% Triton-X100 diluted in PBS for 15 min. Cells were blocked with 5% milk in PBS for 30 min and incubated in primary antibody for 1.5 h at room temperature. Coverslips were washed three times with PBS and incubated in donkey Alexa 488-conjugated anti-mouse IgG2a for 1 h. Coverslips were washed three times with PBS and mounted on slides with Fluoromount G (Southern Biotech).

For chemical long-term potentiation (LTP), hippocampal neurons at DIV 12–14 were equilibrated in artificial cerebrospinal fluid (aCSF) containing 2 mM magnesium (Mg⁺²) and 2 mM calcium (Ca⁺²) at 37^°C in incubator for 30 min. Neurons were washed with PBS and replaced with aCSF containing the treatment buffer (2 mM Ca⁺²; 200 μM glycine; 20 μM bicuculine; 3 μM strychnine), or a vehicle control. Strychnine was diluted in DMSO while glycine and bicuculine were diluted in water, so an equivalent amount of dimethyl sulfoxide (DMSO) or water, respectively, was added as the vehicle control. Neurons were incubated at 37^°C for 5 min for chemical-LTP induction. Coverslips were then transferred to a recovery buffer (aCSF w/Mg⁺²; no drugs) for 20 min at 37^°C. For labeling of synaptic GluA1, neurons were washed 3 times with PBS and incubated with anti-GluA1 antibody against the extracellular N-terminus diluted in PBS with 3% bovine serum albumin (BSA) at 37^°C for 1 h. Cells were then washed with PBS, fixed with 4% PFA, and then permeabilized with 0.1% Triton X-100 for 15 min. Neurons were blocked with 10% BSA for 30 min. Neurons were then stained for total anti-SD4 and total anti-vGlut1 overnight in 3% BSA at 4^°C. After incubation, coverslips were washed 3 times with PBS and incubated in secondary antibodies for each marker for 1 h at room temperature. Neurons were then washed 3 times with PBS and mounted on glass microscope slides for imaging.