Live-cell imaging of receptors around postsynaptic membranes

Abstract

This protocol describes how to image the trafficking of glutamate receptors around excitatory postsynaptic membrane formed on an adhesion protein-coated glass surface. The protocol was developed to clarify how receptors move during the induction of synaptic plasticity. Dissociated neurons are cultured on a coverslip coated with neurexin, which induces the formation of postsynaptic membrane-like structures on the glass surface. A glutamate receptor tagged with a fluorescent protein is then transfected into neurons, and it is observed with total internal reflection fluorescence microscopy. The whole process takes about 3 weeks. Changes in the amount of cell-surface receptors caused by neuronal activities can be quantified, and individual exocytosis events of receptors can be clearly observed around the pseudo-postsynaptic membrane. This protocol has potential applications for studies of movements of membrane proteins around other specialized regions of the cell membrane, such as the inhibitory postsynaptic membrane, the presynaptic membrane or the immunological synapses.

Detection and characterization of individual endocytosis of AMPA‐type glutamate receptor around postsynaptic membrane

“…However, PSLM shows certain critical properties of postsynaptic membrane: clustered distribution of postsynaptic proteins such as PSD95, homer and GluA1‐3, dynamic changes of AMPAR during LTP (Tanaka & Hirano ; Tanaka et al . ). In addition, we report here that there are endocytic zones in peripheries of PSLM as observed around normal synapses (Blanpied et al .…”

“…The image analysis method was similar to previous studies (Tanaka & Hirano ; Tanaka et al . ), but modified. PSLM area was defined as in a previous study (Tanaka & Hirano ).…”

Amyloid‐β oligomers suppress subunit‐specific glutamate receptor increase during LTP

“…Dissociated cells were seeded on poly‐D‐lysine‐ (Merck, P7280) and Neurexin (NRX)‐coated glass in Neurobasal medium (Thermo, 21103‐049) containing 1% penicillin‐streptomycin (Thermo, 15140‐122), 0.25% glutamine (Merck, G6392) and 2% B27 Electro (Thermo, 17504‐044). Detailed procedures for NRX‐coating were described in a previous report [23]. Plasmids were transfected into days in vitro 10–15 neurons with Lipofectamine 2000 (Thermo, 11668‐019).…”

“…Expression vectors for rat GluA1 (flop) or GluA2 (flop) labeled with SEP (super‐ecliptic pHluorin) (GluA1‐SEP or GluA2‐SEP), PSD95 labeled with TagRFPt (PSD95‐RFPt), Neuroligin1 with splice insertion A labeled with HA tag (NLG‐HA) and NRX1β without splice insertion 4 labeled with human immunoglobulin‐Fc region (NRX‐Fc) were prepared as described previously [17,23]. Expression vectors for GluA1‐or GluA2‐SEP (GluA‐SEP), PSD95‐RFPt, and NLG‐HA were transfected into neurons.…”

“…NRX‐Fc vector was transfected into HEK293 cells for purification of NRX‐Fc protein. Detailed methods to purify NRX‐Fc were reported previously [23]. EGFP expression vector was a generous gift from Professor Y.Tagawa (Kagoshima University, Japan).…”