Fig. 3.
Characterization of glutamate receptor composition at Nrx1β-Fc beads by glutamate ionotophoresis and patch-clamp experiment. Neurons co-transfected with Nlg1 and PSD-95-GFP (or Homer1c-GFP) were left in contact with Nrx1β-Fc-coated microspheres for 2–4 h or 24 h, then subjected to whole-cell electrophysiology in the presence of TTX to prevent action potentials and 0 mM Mg to remove the NMDAR block. In some experiments, cells were treated with TTX and APV throughout the bead incubation (24 h). (A) Digital image correlation showing patch-clamp and iontophoresis pipettes, with the corresponding PSD-95-GFP fluorescence. Note that many beads bind to the transfected cell. (B) Magnified view of accumulation of PSD-95-GFP surrounding a Nrx1β-Fc bead and nearby synaptic clusters. (C) Examples of currents elicited upon glutamate application at Nrx1β-Fc beads (trace 1), endogenous PSD-95-GFP clusters (trace 2), or neurite region without PSD-95 accumulation (trace 3), with the membrane potential being clamped at −60 mV. (D) Effect of NMDAR and AMPAR antagonists (CNQX and APV, respectively) on the electrophysiological response of βNrx1β-Fc bead contacts at two contact durations. (E and F) By fitting the curves using a bi-exponential function with two separate time constants, the relative charge carried by AMPARs was computed for various conditions. (E) Synapses identified by PSD-95-GFP or Homer1c-GFP positive puncta. (F) Nrx1β-Fc beads displaying recruitment of PSD-95-GFP or Homer1c-GFP. Data were analyzed by one-way ANOVA and compared two-by-two by Tukey test (*, P < 0.05; ns, not significant).