Depolarization of Hippocampal Neurons Induces Formation of Nonsynaptic NMDA Receptor Islands Resembling Nascent Postsynaptic Densities

Morphometry of islands. A, B, Islands can be identified by their characteristic cytoplasmic density (A, B, arrows) without immunogold labeling. Many islands are not labeled with GluR2 (A) or PSD-95 (B). C, To measure the label that is located on the cytoplasmic side of islands (CaMKII), two vertical lines extending 120 nm into the cytoplasm are drawn to mark the area for measurement as in the case of the PSD complex (Tao-Cheng et al., 2015). The distance between the two vertical lines represents the length of island. The distance of the label was measured from the center of the particle to the outer edge of the plasma membrane (C, white arrows) for all the particles within the marked area. All islands in this figure were from soma. Scale bar, 0.1 µm.

Distribution of NR2B on the plasma membrane of hippocampal neurons. Label for NR2B in 3-week-old dissociated hippocampal neurons prepared by pre-embedding immunogold labeling. A–D, NR2B is concentrated at asymmetric synapses (A, arrowhead; B, high magnification), but is also present in nonsynaptic parts of the plasma membrane, where it can be either in clusters (A, C, large arrows) associated with a cytoplasmic density or as single individual grains (A, D, small arrows). A and B were sampled from dendrites; C and D were sampled from soma. Scale bar, 0.1 µm.

Nonsynaptic NR islands contain cytoplasmic densities (arrows) mimicking the structure of PSDs. A–C, Islands label for different subunits of NMDA receptors as follows: NR2B in A, NR1 in B, and NR2A/B in C (the first two at extracellular epitopes). D1–D3, Serial thin section analysis shows that labels are closely associated with an underlying density manifesting sharp borders. All islands in this figure were sampled from dendrites. Scale bar, 0.1 µm.

Labeling density of NMDA receptor and NR islands on somal plasma membrane after depolarization with high K⁺. A, After high-K⁺ treatment (2 min, 90 mm), the overall labeling intensity for NR2B in somal plasma membranes increases to 126 ± 5% (*) of control values (five experiments, p < 0.01, paired t test). B, After high-K⁺ treatment, the density of NR islands increases to 249 ± 34% (^) of control values (five experiments, p < 0.05, paired t test).

Comparison of NR islands and AMPA receptor patches. A, B, NR2B-labeled islands (A) have a distinct cytoplasmic density (arrowhead), which is lacking in AMPA receptor-labeled patches (B, GluR2 antibody). D, AMPA receptor patches are thought to be exocytosed from cytoplasmic vacuoles containing concentrated receptors (small arrow). C, In contrast, NR2B labels are typically at low concentrations, if present, in clear-membraned vacuoles (small arrows). A, C, and D were samples from dendrites, and B was from soma. Scale bars, 0.1 µm.

Endocytosis of NMDA receptors. A–C, Label for NR2B is sometimes located in clathrin-coated pits (A, B) and the lumens of coated vesicles (C). D, E, Many clathrin-coated pits are not labeled for NR2B. NR-labeled islands (E, arrow) may lie immediately adjacent to a coated pit, but are not endocytosed as an island. Scale bars, 0.1 µm.