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Research ArticleNew Research, Neuronal Excitability

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

Jung-Hwa Tao-Cheng, Rita Azzam, Virginia Crocker, Christine A. Winters and Tom Reese
eNeuro 18 November 2015, 2 (6) ENEURO.0066-15.2015; DOI: https://doi.org/10.1523/ENEURO.0066-15.2015
Jung-Hwa Tao-Cheng
1Electron Microscopy Facility, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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Rita Azzam
1Electron Microscopy Facility, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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Virginia Crocker
1Electron Microscopy Facility, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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Christine A. Winters
2Laboratory of Neurobiology, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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Tom Reese
2Laboratory of Neurobiology, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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Figures

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  • Figure 1.
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    Figure 1.

    NR island. NR islands are defined by patches of somal/dendritic plasma membrane that label for NMDA receptors (NR2B), and are associated with cytoplasmic dense material (arrow). Scale bar, 0.1 µm.

  • Figure 2.
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    Figure 2.

    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.

  • Figure 3.
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    Figure 3.

    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.

  • Figure 4.
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    Figure 4.

    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.

  • Figure 5.
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    Figure 5.

    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).

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    Figure 6.

    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.

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    Figure 7.

    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.

  • Figure 8.
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    Figure 8.

    Islands and PSDs labeled for various PSD-associated proteins. Antibodies and the percentage of islands labeled are listed on the left. Images are arranged based on their laminar distribution at the PSD. Glutamate receptors at the top (NR2B and GluR2) are integral membrane proteins, here labeled with antibodies with extracellular epitopes. Extending successively deeper into the cytoplasm are the MAGUKs (SAP102 and PSD-95) immediately adjacent to the postsynaptic membrane; then GKAP, a binding partner of both PSD-95 and Shank; and a broad band of Shank and Homer, two scaffold proteins that bind to each other. Additionally, CaMKII, a kinase that can bind to NR2B or self-aggregate, is distributed throughout the PSD complex. The laminar localization of these proteins at islands mirrors that at PSDs. Images of islands and PSDs are all from the same sample, and samples are from experiments treated with 2 min high K+. All islands were sampled from soma, and all PSDs were sampled from dendrites. Scale bars, 0.1 µm.

Tables

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    Table 1:

    Effects of stimulation on number of nonsynaptic NMDA receptor islands in neuronal plasma membranes

    Control2 Min high K+2 Min K+ + 2-3 min recovery2 Min K+ + 30 min recovery
    Exp 111.8 (17)38.5 (26)12.5 (40)
    Exp 213.0 (23)35.0 (20)16.0 (25)
    Exp 310.3 (29)68.1 (47)18.5 (27)18.2 (22)
    Exp 45.0 (20)57.7 (26)11.5 (26)16.2 (37)
    Mean ± SEM10.0 ± 1.849.8 ± 7.9*14.2 ± 2.216.2 ± 0.7
    • Data are reported as number of islands per 100 somal profiles (n, Number of neuroanls somal profiles scored).

    • *The number of islands is significantly higher in high K+ vs. control (P < 0.0005), high K+ vs. 2 min K++2-3 min recovery (P < 0.005) and high K+ vs. 2 min K++30 min recovery (P < 0.005); ANOVA with Tukey’s post-test.

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    Table 2:

    Percentage of NR islands labeled for PSD proteins and the ratios of labeling intensity at islands to that at PSDs

    AntibodiesPercentage of islands labeleda (number of exp, number of islands scored) Ratio of labeling intensityb
    NR2B97.5 ± 2.5 (4 exp, 42)1.76 ± 0.13 (3 exp)
    GluR236.3 ± 0.6 (2 exp, 33)1.07 ± 0.24 (2 exp)
    SAP10244.7 ± 4.8 (6 exp, 113)1.02 ± 0.15 (4 exp)
    PSD-9518.3 ± 4.8 (3 exp, 46)0.39 ± 0.08 (3 exp)
    GKAP55.7 ± 15.9 (3 exp, 38)0.70 ± 0.15 (3 exp)
    Shank267.5 ± 12.5 (2 exp, 44)0.60 ± 0.12 (2 exp)
    Homer163.8 ± 6.4 (5 exp, 105)0.59 ± 0.06 (3 exp)
    CaMKII100 (5 exp, 48)1.0 ± 0.14 (3 exp)
    • Data are reported as the mean ± SEM.

    • a Percentage of islands labeled at neuronal somas in 3-week-old dissociated hippocampal cultures after depolarization with high K+. Islands were first identified by the structural characteristic of the cytoplasmic density, and then scored for presence of immunogold labeling with the various antibodies.

    • b Ratio of labeling intensities for each antibody as the number of labels per running micrometer of island divided by number of labels per running micrometer of PSDs in the same experiment.

    • View popup
    Table 3:

    Median distances of label to plasma membrane at islands and PSDs measured from high K+-treated samples

    AntibodyIsland (n, number of particles measured)PSD (n, number of particles measured)
    SAP102Exp 126.7 (19)26.7 (30)
    Exp 226.7 (17)28.3 (18)
    PSD-95Exp 128.3 (14) a 26.7 (158)
    Exp 226.7 (130)
    GKAPExp 135.0 (12)33.3 (215) b
    Exp 230.0 (14)30.0 (72)
    Shank 2Exp 156.7 (56)50.0 (345) b
    Exp 243.3 (53)46.6 (483) b
    Homer 1Exp 153.3 (49)56.7 (120)
    Exp 250.0 (36)53.3 (209)
    CaMKIIExp 153.3 (97)53.3 (113)
    Exp 256.7 (84)60.0 (135)
    • Data are reported as median distance in nanometer (nm).

    • a Pooled from 3 exp.

    • b Data are from the study by Tao-Cheng et al., 2015.

    • There was no statistical difference by Wilcoxon test between islands and PSDs.

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Depolarization of Hippocampal Neurons Induces Formation of Nonsynaptic NMDA Receptor Islands Resembling Nascent Postsynaptic Densities
Jung-Hwa Tao-Cheng, Rita Azzam, Virginia Crocker, Christine A. Winters, Tom Reese
eNeuro 18 November 2015, 2 (6) ENEURO.0066-15.2015; DOI: 10.1523/ENEURO.0066-15.2015

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Depolarization of Hippocampal Neurons Induces Formation of Nonsynaptic NMDA Receptor Islands Resembling Nascent Postsynaptic Densities
Jung-Hwa Tao-Cheng, Rita Azzam, Virginia Crocker, Christine A. Winters, Tom Reese
eNeuro 18 November 2015, 2 (6) ENEURO.0066-15.2015; DOI: 10.1523/ENEURO.0066-15.2015
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Keywords

  • electron microscopy
  • extrasynaptic
  • NMDA receptors
  • postsynaptic density
  • synapse formation

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