Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors - PubMed (original) (raw)

Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors

D V Lissin et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (N-methyl-D-aspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the gamma-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.

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Figures

Figure 1

Figure 1

Epitope-tagged GluR1 and NR1 form functional channels and are targeted to synaptic membranes. (A) (Left) Diagram of the epitope-tagged GluR1 (Upper) and NR1 (Lower). SS, signal sequence. (Right) The inward currents generated by application of glutamate to HEK293 cells expressing Flag-GluR1 (Upper) or HA-NR1 with NR2B (Lower). The currents are blocked by the appropriate subtype-specific antagonist. (B) Examples of the receptor distribution that is observed when hippocampal neurons expressing Flag-GluR1 or HA-NR1 are permeabilized before applying the primary receptor antibodies. (C) (Top) Typical receptor distributions when neurons are not permeabilized and stained for Flag-GluR1 or HA-NR1. (Middle) The distribution of synaptophysin puncta in the same fields. (Bottom) The superimposed images illustrate that the vast majority of Flag-GluR1 and HA-NR1 clusters colocalize with synaptophysin. (D) Quantitation of the percentage of Flag-GluR1 and HA-NR1 clusters that colocalize with synaptophysin (n = 20 for each subtype) indicates that the vast majority of Flag-GluR1 and HA-NR1 clusters are at synapses. (E) Quantitation of the percentage of synaptophysin puncta that colocalize with Flag-GluR1 and HA-NR1 (n = 20 for each subtype) indicate that the majority of synapses are excitatory and contain epitope-tagged receptors. The remaining synapses presumably are inhibitory because 10–30% of synaptophysin puncta colocalized with GAD65 (see Fig. 2_D_). Error bars represent SEM.

Figure 2

Figure 2

Increasing activity causes a decrease in synaptic membrane clusters of Flag-GluR1 but not HA-NR1. (A) Example of the staining patterns that are observed after treatment with picrotoxin. Note that many of the synaptophysin puncta do not colocalize with Flag-GluR1. (B) Quantitation of the percentage of synaptophysin puncta that colocalize with Flag-GluR1 or HA-NR1 in untreated and picrotoxin-treated cultures. Picrotoxin had no effect on HA-NR1 but caused a significant reduction in the synaptic localization of surface clusters of Flag-GluR1 (n = 20 for each condition). (C) The average number of synapses per microscopic field, as defined by synaptophysin staining, was not affected by picrotoxin treatment. (D) The percentage of inhibitory synapses, as measured by GAD65 staining, was not affected by picrotoxin treatment. (E) The percentage of Flag-GluR1 clusters that colocalize with synaptophysin was not affected by picrotoxin treatment. (F) Western blot showing that picrotoxin treatment did not change the level of expression of Flag-GluR1. (G) Total Flag-GluR1 immunoreactivity in the somas of permeabilized cells was not affected by picrotoxin treatment, as measured by integration of total receptor immunoreactivity using NIH Image software. Error bars represent SEM.

Figure 3

Figure 3

The decrease in the surface expression of Flag-GluR1 clusters at synapses is activity- and time-dependent. The Na+ channel toxin tetrodotoxin (A) or the NMDAR antagonist

d

-APV (B) largely prevented the picrotoxin-induced decrease in the proportion of synapses containing surface clusters of Flag-GluR1. Each graph illustrates the percentage of synaptophysin puncta that colocalize with Flag-GluR1. (C) The time course of the picrotoxin-induced effect. The abcissa shows the duration of picrotoxin treatment. Error bars represent SEM.

Figure 4

Figure 4

AMPAR-mediated synaptic currents are decreased by picrotoxin treatment. (A) The ratio of AMPAR- to NMDAR-mediated synaptic currents is decreased in picrotoxin-treated cultures. Examples of the EPSCs (A1) recorded from untreated and picrotoxin-treated cultures and (A2) a summary (n = 5 for untreated cells; n = 6 for picrotoxin-treated group) of the AMPAR-to-NMDAR EPSC ratios obtained from each group. Note that the NMDAR EPSCs were scaled for ease of comparison. [Scale bar represents 25 msec and 15 pA (untreated) or 50 pA (picrotoxin).] (B) The amplitude of mEPSCs is decreased in picrotoxin-treated cultures. Examples of mEPSCs (average of 200–400) (B1) and a summary of all recordings (B2) (n = 6 for control cultures; n = 5 for picrotoxin-treated cultures). (Scale bar represents 10 msec and 2 pA.) Error bars represent SEM.

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