Reciprocal and activity-dependent regulation of surface AMPA and NMDA receptors in cultured neurons

Abstract

Activation of NMDA receptors (NMDARs) can modulate excitatory synaptic transmission in the central nervous system by dynamically altering the number of synaptic AMPA receptors (AMPARs). The surface expression of NMDARs themselves is also subject to modulation in an activity-dependent manner. In addition to NMDAR-induced changes in AMPAR expression, AMPARs have also been found to regulate their own surface expression, independently of NMDARs. However, whether or not AMPARs and NMDARs might reciprocally regulate their surface expression has not previously been systematically explored. We utilized surface biotinylation assays and stimulation protocols intended to selectively stimulate various glutamate receptor subpopulations (e.g. AMPARs vs NMDARs; synaptic vs extrasynaptic). We reveal that activation of synaptic NMDARs increases the surface expression of both NMDAR and AMPAR subunits, while activation of extrasynaptic NMDAR produces the opposite effect. Surprisingly, we find that selective activation of AMPARs reduces the surface expression of not only AMPARs but also of NMDARs. These results suggest that both AMPARs and NMDARs at synaptic sites are subject to modulation by multiple signalling pathways in an activity-dependent way.

Figure 1

Surface expression of GluA1 subunits was modified by hypertonic sucrose treatment. Surface proteins were biotinylated, isolated and immobilized on nitrocellulose membranes for quantitative Western blotting. Treatment with hypertonic sucrose induced a potentiation of surface GluA1 subunits (n = 4). Surface expression of GluN1 (P = 0.955, n = 4), GluN2A (P = 0.836, n = 4) or GluN2B (P = 0.807, n = 4) subunits was unchanged by HSS treatment.

Figure 2

AMPA receptors were involved in the regulation of both AMPAR and NMDAR surface expression. A: Cells were treated for 20 sec with glutamate (100 μM) in the presence of Mg²⁺ (1 mM) and APV (100 μM) to block NMDAR activation. There was a significant reduction in surface GluA1 (P = 0.025, n = 6) GluN1 (P = 0.0.032, n = 6) and GluN2A (P = 0.035, n = 6). GluN2B signals were not significantly reduced (P = 0.368, n = 6) B: When both NMDARs and AMPARs were blocked with APV (100 μM) and GYKI53655 (50 μM), respectively, glutamate (100 μM) failed to cause significant change in the surface signal for GluA1 (P = 0.771, n = 6), GluN1 (P = 0.211, n = 6), GluN2A (P = 0.590, n = 6) and GluN2B (P = 0.339, n = 6). C: Brief (20 sec) application of AMPA in the presence of APV (100 μM), Mg²⁺ (1 mM) and TTX (0.5 μM) caused a decrease in surface GluA1 signal (P = 0.011, n = 6). Interestingly, the surface expression of GluN1 and GluN2A subunits was also reduced (P = 0.013, 0.05, respectively, n = 6). There was no change in surface GluN2B signal (P = 0.511, n = 6). D: The effect of AMPA on AMPAR and NMDAR trafficking was independent of extracellular calcium, since AMPAR activation in Ca²⁺-free solution still caused reduction in surface GluA1 (P = 0.028, n = 6) and GluN2A (P = 0.012, n = 6) subunits.

Figure 3

Activation of synaptic NMDARs increased surface expression of AMPARs and NMDARs. The cells were challenged with HSS in the presence of CNQX (100 μM) to eliminate the opposing effect produced by AMPAR activation. Sucrose-induced release of glutamate produced an increase in GluA1 (P = 0.027, n = 5), GluN1 (P = 0.042, n = 5) and GluN2A (P = 0.028, n = 13) surface signals, while GluN2B surface signal was not affected (P = 0.669, n = 4).

Figure 4

Activation of extrasynaptic NMDA receptors reduced surface AMPA and NMDA receptors. Ca²⁺-free high potassium (50 mM) solution was used to depolarize the cells to release glutamate from nerve terminals in the presence of MK801 (100 μM) and EGTA (10 mM). Under these conditions, synaptic NMDARs will preferentially be activated and then rapidly blocked by the open channel blocker, MK801. The cells were then treated with NMDA (50 μM) for 20 sec which will preferentially activate the extrasynaptic NMDAR population. There was a significant decrease in GluA1 (P = 0.014, n = 5), GluN1 (P = 0.005, n = 5), GluN2A (P = 0.001, n = 5) and GluN2B (P = 0.021, n = 5) surface expression.

Figure 5

Summary diagram illustrating the changes in glutamate receptor surface expression following the selective activation of various subtypes and populations of glutamate receptors. The activation of selected populations of postsynaptic glutamate receptors was achieved as follows: A) predominantly synaptic AMPARs and NMDARs were co-incidently activated by hypertonic sucrose solution (HSS), B) predominantly synaptic NMDARs were activated by HSS in the presence of CNQX, C) the entire complement of surface AMPARs were activated by bath applied AMPA in the presence of APV and Mg²⁺ and D) extrasynaptic NMDARs were activated by applied NMDA after having first blocked synaptic NMDARs with MK-801. Downward arrows superimposed on glutamate receptor subunits illustrate receptor populations activated by each protocol. In all panels, the outcome of the various treatments on the surface expression of glutamate receptors is summarized as either increased (upward arrow), decreased (downward arrow) or unchanged.