Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons

Abstract

We have examined the membrane localization of an AMPA receptor subunit (GluR1) and an NMDA receptor subunit (NR1) endogenously expressed in primary cultures of rat hippocampal neurons. In unstimulated cultures, both GluR1 and NR1 subunits were concentrated in SV2-positive synaptic clusters associated with dendritic shafts and spines. Within 5 min after the addition of 100 microM glutamate to the culture medium, a rapid and selective redistribution of GluR1 subunits away from a subset of synaptic sites was observed. This redistribution of GluR1 subunits was also induced by AMPA, did not require NMDA receptor activation, did not result from ligand-induced neurotoxicity, and was reversible after the removal of agonist. The activation-induced redistribution of GluR1 subunits was associated with a pronounced (approximately 50%) decrease in the frequency of miniature EPSCs, consistent with a role of GluR1 subunit redistribution in mediating rapid regulation of synaptic efficacy. We conclude that ionotropic glutamate receptors are regulated in native neurons by rapid, subtype-specific membrane trafficking, which may modulate synaptic transmission in response to physiological or pathophysiological activation.

Fig. 1.

Glutamate causes a rapid redistribution of GluR1 subunits away from synapses in cultured hippocampal neurons.A, Immunocytochemical localization of GluR1 and SV2 in cultured hippocampal neurons incubated in the absence of added glutamate. A higher magnification image of the region indicated by thebox is displayed in the bottom panels.Arrows in these panels point out an example of a GluR1 puncta colocalized with SV2 immunoreactivity. B, Immunocytochemical localization of GluR1 subunits and SV2 in neurons incubated in the presence of 100 μm glutamate for 15 min. Scale bars, 5 μm. C, Quantitation of the proportion of GluR1-immunoreactive puncta associated with SV2 in control (untreated) and glutamate-treated neurons. D, Quantitation of the proportion of SV2-positive synaptic puncta associated with detectable concentrations of GluR1 immunoreactivity. Data are derived from analysis of a total of 20 independent fields, selected at random and examined in a blinded manner, from three separate experiments. GluR1-positive puncta were defined by a GluR1 immunoreactivity at least twofold greater than background (unclustered regions) in the raw (unprocessed) fluorescence image, as described in Materials and Methods. Bars represent the mean proportion, and error bars represent the SEM from the independent fields (n = 20).

Fig. 2.

NR1 subunits do not undergo detectable redistribution under the same conditions. The same experiments as described in Figure 1 were conducted using anti-NR1 immmunocytochemical staining in control (untreated) (A) and glutamate-treated (B) neurons. Scale bars, 5 μm. Arrows in A and Bpoint out examples of an NR1 puncta colocalized with SV2. Analysis of the proportion of NR1-positive puncta associated with synapses (C) and the number of synapses associated with detectable concentration of NR1 immunoreactivity (D) were determined as in Figure 1.

Fig. 3.

Dual localization of GluR1 and NR1 subunits in the same neurons. Cultured hippocampal neurons were incubated in the absence (A) or presence (B) of 100 μm glutamate for 15 min and then fixed, permeabilized, and processed for immunocytochemical analysis using rabbit anti-GluR1 and mouse anti-NR1, as described in Materials and Methods. Representative fluorescence micrographs obtained under each condition are displayed. Arrows in Aindicate examples of puncta associated with both GluR1 and NR1 immunoreactivity. Open arrows in Bindicate examples of NR1-containing puncta devoid of any detectable GluR1 immunoreactivity, which were observed frequently in glutamate-treated cultures. Scale bars, 5 μm.

Fig. 4.

Rapid redistribution of GluR1 subunits is induced by AMPA and does not require activation of NMDA receptors.A, In control (untreated) neurons, the majority of GluR1 receptor clusters were localized at the periphery of dendritic shafts, consistent with synaptic localization. B, Within 15 min after the addition of 100 μm AMPA to the culture medium, a pronounced redistribution of GluR1 subunits was observed, which was qualitatively identical to that induced by glutamate. Note that most punta are in the center of the dendritic shaft. C, Coapplication of the NMDA receptor antagonist APV (100 μm) together with AMPA did not block the AMPA-induced redistribution of GluR1 subunits, confirming that this redistribution does not require activation of NMDA receptors. Scale bars, 5 μm.

Fig. 5.

Redistribution of GluR1 subunits can be dissociated from ligand-induced neurotoxicity and is reversible after removal of agonist. A, Neurotoxicity in cultured hippocampal neurons was assayed by trypan blue exclusion 24 hr after treatment of cultures for 15 min with 100 μm glutamate or AMPA under conditions identical to those used to induce rapid redistribution of GluR1 subunits. Whereas glutamate caused substantial neurotoxicity, AMPA caused significantly less neurotoxicity [although still significantly more than observed in control (untreated) cultures]. B, AMPA-induced redistribution of GluR1 subunits was reversed within 6 hr after washout of AMPA. Scale bars, 5 μm.

Fig. 6.

Top. Rapid, AMPA-induced redistribution of GluR1 subunits from dendritic spines. A, In control neurons, many clusters of GluR1 subunits (red) colocalized with phalloidin staining (green), consistent with colocalization in dendritic spines.Arrow indicates an example of such a structure, which appears yellow in the merged image. B, After incubation of cultures with 100 μm AMPA for 15 min, many GluR1-containing puncta were observed to be distinct from phalloidin-stained extensions of the dendritic membrane, consistent with a redistribution of GluR1 subunits away from dendritic spines.Arrow indicates an example of such a phalloidin-positive dendritic specialization, observed frequently in AMPA-treated neurons, which was devoid of detectable GluR1 immunoreactivity. Scale bars, 2 μm.

Fig. 7.

Bottom. Analysis of AMPA-induced redistribution of GluR1 subunits in individual dendrites using confocal fluorescence microscopy and three-dimensional image reconstruction. Schematic view of a control neuron (A) and an AMPA-treated neuron (B), showing the scanned region containing an individual dendritic shaft (box).C, D, Representative three-dimensional reconstructions displaying the localization of GluR1 immunoreactivity in dendritic specializations viewed obliquely (as indicated by the schematic) using the maximum projection technique in a control (untreated) neuron and an AMPA-treated (100 μm for 15 min) neuron, respectively. E, F, Collapsed end-on images of the same three-dimensional image reconstructions as in C and D, displayed using the maximum projection technique along thez′-axis, emphasizing the AMPA-induced redistribution of GluR1 subunits (red) away from SV2-positive synaptic sites (green) located at the periphery.

Fig. 8.

The rapid redistribution of GluR1 subunits is associated with decreased frequency of mEPSCs. A, Traces of miniature synaptic events recorded from cultured hippocampal neurons in the presence of lidocaine (10 μm) before (Pre) and after (Post) application of 100 μm AMPA to cells for 15 min, followed by rapid washout.B, Averaged mEPSC (250 events) pretreatment and posttreatment with AMPA from experiment in A show only a slight reduction in amplitude. C, Summary of the effects of AMPA treatment on the mean amplitude and frequency of mEPSCs. Error bars represent SEM (n = 9 cells).