Synaptic scaling requires the GluR2 subunit of the AMPA receptor

Abstract

Two functionally distinct forms of synaptic plasticity, Hebbian long-term potentiation (LTP) and homeostatic synaptic scaling, are thought to cooperate to promote information storage and circuit refinement. Both arise through changes in the synaptic accumulation of AMPA receptors (AMPARs), but whether they use similar or distinct receptor-trafficking pathways is unknown. Here, we show that TTX-induced synaptic scaling in cultured visual cortical neurons leads to the insertion of GluR2-containing AMPARs at synapses. Similarly, visual deprivation with monocular TTX injections results in synaptic accumulation of GluR2-containing AMPARs. Unlike chemical LTP, synaptic scaling is blocked by a GluR2 C-tail peptide but not by a GluR1 C-tail peptide. Knockdown of endogenous GluR2 with an short hairpin RNA (shRNA) also blocks synaptic scaling but not chemical LTP. Scaling can be rescued with expression of exogenous GluR2 resistant to the shRNA, but a chimeric GluR2 subunit with the C-terminal domain swapped with the GluR1 C-terminal domain (GluR2/CT1) does not rescue synaptic scaling, indicating that regulatory sequences on the GluR2 C-tail are required for the accumulation of synaptic AMPARs during scaling. Together, our results suggest that synaptic scaling and LTP use different trafficking pathways, making these two forms of plasticity both functionally and molecularly distinct.

Figure 1.

The GluR2 C-tail, but not the GluR1 C-tail, blocks synaptic scaling. A, Left, Cumulative histograms of mEPSC amplitude from control (n = 15) and TTX-treated (n = 18) neurons (25 events per neuron). Inset, Average mEPSC amplitude for the same conditions. Right, Average mEPSC waveforms for the same conditions. B, Left, Cumulative histograms of mEPSC amplitude for control (n = 10) and TTX-treated (n = 7) neurons transfected with the GluR1CT. Inset, Average mEPSC amplitude for the same conditions. Right, Average mEPSC waveforms for the same conditions. C, Left, Cumulative histograms of mEPSC amplitude for control (n = 9) and TTX-treated (n = 14) neurons transfected with the GluR2CT. Inset, Average mEPSC amplitude for the same conditions. Right, Average mEPSC waveforms for the same conditions. All data here and below are reported as mean ± SEM for the number of neurons indicated; Student's t test, *p < 0.05, **p < 0.01.

Figure 2.

Synaptic scaling induces accumulation of GluR2-containing AMPARs both in vitro and in vivo. A, Left, Example AMPA-mediated currents at −60 mV and +50 mV for a TTX-treated neuron. Right, Rectification index for control (n = 9) and TTX (n = 6). B, Cumulative histogram of mEPSCs from layer 2/3 pyramidal neurons in acute slices from monocular V1, from the control hemisphere (control: n = 27, 50 events per neuron) and from the deprived hemisphere (visually deprived: n = 31, 50 events per neuron). C, Left, Example average mEPSC waveforms before and after wash-in of the selective blocker of GluR2-lacking AMPAR, IEM-1460. Top, Neuron from the control hemisphere; bottom, neuron from the visually deprived hemisphere. D, Top, Average mEPSC amplitude for baseline and IEM-1460 conditions for a control and visually deprived neuron; bottom, average mEPSC amplitude in IEM-1460 expressed as percentage of baseline in same condition ± the propagated SEM for neurons from the control (n = 10) and visually deprived (n = 8) hemispheres.

Figure 3.

RNAi GluR2KD. A, Top, GluR2KD dendrite (green), 1 d after transfection with the shRNA against GluR2, labeled against endogenous GluR2 (red); note nearby untransfected dendrite with normal GluR2 labeling (arrow). Bottom, GluR2KD dendrite (blue) labeled against GluR1 (red) and PSD-95 (green). B, Length density (puncta/micron) of GluR2, GluR1, and PSD-95 puncta in GluR2KD neurons compared with transfection with control construct (n = 13, 11, and 9 for GluR2KD; 21, 14, and 11 for control). C, Example AMPA-mediated currents at −60 mV and +50 mV for control and GluR2KD neurons. D, Rectification index for control (n = 9) and GluR2KD (n = 8) neurons. E–G, mEPSC recordings from control and GluR2KD neurons (n = 10 and 9). *Different from control, p < 0.01. All statistical tests here and below were ANOVAs as appropriate, followed by corrected two-tailed Student's t tests unless noted otherwise.

Figure 4.

GluR1 compensates for GluR2 in GluR2KD neurons. A, Example of control dendrite labeled against endogenous surface GluR1 (top) or GluR3 (bottom) and synapsin; αGluR1 and αGluR3 in red, αsynapsin in green. Scale bar, 2 μm. B, Localization of GluR1 (n = 8) and GluR3 (n = 8) to synapsin. ***GluR3 different from GluR1, p < 0.005. C, Examples of control and GluR2KD dendrites labeled against endogenous surface GluR1 (top) or GluR3 (bottom); αGluR1 and αGluR3 in red, soluble CFP in blue. D, Total intensity of surface GluR1 and GluR3. GluR2KD is expressed as percentage of control for the same condition ± the propagated SEM (GluR1, control: n = 6; GluR2KD: n = 6; GluR3, control: n = 6; GluR2KD: n = 7). *Different from control, p < 0.05. E, Total intensity of total surface GluR3 and GluR3 puncta localized to GluR1 puncta. TTX is expressed as percentage of control for the same condition ± the propagated SEM (control, n = 5; TTX, n = 10). *Different from control, p < 0.05. F, Localization of GluR1 to GluR3 in control (n = 11), GluR2KD (n = 7), and TTX (n = 10) neurons. *Different from control, p < 0.05.

Figure 5.

GluR2KD blocks synaptic scaling. A, Left, Cumulative histogram of mEPSCs amplitude from untransfected neurons in control (n = 14) and TTX-treated (n = 15) conditions (25 events per neuron). Right, Average mEPSC waveforms for the same conditions. B, Left, Cumulative histogram of mEPSC amplitude for control GluR2KD neurons (n = 8), TTX GluR2KD neurons (n = 10), and TTX GluR2KD plus RNAiI (n = 9) neurons. Right, Average mEPSC waveforms for the same conditions. C, Average mEPSC amplitude for indicated conditions, grown in control medium or 24 h TTX. From left to right, n = 14, 8, 15, 10. D, Top, Examples of staining against endogenous GluR1 from control and GluR2KD neurons after 24 h TTX treatment; αGluR1 in red, soluble GFP in green. Bottom, Peak intensity of GluR1 signal in synaptically localized puncta for neurons transfected with a control construct (control) or with the shRNA against GluR2 (GluR2KD); values from TTX-treated neurons expressed as percentage of untreated control (n = 11, 15, 15, 17). Scale bar, 2 μm. *Different from control, p < 0.04; **p < 0.01.

Figure 6.

GluR2KD does not block chemical LTP. A, Average mEPSC waveforms for control and LTP conditions and LTP in GluR2KD neurons. B, Average mEPSC amplitude for the indicated conditions, n = 19, 12, 18, and, 11 respectively. *GluR2KD LTP different from GluR2KD control, p < 0.05; **LTP different from control, p < 0.001; Mann–Whitney test. C, Linear scaling of mEPSC amplitude by synaptic scaling but not by LTP. For both forms of plasticity, mEPSCs were rank ordered, and control values were plotted against potentiated values (either 1 d TTX treatment or chemical LTP protocol). The unity line (gray solid line) represents where points would fall if there were no change in mEPSC amplitude; values above this line indicate an increase in synaptic strength. For synaptic scaling (solid circles), the change in strength is well fit by a linear function with a slope >1 (gray dashed line), indicating a linear scaling of synaptic strength. For LTP (solid squares), the relationship is poorly fit by a linear function (solid black line) but is well fit by an exponential function (dashed black line). D, Top, When the amplitude distributions for TTX are scaled down by the parameters of the linear fit in (C), the distribution closely approximates the control distribution. Bottom, The same procedure for the LTP distribution produces a poor approximation of the control distribution.

Figure 7.

The GluR2 C-tail is required for synaptic scaling. A, Left, Average baseline mEPSC amplitude for the indicated conditions; KD, GluR2KD; RNAiI, RNAi-insensitive GluR2; n = 8, 6, 7, 8, and 8, respectively. B, Average mEPSC amplitude for indicated conditions; TTX is expressed as percentage of control for the same condition ± the propagated SEM (n = 9, 8, and 8, respectively). C, Localization of GluR2 constructs using extracellular GFP tag. Top, GluR2KD plus RNAiI dendrite using antibodies against GFP under nonpermeant conditions (green) to localize surface exogenous RNAiI GluR2 and PSD-95 (red). Bottom, GluR2 KD plus GluR2/CT1 dendrite labeled against GFP (green) to localize surface GluR2/CT1 and PSD-95 (red). D, Average colocalization of RNAiI (n = 9 neurons) and GluR2/CT1 (n = 7 neurons) with PSD-95 in GluR2KD neurons. *Different from control, p < 0.05.