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. 2008 Jul 10;59(1):84-97.
doi: 10.1016/j.neuron.2008.05.014.

Rapid translation of Arc/Arg3.1 selectively mediates mGluR-dependent LTD through persistent increases in AMPAR endocytosis rate

Maggie W Waung  1 , Brad E Pfeiffer, Elena D Nosyreva, Jennifer A Ronesi, Kimberly M Huber
Affiliations

Rapid translation of Arc/Arg3.1 selectively mediates mGluR-dependent LTD through persistent increases in AMPAR endocytosis rate

Maggie W Waung et al. Neuron. .

Abstract

Salient stimuli that modify behavior induce transcription of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and transport Arc mRNA into dendrites, suggesting that local Arc translation mediates synaptic plasticity that encodes such stimuli. Here, we demonstrate that long-term synaptic depression (LTD) in hippocampal neurons induced by group 1 metabotropic glutamate receptors (mGluRs) relies on rapid translation of Arc. mGluR-LTD induction causes long-term increases in AMPA receptor endocytosis rate and dendritic synthesis of Arc, a component of the AMPAR endocytosis machinery. Knockdown of Arc prevents mGluRs from triggering AMPAR endocytosis or LTD, and acute blockade of new Arc synthesis with antisense oligonucleotides blocks mGluR-LTD and AMPAR trafficking. In contrast, LTD induced by NMDA receptors does not persistently alter AMPAR endocytosis rate, induce Arc synthesis, or require Arc protein. These data demonstrate a role for local Arc synthesis specifically in mGluR-LTD and suggest that mGluR-LTD may be one consequence of Arc mRNA induction during experience.

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Figures

Figure 1
Figure 1. Brief mGluR activation induces a persistent and protein synthesis dependent increase in GluR1 endocytosis rate
A, Representative blots of surface (S), internalized (I) (100 µg pulldown) and total (T) GluR1 (10 µg protein) from high-density dissociated hippocampal neuron cultures one hour after DHPG or NMDA treatment in media or in the presence of anisomycin (Aniso). B, Brief DHPG treatment (100 µM; 5 min) results in persistent (60 min) decrease in surface GluR1 and increases in endocytosis rate for GluR1 measured with receptor biotinylation relative to control, or media treated, sister cultures. Preincubation of cultures in anisomycin (20 µM) blocks surface GluR1 decreases and GluR1 endocytosis rate increases. Although application of NMDA (20 µM; 3 min) results in a persistent (60 min) decrease in surface GluR1, it does not persistently alter GluR1 endocytosis rate. N = # cultures per condition is on each bar. Paired t-tests were performed on raw ratio values in treated vs. their respective control, media treated cultures (Supp. Table 1). C, Representative western blots of internalized (I) and total (T) GluR1 in high-density hippocampal cultures. One hour after treatment with media or DHPG, surface receptors were labeled with biotin and allowed to internalize at 37°C for 5,15 or 30 minutes, after which the remaining surface biotin was stripped off and internalized, biotinylated GluR1 was measured. D, Quantification of internalized/total GluR1 levels from data in C reveal that GluR1 endocytosis is not saturated at early time points (5 min) in either control or DHPG-treated cultures and therefore reflects endocytosis rate. N = 5 cultures per condition. A best fit single exponential association function (using the Marquardt and Levenberg method) was used to obtain a τ for endocytosis. E, Representative double-label images of surface (green) and internalized (red) staining of GluR1 in low-density hippocampal neurons. Live cultures were labeled with N-terminal GluR1 antibody one hour after treatment with media (Control), DHPG or NMDA. F, Left: Representative proximal dendrites from images of merged surface and intracellular GluR1 immunostaining. Right: Ratiometric analysis of internal to surface GluR1 puncta number reveal that one hour after treatment, DHPG, but not NMDA, persistently increases the internalization rate constant for GluR1. N = # cells per condition is on each bar. Scale bars = 10 µm. Data pooled from 4 cultures each. For all figures * p < 0.05; ** p < 0.01; ***p< 0.001*.
Figure 2
Figure 2. Activity-regulated cytoskeleton-associated protein (Arc) is rapidly synthesized in dendrites in response to mGluR stimulation
A, DHPG treatment of dissociated hippocampal neurons increases the total area of Arc immunofluorescence in dendrites 10 min after DHPG onset. Pretreatment with anisomycin, but not actinomycin D, blocks the DHPG-induced increases in Arc puncta. Scale bars = 10 and 5 µm. N = # cells each bar. Data from 4–5 cultures/ condition. B1, Local perfusion of DHPG and Alexa488 hydrazide to cultured hippocampal neurons. B2, Quantification of Arc immunofluorescence in the perfused dendritic region. n = 8 dendrites/4 cultures per condition. B3, Representative Arc immunofluorescence in dendritic regions perfused (as indicated by green) with either DHPG alone or DHPG + anisomycin. Scale bars = 10 µm. Two-way ANOVA was used to determine statistical significance. C, Representative western blots of Arc in acute hippocampal slices from mice treated with DHPG in the presence of ACSF or anisomycin (20 µM). Quantitative western blotting for Arc and VCP (valosin containing peptide; used as a loading control) of acute hippocampal slices frozen at 10 min after DHPG onset. Anisomycin blocks DHPG-stimulated rapid increases in Arc. N = # animals is on each bar. D1, Representative Arc immunofluorescence from CA1 regions where a cut (white line) severed dendrites from the pyramidal cell layer. s.p = Str. pyramidale, s.r.= Str. radiaum. β3-Tubulin immunoreactivity indicates the presence of dendrites. Scale bars = 100 and 10 µm. D2, Quantification of Arc immunofluorescence in dendrites that were severed (cut) from the soma or neighboring uncut dendrites. N = # of slices on each bar from 3 rats per condition. E1, Western blot of the synaptoneurosome preparation reveals enrichment of PSD-95 and a reduction in β3-tubulin, GFAP and Histone H3 in comparison to whole homogenate (input) or supernatant (Sup). E2, Western blot (WB) of Arc (using mAb) after Arc immunoprecipitation (using pAb) demonstrates that comparable amounts of Arc was IPed in different conditions. Quantification of 35S Met incorporation into Arc that was immunoprecipitated from synaptoneurosomes. N = # of animals on each bar.
Figure 3
Figure 3. Knockdown of endogenous Arc increases synaptic transmission and surface GluR1 expression
A, Lentivirus expressing a shRNA against Arc (shArc) mRNA in comparison to sister cultures infected with a control lentivirus expressing shRNA against DsRed (shDsRed). Dissociated rat hippocampal neurons were infected at 7 DIV with shArc or shDsRed that co-expresses GFP. 10–12 days postinfection, neurons were fixed, and Arc protein was visualized using immunofluorescence. Scale bar = 10 and 5 µm B, Quantified group data reveal an ~80% knockdown in the total Arc immunofluorescence in dendrites of shArc infected neurons. N = # cells/condition from 2 cultures. C, Representative mEPSCs from shArc and shDsRed infected neurons. Scale bars = 30 pA/100 msec. Neurons expressing shArc exhibited increased mEPSC amplitude, but no difference in mEPSC frequency, input resistance or capacitance in comparison to neurons infected with shDsRed. N= number of cells/condition from 4 cultures. D, E, Representative surface GluR1 on dendrites of neurons infected with either shDsRed or shArc. Scale bars = 10 µm. F, Group data reveal that knockdown of Arc increases basal surface GluR1 in control cultures. N = # cells/condition from 8 cultures.
Figure 4
Figure 4. Endogenous Arc is required for mGluR stimulated AMPAR endocytosis and LTD
A, Representative images of surface GluR1 immunofluorescence in low-density dissociated hippocampal cultures infected with lentivirus expressing shArc or shDsRed treated with media (control), DHPG, or NMDA and fixed 15 min after treatment onset. B, Group data reveal that knockdown of Arc blocks DHPG- induced decreases in surface GluR1. Data pooled from 5 cultures. C, Arc knockdown does not affect NMDA-induced decreases in surface GluR1. N = # cells on each from 3 cultures/condition. D, Representative mEPSCs recorded from neurons infected with shArc or shDsRed before DHPG treatment and 10 min after DHPG washout. Recordings performed in the presence of 1 µM TTX and 100 µM picrotoxin. E, mEPSC frequency 15 minutes after onset of DHPG treatment in shArc and shDsRed infected neurons plotted as a percentage of pre-treatment mEPSC frequency. Group data reveal that Arc knockdown blocks DHPG-induced decreases in mEPSC frequency. N = # cells on each bar from 4 cultures/condition. F, Representative images of internalized GluR1 immunofluorescence in low-density dissociated hippocampal cultures infected with lentivirus expressing shArc or shDsRed. Neurons were live labeled with N-terminal GluR1 antibody immediately after treatment with media (control) or DHPG (100 µM; 5 min) and fixed 15 min later. G, Group data reveal that knockdown of Arc reduces DHPG- induced endocytosis of surface GluR1. Data pooled from 5 cultures. In all images scale bars = 10 µm.
Figure 5
Figure 5. Acute blockade of Arc translation blocks the long-term effects of mGluR activation on GluR1 trafficking
A1, Representative images of Arc immunofluorescence in dissociated hippocampal neurons. Antisense oligonucleotides directed against Arc mRNA, or mismatch oligonucleotides were introduced into 19–21 DIV neurons via a lipid-based delivery system. Neurons were treated with media (control) or DHPG and fixed 10 min after onset of treatment. A2, Quantification of the area of the dendritic Arc fluorescence reveal that either one of two unique Arc antisense oligonucleotides (Arc antisense and Arc antisense 2) block DHPG-induced increases in Arc protein without affecting basal Arc levels (in control mismatch oligo treated cultures). Data from 2 cultures per condition. B1, Representative images of surface GluR1 in neurons treated with antisense or mismatch oligonucleotides 30 min prior to media (control) or DHPG treatment. One hour after DHPG, neurons were fixed and processed for surface GluR1 immunofluorescence. B2, Quantified group data reveal that DHPG fails to induce long-term decreases in surface GluR1 in neurons pretreated with either Arc antisense oligonucleotides, in contrast to neurons treated with mismatch oligonucleotides. Data from 2–3 cultures/condition. C, Representative double-label images of surface (green) and internalized (red) GluR1 in dissociated hippocampal neurons treated with Arc antisense oligo or mismatch oligo. One hour after media (control) or DHPG treatment (100 µM, 5 min), live neurons were labeled with GluR1 antibody. D, Ratiometric quantification of internal to surface GluR1 in the same dendrites reveal that Arc antisense oligonucleotide blocks DHPG-induced, long-term increases in GluR1 endocytosis rate. Data from 2 cultures per condition. In all images scale bars = 10 µm.
Figure 6
Figure 6. Acute blockade of Arc translation blocks mGluR induced LTD in CA1 neurons of acute hippocampal slices
A,B Average normalized EPSC amplitudes of CA1 neurons from acute hippocampal slices recorded with pipettes containing 250 µM mismatch oligonucleotide (A) or Arc antisense oligonucleotide (B). DHPG (100 µM, 5 min) applied to the bath resulted in LTD of EPSC amplitudes in cells filled with mismatch oligonucleotide. In contrast, intracellular introduction of Arc antisense oligonucleotide via patch pipette blocks DHPG-induced LTD. N = # of neurons. C, Representative EPSCs from cells filled with Arc antisense oligonucleotide or mismatch oligonucleotide taken during pre-DHPG baseline (1) or 50 min after LTD induction (2; indicated in A and B). Scale bars = 50 pA/ 10 msec. D, LTD of EPSC amplitude (plotted as a percent of pre-DHPG baseline) at 60 min in Arc antisense oligonucleotide infused neurons that were paired with same day or same slice control neurons infused with mismatch oligonucleotide. Average of 7 experiments is plotted in bold.
Figure 7
Figure 7. Overexpression of Arc in hippocampal neurons increases GluR1 endocytosis and occludes mGluR induced decreases in surface GluR1
A, Representative immunofluorescence of internalized GluR1 in hippocampal neurons infected with lentivirus containing GFP or Arc-GFP (green). B, Quantification of internalized GluR1 puncta reveal that neurons with Arc-GFP expression have increased GluR1 endocytosis rate as compared to neurons expressing GFP only. Data from 3 cultures. C, Representative images of surface GluR1 staining in neurons infected with lentivirus containing GFP or Arc-GFP. Neurons were treated with media (control) or DHPG (100 µM, 5 min) and fixed one hour after onset of treatment. D, Quantification of surface GluR1 puncta number in GFP or Arc-GFP infected neurons treated with media (control) or DHPG reveal that Arc-GFP overexpression decreases basal surface GluR1 levels and blocks DHPG-induced decreases in surface GluR1. Data from 3 cultures. E, Representative images of surface GluR1 staining in neurons infected with lentivirus containing GFP or Arc-GFP. Neurons were treated with media (control) or NMDA and fixed one hour after onset of treatment. F, Quantification of surface GluR1 puncta number in GFP or Arc-GFP infected neurons treated with media (control) or NMDA reveal that Arc-GFP overexpression decreases basal surface GluR1 levels and blocks NMDA-induced decreases in surface GluR1. Data from 3 cultures. In all images scale bars = 10 µm.
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