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. 2010 Oct 19;107(42):18173-8.
doi: 10.1073/pnas.1006546107. Epub 2010 Oct 4.

Arc regulates spine morphology and maintains network stability in vivo

Carol L Peebles  1 , Jong Yoo, Myo T Thwin, Jorge J Palop, Jeffrey L Noebels, Steven Finkbeiner
Affiliations

Arc regulates spine morphology and maintains network stability in vivo

Carol L Peebles et al. Proc Natl Acad Sci U S A. .

Abstract

Long-term memory relies on modulation of synaptic connections in response to experience. This plasticity involves trafficking of AMPA receptors (AMPAR) and alteration of spine morphology. Arc, a gene induced by synaptic activity, mediates the endocytosis of AMPA receptors and is required for both long-term and homeostatic plasticity. We found that Arc increases spine density and regulates spine morphology by increasing the proportion of thin spines. Furthermore, Arc specifically reduces surface GluR1 internalization at thin spines, and Arc mutants that fail to facilitate AMPAR endocytosis do not increase the proportion of thin spines, suggesting that Arc-mediated AMPAR endocytosis facilitates alterations in spine morphology. Thus, by linking spine morphology with AMPAR endocytosis, Arc balances synaptic downscaling with increased structural plasticity. Supporting this, loss of Arc in vivo leads to a significant decrease in the proportion of thin spines and an epileptic-like network hyperexcitability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Arc expression increases spine density and alters spine morphology. (A) Arc (red) localizes to dendritic spines and colocalizes with GFP-actin (green). (Scale bar, 5 μm.) (B–E) Medium density hippocampal neurons at 18–19 DIV were transfected with GFP and Arc or an empty vector and imaged 36–48 h after transfection. Examples of thin and stubby spines are marked by red and green arrows, respectively. (Scale bars, 5 μm.) (C) Arc expression increases spine density. *P < 0.05. (D) Cumulative frequency plots of spine width and length. Arc significantly decreases spine width but does not affect length. Kolmogorov-Smirnov test, P < 0.0001. (E) Arc significantly increases the percentage of thin spines (red arrows in B) and filopodia, and decreases the percentage of stubby spines (green arrows in B). t test, ***P < 0.0005, *P < 0.05. Error bars represent 95% CI. More than 2,500 spines from 64 to 65 dendrites on 12–18 neurons from three separate experiments were analyzed per condition. Measurements were averaged per dendrite. Mush, mushroom; Phyllo, filopodia.
Fig. 2.
Fig. 2.
Arc-mediated GluR1 endocytosis is required for alterations in spine morphology. (A) Deletion of endophilin interaction domain in Arc (Arc Δ91–100) blocks its ability to regulate spine morphology. One-way ANOVA with post hoc Tukey test, F(3.244)= 13.07stubby, 6.536 thin, 0.7452mushroom, 5.304filopodia; **P < 0.01, ***P < 0.001. More than 1,500 spines from 40 dendrites over three experiments per condition were analyzed. (B) Deletion of the dynamin interaction domain alters effect of Arc on spine morphology. Expression of Arc Δ194–215 decreases percentage of thin spines and increases percentage of mushroom spines. t test, **P < 0.005. More than 2,000 spines from 45 dendrites were analyzed in two experiments per condition. Error bars represent 95% CI. (C) Arc expression leads to an increase in the ratio of internal-to-total GluR1 at thin spines. t test, ***P = 0.0001. More than 100 spines of each type over three experiments per condition were analyzed. An example of internal and external surface staining is shown to the right. (D) Arc expression decreases the percentage of spines with surface GluR1. t test, *P = 0.01 (E) Surface GluR1 is specifically decreased in thin spines. t test, **P = 0.0024. More than 3,500 spines from 26 to 27 cells were analyzed per condition over five experiments. (F) Arc Δ91–100 expression does not reduce GluR1 surface expression at thin spines. (Scale bar, 5 μm.)
Fig. 3.
Fig. 3.
Arc−/− mice have decreased spine density and increased spine width. (A) Example of dentate granule Golgi staining from 3-mo-old Arc+/+ and Arc−/− mice. (Scale bars, 5 μm.) Stacks (10-μm) were imaged, and individual spines were measured in their appropriate focal plane. (B and C) Cumulative frequency plots of spine width and length. Arc−/− mice have increased spine width in both CA1 and DG cells (P = 0.006, P = 0.014, respectively). Spine length was also increased in DG cells of Arc−/− mice. P values were determined using Kolmogorov–Smirnov test. (D) Loss of Arc in vivo decreases percentage of thin spines (t test, CA1: P < 0.005, DG: P < 0.05) and increases percentage of mushroom spines (t test, CA1: P < 0.05, DG: P < 0.05). (E) Loss of Arc significantly decreases spine density in CA1 pyramidal cells (t test, P = 0.0053) and DG cells (t test, P < 0.0005). Fourteen dendrites from three animals per genotype were analyzed. Error bars represent 95% CI.
Fig. 4.
Fig. 4.
Increased seizure susceptibility and EEG epileptiform activity in Arc−/− mice. (A) Mice 4–6 mo old were injected i.p. with saline or kainate (17 mg/kg) and analyzed 5 d later. Brain sections were stained for NPY with immunoperoxidase. Arc+/+ (WT) and Arc−/− mice injected with kainate exhibit expected aberrant NPY expression in mossy fibers. This abnormal NPY pattern was also found in saline-treated Arc−/− mice. MF, mossy fibers; ML, DG molecular layer. (B) Quantification of mossy fiber NPY levels in saline-treated mice. au, arbitrary units. One-way ANOVA with post hoc Tukey test, F(2,29) = 3.652; *P < 0.05. (C) Arc−/− mice exhibited shorter seizure latency than either Arc +/− or Arc+/+ (WT) mice (one-way ANOVA: F(2,18) = 5.297; P < 0.05). (D) Arc−/− mice had significantly more severe seizures after two consecutive PTZ doses of 30 mg/kg 6 h apart. Paired t test, *P < 0.05. (E) Chronic EEG recordings reveal frequent generalized cortical interictal spike discharges in Arc−/− and Arc+/− mice. EEG recordings of Arc+/+ (WT) littermate mice revealed normal background cortical activity with no abnormal discharges. Calibration 1 s, 300 mV, electrode montage. LO, left occipital; LP, left parietal; LT, left temporal; RO, right occipital; RP, right parietal; RT, right temporal.
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