A specific requirement of Arc/Arg3.1 for visual experience-induced homeostatic synaptic plasticity in mouse primary visual cortex

Abstract

Visual experience scales down excitatory synapses in the superficial layers of visual cortex in a process that provides an in vivo paradigm of homeostatic synaptic scaling. Experience-induced increases in neural activity rapidly upregulates mRNAs of immediate early genes involved in synaptic plasticity, one of which is Arc (activity-regulated cytoskeleton protein or Arg3.1). Cell biological studies indicate that Arc/Arg3.1 protein functions to recruit endocytic machinery for AMPA receptor internalization, and this action, together with its activity-dependent expression, rationalizes a role for Arc/Arg3.1 in homeostatic synaptic scaling. Here, we investigated the role of Arc/Arg3.1 in homeostatic scaling in vivo by examining experience-dependent development of layer 2/3 neurons in the visual cortex of Arc/Arg3.1 knock-out (KO) mice. Arc/Arg3.1 KOs show minimal changes in basal and developmental regulation of excitatory synaptic strengths but display a profound deficit in homeostatic regulation of excitatory synapses by visual experience. As additional evidence of specificity, we found that the visual experience-induced regulation of inhibitory synapses is normal, although the basal inhibitory synaptic strength is increased in the Arc/Arg3.1 KOs. Our results demonstrate that Arc/Arg3.1 plays a selective role in regulating visual experience-dependent homeostatic plasticity of excitatory synaptic transmission in vivo.

Figure 1.

Arc/Arg3.1 protein expression in mouse primary visual cortex. A, Immunohistochemical labeling of Arc/Arg3.1 (green) and NeuN (red) in L2/3 of mouse visual cortex. Images shown are three-dimensional projections of 20 confocal sections taken at 1 μm z-axis intervals. Top row, NeuN staining in grayscale. Second row, Arc/Arg3.1 staining in grayscale. Third row, Overlay of NeuN and Arc/Arg3.1 in color. Scale bars, 30 μm. Bottom panels, Zoom-in of the areas indicated in the overlay panels. B, Comparison of mean Arc/Arg3.1 staining intensity normalized to that of average NR. ^#p < 0.03, Fisher's PLSD post hoc test after ANOVA. C, Quantification of Arc/Arg3.1 protein level in visual cortex homogenates of mice by immunoblot analysis. An example blot is shown in the top panel. Bottom panel, Quantification of Arc immunoblot signals. ^#p < 0.01, Fisher's PLSD post hoc test after ANOVA.

Figure 2.

Comparison of AMPAR and associated proteins in the visual cortex of Arc/Arg3.1 KO and WT. A, Immunoblot analysis of AMPAR subunits (GluR1 and GluR2/3) and associated proteins (Grip1 and Pick1). Left, Representative immunoblots of WT and KO visual cortex homogenates probed with antibodies against Arc, GluR1 C terminal, GluR2/3 C terminal, Grip1, and Pick1. Right, Quantification of immunoblots. The band intensity was normalized to that of the average WT values to obtain percentage of average WT values. *p < 0.05, t test. B, Surface levels of GluR1 and GluR2 subunits measured by steady-state surface biotinylation in visual cortical slices of WT and KO. Left, Example immunoblots of total homogenate (Input), supernatant (Sup), and biotinylated (Biotin) samples probed with antibodies against GluR1 C terminal (top), GluR2 N terminal (middle), and actin (bottom). The absence of actin in the biotin lanes confirms the specificity of biotinylation of surface proteins. Right, Quantification of surface GluR1 and GluR2. The signals of the input lanes and the biotin lanes were used to calculate the percentage of total GluR1 and GluR2 on the surface (% of total). C, GluR1 and GluR2 levels in the isolated PSD fractions of WT and KO were not significantly different. Left, Representative immunoblots probed with GluR1 C-terminal and GluR2 N-terminal antibodies. Right, Quantification of the immunoblot signals. D, Increase in GluR1 phosphorylation on S831 (left), but not S845 (right), in visual cortex of Arc/Arg3.1 KOs. Top, Representative blots probed with phospho-specific antibodies (P) and GluR1 C-terminal antibody (C). E, No difference in AMPAR I–V relationship measured from L2/3 neurons during L4 stimulation. Left, Example superimposed traces taken at V_h of −60 mV (inward current) and +40 mV (outward current). Right, I–V curve plotting AMPAR current amplitude at different V_h. Open circles, WT; filled circles, KO.

Figure 3.

Comparison of NMDAR function and key synaptic proteins in visual cortex. A, Normal NMDAR-EPSC to AMPAR-EPSC ratio in Arc/Arg3.1 KOs. Evoked EPSCs were measured in L2/3 pyramidal neurons after L4 stimulation. Left, Superimposed representative EPSC traces measured at −70 mV (AMPAR-EPSC) and +40 mV (compound EPSC of AMPAR and NMDAR). The size of AMPAR response was measured as the peak amplitude at −70 mV, and the magnitude of NMDAR response was measured at three times the AMPAR decay time constant (τ) at +40 mV. Right, Comparison of average NMDAR/AMPAR ratio of WT and KO. NMDAR/AMPAR ratio values from individual cells are plotted as small open circles. The average values are shown in filled circles, with error bars denoting SEM. B, Arc/Arg3.1 KOs expressed normal levels of NMDAR subunits NR1, NR2A, and NR2B in the total homogenate of visual cortex. C, The expression levels of postsynaptic proteins SAP97 and PSD95 and a presynaptic protein synaptophysin (Synapto.) were normal in KOs.

Figure 4.

Arc/Arg3.1 KOs display larger basal mEPSCs and mIPSCs in L2/3 pyramidal neurons of visual cortex. A, Comparison of mEPSCs recorded from P23 normal-reared WT and KO. Left, Multiplicative increase in mEPSC amplitude of KO compared with WT. The cumulative probability curve of WT mEPSC amplitudes (black solid line) multiplied with a scaling factor (WT_scaled, scaling factor of 1.1; black dashed line) superimposed with that of KO (gray solid line) (K–S test, p > 0.3). Right, Average mEPSC traces from WT and KO. *p < 0.05, t test. B, Left, Verification that the internal solution used reverses mIPSCs at 0 mV. Each trace was recorded under different holding voltage as indicated. Right, The pharmacologically isolated mIPSCs are blocked by addition of 20 μm bicuculline (+BMI). C, Comparison of mIPSCs recorded from P23 normal-reared WT and KO. Left, Larger mIPSC amplitude in KOs is multiplicative to that of WTs. Cumulative probability curve of mIPSC amplitude of WT_scaled (scaling factor of 1.2; black dashed line) overlapped with that of KO (gray solid line). WT, Black solid line. Right, Average mIPSC traces. *p < 0.03, t test.

Figure 5.

Absence of visual experience-induced homeostatic plasticity of mEPSCs in L2/3 of Arc/Arg3.1 KO visual cortex. A, Bidirectional synaptic scaling in WT. Left, Two days of DR increased mEPSC amplitude, and reexposure to light for 2 h (+2hL) or 1 d (+1dL) reversed this. Middle, Average mEPSC traces from the four groups. Right, No change in mEPSC frequency (ANOVA, F_(3,57) = 0.7, p > 0.5). *p < 0.01, Fisher's PLSD post hoc test. B, Multiplicative scaling up of mEPSCs with DR in WT. The cumulative probability curve of NR_scaled [black dashed line, multiplying mEPSCs from NR (black solid line) with a scaling factor of 1.2] overlapped with that of DR (gray solid line). C, Multiplicative scaling down of mEPSCs with 2 h of light reexposure (+2hL) in WT. The cumulative probability curve of DR_scaled (black dashed line; DR mEPSCs multiplied by a scaling factor of 0.8) overlapped with that of +2hL (gray solid line). DR, Black solid line. D, Visual experience failed to alter mEPSCs in KOs. Left, No change in mEPSC amplitude with DR, +2hL, or +1dL. Middle, Average mEPSC traces from the four groups. Right, No significant change in mEPSC frequency (ANOVA, F_(3,53) = 2.3, p > 0.08). E, No change in mEPSC amplitude distribution between NR and DR KOs. F, No change in mEPSC amplitude distribution between DR and +2hL KOs.

Figure 6.

Arc/Arg3.1 KOs display partial impairment of developmental decrease in mEPSC amplitude in L2/3 visual cortex. A, In normal-reared WTs, a developmental decrease in mEPSC amplitude is accompanied by a developmental increase in mEPSC frequency. Left, Comparison of mEPSC amplitude of P11 and P23 WT (the P23 data are duplicated from Fig. 4A for comparison). A developmental decrease in mEPSC amplitude between P11 and P23 of WT is not multiplicative [K–S test for P23 vs P11_scaled (mEPSCs of P11 multiplied by 0.8), p < 0.0001]. P23, Black solid line; P11, gray solid line; P11_scaled, black dashed line. Middle, Average mEPSC traces from P11 and P23 WT. Right, A developmental increase in mEPSC frequency. Open circles, mEPSC frequency of individual cells; black circles, average mEPSC frequency for each group with error bars. **p < 0.0002, t test. B, In normal-reared Arc/Arg3.1 KOs, mEPSC amplitude at P11 is significantly larger than P23, which is accompanied by an increase in mEPSC frequency. Left, mEPSC amplitude comparison between P11 and P23 KO (the P23 data are duplicated from Fig. 4A for comparison). A non-multiplicative decrease in mEPSC amplitude during development of KO [K–S test P23 vs P11_scaled (=P11 × 0.8), p < 0.002]. P23, Black solid line; P11, gray solid line; P11_scaled, black dashed line. Middle, Average mEPSC traces from P11 and P23 KO. Right, A developmental increase in mEPSC frequency. Open circles, mEPSC frequency of individual cells; black circles, average mEPSC frequency for each group with error bars. **p < 0.001, t test.

Figure 7.

Visual experience-dependent regulation of mIPSCs is not dependent on Arc/Arg3.1. A, Regulation of mIPSCs by visual experience in wild types. Left, Comparison of average mIPSC amplitude between NR, 2 d DR, and 2 d DR reexposed to light for 2 h (+2hL). There was a significant increase in the average mIPSC amplitude in +2hL group compared with NR and DR. Middle, Average mIPSC traces from WT NR, DR, and +2hL groups. Right, Comparison of mIPSC frequency between NR, DR, and +2hL groups. DR significantly decreased mIPSC frequency, which reversed with reexposure to light for 2 h (+2hL). B, Cumulative probability graph of mIPSC amplitudes of WT NR (gray solid line) and DR (black dotted line) groups. Although the average mIPSC amplitude was not different, there was a small but statistically significant difference in the distribution (K–S test, p < 0.01). C, Multiplicative scaling up of mIPSC amplitudes when WT DR are reexposed to light for 2 h (+2hL). The distribution of mIPSC amplitudes of the DR group (black solid line) matched that of the +2hL group (gray solid line) when scaled multiplicatively with a factor of 1.2 (black dotted line) (K–S test, p = 0.3). D, Normal regulation of mIPSCs in Arc/Arg3.1 KOs. Left, The average mIPSC amplitude in the +2hL group is significantly larger than NR or DR. Middle, Average mIPSC traces from KO NR, DR, and +2hL groups. Right, DR decreased mIPSC frequency of KOs, which rapidly reversed by 2 h of light (+2hL). E, Cumulative probability of mIPSC amplitudes of NR (gray solid line) and DR (black dotted line) KOs (K–S test, p < 0.01). F, Similar to WTs, DR Arc/Arg3.1 KOs multiplicatively scaled up mIPSC amplitudes when reexposed to light for 2 h (+2hL). The distribution of mIPSC amplitudes of the DR group (black solid line) matched that of the +2hL group (gray solid line) when multiplied with a scaling factor of 1.1 (black dotted line) (K–S test, p = 0.4). *p < 0.05, Fisher's PLSD post hoc test after ANOVA (p < 0.05).