Spatial exploration-induced Arc mRNA and protein expression: evidence for selective, network-specific reactivation

Abstract

The immediate-early gene Arc is transcribed in neurons that are part of stable neural networks activated during spatial exploratory behaviors. Arc protein has been demonstrated to regulate AMPA-type glutamate receptor trafficking by recruiting endosomal pathways, suggesting a direct role in synaptic plasticity. The purpose of the present study is to examine the fidelity of Arc mRNA translation and the temporal dynamics of behaviorally induced Arc protein expression after rats explore a novel environment. These experiments reveal two waves of Arc protein expression after a single exploration session. In the initial wave, virtually all cells that express Arc mRNA in the hippocampus and parietal cortex also express Arc protein, indicating, at a cellular level, that mRNA transcription and translation are closely correlated from 30 min to 2 h in hippocampal CA and parietal neurons. A second wave of protein expression spans the interval from 8 to 24 h and is also remarkably specific to cells active in the original behavior-induced network. This second wave is detected in a subset of the original active network and displays the novel property that the proportions of Arc-positive neurons become correlated among regions at 24 h. This suggests that the second expression wave is driven by network activity, and the stabilization of circuits reflecting behavioral experience may occur in temporally discrete phases, as memories become consolidated. This is the first demonstration of network-selective translational events consequent to spatial behavior and suggests a role for immediate-early genes in circuit-specific, late-phase synaptic biology.

Figure 1.

Behavioral apparatus and treatment design. A schematic drawing of the behavioral apparatus used for the exploration “treatment” and a list of the intervals intervening between the first and second exposures to the apparatus (ranging from 30 to 1440 min) are shown. Caged refers to the rats killed immediately after being taken from their home cages and is a negative control for behaviorally induced Arc. The animals in the 5 min group explored only once and were killed immediately afterward. This group was used as a negative control for Arc protein expression because at this time point, only Arc mRNA transcription is found in the nucleus.

Figure 2.

Exploration induces Arc protein translation. Sample confocal images of cells expressing Arc protein (red) taken at 25× magnification from CA1 (A, B), CA3 (C, D), DG (E, F), and PCx (G, H) from caged control rats (left column) and from rats that were killed 1 h after exploration (right column) are shown. Nuclei are counterstained with Sytox green. In all regions examined, the exploration rats showed greater numbers of cells with Arc protein than did the caged controls. Scale bar, 100 μm.

Figure 3.

Kinetics of exploration-induced Arc protein expression in CA1, CA3, and PCx. A, Percentage of total cells showing Arc protein (induced by the first exploration) in CA1, CA3, and PCx at all time points studied. All groups were exposed twice to the same environment, except for the caged and 5 min control groups. Caged, n = 10; 5 min, 1, 2, 4 h, n = 8; 0.5, 3, 6, 8, 24 h, n = 6. ^*p < 0.0014 relative to controls; ^#p = 0.04 (not statistically significant after correction for multiple comparisons). B, Percentage of total cells with Arc mRNA foci (induced by the second exploration) in CA1, CA3, and PCx for the caged controls (open circles) and exploration (filled circles) groups. ^*p < 0.001 compared with controls. Cytoplasmic Arc mRNA is not shown in these regions because after 30 min, the Arc mRNA signal decreases to baseline.

Figure 4.

Regression plots comparing the proportions of cells containing Arc protein over the three regions (CA1, CA3, and PCx) and four time points studied. Regression plots between CA1 (x-axis) and CA3 (y-axis) (left column), CA1 and PCx (middle column), and CA3 and PCx (right column) are shown. For simplicity, the time points shown here (other than for caged control) reflect the times at which the expression of Arc protein was statistically above control levels. There were no significant correlations at the 3, 4, and 6 h time points. The correlations in the plots with light gray background are significant at the p < 0.05 level, whereas the correlations in the plot with dark gray background are significant at the p < 0.01 level.

Figure 5.

Kinetics of exploration-induced Arc protein expression in the DG. A, Example of a reconstructed 20 μm flat image, as was used for analysis of Arc expression in granule cells of the DG. Note the sparsity of Arc staining (red) across the entire DG, with the sparsest expression observed in the lower blade. B, Percentage of cells stained for Arc protein in the upper and lower blades of the dentate gyrus at all time points studied. C, Percentage of cells stained for Arc cytoplasmic mRNA in the upper and lower blades of the dentate gyrus at all time points studied. ^*p < 0.0014 compared with controls. Scale bar, 100 μm.

Figure 6.

Arc protein is expressed in the same neurons that express Arc mRNA. A-D, Example confocal images from parietal cortex (nuclei shown in green) taken from a caged control animal (A), an animal killed 5 min after a single exploration session (B), and an animal given two exploration sessions separated by 0.5 h (C) or 8 h (D). The latter two time points correspond to the first appearance of Arc protein in the first and second waves of protein expression, respectively. Arc mRNA intranuclear foci are shown in red, Arc protein is shown in purple, and the colocalization of Arc mRNA and protein is shown in pink (25× magnification; scale bar, 100 μm). E-H, Quantification of the proportions of neurons containing Arc mRNA foci only (red), Arc cytoplasmic protein only (blue), and double-stained neurons with Arc mRNA and protein (green), for the caged, 5 min, and 0.5 and 8 h conditions. To reflect the total proportions of neurons that expressed Arc mRNA and protein, the Double category is added to each of the mRNA and protein counts, indicated by the crosshatched bars. For each group, the total number of cells that expressed Arc protein and also mRNA was also determined (yellow histogram to the right, reflecting colocalization). Compared with caged controls, the 5 min and 8 h groups showed significantly increased numbers of Arc mRNA-expressing cells (^*p < 0.05; ^**p < 0.01, with Bonferroni correction); neither the mRNA nor protein alone is significantly different from caged controls for the 0.5 h group, but the double-stained population is significantly increased from controls (^**p < 0.01); for the 8 h group, the double-labeled cells are also significantly different from controls (^**p < 0.01). The proportions of neurons that showed colocalization of Arc mRNA and protein are statistically greater than caged controls only in the 0.5 and 8 h groups (yellow bars). The results (data not shown) from the 3, 4, and 6 h groups are similar to those from the 5 min group shown; those from the 1 h group (data not shown) are similar to those from the 0.5 h group shown; and those from the 24 h group (data not shown) are similar to those from the 8 h group shown. Note that in the caged control animals, the colocalization is close to chance levels, whereas in the 0.5, 1, 8, and 24 h groups, the colocalization is well above chance (>80%).