Sustained transcription of the immediate early gene Arc in the dentate gyrus after spatial exploration

Abstract

After spatial exploration in rats, Arc mRNA is expressed in ∼2% of dentate gyrus (DG) granule cells, and this proportion of Arc-positive neurons remains stable for ∼8 h. This long-term presence of Arc mRNA following behavior is not observed in hippocampal CA1 pyramidal cells. We report here that in rats ∼50% of granule cells with cytoplasmic Arc mRNA, induced some hours previously during exploration, also show Arc expression in the nucleus. This suggests that recent transcription can occur long after the exploration behavior that elicited it. To confirm that the delayed nuclear Arc expression was indeed recent transcription, Actinomycin D was administered immediately after exploration. This treatment resulted in inhibition of recent Arc expression both when evaluated shortly after exploratory behavior as well as after longer time intervals. Together, these data demonstrate a unique kinetic profile for Arc transcription in hippocampal granule neurons following behavior that is not observed in other cell types. Among a number of possibilities, this sustained transcription may provide a mechanism that ensures that the synaptic connection weights in the sparse population of granule cells recruited during a given behavioral event are able to be modified.

Figure 1.

A, Experimental design used to induce Arc expression with a single spatial exploration experience. All animals explored a novel environment for 5 min and were killed at different times after exploration (5 min, n = 3; 30 min, n = 3; 2 h, n = 3; 6 h, n = 4; 8 h, n = 4; 12 h, n = 4; 24 h, n = 3; caged, n = 4). Caged refers to the animals killed immediately after being taken from their home cages and is a negative control for behaviorally induced Arc. B, Representative confocal image taken with a 40×/1.3 NA objective from the DG granule cell layer stained for the intronic Arc (pre-mRNA) intranuclear foci revealed with Cy3 (red) and the full-length cytoplasmic Arc mRNA probe revealed with FITC (green); the intranuclear staining appears as yellow because the staining is overimposed with green cytoplasmic staining. Note that the cytoplasmic staining is so dense that with only one color it is not reliable for determining whether the cell presents intranuclear foci staining or not; cell body nuclei are counterstained with DAPI (blue). C, The kinetics of exploration-induced Arc mRNA is reported as the percentage of total DG granule cells, which express Arc mRNA; each bar represents the mean proportion of DG cells classified as Arc-positive (foci + cytoplasmic + double) for each treatment group (*p < 0.01 compared with caged, ± SEM). D, Compartment analysis of temporal activity by catFISH of Arc. The Arc FISH staining was found either in the nucleus only (foci), in the cytoplasm only (cytoplasmic), or in both compartments (double). The bars represent the mean proportion of cells, out of the total population of DG neurons, for each classification ± SEM, *p < 0.01 compared with caged controls. E, Proportion of activated cells with foci-only, cytoplasmic-only, or double (those with foci and cytoplasmic) Arc staining, out of the total population of Arc-positive neurons, *p < 0.01 compared with caged controls. The colors in D and E bars refer to the same time point groups indicated in C.

Figure 2.

A, Mean (± SEM) proportion of CA1 pyramidal neurons that present intronic Arc pre-mRNA intranuclear foci staining (foci), Arc mRNA in the cytoplasm (cytoplasmic), and both (double), in the groups of animals killed at different time points after spatial exploration of a novel environment, *p < 0.01. Representative confocal images taken with a 40×/1.3 NA objective of the CA1 region from a caged control animal (B), an animal killed 5 min after spatial exploration (C), and an animal killed 30 min after spatial exploration (D).

Figure 3.

A, Schematic drawing of the experimental design. Animals were allowed to explore a novel environment at different times after an initial injection of Actinomycin D and were killed immediately after the 5 min exploration. Representative confocal images of Arc mRNA expression in the CA1 (B, C) and DG (D, E) regions of the hippocampus taken with a 40×/1.3 NA objective. B, D, Intranuclear Arc mRNA foci were evident 5 min after exploration in control animals injected with vehicle. C, E, No Arc mRNA was evident in animals injected with Actinomycin D 15 min before exploratory behavior. Proportion of CA1 pyramidal neurons (F) and DG granule cell neurons (G) that express Arc mRNA (intranuclear foci) in animals killed immediately after 5 min of spatial exploration, which received Actinomycin D at different time points before the experience (0.15 min, 1 h, 2 h, 8 h; n = 3 each group). Vehicle injection was administered 15 min before exploration and was used as the positive control for behaviorally induced Arc mRNA expression. At this time point Arc mRNA was observed in the nucleus because the behavioral induction began 5 min earlier. Note that the proportion of Arc-expressing cells was significantly higher as compared with caged controls (*p < 0.01) in the 15 min vehicle group and the animals exposed to exploration 8 h after the drug administration. ⁺p < 0.01 compared with vehicle control.

Figure 4.

A, Schematic drawing of the experimental design used. All animals were exposed to a 5 min exploration of a novel environment, then immediately injected with Actinomycin D or vehicle, followed by kill at 30 min, 2 h, or 8 h later. B, Kinetics of Arc mRNA expression in DG neurons of vehicle-treated animals compared with animals injected with Actinomycin D is reported as the percentage of total cells that were classified as Arc-positive; these are cells that express behaviorally induced Arc mRNA (foci, cytoplasmic, and both). Caged refers to the rats killed immediately after being taken from their home cages and is a negative control for behaviorally induced Arc (*p < 0.01 vs caged, ⁺p < 0.01 vs 5 min vehicle). C, Compartment analysis of Arc mRNA-expressing cells either in the nucleus only (foci), in the cytoplasm only (cytoplasmic), or in both compartments (double), *p < 0.01.