Experience-dependent gene expression in the rat hippocampus after spatial learning: a comparison of the immediate-early genes Arc, c-fos, and zif268

Abstract

Neuronal immediate-early gene (IEG) expression is regulated by synaptic activity and plays an important role in the neuroplastic mechanisms critical to memory consolidation. IEGs can be divided into two functional classes: (1) regulatory transcription factors (RTFs), which can broadly influence cell function depending on the "downstream" genes they regulate, and (2) "effector" proteins, which may directly modulate specific cellular functions. The objective of the current study was to determine whether the expression of an effector IEG (Arc) was similar to, or different from, that of two well characterized RTF IEGs (c-fos and zif268) after learning. IEG RNA levels from rats trained in spatial and nonspatial water tasks were determined using RNase protection assays and in situ hybridization. Overall, the regulation of the three IEGs was similar in the hippocampus and the entorhinal and primary visual cortices. Consequently, IEG RNA levels were positively correlated within a structure. By contrast, Arc and zif268 RNA levels were not correlated or only weakly correlated across structures, although c-fos RNA levels were moderately correlated across structures. Arc RNA expression differed from that of zif268 and c-fos in two regards: (1) hippocampal Arc RNA levels were correlated with learning of the hippocampal-dependent spatial, but not hippocampal-independent cued response, water task, and (2) Arc RNA levels in the hippocampus and entorhinal cortex increased after spatial reversal learning relative to an asymptotic performance group. Thus, although the expression of Arc, zif268, and c-fos exhibited many similarities, Arc was most responsive to differences in behavioral task demands.

Fig. 1.

Multiple probe RNase protection assay for the simultaneous detection of Arc, c-fos, and zif268 RNAs. a, Hippocampal RNA levels for all three IEGs increased 45 min after systemic injection of pentylenetetrazole (PTZ) (50 mg/kg, i.p.) relative to caged controls. b, Detection of IEG RNAs in the hippocampus and primary visual cortex of rats trained in different versions of the spatial water task. The data shown here are from the experiment detailed in Results (Figs. 4, 5). See Materials and Methods and Results for full details of the assay and the behavioral groups. The asterisks in a and bindicate a small amount of full-length actin probe that was consistently seen in all samples.

Fig. 2.

Rapid and transient increase in hippocampal IEG RNA levels after spatial water-task training. IEG RNA levels from rats killed at 0.5, 2, and 6 hr after spatial water-task training were compared with caged control levels using the multiple probe RPA. RNA levels for all three IEGs were significantly above control levels at 0.5 hr after training, and c-fos RNA levels were lower than caged control levels 6 hr after training. *p< 0.05 relative to caged (0 hr) group, Mann–Whitney Utest. n = 3–5 rats per group.

Fig. 3.

Hippocampal IEG RNA expression after spatial and cued water-task training. Rats were trained in either the spatial or cued water tasks and killed 30 min later. IEG RNA levels from dorsal hippocampus were analyzed by RPA. A, Acquisition data for rats trained in each task. Performance, as measured by latency to mount the hidden (spatial) or visible (cued) platform, was not different between the two groups (p = 0.24, repeated measures ANOVA). B, Hippocampal IEG RNA levels for rats trained in both tasks were significantly above caged control levels (p < 0.001, Mann–WhitneyU test) but were not different from each other.C, D, Negative correlation between hippocampal Arc RNA levels and performance in the spatial (C), but not cued (D), water task. See Results for additional information. The Spearman correlation coefficient (R_S) is indicated for both cued and spatial trained groups (*p < 0.05).n = 8–9 rats per group.

Fig. 4.

Rapid acquisition of the spatial reversal water task. A, Performance of the seventh session and seventh session-reversal groups during pretraining. Rats were given two training sessions per day, separated by 30 min, for 3 d.B, Final session performance for all experimental groups. Performance between all groups was different on trial 1 (p < 0.01; ANOVA with Fischer'spost hoc analysis). See Materials and Methods and Results for additional details. n = 6 rats per group.

Fig. 5.

Spatial reversal training increasesArc expression in hippocampus and entorhinal cortex. Rats were trained in one of three spatial water tasks (for behavioral data, see Fig. 4) and were killed 30 min later. The experimental groups included caged, first session, seventh session, and seventh session-reversal (n = 6 rats per group). IEG RNA levels for the dorsal hippocampus (A), lateral entorhinal cortex (B), and primary visual cortex (C) were determined by RPA. IEG RNA levels for all water task-trained groups were significantly above caged control levels for each brain region (p < 0.02; Mann–Whitney U test). See Results section for additional details and statistical comparisons. n = 6 rats per group.

Fig. 6.

Increase in Arc RNA expression in the CA1 pyramidal cell layer 30 min after spatial water-task training.a and c show nuclei (blue) and Arc RNA staining (red) for a caged control and a spatial water-task trained rat, respectively.b and d show grayscale images ofArc RNA staining from a andc, respectively. Final magnification is 40×. Scale bars (shown in a and c): 50 μm. See Materials and Methods for experimental details and Table 4 for the summary of the quantitative analyses.

Fig. 7.

Increase in Arc RNA expression in the dentate gyrus and CA3 regions 30 min after spatial water task training. a and c show nuclei (blue) and Arc RNA staining (red) for a caged control and a spatial water task-trained rat, respectively. b and dshow grayscale images of Arc RNA staining froma and c, respectively. Final magnification is 5×. Scale bars (shown in a andc): 200 μm. See Materials and Methods for experimental details and Table 4 for the summary of the quantitative analyses.