Spatial behavior and seizure-induced changes in c-fos mRNA expression in young and old rats

Abstract

The subcellular processes of gene induction and expression in the hippocampus are likely to underlie some of the known age-related impairments in spatial learning and memory. It is well established that immediate-early genes are rapidly and transiently induced in response to neuronal activity and this expression is required for stabilization of durable memories. To examine whether age-related memory impairment might be caused, in part, by differences in the level of cellular activation or subcellular processing, c-fos expression in CA1 pyramidal and dentate gyrus granule cells in the dorsal hippocampus of young and old rats was determined using fluorescence in situ hybridization and reverse transcription polymerase chain reaction. No significant age differences were found in the numbers of pyramidal or granule cells that show c-fos expression; however, c-fos mRNA transcripts were altered in these 2 cell types in aged animals. These findings suggest that though the networks of cells that participate in behavior or seizure-induced activity are largely maintained in aged rats, their RNA transcript levels are altered. This might, in part, contribute to cognitive deficits frequently observed with advancing age.

Fig. 1

There is a significant age-related impairment in the acquisition of the spatial version of the Morris swim task. (A) Older rats exhibited longer corrected integrated path lengths (CIPL) on all 4 training days when compared with the younger rats (* p < 0.05). (B) During the probe trial older animals spent significantly less time in the target quadrant (* p = 0.01) than did the younger rats. (C) On the cued (visible platform) portion of the swim task older and young rats showed improved performance from day 1 to day 2 but there was no significant effect of age on the path lengths (p > 0.05).

Fig. 2

Proportions of CA1 pyramidal cells that express c-fos mRNA by fluorescence in situ hybridization (FISH) and levels of c-fos determined by polymerase chain reaction (PCR) in animals given no behavior treatment. (A and B) Confocal images show differences between the transcription signal of c-fos versus another immediate-early gene (IEG) Arc. Notice the discrete distribution of Arc in the nuclear and cytoplasmic compartments whereas c-fos shows a more diffuse distribution. (C) Similar numbers of pyramidal cells express c-fos mRNA in young and old rats under a caged-control or “resting” condition. (D) Resting c-fos mRNA (caged controls) as determined by reverse transcription (RT)-PCR is significantly lower in old animals relative to young animals (star, p < 0.0001). Confocal images from the hippocampal CA1 region of an old (E) and young (F) rat (magnification × 250). Very few cells express c-fos mRNA in the no behavior treatment condition in either group. Nuclei are counterstained with Sytox (blue) and c-fos mRNA expression is indicated in red (Cy3).

Fig. 3

Proportions of CA1 pyramidal cells that express c-fos mRNA by fluorescence in situ hybridization (FISH) and levels of c-fos mRNA by reverse transcription polymerase chain reaction (RT-PCR) in animals that were given a single 5-minute exploration session (5′ A). (A) The proportions of pyramidal cells (total) that express c-fos mRNA are similar in old and young animals after the 5-minute behavioral exploration treatment. (B) RT-PCR analysis reveals that old animals show a significantly reduced fold increase in c-fos mRNA compared with resting levels and c-fos mRNA levels which are significantly (*=significant) lower in old compared with young rats (star, p < 0.05). (C) CA1 pyramidal cell region illustrating c-fos staining after fluorescence in situ hybridization (confocal image taken at magnification × 250) taken from an old animal. The nuclear counterstain is Sytox (shown in blue), and the c-fos positive staining (red) can be seen within the nuclear compartment. (D) Similar confocal image obtained from a young rat.

Fig. 4

Proportions of CA1 pyramidal cells that express c-fos mRNA by fluorescence in situ hybridization (FISH) and levels of c-fos mRNA by reverse transcription polymerase chain reaction (RT-PCR) from animals that explored a defined environment twice for 5 minutes separated by a 20-minute rest interval (A-20′-A). (A) The proportions of c-fos positive cells activated by this behavior treatment are not significantly different between the 2 age groups. The total proportions reflect cells with nuclear staining (foci), cytoplasmic, or both nuclear and cytoplasmic staining. (B) c-fos mRNA measured with RT-PCR indicates that both old and young animals show a similar fold increase from resting levels and that the relative mRNA levels of c-fos in old animals is significantly lower compared with young animals (star, p < 0.05). (C and D) Confocal images from an old and young rat respectively, illustrate pyramidal cell nuclei (counterstained with Sytox, blue) and c-fos mRNA staining (Cy3; red).

Fig. 5

The proportions of granule cells in young and old rats that express c-fos mRNA by fluorescence in situ hybridization (FISH) and levels of c-fos reverse transcription polymerase chain reaction (RT-PCR) after no behavior treatment (DG no behavior). (A) Similar proportions of granule cells express c-fos mRNA in young and old rats under a caged-control or “resting” condition. (B) No age-related differences in c-fos mRNA as determined by RT-PCR were detected in this behavior control condition. Representative confocal images from the dentrate gyrus of old (C) and young (D) rats. Nuclei are counterstained with sytox (blue) and c-fos mRNA expression in red (Cy3).

Fig. 6

The proportions of granule cells in young and old rats that express c-fos mRNA by fluorescence in situ hybridization (FISH) and levels of c-fos reverse transcription polymerase chain reaction (RT-PCR) after a single 5-minute exploratory behavior treatment (DG 5′-A). (A) No significant age differences were detected in the proportions of granule cells that express c-fos by FISH. (B) Although both age groups showed similar fold change in c-fos mRNA from resting levels, there is a significant difference by RT-PCR in animals that received the single 5-minute behavioral treatment with old rats having lower c-fos mRNA levels relative to young rats. Representative confocal images from the dentate gyrus of old (C) and young (D) rats.

Fig. 7

The proportions of granule cells using fluorescence in situ hybridization (FISH) and levels of c-fos mRNA obtained with reverse transcription polymerase chain reaction (RT-PCR) in animals that explored a defined environment for 5 minutes followed by a rest period in the home cage for 20 minutes and finally were given another 5 minutes exploration session in the same environment (DG A-20′-A). (A) Proportions of c-fos positive cells in the dentate gyrus are similar in young and old animals after 2 exploration periods (star, p > 0.05). (B) Old rats exhibit a significantly reduced fold change in c-fos mRNA level compared with resting levels by RT-PCR and have lower c-fos transcript levels compared with young rats (star, p < 0.05). (C and D) Confocal images of the dentate gyrus (DG) from an old and young rat. Note most of the staining is present in the upper blade (top blade in figure) of DG. Nuclei are shown in blue and c-fos staining in red (Cy3).

Fig. 8

Fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR) data from animals that were given maximal electroconvulsive shock (MECS) treatment and sacrificed after 5 minutes (CA1 5′ MECS). (A) In area CA1 there was no difference in proportions of c-fos–positive cells between age groups. (B) Results from the RT-PCR analysis reveals an age-related decrease (*=significant) in c-fos fold change in older animals (star, p < 0.05) when compared with resting levels and also a decrease in c-fos transcript level relative to young animals. (E) In the dentate gyrus, there is no age-related difference in the numbers of granule cells that express c-fos but old rats show a significant decrease in fold change from resting levels (F) as analyzed with RT-PCR. In addition, old animals have similar relative c-fos mRNA levels compared with young animals because no significant difference in c-fos transcript level was detected by RT-PCR (F). (C and D) Confocal images taken from the CA1 region of an old and young rat respectively; (G and H) are confocal images from the upper blade of an old and young rat, respectively.

Fig. 9

Fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR) data from area CA1 of old and young rats that were given maximal electroconvulsive shock (MECS) and sacrificed either 30 minutes (CA130′ MECS) or 60 minutes (CA160′ MECS) later. FISH reveals that the proportions of cells containing c-fos mRNA are not significantly different between young and old animals at the 30-minute time point (A) but at the 60-minute time point (E) significantly fewer CA1 cells express c-fos mRNA in the old group. RT-PCR indicates that old animals exhibit a similar fold change in c-fos mRNA when compared with resting levels but have a significantly lower relative c-fos mRNA transcript level when compared with young animals at both the 30- and 60-minute intervals (B and F). Panels C (30 minutes) and G (60 minutes) are confocal images from old MECS-treated rats and panels D (30 minutes) and H (60 minutes) are examples from young rats. CA1 pyramidal cells are counterstained with Sytox (blue) and c-fos mRNA is shown in red (Cy3).

Fig. 10

Representative confocal images from the dentate gyrus (DG) 30 minutes after maximal electroconvulsive shock (MECS) (DG 30′ MECS), 60 minutes after MECS (DG 30′ post MECS) images were similar to images shown. (A) Image from an old rat and (B) is from a young animal. c-fos mRNA is shown in red (Cy3) and nuclei are counterstained with Sytox (blue). (C) A lower fold change in old animals compared with resting levels and lower c-fos mRNA transcript levels in old animals relative to young. (D) indicates that older animals at the 1-hour post MECS (60′ post MECS) treatment also have a lower fold change in c-fos mRNA level but express significantly higher c-fos mRNA transcript levels relative to young animals (star, p > 0.05).