Experience-dependent persistent expression of zif268 during rest is preserved in the aged dentate gyrus

Abstract

Background: Aging is typically accompanied by memory decline and changes in hippocampal function. Among these changes is a decline in the activity of the dentate gyrus (DG) during behavior. Lasting memory, however, is thought to also require recapitulation of recent memory traces during subsequent rest - a phenomenon, termed memory trace reactivation, which is compromised in hippocampal CA1 with progressive age. This process has yet to be assessed in the aged DG, despite its prominent role in age-related memory impairment. Using zif268 transcription to measure granule cell recruitment, DG activity in adult and aged animals was assessed both during spatial exploration and as animals remained at rest in the home cage in order to detect potential memory-related replay.

Results: Consistent with the observation of memory trace reactivation in DG, the probability that an individual granule cell transcribes zif268 during rest in the animal's home cage is increased by recent experience in a novel environment. Surprisingly, a comparable increase was observed in the probability of granule cells in the aged DG expressing zif268 during rest. Moreover, no significant age-related difference was observed in the number of granule cells expressing zif268 during rest. Thus, the number and pattern of granule cell expression of zif268 during rest is preserved in aged animals, despite a significant decline in exploration-related zif268 expression.

Conclusions: These data lead to the hypothesis that the input the aged DG receives from backprojections from CA3 (the region widely hypothesized to mediate reactivation) remains functionally intact despite loss of innervation from the perforant path.

Figure 1

The pattern of zif268 expression in the dentate gyrus (DG) is altered by recent experience. Representative montages of confocal images taken across the DG (scale bar = 200 μm) demonstrate that the number of granule cells expressing zif268 in caged control animals (a) taken directly from their home cages is very low (<1%) in both blades of the DG, while spatial exploration (b) induces a robust increase in zif268 transcription localized primarily in the suprapyramidal (SP) blade and not the infrapyramidal (IP) blade. Higher magnification confocal micrographs of the suprapyramidal blade (scale bar = 60 μm) are also shown to illustrate the differences in the pattern of zif268 transcription in the three behavioural conditions implemented in the current study. Relative to caged controls (c), the DG of animals killed immediately following spatial exploration (d, immediate) show significantly more granule cells (~5%) expressing zif268 within the nucleus (short arrow). Animals killed 25 min after exploration (e, delay) show comparable levels of zif268 expression to the immediate group, but the majority of mRNA is within the cytoplasm of granule cells (long arrow). Granule cell nuclei are counterstained with DAPI (blue) and zif268 is labeled with Cy3 (red).

Figure 2

Representative confocal images (scale bar = 10 μm) depict the compartmental expression of IEGs (above) and their temporal relationship to the experiences of the animals used in the experiments described here (below). Cells (counterstained with DAPI, blue) that exhibit high frequency firing of the type that characterize place fields begin to express IEGs such as zif268 (red) within minutes. When this activity occurs 30 minutes prior to the animal being killed (during the time in which both caged control and immediate animals are at rest in the home cage, while delay animals are exploring a novel environment), the transcripts migrate to the cytoplasm where they are apparent as a diffuse cloud (bottom left). In contrast, when this activity occurs within 5 minutes of the animal being killed (during the time in which both caged control and delay animals are at rest in the home cage and immediate animals are exploring a novel environment), the transcripts are visible as bright foci within the nucleus (bottom right). Cells active during both time periods will express zif268 within both cellular compartments (top).

Figure 3

Behavior-induced zif268 expression within the DG. (a) In the suprapyramidal blade, exploration of a novel environment induced zif268 in a comparable number of granule cells in both the immediate (white) and delay (grey) groups that was significantly greater than zif268 expression in caged controls (black). (b) While the number of granule cells expressing zif268 during an episode of rest (i.e., cells transcribing zif268 while animals were in the home cage would correspond to cells expressing cytoplasmic zif268 in the immediate group and all cells expressing nuclear zif268 in the delay group, see Methods) that precedes spatial exploration (immediate) is equivalent to caged controls, significantly more granule cells express zif268 during a rest episode that follows spatial exploration (delay). Note that zif268 expression in caged control animals is depicted solely in the “rest” category since these animals were never removed from the home cage and thus their only experience was comparable to the rest period of both immediate and delay animals. In addition, significantly more zif268 expression occurred in the same cells that were active during exploration (exploration + rest). In the infrapyramidal blade, exploration of a novel environment did not significantly alter the number of granule cells expressing zif268(c) within any cellular compartment or (d) during either epoch (all data are mean ± SEM; *p < 0.05, **p < 0.01 vs. CCs; † p < 0.05, immediate vs. delay).

Figure 4

The behaviorally-induced pattern of zif268 transcription is recapitulated during subsequent rest in the DG. In both immediate (white) and delay (grey) conditions, the percentage of granule cells transcribing zif268 during both exploration and rest (i.e., expressing zif268 within both the nucleus and surrounding cytoplasm) is greater than expected by random chance (black), although significantly more cells are active during both rest and exploration in the delay condition in the suprapyramidal blade (all data are mean ± SEM; *p < 0.01 vs. random chance; † p < 0. 01 immediate vs. delay, ‡ p < 0.05 infrapyramidal vs. suprapyramidal blades).

Figure 5

Aged rats are impaired in the Morris swim task. Analysis of path lengths (a) shows that when the platform was hidden, adult rats (black circle) swam shorter paths to reach the hidden platform than did the aged rats (black square), and this difference was significant on day 4 of training. During trials in which the platform was visible, adult rats (open circle) and aged rats (open square) had comparable path lengths. During the probe trial (b), adult rats (white) spent significantly more time than aged rats (grey) in the quadrant that previously held the platform (target), but not the opposite quadrant (all data are mean ± SEM; *p < 0.05; **p < 0.01, adult vs. aged animals).

Figure 6

The expression of zif268 in the suprapyramidal DG is attenuated with age during exploration but not rest. (a) Compartmental expression of zif268 is depicted for adult (red) and aged (blue) animals that explored a novel environment and those that remained within the home cage (hatched). When zif268 expression in adult and aged animals is compared during each epoch (b) a dramatic decline is apparent in the number of granule cells transcribing zif268 during exploration in aged animals. During the resting period immediately prior to sacrifice, however, no significant difference is seen in the number of zif268+ granule cells. Note that zif268 expression in caged control animals is depicted solely in the “rest” category since these animals were never removed from the home cage and thus their only experience was comparable to the rest period of both adult and aged delay animals. When the percentage of granule cells expressing zif268 during both epochs (exploration + rest) is analyzed relative to probability of co-expression based on chance (c) a significantly higher than chance proportion of cells show zif268 labeling during both epochs in both the adult and aged DG. The comparable number and pattern of zif268 expression during rest is particularly striking given the profound decrease in the number of cells transcribing zif268 during exploration in the aged DG (All data are mean ± SEM; *p < 0.05, **p < 0.01 relative to age-matched caged controls, †p < 0.05, adult vs. aged animals within the same behavioral group; ‡‡ p < 0.01 vs. random chance).