Environmental enrichment restores neurogenesis and rapid acquisition in aged rats

Abstract

Strategies combatting cognitive decline among the growing aging population are vital. We tested whether environmental enrichment could reverse age-impaired rapid spatial search strategy acquisition concomitantly with hippocampal neurogenesis in rats. Young (5-8 months) and aged (20-22 months) male Fischer 344 rats were pair-housed and exposed to environmental enrichment (n = 7 young, 9 aged) or housed individually (n = 7 young, 7 aged) for 10 weeks. After 5 weeks, hidden platform trials (5 blocks of 3 trials; 15 m inter-block interval), a probe trial, and then visible platform trials (5 blocks of 3 trials; 15 m inter-block interval) commenced in the water maze. One week after testing, rats were given 5 daily intraperitoneal bromodeoxyuridine (50 mg/kg) injections and perfused 4 weeks later to quantify neurogenesis. Although young rats outperformed aged rats, aged enriched rats outperformed aged individually housed rats on all behavioral measures. Neurogenesis decreased with age but enrichment enhanced new cell survival, regardless of age. The novel correlation between new neuron number and behavioral measures obtained in a rapid water maze task among aged rats, suggests that environmental enrichment increases their ability to rapidly acquire and flexibly use spatial information along with neurogenesis.

Figure 1. Experiment timeline

Rats were housed individually (n=7 young, n=7 aged) or in pairs and exposed to an enriched environment daily (n=7 young, n=9 aged) for ten weeks. In the 5^th week, rats were trained and tested on hidden platform trials and then visible platform trials 3d later. Beginning one week after testing, rats were injected daily with BrdU (50mg/kg) over 5 days and then perfused 4 weeks later to quantify neurogenesis.

Figure 2. Exposure to an enriched environment subdues age-dependent impairments on hidden and visible platform trials

The rats were trained on hidden platform trials (A), tested on a probe trial administered 15 min after the last hidden platform trial (B) and cued platform trials 3 days later (C). Line graphs depict group means (± S.E.M.) of measures obtained from the YI (white circles), YE (dark gray circles), AI (light gray squares) and AE (black squares) groups. (A) Enrichment enhanced the ability of aged rats to rapidly acquire a spatial search strategy. On all training blocks combined, young rats swam more directly to the hidden platform than aged rats. AI rats swam more circuitous routes to the hidden platform than either AE rats or young rats in either group. B) Probe trial DI scores varied by age and experience. Chi square tests confirmed that the % of rats that performed above or below chance (DI score = 0, dashed line) decreased with age but increased with exposure to enrichment. Specifically, 69% of enriched rats performed above chance whereas only 50% of individually housed rats performed above chance. (C) Previous experience influences performance on visible platform trials. Young rats outperformed aged rats on all training blocks, including the initial training block, likely because they retained procedural information from the spatial task and AE rats outperformed AI rats.

Figure 3. Exposure to an enriched environment reversed the effects of age on neurogenesis

Rats were given five daily injections of BrdU (beginning one week after behavioral testing) and perfused 4 weeks later. The total number of new cells surviving 4 was estimated stereologically using BrdU⁺ cell counts obtained under light microscopy from every 12^th section through the DG. The bar graph depicts group means (± S.E.M.) of total new cell number in the dentate gyri of YI (white bars), YE (dark gray bars), AI (light gray bars) and AE (black bars) rats. (A) Coronal view of the rat brain. The dentate GCL is highlighted in turquoise. (B) Photomicrograph of new (BrdU⁺) cells in the dentate gyrus of an aged rat. Representative examples of ~4 week-old cells labeled with BrdU⁺ (in brown) revealed enzymatically with DAB. (C) Total new cells numbers declined with age but were potentiated by enrichment, regardless of age. More new cells survived ~4 weeks in the dentate gyri of young versus aged and enriched versus individually-housed rats. *p≤0.05 and **p≤0.01.

Figure 4. Fewer new cells expressed mature neuronal phenotypes in the dentate gyri of aged rats

At least 100 BrdU⁺ cells per rat were examined under confocal microscopy (40 × objective with 2.3× digital zoom) to calculate proportions expressing markers of immature (DCX⁺), transitioning (DCX/NeuN⁺) or mature (NeuN⁺) neurons, as well as GFAP⁺ astrocytes or NG2⁺ oligodendrocyte precursors, which were revealed using fluorescent immunohistochemistry. (A,B) Confocal images of new neurons and astrocytes in the dentate gyrus of an adult rat. Representative images of ~4 week old BrdU⁺ cells (in red) that express the neuronal markers DCX (in blue) and/or NeuN (in green; A) or the glial markers GFAP (in blue) or NG2 (in green; B). (C) The proportion of new cells expressing neuronal phenotypes decreased with age and was unaffected by enrichment regardless of age. Mean (± S.E.M.) % of new cells in the dentate gyri of YI (white bars), YE (dark gray bars), AI (light gray bars) and AE (black bars) rats expressing neuronal and glial phenotypes are shown. In all rats, the majority of ~4 week-old cells expressed mature neuronal phenotypes. However, a lower % of new cells expressing mature neuronal phenotypes and a higher % of new cells expressing glial phenotypes was detected in aged versus young rats. No effect of differential experience on the % of new cells expressing either phenotype was detected in young or aged rats. *p≤0.05 and **p≤0.01.

Figure 5. Net neurogenesis declines with age but is increased by exposure to enrichment whereas age-dependent increases in gliogenesis are unaffected by enrichment

Net neurogenesis and gliogenesis was calculated by multiplying total new cell numbers (Fig. 3) by % of new cells expressing neuronal and glial phenotypes (Fig. 4), respectively. The bar graphs depict group mean (± S.E.M.) numbers of neurons (A) or glia (B) in the dentate gyri of YI (white bars), YE (dark gray bars), AI (light gray bars) and AE (black bars) rats. (A) Net neurogenesis declines with age but increases with enrichment, independent of age. Neurogenesis declined with age and was potentiated by exposure to an enriched environment regardless of age. A few weeks of exposure to an enriched environment, therefore, returned levels of hippocampal neurogenesis in aged rats to those observed in young individually-housed rats. (B) Age-dependent increases in gliogenesis are unaffected by exposure to enrichment. We detected a small but significant increase in gliogenesis in aged versus young rats that was unaffected by differential experience. *p≤0.05 and **p≤0.01.

Figure 6. New neuron number correlates with measures of spatial ability

Graphs depict total neuron number plotted against mean pathlengths across hidden platform training blocks (A and C) or against probe trial discrimination index scores (B and D) for YI (white circles), YE (dark gray circles), AI (light gray squares) and AE (black squares) rats. Mean pathlengths correlated negatively with total new neuron number in aged rats (r=−0.56; A). Note that shorter pathlengths indicate better performances. (D) The number of new neurons and discrimination index score are correlated positively in aged rats (r=0.59). * p ≤ 0.05.