Long-term enrichment enhances the cognitive behavior of the aging neurogranin null mice without affecting their hippocampal LTP

Abstract

Neurogranin (Ng), a PKC substrate, is abundantly expressed in brain regions important for cognitive functions. Deletion of Ng caused severe deficits in spatial learning and LTP in the hippocampal CA1 region of mice. These Ng-/- mice also exhibit deficits in the amplification of their hippocampal signaling pathways critical for learning and memory. A short-term exposure to an enriched environment failed to improve their behavioral performances. Here, we showed that a long-term enrichment protocol for the aging mice was beneficial to the Ng-/- as well as Ng+/+ and Ng+/- mice in preventing age-related cognitive decline. Enrichment also caused an increase in the hippocampal CREB level of all three genotypes and Ng level of Ng+/+ and Ng+/- mice, but not that of alphaCaMKII or ERK. Interestingly, hippocampal slices of these enriched aging Ng-/- mice, unlike those of Ng+/+ and Ng+/- mice, did not show enhancement in the high frequency stimulation (HFS)-induced LTP in the CA1 region. It appears that the learning and memory processes in these enriched aging Ng-/- mice do not correlate with the HFS-induced LTP, which is facilitated by Ng. These results demonstrated that long-term enrichment for the aging Ng-/- mice may improve their cognitive function through an Ng-independent plasticity pathway.

Figure 1.

Performances in Barnes maze as affected by environmental enrichment. (A) The graph indicates the escape latency for finding the dark hiding hole for Ng+/+ (Wt), Ng+/− (Het), and Ng−/− (Homo) mice over the successive blocks of trials. Mice received three blocks of trial per day and each block consisted of three trials with different starting locations. The average escape latencies for the last three blocks of trials among the various groups of mice were as follows (mean ± SEM): Wt control, 48.0 ± 0.5 sec; Wt enrich, 36.9 ± 0.8 sec, Het control, 46.5 ± 0.8 sec; Het enrich, 34.4 ± 1.8 sec; Homo control, 52.3 ± 0.5 sec; and Homo enrich, 44.2 ± 1.3 sec. One-way ANOVA pairwise comparison by the Holm–Sidak method showed that both Wt and Het controls were significantly different from Homo control, with P < 0.05 and 0.005, respectively, but Wt control and Het control were not significantly different from each other. Enriched group of all three genotypes were also significantly different from their respective control groups all with P < 0.001. (B) Average escaping time of the first three blocks and the last three blocks of trial. The differences between the starting and ending times represented the progress, and each group of mice made significant progress, all with P < 0.001. For Wt and Het, both of their control and enriched groups made significantly more progress than the Ng−/− mice (all were P < 0.001). The enriched groups of Wt and Het also made more progress than their respective controls, all with P < 0.001. The progress made between Wt and Het; both their control and enriched groups, however, did not differ significantly. (C) During 1 min of the probe trial, the percentage time spent in quadrant 1 (Qd1) where the escape box was located was as follows: Wt control, 30.5 ± 2.3; Wt enrich, 39.0 ± 3.1; Het control, 31.4 ± 2.5; Het enrich, 39.0 ± 2.7; Homo control, 27.8 ± 3.3; and Homo enrich, 34.8 ± 2.9. Their preference scores for the target quadrant over other quadrants for control/enriched mice were 1.4 ± 0.1/2.3 ± 0.4, 1.5 ± 0.2/2.1 ± 0.3, and 1.4 ± 0.2/1.8 ± 0.3 for Ng+/+, Ng+/−, and Ng−/− mice, respectively (a value of 1 signified no preference). Enriched mice seemed to show more preference over their corresponding control mice; however, no significant difference was observed among various genotypes in control or enriched group by t-test comparison.

Figure 2.

Effect of environmental enrichment on the step-down fear conditioning. During conditioning trial, these aged mice were very reluctant to step down and were allowed to remain on platform maximally for 15 min during testing 24 h later. Retention times for control/enrich during conditioning trials were 142.6 ± 37.3/172.6 ± 19.9, 68.2 ± 13.0/180.3 ± 26.0, and 59.6 ± 17.1/147.2 ± 20.1 for Ng+/+, Ng+/−, and Ng−/− mice, respectively; those during testing were, respectively, 674.8 ± 56.7/693.0 ± 52.8, 487.8 ± 71.2/628.7 ± 51.2, and 181.5 ± 45.3/459.2 ± 51.5. All the retention times during testing were significantly different from those during conditioning. Retention times for enriched Ng+/− and Ng−/− were also significantly different from their control both during trial and testing. However, retention times for enriched Ng+/+ mice were not different from control during either trial or testing, and that of enriched Ng+/− showed similar differences in retention time with control in both trial and testing. Thus, the enrichment protocol did not benefit Ng+/+ mice, but exhibited a significant improvement for Ng−/− mice and a positive tendency for Ng+/− mice.

Figure 3.

Performances in radial arm maze as affected by environmental enrichment. Mice were initially habituated in the maze with baits abounding and were trained with four baits constantly at arm number 1, 2, 4, and 7. There were two training trials per day for consecutive 10 d. (A) Progression of time required to retrieve all four baits or if 3 min had elapsed throughout 20 training trials. Averaged times for the last five trials among the various groups of mice were as follows: Wt control, 141.0 ± 2.2 sec; and Wt enrich, 127.2 ± 1.4 sec (Ab); Het control, 143.4 ± 1.8 sec; and Het enrich, 132.7 ± 1.6 sec (Ac); Homo control, 146.9 ± 2.4 sec; and Homo enrich, 129.6 ± 2.6 sec (Ad). Control mice of all three genotypes were similar in their performances (Aa). Enrichment group of each genotype improved their performances significantly over their respective controls with P < 0.001 for all (Ab,c,d). (B) Percentage reference memory errors averaged from the last five trials were as follows: Wt control, 51.7 ± 0.3; Wt enrich, 49.0 ± 1.5; Het control, 50.8 ± 0.3; Het enrich, 54.1 ± 0.5; Homo control, 60.7 ± 0.9; and Homo enrich, 58.1 ± 0.9. Both Wt and Het control mice made significantly less error than that of Homo control with P < 0.001 (Ba). The enrichment protocol did not help these aged mice of any genotype in making less reference memory error (Bb,c,d). (C) Percentage working memory errors averaged from the last five trials were as following: Wt control, 21.3 ± 0.9; Wt enrich, 18.9 ± 1.1; Het control, 21.1 ± 0.4; Het enrich, 21.6 ± 0.6, Homo control, 25.7 ± 0.4; and Homo enrich, 20.7 ± 1.2. Homo control mice made significantly more errors than those of Wt and Het control with P < 0.001 (Ca). After the enrichment, enriched Wt and Het mice did not improve any of their working memory (Cb, Cc), whereas enriched Ng−/− mice made less error compared to their control (P < 0.001) and performed as well as Ng+/+ and Ng+/− mice (Cd).

Figure 4.

Immunoblot analyses for the hippocampal levels of Ng, αCaMKII, CREB, and ERK in the control and enriched mice. The lower portion of nitrocellulose membrane with transferred proteins was analyzed for Ng, and the top portion was analyzed for αCaMKII, CREB, ERK, and actin consecutively after each stripping. (A) Representative immunoblots (one of three) comparing the levels of protein expression in adult Wt mice to those in aged mice of various genotypes. (B) Each set represented one of three immunoblots comparing the levels of protein expression in control and enriched aging Wt, Het, and Hom mice. (C) Bar representations showing relative level of expression in the aging mice after normalizing with actin expression in comparing against adult Wt control. Aging +/− mice expressed only 47.8 ± 2.4% and aging Wt expressed comparable level of Ng comparing to adult Wt. Expressions of αCaMKII and p42 ERK in the aging mice of all genotypes were not significantly different from that of the adult. However, CREB expressions in the aging mice, 80.2 ± 2.3%, 78.8 ± 3.1%, and 85.5 ± 5.6%, respectively, for +/+, +/−, and −/− mice were significantly reduced from that of the adult mice. (D) Bar representations comparing protein expression in enriched mice to those of their respective control mice after normalizing with actin expression. Both enriched +/+ and +/− mice expressed significantly more Ng than their controls. Significant increases in CREB (P < 0.001) were also observed for all enriched mice compared to their controls. However, expressions of αCaMKII and p42 ERK in the enriched mice were not significantly different from their controls.

Figure 5.

Effect of environmental enrichment on LTP in the hippocampal CA1 region. LTP was induced by HFS (100 Hz for 1 sec), and the fEPSP at the CA1 region was recorded for 60 min. The slope of the fEPSP was determined and the steady-state levels of responses among aged Ng+/+ (Wt), Ng+/− (Het), and Ng−/− (Homo) as well as adult (Wt Ad) during the last 10-min blocks were as following (percentage of mean ± SEM of baseline): Wt Ad control, 143.4 ± 1.3%; Wt control, 132.0 ± 0.6%; Wt enrich, 140.0 ± 1.4%; Het control, 118.9 ± 1.1%; Het enrich, 128.6 ± 0.6%; Homo control, 102.1 ± 1.2%; and Homo enrich, 105.6 ± 0.9%. One-way ANOVA pairwise comparisons by Dunn’s method and t-test showed that adult control vs. aged Wt control, Wt control vs. Het control, and both Wt and Het controls vs. Homo control were all significantly different with P < 0.001. Wt and Het enriched vs. their corresponding controls were also significantly different with P < 0.005 and P < 0.001, respectively. Enriched aging Wt achieved almost as good as that of adult control. Both control and enriched Ng−/− showed no LTP under the condition of HFS and they were not significantly different from each other. Representative traces of the fEPSP before (1) and 60 min after (2) HFS are shown with calibration, 1 mV and 5 msec.