Age-related changes in mice behavior and the contribution of lipocalin-2

Abstract

Aging causes considerable changes in the nervous system, inducing progressive and long-lasting loss of physiological integrity and synaptic plasticity, leading to impaired brain functioning. These age-related changes quite often culminate in behavioral dysfunctions, such as impaired cognition, which can ultimately result in various forms of neurodegenerative disorders. Still, little is known regarding the effects of aging on behavior. Moreover, the identification of factors involved in regenerative plasticity, in both the young and aged brain, is scarce but crucial from a regenerative point of view and for our understanding on the mechanisms that control the process of normal aging. Recently, we have identified the iron-trafficking protein lipocalin-2 (LCN2) as novel regulator of animal behavior and neuronal plasticity in the young adult brain. On the other hand, others have proposed LCN2 as a biological marker for disease progression in neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis. Still, and even though LCN2 is well accepted as a regulator of neural processes in the healthy and diseased brain, its contribution in the process of normal aging is not known. Here, we performed a broad analysis on the effects of aging in mice behavior, from young adulthood to middle and late ages (2-, 12-, and 18-months of age), and in the absence of LCN2. Significant behavioral differences between aging groups were observed in all the dimensions analyzed and, in mice deficient in LCN2, aging mainly reduced anxiety, while sustained depressive-like behavior observed at younger ages. These behavioral changes imposed by age were further accompanied by a significant decrease in cell survival and neuronal differentiation at the hippocampus. Our results provide insights into the role of LCN2 in the neurobiological processes underlying brain function and behavior attributed to age-related changes.

Keywords: aging; behavior; cell survival; hippocampus; lipocalin-2.

Figure 1

Body weight increases in aged animals, irrespectively of the genotype. (A) Schematic diagram of the experimental approach and the group of animals used at the respective ages. (B) Body weight measurements at selected ages revealed an age-related effect, with aged animals presenting increased body weight, when compared to genotype-matched 2-months old animals (n = 10–12 mice per group). Data are presented as mean ± SEM, analyzed by two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. ^ΦDenotes differences between young and aged Wt; ^#between young and aged lipocalin-2 (LCN2)-null mice. ^{ΦΦΦΦ, ####}p ≤ 0.0001. EPM, elevated plus maze; CFC, contextual fear conditioning; FST, forced-swim test; MWM, Morris water maze.

Figure 2

Age-related alterations of anxiety-like behaviors in Wt and lipocalin-2 (LCN2)-null mice. (A) Assessment of anxiety-like behavior in the elevated plus maze (EPM) showed an increase in the percentage of time spent in the open arms across age, specifically in LCN2-null mice (n = 10–12 mice per group). (B) General locomotor activity assessed by the total number of entries in the maze revealed no major aging and genotype effects. (C) In the light/dark box test, anxiety-like behavior of LCN2-null mice at 2-months of old is reduced at 18-months of age. (D) Representative progression of EPM performance by the animals during the course of normal aging, evidencing the reduction in anxiety behavior (as an increased time in the EPM open arms) observed in LCN2-null mice. At 12-months of age, Wt mice become more anxious, a phenotype that was lost at 18-months. Data are presented as mean ± SEM, analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. ^#Denotes differences between young and aged LCN2-null mice; *between Wt and LCN2-null mice at each matched age. ^*p ≤ 0.05, ^##p ≤ 0.01, ^####p ≤ 0.0001.

Figure 3

Wt and lipocalin-2 (LCN2)-null mice depressive-like behavior in the course of aging. (A) Depressive-like behavior evaluated as learned helplessness in the forced-swim test (FST) revealed decreased immobility time in older Wt and LCN2-null mice, with null aged mice continuing to present a depressive-like behavior (n = 10–12 mice per group). (B) Mood alterations in older mice were also observed by the latency of time to immobility. (C) Depressive-like behavior in the progress of aging remains in LCN2-null mice, compared to controls, while older Wt animals decreased their immobility time. Data are presented as mean ± SEM, analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. ^ΦDenotes differences between young and aged Wt mice; ^#between differences between young and aged LCN2-null mice; *between Wt and LCN2-null mice at each matched age. ^*p ≤ 0.05, ^##p ≤ 0.01, ^***p ≤ 0.001, ^{ΦΦΦΦ, ####, ****}p ≤ 0.0001.

Figure 4

Spatial learning and memory retrieval and consolidation decreases during normal aging. (A) Analysis of spatial learning and memory in the Morris water maze (MWM) test revealed that all groups learned to find the position of the hidden platform across the 4 days of acquisition. (B) Older Wt mice required more time to find the platform, while lipocalin-2 (LCN2)-null animals presented similar impaired learning curves throughout aging (n = 10–12 mice per group). (C) Cognitive deficits in the course of normal aging, measured by the reduced latency of time in the MWM. (D) Freezing behavior upon re-exposure to conditioning context revealed a major effect of aging in Wt animals, as freezing behavior decreased in older animals. (E) Impaired memory retrieval and consolidation was confirmed by the decreased ratio of discrimination index in aged Wt mice, which was sustained in LCN2-null mice throughout aging (n = 10–12 mice per group). (F) Impaired contextual discrimination along aging in Wt and LCN2-null mice. Data are presented as mean ± SEM, analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. ^ΦDenotes differences between young and aged Wt mice; ^#between differences between young and aged LCN2-null mice; ^*between Wt and LCN2-null mice at each matched age. ^{Φ, #}p ≤ 0.05, ^ΦΦΦΦp ≤ 0.0001, ^***p ≤ 0.001.

Figure 5

Aging reduces hippocampal neurogenesis in both Wt and lipocalin-2 (LCN2)-null animals. (A) Schematic diagram of the BrdU protocol used to label cell survival and neuronal differentiation. (B) Quantification of total number of BrdU⁺ cells in the DG of Wt and LCN2-null mice disclosed a significant effect of aging in cell survival (n = 4–5 mice per group). (C) Percentage of newly born neurons are significantly affected by aging, independently of animal’s genotype. (D) Representative images of BrdU and NeuN staining in the DG of Wt and LCN2-null mice. Data are presented as mean ± SEM, analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. ^ΦDenotes differences between young and aged Wt mice; #between differences between young and aged LCN2-null mice; ^*between Wt and LCN2-null mice at each matched age. ^#,*p ≤ 0.05, ^**p ≤ 0.01, ^ΦΦΦp ≤ 0.001; ^{ΦΦΦΦ, ####}p ≤ 0.0001. IHC, immunohistochemistry.

Figure 6

Schematic representation on the progression of the behavioral dimensions assessed throughout aging, in both Wt and lipocalin-2 (LCN2)-null mice. Comparison of behavioral performances of aged animals with younger ages revealed that aging, in Wt mice, slightly decreased their anxiety state at 18-months (as observed by the increased open arms time), but did not promote depressive-like behaviors (immobility time is decreased). Cognitive domains, both spatial learning and contextual discrimination, was significantly impaired with aging. On the other hand, aged LCN2-null mice sustained their impaired behavior observed already at 2-months of age, specifically in depressive-like behavior and cognitive domains, with the exception of anxiety that was significantly decreased (represented as the increased in the time spent in the open arms).