Characterization of Apathy-Like Behaviors in Mouse Models of Down Syndrome

Abstract

Background:

Apathy is a state of decreased interest, lack of initiative, reduced goal-directed activity and blunted emotional responses. Apathy is one of the most common neuropsychiatric symptoms (NPS) in patients with Alzheimer’s disease (AD) and is also relatively omnipresent in individuals with Down syndrome (DS). Little is known about the apathy-like behaviors in rodent models of AD and DS.

Objective:

This study aimed to characterize apathy-like behaviors with aging in two established DS mouse models: Ts65Dn and Dp16.

Methods:

A battery of behavioral tests including nestlet shredding, marble burying, nest building, and burrowing were performed to examine apathy-like behaviors. Individual z-scores for each mouse for each test, and a composite z-score of apathy-like behavior were analyzed for all mice from these behavioral tests.

Results:

Analysis of individual test results and composite z-score revealed significant apathy-like behaviors in Ts65Dn mice compared to WT controls. In contrast, Dp16 mice did not exhibit significant apathy-like behaviors.

Conclusions:

Our study is the first to characterize apathy-like behaviors in mouse models of DS with aging and highlights the difference between Ts65Dn and Dp16 DS model mice regarding apathy-like manifestations with aging.

Keywords

Alzheimer’s disease apathy-like behavior Down syndrome Dp16 mouse model Ts65Dn

INTRODUCTION

Down syndrome (DS) is the most common cause of intellectual and developmental disabilities, and is caused by total or partial triplication of human chromosome 21 that produces global alterations in gene expression patterns, resulting in a wide array of pathological and clinical manifestations. 1 Over the past few decades, life expectancy for people with DS is rapidly increasing and consequently DS people are at greater risk of developing conditions associated with aging. Notably, majority of DS patients in their 40 s and 50 s develop aging-related dementia syndromes and brain pathologies resembling Alzheimer’s disease (AD). 2 Besides dementia, neuropsychiatric symptoms (NPS) are core features of AD and DS patients. Growing evidence has indicated that apathy, characterized by reduced goal-directed activity and blunted emotional responses, is relatively omnipresent in individuals with AD and DS. Apathy is associated with greater cognitive decline, increased reliance on caregivers to initiate activities, decreased quality of life and increased mortality. 3 Apathetic symptoms in DS individuals at adult age could be an early indicator for the development of AD. 4 Others also found more apathetic symptoms (e.g., loss of interest, social isolation and increased fatigue in daily tasks) in older non-demented DS individuals compared to younger ones, which suggest that apathy is possibly related to normal aging in DS.5,6, 5,6 Currently there are no effective treatments that improve apathy and other NPS in individuals with DS.7 –10

The development of mouse models of DS, involving trisomy of all or part of human chromosome 21 or orthologous mouse genomic regions, are providing important tools for understanding of disease mechanisms and accordingly identification of potential therapeutic targets. Meanwhile, research with DS animal models mainly focus on cognitive symptoms, with NPS including apathy-like behaviors rarely examined.11,12, 11,12 In this study, we characterized apathy-like behaviors in two established DS mouse models, Ts65Dn 13 and Dp16 14 mice at 12–14 months of age, which is equivalent to middle-aged human subjects around 40 years and older.15,16, 15,16 While there is no “gold standard” behavioral test to study apathy, recent studies have proposed using rodent-typical behaviors such as nestlet shredding, 17 marble burying, 17 nest building,18,19, 18,19 and burrowing 20 to model lack of interest and goal-directed behavior in mice. 21 Moreover, these tasks are low-stress and do not rely on learned behavior or memory, thus greatly reducing the confounding factors (related to learning and memory abilities) for interpretation of the experimental results.

MATERIALS AND METHODS

Mice

All mice were housed at Wake Forest School of Medicine Animal Facility. Mice were kept in compliance with the National Institute of Health (NIH) Guide for Care and Use of Laboratory Animals. The facility kept a 12-h light/dark cycle with regular feeding, cage cleaning, and 24-h access to water. Breeder mice were purchased from Jackson Laboratory. The Ts65Dn mouse colony was maintained by crossing Ts65Dn trisomic females (005252) with B6EiC3Sn.BliAF1 males (003647). Ts65Dn mice used in behavioral experiments did not carry the phosphodiesterase 6b (Pde6b) gene mutation associated with retinal degeneration. Dp(16)1Yey (Dp16) mouse colony was maintained by crossing Dp16 heterozygous males (JAX stock # 013530) with C57BL/6J wild type (WT) females (JAX stock # 000664). Genotyping for all mice was determined by standard PCR protocols provided by Jackson Laboratory. All experiments were conducted on male and female 12– 14-month-old mice. For all behavioral test, mice were handled for 5 days before behavioral testing and habituated for at least 1 h prior to experiments and animals were tested by an experimenter blind to genotype. Evaluations were made during the light phase and all animal experiments were performed in accordance with the approval of the Institutional Animal Care and Use Committee at Wake Forest University.

Nestlet shredding

All mice were habituated in the testing room for 1 h prior to start of testing. Group-housed mice were placed individually into a clean mouse cage with bedding and one piece of cotton fiber nestlet (5 cm×5 cm, 5 mm thick) that was pre-weighed and placed on top of the bedding in each cage. Each mouse was left undisturbed in the cage with the nestlet for 30 min. After the test, the nestlet was left in open space overnight to dry and was weighed again. The last weight and the starting weight were used to calculate percentage of nestlet shredded, a smaller number will indicate more severe apathy-like behavior. 17

Marble burying

Group-housed mice were habituated in the testing room 1 h before the test. A standard rat cage (26 cm×48 cm×20 cm) was used with 5 cm layer of unscented mouse bedding material. 15 standard glass toy marbles (assorted styles and colors/15 mm diameter, 5.2 g in weight) were gently put on the surface of the bedding in 3 rows of 5 marbles. Each mouse was gently placed in the cage in a corner away from marbles and allowed to behave freely, undisturbed for 30 min. The percent of total marble volume buried for each marble was recorded and the number of those with >2/3 total volume buried were counted as buried. Fewer marbles buried indicates more severe apathy-like behavior. 17

Nest building

Nest building was performed after the nestlet shredding and marble burying tests. The group-housed mice were habituated with a cotton fiber nestlet (5 cm×5 cm, 5 mm thick) in their home-cage overnight before the day of testing. On the test day, mice were individually-housed in a new cage with fresh, unscented bedding with a piece of clean and pre-weighed nestlet. The mice were allowed to behave freely overnight and the nests were evaluated the following morning. The nest from each mouse was photographed and the nests were graded using scores ranging from 1 (very poor/no nest building) to 5 (optimal nest building) with half-point scores for nests falling between categories according to a previously published scoring scheme by Deacon. 19 Similar to the nestlet shredding test, the amount of nestlet left unshredded was weighed and used to calculate the percentage of unshredded nestlet. A lower nest score and a higher percentage of unshredded nestlet indicates more severe apathy-like behavior.

Burrowing

Group-housed mice were habituated with a burrowing tube filled with food pellets in their home-cage overnight before the day of testing. On the testing day, the mice were individually-housed in a new cage with fresh bedding. A clean burrowing tube was filled with 200 grams of food pellets and placed in each separate cage. For the 2-h burrowing test, the filled burrowing tube was placed in the cage around 2:00– 3:00 pm and the mouse was allowed to behave freely for 2 h. Following the 2-h period, the amount of food left in the tube was weighed and used to calculate the amount burrowed. For the overnight burrowing test, the burrow tube was emptied and refilled with 200 g of new food pellets and placed back in the cage overnight. On the following morning, the amount of food left in the tube was weighed and used to calculate the amount burrowed.20,22, 20,22 Smaller amount burrowed will indicate greater apathy-like behavior.

Open field

Test was performed following a procedure as we previously reported.23,24, 23,24 Briefly, animals were placed in an opaque plastic open field chamber (40×40×40 cm) and permitted to freely explore for 15 min. Time spent in the center and periphery of the chamber were measured and calculated as percentage of the total time. Velocity and distance moved were assessed using EthoVision XT Tracking Software (Noldus Information Technology). Data collection and analysis were performed blinded to genotype.

Composite scores of apathy-like behaviors

Z-scores were calculated for nest shredding, marble burying, nest building (% unshredded and nest building score), 2-h burrowing, and overnight burrowing for each mouse relative to the mean and standard deviation (SD) of WT controls so that changes of±1 z-score unit represented±1 SD of the control sample’s behavior. To reduce the variance and enhance the reliability of our data, we further created a composite score (i.e., a combined z-score for each mouse across all tests). The raw z-scores were corrected so that a higher value was indicative of greater apathy. The composite score for apathy-like behaviors was calculated using the following equation: (z_i (NS) + z_i (MB) + z_i (NB % unshredded) + z_i (NB score) + z_i (2 h burrowing) + z_i (overnight burrowing))/6, which will indicate overall apathy-like behavior severity.21,25, 21,25

Data analysis and statistics

Data were presented as mean±SEM. Summary data are presented as group means with SE bars. Normality of the data were tested using GraphPad Prism (GraphPad Software), and the criteria for parametric testing were met. For comparisons between two groups, a two-tailed independent Student’s t test was performed using GraphPad Prism 10 software. Error probabilities of p < 0.05 were considered statistically significant.

RESULTS

Ts65Dn DS model mice showed apathy-like behaviors

Analysis of nestlet shredding (NS) test revealed that the Ts65Dn mice shredded about twice the amount as WT mice did in 30 min in average (9.1±1.9% in Ts65Dn versus 4.1±1.3% in WT) and we observed a trend towards significant difference (p = 0.051, Fig. 1A). There was no significant difference between the Ts65Dn and WT mice for the marble burying (MB) test, with average 4.1±1.0 and 5.5±1.2 buried marbles for WT and Ts65Dn mice, respectively (Fig. 1B). For the overnight nest building (NB) test, we calculated the percentage of unshredded nestlet and the final nest score based on unshredded amount and the shape of the nest. 19 Strikingly, Ts65Dn mice showed a significant (p < 0.001) higher percentage of unshredded nestlet (Fig. 1C) and a significant (p < 0.001) lower nest building score compared to the WT controls (Fig. 1D). For the 2 h burrowing tests, the Ts65Dn mice burrowed significantly (p < 0.001) less amount of the food pellet compared to WT mice (Fig. 1E). Consistently, Ts65Dn mice had significantly reduced food pellet burrowing in the overnight burrowing test compared to the performance of WT mice (p < 0.001), although Ts65Dn mice appeared to have increased amount of food pellet burrowing compared to the 2 h burrowing best (Fig. 1F). We calculated the z-score for each individual behavioral test and the results were consistent with the analysis described above (Fig. 1 panels below original behavioral analysis). Finally, we analyzed the composite z-score combing raw z score of all 6 behavioral tests (see method) and found significant higher z-score (p < 0.001) in Ts65Dn mice compared to WT mice (Fig. 1G). Additionally, we performed open field (OF) test and did not observe significant difference in locomotor activities (as measured by travel distance and velocity) between Ts65Dn and WT mice (Supplementary Figure 1A, B). Taken together, middle-aged Ts65Dn mice exhibit apathy-like behaviors.

Fig. 1

Apathy-like behaviors in WT and Ts65Dn mice. A) Ts65Dn mice showed a trend of decreased percentage of nestlet shredded in 30 min than WT mice in nestlet shredding (NS) test. B) No difference in numbers of marbles with more than 2/3 total volume buried in marble burying (MB) test. C) Ts65Dn mice showed a significant higher level of unshredded cotton nestlet pad in overnight nest building (NB) test. D) Ts65Dn mice showed a significant lower score of nest building in overnight nest building (NB) test. E) Ts65Dn mice showed a significantly lower burrowing activity at 2 h. F) Ts65Dn mice showed a significantly lower overnight burrowing activity. G) Ts65Dn mice showed significantly higher combined z-score that WT control mice. ^***p < 0.001. n = 8, 8, respectively for WT and Dp16 mice with mixed male and female. Individual z-score of each test is shown in lower panel accordingly.

Dp16 DS model mice did not exhibit apathy-like behaviors

We performed the same battery of behavioral tests for assessment of apathy-like behavior in Dp16 DS model mice. 14 Surprisingly, in all behavioral tests performed, we did not detect any significant differences between Dp16 mice and WT control mice (Fig. 2). Consistently, analysis of individual and composite z-score did not reveal any apathy-like behavioral phenotypes in Dp16 mice (Fig. 2G). In OF test, we did not observe difference between WT and Dp16 mice in travel distance or average velocity (Supplementary Figure 1C, D), indicating normal locomotor function. Thus, middle-aged Dp16 mice do not exhibit apathy-like behaviors.

Fig. 2

Apathy-like behavior tests in WT and Dp16 mice. A) No difference in percentage of nestlet shredded in 30 min in nestlet shredding (NS) test. B) No difference in numbers of marbles with more than 2/3 of total volume buried in marble burying (MB) test. C) No difference in level of unshredded cotton nestlet pad in overnight nest building (NB) test. D) No difference in nest building score between WT and Dp16 mice in overnight nest building (NB) test. E) No difference in burrowing activity at 2 h. F) No difference in overnight burrowing activity. G) There were no significant difference in combined z-score between Dp16 and WT mice. n = 17, 12, respectively, for WT and Dp16 mice with mixed male and female. Individual z-score of each test is shown in lower panel accordingly.

DISCUSSION

Apathy is considered as a prodromal feature of dementia in DS and may be an early indicator of cognitive decline and dementia in DS patients.10,26, 10,26 In this study, we characterized and compared two well-established and widely used DS model mice (Ts65Dn and Dp16) 12 for their apathy-like behaviors. To our knowledge, this is the first time apathy-like behaviors have been examined in DS model mice. Our findings indicate that Ts65Dn mice showed reliable apathy-like behaviors at middle age. In contrast, middle aged Dp16 mice did not show any apparent signs of apathy-like behaviors.

Many neuropsychiatric symptoms, including apathy-like behaviors, remain difficult and challenging to model and evaluate in animals. Since there are no definitive behavioral tests on apathy-like behaviors in mice, especially in DS model mice, we chose to perform a series of different behavioral tests and generate a composite score to evaluate the overall apathy-like behaviors. A similar study using these behavioral tests was recently carried out in an AD mouse model. 21 It is important to note that these tests have traditionally been used to assess other neuropsychiatric behaviors. For instance, nestlet shredding and marble burying tests have previously been used to evaluate compulsive-like and repetitive behaviors. 17 These tests may also be used as a measure of general animal welfare as healthy mice are motivated and interested in completing these tasks. 20 We believe that by performing a battery of tests and the addition of a composite z-score increase the validity of using these tests as a measure of apathy-like behaviors. Indeed, our data confirmed the effectiveness of this composite score method and further indicated the usefulness of these behavior tasks in assessing apathy-like behavior in Ts65Dn and Dp16 DS model mice. Although previous study reported the limitations of marble burying test showing that this test is more related to digging and burrowing in rodents,27,28, 27,28 it was recently used to analyze apathy-like behavior in an AD mouse model. 21 Our study is the first to use marble burying test together with the other behavioral tests to examine apathy-like behaviors in two different DS model mice. Since this is the first time to test this neuropsychiatric behavior in DS mice, we tried to include a comprehensive analysis using selected tests based on literature. 21 Although marble burying test didn’t show any difference between DS mice and WT mice in our study, the overall composite z-score still showed significant difference between Ts65Dn mice and WT mice.

In animals, specific symptoms of apathy-like behavior have been modelled including goal-directed or nest-building behavior and these are seen as indicative of proxies for motivation and daily activities. Nest building can be used for assessing moods and behaviors associated with psychiatric disorders such as depression and schizophrenia. It can also be useful for testing movement disorders such as Parkinson’s disease. 19 Importantly, it was used for testing apathy-like behaviors in several different AD mouse models, including 5xFAD 21 and a tau mouse model of AD. 29 The fact that Ts65Dn mice showed positive apathy-like behavior test results in our nest building test, together with its successful uses in AD model mice, strongly indicate that nest building test is an efficient behavioral test to determine apathy-like behaviors in rodent models. Burrowing in rodents is a sensitive method for detecting behavioral function22,30, 22,30 and it is commonly used for evaluating well-being in mice. 20 It was also recently used for apathy-like behavioral test in a 5xFAD mice model. 21 Notably, the 2 h measure of burrowing test is likely to be more sensitive to individual differences compared to the overnight results 22 and the latter measurement may suffer from a ceiling effect, as many animals will burrow the entire tube contents. Since the apathetic mice are predicted to burrow less compared to the control WT mice, we do not expect to see any significant ceiling effects in these specific tests in both DS model mice. Including both 2 h and overnight burrowing tests are therefore more comprehensive and may reflect any potential individual difference and time dependent behavioral changes. It turned out that both Ts65Dn and Dp16 showed big variance among individual mice in 2 h burrowing test while Ts65Dn mice presented consistent decreased burrowing activity at 2 h and overnight. In contrast, burrowing activities (measured at either 2 h or overnight) were unaltered in Dp16 mice. Increased digging (burrowing) is usually recognized as repetitive behavior due to anxiety- or compulsive-like responses. The reduction of these behaviors can be attributed to various factors. For example, it may be induced by neuropsychological stress or pain. On the other hand, it may be induced by the impaired locomotor activity. We did not observe any changes in the distance moved or average velocity during the open field test (Supplementary Figure 1), indicating normal locomotor ability in both DS model mice.31,32, 31,32 Our data further indicated that reduced burrowing activity in DS mice, especially middle aged Ts65Dn mice is reflective of apathy-like behavior. Representative graphs of the experimental results of nest building test are shown in Supplementary Figure 2. Overall, by performing several different behavioral tests and using a composite score method, we showed that Ts65Dn mice exhibit more reliable apathy-like behavioral deficits with aging compared to the Dp16 mice.

The Ts65Dn and DP16 mice tested in this study were about 12– 14 months old equaling to middle age of human beings. Approximately 40– 80% of persons with DS develop AD-like dementia in their 40 s and 50s15,16, 15,16 and apathy could be an early indicator of cognitive decline and dementia in the DS. It is known that the brains of DS patients share some changes similar to those seen in patients with AD.33 –35 For example, amyloid-β levels are elevated in DS patients throughout life and lowering the amyloid-β levels can alleviate learning and memory deficits in Ts65Dn mice. 36 It is therefore also possible that the manifestation of apathy-like behaviors in DS mice depends on the brain pathophysiology conditions, e.g., amyloid-β levels 21 and tau phosphorylation, 29 which in AD mice have been shown to affect the degree of apathy-like behaviors in previous studies. To the best of our knowledge, none of the existing DS model mice develops aging-related AD brain pathology, i.e., Aβ plaques and tau tangles.37,38, 37,38 Future in-depth studies on brain region-specific pathological changes and molecular biology phenotypes in Ts65Dn mice that are associated with apathy-like behaviors may provide insights into the potential early manifestation of neuropsychiatric behavioral alterations in DS mice and guide future treatment approaches. For instance, previous neuroanatomy study has revealed that apathy is strongly associated with disruption particularly of dorsal anterior cingulate cortex, ventral striatum and connected brain regions and these changes are consistent across clinical disorders and imaging modalities. 39 For DS model mice, it is known that there are widespread and unexpected fundamental differences in behavioral, gene expression and brain development phenotypes between Ts65Dn and Dp16 mice. 40 For example, embryonic and adult gene expression results showed that Ts65Dn brains had considerably more differentially expressed (DEX) genes compared with Dp16 mice. In addition, DEX genes showed little overlap in identity and chromosomal distribution in the two DS models, leading to dissimilarities in affected functional pathways. Perinatal and adult behavioral testing also highlighted differences between the models in their abilities to achieve various developmental milestones and perform hippocampal- and motor-based tasks. Interestingly, Dp16 mice showed no abnormalities in prenatal brain phenotypes, yet they manifested behavioral deficits starting at postnatal day 15 that continued through adulthood. 41 For apathy-like behavior, our findings consistently illustrate unique limitations of each model when studying aspects of brain development and function in DS. It also informs on model selection in future studies investigating how observed neurodevelopmental abnormalities arise, how they contribute to neural behavioral impairment, and when testing therapeutic molecules to ameliorate the intellectual disability associated with DS. Dp16 mice are known to have more aggressive cognitive deficits and synaptic plasticity impairments. 42 Based on literature and our own findings, 43 Ts65Dn (9–12 months old) and Dp16 mice (6– 9 months old) reliably display age-dependent impairments in cognitive function and synaptic plasticity. It is worth mentioning that recent studies indicate phenotype drift and variations in Ts65Dn mice linked to factors such as genetic background and resources of the mouse lines,44,45, 44,45 yet whether these are associated with the apathy-like behaviors detected in Ts65Dn mice still need to be determined. Future studies should also incorporate molecular biology phenotypes which may help elucidating underlying mechanisms associated with related behavioral phenotypes.

It has been indicated that DS occurs more among males than females 46 and male patients display more severe deficits in physical fitness and motor skills. 47 Sex differences (male/female predominant changes) for Dp16 or Ts65Dn may also exist in their apathy-like behaviors. To support this, strain and sex based differences in marble burying behavior have previously been reported. 48 It was also reported that digging is a nuanced motivated behavior, and male and female rodents may perform it differently. 49 The mechanism underlying the sex differences in DS prevalence and development is still unclear, neither is the mechanisms underlying the apathy behavior. We divided the experimental mice by sexes and re-analyzed the combined apathy-like z-score between WT mice and DS mice within each sex group. The results, based on overall z-score analysis, did not reveal sex-based differences in apathy-like behavior (Supplementary Figure 3). Larger number of sample size may help determine any sex difference in the combined z-score and/or individual behavioral test. Moreover, risk factors related to biological sex differences including genetic factor, sex hormones and brain structure deviations between males and females should be put into consideration for future investigation of the topic.

In summary, by performing combined behavioral tests that are usually low-stress and do not rely on learned behavior or memory, we characterized apathy-like behaviors in two different DS model mice at middle age in this study. Ts65Dn mice exhibit apathy-like behavioral phenotype, while Dp16 mice do not. It would be premature to directly correlate apathy-like behavior with pathological progression during aging although both DS mice are known to have cognitive impairment at this age tested in the current study. Therefore, whether and how the apathy-like behavior is associated with the DS pathological and molecular changes in mice still need to be determined and future studies may focus on more in-depth causative mechanism that contributes to the apathy-like behavioral phenotype in different DS mice.

AUTHOR CONTRIBUTIONS

Tao Ma (Conceptualization; Funding acquisition; Investigation; Project administration; Resources; Supervision; Writing – review & editing); Tan Zhang (Formal analysis; Investigation; Project administration; Writing – original draft; Writing – review & editing); Xin Wang (Formal analysis; Investigation; Writing – review & editing); Hannah M. Jester (Formal analysis; Investigation; Writing – review & editing); Xueyan Zhou (Investigation; Methodology; Project administration; Writing – review & editing).

Footnotes

ACKNOWLEDGMENTS

The authors have no acknowledgments to report.

FUNDING

This work was supported by National Institutes of Health grants R01 AG055581, R01 AG056622, and R01 AG073823 (T.M.).

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

Tao Ma is an associate editor of Journal of Alzheimer’s disease but was not involved in the peer-review process of this article nor had access to any information regarding its peer-review.

DATA AVAILABILITY

The data supporting the findings of this study are available on request from the corresponding authors.

The supplementary material is available in the electronic version of this article: .

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