biomedicines Article Sex-Dependent Signatures, Time Frames and Longitudinal Fine-Tuning of the Marble Burying Test in Normal and AD-Pathological Aging Mice Mikel Santana-Santana 1,2 , José-Ramón Bayascas 1,3 and Lydia Giménez-Llort 1,2, * 1 Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; mikel.santana@e-campus.uab.cat (M.S.-S.); joseramon.bayascas@uab.cat (J.-R.B.) 2 Department of Psychiatry and Forensic Medicine, School of Medicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain * Correspondence: lidia.gimenez@uab.cat Abstract: The marble burying (MB) test, a classical test based on the natural tendency of rodents to dig in diverse substrates and to bury small objects, is sensitive to some intrinsic and extrinsic factors. Here, under emerging neuroethological quantitative and qualitative analysis, the MB performance of 12-month-old male and female 3xTg-AD mice for Alzheimer’s disease and age-matched counterparts of gold-standard C57BL6 strain with normal aging unveiled sex-dependent signatures. In addition,  three temporal analyses, through the (1) time course of the performance, and (2) a repeated test  schedule, identified the optimal time frames and schedules to detect sex- and genotype-dependent Citation: Santana-Santana, M.; differences. Besides, a (3) longitudinal design from 12 to 16 months of age monitored the changes Bayascas, J.-R.; Giménez-Llort, L. in the performance with aging, worsening in AD-mice, and modulation through the repeated test. Sex-Dependent Signatures, Time In summary, the present results allow us to conclude that (1) the marble burying test is responsive Frames and Longitudinal to genotype, sex, aging, and its interactions; (2) the male sex was more sensitive to showing the Fine-Tuning of the Marble Burying AD-phenotype; (3) longitudinal assessment shows a reduction in females with AD pathology; (4) Test in Normal and AD-Pathological burying remains stable in repeated testing; (5) the time-course of marbles burying is useful; and (6) Aging Mice. Biomedicines 2021, 9, 994. burying behavior most likely represents perseverative and/or stereotyped-like behavior rather than https://doi.org/10.3390/ anxiety-like behavior in 3xTg-AD mice. biomedicines9080994 Academic Editor: Masaru Tanaka Keywords: neuroethology; behavioral neuroscience methodology; sexual differences; aging; Alzheimer’s disease; comorbidities; phobia; anxiety; OCD (obsessive-compulsive symptoms) Received: 9 July 2021 Accepted: 6 August 2021 Published: 11 August 2021 1. Introduction Publisher’s Note: MDPI stays neutral The behavioral and psychological symptoms associated with dementia (BPSD), in- with regard to jurisdictional claims in cluding neuropsychiatric symptoms (NPS) such as anxiety and phobias, paranoia and published maps and institutional affil- delusion, hallucinations, stereotypes, and other disturbances, are comorbidities manifested iations. in 50–90% of people with Alzheimer’s disease (AD) [1]. These non-cognitive problems affect their quality of life [2], are an important source of distress for patients and care- givers [3], and frequently lead to premature institutionalization [4]. Furthermore, recent studies suggest a distinct distribution of NPS comorbidities among sexes, and therefore Copyright: © 2021 by the authors. there is a need to characterize these differences, elucidate the underlying pathophysiology, Licensee MDPI, Basel, Switzerland. and identify better treatment targets with a gender perspective [5]. At the translational This article is an open access article level, the modeling of BPSD/NPS in basic and preclinical research of AD under the sex distributed under the terms and perspective is also needed to develop better pharmacological and non-pharmacological conditions of the Creative Commons preventive/therapeutical interventions that could be effectively translated into clinical Attribution (CC BY) license (https:// scenarios. In this context, natural species-typical behaviors representing active interaction creativecommons.org/licenses/by/ with the environment are excellent ethological scenes to reflect the interplay of cognitive 4.0/). Biomedicines 2021, 9, 994. https://doi.org/10.3390/biomedicines9080994 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 994 2 of 16 and non-cognitive disturbances induced by normal and AD-pathological aging. In agree- ment with this, we have proven the validity of the 3xTg-AD mice to assess the impact of the disease on naturally occurring executive functions and daily life activities based on species-typical behaviors when interacting with the environment, such as burying behavior [6], and nest-building [7,8]. Burying behavior is commonly measured with the marble burying (MB) test [9], a classical behavioral test employed in rodents that exploit the tendency of these animals to dig in diverse substrates and to bury small objects, such as glass marbles, in a test cage with beddings [10]. Initially, this test was pharmacologically validated for its use to measure anxiety-related behaviors and screening for anxiolytic pharmacological drugs [11,12]. How- ever, controversy exists regarding its specificity as it is also proposed as modeling meaning- less repetitive and perseverative behaviors mimicking psychotic and obsessive-compulsive (OCD) symptoms [13]. Actually, some authors consider that for MB to be regarded as a reliable screening test for a specific assessment of a neuropsychological construct, the introduction of methodological changes or better experimental designs is needed [13–15]. Thus, two-zone configuration, repeated trials, and limitations inherent to MB score and ceiling/floor effects are among the experimental considerations discussed. Additionally, emerging neuroethological analysis of behavior, which integrates the sequence of behav- ioral events in an ethogram, may provide a better understanding of the functional role, its modulation, and underlying mechanisms than classical behavioral analysis. Marble burying behavior is altered in the 3xTg-AD mice. Specifically, it is enhanced in 12-month-old 3xTg-AD male mice, an age mimicking advanced stages of the disease [16,17], can be reversed by risperidone, and be modulated by handling [6,18]. In addition, we have recently proven that at 15-months of age, just 2–3 months of naturalistic isolation, which occurs when congeners die, is enough to exacerbate this behavior despite social lives since they were born, modeling the worsening of OCD described in the current COVID-19 scenario [19]. However, there are still various unresolved questions regarding the effect of sex and age factors on marble burying behavior in normal and AD-pathological aging. First, as in the case in other fields with rodent experimentation [20], the inclusion of female mice in MB testing is not the most common choice [21]. The inclusion of females in animal studies of AD is relevant, even if similar incidence between sexes is found, since risk factors may differentially affect multiple pathways and evolve into different manifestations of NPS and comorbidities [5]. Second, how aging and AD-pathological processes affect burying behavior and the MB profile evolves in a long-term perspective. This is a significant concern due to the intrinsic nature of AD, in which cognitive and psychiatric symptoms are present in early stages and worsen over time as the disease progresses [22]. Hence, to increase the translational value of experimental designs, rather than a transversal comparison of the performance at different age stages, longitudinal studies allow monitoring oof the progression of cognitive and non-cognitive deficits through an AD-pathological life-span. Therefore, the present study aimed to explore further the contribution of sex and aging in the normal and AD-pathological brain in marble burying behavior. We used middle- aged 3xTg-AD mice through a longitudinal study including methodological modifications (two-zone configuration, repeated trials, and time-course counting of marbles buried) to have a better approach to the possible neuropsychiatric constructs involved in their alteration, and we compared them with those presented in their non-transgenic (NTg) counterparts with the gold-standard C57BL/6 strain genetic background. 2. Materials and Methods 2.1. Animals A total number of forty-six 12-month-old male and female mice, homozygous 3xTg- AD (males n = 15, females n = 8) and non-transgenic (NTg, males n = 10, females n = 13) mice on a C57BL/6 background (after embryonic transfer and backcrossing at least 10 gen- erations), established in the Universitat Autònoma de Barcelona [23] were used. The 3xTg-AD mice harboring transgenes were genetically engineered at the University of Cali- mice on a C57BL/6 background (after embryonic transfer and backcrossing at least 10 erations), established in the Universitat Autònoma de Barcelona [23] were used. The AD mice harboring transgenes were genetically engineered at the University of Calif Biomedicines 2021, 9, 994 Irvine, as previously described [16]. Animals were maintained in groups3 of of 163–4 mic cage (Macrolon, 35 × 35 × 25 cm) filled with 5 cm thick layer of clean woodchips that the same used for behavioral testing (Ecopure, Chips6, DateSand, UK; Uniform cro wood fornia Irvine, as previously described [16]. Animals were maintained in groups of 3–4 mice granules with 2.8–1.0 mm chip size) and nesting materials (Kleenex, Art: 0883 per cage (Macrolon, 35 × 35 × 25 cm) filled with 5 cm thick layer of clean woodchips 21 × 20that cm,were White). the sameAllused animals were maintained for behavioral under testing (Ecopure, standard Chips6, laboratory DateSand, UK; Uniformconditio food and water cross-cut woodad granules libitum,with 22 ±2.8–1.0 2°C,mm12 hchip light: size)dark cycle materials and nesting with lights on atArt: (Kleenex, 8:00 a.m 08834060, 21 × relative humidity 50–60%.20 cm, White). All animals were maintained under standard laboratory ◦ conditions of food and water ad libitum, 22 ± 2 C, 12 h light: dark cycle with lights on at 8:00 a.m., and relative humidity 50–60%. 2.2. Experimental Design 2.2. Experimental Design As illustred in Figure 1, animals were behaviorally assessed at middle-ag As illustred in Figure 1, animals were behaviorally assessed at middle-age (12 months months of of age) age) and and re-tested re-tested fourlater four months months laterreached when they whenoldthey agereached old (16 months age In of age). (16 mon age). In thethe AD-genotype, AD-genotype, these these time time points points correspond correspond to advanced to two different two different advanced stages of the s disease with of the disease the the with progressive development progressive of βA and tau development of pathologies βA and tau [17]. pathologies [17]. Figure 1. Fine-tuning of the marble Figure 1. Fine-tuning burying of the marble test test burying by sex, time by sex, timeframe, test–re-test, frame, test–re-test, and and longitudinal longitudinal assessment. assessment. ExperimentalExperimenta design: a 3-day battery design: a 3-dayofbattery behavioral teststests of behavioral consisting consistingof ofaa corner test corner test on on day day 1, a marble 1, a marble burying burying test on daytest on day 2 (MB1), and a2 (MB1), and a repeated test on day repeated test3on(MB2). day 3 (MB2). 2.3. Behavioral Assessments 2.3. Behavioral Assessments Behavioral assessments in the corner test and marble burying test under dim white Behavioral assessments light (20 lx) were conducted in the the during corner test and light phase marble of the burying light: dark test 10 cycle (from under a.m. dim to 1lx) light (20 p.m.). wereTheconducted tests were performed duringinthea counterbalanced light phase of manner, by direct the light: dark observation cycle (from 10 by a trained observer, blind to the genotype, and a camera’s support. All procedures to 1 p.m.). The tests were performed in a counterbalanced manner, by direct observ by a trained observer, blind to the genotype, and a camera’s support. All procedures Biomedicines 2021, 9, 994 4 of 16 were in accordance with the Spanish legislation on the “Protection of Animals Used for Experimental and Other Scientific Purposes” and the EU Directive (2010/63/UE) on this subject. The protocol CEEAH 3588/DMAH 9452 was approved the 8th of March 2019 by the Departament de Medi Ambient i Habitatge, Generalitat de Catalunya. The study complies with the ARRIVE guidelines developed by the NC3Rs and aims to reduce the number of animals used [24]. Day 1—Corner test (CT) was used to evaluate neophobia. The animal was placed in the center of a clean standard home cage filled with woodchip shave bedding and observed for 30 s. We measured the numbers of corners visited (CTc), the latency to perform the first rearing (CTlatR), and the number of rearings (CTr). The ratio of the number of visited corners and rearings variables (Ratio CTc/r) was calculated. Days 2 and 3—Marble burying test (MB1) and re-test (MB2): Nine ceramic marbles were put in a standard home cage (Macrolon, 35 × 35 × 25 cm3 ) with a 5 cm thick layer of clean woodchips. The marbles were placed evenly spaced (three rows of three) in one- quarter of the cage and allowing the mice to avoid interaction with the marbles. Then, the mouse was introduced in the zone without marbles facing the wall and left to interact with the cage freely. A picture of the cage was taken every 5 min to assess the buried marbles’ progress. After 30 min the mice were gently removed from the cage, and the buried marbles were counted. Marbles were counted as buried when their surface was covered at least 90% with bedding material. The number of marbles buried was transformed in a percentage (MBx.y; x, day, y, time of measurement) for further statistical analysis. Twenty-four hours later, animals repeated the test under the same conditions. 2.4. Statistics Statistical analyses were performed using SPSS 23.0 software. In the corner test, the variables recorded were analyzed by a split-plot design with the factors genotype (G), aging (A), sex (S), according to the experimental design G(2)×A(2)×S(2). ANOVA split-plot de- signs analyzed the number of marbles buried with the factors time (T), genotype (G), aging (A), sex (S), and day (D), according to the experimental design T(7)×G(2)×S(2)×A(2)×D(2). Post-hoc comparisons were run with Bonferroni corrections. Both the F and the degrees of freedom values were reported when it was possible. Spearman correlations were made to analyze behavioral correlates between the CT and the MB. Correlation coefficients (r) are indicated. A p-value < 0.05 was considered as statistically significant. Graphics were made with GraphPad Prism 6. Abbreviation: sexAgeMBday·minute (i.e, m12MB2.30, male at 12 months of age, re-test, 30 min). 3. Results 3.1. Corner Test for Neophobia In the corner test (Figure 2), all the variables were sensitive to the aging factor, as the longitudinal analysis showed the reduction of the number of crossings (A, F(1,42) = 80.104; p < 0.001), the number of rearings (A, F(1,42) = 24.564; p < 0.001), the crossings/rearings ratio (A, F(1,42) = 23.903; p < 0.001), and, conversely, the enhancement of the latency of rearing (A, F(1,42) = 18.085; p < 0.001). Moreover, the crossings/rearings ratio was also sensitive to the genotype and aging interaction (G×A, F(1,42) = 18.085; p < 0.001). delayed elicitation of rearing and a lower number of total rearings. Finally, a reduction with aging in the ratio of crossings/rearings was also presented in NTg-females (p = 0.001), Biomedicines 2021, 9, 994 5 of 16 AD-males (p = 0.021) and WT-males (p = 0.001). The post-hoc analysis of these results is also depicted in Table 1. Figure Figure 2.2.Sex Sex andand ageage effects effects in thein the longitudinal longitudinal assessmentassessment in the cornerintest theincorner test mice with in mice normal andwith normal AD-pathological and aging.AD-pathological aging. (A) Number of crossings (A) (B) (CTc); Number Latency of rearing crossings (CTc); (CTlatR); (C)(B) Latency Number of rearing of rearings (CTlatR); (CTr); (D) (C) Ratio CTc/CTr Number of rearings (CTr); (D) Ratio CTc/CTr (CTratio); Factorial analysis: G, genotype (NTg, 3xTg- (CTratio); Factorial analysis: G, genotype (NTg, 3xTg-AD mice); A, aging (12, 16 months of age); S, sex (male, female). p < mice); *AD p