ORIGINAL RESEARCH published: 18 January 2021 doi: 10.3389/fnbeh.2020.611384 Digging Signatures in 13-Month-Old 3xTg-AD Mice for Alzheimer’s Disease and Its Disruption by Isolation Despite Social Life Since They Were Born Lydia Gimenez-Llort 1,2* and Daniel Alveal-Mellado 1,2 1 Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Department of Psychiatry and Forensic Medicine, School of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain The severity of this pandemic’s scenarios will leave significant psychological traces in low resistant and resilient individuals. Increased incidence of depression, anxiety, obsessive-compulsive disorder (OCD), and post-traumatic stress disorder has already been reported. The loss of human lives and the implementation of physical distance Edited by: Valerie J. Bolivar, measures in the pandemic and post-COVID scenarios may have a greater impact Wadsworth Center, United States on the elderly, mostly in those with dementia, as OCD and other neuropsychiatric Reviewed by: symptoms (NPS) are quite prevalent in this population. Modeling NPS in animals relies Tim Karl, in neuroethological perspectives since the response to new situations and traumatic Western Sydney University, Australia Takashi Saito, events, critical for survival and adaptation to the environment, is strongly preserved Nagoya City University, Japan in the phylogeny. In the laboratory, mice dig vigorously in deep bedding to bury food *Correspondence: pellets or small objects they may find. This behavior, initially used to screen anxiolytic Lydia Gimenez-Llort lidia.gimenez@uab.cat activity, was later proposed to model better meaningless repetitive and perseverative behaviors characteristic of OCD or autism spectrum disorders. Other authors found Specialty section: that digging can also be understood as part of the expression of the animals’ general This article was submitted to activity. In the present brief report, we studied the digging ethograms in 13-month-old Individual and Social Behaviors, a section of the journal non-transgenic and 3xTg-AD mice modeling normal aging and advanced Alzheimer’s Frontiers in Behavioral Neuroscience disease (AD), respectively. This genetic model presents AD-like cognitive dysfunction Received: 21 October 2020 and NPS-like phenotype, with high mortality rates at this age, mostly in males. This Accepted: 22 December 2020 Published: 18 January 2021 allowed us to observe the digging pattern’s disruption in a subgroup of 3xTg-AD mice Citation: that survived to their cage mates. Two digging paradigms involving different anxiogenic Gimenez-Llort L and Alveal-Mellado D and contextual situations were used to investigate their behavior. The temporal course (2021) Digging Signatures in and intensity of digging were found to increase in those 3xTg-AD mice that had lost their 13-Month-Old 3xTg-AD Mice for Alzheimer’s Disease and Its Disruption “room partners” despite having lived in social structures since they were born. However, by Isolation Despite Social Life Since when tested under neophobia conditions, this behavior’s incidence was low (delayed), They Were Born. Front. Behav. Neurosci. 14:611384. and the temporal pattern was disrupted, suggesting worsening of this NPS-like profile. doi: 10.3389/fnbeh.2020.611384 The outcomes showed that this combined behavioral paradigm unveiled distinct features Frontiers in Behavioral Neuroscience | www.frontiersin.org 1 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation of digging signatures that can be useful to study these perseverative behaviors and their interplay with anxiety states already present in the AD scenario and their worsening by naturalistic/forced isolation. Keywords: OCD, 3xTg-AD, BPSD, Translational neuroscience, Neurospsychiatric symptoms, Loneliness, isolation, COVID-19 reduced quality of life (Fontenelle et al., 2006; Ruscio et al., 2010). In the current situation, the clinical relevance of OCD has increased, and the consequent worsening of daily life (Fontenelle and Miguel, 2020) has urged the elaboration of guides with international consensus for the management of OCD during COVID-19 and post-COVID-19 scenarios (Fineberg et al., 2020). Among older people, the impact of COVID-19 is devastating (Van Loon et al., 2020), mainly among the frailest living in long- term care homes (Commas-Herrera et al., 2020). In these nursery care settings, most residents are affected by dementia (Seitz et al., 2010), a population that already has higher mortality rates than aged-matched control populations, with males showing deranged neuro-immuno-endocrine system and worse survival than females despite not as bad neuropathological status (van Dijk et al., 1991; Mitchell et al., 2010). On the other hand, despite the current social and working roles can make female GRAPHICAL ABSTRACT | Digging signatures in normal and AD-pathological workers more exposed to COVID-19, the first study on gender aging. Experimental design: 3-days battery of tests to assess digging in two differences in COVID-19 found both genders having the same home cage paradigms (CT, corner test, 1 min; MB, marble burying test with 12 prevalence of the disease, but males being more at risk for marbles), also including an open-field test to control for exploration, anxiety, worse outcomes and death independently of the age (Jin et al., and emotionality. In the present work, 13-month-old NTg (normal aging) and 2020). Dementia care during COVID-19 is a challenge (Wang 3xTg-AD mice (advanced stages of disease) were assessed. et al., 2020). In most home-care centers, the confinement and implementation of physical distance measures, with patients being segregated and relocated in new rooms to protect them INTRODUCTION from the virus, is resulting in social isolation. It is well-known that social vulnerability, frailty, and mortality in older adults is a On the day that the COVID-19 pandemic took one million worrisome triad (Andrew et al., 2008). Social isolation increases people’s life, there is no doubt that the severity of the COVID- the risk of dementia (Wilson et al., 2007) and enhances not only 19 pandemic will leave important psychological traces in low its hallmark cognitive decline but neuropsychiatric disorders, resistant and/or resilient individuals (Farhan and Llopis, 2020). which are already quite prevalent in these patients (Cummings, The first clinical reports already reported increased incidence 1997; Seitz et al., 2010; Zhao et al., 2016). The management of sleep disorders, depression, anxiety, as well as obsessive- of their neuropsychiatric symptoms (NPS) is considered a compulsive disorder (OCD), and post-traumatic stress disorder major issue for the patients’ and caregivers’ quality of life (Banerjee, 2020; Rivera and Carballea, 2020). Since then, the (Kamiya et al., 2014). number of reports on OCD, a complex disorder due to its At the translational level, modeling NPS, also known diagnostic classification and clinical heterogeneity (Ruscio et al., as behavioral and psychological symptoms associated with 2010), has fast increased (Kumar and Somani, 2020; Shafran et al., dementia (BPSD), in rodents relies in neuroethological 2020; Sulaimani and Bagadood, 2020). The American Psychiatry approaches since the response to different kind of situations Association1 defines this mental health disorder as a series of and traumatic events, critical for survival and adaptation to recurring unwanted and distressing thoughts, ideas or sensations the environment, are strongly preserved in the phylogeny (obsessions) and compulsive repetitive behaviors (such as hand (Giménez-Llort et al., 2007). In the wild, burying was first washing, cleaning) or mental acts aimed to decrease the described as a defensive response in rats and considered as resulting distress or according to rules. Pre-pandemic descriptive reflecting the anxiety state of animals (Pinel and Treit, 1978). epidemiology of OCD referred to a prevalence of 3.1% of the In the laboratory, mice dig vigorously in deep bedding to bury general population and the association of this disorder with food pellets or small objects such as marbles (Gyertyan, 1995). significant interference of social interactions, daily activities, and Thus, digging and marble burying have been proposed as simple methods for in vivo identification of biological impacts in 1 APA mice (Broekkamp et al., 1986; Deacon, 2006). This behavior, Obsessive-compulsive disorder https://www.psychiatry.org/patients- families/ocd/what-is-obsessive-compulsive-disorder. initially used to screen anxiolytic activity, was later proposed Frontiers in Behavioral Neuroscience | www.frontiersin.org 2 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation to model meaningless repetitive and perseverative behaviors normal aging and advanced stages of the disease, respectively as compulsive-like characteristics of OCD or autism spectrum (Belfiore et al., 2019). The expression of digging behavior was disorders in rodents (de Brouwer et al., 2020; Mahmood et al., recorded in experimental scenarios where a home-cage with 2020). Also, the animal’s general activity can be a confounding beddings is used, such as the corner test (change to a new factor and, conversely, digging can also be understood as a and clean cage) and the marble tests, also involving different measure of general activity rather than a measure of repetitive or anxiogenic and contextual situations (home-cage with beddings, anxiety-related behavior (Greene-Schloesser et al., 2011; Mitra and without/with objects, respectively). The open-field test was et al., 2017; de Brouwer et al., 2019; Dixit et al., 2020). The included to monitor the animal’s general activity in a classical controversy around the digging behavior and what the paradigm anxiogenic environment without beddings. might measure is open. Spontaneous (non-induced) compulsive-like mice based on MATERIALS AND METHODS bidirectional selection for excessive, repetitive, and perseverant digging behaviors are considered mouse models of OCD Animals exhibiting good face, predictive, and construct validity (Greene- A total number of 43 13-month-old male homozygous 3xTg-AD Schloesser et al., 2011; Mitra et al., 2017). In our precedent studies (n = 28) and non-transgenic (NTg, n = 15) mice on a C57BL/6J with the 3xTg-AD mice, a genetic model of Alzheimer’s disease background (after embryonic transfer and backcrossing at least (Oddo et al., 2003) that presents AD-cognitive dysfunction but 10 generations) established in the Universitat Autònoma de also a conspicuous BPSD-like phenotype (Giménez-Llort et al., Barcelona (Baeta-Corral and Giménez-Llort, 2014) were used 2006, 2007), we showed increased marble-burying in middle- in this study. The 3xTg-AD mice harboring transgenes were aged (12-month-old) male 3xTg-AD mice as compared to age- genetically engineered at the University of California Irvine, matched non-transgenic (NTg) mice with normal aging (Torres- as previously described (Oddo et al., 2003). Animals were Lista et al., 2015). Their marble-burying response was related to maintained in groups of 3–4 mice per cage (Macrolon, 35 × 35 × neophobia and shown to be modulated by repeated handling, 25 cm) filled with 5 cm of clean wood cuttings (Ecopure, Chips6, reversed by chronic treatment with neuroleptic risperidone DateSand, UK; Uniform cross-cut wood granules with 2.8– (Torres-Lista et al., 2015), and modified by chronic caffeine 1.0 mm chip size) and nesting materials (Kleenex, Art: 08834060, (Baeta-Corral et al., 2018). In a social paradigm, elicitation of 21 × 20 cm, White). In the current work, seven of the 28 3xTg- digging behavior was similar among males independently of their AD mice had lost their cage-mates and lived alone in their genotype but early expressed in female 3xTg-AD mice (Torres- cage for 2–3 months. In all the cases, the standard home cages Lista and Giménez-Llort, 2019). In our most recent work, in covered with a metallic grid allow the perception of olfactory and male 3xTg-AD mice under a long-term isolation (Muntsant auditory stimuli from the rest of the colony. All the animals were and Giménez-Llort, 2020a) increased gross and fine motor maintained under standard laboratory conditions of food and activity was recorded, with enhanced nest-building. This is also water ad lib, 22 ± 2◦ C, 12 h light: dark cycle with lights on at a species-specific natural behavior that, when disrupted, some 8:00 am, and relative humidity 50–60%. authors consider a homologous to hoarding in humans with OCD (Warneke, 1993) and modeling compulsive-like behavior in Behavioral Assessment mice (Greene-Schloesser et al., 2011). Recently, during housing Digging and other behaviors were measured in the corner test, routines, we observed that the spontaneous digging behavior open-field test, and marble burying test under dim white light elicited when an animal is transferred to a new home cage (20 lx) during their light phase of the light: dark cycle (from 10 with clean beddings was disrupted in a subgroup of old male am to 1 pm) (see Graphical abstract). 3xTg-AD mice that had recently lost their cage-mates. This Behavioral assessments were performed by direct observation observation was restricted to males and AD-genotype since the by two independent observers blind to the genotype, in a increased mortality rates of male 3xTg-AD mice (Giménez-Llort counterbalanced manner and with the support of a computerized et al., 2008) often lead to some males living alone for such video system. All procedures followed the Spanish legislation on natural reasons. the “Protection of Animals Used for Experimental and Other Given the above studies in 3xTg-AD mice and the current Scientific Purposes” and the EU Directive (2010/63/UE) on clinical reports of increased incidence of OCD and anxiety this subject. The study complies with the ARRIVE guidelines disorders (Banerjee, 2020; Rivera and Carballea, 2020) due to developed by the NC3Rs and aims to reduce the number of strict physical distance measures, a brief translational study animals used (Kilkenny et al., 2010). was designed. The work aimed to confirm the observations of Day 1. Digging in a new home cage only with clean wood derangement of digging patterns in the subgroup of male 3xTg- cuttings was assessed in a corner test (CT). The animal was AD mice that, after 10 months of social life, lost their partners introduced in the cage’s center, and the neophobia response and lived alone during the last 2–3 months. We aimed to define was observed for 1 min. The number of visited corners before the nuances of their digging patterns and their expression under the elicitation of the first rearing (CTcbR) and the latency different anxiogenic conditions, as they are also serving as a of rearing (CTlatR) were noted. Then, the number of visited behavioral tool for current investigations. Therefore, digging corners (CTc) and rearings (CTr) were recorded at 30 and 60 s. patterns were assessed in 13-months-old NTg and the two The ratio of these variables (Ratio CTc/r) was calculated. The subgroups of 3xTg-AD male mice, an age that corresponds to number of digging episodes (>2 s, in order to avoid false-positive Frontiers in Behavioral Neuroscience | www.frontiersin.org 3 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation observations) elicited during 30 and 60 s were noted. Prevalence permanently living in social conditions showed similar values of digging was recorded at the end of the test. in the total number of visited corners and rearings. However, Day 3. The animals were placed in the center of an open-field the behavior of isolated 3xTg-AD mice was significantly reduced (metalwork, beige, 44 × 38 × 10 cm height), and their general to 62% of corners and 48% of rearings, reaching statistical and vertical activity (R, rearings) was recorded for 5 min. The significance the drop in vertical activity [F(2, 40) = 3.765, p = time and distance covered in the center and periphery during 0.0318; post-hoc, p < 0.05 vs. each of the other two groups]. the test were recorded using the VideoTrack analysis system The ratio between horizontal and vertical activity was maintained (ViewPoint Behavior Technology, Lyon, France) (Giménez-Llort equal in all the groups (n = 43, 2.87 ± 0.24). At the end of et al., 1995). Walking speed was calculated as the distance covered the test (60 s), the drop of vertical activity [F(2, 40) = 8.034, p per unit of time. In those animals where distance and time in = 0.0017] was now observable in both subgroups of 3xTg-AD the periphery were zero, zero was given to the walking speed. compared to NTg mice. However, it was more intense in the The number of defecations boli (Def) and presence/absence of isolated 3xTg-AD mice (post-hoc, p < 0.05, 3xTg-AD vs. NTg; urination (Ur) were also noted. Prevalence of rearing, defecation but p < 0.001, isolated 3xTg-AD vs. NTg) that also differed from boli, and urination were recorded at the end of the test. those permanently living in social groups (p < 0.05, isolated Day 12. One week later, animals were assessed in the marble- 3xTg-AD vs. 3xTg-AD). The horizontal activity also showed a burying test (MB). The animal was introduced facing the wall of group effect [F(2, 40) = 4.565, p = 0.0164], but it was due to a home-cage. Twelve glass marbles (1.5 cm diameter, 8 g) were a drop of total visited corners in isolated 3xTg-AD mice (p < evenly spaced (four rows in the cage’s upper area) on a 5 cm thick 0.01, isolated 3xTg-AD vs. the other two groups). The ratio CTc/r layer of clean wood cuttings. The mice were left in the cage with of NTg dropped to 64% while that of both subgroups of 3xTg- marbles for a 30 min period. The evaluation was done twice to AD mice was maintained (n = 28, 2.63 ± 0.26), albeit these record the temporal course at 15 and 30 min. (Santana-Santana differences did not reach statistical significance. and Giménez-Llort, Submitted). Digging in the new home cage Digging behavior at 30 and 60 s in the corner test is illustrated was measured by the level of marbles’ burying: “Buried” (B, in Figure 1B. Prevalence of digging behavior in the new home 100% buried), those left “Intact” (I), and the remaining subset cage at 30 s was scarce, as it was only shown by 1/15 (6.7%) considered as those “Moved or partially buried” (M). NTg mice, 4/21(19%) 3xTg-AD mice, and 1/7 (14%) in the subgroup of isolated 3xTg-AD mice. Thereafter, the behavior Statistics increased in NTg (prevalence 7/15, 47%; incidence 6/15 40%) Results are expressed as mean ± SEM. SPSS 20.0 software was and 3xTg-AD (prevalence 12/21, 57%; incidence 8/21, 33%) used. One way ANOVA followed by post-hoc Duncan’s test mice. In both groups, the ethogram measured as the number was used to compare the three independent groups. Differences of digging episodes showed scarce emergence during the first within the same group were analyzed with a Paired t-test. For 30 s and increased at 60 s (accumulated counts). This temporal categorical variables, Fisher’s exact test with 2 × 2 was used. In all pattern was statistically significant in both groups (NTg mice, the tests, p < 0.05 was considered statistically significant. D60 vs. D30, p < 0.001; 3xTg-AD mice, D60 vs. D30, p < 0.01). In contrast, no new digging behavior episodes were observable RESULTS among isolated 3xTg-AD mice. Figures 2A–E, illustrate the different variables assessed in the As a first step, we investigated the genotype differences, those due open-field test. Except for the distance covered in the central to the three human transgenes. Table 1 summarizes the results of area of the field where all the groups covered a similarly short the 30 variables studied and point at vertical exploratory activity distance [F(2, 212) = 1.894, p = 0.153], all the other variables as the most sensitive variable to show genotype differences, with a showed that the pattern of NTg mice was distinct from that of severe drop of rearing behavior in the periphery. Also, a reduced the two subgroups of 3xTg-AD mice [all F’s(2, 212) > 9.327, number of moved marbles in favor of an increase in those buried p < 0.000131]. In particular, thigmotaxis was different among lead to genotype differences. groups as shown by preference (in time spent, p = 1.16 e-21, To verify our hypothesis that the 3xTg-AD mice that and distance covered p = 1.89 e-19) for the periphery shown recently lost their home-cage partners exhibited different digging by NTg mice, but lost in both groups of 3xTg-AD mice for patterns, the data of 3xTg-AD mice was depicted in two distance and inverted in terms of time (3xTg-AD mice, p = subgroups, according to their most recent housing conditions. 0.00628; isolated 3xTg-AD mice, p = 0.00214) (Figures 2A,B). The results clearly showed that while NTg (n = 15) and 3xTg- This was due to increased freezing behaviors in the center. The AD mice (n = 21) were quite alike in most behaviors, genotype total distance covered by both subgroups of 3xTg-AD mice in differences emerged in the subset of 3xTg-AD mice (n = 7) that the periphery was as scarce as in the center and drastically recently lost their partners. The following paragraphs, illustrated lower than in NTg mice. The distance and time spent allowed in Figures 1–3, show the nuances in the digging patterns elicited to calculate the animals’ averaged walking speed (Figure 2C). in two distinct home-cage settings with beddings. The walking speed was different among groups since both 3xTg- As illustrated in Figure 1A, in all the groups, the ethogram of AD groups walked slower than NTg mice and independently of neophobia in the corner test started with an inspection of the cage the open-field area. The paired analysis indicated that NTg mice (n = 43, 4.18 ± 0.39 visited corners) followed by a first rearing (n walked faster in the center than in the periphery (p < 0.001). = 43, latency: 12.30 ± 1.02 s). At 30 s, NTg and 3xTg-AD mice This was confirmed by their sustained rearing behavior levels Frontiers in Behavioral Neuroscience | www.frontiersin.org 4 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation TABLE 1 | Genotype differences between 13-month-old male 3xTg-AD mice and NTg mice in the corner, open-field, and marble tests. NTg mice n = 15 3xTg-AD mice n = 28 Genotype differences (Mean ± SEM) (Mean ± SEM) 1.Corner test CTcbR (n) 4.64 ± 0.58 4.00 ± 0.53 n.s. CTlatR (n) 13.40 ± 1.90 11.64 ± 1.23 n.s. CTc30 (n) 9.93 ± 0.71 9.36 ± 0.89 n.s. CTr30 (n) 4.33 ± 0.42 3.64 ± 0.44 n.s. Ratio CTc/r30 (index) 2.74 ± 0.55 2.95 ± 0.24 n.s. CTc60 (n) 15.13 ± 0.89 13.14 ± 1.18 n.s. CTr60 (n) 9.60 ± 0.86 5.93 ± 0.66 ** Ratio CTc/r60 (index) 1.77 ± 0.19 2.66 ± 0.26 ** Digging 30” (n of episodes) 0.07 ± 0.07 0.25 ± 0.11 n.s. Digging 60” (n of episodes) 0.80 ± 0.24 0.89 ± 0.26 n.s. 2. Open field test Distance covered in the center (cm) 86.49 ± 10.59 72.07 ± 6.09 n.s. Distance covered in the periphery 266.57 ± 10.99 78.13 ± 9.17 *** (cm) Time spent in the center (s) 15.43 ± 2.31 27.98 ± 1.84 *** Time spent in the periphery (s) 49.84 ± 1.14 16.70 ± 1.76 *** Walking speed in the center (cm/s) 8.81 ± 0.71 3.07 ± 0.23 *** Walking speed in the periphery 5.50 ± 0.23 3.02 ± 0.42 *** (cm/s) R1 (n) 3.20 ± 0.83 0.04 ± 0.01 *** R2 (n) 4.20 ± 1.08 0.43 ± 0.08 ** R3 (n) 4.07 ± 1.05 0.46 ± 0.09 *** R4 (n) 5.00 ± 1.29 0.93 ± 0.18 *** R5 (n) 3.80 ± 0.98 1.00 ± 0.19 *** Total rearings (n) 20.27 ± 5.23 2.86 ± 0.54 *** Defecation (n of boli) 0.33 ± 0.09 4.11 ± 0.78 *** Urination (presence/absence) 0.00 0.74 ± 0.14 *** 3. Marble test I15 (n) 7.20 ± 0.87 6.64 ± 0.73 n.s. M15 (n) 1.33 ± 0.43 1.07 ± 0.37 n.s. B15 (n) 3.47 ± 0.83 4.21 ± 0.67 n.s. I30 (n) 2.80 ± 0.63 3.64 ± 0.71 n.s. M30 (n) 2.20 ± 0.61 0.75 ± 0.23 * B30 (n) 7.00 ± 0.74 7.64 ± 0.75 n.s. Student’s t-test, ***p < 0.001, **p < 0.01, *p < 0.05, n.s. p > 0.05 vs. NTg mice. Corner test: CTcbR, visited corners before the first rearing; CTlatR, latency of rearings in seconds; CTcn, visited corners at n s; CTrn, rearings at n s; Ratio CTc/r n, ratio visited corners divided by rearings at n s of the test; Digging in the corner test at 30 (D30) and 60 (D60) s. Open-field test: Rn, rearings at minute n; Marble test: marbles left intact (I15, I30), half-buried or moved (M15, M30) or completely buried (B15, B30) at 15 and 30 min, respectively. (Figure 2D) through the 5 min of the test. Only 12/21 (57%) The presence of urination was only shown in 3xTg-AD mice (n group-housed 3xTg-AD and 5/7 (71%) isolated 3xTg-AD mice = 24, 0.72 ± 0.09, absent in NTg, p < 0.001), with a prevalence performed rearings, and their number was small. Statistically of 17/21 (81%) in group-housed 3xTg-AD mice and 6/7 (86%) in significant differences in the prevalence were shown between those isolated (both, p < 0.001 vs. NTg mice). 3xTg-AD vs. NTg mice (p < 0.05). As illustrated in Figure 3, digging in the marble test was Emotionality, as measured by defecation and urination, different among groups as measured by the number of marbles was found increased in both subgroups of 3xTg-AD mice as left intact at 15 min [F(2, 40) = 4.283 p = 0.0206]. NTg and 3xTg- compared to NTg mice [both F’s(2, 40) > 16.4, p < 6.78 e-06; AD mice showed similar patterns. However, digging behavior post-hoc p < 0.001 vs. NTg]. Also, 19/21 (90%) grouped and 7/7 in the subgroup of isolated 3xTg-AD mice was increased as (100%) isolated 3xTg-AD defecated during the test, while only measured by a reduced number of marbles left intact (post-hoc 3/15 (20%) NTg mice did so (both, p < 0.001 vs. to NTg mice). isolated 3xTg-AD vs. NTg p = 0.0202; vs. 3xTg-AD p = 0.0086). Frontiers in Behavioral Neuroscience | www.frontiersin.org 5 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation FIGURE 1 | Ethogram and Digging in a new home cage (Corner test). Results are expressed as mean ± SEM. (A) Ethogram in the corner test: CTcbR, visited corners before the first rearing; CTlatR, latency of rearings in seconds; CTcn, visited corners at n s; CTrn, rearings at n s; Ratio CTc/r n, ratio visited corners divided by rearings at n s of the test. (B) Digging in the corner test at 30 (D30) and 60 (D60) s. Statistics: One-way ANOVA followed by post-hoc Duncan’s test, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the NTg group; #p < 0.05 and ##p < 0.01 vs. the 3xTg-AD mice. Paired t-test, aa p < 0.01 and aaa p < 0.001 vs. the same group at 30 s. At the end of the test, differences in the number of marbles half- made to enhance the chances to record digging behavior. The buried o moved were found among groups [F(2, 40) = 3.551, p changes were related to the windows and frames of observation. = 0.0381, post-hoc, 3xTg-AD mice vs. NTg p = 0.0211]. Post-hoc The corner test, performed at 30 s, was prolonged until 60 s. In analysis also detected differences between isolated 3xTg-AD mice the marble test, measures were taken at 15 min, and the end of and 3xTg-AD in the number of marbles left intact at the end of the test to record the temporal progression (Santana-Santana and the test [F(2, 40) = 3.180, p = 0.0523, but post-hoc, isolated 3xTg- Giménez-Llort, Submitted). AD mice vs. 3xTg-AD p = 0.0284] and buried [F(2, 40) = 2.938, The results show the consistency of some behavioral patterns, p = 0.0645, but post-hoc, isolated 3xTg-AD mice vs. NTg p = independently of the genotype or social condition. However, the 0.0429 and vs. 3xTg-AD p = 0.0305]. short battery of tests also allowed to confirm the emergence of disruptions in digging in the 3xTg-AD mice and, among DISCUSSION them, in those that lost their cage-mates. This “natural isolation” subgroup performed worse than expected. As described in the Species-typical behaviors can provide neuroethological tools present work, for the first time, the analysis of the segregated to follow up the animal’s well-being, age/aging and disease data unveiled distinct modulation of digging signatures in terms processes, and modulation by treatments and environmental of prevalence, intensity, and patterns, depending on the context. factors (Oddo et al., 2003; Greene-Schloesser et al., 2011). When tested for neophobia (immediate fear of a new place), the Digging can be spontaneously elicited when animals are prevalence was lower, and the digging pattern was mostly absent. transferred into a new home-cage with clean beddings during However, the activity patterns, thigmotaxis, and emotionality housing routines but can also be reproduced in experimental of both 3xTg-AD mice subgroups did not differ in the open- scenarios such as the corner and the marble tests. In the field test. In this classical test for anxiety, the phenotype of present brief report, two digging paradigms involving different all the 3xTg-AD mice was different from that shown by NTg anxiogenic and contextual situations were used to investigate mice with normal aging. According to the action program by the digging patterns in the 13-month-old males with normal Lát (1973), describing the immediate fearful response and the and genetically induced AD-pathological aging. We confirmed subsequent actions developed by the animal confronted to a the disruptive effects of short isolation (2–3 months) observed new environment, the open-field elicited a severe anxiety/fear- in a subgroup of 3xTg-AD that recently lost their cage-mates like pattern in both subgroups of 3xTg-AD mice. It was after 10 months living in a standard social environment. These characterized by drastically reduced general activity, as measured 3xTg-AD mice that survived to their partners indicated that in terms of time, distance covered, and rearing in the walls. their current social housing conditions modified the patterns, Low performance, with long-lasting periods of freezing in the achieving divergence with NTg mice but also 3xTg-AD mice still center of the field, resulted in an inverted pattern for thigmotaxis. living in social groups. Walking speed patterns were also found disrupted. Emotionality, Elicitation of digging behavior can be more or less as measured by defecation and urination, was increased. conspicuous, depending on many intrinsic and extrinsic Spontaneous animal models with face validity for OCD, such factors, and it is usually under-recorded. Slight methodological as the “BIG mice” (Greene-Schloesser et al., 2011; Mitra et al., adaptations of the corner test and the marble test protocols were 2017) are based on their divergent nesting and marble-burying Frontiers in Behavioral Neuroscience | www.frontiersin.org 6 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation FIGURE 2 | Exploration, Anxiety, and Emotionality in the Open-field test. Results are expressed as mean ± SEM. (A) Distance covered, (B) Time spent, (C) Walking speed, (D) Vertical exploratory activity, (E) Emotionality. Abbreviations: Dc, distance in the center; Dp, distance in the periphery; Tc, time in the center; Tp, time in the periphery; WSc, Walking speed in the center; WSp, walking speed in the periphery; Rn, rearings at minute n; Total R, total number of rearings; Def, defecation boli; Ur, urination. Statistics: One-way ANOVA followed by post-hoc Duncan’s test, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the NTg mice; #p < 0.05. vs. the 3xTg-AD mice, Paired t-test, aa p < 0.01, aaa p < 0.001 vs. the same variable in the center of the open-field test. Fisher’s exact test, +p < 0.05 and + + +p < 0.001 vs. the NTg mice. behaviors, increased compulsive-like digging, with respect to 3xTg-AD mice could not result from locomotor activity since “SMALL mice” counterparts that exhibit risk assessment and this was drastically reduced, whereas it was normal in NTg mice. inhibition of movement in the open-field and the elevated plus- Both subgroups of 3xTg-AD mice showed similar anxiety levels maze. These animals also diverge in the thigmotaxis, and the but differed in the corner and marble tests’ digging signatures. rearing frequency in the periphery is three-fold higher in the In this regard, some pharmacological studies have noted that “BIG mice” compared to the “SMALL.” In the present work in while the dose-dependent reduction in marble-burying could 3xTg-AD mice, the temporal course and intensity of digging be due to several drugs, this effect was not always related to behavior in the marble test were found increased in the isolated their locomotor effects (Jimenez-Gomez et al., 2011). Due to mice. Here, the marble-test experimental setup was similar to this controversy, the open-field or other anxiety tests that also the one used in the corner test except for marble pieces and monitor the general activity are a must for interpretations and the starting position facing the wall. As compared to other discard confounding factors. laboratory protocols, the number of marble pieces was reduced The genetic background is determinant for the expression to 12, and they were allocated in one half of the test cage to of behaviors (Albelda and Joel, 2012). Also, animal models reduce the chance that differences in buried pieces be due to based on overexpression of human transgenes from familial differences in locomotor activity (Nicolas et al., 2006; Thomas forms of the disease are not exempted from limitations and et al., 2009). Still, as shown in the open-field test, the NTg mice shortcomings. The main translational gap, but also a challenge, is were the active animals, similar to “BIG mice,” and both 3xTg-AD the difficulty to fully recapitulate the complexity of neurological mice subgroups exhibited the same anxiety/fear-like behavior. and psychiatric patterns observed at the clinical level in the Therefore, the increased number of pieces buried in isolated human patient (Giménez-Llort et al., 2006, 2007; Kosel et al., Frontiers in Behavioral Neuroscience | www.frontiersin.org 7 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation FIGURE 3 | Digging in a new home cage with small objects (Marble test). Results are expressed as mean ± SEM (A) At 15 and 30 min of the test, the number of marbles left intact (I15, I30), half-buried or moved (M15, M30) or completely buried (B15, B30) is indicated. Statistics: One-way ANOVA followed by post-hoc Duncan’s test, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the NTg mice; #p < 0.05. vs. the 3xTg-AD mice. 2020). Other critical issues refer to genetic engineering since was elicited earlier in the ethogram (on average, at 90 s), in transgene insertions can have phenotypic consequences and agreement with their increased anxious-like profile and shrinking could confound some experiments (Goodwin et al., 2019). The the action program described by Lát (1973). Thus, although the present results used the new Spanish colonies of 3xTg-AD repetitive and compulsive behaviors that characterize the OCD mice established at Universitat Autònoma de Barcelona after are defined as “meaningless,” they are also considered to decrease embryonic transfer on a congenic C57BL/6J background from the resulting distress. progenitors with a hybrid C57BL/6J x 129 Sv genetic background In other animal models of AD such as the APP/PS1, Tg2576, kindly provided by Prof. Frank M LaFerla in 2011. This animal and 5xFAD mice, several authors had reported a social isolation- model, also provided by The Jackson Lab as Stock #004807, induced increase in amyloid pathology (Hsiao et al., 2011; is adapting this genetic strategy, with animals on congenic Huang et al., 2011; Peterman et al., 2020). In contrast, in 3xTg- C57BL/6J or 129S4 expected to be available beginning last AD mice, limited impact was found when long-term isolation January 20192 Concerning our work, we can compare the present was implemented from postweaning to adulthood (Pietropaolo results with those already published in our laboratory using the et al., 2009). However, in our most recent work with these new first colonies of NTg and 3xTg-AD mice in the C57BL/6J x Spanish colonies of 3xTg-AD mice, and also studying naturally 129 Sv genetic background. Those results showed that, at this occurring long-term isolation due to loss of partners in an old age, 3xTg-AD mice’s performance was already disrupted in aged scenario, we found that tau pathology of 3xTg-AD mice the marble test (Torres-Lista et al., 2015). Their pattern was was enhanced, albeit did not reach the statistical significance, similar to that exhibited here by 3xTg-AD mice that lost their probably due to a celing effect (Muntsant and Giménez-Llort, partners. Thus, it seems that in the present work, the C57BL/6J 2020a). Nevertheless, the hippocampal atrophy asymmetry found genetic background induces a delay in the expression of the in human patients with AD (Wachinger et al., 2016) was modeled disruption, but the pattern emerges in the animals that have for the first time in mice and allowed us to show its worsening confronted social isolation in the last 2 months. This would after long-term isolation. Further analysis has also found brain agree with the extended window of observation in the corner differences in the cortical areas (Muntsant and Giménez-Llort, test, allowing to detect of reduced rearing in both subgroups of 2020b). In the present work, the effects of a short-isolation 3xTg-AD mice, with different degrees of statistical significance did not modify the general motor activity pattern nor the (worse in isolated). Furthermore, we recently reported that in the anxiety-like profile of 3xTg-AD mice. However, as we have social interaction test (Torres-Lista and Giménez-Llort, 2019), shown recently (Muntsant and Giménez-Llort, 2020a), a longer the ethogram of 14-month-old male and female 3xTg-AD mice isolation regime induced re-structured negative valence system points at digging as a robust and consistent non-social interaction with the emergence of bizarre behaviors and flight copying-with- behavior. In that context, digging emerged on minute three of stress strategies were found. More importantly, with regards of the test together with self-grooming. In females, the behavior the present work, the long-term period induced a prominent hyperactive pattern in both gross (general motor activity in most 2 Alzforum.org (2020) https://www.alzforum.org/research-models/3xtg. tests) and fine-motor functions (increased nesting-behavior) Frontiers in Behavioral Neuroscience | www.frontiersin.org 8 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation that were correlated to hippocampal tau pathology (Muntsant estimate the impact of a short-isolation regime with regards and Giménez-Llort, 2020a). Activation of cortical and striatal to the emergence or enhancement of compulsive behaviors in regions during the expression of a naturalistic compulsive- Alzheimer’s disease where NPS/BPSD-like symptomatology is like behavior in the rabbit have been described (Cano-Ramírez already present. The prevalence and incidence, the temporal and Hoffman, 2018), so these are also our target areas in our course, and the intensity of this behavior were found to increase current investigations. Excessive nest building has been described in the 3xTg-AD mice that had lost their “room partner” despite as a unique behavioral phenotype in the deer mouse model having lived in social structures for more than 10 months, of obsessive-compulsive disorder (Wolmarans et al., 2016). As since they were born. When they were tested under neophobia mentioned before, psychogenetic bidirectional selection of mice conditions, the incidence of this behavior was smaller (delayed), for nesting behavior is used as an animal model for compulsive- and the pattern of digging was disrupted, resembling that like behaviors, and it is also leading to distinct marble-burying previously reported for worse stages of the disease. Despite (Greene-Schloesser et al., 2011; Mitra et al., 2017). the limitations of a naturalistic isolation (small sample and Thus, despite the current brief report could be considered only affecting one genotype), together with precedent results preliminary, our precedent data describing the consistency of of long-term isolation enhancing nesting-behavior, we present digging in males studied in a social paradigm (Torres-Lista this combined paradigm unveiling distinct features of digging and Giménez-Llort, 2019) and of increased nest-building in signatures as it can be useful to study these perseverative animals under a long-term isolation (Muntsant and Giménez- behaviors, their complex interplay with anxiety states already Llort, 2020a) support the consistent observations during housing present in the AD scenarios and its worsening by naturalistic or routines that lead to this work and the present results showing forced isolation. disruptions in the subgroup under isolation. Still, the limitations of the current work must be noted and are mainly due to the naturalistic scenario, since the natural death of home-cage DATA AVAILABILITY STATEMENT partners at this age only affects to 3xTg-AD mice (Giménez-Llort The raw data supporting the conclusions of this article will be et al., 2008). This fact also limits the sample size of the animals made available by the authors, without undue reservation. under a “naturalistic isolation.” To solve it, in the ongoing investigations, a forced isolation paradigm is being used for both genotypes and including both sexes. Studying digging signatures ETHICS STATEMENT in other paradigms and the impact of isolation at different ages/stages of disease is also a matter of current investigations The animal study was reviewed and approved by CEEAH (Marin-Pardo and Gimenez-Llort, 2020). Universitat Autònoma de Barcelona and DMAH Generalitat Among our different reports on non-pharmacological de Catalunya. preventive/therapeutic interventions in this mouse model, handling was able to modify marble-burying (Torres-Lista et al., AUTHOR CONTRIBUTIONS 2015) and the long-lasting effects of early-life interventions (postnatal handling and environmental enrichment) were LG-L: concept and draft manuscript. Both authors equally also observable in other perseverative behaviors (Torres-Lista contributed to the performance and analysis of the experiments. and Giménez-Llort, 2015). However, in another model, the Both authors revised and approved the final manuscript. 5xTg-AD mice, Petermam et al. showed that prolonged isolation stress accelerated the onset of disease-related pathology despite running wheels and environmental enrichment (Peterman FUNDING et al., 2020). Still, at the clinical level, the beneficial effects of DA-M is recipient of a CONICYT-ANID/73200493 grant. The these interventions foreseen in the context of the pandemic work has been supported by 2017-SGR-1468 and ArrestAD are supported by the literature (Davim et al., 2020). Our H2020 Fet-OPEN-1-2016-2017-737390 to LG-L. preliminary results, with regards to the implementation of a stimulation program to improve both psychological and motor functions in a long-term care center, are encouraging ACKNOWLEDGMENTS (Castillo-Mariqueo et al., 2020), but also indicate that the time frames for intervention seem to be critical and We thank Prof. Frank M. LaFerla Institute for Memory depend on the frailty of aging/AD scenarios (Giménez-Llort, Impairments and Neurological Disorders, Department of 2010). Neurobiology and Behavior, University of California, Irvine, In summary, as a translational neuroscience approach, the USA, for kindly providing the progenitors of the Spanish present brief report in the 3xTg-AD mice can be useful to colonies of 3xTg-AD and NTg mice. Frontiers in Behavioral Neuroscience | www.frontiersin.org 9 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation REFERENCES Fontenelle, L. F., Mendlowicz, M. V., and Versiani, M. (2006). The descriptive epidemiology of obsessive-compulsive disorder. Prog. Neuropsychopharmacol. Albelda, N., and Joel, D. (2012). Animal models of obsessive–compulsive disorder: Biol. Psychiatry 30, 327–337. doi: 10.1016/j.pnpbp.2005.11.001 exploring pharmacology and neural substrates. Neurosci. Biobehav. Rev. 36, Fontenelle, L. F., and Miguel, E. C. (2020). The impact of coronavirus (COVID- 47–63. doi: 10.1016/j.neubiorev.2011.04.006 19) in the diagnosis and treatment of obsessive-compulsive disorder. Depress. Andrew, M. K., Mitnitski, A. B., and Rockwood, K. (2008). Social Anxiety 37, 510–511. doi: 10.1002/da.23037 vulnerability, frailty, and mortality in elderly people. PLoS ONE 3:e2232. Giménez-Llort, L. (2010). The snail and turtle effects: reflections on health doi: 10.1371/journal.pone.0002232 care management in geriatric medicine. Eur. Geriatr. Med. 1:372. Baeta-Corral, R., and Giménez-Llort, L. (2014). Bizarre behaviors and risk doi: 10.1016/j.eurger.2010.10.001 assessment in 3xTg-AD mice at early stages of the disease. Behav. Brain Res. Giménez-Llort, L., Arranz, L., Maté I., and De La Fuente, M. (2008). 258, 97–105. doi: 10.1016/j.bbr.2013.10.017 Gender-specific neuroimmunoendocrine aging in a triple-transgenic 3×Tg- Baeta-Corral, R., Johansson, B., and Giménez-Llort, L. (2018). Long-term AD mouse model for Alzheimer’s disease and its relation with longevity. treatment with low-dose caffeine worsens BPSD-like profile in 3xTg-AD mice Neuroimmunomodulation 15, 331–343. doi: 10.1159/000156475 model of Alzheimer’s disease and affects mice with normal aging. Front. Giménez-Llort, L., Blázquez, G., Cañete, T., Johansson, B., Oddo, S., Tobeña, Pharmacol. 9:79. doi: 10.3389/fphar.2018.00079 A., et al. (2007). Modeling behavioral and neuronal symptoms of Alzheimer’s Banerjee, D. D. (2020). The other side of COVID-19. Impact on obsessive disease in mice: a role for intraneuronal amyloid. Neurosci. Biobehav. Rev. 31, compulsive disorder (OCD) and hoarding. Psychiatry Res. 288:112966. 125–147. doi: 10.1016/j.neubiorev.2006.07.007 doi: 10.1016/j.psychres.2020.112966 Giménez-Llort, L., Blázquez, G., Canete, T., Rosa, R., Vivo, M., Oddo, S., Belfiore, R., Rodin, A., Ferreira, E., Velazquez, R., Branca, C., Caccamo, A., et al. (2006). “Modeling ∼neuropsychiatric symptoms of Alzheimer’s disease et al. (2019). Temporal and regional progression of Alzheimer’s disease-like dementia in 3xTg-AD mice,” in Alzheimer’s Disease: New Advances, eds K. Iqbal, pathology in 3xTg-AD mice. Aging Cell 18:e12873. doi: 10.1111/acel.12873 B. Winblad, and J. Avila, (Englewood, NJ: Medimond), 513–516. Broekkamp, C. L., Rijk, H. W., Joly-Gelouin, D., and Lloyd, K. L. (1986). Major Giménez-Llort, L., Martínez, E., and Ferré S. (1995). Dopamine-independent tranquillizers can be distinguished from minor tranquillizers on the basis and adenosine-dependent mechanisms involved in the effects of N-methyl- of effects on marble burying and swim-induced grooming in mice. Eur. J. d-aspartate on motor activity in mice. Eur. J. Pharmacol. 275, 171–177. Pharmacol. 126, 223–229. doi: 10.1016/0014-2999(86)90051-8 doi: 10.1016/0014-2999(94)00768-3 Cano-Ramírez, H., and Hoffman, K. L. (2018). Activation of cortical and striatal Goodwin, L. O., Splinter, E., Davis, T. L., Urban, R., He, H., Braun, R. E., et al. regions during the expression of a naturalistic compulsive-like behavior in the (2019). Large-scale discovery of mouse transgenic integration sites reveals rabbit. Behav. Brain Res. 35, 168–177. doi: 10.1016/j.bbr.2018.05.034 frequent structural variation and insertional mutagenesis. Genome Res. 29, Castillo-Mariqueo, L., Valenzuela-Garrido, P., and Gimenez-Llort, L. (2020). Santa 494–505. doi: 10.1101/gr.233866.117 Isabel program for psycho-motor function in normal and pathological aging Greene-Schloesser, D. M., Van der Zee, E. A., Sheppard, D. K., Castillo, M. R., during current times of COVID-19 confinement in a long-term care. Int. Gregg, K. A., Burrow, T., et al. (2011). Predictive validity of a non-induced Psychogeriatr. 32, 194–194. doi: 10.1017/S1041610220003245 mouse model of compulsive-like behavior. Behav. Brain Res. 221, 55–62. Commas-Herrera, A., Zalakaín, J., Litwin, C., Hsu, A. T., Lane, N., and Fernández, doi: 10.1016/j.bbr.2011.02.010 J.-L. (2020). ltccovid.org|Mortality Associated With COVID-19 Outbreaks in Gyertyan, I. (1995). Analysis of the marble burying response: marbles serve Care Homes Mortality Associated With COVID-19 Outbreaks in Care Homes: to measure digging rather than evoke burying. Behav. Pharmacol. 6, 24–31. Early International Evidence. Available online at: https://www.ontario.ca/page/ doi: 10.1097/00008877-199501000-00005 howontario-is-responding-covid-19#section-1 (accessed June 7, 2020). Hsiao, Y. H., Chen, P. S., Chen, S. H., and Gean, P. W. (2011). The Cummings, J. L. (1997). The neuropsychiatric inventory: assessing involvement of Cdk5 activator p35 in social isolation-triggered onset of psychopathology in dementia patients. Neurology 48(5 Suppl. 6), S10–S16. early alzheimers disease-related cognitive deficit in the transgenic mice. doi: 10.1212/WNL.48.5_Suppl_6.10S Neuropsychopharmacology 36, 1848–1858. doi: 10.1038/npp.2011.69 Davim, A., Trindade da Silva, L., and Vieira, P. (2020). Environmental enrichment Huang, H. J., Liang, K. C., Ke, H. C., Chang, Y. Y., and Hsieh-Li, H. M. as a strategy to confront social isolation under COVID-19 pandemic. Front. (2011). Long-term social isolation exacerbates the impairment of spatial Behav. Neurosci. (in press). doi: 10.3389/fnbeh.2020.564184 working memory in APP/PS1 transgenic mice. Brain Res. 1371, 150–160. de Brouwer, G., Fick, A., Harvey, B. H., and Wolmarans, W. (2019). A critical doi: 10.1016/j.brainres.2010.11.043 inquiry into marble-burying as a preclinical screening paradigm of relevance Jimenez-Gomez, C., Osentoski, A., and Woods, J. H. (2011). Pharmacological for anxiety and obsessive-compulsive disorder: mapping the way forward. evaluation of the adequacy of marble burying as an animal model Cogn. Affect. Behav. Neurosci. 19, 1–39. doi: 10.3758/s13415-018-00653-4 of compulsion and/or anxiety. Behav. Pharmacol. 22, 711–713. de Brouwer, G., Fick, A., Lombaard, A., Stein, D. J., Harvey, B. H., and Wolmarans, doi: 10.1097/FBP.0b013e32834afebe W. (2020). Large nest building and high marble-burying: two compulsive-like Jin, J. M., Bai, P., He, W., Wu, F., Liu, X. F., Han, D. M., et al. (2020). Gender phenotypes expressed by deer mice (Peromyscus maniculatus bairdii) and their differences in patients with COVID-19: focus on severity and mortality. Front. unique response to serotoninergic and dopamine modulating intervention. Public Health 8:152. doi: 10.3389/fpubh.2020.00152 Behav. Brain Res. 393:112794. doi: 10.1016/j.bbr.2020.112794 Kamiya, M., Sakurai, T., Ogama, N., Maki, Y., and Toba, K. (2014). Factors Deacon, R. M. (2006). Digging and marble burying in mice: simple methods associated with increased caregivers’ burden in several cognitive stages of for in vivo identification of biological impacts. Nat. Protoc. 1, 122–124. Alzheimer’s disease. Geriatr. Gerontol. Int. 14, 45–55. doi: 10.1111/ggi.12260 doi: 10.1038/nprot.2006.20 Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., and Altman, D. G. (2010). Dixit, P. V., Sahu, R., and Mishra, D. K. (2020). Marble-burying behavior test as Improving bioscience research reporting: the ARRIVE guidelines for reporting a murine model of compulsive-like behavior. J. Pharmacol. Toxicol. Methods animal research. PLoS Biol. 8:e1000412. doi: 10.1371/journal.pbio.1000412 102:106676. doi: 10.1016/j.vascn.2020.106676 Kosel, F., Pelley, J. M. S., and Franklin, T. B. (2020). Behavioural and Farhan, R., and Llopis, P. (2020). Psychiatric and neuropsychiatric syndromes and psychological symptoms of dementia in mouse models of Alzheimer’s COVID-19. Lancet Psychiatry. 7:736. doi: 10.1016/S2215-0366(20)30317-5 disease-related pathology. Neurosci. Biobehav. Rev. 112, 634–647. Fineberg, N. A., Van Ameringen, M., Drummond, L., Hollander, E., Stein, doi: 10.1016/j.neubiorev.2020.02.012 D. J., Geller, D., et al. (2020). How to manage obsessive-compulsive Kumar, A., and Somani, A. (2020). Dealing with corona virus anxiety and OCD. disorder (OCD) under COVID-19: a clinician’s guide from the International Asian J. Psychiatr. 51:102053. doi: 10.1016/j.ajp.2020.102053 College of Obsessive Compulsive Spectrum Disorders (ICOCS) and the Lát, J. (1973). The analysis of habituation. Acta Neurobiol. Exp. 33, 771–789. Obsessive-Compulsive and Related Disorders Research Network (OCRN) Mahmood, H. M., Aldhalaan, H. M., Alshammari, T. K., Alqasem, M. A., of the European College of Neuropsychopharmacology. Compr. Psychiatry Alshammari, M. A., Albekairi, N. A., et al. (2020). The role of nicotinic 100:152174. doi: 10.1016/j.comppsych.2020.152174 receptors in the attenuation of autism-related behaviors in a murine BTBR T Frontiers in Behavioral Neuroscience | www.frontiersin.org 10 January 2021 | Volume 14 | Article 611384 Gimenez-Llort and Alveal-Mellado Digging Signatures, Aging, AD, Isolation + tf/J autistic model. Autism Res. Off. J. Int. Soc. Autism Res. 13, 1311–1334. Thomas, A., Burant, A., Bui, N., Graham, D., Yuva-Paylor, L. A., and Paylor, doi: 10.1002/aur.2342 R. (2009). Marble burying reflects a repetitive and perseverative behavior Marin-Pardo, D., and Gimenez-Llort, L. (2020). Olfactory signatures in models of more than novelty-induced anxiety. Psychopharmacology (Berl.) 204, 361–373. aging and Alzheimer’s disease and the effect of social isolation: a translational doi: 10.1007/s00213-009-1466-y neuroscience approach in times of coronavirus pandemic (COVID-19). Int. Torres-Lista, V., and Giménez-Llort, L. (2015). Early postnatal handling Psychogeriatr. 32, 133–133. doi: 10.1017/S1041610220002707 and environmental enrichment improve the behavioral responses of Mitchell, S. L., Miller, S. C., Teno, J. M., Kiely, D. K., Davis, R. B., and Shaffer, 17-month-old 3xTg-AD and non-transgenic mice in the Forced Swim M. L. (2010). Prediction of 6-month survival of nursing home residents with Test in a gender-dependent manner. Behav. Process. 120, 120–127. advanced dementia using ADEPT vs. hospice eligibility guidelines. JAMA J. doi: 10.1016/j.beproc.2015.09.011 Am. Med. Assoc. 304, 1929–1935. doi: 10.1001/jama.2010.1572 Torres-Lista, V., and Giménez-Llort, L. (2019). Vibrating tail, digging, Mitra, S., Bastos, C. P., Chesworth, S., Frye, C., and Bult-Ito, A. (2017). body/face interaction, and lack of barbering: sex-dependent behavioral Strain and sex based characterization of behavioral expressions in signatures of social dysfunction in 3xTg-AD mice as compared to mice non-induced compulsive-like mice. Physiol. Behav. 168, 103–111. with normal aging. J. Alzheimers Dis. 69, 969–977. doi: 10.3233/JAD-1 doi: 10.1016/j.physbeh.2016.11.002 90253 Muntsant, A., and Giménez-Llort, L. (2020a). Impact of social isolation Torres-Lista, V., Lopez-Pousa, S., and Giménez-Llort, L. (2015). Marble-burying is on the behavioral, functional profiles, and hippocampal atrophy enhanced in 3xTg-AD mice, can be reversed by risperidone and it is modulable asymmetry in dementia in times of coronavirus pandemic (COVID- by handling. Behav. Processes 116, 69–74. doi: 10.1016/j.beproc.2015. 19): a translational neuroscience approach. Front. Psychiatry 11:572583. 05.001 doi: 10.3389/fpsyt.2020.572583 van Dijk, P. T., Dippel, D. W., and Habbema, J. D. F. (1991). Muntsant, A., and Giménez-Llort, L. (2020b). Brain atrophy asymmetry in Survival of patients with dementia. J. Am. Geriatr. Soc. 39, 603–610. dementia is worsened by social isolation: a translational neuroscience approach doi: 10.1111/j.1532-5415.1991.tb03602.x in times of coronavirus pandemic (COVID-19). Int. Psychogeriatr. 32, 135–135. Van Loon, A., Rutten, J., Van Buul, L., Joling, K., Smalbrugge, M., and Hertogh, doi: 10.1017/S1041610220002720 C. (2020). Nursing home residents with (suspected) COVID-19 [Factsheet 1: Nicolas, L. B., Kolb, Y., and Prinssen, E. P. (2006). A combined marble burying- Symptoms and course]. locomotor activity test in mice: a practical screening test with sensitivity to Wachinger, C., Salat, D. H., Weiner, M., and Reuter, M. (2016). Alzheimer’s disease different classes of anxiolytics and antidepressants. Eur. J. Pharmacol. 547, neuroimaging I. Whole-brain analysis reveals increased neuroanatomical 106–115. doi: 10.1016/j.ejphar.2006.07.015 asymmetries in dementia for hippocampus and amygdala. Brain 139, Oddo, S., Caccamo, A., Shepherd, J. D., Murphy, M. P., Golde, T. E., Kayed, 3253–3266. doi: 10.1093/brain/aww243 R., et al. (2003). Triple-transgenic model of Alzheimer’s disease with plaques Wang, H., Li, T., Barbarino, P., Gauthier, S., Brodaty, H., Molinuevo, J. L., and tangles: intracellular A and synaptic dysfunction. Neuron 39, 409–421. et al. (2020). Dementia care during COVID-19. Lancet 395, 1190–1191. doi: 10.1016/S0896-6273(03)00434-3 doi: 10.1016/S0140-6736(20)30755-8 Peterman, J. L., White, J. D., Calcagno, A., Hagen, C., Quiring, M., Paulhus, K., et al. Warneke, L. (1993). Anxiety disorders. Focus on obsessive-compulsive disorder. (2020). Prolonged isolation stress accelerates the onset of Alzheimer’s disease- Can. Fam. Physician 39, 1612–1621. related pathology in 5xFAD mice despite running wheels and environmental Wilson, R. S., Krueger, K. R., Arnold, S. E., Schneider, J. A., Kelly, J. F., Barnes, L. enrichment. Behav. Brain Res. 379:112366. doi: 10.1016/j.bbr.2019.112366 L., et al. (2007). Loneliness and risk of Alzheimer disease. Arch. Gen. Psychiatry Pietropaolo, S., Sun, Y., Li, R., Brana, C., Feldon, J., and Yee, B. K. (2009). Limited 64, 234–240. doi: 10.1001/archpsyc.64.2.234 impact of social isolation on alzheimer-like symptoms in a triple transgenic Wolmarans, D., Stein, D. J., and Harvey, B. H. (2016). Excessive nest mouse model. Behav. Neurosci. 123, 181–195. doi: 10.1037/a0013607 building is a unique behavioural phenotype in the deer mouse model Pinel, J. P. J., and Treit, D. (1978). Burying as a defensive response in rats. J. Comp. of obsessive-compulsive disorder. J. Psychopharmacol. 30, 867–874. Physiol. Psychol. 92, 708–712. doi: 10.1037/h0077494 doi: 10.1177/0269881116645554 Rivera, R. M., and Carballea, D. (2020). Coronavirus: a trigger of OCD and illness Zhao, Q. F., Tan, L., Wang, H. F., Jiang, T., Tan, M. S., Tan, L., et al. anxiety disorder? Psychol. Trauma 12:S66. doi: 10.1037/tra0000725 (2016). The prevalence of neuropsychiatric symptoms in Alzheimer’s disease: Ruscio, A. M., Stein, D. J., Chiu, W. T., and Kessler, R. C. (2010). The systematic review and meta-analysis. J. Affect. Disord. 190, 264–271. epidemiology of obsessive-compulsive disorder in the National Comorbidity doi: 10.1016/j.jad.2015.09.069 Survey Replication. Mol. Psychiatry 15, 53–63. doi: 10.1038/mp.2008.94 Santana-Santana, M., and Giménez-Llort, L. Marble burying revisited under the Conflict of Interest: The authors declare that the research was conducted in the neuroethological perspective. (Submitted). absence of any commercial or financial relationships that could be construed as a Seitz, D., Purandare, N., and Conn, D. (2010). Prevalence of psychiatric disorders potential conflict of interest. among older adults in long-term care homes: a systematic review. Int. Psychogeriatr. 22, 1025–1039. doi: 10.1017/S1041610210000608 Copyright © 2021 Gimenez-Llort and Alveal-Mellado. This is an open-access article Shafran, R., Coughtrey, A., and Whittal, M. (2020). Recognising and addressing distributed under the terms of the Creative Commons Attribution License (CC BY). the impact of COVID-19 on obsessive-compulsive disorder. Lancet Psychiatry The use, distribution or reproduction in other forums is permitted, provided the 7, 570–572. doi: 10.1016/S2215-0366(20)30222-4 original author(s) and the copyright owner(s) are credited and that the original Sulaimani, M. F., and Bagadood, N. H. (2020). Implication of coronavirus publication in this journal is cited, in accordance with accepted academic practice. pandemic on obsessive-compulsive-disorder symptoms. Rev. Environ. Health No use, distribution or reproduction is permitted which does not comply with these doi: 10.1515/reveh-2020-0054 [Epub ahead of print]. terms. Frontiers in Behavioral Neuroscience | www.frontiersin.org 11 January 2021 | Volume 14 | Article 611384