Marble-burying is enhanced in 3xTg-AD mice, can be reversed by risperidone and it is modulable by handling

Behavioural Processes 116 (2015) 69–74

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Short report

Marble-burying is enhanced in 3xTg-AD mice, can be reversed by risperidone and it is modulable by handling Virginia Torres-Lista a,b , Secundino López-Pousa c , Lydia Giménez-Llort a,b,∗ a b c

Behavioral Neuroscience Group, Institute of Neuroscience, Universitat Autònoma de Barcelona, Bellaterra, Spain Department of Psychiatry and Forensic Medicine, School of Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain Research Unit and UVaMiD (Memory and Dementia Assessment Unit), Institut d’Assistència Sanitaria, Salt, Spain

a r t i c l e

i n f o

Article history: Received 15 July 2014 Received in revised form 1 May 2015 Accepted 5 May 2015 Available online 6 May 2015 Keywords: Ethogram Translational neuroscience Behavioural and psychological symptoms of dementia (BPSD) Longitudinal study Handling Antipsychotics QoL

a b s t r a c t Translational research on behavioural and psychological symptoms of dementia (BPSD) is relevant to the study the neuropsychiatric symptoms that strongly affect the quality of life of the human Alzheimer’s disease (AD) patient and caregivers, frequently leading to early institutionalization. Among the ethological behavioural tests for rodents, marble burying is considered to model the spectrum of anxiety, psychotic and obsessive–compulsive like symptoms. The present work was aimed to study the behavioural interactions of 12 month-old male 3xTg-AD mice with small objects using the marble-burying test, as compared to the response elicited in age-matched non-transgenic (NTg) mice. The distinction of the classical ‘number of buried marbles’ but also those left ‘intact’ and those ‘changed’ of position of marbles or partially buried (the transitional level of interaction) provided new insights into the modelling of BPSD-like alterations in this AD model. The analysis revealed genotype differences in the behavioural patterns and predominant behaviors. In the NTg mice, predominance was shown in the ‘changed or partially buried’, while interactions with marble were enhanced in 3xTg-AD mice resulting in an increase of marble burying. Besides, genotype-dependent meaningful correlations were found, with the marble test pattern of 3xTg-AD mice being directly related to neophobia in the corner tests. In both genotypes, the increase of burying was reversed by chronic treatment with risperidone (1 mg/kg, s.c.). In 3xTg-AD mice, the repetitive handling of animals during the treatment also exerted modulatory effects. These distinct patterns further characterize the modelling of BPSD-like symptoms in the 3xTg-AD mice, and provide another behavioural tool to assess the benefits of preventive and/or therapeutic strategies, as well as the potential action of risk factors for AD, in this animal model. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Management of the diversity of neuropsychiatric symptoms and behavioural disturbances, quite prevalent among demented patients and occurring from 50 to 90% on those suffering Alzheimer’s disease (AD), is a challenge for clinicians (Ballard and Corbett, 2010). These non-cognitive problems are known to be the most important source of distress and impose a severe burden upon patients and caregivers (i.e. Tan et al., 2005) affecting their quality of life (Shin et al., 2005) and frequently leading to premature institutionalization (Hope et al., 1998). Anxieties and phobias, para-

∗ Corresponding author at: Unitat de Psicologia Mèdica, M5-133, Departament de Psiquiatria i Medicina Legal, Facultat de Medicina, Edifici M, Avinguda Can Domènech, Campus Bellaterra, s/n Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. Tel.: +34 93 5812378; fax: +34 93 5811435. E-mail address: [email protected] (L. Giménez-Llort). http://dx.doi.org/10.1016/j.beproc.2015.05.001 0376-6357/© 2015 Elsevier B.V. All rights reserved.

noia and delusion, hallucinations, stereotyped behaviour and other activity disturbances, aggressiveness, diurnal rhythm alterations, affective disorders are the clusters distinguished by behavioural assessment scales at the clinical level (Reisberg et al., 1987), being commonly referred to as “behavioural and psychological symptoms of dementia (BPSD)”. The complexity of these clinical manifestations has rendered them difficult to model or to find their translational parallel in animals (Giménez-Llort et al., 2007). In spite of being considered an important subject of interest in preclinical pharmacological screening (Anand et al., 2014) still a scarce number of experimental studies address BPSD-like behaviours as compared to the huge literature focused on the cognitive hallmarks of disease (Giménez-Llort et al., 2007). In this context, natural species-typical behaviours that represent active interaction with the environment are excellent ethological scenarios to reflect cognitive and non-cognitive disturbances induced by disease. In agreement with this, we have recently reported impair-

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ment of nesting behaviour in 3xTg-AD mice in an attempt to model the impact of disease on naturally occurring executive functions and daily life activities (Torres-Lista and Giménez-Llort, 2013). In the present work, we have studied other spontaneous natural behaviours exhibited by rodents when they interact with small objects, in this case in an environment that facilitates digging, burrowing and burying (Pinel and Treit, 1978). Since these behaviours are found altered in animal models for some neuropsychiatric diseases, like obsessive compulsive disorder, anxiety and autism spectrum disorders (see below), we analyzed the impact of the Alzheimer’s disease genotype on them as compared to the normal pattern shown by the wild-type strain. We also searched for meaningful correlations with other BPSD-like behaviours expressed in other classical unconditioned tests, its reversal by antipsychotic chronic treatment and the influence of the repeated handling procedure. In the wild, mice dig in the ground to find food, to hoard food, to bury noxious as well as harmless objects, to create a refuge from predators or cold and to make a safe nursery area for the young (Poling et al., 1981; Deacon, 2006). This characteristic behaviour described as ‘defensive behaviour’ (Pinel and Treit, 1978) is considered to reflect the anxiety state of animals (Nicolas et al., 2006). However, in the laboratory, mice dig vigorously in deep bedding such as wood chips and se bedding material to bury food pellets or small objects (i.e. marble pieces) they may find. The ‘Marble-burying test’ is based in this behavioural ethogram and the inhibition of the marble-burying were originally suggested as a screening test for anxiolytic activity (Broekkamp et al., 1986; Njung’e and Handley, 1991). Later it has been proposed to better model meaningless repetitive and perseverative behaviours characteristic of obsessive-convulsive disorder (ODC) (i.e. Thomas et al., 2009) or autism spectrum disorders (ASD) (i.e. Angoa-Pérez et al., 2013). Marble-burying is also reported to have predictive validity for the screening of novel antidepressants (i.e. Nicolas et al., 2006) and antipsychotics (i.e. Bruins Slot et al., 2008) since these compounds also reduce marble burying. Such a wide behavioural profile renders the marble burying test an interesting tool for the study of BPSD-like behaviours characteristic of the advanced stages of Alzheimer’s disease. Therefore, the present study was aimed at characterizing the behavioural response of male triple-transgenic 3xTg-AD mice harbouring familial AD mutations PS1/M146V, A␤APPSwe and tauP301L (Oddo et al., 2003) in the marble burying test (Broekkamp et al., 1986) as behavioural manifestation of BPSD-like symptoms at an age mimicking advanced stages of Alzheimer’s disease (Giménez-Llort et al., 2007). Finally, we studied the effect of chronic administration of a low dose of risperidone, the most frequently prescribed antipsychotic drug in patients with AD (Nobili et al., 2009). This neuroleptic is used for treating associated behavioural symptoms, such as psychosis, psychomotor agitation, aggression, and delusions (Devanand et al., 2012; Wessels et al., 2010). Moreover, risperidone is associated with decrease presence of aggressive behaviour in APP23 mice model for AD (Vloeberghs et al., 2008). On parallel, the influence of the repeated handling procedure during the chronic treatment was also considered, as such a kind a manipulation has been shown to exert anxiolytic effects in rodents (Boix et al., 1988).

2. Materials and methods 2.1. Animals A total number of thirty-two 12-month-old male mice from the Spanish colonies of homozygous 3xTg-AD and NTg mice established in the Medical Psychology Unit, Autonomous University of

Fig. 1. Methodological conditions and spatial setup used for the quantitative and qualitative evaluation of the interactions of the animal with marbles in the Marble Burying Test.

Barcelona (Giménez-Llort et al., 2006) were used in this study (n = 16 each genotype). 3xTg-AD mice harbouring transgenes were genetically engineered at the University of California Irvine, as previously described (Oddo et al., 2003). Animals were maintained in groups of 4 mice per cage (Macrolon, 35 × 35 × 25 cm) filled with 5 cm of clean wood cuttings (JRS Lignocel HBK, Harlan Iberica, SL) under standard laboratory conditions of food and water ad lib, 22 ± 2 ◦ C, 12 h light:dark cycle and relative humidity 50–60%. Behaviour in the (1) corner test, (2) open-field test, and (3) marbleburying test was assessed under dim white light (20 lx) during their light phase of the light:dark cycle (from 10 a.m. to 1 p.m.). Evaluation was performed by both direct observation and analysis of VHS video-tape recorded images blind to the genotype and in agreement with the ARRIVE guidelines by the NCR3Rs and their aim to reduce the number of animals used (Kilkenny et al., 2010). Research was done in accordance with Spanish legislation on “protection of animals used for experimental and other scientific purposes” and the European communities council directive (2010/63/UE) on this subject. 2.2. Apparatus and procedure Neophobia to a new home-cage was assessed in the corner test by introducing the animal into the centre of a standard home-cage and counting the number of visited corners and rearings during a period of 30 s. Latency of the first rearing was also recorded. Immediately after, animals were assessed for locomotor activity and anxiety/emotionality in an open field (woodwork, white, 50 × 50 × 35 cm height). The animals were placed in the centre of the apparatus and observed for 5 min. Horizontal (crossings of 10 × 10 cm squares) and vertical (rearings) locomotor activities, the latency of self-grooming, the number of defecation boli and the presence of urination were also recorded.

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Fig. 2. Marble interaction and other BPSD-like behaviours in 12 month-old 3xTg-AD and NTg male mice (mean ± SEM). n = 16 per group. (A) Corner Test, (B) Open Field Test and (C) Marble Burying Test. Statistics: Genotype effect: Student’s t-test, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. NTg mice. Predominance of behaviours (P): ANOVA ***P < 0.001. Post-hoc Student–Knewman–Keuls, a p < 0.05 vs. intact marble; b p < 0.05 vs. changed of position.

On the next day, animals were assessed in the marble-burying test (Broekkamp et al., 1986). The behavioural interaction of animals with marbles considered not only the number of buried marbles but also the number of marbles left intact and those changed of position or partially buried. In order to reduce confounding factors such as horizontal and vertical locomotor activity or digging (Nicolas et al., 2006; Thomas et al., 2009), the number of pieces of marble was reduced to 10 and allocated in a half of the test cage, as illustrated in Fig. 1. Thereafter, we searched for meaningful correlations between the three variables and with other behavioural variables recorded in classical tests for anxiety such as the corner and open-field test. Thereafter, animals of each genotype were randomly distributed in two treatment groups (n = 8 each group) and handled everyday for 3 weeks for chronic subcutaneous administration of saline or risperidone (1 mg/kg, at 3 p.m.). Risperidone (Janssen–CILAG, Spain) had been previously dissolved in a vehicle of 0.9% saline solution for chronic injection in a volume of 1 ml/kg. The behavioural effects of the antipsychotic treatment were assessed in the marble test using the same procedure, as before, and as compared to saline treated animals. The pieces of marble were thoroughly cleaned after each test. As illustrated in Fig. 1, mice were placed individually in a standard home-cage facing the wall. In this test, the cage contained 10 glass marbles (1 × 1 × 1 cm) evenly spaced (four rows of four, three, two and one marbles per row only in the right area of the cage) on a 5 cm thick layer of clean woodcuttings. The set up was like the one used in the corner test except for the presence of marble pieces and the starting position. The mice were left in the cage with marbles for a 30 min period after which the test was terminated by

removing the mice. Three different kinds of interaction were considered in order to measure the number ‘Buried’ marbles (those 100% buried by wood cuttings), those left ‘Intact’ and the remaining subset considered as those ‘Changed of position’ or partially buried. Parametric statistical analysis is presented to unify all the analysis since those variables requiring non-parametric statistics showed the same statistical differences. Genotype differences were analyzed with Student’s t-test. ANOVA with post-hoc Newman–Keuls were used to evaluate predominance of behaviours. Pearson’s correlation was applied to find meaningful correlations between and within the variables of the behavioural tests. Paired t-test was used to analyse differences in the same animals before and after the respective treatment. p < 0.05 was considered statistical significant. Behavioural responses of age-matched non-transgenic mice (NTg) with the same background (hybrid 129/C57BL6 strain) were used to study these relationships and for comparison with the 3xTg-AD phenotype. 3. Results and discussion At this advanced stages of the disease, presence of BPSD-like symptoms related to anxiety were clearly exhibited by 3xTg-AD mice in both the corner and the open-field test, in agreement with previous reports (i.e. Giménez-Llort et al., 2007; García-Mesa et al., 2011). The most relevant variables are illustrated in Fig. 2. Increased neophobia was shown in the three variables of the corner test (Fig. 2A) as a reduction of visited corners and in longer latency of rearing with consequent reduced number of vertical

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Fig. 3. Meaningful significant Pearson r correlations between measures within the Marble Burying Test and with the Corner and the Open Field tests. **p < 0.01 and *p < 0.05.

exploration. Similarly, in the open-field test, 3xTg-AD mice exhibited reduced total horizontal and vertical exploratory activities as compared to NTg animals. Among the variables related to emotionality, the genotype differences were shown as an increased latency of self-grooming behaviour and increased incidence of urination in 3xTg-AD mice. (Fig. 2B) [all Student t-test, 30 df, *p < 0.05, **p < 0.01 and ***p < 0.001]. The qualitative (three levels of interaction) and quantitative (number) analysis of the marble-burying test showed that in the cages of NTg mice, half of the marbles were found changed of position in the arena or partially buried (Fig. 2C). The other half of marbles were either left intact or buried, in equal parts (25%), what made that the quantitative results in these two variables be undistinguishable [predominance, ANOVA, F(2,47) = 10.96, ***p < 0.001; post-hoc a p < 0.05 vs. intact; b p < 0.05 vs. change of position]. Therefore, in contrast to the standard quantitative evaluation protocol, the consideration of several levels of interaction with small objects enabled to uncover the predominant behaviours. That is, in the standard wild-type strain the most common behavioural interaction did not result in complete burying of the marbles but their change of position or partial burying. On the other hand, the distinction of several levels of interactions can be useful as it allows

to control the level of basal ceiling effect in our sample. This may also help to increase the sensitivity to detect strain or genotype differences and treatment effects in this test (Pinel and Treit, 1978; Poling et al., 1981). Interestingly, in the 3xTg-AD group, a noticeable gradient in the frequency of the three different levels of interactions with marble glasses were found [ANOVA, F(2,47) = 50.51, ***p < 0.001]. Here, burying was the predominant behaviour, followed by change of position and with only 10% of glasses remaining intact [post-hoc, a p < 0.05 vs. intact marble; b p < 0.05 vs. changed of position]. It is important to note that not only burying, but each one of the three levels of marble interactions were able to evidence the existence of genetic differences, with burying and change of position showing equal statistical potency [Student t-test’s: intact, t = 3.363, 30 df, **p < 0.01; changed of position, t = 3.667, 30 df, ***p < 0.001; t = −6.848, 30 df, ***p < 0.001]. This is the first time that an animal model of AD reproduces BPSD-like symptoms (increase) in the marble burying test. The other study assessing transgenic mice for AD in the marble burying test reported that the Tg-APP/PS1 mice buried fewer marbles than its non-transgenic counterparts (Kim et al., 2012). A part of the genetic differences between the two models, in that work results of both male and female genders were pooled

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and that could also contribute to explain the discrepancy with the present results in the 3xTg-AD mice. Meaningful correlations were searched between the three kind of marble interactions and the other behavioural variables recorded in the corner and open-field tests (Fig. 3). The number of marbles left ‘Intact’ only had correlations in the 3xTg-AD genotype [Pearson’s correlation, with visited corners, r = 0.630, **p < 0.01; with rearings, r = 0.523, *p < 0.05, respectively]. The number of marbles that NTg mice ‘Changed’ of position inversely correlated with the number of rearings in the open-field test [Pearson’s correlation, 30 df, r = −0.530, *p < 0.01] what would be feasible with being competing behaviours. In contrast, the number of those that they ‘Buried’ correlated with the latency of rearing in the corner test [Pearson’s correlation, 30 df, r = −0.549, **p < 0.01] as well as the number of rearings [r = 0.520, **p < 0.01] and emotionality (incidence of urination) in the open field test [r = 0.554, **p < 0.01]. No correlations were found for ‘Buried’ marbles in the 3xTg-AD mice. Interestingly, number of marbles ‘Changed’ of position or partially buried, considered as the transitional level of interaction between those left ‘Intact’ and those ‘Buried’, were inversely correlated to the number of one or another, depending on the genotype, NTg and 3xTg-AD, respectively [Pearson’s correlation, 30 df, r = −0.718, **p < 0.01 and r = −0.915, **p < 0.01, respectively]. The correlations do also suggest that the three categories that evaluate the level of interaction are not equally related to other variables of the behavioural profile. These correlations found in NTg mice between burying and the vertical activity provide new insights in the scientific discussion about the sensitivity of burying to assess anxiety or rather model compulsive behaviours. Some authors (i.e. Albelda and Joel, 2012) consider that burying may begin as an appropriate investigative activity but becomes frustrating due to the non-reactive nature of the marble glasses, leading to compulsive burying. As mentioned above, later studies have provided further support for the sensitivity of marble burying to anxiolytic drugs. Thus, the finding that burying was reduced by serotonin reuptake inhibitors and antipsychotics raised the possibility that this behaviour may be related with a model of neophobia, anxiety and meaningless stereotyped behaviour (Broekkamp et al., 1986; Kaurav et al., 2012). Overall, these functional relationships provide feasible interpretations in the understanding of the behaviours developed by 3xTg-AD mice in the marble burying test. The results reinforce the anxiouslike phenotype of this model, first described in our laboratory (Giménez-Llort et al., 2006) and that we have demonstrated to be related to the neuropathological affectation of basolateral amyg˜ et al., 2010). Also, they indicate that it is important to dala (Espana discriminate the different behavioural components (i.e. ‘intact’ and ‘changed of position’) that in the classical marble burying test are considered as a single measure complementary of burying. This distinction allowed us to further identify qualitative and quantitative differences it the behavioural pattern of the 3xTg-AD mice in terms of non-social interactions. In this case, interactions with small objects strongly correlate with its prominent anxious-like profile that extent the ability of this animal model to mimic such BPSD-like symptoms (Giménez-Llort et al., 2007). In a longitudinal approach, marble interaction was assessed again after 3 weeks of chronic treatment with 1 mg/ml, a low dose, of risperidone, a neuroleptic of standard clinical Use in geriatric and AD patients. This allowed us to study the effects of the drug and, on parallel, to do a pharmacological validation of the results obtained in the test. At this low, non-cataleptic dose, risperidone is reported to significantly reduce the number of buried marbles (Bardin et al., 2006). As illustrated in Fig. 4, the genetic differences found before treatment disappeared after chronic treatment, while treatment effect was shown in the number of intact and buried marbles [ANOVA, F(1,31) = 11.656 and F(1,31) = 12.185, respectively,

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Fig. 4. Marble interaction in 12 month-old 3xTg-AD and NTg male mice before and after chronic treatment with risperidone (1 mg/kg, s.c.) (mean ± SEM). n = 8 per group. (A) NTg mice, (B) 3xTg-AD mice. Open bars: Saline treated groups; black bars: Risperidone treated groups. Predominant behaviours is/are pointed out with cercle/elipses. Shift to the L, shift to the left (increase in favour of less interactive behaviours); Shift to the R, shift to the right (increase in favour to more interactive behaviours). The arrows inside the bars illustrate the increase or decrease of a particular marble interaction as compared to its ‘before treatment’ level. ‘Statistics: G, Genotype effect; T, Treatment effect, ANOVA, **p < 0.01 and ***p < 0.001 vs. counterparts. Paired t-test, a p < 0.05 vs. before treatment’.

and both p < 0.01]. In NTg mice, saline induced a shift to the Right on the behavioural pattern, that is, an increase in favour of more interactive behaviours, as shown by the reduction of the number of intact marbles and the increase of buried ones [paired t-test, t = 4,710 and t = −3.036, respectively, df 7 and p < 0.05 in both cases]. As seen in the correlations, the increase was concomitant with an increase of rearings and emotionality, that suggest an overall increase of activity due to repeated treatment with saline. Oppositely, a shift to the left, with reduction of the interactions with marbles was shown in both genotypes after treatment with risperidone [paired t-test, *p < 0.05]. Interestingly, the 3xTg-AD mice also showed a shift to the left on their behavioural pattern by effect of chronic manipulation per se (treatment with saline) although the effect was only shown as a reduction of buried marbles. This effect of chronic saline s.c. injection would be in agreement with the longterm behavioural effects of repetitive handling which is known to reduce anxiety-like behaviours and other measures of emotionality (Boix et al., 1988; Fernández-Teruel et al., 2002). Overall, as a result of the shifts of the behavioural patterns (the predominance of a ‘change of position’ in NTg mice; the predominance of burying in 3xTg-AD mice), genotype effects were completely lost. This also indicates that the benefits were found in the group of animals with higher expression of behavioural interaction with marbles, that is with an anxious-like profile.

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In summary, the present work describes the increase of marbles burying in 3xTg-AD at advanced stages of the disease and it relates it with anxious-like behaviours. Pharmacological reversal of behaviours by chronic administration of a low dose of risperidone was demonstrated in both genotypes. Besides, the modulatory effects of repetitive handling on the activity in the marble test were seen in the 3xTg-AD mice. The results present the marble burying response of 3xTg-AD mice as an additional tool for translational evaluation of BPSD-like behaviours in relation to preventive and/or therapeutical strategies targeted at Alzheimer’s disease. It may also have a role in the evaluation of the potential risk factors for the disease. Acknowledgements This study was supported by Instituto de Salud Carlos III: ISC3 PI10/00283 and ‘Conveni de Col·laboració UAB-IAS, 2009’. We belong to RETICEF, Red de Envejecimiento y Fragilidad (Thematic Network of Collaborative Research on Ageing and Frailty) RD12/0043/0018. We thank Helga Rivas for her skilful assistance. We thank Prof. Frank M LaFerla Institute for Memory Impairments and Neurological Disorders, Department of Neurobiology and Behavior, University of California, Irvine, USA, for kindly providing after MTA to L.G-LL. the progenitors of the Spanish colonies. References Albelda, N., Joel, D., 2012. Animal models of obsessive–compulsive disorder: exploring pharmacology and neural substrates. Neurosci. Biobehav. Rev. 36, 47–63. Anand, R., Gill, K.D., Mahdi, A.A., 2014. Therapeutics of Alzheimer’s disease: past, present and future. Neuropharmacology 76, 27–50. Angoa-Pérez, M., Kane, M.J., Briggs, D.I., Francescutti, D.M., Kuhn, D.M., 2013. Marble burying and nestlet shredding as tests of repetitive, compulsive-like behaviors in mice. J. Vis. Exp. 82, e50978. Ballard, C., Corbett, A., 2010. Management of neuropsychiatric symptoms in people with dementia. CNS Drugs 24, 729–739. Bardin, L., Kleven, M.S., Barret-Grévoz, C., Depoortère, R., Newman-Tancredi, A., 2006. Antipsychotic-like vs cataleptogenic actions in mice of novel antipsychotics having D2 antagonist and 5-HT1A agonist properties. Neuropsychopharmacology 31, 1869–1879. ˜ A., 1988. The anxiolytic action of Boix, F., Fernández-Teruel, A., Tobena, benzodiazepines is not present in handling-habituated rats. Pharmacol. Biochem. Behav. 31, 541–546. Broekkamp, C.L., Rijk, H.W., Joly-Gelouin, D., Lloyd, K.L., 1986. Major tranquillizers can be distinguished from minor tranquillizers on the basis of effects on marble burying and swim-induced grooming in mice. Eur. J. Pharmacol. 126, 223–229. Bruins Slot, L.A., Bardin, L., Auclair, A.L., Depoortere, R., Newman-Tancredi, A., 2008. Effects of antipsychotics and reference monoaminergic ligands on marble burying behavior in mice. Behav. Pharmacol. 19, 145–152. Deacon, R.M., 2006. Digging and marble burying in mice: simple methods for in vivo identification of biological impacts. Nat. Protoc. 1, 122–124. Devanand, D.P., Mintzer, J., Schultz, S.K., Andrews, H.F., Sultzer, D.L., de la Pena, D., Gupta, S., Colon, S., Schimming, C., Pelton, G.H., Levin, B., 2012. Relapse risk after discontinuation of risperidone in Alzheimer’s disease. N. Engl. J. Med. 367, 1497–1507. ˜ J., Giménez-Llort, L., Valero, J., Minano, ˜ Espana, A., Rábano, A., Rodriguez-Alvarez, J., LaFerla, F.M., Saura, C.A., 2010. Intraneuronal beta-amyloid accumulation in the amygdala enhances fear and anxiety in Alzheimer’s disease transgenic mice. Biol. Psychiatry. 67, 513–521.

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