Genes, Brain and Behavior (2015) 14: 145–157

doi: 10.1111/gbb.12192

Decreased aggression and increased repetitive behavior in Pten haploinsufficient mice A. E. Clipperton-Allen and D. T. Page∗ Department of Neuroscience, The Scripps Research Institute, Jupiter, FL, USA *Corresponding author: D. T. Page, Department of Neuroscience, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA. E-mail: [email protected]

Aggression is an aspect of social behavior that can be elevated in some individuals with autism spectrum disorder (ASD) and a concern for peers and caregivers. Mutations in Phosphatase and tensin homolog (PTEN), one of several ASD risk factors encoding negative regulators of the PI3K–Akt–mTOR pathway, have been reported in individuals with ASD and comorbid macrocephaly. We previously showed that a mouse model of Pten germline haploinsufficiency (Pten+/− ) has selective deficits, primarily in social behavior, along with broad overgrowth of the brain. Here, we further examine the social behavior of Pten+/− male mice in the resident–intruder test of aggression, using a comprehensive behavioral analysis to obtain an overall picture of the agonistic, non-agonistic and non-social behavior patterns of Pten+/− mice during a free interaction with a novel conspecific. Pten+/− male mice were involved in less aggression than their wild-type littermates. Pten+/− mice also performed less social investigation, including anogenital investigation and approaching and/or attending to the intruder, which is consistent with our previous finding of decreased sociability in the social approach test. In contrast to these decreases in social behaviors, Pten+/− mice showed increased digging. In summary, we report decreased aggression and increased repetitive behavior in Pten+/− mice, thus extending our characterization of this model of an ASD risk factor that features brain overgrowth and social deficits. Keywords: Aggression, autism, Pten, repetitive behavior, resident–intruder Received 19 August 2014, revised 25 November, 23 september and 16 october 2014, accepted for publication 1 December 2014

Autism spectrum disorder (ASD) is characterized by social behavior and communication deficits, and repetitive, stereotyped, restricted patterns of behavior (APA 2005, 2013; WHO 1992). This neurodevelopmental disorder is highly heritable (∼2:1 concordance ratio in monozygotic/dizygotic twins; Bohm et al. 2013), sexually dimorphic (∼80% male; Perou et al. 2013) and has a prevalence of ∼1–2% in the general population (Perou et al. 2013).

Aggression, a frequent concern in the treatment of individuals with ASD, is associated with increased likelihood of admission to long-term residential facilities, decreased quality of life and increased stress in parents, siblings, peers and caregivers (Gardner & Moffatt 1990; Lakin 1983; Lecavalier et al. 2006; Matson & Nebel-Schwalm 2007; Tomanik et al. 2004). Although prevalence estimates for aggression in ASD are complicated by the variety of scales and methodologies used across different studies (reviewed in Matson & Nebel-Schwalm 2007), up to 70% of individuals with ASD may show aggression toward others (Hartley et al. 2008; Holden & Gitlesen 2006; Kanne & Mazurek 2011; Maskey et al. 2013; McClintock et al. 2003). However, the neurobiological mechanisms underlying aggression in ASD are unclear. Many genetic risk factors for ASD have been identified, including several phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)–Protein kinase B–mammalian target of rapamycin (mTOR) pathway negative regulators (e.g. TSC1/2, NF1, FMR1, PTEN; Abrahams & Geschwind 2008; Bourgeron 2009; de Vries 2010). Mutations in Phosphatase and tensin homolog (PTEN) have been reported in ASD individuals with head circumference >2 standard deviations above normal (macrocephaly); prevalence estimates range from 7% to 17% in this population (Butler et al. 2005; Buxbaum et al. 2007; Klein et al. 2013; McBride et al. 2010; Varga et al. 2009). We have previously reported that Pten germline haploinsufficient (Pten+/− ) mice show surprisingly specific behavioral deficits given their widespread brain overgrowth (Clipperton-Allen & Page 2014; Page et al. 2009). Pten+/− mice of both sexes show deficits in social behavior, and males perform more repetitive behavior (marble burying). Sex-specific phenotypes in several behaviors associated with ASD comorbidities, including mood, anxiety, circadian activity and emotional learning, were also found. Taken together, the broad brain overgrowth and selective behavioral impairments suggest that brain regions and/or constituent cell types involved in social behavior are more sensitive and less adaptable to changes in brain growth trajectory (Clipperton-Allen & Page 2014). To investigate whether the observed deficits in social approach and recognition extend to differences in behavior during a free social interaction task, we tested male Pten+/− and wild-type (Pten+/+ ) littermates on a standard laboratory test of aggression, the resident–intruder paradigm, in which a resident mouse has a novel conspecific intruder placed into its home cage, and aggressive behavior is measured (e.g. Clipperton-Allen et al. 2010, 2011; Guillot & Chapouthier 1996; Holmes et al. 2002). We used a comprehensive behavioral analysis to determine the effects of Pten haploinsufficiency on agonistic, non-agonistic and non-social behavior, and to obtain an overall picture of the behavioral

© 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society

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Clipperton-Allen and Page Table 1: Descriptions of scored behaviors Behavior Mutual agonistic behaviors Aggressive postures Reciprocal attacks Agonistic behaviors delivered by resident Resident-initiated fighting Attacks delivered Dominant behavior Chasing the intruder Agonistic behaviors delivered by intruder Intruder-initated fighting Attacks received Submissive behavior

Avoidance of the intruder Defensive upright posturing Non-agonistic social behaviors Social inactivity Oronasal investigation Body investigation Anogenital investigation Stretched approaches Approaching and/or attending to the intruder Non-social behaviors Horizontal exploration Vertical exploration Digging Solitary inactivity Self-grooming Abnormal stereotypies

Description Physical attacks which include box/wrestle, offensive and defensive postures, lateral sideways threats and tail rattle. Physical attacks with a locked fight, including tumbling, kick-away and counterattack, where the attacker cannot be identified. Multiple consecutive physical attacks, including dorsal/ventral bites, kick-away, tumbling and counterattack, initiated by the resident. Count of, and latency to, individual physical attacks by the resident, including bites to dorsal/ventral regions. The resident mouse is in control; includes pinning of the intruder, aggressive grooming, crawling over or on top, and mounting attempt; reciprocal to ‘submissive behavior’. The resident mouse actively follows, or pursues and chases the intruder; reciprocal to ‘avoidance of the intruder’. Multiple consecutive physical attacks, including dorsal/ventral bites, kick-away, tumbling and counterattack, initiated by the intruder. Count of, and latency to, individual physical attacks by the intruder, including bites to dorsal/ventral regions. The intruder is in control; includes resident crawls under, supine posture (ventral side exposed), prolonged crouch and any other behavior in which the intruder is dominant (e.g., the intruder pins, aggressive grooming of the resident); reciprocal to ‘dominant behavior’. The resident withdraws and runs away from the intruder while the intruder is chasing; reciprocal to ‘chasing the intruder’. Species-typical defensive behavior; upright with the head tucked and the arms ready to push away. Includes sit/lie/sleep together. Active sniffing of the intruder’s oronasal area. Active sniffing of the intruder’s body. Active sniffing of the intruder’s anogenital region. Risk assessment behavior; back feet do not move and front feet approach the intruder. Only frequency and latency were measured. Often from across the cage; the resident’s attention is focused on the intruder, head tilted toward the intruder and/or movements toward the intruder; this becomes ‘chasing the intruder’ once along the tail or investigation if within 1.5 cm of the intruder. Movement around the cage, includes active sniffing of air, ground. Movement to investigate upwards, both front feet off the ground; includes sniffing, wall leans and lid chews (less than 3). Rapid stereotypical movement of forepaws in the bedding. No movement; includes sit, lie down and sleep. Rapid movement of forepaws over facial area and along body. ‘Strange’ behaviors, including spinturns, repeated jumps/lid chews/head shakes (more than 3).

patterns of Pten+/− mice during interaction with a novel conspecific.

Materials and methods Subjects Mice of the B6.129-Ptentm1Rps line (Podsypanina et al. 1999) were obtained from the repository at the National Cancer Institute at Frederick, where they were already backcrossed onto a congenic C57BL/6J background by the Donating Investigator. The line has been maintained by backcrossing to C57BL/6J mice for more than 10 generations. Mice used in this study were generated by crossing Ptentm1Rps/+ (Pten+/− ) mice with wild-type (Pten+/+ ) mice.

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From weaning until 7 days before testing, mice were housed in groups of 4–5 in clear polyethylene cages (19.1 × 29.2 × 12.7 cm3 ; Allentown Inc., Allentown, NJ, USA) on ventilated racks (Model No. MD75JU160MVPSHR, Allentown Inc.) and provided with 1∕4′′ corncob bedding, nestlets and ad libitum water and food (Teklad Global 18% Protein Extruded Rodent Diet 2920X, Harlan Laboratories, Indianapolis, IN, USA). Seven days prior to testing, experimental mice were separated and housed individually, with no cage changes but under otherwise normal conditions. This isolation allowed test mice to establish a territory, and increased the likelihood of attack (e.g. Dahlhaus & El-Husseini 2010; Ginsburg & Allee 1942; Uhrich 1938), which was particularly important because C57BL/6 males are less aggressive than several other inbred and outbred strains (Guillot & Chapouthier 1996; Parmigiani et al. 1999; Peters et al. 1972). Male Pten+/+ intruder mice [postnatal day 60 (P60) to P90] were housed in Genes, Brain and Behavior (2015) 14: 145–157

Aggression and repetitive behavior in Pten+/− mice Table 2: Descriptions of categories and composite behaviors Behavior

Description

Total activity

Total duration, frequency and latency to the first of all active behaviors, both social and non-social. Excluded from this composite behavior are inactive alone, inactive together and self-grooming. Total duration, frequency and latency to the first of any behavior involving both the resident and the intruder, including aggressive postures, reciprocal attacks, resident-initiated fighting, attacks delivered, dominant behavior, chasing the intruder, intruder-initiated fighting, attacks received, avoidance of the intruder, submissive behavior, defensive upright posturing, social inactivity, oronasal investigation, body investigation, anogenital investigation, stretched approaches and approaching and/or attending to the intruder. Total duration, frequency and latency to the first of aggressive postures, reciprocal attacks, resident-initiated fighting, attacks delivered, dominant behavior, chasing the intruder, intruder-initiated fighting, attacks received, avoidance of the intruder, submissive behavior and defensive upright posturing. This composite behavior represents the overall levels of agonism present in the resident–intruder interactions and does not indicate the direction of the agonistic behavior (i.e., whether agonistic behavior is directed toward the resident or toward the intruder). Total duration, frequency and latency to the first of resident-initiated fighting, attacks delivered, dominant behavior and chasing the intruder. Total duration, frequency and latency to the first of intruder-initiated fighting, attacks received, avoidance of the intruder, submissive behavior and defensive upright posturing. Agonistic behavior delivered minus agonistic behavior received. A negative score indicates that the resident was the submissive animal in the pair, while a positive score signifies that the resident was the dominant mouse. Total duration, frequency and latency to the first of oronasal investigation, body investigation, anogenital investigation, stretched approaches and approaching and/or attending to the intruder. Total duration, frequency and latency to the first of any behavior involving only the resident, including horizontal exploration, vertical exploration, digging, solitary inactivity, self-grooming and abnormal stereotypies. Total duration, frequency and latency to the first of horizontal exploration, vertical exploration and digging. Total duration, frequency and latency to the first of solitary inactivity and self-grooming.

Total social behaviors

Total agonistic behaviors

Agonistic behaviors delivered Agonistic behaviors received Dominance score

Social investigation

Total non-social behaviors

Non-social locomotor behaviors Non-social non-locomotor behaviors

groups of four throughout the experiment. Male Pten+/− and Pten+/+ mice were tested during the dark (active) phase of the 12:12 h reversed light/dark cycle (lights on at 2230 h) in adulthood (P82 to P112). Heavier animals are typically dominant in these interactions (e.g. Ginsburg & Allee 1942; Uhrich 1938), but as we have previously shown that Pten+/− mice have normal body weight (Clipperton-Allen & Page 2014; Page et al. 2009), it is unlikely that weight discrepancies between genotypes affected the test. Furthermore, intruders did not grossly differ in size, weight or age from each other or from the residents. While both sexes show agonistic behavior (e.g., Clipperton-Allen et al. 2010, 2011; Uhrich 1938), overt aggression, including attacks and aggressive postures, is almost exclusively performed by males, especially in the background strain of our germline Pten+/− mice, C57BL/6 (Parmigiani et al. 1999). As these are the typical measures of agonistic behavior studied in the mouse resident–intruder test, and because of the broader social deficits in Pten+/− males, as well as to sex differences in aggression (Eagly & Steffen 1986; Knight et al. 1996; Potegal & Archer 2004) and in ASD (Perou et al. 2013), only male Pten+/− and Pten+/+ mice were tested. All research was approved by The Scripps Research Institute’s Institutional Animal Care and Use Committee and conducted in accordance with National Institutes of Health and Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines.

Procedure At least 1 h before testing, all residents and intruders were moved into the testing room and left undisturbed. In their active (dark) phase, Genes, Brain and Behavior (2015) 14: 145–157

each resident mouse had a Pten+/+ male intruder placed into his home cage under red light conditions. Intruders were painted with non-toxic white block printing ink (3503 – White water soluble ink, Speedball, Statesville, NC, USA) to assist in identification during behavioral scoring. The mice were left undisturbed to freely interact for 15 min while being videotaped from above through clear acrylic lids.

Behavioral analysis In addition to overt attacks or fighting, other agonistic, non-agonistic and non-social behaviors are being increasingly included in the analysis of the resident–intruder paradigm (e.g., Bortolato et al. 2013; Clipperton-Allen et al. 2010, 2011; Meng et al. 2011; Pietropaolo et al. 2004; Pobbe et al. 2010, 2012; Scattoni et al. 2011; Wang et al. 2011). Thus, the recorded social interactions were scored for 23 behaviors based on the ethogram by Grant and Mackintosh (1963; see Table 1 for behavior descriptions, modified from Clipperton et al. 2008; Clipperton-Allen et al. 2010, 2011) by an expert observer, who was unaware of the animals’ genotype, using The Observer XT 10 Video Analysis software (Noldus Information Technology, Wageningen, the Netherlands). Behavioral analysis was focused on the resident (experimental) mouse, with the behavior of the intruder collected only in relation to the behavior of the resident and in reciprocal pairs of behaviors (Clipperton-Allen et al. 2010, 2011). Typically, dominant behavior on the part of one mouse was met by submissive behavior by the other. To gain an overall impression of the animals’ behavior, individual behaviors were also combined to form 10 categories of individual behaviors (see Table 2, modified from Clipperton et al. 2008;

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Figure 1: Agonistic behaviors performed by the resident toward the intruder during the 15-min resident–intruder test. (a) Dominance score (frequency); (b) delivered agonistic behavior (frequency); (c–e) attacks delivered (frequency, c; frequency by 5-min intervals, d; latency, e); (f–h) resident-initiated fighting (duration, f; frequency by 5-min intervals, g; latency, h). Means + SEM shown; Pten+/+ , yellow bars and triangles; Pten+/− , blue bars and triangles. Gray dotted line indicates the maximum latency of 900 seconds (i.e., behavior was not performed). *Significant difference between Pten+/− and Pten+/+ male mice, P < 0.05. #Significant difference from zero (no difference in delivered and received aggression), P < 0.01. ##Significant difference from zero, P < 0.001. Significant effect of time in Pten+/+ mice, P < 0.05. Clipperton-Allen et al. 2010, 2011). These categories were calculated by adding together the duration or frequency of each individual behavior to reach the composite score; for latency measures, the latency to the first behavior performed in a category was used. The composite scores provided insight into whether mice of different genotypes show phenotypic alterations in specific individual behaviors or general categories of behavior. This is particularly important for agonistic behaviors, as the total agonistic behavior category includes types of aggression-related behavior with divergent functional implications (e.g., dominance behaviors intended to establish a dominance hierarchy vs. overt attacks aimed at establishing an exclusive territory; Miczek et al. 2001; Scott & Fredericson 1951). Total agonistic behavior also included agonistic behaviors performed by the resident, the intruder or both (in cases in which the aggressor could not be identified). To determine which animal of the pair was dominant, a ‘dominance score’ was calculated by determining the difference

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between the amount of agonistic behavior delivered and agonistic behavior received by the resident (see Tables 1 and 2 for descriptions), with a high positive score indicating that the resident mouse was dominant (delivering the majority of the agonistic behavior and receiving very little), while a negative score indicated a submissive resident.

Statistical analysis Planned comparisons between genotypes (across the 15 min test) were performed for the frequency, duration and latency of each behavior (a latency of 900 seconds was assigned if a behavior was not performed) using independent-sample t-tests, or non-parametric Mann–Whitney U tests if the assumption of equality of variance was violated (Levene’s test). One-sample t-tests were used to determine if the dominance score was significantly different from Genes, Brain and Behavior (2015) 14: 145–157

Aggression and repetitive behavior in Pten+/− mice Table 3: Mixed-model ANOVA main effects of time and genotype on aggressive behavior Behavior

Main effect

Statistics

Dominance score

Time (duration) Time (frequency) Genotype (frequency) Time (duration) Genotype (frequency) Time (frequency) Genotype (frequency) Time (frequency) Genotype (duration) Genotype (frequency) Time (duration) Time (frequency) Time (duration) Time (duration) Genotype (frequency) Time (frequency) Time (duration) Genotype (frequency) Genotype (frequency) Time (duration) Time (frequency)

F 2,40 = 8.43, P = 0.001 F 2,40 = 3.34, P = 0.046 F 1,20 = 5.26, P = 0.033 F 2,40 = 5.99, P = 0.005 F 1,20 = 6.42, P = 0.020 F 2,40 = 3.50, P = 0.040 F 1,20 = 5.41, P = 0.031 F 2,40 = 3.45, P = 0.041 F 1,20 = 5.03, P = 0.036 F 1,20 = 4.58, P = 0.045 F 2,40 = 6.45, P = 0.004 F 2,40 = 6.02, P = 0.005 F 2,40 = 4.26, P = 0.021 F 2,40 = 3.92, P = 0.028 F 1,20 = 7.02, P = 0.015 F 2,40 = 3.37, P = 0.045 F 2,40 = 9.19, P = 0.001 F 1,20 = 6.20, P = 0.022 F 1,20 = 6.18, P = 0.022 F 2,40 = 7.77, P = 0.001 F 2,40 = 7.15, P = 0.002

Agonistic behavior delivered Attacks delivered Resident-initiated fighting

Dominant behavior Chasing the intruder Total agonistic behavior Aggressive postures Agonistic behavior received Attacks received Avoidance of the intruder

zero. Additionally, because behavior can vary across the 15-min test, the social interaction was divided into three 5-min intervals, and mixed-model analyses of variance (ANOVAs) were used, with time as the within-subjects factor (0–5, 5–10, 10–15 min) and genotype as the between-subjects factor (Pten+/+ , Pten+/− ); the duration, frequency and latency measures of each behavior or category were the dependent variables. If a time × genotype interaction was found, within-subject ANOVAs (effect of time on each genotype) and t-tests (effect of genotype on each time) were used for post hoc comparisons. All analyses were performed using PASW Statistics 18 (IBM Corporation, Armonk, NY, USA), with significance set at P < 0.05. If statistics were not significant, results are not shown.

Results Under natural conditions, male mice will often establish and maintain an exclusive territory or dominate a shared territory (Miczek et al. 2001, 2007; see Latham & Mason 2004 for a review), and in male laboratory mice, intruders are consistently attacked (e.g., Clipperton-Allen et al. 2010, 2011; Ginsburg & Allee 1942; Parmigiani et al. 1999; Peters et al. 1972; Uhrich 1938). In addition to fighting and overt aggression, which are the most common metrics of the resident–intruder test, we also included other measures of ‘agonistic’ behavior, which include both the aggression and the reaction to it, and non-aggressive (social or non-social) behaviors, to gain a more complete profile of Pten+/− mouse behavior upon presentation of a novel, same-sex intruder.

Aggressive behavior Overall, Pten+/− mice performed, and received, less overt aggression than Pten+/+ mice, indicating a broad decrease Genes, Brain and Behavior (2015) 14: 145–157

in aggressive behavior (see Fig. 1), while leaving non-overt agonistic behavior largely unchanged. Pten+/− mice had lower dominance scores than their Pten+/+ littermates (U = 24.50, z = −2.37, P = 0.018; see Fig. 1a), although both genotypes did show significant dominance over the intruders, with a dominance score significantly greater than zero (Pten+/+ : t(10) = 5.16, P < 0.001; Pten+/− : t(10) = 3.55, P = 0.005; see Fig. 1a). Pten+/− mice performed fewer Agonistic Behaviors Delivered to the intruder (U = 24.00, z = −2.40, P = 0.016; see Fig. 1b); this was due to reduced performance of individual overt aggressive behaviors. Specifically, they performed fewer discrete attacks delivered (U = 36.00, z = −2.05, P = 0.040; see Fig. 1c) and spent less time in resident-initiated fighting (U = 36.00, z = −2.05, P = 0.040; see Fig. 1f), with a longer latency to both behaviors (attacks delivered: U = 36.00, z = −2.05, P = 0.040; resident-initiated fighting: U = 36.00, z = −2.05, P = 0.040; see Fig. 1e,h). Time × genotype interactions were also found in the mixed-model ANOVAs for the number of attacks delivered (F 2,40 = 3.55, P = 0.038; see Fig. 1d) and resident-initiated fighting (F 2,40 = 3.50, P = 0.040; see Fig. 1g); post hoc tests showed that Pten+/+ , but not Pten+/− , males increased these behaviors across the trial (attacks delivered: F 2,20 = 3.53, P = 0.049; resident-initiated fighting: F 2,20 = 3.48, P = 0.050; see Fig. 1d,g). Main effects of the mixed-model ANOVA are shown in Table 3. This reduction in aggression delivered by Pten+/− mice was reflected in fewer total agonistic behaviors across the trial (U = 19.00, z = −2.73, P = 0.006; see Fig. 2a). Mutual aggression was also reduced in Pten+/− males, with less aggressive postures (U = 33.00, z = −2.46, P = 0.014; see Fig. 2b)

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and reciprocal attacks (U = 38.50, z = −2.15, P = 0.032; see Fig. 2c); they also showed a longer latency to both behaviors (aggressive postures: U = 33.00, z = −2.46, P = 0.014; reciprocal attacks: U = 38.50, z = −2.15, P = 0.032; see Fig. 2e,f). Furthermore, mixed-model ANOVA revealed a time × genotype interaction for the number of aggressive postures performed (F 2,40 = 3.37, P = 0.045; see Fig. 2d), although post hoc tests revealed no significant differences between genotypes at any time point, or any effects of time in either Pten+/+ or Pten+/− mice. Main effects of the mixed-model ANOVAs are shown in Table 3. Interestingly, intruders interacting with Pten+/− mice also performed less agonistic behavior, as Pten+/− males experienced fewer agonistic behaviors received (U = 30.50, z = −1.98, P = 0.048; see Fig. 3a). As in the case of agonistic behaviors performed by the resident, these decreases were specific to overt aggressive behaviors: Pten+/− mice experienced fewer attacks received (U = 30.50, z = −2.38, P = 0.017; see Fig. 3b), as well as less intruder-initiated fighting (U = 38.50, z = −2.15, P = 0.032; see Fig. 3d), and

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Figure 2: Agonistic behaviors where both resident and intruder are involved, and/or where the instigator is unclear. (a) Total agonistic behavior (frequency); (b, d, e) aggressive postures (duration, b; frequency by 5-min intervals, d; latency, e); (c, f) reciprocal attacks (duration, c; latency, f). Means + SEM shown; Pten+/+ , yellow bars and triangles; Pten+/− , blue bars and triangles. Gray dotted lines indicate the maximum latency of 900 seconds (i.e., behavior was not performed). *Significant difference between Pten+/− and Pten+/+ males, P < 0.05.

the latency to both behaviors was also increased (attacks received: U = 34.00, z = −2.10, P = 0.035; intruder-initiated fighting: U = 38.50, z = −2.15, P = 0.032; see Fig. 3c,e). This may be due, in part, to the decreased agonistic behavior delivered by the resident, as aggression in intruders is often in response to that of the residents (Ginsburg & Allee 1942). Main effects of the mixed-model ANOVAs are shown in Table 3.

Non-aggressive behaviors We found that Pten+/− mice switched between behaviors (‘behavioral shifting’; Clipperton-Allen et al. 2010, 2011) less often than Pten+/+ mice (total activity frequency: t(20) = 2.19, P = 0.041; see Fig. 4a), but spent the same amount of time performing active behaviors. This is consistent with our previous result, which showed that Pten+/− mice do not differ from Pten+/+ mice in overall activity in the open field test (Clipperton-Allen & Page 2014). Pten+/− mice were also less social, showing lower total social behaviors (t(20) = 2.92, P = 0.008; see Fig. 4b). Time × genotype Genes, Brain and Behavior (2015) 14: 145–157

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Figure 3: Agonistic behaviors performed by intruder. (a) Agonistic behavior received (frequency); (b, c) attacks received (frequency, b; latency, c); (d, e) intruder-initiated fighting (duration, d; latency, e). Means + SEM shown; Pten+/+ , yellow bars; Pten+/− , blue bars. Gray dotted line indicates the maximum latency of 900 seconds (i.e. behavior was not performed). *Significant difference between Pten+/− and Pten+/+ males, P < 0.05.

interactions were found for the amount (F 2,40 = 4.40, P = 0.019) and number (F 2,40 = 3.51, P = 0.039) of total social behaviors (see Fig. 4e,f); post hoc tests indicated that Pten+/− , but not Pten+/+ , males showed a decrease in the duration (F 2,20 = 10.16, P = 0.001) and frequency (F 2,20 = 16.96, P < 0.001) of total social behaviors over time, specifically from the beginning of the trial to the latter two time bins (all P < 0.011; see Fig 4e,f). Post hoc tests also revealed that Pten+/− spent less time performing total social behaviors at 0–5 min (t(17.4) = 2.40, P = 0.024) and 10–15 min (t(20) = 3.43, P = 0.003; see Fig 4f) and performed fewer total social behaviors at all three time points (0–5 min: t(20) = 2.96, P = 0.008; 5–10 min: t(14.6) = 2.69, P = 0.017; 10–15 min: t(10.8) = 2.68, P = 0.022; see Fig. 4e). This decrease in total social behaviors is due to decreased social investigation by Pten+/− mice (t(20) = 2.70, P = 0.014; see Fig. 4c), as well as to the decreased agonistic behavior described above. This effect was largely driven by reduced anogenital investigation across the trial (t(20) = 2.38, P = 0.027; see Fig. 4d). Additionally, a time × genotype interaction was found for body investigation frequency (F 2,40 = 5.32, P = 0.009; see Fig. 4h), which post hoc tests indicated was due to Pten+/− mice performing significantly fewer body investigations at 10–15 min (t(20) = 2.20, P = 0.040); both Pten+/+ and Pten+/− males also showed a decrease in this behavior over time (Pten+/+ : F 2,20 = 9.61, P = 0.001; Pten+/− : F 2,20 = 28.35, P < 0.001), with differences between all time points in the Pten+/− mice, and all time points except between 5–10 and 10–15 min in the Pten+/+ group (all P < 0.041; see Fig 4h). Pten+/− mice also Genes, Brain and Behavior (2015) 14: 145–157

showed decreased social interaction by approaching and/or attending to the intruder less often (U = 19.50, z = −2.69, P = 0.007; see Fig. 4g), consistent with our previous results on a social approach assay (Clipperton-Allen & Page 2014). Digging was the only individual non-social behavior that Pten+/− mice performed more than Pten+/+ mice (U = 29.50, z = −2.04, P = 0.042; see Fig. 5a). This was reflected in increased total non-social behaviors overall (t(20) = 2.92, P = 0.008; see Fig. 5b), and in a time × genotype interaction for these behaviors (F 2,40 = 4.40, P = 0.019; see Fig. 5c). Post hoc tests indicated that Pten+/− , but not Pten+/+ , males showed an increase in total non-social behaviors over time (F 2,20 = 10.16, P = 0.001), with differences between 0–5 and both 5–10 and 10–15 min (all P < 0.010; see Fig. 5c). Post hoc tests also revealed that Pten+/− mice spent more time performing total non-social behaviors than Pten+/+ mice at 0–5 min (t(17.4) = 2.47, P = 0.024) and 10–15 min (t(20) = 3.43, P = 0.003). Main effects of the mixed-model ANOVAs are shown in Table 4.

Discussion The results presented here extend our knowledge of Pten haploinsufficient mice – a model of a risk factor for ASD with macrocephaly – by demonstrating that Pten+/− males display reduced aggression, as well as increased repetitive behavior, compared with Pten+/+ mice. Pten+/− male mice were involved in less attacking, both delivered to and received from the intruder, which was reflected in decreased agonistic

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Figure 4: Non-agonistic social behaviors. (a) Behavioral shifting (total activity frequency); (b, e, f) total social behavior (duration, b; frequency by 5-min intervals, e; duration by 5-min intervals, f); (c) social investigation (duration); (d) anogenital investigation (duration); (g) approach and/or attend to the intruder (frequency); (h) body investigation (frequency by 5-min intervals). Means + SEM shown; Pten+/+ , yellow bars and triangles; Pten+/− , blue bars and triangles. *Significant difference between Pten+/− and Pten+/+ males, P < 0.05. **Significant difference between Pten+/− and Pten+/+ males, P < 0.01. Significant effect of time in Pten+/+ mice, P < 0.01. Significant effect of time in Pten+/− mice, P < 0.01. Significant effect of time in Pten+/− mice, P < 0.001. Significant difference between time points, P < 0.05. Significant difference between time points, P < 0.01. Significant difference between time points, P < 0.001.

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behavior (delivered, received and total). While the observed effects on aggression, which are in the opposite direction to the increased aggression observed in humans with ASD, are somewhat surprising, several other mouse models of ASD also show decreased aggression (e.g., Fmr1-KO, En2-KO; Cheh et al. 2006; Dahlhaus & El-Husseini 2010; Spencer et al. 2011). This suggests that the direction of effect for dysregulation of aggression may be species specific. Alternatively, the difference may relate to a qualitative difference in aggression toward a stranger as opposed to a familiar individual: the Genes, Brain and Behavior (2015) 14: 145–157

resident–intruder assay measures the aggressive (and other) behavioral response to an intruder entering the home cage of the resident, whereas the majority of reports of increased aggression in ASD are toward teachers and/or caregivers (e.g., Hartley et al. 2008; Lecavalier 2006; Lecavalier et al. 2006; Maskey et al. 2013) or unspecified targets (e.g., Holden & Gitlesen 2006; McDougle et al. 2003; Niditch et al. 2012; Robb 2010). Some studies have found that the presence of a stranger leads to minimal social interaction (Hobson & Lee 1998; Lord 1993) and increased stereotypic, repetitive behavior (Hutt & Hutt 1965), which is consistent with our current findings that Pten+/− mice showed decreased social interaction and aggressive behavior and increased repetitive behavior. Pten+/− mice also showed less social investigation, particularly of the anogenital region, and approached and/or attended to the intruder less than Pten+/+ mice. Together, these behavioral differences resulted in less social behavior overall. We do not anticipate that this decrease is caused by an overall change in activity levels, given that Pten+/− mice do not differ from wild-type controls in time spent in active behaviors in the present study nor do they differ in locomotor activity in an open field test (Clipperton-Allen & Page 2014). Correspondingly, Pten+/− mice performed more of the non-social behavior digging. Pten+/− mice appear to have performed digging at higher levels, instead of the higher levels of agonistic and non-agonistic social behaviors performed by Pten+/+ mice in the current test. This is consistent with our previous finding that Pten+/− males buried more marbles in the marble burying test (Clipperton-Allen & Page 2014); indeed, it has been shown that marbles are not required to elicit digging, but simply serve as a useful metric for this repetitive, stereotyped behavior, which would occur regardless of their presence (Gyertyán 1995; Thomas et al. 2009). The ethological analysis used in this study has allowed us to observe that Pten+/− mice show both decreased social investigatory and agonistic behaviors and repetitive behaviors in the same assay. Although our data do not indicate a relationship exists between these behaviors, increased aggression has been associated with increased repetitive behaviors in individuals with ASD (Kanne & Mazurek 2011); although the effects are not in the same direction as our results, this suggests that these two abnormal behaviors may be linked. Similarly, there may be a relationship between the performance of repetitive behaviors and social situation-induced anxiety in ASD; children with ASD may perform more repetitive behaviors in a social context (Baron-Cohen 1989), and some self-reports confirm that repetitive behavior reduces anxiety (Turner 1999). The current results complement the findings of our previous, more extensive behavioral phenotyping of the germline Pten+/− line (Clipperton-Allen & Page 2014). Notably, male Pten+/− mice were less anxious than their Pten+/+ littermates (Clipperton-Allen & Page 2014). Aggression has been associated with anxiety in humans with (e.g., Niditch et al. 2012; Pugliese et al. 2013) and without ASD (e.g., Frick et al. 1999; Hatfield & Dula 2014; Marsee et al. 2008; Sareen et al. 2004; Tsiouris et al. 2011), and it has previously been suggested that more aggressive male mice may also be more anxious (Ferrari et al. 1998; Guillot & Chapouthier 1996).

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Clipperton-Allen and Page Table 4: Mixed-model ANOVA main effects of time and genotype on non-aggressive behaviors Behavior

Main effect

Statistics

Total activity

Time (duration) Genotype (frequency) Time (duration) Genotype (duration) Genotype (frequency) Time (duration) Time (frequency) Genotype (duration) Genotype (frequency) Time (duration) Time (frequency) Genotype (duration) Genotype (frequency) Time (duration) Time (frequency) Time (duration) Time (frequency) Time (duration) Time (frequency) Genotype (frequency) Time (frequency) Time (duration) Time (frequency) Genotype (duration) Genotype (frequency) Time (duration) Genotype (duration) Time (frequency) Time (duration) Time (frequency) Time (frequency) Time (duration) Time (frequency) Time (duration) Time (frequency)

F 2,40 = 24.75, P < 0.001 F 2,40 = 4.79, P = 0.041 F 2,40 = 5.40, P = 0.008 F 1,20 = 8.56, P = 0.008 F 1,20 = 10.28, P = 0.004 F 2,40 = 56.02, P < 0.001 F 2,40 = 44.63, P < 0.001 F 1,20 = 7.31, P = 0.014 F 1,20 = 14.03, P = 0.001 F 2,40 = 15.76, P < 0.001 F 2,40 = 14.35, P < 0.001 F 1,20 = 5.66, P = 0.027 F 1,20 = 5.60, P = 0.028 F 2,40 = 22.27, P < 0.001 F 2,40 = 27.83, P < 0.001 F 2,40 = 7.83, P = 0.001 F 2,40 = 9.09, P = 0.001 F 2,40 = 26.53, P < 0.001 F 2,40 = 9.49, P < 0.001 F 1,20 = 8.81, P = 0.008 F 2,40 = 13.72, P < 0.001 F 2,40 = 6.91, P = 0.003 F 2,40 = 7.21, P = 0.002 F 1,20 = 7.28, P = 0.014 F 1,20 = 7.50, P = 0.013 F 2,40 = 5.39, P = 0.008 F 1,20 = 8.55, P = 0.008 F 2,40 = 4.66, P = 0.015 F 2,40 = 14.12, P < 0.001 F 2,40 = 11.06, P < 0.001 F 2,40 = 5.12, P = 0.011 F 2,40 = 24.71, P < 0.001 F 2,40 = 31.23, P < 0.001 F 2,40 = 25.37, P < 0.001 F 2,40 = 31.93, P < 0.001

Total social behaviors

Social investigation

Anogenital investigation

Body investigation Oronasal investigation Approaching and/or attending to the intruder

Stretched approaches Digging

Total non-social behaviors Non-social locomotor behaviors Horizontal exploration Vertical exploration Non-social non-locomotor behaviors Self-grooming

Several of the behavioral abnormalities we have observed in Pten+/− mice (Clipperton-Allen & Page 2014) are also consistent with the results of other ASD mouse models; for example, Fmr1-KO mice fail to prefer a social stimulus in the three-chamber test and may show decreased anxiety, in addition to decreased threats and minimal aggression toward an intruder (Dahlhaus & El-Husseini 2010; Spencer et al. 2011). En2-KO mice also show decreased social investigation in juvenile interactions (Brielmaier et al. 2012) and decreased aggression (Cheh et al. 2006), as well as increased serotonin (5-HT) in the cerebellum (Cheh et al. 2006), suggesting that the 5-HT system may mediate these effects. Consistent with this observation, the 5-HT system is implicated in the pathogenesis and treatment of ASD (Bartlett et al. 2005; Benvenuto et al. 2013; Farmer et al. 2013; Gabriele et al., 2014), and mice with loss of function mutations in Slc6a4 (Holmes et al. 2002), which have elevated extracellular 5-HT (Mathews et al. 2004; Montañez et al. 2003), or that received chronic treatment with the selective

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serotonin reuptake inhibitor fluoxetine (reviewed in Olivier et al. 2011), show decreased aggression. Correspondingly, several other mouse models show increased aggression and decreased 5-HT signaling (Gordon & Hen 2004; Holmes 2008; Lesch et al. 2012; Mosienko et al. 2012). The 5-HT system has also been shown to interact with Pten and the PI3K–Akt–mTOR pathway (Cowen 2007; Ji et al. 2006), and we have previously shown that Slc6a4 haploinsufficiency can exacerbate brain overgrowth and social behavior deficits in Pten+/− mice (Page et al. 2009). The currently observed decrease in social investigation and approaching and/or attending to the intruder is also consistent with our previous findings, where male Pten+/− mice were impaired on multiple social behavior tests. Specifically, male Pten+/− mice showed no social preference in the three-chamber social approach test in one location (Clipperton-Allen & Page 2014), but normal preference in another (Page et al. 2009). Additionally, these mice failed to habituate to a juvenile conspecific in a test of social recognition (Clipperton-Allen & Page 2014). Genes, Brain and Behavior (2015) 14: 145–157

Aggression and repetitive behavior in Pten+/− mice

Together with our previous results (Clipperton-Allen & Page 2014), we have found that male germline Pten haploinsufficient mice show reduced social and agonistic behavior and increased repetitive behavior. Additionally, germline Pten haploinsufficient mice are a useful model for investigating the effects of mutations in the PI3K–Akt–mTOR pathway and their relation to core symptoms and comorbid disorders of ASD. Male Pten+/− mice show decreased social interest across several paradigms, as well as increased repetitive behavior, thus recapitulating core criteria for ASD. Similarly, Pten+/− males also show phenotypes relevant to several ASD comorbidities, including anxiety and depression-like behaviors. Thus, these mice may be a useful model for understanding the relationship between social behavioral deficits and repetitive behavior.

References Abrahams, B.S. & Geschwind, D.H. (2008) Advances in autism genetics: on the threshold of a new neurobiology. Nat Rev Genet 9, 341–355. American Psychiatric Association (2005) Diagnostic and Statistical Manual of Mental Disorders: DSM-IV-TR. American Psychiatric Publishing, Arlington, VA. American Psychiatric Association (2013) Diagnostic and Statistical Manual of Mental Disorders: DSM-5. American Psychiatric Association, Arlington, VA. Baron-Cohen, S. (1989) Do autistic children have obsessions and compulsions. Br J Clin Psychol 28, 193–200. Bartlett, C.W., Gharani, N., Millonig, J.H. & Brzustowicz, L.M. (2005) Three autism candidate genes: a synthesis of human genetic analysis with other disciplines. Int J Dev Neurosci 23, 221–234. Benvenuto, A., Battan, B., Porfirio, M.C. & Curatolo, P. (2013) Pharmacotherapy of autism spectrum disorders. Brain Dev 35, 119–127. Bohm, H.V., Stewart, M.G. & Healy, A.M. (2013) On the Autistic Spectrum Disorder concordance rates of twins and non-twin siblings. Med Hypotheses 81, 789–791. Bortolato, M., Godar, S.C., Alzghoul, L., Zhang, J., Darling, R.D., Simpson, K.L., Bini, V., Chen, K., Wellman, C.L., Lin, R.C.S. & Shih, J.C. (2013) Monoamine oxidase A and A/B knockout mice display autistic-like features. Int J Neuropsychopharmacol 16, 869–888. Bourgeron, T. (2009) A synaptic trek to autism. Curr Opin Neurobiol 19, 231–234. Brielmaier, J., Matteson, P.G., Silverman, J.L., Senerth, J.M., Kelly, S., Genestine, M., Millonig, J.H., DiCicco-Bloom, E. & Crawley, J.N. (2012) Autism-relevant social abnormalities and cognitive deficits in Engrailed-2 knockout mice. PLoS One 7, e40914. Butler, M., Dasouki, M., Zhou, X.-P., Talebizadeh, Z., Brown, M., Takahashi, T., Miles, J., Wang, C., Stratton, R., Pilarski, R. & Eng, C. (2005) Subset of individuals with autism spectrum disorders and extreme macrocephaly associated with germline PTEN tumour suppressor gene mutations. J Med Genet 42, 318–321. Buxbaum, J.D., Cai, G., Chaste, P., Nygren, G., Goldsmith, J., Reichert, J., Anckarsäter, H.R., Rastam, M., Smith, C.J., Silverman, J.M., Hollander, E., Leboyer, M., Gillberg, C., Verloes, A. & Betancur, C. (2007) Mutation screening of the PTEN gene in patients with autism spectrum disorders and macrocephaly. Am J Med Genet B Neuropsychiatr Genet 144B, 484–491. Cheh, M.A., Millonig, J.H., Roselli, L.M., Ming, X., Jacobsen, E., Kamdar, S. & Wagner, G.C. (2006) En2 knockout mice display neurobehavioral and neurochemical alterations relevant to autism spectrum disorder. Brain Res 1116, 166–176. Clipperton, A.E., Spinato, J.M., Chernets, C., Pfaff, D.W. & Choleris, E. (2008) Differential effects of estrogen receptor alpha and beta specific agonists on social learning of food preferences in female mice. Neuropsychopharmacology 33, 2362–2375. Genes, Brain and Behavior (2015) 14: 145–157

Clipperton-Allen, A.E. & Page, D.T. (2014) Pten haploinsufficient mice show broad brain overgrowth but selective impairments in autism-relevant behavioral tests. Hum Mol Genet 23, 3490–3505. Clipperton-Allen, A.E., Cragg, C.L., Wood, A.J., Pfaff, D.W. & Choleris, E. (2010) Agonistic behavior in males and females: effects of an estrogen receptor beta agonist in gonadectomized and gonadally intact mice. Psychoneuroendocrinology 35, 1008–1022. Clipperton-Allen, A.E., Almey, A., Melichercik, A., Allen, C.P. & Choleris, E. (2011) Effects of an estrogen receptor alpha agonist on agonistic behaviour in intact and gonadectomized male and female mice. Psychoneuroendocrinology 36, 981. Cowen, D.S. (2007) Serotonin and neuronal growth factors – a convergence of signaling pathways. J Neurochem 101, 1161–1171. Dahlhaus, R. & El-Husseini, A. (2010) Altered neuroligin expression is involved in social deficits in a mouse model of the fragile X syndrome. Behav Brain Res 208, 96–105. Eagly, A.H. & Steffen, V.J. (1986) Gender and aggressive behavior: a meta-analytic review of the social psychological literature. Psychol Bull 100, 309–330. Farmer, C., Thurm, A. & Grant, P. (2013) Pharmacotherapy for the core symptoms in autistic disorder: current status of the research. Drugs 73, 303–314. Ferrari, P.F., Palanza, P., Parmigiani, S. & Rodgers, R.J. (1998) Interindividual variability in Swiss male mice: relationship between social factors, aggression, and anxiety. Physiol Behav 63, 821–827. Frick, P.J., Lilienfeld, S.O., Ellis, M., Loney, B. & Silverthorn, P. (1999) The association between anxiety and psychopathy dimensions in children. J Abnorm Child Psychol 27, 383–392. Gabriele, S., Sacco, R. & Persico, A.M. (2014) Blood serotonin levels in autism spectrum disorder: a systematic review and meta-analysis. Eur Neuropsychopharmacol 24, 919–929. Gardner, W.I. & Moffatt, C.W. (1990) Aggressive behaviour: definition, assessment, treatment. Int Rev Psychiatry 2, 91–100. Ginsburg, B. & Allee, W.C. (1942) Some effects of conditioning on social dominance and subordination in inbred strains of mice. Physiol Zool 15, 485–506. Gordon, J.A. & Hen, R. (2004) The serotonergic system and anxiety. Neuromolecular Med 5, 27–40. Grant, E.C. & Mackintosh, J.H. (1963) A comparison of the social postures of some common laboratory rodents. Behaviour 21, 246–259. Guillot, P.-V. & Chapouthier, G. (1996) Intermale aggression and dark/light preference in ten inbred mouse strains. Behav Brain Res 77, 211–213. Gyertyán, I. (1995) Analysis of the marble burying response: marbles serve to measure digging rather than evoke burying. Behav Pharmacol 6, 24–31. Hartley, S.L., Sikora, D.M. & McCoy, R. (2008) Prevalence and risk factors of maladaptive behaviour in young children with Autistic Disorder. J Intellect Disabil Res 52, 819–829. Hatfield, J. & Dula, C.S. (2014) Impulsivity and physical aggression: examining the moderating role of anxiety. Am J Psychol 127, 233–243. Hobson, R.P. & Lee, A. (1998) Hello and goodbye: a study of social engagement in autism. J Autism Dev Disord 28, 117–127. Holden, B. & Gitlesen, J.P. (2006) A total population study of challenging behaviour in the county of Hedmark, Norway: prevalence, and risk markers. Res Dev Disabil 27, 456–465. Holmes, A. (2008) Genetic variation in cortico-amygdala serotonin function and risk for stress-related disease. Neurosci Biobehav Rev 32, 1293–1314. Holmes, A., Murphy, D.L. & Crawley, J.N. (2002) Reduced aggression in mice lacking the serotonin transporter. Psychopharmacology (Berl) 161, 160–167. Hutt, C. & Hutt, S.J. (1965) Effects of environmental complexity on stereotyped behaviours of children. Anim Behav 13, 1–4. Ji, S.-P., Zhang, Y., Van Cleemput, J., Jiang, W., Liao, M., Li, L., Wan, Q., Backstrom, J.R. & Zhang, X. (2006) Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse. Nat Med 12, 324–329.

155

Clipperton-Allen and Page Kanne, S.M. & Mazurek, M.O. (2011) Aggression in children and adolescents with ASD: prevalence and risk factors. J Autism Dev Disord 41, 926–937. Klein, S., Sharifi-Hannauer, P. & Martinez-Agosto, J.A. (2013) Macrocephaly as a clinical indicator of genetic subtypes in autism. Autism Res 6, 51–56. Knight, G.P., Fabes, R.A. & Higgins, D.A. (1996) Concerns about drawing causal inferences from meta-analyses: an example in the study of gender differences in aggression. Psychol Bull 119, 410–421. Lakin, K.C. (1983) New admissions and readmissions to a national sample of public residential facilities. Am J Ment Defic 88, 13–20. Latham, N. & Mason, G. (2004) From house mouse to mouse house: the behavioural biology of free-living Mus musculus and its implications in the laboratory. Appl Anim Behav Sci 86, 261–289. Lecavalier, L. (2006) Behavioral and emotional problems in young people with pervasive developmental disorders: relative prevalence, effects of subject characteristics and empirical classification. J Autism Dev Disord 36, 1101–1114. Lecavalier, L., Leone, S. & Wiltz, J. (2006) The impact of behaviour problems on caregiver stress in young people with autism spectrum disorders. J Intellect Disabil Res 50, 172–183. Lesch, K.-P., Araragi, N., Waider, J., van den Hove, D. & Gutknecht, L. (2012) Targeting brain serotonin synthesis: insights into neurodevelopmental disorders with long-term outcomes related to negative emotionality, aggression and antisocial behaviour. Philos Trans R Soc Lond B Biol Sci 367, 2426–2443. Lord, C. (1993) The complexity of social behaviour in autism. In Baron-Cohen, S., Tager-Flusberg, H. & Cohen, D.J. (eds), Understanding Other Minds: Perspectives from Autism. Oxford University Press, Oxford, pp. 292–316. Marsee, M.A., Weems, C.F. & Taylor, L.K. (2008) Exploring the association between aggression and anxiety in youth: a look at aggressive subtypes, gender, and social cognition. J Child Fam Stud 17, 154–168. Maskey, M., Warnell, F., Parr, J.R., Le Couteur, A. & McConachie, H. (2013) Emotional and behavioural problems in children with autism spectrum disorder. J Autism Dev Disord 43, 851–859. Mathews, T.A., Fedele, D.E., Coppelli, F.M., Avila, A.M., Murphy, D.L. & Andrews, A.M. (2004) Gene dose-dependent alterations in extraneuronal serotonin but not dopamine in mice with reduced serotonin transporter expression. J Neurosci Methods 140, 169–181. Matson, J.L. & Nebel-Schwalm, M. (2007) Assessing challenging behaviors in children with autism spectrum disorders: a review. Res Dev Disabil 28, 567–579. McBride, K.L., Varga, E.A., Pastore, M.T., Prior, T.W., Manickam, K., Atkin, J.F. & Herman, G.E. (2010) Confirmation study of PTEN mutations among individuals with autism or developmental delays/mental retardation and macrocephaly. Autism Res 3, 137–141. McClintock, K., Hall, S. & Oliver, C. (2003) Risk markers associated with challenging behaviours in people with intellectual disabilities: a meta-analytic study. J Intellect Disabil Res 47, 405–416. McDougle, C.J., Stigler, K.A. & Posey, D.J. (2003) Treatments of aggression in children and adolescents with autism and conduct disorder. J Clin Psychiatry 64, 16–25. Meng, L., Lu, L., Murphy, K.M., Yuede, C.M., Cheverud, J.M., Csernansky, J.G. & Dong, H. (2011) Neuroanatomic and behavioral traits for autistic disorders in age-specific restricted index selection mice. Neuroscience 189, 215–222. Miczek, K.A., Maxson, S.C., Fish, E.W. & Faccidomo, S. (2001) Aggressive behavioral phenotypes in mice. Behav Brain Res 125, 167–181. Miczek, K.A., Faccidomo, S.P., Fish, E.W. & DeBold, J.F. (2007) Neurochemistry and molecular neurobiology of aggressive behavior. In Lajtha, A. & Blaustein, J.D. (eds), Handbook of Neurochemistry and Molecular Neurobiology. Springer US, pp. 285–336. Montañez, S., Owens, W.A., Gould, G.G., Murphy, D.L. & Daws, L.C. (2003) Exaggerated effect of fluvoxamine in heterozygote serotonin transporter knockout mice. J Neurochem 86, 210–219.

156

Mosienko, V., Bert, B., Beis, D., Matthes, S., Fink, H., Bader, M. & Alenina, N. (2012) Exaggerated aggression and decreased anxiety in mice deficient in brain serotonin. Transl Psychiatry 2, e122. Niditch, L.A., Varela, R.E., Kamps, J.L. & Hill, T. (2012) Exploring the association between cognitive functioning and anxiety in children with autism spectrum disorders: the role of social understanding and aggression. J Clin Child Adolesc Psychol 41, 127–137. Olivier, J.D.A., Blom, T., Arentsen, T. & Homberg, J.R. (2011) The age-dependent effects of selective serotonin reuptake inhibitors in humans and rodents: a review. Prog Neuropsychopharmacol Biol Psychiatry 35, 1400–1408. Page, D.T., Kuti, O.J., Prestia, C. & Sur, M. (2009) Haploinsufficiency for Pten and Serotonin transporter cooperatively influences brain size and social behavior. Proc Natl Acad Sci U S A 106, 1989–1994. Parmigiani, S., Palanza, P., Rodgers, J. & Ferrari, P.F. (1999) Selection, evolution of behavior and animal models in behavioral neuroscience. Neurosci Biobehav Rev 23, 957–970. Perou, R., Bitsko, R.H., Blumberg, S.J., Pastor, P., Ghandour, R.M., Gfroerer, J.C., Hedden, S.L., Crosby, A.E., Visser, S.N., Schieve, L.A., Parks, S.E., Hall, J.E., Brody, D., Simile, C.M., Thompson, W.W., Baio, J., Avenevoli, S., Kogan, M.D. & Huang, L.N. (2013) Mental health surveillance among children – United States, 2005–2011. MMWR Recomm Rep 62, 1–35. Peters, P.J., Bronson, F.H. & Whitsett, J.M. (1972) Neonatal castration and intermale aggression in mice. Physiol Behav 8, 265–268. Pietropaolo, S., Branchi, I., Cirulli, F., Chiarotti, F., Aloe, L. & Alleva, E. (2004) Long-term effects of the periadolescent environment on exploratory activity and aggressive behaviour in mice: social versus physical enrichment. Physiol Behav 81, 443–453. Pobbe, R.L.H., Pearson, B.L., Defensor, E.B., Bolivar, V.J., Blanchard, D.C. & Blanchard, R.J. (2010) Expression of social behaviors of C57BL/6J versus BTBR inbred mouse strains in the visible burrow system. Behav Brain Res 214, 443–449. Pobbe, R.L.H., Pearson, B.L., Blanchard, D.C. & Blanchard, R.J. (2012) Oxytocin receptor and Mecp2308/Y knockout mice exhibit altered expression of autism-related social behaviors. Physiol Behav 107, 641–648. Podsypanina, K., Ellenson, L.H., Nemes, A., Gu, J., Tamura, M., Yamada, K.M., Cordon-Cardo, C., Catoretti, G., Fisher, P.E. & Parsons, R. (1999) Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc Natl Acad Sci U S A 96, 1563–1568. Potegal, M. & Archer, J. (2004) Sex differences in childhood anger and aggression. Child Adolesc Psychiatr Clin N Am 13, 513–528. Pugliese, C.E., White, B.A., White, S.W. & Ollendick, T.H. (2013) Social anxiety predicts aggression in children with ASD: clinical comparisons with socially anxious and oppositional youth. J Autism Dev Disord 43, 1205–1213. Robb, A.S. (2010) Managing irritability and aggression in autism spectrum disorders in children and adolescents. Dev Disabil Res Rev 16, 258–264. Sareen, J., Stein, M.B., Cox, B.J. & Hassard, S.T. (2004) Understanding comorbidity of anxiety disorders with antisocial behavior: findings from two large community surveys. J Nerv Ment Dis 192, 178–186. Scattoni, M.L., Ricceri, L. & Crawley, J.N. (2011) Unusual repertoire of vocalizations in adult BTBR T+tf/J mice during three types of social encounters. Genes Brain Behav 10, 44–56. Scott, J.P. & Fredericson, E. (1951) The causes of fighting in mice and rats. Physiol Zool 24, 273–309. Spencer, C.M., Alekseyenko, O., Hamilton, S.M., Thomas, A.M., Serysheva, E., Yuva-Paylor, L.A. & Paylor, R. (2011) Modifying behavioral phenotypes in Fmr1KO mice: genetic background differences reveal autistic-like responses. Autism Res 4, 40–56. Thomas, A., Burant, A., Bui, N., Graham, D., Yuva-Paylor, L.A. & Paylor, R. (2009) Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology (Berl) 204, 361–373. Genes, Brain and Behavior (2015) 14: 145–157

Aggression and repetitive behavior in Pten+/− mice Tomanik, S., Harris, G.E. & Hawkins, J. (2004) The relationship between behaviours exhibited by children with autism and maternal stress. J Intellect Dev Disabil 29, 16–26. Tsiouris, J.A., Kim, S.Y., Brown, W.T. & Cohen, I.L. (2011) Association of aggressive behaviours with psychiatric disorders, age, sex and degree of intellectual disability: a large-scale survey. J Intellect Disabil Res 55, 636–649. Turner, M. (1999) Annotation: repetitive behavior in autism: a review of psychological research. J Child Psychol Psychiatry 40, 839–849. Uhrich, J. (1938) The social hierarchy in albino mice. J Comp Psychol 25, 373–413. Varga, E.A., Pastore, M., Prior, T., Herman, G.E. & McBride, K.L. (2009) The prevalence of PTEN mutations in a clinical pediatric cohort with autism spectrum disorders, developmental delay, and macrocephaly. Genet Med 11, 111–117. de Vries, P.J. (2010) Targeted treatments for cognitive and neurodevelopmental disorders in tuberous sclerosis complex. Neurotherapeutics 7, 275–282. Wang, X., McCoy, P.A., Rodriguiz, R.M., Pan, Y., Je, H.S., Roberts, A.C., Kim, C.J., Berrios, J., Colvin, J.S., Bousquet-Moore, D.,

Genes, Brain and Behavior (2015) 14: 145–157

Lorenzo, I., Wu, G., Weinberg, R.J., Ehlers, M.D., Philpot, B.D., Beaudet, A.L., Wetsel, W.C. & Jiang, Y.-H. (2011) Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. Hum Mol Genet 20, 3093–3108. World Health Organization (1992) The ICD-10 Classification of Mental and Behavioural Disorders: Clinical Descriptions and Diagnostic Guidelines. World Health Organization, Geneva.

Acknowledgments We thank Mrs Trina L. Kemp for invaluable administrative assistance and Dr Julien Séjourné for useful advice on an early version of this manuscript. This work was supported by gift funds from Ms Nancy Lurie Marks and The American Honda and Children’s Healthcare Charity Inc, as well as startup funds from The Scripps Research Institute.

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