Abstract

Accelerated head and brain growth (macrocephaly) during development is a replicated biological finding in a subset of individuals with autism spectrum disorder (ASD). However, the relationship between brain overgrowth and the behavioral and cognitive symptoms of ASD is poorly understood. The PI3K–Akt–mTOR pathway regulates cellular growth; several genes encoding negative regulators of this pathway are ASD risk factors, including PTEN. Mutations in PTEN have been reported in individuals with ASD and macrocephaly. We report that brain overgrowth is widespread in Pten germline haploinsufficient (Pten+/−) mice, reflecting Pten mRNA expression in the developing brain. We then ask if broad brain overgrowth translates into general or specific effects on the development of behavior and cognition by testing Pten+/− mice using assays relevant to ASD and comorbidities. Deficits in social behavior were observed in both sexes. Males also showed abnormalities related to repetitive behavior and mood/anxiety. Females exhibited circadian activity and emotional learning phenotypes. Widespread brain overgrowth together with selective behavioral impairments in Pten+/− mice raises the possibility that most brain areas and constituent cell types adapt to an altered trajectory of growth with minimal impact on the behaviors tested in our battery; however, select areas/cell types relevant to social behavior are more vulnerable or less adaptable, thus resulting in social deficits. Probing dopaminergic neurons as a candidate vulnerable cell type, we found social behavioral impairments in mice with Pten conditionally inactivated in dopaminergic neurons that are consistent with the possibility that desynchronized growth in key cell types may contribute to ASD endophenotypes.

INTRODUCTION

Autism spectrum disorder (ASD) is a neurodevelopmental disorder present in ∼1% of the population, with high sexual dimorphism (∼80% male) and heritability (∼90% concordance in monozygotic twins) (1). The diagnostic criteria for ASD include deficits in social behavior and communication, and restricted, repetitive, stereotyped patterns of behavior (2–4). Macrocephaly and microcephaly, head circumferences of more or less than two standard deviations (SD) from normal, respectively, are present in a subset of individuals with ASD. Determination of the precise frequency and trajectory of altered head/brain growth in ASD is an active area of investigation (5–13). Indeed, it has been hypothesized that an altered trajectory of brain growth, especially early overgrowth, contributes to the pathophysiology of ASD (14). However, we lack a mechanistic understanding of how changes in the rate of brain growth relates to the behavioral and cognitive symptoms of ASD and the function of underlying neural circuitry.

Phosphatase and tensin homolog (PTEN), a gene located at chromosome 10q23, is candidate risk gene for ASD and macrocephaly based on multiple studies (15–25). The protein and lipid phosphatase encoded by PTEN is a canonical negative regulator of the PI3K–Akt–mTOR pathway (26), and is involved in a wide variety of cellular processes relevant to brain growth and circuit function, from proliferation and differentiation of neural progenitors to synaptic transmission (27–38; reviewed in 39, 40). Mutations in other regulators of the PI3K-Akt-mTOR pathway, including TSC1/2, NF1, FMR1 and MECP2, have also been identified as ASD risk factors (41–43). Studying the mechanisms by which mutations in PTEN affect dynamics of brain growth and the behavioral and cognitive symptoms of ASD and comorbid disorders, is therefore likely to provide insight into multiple risk factors involved in this pathway.

PTEN mutations are more frequent in individuals with both an ASD diagnosis and macrocephaly (estimated prevalence 7–17%), particularly in those with extreme macrocephaly (head circumference >3 SD above the mean) (16, 17, 21, 25). Individuals with PTEN hamartoma tumor syndromes, which involve the development of benign tumor-like malformations (hamartomas), have a high frequency of macrocephaly and are sometimes comorbid with ASD (15, 18, 19). Germline Pten haploinsufficient (Pten+/−) mice, which approximate genomic lesions found in humans who are PTEN haploinsufficient, show an increase in total brain mass, as well as impaired social behavior and sensorimotor gating (44). It is not known if this overgrowth translates into broader effects on behavior in these mice, or if effects are restricted to specific domains and vulnerable neural systems.

To address this question, we tested behaviors relevant to ASD core symptoms in Pten+/− mice, including repetitive, stereotyped behavior (marble burying test), and social behavior (social approach, novelty, and recognition), to replicate and extend our previous findings (44).We also assayed behaviors relevant to disorders commonly comorbid with ASD, including mood disorders (∼60%) (45), anxiety disorders (∼80%) (45), intellectual disability (∼60%) (46) and circadian rhythm and/or sleep disorders (∼53% in ASD children compared with 32% in typically developing children) (47). Tests included tail suspension and forced swim tests (mood disorders), dark–light emergence and open field tests (anxiety), trace fear conditioning (emotional learning and memory), circadian rhythm and activity (sleep disorders), and hotplate and rotarod tests (sensorimotor abilities).

RESULTS

Brain mass in Pten haploinsufficient mice

To validate Pten haploinsufficient mice as a model of absolute and relative brain overgrowth, the brains of a random subset of adult mice tested in this study were extracted and weighed. Planned comparisons showed that Pten+/− mice of both sexes had significantly greater absolute and relative brain masses (all t > 5.96, all P < 0.001; see Fig. 1A–D) than wild-type (WT) littermates, but did not differ in body size (see Fig. 1B; Table 1 contains additional statistics). This is consistent with our previous report of elevated brain mass in Pten+/− mice (44), as well as reports of an association between macrocephaly, ASD and PTEN mutations in humans. To determine whether the overgrowth in Pten haploinsufficient brains is localized to a particular discreet gross neuroanatomical region or whether the overgrowth occurs across regions, we also carried out dissection of brains from adult WT and adult Pten+/− male mice and found overgrowth in anterior and posterior halves of the cerebral cortex, in the cerebellum, and in the remainder of the brain left over after dissection (Fig. 1E). This indicates that the overgrowth that results from Pten haploinsufficiency is widespread at the gross neuroanatomical level.

Table 1.

Between-subjects and mixed-model analyses of variance (ANOVAs) statistics for non-social tests of Ptentm1Rps mice

Analysis Effect Statistics 
Brain mass 
 2 × 2 ANOVA: body weight Sex F(1,95) = 149.38, P < 0.001 (M > F) 
 2 × 2 ANOVA: brain weight Genotype F(1,95) = 188.70, P < 0.001 (WT < Pten+/−
 2 × 2 ANOVA: brain weight as % of body weight Sex F(1,95) = 133.58, P < 0.001 (M < F) 
Genotype F(1,95) = 63.15, P < 0.001 (WT < Pten+/−
Tail Suspension Test 
 2 × 2 ANOVA: immobility Sex × Genotype F(1,47) = 10.43, P = 0.002 
Post hoc: t(23) = 3.26, P = 0.003 (M < F in WT mice) 
Forced swim test 
 2 × 2 ANOVA: immobility Sex F(1,44) = 4.13, P = 0.048 (M > F) 
Dark–light emergence test 
 2 × 2 ANOVA: chamber crossings Sex F(1,53) = 12.70, P = 0.001 (M < F) 
Open field test 
 2 × 2 ANOVA: center time (inverse of thigmotaxis) Sex F(1,44) = 6.46, P = 0.015 (M > F) 
Trace fear conditioning 
 2 × 2 × 4 ANOVA: freezing Sex × Genotype F(1,47) = 7.80, P = 0.008 
  Sex × Genotype × Test F(3,141) = 4.14, P = 0.008 
Post hoc: training baseline versus cue test, cue baseline versus context test, all P < 0.008 
Test F(3,141) = 124.90, P < 0.001 
 2 × 4 ANOVA: freezing in females Genotype F(1,22) = 6.35, P = 0.020 (WT > Pten+/−
Test F(3,66) = 44.30, P < 0.001 
Genotype × Test F(3,66) = 4.56, P = 0.006 
 2 × 4 ANOVA: freezing in males Test F(3,75) = 86.76, P < 0.001 
 2 × 2 ANOVA: distance moved (training baseline) Sex F(1,47) = 4.10, P = 0.048 (M < F) 
 2 × 2 ANOVA: freezing (context test) Sex × Genotype F(1,47) = 7.07, P = 0.011 
Post hoc: t(24) = 3.04, P = 0.006 (M > F in Pten+/− mice) 
 2 × 2 ANOVA: freezing (cue test) Sex × Genotype F(1,47) = 4.54, P = 0.038 
Post hoc: t(24) = 2.25, P = 0.034 (M > F in Pten+/− mice) 
Circadian Rhythm and Activity 
 2 × 2 × 6 ANOVA: rotations Sex F(1,33) = 77.82, P < 0.001 (M < F) 
Sex × Genotype F(1,33) = 15.27, P < 0.001 
Post hoc: F(1,17) = 70.00, P < 0.001 (M < F in WT mice); F(1,16) = 14.77, P = 0.001 (M < F in Pten+/− mice) 
Time F(5,165) = 39.27, P < 0.001 
 2 × 2 × 6 ANOVA: bouts of activity Sex F(1,34) = 15.30, P < 0.001 (M > F) 
Genotype F(1,34) = 4.58, P = 0.040 (WT < Pten+/−
Time F(5,170) = 86.48, P < 0.001 
Sex × Time F(5,170) = 8.45, P < 0.001 
 Genotype effects on rotations in females Light phase day 3 t(16) = 2.21, P = 0.042 (WT > Pten+/−
Dark phase day 2 t(16) = 2.65, P = 0.017 (WT > Pten+/−
 Genotype effects on bouts of activity in females Dark phase day 3 t(16) = 2.16, P = 0.045 (WT < Pten+/−
Analysis Effect Statistics 
Brain mass 
 2 × 2 ANOVA: body weight Sex F(1,95) = 149.38, P < 0.001 (M > F) 
 2 × 2 ANOVA: brain weight Genotype F(1,95) = 188.70, P < 0.001 (WT < Pten+/−
 2 × 2 ANOVA: brain weight as % of body weight Sex F(1,95) = 133.58, P < 0.001 (M < F) 
Genotype F(1,95) = 63.15, P < 0.001 (WT < Pten+/−
Tail Suspension Test 
 2 × 2 ANOVA: immobility Sex × Genotype F(1,47) = 10.43, P = 0.002 
Post hoc: t(23) = 3.26, P = 0.003 (M < F in WT mice) 
Forced swim test 
 2 × 2 ANOVA: immobility Sex F(1,44) = 4.13, P = 0.048 (M > F) 
Dark–light emergence test 
 2 × 2 ANOVA: chamber crossings Sex F(1,53) = 12.70, P = 0.001 (M < F) 
Open field test 
 2 × 2 ANOVA: center time (inverse of thigmotaxis) Sex F(1,44) = 6.46, P = 0.015 (M > F) 
Trace fear conditioning 
 2 × 2 × 4 ANOVA: freezing Sex × Genotype F(1,47) = 7.80, P = 0.008 
  Sex × Genotype × Test F(3,141) = 4.14, P = 0.008 
Post hoc: training baseline versus cue test, cue baseline versus context test, all P < 0.008 
Test F(3,141) = 124.90, P < 0.001 
 2 × 4 ANOVA: freezing in females Genotype F(1,22) = 6.35, P = 0.020 (WT > Pten+/−
Test F(3,66) = 44.30, P < 0.001 
Genotype × Test F(3,66) = 4.56, P = 0.006 
 2 × 4 ANOVA: freezing in males Test F(3,75) = 86.76, P < 0.001 
 2 × 2 ANOVA: distance moved (training baseline) Sex F(1,47) = 4.10, P = 0.048 (M < F) 
 2 × 2 ANOVA: freezing (context test) Sex × Genotype F(1,47) = 7.07, P = 0.011 
Post hoc: t(24) = 3.04, P = 0.006 (M > F in Pten+/− mice) 
 2 × 2 ANOVA: freezing (cue test) Sex × Genotype F(1,47) = 4.54, P = 0.038 
Post hoc: t(24) = 2.25, P = 0.034 (M > F in Pten+/− mice) 
Circadian Rhythm and Activity 
 2 × 2 × 6 ANOVA: rotations Sex F(1,33) = 77.82, P < 0.001 (M < F) 
Sex × Genotype F(1,33) = 15.27, P < 0.001 
Post hoc: F(1,17) = 70.00, P < 0.001 (M < F in WT mice); F(1,16) = 14.77, P = 0.001 (M < F in Pten+/− mice) 
Time F(5,165) = 39.27, P < 0.001 
 2 × 2 × 6 ANOVA: bouts of activity Sex F(1,34) = 15.30, P < 0.001 (M > F) 
Genotype F(1,34) = 4.58, P = 0.040 (WT < Pten+/−
Time F(5,170) = 86.48, P < 0.001 
Sex × Time F(5,170) = 8.45, P < 0.001 
 Genotype effects on rotations in females Light phase day 3 t(16) = 2.21, P = 0.042 (WT > Pten+/−
Dark phase day 2 t(16) = 2.65, P = 0.017 (WT > Pten+/−
 Genotype effects on bouts of activity in females Dark phase day 3 t(16) = 2.16, P = 0.045 (WT < Pten+/−

N.B. 2 × 2 ANOVAs are sex × genotype analyses; 2 × 2 × 4 ANOVAs analyze effects of sex, genotype and test; 2 × 4 ANOVAs are analyses of genotype and time or test; 2 × 2 × 6 ANOVAs analyze each phase of the L:D cycle for 3 days within-subjects, as well as sex and genotype. Directions of main effects are indicated where possible.

Figure 1.

Germline Pten+/− mice are a model of absolute and relative brain overgrowth, reflecting broad expression of Pten mRNA in the developing mouse and human brain. (A) Representative WT brain (left), representative Pten+/− brain (middle), and overlay of representative WT (right) over Pten+/− brains (white with red outline); (B) body mass for WT and Pten+/− mice; (C) brain mass for WT and Pten+/− male and female mice; (D) brain as a percent of the body mass for WT and Pten+/− male and female mice; (E) mass of dissected brain regions from adult WT and Pten+/− male mice; (F) Images of the brains of wild type mice stained using in situ hybridization (ISH) probes to Pten; (G) comparison of mouse Pten ISH expression intensity across brain regions and developmental timepoints (data for F–G collated from http://developingmouse.brain-map.org); (H) comparison of human PTEN mRNA expression levels across brain regions and developmental timepoints (developmental transcriptome data for H collated from www.brainspan.org). PCW, postconceptional weeks; RPKM, reads per kilobase of exon model per million mapped reads. **indicates a significant difference between WT and Pten+/− mice, P < 0.001; *indicates a significant difference between WT and Pten+/− mice, P < 0.01.

Figure 1.

Germline Pten+/− mice are a model of absolute and relative brain overgrowth, reflecting broad expression of Pten mRNA in the developing mouse and human brain. (A) Representative WT brain (left), representative Pten+/− brain (middle), and overlay of representative WT (right) over Pten+/− brains (white with red outline); (B) body mass for WT and Pten+/− mice; (C) brain mass for WT and Pten+/− male and female mice; (D) brain as a percent of the body mass for WT and Pten+/− male and female mice; (E) mass of dissected brain regions from adult WT and Pten+/− male mice; (F) Images of the brains of wild type mice stained using in situ hybridization (ISH) probes to Pten; (G) comparison of mouse Pten ISH expression intensity across brain regions and developmental timepoints (data for F–G collated from http://developingmouse.brain-map.org); (H) comparison of human PTEN mRNA expression levels across brain regions and developmental timepoints (developmental transcriptome data for H collated from www.brainspan.org). PCW, postconceptional weeks; RPKM, reads per kilobase of exon model per million mapped reads. **indicates a significant difference between WT and Pten+/− mice, P < 0.001; *indicates a significant difference between WT and Pten+/− mice, P < 0.01.

Expression of Pten in the developing and adult mouse and human brain

We predicted that the widespread brain overgrowth in Pten+/− mice reflects the expression of Pten in the developing brain. Previous studies that have examined the expression of Pten mRNA or protein at select timepoints and/or in select brain regions provide evidence consistent with this possibility (33, 48–50). To obtain a global view of the spatiotemporal pattern of Pten expression across brain structures and developmental timepoints, we carried out an informatics analysis of Pten mRNA levels in the developing (E11.5, E13.5, E15.5, E18.5, P4, P14 and P28) and adult (P56) mouse brain using the Allen Brain Atlas (www.brain-map.org, last accessed on 1 December 2013) in situ hybridization data set (51). For any given stage of development, the expression level of Pten mRNA is similar across brain regions (Fig. 1F and G). We also examined the expression of PTEN in the human brain using the BrainSpan Atlas of the Developing Human Brain Developmental Transcriptome data set (www.brainspan.org, last accessed on 1 December 2013). The expression of PTEN mRNA is broad across brain regions for any given developmental stage in humans (Fig. 1H), similar to the mouse. Thus, based on both widespread mRNA expression and brain overgrowth, one might predict that the social behavioral deficits previously reported in Pten haploinsufficient mice (44) would occur in the context of wide-ranging behavioral deficits. To test this possibility, we employed a battery of behavioral assays relevant to ASD and comorbid disorders.

Tests related to ASD core symptoms

Three-chamber social approach and social novelty

As impaired social behavior is a key diagnostic criterion for autism, we tested Pten haploinsufficient mice and their WT littermates on the three-chamber social approach assay (52). In addition to the social approach phase, which involved choosing between a chamber containing an empty cage and one with a cage containing a novel social stimulus, we included a social novelty assay, where a novel stimulus was placed into the previously empty cage, to determine if there were abnormalities in the preference for a novel social stimulus over a familiar one (see Fig. 2A).

Figure 2.

Results of three-chamber social approach and social novelty test (A–C) and social recognition test (D–F) in Pten+/− mice. (A) Organization of three-chamber social approach and social novelty test; (B) three-chamber social approach and social novelty in females; (C) three-chamber social approach and social novelty in males; (D) organization of social recognition assay; (E) investigation of stimulus mouse in social recognition assay across trials; (F) habituation and dishabituation. *indicates a significant difference between the mouse chamber and the object chamber or between the novel and familiar mouse chambers, P < 0.05. **indicates a significant difference between the novel and familiar mouse chambers, P < 0.01. #indicates significant habituation or dishabituation, P < 0.05. ##indicates significant habituation or dishabituation, P < 0.01. ###indicates significant habituation or dishabituation, P < 0.001. juv., juvenile; ITI, intertrial interval; hab., habituation trial; dishab., dishabituation trial.

Figure 2.

Results of three-chamber social approach and social novelty test (A–C) and social recognition test (D–F) in Pten+/− mice. (A) Organization of three-chamber social approach and social novelty test; (B) three-chamber social approach and social novelty in females; (C) three-chamber social approach and social novelty in males; (D) organization of social recognition assay; (E) investigation of stimulus mouse in social recognition assay across trials; (F) habituation and dishabituation. *indicates a significant difference between the mouse chamber and the object chamber or between the novel and familiar mouse chambers, P < 0.05. **indicates a significant difference between the novel and familiar mouse chambers, P < 0.01. #indicates significant habituation or dishabituation, P < 0.05. ##indicates significant habituation or dishabituation, P < 0.01. ###indicates significant habituation or dishabituation, P < 0.001. juv., juvenile; ITI, intertrial interval; hab., habituation trial; dishab., dishabituation trial.

WT females spent significantly more time with the social stimulus during social approach, and the novel social stimulus during social novelty (all t > 2.40, all P < 0.034; see Fig. 2B), while Pten+/− females showed no significant differences in chamber time. WT male mice showed the same pattern as WT females, spending significantly more time in the chambers of the social and novel social stimuli during social approach and social novelty, respectively (all t > 3.08, all P < 0.010; see Fig. 2C). Pten+/− males did not show significant differences in chamber time in either social approach or social novelty (see Fig. 2C). There were no significant genotype differences in distance traveled or velocity in either the social approach or social novelty phases (data not shown). Additional statistics are in Table 2.

Table 2.

Between-subjects and mixed-model analyses of variance (ANOVAs) statistics for social behavior tests of Ptentm1Rps and DAT-Cre+/−; PtenloxP/loxP mice

Analysis Effect Statistics 
Social recognition 
 2 × 2 × 5 ANOVA: social investigation Test F(4,196) = 16.61, P < 0.001 
 2 × 2 ANOVA: habituation (H1 investigation – H4 investigation) Sex × Genotype F(1,49) = 4.60, P = 0.037 
Post hoc: t(30) = 2.46, P = 0.020 (M < F in Pten+/− mice) 
Three-chamber social approach and social novelty in DAT-Cre; PtenloxP/loxP mice 
 2 × 2 ANOVA: distance traveled (social approach, total) Sex × Genotype F(1,60) = 6.01, P = 0.017 
Post hoc: t(30) = 2.26, P = 0.032 (M < F in DAT-Pten-cKO mice) 
Analysis Effect Statistics 
Social recognition 
 2 × 2 × 5 ANOVA: social investigation Test F(4,196) = 16.61, P < 0.001 
 2 × 2 ANOVA: habituation (H1 investigation – H4 investigation) Sex × Genotype F(1,49) = 4.60, P = 0.037 
Post hoc: t(30) = 2.46, P = 0.020 (M < F in Pten+/− mice) 
Three-chamber social approach and social novelty in DAT-Cre; PtenloxP/loxP mice 
 2 × 2 ANOVA: distance traveled (social approach, total) Sex × Genotype F(1,60) = 6.01, P = 0.017 
Post hoc: t(30) = 2.26, P = 0.032 (M < F in DAT-Pten-cKO mice) 

NB: 2 × 2 ANOVAs are sex × genotype analyses; 2 × 2 × 5 ANOVAs analyze effects of sex, genotype and time. Directions of main effects are indicated where possible.

Our current results thus replicated our previous finding of social deficits in Pten+/− females, indicating that these are reliable phenotypes despite different rearing environments and testing apparati. Under both conditions, Pten+/− females consistently showed no significant social preference (44), even though the magnitude of this effect was relatively subtle (e.g., P = 0.027 in Pten+/+ versus P = 0.142 in Pten+/−). In the current experiment, Pten+/− females also displayed an impaired preference for social novelty, which had not been previously tested. Male Pten+/− mice did not show a significant social or novel social preference, in contrast to our previous findings (44), possibly due to different testing conditions, a steeper decline in social preference across the assay, development of a social aversion, or increased interest in the non-social stimulus.

Social recognition

In addition to female social approach deficits, we have previously found impaired social recognition, as measured by habituation in an extension of the three-chamber social approach task, in Pten+/− males (44). To explore this, we examined juvenile conspecific recognition in Pten+/− and WT mice by repeatedly presenting the same stimulus mouse (see Fig. 2D); decreased social investigation across four presentations denoted recognition (H1–H4; habituation). An increase in investigation from the last habituation trial during the presentation of a novel mouse (H5) indicated dishabituation, and was an important control for general habituation to the testing situation. Additionally, this task included a built-in measure of social interest, which was observed in H1.

Planned comparisons showed no significant differences between genotypes in initial social interest (H1), habituation, or dishabituation. However, Pten+/− males showed dishabituation [t(12) = 2.71, P = 0.019], but no habituation (see Fig. 2E and F); it should be noted, though, that the initial investigation at H1 was lower than investigation at dishabituation (H5). Females of both genotypes and WT males significantly habituated and dishabituated (all t > 2.71, all P < 0.017; see Fig. 2E and F). See Table 2 for additional statistics.

While normal social recognition (habituation followed by dishabituation) was found in females of both genotypes and male WT mice, Pten+/− males failed to habituate to a social stimulus. This is consistent with our previous results, despite different environments, apparati, stimuli, and test paradigms (44). Interestingly, unlike the social approach and social novelty tests, females of both genotypes were socially equivalent; both showed habituation and dishabituation, as did WT males; furthermore, all mice showed equivalent initial social interest in H1.

Marble burying test

In addition to social behavioral and communication deficits, the other core symptom of ASD is restricted, repetitive and stereotyped patterns of behavior (2–4). We assessed this type of behavior using the marble burying test (53). Planned comparison t-tests found that Pten+/− males buried significantly more marbles than WT male mice [t(22) = 2.10, P = 0.048; see Fig. 3], but showed no difference between Pten+/− and WT females. These results indicate that Pten+/− males exhibit more repetitive, stereotyped behavior than WT males, and that females show no genotype differences on this task.

Figure 3.

Marble burying test in Pten+/− mice. (A) Initial setup of marbles in cage; (B) percentage of marbles buried at least two-thirds after 30 min. *indicates a significant difference between WT and Pten+/− male mice, P < 0.05.

Figure 3.

Marble burying test in Pten+/− mice. (A) Initial setup of marbles in cage; (B) percentage of marbles buried at least two-thirds after 30 min. *indicates a significant difference between WT and Pten+/− male mice, P < 0.05.

Tests of mood disorder-like behaviors

Tail suspension test

Owing to the frequent comorbidity of depression and other mood disorders with ASD (45), we used the tail suspension test to determine whether Pten+/− mice showed a depression-like phenotype. In this paradigm, depression-like behavior is operationally defined as increased immobility. Planned comparisons revealed that Pten+/− males were significantly more immobile than WT males [t(25) = 3.11, P = 0.005; see Fig. 4A], but females did not differ by genotype. See Table 1 for additional statistics.

Figure 4.

Tests of depression-like (A and B) and anxiety-like behavior (C–H) in Pten+/− mice. (A) Tail suspension test; (B) forced swim test; (C, E, G) dark–light emergence test; (D, F, H) open field test. *indicates a significant difference between WT and Pten+/− male mice, P < 0.05. **indicates a significant difference between WT and Pten+/− males, P < 0.01. indicates a significant difference between WT male and female mice, P < 0.01. #indicates a significant difference between chambers, P < 0.05. ###indicates a significant difference between chambers or between the center and perimeter regions, P < 0.001.

Figure 4.

Tests of depression-like (A and B) and anxiety-like behavior (C–H) in Pten+/− mice. (A) Tail suspension test; (B) forced swim test; (C, E, G) dark–light emergence test; (D, F, H) open field test. *indicates a significant difference between WT and Pten+/− male mice, P < 0.05. **indicates a significant difference between WT and Pten+/− males, P < 0.01. indicates a significant difference between WT male and female mice, P < 0.01. #indicates a significant difference between chambers, P < 0.05. ###indicates a significant difference between chambers or between the center and perimeter regions, P < 0.001.

Forced swim test

The second rodent test of depression, the forced swim test, also uses immobility as a measure of depression-like behavior. Planned comparisons showed that Pten+/− males exhibited more immobility than WT males throughout the test [t(17.4) = 2.38, P = 0.029], particularly from 2 to 6 min [t(21) = 2.28, P = 0.033; see Fig. 4B], but no effects of genotype were found in females. Additional statistics are in Table 1.

Results of both the forced swim test and the tail suspension test indicate that Pten+/− males show more depression-like behavior than their WT littermates, while no differences were observed between genotypes for female mice.

Tests of anxiety-like behaviors

Dark–light emergence test

As ASD is also frequently comorbid with anxiety disorders (45), we investigated anxiety-like behavior in the Pten+/− mice using the dark–light emergence assay. This test assesses a mouse's willingness to explore a brightly lit open area, which is anxiogenic (likely due to increased vulnerability to predators), as opposed to remaining in a ‘safe’, dark, covered environment. Thus, less time spent in the brightly lit open chamber and a longer latency to enter this chamber indicates more anxiety-like behavior (54).

Planned comparisons revealed that Pten+/− males spent significantly more time in the light chamber, made more crossings between chambers and had a shorter latency to enter the light chamber than WT males (all t > 2.26, all P < 0.031; see Fig. 4C and E). All groups showed a significant preference for the dark chamber over the light chamber (all t > 2.76, all P < 0.023; see Fig. 4G), verifying that the light chamber was aversive, as expected. Table 1 contains additional statistics.

Open field test

In the open field test, increased thigmotaxis (the tendency of mice to avoid the center of the arena and instead occupy its corners and sides) indicated increased anxiety, while total distance moved and velocity measured activity.

Planned comparisons found that Pten+/− males spent more time in and made more entries into the center of the open field than WT males (all t > 2.18, all P < 0.041; see Fig. 4D). All groups spent the significant majority of time in thigmotaxis (all t > 20.60, all P < 0.001; see Fig. 4F), confirming the expected avoidance of the center of the open field.

No genotype differences were found for velocity or distance traveled (see Fig. 4H). This indicates that impaired locomotor performance does not account for results on this or other tests. Some sex differences were also observed, but there were no interactions by genotype (see Table 1 for these and additional statistics).

Both dark–light emergence and open field assays found that Pten+/− male mice exhibited less anxiety than WT males, while still avoiding the light chamber or center of the open field and showing no differences in locomotion. Female mice showed no genotype differences.

Emotional learning

Trace fear conditioning

In addition to having a greater likelihood of mood and anxiety disorders, individuals with ASD may be less responsive to emotional stimuli and show differences in conditioned fear (55– 60), and often show comorbid intellectual disabilities (46). We therefore tested Pten+/− mice and their WT littermates on trace fear conditioning, which involves learning, memory, attentional and emotional components. An increase in freezing from baseline to test indicates that mice have learned to associate a neutral stimulus (tone or context) with an aversive one (footshock).

Planned comparisons showed no genotype differences in freezing during any phase in males (see Fig. 5D and F), and significant effects of genotype in females, with Pten+/− mice freezing less during the context and cue tests (all t > 2.26, all P < 0.034; see Fig. 5D and E). All groups showed significant effects of test on freezing behavior (all t > 10.45, all P < 0.001; see Fig. 5D). Post hoc tests revealed differences between training baseline and context test (all P < 0.001; see Fig. 5D), and between cue baseline and cue test (all P < 0.009; see Fig. 5D), indicating that all mice learned both contextual and cued associations. Additional statistics are shown in Table 1.

Figure 5.

Trace fear conditioning in Pten+/− mice. (A) Training paradigm; (B) chamber for training and context test; (C) ‘disguised’ chamber for cue baseline and cue test: chambers were disguised by altering their shape, size, color, and texture using opaque white inserts (triangular shape with two white walls of 30.3 cm and the wall containing the door measuring 24.5 cm, lights off in both room and box, smooth white floor instead of grid), as well as sound (no fans) and odor [cleaned with 70% isopropyl alcohol (Fisher Scientific, Pittsburgh, PA, USA) instead of EtOH, orange extract in box (McCormick Pure Orange Extract, McCormick & Company, Inc., Sparks, MD, USA)]. Mice were also moved from the colony room in a clear plastic box (35 × 41 × 17.5 cm), draped and containing a novel type of bedding material, instead of from a holding room in their home cage, and were placed in different chambers (i.e., different locations) than previously used; (D) freezing as percent of time by phase; (E) freezing as a percent of time in females; (F) freezing as a percent of time in males. *indicates a significant difference between WT and Pten+/− females, P < 0.05.

Figure 5.

Trace fear conditioning in Pten+/− mice. (A) Training paradigm; (B) chamber for training and context test; (C) ‘disguised’ chamber for cue baseline and cue test: chambers were disguised by altering their shape, size, color, and texture using opaque white inserts (triangular shape with two white walls of 30.3 cm and the wall containing the door measuring 24.5 cm, lights off in both room and box, smooth white floor instead of grid), as well as sound (no fans) and odor [cleaned with 70% isopropyl alcohol (Fisher Scientific, Pittsburgh, PA, USA) instead of EtOH, orange extract in box (McCormick Pure Orange Extract, McCormick & Company, Inc., Sparks, MD, USA)]. Mice were also moved from the colony room in a clear plastic box (35 × 41 × 17.5 cm), draped and containing a novel type of bedding material, instead of from a holding room in their home cage, and were placed in different chambers (i.e., different locations) than previously used; (D) freezing as percent of time by phase; (E) freezing as a percent of time in females; (F) freezing as a percent of time in males. *indicates a significant difference between WT and Pten+/− females, P < 0.05.

Pten+/− females showed less fear than WT females in response to shock-associated context and cue, indicating a potential deficit in emotional learning and memory. Males did not show any genotype differences on this assay.

Circadian rhythm and activity

Parental reports estimate that 50–80% of children with ASD have sleep difficulties, compared with 9–50% of age-matched control children (47). These difficulties include problems with sleep initiation and maintenance, less sleep, unstable sleep and irregular sleep–wake patterns (47, 61–63). Thus, we investigated activity patterns of Pten haploinsufficient mice on a 12:12 light/dark (L:D) schedule, and assessed the free-running circadian rhythm of these animals during constant darkness (D:D).

Light : dark schedule

Planned comparisons between genotypes showed that female Pten+/− mice made fewer wheel rotations than WT females throughout the L:D schedule, as well as across light phases (all t > 2.21, all P < 0.043; see Fig. 6A and B). During the dark phases of the L:D cycle, Pten+/− females performed fewer rotations and more continuous bouts of activity (all t > 2.16, all P < 0.046; see Fig. 6B and D). Planned comparisons between genotypes in males found fewer differences: Pten+/− mice made more rotations during the light phase of Day 3, which was sufficient to drive an increase in rotations across light phases (all F > 6.01, all P < 0.026; see Fig. 6A), but no male genotype differences were observed for bouts of activity (see Fig. 6B). Additional statistics can be found in Table 1.

Figure 6.

Circadian rhythm and activity during 12:12 h light/dark (L:D; A–D) and constant dark (D:D; E–I) schedules in Pten+/− mice. (A and B) Wheel rotations during L:D; (C and D) bouts of activity during L:D; (E) average tau during D:D. (F–I) Representative actograms: (F) WT female; (G) Pten+/− female; (H) WT male; (I) Pten+/− male. *indicates a significant difference between WT and Pten+/− females, P < 0.05. **indicates a significant difference between WT and Pten+/− females, P < 0.01. ¤indicates a significant difference between WT and Pten+/− males, P < 0.05. ##indicates a significant difference between light and dark phases, P < 0.01. ###indicates a significant difference between light and dark phases, P < 0.001.

Figure 6.

Circadian rhythm and activity during 12:12 h light/dark (L:D; A–D) and constant dark (D:D; E–I) schedules in Pten+/− mice. (A and B) Wheel rotations during L:D; (C and D) bouts of activity during L:D; (E) average tau during D:D. (F–I) Representative actograms: (F) WT female; (G) Pten+/− female; (H) WT male; (I) Pten+/− male. *indicates a significant difference between WT and Pten+/− females, P < 0.05. **indicates a significant difference between WT and Pten+/− females, P < 0.01. ¤indicates a significant difference between WT and Pten+/− males, P < 0.05. ##indicates a significant difference between light and dark phases, P < 0.01. ###indicates a significant difference between light and dark phases, P < 0.001.

Constant dark schedule

No significant effects of sex, genotype or their interaction on free-running circadian rhythm (tau) were found (see Fig. 6E–I).

While we observed no significant change in tau phenotype, Pten+/− females were less active than their WT counterparts during the light phase, dark phase and throughout the L:D schedule, and had more bouts of activity during the dark phase than the WT females, suggesting that, in addition to the decrease in overall activity, their activity was less sustained and continuous. Male Pten+/− mice were more active than WT males during the light phase, but did not differ in number of bouts. All mice were more active during the dark phase than during the light phase.

Motor coordination and nociception

Hotplate test

Decreased pain sensitivity could explain Pten+/− females' deficits in trace fear conditioning. To rule this out, mice were tested on the hotplate; a longer latency to lick a hind paw indicated reduced nociception. As planned comparisons revealed no genotype differences, and no significant effects of genotype, sex or their interaction were found (see Fig. 7A), differences in nociception are unlikely to account for our results.

Figure 7.

Sensorimotor assays: nociception (A) and motor learning and behavior (B) in Pten+/− mice. (A) Hotplate test; (B) rotarod test.

Figure 7.

Sensorimotor assays: nociception (A) and motor learning and behavior (B) in Pten+/− mice. (A) Hotplate test; (B) rotarod test.

Rotarod test

To verify that there were no balance, motor behavior, or motor learning impairments, which could confound the results of the circadian wheel-running assay, mice were tested on the rotatod. A shorter latency to fall indicated impaired motor coordination and balance, and increased fall latency on later trials indicated motor learning of the task. All groups showed significant improvement across the three tests (all F > 5.77, all P < 0.010; see Fig. 7B), indicating normal motor learning. Planned comparisons found no genotype differences within each sex, and there were no significant effects of genotype, sex or their interaction on the latency to fall on any or across all tests.

Social behavior in mice with conditional inactivation of Pten in dopaminergic neurons

Three-chamber social approach and social novelty

Our results indicate that germline Pten haploinsufficiency leads to surprisingly specific behavioral impairments. This suggests that distinct neural circuits and constituent cell types, including those involved in social behavior, may be more vulnerable or less adaptable to changes in patterns of growth caused by mutations in Pten. The dopamine (DA) system is a likely candidate: it is implicated in many of the behaviors altered in Pten+/− mice (e.g. social behavior, repetitive behavior, aversive conditioning, anxiety and depression) (64–73), and decreased DA activity has also been observed in the medial prefrontal cortices of autistic children (74). Therefore, we used mice in which Pten was conditionally inactivated in DA neurons using the Cre-loxP system to investigate the role of Pten in DA neurons on social behavior. Importantly, it has previously been shown that dopaminergic (DA) neurons are overgrown (via both hypertrophy and hyperplasia) in animals in which Pten has been conditionally deleted using a Cre line that expresses in the pattern of Dopamine transporter (also known as Slc6a3 or DAT) (75). Thus, Slc6a3tm1.1(cre)Bkmn/WT; Ptentm1Hwu/tm1Hwu (referred to here as DAT-Cre+/−;PtenloxP/loxP) mice and their Cre-negative control (DAT-Cre−/−;PtenloxP/loxP) littermates were assayed on the three-chamber social approach and social novelty assay to test whether the behavioral phenotypes observed in Pten+/− mice may be due to modulation of Pten in DA neurons.

Control females spent significantly more time with the social stimulus during social approach [t(17) = 2.52, P = 0.022], but showed no preference for the novel social stimulus during social novelty, and DAT-Cre+/−; PtenloxP/loxP females showed no social or social novel stimulus preference (see Fig. 8A). Both control and DAT-Cre+/−; PtenloxP/loxP males spent significantly more time with the social stimulus during social approach (all t > 3.08, all P < 0.010), while control [t(13) = 2.60, P = 0.022], but not DAT-Cre+/−; PtenloxP/loxP males, significantly preferred the chamber containing the novel social stimulus during social novelty (see Fig. 8B). DAT-Cre+/−; PtenloxP/loxP males also traveled significantly shorter distances during the social approach phase [t(26) = 2.54, P = 0.017; see Fig. 8C]. Additional statistics are in Table 1.

Figure 8.

Results of three-chamber social approach and social novelty test in DAT-Cre+/−; PtenloxP/loxPmice. (A) Three-chamber social approach and social novelty in females; (B) three-chamber social approach and social novelty in males; (C) distance traveled by across social approach and social novelty. *indicates a significant difference between the mouse chamber and the object chamber, P < 0.05. **indicates a significant difference between the mouse chamber and the object chamber or between the novel and familiar mouse chambers, P < 0.01. #indicates a significant difference between DAT-Cre−/−; PtenloxP/loxP and DAT-Cre+/−; PtenloxP/loxP males, P < 0.05. indicates a significant difference between male and female DAT-Cre+/−; PtenloxP/loxP mice, P < 0.05.

Figure 8.

Results of three-chamber social approach and social novelty test in DAT-Cre+/−; PtenloxP/loxPmice. (A) Three-chamber social approach and social novelty in females; (B) three-chamber social approach and social novelty in males; (C) distance traveled by across social approach and social novelty. *indicates a significant difference between the mouse chamber and the object chamber, P < 0.05. **indicates a significant difference between the mouse chamber and the object chamber or between the novel and familiar mouse chambers, P < 0.01. #indicates a significant difference between DAT-Cre−/−; PtenloxP/loxP and DAT-Cre+/−; PtenloxP/loxP males, P < 0.05. indicates a significant difference between male and female DAT-Cre+/−; PtenloxP/loxP mice, P < 0.05.

DISCUSSION

We have demonstrated that brain overgrowth occurs broadly across brain regions in germline Pten+/− mice, likely reflecting the expression of Pten across anatomical regions and time points in the developing brains of mice and humans. However, we have also shown that the behavioral phenotypes present in germline Pten+/− mice are selective (see Table 3 for summary of results). This finding opens the door for exploring the possibility that most brain areas and constituent cell types adapt to an altered pattern of growth with limited impact on behavior, while key areas and cell types relevant to social behavior are more vulnerable or less adaptable, thus resulting in social deficits. Consistent with this idea, mice with conditional inactivation of Pten in the pattern of Dopamine transporter (DAT-Cre+/−; PtenloxP/loxP), which have hypertrophy and hyperplasia of DA neurons (75) (i.e., desynchronized growth of a cell type important for social behavior), display social deficits in our hands.

Table 3.

Summary of behavioral test results in Ptentm1Rps and DAT-Cre+/−; PtenloxP/loxP mice

 
Test Males Females 
Ptentm1Rps mice 
Brain mass (absolute, relative) ↑ ↑ 
Social approach ↓ ↓ 
Social novelty ↓ ↓ 
Social recognition (habituation) ↓ – 
Marble burying test ↑ – 
Tail suspension test (‘depression’) ↑ – 
Forced swim test (‘depression’) ↑ – 
Dark–light emergence (‘anxiety’) ↓ – 
Open field test (‘anxiety’) ↓ – 
Trace fear conditioning – ↓ 
Circadian rhythm/activity (tau during D:Da– – 
Circadian rhythm/activity (activity in L:Dbc ↓ 
Hotplate test – – 
Rotarod test – – 
DAT-Cre+/−; PtenloxP/loxP Mice 
Social approach – ↓ 
Social novelty ↓ d 
 
Test Males Females 
Ptentm1Rps mice 
Brain mass (absolute, relative) ↑ ↑ 
Social approach ↓ ↓ 
Social novelty ↓ ↓ 
Social recognition (habituation) ↓ – 
Marble burying test ↑ – 
Tail suspension test (‘depression’) ↑ – 
Forced swim test (‘depression’) ↑ – 
Dark–light emergence (‘anxiety’) ↓ – 
Open field test (‘anxiety’) ↓ – 
Trace fear conditioning – ↓ 
Circadian rhythm/activity (tau during D:Da– – 
Circadian rhythm/activity (activity in L:Dbc ↓ 
Hotplate test – – 
Rotarod test – – 
DAT-Cre+/−; PtenloxP/loxP Mice 
Social approach – ↓ 
Social novelty ↓ d 

aD:D constant dark schedule.

bL:D schedule of 12:12 h light/dark.

cOnly in light phase.

dNo social novelty preference in control females.

Germline Pten+/− mice of both sexes showed social impairments; both males and females showed no social or social novelty preference in the three-chamber social approach and social novelty assay. Additionally, germline Pten+/− males failed to habituate during the social recognition test. Thus, females appear to show decreased interest in social stimuli, but intact social recognition, while males exhibit social recognition impairments and deficits in social interest. These data support the idea of distinction between social recognition, as defined by habituation/dishabituation, and a preference for a social or novel social stimulus, as tested in the three-chamber assay. Similar results have been found using oxytocin knockout mice, which show impaired social recognition (76) but intact social approach behavior (77).

Male Pten+/− mice also exhibited increased repetitive (marble burying) and depression-like behavior, and minimal effects on circadian rhythmicity and activity; no differences were found on trace fear conditioning, rotarod or hotplate tests. Interestingly, Pten+/− male mice also showed decreased anxiety-like behavior. Although this contrasts with reports of elevated anxiety in some individuals with ASD (45), this does indicate dysregulation of anxiety-like behavior in these mice. Pten+/− female mice showed a different pattern of results, with circadian activity and trace fear conditioning phenotypes as well as decreased social interest, but no differences from WT on any other test. These sexually dimorphic patterns of impairment are not surprising, given both the unequal frequency of ASD between sexes (1) and the well-documented involvement of the Pten-PI3K–Akt–mTOR pathway in both androgen and estrogen receptor signaling (78–83). Indeed, investigating the biological basis of these differences may provide insight into underlying causes of sex differences in ASD.

The abnormal behavior shown by germline Pten+/− mice (summarized in Table 3) appears to be largely restricted to social behavior, with sex-specific impairments in repetitive behavior, mood and anxiety (males), and possibly circadian rhythmicity and emotional memory (females). This suggests that the behavioral effects of Pten haploinsufficiency are more subtle than the broad expression and cellular function of Pten would predict. Consistent with this, a recent report of cognitive testing in humans with germline PTEN mutations has shown a broad range of intact intellectual abilities in this population (84). This raises the question of how mutations in Pten, a general regulator of growth, can have relatively selective effects on behavior and cognition. As a way to explore this question, we hypothesize that one effect of Pten mutations may be to desynchronize the normal pattern of growth in key cell types relevant for social behavior, thus diverting the downstream connectivity and synaptic function of circuitry underlying social behavior toward a pathological state.

DA neurons are one cell type suitable for testing this hypothesis. The DA system is known to influence social behavior, along with other behaviors altered in germline Pten+/− mice (64, 85). There is evidence that Pten acts in DA neurons of the ventral tegmental area (VTA) to regulate the activity of serotonin 2c receptors (5-HT2cR) (86). The 5-HT2cR activity in turn modulates the activity of VTA DA neurons (87); these neurons project to the nucleus accumbens and the prefrontal cortex (88), areas that have been implicated in social interaction and social behavior (89, 90). We have previously shown that haploinsufficiency for serotonin transporter (Slc6a4), which is presumed to elevate levels of synaptic serotonin, exacerbates social deficits in Pten haploinsufficient mice (44). Furthermore, decreased DA activity has also been observed in the medial prefrontal cortices of autistic children (74), further supporting this as a candidate circuit (91).

Consistent with this possibility, female DAT-Cre+/−; PtenloxP/loxP mice failed to show a preference for a social stimulus in the social approach assay and male DAT-Cre+/−; PtenloxP/loxP mice demonstrated an impaired preference for social novelty. While DAT-Cre+/−; PtenloxP/loxP females were also impaired in social novelty, the interpretation of this result is unclear since control females in this experiment did not show a preference for the novel stimulus. This is in contrast to social novelty results in germline Pten+/− females and WT controls, where WT animals showed a significant preference for the novel social stimulus and mutants did not. In males, both germline Pten+/− and DAT-Cre+/−; PtenloxP/loxP mice showed deficits in the social novelty assay; however, DAT-Cre+/−; PtenloxP/loxP males did not show the same deficits in the social approach assay found in germline Pten+/− males. While the reason for these differences in social behavioral results between germline Pten+/− and DAT-Cre+/−; PtenloxP/loxP mice may be related to the nature of the genetic manipulations involved, another possible explanation is that germline Pten+/− mice were tested in the dark, active phase of the cycle, while DAT-Cre+/−; PtenloxP/loxP were tested in the light, inactive phase. Additionally, male DAT-Cre+/−; PtenloxP/loxP mice also showed reduced locomotor activity during the social approach; previous research has not found such impairments, although the sex of these mice was not specified (92). Thus, this could be because of pooling the sexes, as we found that female DAT-Cre+/−; PtenloxP/loxP mice showed more locomotion than their male counterparts during social approach. These interesting results merit further study to determine whether these deficits generalize to other social assays. Research is also required to determine whether they replicate the effects of germline haploinsufficiency for Pten on other behavioral tasks, such as repetitive behavior, anxiety-like behavior and learning and memory.

The behavioral results, taken together with evidence supporting an interaction of the Pten-PI3K-Akt-mTOR and serotonin pathways (44, 86), also implicate serotonergic neurons and postsynaptic targets. Serotonin signaling is known to influence social behavior, along with repetitive or stereotypic behavior, mood and anxiety, learning and memory, and circadian rhythms (reviewed in 93). This is further supported by the treatment of ASD patients with serotonergic drugs (reviewed in 94, 95). An additional possible cell type is oxytocinergic neurons; the involvement of oxytocin in social behavior is well documented (89, 96, 97), and it has also been shown to affect depression- and anxiety-like behaviors (98). ASD patients treated with oxytocin have also shown improvements [see (94) for a review]. Future experiments will test the effect of Pten mutations in these and select other candidate cell types to help elucidate the relationship between Pten-regulated growth, synaptic connectivity/plasticity and the development of behavior and cognition, especially as it relates to autism.

CONCLUSIONS

Pten germline haploinsufficiency in mice, which causes a profound and consistent brain overgrowth and which approximates a risk factor in humans with PTEN hamartoma syndromes, ASD and other disorders, results in surprisingly specific effects on behaviors relevant to ASD and comorbid disorders. Social deficits were observed in both sexes. Males also showed repetitive behavior, increased depression-like behavior and decreased anxiety, while females exhibited abnormal circadian rhythm and activity and impaired emotional learning. The limited behavioral impairments observed suggest that specific cell types or neural systems may be particularly vulnerable to mutations in Pten, such as the DA system. Mice in which Pten had been inactivated in DA neurons showed impaired social approach behavior in the three-chamber test. Future research involving additional social assays will expand on the nature of this impairment, and will address which social and non-social phenotypes are associated with disrupted Pten function in this and other specific cell types and/or neural systems.

MATERIALS AND METHODS

Informatics analysis of Pten expression in the developing mouse and human brain

The source data for the analysis of Pten expression in the developing mouse brain were from the Allen Brain Atlas (www.brain-map.org, last accessed on 1 December 2013) in situ hybridization data set (51). The analysis of PTEN expression in the developing human brain was based on data from the BrainSpan Atlas of the Developing Human Brain Developmental Transcriptome data set (www.brainspan.org, last accessed on 1 December 2013).

Subjects

Mice of the B6.129-Ptentm1Rps strain (99) (from the National Cancer Institute, backcrossed to congenicity in the C57BL/6 background for 10 generations) were crossed with C57BL/6J mice, thus producing Ptentm1Rps/+(Pten haploinsufficient or Pten+/−) and WT (Pten+/+) offspring.

To conditionally inactivate Pten in DA neurons, we used the Cre-loxP system. We used the B6.SJL-Slc6a3tm1.1(cre)Bkmn/J strain (100), which expresses Cre in the pattern of Dopamine transporter (DAT), and the B6.129S4-Ptentm1Hwu/J strain (101), in which exon 5 of Pten is flanked by loxP sites. Both lines were from the Jackson Laboratory and backcrossed to congenicity in the C57BL/6 background for at least 10 generations. Slc6a3tm1.1(cre)Bkmn/+; Ptentm1Hwu/tm1Hwu (DAT-Cre+/−; PtenloxP/loxP) mice and Cre-negative (DAT-Cre−/−; PtenloxP/loxP) littermate controls for use in testing were generated by crossing Slc6a3tm1.1(cre)Bkmn/+; Ptentm1Hwu/tm1Hwu with Ptentm1Hwu/tm1Hwu mice.

Mice were housed in groups of 2–5 in clear polyethylene cages (19.1 × 29.2 × 12.7 cm; Allentown Inc., Allentown, NJ, USA) on ventilated racks (Model no. MD75JU160MVPSHR, Allentown Inc., Allentown, NJ, USA) and provided with 1/4′′ corncob bedding, nestlets, and food (Teklad Global 18% Protein Extruded Rodent Diet 2920X) and tap water ad libitum.

Pten+/− and WT mice of both sexes were tested in adulthood [postnatal day 49 (P49) to P142] during the dark (active) phase of a 12:12 h reversed light–dark cycle (lights on at 2230 hours). DAT-Cre+/−; PtenloxP/loxP and control mice of both sexes were tested in adulthood (P48–P106) during the light phase of a 12:12 h non-reversed L:D cycle (lights on at 0700 hours). Separate cohorts of mice were run through the following batteries:

  • Group 1: Tail suspension test, rotarod test, trace fear conditioning

  • Group 2: Tail suspension test, rotarod test, trace fear conditioning, hotplate test

  • Group 3: Dark–light emergence, open field test, hotplate test, circadian rhythm and activity

  • Group 4: Forced swim test

  • Group 5: Social recognition

  • Group 6: Three-chamber social approach + social novelty

  • Group 7: Social recognition, marble burying

  • Group 8 (DAT-Cre; PtenloxP/loxP mice): Three-chamber social approach + social novelty

All research was conducted in accordance with National Institutes of Health and Association for Assessment and Accreditation of Laboratory Animal Care guidelines and approved by The Scripps Research Institute's Institutional Animal Care and Use Committee.

Behavioral tests

Unless specified, behavioral testing took place under red-light conditions, and mice were moved to a holding room in the behavioral testing area at least 1 h prior to the beginning of the assay. Apparati were cleaned with 70% ethanol (EtOH; Sigma-Aldrich, St. Louis, MO, USA), 1% Micro-90 (International Products Corporation, Burlington, NJ, USA) and/or quatricide (2 oz/gallon; Pharmacal Research Laboratories, Inc., Waterbury, CT, USA), unless otherwise stated. Manual scoring was performed by a trained observer blind to sex and genotype; automatic scoring was performed using the Ethovision XT video tracking system (Noldus, Wageningen, The Netherlands). Mice were typically tested in batteries, including both established tests and those under development, with at least 3–7 days between assays. Details of these paradigms are listed below.

Three-chamber social approach + social novelty

Mice (Pten+/+ females, n = 13; Pten+/− females, n = 12; Pten+/+ males, n = 13; Pten+/− males, n = 10; DAT-Cre−/−; PtenloxP/loxP females, n = 18; DAT-Cre+/−; PtenloxP/loxP females, n = 18; DAT-Cre−/−; PtenloxP/loxP males, n = 14; DAT-Cre+/−; PtenloxP/loxP females, n = 14) were tested as previously described (52) under white-light conditions. Briefly, test mice were each placed into the center of a clear acrylic arena (59 × 39 × 22 cm) that was divided into three equal compartments (each 19.5 × 39 × 22 cm). Testing consisted of three phases (see Fig. 2A): 5 min acclimation to the empty arena, 10 min sociability testing [choice between two acrylic cages (18 clear vertical bars of 0.6 cm in diameter, clear lid and black floor of 18.1 cm in diameter), one containing a novel, same-sex conspecific (location counterbalanced across mice)], and 10 min social novelty testing (novel, same-sex conspecific placed in the previously empty cage; see Fig. 2A). Cages and chambers were cleaned with Micro-90 and/or quatricide and paper towel-dried between mice. Time spent in each chamber was recorded by Ethovision.

Social recognition

The social recognition test took place in a home cage-like environment in which test mice (Pten+/+ females, n = 16; Pten+/− females, n = 13; Pten+/+ males, n = 11; Pten+/− males, n = 13) had been housed alone for 2 h (102). The procedure was modified from Ref. (103); briefly, stimuli were same-sex, juvenile (P21–P28) conspecifics contained in an acrylic tube (7.25 cm in diameter, 12.5 cm tall) with holes in the bottom (3 offset rows of twelve 4 mm-diameter holes). Tests consisted of 5 min stimulus presentations separated by 10 min intertrial intervals; the first four presentations were of the same mouse, with the fifth using a novel juvenile (see Fig. 2E). Investigation was manually scored from video, and mice spending <10 s investigating the stimulus during the first habituation (distributed across groups) were removed from the analysis.

Marble burying

Under white-light conditions, mice (Pten+/+ females, n = 12; Pten+/− females, n = 12; Pten+/+ males, n = 12; Pten+/− males, n = 12) were placed individually in a home-cage-like environment with 5 cm of 1/4″ corncob bedding and 20 black marbles (14.3 mm in diameter) arranged in a 4 × 5 matrix (see Fig. 3A), and left undisturbed for 30 min. The number of marbles that were at least two-thirds buried at the end of the trial were counted (53).

Tail suspension test

Mice (Pten+/+ females, n = 12; Pten+/− females, n = 12; Pten+/+ males, n = 13; Pten+/− males, n = 14) were suspended from a hook with medical tape attached ∼2 cm from the tail tip for 6 min, and immobility was recorded with Ethovision using a 7% immobility threshold.

Forced swim test

Under white light, mice (Pten+/+ females, n = 13; Pten+/− females, n = 12; Pten+/+ males, n = 12; Pten+/− males, n = 11) were placed into a 2L Pyrex beaker (No. 1000, Corning, Tewksbury, MA, USA) containing 14 cm of 30°C water. Water temperature was maintained by a heating pad on the low setting (Walgreens Heating Pad Model 533-912, Walgreens, Deerfield, IL, USA). Time spent immobile during the 6 min test was manually recorded. Following testing, mice were placed in a clean cage to recover and dry off; one corner of this cage was under a surgical light (Amsco Examiner 10, Steris, Mentor OH) for additional heat, and reached ∼29°C. Mice were then returned to their home cages.

Dark–light emergence

Mice (Pten+/+ females, n = 12; Pten+/− females, n = 10; Pten+/+ males, n = 19; Pten+/− males, n = 16) were tested in two chambers of the three-chamber apparatus. The dark chamber had black inserts on the walls and floor, a black lid, and illumination of ∼0–1 lux, while the light side had clear side walls, one white wall separating it from the unused third chamber (opposite the dark chamber), a light gray floor, no lid, and was illuminated to ∼600 lux with a lamp. Mice were placed into the dark chamber and allowed to explore for 5 min. Latency to enter the light compartment, duration of time in each compartment, and number of crossings between chambers were manually scored from video.

Open field test

Each mouse (Pten+/+ females, n = 12; Pten+/− females, n = 12; Pten+/+ males, n = 12; Pten+/− males, n = 12) was placed in the center of the open field arena (43.8 × 43.8 × 32.8 cm) under ∼240 lux for 5 min. Total distance moved, velocity and measures (time and number of entries) of center and thigmotaxis (occupying the corners and sides of the open field) time were recorded automatically.

Trace fear conditioning

The trace fear conditioning paradigm consisted of four phases: training, context test, cue baseline, and cue test. During training, each mouse (Pten+/+ females, n = 12; Pten+/− females, n = 12; Pten+/+ males, n = 13; Pten+/− males, n = 14) was placed into a Phenotyper chamber (29.2 × 29 × 30.5 cm, Pten-T10/N, Noldus Information Technology, Wageningen, The Netherlands) equipped with an electrified floor constructed of 32 steel bars (0.3 cm in diameter, spaced 1 cm apart), a speaker, and clear walls, each inside noise-attenuating boxes with fans on (59.2 × 40.5 × 55.7 cm, Med Associates Inc., St. Albans, VT, USA). White lights were on, resulting in illumination of ∼245 lux in each chamber. Training consisted of a 2 min baseline followed by five tone–interval–shock sequences (20 s long 85 dB white noise ‘tone’, 20 s interval, 2 s 0.5 mA footshock) at variable intersequence intervals of 150–200 s (see Fig. 5A). Testing occurred 24 h later; mice were tested for contextual conditioning by returning to their training chambers for 5 min, and for cue conditioning at least 1 h later by being placed into a novel environment for a 3 min baseline followed by a 3 min tone presentation (see Fig. 5B and C). Freezing behavior was automatically recorded during training and testing.

Rotarod test

Mice (Pten+/+ females, n = 13; Pten+/− females, n = 12; Pten+/+ males, n = 12; Pten+/− males, n = 11) were placed on a rotating rod ∼10.5 cm in circumference (ENV-577M, Med Associates Inc., St. Albans, VT, USA); the speed of rotation gradually increased from 4 to 40 rpm over a 5 min period. Mice received three trials, spaced at least 1 h apart. Their latency to fall, both over time and averaged across tests, was measured.

Hotplate test

Each mouse (Pten+/+ females, n = 12; Pten+/− females, n = 12; Pten+/+ males, n = 12; Pten+/− males, n = 13) was gently placed on a hotplate (Hot/Cold Plate 35100, Ugo Basile, Comerio, Italy) maintained at 55°C and surrounded by a clear cylinder, where it remained for 45 s or until it licked a rear paw.

Circadian rhythm and activity

Mice (Pten+/+ females, n = 10; Pten+/− females, n = 8; Pten+/+ males, n = 9; Pten+/− males, n = 10) were individually housed for 10 days in cages (36 × 20.5 × 14 cm, Allentown Inc., Allentown NJ) containing a running wheel (5.5 cm wide and 11 cm in diameter), 1/8″ corncob bedding, white noise, and ad libitum access to food and water. The light cycle was unchanged from that of the colony room for the first 3 days (L:D schedule), and mice were then housed in constant darkness for 7 days (D:D schedule). The frequency of wheel rotations were constantly recorded using ClockLab (Actimetrics, Wilmette, IL, USA) as a measure of activity and wakefulness. During the L:D schedule, wheel rotations and ‘bouts’ of activity (continuous rotations) were analyzed overall, by phase (light or dark), and by day. The free-running circadian rhythm (tau) was assessed during the D:D schedule.

Statistical analysis

Planned comparisons between genotypes within each sex and/or time were performed for all assays using independent-sample t-tests, as we have previously observed sex-specific phenotypes in Pten+/− mice (44). Additional statistics are listed in Tables 1 and 2. Tail suspension, forced swim, dark–light emergence, open field, marble burying, rotarod and hotplate tests were analyzed with 2 (sex: male or female) × 2 (genotype: Pten+/− or WT) between-subjects analyses of variance (ANOVAs). Within-group comparisons were used to analyze chamber preferences for the three-chamber social approach plus social novelty test (time in mouse chamber versus time in empty chamber; time in novel mouse chamber versus time in familiar mouse chamber), preference for dark chamber in the dark–light emergence test (time in dark chamber versus time in light chamber), and preference for thigmotaxis in the open field test (time in thigmotaxis versus time in center). Mixed model ANOVAs were used to analyze data from social recognition [2 (sex) × 2 (genotype) × 5 (test)], trace fear conditioning [2 (sex) × 2 (genotype) × 4 (phase: training baseline (0–2 min), context test, cue test baseline, cue test)], and circadian rhythm and activity [2 (sex) × 2 (genotype) × phase (light or dark; collapsed over 3 days and individually)] assays. Post hoc tests consisted of Tukey's or Bonferroni corrected t-tests as appropriate. In all cases, normality was assessed using Levene's and/or Mauchly's tests to ensure appropriate statistical tests were used. All statistics were performed using PASW 18 (IBM Corporation, Armonk, NY, USA), with significance set at P < 0.05. Statistics for non-significant results are not shown.

FUNDING

We are grateful for gift funds from Mrs Nancy Lurie Marks and startup funds from The Scripps Research Institute for support.

ACKNOWLEDGEMENTS

We thank Dr Alicia Faruzzi Brantley for behavioral testing advice and for supervising the Mouse Behavior Core at The Scripps Research Institute, as well as Dr Clemence Girardet for valuable advice on behavioral tests included in this paper, and Dr Oscar Diaz-Ruiz for assistance with testing of the DAT-Cre+/−; PtenloxP/loxP mice. We also thank Mrs Trina L. Kemp for invaluable administrative assistance, and Dr Julien Séjourné and Mr Craig P. Allen for useful advice on an early version of this manuscript. The BrainSpan Atlas of the Developing Human Brain (©2012 Allen Institute for Brain Science) is available from http://brainspan.org. The Allen Developing Mouse Brain Atlas (©2012 Allen Institute for Brain Science) is available from http://developingmouse.brain-map.org.

Conflict of Interest statement. None declared.

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