Home-cage hypoactivity in mouse genetic models of autism spectrum disorder

Abstract

Genome-wide association and whole exome sequencing studies from Autism Spectrum Disorder (ASD) patient populations have implicated numerous risk factor genes whose mutation or deletion results in significantly increased incidence of ASD. Behavioral studies of monogenic mutant mouse models of ASD-associated genes have been useful for identifying aberrant neural circuitry. However, behavioral results often differ from lab to lab, and studies incorporating both males and females are often not performed despite the significant sex-bias of ASD. In this study, we sought to investigate the simple, passive behavior of home-cage activity monitoring across multiple 24-h days in four different monogenic mouse models of ASD: Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 knockout mice. Relative to sex-matched wildtype (WT) littermates, we discovered significant home-cage hypoactivity, particularly in the dark (active) phase of the light/dark cycle, in male mice of all four ASD-associated transgenic models. For Cntnap2^−/− and Pcdh10^+/− mice, these activity alterations were sex-specific, as female mice did not exhibit home-cage activity differences relative to sex-matched WT controls. These home-cage hypoactivity alterations differ from activity findings previously reported using short-term activity measurements in a novel open field. Despite circadian problems reported in human ASD patients, none of the mouse models studied had alterations in free-running circadian period. Together, these findings highlight a shared phenotype across several monogenic mouse models of ASD, outline the importance of methodology on behavioral interpretation, and in some genetic lines parallel the male-enhanced phenotypic presentation observed in human ASDs.

1. Introduction

Autism spectrum disorder (ASD) has an estimated prevalence of 1 in 59 children and is substantially sex-biased, with males 4 times more likely than females to receive an ASD diagnosis (Baio et al., 2018). Core features of ASD include repetitive behaviors, abnormal sensory processing, and deficits in communication, social interaction, and cognition (American Psychiatric Association, 2013). Mouse genetic models have been an invaluable tool for studying electrophysiological, circuit, and molecular mechanisms contributing to ASD (see Bey & Jiang, 2014; Ellegood & Crawley, 2015; Hulbert & Jiang, 2016 for recent reviews). In rodent models of ASD, behavioral assays have been developed to assess social interaction, repetitive behaviors, communication, and cognition (Chang, Cole, & Costa, 2017; Silverman, Yang, Lord, & Crawley, 2010). However, behavioral methodologies often vary between labs, and even when behavioral procedures are systematized as rigorously as possible, results within the same genetic line may differ from lab to lab, confounding interpretation (Crabbe, Wahlsten, & Dudek, 1999; Wahlsten et al., 2003).

Genome-wide association and whole exome sequencing studies have identified numerous monogenic risk factors for ASD (De Rubeis & Buxbaum, 2015; Persico & Napolioni, 2013; Willsey & State, 2015), and monogenic mouse models displaying both face and construct validity have been created (Bey & Jiang, 2014; Ellegood & Crawley, 2015; Hulbert & Jiang, 2016). In the present study, we investigated four monogenic mouse models of ASD: Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 knockout (KO). SH3 and multiple Ankyrin repeat domains 3 (Shank3) is a scaffolding protein in the postsynaptic density whose deletion contributes to Phelan-McDermid syndrome, intellectual disability, and ASD-like behaviors in humans (Durand et al., 2007), and social deficits and repetitive behaviors in mice (Peça et al., 2011). Contactin associated protein-like 2 (CNTNAP2) is a member of the neurexin superfamily and is associated with potassium channels, myelination, and neuron-glia interaction (Poliak et al., 1999). Its deletion results in cortical dysplasia-focal epilepsy, seizures, and ASD in humans (Strauss et al., 2006), and seizures, social deficits, motor stereotypies, communication abnormalities, and interneuron reductions in mice (Peñagarikano et al., 2011). Protocadherin-10 is a member of the cadherin superfamily of cell-adhesion molecules whose reduction has been associated with ASD in human genome-wide association studies (Bucan et al., 2009; Morrow et al., 2008), and altered communication, male-specific social deficits, and dendritic spine morphology alterations of the lateral/basolateral amygdala in Pcdh10^+/− mice (Schoch et al., 2017). Fmr1 encodes for the Fragile X mental retardation protein and is one of the leading single-gene causes of autism and intellectual disability (Hagerman, Rivera, & Hagerman, 2008; Turner, Webb, Wake, & Robinson, 1996). Fragile X patients display intellectual impairment, social deficits, communication problems, anxiety, hyperarousal, and hyperactivity (Garber, Visootsak, & Warren, 2008; Yu & Berry-Kravis, 2014), and Fmr1 KO mice have learning deficits, abnormal social behavior, anxiety, altered dendritic spine development, and aberrant cortical synchrony (The Dutch-Belgium Fragile X Consortium, 1994; Comery et al., 1997; Spencer, Alekseyenko, Serysheva, Yuva-Paylor, & Paylor, 2005; Gonçalves, Anstey, Golshani, & Portera-Cailliau, 2013).

In this study, we quantified home-cage activity and circadian rhythms across multiple 24-h days in Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO mice. These behaviors were chosen because they are relatively high-throughput, they are objective and easily quantifiable, and they are passive, allowing for consistent, controlled, and comparable data between groups. Moreover, activity and circadian problems are commonly reported in ASD. An estimated 30–70% of ASD patients exhibit ADHD symptomology (Antshel, Zhang-James, Wagner, Ledesma, & Faraone, 2016; Davis & Kollins, 2012), with one-third exhibiting hyperactivity (Carlsson et al., 2013). Other studies have reported significantly reduced physical activity or hypoactivity in ASD children (Pan, 2008; Posserud, Hysing, Helland, Gillberg, & Lundervold, 2016). Sleep problems such as increased latency to sleep, early morning waking, and altered melatonin profiles are frequently reported in ASD, suggesting possible circadian rhythm abnormalities (Glickman, 2010; Kulman et al., 2000; Melke et al., 2008; Mulder et al., 2010; Ritvo et al., 1993; Tordjman, Anderson, Pichard, Charbuy, & Touitou, 2005).

Previous studies investigating activity levels in these monogenic mouse models of ASD have utilized brief (1 h or less) open field tests. Cntnap2^−/− mice have been reported as hyperactive in short (< 1 h) activity monitoring sessions (Brunner et al., 2015; Peñagarikano et al., 2011). Likewise, Fmr1 KO mice have been described as hyperactive in the open field in numerous publications (The Dutch-Belgium Fragile X Consortium, 1994; Peier et al., 2000; Mineur, Sluyter, de Wit, Oostra, & Crusio, 2002; Restivo et al., 2005; Spencer et al., 2005; Dahlhaus & El-Husseini, 2010; Yuskaitis et al., 2010; Liu, Chuang, & Smith, 2011; Gholizadeh, Arsenault, Xuan, Pacey, & Hampson, 2014; Uutela et al., 2014; Ding et al., 2014). Shank3b^−/− mice have previously been reported to be hypoactive (Bidinosti et al., 2016; Copping et al., 2017; Dhamne et al., 2017; Kouser et al., 2013; Lee et al., 2015; Mei et al., 2016), however numerous other labs have reported no open field activity differences between Shank3b^−/− males and WT controls (Drapeau, Dorr, Elder, & Buxbaum, 2014; Duffney et al., 2015; Jaramillo et al., 2016; Peça et al., 2011). In some instances investigating both Shank3b^−/− males and females in the open field, males were found to be hypoactive, but females were not (Wang et al., 2011; Yang et al., 2012). Activity has not previously been reported in Pcdh10^+/− mice except during social behavior (Schoch et al., 2017), but we have not observed activity differences in the open field (data not shown). We speculated that activity results recorded in the home-cage might differ from, or even contradict, previous activity reports acquired from the novel open field area.

Given that basal activity alterations can confound many behavioral assays utilized to investigate ASD-like phenotypes in rodents, including social interaction, repetitive behaviors, and cognitive tasks, it is important to investigate and consider the discrepancies between undisturbed home-cage activity and open field activity measurements, which may be impacted by anxiogenic or stimulating elements induced by either handling, or the novelty and brevity of the open field arena. The purposes of the present study were to investigate and compare home-cage activity and free-running circadian periods in multiple ASD models for the first time using consistent methodologies in the same lab environment, to examine both males and females given the pronounced sex-bias in ASD diagnoses (Baio et al., 2018), and to compare findings with published activity results— specifically highlighting the disparities between home-cage activity across multiple diurnal days and brief activity monitoring in a novel environment.

2. Methods

2.1. Animals

B6.129-Shank3^tm2Gfng/J (Stock #017688) heterozygous males and females with exons 13–16 of Shank3 replaced with a neo cassette were purchased from The Jackson Laboratory and mated together to generate Shank3b^−/− offspring and Wildtype (WT) controls as previously described (Peça et al., 2011). Heterozygous B6.129(Cg)-Cntnap2^tm1Pele/ J males and females with exon 1 of Caspr2 replaced with a neo cassette were backcrossed to C57BL/6J for 10–12 generations, purchased from The Jackson Laboratory (Stock #017482), and mated together as previously described to produce experimental Cntnap2^−/− males and females and WT controls (Peñagarikano et al., 2011; Poliak et al., 2003). Pcdh10^+/− mice in which the first exon of Pcdh10 was replaced with a lacZ-neo cassette were created by Lexicon Pharmaceuticals, Inc. (Basking Ridge, NJ) and backcrossed for more than 15 generations as previously described (Schoch et al., 2017; Uemura, Nakao, Suzuki, Takeichi, & Hirano, 2007). Male Pcdh10^+/− mice were mated with C57BL/6J female mice purchased from the Jackson Laboratory (Stock #000664) to produce heterozygous Pcdh10^+/− experimental animals and Pcdh10^+/+ wildtype controls. B6.129P2-Fmr1^tm1Cgr/J female mice with exon 5 of Fmr1 replaced with a neo cassette were generated as previously described (The Dutch-Belgium Fragile X Consortium, 1994), backcrossed with C57BL/6J for many generations, and purchased from The Jackson Laboratory (Stock #003025) and mated with C57BL/6J (stock #000664) males to generate Fmr1 KO mice. Because Fragile X is an X-linked condition, and females display much milder and more variable symptoms than males due to random X-inactivation (Loesch, Huggins, & Hagerman, 2004; Yu & Berry-Kravis, 2014), only male Fmr1 KO mice were studied. Mice for all lines were weaned at 21 days old in cages of 3–5 sex-matched littermates. All mice were between 2 and 4.5 months old at the beginning of activity monitoring experimentation. Cohorts comprised of sex-matched littermates were used for all experimental groups. For all seven comparisons made in this study (male and female Shank3b^−/−, Cntnap2^−/−, and Pcdh10^+/− mice and male Fmr1 KO mice), there were no differences in age between WT and sex-matched mutants (all p > 0.15). Age did not correlate with activity for any of the individual groups, with the lone exception of a negative correlation between age and activity for Shank3b WT males in the horizontal (Pearson’s r = −0.716, p = 0.020), but not vertical axis (p = 0.790). For the group comparison involving both WT and Shank3b^−/− male mice, there was no correlation between age and activity (p = 0.134). All animals were maintained on a 12-h light: 12-h dark cycle (lights on at 7:00 am), except where indicated otherwise for circadian studies in constant darkness, with food and water provided ad libitum. All experiments were approved by the University of Pennsyl-vania Institutional Care and Use Committee (IACUC protocol 804407) and conducted in accordance to National Institute of Health guidelines.

2.2. Activity monitoring

Activity monitoring was performed as previously described (Angelakos et al., 2016). Mice were single housed within individual noise- and light-attenuating chambers (22″ × 16″ × 19″, Med Associates, St. Albans, VT) equipped with a 250 lx light source (80 lx at cage floor), ventilation fan, and an infrared beam break system which surrounded the mouse cage on all four sides and provided a high-resolution scaffold of infrared beams and detectors (Opto M3, Columbus Instruments, Columbus, OH). Infrared beams were spaced 0.5″ apart and provided two horizontal grids at 0.75″ and 2.75″ from the cage floor to quantify horizontal and vertical (rearing) activity, respectively. Mice were allowed to acclimate to the chambers for one week before data collection. Following acclimation, activity counts (beam breaks) were tabulated in 10-second intervals in both the XY (horizontal) and Z (vertical) direction continuously for 7 days in 12-h light: 12-h dark (12 h:12 h LD). Activity counts were pooled into 1-h bins across the entire diurnal cycle and averaged over the course of the 7 days. After 7 days of activity monitoring in 12 h:12 h LD, lights were switched off for 2 consecutive weeks of constant darkness (DD) and activity counts were compiled in 1-min intervals over the course of DD. Actogram generation and circadian period (tau) estimation were automated by Clocklab software (Actimetrics). Tau was calculated by Clocklab software as the slope of activity onset from day 2 to day 14 of the DD actogram. Day 1 of DD was discarded from circadian and activity analyses as all mice received a cage change on this day. Peak activity during DD was calculated by binning activity counts into every possible 1-h bin on a sliding scale and then averaging the bins with maximum activity count from each calendar day during the DD period. For Pcdh10^+/− and Fmr1 KO groups, only a subset of mice underwent circadian monitoring in constant darkness (see Fig. 4 for numbers).

2.3. Statistics

All statistical analysis was performed using SPSS for Windows (V.24.0). To analyze home-cage activity (1-h bins), Mixed Design ANOVAs were utilized with genotype as the between-subjects factor (WT or transgenic) and time as the within-subjects factor. Post hoc multiple comparisons were performed using Bonferroni’s adjustment for multiple comparisons. Where the assumption of sphericity was violated, Greenhouse-Geisser corrected F values are given. Multivariate ANOVAs (MANOVAs) were performed to analyze activity counts in the light cycle and dark cycle, with alphas corrected for multiple ANOVAs and set at α = 0.05/2, followed by post hoc Bonferroni’s adjustment for multiple comparisons. Student’s T-test was used to compare 24-h activity counts, activity counts during habituation, circadian tau values, and peak activity counts during DD between WT and transgenic mice. One-way ANOVAs compared activity levels of transgenic animals to “reference” activity levels averaged across all sex-matched WT animals. Pearson’s r was used to correlate animal age with activity levels.

3. Results

3.1. Home-cage hypoactivity in male Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO mice

Home-cage beam break counts were tabulated over 7 days of continuous monitoring in 12 h:12 h LD in male experimental mice and sex-matched WT littermate controls of the four transgenic models of autism spectrum disorder: Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO. In the horizontal (XY) direction, compared to WT, ASD-associated genetic deletion resulted in decreased home-cage activity for male Shank3b^−/− (main effect of genotype; F(1,18) = 14.466, p = 0.001, Fig. 1a), male Cntnap2^−/− (time*genotype interaction: F (23,467) = 4.409, p = 0.003, Fig. 1c), male Pcdh10^+/− (main effect of genotype: F(1,20) = 10.562, p = 0.004, Fig. 1e), and male Fmr1 KO mice (main effect of genotype: F(1,25) = 8.125, p = 0.009, Fig. 1g). Analysis by light/dark cycle revealed significantly reduced activity specifically in the dark (active) phase for Shank3b^−/− (MANOVA, F (2,17) = 8.093, p = 0.003, Fig. 1b), Pcdh10^+/− (MANOVA, F (2,19) = 6.331, p = 0.008, Fig. 1f), and Fmr1 KO males (MANOVA: F (2,24) = 26.801, p < 0.000001, Fig. 1h), relative to WT littermates. The magnitude of activity differences between male WT and ASD model littermates was even larger in the vertical (rearing) direction for all lines studied (Supplementary Fig. 1). Thus, mutation in four different ASD-associated genes resulted in decreased home-cage ambulatory and rearing activity in male mutant mice relative to sex-matched controls.

Fig. 1.

Male Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO mice are hypoactive in the home-cage relative to sex-matched WT controls. (A,B) Shank3b^−/− males exhibit significantly reduced horizontal activity compared to WT littermates. Expressed in 1-h bins (A) and by light/dark cycle (B). (C,D) Cntnap2^−/− males have decreased horizontal activity compared to WT controls. Expressed in 1-h bins (C) and by light/dark cycle (D). (E,F) Pcdh10^+/− males display significantly less horizontal activity than WT littermates. Expressed in 1-h bins (E) and by light/dark cycle (F). (G,H) Fmr1 KO males demonstrate significantly lower horizontal activity than WT controls. Expressed in 1-h bins (G) and by light/dark cycle (H). Mean ± standard error of the mean (s.e.m.) *p < 0.05, **p < 0.01, ***p < 0.001.

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.nlm.2019.02.010.

3.2. Shank3b^−/− female mice exhibit home-cage hypoactivity, but female Cntnap2^−/− and Pcdh10^+/− have normal activity levels

Because ASD is a male-biased disorder and females often exhibit less severe symptomology than males, even under the same genetic insult (Jacquemont et al., 2014; Robinson, Lichtenstein, Anckarsäter, Happé, & Ronald, 2013; Werling & Geschwind, 2013), we also quantified home-cage activity behavior in female mice and sex-matched littermates to look for potential sex-specific deficits. Female Fmr1 KO mice were not studied because Fragile X is an X-linked condition and phenotypes in females are less severe, more variable, and less predictable than males due to random X-inactivation (Loesch et al., 2004; Yu & Berry-Kravis, 2014). Similar to Shank3b^−/− male mice, Shank3b^−/− females exhibited robust home-cage hypoactivity relative to WT (main effect of genotype: F(1,18) = 20.447, p < 0.001, Fig. 2a). Analysis by light/dark cycle indicated that Shank3b^−/− females were less active than WT females in both the light and dark phases (MANOVA, F (2,17) = 9.699, p = 0.002, Fig. 2b). Interestingly, unlike male Cntnap2^−/− and Pcdh10^+/− mice, there were no activity differences relative to sex-matched WT littermates in female Cntnap2^−/− mice (F (1,19) = 0.033, p = 0.86, Fig. 2d) or female Pch10^+/− mice (F (1,20) = 1.960, p = 0.18, Fig. 2f). These female activity findings were similar in rearing behavior (Supplementary Fig. 2). Altogether, activity alterations were male-specific for two of the four ASD-associated mouse models studied, consistent with heightened ASD symptomology in males compared to females given the same genetic insult (Jacquemont et al., 2014; Robinson et al., 2013).

Fig. 2.

Female Shank3b^−/− mice are hypoactive, but female Cntnap2^+/− and Pcdh10^+/− display no home-cage activity differences, relative to sex-matched WT controls. (A,B) Shank3b^−/− females exhibit significantly reduced horizontal activity compared to WT littermates. Expressed in 1-h bins (A) and by light/dark cycle (B). (C,D) Cntnap2^−/− females have no differences in horizontal activity compared to WT controls. Expressed in 1-h bins (C) and by light/dark cycle (D). (E,F) Pcdh10^+/− females display no differences in horizontal activity in comparison to WT littermates. Expressed in 1-h bins (E) and by light/dark cycle (F). Mean ± standard error of the mean (s.e.m.) *p < 0.05, **p < 0.01, ***p < 0.001.

3.3. Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO mice are not hypoactive during the habituation phase

Activity monitoring experiments are typically performed in a novel open field arena and are brief in nature (usually 1 h or less). Because our activity monitoring cages differ in dimension, environment, and number of cohabitants from the group-housed home-cages that the mice are reared in, we can capture some of this novelty by quantifying activity levels during the initial habituation phase in our activity monitoring chambers. Activity measurements were made during the first 10 minutes, 30 minutes, and 1 h in the activity monitoring chambers, coinciding with common time intervals used for open field experiments. In comparison to sex-matched wildtype littermates, Shank3b^−/− male and female mice were hypoactive in the XY-axis during the first 30 min (Student’s t-test, male: p = 0.017, female: p = 0.013; Fig. 3b) and 1 h (Student’s t-test, male: p = 0.011, female: p = 0.007; Fig. 3c) in the activity monitoring system. For male Cntnap2^−/−, Pcdh10^+/− and Fmr1 KO mice, however, there were no activity differences during the habituation phase between transgenic mice and WT controls (p > 0.180 for all comparisons; Fig. 3d–l), in contrast to the hypoactivity observed following acclimation to the home-cage (Fig. 1). There were also no activity differences between Cntnap2^−/− and Pcdh10^+/− female mice and WT littermates during habituation (p > 0.131 for all comparisons; Fig. 3d–i). Activity findings in the vertical direction during habituation were similar to those observed in the horizontal direction (Supplementary Fig. 3). The lack of activity differences between male Cntnap2^−/−, Pcdh10^+/− and Fmr1 KO mice and their WT counterparts during habituation highlights the differences between home-cage activity monitoring and short-term activity monitoring in a novel environment.

Fig. 3.

Shank3b^−/− mice are hypoactive during habituation to the activity monitors, but Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO mice exhibit no activity differences in the habituation phase relative to sex-matched WT littermates. (A–C) Shank3b^−/− males and females have reduced horizontal activity in the first 30 min (B) and 1h (C) of activity monitoring in comparison to sex-matched WT controls. D-F) There are no differences in activity counts between Cntnap2^−/− mice and WT during the first 10 min (D), 30 min (E), or 1h (F) of habituation. (G–I) Pcdh10^+/− male and female mice display similar activity levels during the first hour of habituation compared to sex-matched WT littermates. (J–L) There are no differences in activity counts during the habituation phase between Fmr1 KO males and WT controls. Mean ± standard error of the mean (s.e.m.) *p < 0.05, **p < 0.01.

3.4. Circadian rhythms are unaltered in the four ASD mouse models

Circadian issues are common in ASD patients, who often have difficulties falling asleep and have more frequent night awakenings than the control population (Glickman, 2010; Richdale & Prior, 1995). Because of this, we investigated intrinsic circadian period of the ASD-associated transgenic mouse lines. Following two weeks of continuous activity monitoring, lights were shut off and mice were allowed to free-run in continuous darkness for two weeks. Despite activity alterations found in the ASD mouse models, there were no differences in free-running circadian period between male and female transgenic mice and sex-matched littermates for any of the four ASD-associated lines studied (Student’s t-test, p > 0.25 for all comparisons, Fig. 4a–d). Moreover, activity patterns found in 12 h:12 h LD remained consistent in constant darkness (DD), with the exception of Fmr1 KO mice not being hypoactive in the horizontal axis relative to WT controls. Male Shank3b^{−/ −} (Student’s t-test, horizontal: p = 0.0005; Fig. 4e, vertical: p = 0.00003; Fig. 4i), Cntnap2^−/− (horizontal: p = 0.26; Fig. 4f, vertical: p = 0.014; Fig. 4j), Pcdh10^+/− (horizontal: p = 0.018; Fig. 4g, vertical: p = 0.030; Fig. 4k), and Fmr1 KO (horizontal: p = 0.99; Fig. 4h, vertical: p = 0.024; Fig. 4l) mice displayed lower daily peak activity in DD than sex-matched WT controls. Female Shank3b^−/− mice were hypoactive in DD relative to sex-matched WT (horizontal: p = 0.0007; Fig. 4e, vertical: p = 0.0006; Fig. 4i), but female Cntnap2^−/− (horizontal: p = 0.41; Fig. 4f, vertical: p = 0.55; Fig. 4j) and Pcdh10^+/− (horizontal: p = 0.58; Fig. 4g, vertical: p = 0.61; Fig. 4k) mice displayed no peak activity differences compared to WT littermates.

Fig. 4.

Normal free-running circadian periods and hypoactivity in Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO mice during constant darkness. A-D) Relative to sex-matched WT controls, there are no differences in free-running circadian period (tau) in (A) Shank3b^−/− males (23.60 ± 0.02 h, WT: 23.64 ± 0.02 h; p = 0.25) or females (23.62 ± 0.03 h, WT: 23.67 ± 0.02 h; p = 0.25), (B) Cntnap2^−/− males (23.61 ± 0.02 h, WT: 23.61 ± 0.02 h; p =0.93) or females (23.65 ± 0.04 h, WT: 23.64 ± 0.03 h; p= 0.64), (C) Pcdh10^+/− males (23.53 ± 0.04 h, WT: 23.54 ± 0.08 h; p= 0.87) or females (23.51 ± 0.05 h, WT: 23.50 ± 0.03 h; p =0.83), or (D) Fmr1 KO males (23.67 ± 0.02 h, WT: 23.64 ± 0.02 h; p =0.26). (E–L) Peak activity patterns during DD (1-h bins) are similar to activity patterns observed during 12h:12 h LD. (E,I) Shank3b^−/− male and female mice have lower peak activity in DD than sex-matched WT controls. (F,J) Relative to sex-matched WT in DD, male Cntnap2^−/− mice are not hypoactive in the (F) horizontal axis, but are hypoactive in the (J) vertical direction. Female Cntnap2^−/− mice are not hypoactive relative to sex-matched WT in DD. (G,K) Pcdh10^+/− males but not females have lower peak activity levels in DD compared to WT. (H,L) Fmr1 KO males do not have altered peak activity levels in the (H) XY-axis, but display reduced peak activity in the (L) Z-axis during DD in comparison to WT littermates. Mean ± standard error of the mean (s.e.m.) *p < 0.05, **p < 0.01, ***p < 0.001.

4. Discussion

In this study, we performed home-cage activity monitoring and circadian behavioral analysis in four monogenic mouse models of ASD. Male mice of all four lines studied— Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO—exhibited home-cage hypoactivity, particularly in the dark (active) phase of the diurnal cycle, relative to sex-matched WT controls. Our findings present a shared phenotype across multiple mouse models of ASD, performed in one lab using consistent methodologies. Activity alterations (both hyperactivity and hypoactivity) are commonly reported in ASD (Carlsson et al., 2013; Posserud et al., 2016), and the mutual phenotype of hypoactivity observed in these monogenic mouse lines may suggest common neural underpinnings. One possibility may be aberrant corticostriatal circuitry, which plays a role in coordinated movement and is believed to be one of the major structural alterations contributing to repetitive behaviors and ASD (Delmonte, Gallagher, O’Hanlon, McGrath, & Balsters, 2013; Hollander et al., 2005; Langen et al., 2012; Mosconi et al., 2009; Shepherd, 2013). Altered corticostriatal connectivity and neural firing have previously been reported in Shank3b^−/− mice (Peça et al., 2011; Peixoto, Wang, Croney, Kozorovitskiy, & Sabatini, 2016), Cntnap2^−/− mice (Peñagarikano et al., 2011; Selimbeyoglu et al., 2017), Fmr1 KO mice (Centonze et al., 2008; Lai, Lerch, Doering, Foster, & Ellegood, 2016), and Fragile X humans (Dennis & Thompson, 2013; Haas et al., 2009). Pcdh10 has been shown to be involved in striatal axon growth (Uemura et al., 2007), and corticostriatal alterations have also been reported in other mouse models of ASD, including neuroligin-1 KO and BTBR mice (Blundell et al., 2010; Ellegood, Babineau, Henkelman, Lerch, & Crawley, 2013). In contrast to the hypoactivity observed in the present study, other mouse models of ASD including the 16p11.2 del/+ chromosomal copy number variation mouse model (Angelakos et al., 2016; Arbogast et al., 2016; Horev et al., 2011; Portmann et al., 2014) and the Ptchd1^Y/- mouse model of ASD (Ung et al., 2017; Wells, Wimmer, Schmitt, Feng, & Halassa, 2016) exhibit robust hyperactivity in the home-cage. 16p11.2 del/+ mice display cellular and molecular alterations in the striatum and deficits in striatum-dependent behaviors (Grissom et al., 2017; Portmann et al., 2014), and PTCHD1 expression is enriched in the cortex and striatum (Ung et al., 2017). Together, these results suggest that hypoactivity is not a defining characteristic of ASD mouse models, but rather, activity alterations in both directions are commonly observed in various rodent models of ASD, as well as in humans with ASD. Future studies will need to determine if misregulated corticostriatal circuits underlie these activity alterations.

4.1. Cntnap2^−/− and Pcdh10^+/− mice may be useful models for studying sex differences in ASD

For Cntnap2^−/− and Pcdh10^+/− mice, home-cage hypoactivity was sex-specific, as female mice of these lines displayed activity levels similar to sex-matched WT littermates. Male-enhanced activity alterations in these lines parallel sex differences in phenotypic presentation in human ASD patients (Werling & Geschwind, 2013) and supports findings that females require a larger mutational burden than males to manifest similar ASD-related symptomology (Jacquemont et al., 2014; Robinson et al., 2013). Cntnap2^−/− mice have previously been shown to have male-specific reductions in visually-evoked cortical activity (Townsend & Smith, 2017), and male but not female mice exposed to a Cntnap2-reactive antibody in utero display ASD-associated behavioral deficits and structural alterations in the cortex and hippocampus (Brimberg et al., 2016). Relative to sex-matched WT littermates, Pcdh10^+/− male mice exhibit significantly reduced social approach behavior, whereas female Pcdh10^+/− mice do not (Schoch et al., 2017). These findings, in concert with the male-specific activity reductions outlined in the present study, suggest that Cntnap2^−/− and Pcdh10^+/− mice may serve as informative genetic models for investigating sex differences in ASD.

4.2. Home-cage activity dissociates from activity observed in a novel arena

Another main conclusion from this study is the importance of dissociating continuous, undisturbed home-cage activity from novelty-induced, brief activity measurements that may contain anxiogenic elements, such as those obtained in an open field test. Activity findings reported in the open field and during the habituation phase of our study do not necessarily match our findings in the acclimated home-cage. Cntnap2^−/− and Fmr1 KO mice have been reported to be hyperactive in the open field (The Dutch-Belgium Fragile X Consortium, 1994; Peier et al., 2000; Mineur et al., 2002; Restivo et al., 2005; Spencer et al., 2005; Dahlhaus & El-Husseini, 2010; Yuskaitis et al., 2010; Liu et al., 2011; Peñagarikano et al., 2011; Gholizadeh et al., 2014; Uutela et al., 2014; Brunner et al., 2015) and displayed no activity differences during the habituation phase in our task (Fig. 3), but longer activity studies across multiple undisturbed days corroborate our findings of hypoactivity in Cntnap2^−/− (Thomas, Schwartz, Saxe, & Kilduff, 2016) and Fmr1 KO male mice (Bonasera, Chaudoin, Goulding, Mittek, & Dunaevsky, 2017). Although it is difficult to make direct comparisons between results in the open field, which contains no bedding and is 1.5–10 × larger in area than the 4.5″ × 8.5″ home-cages we use in our study, it is clear that activity patterns in a short-duration novel environment greatly differ from activity patterns in the undisturbed home-cage.

4.3. Limitations of circadian analysis in C57BL/6J mice

Some limitations exist in our study. Circadian alterations have been reported in ASD patients (Glickman, 2010), but none of the ASD mouse models studied showed changes in circadian periodicity or activity in DD (Fig. 4). These circadian abnormalities have been speculated to be related to altered melatonin synthesis and concentration in those with ASD compared to typically developing controls (Bourgeron, 2007; Kulman et al., 2000; Melke et al., 2008; Mulder et al., 2010; Nir et al., 1995; Ritvo et al., 1993; Tordjman et al., 2005). However, due to a mutation in the melatonin synthesis pathway, C57BL/6J mice have significantly attenuated melatonin production (Roseboom et al., 1998). Although augmentation of melatonin protein in C57BL/6J mice via outcrossing to a melatonin-proficient strain results in no alterations in the circadian rhythm (Kasahara, Abe, Mekada, Yoshiki, & Kato, 2010), given the link between altered melatonin production and ASD, future circadian experiments should be performed using transgenic ASD model mice on a melatonin-proficient strain, such as CBA mice (Kennaway, Voultsios, Varcoe, & Moyer, 2002).

4.4. Additional limitations

Although home-cage hypoactivity was demonstrated across the various monogenic ASD mouse models utilized in this study, the nature of the hypoactive behavior was not observable. Future studies video tracking the movement patterns of these mouse models may prove useful for identifying potential stereotypies or behavioral abnormalities underlying the hypoactive phenotypes. Another possible limitation of our study is the variance in wildtype activity between sex-matched mice of different genetic lines, confounding direct comparison between transgenic groups. One possible explanation for the inconsistencies in wildtype activity across groups may be that this simply reflects inherent variability within C57BL/6J mice. However, other possibilities are likely to play a role. Although none of the ASD-associated genes studied are known to be imprinted (http://www.geneimprint.com/), the possibility remains that parental origin of the mutant allele impacts behavioral phenotypes of the progeny. Pcdh10 was transmitted via the paternal allele in accordance with previous studies using this mouse model (Uemura et al., 2007; Schoch et al., 2017) so that offspring would be reared by WT mothers. Out of necessity, however, experimental animals for the other three lines were generated as the offspring of transgenic mothers. It is entirely possible that mutations in Shank3b, Caspr2, or Fmr1 impact maternal behavior, which is known to affect both development and subsequent behavior of the offspring (Branchi & Cirulli, 2014), but these would have to act in the heterozygous state. For these reasons, investigating how maternally-transmitted Pcdh10 impacts the development and behavior of Pcdh10^+/− mice, which is hitherto unknown, would be an interesting future avenue of research. Another possible explanation for the variance in WT activity levels is that age/body weight was not strictly controlled between groups. Importantly, cohorts of sex-matched littermates were used for all within-genotype direct comparisons performed in this study. Slight variations in age or body weight between genotypes, however, may contribute to the between-group discrepancies in WT activity levels. Although comparisons utilizing sex-matched littermates, which eliminate environmental variables, are strongly preferred, we also re-analyzed activity counts after collapsing wildtype activity levels across the different genotypes to create “reference” wildtype values. Relative to the “reference” WT value, male Fmr1 KO mice were hypoactive in the vertical axis, but were no longer hypoactive in the horizontal axis. Apart from this, all activity findings utilizing “reference” WT values were the same as the findings directly comparing the transgenic animals to sex-matched WT littermates (Supplementary Fig. 4).

4.5. Conclusions and future directions

This study revealed a common home-cage hypoactivity phenotype, relative to WT, in male mice of four different monogenic mouse models of ASD. While human Fragile X patients and those with CNTNAP2 mutation are hyperactive (Baumgardner, Reiss, Freund, & Abrams, 1995; Strauss et al., 2006; Sullivan et al., 2006; Tranfaglia, 2011), the consistent phenotype of activity alterations in ASD patients and mouse models may point to common aberrant neural circuity. Future studies including other activity-dependent behavioral tasks such as tests of motivation/depression, or involving brain region-specific genetic rescue in our current activity setup, may help elucidate the neural circuitry underlying the hypoactivity observed in these mice. Given that ASD is a neurodevelopmental disorder, future investigation tracking home-cage activity patterns across development may reveal critical developmental timepoints and may shed further light onto potential mechanisms underlying the activity alterations. Together, this shared phenotype between Shank3b^−/−, Cntnap2^−/−, Pcdh10^+/−, and Fmr1 KO mice helps validate these monogenic models of ASD and may assist in pinpointing structural and mechanistic deficits common between humans with ASD and rodent genetic models.