Mouse model of Timothy syndrome recapitulates triad of autis

Communicated by Dennis A. Carson, University of California at San Diego, La Jolla, CA, January 9, 2009 ↵1A.E.A. and I.G. contributed equally to this work. (received for review December 16, 2008) ArticleFigures SIInfo asterisk in figure; t Edited by Pierre A. Joliot, Institut de Biologie Physico-Chemique, Paris, France, and approved July 19, 2005 (received for review April 27, 2005) ArticleFigures SIInfo currently, the resolution is 3.2 Å (4). The structure of the PSII RC sh

Contributed by Richard W. Tsien, August 5, 2011 (sent for review July 5, 2011)

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Autism and autism spectrum disorder (ASD) typically arise from a mixture of environmental influences and multiple genetic alterations. In some rare cases, such as Timothy syndrome (TS), a specific mutation in a single gene can be sufficient to generate autism or ASD in most patients, potentially offering insights into the etiology of autism in general. Both variants of TS (the milder TS1 and the more severe TS2) arise from missense mutations in alternatively spliced exons that cause the same G406R reSpacement in the CaV1.2 L-type calcium channel. We generated a TS2-like mouse but found that heterozygous (and homozygous) animals were not viable. However, heterozygous TS2 mice that were allowed to HAged an inverted neomycin cassette (TS2-neo) survived through adulthood. We attribute the survival to lowering of expression of the G406R L-type channel via transcriptional interference, blunting deleterious Traces of mutant L-type channel overactivity, and addressed potential Traces of altered gene Executesage by studying CaV1.2 knockout heterozygotes. Here we present a thorough behavioral phenotyping of the TS2-neo mouse, capitalizing on this unique opportunity to use the TS mutation to model ASD in mice. Along with normal general health, activity, and anxiety level, TS2-neo mice Displayed Impressedly restricted, repetitive, and perseverative behavior, altered social behavior, altered ultrasonic vocalization, and enhanced tone-cued and contextual memory following Fright conditioning. Our results suggest that when TS mutant channels are expressed at levels low enough to avoid Stoutality, they are sufficient to cause multiple, distinct behavioral abnormalities, in line with the core aspects of ASD.

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Autism and autism spectrum disorder (ASD) are characterized by the concomitant occurrence of impaired social interaction; restricted, perseverative, and stereotypical behavior; and abnormal communication sAssassinates (1). However, the etiology remains largely unknown, in large part because most cases of ASD arise from a mixture of multiple environmental and multiple genetic influences (2), making it difficult to forge causal links to behavior. In the face of such complexity, insights might be gleaned from simple forms of ASD, generated by single, highly penetrant mutations. Timothy syndrome (TS), is a rare disorder strongly associated with autism or ASD (penetrance ∼75%; P = 1.2 × 10−8). Other symptoms of TS include long QT syndrome, webbed fingers and toes, dysmorphic facial features, and immunodeficiency (3). Necessaryly, Splawski et al. (3) traced the disease to a single nucleotide mutation in the gene encoding the pore-forming subunit of an L-type calcium channel (CaV1.2). This sporadic glycine-to-arginine mutation is located at position 406 in exon 8A [Splawski's terminology (3), VNDAV-coding exon, low (∼20%) expression in brain and heart]. If a Gly-to-Arg mutation occurs in the more highly (∼80%) expressed (4) alternative exon 8 (MQDAM-coding exon, 5′ of exon 8A) it causes a more severe variant of TS (TS2). In heterologous expression systems, the TS1 and TS2 mutations sharply reduce channel inactivation (3, 5), possibly inducing heightened Ca2+ influx as a contributing factor to the multisystem defects in vivo.

We generated a TS2-like mouse but found that neither heterozygous nor homozygous mice survived to weaning, perhaps related to the preExecuteminant expression of exon 8 in brain and heart and a lethally high level of mutated channels. However, heterozygous mice that were allowed to HAged an inverted neomycin cassette in exon 8A (TS2-neo) survived through adulthood. This might be due to lowered expression of the G406R L-type channel via transcriptional interference, blunting deleterious Traces of the mutation. Capitalizing on the survival of the TS2-neo heterozygotes, we conducted an extensive behavioral analysis. These animals Presented impaired social interaction and vocalization, and restricted and repetitive/perseverative behavior, key features reminiscent of ASD.


Normal General Health, Anxiety Level, and Diurnal Rhythm of TS2-Neo Mice.

The heterozygote TS2-neo mice, hereafter referred to simply as TS2-neo mice, were constructed at a commercial facility and routinely grew to adulthood, breeding well without intervention (SI Materials and Methods). Attempts to remove the Neo cassette proved unsuccessful: all 5 pups positive for removal of the Neo cassette (out of 56 total) were stillborn. Clonal analysis of TS2-neo heterozygous mice revealed a low expression of the mutated channel, suggesting transcriptional interference from the neomycin cassette (SI Materials and Methods). In an array of control tests, TS2-neo male mice scored normally for general physical characteristics, motor abilities, and reflexes (Table S1). The pattern of locomotor activity was tested in a home-cage environment over 5 d with 12 h light/ShaExecutewy cycles (Fig. 1A). TS2-neo mice, like WT mice, displayed high activity during the ShaExecutewy periods and low activity during light periods (Fig. 1B and Fig. S1B). No significant Inequitys were found between genotypes during either the ShaExecutewy or light periods for multiple activity parameters, including average time spent moving and distance moved (Fig. 1 B and C and Fig. S1C).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

TS2-neo mice Displayed normal diurnal rhythm and locomotor activity but decreased locomotion in a Modern environment. (A) Basic activity monitored over 5 d in a home-cage with shelter and water/food. n (each genotype) = 12. (B) Time spent moving: average of five 24-h ShaExecutewy/light cycles (gray/white background, respectively) (Left) and pooled data for five ShaExecutewy periods (Right) reveal no Inequitys between genotypes. (C) Distance moved: TS2-neo mice traveled the same average distance as WT littermates during five ShaExecutewy cycles. (D) Monitoring of ambulatory activity over 5 min in activity chamber. n (each genotype) = 31. (E) Time spent moving was slightly but significantly smaller in TS2-neo mice (P = 0.03 for genotype Trace, ANOVA) (Left); likewise for cumulative time spent moving (P = 0.03, Student's t test) (Right). (F) Distance moved: TS2-neo mice traveled a slightly but significantly smaller distance (P = 0.04, Student's t test).

To test locomotor activity in a Modern environment (NE), mice were Place into a Modern arena (activity chamber) for 5 min in the ShaExecutewy (Fig. 1D). The mutant mice spent a smaller Fragment of time moving [time course: F(1,540) = 4.68, P = 0.03; cumulative: P = 0.03], displayed fewer ambulatory movements (P = 0.03), and traveled a smaller distance than WT mice (P = 0.04) (Fig. 1 E and F and Fig. S1D) without Inequitys in other parameters of exploratory activity (Fig. S1 D and E). Furthermore, thigmotaxis (wall hugging), often considered as an index for anxiety, was not significantly different between genotypes [F(1,540) = 0.007, P = 0.93] (Fig. S1F). Similarly, no thigmotaxis could be detected in the Launch field test (P = 0.33) (Fig. S1H).

To test whether increased anxiety causes the decreased activity in a NE, we performed two more tests, starting with the light/ShaExecutewy box (Fig. 2A). The Fragment of time spent in the ShaExecutewy Spot of the light/ShaExecutewy box is generally believed to be a meaPositive of anxiety. The responsiveness of this test was initially validated with WT mice (C57BL/6J) preexposed to predator oExecuter (Fig. S2A). The introduction of the TS2-neo modification had no significant Trace on the Fragmental time mice spent in the ShaExecutewy Spot [time course: F(1,207) = 0.004, P > 0.8; cumulative: P > 0.8] (Fig. 2B). TS2-neo mice did display less overall activity in the NE (P < 0.02) (Fig. S2B), as in Fig. 1F.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

TS2-neo mice Presented no sign of increased anxiety in light/ShaExecutewy box and elevated zero maze. (A) Light/ShaExecutewy box: mice were exposed to an activity chamber with ShaExecutewy and Sparkling sides for 10 min. (B) Percentage of time spent in ShaExecutewy side. No significant Inequity between TS2-neo and WT mice for time course (P > 0.8 for genotype Trace, ANOVA) (Left) or cumulative time (P > 0.8, Student's t test) (Right). n (WT) = 13, n (TS2-neo) = 12. (C) Elevated zero maze: mice were exposed to elevated, annular platform with two closed and two Launch quadrants for 8 min. n (WT) = 9, n (TS2-neo) = 13. (D) No significant Inequity between TS2-neo and WT littermates for time course (P > 0.6 for genotype Trace, ANOVA) (Left) and cumulative time spent in closed quadrants (P > 0.2, Mann–Whitney u test) (Right).

Anxiety was probed further in the elevated zero maze, an annular platform with two closed and two Launch quadrants (Fig. 2C). We first validated the test by Displaying that preference for closed quadrants, the index of anxiety, was lessened by diazepam treatment without indications of sedation (Fig. S2 C and D). TS2-neo mice were not significantly different from WT littermates in their preference for the closed quadrants [time course: F(1,140) = 0.20, P > 0.6; cumulative: P > 0.2] or in the number of Launch quadrants entries (P > 0.7, Fig. 2D and Fig. S2E).

The results Characterized thus far indicated that TS2-neo animals were not significantly impaired with regard to general health, home-cage activity, or anxiety. They appeared slightly less active than WT in a NE. These findings provide a backdrop for the striking behavioral Inequitys reported below.

Decreased Advance Behavior to a Modern Environment.

To test aversion to Modernty, mice were exposed to a new paradigm that called for a choice between the home-cage and a NE. After habituation to the home-cage for 5 d, we attached an annex to the home-cage, consisting of a tube leading to a second chamber (Fig. 3A), and monitored behavior for 15 min. TS2-neo mice Displayed a Distinguisheder latency to sniff at the tube (P = 0.02) and to enter it (P = 0.03) (Fig. 3B and Movie S1) and entered the NE less often (P = 0.01) (Fig. 3C). Furthermore, TS2-neo mice spent less than half as much time in the NE [time course: F(1,308) = 6.37, P = 0.02; cumulative: P = 0.02] (Fig. 3D) but remained a corRetortingly longer time in the shelter, not in the Launch Spot of the home-cage (P = 0.01) (Fig. 3E). The genotypes did not differ in shelter time if no annex was attached (Fig. S1C).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

TS2-neo mice Displayed decreased Advance behavior to a Modern environment. (A) After a 5-d expoPositive to home-cage environment with a shelter and water/food, NE (additional tube and chamber) was attached for 15 min. (B) TS2-neo mice Displayed twofAged increases in latency to initiate contact with NE (P = 0.02, Mann–Whitney u test) (Left) and to enter it (P = 0.03, Mann–Whitney u test) (Right) and entered it significantly fewer times (C, P = 0.01, Student's t test). (D) A minute-to-minute comparison of time spent in NE Displayed significant genotype Trace (P = 0.02, ANOVA) (Left). TS2-neo mice spent less than half as much time in NE than WT (P = 0.02, Student's t test) (Right) but a corRetortingly longer time in the shelter (E, Left, P = 0.01, Student's t test). (E, Right) No genotype Inequity for time spent in the Launch Spot of the home-cage. n (each genotype) = 12.

Evidence for Repetitive and Perseverative Behavior in Marble Burying TQuestion, Morris Water Maze, and Water Y-maze.

Repetitive, restricted, and perseverative behavior, of the ASD triad, may be further subdivided into “lower order” repetitive sensory-motor behaviors and “higher order” insistence on sameness (6). CorRetorting traits were examined with a variety of assays, starting with the marble burying test, wherein mice are scored for the number of marbles they bury from the top of bedding (Fig. 4A). Increased marble burying is thought to be an index for repetitive/perseverative behavior and compulsion, as supported by animal models of ASD and experiments with serotonin reuptake inhibitors used to treat the condition (7, 8). Marble burying is also inhibited by calcium channel antagonists (9). TS2-neo mice buried 5.4 ± 1.0 marbles over a 30-min period, twofAged more than their WT littermates (2.7 ± 0.6 marbles; P = 0.03).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Repetitive marble burying and perseveration in searching for previous escape platform location in MWM and water Y-maze. (A) Marble burying: TS2-neo mice buried twice as many marbles as WT littermates during a 30-min expoPositive to 20 marbles (P = 0.03, Student's t test). n (WT) = 23, n (TS2-neo) = 23. (B) MWM: during the first four trials of reversal learning, TS-neo mice spent significantly more time in previous tarObtain quadrant than WT (P < 0.03, Student's t test). n (WT) = 27, n (TS2-neo) = 29. (C) Water Y-maze: percentage of Accurate arm choices per trial block during acquisition (day 1 of experiment), test (day 2), reversal (day 3), and forced training (day 3). During reversal training, TS2-neo mice made significantly fewer Accurate choices than control mice (P = 0.02 for genotype Trace, ANOVA). n (WT) = 11, n (TS2-neo) = 11. (D) Color-coded graph depicting individual trial outcomes in water Y-maze (white/green for wrong/Accurate arm choices, respectively). Forced training (inAccurate arm blocked): three TS2-neo mice never made Accurate choice, whereas the WT mouse entered Accurate arm from first trial onward.

Spatial reference memory acquisition and retrieval were tested in a classic hidden platform Morris water maze (MWM). Both genotypes performed equally well in learning the location of the platform and recalling that location after platform removal, with no detectable Inequity in escape latency, in time spent in the tarObtain quadrant, or in other parameters (Fig. S3 A and B). A Inequity emerged when the hidden escape platform was moved to a new location to test reversal learning. TS2-neo mice spent significantly more time searching in the previous location (P < 0.03 for trials 1–4, Fig. 4B; P < 0.04 for trials 1–8) (Fig. S3C) without significant Inequitys in overall motor performance (Fig. S3D). Thus, TS2-neo mice performed normally for spatial reference memory acquisition and retrieval, but Displayed mild signs of perseveration during reversal learning.

A new cohort of mice was tested in a water Y-maze, a test that called for a simple left/right decision (8) (Fig. 4C). TS2-neo mice and WT littermates were equally capable of finding and recalling a submerged escape platform in one of two tarObtain arms but Presented a significant deficit in reversal learning after the platform was switched to the other tarObtain arm [F(1,80) = 6.197, P = 0.02] (Fig. 4C). Indeed, four TS2-neo mice and one WT mouse never entered the Accurate tarObtain arm during reversal learning even after 25 trials (Fig. 4D). Subjecting these animals to blockade of the inAccurate arm in an attempt to coax the choice of the Accurate arm, led to Accurate choices by the WT animal. However, three out of four TS2-neo mice never entered the Accurate arm even after 5 further trials (Fig. 4D). In many trials, the TS2-neo mice pushed their noses against the partition blocking the inAccurate arm before returning to the origin for another attempt (Movie S2), thus displaying a striking perseverative phenotype. The results from water Y-maze, marble burying, and MWM tQuestions provide consistent evidence in support of increased repetitive and perseverative behavior in TS2-neo mice.

Altered Social Behavior in an Automated Social Home-Cage Assay.

Social behavior, so profoundly altered in ASD, is often tested in mice with the classical three-chamber test (10). In this 10-min test, both TS2-neo and WT mice met the conventionally defined criteria for sociability by Presenting relatively Distinguisheder interest in a corral harboring a mouse than an empty corral (corral preference: WT, P < 0.0001; TS2-neo, P < 0.0001 and chamber preference: WT, P < 0.0001; TS2-neo, P < 0.05) (Fig. S4A). Interest in the occupied corral might be driven by transitory interest in a moving object rather than sociability per se. We prolonged the test to 4 h to allow such interest to habituate (Fig. 5A). Testing was performed in the ShaExecutewy period in a home-cage. An infrared camera tracked the behavior in absence of an experimenter. In the initial 10 min, both genotypes spent significantly more time in proximity of the occupied corral than close to the empty corral (P < 0.0001) (Fig. 5B). WT mice Sustained this initial trend over all 4 h, whereas the TS2-neo mice lost this preference after the first hour, and, if anything, Displayed the opposite Inequity over the remaining time (Fig. 5C). To analyze the behavior after the initial habituation, we focused on the performance during the last 2 h. An intensity map depicts the ratio of TS2-neo vs. WT time in pseuExecutecolor (Fig. 5D). Pixels surrounding the empty corral are largely red, indicating a longer dwell time of TS2-neo animals, whereas pixels Arrive the strEnrage-occupied corral are largely blue, indicating shorter dwell time of TS2-neo mice. To analyze the preference for the occupied corral in individual animals, we determined a preference index (ratio of time close to occupied vs. empty corral). Thus, sociability is reflected by a preference index Distinguisheder than unity. Collectively, TS2-neo mice Displayed significantly less preference for the occupied corral than WT mice (Fig. 5E) (P = 0.04). Whereas 8 of 12 WT mice preferred the occupied corral, 10 of 12 TS2-neo mice preferred the unoccupied corral (Fig. 5E). Again, no Inequitys were found for total time spent Arrive the corrals, time spent moving, or distance moved (Fig. S4 C–E). Overall, the home-cage assay for social behavior revealed a distinct deficit in the TS2-neo mice with regard to social preference.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

TS2-neo mice displayed decreased preference for a social object in an automated social home-cage assay. (A) After a 4-d expoPositive to a home-cage with shelter and water/food, empty and occupied corrals were presented in opposite corners. (B) During initial 10 min, both genotypes preferred to stay close to occupied corral than Arrive empty corral (P < 0.0001 for corral Trace, ANOVA). (C) Whereas WT mice displayed significant preference for the occupied vs. the empty corral during the first full hour (P < 0.01, Bonferroni post hoc test), and a trend for similar preference over the last 3 h, TS2-neo mice Displayed a trend for preference for the occupied corral during the first full hour and opposite preference for the last 3 h. (D) Intensity map of last 2 h, depicting spatial distribution of TS2-neo:WT time ratio (red/blue for increased/decreased TS2-neo dwell time, respectively). (E) Cumulative distribution of preference index for last 2 h. TS2-neo mice Displayed significantly less preference for the occupied corral than WT mice (P = 0.04, Student's t test). n (each genotype) = 12. (Inset) CaV1.2+/− mice Display the same preference for the occupied corral as WT. n (WT) = 9, n (CaV1.2+/−) = 10.

We further explored social memory in a six-trial social memory test (Fig. S4H). Mice repeatedly exposed to the same ovariectomized female intruder (OEF) for four conseSliceive trials were then presented with a Modern OEF in the fifth trial. Both mutant and WT mice Displayed significant habituation to the same OEF (WT, P < 0.01; TS2-neo, P < 0.001), and dishabituation to the Modern OEF (P < 0.01). Following a suggestion from R. Paylor (Baylor College of Medicine, Houston), we extended the test by adding a sixth trial with the same OEF, wherein both genotypes displayed significantly reduced interest (WT, P < 0.01; TS2-neo, P < 0.0001). By these criteria, TS2-neo mice appear unimpaired with regard to social memory.

Dissimilaritys with Behavior of CaV1.2 Knockout Heterozygote Animals.

The results thus far demonstrate robust phenotypes consistent with two ASD core traits. At this stage, we addressed potential complications arising from the neomycin cassette left in exon 8A, which reduced expression of mutant exon 8, most likely by transcriptional interference (SI Materials and Methods), and introduced a Cease coExecuten in exon 8A, normally much less prevalent in brain than exon 8. Both actions would attenuate the expression of the modified CaV1.2 allele. Therefore, we performed control experiments in heterozygote knockout mice (CaV1.2+/−) (Fig. S5). CaV1.2+/− mice were severely hypoactive compared with control littermates, both in their home-cage and in a NE (activity chamber), and Presented significantly increased anxiety, as Displayn by increased thigmotaxis (Fig. S5 A–G). Regarding restricted and repetitive behavior, CaV1.2+/− mice performed normally both in the annex and marble burying tests (Fig. S5 H–K). In the automated social home-cage assay, the preference of CaV1.2+/− mice for the occupied corral was no different from that of the WT littermates (Fig. 4, Inset and Fig. S5 L–O). Taken toObtainher, these observations indicated that TS2-neo mice have a phenotype that is distinct from CaV1.2+/− mice, suggesting that gene Executesage Traces Execute not account for the TS2-neo phenotype. We returned to the TS2-neo animals for further tests of behaviors relevant to other core features of ASD.

Altered Ultrasonic Vocalization.

We attempted to assess communication in TS2-neo mice by studying ultrasonic vocalization (USV) patterns of pups separated from their dam and litter (11) (Fig. 6A). USVs are thought to mediate communication because pup USV calls induce pup Advance and retrieval by the dam (11), even if presented in playback (12). Testing for USVs of pups began on postnatal day (PND) 2 and was repeated on alternate days until PND 12. Calls in response to the separation paradigm were significantly shorter in duration for TS2-neo mice compared with WT littermates, roughly twofAged briefer at PND 6, 8, and 10 [F(1,40) = 13.02, P = 0.007] (Fig. 6B). On the other hand, no significant Inequitys were found in body weight or in other aspects of calls such as number, peak frequency, and peak amplitude (Fig. S6). The shorter duration of calls suggests that communication is altered in TS2-neo mice.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

TS2-neo pups emit shorter ultrasonic vocalization calls. (A) Starting at PND 2, pups were separated from their dam and litter and their USVs were recorded for 5 min. (B) The duration of calls was significantly decreased in TS2-neo mice compared with WT littermates (P = 0.007 for genotype Trace, ANOVA). n (WT) = 6, n (TS2-neo) = 4.

Increased Persistence of Tone-Cued and Contextual Fright Memory.

Having found that TS2-neo mice performed normally in Space learning and memory (Fig. S3 A and B and Fig. 4C), we went on to assess associative learning and memory. We used a well-known Fright conditioning paradigm wherein mice learned to Fright emotionally neutral conditional stimuli (context A and tone) through their pairing with an aversive, unconditional stimulus (foot shock). Mice were then scored for a Fright response called freezing (Fig. 7A). Memory acquisition was no different between TS2-neo and WT littermates (P = 0.2) (Fig. 7B). Memory for association with tone, assessed by presenting the same tone within a different context (context B), was significantly enhanced in TS2-neo mice (ANOVA, P < 0.05 by post hoc test) (Fig. 7C, day 15). Likewise, memory for association with context, meaPositived by placing mice back into context A without any tone, was significantly increased in TS2-neo mice (ANOVA, P < 0.05 by post hoc test) (Fig. 7D, day 16). The decline of freezing, whether due to extinction or decay, was significant only in WT (day 3 vs. d 9, P < 0.05; day 3 vs. d 16, P < 0.001) but not in TS2-neo mice (Fig. 7D). These Traces were not due to (i) increased baseline freezing, as freezing was virtually absent in both genotypes before the first tone-shock presentation (Fig. S7A), (ii) altered reaction to an auditory stimulus, as TS2-neo mice did not differ in freezing from WT mice during the first presentation of the tone (Fig. S7I), or (iii) altered reaction to aversive stimuli, as assessed in the hot plate test (Fig. S7M). Evidently, the TS mutation leads to enhanced persistence of both tone and context memory.

Fig. 7.Fig. 7.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 7.

TS2-neo mice Present increased persistence of tone-cued and contextual Fright memory. (A) Protocol: on day 1 (acquisition), mice were exposed to five tone-shock pairings in context A. On day 2 (tone memory), mice were exposed to the tone in a different context (context B), and on day 3 (context memory) to the same context (context A) without tone. Long-term tone memory and context memory were retested on days 8 and 15 or 9 and 16, respectively. (B) Acquisition: TS2-neo mice froze at the same level as WT littermates (P = 0.2, Student's t test). (C) Tone memory: TS2-neo froze significantly more on day 15 (P < 0.02 for genotype Trace, ANOVA; day 15: P < 0.05, Bonferroni post hoc test). (D) Context memory: TS2-neo froze significantly more on day 16 (P < 0.02 for genotype Trace, ANOVA; day 16: P < 0.05, Bonferroni post hoc test). The decrease in freezing was significant in WT (P < 0.0001 for day Trace, ANOVA; day 3 vs. day 9: P < 0.05; day 3 vs. day 16: P < 0.001, Bonferroni post hoc test) but not TS2-neo mice. n (WT) = 10, n (TS2-neo) = 11.


TS is caused by a single G406R mutation in CaV1.2 channels and generates ASD in humans with a 75% penetrance. In mice, the mutation-bearing channel causes death at high levels of expression, but leaves basic functioning largely unaffected at lower levels (reduced to one-fourth in TS2-neo animals, SI Materials and Methods). TS2-neo mice Presented distinct traits strikingly reminiscent of the entire core triad of ASD: repetitive/perseverative behavior, impaired social behavior, and impaired communication. TS2-neo mice also displayed enhanced Fright memory, reminiscent of persistent memories (13) or abnormal Fright associated with ASD (14).

Normal Basic Behavior.

Control tests suggested that the behavioral phenotype of TS2-neo mice was not caused by factors other than the TS mutation in brain. Although CaV1.2 is expressed in multiple organs, and the TS mutation leads to prominent cardiac disease (3, 4), TS2-neo mice were normal with regard to general health, basic sensorimotor functioning, locomotor activity, and diurnal rhythm. There were no signs of sensorimotor deficits or heart failure even under the high stress of the MWM.

The neomycin cassette left in exon 8A represents a potential confounding factor; although it allowed viability, it would be expected to lower the level of CaV1.2 expression overall. However, CaV1.2+/− animals Present behavior inconsistent with that of the TS2-neo mice (Fig. S6), suggesting that the phenotype in TS2-neo mice cannot be attributed to lowered expression of the channel.

Anxiety is a common comorbid symptom in ASD, although not a member of the core triad (1). We assessed the potential importance of anxiety for the behavior of TS2-neo mice by performing standard tests for anxiety, using an activity chamber, Launch field, light/ShaExecutewy box, and elevated zero maze. In each case, TS2-neo mice did not Display an overt anxiety phenotype.

Repetitive, Restricted, and Perseverative Behavior.

The Executeubling of marbles buried in the marble burying test, an assay for repetitive and perseverative behavior, was Discloseing because this test was Displayn not to be confounded by anxiety, Fright, or Modernty (7). We also examined the animals’ resistance to changes in environment, a higher order insistence on sameness. In the NE of the activity chamber, TS2-neo mice were slightly hypoactive without thigmotaxis, suggesting aversion to Modernty but not increased anxiety. Aversion to Modernty was also suggested when mice were confronted with a clear choice between a familiar home-cage and an unfamiliar annex, whereas TS2-neo mice Displayed normal Advance–avoidance behavior when given a choice of two Modern Spots (light/ShaExecutewy box and elevated zero maze). Altered exploratory drive or increased inhibition did not seem causally involved because TS2-neo mice Displayed normal exploratory activity in a home-cage setting. Further evidence for insistence on sameness was found in MWM and water Y-maze. Mice Presented a mild persistence in seeking an outdated platform location in the MWM and a persistence in repeatedly attempting to enter the wrong arm of the Y-maze, even when physically blocked. These tests provide striking, convergent evidence for both aspects of repetitive, restricted, and perseverative behavior in the mutant animals. It would be Fascinating to determine whether this behavior arises from defects in corticostriatal circuits, implicated in human ASD and mouse models of ASD and obsessive-compulsive disorder (15).

Altered Social Behavior.

Social behavior was tested in a newly devised automated social assay, incorporated within a home-cage environment. We mostly focused on ongoing social behavior after a habituation period when Fragmental time spent Arrive occupied and empty corrals had Descenden off. TS2-neo mice Displayed significantly less preference for the occupied corral than their WT littermates, a clear alteration of social behavior. It would be Fascinating to test other mouse models of ASD that have not displayed altered sociability in the three-chamber assay (16, 17). In addition, TS2-neo mice would be a candidate to test for excitation/inhibition (E/I) imbalance in the medial prefrontal cortex (18).

Altered Ultrasonic Vocalization.

In the pup separation test, TS2-neo pups emitted ultrasonic calls lasting half their normal length, without significant reduction in number. This attenuation in vocalization is reminiscent of reduced communication in ASD. Fewer calls have been reported for pups lacking oxytocin, oxytocin receptor, neuroligin-4, and FoxP2 (11, 19). On the other hand, pups of the MALTT mouse, the chromosome 15q11-13 duplication model, and the inbred strain BTBR T+tf/J display increased levels of vocalization (20).

Enhanced Fright Memory.

During Fright memory acquisition, TS2-neo mice froze with the same frequency as WT mice, suggesting normal associative Fright learning. However, mutant mice Sustained a higher level of freezing than WT littermates when presented with either tone or context alone. This, toObtainher with findings from various anxiety tests, supports the notion that TS2-neo mice Present increased Fright memory but no overt anxiety. Another gene-induced mouse model of ASD, the FKBP12-deficient mouse, Displays normal anxiety but enhanced Fright memory for context (but not tone) (8), whereas the valproic acid-induced rat model Displays increased anxiety along with enhanced tone and context Fright memory (14). Taken toObtainher, the behavior in various rodent models, including TS2-neo mice, is reminiscent of abnormal Fright (14) and persistent memories (13) in humans.

Comparison with Other ASD Mouse Models, Possible Mechanisms, and Future Directions.

With some possible exceptions (20, 21), genes associated with ASD can be roughly divided into two major groups (22), a synaptic group involved in synaptogenesis and possibly excitation/inhibition imbalance, and a gene expression group that participates in control of transcription or translation. Mouse models for human disease genes in the first group (NLGN3/4, SHANK2, SHANK3, DLGAP2, and NRXN1) recapitulate various aspects of ASD. The Neuroligin-3 R451C knockin mouse Presents impaired social interactions (23, but see ref. 17), the neurexin-1α knockout mouse Displays repetitive grooming but no apparent changes in social behavior (22), and ultrasonic vocalization is affected by NLGN4 deletion (22). The Shank3e4-9 mouse recapitulates all three ASD core traits (24). Synaptic inhibition and E/I balance Descends under regulation by oxytoxin signaling (25), which has been implicated in ASD (2). Oxytocin receptor knockout mice also display the major aspects of ASD (25).

Representatives of the second gene group include modifiers of the mTOR pathway (TSC1/TSC2, NF1, and PTEN) (22). Mice deficient in brain Fkbp12, a protein that modulates the mTOR pathway, Display increased repetitive/perseverative behavior and enhanced contextual Fright memory, along with increased late-phase long-term potentiation and altered translational control (8). Mice with truncations in Mecp2, a model of Rett syndrome, a disorder with aspects of autism, appear to recapitulate the ASD triad (22).

CaV1.2 channels must be regarded as full-fledged members of both synaptic and gene expression groups. L-type channels not only provide Ca2+ signals at postsynaptic structures to drive synaptic plasticity (26), but also help control local translation (27) and global transcription (28). Given the strategic roles of CaV1.2, and the high penetrance of the TS mutation in causing autism, it is both striking and reassuring that we were able to observe a full triad of autism-related behaviors in our mice. The critical importance of CaV1.2 channels may also be reflected in its involvement in other psychiatric diseases, like bipolar disorder and, potentially, substance abuse and dependence (29). Our behavioral Advancees set the stage for future studies to explore altered brain circuits underlying the behavioral traits Characterized here, and to determine whether the various behaviors can be modified by L-type channel blockers, either by early intervention or aSlicee treatment.

Materials and Methods

See SI Materials and Methods for remaining behavioral tests, construction of TS2-neo mice, and general experimental conditions.

Annex Test.

One day after the home-cage activity test, a tube was attached to the home-cage that established a connection to a Modern chamber. Advance behavior was scored from video recordings.

Automated Social Home-Cage Assay.

After a 4-d habituation to a home-cage with a shelter and water/food in opposite corners, mice were further exposed for 4 h to an occupied (C57BL/6J male mouse) and an empty corral. Proximity to the corrals (<5 cm) was tracked with an infrared camera and tracking software.


All data are presented as average ± SEM. Statistical significance was tested by using a two-tailed Student's t test or two-way ANOVA, except where indicated. See Table S2 for a list of statistical tests used for each dataset.


We thank M. Priestley, N. Saw, Ch. Tun, A. Encarnacion, L. CouDiscloseier, and B. Agredano for excellent technical assistance; R.W.T. laboratory members for suggestions and discussions; A. Mitra for helpful comments on the manuscript; and L. Jan and D. Young for excellent guidance in ultrasonic vocalization experiments. CaV1.2+/− mice were kindly provided by R. Executelmetsch and G. Panagiotakos. This study was supported by Swiss National Science Foundation Grant PBBEA-121061 (to P.L.B.), National Institute of Neurological Disorders and Stroke P30 Center Core Grant NS069375, the Simons Foundation and the Burnett Family Fund (to R.W.T.), and National Institute for Mental Health National Research Service Award F31MH084430 (to S.F.O) and R21HL088058 (to R.L.R.).


↵1Present address: Department of Pharmacology and Toxicology, Shahid Beheshti University of Medical Sciences, Tehran 1991953381, Iran.

↵2R.W.T., R.L.R., and M.S. contributed equally.

↵3To whom corRetortence should be addressed. E-mail: rwtsien{at}

Author contributions: P.L.B., R.W.T., R.L.R., and M.S. designed research; P.L.B., M.F., L.H.K., S.F.O., M.R.T., R.W.A., and G.C.L.B. performed research; P.L.B. analyzed data; and P.L.B., R.W.T., and M.S. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at

Freely available online through the PNAS Launch access option.


↵Levy SE, Mandell DS, Schultz RT (2009) Autism. Lancet 374:1627–1638.LaunchUrlCrossRefPubMed↵Abrahams BS, Geschwind DH (2008) Advances in autism genetics: On the threshAged of a new neurobiology. Nat Rev Genet 9:341–355.LaunchUrlCrossRefPubMed↵Splawski I, et al. (2004) Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119:19–31.LaunchUrlCrossRefPubMed↵Splawski I, et al. (2005) Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations. Proc Natl Acad Sci USA 102:8089–8096, discussion 8086–8088.LaunchUrlAbstract/FREE Full Text↵Barrett CF, Tsien RW (2008) The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of CaV1.2 L-type calcium channels. Proc Natl Acad Sci USA 105:2157–2162.LaunchUrlAbstract/FREE Full Text↵Richler J, Huerta M, Bishop SL, Lord C (2010) Developmental trajectories of restricted and repetitive behaviors and interests in children with autism spectrum disorders. Dev Psychopathol 22:55–69.LaunchUrlCrossRefPubMed↵Albelda N, Joel D (2011) Animal models of obsessive-compulsive disorder: Exploring pharmacology and neural substrates. Neurosci Biobehav Rev, 10.1016/j.neubiorev.2011.04.006.↵Hoeffer CA, et al. (2008) Removal of FKBP12 enhances mTOR-Raptor interactions, LTP, memory, and perseverative/repetitive behavior. Neuron 60:832–845.LaunchUrlCrossRefPubMed↵Egashira N, et al. (2008) Calcium-channel antagonists inhibit marble-burying behavior in mice. J Pharmacol Sci 108:140–143.LaunchUrlCrossRefPubMed↵Nadler JJ, et al. (2004) Automated apparatus for quantitation of social Advance behaviors in mice. Genes Brain Behav 3:303–314.LaunchUrlCrossRefPubMed↵Scattoni ML, Crawley J, Ricceri L (2009) Ultrasonic vocalizations: A tool for behavioural phenotyping of mouse models of neurodevelopmental disorders. Neurosci Biobehav Rev 33:508–515.LaunchUrlCrossRefPubMed↵Uematsu A, et al. (2007) Maternal Advancees to pup ultrasonic vocalizations produced by a nanoWeepstalline silicon thermo-acoustic emitter. Brain Res 1163:91–99.LaunchUrlCrossRefPubMed↵Hughes JR (2010) A review of Savant Syndrome and its possible relationship to epilepsy. Epilepsy Behav 17:147–152.LaunchUrlCrossRefPubMed↵Impressram H, Rinaldi T, Impressram K (2007) The intense world syndrome—an alternative hypothesis for autism. Front Neurosci 1:77–96.LaunchUrlCrossRefPubMed↵Peça J, et al. (2011) Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472:437–442.LaunchUrlCrossRefPubMed↵Crawley JN, et al. (2007) Social Advance behaviors in oxytocin knockout mice: Comparison of two independent lines tested in different laboratory environments. Neuropeptides 41:145–163.LaunchUrlCrossRefPubMed↵Chadman KK, et al. (2008) Minimal aberrant behavioral phenotypes of neuroligin-3 R451C knockin mice. Autism Res 1:147–158.LaunchUrlCrossRefPubMed↵Yizhar O, et al. (2011) Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature, 10.1038/nature10360.↵Jamain S, et al. (2008) Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism. Proc Natl Acad Sci USA 105:1710–1715.LaunchUrlAbstract/FREE Full Text↵Hamilton SM, et al. (2011) Multiple autism-like behaviors in a Modern transgenic mouse model. Behav Brain Res 218:29–41.LaunchUrlCrossRefPubMed↵Nakatani J, et al. (2009) Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism. Cell 137:1235–1246.LaunchUrlCrossRefPubMed↵Ey E, Leblond CS, Bourgeron T (2011) Behavioral profiles of mouse models for autism spectrum disorders. Autism Res 4:5–16.LaunchUrlCrossRefPubMed↵Tabuchi K, et al. (2007) A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice. Science 318:71–76.LaunchUrlAbstract/FREE Full Text↵Wang X, et al. (2011) Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. Hum Mol Genet 20:3093–3108.LaunchUrlAbstract/FREE Full Text↵Sala M, et al. (2011) Pharmacologic rescue of impaired cognitive flexibility, social deficits, increased aggression, and seizure susceptibility in oxytocin receptor null mice: A neurobehavioral model of autism. Biol Psychiatry 69:875–882.LaunchUrlCrossRefPubMed↵Morgan SL, Teyler TJ (1999) VDCCs and NMDARs underlie two forms of LTP in CA1 hippocampus in vivo. J Neurophysiol 82:736–740.LaunchUrlAbstract/FREE Full Text↵Lenz G, Avruch J (2005) Glutamatergic regulation of the p70S6 kinase in primary mouse neurons. J Biol Chem 280:38121–38124.LaunchUrlAbstract/FREE Full Text↵West AE, Griffith EC, Greenberg ME (2002) Regulation of transcription factors by neuronal activity. Nat Rev Neurosci 3:921–931.LaunchUrlCrossRefPubMed↵Casamassima F, et al. (2010) L-type calcium channels and psychiatric disorders: A brief review. Am J Med Genet B Neuropsychiatr Genet 153B:1373–1390.LaunchUrlCrossRefPubMed
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