Human Molecular Genetics Advance Access originally published online on May 21, 2007
Human Molecular Genetics 2007 16(15):1802-1813; doi:10.1093/hmg/ddm128
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Rai1 deficiency in mice causes learning impairment and motor dysfunction, whereas Rai1 heterozygous mice display minimal behavioral phenotypes
1 Department of Molecular and Human Genetics, 2 Department of Pathology, 3 Department of Neurology, 4 Department of Pediatrics, 5 Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA and 6 Texas Children's Hospital, Houston, TX, USA
* To whom correspondence should be addressed at: Department of Molecular and Human Genetics, Baylor College of Medicine, Room 604B, One Baylor Plaza, Houston, TX 77030-3498, USA. Tel: +1 7137986530; Fax: +1 7137985073; Email: jlupski{at}bcm.tmc.edu
Received April 2, 2007; Accepted May 4, 2007
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ABSTRACT |
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Smith–Magenis syndrome (SMS) is associated with an
3.7 Mb common deletion in 17p11.2 and characterized by its craniofacial and neurobehavioral abnormalities. The reciprocal duplication leads to dup(17)(p11.2p11.2) associated with the Potocki–Lupski syndrome (PLS), a neurological disorder whose features include autism. Retinoic acid induced 1 (RAI1) appears to be responsible for the majority of clinical features in both SMS and PLS. Mouse models of these syndromes harboring an 2 Mb chromosome engineered deletion and duplication, respectively, displayed abnormal locomotor activity and/or learning deficits. To determine the contribution of RAI1 in the neurobehavioral traits in SMS, we performed a battery of behavioral tests on Rai1 mutant mice and the Df(11)17–1/+ mice that have a small deletion of 590 kb. The mice with the small deletion were hypoactive like the large deletion mice and they also showed learning deficits. The Rai1+/– mice exhibited normal locomotor activity. However, they had an abnormal electroencephalogram with overt seizure observed in a subset of mice. The few surviving Rai1–/– mice displayed more severe neurobehavioral abnormalities including hind limb clasping, overt seizures, motor impairment and context- and tone-dependant learning deficits. X-gal staining of the Rai1+/– mice suggests that Rai1 is predominantly expressed in neurons of the hippocampus and the cerebellum. Our results suggest that Rai1 is a critical gene in the central nervous system functioning in a dosage sensitive manner and that the neurobehavioral phenotype is modified by regulator(s) in the 590 kb genomic interval, wherein the major modifier affecting the craniofacial penetrance resides. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Smith–Magenis syndrome (SMS, MIM 18829) is a multiple congenital abnormalities/mental retardation disorder with characteristic craniofacial and neurobehavioral manifestations (1–4). The minimum prevalence of SMS is 1 in 25 000 births (5). Individuals with SMS usually have low adaptive functioning with relative strengths in socialization and relative weakness in daily living skills (6), mild-to-moderate mental retardation (1) and display a characteristic neurobehavioral pattern of stereotypes, self-injurious and maladaptive behaviors (4). More than 70% of SMS cases harbor an
3.7 Mb common deletion mediated by non-allelic homologous recombination using flanking low copy repeats as substrates (7).
A critical region for SMS (SMCR) was confined to an 1.1 Mb genomic interval by breakpoint junction analysis in SMS patients with unusual smaller-sized deletions (8). Twenty genes were found in the SMCR, 19 of which are also found in the syntenic 32–34 cM region of mouse chromosome 11 (8). The order and orientation of each of these 19 genes are conserved between humans and mice (8,9). Mutations in a gene within the SMCR, retinoic acid induced 1 (RAI1), were identified in nine patients with typical SMS features but no FISH detectable deletions (10–13). These patients displayed characteristic SMS craniofacial and neurobehavioral phenotypes indicating that RAI1 is the major causative gene. Studies on mouse models (14) suggest that RAI1 is likely the dosage sensitive gene responsible for clinical features in the Potocki–Lupski (PLS) MIM610883 dup(17)(p11.2p11.2) syndrome, a neurobehavioral disorder with autistic features that is caused by reciprocal duplication of the same region commonly deleted in the SMS (15–17).
RAI1 contains a polymorphic CAG repeat that encodes a polyglutamine stretch in its N-terminus. RAI1 has been implicated in neurobehavioral traits as evidenced by the findings that the length of the CAG repeats has been associated with two neurological diseases; modification of the age at onset for spinocerebellar ataxia type 2 (SCA2) (18) and response to neuroleptic drugs in patients with schizophrenia (19). A single plant homeo domain (PHD) zinc finger, frequently observed in chromatin remodeling transcriptional regulators, is present in the C-terminus of both human and mouse RAI1 (10). Murine Rai1 has only two glutamines instead of a polyglutamine stretch, but it shares 86% similarity with its human homologue. The expression of Rai1, previously named GT1, was stimulated by the treatment of retinoic acid in embryonic carcinoma cells and it is expressed in neurons specifically in the brain (20). During embryogenesis, Rai1 is predominantly expressed in the epithelial cells involved in organogenesis (21).
To further understand the etiology of SMS through animal studies, we have constructed several mouse models of SMS and PLS: the Df(11)17/+ mice (Del mutant) harboring a large 2 Mb deleted region syntenic to the common SMS deletion, whereas the Dp(11)17/+ mice (Dup mutant) contain a reciprocal duplication created by chromosome engineering (22), the Df(11)17–4/+ mice having a deletion of 1 Mb (23), the Df(11)17–1/+ mice having a small deletion of 590 kb (24), and Rai1 deficient mice containing an insertionally inactivated allele generated by gene targeting (21). About 25 genes were deleted in the large deletion mice, whereas only 11 genes were deleted in the mice with the small or 590 kb deletion. We have characterized and compared the craniofacial features and growth of these mouse models at the N2 generation. Table 1 summarizes the phenotypes in different mouse models.
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Del mutant mice partially recapitulate the SMS features, exhibiting craniofacial abnormalities, seizures and obesity. In addition, the Del mutants displayed hypoactivity and abnormal circadian rhythm, while the Dup mutants that have a reciprocal duplication were hyperactive and have impaired contextual fear conditioning. Similar craniofacial defects and obesity were also observed in three lines of mice harboring a smaller deletion (
590 kb) (24) and the mice with inactivation of the Rai1 gene by gene targeting (21). Importantly, in a congenic C57BL/6 background, Del mice and the smaller deletion mice had a similar penetrance, whereas that of Rai1+/– mice was significantly lower, which suggested the existence of a major genetic modifier(s) in the 590 kb interval surrounding Rai1 affecting the craniofacial penetrance (23). It is unknown how the dosage insufficiency of Rai1 affects the mammalian behaviors and what the contribution of Rai1 is in the behavioral phenotypes observed in the Del mouse models.
To explore the role of RAI1 in SMS and its function in the nervous system, we performed a battery of behavioral tests on both the mice with a small590 kb deletion and Rai1+/– mice and compared their behaviors with those observed in the mice with a large 2 Mb deletion in the same genetic background. We also evaluated the behaviors of the Rai1–/– mice and the expression profile of Rai1 in the nervous system. In addition, we examined the clinically observed seizures in the Rai1 mutant mice by electrophysiological methods using video-electroencephalograph (VEEG). Our data indicated that Rai1 is strongly expressed in a subset of neurons in the brain and is required for motor function and learning ability. In addition, our results suggest that other major genetic modifier(s) for the behavioral phenotype in SMS resides in the 590 kb genomic interval deleted in the small deletion mice.
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RESULTS |
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Rai1 is strongly expressed in the central nervous system
We performed X-gal staining on frozen sections prepared from the brains of postnatal Rai1+/– mice that have a lacZ reporter gene knocked-into the Rai1 gene. Blue-staining resulting from the inserted ß-galactosidase, indicative of Rai1 gene expression, was detected throughout the brain in low level and predominantly in the hippocampus and cerebellum (Fig. 1A), consistent with its in situ hybridization expression profile from the Allen Brain Atlas. In the hippocampus, the expression was observed in areas CA1, CA2, CA3 and the dentate gyrus (Fig. 1B). In the cerebellum, the expression was seen in the Purkinje layer (Fig. 1C). Other sites of strong expression include the striatum, neurons in the cerebral cortex, the nucleus accumbens, the epithelial membrane lining the ventricles of the brain, the ependyma and the choroid plexus (Fig. 1D–F). Expression of Rai1 was also observed in the endothelial cells of blood vessels throughout the brain (Fig. 1F). In P7 and P20 stages, Rai1 is expressed in a broader pattern in the developing brains and strong expression in the hippocampus and cerebellum was also observed (Fig. 1G and H). To examine which cells were stained in the central nervous system, we visualized neurons using anti-NeuN antibody and Rai1 expression with anti-lacZ antibody. We found that the NeuN expressing cells also express lacZ, suggesting that Rai1 is expressed only in the neurons (Fig. 1I–K).
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In addition to the brain, expression of Rai1 was also observed in the thymus, bronchioles of the lung, collecting tubules in the kidney, the testis, the epididymis, the ovary, hair follicles in the skin, the tongue (Fig. 2). The mandibular gland and the intestine were well stained (data not shown). Rai1 expression is low in the heart, the liver and the spleen with very few subsets of expressing cells. In adult mice, the central canal and the posterior horn of gray matter of the spinal cord were blue stained (Fig. 2M). The central canal is filled with the cerebrospinal fluid and Rai1 is likely expressed in the ependymal cells similar to its expression in the ependyma in the brain. Rai1 is well expressed in the developing spinal cord (Fig. 2N). In summary, Rai1 is broadly expressed in multiple organs and tissues with variant expression levels in a subtype of tissues and cells.
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Behavioral activities in mice with a small
590 kb deletion and Rai1+/– miceThe behavioral tests that showed significant differences between the Df(11)17/+ or Dp(11)17/+ mice and their wild-type littermates include the open-field test, light-dark test, prepulse inhibition of the acoustic startle response and the conditioned fear test (25). To understand the contribution of Rai1 to the behavioral abnormalities, we performed the same behavioral tests on male mice with a small deletion, Df(11)17–1, and on the Rai1+/– male mice in a mixed background at the N2 (75% C57BL/6 and 25% 129SvEv) generation; a genetic background that is consistent with the Del mutants tested previously (25). We performed behavioral tests only on male mice because the behavioral phenotypes have better penetrance in male Del or Dup mice than in female mutant mice (22).
The open-field test was performed to measure locomotor activity and anxiety-related responses (26). In a novel arena, similar to the Df(11)17 mice (25), the mice with the small deletion were hypoactive; they spent significantly less time moving (P < 0.05) and traveled a slightly shorter distance (P = 0.17) (Fig. 3A and B). They reared more than wild-type mice (P = 0.05) (Fig. 3C) and spent a higher proportion of their exploration in the center of the open field compared with the wild-type mice (P < 0.05) (Fig. 3D). In contrast, travel distance, travel time and center/total distance ratio of the Rai1+/– mutants were similar to that of the wild-type mice (Fig. 3A, B and D). The only significant difference between the Rai1+/– mutants and wild-type mice is that the vertical activity (rearing) was significantly increased in the Rai1+/– mice (Fig. 3C).
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Individuals with SMS display different levels of cognitive deficits. We evaluated associative learning and memory in mice by the conditioned fear test, a fear-based response using a Pavlovian paradigm. The context-dependent fear conditioning is to test whether animals learned to associate a particular environment (context) with an aversive stimulus (an electric foot shock). In tone-dependent (cued) fear conditioning, the animals were tested to examine if they learned the association of an auditory stimulus with an aversive stimulus. The fear response to the presence of context or tone was a conditional freezing behavior characterized by a motionless, crouching posture and was assessed at 10 s intervals. The mice with a small deletion showed significantly less freezing without the conditioned stimulus (context test) (P < 0.05) and in the presence of conditioned stimulus (CS test) (P < 0.05) (Fig. 4). The Rai1+/– mutants displayed levels of freezing behavior comparable with wild-type in both contextual and auditory cued conditioned fear tests (Fig. 4) consistent with the previous finding (14). Reduced freezing activity has been reported in the Dup mice in the context test but not in the CS test. However, the Del mice showed normal freezing in both context and CS tests (14,25).
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Prepulse inhibition is a test to assess sensorimotor gating. For the maximum startle response, Dup mutant mice startled significantly less than wild-type mice (25). In contrast, the smaller deletion mice startled significantly more than wild-type mice (P < 0.05), whereas there was no significant difference between Rai1+/– and wild-type mice (Supplementary Material, Fig. S1). There were no significant differences in the levels of prepulse inhibition for all genotypes (data not shown). Similar prepulse inhibition test results for Rai1+/– mice were reported previously (14).
The light–dark exploration test is used to assess anxiety-related responses. Similar to the Del mice with the large 2 Mb deletion, there were no significant differences in the mice with small deletion and the Rai1+/– mutants compared with wild-type mice in the light–dark test (Supplementary Material, Fig. S2).
Motor dysfunction and learning deficits in Rai1–/– mice
We previously reported that the vast majority of the Rai1–/– mice died during embryonic development (21). To explore the possibility of behavioral studies on Rai1–/– mice, we conducted large-scale breeding. We found that the body weight of the Rai1–/– mutants (female, N = 4) that survived is similar to wild-type mice at 3 months of age regardless of postnatal growth retardation (Fig. 5A). Approximately 10% of homozygous mutants at ages over 4 months displayed kyphosis (Fig. 5E and F). Microscopic studies on formalin fixed paraffin sections stained with hematoxylin and eosin revealed normal histology of the brain. The pyramidal cell layer in Ammon's horn and the granule cell layer in the dentate gyrus were well defined in the hippocampus of adult Rai1–/– mutants (data not shown).
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When suspended by the tail, all the wild-type mice held their hind legs widely apart. We observed that all the Rai1–/– mutants consistently displayed hind limb clasping when suspended, which remained even after 3 months of age (Fig. 5B–D). They brought their hind limbs together (Fig. 5C) and some of them brought all four limbs together (Fig. 5D).
The hind limb clasping in the Rai1–/– mutants prompted us to evaluate their motor function using a battery of objective tests. Mice were evaluated on a rotating rod apparatus (rotarod) in which the speed of rotation was increased linearly from 4 to 40 rpm over 4 min. Both Rai1+/– and Rai1–/– mutants exhibited improvement in fall-off time during the 4-day period of testing and the mutants remained on the rotating rod for a time similar to wild-type mice (Fig. 6A). In the vertical pole test, mice were placed at the end of a horizontal pole and then the pole was lifted to a vertical position with mice at the top of the pole. Rai1–/– mice fell off the pole more readily and frequently than wild-type and heterozygous littermates significantly (P = 0.009) (Fig. 6B). We also analyzed the ability of mice to hold on to a wire with their forepaws in a wire suspension test. Whereas wild-type and heterozygous mice generally suspended in the wire for 30–60 s, Rai1–/– mice tended to fall off in <10 s (P < 0.01) (Fig. 6C). The ability of mice to remain on a thin, horizontal wooden dowel was tested. The wild-type and heterozygous mice were able to remain on the dowel for the maximum of 60 s. In contrast, the Rai1–/– mice lost their balance and fell off the dowel rapidly and usually in 11 s (P < 0.001) (Fig. 6D). These tests suggest that Rai1–/– mice have motor dysfunction.
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Similar to the Rai1+/– mutants, there was no significant difference detected in the homozygous mutants for open-field test, light–dark test and prepulse inhibition with the exception that the Rai1–/– mutants showed significantly increased latency to enter as assessed in the light–dark test (data not shown). The levels of prepulse inhibition were similar between Rai1–/– mutants and wild-type mice, indicating that the sensorimotor gating appeared normal in the mutants. Interestingly, the Rai1 homozygous mutants showed defective context-dependent as well as tone-dependent learning as evaluated by the conditioned fear tests (Fig. 7). Compared with the wild-type mice, the Rai1–/– mice demonstrated substantially less freezing behavior when placed in the test chamber without the conditioned stimulus (context test) (P < 0.01, in males only) and when in a modified chamber in the presence of the conditioned stimulus (P < 0.05, in both males and females) (Fig. 7). The decreased freezing behavior was also observed in the Dup mice and mice with the small deletion but not in Del mice and the Rai1+/– mice (Fig. 4) (25).
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Seizures in the Rai1 mutant mice
Overt seizures were observed in about one-third of Rai1–/– mutants after 3 months of age. Prolonged VEEG monitoring revealed an abnormal wake and sleep background punctuated by generalized spikes, multiple spikes and sharp waves. It was also noted that some of the events of sudden behavioral arrest correlated with electrographic seizures heralded by a generalized high voltage spike and slow wave complex followed by a 20-s long run of generalized 2.5–4 Hz spike and slow waves that evolved into a prolonged epoch of frontally dominant 6 Hz sharp waves (Fig. 8B). Overt generalized clonic–tonic–clonic seizures were observed as well. The electrographic patterns were characterized by an initial onset of 6–7 Hz sharp waves that correlated with behavioral arrest. As the sharp waves gave way to generalized rhythmic 3 Hz spike and slow waves, an initial inactivity progressed into whole body clonic–tonic–clonic seizure lasting on average 30 seconds. Approximately 2% of Rai1+/– mice displayed overt seizures when they were over 4 months of age. However, even for the heterozygous mice without electrographic seizures recorded, VEEG showed an abnormal awake and sleep baseline electroencephalogram characterized by frequent epileptiform discharges (Fig. 8C and D).
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DISCUSSION |
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We have genetically manipulated mice and generated several different models for SMS including mice with different deletions and mice with targeted disruption in the single Rai1 gene. Previous studies on body weight and craniofacial features indicated that Rai1+/– mice recapitulate the features of obesity and craniofacial abnormalities in mice with large or small deletions, indicating that dosage insufficiency of RAI1 contributes to these features in SMS. Herein we characterized the behavioral features of mice with the small deletion, Rai1+/– mice and Rai1–/– mice, and compared these features with those of the large deletion mice and Rai1+/– mice reported previously. Our data indicate that mice with the small deletion showed the behavioral abnormalities as observed in the large deletion mice and the Rai1–/– mice display abnormalities in complex traits including motor functions and learning ability, whereas minimal behavioral abnormalities were observed in the Rai1+/– mice.
Del mice were hypoactive while the mice with reciprocal duplication were hyperactive (27). Hypoactivity was also observed in the small deletion mice in this study as indicated by significantly reduced travel time in the open field test. Thus, the 590 kb interval deleted in the small deletion mice contains the gene(s) or regulatory elements responsible for the abnormal locomotor activity in the Del mice. In the open-field test, the travel distance and travel time for the Rai1+/– mice were comparable with those in the wild-type littermates, suggesting that one copy of Rai1 is enough for normal locomotive activity in the N2 generation. However, a previous study showed that the Rai1+/– mice generated by mating Dp(11)17/+ mice in a C57BL/6 background with Rai1+/– mice in a mixed C57BL/6 and 129SvEv genetic background (F2 generation) exhibited significantly decreased total travel distance and thus were hypoactive (14). This discrepancy suggests that variation in different genetic background substantially affects the penetrance and expressivity of behavioral phenotypes due to Rai1 dosage effect. Of note, the Rai1+/– mice did display some neurobehavioral phenotypes even in the N2 background. The significantly increased exploratory vertical activity observed in N2 background herein is also observed in that study (14). In addition, ~2% of Rai1+/– mice displayed overt seizures and an abnormal electroencephalograph (EEG) was observed in the Rai1+/– mice. Therefore, loss of one copy of Rai1 compromises normal neural development and we propose that Rai1 is at least partially responsible for the neurobehavioral phenotypes in the deletion mice.
We previously demonstrated that Rai1 is dosage dependant in skeletal development; the skeletal abnormalities in Rai1–/– mice are much more severe than those observed in Rai1+/– mice (21). Rai1 gene copy number variation as a cause of behavioral traits has been explored in a study to rescue the phenotypes in Dup mice through restoration of the normal disomic Rai1 gene dosage in compound heterozygous mice carrying a Dp(11)17 along with a null allele of Rai1 (14). By examining the behavioral traits in the Rai1+/– and Rai1–/– mice, we provide direct evidence to show that the more reduced the copy number of Rai1 in a mouse, the more severe the behavioral abnormalities including the learning disability, motor deficiency, and seizures. The deficits in both context associative and tone-dependent learning in conditioned fear tests were also observed in the small deletion mice and Rai1–/– mice but not in the Rai1+/– mice. One possible explanation for the more severe behavioral phenotypes in the deletion mice compared with the heterozygous knockout mice is that the expression of Rai1 from the remaining normal allele in the small deletion mice is less than that in the Rai1+/– mice because of potential trans-regulatory elements in the deleted region. The reduced penetrance of craniofacial abnormalities in mice with the smaller deletion and Rai1+/– is also consistent with this interpretation (23). The contextual learning deficit is sensitive to lesions in the hippocampus where Rai1 showed predominant expression in the neurons of both the cornu ammonis and dentate gyrus. Thus, the deficit in learning and memory may result from a hippocampus defect. Evaluation of long-term potentiation and long-term depression in hippocampal slices is necessary for delineation of the cellular mechanisms (28).
In the same N2 generation, the small deletion mice exhibited defective locomotor activity whereas the Rai1+/– mice were comparable with the wild-type. The Rai1 allele in the knockout mice is a functional null allele and not likely a hypomorphic allele because sequences that encode the functional domains of nuclear localization and the PHD domain were included within the 4 kb deletion in the targeted allele (21). Furthermore, RT–PCR for Rai1 using primers to the deleted and downstream regions revealed no transcripts (21); the absence of Rai1 gene expression was also observed with whole genome expression arrays of Rai1–/– embryos (data not shown). Thus, we cannot exclude the involvement of modifiers located within the small deletion interval in contributing to behavioral traits. A previous study on the craniofacial abnormalities in SMS mouse models suggested that trans regulator(s) other than the Rai1 in the SMS region can modify the penetrance of the craniofacial phenotype (23). Similarly, gene(s) or regulatory elements other than the Rai1 coding sequences are involved in the behavioral phenotypes. The 11 genes other than the Rai1 in the small deletion interval include Rasd1 encoding a small G protein that has been shown to be a critical modulator in the circadian timekeeping system in mice (29). Behavioral studies on mice with these genes disrupted, with careful evaluation of the heterozygous mutants and double knockout of Rai1 and individual genes in the small deletion, may help clarify the functions of these genes in behavior as assessed by objective testing.
It is interesting to note that impaired fear conditioning was present in the Df(11)17–1 mice with 590 kb deletion and Rai1–/– mice but not in the Del mice with 2 Mb deletion although the 590 kb deletion is located within the 2 Mb region and the Df(11)/17–1 mice and the Del mice were evaluated in the same background. During the training stage of the conditioned fear test, when a mild foot shock was applied the mice with the smaller deletion and the Rai1–/– mice manifested the same response as the wild-type mice such as running, jumping and vocalizing, suggesting that pain sensitivity of these mice is similar to the wild-type mice. Our previous studies showed that compound heterozygous Del/Dup mice have normal body weight and normal craniofacial features and are normal in the conditioned fear test (14), suggesting that the related phenotypes in the Del and Dup are due to gene dosage effects. Importantly, the restoration of the normal disomic Rai1 gene dosage is sufficient to rescue the physical and behavioral phenotypes observed in Del mice (14) indicating that Rai1 is the dosage sensitive gene. Therefore, the impaired fear conditioning in the mice with smaller deletion is mainly caused by dosage insufficiency of Rai1 plus the effect of genetic modifiers within the 590 kb. This phenotype was inhibited in the Del mice by the deleted region outside of the 590 kb interval. Examination of the behavioral phenotypes in the compound heterozygous mice with the smaller deletion and Dup allele may help further elucidate this regional interaction.
In summary, our behavioral studies suggest that Rai1 is a dosage sensitive gene required for normal behavioral function and Rai1 copy number variation affects mouse behaviors with minimal behavioral abnormalities for one copy loss and severe motor and learning deficits for complete loss of Rai1. Gene(s) other than the Rai1 or regulatory elements in the 590 kb deleted in the small deletion mice modify the behavioral traits.
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MATERIALS AND METHODS |
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Testing animals
Mice were housed under constant temperature, humidity and a light/dark cycle of 12 h (light 7:00 AM to 7:00 PM). All animal studies were conducted under protocols approved by the Baylor Institutional Animal Care and Use Committee and followed NIH guidelines. We separated the behavioral tests into two independent studies. Mice for both studies were around 3 months of age at the beginning of the testing. Experiments were conducted by an experimenter blinded to the genotypes of the mice. One study is to compare the behaviors of smaller deletion mice and the Rai1+/– mice with that of the large deletion mice. Mice were tested in the mixed (75% C57BL/6 and 25% 129SvEv) genetic background at the N2 generation. Twenty-one small deletion males and 20 wild-type male littermates were evaluated in the open-field, light–dark, prepulse inhibition of the startle response and conditioned fear test. The same battery of tests was performed on 19 Rai1+/– males and 19 wild-type male littermates.
Another study sought to explore the behavioral defects in the Rai1–/– mutants. Mice were maintained on a mixed (50% C57BL/6 and 50% 129SvEv) background. Mice with different genotypes were housed with their littermates but with males and females separately. Data used for statistical analysis are from tests of 8 (5 females and 3 males) Rai1–/– mutants, 15 (5 females and 10 males) Rai1+/– mutants and 12 (6 females and 6 males) wild-type mice.
Motor function test
Motor skills were evaluated by rotating rod test, vertical pole test, suspended wire test and dowel test. For the rotating rod test, mice were placed on a rotating rod (model 7650 Rota-rod, Ugo Basile) that accelerated from 3 to 30 rpm. The mice were given one training trial 2 h before the test. For four consecutive days, four trials were performed per day with 45–60 min interval between trials. The maximum duration of each trial was 10 min. The time that the mice fell off the rod was recorded. For the vertical pole test, mice were placed on one end of a pole (1.9 cm diameter, 43 cm length). The pole was shifted to a vertical position. A score was given based on the position of the pole and the time that the mice remained on the pole (30). The wire test was implemented by suspending mice with their forepaws on a 2 mm wire, and the amount of time they stayed on the wire was recorded in a 60 s interval. For the dowel test, mice were placed in the center of a horizontal wooden dowel (0.9 cm diameter) suspended between two platforms. The time that the mice remained on the dowel was recorded in a 60 s interval.
Conditioned fear test
The fear conditioning was conducted in a test chamber (26 x 22 x 18 cm) with a grid floor used to deliver an electric foot shock. The chamber was placed inside a sound attenuating chest (56 x 38 x 36 cm) with a peep hole on one side of the door to allow the observation of mouse behaviors. The chamber was cleaned with 100% ethanol before each mouse was placed inside. Freezing was assessed as 1 (freezing posture) or 0 (no freezing posture) and the scores were averaged over 10 s intervals and converted to a percentage score. One mouse was placed in the chamber for 2 min to get familiar with the chamber. An 80 dB white noise was present for 30 s, which serves as the conditioned stimulus (CS). A mild foot shock (2 s, 0.5 mA) was followed, which serves as the unconditioned stimulus (US). After 2 min, another CS–US pair was provided. The mouse was placed back to its home cage after 30 s. Twenty-four hours later, the mouse was placed in the test chamber and freezing behavior was recorded every 10 s for 5 min (context test). For auditory CS test, mice were placed in the same chamber with environment and contextual cues alternated by changing light color, space size, smell and floor. Freezing was recorded for 3 min without CS (pre-CS phase) and for another 3 min with the continuous auditory CS (CS phase).
Open-field test, light–dark exploration and prepulse inhibition
Mice were placed in the center of an open-field space (40 x 40 x 30 cm) and allowed to explore freely for 30 min. Activities were quantitated by a computer-operated Digiscan optical animal activity system (RXYZCM, Accuscan Electronics). Data were collected over 2 min intervals during a 30 min test session. Light–dark exploration test and startle and prepulse inhibition of the startle were performed as described (25).
EEG recordings
Mice were anesthetized with avertin, and teflon-coated silver wire electrodes (0.005 in diameter) were implanted bilaterally into the subdural space over the frontal, temporal parietal and occipital cortexes at least 24 h before recording. The electrodes were soldered to a microminiature connector. EEG activity was recorded on mice moving freely in the test cage during random 2–24 h samples for 7–10 days using a digital VEEG (Stellate Harmonie).
Histology, immunohistochemistry and X-gal staining
Adult mice were anesthetized with avertin and the brains were dissected and fixed with 10% formalin for at least 24 h. The tissues were then washed, embedded in paraffin, sectioned and stained with hematoxylin and eosin. For immunohistochemistry, brains were cut sagittally in half and embedded in frozen tissue matrix (optimal cutting temperature compound OCT, tissue-Tek). Sections of 15 µm were sliced using a cryostat (Microm HM500) and stored in –80°C. Sections were fixed in 1:1 acetone: methanol for 10 min. Rabbit polyclonal anti-beta-galactosidase antibody (Cortex Biochem, San Leandro, CA) was diluted at 1:200 in 10% goat serum-PBS. Mouse anti-Neuronal Nuclei (NeuN) monoclonal antibody (Chemicon, Temecula, CA) was diluted at 1:100. The secondary antibodies are Goat anti-rabbit Alexa Fluor 594, and Rabbit anti-mouse Alexa Fluor 488 (Molecular probes, Carlsbad, CA) diluted at 1:200. Sections were mounted in SlowFade Antifade (Molecular Probes, Carlsbad, CA). For X-gal staining, frozen sections were air-dried for 5 min and stained as described before (21) with the exception that the staining was performed in 37°C overnight in darkness. After staining, the sections were washed and counter-stained with fast red.
Statistical analysis
Except for the pole test, behavior data were analyzed using ANOVA with appropriate follow-up comparisons of the main effect. The pole test data were analyzed using the non-parametric Mann–Whitney U test.
Web resources
Accession numbers and URLs for data presented herein are as follows:
On line Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/.
Allen Brain Atlas (ABA), http://www.brain-map.org/welcome.do/.
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Supplementary Material is available at HMG Online.
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ACKNOWLEDGEMENTS |
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We thank Drs L. Luo and K. Walz for critical reviews. This work was supported in part by grants from the National Institute for Dental and Craniofacial Research (R01 DEO15210), the Mouse Physiology Core and the Neurobehavioral Core of the Baylor Mental Retardation and Developmental Disabilities Research Center (P30 HD 24064) and a grant from the March of Dimes (J.R.L. and W.B.).
Conflict of Interest statement. None declared.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Bi W., Ohyama T., Nakamura H., Yan J., Visvanathan J., Justice M.J., Lupski J.R. Inactivation of Rai1 in mice recapitulates phenotypes observed in chromosome engineered mouse models for Smith–Magenis syndrome. Hum. Mol. Genet. (2005) 14:983–995.
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Walz K., Caratini-Rivera S., Bi W., Fonseca P., Mansouri D.L., Lynch J., Vogel H., Noebels J.L., Bradley A., Lupski J.R. Modeling del(17)(p11.2p11.2) and dup(17)(p11.2p11.2) contiguous gene syndromes by chromosome engineering in mice: phenotypic consequences of gene dosage imbalance. Mol. Cell. Biol. (2003) 23:3646–3655.
[Abstract/Free Full Text] - Yan J., Bi W., Lupski J.R. Penetrance of craniofacial anomalies in mouse models of Smith–Magenis syndrome is modified by genomic sequence surrounding Rai1—not all null alleles are alike. Am. J. Hum. Genet. (2007) 80:518–525.[CrossRef][Web of Science][Medline]
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Yan J., Keener V.W., Bi W., Walz K., Bradley A., Justice M.J., Lupski J.R. Reduced penetrance of craniofacial anomalies as a function of deletion size and genetic background in a chromosome engineered partial mouse model for Smith–Magenis syndrome. Hum. Mol. Genet. (2004) 13:2613–2624.
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Shahbazian M., Young J., Yuva-Paylor L., Spencer C., Antalffy B., Noebels J., Armstrong D., Paylor R., Zoghbi H. Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3. Neuron (2002) 35:243–254.[CrossRef][Web of Science][Medline]
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