Behavioural phenotyping assays for mouse models of autism
(Full-text capture 2026-09-21; web artifacts lightly stripped; truncated.)
Jill L Silverman
*Laboratory of Behavioral Neuroscience, Intramural Research Program, National Institute of Mental Health, Porter Neuroscience Research Center Building 35 Room 1C903, Bethesda, Maryland 20892-3730, USA
*, Mu Yang
Mu Yang
*Laboratory of Behavioral Neuroscience, Intramural Research Program, National Institute of Mental Health, Porter Neuroscience Research Center Building 35 Room 1C903, Bethesda, Maryland 20892-3730, USA
Catherine Lord
‡Autism and Communication Disorders Center, University of Michigan, Ann Arbor, Michigan 48109-5406, USA
Jacqueline N Crawley
*Laboratory of Behavioral Neuroscience, Intramural Research Program, National Institute of Mental Health, Porter Neuroscience Research Center Building 35 Room 1C903, Bethesda, Maryland 20892-3730, USA
*
- Author information
- Copyright and License information
*Laboratory of Behavioral Neuroscience, Intramural Research Program, National Institute of Mental Health, Porter Neuroscience Research Center Building 35 Room 1C903, Bethesda, Maryland 20892-3730, USA
‡Autism and Communication Disorders Center, University of Michigan, Ann Arbor, Michigan 48109-5406, USA
✉
Correspondence to J.N.C. crawleyj@mail.nih.gov
© 2010 Macmillan Publishers Limited. All rights reserved
PMCID: PMC3087436 NIHMSID: NIHMS292369 PMID: 20559336
The publisher’s version of this article is available at Nat Rev Neurosci
Abstract
Autism is a heterogeneous neurodevelopmental disorder of unknown aetiology that affects 1 in 100–150 individuals. Diagnosis is based on three categories of behavioural criteria: abnormal social interactions, communication deficits and repetitive behaviours. Strong evidence for a genetic basis has prompted the development of mouse models with targeted mutations in candidate genes for autism. As the diagnostic criteria for autism are behavioural, phenotyping these mouse models requires behavioural assays with high relevance to each category of the diagnostic symptoms. Behavioural neuroscientists are generating a comprehensive set of assays for social interaction, communication and repetitive behaviours to test hypotheses about the causes of austism. Robust phenotypes in mouse models hold great promise as translational tools for discovering effective treatments for components of autism spectrum disorders.
Autism is a complex neurodevelopmental disorder with extraordinarily high heritability. Concordance between monozygotic twins reaches 90% for autism spectrum disorders (ASDs), as compared with less than 10% for dizygotic twins and siblings, and approximately 0.6–1.0% occurrence in the general population, along with a 4:1 male:female ratio1– 5. The number of reported cases of autism has risen rapidly over the past decade, largely due to better diagnostic instruments and public awareness, although environmental causes and gene–environment interactions are also under investigation6, 7. Considerable efforts are now focused on understanding the genetic causes of autism (see `Further Information’) and using the genetic findings to select rational targets for effective treatments. Large international consortia are conducting linkage analyses to identify chromosomal loci and association and whole-genome scans to discover candidate genes. Rare variants in candidate genes have been reported, both de novo and familial, as well as copy number variants and epigenetic factors8– 10. Strong evidence indicates that functionally interrelated mechanisms underlie the disorder. Synaptic development genes implicated in autism include neurexins, neuroligins, shanks, reelin, integrins, cadherins and contactins. However, each candidate gene mutation occurs in only a few individuals with autism1, 3, 9– 17. Signalling, transcription, methylation and neurotrophic genes implicated in ASDs include phosphatase and tensin homologue ( PTEN), MET, engrailed 2 ( EN2), methyl-CpG-binding protein 2 ( MECP2), fragile X mental retardation 1 ( FMR1), tuberous sclerosis 2 ( TSC2), calcium channel, voltage-dependent, L type, alpha 1C ( CACNA1C), ubiquitin ligase E3A ( UBE3A), Ca2+-dependent activator protein for secretion 2 ( CADPS2) and brain-derived neurotrophic factor ( BDNF)1, 10, 18– 25. Neurotransmission genes, including the serotonin transporter, oxytocin and vasopressin receptors and GABA (γ-aminobutyric acid) receptor subunit β3, have been repeatedly associated with autism or highly implicated in social and affiliative behaviours impaired in autism1, 26, 27. Copy number variants include chromosomal duplications at 15q11–13 and 17p11.2 and deletions at 16p11.2 and 22q13.3 ( Refs 8, 10, 15, 28– 32).
One compelling approach to test hypotheses about the many candidate genes for autism is to generate analogous mutations in the mouse genome and evaluate the mutant line for phenotypes analogous to the symptoms of autism33– 35. Effective animal models should incorporate face validity (strong analogies to the endophenotypes of the human syndrome), construct validity (the same biological dysfunction that causes the human disease, such as a gene mutation or anatomical abnormality) and predictive validity (analogous response to treatments that prevent or reverse symptoms in the human disease)36, 37. Mouse models have been generated with chromosomal deletions and with knockout and humanized knock-in mutations in many of the candidate genes detected in subsets of individuals with ASDs1, 25, 29, 38– 68. Mouse models with construct validity are being used to evaluate hypotheses about both genetic and environmental causes of autism, including single gene polymorphisms, copy number variants, epigenetic modifications, environmental toxins, prenatal infections, immune dysfunctions and mitochondrial abnormalities22, 63, 69– 77. Hypotheses about multiple risk genes and gene–environment interactions are tested in mouse models that incorporate construct validity for two or more hypothesized causes, using the same behavioural assays as read-outs. Naturally occurring phenotypic differences among inbred mouse strains have been successfully utilized to identify model systems with high face validity and cost efficiency78– 91. Phenotypes with strong face validity provide ideal translational tools for evidence-based treatment discovery22, 63, 73– 77, 88, 92. TABLE 1 presents examples of genetic mouse models displaying behavioural phenotypes that are relevant to the three diagnostic criteria for autism25, 29, 38– 41, 43– 68, 78– 87, 89– 91.
Table 1.
Examples of autism-relevant behaviours in genetic mouse models of autism spectrum disorders
| Mouse model | Genetic characteristics | Behavioural phenotypes relevant to the symptoms of autism * |
|---|---|---|
| Nlgn4 | Null mutation in the murine orthologue of the human NLGN4 gene43 | - Reduced reciprocal social interactions43 - Low sociability43 - Lack of preference for social novelty43 - Reduced ultrasonic vocalizations43 |
| Nlgn3 | Homozygous mutation of humanized R451C mutation of the Nlgn3 gene44, 45 | - No genotype differences in reciprocal social interactions44, 45 - No genotype differences in sociability44, 45 - No genotype differences in preference for social novelty44 - Reduced ultrasonic vocalizations44 |
| Null mutation in the murine orthologue of the human NLGN3 gene41 | - No genotype differences in reciprocal social interactions41 - Reduced preference for social novelty41 | |
| Neurexin 1α | Null mutation in the murine neurexin 1α generated by deleting the first exon of the gene46 | - No genotype differences in reciprocal social interactions46 - No genotype differences in sociability46 - Impaired nest-building behaviour46 - Increased repetitive self-grooming46 |
| Nlgn1 | Null mutation in the murine orthologue of the human NLGN1 gene47 | - No genotype differences in reciprocal social interactions47 - No genotype differences in sociability47 - No genotype differences in preference for social novelty47 - Impaired nest-building behaviour47 |
| Pten | Conditional null mutation, inactivated in neurons of the cortex and hippocampus, mouse orthologue of the human PTEN gene68 | - Reduced reciprocal social interactions68 - Low sociability68 - Impaired nest-building behaviour68 - Impaired social recognition68 |
| Pten haploinsufficent mutant line in which exon 5, and thus the core catalytic phosphatase domain, is deleted48 | - Low sociability in females48 | |
| En2 | Null mutation in the murine orthologue of the human EN2 gene49, 50 | - Reduced reciprocal social interactions49 - Increased repetitive self-grooming49 - No genotype differences in sociability, confounded by low activity levels50 |
| 15q11–13 | Duplication in the genomic region on the mouse chromosome 7 homologous to the human genomic region 15q11–13 ( REF. 29) | - Low sociability29 - Ultrasonic vocalizations elevated in pups and reduced in adults29 - Impaired reversal learning29 |
| 17p11.2 | Duplication in the genomic region of murine chromosome 11 homologous to the human genomic region 17p11.2 ( REF. 51) | - Low sociability51 - No genotype differences in preference for social novelty51 - Impaired nest-building behaviour51 |
| Gabrb3‡ | Null mutation in the murine orthologue of the human GABRB3 gene52 | - Low sociability‡ ( REF. 52) - Lack of preference for social novelty‡ ( REF. 52) - Repetitive stereotyped circling patterns‡ ( REF. 52) - Impaired nest-building behaviour‡ ( REF. 52) |
| Slc6a4 | Null mutation in the murine orthologue of the human serotonin transporter ( SLC6A4) gene50 | - Low sociability50 - Lack of preference for social novelty50 |
| Haploinsufficient mutant line of the human serotonin transporter SLC6A gene48 | - Impaired social recognition48 | |
| Oxt | Null mutation in the murine Oxt gene generated by either a deletion in the first exon40, 53, 54 or by deletions in the last two exons40 | - Impaired social recognition53 - Reduced pup ultrasonic vocalizations54 - No genotype differences in sociability40 - No genotype differences in preference for social novelty40 |
| Avpr1b | Null mutation of the murine vasopressin receptor 1b Avpr1b gene55, 56 | - Impaired social recognition55 - Reduced pup ultrasonic vocalizations56 |
| Mecp2 | Heterozygous mutation in methyl-CpG-binding protein 2 ( REFS 39, 57, 58, 59) | - Hindlimb clasping57, 58 - Social avoidance39 - Impaired social recognition59 - Reduced social interest in an arena59 |
| Fmr1 | Null mutant mouse with a targeted mutation in the Fmr1 gene in three genetic backgrounds: C57BL/6J38, 50, 60, 61; hybrid of FVB/NJ × C57BL/6J62; and FVB/N-129/OlaHsd50 | - Increased social approach60, 61 - Reduced reciprocal social interactions38 - No genotype differences in sociability62 - No genotype differences in preference for social novelty62 - Low sociability dependent on genetic background50 - No genotype differences in preference for social novelty50 |
| Tsc | Heterozygous mutation that replaces the second exon in the Tsc2 gene63 | - No genotype differences in sociability63 |
| Heterozygous mutation generated by replacing exons 6–8 in the Tsc1 gene65 | - Reduced reciprocal social interactions65 - Impaired nest-building behaviour65 | |
| Foxp2 | Homozygous and heterozygous mutations in the mouse homologue of the FOXP2 gene64 Knock-in mice for the mouse homologue of FOXP2 ( REF. 67) | - Reduced pup ultrasonic vocalizations64, 67 |
| Fgf17 | Null mutation in the murine Fgf17 gene generated by deletion of the sites that encode the signal peptide66 | - Reduced reciprocal social interactions66 - Lack of preference for social novelty66 - Reduced pup ultrasonic vocalizations66 |
| Cadps2 | Null mutation in murine orthologue of the Cadps2 gene25 | - Reduced reciprocal social interactions25 |
| BTBR | BTBR T + tf/J (BTBR strain) is a genetically homogenous inbred strain that displays behavioural traits with face validity to all three diagnostic symptoms of autism | - Reduced reciprocal social interactions78, 81, 90, 91, 111 - Low sociability81, 83, 88, 90, 91, 111 - Increased repetitive self-grooming81, 88, 90, 111 - Reduced social transmission of food preference81 - Ultrasonic vocalizations elevated in pups and reduced in adults87, 89 - Unusual ultrasonic vocalization call categories in pups and adults87, 135 |
| BALB | BALB/cJ and BALB/cByJ are genetically homogenous inbred strains that display relatively low social behaviour in various settings, reduced ultrasonic vocalizations and reduced empathy-like behaviour | - Low sociability79, 83 - No genotype differences in preference for social novelty83 - Reduced reciprocal social interactions84 - Reduced ultrasonic vocalizations in adolescent same-sex social interaction84 - Reduced place-conditioned social reward85 - Reduced social learning during social distress‡ ( REFS 80, 145) |
| C58/J | C58/J is a genetically homogenous inbred strain that displays low sociability, primarily in males, and high levels of two distinct repetitive behaviours that emerge early in development | - High level of repetitive motor stereotypies82, 86 - Low sociability82, 86 - Increased repetitive self-grooming86 |
*
Behavioural tests are described in the main text.
‡
Phenotypes of survivors.
Avpr1b, arginine vasopressin receptor 1b; Cadps2, Ca2+-dependent activator protein for secretion 2; En2, engrailed 2; Fgf17, fibroblast growth factor 17; Fmr1, fragile × mental retardation syndrome 1; Foxp2, forkhead box protein 2; Gabrb3, gamma-aminobutyric acid A receptor, subunit beta 3; Mecp2, methyl-CpG-binding protein 2; Nlgn, neuroligin; Oxt, oxytocin; Pten, phosphatase and tensin homologue; Slc6a4, solute carrier 6 member 4; Tsc, tuberous sclerosis.
How do we model the symptoms of autism in mice?
Designing mouse behavioural tasks that are relevant to human mental disorders presents a daunting challenge. Symptoms may be uniquely human and are often inherently variable. Autism diagnosis is currently based on purely behavioural criteria, as no consistent biological markers have yet been identified2, 93– 98. Until now, DSM-IV99, the diagnostic manual of the American Psychiatric Association, and ICD-10100, the diagnostic manual of the World Health Organization, have required the presence of core elements in three specific categories: abnormal reciprocal social interactions, which include reduced interest in peers and difficulty maintaining social interaction, and failure to use eye gaze and facial expressions to communicate efficiently; impaired communication, which generally presents as language delays, deficits in language comprehension and response to voices, stereotyped or literal use of words and phrases, poor pragmatics (knowing how and when to use language) and lack of prosody, resulting in monotone or exaggerated speech patterns; and repetitive behaviours, which include motor stereotypies, repetitive use of objects, compulsions and rituals, insistence on sameness, upset to change and unusual or very narrow restricted interests. Proposed DSM-V revisions may merge the first two criteria into a more general social-communication factor that includes lack of social reciprocity and deficits in nonverbal and verbal communication, beginning in early childhood.
Based on extensive advice generously contributed by autism clinical experts, behavioural neuroscientists are engaged in generating new mouse behavioural tasks and in refining existing paradigms from the behavioural neuroscience literature that maximize face validity to each of the core symptoms. Here, we review the tests that have proven most useful, along with the essential control measures, for the triad of diagnostic features of autism. Neuroanatomical, biochemical, electrophysiological and genetic similarities between mice and humans support the use of mouse models to further our understanding of biological mechanisms underlying the behavioural manifestations of autism. Similar responses to pharmacological treatments in mice and humans encourage the use of well-validated mouse models in the discovery of effective therapeutics for ASD.
Assays for social interaction abnormalities in mice
Mus musculus is a social species that engages in high levels of reciprocal social interactions, communal nesting, sexual and parenting behaviours, territorial scent marking and aggressive behaviours101– 105. A variety of social assays have been described in the behavioural neuroscience literature34, 37. The examples described below were designed to maximize relevance to the types of social deficits that are specific to autism.
Reciprocal social interactions
Fine-grained measures of interactions between pairs or groups of juvenile or adult mice placed together in standard cages or specialized arenas provide the most detailed insights into reciprocal social interactions. Parameters routinely evaluated include nose-to-nose sniffing, nose-to-anogenital sniffing, following, pushing past each other with physical contact, crawling over and under each other with physical contact, chasing, mounting and wrestling78, 81, 90, 103, 106. Parameters are scored from videotapes by investigators, using data sheets or event-recording software. Automated videotracking systems have also been used to score social interactions between two mice41, 107. The experimental design, including the specific parameters scored, session duration, time of day, prior social isolation, environmental enrichment and pair composition by age, sex and strain, is optimized to meet the goals of the experiment. Repeated testing of the same mice is usually possible; this allows researchers to evaluate trajectories across the neurodevelopmental stages of pup, juvenile, young adult and older adult. FIG. 1 and Supplementary information S1 (movie) illustrate reciprocal social interactions in mice.
Figure 1. Reciprocal social interactions.
a | The Noldus PhenoTyper 3000 apparatus containing two unfamiliar juvenile male C57BL/6J (B6) mice engaged in social interaction. b | Nose-to-nose sniffing between two unfamiliar juvenile male B6 mice. A video camera records the 10-minute session. A human observer, uninformed of the treatment condition, scores parameters of social interaction and non-social exploration of the arena using Noldus Observer event-recording software. Social parameters scored include following (one mouse walks closely behind the other, keeping pace) and push–crawl (physical contact includes pushing the snout or head underneath the partner’s body, squeezing between the partner and the arena wall or floor, and crawling over or under the partner’s body). Non-social parameters include self-grooming (the mouse grooms its face and body regions in a normal sequential pattern) and arena exploration (walking around the arena, sniffing the walls, floor and bedding, and digging in the bedding). Detailed scoring methods are described in Refs 44, 81, 90, 91, 111. c | Representative data for reciprocal social interactions in pairs of juvenile males of two high-sociability inbred strains of mice, B6 and FVB/Ant, and a low-sociability strain, BTBR T+tf/J (BTBR). BTBR mice exhibited lower levels of following and push–crawl and higher levels of self-grooming and arena exploration than B6 mice, as previously reported81, 90, 111. FVB/Ant exhibited high levels of following and push–crawl similar to B6, low self-grooming similar to B6, and arena exploration similar to BTBR. These data further support the interpretation of a specific social deficit and unusual repetitive behaviour in BTBR mice. n = 12 B6 mice, 16 FVB/Ant mice and 12 BTBR mice. * p < 0.05 compared with B6 mice.
Social approach
Simpler, automated measures of direct social approach offer more standardized, higher-throughput assays, although fewer details of reciprocal interactions are captured. We developed an automated three-chambered social approach task, which scores time spent in a side chamber with a novel mouse versus time spent in a side chamber with a non-social novel object, an inverted wire pencil cup44, 81, 90, 108, 109. Sociability is defined as the subject mice spending more time in the chamber containing the novel target mouse than in the chamber containing the inanimate novel object. The wire cup serves as the novel object on one side and as the container control for novel object plus novel mouse on the other side. With the target novel mouse contained, the social approach is initiated by the subject mouse only. The widely spaced wire bars of the container permit olfactory, visual, auditory and some tactile contact while preventing aggressive and sexual interactions, thus ensuring a pure measure of simple interest in approaching and remaining in physical proximity to another.
Our photocell-equipped apparatus uses infrared beams embedded in the partitions between compartments40, 44, 81, 83, 90, 91, 109– 111. As the subject mouse moves between the three compartments, beam-breaks are recorded by the software and converted to time the mouse spends in each compartment and number of entries into each compartment. To provide a corroborative and more specific measure of social investigation during the test session, an observer scores time spent sniffing the novel mouse and time spent sniffing the novel object from session videotapes or in real time. The number of entries between compartments provides an independent measure of general exploratory locomotion. Mice can be tested more than once in this task — for example, at different ages to follow developmental trajectories. Videotracking software systems have been successfully used with the three-chambered apparatus, as well as observer scoring from videotapes43, 48, 77, 107. FIG. 2 and Supplementary information S2 (movie) illustrate the automated social approach test in mice.
Figure 2. Automated three-chambered social approach.
a | The test apparatus, a rectangular, three-chambered box made of clear polycarbonate44, 81, 82, 83, 90, 91, 109, 111. Retractable doorways built into the two dividing walls control access to the side chambers. Entries into each chamber are automatically detected by photocells embedded in the doorways. The number of entries and time spent in each chamber are tallied by the software. The test session begins with a 10-minute habituation session in the centre chamber only, followed by a 10-minute habituation session with access to all 3 empty chambers. If an innate side preference for either the right or left chamber is detected during the habituation session, the testing environment is reorganized to equalize light levels, nearby objects, and so on. The subject is then briefly confined to the centre chamber while a novel object (an inverted stainless steel wire pencil cup) is placed in one of the side chambers. A novel mouse, previously habituated to the enclosure, is placed in an identical wire cup located in the other side chamber. A weighted plastic cup is placed on the top of each inverted wire cup to prevent the subject from climbing on top. The side chambers containing the novel object and the novel mouse are alternated between left and right across subjects. After the novel object and the novel mouse are positioned, the two side doors are simultaneously lifted and the subject is allowed access to all three chambers for 10 minutes. In addition to the automatically tallied time spent in each chamber and entries into each chamber, an observ
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