Female mice prenatally exposed to valproic acid exhibit complex and prolonged social behavior deficits.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized mainly by deficits in social communication and stereotyped and restricted behavior and interests with a male to female bias of 4.2/1. Social behavior in ASD animal models is commonly analyzed in males, and seldomly in females, using the widely implemented three-chambers test procedure. Here, we implemented a novel procedure, the Live Mouse Tracker (LMT), combining artificial intelligence, machine learning procedures and behavioral measures. We used it on mice that were prenatally exposed to valproic acid (VPA) (450 mg/kg) at embryonic day 12.5, a widely recognized and potent ASD model that we had previously extensively characterized. We focused on female mice offspring, in which social deficits have been rarely documented when using the 3-CT procedure. We recorded several parameters related to social behavior in these mice, continuously for three days in groups of four female mice. Comparisons were made on groups of 4 female mice with the same treatment (4 saline or 4 VPA) or with different treatments (3 saline and 1 VPA). We report that VPA females show several types of social deficits, which are different in nature and magnitude in relation with time. When VPA mice were placed in the LMT alongside saline mice, their social deficits showed significant improvement as early as 1 h from the start of the experiment, lasting up to 3 days throughout the duration of the experiment. Our findings suggest that ASD may be underdiagnosed in females. They also imply that ASD-related social deficits can be ameliorated by the presence of typical individuals.

A systematic review of the development and application of home cage monitoring in laboratory mice and rats.

BACKGROUND: Traditionally, in biomedical animal research, laboratory rodents are individually examined in test apparatuses outside of their home cages at selected time points. However, the outcome of such tests can be influenced by various factors and valuable information may be missed when the animals are only monitored for short periods. These issues can be overcome by longitudinally monitoring mice and rats in their home cages. To shed light on the development of home cage monitoring (HCM) and the current state-of-the-art, a systematic review was carried out on 521 publications retrieved through PubMed and Web of Science. RESULTS: Both the absolute (~ × 26) and relative (~ × 7) number of HCM-related publications increased from 1974 to 2020. There was a clear bias towards males and individually housed animals, but during the past decade (2011-2020), an increasing number of studies used both sexes and group housing. In most studies, animals were kept for short (up to 4 weeks) time periods in the HCM systems; intermediate time periods (4-12 weeks) increased in frequency in the years between 2011 and 2020. Before the 2000s, HCM techniques were predominantly applied for less than 12 h, while 24-h measurements have been more frequent since the 2000s. The systematic review demonstrated that manual monitoring is decreasing in relation to automatic techniques but still relevant. Until (and including) the 1990s, most techniques were applied manually but have been progressively replaced by automation since the 2000s. Independent of the year of publication, the main behavioral parameters measured were locomotor activity, feeding, and social behaviors; the main physiological parameters were heart rate and electrocardiography. External appearance-related parameters were rarely examined in the home cages. Due to technological progress and application of artificial intelligence, more refined and detailed behavioral parameters have been investigated in the home cage more recently. CONCLUSIONS: Over the period covered in this study, techniques for HCM of mice and rats have improved considerably. This development is ongoing and further progress as well as validation of HCM systems will extend the applications to allow for continuous, longitudinal, non-invasive monitoring of an increasing range of parameters in group-housed small rodents in their home cages.

Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice.

Autism is characterized by atypical social communication and stereotyped behaviors. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1-2% of patients with autism and intellectual disability, but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterized the behavior of Shank3 Δ11/Δ11 mice from 3 to 12 months of age. We observed decreased locomotor activity, increased stereotyped self-grooming and modification of socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEGs). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g., Grm2, Dlgap1), G-protein-signaling pathways (e.g., Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g., Gad2). Downregulated and upregulated genes were enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN), respectively. Several DEGs (Cnr1, Gnal, Gad2, and Drd4) were reported as striosome markers. By studying the distribution of the glutamate decarboxylase GAD65, encoded by Gad2, we showed that the striosome compartment of Shank3 Δ11/Δ11 mice was enlarged and displayed much higher expression of GAD65 compared to wild-type mice. Altogether, these results indicate altered gene expression in the striatum of Shank3-deficient mice and strongly suggest, for the first time, that the excessive self-grooming of these mice is related to an imbalance in the striatal striosome and matrix compartments.

Day-to-day spontaneous social behaviours is quantitatively and qualitatively affected in a 16p11.2 deletion mouse model.

Background: Autism spectrum disorders affect more than 1% of the population, impairing social communication and increasing stereotyped behaviours. A micro-deletion of the 16p11.2 BP4-BP5 chromosomic region has been identified in 1% of patients also displaying intellectual disabilities. In mouse models generated to understand the mechanisms of this deletion, learning and memory deficits were pervasive in most genetic backgrounds, while social communication deficits were only detected in some models. Methods: To complement previous studies, we itemized the social deficits in the mouse model of 16p11.2 deletion on a hybrid C57BL/6N × C3H.Pde6b+ genetic background. We examined whether behavioural deficits were visible over long-term observation periods lasting several days and nights, to parallel everyday-life assessment of patients. We recorded the individual and social behaviours of mice carrying a heterozygous deletion of the homologous 16p11.2 chromosomic region (hereafter Del/+) and their wild-type littermates from both sexes over two or three consecutive nights during social interactions of familiar mixed-genotype quartets of males and of females, and of same-genotype unfamiliar female pairs. Results: We observed that Del/+ mice of both sexes increased significantly their locomotor activity compared to wild-type littermates. In the social domain, Del/+ mice of both sexes displayed widespread deficits, even more so in males than in females in quartets of familiar individuals. In pairs, significant perturbations of the organisation of the social communication and behaviours appeared in Del/+ females. Discussion: Altogether, this suggests that, over long recording periods, the phenotype of the 16p11.2 Del/+ mice was differently affected in the locomotor activity and the social domains and between the two sexes. These findings confirm the importance of testing models in long-term conditions to provide a comprehensive view of their phenotype that will refine the study of cellular and molecular mechanisms and complement pre-clinical targeted therapeutic trials.

LMT USV Toolbox, a Novel Methodological Approach to Place Mouse Ultrasonic Vocalizations in Their Behavioral Contexts-A Study in Female and Male C57BL/6J Mice and in Shank3 Mutant Females.

Ultrasonic vocalizations (USVs) are used as a phenotypic marker in mouse models of neuropsychiatric disorders. Nevertheless, current methodologies still require time-consuming manual input or sound recordings clean of any background noise. We developed a method to overcome these two restraints to boost knowledge on mouse USVs. The methods are freely available and the USV analysis runs online at https://usv.pasteur.cloud. As little is currently known about usage and structure of ultrasonic vocalizations during social interactions over the long-term and in unconstrained context, we investigated mouse spontaneous communication by coupling the analysis of USVs with automatic labeling of behaviors. We continuously recorded during 3 days undisturbed interactions of same-sex pairs of C57BL/6J sexually naive males and females at 5 weeks and 3 and 7 months of age. In same-sex interactions, we observed robust differences between males and females in the amount of USVs produced, in the acoustic structure and in the contexts of emission. The context-specific acoustic variations emerged with increasing age. The emission of USVs also reflected a high level of excitement during social interactions. We finally highlighted the importance of studying long-term spontaneous communication by investigating female mice lacking Shank3, a synaptic protein associated with autism. While the previous short-time constrained investigations could not detect USV emission abnormalities, our analysis revealed robust differences in the usage and structure of the USVs emitted by mutant mice compared to wild-type female pairs.

Testosterone Increases the Emission of Ultrasonic Vocalizations With Different Acoustic Characteristics in Mice.

Testosterone masculinizes male sexual behavior through an organizational and activational effects. We previously reported that the emission of ultrasonic vocalizations (USVs) in male mice was dependent on the organizational effects of testosterone; females treated with testosterone in the perinatal and peripubertal periods, but not in adults, had increased USV emissions compared to males. Recently, it was revealed that male USVs have various acoustic characteristics and these variations were related to behavioral interactions with other mice. In this regard, the detailed acoustic characteristic changes induced by testosterone have not been fully elucidated. Here, we revealed that testosterone administered to female and male mice modulated the acoustic characteristics of USVs. There was no clear difference in acoustic characteristics between males and females. Call frequencies were higher in testosterone propionate (TP)-treated males and females compared to control males and females. When the calls were classified into nine types, there was also no distinctive difference between males and females, but TP increased the number of calls with a high frequency, and decreased the number of calls with a low frequency and short duration. The transition analysis by call type revealed that even though there was no statistically significant difference, TP-treated males and females had a similar pattern of transition to control males and females, respectively. Collectively, these results suggest that testosterone treatment can enhance the emission of USVs both in male and female, but the acoustic characteristics of TP-treated females were not the same as those of intact males.

Dlx5 and Dlx6 expression in GABAergic neurons controls behavior, metabolism, healthy aging and lifespan.

Dlx5 and Dlx6 encode two homeobox transcription factors expressed by developing and mature GABAergic interneurons. During development, Dlx5/6 play a role in the differentiation of certain GABAergic subclasses. Here we address the question of the functional role of Dlx5/6 in the mature central nervous system. First, we demonstrate that Dlx5 and Dlx6 are expressed by all subclasses of adult cortical GABAergic neurons. Then we analyze VgatΔDlx5-6 mice in which Dlx5 and Dlx6 are simultaneously inactivated in all GABAergic interneurons. VgatΔDlx5-6 mice present a behavioral pattern suggesting reduction of anxiety-like behavior and obsessive-compulsive activities, and a lower interest in nest building. Twenty-month-old VgatΔDlx5-6 animals have the same size as their normal littermates, but present a 25% body weight reduction associated with a marked decline in white and brown adipose tissue. Remarkably, both VgatΔDlx5-6/+ and VgatΔDlx5-6 mice present a 33% longer median survival. Hallmarks of biological aging such as motility, adiposity and coat conditions are improved in mutant animals. Our data imply that GABAergic interneurons can regulate healthspan and lifespan through Dlx5/6-dependent mechanisms. Understanding these regulations can be an entry point to unravel the processes through which the brain affects body homeostasis and, ultimately, longevity and healthy aging.

Real-time analysis of the behaviour of groups of mice via a depth-sensing camera and machine learning.

Preclinical studies of psychiatric disorders use animal models to investigate the impact of environmental factors or genetic mutations on complex traits such as decision-making and social interactions. Here, we introduce a method for the real-time analysis of the behaviour of mice housed in groups of up to four over several days and in enriched environments. The method combines computer vision through a depth-sensing infrared camera, machine learning for animal and posture identification, and radio-frequency identification to monitor the quality of mouse tracking. It tracks multiple mice accurately, extracts a list of behavioural traits of both individuals and the groups of mice, and provides a phenotypic profile for each animal. We used the method to study the impact of Shank2 and Shank3 gene mutations-mutations that are associated with autism-on mouse behaviour. Characterization and integration of data from the behavioural profiles of Shank2 and Shank3 mutant female mice revealed their distinctive activity levels and involvement in complex social interactions.

Shank2 Mutant Mice Display Hyperactivity Insensitive to Methylphenidate and Reduced Flexibility in Social Motivation, but Normal Social Recognition.

Mouse models of autism can be used to study evolutionarily conserved mechanisms underlying behavioral abnormalities in social communication and repetitive behaviors. SHANK genes code for synaptic scaffolding proteins at excitatory synapses and mutations in all SHANK genes have been associated with autism. Here, we present three behavioral aspects of the mutant mice deleted for exon 16 in Shank2. First, we treated Shank2 mutant mice with methylphenidate to rescue the hyperactivity. Our failure to do so suggests that the hyperactivity displayed by Shank2 mutant mice is not related to the one displayed by the typical mouse models of hyperactivity, and might be more closely related to manic-like behaviors. Second, by testing the effect of group housing and social isolation on social interest, we highlighted that Shank2 mutant mice lack the typical flexibility to modulate social interest, in comparison with wild-type littermates. Finally, we established a new protocol to test for social recognition in a social context. We used this protocol to show that Shank2 mutant mice were able to discriminate familiar and unknown conspecifics in free interactions. Altogether, these studies shed some light on specific aspects of the behavioral defects displayed by the Shank2 mouse model. Such information could be used to orient therapeutic strategies and to design more specific tests to characterize the complex behavior of mouse models of autism.

Genetic identification of a hindbrain nucleus essential for innate vocalization.

Vocalization in young mice is an innate response to isolation or mechanical stimulation. Neuronal circuits that control vocalization and breathing overlap and rely on motor neurons that innervate laryngeal and expiratory muscles, but the brain center that coordinates these motor neurons has not been identified. Here, we show that the hindbrain nucleus tractus solitarius (NTS) is essential for vocalization in mice. By generating genetically modified newborn mice that specifically lack excitatory NTS neurons, we show that they are both mute and unable to produce the expiratory drive required for vocalization. Furthermore, the muteness of these newborns results in maternal neglect. We also show that neurons of the NTS directly connect to and entrain the activity of spinal (L1) and nucleus ambiguus motor pools located at positions where expiratory and laryngeal motor neurons reside. These motor neurons control expiratory pressure and laryngeal tension, respectively, thereby establishing the essential biomechanical parameters used for vocalization. In summary, our work demonstrates that the NTS is an obligatory component of the neuronal circuitry that transforms breaths into calls.

Behavioural Phenotypes and Neural Circuit Dysfunctions in Mouse Models of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental condition primarily characterised by alterations in social interaction and communication combined with the presence of restricted interests and stereotyped behaviours. Mutations in several genes have been associated with ASD resulting in the generation of corresponding mouse models. Here, we focus on the behavioural (social and stereotyped behaviours), functional and structural traits of mice with mutations in genes encoding defined synaptic proteins including adhesion proteins, scaffolding proteins and subunits of channels and receptors. A meta-analysis on ASD mouse models shows that they can be divided into two subgroups. Cluster I gathered models highly impaired in social interest, stereotyped behaviours, synaptic physiology and protein composition, while Cluster II regrouped much less impaired models, with typical social interactions. This distribution was not related to gene families. Even within the large panel of mouse models carrying mutations in Shank3, the number of mutated isoforms was not related to the severity of the phenotype. Our study points that the majority of structural or functional analyses were performed in the hippocampus. However, to robustly link the structural and functional impairments with the behavioural deficits observed, brain structures forming relevant nodes in networks involved in social and stereotyped behaviours should be targeted in the future. In addition, the characterisation of core ASD-like behaviours needs to be more detailed using new approaches quantifying the variations in social motivation, recognition and stereotyped behaviours.

mouseTube - a database to collaboratively unravel mouse ultrasonic communication.

Ultrasonic vocalisation is a broadly used proxy to evaluate social communication in mouse models of neuropsychiatric disorders. The efficacy and robustness of testing these models suffer from limited knowledge of the structure and functions of these vocalisations as well as of the way to analyse the data. We created mouseTube, an open database with a web interface, to facilitate sharing and comparison of ultrasonic vocalisations data and metadata attached to a recording file. Metadata describe 1) the acquisition procedure, e.g., hardware, software, sampling frequency, bit depth; 2) the biological protocol used to elicit ultrasonic vocalisations; 3) the characteristics of the individual emitting ultrasonic vocalisations ( e.g., strain, sex, age). To promote open science and enable reproducibility, data are made freely available. The website provides searching functions to facilitate the retrieval of recording files of interest. It is designed to enable comparisons of ultrasonic vocalisation emission between strains, protocols or laboratories, as well as to test different analysis algorithms and to search for protocols established to elicit mouse ultrasonic vocalisations. Over the long term, users will be able to download and compare different analysis results for each data file. Such application will boost the knowledge on mouse ultrasonic communication and stimulate sharing and comparison of automatic analysis methods to refine phenotyping techniques in mouse models of neuropsychiatric disorders.

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication.

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies for modeling the genetic bases of vocal communication deficits. Characterizing the vocal behavior of mouse models carrying mutations in genes associated with neuropsychiatric disorders such as autism spectrum disorders will help to understand the mechanisms leading to social communication deficits. We provide here protocols to reliably elicit ultrasonic vocalizations in pups and in adult mice. This standardization will help reduce inter-study variability due to the experimental settings. Pup isolation calls are recorded throughout development from individual pups isolated from dam and littermates. In adulthood, vocalizations are recorded during same-sex interactions (without a sexual component) by exposing socially motivated males or females to an unknown same-sex conspecific. We also provide a protocol to record vocalizations from adult males exposed to an estrus female. In this context, there is a sexual component in the interaction. These protocols are established to elicit a large amount of ultrasonic vocalizations in laboratory mice. However, we point out the important inter-individual variability in the vocal behavior of mice, which should be taken into account by recording a minimal number of individuals (at least 12 in each condition). These recordings of ultrasonic vocalizations are used to evaluate the call rate, the vocal repertoire and the acoustic structure of the calls. Data are combined with the analysis of synchronous video recordings to provide a more complete view on social communication in mice. These protocols are used to characterize the vocal communication deficits in mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders. More ultrasonic vocalizations recordings can also be found on the mouseTube database, developed to favor the exchange of such data.

Social communication in mice--are there optimal cage conditions?

Social communication is heavily affected in patients with neuropsychiatric disorders. Accordingly, mouse models designed to study the mechanisms leading to these disorders are tested for this phenotypic trait. Test conditions vary between different models, and the effect of these test conditions on the quantity and quality of social interactions and ultrasonic communication is unknown. The present study examines to which extent the habituation time to the test cage as well as the shape/size of the cage influence social communication in freely interacting mice. We tested 8 pairs of male mice in free dyadic social interactions, with two habituation times (20 min and 30 min) and three cage formats (rectangle, round, square). We tested the effect of these conditions on the different types of social contacts, approach-escape sequences, follow behavior, and the time each animal spent in the vision field of the other one, as well as on the emission of ultrasonic vocalizations and their contexts of emission. We provide for the first time an integrated analysis of the social interaction behavior and ultrasonic vocalizations. Surprisingly, we did not highlight any significant effect of habituation time and cage shape/size on the behavioral events examined. There was only a slight increase of social interactions with the longer habituation time in the round cage. Remarkably, we also showed that vocalizations were emitted during specific behavioral sequences especially during close contact or approach behaviors. The present study provides a protocol reliably eliciting social contacts and ultrasonic vocalizations in adult male mice. This protocol is therefore well adapted for standardized investigation of social interactions in mouse models of neuropsychiatric disorders.

Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: a gradient of severity in cognitive impairments.

SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.

Olfactory preference conditioning changes the reward value of reinforced and non-reinforced odors.

Olfaction is determinant for the organization of rodent behavior. In a feeding context, rodents must quickly discriminate whether a nutrient can be ingested or whether it represents a potential danger to them. To understand the learning processes that support food choice, aversive olfactory learning and flavor appetitive learning have been extensively studied. In contrast, little is currently known about olfactory appetitive learning and its mechanisms. We designed a new paradigm to study conditioned olfactory preference in rats. After 8 days of exposure to a pair of odors (one paired with sucrose and the other with water), rats developed a strong and stable preference for the odor associated with the sucrose solution. A series of experiments were conducted to further analyze changes in reward value induced by this paradigm for both stimuli. As expected, the reward value of the reinforced odor changed positively. Interestingly, the reward value of the alternative odor decreased. This devaluation had an impact on further odor comparisons that the animal had to make. This result suggests that appetitive conditioning involving a comparison between two odors not only leads to a change in the reward value of the reinforced odor, but also induces a stable devaluation of the non-reinforced stimulus.

Heterogeneous pattern of selective pressure for PRRT2 in human populations, but no association with autism spectrum disorders.

Inherited and de novo genomic imbalances at chromosome 16p11.2 are associated with autism spectrum disorders (ASD), but the causative genes remain unknown. Among the genes located in this region, PRRT2 codes for a member of the synaptic SNARE complex that allows the release of synaptic vesicles. PRRT2 is a candidate gene for ASD since homozygote mutations are associated with intellectual disability and heterozygote mutations cause benign infantile seizures, paroxysmal dyskinesia, or hemiplegic migraine. Here, we explored the contribution of PRRT2 mutations in ASD by screening its coding part in a large sample of 1578 individuals including 431 individuals with ASD, 186 controls and 961 individuals from the human genome Diversity Panel. We detected 24 nonsynonymous variants, 1 frameshift (A217PfsX8) and 1 in-frame deletion of 6 bp (p.A361_P362del). The frameshift mutation was observed in a control with no history of neurological or psychiatric disorders. The p.A361_P362del was observed in two individuals with autism from sub-Saharan African origin. Overall, the frequency of PRRT2 deleterious variants was not different between individuals with ASD and controls. Remarkably, PRRT2 displays a highly significant excess of nonsynonymous (pN) vs synonymous (pS) mutations in Asia (pN/pS = 4.85) and Europe (pN/pS = 1.62) compared with Africa (pN/pS = 0.26; Asia vs Africa: P = 0.000087; Europe vs Africa P = 0.00035; Europe vs Asia P = P = 0.084). We also showed that whole genome amplification performed through rolling cycle amplification could artificially introduce the A217PfsX8 mutation indicating that this technology should not be performed prior to PRRT2 mutation screening. In summary, our results do not support a role for PRRT2 coding sequence variants in ASD, but provide an ascertainment of its genetic variability in worldwide populations that should help researchers and clinicians to better investigate the role of PRRT2 in human diseases.

Assessing behavioural and cognitive domains of autism spectrum disorders in rodents: current status and future perspectives.

The establishment of robust and replicable behavioural testing paradigms with translational value for psychiatric diseases is a major step forward in developing and testing etiology-directed treatment for these complex disorders. Based on the existing literature, we have generated an inventory of applied rodent behavioural testing paradigms relevant to autism spectrum disorders (ASD). This inventory focused on previously used paradigms that assess behavioural domains that are affected in ASD, such as social interaction, social communication, repetitive behaviours and behavioural inflexibility, cognition as well as anxiety behaviour. A wide range of behavioural testing paradigms for rodents were identified. However, the level of face and construct validity is highly variable. The predictive validity of these paradigms is unknown, as etiology-directed treatments for ASD are currently not on the market. To optimise these studies, future efforts should address aspects of reproducibility and take into account data about the neurodevelopmental underpinnings and trajectory of ASD. In addition, with the increasing knowledge of processes underlying ASD, such as sensory information processes and synaptic plasticity, phenotyping efforts should include multi-level automated analysis of, for example, representative task-related behavioural and electrophysiological read-outs.

The Autism ProSAP1/Shank2 mouse model displays quantitative and structural abnormalities in ultrasonic vocalisations.

Mouse ultrasonic vocalisations have been often used as a paradigm to extrapolate vocal communication defects observed in patients with autism spectrum disorders (ASD). The role of these vocalisations as well as their development, structure and informational content, however, remain largely unknown. In the present study, we characterised in depth the emission of pup and adult ultrasonic vocalisations of wild-type mice and their ProSAP1/Shank2(-/-) littermates lacking a synaptic scaffold protein mutated in ASD. We hypothesised that the vocal behaviour of ProSAP1/Shank2(-/-) mice not only differs from the vocal behaviour of their wild-type littermates in a quantitative way, but also presents more qualitative abnormalities in temporal organisation and acoustic structure. We first quantified the rate of emission of ultrasonic vocalisations, and analysed the organisation of vocalisations sequences using Markov models. We subsequently measured duration and peak frequency characteristics of each ultrasonic vocalisation, to characterise their acoustic structure. In wild-type mice, we found a high level of organisation in sequences of ultrasonic vocalisations, suggesting a communicative function in this complex system. Very limited significant sex-related variations were detected in their usage and acoustic structure, even in adult mice. In adult ProSAP1/Shank2(-/-) mice, we found abnormalities in the call usage and the structure of ultrasonic vocalisations. Both ProSAP1/Shank2(-/-) male and female mice uttered less vocalisations with a different call distribution and at lower peak frequency in comparison with wild-type littermates. This study provides a comprehensive framework to characterise abnormalities of ultrasonic vocalisations in mice and confirms that ProSAP1/Shank2(-/-) mice represent a relevant model to study communication defects.